section,content,source,topic,year
Summary of the Hallmarks of Aging,"Aging is driven by hallmarks fulfilling the following three premises: (1) their age-associated manifestation, (2) the acceleration of aging by experimentally accentuating them, and (3) the opportunity to decelerate, stop, or reverse aging by therapeutic interventions on them. We propose the following twelve hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. These hallmarks are interconnected among each other, as well as to the recently proposed hallmarks of health, which include organizational features of spatial compartmentalization, maintenance of homeostasis, and adequate responses to stress.",Cell,Hallmarks of Aging,2022
Genomic Instability and DNA Damage Sources,"Genome integrity and stability are pervasively threatened by exogenous chemical, physical, and biological agents, as well as by endogenous challenges such as DNA replication errors, chromosome segregation defects, oxidative processes, and spontaneous hydrolytic reactions. The wide range of genetic lesions caused by these extrinsic or intrinsic sources of damage include point mutations, deletions, translocations, telomere shortening, single- and double-strand breaks, chromosomal rearrangements, defects in nuclear architecture, and gene disruption caused by the integration of viruses or transposons. All these molecular alterations and the resulting genomic mosaicism may contribute to both normal and pathological aging.",Cell,Genomic Instability,2022
DNA Repair Networks and Age-Related Decline,"Accordingly, organisms have evolved a complex array of DNA repair and maintenance mechanisms to deal with the damage inflicted to nuclear and mitochondrial DNA (mtDNA) and to ensure the appropriate chromosomal architecture and stability. These DNA repair networks lose efficiency with age, which accentuates the accumulation of genomic damage and the ectopic accumulation of DNA in the cytosol.",Cell,Genomic Instability,2022
Somatic Mutations and Genomic Alterations in Aging,"Cells from aged humans and model organisms accumulate somatic mutations at nuclear DNA. Other forms of damage, such as chromosomal aneuploidy and copy-number variations, are also associated with aging. All these DNA alterations may affect essential genes and transcriptional pathways, resulting in dysfunctional cells that may finally compromise tissue and organismal homeostasis. This is especially relevant when DNA damage impacts on stem cells, hampering their role in tissue renewal or leading to their exhaustion, which in turn promotes aging and increases susceptibility to age-related pathologies. The mutational burden in histologically normal human tissues is remarkable. For example, normal esophageal epithelium cells from young individuals already display hundreds of mutations and may carry more than 2,000 mutations per cell by middle age. The accumulation of DNA mutations throughout life is likely tolerated because of the excessive energetic cost of the complete repair of all genomic damages caused by exogenous and endogenous challenges. Consequently, cells favor survival over genomic integrity.",Cell,Genomic Instability,2022
"Mutation Rates, DNA Repair, and Longevity Correlation","These data also suggest that similar to carcinogenesis, driver mutations alone may not be sufficient to accelerate aging because they require a permissive microenvironment created by non-mutagenic promoting factors to become penetrant. Comparative analysis of the mutational landscape across mammalian species has shown that species-specific somatic mutation rate is inversely correlated with lifespan. To date, there is no clear evidence that the normal rate of mutation fixation is responsible for aging, but numerous studies have shown that DNA repair deficiencies have the potential to cause aging. Thus, alterations in DNA repair mechanisms accelerate aging in mice and underlie several human progeroid syndromes. Conversely, transgenic mice overexpressing the mitotic checkpoint kinase BubR1 exhibit an extended healthy lifespan. Moreover, studies in humans and other long-lived species have shown that enhanced DNA repair mechanisms coevolve with increased longevity.",Cell,Genomic Instability,2022
SIRT6 and DNA Repair Enhancement,"Sirtuin-6 (SIRT6) may play a major role in this differential reparative efficiency across species. Overexpression of SIRT6 in mice reduces genomic instability, improves double-strand break repair, and extends lifespan, although other explanations, such as improved glucose metabolism and restoration of energy homeostasis, have been proposed to explain the prolongevity effects of SIRT6. Notably, recent work has shown that small-molecule activation of 8-oxoguanine DNA glycosylase 1 increases oxidative DNA damage repair and may have therapeutic applications in the context of aging and other processes linked to excessive oxidative damage. These findings suggest that interventions aimed at reducing the mutational load of nuclear DNA or at enhancing or rerouting its repair mechanisms may delay aging and the onset of age-related diseases, but further causal evidence in this regard is still missing.",Cell,Genomic Instability,2022
Mitochondrial DNA Instability and Aging,"Genomic instability affecting mtDNA may contribute to aging and age-related pathologies. mtDNA is strongly impacted by aging-associated mutations and deletions due to its high replicative index, the limited efficiency of its repair mechanisms, its oxidative microenvironment, and the lack of protective histones embracing this small DNA molecule. Somatic mtDNA alterations increase across human tissues during aging, but it remains unclear whether this increase truly impacts the aging process at the functional level. The causal implication of mtDNA mutations in driving aging has been difficult to assess because of “heteroplasmy,” which implies the co-existence of mutated and wild-type genomes within the same cell. However, deep-sequencing of aged cells revealed that their mtDNA mutational load may substantially increase through clonal expansion events. The accelerated expansion of mitochondrial mutations with age has also been observed in both primate oocytes and somatic tissues, as well as in lymphoblasts from patients with neurodegenerative diseases.",Cell,Mitochondrial DNA,2022
Sources and Implications of mtDNA Mutations,"Of note, ultra-sensitive sequencing indicates that most mtDNA mutations in aged cells arise from replication errors caused by mtDNA polymerase γ rather than from oxidative stress. Preliminary evidence that mtDNA mutations might be directly involved in aging and age-related pathologies was provided by human disorders that are caused by mtDNA damage and partially phenocopy aging. Further causative evidence has arisen from studies on mice deficient in DNA polymerase γ that exhibit accelerated aging and reduced lifespan associated with deletions rather than point mutations in mtDNA. Overall, these data suggest that the avoidance, attenuation, or correction of mtDNA mutations might contribute to extend healthspan and lifespan. Nevertheless, as in the case of nuclear DNA mutations, experimental evidence demonstrating deceleration of aging by gain of function in mtDNA repair mechanisms is still largely missing.",Cell,Mitochondrial DNA,2022
Nuclear Architecture and Genome Stability,"Defects in the nuclear lamina, which constitutes a scaffold for tethering chromatin and protein complexes, can generate genome instability. Accelerated aging syndromes such as the Hutchinson-Gilford and the Néstor-Guillermo progeria syndromes (HGPS and NGPS, respectively) are caused by mutations in genes LMNA and BANF1 encoding protein components of nuclear lamina. Alterations of the nuclear lamina and production of an aberrant prelamin A isoform called progerin are also characteristics of normal human aging, and lamin B1 levels decline during cellular senescence. Animal and cellular models have facilitated the identification of the response mechanisms and stress pathways elicited by nuclear lamina aberrations caused by aging and progeria, including activation of tumor suppressor protein p53 (TP53), deregulation of the somatotrophic axis, and attrition of adult stem cells.",Cell,Nuclear Architecture,2022
Therapeutic Approaches for Progeria and Nuclear Lamina Defects,"The causal implication of nuclear lamina abnormalities in premature aging has been corroborated by the observation that decreasing prelamin A or progerin levels delays the onset of progeroid features and extends lifespan in mouse models of HGPS. This can be achieved by systemic injection of antisense oligonucleotides, farnesyltransferase inhibitors, a combination of statins and aminobisphosphonates, restoration of the somatotrophic axis, or blockade of NF-kB signaling. Some of these interventions have been already approved for use in progeria patients. Moreover, gene editing strategies have been recently developed to correct LMNA mutations in cells from HGPS patients and in animal models of this disease. Hopefully, these approaches will be clinically implemented for the future treatment of progeria, but to date, no evidence is available showing that reducing progerin would delay normal aging.",Cell,Nuclear Architecture,2022
Mechanisms and Consequences of Telomere Attrition,"DNA damage at the end of chromosomes (telomeres) contributes to aging and age-linked diseases. Replicative DNA polymerases are unable to complete the copy of telomere regions of eukaryotic DNA. Accordingly, after several rounds of cell division, telomeres undergo a substantial shortening that induces genomic instability and finally leads to either apoptosis or cell senescence. These deleterious effects can be prevented by the reverse-transcriptase activity of telomerase, an active ribonucleoprotein that elongates telomeres to maintain their adequate length. However, most mammalian somatic cells do not express telomerase, which leads to the progressive and cumulative erosion of telomere sequences from chromosome ends throughout life. There are several examples in which telomere attrition attenuates carcinogenesis through limiting the replicative lifespan of malignant cells. Hence, in contrast to genomic instability which unambiguously favors oncogenesis, telomere attrition may antagonize malignancy. For this reason, we consider telomere attrition as a hallmark of aging that is separable from genomic instability.",Cell,Telomere Attrition,2022
Telomerase Deficiency and Age-Related Diseases,"Telomerase deficiency in humans is associated with premature development of diseases such as pulmonary fibrosis, aplastic anemia, and dyskeratosis congenita, all of which hamper the regenerative capacity of the affected tissues. Telomere shortening is also observed during normal aging in many different species, including humans and mice. The telomeric attrition rate is influenced by age, genetic variants, lifestyle, and social factors; depends on the proliferative activity of the affected cells; and predicts lifespan in a wide variety of species. Telomere uncapping can also result from deficiencies in shelterins, a group of proteins that block the DNA damage response at chromosome ends and modulate telomere length. Several loss-of-function models for shelterin components indicate a decline of tissue regenerative capacity and accelerated aging, even in the presence of telomeres with a normal length.",Cell,Telomere Attrition,2022
Experimental Evidence and Telomerase Reactivation,"Genetically modified animal models have revealed causal links between telomere attrition, cellular senescence, and organismal aging. Mice with shortened or lengthened telomeres exhibit decreased or increased lifespan, respectively. Notably, the premature aging of telomerase-deficient mice can be reverted when telomerase is genetically reactivated. Moreover, normal aging can be delayed in mice by pharmacological activation or systemic viral transduction of telomerase, whereas mice with hyperlong telomeres show increased lifespan and metabolic health improvement. Likewise, mice engineered to maintain physiological levels of telomerase in adult neurons preserve the survival of these cells and maintain cognitive function in Alzheimer’s disease models. Thus, aging can be modulated by telomerase activation.",Cell,Telomere Attrition,2022
Telomerase Activation and Therapeutic Potential,"In humans, many studies have provided evidence for causal associations between short telomere length and age-related diseases. In particular, generation of mouse models with short telomeres has demonstrated that telomeric attrition is at the origin of telomere syndromes and prevalent age-associated diseases, such as pulmonary and kidney fibrosis. These links between telomere dynamics and organismal aging have resulted in the design of new interventions to delay aging and age-related diseases. As an example, telomerase activation using a gene therapy strategy has shown therapeutic effects on mouse models of pulmonary fibrosis and aplastic anemia.",Cell,Telomere Attrition,2022
Epigenetic Alterations and Their Role in Aging,"The large variety of epigenetic changes that contribute to aging include alterations in DNA methylation patterns, abnormal post-translational modification of histones, aberrant chromatin remodeling, and deregulated function of non-coding RNAs (ncRNAs). These regulatory and often reversible changes impact on gene expression and other cellular processes, resulting in the development and progression of several age-related human pathologies, such as cancer, neurodegeneration, metabolic syndrome, and bone diseases. A vast array of enzymatic systems is involved in the generation and maintenance of epigenetic patterns. These enzymes include DNA methyltransferases, histone acetylases, deacetylases, methylases, and demethylases, as well as protein complexes implicated in chromatin remodeling or in ncRNA synthesis and maturation.",Cell,Epigenetic Alterations,2022
Age-Related Changes in DNA Methylation,"The human DNA methylation landscape accumulates multiple changes with the passage of time. Early studies described an age-associated global hypomethylation, but further analyses revealed that specific loci, including those of several tumor suppressor genes and Polycomb target genes, are hypermethylated with age. Cells from patients and mice with progeroid syndromes also exhibit DNA methylation changes that partially recapitulate those found in normal aging. The functional consequences of most of these age-related epimutations are uncertain, as the majority of changes affect introns and intergenic regions.",Cell,DNA Methylation,2022
Epigenetic Clocks and Longevity Interventions,"Epigenetic clocks based on DNA methylation status at selected sites have been introduced to predict chronological age and mortality risk as well as to evaluate interventions that may extend human lifespan. This has been demonstrated with protocols aimed at thymus regeneration, which resulted in improved risk indices for many age-related diseases and a mean epigenetic age approximately 1.5 years less than baseline after 1 year of treatment. Moreover, predictions of human morbidity and mortality showed a 2-year decrease in epigenetic versus chronological age, which persisted 6 months after discontinuing treatment. Likewise, α-ketoglutarate supplementation for 7 months turned back the epigenetic clock by 8 years. In summary, DNA methylation changes are associated with aging, but there is no definitive evidence that they actually cause aging. Further studies will be necessary to demonstrate that defective maintenance of DNA methylation produces accelerated aging and that improved fidelity in maintenance of DNA methylation patterns extends longevity. It will also be necessary to identify the molecular drivers responsible for the modulation of changes occurring in the aged human methylome.",Cell,DNA Methylation,2022
Histone Modifications and Aging,"Global loss of histones and tissue-dependent changes in their post-translational modifications are also closely linked to aging. Increased histone expression extends lifespan in Drosophila, whereas increased histone H4K16 acetylation or H3K4 trimethylation and decreased levels of H3K9 or H3K27 trimethylation are found in fibroblasts from aged individuals and progeroid patients. These histone modifications can lead to transcriptional changes, loss of cellular homeostasis, and age-associated metabolic decline. Of note, loss of heterochromatic marks at telomeres has been shown to lead to telomere lengthening.",Cell,Histone Modifications,2022
Histone-Modifying Enzymes and Longevity Regulation,"Histone demethylases modulate lifespan by targeting components of key longevity routes such as the insulin/insulin growth factor-1 (IGF-1) signaling pathway. Other histone-modifying enzymes such as members of the SIRT family of protein deacetylases and ADP-ribosyltransferases also contribute to healthy aging. Transgenic overexpression of SIRT1 improves genomic stability and metabolic efficiency during aging in mice, although without increasing longevity. Overexpression of mitochondrial SIRT3 reverses the regenerative capacity lost in aged hematopoietic stem cells (HSCs) and can mediate the beneficial effects of dietary restriction in longevity. Similarly, Sirt6 ablation in mice results in accelerated aging, whereas Sirt6 overexpression extends lifespan. The underlying mechanisms derive from the fact that Sirt6 is a multitask protein with ability to interconnect chromatin dynamics with metabolism and DNA repair. Finally, Sirt7 deficiency induces global genomic instability, metabolic dysfunctions, and premature aging.",Cell,Histone Modifications,2022
Therapeutic Modulation of Histone Modifiers,"Together, these findings are consistent with the idea that a decrease in deacetylase activity would result in chromatin relaxation, increased exposure to DNA damaging agents, and enhanced genomic instability. Conversely, genetic inactivation of the histone acetyltransferase KAT7 in human stem cells decreases histone H3K14 acetylation and alleviates cell senescence features. Moreover, intravenous injection of lentiviral vectors encoding Cas9/sg-Kat7 ameliorates hepatocyte senescence and liver aging and extends lifespan in both normal and progeroid mice. Inhibitors of histone acetyltransferases also ameliorate the premature aging phenotype and extend lifespan of progeroid mice, whereas histone deacetylase activators promote longevity in part via upregulation of SIRT1 activity. Together, these findings suggest that histone-modifiers should be further explored as part of therapeutic strategies against age-associated cognitive decline, although it is still unclear whether these interventions influence aging and longevity through purely epigenetic mechanisms, by impinging on DNA repair and genome stability or via transcriptional alterations affecting metabolic or signaling pathways.",Cell,Histone Modifications,2022
Chromatin Remodeling and Epigenetic Regulation in Aging,"Besides DNA- and histone-modifiers, several chromosomal proteins and chromatin remodeling factors, such as the heterochromatin protein 1a (HP1a) and Polycomb group proteins which are implicated in genomic stability and DNA repair, may modulate aging. Alterations in these epigenetic factors result in profound changes in chromatin architecture, including global heterochromatin loss and redistribution, which are common events in aged cells.",Cell,Chromatin Remodeling,2022
Heterochromatin Maintenance and Longevity,"The causal relevance of these chromatin alterations in aging has been largely studied in invertebrates in which loss-of-function mutations in HP1a decrease longevity, whereas its overexpression expands healthspan and lifespan. Similar studies in mammals are still limited, but most studies indicate that heterochromatin relaxation contributes to aging and aging-related pathologies, whereas maintenance of heterochromatin promotes longevity. For example, loss of PIN1—a prolyl isomerase essential to preserve heterochromatin—is associated with premature aging and neurodegeneration in different species from Drosophila to mammals. Nevertheless, experiments aimed at extending vertebrate longevity by gain of function of chromatin remodeling factors are still missing.",Cell,Chromatin Remodeling,2022
Non-Coding RNAs and Their Role in Aging,"The large and growing universe of ncRNAs, including lncRNAs (such as telomeric RNAs or TERRA), microRNAs (miRNAs), and circular RNAs, has emerged as epigenetic factors with ability to influence aging. ncRNAs modulate healthspan and lifespan by post-transcriptional targeting of components of longevity networks or by regulating stem cell behavior. A circular RNA mediates the effect of the insulin/IGF-1 signaling pathway on Drosophila lifespan, but most studies have focused on miRNAs, and there is still debate on the extent to which other ncRNAs may derive from transcriptional noise, with their regulatory roles in human physiology and pathology only circumscribed to few specific cases.",Cell,Non-Coding RNAs,2022
MicroRNAs as Modulators of Longevity,"Gain- and loss-of-function studies first confirmed the capacity of several miRNAs to modulate longevity in invertebrates. Subsequent studies in mice have provided causal evidence on the functional relevance of miRNAs in aging. For example, miRNA-188-3p expression is upregulated in skeletal endothelium during aging and contributes to vascular problems associated with the passage of time. Depletion of miR-188 in mice alleviates the age-related decline in beneficial bone capillary subtypes, whereas endothelial-specific overexpression of this miRNA decreases bone mass and delays bone regeneration. Conversely, depletion of miR-455-3p in mice exhibits deleterious effects on mitochondrial dynamics, cognitive behavior, and lifespan, whereas its overexpression preserves these functions and extends lifespan. Overall, these findings suggest that miRNAs may causally contribute to aging and aging-related pathologies and represent potential therapeutic targets for delaying or ameliorating these conditions.",Cell,Non-Coding RNAs,2022
Retrotransposon Derepression and Aging Mechanisms,"Recent studies have unveiled the role of retrotransposons in aging of complex metazoans, including humans. These retrotransposable elements are mobile genetic units that can move from one genomic location to another, using a molecular mechanism that involves an RNA intermediate. Retrotransposons consist of long interspersed nuclear elements (LINEs), which encode the required proteins for retrotransposition, and SINEs, which are short, non-coding RNAs that hijack the LINE protein machinery. Retrotransposons are reactivated in senescent cells and during lifetime and generate deleterious effects through genetic and epigenetic changes or by activation of immune pathways triggered after identification of retrotransposon nucleic acids as foreign DNA. Mechanistically, epigenetic derepression of LINE-1 RNA inhibits the epigenetic reader Suv39H1,2 resulting in global reduction of H3K9me3 and heterochromatin, whereas reverse transcription of LINE-1 RNA results in double-stranded cDNA that activates the cGAS/STING/interferon pathway.",Cell,Retrotransposons,2022
Therapeutic Targeting of Retrotransposons for Longevity,"Treatments with nucleoside reverse-transcriptase inhibitors (NRTIs), which suppress or attenuate retrotransposition, extend lifespan of Sirt6-null mice and improve healthspan, ameliorating bone and muscle phenotypes. Likewise, treatment of aged wild-type mice with NRTIs reduces the levels of DNA damage markers. Moreover, in vivo targeting of retrotransposons with antisense oligonucleotides increases the lifespan of progeroid mice. Notably, a rare SIRT6 variant in centenarians is a stronger suppressor of LINE1 retrotransposons, enhances genome stability, and can more robustly kill cancer cells than wild-type SIRT6. Collectively, these findings suggest that retrotransposons causally contribute to the aging process and that interventions that oppose retrotransposon activity might improve healthy longevity. Further clinical studies in aged populations with drugs targeting the different functions of retrotransposons may delineate novel intervention strategies on aging and aging-related pathologies.",Cell,Retrotransposons,2022
Gene Expression Changes During Aging,The mechanisms underlying the effects of all the above epigenetic factors converge at the modulation of gene expression levels. Aging causes an increase of the transcriptional noise and an aberrant production and maturation of many mRNAs. Microarray-based comparisons of young and old tissues from human and other species have identified age-related transcriptional signatures that result from epigenetic changes occurring during aging. Environmental exposures also cause alterations in gene regulation via DNA methylation alterations and histone modifications and promote aging-related epigenetic changes including the acceleration of epigenetic clocks.,Cell,Gene Expression,2022
Transcriptomic Shifts and Therapeutic Implications,"Single-cell transcriptomic and plasma proteomics of multiple cell types and organs at several ages across the entire mouse lifespan have unveiled remarkable gene expression shifts during aging. These changes specially affect certain biological processes, such as inflammation, protein folding, extracellular matrix (ECM) regulation, and mitochondrial function, which are widely deregulated in aging. The common expression patterns observed during aging in different tissues may help to guide future interventions aimed at improving healthspan and lifespan. Likewise, the observed decline in transcriptional and post-transcriptional efficiency and fidelity in the course of aging, and its negative consequences on the proteome health may also open new opportunities for prolongevity strategies.",Cell,Gene Expression,2022
Loss of Proteostasis and Age-Related Diseases,"Aging and several age-related morbidities, such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and cataract, are associated with impaired protein homeostasis or proteostasis, leading to the accumulation of misfolded, oxidized, glycated, or ubiquitinylated proteins that often form aggregates as intracellular inclusion bodies or extracellular amyloid plaques.",Cell,Loss of Proteostasis,2022
Proteostasis Collapse and Translation Accuracy,"Intracellular proteostasis can be disrupted due to the enhanced production of erroneously translated, misfolded, or incomplete proteins. Genetic manipulation of the ribosomal protein RPS23 to improve the accuracy of RNA-to-protein translation extends lifespan in Schizosaccharomyces pombe, Caenorhabditis elegans, and Drosophila melanogaster, whereas a mutation in RPS9 that favors error-prone translation causes premature aging in mice. Another mechanism driving the collapse of the proteostasis network resides in slowed translation elongation and cumulative oxidative damage of proteins, increasingly distracting the chaperones from folding healthy proteins required for cellular fitness. In addition, numerous age-related neurodegenerative diseases including ALS and Alzheimer can be caused by mutations in proteins that render them intrinsically prone to misfolding and aggregation, hence saturating the mechanisms of protein repair, removal, and turnover that are required for maintenance of the healthy state.",Cell,Loss of Proteostasis,2022
Quality Control Failure and Proteasome Decline,"The proteostasis network also collapses when mechanisms assuring quality control fail, for instance, due to reduced function of the unfolded protein response (UPR) in the endoplasmic reticulum (ER), when stabilization of correctly folded proteins is compromised, or when mechanisms for the degradation of proteins by the proteasome or the lysosome become insufficient. Reduction of proteasome activity has been observed in aged organs including the brain of the short-lived fish Nothobranchius furzeri. Moreover, some mono-ubiquitinylated proteins accumulate in aging tissues from flies, mice, monkeys, and humans, as documented for histone 2A.",Cell,Loss of Proteostasis,2022
Lysosomal Degradation and Autophagy in Proteostasis,"The degradation of proteins by the lysosome can be achieved in a specific fashion, through chaperone-mediated autophagy (CMA), wherein proteins exposing a pentapeptide motif resembling KFERQ first bind to heat shock protein HSC70 and then to lysosome-associated membrane protein type 2A (LAMP2A), which facilitates the translocation of the client protein into the lumen of the lysosome. Hepatic LAMP2A expression declines with age in mice, and its transgenic re-expression reduces liver aging. Protein aggregates can also be removed by macroautophagy upon their inclusion in two-membrane vesicles, the autophagosomes, for their later fusion with lysosomes. Since autophagosomes can envelop non-proteinaceous structures, this process will be discussed separately from proteostasis in the next hallmark section (disabled macroautophagy). Nonetheless, stimulation of autophagy constitutes a valid strategy for the elimination of intracellular protein aggregates.",Cell,Loss of Proteostasis,2022
Proteostasis Disruption and Accelerated Aging,"Perturbation of general proteostasis accelerates aging. For example, feeding Drosophila melanogaster with advanced glycation end products (AGEs) or lipofuscin (an aggregate of covalently cross-linked proteins, sugars, and lipids) causes the accumulation of AGE-modified and carbonylated proteins with a reduction of healthspan and lifespan that is further accentuated upon knockdown of the lysosomal protease cathepsin D. Loss of the protease ZMPSTE24 abolishes the normal proteolytic maturation of prelamin A and causes a progeroid syndrome in mice, phenocopying that observed in humans with loss-of-function mutations of ZMPSTE24. In mice, knockout of LAMP2A (essential for chaperone-mediated autophagy, CMA) in neurons profoundly affects the proteome, yielding similar changes as found in Alzheimer patients. Indeed, inhibition of CMA in mice exacerbates experimental Alzheimer’s disease, whereas its stimulation by a pharmacological CMA activator attenuates the pathology.",Cell,Loss of Proteostasis,2022
Experimental Restoration of Proteostasis and Longevity,"Experimental amelioration of proteostasis can retard the aging process. Intranasal application of recombinant human HSP70 protein to mice enhances proteasome activity, reduces brain lipofuscin levels, enhances cognitive functions, and extends lifespan. Similarly, administration of the chemical chaperone 4-phenylbutyrate to aged mice reduces ER stress in the brain and improves cognition. In nematodes and flies, transfection-enforced overexpression of isolated proteasome subunits improves proteostasis and increases lifespan. In mice, stimulation of CMA by transgenic expression of LAMP2A in hematopoietic stem cells improves the survival of the targeted cell populations, in line with the observation that pharmacological enhancement of CMA attenuates Alzheimer’s pathology and arteriosclerosis. Hence, activation of CMA may constitute a valid strategy for delaying the aging process.",Cell,Loss of Proteostasis,2022
Integrated Stress Response and Longevity Mechanisms,"A phase 3 clinical trial has revealed that in patients with recent ALS diagnosis, administration of the antihypertensive guanabenz inhibits progression to the life-threatening bulbar stage. Guanabenz may act to stimulate the phosphorylation (or to inhibit the dephosphorylation) of eukaryotic translation initiation factor 2α (eIF2α), which occurs in the context of the integrated stress response (ISR) as part of the unfolded protein response (UPR), although it remains under debate to what extent the actions of guanabenz are mediated by the stimulation of the ISR. Importantly, eIF2α phosphorylation causes a switch from 5′ cap-dependent to 5′ cap-independent RNA translation, which is enhanced by several longevity-extending manipulations. Moreover, eIF2α phosphorylation is essential for the induction of stress granules, which are required for longevity extension by dietary restriction in worms. Finally, eIF2α phosphorylation is indispensable for the induction of autophagy, which is a major anti-aging mechanism, suggesting a crosstalk between UPR and autophagy in prolongevity pathways. Future studies must determine whether the capacity of guanabenz to attenuate neurodegeneration is mediated via ISR stimulation or alternative mechanisms. Indeed, it has been proposed that inhibitors of ISR might also be used for the treatment of neurodegenerative diseases.",Cell,Loss of Proteostasis,2022
Disabled Macroautophagy and Aging,"Macroautophagy (that we will refer to as 'autophagy') involves the sequestration of cytoplasmic material in two-membrane vesicles, the autophagosomes, which later fuse with lysosomes for the digestion of luminal content. Thus, autophagy is not only involved in proteostasis but also affects non-proteinaceous macromolecules (such as ectopic cytosolic DNA, lipid vesicles, and glycogen) and entire organelles (including dysfunctional mitochondria targeted by 'mitophagy,' and other organelles leading to 'lysophagy,' 'reticulophagy,' or 'pexophagy'), as well as invading pathogens ('xenophagy'). An age-related decline in autophagy constitutes one of the most important mechanisms of reduced organelle turnover, justifying its discussion as a new hallmark of aging. As a note of caution, genes and proteins that participate in the autophagic process are also involved in alternative degradation processes such as LC3-associated phagocytosis of extracellular material, and the extrusion of intracellular waste (e.g., dysfunctional mitochondria) in the form of exospheres for their subsequent removal by macrophages. That said, there is strong evidence that the core process of autophagy is relevant to aging.",Cell,Disabled Macroautophagy,2022
Autophagy Decline and Its Impact on Aging,"In humans, the expression of autophagy-related genes, such as ATG5, ATG7, and BECN1, declines with age. CD4+ T lymphocytes isolated from the offspring of parents with exceptional longevity show enhanced autophagic activity compared with age-matched controls. Decreased autophagy in circulating B and T lymphocytes from aging donors is accompanied by a reduction of the pro-autophagic metabolite spermidine. Similarly, in rodents, a progressive deterioration of autophagy has been described for some organs, supporting the idea that autophagic flux is compromised with age. Reduction of autophagic flux may participate in the accumulation of protein aggregates and dysfunctional organelles, reduced elimination of pathogens, and enhanced inflammation because autophagy eliminates proteins involved in inflammasome activation and their upstream triggers.",Cell,Disabled Macroautophagy,2022
Genetic Inhibition of Autophagy and Premature Aging,"Genetic inhibition of autophagy accelerates the aging process in model organisms. This process is partially reversible, as illustrated in mice in which Atg5 is downregulated by a doxycycline-inducible shRNA. Atg5 knockdown causes the premature degeneration and senescence of multiple organ systems leading to premature death. Upon withdrawal of doxycycline, autophagy restoration is accompanied by attenuated systemic inflammation and segmental reduction of aging. Of note, in this model, the transient inhibition of autophagy is followed by a major increase in the incidence of malignancies. Hence, autophagy apparently acts as a tumor-suppressive mechanism, which may involve cell-autonomous processes and cancer immunosurveillance. In patients, loss-of-function mutations of genes that regulate or execute autophagy have been causally linked to a broad spectrum of cardiovascular, infectious, neurodegenerative, metabolic, musculoskeletal, ocular, and pulmonary disorders, many of which resemble premature aging at the histopathological and functional levels.",Cell,Disabled Macroautophagy,2022
Autophagy Activation and Lifespan Extension,"There is ample evidence that stimulation of autophagic flux increases healthspan and lifespan in model organisms. For example, increasing autophagy solely in the enterocytes of the intestine increases Drosophila lifespan. In mice, transgenic overexpression of Atg5 under the control of a ubiquitously expressed promoter is sufficient to extend lifespan and to improve metabolic health and motor function. Moreover, a knockin mutation of beclin 1 (Becn1F121A/F121A) to reduce its inhibition by Bcl-2 causes an increase in autophagic flux, as well as an extension of lifespan. This effect is coupled to a reduction of age-associated pathologies and spontaneous tumorigenesis, as well as to increased neurogenesis.",Cell,Disabled Macroautophagy,2022
Spermidine and EP300-Linked Autophagy Enhancement,"Oral supplementation of spermidine to mice induces autophagy in multiple organs and extends longevity by up to 25%, accompanied by reduced cardiac aging. This effect is lost upon cardiomyocyte-specific knockout of Atg7, suggesting that it relies on autophagy. Mechanistically, the pro-autophagic effects of spermidine have been linked to an inhibition of the acetyl transferase EP300 (resulting in reduced acetylation of several core autophagy proteins) or to the hypusination of eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Among these factors, EP300 is the target of the longevity-enhancing drugs nordihydroguairaretic acid and salicylate. Pharmacological inhibition of EP300 with C646 mimics the stimulatory effects of spermidine on autophagy and cancer immunosurveillance. When circulating B lymphocytes or CD8+ T cells from aged human donors are cultured in the presence of spermidine, the cells recover juvenile levels of TFEB and eIF5A, coupled to a normalization of autophagic flux. Moreover, in Drosophila, hypusination deficiency due to a heterozygous mutation or knockdown of deoxyhypusine synthase abolished lifespan extension by spermidine supplementation.",Cell,Disabled Macroautophagy,2022
Autophagy-Linked Metabolic and Therapeutic Pathways,"Deoxyhypusine synthase deficiency in murine T cells triggers severe intestinal inflammation coupled to epigenetic remodeling and rewiring of the tricarboxylic acid cycle, whereas spermidine treatment of wild-type mice protects against colitis and colon carcinogenesis. Hence, both EP300 inhibition and eIF5A hypusination appear plausible targets to explain the in vivo effects of spermidine. Pharmacological agents that induce mitophagy and have a positive impact on murine healthspan include NAD+ precursors (such as nicotinamide, nicotinamide mononucleotide, and nicotinamide riboside) and urolithin A. Clinical trials have demonstrated the efficacy of NAD+ precursors in the chemoprevention of non-melanoma skin cancer, in reversing insulin resistance in prediabetic women, and in reducing neuroinflammation in patients with Parkinson’s disease. Moreover, a phase 3 trial has revealed the capacity of urolithin A to improve muscle strength and to reduce C-reactive protein (CRP).",Cell,Disabled Macroautophagy,2022
Nutrient-Sensing Networks and Aging,"The nutrient-sensing network is highly conserved in evolution. It includes extracellular ligands, such as insulins and IGFs, the receptor tyrosine kinases with which they interact, as well as intracellular signaling cascades. These cascades involve the PI3K-AKT and the Ras-MEK-ERK pathways, as well as transcription factors, including FOXOs and E26 factors, which transactivate genes involved in diverse cellular processes. The mechanistic target of rapamycin (MTOR) complex-1 (MTORC1) responds to nutrients, including glucose and amino acids, and to stressors such as hypoxia and low energy to modulate the activity of numerous proteins including transcription factors such as SREBP and TFEB. This network is a central regulator of cellular activity, including autophagy, mRNA and ribosome biogenesis, protein synthesis, glucose, nucleotide and lipid metabolism, mitochondrial biogenesis, and proteasomal activity. Network activity responds to nutrition and stress status by activating anabolism if nutrients are present and stress is low, or by inducing cellular defense pathways in response to stress and nutrient shortage. Genetically reduced activity of components of the nutrient-sensing network can increase lifespan and healthspan in diverse animal models. Moreover, genetic association studies in humans have implicated the FOXO3 transcription factor and genetic variants encoding components of the network in human longevity. Epigenetic age is also associated with nutrient-sensing in human cells.",Cell,Deregulated Nutrient-Sensing,2022
Somatotrophic Axis and Longevity Regulation,"In youth, activity of this signaling network functions to promote beneficial anabolic processes, but during adulthood, it acquires pro-aging properties. The somatotrophic axis—the first one historically implicated in the control of aging—is a growth-stimulatory cascade that, at its apex, involves growth hormone (GH) produced by the hypophysis. GH acts on the GH receptor of hepatocytes to stimulate the secretion of IGFs, in particular IGF1, which promotes growth and development via the IGF1R to stimulate trophic signals through activation of PI3K-AKT and the MTORC1 network. In multiple model organisms, spontaneous or engineered mutations of this pathway enhance lifespan and retard facets of age-associated deterioration. Innate defects in the somatotrophic axis cause dwarfism, but inhibition of this axis from early adulthood has beneficial effects on organismal health.",Cell,Deregulated Nutrient-Sensing,2022
ALK Pathway and Metabolic Aging,"Another signaling pathway involved in nutrient-sensing relies on the receptor tyrosine kinase ALK, which, in mice, is induced in the hypothalamus by feeding and responds to the ligands augmentor α and β (Auga and Augb). In Drosophila, knockdown of ALK decreases triglyceride levels and the expression of several insulin-like peptides, whereas genetic or pharmacological inhibition of ALK extends healthspan and lifespan, mostly in females. In mice, body-wide or hypothalamus-specific deletion of ALK, as well as double knockout of Auga and Augb, promotes resistance against diet-induced obesity, and in humans, a loss-of-function mutation of ALK is associated with leanness. Hence, this pathway may offer additional targets for interventions on metabolic aging.",Cell,Deregulated Nutrient-Sensing,2022
Rapamycin and Nutrient-Sensing Therapeutics,"Drugs targeting diseases such as cancer and metabolic disease often engage the nutrient-sensing network, thus such drugs are candidates for repurposing as geroprotectors. Rapamycin and rapalogs, which disrupt the MTORC1 complex, have proved to extend lifespan in model organisms even with treatment starting late in adulthood. In mice, rapamycin can increase diverse aspects of health, although it exacerbates some age-related traits such as cataract, and it is protective in models of neurodegenerative and other age-related diseases. Elderly humans are susceptible to viral respiratory infections. Pre-treatment with MTORC1 inhibitors increased the immune response of elderly volunteers to immunization against influenza and reduced viral respiratory infections in the ensuing winter, thus pointing to a potential strategy for reverting age-related immunosenescence.",Cell,Deregulated Nutrient-Sensing,2022
Nutrient Sensing and Dietary Regulation of Aging,"Diet is one of the most practical targets for interventions into human aging. Mechanistically, overnutrition: (1) triggers intracellular nutrient sensors, such as MTORC1 (activated by leucine and other amino acids), and the acetyltransferase EP300 (activated by acetyl coenzyme A); (2) inhibits sensors that detect nutrient scarcity, such as AMP-activated kinase (AMPK) and the deacetylases SIRT1 and SIRT3 (which respond to NAD+); and (3) abolishes catabolic reactions (glycogenolysis, proteolysis for gluconeogenesis, and lipolysis coupled to ketogenesis) with consequent suppression of adaptive cellular stress responses, including autophagy, antioxidant defense, and DNA repair. Conversely, fasting and dietary restriction inhibit MTORC1 and EP300; activate AMPK, SIRT1, and SIRT3; and stimulate adaptive cellular stress responses as they suppress the somatotrophic axis and extend longevity in multiple model organisms including primates.",Cell,Deregulated Nutrient-Sensing,2022
Dietary Restriction and Intermittent Fasting in Longevity,"Nutrient sensors constitute targets for potential longevity drugs, but health benefits and extended lifespan might also be achieved by dietary restrictions. Mechanistically, this is possible via reduction of overall caloric intake, manipulation of the dietary composition, or time-restricted feeding. Dietary restriction regimens are particularly successful in extending lifespan in male C57BL/6J mice, if the animals are completely deprived from nutrients during daytime. However, dietary restriction regimens do not extend lifespan in all mouse strains, supporting the contention that they must be adapted to the genetic makeup of each individual. In humans, clinical assays based on dietary restriction are complicated by poor compliance, yet suggest positive effects on immunity and inflammation. Intermittent fasting (e.g., 1 day without nutrients, followed by 1 day of ad libitum feeding) can avoid long-term weight loss induced by caloric restriction, yet increases lifespan in mice and improves biomarkers of health in clinical trials. Life time extension of a similar intermittent fasting regimen in flies has been attributed to the nighttime-specific upregulation of autophagy-stimulatory genes, but this has not yet been investigated in mammals.",Cell,Deregulated Nutrient-Sensing,2022
"Ketogenic Diet, Fasting, and Metabolic Benefits","Rapamycin-induced longevity extension (which in flies partially depends on autophagy induction) can be obtained by constant long-term exposure, as well as by intermittent regimens, suggesting that pulsatile inhibition of this axis is sufficient to obtain the benefits of lifespan extension. The optimal interval for such intermittent treatments has not yet been determined for clinical use, although partial caloric restriction for 4–7 days every 3–4 weeks may be sufficient to improve metabolic syndrome and anticancer immunosurveillance. Another potentially beneficial regimen is ketogenic diet, which is a low-carbohydrate, high-fat, and adequate protein diet. Both fasting and ketogenic diet increase the production of ketone bodies (in particular 3-hydroxybutyrate), which are synthesized from acetyl coenzyme A in the liver in an autophagy-dependent fashion, can reach millimolar concentrations in the plasma and replace glucose as an essential fuel, for instance, for the maintenance of brain function. Permanent but not cyclic administration of 3-hydroxybutyrate in the drinking water increases lifespan and healthspan in mice. This strongly suggests that this ketone body mediates some of the beneficial effects of ketogenic diet. Mechanistically, 3-hydroxybutyrate induces vasodilatation and activates immune responses acting on GTP protein coupled receptor 109A, whereas it directly inhibits the NLRP3 inflammasome, indicating a potential pleiotropic mode of action.",Cell,Deregulated Nutrient-Sensing,2022
Mitochondrial Dysfunction and Aging Mechanisms,"Mitochondria are not only the powerhouses of the cell but also constitute latent triggers of inflammation when reactive oxygen species (ROS) or mitochondrial DNA (mtDNA) leak out of the organelle, causing activation of inflammasomes or cytosolic DNA sensors, respectively, and cell death when activators of caspases, nucleases, or other lethal enzymes are released from the intermembrane space. With aging, mitochondrial function deteriorates due to multiple intertwined mechanisms including the accumulation of mtDNA mutations, deficient proteostasis leading to the destabilization of respiratory chain complexes, reduced turnover of the organelle, and changes in mitochondrial dynamics. This situation compromises the contribution of mitochondria to cellular bioenergetics, enhances the production of ROS, and may trigger accidental permeabilization of mitochondrial membranes causing inflammation and cell death. Logically, the function of mitochondria is primordial for the maintenance of health, and its progressive deterioration contributes to the aging phenotype.",Cell,Mitochondrial Dysfunction,2022
Healthspan Extension via Mitochondrial Function Enhancement,"Healthspan-extending interventions can stimulate the function of mitochondria. For instance, placebo-controlled trials have revealed positive effects of L-carnitine supplementation on both pre-frail subjects and elderly men. The effect is possibly mediated by counteracting age-related declining L-carnitine levels which may limit fatty acid oxidation by mitochondria. Paradoxically, in model organisms, lifespan can be improved by compromising mitochondrial function, which induces a hormetic response ('mitohormesis'), provided that this inhibition is partial and occurs early during development. In C. elegans, partial inhibition of mitochondrial protein synthesis or import enhances lifespan through a mechanism involving the mitochondrial UPR (UPRmt). In Drosophila, muscle-specific knockdown of complex I subunit NDUFS1/ND75 extends longevity in an UPRmt-dependent fashion. Mild inhibition of mitochondrial ATP synthesis with TPP-thiazole can improve metabolic health in aging mice, reducing visceral fat and improving glucose tolerance, mitochondrial quality, and oxidative metabolism.",Cell,Mitochondrial Dysfunction,2022
Mitochondrial Uncoupling and Pharmacological Interventions,"Partial uncoupling of hepatic mitochondria by means of a controlled release mitochondrial protonophore (CRMP) also reverses age-related metabolic syndrome in mice with high-fat diet-induced obesity. In non-human primate models including spontaneously obese rhesus macaques and high-fat, high-fructose-fed cynomolgus macaques, CRMP reverses signs of metabolic syndrome and improves fatty acid oxidation. These effects are coupled to a reduction of hepatic acetyl-coenzyme A levels, a phenomenon known to stimulate autophagy. Protonophores induce mitophagy, which might explain their positive effects on metabolism as well. Metformin, an antidiabetic considered as a weak complex I inhibitor, has been discussed as a possible anti-aging drug. However, thus far, there is no evidence that challenging mitochondria can increase healthspan or lifespan in humans.",Cell,Mitochondrial Dysfunction,2022
Mitochondrial Membrane Permeability and Therapeutic Strategies,"Increased mitochondrial membrane permeability (MMP) due to the absence of serum/glucocorticoid regulated kinase-1 decreases lifespan, which is further compromised when autophagy is enhanced but normalized when autophagy is inhibited by knockdown of essential autophagy-relevant genes in C. elegans. Hence, MMP may constitute a life-threatening condition that is aggravated by autophagy. A modified tetrapeptide, elamipretide, has been developed to target cardiolipin in the inner mitochondrial membrane (IMM) and then turned out to bind to the IMM protein adenine-nucleotide translocator-1 to inhibit the mitochondria permeability transition, which is one particular mechanism leading to MMP. Elamipretide has positive effects on multiple aging-related phenotypes in mice and has yielded positive results in a clinical trial on patients with Barth syndrome. It will be important to understand whether elamipretide can be advantageously combined with other lifespan-enhancing drugs including autophagy enhancers. In addition to these works, there are also several preclinical and clinical studies evaluating the potential beneficial effects of the antioxidant lipophilic cations MitoQ and SkQ1. Further research will define the utility of all these compounds in the context of other interventions aimed at ameliorating age-associated mitochondrial dysfunctions.",Cell,Mitochondrial Dysfunction,2022
Mitochondrial Microproteins and Aging,"Plasma levels of the microprotein humanin, which is encoded by mtDNA, decline with age. However, centenarians and their offspring exhibit high levels of humanin. Notably, humanin levels negatively correlate with IGF1 in humans and treatment of patients with GH-insufficiency, with GH or IGF1, reduces circulating humanin. Transgenic expression of humanin in C. elegans extends longevity through autophagy induction, and treatment of middle-aged mice with the humanin analog HNG improves metabolic healthspan and reduces systemic inflammation. Another mtDNA-encoded microprotein, MOTS-c, declines with age but can be induced by exercise. MOTS-c favors the production of the metabolite 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), which acts as an endogenous AMPK agonist, thereby preventing age-dependent and high-fat-diet-induced insulin resistance, as well as diet-induced obesity. Hence, mitochondrial microproteins emerge as potential anti-aging factors that link organellar function to organismal homeostasis.",Cell,Mitochondrial Dysfunction,2022
Overview of Cellular Senescence and Aging,"Cellular senescence is a response elicited by acute or chronic damage. In humans, senescent cells accumulate in multiple tissues at different rates, from 2- to 20-fold when comparing young (<35 years) to old (>65 years) healthy donors, mainly affecting fibroblasts, endothelial cells, and immune cells, although all cell types can undergo senescence during aging, a process that is triggered at least in part by telomere shortening with aging. In fact, even post-mitotic or slowly proliferating tissues, such as the brain or the heart, may harbor senescent cells. In addition, focal or tissue-specific accumulation of senescent cells occurs in many diseases. The most compelling evidence for the causal role of cellular senescence in aging is that continued genetic or pharmacological elimination of senescent cells extends the healthspan and longevity of naturally aged mice. Also, genetic or pharmacological elimination of senescent cells is therapeutic in many diseases modeled in mice, and at least three clinical trials have been completed and fifteen clinical trials are ongoing or planned to target senescence for a variety of indications.",Cell,Cellular Senescence,2022
Triggers and Mechanisms of Cellular Senescence,"The types of damage that trigger primary senescence include oncogenic signaling, genotoxic damage, critically short telomeres, mitochondrial damage, viral or bacterial infection, oxidative damage, nutrient imbalance, and mechanical stress. In addition, secondary or paracrine senescence can be triggered by extracellular mediators of inflammation and fibrosis including CCL2, IL-1β, IL-6, IL-8, and TGF-β. There is evidence suggesting that primary and secondary senescence differ in relevant biological aspects, but the molecular basis of this distinction remains elusive. Historically, the most salient feature of cellular senescence is a stable proliferative arrest mediated by the activation of the tumor suppressors TP53 and CDKN2A/p16, and their downstream effectors CDKN1A/p21 and retinoblastoma-1 (RB1) family proteins, respectively. Together, these proteins inhibit cyclin-dependent kinases (CDKs) and transcriptional activators (E2F family) that drive the cell cycle. Another important event during senescence is the depletion of lamin B1 from the nuclear envelope, resulting in the loss of lamin-associated heterochromatin and de novo formation of heterochromatin rich in H3K9me3, a process that can be visualized as HP1α foci or senescence-associated heterochromatin foci (SAHFs).",Cell,Cellular Senescence,2022
Biological Roles and Reversibility of Senescence,"The net result of these processes is a long-term and viable proliferative arrest with a low rate of spontaneous escape. Depending on their molecular makeup, cancer cells exposed to genotoxic therapy may undergo a canonical senescence response with a highly stable cell cycle arrest, a senescence-like response with a highly reversible arrest, or can even completely bypass senescence. Of note, senescence also plays a role during embryogenesis in the programmed elimination of specific cells and structures, indicating that this process has beneficial as well as deleterious roles depending on context.",Cell,Cellular Senescence,2022
Senescence and Human Diseases,"Cellular senescence is implicated in multiple non-proliferative diseases, including lung fibrosis, kidney diseases, liver steatosis, obesity-associated metabolic syndrome, type I and II diabetes, atherosclerosis, as well as Alzheimer’s and Parkinson’s diseases. The pathogenic role of cellular senescence in these diseases can be explained by the senescence-associated secretory phenotype (SASP). SASP results from three features of senescent cells: (1) the transcriptional derepression of endogenous retroviruses, most notably LINE-1, which causes cytosolic leakage of double-stranded DNA and activates the cGAS/STING and TLR pathways; (2) the mitochondrial overproduction of ROS; and (3) the perturbation of the autophagy-lysosomal system leading to an expansion of lysosomal content that facilitates the histochemical detection of lysosomal senescence-associated beta-galactosidase (SABG).",Cell,Cellular Senescence,2022
SASP Signaling Pathways and Consequences,"SASP is highly heterogeneous, depending on the cell type-specific activation of innate immunity signaling pathways (cGAS/STING, TLRs, and NLRPs), mTORC1, and transcription factors (NF-κB, CBPs, GATA4, and others). SASP usually has simultaneous and partially conflicting consequences on the microenvironment: (1) to recruit and activate immune cells through the secretion of chemokines (CCL2, CXCL2, and CXCL3) and cytokines (IL-1β, IL-2, IL-6, and IL-8); (2) to suppress the immune system through the secretion of TGF-β; (3) to trigger fibroblast activation and collagen deposition through pro-fibrotic factors (TGF-β, IL-11, and PAI1); (4) to remodel the ECM through the secretion of matrix metalloproteases; (5) to trigger the activation and proliferation of progenitor cells through the secretion of growth factors (EGF and PDGF); and (6) to trigger paracrine senescence in neighboring cells (TGF-β, TNF-α, and IL-8). In many diseases, the net effect of SASP is chronic inflammation and progressive fibrosis.",Cell,Cellular Senescence,2022
Markers and Biological Roles of Cellular Senescence,"Although there is not a single unequivocal marker of cellular senescence, this process can be identified by the co-existence of a combination of features that, together, are specific and provide a molecular definition to the phenomenon: (1) lysosomal expansion, detectable by SABG; (2) upregulation of CDK inhibitors, particularly p16 and/or p21; (3) loss of LMNB1 from the nuclear envelope; (4) loss of the chromatin component HMGB1 from the nucleus and its extracellular release as an alarmin; (5) heterochromatic foci, visualized as HP1γ nuclear foci or SAHFs; (6) high levels of ROS; (7) exacerbated DNA damage, visualized as γH2AX nuclear foci; and (8) high levels of SASP factors, notably IL-6, TGF-β, PAI1, and others. Cellular senescence is a potent tumor suppressor mechanism, but mounting evidence has linked it to tissue repair processes in which senescent cells promote localized fibrosis and the recruitment of immune cells that then remove damaged and senescent cells. Tissue repair can thus be considered a two-step process: cellular senescence followed by immune recruitment and immune clearance of senescent cells. The pathological consequences of senescence only become visible when immune clearance fails, leading to the accumulation of senescent cells and chronic fibrosis.",Cell,Cellular Senescence,2022
Senolytic Therapies and Mechanisms of Action,"The strong association between cellular senescence and multiple pathologies has spurred the search for small chemical compounds that selectively kill senescent cells and that are referred to as senolytics. Senolysis (elimination of senescent cells) is very different from the cancellation of the senescence response, which can result, for example, from mutation of p16 or p21. Senolysis does not prevent the execution of senescence but rather recapitulates the natural immune clearance of senescent cells. In support of this, mice subjected to long-term genetic-induced or pharmacologically induced senolysis present extended longevity without increased cancer incidence or signs of defective tissue repair. The number of senolytic therapies is still limited, but some have been extensively used in preclinical models of disease, as exemplified by navitoclax, dual treatment with dasatinib and quercetin (D/Q), fisetin, cardiac glycosides, and others.",Cell,Cellular Senescence,2022
Molecular Targets and Senolytic Compounds,"The survival and apoptotic resistance of senescent cells strongly depends on the BCL2 family of proteins, especially BCLXL, but also BCL2 and BCLW. This renders senescent cells highly vulnerable to navitoclax, which targets these three proteins. Navitoclax has been evaluated in clinical trials for antitumor activity and it is expected that this drug (or derivatives lacking toxicity on platelets) will enter clinical trials for senescence-associated diseases. Other potential senolytic treatments such as D/Q and fisetin are approved for human use and are being tested in various clinical trials for multiple indications. The mechanistic basis for their action remains unclear. Dasatinib is a promiscuous kinase inhibitor, and quercetin and fisetin are natural flavonoids with multiple targets. D/Q has been tested in clinical trials with promising results in the case of lung and kidney fibrosis.",Cell,Cellular Senescence,2022
Emerging Immunological and Chemical Senolytics,"Cardiac glycosides inhibit the plasma membrane Na+/K+-ATPase present in all cells causing a cationic imbalance and lowering the intracellular pH. The mechanism of senolysis by cardiac glycosides is likely connected to the vulnerability of senescent cells to low intracellular pH. Thus, chemical inhibition of glutaminase deprives cells of a mechanism to counteract low pH and results in senolysis. All the above-discussed senolytic compounds exert therapeutic activity in a wide range of murine disease models associated with senescence. Senolysis can also be achieved by immunological approaches that target proteins appearing on the surface of senescent cells. In particular, antibodies directed against the glycoprotein NMB (GPNMB) and CAR T cells directed against the receptor uPAR attenuate senescence-associated disease models in mice.",Cell,Cellular Senescence,2022
Therapeutic Outlook for Senescence Targeting,"In summary, cellular senescence is an important response to stress and damage that, in normal physiology, is followed by immune clearance, but that upon aging or chronic damage fails to be eliminated by immune mechanisms and hence is pathogenic due to the abundant secretion of pro-inflammatory and pro-fibrotic factors. Therapeutic strategies aimed at killing senescent cells have been extensively explored in animal models and are now in clinical trials.",Cell,Cellular Senescence,2022
Stem Cell Exhaustion and Aging,"Aging is associated with reduced tissue renewal at steady state, as well as with impaired tissue repair upon injury, with each organ having its own strategy for renewal and repair. For example, in skeletal muscle, one single-cell type, the satellite cell, is placed at the apex of a unipotent and unidirectional hierarchy, both for renewal and repair. In skin epidermis, which is characterized by high renewal and exposure to injury, there are multiple stem cell niches, particularly in association with the hair follicles, each one generating its progeny and territory. However, upon injury, multiple cells can acquire stem cell properties and subvert territorial boundaries. Other organs like liver, lung, or pancreas exhibit rather low renewal rates under normal conditions, contrasting with the acquisition of stem cell properties including proliferation and multipotency by different cell types.",Cell,Stem Cell Exhaustion,2022
Injury-Induced Plasticity and Tissue Repair,"Tissue repair is believed to rely to a large extent on injury-induced cellular de-differentiation and plasticity. For example, in the intestine, brain, and lung, injury induces de-differentiation of non-stem cells, which reactivates normally silent embryonic and stemness transcription programs, thus acquiring the plasticity needed for tissue repair. Injury-induced plasticity (and its progressive loss with aging) may be more relevant for aging than the plasticity of resident stem cells under normal homeostatic conditions. Stem and progenitor cells are all subject to the same hallmarks of aging as are cells without stem potential, and for this reason, the abundant literature about the impact of each hallmark of aging on stem cell function is not discussed here.",Cell,Stem Cell Exhaustion,2022
Cellular Reprogramming and Restoration of Stem Cell Function,"A general strategy to counter the decline of stem cell function with aging is based on the concept of cellular reprogramming. This process is thought to act in a cell-autonomous manner on multiple cell types; however, its impact on stem and progenitor cells is considered of higher relevance because of its long-term impact. Cellular reprogramming has emerged as a promising approach to restore tissue regeneration capacity and mitigate the decline of stem cell function that occurs with aging.",Cell,Stem Cell Exhaustion,2022
Cellular Reprogramming and Pluripotency,"Cellular reprogramming toward pluripotency consists in the conversion of adult somatic cells into embryonic pluripotent cells (known as induced pluripotent stem cells or iPSCs) by the concomitant action of four externally transduced transcription factors, namely, OCT4, SOX2, KLF4, and MYC (OSKM). The process of reprogramming usually requires several weeks during which cells first lose their differentiated phenotype by transcriptional repression of cell identity genes and subsequently transactivate pluripotency genes. Full reprogramming not only implies a change of cellular identity but also cellular rejuvenation, characterized by a number of aging features that are reset to the embryonic state, as indicated by p16 reduction, extension of telomeres, and resetting of the DNA methylation clock. Interestingly, rejuvenation occurs in a progressive fashion starting shortly after the initiation of de-differentiation.",Cell,Stem Cell Exhaustion,2022
Partial Reprogramming and Cellular Rejuvenation,"It is possible to initiate reprogramming with OSKM, interrupt the process at an intermediate state, and allow cells to return to their original identity. This transient cellular perturbation, variously known as partial, transient, or intermediate reprogramming, is able to rejuvenate cellular markers of aging such as the DNA methylation clock, DNA damage, epigenetic patterns, and aging-associated changes in the transcriptome, both in vitro and in vivo. Therefore, it can be proposed that the processes of de-differentiation and rejuvenation are coupled. Specifically, de-differentiation implies the erasure of epigenetic and transcriptional programs, and this may also erase aging-associated alterations. Upon interruption of partial reprogramming, cells re-establish their original epigenetic and transcriptional status in a process of re-differentiation that does not re-establish the erased aging-associated changes and therefore resets the epigenome and transcriptome to a younger state.",Cell,Stem Cell Exhaustion,2022
Tissue Rejuvenation Through Transient Reprogramming,"Transient reprogramming in mice confers repair capacity to old tissues so that a subsequent damage is repaired as efficiently as in young individuals. This increased repair capacity has been shown for models of tissue damage in the endocrine pancreas, skeletal muscle, nerve fibers, eye, skin, heart, and liver. Also, tissue dysfunctions characteristic of natural aging, such as reduced visual acuity and the loss of adult neurogenesis in the hippocampus and long-term memory, can be partially reversed by transient reprogramming. There are a few instances in which transient reprogramming is beneficial also during the process of tissue repair, such as in traumatic brain injury and skin wound healing. Finally, the lifespan of progeroid mice can be extended by transient reprogramming, although extension of longevity by OSKM has not yet been reported for wild-type mice.",Cell,Stem Cell Exhaustion,2022
Natural Tissue Repair and Reprogramming Convergence,"Partial reprogramming recapitulates features of natural tissue repair. In both cases, cells undergo a transient process of de-differentiation, acquisition of embryonic and progenitor features, and subsequent re-differentiation. Thus, de- and re-differentiation could explain tissue rejuvenation, in line with the observation that transient de-differentiation of myocytes, followed by their re-differentiation, induces rejuvenation of the transcriptome. The natural process of tissue repair may imply some degree of cellular rejuvenation, in accord with the finding that the epigenetic methylation clock accelerates soon after tissue injury and partially reverses during tissue repair. Moreover, tissue damage reportedly creates a tissue microenvironment that is highly permissive for IL-6-driven reprogramming. Finally, cyclic expression of the transcription factor FOXM1 extends the longevity of progeroid mice and wild-type mice. Although the detailed mechanism is still unexplored, FOXM1 is induced in the kidney upon injury and participates in triggering de-differentiation and proliferation of tubular epithelial cells during the repair process. Thus, several features of natural tissue repair and artificial reprogramming may converge, perhaps allowing refinement of strategies for restoring repair capacity in aging tissues.",Cell,Stem Cell Exhaustion,2022
Altered Intercellular Communication in Aging,"Aging is coupled to progressive alterations in intercellular communication that increase systemic noise and compromise homeostatic and hormetic regulation. These changes lead to deficiencies in neural, neuroendocrine, and hormonal signaling pathways, including the adrenergic, dopaminergic, insulin/IGF1-based, and renin-angiotensin systems, as well as sex hormones, consistent with the loss of reproductive functions. Although the primary causes of such alterations are cell intrinsic, as well documented for the senescence-associated secretory phenotype (SASP), these derangements in intercellular communication ultimately form a hallmark of aging on their own.",Cell,Altered Intercellular Communication,2022
Systemic Effects and Meta-Cellular Hallmarks,"Alterations in intercellular communication bridge the cell-intrinsic hallmarks to meta-cellular hallmarks, including the chronification of inflammatory reactions, the decline of immunosurveillance against pathogens and premalignant cells, and disruptions in bidirectional communication between the human genome and the microbiome, resulting in dysbiosis. These processes contribute to a global loss of physiological coordination during aging.",Cell,Altered Intercellular Communication,2022
Research Directions and Mechanistic Insights,"A number of studies have focused on identifying blood-borne systemic factors with pro-aging or pro-longevity properties, analyzing diverse communication systems between cells, and evaluating the functional relevance of extracellular matrix (ECM) disruption during aging. These investigations aim to understand how aging-related changes in intercellular signaling contribute to systemic deterioration and explore interventions that could restore youthful communication networks.",Cell,Altered Intercellular Communication,2022
Pro-Aging Blood-Borne Factors,"A single transfusion of old blood induces features of aging in young mice within a few days, while dilution of old mouse blood with saline containing 5% albumin rejuvenates multiple tissues. This demonstrates the existence of circulating factors that promote aging. Among these pro-aging blood-borne factors, CCL11 (eotaxin) and β2-microglobulin reduce neurogenesis, IL-6 and TGF-β impair hematopoiesis, and complement factor C1q compromises muscle repair. Theoretically, neutralizing these factors could yield anti-aging effects. Several of these molecules are secreted as part of the senescence-associated secretory phenotype (SASP) and may contribute to ‘contagious aging,’ which also involves extracellular vesicles. Therefore, compounds known as senomorphics may suppress SASP activity and slow down aging.",Cell,Altered Intercellular Communication,2022
Anti-Aging Blood-Borne Factors,"Soluble factors present in the blood of young mice effectively restore renewal and repair capacity in old mice. Heterochronic parabiosis experiments and single-cell transcriptomics confirm that young blood rejuvenates multiple tissues and restores age-related declines in gene expression, especially mitochondrial genes involved in the electron transport chain. Identified rejuvenating factors include the chemokine CCL3/MIP-1α, which revitalizes hematopoietic stem and progenitor cells; the metalloproteinase inhibitor TIMP2, which rejuvenates the hippocampus; and the anti-inflammatory cytokine IL-37, which improves exercise endurance and metabolism in aged mice. Additionally, GDF11 rejuvenates muscle, brain, and endocrine pancreas but can also have profibrotic side effects. Overexpression of VEGF enhances liver and muscle repair, improves general health, and extends average lifespan by 40%, highlighting the potential of blood-borne signaling factors as anti-aging interventions.",Cell,Altered Intercellular Communication,2022
Long-Range and Short-Range Communication Systems,"The central nervous system regulates multiple aspects of aging across peripheral organs, demonstrating that brain-specific genetic manipulations—such as overexpression of SIRT1 or UCP1, or knockout of IKBKB and TRPV1—can extend mouse lifespan. The exact mechanisms underlying these long-range regulatory effects remain unclear. In addition to long-distance control, intercellular communication also occurs through short-lived extracellular molecules (including reactive oxygen species, nitric oxide, nucleic acids, prostaglandins, and other lipophilic messengers), soluble tissue-derived factors such as adipokines, baptokines, cardiokines, hepatokines, and myokines (including exerkines from muscle activity), as well as membrane-bound ligands like IL-1α and direct cell-to-cell contacts via tight and gap junctions. These diverse communication pathways are frequently altered with aging and are under investigation for their potential pro- and anti-aging effects.",Cell,Altered Intercellular Communication,2022
Extracellular Matrix (ECM) and Aging,"Aging leads to extensive damage in the extracellular matrix (ECM), including accumulation of advanced glycation end products (AGEs), carbonylation, carbamylation, elastin fragmentation, and collagen crosslinking, collectively resulting in tissue fibrosis ('fibroaging'). This process is driven in part by excessive TGF-β signaling and nuclear translocation of mechanotransducers YAP and TAZ, which activate pro-fibrotic genes such as transglutaminase-2, lysyl oxidase (LOX), and LOX-like enzymes. Increased ECM stiffness further exacerbates senescence and fibrosis, as senescent cells secrete matrix metalloproteases that degrade the ECM and release damage-associated molecular patterns, thereby activating inflammatory and pro-fibrotic pathways. Stiffness also promotes WNT signaling, which interacts with NOTCH, RAS, TGF-β/SMAD, and Hedgehog/GLI pathways to drive fibroblast activation and fibrosis. Mechanosensing via the ion channel PIEZO1 contributes to age-related decline of progenitor cell function.

Causal studies confirm ECM stiffness as a key aging driver and identify rejuvenation strategies. Inhibition of Piezo1 restores youthful function in oligodendrocyte progenitors in aged mice, while maintaining YAP activity prevents aging phenotypes via suppression of cGAS-STING signaling. Conversely, mutations preventing collagen degradation accelerate vascular senescence and aging. Rare variants in COL25A1, a brain-specific collagen, are associated with protection against Alzheimer’s disease. ECM derived from young fibroblasts rejuvenates aged cells, and ECM compounds like chondroitin sulfate and hyaluronic acid extend lifespan in nematodes. Expression of human hyaluronidase TMEM2 promotes ER stress resistance and longevity through p38/ERK MAPK modulation. In mice, enhancing chondroitin 6-sulfotransferase improves memory and delays brain aging, while human studies link oral glucosamine/chondroitin intake with lower all-cause mortality, although causality through ECM restoration remains to be confirmed.",Cell,Altered Intercellular Communication,2022
Chronic Inflammation (Inflammaging),"Aging is accompanied by a persistent low-grade inflammatory state known as 'inflammaging,' characterized by systemic and localized manifestations such as arteriosclerosis, neuroinflammation, osteoarthritis, and intervertebral disc degeneration. Circulating levels of pro-inflammatory cytokines and biomarkers, including C-reactive protein (CRP), increase with age, and elevated IL-6 levels in plasma serve as predictive biomarkers of all-cause mortality in elderly populations. Parallel to this chronic inflammation, immune function deteriorates, as revealed by single-cell and high-dimensional profiling of myeloid and lymphoid populations in humans and mice. Notably, a population of age-associated T cells (Taa cells)—exhausted memory T cells producing granzyme K—emerges and promotes pro-inflammatory signaling. Aging also leads to a skewed T cell balance, with hyperactivation of TH1 and TH17 subsets, impaired immunosurveillance (reducing clearance of senescent, infected, and malignant cells), increased autoimmunity due to loss of self-tolerance, and reduced tissue repair and barrier maintenance. These immune alterations synergize to drive systemic inflammation, fueling both degenerative and age-related diseases.",Cell,Altered Intercellular Communication,2022
Links Between Inflammation and Other Aging Hallmarks,"Chronic inflammation ('inflammaging') arises from multiple interconnected mechanisms associated with other hallmarks of aging. Genomic instability, mitochondrial dysfunction, impaired autophagy, and senescence all contribute to inflammatory signaling. For example, cytosolic translocation of nuclear and mitochondrial DNA activates DNA-sensing pathways when autophagy fails to remove ectopic DNA. Genomic instability promotes clonal hematopoiesis of indeterminate potential (CHIP), expanding pro-inflammatory myeloid populations that drive cardiovascular aging. Common CHIP mutations affect epigenetic modifiers such as DNMT3 and TET2; TET2 loss enhances IL-1β and IL-6 production, increasing cardiovascular disease risk, which can be mitigated by IL-1β neutralization or IL-6 receptor loss-of-function.

Inflammation can also result from epigenetic dysregulation, proteostasis collapse, and impaired autophagy. Overactivation of nutrient-sensing pathways, including the GH/IGF1/PI3K/AKT/mTORC1 axis, enhances inflammatory signaling. The senescence-associated secretory phenotype (SASP) further fuels inflammation by releasing cytokines, growth factors, and proteases, while accumulation of extracellular debris and pathogens—poorly cleared due to immune exhaustion—sustains chronic immune activation. Aging-associated thymic involution leads to diminished thymopoiesis and T-cell repertoire diversity, reducing adaptive immunity and promoting systemic inflammation. Caloric restriction (CR) improves thymic function in humans, and deletion of the CR-downregulated gene PLA2G7 in mice prevents thymic atrophy. Finally, disturbances in circadian rhythms and intestinal barrier dysfunction exacerbate inflammaging, linking systemic inflammation to metabolic and immune decline.",Cell,Chronic Inflammation and Hallmarks of Aging,2023
Anti-Inflammatory and Anti-Aging Interventions,"Inflammation functions both as a hallmark of aging and a key driver of other hallmarks, meaning that interventions targeting immune and inflammatory pathways can broadly influence lifespan and healthspan. Experimental models show that immune dysfunction alone can accelerate systemic aging: for instance, T cell-specific deletion of the mitochondrial transcription factor TFAM induces premature cardiovascular, cognitive, and metabolic decline, which can be partially reversed by TNF-α inhibition (etanercept). Similarly, hematopoietic deletion of the DNA repair enzyme ERCC1 in mice causes immunosenescence and systemic aging, alleviated by the senolytic compound fisetin. These findings indicate that immune cell aging is not just a symptom but a cause of organismal aging.

Several anti-inflammatory treatments have demonstrated potent anti-aging effects. TNF-α blockade prevents sarcopenia and improves cognition, while inhibition of interferon signaling (via IFNAR1 knockout) restores immune balance in aged lungs. Prostaglandin E2 receptor EP2 inhibition in myeloid cells also enhances cognition in old mice. Moreover, suppression of inflammasome activity—particularly via NLRP3 deletion or inhibition—improves metabolic health, glucose regulation, and motor performance, extending lifespan. Pharmacological inhibitors of NLRP3 and caspase-1 show promising preclinical outcomes in both normal and accelerated aging models.

The anti-IL-1β monoclonal antibody canakinumab represents one of the most clinically validated anti-aging interventions. In the CANTOS trial, patients with a history of myocardial infarction treated with canakinumab exhibited not only reduced cardiovascular events but also lower incidence of diabetes, hypertension, and lung cancer. Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin may confer protective effects against cardiovascular and gastrointestinal cancers, though long-term trials in the elderly have shown limited benefit, suggesting that earlier or combinatorial interventions may be more effective. Overall, these findings position immune and inflammatory modulation as a viable strategy to counteract systemic aging and extend healthspan.",Cell,Chronic Inflammation and Anti-Aging Therapies,2023
Dysbiosis and Aging,"The gut microbiome plays a central role in host physiology, influencing nutrient digestion and absorption, immune defense, and the synthesis of vital metabolites such as vitamins, amino acid derivatives, secondary bile acids, and short-chain fatty acids (SCFAs). Beyond the gastrointestinal tract, gut microbes communicate with distant organs—including the brain and peripheral nervous system—through metabolic, immune, and neural signaling, thereby maintaining systemic homeostasis. Aging disrupts this finely tuned bacteria-host communication, leading to a state known as dysbiosis. Dysbiosis involves reduced microbial diversity, altered microbial composition, and imbalanced production of microbial metabolites. These changes contribute to various pathological conditions including obesity, type 2 diabetes, cardiovascular diseases, ulcerative colitis, neurological disorders, and cancer. The recognition of the gut microbiota as a modulator of systemic aging has thus spurred growing research into how microbial shifts both reflect and accelerate the aging process.",Cell,Dysbiosis and Aging,2023
Microbiota Alterations in Aging,"The gut microbiome exhibits significant variability among individuals due to genetic background, diet, lifestyle, and environmental factors, complicating efforts to link specific microbial changes to age-related diseases. Nonetheless, large-scale studies and meta-analyses have revealed reproducible patterns of microbiome alterations during aging. After stabilizing in adulthood, microbial diversity and community structure gradually decline with age, leading to decreased ecological resilience. Research on centenarians has consistently shown a reduction in core taxa such as *Bacteroides* and *Roseburia*, alongside an enrichment of genera like *Bifidobacterium* and *Akkermansia*, which are thought to promote longevity.

Recent large-cohort analyses involving more than 9,000 individuals aged 18–101 revealed that gut microbiomes become increasingly individualized with age—a phenomenon correlated with beneficial microbial metabolites involved in immune regulation, inflammation control, and aging modulation. Healthy older adults show continued diversification toward unique microbiome profiles, whereas unhealthy individuals exhibit reduced divergence, often retaining higher levels of *Bacteroides*, a pattern linked to decreased survival. However, results across studies are not fully concordant. For instance, the ELDERMET study found increased dominance of *Bacteroides*, *Alistipes*, and *Parabacteroides* in elderly individuals, with shifts correlating to frailty, cognitive decline, depression, and inflammation. Cross-ethnic studies have further identified shared microbiome aging trajectories, including a decline in sex-specific microbial differences and higher abundances of beneficial bacteria like *Akkermansia* in older adults.

Despite variability, a convergence emerges in microbiota-derived plasma metabolites. Levels of tryptophan-derived indoles and phenylalanine/tyrosine fermentation products—such as p-cresolsulfate, phenylacetylglutamine, and p-cresol glucuronide—serve as metabolic signatures of aging. In frail elderly individuals, higher fecal p-cresol levels correlate with increased frailty, while elevated plasma indoles associate with improved physical fitness. In mice, indoles extend healthspan and lifespan by activating the aryl hydrocarbon receptor and reducing inflammation. Functional analyses of centenarian microbiomes show enrichment in species like *Alistipes putredinis* and *Odoribacter splanchnicus*, which produce unique secondary bile acids such as isoallo-lithocholic acid—potent antimicrobials that protect against multidrug-resistant pathogens like *Clostridioides difficile* and *Enterococcus faecium*. These findings suggest that microbial metabolites and bile acid profiles in long-lived individuals contribute to intestinal homeostasis, resilience to infection, and overall systemic health.",Cell,Gut Microbiota Alterations and Aging,2023
Gut Dysbiosis in Progeria and Longevity,"Multiomics studies in pathological aging have revealed that two different mouse models of progeria exhibit intestinal dysbiosis mainly characterized by an increase in the abundance of Proteobacteria and Cyanobacteria and a decrease in levels of Verrucomicrobia. Consistent with these findings, human progeria patients with HGPS or NGPS also show intestinal dysbiosis, whereas long-lived humans exhibit a substantial reduction in Proteobacteria and a significant increase in Verrucomicrobia. The causal implications of these changes were demonstrated in vivo by fecal microbiota transplantation (FMT). FMT from wild type to progeroid mice recipients enhanced healthspan and lifespan in both accelerated-aging models, whereas administration of the verrucomicrobium Akkermansia muciniphila was also sufficient to obtain such effects. Conversely, FMT from progeroid donors to wild-type recipients induced detrimental metabolic alterations. Restoration of secondary bile acids and other metabolites depleted in progeroid mice phenocopied the beneficial effects of reestablishing a healthy microbiome.",Cell,Fecal Microbiota Transplantation,2023
FMT and Systemic Inflammation in Aging,"FMT also revealed the causative role of gut dysbiosis in the chronic systemic inflammation and the decline in adaptive immunity associated with aging and age-related diseases. Transfer of the gut microbiota from old mice to young germ-free mice triggered inflammatory responses characterized by enhanced CD4+ T cell differentiation in spleen, upregulation of inflammatory cytokines, and increased circulation of inflammatory factors of bacterial origin. FMT also provided evidence for the implication of the gut microbiota in the maintenance of brain health and immunity during aging. Microbiota from young mice donors reversed aging-associated differences in hippocampal metabolites and brain immunity and ameliorated age-associated impairments in cognitive behavior when transplanted into an aged host.",Cell,Fecal Microbiota Transplantation,2023
Heterochronic FMT and Immune Rejuvenation,"These works open the possibility of manipulating the gut microbiota with pre-, pro-, and post-biotics to rejuvenate the immune system and the aging brain. Heterochronic fecal transfers confirmed the causal link between age-dependent changes in microbial composition and a decline in the function of the host immune system. Indeed, the defective germinal center reaction in Peyer’s patches of aged mice can be rescued by FMT from younger animals without affecting germinal center reactions in peripheral lymph nodes. Finally, FMT from young donor mice improves ovarian function and fertility in aged mice. These beneficial effects are associated with an improvement in the immune microenvironment of aged ovaries, with decreased macrophages and macrophage-derived multinucleated giant cells, reduced levels of pro-inflammatory IFNg, and increased abundance of the anti-inflammatory cytokine IL-4.",Cell,Fecal Microbiota Transplantation,2023
Probiotic Interventions and Cognitive Aging,"The probiotic Lactobacillus plantarum GKM3 promotes longevity and alleviates age-related cognitive impairment in the SAMP8 mouse model of accelerated aging. Interventions on gut microbiota composition also restored the age-linked decline in microglial maturation and function which causes altered brain plasticity and promotes neurodegeneration. Recolonization experiments or administration of gut microbiota metabolites, such as SCFAs, prevented the age-associated decline of beneficial Bifidobacterium, increased Akkermansia abundance, and restored microglial function in middle-aged mice.",Cell,Gut Microbiota,2023
Caloric Restriction and SCFA-Producing Bacteria,"Caloric restriction diets induce structural changes of the gut microbiome increasing the abundance of Lactobacillus and other species that influence healthy aging. The gut microbiota-induced inflammaging and the consequent increase in insulin resistance can also be reversed by restoring abundance of beneficial SCFA-producing bacteria, such as A. muciniphila, in aged mice and macaques. Similarly, a randomized, double-blind, placebo-controlled pilot study in overweight/obese insulin-resistant volunteers showed that oral administration of pasteurized A. muciniphila improved insulin sensitivity, reduced insulinemia, and plasma total cholesterol levels.",Cell,Gut Microbiota,2023
Restoring Youthful Microbiome for Longevity,"Collectively, these results underscore the causal links between aging and dysbiosis and suggest that interventions aimed at restoring a youthful microbiome may extend healthspan and lifespan.",Cell,Gut Microbiota,2023
Interconnection of Hallmarks of Aging,"All the 12 hallmarks of aging are strongly related among each other. For example, genomic instability (including that caused by telomere shortening) crosstalks to epigenetic alterations (e.g., through the loss-of-function mutation of epigenetic modifiers such as TET2), loss of proteostasis (e.g., due to the production of mutated, misfolded proteins), disabled macroautophagy (e.g., through the capacity of autophagy to remove supernumerary centrosomes, extranuclear chromatin, and cytosolic DNA), deregulated nutrient-sensing (e.g., because SIRT6 is an NAD+ sensor involved in DNA repair but also responding to nutrient scarcity), mitochondrial dysfunction (e.g., due to the mutation of mtDNA), cellular senescence (e.g., because DNA damage triggers senescence), altered intercellular communication (e.g., by hampering activation of communication pathways), chronic inflammation (e.g., because CHIP and leakage of DNA into the cytosol induce inflammation), and dysbiosis (e.g., because mutations in intestinal cells favors dysbiosis, whereas specific microbial proteins and metabolites act as mutagens). Similar functional relationships can be listed for most if not all hallmarks of aging, illustrating their formidable interconnectivity.",Cell,Hallmarks of Aging,2023
Multitarget Anti-Aging Interventions,"This entanglement is also visible at the level of experimental anti-aging interventions that often simultaneously target several hallmarks. Thus, SIRT activators including NAD+ precursors attenuate genomic instability (via DNA repair), epigenetic alterations (via histone deacetylation), loss of proteostasis (via the removal of protein aggregates), disabled macroautophagy (via autophagy enhancement), deregulated nutrient-sensing (via activation of nutrient scarcity sensors), and mitochondrial dysfunction (via an increase in mitophagy-dependent quality control). Spermidine complexes to DNA (hence counteracting genomic instability), affects translation (avoiding loss of proteostasis), stimulates macroautophagy, reverses lymphocyte senescence, prevents the exhaustion of muscle stem cells, maintains circadian rhythms, suppresses inflammation, stimulates cancer immunosurveillance, and is produced by intestinal bacteria. Metformin has a pleiotropic mode of action including induction of autophagy, activation of the nutrient scarcity sensor AMPK, inhibition of mitochondrial respiration, alleviation of adipocyte senescence, suppression of inflammation, and favorable shifts in the gut microbiota. Similarly, maintenance of eubiosis by oral supplementation of A. muciniphila stimulates intestinal autophagy, reduces metabolic syndrome, dampens inflammation, and enhances anticancer immune responses.",Cell,Hallmarks of Aging,2023
"Primary, Antagonistic, and Integrative Hallmarks","Although each of the 12 hallmarks of aging can be targeted one by one, yielding tangible benefits for healthspan and lifespan, there is some kind of hierarchy among them. The primary hallmarks, which reflect damages affecting the genome, telomeres, epigenome, proteome, and organelles, progressively accumulate with time and unambiguously contribute to the aging process. The antagonistic hallmarks, which reflect responses to damage, play a more nuanced role in the aging process. For example, trophic signaling and anabolic reactions activated by nutrient-sensing have beneficial actions in youth but are largely pro-ageing later on. In an archetypal case of antagonistic pleiotropy, the nutrient-sensing network contributes to organ development until young adulthood but plays a detrimental role beyond this stage. Additionally, reversible and low-dose mitochondrial dysfunction can stimulate beneficial counterreactions (via mitohormesis), whereas limited and spatially confined levels of cellular senescence contribute to the suppression of oncogenesis and improve wound healing. Finally, the integrative hallmarks arise when the accumulated damage inflicted by the primary and antagonistic hallmarks cannot be compensated any more, resulting in stem cell exhaustion, intercellular communication alterations including ECM damage, chronic inflammation, and dysbiosis, which together dictate the pace of aging.",Cell,Hallmarks of Aging,2023
Eight Hallmarks of Health and Systemic Integration,"Recently, we postulated the existence of eight hallmarks of health, which include organizational features of spatial compartmentalization (integrity of barriers and containment of local perturbations), maintenance of homeostasis over time (recycling and turnover, integration of circuitries, and rhythmic oscillations), and an array of adequate responses to perturbation (homeostatic resilience, hormetic regulation, and repair and regeneration). Aging is linked to progressive deterioration of these hallmarks of health, implying an incapacity to maintain spatial compartmentalization, assure long-term homeostasis, and respond to stress through repair, regeneration, and hormetic regulation. This decline affects all strata of organismal organization—molecular, organelle, cellular, organ, systemic, and meta-organismal (including the microbiota). Thus, the 12 hallmarks of aging are interconnected to the eight hallmarks of failing health and the eight strata of organismal organization, creating a multidimensional space of interactions that may explain key features of aging.",Cell,Hallmarks of Aging,2023
Heterochronic Parabiosis and Systemic Regulation of Aging,"Heterochronic parabiosis experiments, in which the vascular systems of young and old mice are connected, illustrate the importance of systemic regulatory factors such as hormones and circulating cells in the aging process. This has been characterized at the single-cell transcriptomic level, revealing how young systemic environments can rejuvenate older tissues and vice versa. Aging thus integrates cell-autonomous and non-cell-autonomous mechanisms, as shown in Drosophila (where intestinal autophagy extends lifespan) and in mice (where injection of senescent fibroblasts induces osteoarthritis). These results suggest that pro-aging and anti-aging mechanisms are communicated among different cell types, explaining why normal aging affects multiple organs synchronously, unlike pathological aging where specific diseases manifest locally and early. The distinction between normal and pathological aging remains debated, and some progeroid syndromes show incomplete or segmental aging.",Cell,Hallmarks of Aging,2023
Strategies for Human Longevity Interventions,"With the progress of longevity strategies in mammalian models and early clinical trials, it becomes crucial to design rational interventions for human aging. Strategies may involve avoidance of age-accelerating environmental factors (pollution, stress, inactivity, poor diets), adoption of health-promoting lifestyles (diet, exercise, sleep, social activities), administration of pleiotropic drugs (e.g., NAD+ precursors, metformin, spermidine, MTORC1 inhibitors), or specific medical interventions such as pharmacological, genetic, cell-based, or bioengineering therapies. Combination regimens might maximize benefits while minimizing side effects. Personalization based on genetic, epigenetic, metabolomic, or phenotypic aging clock data may further enhance efficacy. However, aging is not yet an approved medical target, so initial clinical trials must focus on preventing or mitigating age-related diseases rather than aging itself. The outcomes of upcoming phase 3 trials will determine the trajectory of geroscience and the development of effective interventions for healthy longevity.",Cell,Hallmarks of Aging,2023
The Central Role of DNA Damage in the Ageing Process,"Ageing is a complex, multifaceted process leading to widespread functional decline affecting every organ and tissue. Remarkably, it is still unknown if ageing has a unifying causal mechanism or is grounded in multiple sources. Phenotypically, the ageing process is associated with a wide variety of features at the molecular, cellular and physiological level, e.g., genomic and epigenomic alterations, loss of proteostasis, declining overall cellular and sub-cellular function, deregulation of signaling systems. However, the relative importance, mechanistic interrelationships and hierarchical order of those ageing features have not been clarified. Here, we synthesize accumulating evidence that DNA damage affects most if not all aspects of the ageing phenotype making it a most likely unifying cause of ageing. Hence, targeting DNA damage and its mechanistic links with the ageing phenotype will provide a logical rationale for developing interventions to counteract age-related dysfunction and disease in concert.",Nature Reviews Molecular Cell Biology,DNA Damage and Aging,2021
The Ultimate Cause of Ageing,"There is wide agreement that ageing in metazoa is ultimately caused by the declining force of natural selection, once genes have been passed on to the next generation. Hence, mutations that only have adverse effects late in life, are not eliminated by purifying selection and therefore allowed to accumulate in the germline. Pleiotropic mutations with beneficial effects before, but adverse effects after reproduction, are even positively selected. The consequences of accumulation of such germline mutations only become evident when lifespan is no longer curtailed by extrinsic sources of early mortality, as with modern humans or animals kept in protective environments, explaining the steep rise in multimorbidity at advanced age.",Nature Reviews Molecular Cell Biology,Evolutionary Basis of Aging,2021
Evolutionary Logic and Proximate Causes of Ageing,"While the evolutionary logic of ageing is clear, surprisingly little is known about its proximate causes, even though ageing is the source of most chronic diseases and the main burden for healthcare in advanced societies world-wide. Does ageing have a sheer infinite number of origins, as predicted by evolutionary theory, or could there be one ancestral cause present from the beginning that with increasing complexity of life was later joined by many secondary causes? In an attempt to better understand ageing, a number of processes that causally contribute to pathologies occurring at old age have been identified. In this perspective we show how the main features of the ageing phenotype, causally and mechanistically, converge onto one factor: DNA damage, rendering this a strong candidate as the primary cause of ageing.",Nature Reviews Molecular Cell Biology,DNA Damage and Aging,2021
The Ultimate Cause of Ageing,"There is wide agreement that ageing in metazoa is ultimately caused by the declining force of natural selection, once genes have been passed on to the next generation. Hence, mutations that only have adverse effects late in life, are not eliminated by purifying selection and therefore allowed to accumulate in the germline. Pleiotropic mutations with beneficial effects before, but adverse effects after reproduction, are even positively selected. The consequences of accumulation of such germline mutations only become evident when lifespan is no longer curtailed by extrinsic sources of early mortality, as with modern humans or animals kept in protective environments, explaining the steep rise in multimorbidity at advanced age.",Nature Reviews Molecular Cell Biology,Evolutionary Basis of Aging,2021
Evolutionary Logic and Proximate Causes of Ageing,"While the evolutionary logic of ageing is clear, surprisingly little is known about its proximate causes, even though ageing is the source of most chronic diseases and the main burden for healthcare in advanced societies world-wide. Does ageing have a sheer infinite number of origins, as predicted by evolutionary theory, or could there be one ancestral cause present from the beginning that with increasing complexity of life was later joined by many secondary causes? In an attempt to better understand ageing, a number of processes that causally contribute to pathologies occurring at old age have been identified. In this perspective we show how the main features of the ageing phenotype, causally and mechanistically, converge onto one factor: DNA damage, rendering this a strong candidate as the primary cause of ageing.",Nature Reviews Molecular Cell Biology,DNA Damage and Aging,2021
Genome Instability at Dysfunctional Telomeres,"The discovery in the late 1980s that S. cerevisiae “ever shorter telomeres” (EST1) mutants undergo replicative senescence has popularized the concept that progressive telomere shortening drives the ageing process. In mammals, telomeres consist of thousands of TTAGGG repeats covered by the shelterin complex that facilitates formation of a lariat-like T-loop, and thereby hides the telomeric end preventing activation of the DDR sensors. Due to incomplete lagging strand synthesis during DNA replication, the number of repeats decreases with each cell division. In the germline and in some somatic stem cells this loss is compensated by telomerase, which is silenced in most somatic cells during early development, restricting the number of cell divisions until telomeres become critically short.",Nature Reviews Molecular Cell Biology,Telomere Shortening and Aging,2021
Telomere Shortening and Cellular Senescence,"An unprotected telomere resembles a persistent DNA double strand break (DSB) triggering chronic DDR activation resulting in replicative senescence. Even a single DSB suffices to cause full-blown cell cycle blockade. The pathogenicity of telomere shortening in ageing is an antagonistic pleiotropic effect of a trait that must have been selected for its early benefits such as limiting unrestrainted proliferation and hence tumor formation. Genetic defects in telomere maintenance cause human telomeropathies, including dyskeratosis congenita, aplastic anemia, and pulmonary and liver disease exhibiting multiple progeroid features.",Nature Reviews Molecular Cell Biology,Telomere Dysfunction,2021
Telomerase Deficiency and Telomere Dynamics,"In mice, segmental premature ageing only manifest in telomerase mutants after several generations, likely because their particularly long telomeric repeats take several generations to become critically shortened and thus dysfunctional. The estimated telomere length in bulk human tissues does not suggest that on average telomeres become critically short in normal ageing, even at old age. However, progressive telomere shortening might alter expression of specific subtelomeric genes, the in vivo relevance of which during ageing is yet to be determined.",Nature Reviews Molecular Cell Biology,Telomerase and Aging,2021
DNA Damage-Induced Epigenetic Alterations,"The epigenome is comprised of DNA methylation and many histone modifications and is unstable over the lifetime of somatic cells. Some changes are similar between cells in a tissue and are likely adaptive or programmed, others are progressive and/or stochastic, similar to DNA damage and mutations, contributing to intercellular heterogeneity, possibly with important functional consequences. Chromatin modifications include phosphorylation, methylation, acetylation, ubiquitination, sumoylation, citrullination, and polyADPribosylation (PAR), most of which are also part of the DDR. Age-dependent chromatin modifications include loss of histones and increased “fuzziness” of nucleosomes, linked with local and global chromatin remodeling, an imbalance of activating and repressive histone modifications, and transcriptional changes. In humans and experimental animals, diverse sets of age-related alterations in DNA methylation in various tissues have been found to strongly correlate with chronological age and are now used as epigenetic clocks. Because such clocks tick similarly from cell to cell, the underlying CpG methylation statuses likely reflect adaptive changes.",Nature Reviews Molecular Cell Biology,Epigenetic Alterations in Aging,2021
DNA Damage as a Driver of Epigenetic Changes,"Increasing evidence suggests that DNA damage is a major driver of age-associated epigenetic changes. The DNA methyltransferase Dnmt1 localizes to sites of DNA repair, and many chromatin remodelers regulate the assembly of distinct repair machineries, lesion removal, and restoration of the original chromatin state, which may leave epigenetic marks. For example, after the repair of transcription-blocking lesions in C. elegans, H3K4me2 deposition facilitates the resumption of transcription of genes regulating protein biosynthesis and homeostasis and consequently promotes longevity. The DDR in human cells leads to loss of H3K27me3, promoting cellular senescence. The phosphorylated histone variant γH2AX forms foci at the site of DSBs. Such foci accumulate in various mouse tissues with ageing, indicative of persistent chromatin alterations resulting from DNA damage. ‘DNA segments with chromatin alterations reinforcing senescence’ (DNA-SCARS) have been found enriched in senescent cells, exemplifying persistent local chromatin changes due to irreparable DNA lesions.",Nature Reviews Molecular Cell Biology,DNA Damage and Epigenetic Remodeling,2021
Epigenetic Consequences of DNA Repair and DDR,"In cell lines it has been demonstrated that DNA methylation patterns are altered during homologous recombination (HR) repair, followed by further modification weeks later by base excision repair-mediated transcription-associated demethylation. Poly-ADP-ribosylation of histones and the Poly-ADP-Ribose polymerase 1 (PARP1) itself facilitates repair of single-strand breaks, serving as a landing platform for proteins in base excision repair. PARylation severely reduces cellular NAD+ pools, which may trigger apoptosis or may indirectly inhibit Sirtuin proteins, which in turn affect genome-wide chromatin acetylation, ageing, and DNA repair and trigger gene expression changes that resemble those observed in ageing mouse brain.",Nature Reviews Molecular Cell Biology,PARP1 and Sirtuins in Epigenetic Aging,2021
Epigenetic Heterogeneity and Aging,"It is thus plausible that continuous DNA damage induction and repair for tens of thousands of lesions daily leave epigenetic marks and thereby contribute to intercellular epigenetic heterogeneity in ageing, particularly since somatic cells do not have to function forever and epigenetic memory is erased in the germline at the start of the next generation. Consistent with these ideas, transcription in aged cells appears far more variable than in young cells. Hence, the DDR likely is a primary cause of epigenetic changes that lead to deterioration of control of gene expression, which in turn contributes to somatic heterogeneity and time-dependent overall functional decline.",Nature Reviews Molecular Cell Biology,Epigenetic Instability in Aging,2021
DNA Damage-Induced Proteostatic Stress,"Proteostatic pathways control the synthesis, folding and degradation of proteins. Several age-related diseases are associated with protein misfolding and aggregation such as Alzheimer (AD) and Parkinson disease (PD). Misfolded proteins can arise when structural alterations affect solubility, thus causing protein aggregates, e.g. upon oxidative, heat, or endoplasmic reticulum stress. Multiple lines of evidence link DNA damage to proteostatic stress. Children with the premature ageing condition Cockayne syndrome (CS), which is caused by a defect in transcription-coupled repair (TCR), show neurofibrillary tangles in the cerebellar cortex occurring decades earlier than in familial early-onset AD. Defective TCR accelerates neurodegeneration in a C. elegans model for CS thus further underlining the ancestral role of DNA damage in driving age-related neuronal pathology. DNA damage and altered expression and activity of DNA repair genes have been implicated in the pathogenesis of AD and other dementias, such as reduced nucleotide excision repair (NER) efficiency in human PD. Several DNA repair mechanisms, particularly mismatch repair, are involved in the repeat expansion underlying Huntington disease and vice versa mutant huntingtin has been linked to defects in repairing transcription-associated DNA strand breaks.",Nature Reviews Molecular Cell Biology,Proteostasis and DNA Damage,2021
Transcriptional Stress and Protein Aggregation,"DNA damage could trigger proteostatic stress, for example, through increased stalling of transcription (transcriptional stress) or (epi)mutation-mediated transcriptional noise. This likely affects assembly, stoichiometry, proper folding and functioning of protein complexes, triggering proteostatic stress and aggregation. Single cell sequencing of human neurons has confirmed that somatic mutations increase during ageing and do so at a higher rate in cells from patients with neurodegenerative diseases. Stochastic transcription-blocking DNA lesions accumulating in post-mitotic tissues such as neurons, which do not dilute DNA damage by replication, likely cause the genome-wide reduced expression preferentially of large genes observed during natural ageing and in an accelerated fashion in progeroid NER/TCR-deficient mice. These DNA-damage-driven mechanisms would explain the decoupling of transcription and protein expression and loss of stoichiometry of protein complexes noted during ageing in different species, thus creating proteotoxic stress and protein aggregates.",Nature Reviews Molecular Cell Biology,Transcriptional Stress and Aging,2021
Proteostasis Pathways and DNA Damage Response,"Defects in chaperones, the ubiquitin proteasome system and autophagy can result in accumulation of misfolded proteins. The DDR itself can strain the proteostatic machineries and IRE1α and transcription factor XBP1 – both key regulators of the endoplasmic reticulum unfolded protein response (UPRER) – are induced in DNA repair defective progeroid mice. Also autophagy is induced by DNA damage signaling and is indeed required for survival amid persistent DNA damage. When unrepaired DNA lesions drive cells into senescence they exert a chronic senescence-associated secretory phenotype; which is thought to strain the UPRER. In contrast, calorie restriction reduces transcription stress and simultaneously alleviates the UPRER, providing a direct link between DNA-damage-driven transcription stress and proteostatic stress. Taken together, these observations support a central role of DNA damage and (epi)mutations as major causes of proteotoxic stress with age.",Nature Reviews Molecular Cell Biology,DNA Damage and Proteostasis Decline,2021
Mitochondrial Dysfunction and Aging,"As the organelles that regulate energy and metabolic homeostasis, mitochondria have since long been associated with ageing, mostly as main source of ROS and linked with ageing diseases, such as Parkinson disease (PD) and sarcopenia. The primary cause of mitochondrial dysfunction has often been sought in ROS-induced damage to mitochondria’s own genome, which measuring less than 17 Kb is infinitely smaller than the 3 billion bp of its nuclear counterpart but is present in multiple copies in each of the thousands of organelles in a typical mammalian cell.",Nature Reviews Molecular Cell Biology,Mitochondrial Dysfunction,2021
Somatic mtDNA Mutations and Aging Phenotypes,"The most popular hypothesis to explain age-related mitochondrial dysfunction is accumulation of somatic mutations in the mitochondrial genome, as a consequence of errors during replication and the lack of most of the sophisticated repair pathways active in the nucleus. Mice expressing a proofreading-deficient mitochondrial DNA polymerase (POLG) have greatly elevated mtDNA mutations and display multiple symptoms of premature ageing. Increased mtDNA mutations have been correlated to loss of Cytochrome C oxidase (COX) activity in aged human skeletal muscle fibers, substantia nigra and hippocampus of normally aged human brain, and various other tissues. However, it is unclear if the frequency of such mtDNA mutations reaches functionally important levels with natural age to ever cause phenotypic effects. More advanced methods, such as digital PCR, indicated fairly low frequencies of mtDNA deletions and ultra-deep sequencing did not show an age-dependent increase of mutations in wild type mice and instead suggested that most somatic mtDNA mutations originate from replication errors during development.",Nature Reviews Molecular Cell Biology,mtDNA Mutations and Aging,2021
Nuclear DNA Damage and Mitophagy,"An important connection between nuclear DNA damage and mitochondrial dysfunction implicates mitophagy, the selective degradation of mitochondria by autophagy. High levels of nuclear DNA damage, e.g. in cells from aged organisms or DNA repair mutants, lead to prolonged activation of PARP1, a DNA break sensor that upon activation consumes large amounts of NAD+. Inhibition of PARP or supplementation of NAD+ was reported to alleviate some premature ageing phenotypes associated with defects in DNA repair by restoring mitochondrial function and mitophagy.",Nature Reviews Molecular Cell Biology,Mitophagy and DNA Damage,2021
DNA Damage Effects on Mitochondrial Function,"Hence, while the role of mtDNA mutations remains subject to debate, aspects that are not yet well explored are the effect of DNA damage itself (as opposed to mutations) on mitochondrial DNA replication and transcription and damage to the over 1000 mitochondrial genes in the nuclear genome.",Nature Reviews Molecular Cell Biology,DNA Damage and Mitochondria,2021
Stem Cell Exhaustion and DNA Damage,"Somatic stem cell exhaustion has two components, decline of stem cell number and reduced functional capacity. Different stem cells utilize distinct DDR mechanisms: for instance, quiescent hematopoietic stem cells (HSCs) and hair follicle stem cells (HFSCs) employ fast but less accurate non-homologous end-joining (NHEJ), while cycling HSCs and intestinal stem cells prefer accurate homologous recombination (HR) or in case of too extensive damage opt for apoptosis, as do embryonic stem cells. In contrast, irreparable damage drives melanocyte stem cells and aged HFSCs into premature differentiation thereby clearing the stem cell pool. Accumulation of DNA damage has been observed in human and mouse HSCs as well as in muscle, intestinal, mesenchymal, neural, skin, and germ stem cells. Various DNA repair deficiencies trigger stem cell exhaustion. Muscle-forming satellite cells in progeroid Ercc1 repair mutant mice were incapable of following the regular proliferation and differentiation programs, and third-generation telomerase-deficient mouse mutants display stem cell insufficiencies in the hematopoietic system, gut, skin and testis.",Nature Reviews Molecular Cell Biology,DNA Damage and Stem Cell Aging,2021
DNA Damage Accumulation in Hematopoietic Stem Cells,"The underlying role of DNA damage has been particularly well documented in HSCs. During ageing, HSCs expand in number but decline in pluripotency, skewing towards the myeloid lineage. DNA damage increases in aged HSCs likely from replication stress. As most adult stem cells, HSCs reside predominantly in a quiescent state, which offers some protection from endogenous genotoxic stress such as metabolic ROS, but their extended time for accumulating DNA lesions and use of error-prone NHEJ increase mutagenesis. Defective DNA repair limits HSC functionality in ageing and progeroid mice. Thus, time-dependent accumulation of stochastic DNA damage severely hampers stem cell functionality, increasing mutations during human HSC ageing, impairing functional properties, promoting clonal expansion of positively selected somatic mutations resulting in loss of clonal diversity or raising the potential for oncogenic transformation. Age-dependent accumulation of somatic mutations has indeed been observed in various cell types, such as satellite cells in humans that acquire on average 13 somatic mutations per year.",Nature Reviews Molecular Cell Biology,Hematopoietic Stem Cell DNA Damage,2021
Niche-Dependent Stem Cell Exhaustion and DDR,"Also the non-cell-autonomous DDR can compromise the stem cell niche and promote stem cell exhaustion. Genome instability amid dysfunctional telomere maintenance or Sirt6 deficiency results in niche-dependent defects in hematopoietic stem cells. Notch signaling by the niche regulates the level of p53 in muscle stem cells via Mdm2 repression. With increasing age, fading niche support drives these cells into cell death via mitotic catastrophe upon activation. In C. elegans somatic niche cells regulate the DDR in germ stem cells via FGF-like signaling and a similar niche regulation of the p53-mediated DDR was observed in mouse HFSCs.",Nature Reviews Molecular Cell Biology,Stem Cell Niche and DDR,2021
Conclusions on DNA Damage-Driven Stem Cell Exhaustion,"In conclusion, accumulating DNA damage is increasingly recognized to drive stem cell exhaustion during ageing through a combination of apoptosis, premature differentiation, cytostatic DNA damage checkpoint signaling, accumulation of mutations, and DNA damage-driven alterations in intercellular communication affecting stem cell niches.",Nature Reviews Molecular Cell Biology,DNA Damage and Stem Cell Exhaustion,2021
Systemic Effects of DNA Damage on Aging,"The importance of signal transduction mechanisms in ageing has become evident since the paradigm-shifting discovery of lifespan-extending mutations in insulin-like signaling (IIS) in C. elegans. Consequently, several signaling systems have been shown to regulate longevity in species ranging from yeast to mammals. Interventions such as calorie restriction (CR) at least in part exert their anti-ageing effects by inhibiting signaling cascades such as IIS and the mTOR pathways. In contrast, inflammatory signaling is thought to promote a range of age-related pathologies.",Nature Reviews Molecular Cell Biology,Systemic Effects of DNA Damage,2021
DNA Damage Response and Inflammation,"The DDR is a potent activator of inflammatory responses. This is literally obvious in the response to UV-induced DNA damage in the skin where inflammation is counteracted by systemic immunosuppression triggered by Langerhans cells migrating from the skin to the lymph nodes to activate regulatory T cells. DNA-damage-induced senescent cells exert complex non-cell-autonomous effects, which senolytics aim to curb. DNA damage triggers innate immune responses that in C. elegans regulate systemic stress signaling. Inflammatory responses have also been observed in DNA-repair-deficient progeroid mice, which at the same time attenuate the somatotrophic (including IIS), thyrotrophic, and lactotrophic hormonal axes, as an anti-aging response, which resembles CR and IGF-1R and other dwarf mutant mice that are long-lived.",Nature Reviews Molecular Cell Biology,Inflammation and DDR,2021
Hormonal and Metabolic Responses to DNA Damage,Unrepaired transcription-blocking lesions suppress IGF-1 signaling in mouse and human cells resulting in elevated stress resistance. In C. elegans IIS attenuation enhanced tissue maintenance amid DNA damage accumulation through the activation of the FOXO transcription factor DAF-16. The paradoxical similarity between responses triggered by DNA damage and interventions delaying ageing suggested that a systemic DDR triggers a ‘survival response’ to counteract the detrimental consequences of DNA damage.,Nature Reviews Molecular Cell Biology,DNA Damage and Metabolic Signaling,2021
DDR and Systemic Communication in Aging,"Taken together, the DDR exerts multiple effects on age-related alterations in local and systemic communication mechanisms by affecting inflammatory and key endocrine signaling components that impact the ageing process.",Nature Reviews Molecular Cell Biology,DDR and Systemic Aging Mechanisms,2021
Nutritional Interventions and Genome Stability,"Nutritional interventions impact ageing and lifespan throughout the animal kingdom. Initially observed in the 1930s in rats, calorie restriction (CR) – reduced calorie intake without malnutrition – is the most robust universal health- and lifespan-promoting intervention in species ranging from yeast to mammals. It is thought that CR exerts its lifespan-extending effects through specific nutrient sensing pathways, including insulin-like signaling (IIS), Sirtuins, and the AMP-activated protein kinase (AMPK) regulated mammalian target of rapamycin (mTOR) pathway. In addition to the IIS attenuation in DNA-repair-deficient progeroid mice and worms discussed above, the DDR kinase ATM phosphorylates several key proteins of the IIS–mTOR pathways after DNA damage.",Nature Reviews Molecular Cell Biology,Calorie Restriction and Genome Stability,2021
Calorie Restriction and DNA Repair Enhancement,"CR dramatically delays premature ageing in DNA repair mutant mice likely by decreased levels of ROS and other reactive compounds leading to reduced DNA damage levels. Longevity-promoting changes in nutrient sensing pathways can also stimulate DNA repair itself, suggesting that some of the observed health benefits in normal ageing could be due to improved genome maintenance. mTOR inhibition by rapamycin in vivo, which extends lifespan, increases levels of the DNA repair protein O-6-methylguanine-DNA methyltransferase (MGMT). CR also activates Sirt1 and AMPK, promoting DNA damage repair and signaling as an epigenetic regulator and increasing NER capacity, respectively.",Nature Reviews Molecular Cell Biology,DNA Repair and Nutrient Signaling,2021
Nutrient Sensing Pathways and DNA Damage Regulation,"The protein kinase AKT, a central positive regulator of various nutrient sensing pathways, negatively regulates DNA repair and inhibits key DDR factors including Chk1, Topbp1, and p53. FOXO3a, which is activated by reduced IIS, promotes the binding of TIP60 with ATM, optimizing ATM activation after DNA damage. In summary, abundant evidence indicates that DNA damage affects key signaling mechanisms – by impinging on IIS, Sirtuins, AMPK and mTOR – that regulate lifespan and elicit anti-ageing effects of calorie restriction in model organisms.",Nature Reviews Molecular Cell Biology,Nutrient Sensing and DNA Damage Response,2021
Is DNA Damage the Primary Cause of Ageing?,"Spontaneous DNA damage thus impinges on all major aspects of the ageing phenotype. Some of the physiological alterations in turn boost genome instability thus amplifying the deterioration of homeostasis during ageing. The strong mechanistic link of DNA damage with ageing, and the role of DNA as the primary template for all cellular functions, make it a major candidate as the primary cause of ageing. However, at least three important arguments against this conclusion should be addressed.",Nature Reviews Molecular Cell Biology,DNA Damage as the Primary Cause of Aging,2021
Argument 1: DNA Repair and Lifespan Extension,"First, if DNA damage is central to the ageing process, one would expect that improving DNA repair extends lifespan and evidence for this is scarce. However, it is important to realize that DNA damage is comprised of a plethora of distinct chemical alterations, the repair of which does not depend on one gene and not even on one pathway. Instead DNA repair involves at least seven well-balanced multi-enzyme core pathways and many more accessory processes that encompass hundreds of genes, many of which have other roles as well. Hence, the function of DNA repair as a longevity assurance system cannot be generally improved by simply upregulating the activity of one or few genes. It took evolution millions of years improving DNA repair in long-lived species, such as primates. Moreover, apart from DNA repair per se, cellular systems affecting DNA damage generation and outcome, such as metabolism, anti-oxidant defense, cell death, senescence, and mutagenesis are relevant as well.",Nature Reviews Molecular Cell Biology,DNA Repair and Longevity,2021
Argument 2: Measuring DNA Damage and Mutations,"Second, reliable quantification of spontaneous DNA damage in animal or human tissues appears technically extremely difficult hampering efforts to show an age-related increase to levels that likely impair cellular function and explain age-related pathologies. However, DNA mutations, a consequence of erroneous DNA repair, can now be accurately determined and have been shown to accumulate with age in humans and mice in a tissue-specific manner. Nevertheless, while there is no doubt that accumulating mutations cause cancer and possibly increased cancer risk with age, it is as yet unknown if their frequency is high enough to account for the loss of tissue function and increased disease risk at old age. However, besides causing mutations, accumulating DNA damage also interferes with gene expression and replication causing replication and transcription stress, senescence, functional decline and cell death, all main drivers of ageing.",Nature Reviews Molecular Cell Biology,DNA Mutations and Aging,2021
Argument 3: GWAS and Genetic Associations with Aging,"A third, more recent argument against DNA damage-centric ageing theories is the dearth of DNA repair genes emerging from genome-wide association studies (GWAS) of ageing-related diseases or extreme longevity. However, the utter complexity of the genetics of ageing and longevity makes it highly unlikely to find genetic association with common variants in generally underpowered studies. Extreme longevity is rare and individual age-related diseases often involve genes not necessarily related to systemic ageing, e.g., lipoprotein genes. Nevertheless, in a meta-analysis of over 400 GWAS of five major categories of age-related diseases genome maintenance pathways were found, and genome maintenance was also the top pathway found associated with the age of natural menopause. Age of natural menopause is strongly linked with a wide variety of ageing-pathologies, including cardiovascular disease, type II diabetes and osteoporosis, and importantly with longevity. These findings are consistent with the observation in both humans and mice that the vast majority of rare genetic progeroid syndromes, where multiple ageing-associated diseases develop early in life, are caused by mutations in DNA repair genes.",Nature Reviews Molecular Cell Biology,GWAS Evidence and DNA Repair,2021
Evidence Supporting DNA Damage as the Main Driver of Aging,"Hence, while not invalid, all three arguments against a major role of DNA damage in ageing are unconvincing in view of the sheer complexity of DNA repair processes and the abundant evidence that only DNA repair dysfunction, not defects in proteostasis, antioxidant defense, immune response or any other physiological defense system, is associated with systemic premature ageing. Based on all the evidence, DNA damage is by far the most likely molecular driver of ageing. DNA damage and the DDR lead to broad cellular and physiological endpoints that can explain the entire spectrum of ageing phenotypes, from atrophy to inflammation and cancer. This understanding is far from new: It is known since the 1940s that rodents exposed to radiation show multiple symptoms of premature ageing and the first proposals that DNA damage was the main driver of ageing stem from the 1960s. More recently, the validity of these old observations was dramatically underscored by the notion that the long-term consequences of DNA-targeting chemo- and radiotherapies of cancer are accelerated, multi-organ ageing.",Nature Reviews Molecular Cell Biology,DNA Damage and Aging Mechanisms,2021
Evolutionary Perspective on DNA Damage and Aging,"The causal relationship between DNA damage and ageing may go back in evolution to the first replicators. When DNA became the genetic material, it was already far more stable than RNA, the presumed initial carrier of genetic information. The subsequent increased length of DNA templates put a premium on faithful replication and repair, which became prerequisites for rejuvenation amid the intrinsic instability of nucleic acids even during early evolution when life was not much more than compartmentalized DNA and well before the various homeostatic alterations of ageing discussed here had evolved. Hence, DNA damage as a primary cause of ageing has probably been with us since the origin of life.",Nature Reviews Molecular Cell Biology,Evolutionary Origins of DNA Damage and Aging,2021
Future Prospects for Targeting DNA Damage in Aging,"Time-dependent accumulation of DNA damage of endogenous and exogenous origin and its consequences progressively hamper cellular functionality and increase susceptibility to develop the chronic ailments of ageing. Interventions that aim at alleviating the root cause of ageing-associated multimorbidity should therefore be targeted at restoring genome integrity by reducing DNA damage and augmenting DNA repair. Reducing exogenous DNA damage for example through UV protection and avoidance of tobacco smoking has already proven to lower ageing-associated disease risks. Dietary interventions might be able to reign in some endogenous DNA damage sources, but the majority of spontaneous lesions will inevitably occur. Augmenting DNA repair has remained a great challenge due to the intricate complexity of repair machineries.",Nature Reviews Molecular Cell Biology,Future Directions in DNA Damage and Aging,2021
Enhancing DNA Repair and Genome Stability,"An exception are the highly lesion-specific photolyase repair enzymes, active in many species but not placental mammals. Ectopic expression of this enzyme is indeed sufficient to prevent UV-induced carcinogenesis in mice. However, those one-enzyme reactions are incapable of repairing the myriad of different lesions that require more sophisticated repair systems. Master regulators of DNA repair affecting multiple DNA repair systems have thus far remained elusive but might await discovery. Genetic screens using model organisms might be very suitable for the pursuit of such mechanisms augmenting genome stability.",Nature Reviews Molecular Cell Biology,DNA Repair Enhancement Strategies,2021
Towards Mechanistic Understanding and Therapeutic Outlook,"Since the initial proposals that DNA damage was the main cause and DNA repair the main determinant of ageing, and the subsequent discovery that DNA repair defects can accelerate the development of a wide range of age-related pathologies, great strides have been made in unraveling the mechanistic links between DNA damage and nearly every aspect of the ageing process. Venturing further into the mechanisms through which DNA damage affects each of the major processes that causally contribute to pathologies occurring at old age opens perspectives to tackle the ageing process at its causal roots and thus counteract all ageing-associated diseases simultaneously.",Nature Reviews Molecular Cell Biology,Mechanistic and Therapeutic Perspectives,2021
Autophagy in Aging and Longevity,"Our understanding of the process of autophagy and its role in health and diseases has grown remarkably in the last two decades. Early work established autophagy as a general bulk recycling process which involves the sequestration and transport of intracellular material to the lysosome for degradation. Currently, autophagy is viewed as a nexus of metabolic and proteostatic signalling that can determine key physiological decisions from cell fate to organismal lifespan. Here, we review the latest literature on the role of autophagy and lysosomes in stress response and longevity. We highlight the connections between autophagy and metabolic processes, the network associated with its regulation, and the links between autophagic dysfunction, neurodegenerative diseases, and aging.",Autophagy in aging and longevity,Autophagy and Longevity,2021
Introduction to Autophagy,"Autophagy is a conserved intracellular process that encapsulates and delivers macromolecular cargoes such as proteins and organelles to lysosomes for subsequent degradation. Three forms of autophagy have been characterized based on distinct mechanisms of cargo delivery to lysosomes: (1) Macroautophagy, whereby double-membraned vesicles called autophagosomes engulf autophagic cargo and direct them to lysosomes upon fusion; (2) Chaperone-mediated autophagy (CMA), where cargo is translocated into the lysosomal lumen by recognition of their KFERG-like domain by cytosolic chaperones such as heat shock cognate 70 kDa proteins; and (3) Microautophagy, by which cargo enters lysosomes through membrane invaginations.",Autophagy in aging and longevity,Autophagy Mechanisms,2021
Macroautophagy and Core Machinery,"Overall, autophagy can direct either degradation of bulk or selective cargo. Examples of the latter include the selective breakdown of protein aggregates (aggrephagy), mitochondria (mitophagy), lysosomes (lysophagy), and ribosomes (ribophagy). The most studied form of autophagy, macroautophagy, is regulated by a core autophagic machinery of key autophagy-related (ATG) proteins. The process is initiated by the Unc-51-like kinase 1 (ULK1)/ATG13/ATG101/FIP200 initiation complex in response to low cellular nutrient or energy levels, which recruits the Beclin1/ATG14/VPS15/VPS34 Class III PI3K complex to generate phosphatidylinositol 3-phosphate (PI3P) for nucleation and formation of the double-membraned preautophagosomal phagophore at the autophagic cargo site.",Autophagy in aging and longevity,Macroautophagy and ATG Proteins,2021
Phagophore Expansion and Selective Autophagy,"Subsequent phagophore expansion requires the ATG5/ATG12/ATG16L complex and the ATG8 family of microtubule-associated proteins LC3 and GABARAP. The ATG5/ATG12/ATG16L complex promotes the conjugation of ATG4-cleaved LC3/GABARAP (LC3-I) to phosphatidylethanolamine (PE) to form LC3-II, which integrates into phagophore membranes. The phagophore expands until it eventually engulfs the autophagic cargo in an autophagosome. In selective autophagy, delivery of specific autophagic cargoes into LC3-containing phagophores or autophagosomes is mediated through the interaction of cargo-bound autophagy receptors with LC3 via their LC3-interacting motifs. A well-studied example is SQSTM1/P62, a selective autophagy receptor that binds ubiquitinated protein aggregates and delivers them for aggrephagy clearance.",Autophagy in aging and longevity,Phagophore Formation and Selective Autophagy,2021
Autophagy Dysfunction and Aging,"While P62 mediates several types of 'phagies', other autophagy receptors mediating organelle-specific selective autophagy have been uncovered. Age-related conditions such as neurodegenerative diseases are known to be caused by or correlated with mutations and dysregulation of ATG proteins, selective autophagy, and their receptors. To circumvent such diseases of aging, efforts to pharmacologically modulate autophagy are at the forefront of multiple research programs in academia and the pharmaceutical industry. This review highlights the latest links between autophagy and metabolism, recent elucidation of transcriptional regulatory mechanisms governing autophagy, and emerging evidence of the impact that dysfunctional autophagy has on neurodegenerative diseases and aging.",Autophagy in aging and longevity,Autophagy Dysfunction and Aging,2021
Nutrient Sensing Governing Autophagy Induction,"The regulation of autophagy is mediated by environmental cues such as nutrient levels and external stressors like hypoxia or heat through a complex network of proteins and signalling pathways. Nutrient-sensing pathways such as adenosine monophosphate-activated kinase (AMPK) and mechanistic target of rapamycin (mTOR) act in an opposing manner to modulate autophagy. Nutrient-depleted conditions driving a high AMP/ATP ratio induce autophagy by activating AMPK, which in turn phosphorylates and activates ULK1, the serine/threonine protein kinase activator of autophagy. Conversely, detection of elevated amino acids by mTORC1, the nutrient-sensing complex containing mTOR at the lysosomal membrane, promotes cell proliferation, biomass production, and represses autophagy by suppressing ULK1. Under nutrient-deprived conditions or by pharmacological inhibition, mTOR inactivation relieves ULK1 suppression and induces autophagy. These findings suggest a systematic integration of metabolism by mTOR, counter-balanced by cellular energy state sensing via AMPK to satisfy homeostatic demands.",Autophagy in aging and longevity,Nutrient Sensing and Autophagy,2021
"mTOR, AMPK, and Aging","Dysfunctional regulation of the mTOR and AMPK signalling pathways can contribute to organismal aging by impairing autophagy and by giving rise to cellular vulnerabilities conducive for the development of cancer, metabolic disease, and neurodegeneration. Reducing mTORC1 function is sufficient to extend lifespan in yeast, worms, flies, and mice. As mTORC1 activity is modulated at the lysosomal membrane, the lysosome has emerged as a key organelle integrating decisions pertaining to metabolic fate and macromolecule recycling. Proper lysosomal function, essential for efficient degradation of autophagic cargoes, declines with age, particularly in organs such as the liver, brain, and muscle. Many lysosomal storage diseases (LSDs) share similar phenotypes with aging tissues, such as aberrant mTOR activity and compromised autophagic flux, suggesting that aging may reflect LSD-like conditions preventing cells from proper degradation and nutrient signalling.",Autophagy in aging and longevity,mTOR and AMPK in Aging,2021
"Autophagy, Caloric Restriction, and Metabolic Homeostasis","One of the benefits of functional autophagy on metabolic homeostasis is its ability to provide cells with precursors for anabolic and energy-demanding pathways under nutrient-restricted conditions. Autophagy is upregulated during caloric restriction (CR), which leads to lifespan extension across species and reduces the incidence of age-related disorders. Mice subjected to intermittent fasting show activation of autophagy in liver, fat, brain, and muscle, leading to lower blood glucose and lipid levels. CR studies in non-human primates have shown lifespan improvements and delayed onset of age-related disorders. Pharmacological mimetics of CR, such as fisetin and metformin, offer neuroprotection against aging-related oxidative stress with increased autophagy gene expression via the AMPK pathway.",Autophagy in aging and longevity,Caloric Restriction and Autophagy,2021
Exercise-Induced Autophagy and Longevity,"Aging mice subjected to cyclic ketogenic diets exhibit extended lifespan and healthspan, likely through increased fatty acid oxidation and decreased TOR and insulin signalling. Organisms can survive extended starvation by inducing organ-specific autophagy in the liver, pancreas, kidney, and muscle while sparing the brain. Breakdown of adipose tissue and muscle sustains the liver during starvation to export glucose and ketone bodies for brain metabolism. Blood glucose levels are maintained during starvation by AMPK activation of muscle autophagy, and aging-induced myopathy and mitochondrial dysfunction are accelerated if AMPK modulation is lost. Exercise-induced autophagy mediates beneficial metabolic effects in neurodegeneration, neurogenesis, and cognition. Moderate running extends lifespan, in part through autophagy induction and other metabolic and physiological mechanisms. Understanding how nutrient restriction- and exercise-induced autophagy influence lifespan remains a fertile field of aging research.",Autophagy in aging and longevity,Exercise and Autophagy in Longevity,2021
Nutrient Sensing Governing Autophagy Induction,"The regulation of autophagy is mediated by environmental cues such as nutrient levels and external stressors like hypoxia or heat through a complex network of proteins and signalling pathways. Nutrient-sensing pathways such as adenosine monophosphate-activated kinase (AMPK) and mechanistic target of rapamycin (mTOR) act in an opposing manner to modulate autophagy. Nutrient-depleted conditions driving a high AMP/ATP ratio induce autophagy by activating AMPK, which phosphorylates and activates ULK1, a serine/threonine protein kinase that initiates autophagy. Conversely, elevated amino acid levels activate mTORC1, a nutrient-sensing complex at the lysosomal membrane, promoting cell proliferation and repressing autophagy by inhibiting ULK1. Under nutrient-deprived conditions or pharmacological inhibition, mTOR inactivation relieves ULK1 suppression and induces autophagy. Beyond amino acids, glucose and cholesterol also modulate lysosomal mTOR activation, highlighting a systematic integration of metabolic cues through mTOR and AMPK balance to sustain homeostasis.",Autophagy in aging and longevity,Nutrient Signaling and Autophagy Regulation,2021
mTOR and AMPK in Aging and Lysosomal Function,"Dysfunctional regulation of mTOR and AMPK signaling pathways contributes to organismal aging by impairing autophagy and predisposing cells to cancer, metabolic diseases, and neurodegeneration. Reduced mTORC1 function extends lifespan in yeast, worms, flies, and mice. Since mTORC1 activity is modulated at the lysosomal membrane, the lysosome emerges as a central organelle integrating metabolic fate and macromolecule recycling. Proper lysosomal function is essential for autophagic degradation but declines with age in organs such as the liver, brain, and muscle. Lysosomal storage diseases (LSDs) exhibit phenotypes similar to aged tissues, including aberrant mTOR signaling and impaired autophagic flux. Therefore, aging may represent an LSD-like state where cells fail to degrade macromolecules efficiently and maintain nutrient signaling.",Autophagy in aging and longevity,"mTOR, AMPK, and Lysosomal Aging",2021
"Caloric Restriction, Autophagy, and Longevity","Functional autophagy contributes to metabolic homeostasis by supplying precursors for anabolic and energy-demanding pathways during nutrient scarcity. Autophagy is upregulated during caloric restriction (CR), which extends lifespan across species and reduces age-related disorders. Intermittent fasting in mice activates autophagy in liver, fat, brain, and muscle, lowering blood glucose and lipid levels. CR studies in primates demonstrate improved lifespan and delayed onset of age-related conditions. Pharmacological CR mimetics, such as fisetin and metformin, enhance autophagy gene expression via AMPK activation and protect against oxidative stress. Similarly, cyclic ketogenic diets in aging mice extend lifespan and healthspan by increasing fatty acid oxidation, activating PPARα, and suppressing TOR and insulin signaling.",Autophagy in aging and longevity,Caloric Restriction and Autophagy,2021
Exercise-Induced Autophagy and Systemic Effects,"Organisms survive prolonged starvation by organ-specific induction of autophagy, particularly in liver, pancreas, kidney, and muscle, while sparing the brain. During starvation, adipose tissue and muscle degradation sustains liver function to export glucose and ketone bodies, fueling the brain. Blood glucose levels are maintained via AMPK-mediated muscle autophagy, and disruption of this regulation accelerates myopathy and mitochondrial dysfunction with age. Exercise-induced autophagy promotes metabolic health, neurogenesis, and cognitive function. Moderate running extends lifespan through autophagy activation alongside metabolic, cardiovascular, musculoskeletal, and neuroprotective benefits. Understanding how nutrient restriction and exercise-induced autophagy synergistically influence human longevity remains a central pursuit in aging research.",Autophagy in aging and longevity,Exercise and Autophagy-Mediated Longevity,2021
Metabolic Integration of Autophagy Protein Function,"The activation of autophagy initiates an energetically demanding process that is tightly regulated by the localization, accessibility, and proper modification of ATGs and associated proteins. Many proteins that regulate autophagy undergo post-translational modifications (PTMs), including phosphorylation, ubiquitination, acetylation, O-linked attachment of β-N-acetyl-glucosamine (O-GlcNAc), and thiol modifications. LC3 is a well-studied and key autophagy protein subjected to PTMs that determine its correct localization to the autophagosome membrane. Under nutrient-depleted conditions, nuclear LC3 must be deacetylated, translocated to the cytoplasm, and lipidated for autophagy to progress.",Autophagy in aging and longevity,Autophagy Protein Regulation,2021
Post-Translational Modifications Regulating LC3 Dynamics,"LC3 phosphorylation, catalyzed by protein kinase A and the Hippo kinases STK3/STK4, regulates its lipidation and incorporation into maturing autophagosomes. The lipidated form of LC3 (LC3-II) is elevated in old mice, possibly due to AMPK activity. However, the mechanisms underlying LC3 dynamics in aging are not fully understood. Evidence suggests that compromised lysosomal capacity may contribute, as mature LC3-II-containing autophagosomes accumulate in older animals and remain undegraded, indicating an age-associated decline in autophagic flux.",Autophagy in aging and longevity,LC3 Phosphorylation and Aging,2021
Selective Autophagy of Organelles and Metabolic Proteins,"Autophagy can directly influence metabolism by degrading organelles responsible for energy production, such as mitochondria, or those that store energy, like lipid droplets. Selective autophagy of cargo is mediated by receptors that recognize organelles or macromolecules designated for degradation. Mitophagy involves the autophagic turnover of old or dysfunctional mitochondria, preventing the build-up of reactive oxygen species (ROS) under prolonged hypoxia. Mitochondrial biogenesis is controlled by PGC1α, while clearance through mitophagy is regulated by PARKIN-mediated ubiquitination and high NAD+/NADH ratios. The balance between mitophagy and mitochondrial biogenesis is essential for longevity, as damaged mitochondria accumulation accelerates aging. Mitochondrial network dynamics also play a key role in lifespan extension through caloric restriction and AMPK activation.",Autophagy in aging and longevity,Mitophagy and Energy Metabolism,2021
Mitophagy and Neurodegenerative Diseases,"Compromised mitophagy is a hallmark of Parkinson’s disease (PD), where damaged mitochondria accumulation renders neurons vulnerable. In Alzheimer’s disease (AD), impaired mitophagy is characterized by reduced levels of phosphorylated TBK1 and ULK1, and cognitive decline can be rescued by restoring mitophagy. These findings suggest that mitochondrial dysfunction and impaired energy metabolism contribute to AD pathogenesis. The initiation, amplitude, and duration of mitophagy are modulated by mitochondrial complex I activity, where oxidative phosphorylation increases autophagy amplitude while glycolysis dampens it. mTOR inhibitors and glucose starvation can induce mitophagy while preserving complex I function. Thus, enhancing mitophagy while maintaining proper mitochondrial biogenesis and dynamics represents a promising approach to delay age-related diseases.",Autophagy in aging and longevity,Mitophagy and Neurodegeneration,2021
Lipophagy and Lysosomal Lipid Metabolism,"Lipophagy, the autophagic breakdown of lipid droplets, plays a crucial role in maintaining metabolic balance. While the receptor for lipophagy remains unknown, lysosomal lipid degradation is essential for lifespan extension in longevity models. Lysosomal lipid signaling can influence nuclear hormone receptor activity, affecting lipid metabolism and lifespan, and can also modulate mitochondrial activity. Non-cell autonomous regulation of lipophagy has been observed, where autophagy activation in hypothalamic POMC neurons triggers lipophagy in brown adipose tissue (BAT) and liver. Conversely, knockout of Atg7 in POMC neurons blocks this effect. This CNS–periphery autophagic communication offers potential targets for treating metabolic disorders, fatty liver disease, and neurodegenerative conditions.",Autophagy in aging and longevity,Lipophagy and Metabolic Regulation,2021
Chaperone-Mediated Autophagy and Metabolic Control,"Chaperone-mediated autophagy (CMA), regulated by the lysosomal protein LAMP2A, selectively degrades proteins involved in metabolic pathways such as glycolysis, lipolysis, and lipogenesis. CMA dynamically modulates energy flux through these pathways. Loss of CMA leads to increased glycolysis and lipogenesis, decreased lipolysis, and reduced ATP levels in mouse livers. These findings raise the question of whether non-selective macroautophagy also regulates metabolic enzyme abundance and activity. Overall, the connections between autophagy and metabolism emphasize the need for systematic proteomic, metabolomic, and tissue-specific analyses to better understand autophagic and metabolic decline during aging.",Autophagy in aging and longevity,Chaperone-Mediated Autophagy and Metabolism,2021
"Transcriptional, Epigenetic, and Post-Transcriptional Regulation of Autophagy","Transcriptional and epigenetic regulation of autophagy (ATG) genes are key mechanisms that impact physiology and lifespan. Several transcription factors (TFs) regulate the expression of core ATG genes involved in all stages of autophagy, from phagophore initiation to cargo degradation in autolysosomes. ATG gene transcription is tightly controlled by nutrient and energy-sensing pathways. TFs themselves are regulated through phosphorylation, which determines their nuclear localization and transcriptional activity.",Autophagy in aging and longevity,Regulation of Autophagy Genes,2021
TFEB as a Master Regulator of Autophagy and Lysosomal Biogenesis,"The transcription factor EB (TFEB), a member of the MITF family, serves as a master regulator of autophagy and lysosomal biogenesis. Under nutrient-rich conditions, TFEB is phosphorylated by mTOR and ERK at lysosomal membranes and retained in the cytoplasm through binding to 14–3–3 proteins. Nutrient depletion inhibits mTOR and activates calcineurin, resulting in TFEB dephosphorylation and nuclear translocation. TFEB activation also occurs under ER and mitochondrial stress. Exportin-1 (XPO1) mediates TFEB nuclear export, and its inhibition promotes TFEB nuclear localization, autophagy gene induction, and lifespan extension. Both mTORC1 and mTORC2 regulate TFEB localization through phosphorylation, while TFEB acetylation is necessary for full transcriptional activation. TFEB has emerged as a major target for therapeutic autophagy induction in neurodegenerative diseases, metabolic disorders, and aging.",Autophagy in aging and longevity,TFEB Regulation and Longevity,2021
FOXO Transcription Factors in Autophagy and Longevity,"The FOXO family of transcription factors, including FOXO1 and FOXO3, induce autophagy by upregulating core ATG genes, thereby promoting autophagic flux and lifespan extension. Conversely, FOXK1 inhibits autophagy by competing with FOXO3 for promoter binding. FOXO TFs are inhibited by the insulin/insulin-like growth factor signaling (IIS) pathway through AKT-mediated phosphorylation, which sequesters them in the cytoplasm. As longevity factors, FOXO proteins are crucial in IIS pathway mutants where autophagy contributes to extended lifespan. Other regulators such as GATA-1, XBP1, JNK, and REST also influence FOXO activity, while FOXO and TFEB can co-regulate target genes that enhance oxidative stress resistance and longevity, highlighting extensive transcriptional crosstalk.",Autophagy in aging and longevity,FOXO and Autophagy Regulation,2021
Additional Transcriptional Regulators of Autophagy,"Besides FOXO and TFEB, several other transcription factors directly regulate ATG gene expression, including TP53, CREB, E2F1, NFKB, ZKSCAN, NRF2, PPARα, CEBP, XBP1, HIF1, JUN, and FXR. E2F1, HIF1, and JUN generally activate ATG gene expression, while FXR acts as a repressor and NFKB displays dual roles depending on context. The coordinated action of these TFs may enhance autophagy through selective transactivation of gene subsets. Understanding the interplay among these regulators and their associated epigenetic modifications is critical for elucidating how autophagy is fine-tuned during aging and stress adaptation.",Autophagy in aging and longevity,Autophagy Gene Transcription Factors,2021
Epigenetic Regulation of Autophagy Genes,"Histone-modifying enzymes regulate post-translational modifications (PTMs) on histone proteins, determining chromatin condensation and subsequent transcriptional activity of DNA. Autophagy genes are influenced by epigenetic modifications such as histone acetylation and methylation. The acetyltransferase KAT8/hMOF and its associated histone mark H4K16ac regulate ATG gene expression, with autophagy induction correlating with their downregulation. Another acetylation mark, H3K56ac, modulates cell growth through the yeast TORC1 ortholog of mTORC1, with mutants displaying hypersensitivity to rapamycin. Enrichment of activating marks H3K56ac and H3K27ac at ATG gene loci under nutrient deprivation suggests their involvement in autophagy activation.",Autophagy in aging and longevity,Histone Acetylation and Autophagy,2021
Histone Methylation and Nutrient-Dependent Autophagy,"Histone methylation regulates autophagy in both activating and repressive ways depending on the residue modified. Autophagy induction is linked to decreased transcriptionally active H3K4me3 and increased repressive H4K20me3 marks. Under glucose deprivation, autophagy stimulation correlates with increased H3R17me2 through the methyltransferase CARM1, which co-activates TFEB-driven autophagy. Persistent starvation stabilizes CARM1 by preventing its degradation, enhancing TFEB activity. Conversely, repressive methylation such as H3K9me2 suppresses LC3B and WIPI1 expression, while histone methyltransferase G9A and BRD4 repress TFEB-independent autophagic programs. Nutrient availability influences these modifications, as starvation-induced BRD4 dissociation from chromatin occurs via AMPK and SIRT1 signaling.",Autophagy in aging and longevity,Histone Methylation and Autophagy Control,2021
DNA Methylation and Other Epigenetic Modifications in Autophagy,"DNA methylation, catalyzed by DNA methyltransferases (DNMTs), represses autophagy gene expression. In macrophages from aged mice, increased DNMT2-mediated hypermethylation of ATG5 and LC3B promoters suppresses ATG gene transcription, which can be restored by DNMT2 inhibition. Other less-explored epigenetic marks, such as monoubiquitination of histone H2B (H2Bub1), also influence autophagy. Starvation-induced deubiquitination via USP44 reduces H2Bub1 levels, promoting autophagy gene expression. Collectively, these studies reveal that diverse epigenomic modifications accompany autophagy induction, though their precise roles in cellular homeostasis and organismal aging remain to be fully understood.",Autophagy in aging and longevity,DNA Methylation and Epigenetic Regulation of Autophagy,2021
Post-Transcriptional Regulation of Autophagy,"Various RNAs and RNA-binding proteins (RBPs) regulate autophagy-related gene (ATG) mRNAs post-transcriptionally, either directly or indirectly through transcription factor modulation. MicroRNAs (miRNAs) can repress the expression of autophagy-related genes at multiple stages. For example, miR128 inhibits TFEB, impairing TFEB-activated ATG gene transcription. Early autophagy components such as ULK1 and Beclin1 are targeted by miR30a, miR30b, and miR471–5p, while ATG12, ATG5, ATG7, and LC3 are regulated by miR30b, miR30c, miR130a, miR471–5p, and miR101. The autophagy receptor P62/SQSTM1 is targeted by miR17, miR20, miR93, miR106, and miR372, whereas lysosomal genes such as LAMP2 and SUMF1 are regulated by miR207 and miR95. Understanding these miRNA networks is crucial for evaluating their functional and therapeutic potential in modulating autophagy.",Autophagy in aging and longevity,miRNA Regulation of Autophagy,2021
Long Non-Coding RNAs and Autophagy Modulation,"Long non-coding RNAs (lncRNAs), non-coding transcripts over 200 bases, have emerged as key regulators of autophagy. The lncRNA H19 inhibits autophagy under high-glucose conditions by repressing the GTPase DIRAS3, which affects the PI3K/AKT/mTOR pathway and BECLIN1 and ATG7 expression in diabetic cardiomyopathy. Conversely, H19 can induce autophagic cell death in cerebral ischemia by inhibiting DUSP5. Other lncRNAs such as NBR2, MEG3, HOTAIR, PTENP1, MALAT1, and H19 regulate autophagy across different stages. The lncRNA NEAT1, upregulated in Parkinson’s disease models, promotes autophagy by stabilizing PINK1 and enhancing mitophagy. Additional lncRNAs including GAS5, CAIF, and DICER1-AS1 modulate autophagy in various pathologies such as cancer, myocardial infarction, and osteosarcoma, respectively, implicating lncRNA dysregulation in disease-associated autophagy changes.",Autophagy in aging and longevity,lncRNA Regulation of Autophagy,2021
RNA-Binding Proteins and Autophagy Control,"RNA-binding proteins (RBPs) influence autophagy by regulating RNA stability, splicing, and translation. The RBP HuD enhances ATG5 mRNA expression in pancreatic β cells, promoting autophagosome formation. In contrast, tristetraprolin suppresses Beclin1 and LC3-II, reducing autophagy and increasing cell death in lung adenocarcinoma. TDP-43, an aggregation-prone RBP implicated in ALS and FTD, destabilizes the mTORC1 adaptor protein RAPTOR when depleted, leading to decreased mTOR activity, TFEB nuclear translocation, and increased autophagy. However, TDP-43 depletion also disrupts autophagosome-lysosome fusion in a dynactin 1-dependent manner, impairing autophagic flux. These findings underscore the complex and dual roles of RBPs in autophagy regulation and highlight their relevance in neurodegenerative diseases.",Autophagy in aging and longevity,RNA-Binding Proteins and Autophagy,2021
Transgenerational Inheritance of Starvation Resistance,"The transgenerational inheritance of stress resistance is an emerging area of study with implications for autophagy regulation. In C. elegans, heritable small RNAs have been shown to persist for three generations following starvation. Similarly, epigenetic modifications involving the H3K4me3 regulatory complex promote lifespan extension across three generations and confer stress-induced survival advantages. During acute starvation, AMPK prevents histone methyltransferases from methylating H3K4, ensuring that chromatin marks are not established until nutrient levels are restored. In mammals, DNA methylation at 5-methylcytosine is linked to transgenerational responses to high-fat diets, while in nematodes, 6-methyladenine methylation correlates with mitochondrial stress responses.",Autophagy in aging and longevity,Transgenerational Starvation Resistance,2021
Epigenetic Transmission and Autophagy Regulation,"These findings highlight the critical role of germline-soma communication in transmitting stress signals across generations. However, it remains unclear whether the inheritance of stress responses directly modulates autophagy in descendants. The interplay between epigenetic and transcriptional modulation of autophagy genes represents a fundamental mechanism of autophagy regulation. Understanding how aging disrupts these mechanisms may provide insight into the dysregulation of autophagy observed in metabolic syndromes and neurodegenerative diseases.",Autophagy in aging and longevity,Epigenetic Inheritance and Autophagy,2021
Autophagy Impairment in Aging and Neurodegeneration,"Autophagic function declines with age, contributing to the accumulation of proteotoxic damage and reduced proteostasis, which are hallmarks of neurodegenerative diseases (NDs). Genes involved in the autophagy-lysosomal pathway are essential for lifespan extension across species, and their dysfunction is linked to diseases such as Alzheimer’s (AD), Parkinson’s (PD), Huntington’s (HD), amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). Mutations in autophagy-related genes like SQSTM1, PINK1, and Parkin disrupt selective degradation of protein aggregates or mitochondrial quality control, accelerating disease pathogenesis. Lysosomal dysfunction, such as that caused by PSEN1 mutations in familial AD, compromises autophagic degradation, while epigenetic alterations like reduced H4K16ac in AD brains indicate transcriptional dysregulation of autophagy genes. Together, these findings underscore the link between autophagy decline and age-associated neurodegeneration.",Autophagy in aging and longevity,Autophagy and Neurodegenerative Diseases,2021
Pathological and Genetic Evidence of Autophagy Dysfunction,"Genetic studies reveal that autophagy-related mutations drive ND pathology by impairing autophagosome formation or flux. For example, SQSTM1 mutations in ALS and FTLD interfere with selective clearance of protein aggregates, while PINK1 and Parkin mutations in PD disrupt mitophagy, reinforcing the importance of mitochondrial homeostasis in disease. Pathological observations further show autophagosome accumulation in AD neurons, suggesting blocked autophagic flux. However, limitations in clinical tools make it challenging to directly measure autophagy dynamics in vivo. At the molecular level, ND-associated mutant proteins can directly inhibit autophagy: α-synuclein in PD sequesters TFEB, preventing lysosomal gene activation; mutant Huntingtin in HD disrupts miRNA function and Beclin1 protection; and polyQ-expanded proteins like ataxin-3 compromise autophagy induction by destabilizing Beclin1.",Autophagy in aging and longevity,Genetic and Molecular Autophagy Impairments,2021
Cross-Talk Between Autophagy Pathways and ND Proteins,"Beyond macroautophagy, other autophagic mechanisms such as chaperone-mediated autophagy (CMA) and microautophagy are also disrupted in neurodegenerative contexts. Mutant tau proteins, depending on their biochemical properties, can alter their uptake and degradation through macroautophagy, CMA, and endosomal microautophagy (eMI). This highlights the complex interactions between ND-associated proteins and autophagic pathways, emphasizing the need for targeted therapeutic strategies that account for pathway-specific dysfunctions. Disrupted autophagy exacerbates proteotoxic stress, leading to a vicious cycle of aggregate accumulation and neuronal death.",Autophagy in aging and longevity,Tauopathies and Autophagy Pathway Interactions,2021
Selective Neuronal Vulnerability and Cell-Type Specific Roles of Autophagy,"Autophagy dysfunction contributes to selective neuronal vulnerability observed in different neurodegenerative diseases. Neuronal populations vary in their levels of autophagic activity and in susceptibility to its failure. Autophagy is also compartmentalized within neurons, playing specialized roles in synaptic maintenance and axonal transport. Additionally, impaired autophagy in glial cells can indirectly affect neuronal homeostasis, amplifying neurodegeneration. These findings suggest that future therapeutic interventions must consider both neuron- and cell-type-specific autophagy regulation to effectively mitigate disease progression.",Autophagy in aging and longevity,Selective Neuronal Vulnerability in ND,2021
Impaired Nucleocytoplasmic Transport and Loss of Nuclear Integrity in Autophagy Decline,"Efficient nucleocytoplasmic transport of autophagy-regulating transcription factors such as TFEB via RanGTP-dependent importins and exportins is essential for maintaining autophagic activity. With aging, nuclear pore complexes (NPCs) deteriorate, causing nuclear leakiness in post-mitotic cells like neurons and impairing TFEB nuclear retention, leading to reduced autophagy. Stress conditions such as heat shock, oxidative stress, and UV irradiation further inhibit nuclear import by blocking the recycling of importin α to the cytoplasm, disrupting nuclear transport and autophagy regulation. These findings underscore that age-related nuclear transport inefficiencies can significantly contribute to the decline in autophagic capacity.",Autophagy in aging and longevity,Nucleocytoplasmic Transport and Autophagy,2021
Nuclear Transport Disruption in Neurodegenerative Diseases,"Neurodegenerative disease (ND)-associated proteins can directly disrupt nucleocytoplasmic transport by mislocalizing nuclear pore components and transport factors. In ALS and FTD caused by C9ORF72 mutations, phase-separated stress granules sequester nucleocytoplasmic transporters in the cytoplasm, inhibiting their nuclear functions. Mutant tau in FTD alters microtubule dynamics and deforms the nuclear membrane, while interactions between tau and nuclear pore protein Nup98 promote tau fibrillization. These alterations lead to impaired nuclear pore function, abnormal protein localization, and compromised autophagic gene regulation, exacerbating proteotoxic stress.",Autophagy in aging and longevity,Neurodegeneration and Nuclear Transport Defects,2021
Therapeutic Implications of Restoring Nuclear Transport,"Emerging evidence suggests that restoring nucleocytoplasmic transport could mitigate proteotoxicity in neurodegeneration. Importin proteins, recently identified to possess disaggregase activity, can counteract cytosolic aggregation of misfolded proteins such as FUS. Moreover, pharmacological modulation of nuclear export pathways using inhibitors like exportin-1 (XPO1/CRM1) blockers may restore nuclear localization of autophagy-inducing transcription factors like TFEB. Together, these findings emphasize that maintaining nuclear integrity and proper protein partitioning between the nucleus and cytoplasm is vital for preserving autophagic dynamics and overall proteome stability in aging and disease.",Autophagy in aging and longevity,Therapeutic Restoration of Nuclear Transport,2021
Emerging Links Between Phase Separation and Autophagy,"Aberrant phase separation processes, such as the cytoplasmic sequestration of nucleocytoplasmic transporters by stress granules, can impair autophagy by blocking the nuclear import of autophagy-inducing transcription factors like TFEB. This connection highlights the interplay between phase separation and autophagic dysfunction, particularly in neurodegenerative diseases (NDs). Proteins associated with ALS and FTD, including the RNA-binding protein TDP-43, exhibit phase condensate behavior that affects autophagy regulation. Similarly, other RNA-binding proteins (RBPs) such as FUS, HNRNPA2B1, HNRNPA1, ATXN1, and ATXN2, along with key autophagy regulators, are capable of mediating phase separation, linking these molecular processes to the pathology of aging and NDs.",Autophagy in aging and longevity,Phase Separation and Autophagy,2021
Mechanistic Interplay Between Stress Granules and Autophagy,"Experimental evidence shows that phase-separated condensates can evade autophagic degradation. For example, heat stress-induced PGL granules in C. elegans embryos become resistant to autophagy via mTORC1 activation. Conversely, the autophagy receptor P62 promotes phase separation of ubiquitin-positive proteins, facilitating their clearance through aggrephagy as a compensatory response to proteasome malfunction. These findings suggest that phase separation can either hinder or enhance autophagy depending on the context, revealing a complex regulatory relationship.",Autophagy in aging and longevity,Stress Granules and Autophagy Crosstalk,2021
Pathological Transitions and Aging Implications,"The dynamic nature of phase-separated condensates allows them to transition from liquid-like to solid or aggregated states, contributing to pathological protein accumulation. Understanding the spatiotemporal and mechanistic details of these transitions will be critical for elucidating how they progressively disrupt autophagy and proteostasis during aging and in neurodegenerative diseases. Future research into phase separation dynamics may uncover novel therapeutic targets to restore autophagy and prevent the buildup of toxic protein aggregates.",Autophagy in aging and longevity,Phase Separation in Aging and Disease,2021
Definitions and Concepts of Ageing,"As humans, we are all ageing, but the exact definition of ageing is still heavily debated. However, in the field of ageing biology, most scientists would agree that the dysregulation of biological mechanisms affecting the genome, proteome or any other biological molecule is part of the definition. There is also discordance in the definition of the phenotypes that are commonly used to define and study the ageing process. Lifespan is defined as the number of years lived. Yet, for the definitions of healthspan and longevity, several slightly varying interpretations exist in the literature as the traits are more subjective and less easy to quantify. Healthspan is often defined as the number of years lived in good health or the number of years lived before the occurrence of any major debilitating disease. For the phenotype longevity, survival to an exceptionally old age is used, which is either defined as survival above a defined age, for example 95 years, or belonging to a birth-cohort-specific survival percentile, for example 10% longest lived. It will be a challenge for the field to delineate these definitions in the future to generate data that is comparable between different studies.",Genetics of Human Longevity: From Variants to Genes to Pathways,Definitions of Ageing and Longevity,2024
Ageing as a Risk Factor for Disease,"Fortunately, the field of ageing biology agrees that ageing itself represents the major risk factor for the occurrence of most debilitating chronic diseases, such as cardiovascular diseases, metabolic diseases – for example type 2 diabetes – and neurological impairments, including Alzheimer’s disease. Furthermore, ageing increases the risk of suffering from severe outcomes from infections as the function of the immune system declines with increasing age, resulting in ineffectiveness in fighting pathogens and detecting and destroying cancerous cells. Unfortunately, this decrease in immune function also results in a decreased effectiveness of vaccines that therefore offer less protection in aged individuals.",Genetics of Human Longevity: From Variants to Genes to Pathways,Ageing and Disease Risk,2024
Healthspan vs Lifespan and the Importance of Healthy Ageing,"Many of us would like to live a long life, but, currently, that often means spending the last decade or two of one’s life suffering from multiple debilitating diseases. Consequently, the advancements in medical care to prevent death due to chronic diseases (i.e. cancer and type 2 diabetes) or infections (i.e. through improved hygiene and the development of vaccines and antibiotics) and the subsequent increase in lifespan now urgently need to be matched by major advancements that increase healthspan. The most eminent challenge of the ageing field is to identify how we can improve health during ageing to enable people to live long and productive lives without posing a burden on the healthcare system. Thus, the incentive of current studies should not be to increase lifespan but to focus on the extension of healthspan. To this end, we need to utilize all possible tools to investigate how we can manipulate the healthspan of an individual via, for example, the environment, nutrition, microbiome and physical activity (i.e. exercise habits). Although some of these interventions may work in broad populations, the success of other interventions may depend on an individual’s genetic makeup. Consequently, a personalized approach might be necessary to address the challenges of an ageing population.",Genetics of Human Longevity: From Variants to Genes to Pathways,Healthspan and Personalization,2024
Studying Long-Lived Individuals to Understand Healthy Ageing,"The traditional approach in medicine is to study the unhealthy individuals within the population to find out what contributes to, or even causes, their disease and develop a cure or treatment for this particular disease. However, it might be more beneficial to study the other side of the spectrum – in this case, the extremely healthy and long-lived. This approach may enable us to understand what we can do to not only cure diseases but also push the entire population to stay healthy in old age. Exceptionally long-lived individuals often delay or even escape the onset of most age-related diseases, thus making longevity an interesting naturally occurring case study for healthy ageing within the human population. Moreover, longevity is assumed to be a heritable trait. Studying the genetic make-up of the exceptionally long-lived may thus help us uncover the underlying molecular mechanisms that could be targeted to improve healthspan.",Genetics of Human Longevity: From Variants to Genes to Pathways,Exceptional Longevity and Heritability,2024
Focus and Scope of the Review,"The focus of this review will be on mechanisms shared between humans and model organisms to understand what we can learn from the genetics of longevity. First, we will discuss the findings and limitations of the genetic studies performed in humans, with a focus on studies that included an extensive and well-defined study population (n ≥ 200 long-lived individuals). Second, we will describe how we can utilize model organisms and the evolved differences in the natural lifespan among them (with a focus on longevity, not median lifespan, in the context of the species) to further deepen our understanding of ageing and longevity in humans.",Genetics of Human Longevity: From Variants to Genes to Pathways,Review Focus and Methodology,2024
Heritability of Longevity,"Studies of long-lived families reveal that longevity can be inherited as a genetic trait. Although the heritability of longevity has not yet been estimated, as a placeholder, the heritability of lifespan – determined in twin and pedigree-based studies – is estimated to be between 10% and 30%. The success of studies investigating the genetic component of longevity is dependent on the selection criteria used to select long-lived cases. However, because the meaning of longevity as a trait is not well defined, the use of heterogeneous study cohorts to investigate longevity unfortunately complicates the interpretation of the results. Based on the analysis of two large family-based datasets, the most refined selection criteria for individuals to be considered exceptionally long-lived is that they belong to the top 10% of survivors of their birth cohort and have additional family members who meet the same criteria. These selection criteria were further expanded by taking into account that the newly defined longevity phenotype is inherited in at least 20% of family members for at least two subsequent generations and used to create a ‘longevity relatives count’ score. An alternative longevity score that has been used in the field is the ‘family longevity selection score’. However, thus far, both scores have only been applied to a limited number of populations, and more research is needed to prove their usability for the selection of genetically enriched long-lived cases.",Genetics of Human Longevity: From Variants to Genes to Pathways,Heritability of Longevity,2024
Current Approaches to Studying the Genetics of Longevity in Humans,"Most genetic differences between humans are caused by single-nucleotide polymorphisms (SNPs), whereas other, less common, changes can affect multiple nucleotides, for example insertions. The primary aim of genetic studies of exceptionally long-lived individuals is to detect genetic variants that are significantly enriched or depleted in their genome in comparison to controls from the general population. The earliest genetic studies of longevity employed a hypothesis-driven approach and focused on investigating SNPs in specific genes or pathways that were previously linked to lifespan regulation in model organisms or age-related diseases in humans. The development of genotyping arrays enabled the investigation of thousands of SNPs in multiple genes at the same time using more unbiased hypothesis-free approaches. Recent advances in sequencing technologies now allow for the investigation of the whole exome or even the entire genome. The most extensive genetic studies of human longevity have been performed in cohorts from populations of European, East Asian and Ashkenazi Jewish ancestry, as summarized in Table 1. In the following, we will elaborate on some of the findings from the most rigorous studies that were more likely to identify relevant SNPs, loci and genes due to the stringent inclusion criteria of long-lived cases. An overview of all genetic variants and genes associated with human longevity is available in the LongevityMap database (http://genomics.senescence.info/longevity/).",Genetics of Human Longevity: From Variants to Genes to Pathways,Genetic Studies of Longevity,2024
Candidate Gene– and Pathway–Based Studies Overview,"Candidate gene– or pathway–based studies are hypothesis-driven investigations of the association between genetic variants in a gene, or multiple genes within a pathway, with a given phenotype, such as longevity. The selection of the genes is usually informed by prior knowledge of the connection between the gene and the phenotype, that is from studies in model organisms. Candidate gene–based studies were the first studies conducted to investigate the genetics of human longevity, as this approach is relatively inexpensive and methodically less challenging than hypothesis-free approaches given that only the variants of interest need to be measured.",Genetics of Human Longevity: From Variants to Genes to Pathways,Candidate Gene and Pathway Studies,2024
Gene-Based Longevity Studies,"One of the earliest candidate gene–based studies of human longevity was performed by Schachter et al., who were able to identify an association of genetic variation in the APOE locus with longevity. Until today, this is the best replicated longevity locus, and its association has been confirmed in many different study populations. There are two main genetic variants at this locus implicated in longevity: ApoE ε2 (rs7412), which is enriched in long-lived individuals, and ApoE ε4 (rs429358), which is depleted in long-lived individuals. The only other longevity-associated locus that has been replicated in several independent studies is FOXO3, which was first identified by Willcox et al. and later replicated by several other groups. Additional loci have been identified using candidate gene–based studies (see the LongevityMap database for an overview), but these could so far not be replicated in independent studies and/or populations.",Genetics of Human Longevity: From Variants to Genes to Pathways,Gene-Based Longevity Studies,2024
Pathway-Based Longevity Studies and Key Findings,"As genetic variants within different genes that belong to the same pathway may result in a shared downstream effect, the candidate gene–based approach was subsequently broadened to investigate multiple genes within, or even entire, lifespan-associated pathways. The pathways that were found to be relevant and were further investigated are as follows: (1) insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS), (2) mTOR signalling, (3) DNA-damage and repair, and (4) telomere maintenance. The first study of this kind by Deelen et al. focused on 1021 SNPs in 68 genes assigned to the IIS pathway and 88 SNPs in 13 genes that are part of the telomere maintenance pathway to investigate the synergistic effect of the variants on longevity. Both pathways showed significant association with longevity. The association of the IIS pathway was distributed across nine genes (AKT1, AKT3, FOXO4, IGF2, INS, PIK3CA, SGK, SGK2 and YWHAG), whereas the gene POT1 by itself was sufficient to explain the association of the telomere maintenance pathway with longevity.",Genetics of Human Longevity: From Variants to Genes to Pathways,Longevity Pathways and SNP Associations,2024
DNA Repair and SNP–SNP Interaction Studies,"Another extensive gene-set analysis study by Debrabant et al. was focused on an in-depth investigation of the DNA-damage response and repair pathway by testing the association of 592 SNPs in 77 genes – that can be categorized into 9 sub-pathways – with longevity. Although none of the tested associations passed adjustment for multiple testing, and the findings could not be replicated in an independent dataset, it is worth mentioning that the base excision repair sub-pathway was nominally associated with longevity due to genetic variation in several genes, whereas the nominally significant association of the homologous recombinational repair and RecQ helicase activities pathways was primarily the result of association of only one gene (RAD52 and WRN, respectively). The most recent pathway-based study, which used a SNP–SNP interaction approach, was performed on 1058 SNPs in 140 genes by Dato et al. The focus of this study was on the IIS, DNA repair and pro/antioxidant pathways. They identified several SNP–SNP interactions associated with longevity, and the strongest ones involved genetic variants in TP53 (indicating an interaction between the DNA repair and pro/antioxidant pathways) and GHSR (indicating an interaction between the IIS and DNA repair pathways).",Genetics of Human Longevity: From Variants to Genes to Pathways,DNA Repair and SNP Interactions,2024
Linkage Studies in Longevity Research,"With the development of genotyping arrays, the focus of the field subsequently shifted to hypothesis-free approaches – such as linkage studies – to identify genetic loci that are associated with longevity. Linkage studies of longevity are aimed at the identification of chromosomal regions that show cosegregation within long-lived family members and thus require a family-based study design. Contrary to the initial expectations, only very few loci were identified through such studies, which is likely due to their relatively small sample size. In addition, none of the identified loci did, thus far, replicate between studies.",Genetics of Human Longevity: From Variants to Genes to Pathways,Linkage Studies,2024
Genome-Wide Association Studies of Longevity,"Another hypothesis-free approach that is now often used in the field is the identification of SNPs using genome-wide association studies (GWAS). GWAS that used longevity as the phenotypic read-out were able to identify several variants that are associated with this trait. However, the only genetic variants that are successfully replicated between independent studies and populations are the ones located in the APOE locus, which were already identified using candidate gene studies. Robust identification of additional variants is currently simply hampered by the limited number of cases – that is long-lived individuals – available for genetic studies. GWAS of longevity are further complicated by the absence of a proper control cohort, as the matching control individuals (i.e. those from the same birth cohorts as the cases) have already died before being collected. As a workaround to address some of these hurdles, and to further increase the number of detected variants, several recent studies combined genetic studies on longevity with those of other ageing-related phenotypes.",Genetics of Human Longevity: From Variants to Genes to Pathways,Genome-Wide Association Studies (GWAS),2024
Polygenic Score Analyses in Longevity,"In addition to the study of SNPs, several studies have looked at polygenic scores (PGSs) to determine the enrichment or depletion of age-related disease-associated variants in long-lived individuals. Early studies showed that long-lived individuals have a similar burden of common disease-associated genetic variants, but more recent studies have shown a consistent depletion of PGSs for Alzheimer’s disease. Hence, this could partly explain why many long-lived individuals remain cognitively healthy up until very high ages. However, it is still very likely that the extreme lifespan of long-lived individuals is primarily determined by variants promoting health, given that sequencing studies show that long-lived individuals seem to carry many pathogenic variants.",Genetics of Human Longevity: From Variants to Genes to Pathways,Polygenic Scores and Longevity,2024
Future Directions in Genetic Studies of Longevity,"Although the number of genetic studies has dramatically increased over the past decades, the number of identified genes that are reliably associated with human longevity has stagnated. Only two genes (APOE and FOXO3) have consistently been linked to longevity in studies of different designs and using data from different populations. Even efforts to combine the data from different studies – that is meta-analyses – were not able to significantly increase the number of longevity-associated genes. The steady increase in the ratio of sequenced long-lived individuals over identified variants supports the hypothesis that the genetic component of human longevity is likely not encoded by common genetic variants with large effect sizes, but rather by rare variants that either enable longevity autonomously or in concert.",Genetics of Human Longevity: From Variants to Genes to Pathways,Future of Longevity Genetics,2024
Strategies for Studying Rare Variants in Longevity,"Given that rare variants can hardly be detected with our current approaches, the field needs to adapt its strategy to be able to study their effects on longevity. One way forward would be to massively increase the sample size of the genetic studies to allow the detection of variants with a small effect size, which would only be possible by setting up new longevity studies. Although this effort might eventually lead to success, this strategy is not practicable given the high costs to set up such studies and the limited number of individuals who are currently considered long-lived. Revisiting the candidate gene– or pathway–based approach offers an alternative. However, instead of focusing on common variants (as has been done previously), these studies should focus on the identification of rare variants in genes and pathways that have been associated with lifespan regulation in model organisms.",Genetics of Human Longevity: From Variants to Genes to Pathways,Rare Variant Analysis Strategies,2024
Functional Characterization of Longevity-Associated Variants,"The most critical step is to restrict the number of variants to be considered for subsequent experimental analyses to variants located in the most promising genes. The selection of such genes can be accomplished by only including genes belonging to pathways that are associated with longevity in multiple model organisms. As the identified variant will be rare and the association of the variants with longevity is hard to test, the causality between the variant and longevity must be confirmed in the laboratory. Hence, it is advisable to only focus on variants in regions of the genome that are of known function and can therefore be reliably investigated (e.g. exons or enhancer and suppressor sequences). In the case of variants residing in exons, it would also be sensible to only investigate protein-altering variants to improve the likelihood of a functional consequence of the variant. We will next discuss some results from studies that already successfully applied a functional characterization approach.",Genetics of Human Longevity: From Variants to Genes to Pathways,Functional Characterization of Variants,2024
In Vitro Characterization of Genetic Variants,"Genetic association studies offer a fantastic tool to identify the mechanisms that facilitate longevity. However, most studies stay short of exploring the actual functional consequence of genetic variants and, consequently, establishing causality. Given that many of the identified variants cannot be confirmed in independent studies and populations of different ethnicities, one might wonder if they are indeed causal. Fortunately, previous studies have shown that functional characterization of the effects of longevity-associated genetic variants is possible in vitro.",Genetics of Human Longevity: From Variants to Genes to Pathways,Functional Characterization of Genetic Variants,2024
Functional Studies of Common Longevity-Associated Variants,"The common longevity-associated variants that were so far functionally characterized are the ApoE ε2- and ε4-defining variants rs7412 and rs429358, respectively, rs2802292, rs12206094 and rs4946935, all located in the intronic region of FOXO3, as well as several regulatory variants in PRKCH, CLU and NFKBIA. The investigation of intronic variants poses challenges and limitations, as intronic regions are usually less conserved between species, impeding the possibility of using in vivo models to functionally investigate these variants. The functional study of two of the common intronic variants in FOXO3 – rs12206094 and rs4946935 – was consequently performed by transiently introducing a luciferase reporter construct under the control of the variant-specific FOXO3 promoter sequence into the human Panc1 and Jurkat cell lines. Both variants seem to increase the expression of FOXO3 in normal cell culture conditions but result in a decrease in expression upon IGF-1, but not insulin, stimulation. To determine the function of rs2802292, Grossi et al. introduced the variant into the near-haploid human embryonic stem cell line HAP-1 via CRISPR-Cas9 and showed that this variant increases FOXO3 expression, which is further enhanced by exposure to stressful conditions due to the creation of a binding site for the heat shock transcription factor HSF1.",Genetics of Human Longevity: From Variants to Genes to Pathways,Common Longevity Variants,2024
Functional Validation of Rare Longevity-Associated Variants,"Experimental validation of rare variants is even more important than for common variants due to the absence of statistical power to associate them with longevity. This approach has already successfully been applied in the case of two rare variants in the coding region of the IGF1R gene – rs777765504 (Ala67Thr) and rs34516635 (Arg437His). The investigation of the IGF1R variants in Igf1r null mouse embryonic fibroblasts by a rescue approach showed that the variants lead to reduced activity of IGF1R and downstream signalling (phosphorylation of AKT) upon IGF1 stimulation, resulting in reduced transcript levels of IGF1 signalling-induced genes. A reduction in IIS pathway activity has previously been linked to lifespan extension in model organisms, supporting the conclusion that the identified rare variants in IGF1R may indeed be causal in extending lifespan. A similar approach has been successfully used to show a functional effect of rs183444295 (Ala313Ser) and rs201141490 (Asn308Lys) in SIRT6. However, it has to be noted that these variants were not significantly enriched in long-lived individuals, making it less convincing that they contributed to the longevity of their carriers.",Genetics of Human Longevity: From Variants to Genes to Pathways,Rare Longevity Variants,2024
In Vivo Characterization of Genetic Variants,"The above-mentioned functional studies pave the way for further investigations of other longevity-associated common variants, as well as rare genetic variants identified from whole-genome and exome sequencing of long-lived individuals. Given that some of these newly identified variants might be conserved in model organisms, investigation of their in vivo effect could further elucidate whether the variants can indeed induce a healthier ageing phenotype and an extension in lifespan. The first studies using this approach are currently underway.",Genetics of Human Longevity: From Variants to Genes to Pathways,In Vivo Characterization of Variants,2024
Supporting Evidence from Studies in Model Organisms,"To improve our selection of the most promising lifespan-associated genes, we need to combine all possible information from studies in model organisms, with a focus on evolutionarily conserved pathways, and ideally supporting evidence from human studies. This knowledge can subsequently be used to screen the genetic data obtained from long-lived individuals to identify rare genetic variants in these candidate genes. In the past decades, studies in model organisms have investigated various genes and their potential to increase lifespan. This research was initiated by the landmark study by Kenyon et al., who showed that mutations in daf-2 – the mammalian homolog of the insulin/IGF-1 receptor family – can more than double the lifespan of nematode worms (Caenorhabditis elegans). This finding that genetic manipulations of genes involved in the IIS pathway result in lifespan extension was subsequently replicated in fruit flies (Drosophila melanogaster) and mice (Mus musculus), and opened up the field for investigation of other conserved pathways and their potential to regulate lifespan.",Genetics of Human Longevity: From Variants to Genes to Pathways,Model Organism Studies,2024
Pathways Enriched for Longevity-Associated Genes in Model Organisms,"To methodically determine the most relevant and conserved longevity-associated pathways that are based on experimental approaches, the longevity-associated genes for mice, fruit flies and nematode worms were collected from GenAge. All ‘pro-longevity’ and ‘anti-longevity’ genes were used, and only unannotated genes were excluded. The gene list was uploaded to Gene Ontology, and the annotation dataset PANTHER Pathways was employed to enrich for pathways. The significantly enriched pathways were compared between the three organisms and reduced to the pathways that were found to be significantly enriched in all of them. Based on the pathway enrichment of the longevity-associated genes, the following overarching pathways could be identified: (1) cellular stress, (2) IIS, (3) endothelin signalling, (4) apoptosis signalling and (5) immune function. It should be noted that the main limitation of this analysis is that many of the lifespan studies in model organisms – especially in mice – are biased, as they were based on initial findings in lower organisms. Hence, pathways that are only relevant for higher organisms are not picked up using this approach.",Genetics of Human Longevity: From Variants to Genes to Pathways,Conserved Longevity Pathways,2024
Comparative Genomics and Evolutionary Insights into Longevity,"Many genes that are essential for the function of an organism have evolved in a shared ancestral organism and are thus largely conserved across species. We assume that genes – or at least the underlying molecular mechanisms and pathways – that govern ageing also follow this pattern. Hence, we can utilize comparative biology to learn more about human longevity. An example of this is provided by the IIS pathway. The investigation of variation in maximum lifespan between closely related animals may thus enable us to determine common mechanisms inherent to multiple long-lived species. Based on a comparative genomic study among mammals, reptiles and amphibia, de Magalhães and Toussaint concluded that the type of ageing observed in humans likely evolved early in mammalian evolution, probably as a side effect of another mammalian-specific trait, and should therefore obey similar mechanisms in all mammals. The extended lifespan observed in some mammals – such as naked mole rats, elephants, bats and whales – is likely caused by adaptations that were independently attained in these species. Consequently, shared biological adaptations that increased the lifespan of other mammals might also translate to humans.",Genetics of Human Longevity: From Variants to Genes to Pathways,Comparative Genomics and Longevity,2024
Evolutionary Theories and Adaptations of Longevity,"Genes that have the capacity to increase lifespan are assumed to be more conserved in long-lived species. However, in the context of evolution, it would also make sense that the perturbation of genes that are highly conserved in long-lived species is so stringently conserved because these genes cannot tolerate perturbation and, as a consequence, would shorten lifespan if mutated. Another hypothesis is that ageing is conserved in mammals, and as longevity is positively correlated with the complexity of the intraspecies relationships – meaning that in social species living in groups, longevity provides a fitness benefit to the survival of the population – it is consequently an adaptation. In this context, shared or similar variations in genes of long-lived species that are otherwise conserved in all mammals are of interest. If longevity is indeed an adaptive mechanism, then the focus of the field should shift to genes that are rapidly evolving as a consequence of positive selection. The findings from comparative genomic studies that investigated different mammals at the family or species level that evolved longevity in comparison to their short-lived cousins are summarized in Table 6.",Genetics of Human Longevity: From Variants to Genes to Pathways,Evolutionary Adaptations of Longevity,2024
Most Prominent Longevity-Associated Candidate Pathways for Future Studies,"Condensing all longevity-associated genes down to the ones that were implicated by at least two of the above-described approaches in either humans or model organisms results in a list of 18 genes. These remaining genes can be assigned to five overarching pathways: (1) IIS, (2) DNA-damage response and repair, (3) immune function, (4) cholesterol metabolism and (5) telomere maintenance. Eight of the 16 genes – namely AKT, FOXO, IGF, INSR, IRS, PIK3C, RPS6KB/S6K and SHC – can be grouped as part of the IIS pathway, although some of these genes – such as AKT and PIK3C – are also part of other pathways, such as immune function. The remaining genes are characteristic for the four other pathways – with BLM, ERCC, RAD and XRCC being part of the DNA-damage response and repair, and IL6 and ADCY being genes relevant to immune function. Finally, APOE and POT1 belong to the pathways of cholesterol metabolism and telomere maintenance, respectively.",Genetics of Human Longevity: From Variants to Genes to Pathways,Candidate Longevity Pathways,2024
The Importance of the IIS Pathway in Human Longevity,"Based on this summary, the IIS pathway is the most interesting to investigate further in humans as it comprises multiple genes that are directly linked to longevity. Previous candidate pathway studies based on common, as well as rare, genetic variants have already provided some evidence that the IIS pathway is indeed relevant to human longevity. Consequently, further investigation of this pathway – including a focus on rare genetic variants coming from exceptionally long-lived humans – would likely prove fruitful. Furthermore, these findings are supported by several studies that have shown that non-genetic interventions – for example dietary restriction or pharmacological treatment (i.e. rapamycin) – can extend lifespan in model organisms through manipulation of the IIS pathway. All genes that were identified in Table 7 are highlighted in the simplified version of the IIS pathway.",Genetics of Human Longevity: From Variants to Genes to Pathways,IIS Pathway and Longevity,2024
Other Pathways Relevant to Longevity and Lifespan Regulation,"Although the genetic evidence based on the current literature is weaker, the other four pathways could also be of interest in future model organism- and human-based studies. There is some evidence for the involvement of these pathways in the regulation of lifespan across species, including human longevity. It is clear from the literature that manipulation of the DNA-damage response and repair, the immune function and telomere maintenance pathways can result in changes in lifespan. Cholesterol metabolism, on the other hand, is strongly linked to human longevity via APOE. This pathway is unique in that no model organism-based genetic study has implicated cholesterol metabolism in the regulation of lifespan, which is likely due to the unique role the different APOE isoforms play in humans.",Genetics of Human Longevity: From Variants to Genes to Pathways,Alternative Longevity Pathways,2024
"Nutrition, Longevity, and Disease: Overview","Diet as a whole, encompassing food composition, calorie intake, and the length and frequency of fasting periods, affects the time span in which health and functional capacity are maintained. Aging and nutrition studies in simple organisms, rodents, monkeys, and humans link longevity to conserved growth and metabolic pathways and outline their role in aging and age-related disease. This research focuses on feasible nutritional strategies shown to delay aging and/or prevent diseases through epidemiological, model organism, clinical, and centenarian studies, while emphasizing the importance of avoiding malnourishment and frailty.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Nutrition and Longevity Overview,2022
Defining a Longevity Diet,"The findings from aging and nutrition studies are integrated to define a longevity diet based on a multi-pillar approach adjusted for age and health status to optimize lifespan and healthspan in humans. This approach considers the balance between nutrient intake, fasting frequency, and overall caloric consumption to promote metabolic resilience, delay aging, and prevent age-associated diseases while maintaining optimal physiological function.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Longevity Diet Framework,2022
Introduction: Food as Medicine and the Foundation of Longevity,"In 440 BCE, the Greek physician Hippocrates said, 'Let food be thy medicine and let thy medicine be food.' His wisdom has proven true since we now know that altering the level, type, and timing of food consumption (i.e., fasting) is perhaps the most potent, feasible, and safest intervention to improve health, extend longevity, and extend the time in which health and functional capacity are maintained (i.e., healthspan) in species ranging from bacteria to humans. The fundamental relationships between nutrients and cellular responses are conserved from unicellular microorganisms to humans. However, despite extensive research, the type, quantity, and combination of nutrients that optimize healthy longevity remain highly controversial. Increasing evidence suggests that in humans, nutrition must be adjusted to age, sex, genetics, and metabolic risk status of an individual and that tailoring specific dietary recommendations is essential for full beneficial effects to be realized. Understanding and harnessing these evolutionarily conserved mechanisms, in addition to personalizing dietary interventions, will be key to optimizing human healthspan and longevity.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Introduction to Nutrition and Longevity,2022
Nutrition and Delayed Aging in Short-Lived Species,"The pace of aging can be altered by inhibiting the function of nutrient-responsive genes and pathways or altering the quantity, type of nutrients, and feeding patterns that regulate them. Evidence from studies in yeast, worms, and fruit flies demonstrates that nutritional modulation of longevity operates through metabolic and growth regulatory pathways that are key influences on healthspan. These conserved mechanisms play central roles in aging regulation. Insights gleaned from studies of short-lived species provide the foundation for the fundamental biology of longevity and for understanding how different nutrients and their levels impact molecular processes vital to maintaining health with advancing age.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Nutrition and Aging in Model Organisms,2022
Yeast as a Model for Aging Research,"Aging in yeast is assessed either by measuring survival of nondividing cells (chronological lifespan) or the replicative capacity of individual mother cells (replicative lifespan). The quantity and type of nutrients available are at the center of the regulation of virtually every stage of the life history of simple organisms. Sugars and specific amino acids have strong effects in regulating both stress resistance and longevity pathways in yeast. In Saccharomyces cerevisiae laboratory strains, nutrients are provided in the form of carbohydrates, proteins, and lipids. The presence of glucose activates the yeast Ras-adenylate cyclase (AC)-PKA pathway, whereas amino acids regulate the Pkh/PDK and Tor-Sch9/S6K pathways. Mutations that decrease the activity of either the Tor-Sch9/S6K or Ras-AC-PKA pathways extend lifespan and healthspan, accompanied by the activation of stress resistance transcription factors Msn2/Msn4, increased antioxidant enzyme expression, reduced DNA damage, and an extended reproductive period. Genetic mutations in both pathways have additive effects on lifespan, indicating multiple routes to harness growth pathways to extend longevity.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Yeast Aging Mechanisms,2022
Molecular Mechanisms of Caloric Restriction in Yeast,"Recent studies are defining the molecular impact of diet composition and fasting on aging. Yeast studies of caloric restriction (CR) usually involve lowering the availability of sugars (e.g., from 2% to 0.5% glucose) or nitrogen sources (e.g., amino acid restriction). Genetic studies of nitrogen restriction indicate that autophagy, mitochondrial function, translation, RNA processing, and the stress response are all important in conferring longevity. Although restriction of different carbon sources (e.g., glucose, galactose) can have different effects on lifespan extension, the shared key pathways associated with longevity involve the regulation of glycolysis and the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, lipid metabolism, oxidative stress, DNA damage, apoptosis, and autophagy. Mechanistically, longevity extension is linked to increased stress resistance, altered redox metabolism, and potentially increased engagement of lipid and peroxisomal metabolism. Thus, aging studies in this unicellular eukaryote model reveal interconnections among stress and nutrient signaling pathways and indicate that signal transduction pathways activated by glucose and amino acids reduce stress resistance and accelerate metabolism and growth to shorten lifespan.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Caloric Restriction in Yeast,2022
Insulin Signaling and Longevity Regulation in Worms,"In the simple nematode Caenorhabditis elegans, the insulin signaling pathway influences longevity using key players similar to those in yeast, including the insulin receptor (IR) homolog Daf-2, AKT, TOR, and the stress resistance forkhead transcription factor (FOXO) Daf-16. Genetic studies on longevity regulation in worms also implicate stress signaling, mitochondrial function, metabolic adaptation, nuclear receptor signaling, translation regulation, and immune modulation. Some of the first longevity genes identified in worms (age-1 and clk-1) were linked to insulin, growth signaling, and mitochondrial function. These mutants were later associated with the mitochondrial unfolded protein response (mitoUPR), which sensitizes the innate immune response via stress response signaling. Several studies indicate that the mechanisms behind longevity conferred by dietary restriction (DR) and by reduction in insulin-like signaling are similar but not equivalent.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Insulin Signaling and Longevity in Worms,2022
Dietary Restriction and Fasting Mechanisms in Worm Longevity,"In worms, dietary restriction (DR) is often accomplished by food dilution, as the animals live on their food source, a bacterial infused layer. The term DR rather than caloric restriction (CR) is used in worm studies because actual calorie intake of individuals is not quantified. DR, which is effective in extending longevity in worms, recruits many of the same genes identified by genetic screens as modulators of longevity, including those in growth signaling, proteostasis, the stress response, and metabolic pathways. Fasting induces pathways involved in proteostasis through stress response signaling factors and protects against the disruption of proteostasis, suggesting a protective metabolic state. Mitochondrial and peroxisomal function have also been implicated in worm longevity regulation by DR. Remodeling of mitochondrial architecture is required for longevity, and peroxisomal involvement reflects greater reliance on lipid fuel utilization during nutrient deprivation.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Dietary Restriction and Proteostasis in Worms,2022
"Gene Regulation, Proteostasis, and Autophagy in Worm Longevity","Changes in gene activation or repression are key to implementing the longevity program, but other regulatory mechanisms are involved. The metabolic switch to lipid-based metabolism with dietary restriction involves changes to gene expression via RNA processing. Regulation of global protein homeostasis (proteostasis) is also important in dietary restriction mechanisms; while global translation is diminished, subsets of transcripts are preferentially translated, indicating a targeted rather than generalized reduction of protein synthesis. Genetic strategies to augment autophagy extend longevity in worms, highlighting the importance of recycling and removal of damaged proteins. The protective effect of autophagy is linked to mitochondrial function, indicating a role for metabolism-regulated proteostasis pathways. The proteasome system, which degrades damaged proteins, declines with age but can be rescued by genetic strategies mimicking dietary restriction or dampening growth signaling. Overall, worm aging studies reveal complex interactions between growth, metabolism, and proteostasis in the regulation of longevity.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Autophagy and Proteostasis in Worm Longevity,2022
Insulin-Like Signaling and Longevity in Flies,"There is substantial evidence that reduced insulin-like signaling extends longevity in the fruit fly Drosophila melanogaster. Factors including the fly homologs of insulin receptor substrate (Chico), AKT, and forkhead transcription factor (dFOXO) are established longevity regulatory factors. Pharmacological strategies to reduce growth signaling are effective in enhancing fly lifespan. The fly model is particularly advantageous due to the ability to study large numbers of organisms and the availability of powerful genetic tools, allowing in-depth exploration of genetic and nutrient interactions in longevity regulation. Studies in flies reveal interactions between genetics and diet that impact longevity. Metabolic hubs linked to longevity across genetic backgrounds include glycolytic and gluconeogenic intermediates such as phosphoenolpyruvate, amino acids like threonine and arginine, and alpha-ketoglutarate, a key factor in the TCA cycle, transamination reactions, and epigenetic gene regulation.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Insulin Signaling and Longevity in Flies,2022
Metabolic Regulation and Nutrient Balance in Fly Longevity,"Flies fed citrate or beta hydroxybutyrate, a component of ketone bodies, are healthier and live longer, linking the TCA cycle and ketogenesis to longevity programs independently of other effects of fasting. Macronutrient balance plays a critical role in lifespan regulation, with both very low and very high protein levels negatively impacting survival. These findings align with similar results observed in mice and humans. Dietary restriction (DR) is implemented in flies by diluting the diet and acts in part independently of insulin-like signaling pathways, at least for the upstream longevity mechanisms. Genetic differences among strains affect the ability of DR to increase survival. Transcriptional analysis identifies phases of response to DR, beginning with activation of oxidative metabolism, followed by stress signaling and lipid metabolism, and later autophagy, stress, and a metabolic shift toward fatty acid oxidation (FAO) and gluconeogenic gene expression.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Dietary Restriction and Metabolic Regulation in Flies,2022
"Fasting, Proteostasis, and Age-Specific Interventions in Flies","Intermittent fasting (IF) effectively delays aging in adult flies, but the animals need to be switched back to ad libitum feeding at older ages, highlighting the need for age-specific dietary interventions. IF suppresses the age-related decline in proteostasis-related pathways and affects both stress response and inflammation. It also preserves the integrity of gene expression regulation and has additive effects when combined with dietary restriction. Fasting in flies induces the cAMP-responsive transcription factor CREB, known for its role in metabolic regulation, as well as in inflammatory and immune pathways. Time-restricted feeding (TRF) is also beneficial and is associated with depletion of ectopic lipid stores. These findings emphasize that different fasting regimens can interact with metabolic and signaling pathways to enhance healthspan and longevity in simple organisms.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Fasting and Proteostasis in Fly Longevity,2022
Take-Home Message from Short-Lived Species,"Studies in short-lived species are invaluable for advancing the field of nutrition and aging research. It is clear that genes regulate the health and longevity of these organisms and that many of the key aging genetic pathways are regulated by nutrient levels and composition. Studies in simple organisms also indicate that genes play a role in how an individual organism responds to nutritional cues to promote health and longevity. There are complex interactions between nutrient composition and the engagement of longevity pathways. Furthermore, the age of onset influences diet efficacy, a feature also evident in mammalian studies. In short-lived species, aging appears to be regulated through inhibition of growth and alteration of metabolic pathways. Mechanisms associated with fasting, including greater stress resistance, reliance on lipid fuel use, and activation of proteostatic mechanisms, are shared features of delayed aging. A substantial body of evidence indicates that cellular processes including mitochondrial energy metabolism, autophagy, and the stress response are likely causal in implementing longevity induced by diet manipulation. Importantly, these signatures are at least partially conserved in mammals.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Take-Home Message from Short-Lived Species,2022
Nutrient Response Pathways in Mammals,"In mammals, specific nutrients influence genetic pathways that regulate aging and disease, pointing to conserved mechanisms of longevity across species. Within non-restrictive feeding strategies, diets with increased protein and certain amino acids, such as methionine, elevate growth hormone (GH) and insulin-like growth factor 1 (IGF-1) signaling, thereby shortening lifespan. For example, reducing protein intake from 18% to 7% of calories in mice decreased IGF-1 levels by over 30% and doubled levels of IGFBP1, an inhibitor of IGF-1 signaling. Similarly, humans on high-protein diets have significantly higher circulating IGF-1 levels. Genetic studies in mice demonstrate that deficiencies in GH or its receptor (GHR) extend lifespan by 35%–50%, while deficiencies in growth hormone releasing hormone receptor (GHRHR) extend lifespan by 20%–25%, highlighting the GHRH-GH-GHR axis as a key regulator of aging and lifespan. These deficiencies result in reduced circulating IGF-1 and insulin levels, contributing to extended longevity.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Protein-Endocrine Axis and Longevity in Mammals,2022
Growth Hormone and IGF-1 Signaling in Lifespan Regulation,"The relative contributions of insulin, IGF-1, and GHR signaling to lifespan extension in GH- or GHR-deficient mice remain unclear, but reduced activation of these pathways correlates with protection from age-related diseases. In GHR-deficient mice, tumor incidence decreased from 83.3% in wild-type animals to 42.1%, with complete protection from adenocarcinomas. These mice are also resistant to insulin resistance and age-related cognitive decline. Insulin and IGF-1 activate their receptors and the downstream IRS-PI3K-AKT and TOR-S6K pathways, which regulate growth and metabolism. Mice lacking one copy of the IGF-1R gene live 16%–33% longer, with females showing stronger effects, while IRS-1 mutants exhibit 16%–30% lifespan extension. Knockout of the S6K gene or treatment with the TOR-S6K inhibitor rapamycin beginning in middle age also extends longevity. These findings align with observations in yeast, flies, and worms, where reduced TOR-S6K or insulin-like signaling enhances lifespan and healthspan.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",GH/IGF-1 Signaling and Aging Mechanisms,2022
Human Evidence for GH and IGF-1 Pathway Modulation,"Studies in humans mirror the longevity benefits of reduced GH-IGF-1 signaling observed in animal models. Individuals with growth hormone receptor deficiency (GHRD), such as those in Ecuador, exhibit extremely low cancer incidence and rare cases of diabetes despite high obesity prevalence, attributed to increased insulin sensitivity. These individuals also maintain cognitive performance comparable to younger controls. The effects of GHRD on obesity, insulin sensitivity, diabetes, cancer protection, and cognitive preservation are consistent with observations in GHRD mice, reinforcing the conserved role of the GH-IGF-1 axis in modulating aging and disease resistance across species.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Human Evidence for GH/IGF-1 Pathway in Longevity,2022
The Sugar-Endocrine Axis in Aging,"In addition to the protein-endocrine axis, sugars play a central role in signaling pathways that accelerate the aging process. As observed in Saccharomyces cerevisiae, glucose may contribute to mammalian aging both by increasing insulin release and by directly activating certain pro-aging pathways. In rat cardiomyocytes, glucose restriction induces activation of the early growth response protein 1 (Egr1) transcription factor, the mammalian ortholog of yeast Msn2/Msn4, through regulation of protein kinase A (PKA) and AMP-activated protein kinase (AMPK). Glucose can also activate mTORC1, which senses dihydroxyacetone phosphate (DHAP), a glycolytic metabolite. These pathways collectively suggest that excess glucose availability promotes cellular processes that accelerate aging.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Sugar-Endocrine Axis and Aging,2022
Genetic and Molecular Evidence of Sugar-Induced Aging,"Mutations that disrupt glucose-associated signaling pathways are linked to increased longevity and reduced morbidity in mammals. For instance, disruption of adenylyl cyclase (AC) type 5, predominantly expressed in the heart and brain, increased median lifespan by 30% and protected mice from cardiomyopathy, possibly through reduced oxidative damage, paralleling findings in yeast. Similarly, disruption of the RIIb subunit of PKA in male mice extends lifespan, lowers fasting glucose and insulin levels, and protects against left ventricular hypertrophy. These studies indicate that modulating glucose-sensing and cAMP-dependent pathways can mitigate age-related pathologies and promote longevity.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Glucose Signaling and Longevity Mechanisms,2022
Caloric Restriction in Rodents,"There is a large literature on the beneficial impact of caloric restriction (CR) on indices of health, including the onset and progression of multiple age-related diseases and conditions in rodents. The mechanisms responsible for the effects of CR on longevity and disease involve nutrient-responsive signaling genes, although additional mechanisms, including the prevention of insulin resistance and metabolic diseases, are also involved. Decreased adiposity is a hallmark of CR in rodents and is associated with changes in fat function, including the secretion of protein and lipid factors associated with metabolic homeostasis. Not all strains respond equivalently to the same imposed degree of restriction, and sex dimorphism has become a focal point in nutritional studies of aging. Despite a dampened growth response in rodents on CR, the immune response is often improved, though it remains to be determined whether CR increases susceptibility to infectious diseases. A major question in rodent studies is whether the benefits of CR can be replicated through manipulation of diet composition or timing of feeding, as evidence suggests potential interactions between these factors. As the literature on CR in rodents has been extensively reviewed, this review focuses on the effects of CR in monkeys and humans.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Caloric Restriction in Rodents,2022
Caloric Restriction in Monkeys,"Nonhuman primates are highly translational models for biomedical research, and the rhesus monkey (Macaca mulatta) is one of the best characterized for aging studies. Two major long-term studies of CR were conducted over 30 years—one at the Wisconsin National Primate Research Center and the other at the National Institute on Aging (NIA) intramural program. Although early results appeared contradictory, later comparisons revealed that monkeys which weighed less and ate less lived longer and remained healthier later in life. The hallmarks of CR in monkeys mirror those found in mouse studies: lower adiposity, lower fasting glucose and insulin, greater insulin sensitivity, and improved lipid profiles. These same metabolic improvements were also observed in short-term human CR trials. MRI studies indicate delayed brain aging in CR monkeys, characterized by preservation of gray and white matter and improved insulin sensitivity. CR also delayed sarcopenia, preserved muscle mass, prevented declines in physical activity, and reduced metabolic cost of movement. Furthermore, CR preserved the T cell repertoire, countering immune aging linked to disease vulnerability. In skeletal muscle, CR enhanced gene expression related to energy metabolism and proteostasis, while reducing immune and inflammatory gene activity. In hepatic tissue, CR increased genes related to oxidative phosphorylation, lipid metabolism, and proteostasis, and downregulated inflammatory pathways. Serum metabolomics revealed conserved biological responses to CR between rodents and monkeys, including enrichment of ketone bodies, fatty acids, and fasting-associated metabolites such as succinate, glutamine, and lactate. These findings suggest that the biology of CR is at least partially conserved from mice to nonhuman primates.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Caloric Restriction in Monkeys,2022
Caloric Restriction in Humans,"The NIH/NIA-sponsored 2-year CALERIE study demonstrated that the systemic hallmarks of caloric restriction (CR) observed in rodents and monkeys are largely recapitulated in humans. CR induced a loss of total body weight and a reduction in adiposity, resulting in fat-free mass being higher as a percentage of total body mass in individuals on the CR regimen. CR was associated with greater insulin sensitivity, lower cardiovascular disease risk scores, and improved biomarkers of liver health. Analysis of clinical and plasma biomarkers from CALERIE subjects indicates that the pace of aging is delayed, a finding later corroborated using methylation clock analysis. The similarities between human, nonhuman primate, and rodent responses to CR argue for strong conservation of the underlying mechanisms through which CR impacts mammalian health and longevity.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Caloric Restriction in Humans,2022
The Biology of Mammalian Caloric Restriction,"Despite species specificity in how aging manifests and mortality determinants, the cellular biology underlying caloric restriction (CR) is conserved. Key processes promoting healthier physiological states include autophagy, proteostasis, energy metabolism and the switch to lipid fuel usage, changes in growth signaling and protein synthesis pathways, and engagement of gene regulatory mechanisms such as RNA processing—all linked to longevity regulation in short-lived species. Longevity is generally associated with reduced growth pathway activity and a shift toward metabolic patterns typical of fasting responses. These changes coincide with reduced inflammation without compromising immune function, offering protection against diseases such as cancer, cardiovascular disease, Alzheimer’s, and autoimmune disorders. However, diet timing, sex, and individual metabolic and genetic backgrounds all influence CR efficacy. Although genetic effects on CR outcomes are less pronounced in outbred species like humans than in inbred laboratory animals, genetics remain an important consideration in optimizing dietary interventions. The overarching conclusion from aging and nutrition research is that while no single dietary pattern fits all, specific nutritional strategies can optimize health and longevity.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Biological Mechanisms of Caloric Restriction,2022
Time-Restricted Eating (TRE) in Humans,"There is growing evidence supporting the beneficial effects of time-restricted eating (TRE) in humans. Most clinical trials have focused on weight loss and correction of metabolic impairments in subjects with obesity, metabolic syndrome, or type 2 diabetes (T2D). Common TRE regimens involve an 8–10 hour daily eating window, with durations ranging from 4 to 12 weeks, and in some cases, TRE is practiced only 5 days per week. Nearly all studies report reductions in body weight, adiposity, or waist circumference. Several studies also show improvements in circulating markers linked to cardiovascular disease, although not all trials demonstrate consistent benefits. Improvements in glucose regulation are less commonly observed, except in specific studies. For example, in healthy-weight individuals, TRE lowered glucose levels without altering cardiovascular disease indices. More stringent regimens with 6-hour eating windows improve insulin sensitivity but are more difficult to sustain.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Time-Restricted Eating in Humans,2022
Clinical and Epidemiological Evidence on TRE,"Epidemiological evidence on TRE is mixed. Longer fasting periods involving breakfast skipping have been consistently associated with higher mortality, particularly from cardiovascular disease. Alternate-day fasting for 4 weeks has been shown to improve cardiovascular markers, reduce trunk fat, improve the fat-to-lean ratio, and increase beta-hydroxybutyrate levels even on non-fasting days. Overall, TRE demonstrates metabolic and cardiovascular benefits in both rodents and humans, but compliance challenges and potential side effects suggest that an 11–12 hour daily eating period may be the most practical and sustainable option until further research establishes the safety and efficacy of shorter fasting durations.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Clinical Evidence and Safety of TRE,2022
Periodic Fasting and Fasting-Mimicking Diets in Humans,"In humans, markers and risk factors for aging and age-related diseases—including IGF-1, insulin, glucose, insulin resistance, HbA1c, C-reactive protein (CRP), hypertension, and high cholesterol—can be influenced by both dietary composition and fasting periods. Intermittent fasting (IF) regimens, such as time-restricted eating (TRE), alternate-day fasting, or two-day-per-week fasting, require frequent and long-term adherence, and only certain types are both effective and free from adverse effects. Periodic fasting (PF) cycles are emerging as an alternative to IF. Unlike IF, PF does not require continuous restriction but involves longer fasting periods of 2 to 4 consecutive days. The advantage of PF is that it can be practiced less frequently—typically once or twice per month—and still confer long-term health benefits that persist months after fasting cycles have ended. PF can also be used therapeutically, applied intermittently to treat or manage specific conditions such as cancer.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Periodic Fasting and Longevity,2022
Development and Purpose of Fasting-Mimicking Diets (FMDs),"While water-only fasting lasting three or more days is feasible, its extreme nature raises safety and compliance concerns, especially for relatively healthy individuals who lack strong medical motivation. Early clinical trials of water-only fasting in cancer patients progressed slowly due to the difficulty of the intervention and skepticism among oncologists. To overcome these limitations and enhance fasting benefits, fasting-mimicking diets (FMDs) were developed. FMDs are specifically formulated nutritional regimens designed to reproduce the physiological effects of prolonged fasting while providing essential nutrients. These diets have been tested in both animal models and human clinical studies, showing potential for improving metabolic health, reducing disease risk factors, and promoting longevity through mechanisms similar to water-only fasting.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Fasting-Mimicking Diets in Aging and Disease,2022
Periodic Fasting and Fasting-Mimicking Diets (FMDs) in Rodents,"Fasting-mimicking diets (FMDs) are plant-based, low-calorie, low-protein, low-sugar, and high-fat nutritional formulations designed to replace water-only fasting while maintaining or exceeding its physiological benefits. These diets modulate key fasting-related markers such as IGF-1, IGFBP1, glucose, and ketone bodies. As part of an emerging nutri-technology field, FMDs aim to use specific ingredients and complex food compositions as therapeutic interventions that can complement or substitute pharmacological treatments. In mouse models, periodic FMD cycles demonstrate protective effects across a broad spectrum of diseases. They improve outcomes in type 1 and type 2 diabetes models, prevent premature death associated with high-fat/calorie diets, reduce autoimmune disease symptoms and pathology, lower tumor incidence and progression, and extend lifespan. In humans, FMD cycles also lead to sustained improvements in disease markers—including IGF-1 and leptin—lasting for weeks after resuming a normal diet, mirroring the results seen in mice.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Fasting-Mimicking Diets in Rodents,2022
Mechanisms and Regenerative Effects of FMDs in Mice,"The protective and rejuvenating effects of fasting-mimicking diet (FMD) cycles in rodents involve mechanisms similar to those observed in caloric restriction, including reduced adiposity, improved insulin sensitivity, and lowered inflammation. FMD triggers the activation of stem cells and developmental-like programs across multiple tissues, with the re-feeding phase after fasting appearing central to regenerative outcomes. Consistent with these effects, FMD reduces autoimmune cell activity, promotes remyelination, and decreases pathology in mouse models of multiple sclerosis. In leptin receptor-deficient (db/db) mice, representing type 2 diabetes, FMD lowers insulin resistance and restores metabolic balance. In type 1 diabetes models, FMD induces a pancreatic gene expression pattern reminiscent of embryonic development, reversing beta cell depletion and restoring insulin production. Monthly 5-day FMD cycles in mice on high-fat diets lower body fat, improve cardiac function, reduce cholesterol, and restore lifespan to that of standard-diet mice. FMD cycles of 4 days also extend lifespan, delay tumor formation, and mitigate cognitive decline when initiated at middle age. Both CR and FMD protect against cancer in xenograft models, suggesting overlapping anti-tumor mechanisms.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Mechanisms and Regenerative Effects of FMDs,2022
Metabolite Signaling and Fasting-Induced Longevity,"Metabolite signaling plays an integral role in fasting biology. Studies show that enhanced mitochondrial activity mediated by fasting-related metabolites—such as ketone bodies and tricarboxylic acid (TCA) cycle intermediates—contributes to longevity and tissue regeneration. Administration of alpha-ketoglutarate, a TCA intermediate, extends lifespan in mice, suggesting that mitochondrial stimulation, increased stress resistance, and anti-inflammatory responses are key components of the fasting-mimicking diet’s longevity benefits. These findings underscore the potential for metabolic signaling molecules to mimic or amplify the physiological benefits of periodic fasting and FMD regimens.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Metabolite Signaling in Fasting Biology,2022
Periodic Fasting and Fasting-Mimicking Diets (FMDs) in Humans,"In humans, periodic fasting (PF) and fasting-mimicking diets (FMDs) have been evaluated both in healthy individuals and as therapeutic interventions for disease management. In a randomized crossover study involving 100 participants, 71 subjects received three monthly 5-day FMD cycles. The intervention led to significant reductions in body weight, trunk and total body fat, blood pressure, and circulating IGF-1 levels. A post hoc analysis revealed further benefits among participants with elevated baseline risk factors, including reductions in fasting glucose, triglycerides, total and low-density lipoprotein cholesterol, and C-reactive protein levels. These findings suggest that FMD cycles confer metabolic and cardiovascular benefits, particularly in individuals with pre-existing metabolic risk.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Clinical Effects of FMDs in Healthy Individuals,2022
Fasting-Mimicking Diets in Cancer Treatment,"Several clinical studies have investigated the role of fasting-mimicking diets (FMDs) in cancer therapy. A randomized trial involving 125 women with breast cancer found that FMD enhanced the efficacy of chemotherapy, improving both clinical and pathological responses, even when most participants completed only two dietary cycles. Another feasibility study with 36 breast cancer patients combined FMD with hormone therapy and demonstrated that the regimen was safe, improved biomarkers and risk factors associated with cancer progression, and did not cause loss of muscle mass or function. These results indicate that FMD can act as a supportive, non-pharmacological intervention to enhance the therapeutic response and mitigate treatment-related metabolic stress in oncology settings.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",FMDs and Cancer Therapy Outcomes,2022
Overall Therapeutic Potential of FMD Cycles,"Fasting-mimicking diet (FMD) cycles have been consistently associated with anti-inflammatory, metabolic, and regenerative effects in both animal and human studies. The beneficial physiological changes induced by FMDs, including reductions in inflammation and metabolic risk markers, can persist for months after completion of the cycles. This suggests that even infrequent implementation—approximately 3 to 4 times per year—may yield clinically meaningful benefits for disease prevention and treatment. Moreover, these interventions can complement long-term improvements in daily dietary habits without requiring continuous restriction, offering a practical and sustainable strategy for enhancing human healthspan and longevity.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Long-Term Benefits and Clinical Applications of FMDs,2022
Macronutrient Composition and Levels in Longevity,"The influence of nutrition on lifespan and age-related disease is well established, though consensus on the optimal diet for promoting healthspan remains elusive. Nutrient response mechanisms affecting longevity are highly conserved across species, from simple organisms to humans, enabling cross-species insights into effective dietary strategies. Optimal nutrition must be tailored not only to general principles but also to individual variables including age, sex, genetics, lifestyle, and current health status. Future advances in multi-omic profiling and artificial intelligence are expected to refine personalized nutritional therapies. However, it is already clear that diets promoting central adiposity drive insulin resistance and elevate risks for diabetes, cancer, and neurodegenerative diseases in both rodents and humans.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Macronutrient Composition and Personalized Nutrition,2022
High-Calorie Diets and Their Effects on Healthspan,"In rodents and humans, calorie intake exceeding energy expenditure triggers lipogenesis, fat accumulation, and obesity, major contributors to age-related diseases. Excess glucose is converted to triglycerides in the liver and transported to adipose and muscle tissues via very-low-density lipoproteins (VLDL). While genetic factors modulate dietary responses, diets high in saturated fats and sugars consistently promote obesity, insulin resistance, hypercholesterolemia, and shortened lifespan. In rodent models, high-fat diets containing 40–60% of calories from fat—often accompanied by high sugar content—are sufficient to induce these pathologies. Similarly, in humans, daily calorie consumption has risen approximately 20% (∼425 kcal/day) since 1970, with the Western diet characterized by elevated levels of sugars, starches, saturated fats, and proteins. This dietary pattern leads to increased insulin, blood glucose, IGF-1, and lipid levels, which activate pro-aging pathways while fostering metabolic dysfunction and obesity. Together, these effects accelerate aging and drive the onset of chronic diseases independently of chronological age.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",High-Calorie Diets and Aging Mechanisms,2022
Low-Carbohydrate and Ketogenic Diets Overview,"Low-carbohydrate diets in humans typically restrict carbohydrate intake to 50–60 g per day, replacing the remaining calories primarily with fats and moderate to high levels of protein. The ketogenic diet (KD), first described by Dr. Wilder in 1921 as a fasting-mimicking therapy for epilepsy, was designed to induce ketosis by limiting carbohydrate intake to less than 15 g per day, providing about 1 g of protein per kilogram of body weight, and deriving the rest of the calories from fat. The modern adaptation of the KD, popularized in the 1970s by Robert Atkins, allowed for higher protein intake while maintaining low carbohydrate consumption. However, the widespread adoption of modified KDs often led to inclusion of Western diet ingredients, such as processed fats and animal products, which may negate potential health benefits.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Introduction to Ketogenic and Low-Carbohydrate Diets,2022
Ketogenic Diet Studies in Rodents,"In animal studies, the ketogenic diet (KD) modestly increases lifespan and enhances metabolic, physical, and cognitive function when applied to adult mice. Cyclic KD regimens further improve metabolic health and cognitive performance. KD has been shown to improve cerebrovascular function in mice and enhance cognition in rat models via modulation of brain metabolite transport systems. In Alzheimer’s disease models, KD and exogenous ketone body supplementation reduce amyloid plaque burden and improve cognitive function, likely through enhanced mitochondrial activity in hippocampal neurons. Mechanistically, KD induces autophagy in the liver, a critical step for ketone body synthesis, through the activation of PPARα and autophagic clearance of inhibitory complexes. These findings highlight the interconnected roles of lipid metabolism, autophagy, and stress resistance in mediating the health and longevity benefits of KD. However, since many KD formulations involve low protein intake, some observed benefits may partially result from protein restriction. Future research should evaluate how different fat sources—animal versus plant-based—modulate KD’s effects on aging and disease.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Ketogenic Diets in Rodent Models of Longevity,2022
Human Studies and Epidemiological Evidence on Low-Carb Diets,"In humans, ketogenic and low-carbohydrate diets have been extensively studied for weight loss and metabolic health. Meta-analyses show that low-carbohydrate and ketogenic diets are not more effective than balanced low-calorie or low-fat diets in reducing BMI or improving cholesterol and triglyceride profiles. Large-scale epidemiological studies provide mixed results regarding mortality outcomes. A study tracking over 129,000 adults for two decades found that low-carbohydrate diets based on animal sources were associated with higher all-cause and cancer mortality, while those emphasizing plant-based sources were linked to lower mortality rates. Specifically, men on animal-based low-carb diets exhibited a 66% increased cancer mortality risk, while women showed a 26% increase. Another meta-analysis of over 432,000 participants revealed that both very low (<40% of energy) and very high (>70% of energy) carbohydrate intake increased mortality risk compared to moderate intake (50–55%). Mortality risk rose by more than 50% in groups consuming less than 20% of energy from carbohydrates. Importantly, replacing carbohydrates with plant-derived proteins and fats reduced mortality risk, whereas replacing them with animal-derived sources increased it. These findings underscore the importance of macronutrient source and balance, suggesting that moderate, plant-based low-carbohydrate diets may represent a more sustainable and health-promoting alternative to strict ketogenic regimens.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Human Studies and Mortality Risk in Low-Carb Diets,2022
Low-Protein and Amino Acid Diets in Rodents,"A comprehensive study in mice comparing 25 diets varying in fat, protein, and carbohydrate content revealed that low-protein, high-carbohydrate diets were the most beneficial for longevity and health, though the effects varied between sexes. Follow-up studies demonstrated that low-protein diets improved cognition and preserved neuronal architecture, likely through activation of nutrient signaling pathways in the hippocampus. Conversely, very-low-protein diets reduced food intake due to altered hypothalamic signaling. Methionine restriction (MR) has been shown to increase lifespan in mice and may enhance cancer treatment efficacy when combined with standard therapies. MR activates the liver-derived hormone FGF21, which mediates beneficial effects on metabolism, adipose remodeling, and inflammation. Lifelong restriction of branched-chain amino acids (BCAAs) reduces adiposity and extends lifespan in male but not female mice, indicating sex-dependent differences in response. Overall, protein or amino acid restriction reduces IGF-1 signaling and promotes longevity through mechanisms conserved from yeast to mammals.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Protein Restriction and Longevity in Rodents,2022
Protein Restriction and Growth Signaling in Humans,"In humans, caloric restriction alone does not significantly lower IGF-1 levels unless accompanied by reduced protein intake. Similar to rodents, low-protein diets suppress pro-growth signaling both upstream (GHRH, GH) and downstream (mTOR, S6K) of IGF-1. This leads to lower insulin levels and improved insulin sensitivity. However, the relationship between protein intake, mortality, and longevity is age-dependent. For individuals aged 65 or younger, consuming more than 20% of calories from protein is associated with a 75% increase in overall mortality and a 400% increase in cancer mortality compared to diets with less than 10% of calories from protein. These associations disappear in individuals aged 66 and older, suggesting that protein restriction may lose its protective effect with age due to diminished inhibition of pro-aging pathways. These findings imply that dietary protein needs should shift with age to maintain metabolic balance and longevity benefits.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions","Protein Intake, IGF-1 Signaling, and Age-Dependent Effects",2022
Protein Source and Mortality Correlations,"Epidemiological data indicate that not only the quantity but also the source of dietary protein influences mortality and longevity. Re-analysis of low-carbohydrate diet studies suggests that the higher mortality observed in low-carb groups may be partly due to elevated protein intake, particularly from animal sources. In one study, the lowest-carbohydrate group consumed 22.3% of energy from proteins versus 15% in the high-carbohydrate group, corresponding to higher all-cause, cardiovascular, and cancer mortality. In contrast, participants consuming vegetable-based low-carbohydrate diets, which maintained moderate protein levels (~18.7% of energy intake), exhibited reduced all-cause and cardiovascular mortality. These findings emphasize that animal-derived proteins are strongly associated with age-related mortality and chronic disease risk, while plant-based proteins appear protective. They also highlight the importance of macronutrient balance and suggest that the efficacy of dietary interventions depends on both composition and the individual's age range.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Animal vs. Plant Protein and Mortality Outcomes,2022
Low-Fat and High-Fat Diets,"For decades, low-fat diets have been promoted to reduce obesity and cardiovascular risk. However, despite a reduction in dietary fat consumption in the United States, obesity rates have continued to rise, suggesting that excessive calorie intake and poor diet composition are more critical factors than fat consumption alone. A major randomized trial of 7,447 participants at high cardiovascular risk compared a Mediterranean diet enriched with extra-virgin olive oil or mixed nuts to a low-fat control diet. The study found that the risk of major cardiovascular events was approximately 30% lower in the Mediterranean diet groups, which included healthy unsaturated fats, compared to the low-fat group. These findings are consistent with epidemiological data showing that diets high in animal fat and protein increase mortality, while those high in plant-based fats and carbohydrates reduce disease risk. The overall consensus is that an optimal diet includes relatively high carbohydrate intake balanced with healthy fat sources—particularly from plants—rather than strict fat restriction.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Low-Fat vs. High-Fat Diets and Cardiovascular Health,2022
Vegan and Vegetarian Diets,"Vegan and vegetarian dietary patterns differ significantly in their health effects. Studies show that pesco-vegetarians—those who include fish but exclude other animal products—have reduced overall mortality compared to regular meat eaters. Vegan diets, while associated with lower risks of cancer, hypertension, and diabetes, are also linked to a 43% higher risk of all fractures and a 2.3-fold higher risk of hip fractures compared to non-vegans. This frailty may result from deficiencies in certain amino acids and overall protein intake. Data from the EPIC-Oxford study revealed that 16.5% of vegan men and 8.1% of vegan women consume less protein than recommended levels. The reliance on legumes as the primary protein source in vegan diets contributes to low intake of methionine and other essential amino acids. In summary, while vegan diets confer significant protection against metabolic and age-related diseases, vegetarian and pesco-vegetarian diets may offer superior long-term health outcomes by preventing protein and amino acid deficiencies that can lead to frailty.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions","Vegan, Vegetarian, and Pesco-Vegetarian Diets in Longevity",2022
A Multi-Pillar Approach for Nutrition and Healthspan,"Evidence from aging and nutrition research highlights the importance of a hypothesis-driven, multi-disciplinary framework to define dietary patterns that promote healthy longevity. Epidemiological studies alone can be misleading, as they often oversimplify complex relationships between nutrients and mortality. For instance, while low IGF-1 levels have been linked to increased mortality in some studies, others show that both low and high IGF-1 levels correlate with higher mortality, suggesting that maintaining mid-range IGF-1 levels is optimal for health. Thus, epidemiology should serve as one of four complementary pillars: (1) basic research on lifespan and healthspan mechanisms, (2) carefully controlled clinical trials, (3) studies of long-lived populations, and (4) epidemiological data assessing nutrient ranges and risk factors while considering age, sex, and metabolic status.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Framework for Nutritional Longevity Research,2022
The Longevity Diet – Principles and Composition,"The longevity diet, derived from studies across species and populations, emphasizes a normocaloric dietary pattern that supports lifespan and healthspan with minimal side effects. This diet is characterized by a moderate-to-high carbohydrate intake, low but sufficient protein intake (mostly plant-based), and healthy fat sources—primarily from plants. Regular inclusion of fish and other pesco-vegetarian proteins is recommended. For example, animal products accounted for about 1% of the traditional Okinawan diet and were also consumed occasionally in Sardinian and Loma Linda populations known for exceptional longevity. Mechanistically, this diet reduces the activity of pro-aging pathways involving GHR, IGF-1, insulin, and TOR-S6K signaling. In individuals over 65, however, excessive protein restriction may contribute to frailty due to muscle and lean mass loss, as IGF-1 levels are already naturally lower with age. Therefore, balanced protein intake becomes crucial for the elderly to maintain muscle health while minimizing age-related disease risk.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Longevity Diet Composition and Mechanisms,2022
Macronutrient Ratios and Dietary Variations,"The longevity diet typically includes approximately 30% of energy intake from fat, primarily from plant-based and unsaturated sources such as olive oil and nuts, consistent with evidence from Mediterranean-style diets. While traditional Okinawan diets were lower in fat, both patterns are effective variations that promote health and longevity. The diet also emphasizes complex carbohydrates to provide sustained energy without increasing insulin or activating glucose-related pro-aging pathways. Fats appear to have less detrimental effects on aging than protein or sugar due to their involvement in fasting metabolism and ketone body generation. Modeling studies support these findings: transitioning from a Western diet to one rich in legumes, whole grains, and nuts while reducing red and processed meats could increase life expectancy by approximately 10.7 years for women and 13 years for men if adopted at age 20, or by about 8 years when started at age 60.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Macronutrient Balance and Longevity Outcomes,2022
Practical Guidelines and Fasting Integration,"To maximize benefits and prevent malnutrition, especially in adults over 65, the longevity diet should be carefully designed to maintain adequate bone density, muscle mass, and blood health. It should ideally incorporate a 12–13-hour daily fasting window, which has been shown to be both safe and effective. Periodic fasting-mimicking diet (FMD) cycles between ages 18 and 70 can further enhance metabolic health by reversing insulin resistance and lowering IGF-1, blood pressure, cholesterol, and inflammation in at-risk individuals. Rather than adhering to a fixed calorie limit, individuals should tailor daily intake based on BMI (<25), body composition, and waist circumference. Overall, the longevity diet serves as a preventive healthcare strategy—reducing morbidity and sustaining vitality into old age when combined with regular physical activity and appropriate fasting intervals.","Nutrition, Longevity and Disease: From Molecular Mechanisms to Interventions",Practical Longevity Diet Implementation,2022
Defining Exceptional Longevity and Successful Aging,"The definition of exceptional longevity is inherently arbitrary but should incorporate distinctions between chronological and biological age, as well as the preservation of functional capacity. Two core assumptions underpin successful aging: (1) an individual’s biological age is lower than their chronological age, and (2) functional status is either preserved or its decline significantly delayed. Exceptionally long-lived individuals naturally tend to meet both conditions at least during key periods of their lifespan. Evidence from literature spanning 1980 to 2018 indicates that exceptional longevity arises from a dynamic interaction of genetic, environmental, cultural, and geographic mechanisms. Studying centenarians and other long-lived populations helps to clarify how these factors influence the pace of aging, maintain physiological homeostasis, and promote resilience. Furthermore, such research provides insights into biomarkers of aging, potential interventions to slow age-related decline, and strategies to define and measure successful aging.",Exceptional Human Longevity,Definition and Mechanisms of Successful Aging,2023
Centenarians – Demographics and Health Characteristics,"Centenarians represent one of the fastest-growing demographic groups in developed nations. In 1995, the probability of surviving from birth to age 100 was approximately 1 in 20 million across human history but had risen to 1 in 50 for females in low-mortality countries such as Japan and Sweden. By 2009, this likelihood further increased to roughly 1 in 2. Today, about 1 in 5000 individuals in the United States is a centenarian, and this prevalence is expected to rise markedly in the coming decades. Although life expectancy and median lifespan have dramatically increased over the past century, the maximum recorded human lifespan—around 122 years (Jeanne Calment)—has remained relatively constant, suggesting a possible biological limit to longevity. However, gradual future increases cannot be ruled out.",Exceptional Human Longevity,Demographic Trends and Maximum Lifespan,2023
Disease Onset and Health Profiles of Centenarians,"The onset of common age-associated diseases among centenarians is highly variable, reflecting significant heterogeneity in aging. Approximately 24% of male and 43% of female centenarians are diagnosed with one or more age-related diseases before 80 years of age, while 43% of both sexes reach 80 without major illness. Notably, 15% of females and 30% of males at age 100 have no recorded disease diagnosis. These findings indicate that delayed disease onset, rather than complete avoidance, is a key contributor to exceptional longevity. Despite aging, about 25% of centenarians remain cognitively intact, and among those who develop cognitive decline, symptoms typically appear only around age 92. Some centenarians even show neuropathological markers of Alzheimer’s disease without clinical dementia, highlighting remarkable neural resilience. Cancer tends to develop much later than in the general population, and supercentenarians (≥110 years) often experience minimal vascular disease and retain functional independence or require only limited assistance.",Exceptional Human Longevity,"Healthspan, Disease Resistance, and Cognitive Resilience",2023
Geographic Clustering of Exceptionally Long-Lived Individuals,"Clusters of exceptionally long-lived populations around the world highlight the strong influence of environmental and lifestyle factors on human longevity. Okinawans in Japan practice 'hara hachi bu'—eating until 80% full—and follow a colorful, plant-rich 'rainbow diet' in which soy is the primary protein source. Their caloric intake is significantly lower than average, leading to a lean body mass (around 20 kg/m² BMI) and suggesting that mild caloric restriction contributes to their extended lifespan. Additionally, Okinawans display a slower decline in dehydroepiandrosterone (DHEA) levels, a hormonal marker associated with longevity, similar to trends seen in caloric restriction experiments in animals.",Exceptional Human Longevity,Okinawan Longevity and Caloric Restriction,2023
Longevity in Sardinia and Genetic Isolation,"In Ovodda, Sardinia, men exhibit exceptional longevity, with many living significantly longer than their counterparts elsewhere. Interestingly, Sardinians who emigrated in early or middle adulthood still tend to achieve extreme old age, indicating a strong genetic component possibly due to lineage from a small number of original settlers and long-term population isolation. This may have resulted in unique genetic traits conducive to longevity. The case of Jiroemon Kimura, who lived to 116 and had long-lived siblings, further suggests a hereditary contribution to exceptional lifespan, though he attributed his longevity to modest meal sizes and balanced lifestyle habits.",Exceptional Human Longevity,Sardinian and Genetic Factors in Longevity,2023
Seventh-Day Adventists and Lifestyle-Based Longevity,"Seventh-Day Adventists, primarily located in Loma Linda, California, are among the longest-living populations in the United States, with average lifespans 5–10 years longer than the national mean. Their faith-based lifestyle emphasizes abstinence from alcohol and tobacco, a largely vegetarian diet, and strong spiritual and community engagement. Regular participation in religious activities—regardless of faith—has been associated with increased lifespan, likely mediated by reduced stress and lower levels of circulating stress hormones. This holistic integration of diet, spirituality, and social cohesion represents a model of health-promoting living.",Exceptional Human Longevity,Seventh-Day Adventists and Lifestyle Longevity,2023
Other Longevity Hotspots and Shared Behavioral Patterns,"Additional regions exhibiting exceptional longevity include the Nicoya Peninsula in Costa Rica and the island of Ikaria in Greece. Across these geographically distinct 'blue zones,' several common themes emerge: consumption of primarily plant-based diets with moderate caloric intake, regular physical activity integrated into daily routines, strong social and family support systems, and a sense of purpose or meaning in life. These shared behavioral and environmental factors collectively contribute to enhanced healthspan and reduced disease burden, reinforcing the view that longevity is not solely genetic but heavily shaped by lifestyle and community context.",Exceptional Human Longevity,Global Longevity Hotspots and Common Lifestyle Factors,2023
Trends in Longevity – Global Patterns and Implications,"Modern longevity trends reveal that while human life expectancy has increased dramatically across the globe, the quality of those added years remains a critical concern. Japan stands as a leading example of sustained gains in longevity, yet similar demographic shifts are emerging in other developed and developing nations. A central issue accompanying extended lifespan is whether these additional years will be characterized by health or disability. While longer life expectancy reflects major public health successes—such as improved sanitation, nutrition, and immunization that have dramatically reduced infant and early-life mortality—these improvements largely safeguard against premature death rather than directly altering intrinsic biological aging processes. Thus, the rise in median lifespan primarily mirrors the reduction of external mortality risks rather than a fundamental slowing of aging.",Exceptional Human Longevity,Global Longevity Trends and Healthspan Concerns,2023
Demographics of Longevity,"As of 2015, Japan, Germany, Italy, Greece, Finland, and Sweden had the world’s oldest populations, with projections indicating a continued longevity shift toward Asian nations by 2050, particularly Japan, South Korea, Hong Kong, and Taiwan. At age 65, Japanese women and men could expect an additional 25.2 and 20.0 years of life, respectively. Historical data show that Japanese female life expectancy increased steadily by about 3 months per year over the past 160 years, a trend unparalleled in human history. However, across countries with the highest life expectancies, the proportion of healthy life years varies widely—from 25% to 75% of total post-65 life expectancy. Notably, Norway, Sweden, and Iceland consistently report the greatest number of healthy years lived after 65, reflecting not only genetic and healthcare advantages but also social and environmental conditions that promote active aging.",Exceptional Human Longevity,Demographics and Healthy Life Expectancy,2023
Compression of Morbidity – Models of Extended Longevity,"The concept of 'compression of morbidity' explores how increased lifespan interacts with the timing and duration of disease and disability in aging populations. Three main models describe these dynamics: (1) life extension without delaying disease onset, where additional years are accompanied by prolonged morbidity; (2) parallel shifts of both disease onset and lifespan, resulting in no change in total morbidity duration; and (3) delayed disease onset with extended lifespan, where morbidity occupies a smaller proportion of total life—thus achieving 'compression of morbidity.' This third model, proposed by Fries, suggests that chronic illnesses begin later in life, and the delay in their onset exceeds the gain in total lifespan, leading to reduced cumulative disability and suffering in old age.",Exceptional Human Longevity,Compression of Morbidity Models,2023
Empirical Evidence for Compression of Morbidity,"Data trends support the notion that disability and morbidity can indeed be compressed in aging populations. U.S. studies have shown a decline in disability prevalence of about 2% per year, alongside a 1% annual reduction in mortality rates, consistent with Fries’ hypothesis. During the 1980s and 1990s, the proportion of elderly individuals requiring assistance with activities of daily living (ADLs) decreased substantially. Some research extending into the early 21st century continued to show declines, while other analyses suggested a plateau in these improvements. Moreover, between 1984 and 2010, mobility and mental health impairments also decreased among older adults. However, given that individuals aged 85 and older now represent the fastest-growing segment of the U.S. population, the compression of morbidity may be less evident at extreme old ages. In supercentenarians, delays in both physical and cognitive impairment are observed, indicating that even near the biological limits of human lifespan, disease onset continues to shift later into life.",Exceptional Human Longevity,Disability Trends and Morbidity Delay Evidence,2023
Why Are Some Individuals Long-Lived? – Overview,"The determinants of exceptional human longevity involve a complex interplay of genetic, environmental, and behavioral factors, as well as sex-specific differences and biological resiliency. Twin studies and familial patterns have been instrumental in distinguishing hereditary influences from environmental ones. While genetics confer a baseline potential for extended lifespan, environmental and lifestyle factors—including diet, social connections, and stress resilience—play dominant roles in realizing that potential. Resiliency, defined as the ability to adapt to stressors and maintain physiological equilibrium with age, is increasingly recognized as a central feature of individuals who achieve exceptional longevity.",Exceptional Human Longevity,Determinants of Human Longevity,2023
Genetic Influences on Longevity,"The genetic contribution to human longevity is supported by strong empirical evidence. Monozygotic twins exhibit more similar lifespans than dizygotic twins, and centenarians often have long-lived family members. Jeanne Calment, who lived to 122, had numerous long-lived relatives, particularly on her paternal side, suggesting her extreme longevity had a substantial hereditary basis. Specific genetic polymorphisms are associated with prolonged lifespan, notably within the APOE gene, where the ε4 allele—linked to lipid metabolism and Alzheimer’s risk—is less frequent among centenarians. Other genes implicated include those in the insulin/IGF-1 signaling pathway, cholesteryl ester transfer protein (CETP), anti-inflammatory cytokines like IL-10, RNA-editing enzymes, and stress response genes such as HSP70. Furthermore, genome-wide association studies continue to uncover novel variants correlated with long life, reinforcing that longevity is polygenic and involves networks of metabolic and repair pathways.",Exceptional Human Longevity,Genetic Basis of Longevity,2023
Gene Conservation and Functional Longevity Pathways,"Genes associated with extended lifespan appear to be evolutionarily conserved among long-lived species. For example, the BRCA1 gene and the growth hormone receptor (GHR) gene show strong correlations with maximal lifespan across mammals. In humans, a specific GHR exon 3 deletion variant increases growth hormone sensitivity, and homozygosity for this deletion is linked to approximately 10 additional years of life, particularly benefiting males. Interestingly, while antioxidant defense genes such as superoxide dismutase are essential for cellular protection, polymorphisms in these genes have not been consistently linked to longevity, suggesting that systemic regulation of stress response and metabolic balance plays a larger role than isolated antioxidant mechanisms.",Exceptional Human Longevity,Gene Conservation and Functional Adaptations,2023
Environmental and Behavioral Influences,"Despite compelling genetic associations, environmental and behavioral factors exert a stronger overall influence on human longevity. Twin studies indicate that only about 25% of the variance in adult lifespan can be attributed to genetics, leaving the majority shaped by external factors such as diet, physical activity, social environment, and healthcare. These findings underscore that long life is not predetermined but can be significantly extended through health-promoting behaviors and environments. Populations with high longevity—such as those in Okinawa, Sardinia, and Loma Linda—illustrate how cultural norms like moderate caloric intake, plant-based diets, social engagement, and low stress collectively contribute to exceptional lifespan.",Exceptional Human Longevity,Environmental and Lifestyle Factors in Longevity,2023
Sex Differences in Longevity – Overview,"Across virtually all human populations, women consistently outlive men. This pattern is evident from birth—female infants have higher survival rates—and continues throughout life. In the United States, beginning at approximately age 55, the ratio of women to men doubles by age 75 and doubles again by age 85 and older. These demographic patterns underscore a biological and possibly evolutionary advantage in female longevity, though they also highlight differences in morbidity and functional status between the sexes in later life.",Exceptional Human Longevity,Sex Differences in Human Longevity,2023
Evolutionary and Reproductive Influences,"Evolutionary perspectives suggest that selection pressures favoring female longevity may be tied to reproductive success and caregiving. Middle-aged mothers tend to live longer, and later maternal age at the birth of the last child correlates with increased lifespan. The 'grandmother hypothesis' posits that the survival advantage of older women may enhance the survival and reproductive success of their descendants by providing caregiving and social support, particularly through maternal grandmothers. Additionally, caregiving behavior—observed even in nonhuman primates—is associated with greater longevity, suggesting an adaptive benefit for extended postreproductive life.",Exceptional Human Longevity,Evolutionary and Reproductive Theories of Longevity,2023
Sociodemographic and Biological Factors,"Several sociocultural and biological factors may contribute to the sex gap in lifespan. Offspring of younger mothers tend to live longer, whereas paternal age appears to have minimal impact. For men, occupational history and genetic relatedness to other male centenarians are predictive of longevity, while for women, environmental stability—such as shared living conditions with long-lived partners—plays a more substantial role. Physiologically, women’s typically shorter stature and lower growth hormone (GH) secretion may be linked to slower aging and longer lifespan, reflecting trade-offs between growth and longevity seen across species.",Exceptional Human Longevity,Sociodemographic and Hormonal Contributors to Longevity,2023
Functional Status and Aging Quality,"Despite their longer lifespans, women often experience greater morbidity and functional decline compared to men in older age. Studies show that while women live longer, men maintain better physical and functional status in advanced years. This discrepancy illustrates a key paradox in longevity research: female survival advantage does not necessarily translate into superior healthspan. Understanding the biological and environmental underpinnings of this disparity remains a central challenge in gerontology.",Exceptional Human Longevity,Longevity and Functional Status Differences Between Sexes,2023
Resiliency and Successful Aging,"Resiliency—the capacity to withstand, adapt to, or recover from various stressors—is increasingly recognized as a defining factor of successful aging and exceptional longevity. It encompasses both physiological and psychological dimensions, influencing how individuals respond to biological challenges, disease processes, and environmental stress. Although the specific mechanisms of resiliency remain poorly defined and may vary across organ systems, evidence suggests that resilient individuals maintain homeostatic balance and functional capacity even in the face of age-related decline or chronic disease.",Exceptional Human Longevity,Resiliency and Longevity Mechanisms,2023
Physical and Physiologic Resilience,"Animal studies indicate that physical activity may enhance resilience more effectively than caloric restriction, improving the body’s capacity to handle metabolic, inflammatory, and oxidative stress. Physiologic resilience refers to the maintenance of homeostasis across multiple systems, counteracting declines in muscular strength, sleep quality, skeletal health, and cognitive function. This cross-system adaptability may explain why some centenarians live well beyond 100 years despite the onset of chronic diseases before age 80—suggesting that resilience, rather than disease absence, is key to extended survival and healthspan.",Exceptional Human Longevity,Physiologic and Functional Resilience,2023
Psychological and Social Resilience,"Psychological resiliency—the ability to cope effectively with emotional stress, maintain optimism, and recover from adversity—plays a complementary role in promoting longevity. Reduced depression, sustained cognitive engagement, and strong social relationships contribute to adaptive stress responses that buffer against behavioral and neurobiological decline. Frameworks for studying psychological and social resiliency in aging populations increasingly emphasize the interplay between emotional regulation, community belonging, and physiological stress responses, suggesting that mental resilience is deeply intertwined with biological longevity.",Exceptional Human Longevity,Psychological and Social Resilience in Aging,2023
How Is Exceptional Longevity Achieved – Overview,"Exceptional human longevity arises from a combination of biological, behavioral, and environmental strategies that support sustained healthspan. Empirical evidence highlights the roles of dietary moderation, regular physical activity, avoidance of harmful behaviors such as smoking and excessive alcohol consumption, maintenance of social relationships, and psychological engagement in extending life expectancy. Poor health behaviors are estimated to contribute to nearly 40% of preventable deaths in the United States, underscoring the power of lifestyle modification in promoting both lifespan and healthspan.",Exceptional Human Longevity,Mechanisms and Strategies for Longevity,2023
Caloric Restriction and Longevity,"Caloric restriction (CR) is one of the most robust and reproducible interventions for extending lifespan in model organisms, from yeast and worms to rodents and primates. Studies indicate that a 30–60% reduction in caloric intake can increase mean and maximum lifespan, delay the onset of metabolic and neurodegenerative diseases, and reduce cancer incidence. In rhesus monkeys, long-term CR lowered the risk of diabetes, cardiovascular disease, and brain atrophy. In humans, the CALERIE trial demonstrated that a 2-year 11.7% caloric reduction slowed biological aging based on molecular biomarkers, independent of weight loss. However, the feasibility of long-term CR in free-living humans remains limited, suggesting that understanding the underlying molecular mechanisms—such as reduced insulin/IGF-1 signaling and improved mitochondrial efficiency—may help identify more practical interventions.",Exceptional Human Longevity,Caloric Restriction and Metabolic Health,2023
Physical Activity and Body Weight,"Regular physical activity is a cornerstone of healthy aging and increased life expectancy. Exercise improves cardiovascular function, insulin sensitivity, and cognitive performance, even without significant changes in body weight. Epidemiologic studies reveal that maintaining a body mass index (BMI) between 20.0 and 24.9 kg/m² confers the lowest mortality risk, while obesity and underweight are linked to higher all-cause mortality. Vigorous walking or equivalent activity of around 450 minutes per week can extend life expectancy by approximately 4.5 years. Moreover, combining normal weight with consistent activity yields up to 7.2 additional years of life compared to sedentary obesity. These findings highlight that regular movement—not merely calorie balance—is a dominant driver of survival, particularly beyond age 74.",Exceptional Human Longevity,Exercise and Longevity,2023
Social Engagement and Psychological Well-Being,"Longevity is also deeply influenced by social and psychological factors. Strong social connections, purposeful living, and cognitive engagement correlate with reduced mortality and improved mental health in aging populations. The absence of close social relationships increases mortality risk to a degree comparable with smoking, while a strong sense of life purpose predicts better cognitive resilience and functional independence. Even short-term interventions that promote social or productive engagement have been shown to enhance memory and well-being. These findings suggest that social and psychological enrichment is a critical yet often underappreciated determinant of healthy aging and exceptional longevity.",Exceptional Human Longevity,Social and Psychological Determinants of Longevity,2023
Integration of Lifestyle Strategies,"Taken together, the evidence supports a multidimensional approach to achieving exceptional longevity: moderate caloric intake without malnutrition, regular physical activity, maintenance of healthy body weight, abstinence from harmful substances, and sustained social and cognitive engagement. These behaviors are consistently observed in populations with high concentrations of centenarians, such as Okinawa, Sardinia, and Loma Linda. Collectively, they represent practical, evidence-based pathways to extend not only lifespan but also the period of life lived in good health and independence.",Exceptional Human Longevity,Integrated Lifestyle Model of Longevity,2023
Measuring Successful Aging – Overview,"Accurately measuring successful aging requires the identification of reliable biomarkers that reflect biological rather than chronological age. Such biomarkers could enable researchers to estimate life expectancy, evaluate anti-aging interventions, and standardize research methods across studies. To qualify as a biomarker of aging, a measure should correlate quantitatively with age, remain unaffected by disease processes, be independent of confounding metabolic or nutritional influences, and change predictably with interventions that alter the rate of aging.",Exceptional Human Longevity,Definition and Criteria for Aging Biomarkers,2023
Criteria and Challenges in Biomarker Validation,"Although minimal validation criteria exist, they require refinement to account for the overlap between primary aging and chronic disease. For example, biomarkers altered during caloric restriction or pharmacologic interventions targeting senescence could still accurately reflect biological aging processes. However, distinguishing between changes due to aging itself versus superimposed disease or environmental factors remains a major challenge. Researchers must also recognize that aging affects tissues and organs heterogeneously—biochemical alterations, impaired organ function, or maladaptive responses to stress may vary significantly within individuals and across populations.",Exceptional Human Longevity,Validation and Complexity of Aging Biomarkers,2023
Physiologic and Structural Characteristics of Aging,"Common physiological features associated with aging include increased post-maturation mortality, accumulation of molecular damage (such as lipofuscin and cross-linked proteins), and reduced adaptive capacity of organs like the kidneys and liver. Functional impairments such as decreased cardiovascular response to exercise and heightened disease incidence (e.g., osteoporosis, Alzheimer’s disease) are also frequent. However, because these changes vary widely among individuals and organs, they cannot be universally applied as definitive indicators of primary aging. Moreover, eliminating leading causes of mortality such as cancer or atherosclerosis would increase life expectancy by only about a decade, underscoring that aging itself remains the primary limiting factor.",Exceptional Human Longevity,Physiological Hallmarks and Mortality Trends in Aging,2023
Candidate Biomarkers and Their Limitations,"Proposed biomarkers of primary aging and longevity include physiological measures such as gait speed, grip strength, and the ability to perform activities of daily living (ADLs). Biochemical markers—such as those used in clinical tests for glucose, lipid metabolism, or inflammation—can be integrated to estimate biological age. System-specific indicators, for instance, those reflecting cardiovascular or cognitive health, serve as useful surrogates for overall functional integrity. Nonetheless, each measure has limitations and may be influenced by disease or environmental context. The inclusion of centenarians in biomarker studies is particularly valuable, as they exemplify both the pathways to long life and the heterogeneity among the exceptionally aged.",Exceptional Human Longevity,Candidate Biomarkers and Research Implications,2023
Early-Life Biomarker Studies and Biological Aging,"Measuring biological aging in younger populations helps reduce confounding from chronic diseases. The Dunedin Study, which has followed a 1972–1973 birth cohort in New Zealand, assessed multiple dimensions—balance, grip strength, motor and cognitive function, facial aging, and laboratory biomarkers—to estimate biological age. Results revealed significant inter-individual variability: some individuals aged biologically faster or slower than their chronological peers. Such findings highlight that biological aging occurs on a spectrum even among disease-free young adults and may predict later-life health outcomes, providing a promising avenue for early intervention and personalized longevity strategies.",Exceptional Human Longevity,Longitudinal and Early-Life Biomarker Research,2023
Implications of Exceptional Longevity – Overview,"Individuals who achieve exceptional longevity often experience compression of morbidity, demonstrating that longer life expectancy does not necessarily entail prolonged disability. These individuals provide valuable insights into lifestyle strategies that promote healthspan extension and independence, and suggest that healthy aging can occur alongside extended lifespan. Their biological and functional profiles help inform medical approaches for assessing biological age and optimizing preventive health strategies across the lifespan.",Exceptional Human Longevity,Compression of Morbidity and Healthy Aging,2023
Biomarkers and Biological Age Assessment,"The identification and validation of biomarkers linked to exceptional longevity enable more accurate estimation of biological age and individualized health risk. Several algorithms have been proposed to estimate biological age by integrating biochemical, molecular, and functional measurements. Models that combine physiological performance metrics—such as grip strength or mobility—with clinical biomarkers may offer the most predictive accuracy. However, discrepancies between individual-level and population-level assessments remain a challenge, underscoring the need for longitudinal studies to refine personal biological age estimation.",Exceptional Human Longevity,Biological Age Estimation and Longevity Biomarkers,2023
Health Screening and Life Expectancy,"Traditional health screening protocols are largely based on chronological age, which can lead to inappropriate over-screening of those with limited life expectancy and under-screening of biologically younger individuals. Incorporating biological age and individualized life expectancy into screening frameworks could enhance preventive care efficiency. Evidence from cancer screening studies indicates that screening offers little or no benefit when life expectancy is less than five years, highlighting the importance of tailoring preventive measures to biological, rather than chronological, aging trajectories.",Exceptional Human Longevity,Personalized Health Screening in Aging Populations,2023
Individualized Longevity Prediction Tools,"Validated mortality indices and life expectancy calculators that incorporate patient-specific risk factors—such as comorbidities, functional performance, and biomarker data—offer a pathway toward personalized preventive medicine for older adults. These models can help clinicians determine the appropriateness of interventions like cancer screening, cardiovascular risk management, and other age-related monitoring. The integration of such tools into clinical practice supports precision healthcare and aligns screening and treatment decisions with an individual’s biological rather than chronological age.",Exceptional Human Longevity,Predictive Models and Longevity Estimation Tools,2023
Epigenetics and Aging – Overview,"Aging is accompanied by extensive, progressive alterations to the epigenetic landscape that occur in both dividing and nondividing cells. These changes include global reductions in core histone levels, altered DNA methylation patterns, posttranslational histone modifications, and dysregulation of noncoding RNAs. Such modifications affect chromatin structure and accessibility, leading to widespread transcriptional dysregulation, genomic instability, and activation of transposable elements. Together, these molecular changes play a central role in the biological aging process.",Epigenetics and Aging,Epigenetic Mechanisms of Aging,2023
Epigenetic Remodeling and Gene Expression,"Epigenetic aging results in altered chromatin architecture that disrupts normal gene expression programs. As chromatin becomes more open or heterochromatin domains erode, cells exhibit aberrant activation of silenced genes and suppression of genes required for homeostasis. This remodeling also reactivates transposable elements, which contributes to genomic instability. The decline in histone production and replacement with histone variants further modifies nucleosome dynamics, reinforcing the age-associated transcriptional imbalance.",Epigenetics and Aging,Chromatin Structure and Gene Expression in Aging,2023
Environmental and Transgenerational Effects,"Environmental factors such as diet, stress, and toxin exposure can induce epigenetic modifications that influence lifespan. Remarkably, some epigenetic states are heritable across generations, meaning that parental environmental exposures or dietary habits can shape offspring longevity. This transgenerational inheritance underscores that lifespan is not strictly genetically determined but is largely influenced by dynamic, reversible epigenetic processes that respond to environmental cues.",Epigenetics and Aging,Environmental and Transgenerational Epigenetics,2023
Therapeutic Implications,"The reversibility of epigenetic marks offers promising therapeutic avenues for delaying aging and treating age-associated diseases such as cancer, neurodegeneration, and metabolic disorders. Modulators of DNA methyltransferases, histone deacetylases, and other epigenetic enzymes can extend lifespan in model organisms. These findings suggest that targeted epigenetic therapies, potentially combined with dietary interventions, may restore youthful gene expression patterns and promote longevity in humans.",Epigenetics and Aging,Epigenetic Therapies and Longevity,2023
Epigenetics and Aging – Introduction,"Aging is a complex, multifactorial biological process characterized by a gradual, time-dependent decline in physiological function. It heightens vulnerability to diseases such as cancer, diabetes, cardiovascular disorders, and neurodegenerative conditions. While organismal aging reflects systemic deterioration, cellular senescence—irreversible growth arrest in response to oncogenic or stress signals—acts as a protective mechanism against tumorigenesis and also participates in tissue remodeling during development and repair.",Epigenetics and Aging,Overview of Aging and Senescence,2023
Epigenetic Mechanisms and Aging Hallmarks,"Among the established hallmarks of aging, epigenetic alterations are recognized as a central driver of functional decline. Epigenetic regulation encompasses heritable yet reversible modifications that do not alter DNA sequence but modulate chromatin accessibility, gene expression, and genomic stability. The epigenome translates genetic information into functional phenotypes, connecting genotype to phenotype through processes such as DNA methylation, histone modification, and noncoding RNA regulation.",Epigenetics and Aging,Role of Epigenetic Alterations in Aging,2023
Epigenetic Drift and Variability in Lifespan,"Epigenetic variability, or 'epigenetic drift,' contributes significantly to lifespan differences even among genetically identical individuals. Studies on monozygotic twins suggest that genetic factors explain only 20–30% of lifespan variation, with the remainder attributed to environmental influences on epigenetic patterns. Similarly, in honeybees, identical genomes produce long-lived queen bees and short-lived worker bees due to diet-induced epigenetic modifications. This drift drives transcriptional variability and genomic instability, hallmarks of aging at the cellular level.",Epigenetics and Aging,Epigenetic Drift and Lifespan Variation,2023
Therapeutic Implications and Reversibility,"Unlike fixed genetic mutations, epigenetic changes are enzymatically regulated and reversible, offering potential therapeutic targets for delaying aging and mitigating age-related diseases. Understanding how external and internal factors shape the epigenome can enable the development of interventions that restore youthful epigenetic states. This reversibility makes epigenetic modulation an attractive strategy for healthspan extension and age-associated disease prevention.",Epigenetics and Aging,Reversibility and Therapeutic Potential of Epigenetic Changes,2023
Types of Epigenetic Information,"The epigenome comprises multiple layers of regulatory information, including DNA methylation, histone presence and modification, chromatin remodeling, histone variants, and the expression of noncoding RNAs. These elements collectively determine gene accessibility and function, influencing the identity and behavior of all cell types. Each of these epigenetic mechanisms contributes to the aging process, shaping cellular fate, tissue integrity, and organismal longevity through dynamic control of chromatin organization and gene regulation.",Epigenetics and Aging,Epigenetic Layers and Their Role in Aging,2023
Chromatin Structure and Aging,"Chromatin, the primary carrier of epigenetic information, plays a central role in aging. Its fundamental unit, the nucleosome, consists of DNA wrapped around histone octamers (H2A, H2B, H3, and H4), with linker histones and nonhistone proteins such as HP1 organizing higher-order structures like heterochromatin. Chromatin architecture controls all nuclear processes—including replication, transcription, recombination, and DNA repair—by modulating DNA accessibility. Age-associated alterations in chromatin packing and histone dynamics lead to impaired genome stability and aberrant gene expression, key drivers of cellular aging.",Epigenetics and Aging,Chromatin Remodeling and Genome Stability,2023
Model Organisms in Aging Research,"Due to the long lifespan of mammals, aging studies often rely on model organisms with shorter lifespans such as yeast (Saccharomyces cerevisiae), worms (Caenorhabditis elegans), flies (Drosophila melanogaster), and vertebrate models like mice, zebrafish, naked mole rats, and African turquoise killifish. Yeast models have been pivotal in uncovering conserved aging pathways and genes that regulate lifespan extension. These organisms enable researchers to dissect fundamental epigenetic mechanisms of aging conserved across evolution, providing insight into molecular pathways relevant to humans.",Epigenetics and Aging,Experimental Models of Epigenetic Aging,2023
Human Models of Accelerated Aging,"Hutchinson–Gilford progeria syndrome (HGPS) and Werner syndrome are rare human disorders characterized by premature aging phenotypes caused by mutations in DNA repair genes or the LMNA gene encoding A-type lamin. These mutations lead to chromatin disorganization and genomic instability, mimicking key aspects of physiological aging. Although patients with these syndromes display segmental aging affecting multiple tissues, they serve as valuable models for studying the molecular and epigenetic deterioration associated with natural aging.",Epigenetics and Aging,Progeria Syndromes and Chromatin Disorganization,2023
Conserved Epigenetic Aging Mechanisms,"Across species—from unicellular yeast to complex mammals—the epigenome undergoes progressive loss of structure and function with age. This deterioration manifests as disrupted chromosomal architecture, loss of genomic integrity, and altered transcriptional landscapes. The conservation of these changes across diverse models highlights universal principles underlying aging. Current research aims to synthesize these findings to better understand how epigenetic reprogramming could restore youthful function and inform future therapeutic strategies for aging and age-related diseases.",Epigenetics and Aging,Conservation and Future Directions in Epigenetic Aging,2023
The Heterochromatin Loss Model of Aging – Overview,"The heterochromatin loss model of aging proposes that aging is driven by the progressive loss of heterochromatin, leading to disruption of nuclear architecture and altered expression of genes within formerly silenced regions. This decay of heterochromatin integrity has been observed across species from yeast to humans. Experimental evidence indicates that accelerating heterochromatin loss shortens lifespan, while restoring heterochromatin structure can extend it, establishing chromatin organization as a central determinant of longevity.",Epigenetics and Aging,Heterochromatin Loss Model of Aging,2023
Role of Sirtuins and Histone Deacetylation,"Heterochromatin silencing depends on the removal of histone acetylation within repressive chromatin regions. Histone deacetylases (HDACs), including the conserved sirtuin family, play critical roles in maintaining heterochromatin and lifespan. In yeast, deletion of the HDAC gene SIR2 shortens lifespan, while its overexpression extends it. Similarly, mammalian SIRT1 deacetylates histones and transcriptional regulators, preserving genome integrity. Redistribution of SIRT1 from silenced loci to DNA damage sites mirrors aging-associated gene expression changes, emphasizing its role in genome maintenance and heterochromatin stability.",Epigenetics and Aging,Sirtuins and Lifespan Regulation,2023
Experimental Evidence Across Species,"Loss of silencing at ribosomal DNA (rDNA) loci promotes genomic instability and accelerates aging in yeast, providing a mechanistic link between chromatin architecture and lifespan. Comparable findings have been observed in mammals, where repression of repetitive elements by SIRT1 is vital for genome stability. Studies in progeria and Werner syndrome—both marked by disorganized chromatin and loss of heterochromatin marks—further support that chromatin deterioration contributes to accelerated aging. These disorders mirror several molecular features of natural aging, although it remains debated how fully they recapitulate physiological aging.",Epigenetics and Aging,Experimental Validation in Yeast and Humans,2023
Senescence-Associated Heterochromatin Foci and Paradox Resolution,"An apparent paradox of the heterochromatin loss model arises from the coexistence of global heterochromatin loss with the formation of senescence-associated heterochromatin foci (SAHF) in aging and senescent cells. SAHF are dense chromatin domains that repress proliferation-promoting genes. Recent studies using Hi-C and FAIRE analyses propose a two-step model to reconcile this paradox: first, global heterochromatin decondensation occurs, followed by the reformation of heterochromatin at formerly euchromatic regions. This 'heterochromatin redistribution' suggests that aging involves both loss and reorganization of heterochromatin structure.",Epigenetics and Aging,Heterochromatin Redistribution and SAHF Formation,2023
Global Histone Protein Reduction During Aging – Overview,"Beyond the heterochromatin loss model, aging is now understood to involve a global reduction of core histone proteins across the genome. This decline in histone abundance has been observed in multiple organisms, including yeast, worms, and human cells. In budding yeast, approximately half of the core histones are lost during replicative aging, leading to widespread chromatin decompaction and increased global transcription. These findings reveal that histone loss itself can act as a causal factor in aging by disrupting chromatin structure and transcriptional regulation.",Epigenetics and Aging,Global Histone Reduction and Aging Mechanisms,2023
Mechanisms of Histone Loss and Lifespan Extension,"In yeast, histone protein levels decline during aging due to reduced synthesis rather than reduced mRNA abundance. Supplying extra histone H3 and H4 proteins through overexpression, deletion of histone gene repressors (such as Hir), or inhibition of histone degradation pathways (via Tom1 deletion) significantly extends lifespan. This effect occurs independently of calorie restriction, a well-established longevity intervention, suggesting histone maintenance defines a distinct pro-longevity pathway. Similarly, lithium exposure in worms correlates with increased histone gene expression and lifespan extension.",Epigenetics and Aging,Histone Regulation and Lifespan Pathways,2023
Cross-Species Evidence for Histone Decline,"Reduced histone protein levels have been detected in aged worms, senescent human fibroblasts, and human diploid primary cells undergoing replicative senescence. In humans, this reduction appears to result from telomere shortening that triggers DNA damage responses, impairing new histone synthesis. These findings suggest that histone decline is a conserved hallmark of aging across species, potentially explaining how telomere attrition and chromatin deterioration jointly limit cellular replicative potential.",Epigenetics and Aging,Conservation of Histone Loss Across Species,2023
Consequences of Histone Depletion on Chromatin Architecture,"Histone depletion profoundly alters chromatin structure by reducing nucleosome occupancy and increasing nucleosomal fuzziness—less precise positioning along DNA sequences. This chromatin relaxation results in inappropriate DNA accessibility and global transcriptional deregulation. In yeast, overexpression of H3 and H4 partially restores nucleosome organization and corrects transcriptional imbalances, suggesting that histone abundance directly governs chromatin stability. Whether this compensatory mechanism extends to multicellular organisms remains an open question in aging research.",Epigenetics and Aging,Chromatin Landscape Alterations from Histone Loss,2023
Genomic Instability and Chromatin Relaxation During Aging,"Chromatin relaxation, resulting from heterochromatin and histone loss, contributes to genomic instability during aging. In aged yeast, reduced nucleosome occupancy leads to increased DNA damage, higher rates of chromosomal rearrangements, mitochondrial DNA insertions into the nuclear genome, and elevated retrotransposition. Partial restoration of histone H3 and H4 levels reverses these defects, indicating that histone loss directly drives transcriptional dysregulation and genome instability. Accumulation of DNA damage and formation of γ-H2AX foci have been consistently observed across aged mice, senescent human cells, and patients with premature aging syndromes, suggesting that DNA damage accumulation is a conserved hallmark of aging.",Epigenetics and Aging,Genomic Instability and Chromatin Relaxation,2023
DNA Damage and Repair in Aging,"While DNA damage increases with age, its precise role as a causal factor remains under investigation. In yeast, somatic mutation accumulation does not appear to directly cause aging, suggesting that age-related DNA damage may arise from either increased susceptibility of open chromatin to damage or reduced repair capacity in old cells. Comparative transcriptomic analyses of DNA repair genes in long-lived versus short-lived species indicate that longevity correlates with higher expression of specific DNA repair pathways, implying superior genome maintenance mechanisms in long-lived organisms.",Epigenetics and Aging,"DNA Damage, Repair, and Longevity",2023
Retrotransposon Activation and the 'Aging by Transposition' Model,"Heterochromatin decay during aging leads to reactivation of endogenous retrotransposable elements, which are normally silenced in young cells. Loss of heterochromatin allows these elements to be transcribed and reinserted into new genomic locations, disrupting genome integrity and cellular homeostasis. This phenomenon, observed across yeast, worms, flies, mice, and human cells, forms the basis of the 'aging by transposition' model. Experimental evidence shows that histone overexpression suppresses retrotransposition in aged yeast, while calorie restriction reduces retrotransposon activity in aged mice. These findings suggest that maintaining chromatin repression of mobile elements is critical for genome stability and longevity.",Epigenetics and Aging,Aging by Transposition Hypothesis,2023
Molecular Regulation of Retrotransposon Silencing,"SIRT6, a histone deacetylase and ADP-ribosylase, is crucial for repressing LINE-1 retrotransposons through ADP-ribosylation of KAP1, which promotes interaction with HP1 and packaging into heterochromatin. During aging, SIRT6 is redistributed from retrotransposon loci to DNA breaks, leading to derepression and activation of transposable elements. In human stem cell models, transcriptional activation of SINE/Alu elements induces DNA damage and cellular senescence, while suppressing Alu transcripts reverses senescence and DNA damage markers. This demonstrates a direct causal relationship between retrotransposon activity and aging-associated cellular decline.",Epigenetics and Aging,SIRT6 and Retrotransposon Silencing,2023
"Transposition, Aging, and Disease","Retrotransposon activation is not limited to aging but also occurs during cancer and neurodegeneration, both strongly age-associated conditions. In these contexts, increased retrotransposition contributes to genomic rearrangements and cellular dysfunction. The convergence of chromatin relaxation, DNA damage, and transposable element activation underscores their interdependence as drivers of aging. Defining the causal sequence of these molecular events remains a critical challenge for future research aimed at understanding the initiation and progression of aging in healthy individuals.",Epigenetics and Aging,Retrotransposition in Aging and Disease,2023
Histone Variant Dynamics and Aging Overview,"In addition to canonical histones H2A, H2B, H3, and H4, nonallelic histone variants perform specialized roles in chromatin remodeling and gene regulation. During aging, the expression and deposition of these variants undergo significant changes that influence chromatin dynamics, transcription, and cellular senescence. Unlike the global loss of core histones observed with aging, histone variant changes are more heterogeneous and organism-specific, suggesting distinct contributions of each variant to the aging process.",Epigenetics and Aging,Histone Variants and Aging,2023
Histone H3.3 Accumulation and Senescence Induction,"The histone H3 variant H3.3, which is incorporated into chromatin independently of DNA replication, becomes the dominant H3 isoform in senescent human cells. In contrast, replication-dependent H3.1 declines during aging. Overexpression of H3.3 or its N-terminally cleaved form, H3.3cs1, induces cellular senescence even without oncogenic stress, indicating that excessive H3.3 incorporation contributes to the senescence phenotype. Deposition of H3.3cs1 is mediated by histone chaperones ASF1a, UBN1, and likely HIRA. Consistently, in aged mouse brains, H3.3 accumulation accompanies cognitive and transcriptional regulation changes, highlighting its role in neuronal plasticity and chromatin maintenance.",Epigenetics and Aging,H3.3 Variant and Cellular Senescence,2023
Functional Role of HIRA and Yeast Correlations,"Deletion of the yeast homolog of the HIRA complex extends replicative lifespan by increasing histone gene expression, thereby counteracting histone loss during aging. Although HIRA promotes H3.3 deposition in senescent mammalian cells, its loss in yeast exerts pro-longevity effects through a distinct mechanism—elevating core histone levels. These observations underscore the context-dependent roles of histone chaperones and histone variant deposition in lifespan regulation across species.",Epigenetics and Aging,HIRA Complex and Lifespan Regulation,2023
H2A Variant MacroH2A and Chromatin Reorganization,"Among H2A variants, macroH2A has been most strongly associated with aging. MacroH2A is enriched in senescence-associated heterochromatin foci (SAHF), traditionally linked to transcriptional repression. Its levels increase with replicative senescence in fibroblasts and with organismal aging in mice and primates. However, macroH2A1 also promotes transcriptional activation of senescence-associated secretory phenotype (SASP) genes following oncogenic stress, driving a feedback loop that amplifies senescence signaling. Reactive oxygen species and DNA damage trigger ATM-dependent removal of macroH2A1 from SASP loci, shifting it back toward repressive SAHF regions. This redistribution underscores macroH2A1’s dual regulatory roles in transcription and chromatin compaction during aging.",Epigenetics and Aging,MacroH2A Variant and Chromatin Dynamics,2023
Histone Variant Remodeling as a Hallmark of Senescence,"Overall, histone variant remodeling—characterized by accumulation of H3.3 and redistribution of macroH2A1—emerges as a hallmark of chromatin aging. These changes contribute to transcriptional deregulation, reinforce senescence-associated chromatin states, and influence cellular plasticity. The interplay between histone variant turnover, proteolytic processing, and chaperone-mediated deposition defines a new regulatory layer of chromatin maintenance and senescence progression.",Epigenetics and Aging,Histone Variant Remodeling in Senescence,2023
Overview of Histone Modifications and Aging,"Histones undergo numerous posttranslational modifications (PTMs) such as acetylation, methylation, and ubiquitination, which regulate DNA accessibility and chromatin architecture. These modifications, added or removed by specific enzymes, orchestrate essential cellular processes including transcription, DNA repair, and replication. During aging, alterations in the abundance and activity of histone-modifying enzymes disrupt epigenomic balance, contributing to transcriptional drift, genomic instability, and functional decline. Among the histone PTMs most closely linked to aging are acetylation and methylation on lysine residues, which have been shown to either promote or delay aging depending on their site and abundance.",Epigenetics and Aging,Histone Modifications and Longevity,2023
Histone Acetylation: H3K56Ac and H4K16Ac,"Two key histone acetylation marks, H3K56Ac and H4K16Ac, exhibit opposing trends during aging. In yeast, H3K56 acetylation decreases with age, while H4K16 acetylation increases. Balanced levels of H3K56Ac are essential for longevity—both loss and hyperacetylation reduce lifespan by impairing chromatin assembly, DNA replication, and transcriptional regulation. Deletion of histone deacetylases Hst3/Hst4 shortens lifespan, whereas moderate overexpression extends it. Conversely, increased H4K16Ac, driven by declining Sir2 activity, promotes aging by disrupting telomeric chromatin structure. Overexpression of Sir2 or deletion of the H4K16 acetyltransferase SAS2 extends lifespan, linking chromatin acetylation balance to the heterochromatin loss model of aging.",Epigenetics and Aging,Histone Acetylation in Aging,2023
Role of HATs and HDACs in Lifespan Regulation,"Beyond Sir2, multiple histone acetyltransferases (HATs) and deacetylases (HDACs) influence aging. Deletion of NuA4 HAT complex components extends yeast lifespan, potentially through effects on both histone and nonhistone substrates involved in metabolic and mTOR pathways. Similarly, deletion of Rpd3, an HDAC complex component, extends lifespan in yeast and flies. These enzymes modulate diverse signaling pathways, indicating that global histone acetylation dynamics have pleiotropic effects on longevity. In mammals, loss of histone acetylation in repetitive DNA regions of aged brains correlates with reduced chromatin integrity and memory decline. Restoring H4K12 acetylation rescues learning and transcriptional activity, highlighting a role for acetylation in maintaining cognitive function during aging.",Epigenetics and Aging,HAT and HDAC Enzymes in Longevity,2023
Histone Methylation Patterns During Aging,"Aging alters key methylation marks across histones, particularly H3K4me3, H3K9me3, H3K27me3, and H3K36me3. Generally, repressive methylation marks decline while activating marks rise, reflecting chromatin decompaction. In flies and mammals, reductions in HP1 and H3K9me2 accompany heterochromatin loss, while tissue-specific increases in H3K9me3 occur in aged fly heads. In HGPS fibroblasts, decreased H3K9me3 and H3K27me3 with elevated H4K20me3 illustrate the reorganization of heterochromatin. These shifts collectively indicate a transition toward a more transcriptionally permissive chromatin state during aging.",Epigenetics and Aging,Histone Methylation Dynamics,2023
Methyltransferases and Demethylases Regulating Lifespan,"RNAi and genetic studies in C. elegans and Drosophila identified key methyltransferases (SET-2, ASH-2, SET-9) and demethylases (RBR-2, UTX-1) that modulate lifespan. Reduction of H3K4me3 through loss of SET-2 or ASH-2 extends lifespan, while depletion of RBR-2 shortens it—demonstrating that reduced active methylation can enhance longevity. Conversely, inhibition of the H3K27me3 demethylase UTX-1 extends lifespan through insulin signaling pathways. In contrast, excessive H3K27me3 or Polycomb repressive complex mutations can shorten lifespan, emphasizing the importance of balanced chromatin repression. In progeria models, elevated H3K9me3 and SUV39H1 impede DNA repair and accelerate aging, whereas depletion of SUV39H1 restores repair efficiency and extends lifespan.",Epigenetics and Aging,Histone Methyltransferases and Longevity,2023
H3K36 Methylation and Transcriptional Stability,"H3K36me3 plays a protective role against transcriptional noise and aging-related gene expression drift. In yeast, loss of the methyltransferase SET2 shortens lifespan, while deletion of the demethylase RPH1 extends it. Reduced H3K36me3 in aged cells leads to a more open chromatin structure and activation of cryptic promoters. In C. elegans and Drosophila, genes with stable expression during aging display high H3K36me3 enrichment, suggesting a conserved function in maintaining transcriptional fidelity. Loss of H3K36me3 correlates with lifespan shortening, while interventions that suppress transcriptional drift extend lifespan, supporting the role of epigenetic stability in longevity.",Epigenetics and Aging,H3K36me3 and Transcriptional Drift,2023
Histone Ubiquitination and Longevity,"Histone ubiquitination also contributes to lifespan control. In yeast, deletion of components of the deubiquitinase (DUB) module of the SAGA complex extends lifespan, indicating that reduced DUB activity promotes longevity. This effect is independent of acetylation activity but interacts with Sir2-mediated pathways. Because histone ubiquitination regulates both transcription and DNA repair, understanding its specific contributions to aging could reveal conserved mechanisms in multicellular organisms. Future research will determine whether the lifespan-extending effects of altered histone ubiquitination in yeast translate to higher eukaryotes.",Epigenetics and Aging,Histone Ubiquitination and Lifespan,2023
Overview of Nucleosome Remodeling and Aging,"Nucleosome remodeling is a dynamic process regulated by ATP-dependent chromatin remodeling complexes that reposition, evict, or restructure nucleosomes to facilitate DNA-based processes such as transcription, replication, and repair. These complexes, which include families like SWI/SNF, ISWI, CHD, and INO80, function in concert with histone modifiers and chaperones to maintain chromatin plasticity. During aging, dysfunction or loss of nucleosome remodelers disrupts chromatin structure, leading to impaired DNA repair, increased genome instability, and altered transcriptional regulation—key hallmarks of aging.",Epigenetics and Aging,Nucleosome Remodeling and Aging,2023
NURD Complex Dysfunction in Aging and Progeria,"The Nucleosome Remodeling and Deacetylase (NURD) complex is an evolutionarily conserved ATP-dependent chromatin remodeler involved in chromatin compaction and transcriptional repression. Studies in Hutchinson–Gilford progeria syndrome (HGPS) and aged human cells show substantial loss of NURD components, including RBBP4 and RBBP7. Their depletion in cell culture leads to chromatin disorganization and persistent DNA damage, mirroring features of both physiological and premature aging. These findings suggest that loss of NURD subunits precedes DNA damage accumulation, implying that chromatin remodeling defects are primary drivers of genomic instability during aging.",Epigenetics and Aging,NURD Complex and Genome Stability,2023
LET-418/Mi2 and Lifespan Regulation in C. elegans,"In C. elegans, the NURD catalytic subunit homolog LET-418/Mi2 acts as a regulator of lifespan. Loss of let-418 function extends lifespan and enhances resistance to environmental stressors via the DAF-16/FOXO transcription factor pathway. Genetic interactions indicate that LET-418 may regulate longevity through the germ cell loss pathway, a mechanism conserved across species including flies and plants. These results link NURD complex remodeling activity to organismal longevity control through stress adaptation and FOXO-mediated gene regulation.",Epigenetics and Aging,LET-418/Mi2 and DAF-16 Longevity Pathway,2023
ISW2 Remodeler and Caloric Restriction Pathways in Yeast,"Genome-wide deletion screens in budding yeast identified the ISW2 ATP-dependent nucleosome remodeler as a negative regulator of lifespan. Deletion of ISW2 extends replicative lifespan through a pathway parallel but distinct from the classical TOR inhibition pathway activated by caloric restriction (CR). ISW2 loss upregulates stress response genes, including RAD51, which promotes homologous recombination (HR)-mediated DNA repair. Overexpression of RAD51 alone extends lifespan, demonstrating that enhanced DNA repair capacity contributes to ISW2-associated longevity.",Epigenetics and Aging,ISW2 and Stress-Response-Mediated Longevity,2023
SWI/SNF Complex and FOXO-Dependent Lifespan Extension,"The SWI/SNF chromatin remodeling complex acts as a crucial cofactor for DAF-16/FOXO-driven transcriptional activation in C. elegans. RNAi knockdown of SWI/SNF subunits abolishes FOXO-mediated lifespan extension, demonstrating that remodeling activity is required for the expression of pro-longevity genes. This cooperative regulation highlights the intersection between chromatin remodeling and transcriptional control of stress-resistance pathways in aging.",Epigenetics and Aging,SWI/SNF and FOXO Regulation of Longevity,2023
CHD1 Deletion and Lifespan Extension,"Deletion of CHD1, another ATP-dependent nucleosome remodeler, extends replicative lifespan in yeast. This finding, derived from a large-scale analysis of nearly 4700 yeast deletion mutants, suggests that certain chromatin remodelers may act as lifespan limiters. Given the high conservation of CHD family proteins across eukaryotes, these results raise the possibility that modulating CHD1 or related remodeler activity could influence aging and longevity in mammals.",Epigenetics and Aging,CHD1 and Replicative Lifespan,2023
Summary of Remodeling Complexes in Longevity,"Collectively, evidence across model organisms supports the view that ATP-dependent nucleosome remodelers have dual roles in aging—some promote genomic stability and healthy chromatin maintenance (e.g., NURD), while others constrain lifespan when overactive (e.g., ISW2, CHD1). These complexes influence aging through pathways involving DNA repair efficiency, stress response, and transcriptional regulation. Understanding their balance between chromatin accessibility and compaction may offer new therapeutic strategies for maintaining genome integrity and delaying aging.",Epigenetics and Aging,Chromatin Remodeling Complexes and Longevity,2023
Overview of DNA Methylation and Aging,"DNA methylation, the covalent addition of a methyl group to cytosine residues within CpG dinucleotides, is one of the most studied epigenetic modifications linked to aging. In young cells, promoter CpG methylation silences gene expression through heterochromatin formation, whereas active promoters remain unmethylated. During aging, the DNA methylation landscape becomes increasingly disordered due to stochastic epigenetic drift driven by environmental and replication-associated factors. Nonetheless, some methylation changes occur directionally and reproducibly at specific genomic loci, suggesting a regulated, mechanistic role for methylation in the aging process.",Epigenetics and Aging,DNA Methylation Overview,2023
Epigenetic Drift and Environmental Influence,"As organisms age, identical genomes diverge epigenetically due to environmental exposures and random errors in methylation maintenance, a phenomenon termed 'epigenetic drift.' In monozygotic twins, DNA methylation profiles become increasingly distinct with age. Environmental factors, such as diet, further influence methylation patterns—as observed in honeybee caste differentiation, where dietary inputs modulate lifespan and phenotype through methylation of developmental genes. Experimental silencing of Dnmt3 in bees reproduces these methylation shifts, highlighting the environment's capacity to reshape the methylome and lifespan.",Epigenetics and Aging,Epigenetic Drift and Environmental Factors,2023
Global Hypomethylation and Genome Instability,"Aging is broadly associated with genome-wide DNA hypomethylation, particularly in repetitive DNA regions and transposable elements. Loss of methylation at these sites compromises heterochromatin integrity and promotes retrotransposition, contributing to genomic instability. This hypomethylation correlates with declining expression of DNMT1, the maintenance DNA methyltransferase. Supporting this, Dnmt1 haploinsufficient mice exhibit age-related cognitive decline, linking DNMT1 loss to functional aging. In addition, specific promoters such as ITGAL and IL17RC undergo demethylation during aging, leading to immune dysregulation and autoimmunity.",Epigenetics and Aging,Global DNA Hypomethylation and Instability,2023
Regional Hypermethylation and Gene Repression,"Alongside global hypomethylation, aging also induces hypermethylation at specific CpG sites, particularly within CpG islands of promoter regions. These targeted methylation gains repress gene expression, often affecting developmental and tumor-suppressor genes. Age-associated hypermethylation is frequently enriched at Polycomb group (PcG) target genes marked by H3K27me3 and H3K4me3, reflecting epigenetic reprogramming at bivalent promoters. Such selective hypermethylation may serve as a compensatory mechanism to silence transcriptionally active regions during chromatin relaxation in aging cells.",Epigenetics and Aging,CpG Hypermethylation and Gene Repression,2023
Tissue-Specific and Functional DNA Methylation Patterns,"Age-dependent methylation changes vary across tissues and genomic contexts. Whole-genome analyses reveal that while CpG islands show consistent methylation alterations, non-island regions exhibit tissue-specific changes. For example, pancreatic β cells experience hypomethylation at enhancer regions due to impaired recognition by DNA methyltransferases, altering transcriptional networks. However, in hematopoietic stem cells and blood, correlations between methylation changes and gene expression remain weak, implying complex regulatory interdependencies.",Epigenetics and Aging,Tissue-Specific DNA Methylation Changes,2023
Epigenetic Clocks and Age Prediction,"Consistent and reproducible methylation changes at specific CpG sites across individuals have enabled the development of 'epigenetic clocks'—predictive biomarkers that estimate biological age from DNA methylation patterns. For instance, age prediction can be achieved with high accuracy using methylation data from as few as three CpG sites in blood DNA. These clocks highlight the utility of DNA methylation as a quantitative measure of aging rate and health status, reflecting cumulative environmental and biological influences.",Epigenetics and Aging,Epigenetic Clocks and Biomarkers,2023
DNA Methylation–Histone Modification Interactions,"DNA methylation changes during aging are closely intertwined with histone modification dynamics. Regions gaining DNA methylation often coincide with bivalent histone marks (H3K4me3 and H3K27me3) at poised promoters, whereas hypomethylated regions align with active enhancer marks (H3K9Ac, H3K27Ac). Replicative senescence also induces predictable DNA methylation changes, sufficient to estimate cellular age or population doublings. Altered methylation at SIRT1-regulated loci further suggests that sirtuins may coordinate chromatin deacetylation and methylation during aging.",Epigenetics and Aging,Crosstalk Between DNA Methylation and Histone Marks,2023
"DNA Methylation, Cancer, and Aging Parallels","The DNA methylation landscape of aging mirrors that of tumorigenesis—characterized by global hypomethylation and promoter-specific hypermethylation. Hypomethylation of repetitive elements contributes to genomic instability, while hypermethylation of tumor-suppressor promoters facilitates silencing and predisposes cells to transformation. Polycomb target genes exhibit hypermethylation in both aging and cancer, reinforcing the mechanistic link between epigenetic drift and carcinogenesis. Understanding how DNA methylation deteriorates with age may thus provide critical insights into the molecular origins of age-related cancers.",Epigenetics and Aging,DNA Methylation and Cancer Risk,2023
Overview of Noncoding RNAs in Aging,"Noncoding RNAs (ncRNAs) encompass a vast and functionally diverse group of RNA molecules that do not encode proteins but exert powerful regulatory control over gene expression, chromatin structure, and genome organization. Deep sequencing studies reveal that 60–90% of the human genome is transcribed, generating a wide range of short and long ncRNAs. These RNAs modulate transcriptional and post-transcriptional processes and are now recognized as crucial players in aging, influencing lifespan, senescence, and susceptibility to age-related diseases such as cancer, neurodegeneration, and cardiovascular disorders.",Epigenetics and Aging,Overview of ncRNAs in Aging,2023
ncRNA Conservation and rDNA Regulation,"ncRNAs are evolutionarily conserved across species, including simple eukaryotes like yeast. In Saccharomyces cerevisiae, ncRNA transcription from normally silent rDNA loci shortens lifespan by destabilizing rDNA repeats, while mutations that suppress these ncRNAs extend longevity. Similarly, in mammalian systems, aberrant expression of ncRNAs derived from repetitive Alu elements promotes senescence in human stem cells, while their knockdown reverses this phenotype. These findings demonstrate that ncRNA-mediated control of repetitive and ribosomal DNA regions serves as a conserved longevity determinant across species.",Epigenetics and Aging,Evolutionary Conservation of ncRNA Function,2023
Small Noncoding RNAs and Dicer Decline,"Aging in multiple organisms, including worms, mice, and humans, is accompanied by a decline in Dicer levels, an RNase III enzyme essential for small ncRNA (sncRNA) processing. Reduced Dicer expression leads to impaired microRNA (miRNA) biogenesis and early cellular senescence. Caloric restriction (CR) restores Dicer levels and reverses sncRNA deficits in mice, indicating that miRNA processing capacity is tightly linked to longevity. In aged human adipocytes, decreased Dicer mirrors these findings, supporting a conserved role for sncRNA dysregulation in mammalian aging.",Epigenetics and Aging,Dicer and Small RNA Processing in Aging,2023
MicroRNAs and Lifespan Regulation,"MicroRNAs (miRNAs) modulate gene expression post-transcriptionally and play pivotal roles in lifespan regulation. In C. elegans, the miRNA lin-4 extends lifespan by repressing its pro-aging target lin-14. Conversely, lin-4 loss shortens lifespan, while lin-14 knockdown increases it, demonstrating their antagonistic control of longevity through the insulin/IGF-1 pathway involving DAF-2 and DAF-16/FOXO. Approximately one-fourth of C. elegans miRNAs show age-dependent expression changes, with both pro- and anti-aging effects. miRNAs are also implicated in mammalian aging, though tissue specificity often obscures global patterns of regulation.",Epigenetics and Aging,miRNAs and Longevity Mechanisms,2023
miRNAs and Neurodegenerative Aging,"In humans and animal models, miRNAs play critical roles in brain aging and neurodegeneration. The miR-34 family, elevated in Alzheimer’s disease brains and aged fly and worm neurons, targets key anti-aging genes such as SIRT1 and BCL2, promoting neuronal decline. Similarly, miR-144 overexpression in aged brains suppresses neuroprotective factors, exacerbating cognitive impairment. These miRNAs contribute to neuronal senescence and apoptosis, highlighting the broader role of small ncRNAs in age-associated brain dysfunction and neurodegenerative disorders.",Epigenetics and Aging,miRNAs in Neurodegeneration,2023
Long Noncoding RNAs and Epigenetic Regulation,"Long noncoding RNAs (lncRNAs) regulate transcription through direct interactions with chromatin and chromatin-modifying complexes. The lncRNA Gas5 is upregulated in aged mouse brains and correlates with impaired learning and memory. Another example, H19 lncRNA, participates in the IGF2/H19 imprinted locus, repressing an imprinted gene network via methyl-CpG–binding domain protein 1. Loss of imprinting at this locus during aging reactivates silenced genes and is linked to prostate cancer risk, illustrating the epigenetic connection between aging and carcinogenesis.",Epigenetics and Aging,lncRNAs and Epigenetic Control of Aging,2023
lncRNAs in Senescence and Neurological Aging,"Several lncRNAs influence major senescence pathways, including the p53/p21 axis. These RNAs show differential expression between young and senescent fibroblasts, suggesting a role in the onset of cellular senescence. In neurodegenerative conditions such as Huntington’s disease, altered lncRNA profiles are observed, potentially modulating chromatin structure and transcriptional regulation in affected neurons. These findings indicate that lncRNA-mediated transcriptional reprogramming is a common feature of age-related neurological decline.",Epigenetics and Aging,lncRNAs in Senescence and Neurodegeneration,2023
ncRNAs in Heterochromatin and Genome Architecture,"Beyond transcriptional control, ncRNAs actively participate in chromatin organization. Products of the RNA interference (RNAi) pathway, such as small interfering RNAs, direct histone-modifying enzymes to repetitive DNA regions, promoting heterochromatin formation. Other ncRNAs help maintain three-dimensional genome architecture by functioning as boundary elements that prevent heterochromatin spreading. Although aging-related alterations in these structural ncRNAs are not yet fully characterized, they likely contribute to age-associated chromatin relaxation and genomic instability.",Epigenetics and Aging,ncRNAs in Chromatin Architecture,2023
Overview of Transgenerational Epigenetic Inheritance and Aging,"Traditional genetics posits that heritable traits are transmitted through DNA sequence, with epigenetic marks being reset during germline formation. However, growing evidence across plants, animals, and humans suggests that certain epigenetic modifications can bypass this reset and be transmitted across generations. These transgenerational epigenetic effects can influence aging and longevity, demonstrating that life span is not determined solely by genetic variation but also by inherited epigenetic states established in parental generations.",Epigenetics and Aging,Transgenerational Epigenetic Inheritance,2023
Histone Methylation and Heritable Longevity in C. elegans,"In C. elegans, deficiencies in components of the H3 K4me3 methyltransferase complex—specifically ASH-2, WDR-5, or SET-2—extend life span not only in the directly affected individuals but also in three subsequent generations of genetically normal descendants. Although the offspring lack the original methyltransferase mutations, they retain the extended life span phenotype, suggesting inheritance of a persistent transcriptional program rather than direct histone methylation patterns. This discovery represents one of the first demonstrations that longevity can be epigenetically inherited across generations via altered gene expression.",Epigenetics and Aging,Heritable Longevity via H3K4me3 Regulation,2023
Mechanisms Underlying Transgenerational Longevity,"Microarray analyses of descendants from H3 K4me3-deficient parents reveal persistent changes in gene expression, even after normal histone methylation levels are restored. This suggests that heritable longevity results from stable transcriptional reprogramming or retention of secondary epigenetic features, such as noncoding RNA expression or chromatin accessibility. Although the specific downstream pathways remain unclear, these findings support the existence of 'epigenetic memory' that influences lifespan across generations without direct DNA sequence alteration.",Epigenetics and Aging,Mechanisms of Epigenetic Memory,2023
Parental Environmental Effects on Offspring Metabolism,"Transgenerational inheritance is not limited to chromatin modifications. In mammals, environmental exposures can shape offspring physiology via epigenetic reprogramming of germ cells. A landmark study on diet-induced obesity demonstrated that male mice fed a high-fat diet produced offspring with increased susceptibility to obesity and insulin resistance, even when conception occurred through in vitro fertilization. This confirms that epigenetic alterations in parental gametes, rather than behavioral or uterine factors, can transmit metabolic traits across generations.",Epigenetics and Aging,Parental Diet and Epigenetic Inheritance,2023
Evolutionary Implications of Transgenerational Epigenetics,"The discovery that environmental exposures and chromatin modifications in one generation can influence lifespan and disease susceptibility in subsequent generations suggests a mechanism for adaptive evolution beyond genetic mutation. Through such transgenerational epigenetic inheritance, ancestral environmental conditions—such as diet, stress, or toxin exposure—may shape the aging trajectories and metabolic fitness of descendants. This paradigm challenges the traditional boundaries of heredity, positioning epigenetic memory as a key driver of both aging and evolutionary adaptation.",Epigenetics and Aging,Evolutionary Role of Transgenerational Epigenetics,2023
Epigenetic Basis of Life Span–Extending Regimens,"Epigenetic alterations represent one of the most modifiable hallmarks of aging. Unlike other molecular aging mechanisms, they can be targeted through genetic, environmental, or pharmacological interventions. Life span–extending strategies often act by modulating DNA methylation, histone modification, or chromatin remodeling. Because these mechanisms are reversible, interventions that modify epigenetic enzymes—such as DNA methyltransferases (DNMTs), histone deacetylases (HDACs), or sirtuins—offer promising routes to delay aging and prevent age-related diseases like cancer and neurodegeneration.",Epigenetics and Aging,Epigenetic Mechanisms in Longevity,2023
Caloric Restriction (CR) and Epigenetic Modulation,"Caloric restriction (CR) is the most robust, reproducible intervention to extend lifespan across species—from yeast to primates. CR influences the epigenome by modulating DNA methylation at specific genomic loci and altering histone modification patterns. It may enhance the expression or activity of DNA methyltransferases DNMT1 and DNMT3B, thereby stabilizing methylation homeostasis. CR also affects histone acetylation dynamics through activation of class III HDAC enzymes such as Sir2 and its mammalian ortholog SIRT1, leading to transcriptional repression of pro-aging genes. Transcriptome studies reveal that CR prevents age-associated gene expression changes and induces neuroprotective expression profiles in the brain.",Epigenetics and Aging,Caloric Restriction and Epigenetic Regulation,2023
CR Mimetics: Pharmacological Induction of Longevity Pathways,"Given the difficulty of long-term CR adherence in humans, 'CR mimetics' aim to reproduce CR’s molecular and epigenetic effects without reducing calorie intake. Many of these compounds target sirtuins, which link nutrient sensing to chromatin state. Resveratrol, a plant-derived polyphenol, activates SIRT1 and induces gene expression changes resembling those under CR. Synthetic sirtuin-activating compounds (STACs), such as SRT1720 and SRT2104, further extend lifespan in obese mice and improve metabolic health. The structural elucidation of SIRT1–resveratrol complexes provides a blueprint for developing next-generation activators capable of enhancing deacetylation of native histone and transcription factor substrates.",Epigenetics and Aging,Sirtuin Activation and CR Mimetics,2023
Metformin as an Epigenetic Longevity Drug,"Metformin, a widely prescribed antidiabetic drug, mimics key aspects of caloric restriction. It enhances insulin sensitivity, regulates carbohydrate metabolism, and activates AMP-activated protein kinase (AMPK). Emerging evidence suggests that metformin also modulates epigenetic regulators: human subjects treated with metformin display increased SIRT1 levels, implicating a potential deacetylation-driven mechanism in its anti-aging effects. Epidemiological studies reveal that diabetic patients on metformin exhibit not only reduced mortality compared to untreated diabetics but even longer average lifespan than healthy controls, underscoring its potential as a safe, accessible lifespan-extending compound.",Epigenetics and Aging,Metformin and Epigenetic Modulation,2023
Spermidine and Histone Acetylation Control,"Spermidine, a naturally occurring polyamine, extends lifespan across multiple organisms by directly inhibiting histone acetyltransferases (HATs). This inhibition maintains histone H3 in a hypoacetylated state, reducing global acetylation and promoting cellular stress resistance. Spermidine supplementation enhances autophagy and reduces oxidative damage in aging models, consistent with its ability to preserve chromatin integrity. Dietary administration of spermidine in mice mitigates age-related oxidative stress and tissue degeneration, highlighting histone acetylation control as a key mechanism for lifespan extension.",Epigenetics and Aging,Spermidine and Histone Acetylation,2023
"Acetyl-CoA Levels, Autophagy, and Longevity","Cellular acetyl-CoA availability links metabolism to epigenetic regulation. Depletion of acetyl-CoA—via fasting, dietary restriction, or pharmacological inhibition—reduces histone acetylation, inducing autophagy and extending lifespan. Conversely, high acetyl-CoA levels promote histone hyperacetylation through HATs such as p300, suppressing autophagy and accelerating aging. Studies in flies and mammals confirm that reduced acetyl-CoA triggers autophagy-associated longevity. Sir2/SIRT1-mediated deacetylation of histone H4 K16Ac plays a central role in this process, as low H4 K16Ac enhances autophagic flux and cellular rejuvenation. Thus, acetyl-CoA metabolism integrates nutrient status with chromatin-mediated longevity control.",Epigenetics and Aging,"Acetyl-CoA, Histone Acetylation, and Autophagy",2023
Autophagy as a Common Mechanism of Longevity,"Autophagy induction represents a convergent mechanism among multiple longevity-promoting interventions, including CR, sirtuin activation, acetyl-CoA depletion, and polyamine supplementation. Epigenetically, autophagy is regulated by histone acetylation and SIRT1-mediated deacetylation of key transcriptional activators such as FOXO. These pathways promote clearance of damaged organelles and proteins, maintaining proteostasis and genomic integrity. The upregulation of autophagy through epigenetic and metabolic reprogramming is increasingly viewed as a central node in the biology of healthy aging.",Epigenetics and Aging,Autophagy and Epigenetic Longevity,2023
Senolytic Strategies and Epigenetic Enzyme Targets,"Recent advances highlight the removal of senescent cells as another viable approach to improve health span and life span. Senolytic agents, which selectively eliminate senescent cells, alleviate age-related dysfunctions and chronic inflammation. Given that cellular senescence is maintained through extensive epigenetic remodeling—such as heterochromatin formation and stable gene repression—targeting specific epigenetic enzymes involved in maintaining senescence (e.g., HDACs, DNMTs, or histone methyltransferases) presents an emerging therapeutic strategy for pharmacological rejuvenation.",Epigenetics and Aging,Senolytics and Epigenetic Therapeutics,2023
Epigenetic Perspective on Aging,"Aging is a multifaceted biological process resulting from cumulative molecular damage and dysregulated signaling across time. Through the lens of epigenetics, it is increasingly evident that many longevity-determining factors operate by modifying the epigenome. Young cells maintain compact chromatin and tightly controlled gene expression, while aged cells exhibit disrupted chromatin architecture, increased DNA accessibility, and dysregulated ncRNA expression. These alterations collectively push cells beyond a threshold of stability, leading to permanent cell cycle arrest or senescence. Crucially, because epigenetic mechanisms are reversible, they offer opportunities to reprogram aged cells toward a more youthful state.",Epigenetics and Aging,Epigenetic Landscape of Aging,2023
Challenges in Identifying Causal Aging Mechanisms,"Distinguishing between causal and correlative molecular changes remains a major challenge in aging research. Many pathways implicated in aging are interconnected, making it difficult to separate pro-aging effects from secondary, adaptive, or bystander phenomena. A key future goal is to map the hierarchical relationships among signaling and epigenetic pathways to clarify which alterations truly drive aging and which simply accompany it. This will require controlled gain- and loss-of-function studies across multiple species and tissues to move beyond correlational observations.",Epigenetics and Aging,Causality in Epigenetic Aging Pathways,2023
Technological and Experimental Advances,"The integration of next-generation sequencing, single-cell epigenomics, and functional genomics is rapidly enhancing our ability to dissect the molecular architecture of aging. Comprehensive gene deletion studies in model organisms such as yeast have already identified hundreds of genes influencing replicative lifespan, though many remain uncharacterized. Combining large-scale genomic screening with precise molecular assays will help unravel how specific chromatin regulators, noncoding RNAs, and metabolic cues collectively shape longevity outcomes.",Epigenetics and Aging,Advances in Epigenetic Aging Research,2023
Epigenetic Rejuvenation and Therapeutic Opportunities,"The reversibility of epigenetic states underpins the concept of 'resetting the aging clock.' By restoring youthful chromatin configurations or rebalancing key histone modifications, it may be possible to rejuvenate cellular function. Epigenetic drugs and dietary interventions could target multiple age-related diseases simultaneously, providing a holistic approach to health span improvement. However, due to the high interconnectivity of epigenetic networks, such interventions must be carefully optimized to avoid off-target effects that might disrupt essential regulatory systems.",Epigenetics and Aging,Epigenetic Rejuvenation Strategies,2023
Future Directions and Outlook,"The field’s future lies in achieving a systems-level understanding of how epigenetic, transcriptional, and metabolic changes interact to determine aging trajectories. Researchers aim to identify precise epigenetic biomarkers that distinguish causal mechanisms from correlative changes and to test interventions capable of reprogramming aged cells. A deeper hierarchical understanding of the epigenome’s influence on longevity will enable the rational design of therapies to restore youthful gene expression, prevent age-related pathologies, and extend both life span and health span.",Epigenetics and Aging,Future of Epigenetic Aging Research,2023
Definition and Scope of Aging and Healthspan,"Aging is defined as the gradual loss of molecular fidelity following sexual maturity, ultimately leading to functional decline, disease, and death. The rate of aging is inversely correlated with mean lifespan and serves as the principal risk factor for cancer, neurodegeneration, and cardiovascular disease. Healthspan, in contrast, represents the period of life free from chronic disease and functional impairment. Biogerontology aims to uncover mechanisms to extend both lifespan and healthspan through genetic, metabolic, and epigenetic modulation.",Epigenetic Mechanisms of Longevity and Aging,Aging and Healthspan Definitions,2016
Model Organisms in Aging Research,"Animal models have been central to deciphering the biological mechanisms underlying aging. Yeast models have helped define replicative and chronological lifespan, while short-lived invertebrates such as worms (C. elegans) and flies (Drosophila) have provided insights into conserved genetic and metabolic pathways. Vertebrate models like mice bridge the gap between these systems and human physiology but are limited by their relatively long lifespans. The African turquoise killifish (Nothobranchius furzeri) provides a promising short-lived vertebrate model that exhibits human-like age-related pathology, enabling more efficient lifespan and healthspan studies.",Epigenetic Mechanisms of Longevity and Aging,Model Systems in Longevity Research,2016
Genetic and Non-Genetic Factors Influencing Longevity,"Both genetic mutations and non-genetic factors contribute significantly to lifespan variation. Non-genetic interventions such as calorie restriction, reduced basal metabolic rate, enhanced stress response, balanced mitochondrial protein synthesis, and lowered fertility have been correlated with extended lifespan. These findings suggest that environmental and metabolic inputs modulate aging through 'epi-genetic' pathways that regulate gene expression and chromatin structure, highlighting the interplay between genetics, metabolism, and epigenetics in determining longevity.",Epigenetic Mechanisms of Longevity and Aging,Determinants of Longevity,2016
Chromatin Dynamics and Epigenetic Regulation in Aging,"Chromatin, composed of DNA wrapped around histone proteins, governs transcriptional accessibility through dynamic structural modifications. Epigenetic factors—including DNA methyltransferases, histone-modifying enzymes, and chromatin remodelers—regulate long-term gene expression states. Aging is associated with global histone loss, chromatin remodeling, imbalanced histone modifications, and nuclear reorganization. In mammals, age also brings genome-wide and locus-specific DNA methylation changes, altered heterochromatin domains, and transcriptional dysregulation, suggesting that chromatin remodeling is both a marker and mediator of aging.",Epigenetic Mechanisms of Longevity and Aging,Chromatin and Epigenetic Remodeling in Aging,2016
Causality and Complexity in Epigenetic Aging,"It remains unresolved whether changes in epigenetic enzyme activity drive longevity-related gene expression or whether the reverse is true. In simple organisms, single mutations in histone modifiers or DNA methylation enzymes can dramatically extend lifespan, indicating direct epigenetic control. However, in complex multicellular organisms with redundant enzyme systems, aging likely results from large-scale, integrated responses to environmental stress and nutrient fluctuations rather than single-gene effects. Understanding these interactions is crucial for defining causal relationships between epigenetic alterations and aging phenotypes.",Epigenetic Mechanisms of Longevity and Aging,Causality Between Epigenetics and Aging,2016
Scope and Resources for Epigenetic Aging Research,"This review consolidates findings across model systems to highlight the central role of epigenetic regulation in aging. It provides comparative summaries of epigenetic mechanisms in various species, outlines advantages and limitations of experimental models, and compiles cutting-edge methodologies and data repositories for aging research. Notably, advances such as large-scale epigenomic profiling and CRISPR-based mutagenesis are accelerating the ability to dissect causal pathways in longevity and identify potential therapeutic targets to modulate aging.",Epigenetic Mechanisms of Longevity and Aging,Epigenetic Research Tools and Resources,2016
General Loss of Histones as a Mechanism of Aging,"A consistent epigenetic hallmark of aging, observed from yeast to humans, is the global reduction in histone levels. In replicatively aged yeast, MNase-seq analyses have revealed approximately 50% nucleosome loss across the genome, leading to increased nucleosome 'fuzziness' and destabilized chromatin organization. Overexpression of histones in yeast extends lifespan, highlighting the importance of maintaining histone dosage for genomic stability and longevity. These findings suggest that histone depletion contributes causally to age-associated chromatin relaxation and transcriptional dysregulation.",Epigenetic Mechanisms of Longevity and Aging,Histone Loss in Aging,2016
Mechanisms of Histone Loss,"Histone loss during aging appears to result from both transcriptional and post-translational mechanisms. Reduced histone gene expression accompanies cell-cycle exit and senescence, while degradation pathways such as autophagy may further deplete histone stores. In yeast, piecemeal microautophagy of the nucleus may contribute to histone degradation, although direct evidence is lacking. In human senescent fibroblasts, cytoplasmic chromatin fragments containing DNA and repressive histone marks are shed and degraded via lysosomes, suggesting an autophagic clearance route for chromatin components.",Epigenetic Mechanisms of Longevity and Aging,Mechanisms Underlying Histone Depletion,2016
Histone Loss in Human and Non-Dividing Cells,"While histone loss is evident in dividing cells undergoing replicative senescence, it remains uncertain whether similar chromatin depletion occurs in non-dividing, post-mitotic cells such as neurons. Since these cells do not experience the replication-linked histone dilution seen in dividing systems, their chromatin maintenance mechanisms may differ fundamentally. Determining whether histone loss contributes to functional decline in long-lived tissues like the brain is an important open question in the study of organismal aging.",Epigenetic Mechanisms of Longevity and Aging,Histone Loss in Post-Mitotic Tissues,2016
Future Directions in Histone Dynamics Research,"Future research aims to map genomic regions exhibiting localized histone loss ('pockets') using advanced single-cell sequencing and chromatin profiling methods. Investigating the signaling pathways that trigger histone depletion and identifying alternative histone isoforms expressed during senescence may reveal mechanisms of chromatin adaptation in non-dividing cells. Alternative histones, produced through splicing variants that enable polyadenylation and stabilization, may maintain chromatin plasticity in senescent or differentiated cells and represent a distinct epigenetic signature of aging.",Epigenetic Mechanisms of Longevity and Aging,Future Directions in Histone and Chromatin Aging Research,2016
Imbalance of Activating and Repressive Histone Modifications,"Histone modifications, particularly the activating mark H3K4me3 and the repressive mark H3K27me3, play central roles in regulating lifespan across multiple species. In C. elegans, components of the ASH-2/WDR-5/SET-2 complex that mediate H3K4 trimethylation influence longevity—knockdown of methyltransferase subunits extends lifespan, while knockdown of the demethylase rbr-2 shortens it. Conversely, rbr-2 overexpression lengthens lifespan, underscoring that balanced H3K4me3 levels are essential for epigenetic control of longevity. These lifespan effects are germline-dependent and transgenerational, indicating heritable epigenetic mechanisms in aging.",Epigenetic Mechanisms of Longevity and Aging,Histone Methylation and Lifespan Regulation,2016
Contrasting Roles of H3K27me3 Across Species,"The repressive modification H3K27me3, catalyzed by the PRC2 complex and removed by UTX-1, shows species-specific effects on lifespan. In worms, decreased H3K27me3 levels correlate with aging, while increased levels—achieved via utx-1 knockdown—extend lifespan in an insulin-dependent, germline-independent manner. However, in flies and mammals, heterozygous mutations in the H3K27 methyltransferase E(Z)/EZH2 paradoxically lengthen lifespan, suggesting that local gene-specific effects may outweigh global chromatin trends. Thus, while aging often involves a gain of activating marks and loss of repressive ones, exceptions across species reflect complex regulatory dynamics.",Epigenetic Mechanisms of Longevity and Aging,H3K27me3 and Cross-Species Differences,2016
Histone Modification Patterns in Flies and Vertebrates,"In Drosophila, overexpression of the H3K4 demethylase Lid extends lifespan, while its knockdown reduces male lifespan by nearly 20%. Interestingly, in contrast to worms, mutations that reduce H3K27me3 through PRC2 disruption also extend lifespan by derepressing target genes like Abd-B and Odc1. Aging flies exhibit a global loss of activating marks such as H3K4me3 and H3K36me3 and a gain of repressive marks like H3K9me3. Similar upregulation of H3K27me3 occurs in the short-lived killifish, suggesting evolutionary divergence in histone modification signatures during aging.",Epigenetic Mechanisms of Longevity and Aging,Histone Modification Trends in Flies and Vertebrates,2016
Histone Acetylation and Small Molecule Modulators of Longevity,"Global histone acetylation levels are closely linked to aging and longevity. The polyamine spermidine inhibits histone acetyltransferases (HATs), maintaining hypoacetylated chromatin. Spermidine levels decline with age in multiple organisms, and supplementation extends lifespan in yeast, worms, flies, and human cells. This extension correlates with enhanced stress tolerance and upregulated autophagy through histone acetylation changes at stress-response genes, illustrating how small-molecule modulation of histone acetylation can mimic calorie restriction and promote longevity.",Epigenetic Mechanisms of Longevity and Aging,Histone Acetylation and Longevity Modulators,2016
Senescence-Associated Chromatin Remodeling in Humans,"In mammalian cells, senescence induces profound chromatin reorganization, including formation of senescence-associated heterochromatin foci (SAHF), which contain repressive histone marks, macroH2A, and HMGA proteins. Genome-wide analyses show that up to 30% of chromatin is reorganized in senescent cells, forming large H3K4me3 and H3K27me3-enriched 'mesas' over lamin-associated domains (LADs) and corresponding 'canyons' of H3K27me3 loss elsewhere. These changes are associated with lamin B1 depletion, autophagic degradation of repressive histones, and increased promoter H4K16 acetylation maintained by the HIRA histone chaperone and variant H3.3 deposition.",Epigenetic Mechanisms of Longevity and Aging,Senescence and Chromatin Reorganization,2016
"Ubiquitylation, Sumoylation, and Chromatin Stability","Beyond methylation and acetylation, chromatin-associated modifications like ubiquitylation and sumoylation also influence aging. In yeast, deletion of SAGA/SLIK deubiquitinase subunits such as Sgf73 extends lifespan through enhanced silencing of telomeric genes and stabilization of compact chromatin. In higher organisms, senescent cells accumulate ubiquitylated and sumoylated proteins, including histone H2A, telomerase, and p53, promoting proteasomal degradation. Overexpression of SUMO induces premature aging in worms, and elevated SUMO levels are observed in aged rats, linking these small-peptide modifications to senescence pathways involving PML nuclear bodies and p53/pRb signaling.",Epigenetic Mechanisms of Longevity and Aging,Ubiquitylation and Sumoylation in Aging,2016
Global Alterations in Histone Modifications During Aging,"Across multiple organisms, aging correlates with profound shifts in histone modification patterns. These include the loss of repressive marks and gain of activating marks, although trends may vary locally depending on chromatin context and model organism. In hematopoietic stem cells, for example, H3K4me3 peaks broaden with age, particularly over self-renewal genes, while H3K27me3 peaks remain constant but expand in width. Such imbalances in histone modifications may either drive degenerative phenotypes or serve protective functions, such as preserving genomic integrity, underscoring the dual and context-dependent roles of chromatin remodeling in aging.",Epigenetic Mechanisms of Longevity and Aging,Global Histone Modification Patterns in Aging,2016
Transcriptional Deregulation as a Consequence of Epigenetic Change,"In replicatively aged yeast, global loss of histones leads to widespread transcriptional amplification across the genome. This occurs because reduced nucleosome density increases chromatin accessibility, allowing more frequent engagement of transcriptional machinery. Genes most upregulated with age already display distinct chromatin features in young cells—such as poorly phased nucleosomes, lack of nucleosome-depleted regions at promoters, and enrichment for TATA boxes and repressive factors like Asf1 and Tup1. During aging, these same genes experience further nucleosome loss at promoters, resulting in exaggerated transcriptional activation. Restoration of histone levels suppresses this effect, confirming that histone depletion directly drives transcriptional deregulation in aged cells.",Epigenetic Mechanisms of Longevity and Aging,Transcriptional Amplification from Histone Loss,2016
Upregulation of Intragenic Cryptic Transcripts Due to Reduced Histone Methylation over Gene Bodies,"Histone H3K36 methylation plays a key role in maintaining transcriptional fidelity and regulating lifespan. In replicatively aged yeast, loss of H3K36me3—caused by deletion of the Set2 methyltransferase—results in the emergence of intragenic cryptic transcripts and shortened lifespan. Conversely, deletion of the demethylase Rph1 extends lifespan by approximately 30%, likely by suppressing the production of spurious transcripts originating within gene bodies. This mechanism is conserved across species: in worms, loss of the Set2 homolog MET-1 shortens lifespan, while deletion of the demethylase JMJD-2 has pro-longevity effects. H3K36me3 thus acts as a stabilizing chromatin mark that prevents transcriptional noise and preserves gene expression precision during aging. Its reduction disrupts transcriptional integrity, contributing to the molecular drift associated with aging.",Epigenetic Mechanisms of Longevity and Aging,H3K36 Methylation and Cryptic Transcription in Aging,2016
Transcriptional Noise Due to DNA Damage,"Aging is associated with increased transcriptional noise, characterized by stochastic fluctuations in gene expression. Single-cell transcriptomic analyses of cardiomyocytes from aged mice reveal substantial variability in expression levels of key housekeeping genes compared to young controls. Similarly, mouse embryonic fibroblasts exposed to the DNA-damaging agent hydrogen peroxide exhibit elevated transcriptional noise, implicating DNA damage as a primary driver. However, this increase occurs as a delayed response, likely resulting from persistent genomic alterations such as mutations or epigenetic remodeling rather than transient oxidative damage. Notably, in proliferative hematopoietic cells—including stem cells, granulocytes, and naive lymphocytes—this effect is minimal, though older cells tend to show generally higher transcript abundance. These differences reflect tissue-specific mutation burdens and suggest that age-related transcriptional deregulation contributes to functional decline predominantly in long-lived, post-mitotic cells.",Epigenetic Mechanisms of Longevity and Aging,Transcriptional Noise and DNA Damage,2016
Altered Transcriptional Programs,"A hallmark of aging is large-scale reprogramming of transcriptional profiles, most notably observed in senescent human cells. These cells exhibit extensive epigenetic remodeling that drives activation of cellular defense and inflammatory genes comprising the senescence-associated secretory phenotype (SASP). While SASP helps promote immune-mediated clearance of pre-malignant cells, chronic activation contributes to tissue degeneration and inflammation. Loss of the repressive histone mark H3K27me3 correlates with increased SASP gene expression, whereas inhibition of the H3K4 methyltransferase MLL1 reduces SASP indirectly by repressing pro-proliferative and DNA damage response genes. In aged hematopoietic stem cells (HSCs), similar epigenetic changes accompany transcriptional shifts toward inflammatory and adhesion-related genes, alongside downregulation of DNA repair and chromatin remodeling pathways. These alterations reduce regenerative potential and promote myeloid lineage bias. Additionally, aged and senescent cells show aberrant activation of repetitive elements such as LINEs, SINEs, and LTRs, likely due to heterochromatin erosion. Collectively, these findings highlight how epigenetically driven transcriptional instability underlies both cellular senescence and organismal aging, suggesting that restoring transcriptional balance could enhance longevity and tissue function.",Epigenetic Mechanisms of Longevity and Aging,Epigenetic Reprogramming and Transcriptional Alterations in Aging,2016
Site-Specific Losses and Gains of Heterochromatin,"Heterochromatin, traditionally considered stable throughout life, undergoes both losses and localized gains during aging and senescence. Constitutive heterochromatin—typically found at telomeres, centromeres, and pericentromeric regions—progressively deteriorates due to telomere shortening, nuclear envelope breakdown, and boundary transcriptional changes. In yeast, deregulation of heterochromatin components such as Sir2 leads to loss of silencing at the rDNA locus, MAT locus, and subtelomeric regions, contributing to aging via accumulation of extrachromosomal rDNA circles (ERCs). Overexpression of Sir2 or its activators extends lifespan, while elevated H4K16 acetylation with age opposes this effect. In mammalian systems, senescence is characterized by disorganized constitutive heterochromatin, global DNA hypomethylation, and loss of repressive marks such as H3K9me3 and H3K27me3 due to decreased EZH2 activity. Reduced H3K27me3 derepresses p16INK4a, a major senescence marker, whereas EZH2 overexpression prolongs cellular lifespan by maintaining heterochromatin integrity.

Paradoxically, senescent fibroblasts exhibit localized heterochromatin gains in the form of senescence-associated heterochromatin foci (SAHFs), enriched in γ-H2AX, HP1γ, H3K9me3, macroH2A, and HMGA proteins. SAHFs arise through sequential assembly by the HUCA histone chaperone complex (HIRA, ASF1a, CABIN1, UBN1) and subsequent recruitment of structural proteins. These foci are believed to form in late-replicating euchromatic regions, potentially serving as compensatory mechanisms to contain transcriptional noise and preserve genome stability. Collectively, aging entails widespread heterochromatin disorganization alongside targeted remodeling events that modulate gene expression, genomic integrity, and ultimately, cellular longevity.",Epigenetic Mechanisms of Longevity and Aging,Heterochromatin Remodeling in Aging and Senescence,2016
Alteration in DNA Methylation Levels and Patterns with Age,"DNA methylation, primarily occurring on the 5-carbon of cytosine residues in CpG dinucleotides, is one of the best-characterized epigenetic modifications associated with aging. Across multiple species, aging is marked by a combination of global DNA hypomethylation and localized promoter hypermethylation. While non-mammalian models such as yeast lack cytosine methylation and worms and flies possess minimal levels, alternative modifications like N6-adenine methylation (6mA) have been discovered in these organisms, potentially serving analogous roles in transposon repression and genome stability. In flies, overexpression of the DNA methyltransferase Dnmt2 extends lifespan, whereas its loss shortens it, highlighting a conserved link between methylation and longevity.

In mammals, DNA methylation is mediated by DNMT1 (maintenance) and DNMT3A/3B (de novo) methyltransferases, with demethylation regulated by TET enzymes. During aging, global loss of 5-methylcytosine (5mC) occurs predominantly in repetitive, heterochromatic regions, while local hypermethylation accumulates at gene promoters. Mouse studies demonstrate progressive hepatic 5mC decline with age and altered DNMT1 expression in long-lived Ames dwarf mice, suggesting hormonal and metabolic control over methylation maintenance. Human epigenome analyses reveal similar trends, with promoter CpGs gaining methylation and intergenic regions losing it. Large-scale methylation studies have led to the discovery of DNA methylation ‘clocks’—notably Horvath’s clock—based on methylation patterns at specific CpG sites that accurately predict biological age across tissues.

Collectively, these data support a model wherein aging involves widespread DNA hypomethylation contributing to genomic instability, coupled with focal promoter hypermethylation that alters transcription of age-related genes. These methylation shifts mirror the concurrent imbalance in histone modifications, underscoring a coordinated epigenetic remodeling process that influences lifespan and healthspan.",Epigenetic Mechanisms of Longevity and Aging,Age-Associated DNA Methylation Changes and Epigenetic Clocks,2016
Nutrient Signaling and Its Effects on the Chromatin,"Nutrient availability is a central regulator of lifespan across species, exerting its effects through evolutionarily conserved pathways that converge on chromatin and epigenetic regulation. Nutrients such as glucose, amino acids, and growth factors are sensed by molecular networks including glucose sensors, the TOR (target of rapamycin) pathway, insulin/IGF signaling, and sirtuin proteins—all of which modulate chromatin structure and gene expression. These pathways integrate environmental and metabolic inputs to orchestrate genome-wide epigenetic responses that promote or inhibit longevity. Beyond nutrients, other environmental cues such as circadian rhythms, hormonal fluctuations, and physical activity similarly reshape the epigenome, impacting cellular metabolism, repair, and resilience.

Among these, nutrient signaling represents the most extensively characterized link between environmental factors and chromatin regulation. Caloric intake, nutrient-sensing pathways, and metabolic intermediates can influence histone acetylation, methylation, and DNA methylation through changes in the cellular pools of cofactors such as NAD⁺, acetyl-CoA, and SAM (S-adenosylmethionine). These changes modulate chromatin accessibility and transcriptional programs governing stress resistance, energy metabolism, and cellular maintenance—key determinants of lifespan and healthspan. Consequently, nutrient-driven epigenetic reprogramming acts as a dynamic interface between metabolism and longevity, forming the molecular basis for dietary and pharmacological interventions that target aging.",Epigenetic Mechanisms of Longevity and Aging,Nutrient Signaling and Epigenetic Regulation of Longevity,2016
Glucose Sensing and the Ras/AC/PKA Pathway,"In yeast, glucose availability is sensed primarily through the Ras/adenylate cyclase (AC)/protein kinase A (PKA) signaling cascade, which plays a pivotal role in regulating lifespan via epigenetic and metabolic mechanisms. Deletion of key components such as Ras2 or adenylate cyclase significantly extends chronological lifespan, partly through enhanced oxidative stress protection and activation of stress-response pathways. The Ras and TOR pathways intersect at the stress regulon SOD2 and are modulated by glucose-repressible kinase Rim15 and transcription factors Msn2/4 and Gis1, linking nutrient sensing to antioxidant defense and energy homeostasis. Notably, calorie restriction synergizes with mutations in RAS2 and SCH9 (a TOR pathway component) to achieve up to a tenfold lifespan extension, underscoring the partial independence and additive effects of these longevity pathways.

Epigenetically, several downstream regulators in this pathway influence chromatin state and transcriptional precision. The Gis1-related protein Rph1, an H3K36 demethylase, modulates replicative lifespan and stationary-phase gene expression. Deletion of GIS1 shortens lifespan by promoting Acs2-mediated histone acetylation, whereas deletion of RPH1 extends lifespan through mitochondrial-to-nuclear signaling that represses subtelomeric gene expression. These mutations produce distinct transcriptional profiles relative to wild-type yeast, indicating that glucose signaling interfaces with large-scale epigenetic remodeling. Collectively, the Ras/AC/PKA pathway exemplifies how nutrient sensing directly shapes chromatin dynamics and longevity through coordinated regulation of histone modifiers and metabolic enzymes.",Epigenetic Mechanisms of Longevity and Aging,"Glucose Sensing, Ras/AC/PKA Pathway, and Epigenetic Regulation of Lifespan",2016
"Calorie Restriction, the TOR Pathway and Histone Acetylation","The Target of Rapamycin (TOR) pathway serves as a central hub linking nutrient availability to chromatin regulation and lifespan. In yeast, the TOR kinase forms two complexes: the rapamycin-sensitive TORC1 (Tor1/2, Lst8, Kog1, Tco89) and the rapamycin-insensitive TORC2 (Tor2, Avo1–3, Lst8). TORC1 primarily governs ribosome biogenesis, translation, autophagy, and cell growth through Sch9 kinase signaling. Deletion of TOR1 or SCH9, or treatment with low doses of rapamycin, markedly extends both replicative and chronological lifespan, demonstrating that nutrient sensing via TORC1 is a critical determinant of longevity.

TORC1 inhibition induces extensive chromatin remodeling. Under nutrient-rich conditions, TORC1 signaling recruits the Esa1-histone acetyltransferase complex to ribosomal protein (RP) gene promoters, increasing H4 acetylation and transcription. Rapamycin or starvation suppresses TORC1 activity, displacing Esa1 and recruiting the Rpd3–Sin3 histone deacetylase complex, which deacetylates H4K5/K12 and compacts chromatin to repress ribosomal biogenesis. Moreover, TORC1 inhibition reduces H3K56 acetylation globally and at rDNA loci—a modification essential for open chromatin and rRNA transcription. Mutations in H3K56 or its modifying enzymes (Rtt109, Hst3/4) disrupt DNA repair and shorten lifespan, underscoring the importance of dynamic H3K56 acetylation for genomic stability and longevity.

In multicellular organisms, TOR signaling similarly integrates nutrient status with epigenetic regulation. The worm homolog CeTOR (LET-363) forms TORC1 and TORC2 complexes analogous to yeast. Lifespan extension arises from combined mutations in TOR downstream effectors (RSKS-1/Sch9) and insulin signaling components (DAF-2), revealing synergistic crosstalk between these pathways. In Drosophila, lifespan is extended by overexpression of TSC1/2, PTEN, FOXO, or 4E-BP, or by pharmacological TOR inhibition via rapamycin, with protective effects on cardiac and neuronal function. Collectively, TORC1 suppression and reduced histone acetylation remodel chromatin to favor stress resistance, autophagy, and metabolic efficiency—hallmarks of calorie restriction-induced longevity.",Epigenetic Mechanisms of Longevity and Aging,"TOR Pathway, Calorie Restriction, and Histone Acetylation in Longevity",2016
Calorie Restriction and the Insulin Signaling Pathway,"The insulin and insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway is one of the most evolutionarily conserved regulators of lifespan and is tightly connected to nutrient sensing and chromatin regulation. In *C. elegans*, insulin/IGF-1 signals through the receptor DAF-2, activating phosphatidylinositol-3-kinase (PI3K) and AKT to phosphorylate and inactivate the FOXO transcription factor DAF-16. Under calorie restriction or reduced insulin signaling, DAF-16 translocates to the nucleus to activate stress-resistance and longevity-associated genes. Mutations in *age-1* (PI3K) and *daf-2* double lifespan, while *daf-16* loss abolishes this effect, confirming DAF-16 as the central effector of IIS-mediated longevity. High glucose intake, conversely, suppresses DAF-16 activity and accelerates aging. Additionally, AMPK phosphorylates DAF-16 under low-energy conditions, modulating transcriptional outcomes independent of nuclear localization.

Epigenetically, DAF-16 directly interacts with the SWI/SNF chromatin remodeling complex, recruiting it to target promoters to activate transcriptional programs that promote dauer formation, oxidative stress resistance, and extended lifespan. This demonstrates a direct molecular interface between nutrient sensing, chromatin remodeling, and transcriptional regulation. Similar mechanisms exist in higher organisms: reduced insulin signaling through mutations in insulin receptors (*InR*), or their substrates (*Chico*, *Lnk*), significantly extend lifespan in *Drosophila*. In mammals, dwarf mouse models such as Ames, Snell, Laron, and Little mice show reduced growth hormone (GH) and IGF-1 signaling, resulting in smaller body size but extended lifespan. These models highlight the correlation between low IGF-1 levels and delayed aging.

Pharmacologically, metformin—a long-established antidiabetic drug—mimics calorie restriction by reducing hepatic glucose output, increasing insulin sensitivity, inhibiting mitochondrial complex I, and activating AMPK. Animal studies show metformin improves both lifespan and healthspan, prompting the FDA-approved TAME (Targeting Aging with Metformin) trial to evaluate it as an anti-aging therapy. Collectively, reduced insulin/IGF-1 signaling exerts its pro-longevity effects through transcriptional and chromatin-level reprogramming mediated by DAF-16/FOXO and associated epigenetic cofactors, linking metabolic cues directly to genome stability and lifespan extension.",Epigenetic Mechanisms of Longevity and Aging,"Insulin/IGF-1 Signaling, Calorie Restriction, and Epigenetic Regulation of Longevity",2016
Metabolism and Sirtuin Activation,"The diverse environmental stimuli (including nutrients) alter the metabolic activity and supply of cofactors and substrates within cells. Cells can sense this altered energy status by, for example, assessing NAD+ levels that are reduced with age. Sirtuins are NAD-dependent deacetylases implicated in aging in multiple model systems (Longo and Kennedy, 2006). Calorie restriction induces an oxidative metabolic state manifested in high NAD+ levels and, consequently, high Sirtuin activity. Overexpression of yeast Sir2 and Sir2 protein homologs (SIR-2.1 in worms, SIRT2 in flies, and SIRT1 in mammals) extend lifespan (Herranz et al., 2010; Kaeberlein et al., 1999; Kim et al., 1999; Rogina and Helfand, 2004; Tissenbaum and Guarente, 2001); however, only yeast Sir2 has been strongly connected to calorie restriction pathways.",Epigenetic Mechanisms of Longevity and Aging,Sirtuins,2016
Epigenetic Role of Sir2 and STACs in Longevity,"Importantly, Sir2 is a histone deacetylase and regulates lifespan in yeast through multiple epigenetic mechanisms impacting heterochromatin, ERCs, H4K16ac, and subtelomeric gene expression as discussed above (Dang et al., 2009). Worms grown in presence of sirtuin activating compounds (STACs) like resveratrol (a plant polyphenol) show a slight (10%), but consistent, increase in lifespan (Wood et al., 2004). Boosting NAD+ levels by genetic or pharmacological means also extends worm and mouse lifespan (Mouchiroud et al., 2013; Zhang et al., 2016). Two models for Sir2 action have been found (1) in yeast, in which Sir2 may stimulate heterochromatin formation or reduce H4K16 acetylation at subtelomeric loci to increase lifespan, and (2) in mammals, in which it may directly deacetylate FOXO transcription factors and regulate its target genes (Brunet et al., 2004; Daitoku et al., 2004; Frescas et al., 2005).",Epigenetic Mechanisms of Longevity and Aging,Sirtuins,2016
Histone Deacetylases and Lifespan in Flies,"The effect of H4K16 deacetylation on fly lifespan is more complicated as different HDACs have disparate effects on lifespan. As in yeast and worms, Sir2 overexpression and STACs such as resveratrol and fisetin extend lifespan in flies acting via the calorie restriction pathway (Wood et al., 2004). However, haploinsufficiency of Rpd3, another HDAC targeting H4K16, also results in lifespan extension (Rogina et al., 2002). Although the RPD3 findings are similar to yeast, Drosophila longevity is not mediated through heterochromatic gene silencing. Together, these data suggest that, in flies, different deacetylases (SIR2 and RPD3) function in opposite ways at euchromatin to affect longevity by influencing specific gene expression patterns (Frankel and Rogina, 2005). The disparity may stem from the multiple substrates targeted by these proteins but nevertheless underscores the importance of specific gene regulation in longevity, which as discussed above, may be a more dominant mechanism regulating lifespan in some models.",Epigenetic Mechanisms of Longevity and Aging,Histone Deacetylases,2016
Acetyl-CoA and Epigenetic Modifications in Aging,"Another metabolite, acetyl-CoA, increases in aged flies, contributing to increased acetylation of H4K12 and key metabolic enzymes. Targeted reduction of ATP citrate lyase, an acetyl-CoA-producing enzyme and the acetyltransferase for H4K12, Chameau, promotes longevity (Peleg et al., 2016). However, the exact role of acetylation in limiting lifespan in flies is still unclear. For one, different deacetylases, SIR2 and RPD3, have different effects on lifespan. Second, increased H3K9ac achieved by mutation of Su(var) 2-1 has no effect on lifespan (Frankel and Rogina, 2005). Third, administration of different HDAC inhibitors like sodium butyrate, TSA, PBA, and SAHA results in different changes in mean lifespan. The dosage, time of administration, and genetic background of flies used are critical to observing lifespan changes (McDonald et al., 2013). Thus, more work will be required to determine the key epigenetic changes underlying fly lifespan modulation.",Epigenetic Mechanisms of Longevity and Aging,Acetyl-CoA,2016
Calorie Restriction and Vertebrate Longevity,"The hallmark study that first reported mean and maximum lifespan extension by calorie restriction in a vertebrate model was conducted in rats (McCay et al., 1989). In mice, calorie restriction intervention can be initiated at weaning (3–6 weeks), 1 year, or even up to 19 months to see health benefits. Adult-initiated calorie restriction (1 year) increases mean but not maximum lifespan (Weindruch and Walford, 1982). Calorie restriction initiated in older mice (19 months) has significant effects on mean and maximum lifespans in addition to reducing cancer incidence (Dhahbi et al., 2004). Late-stage benefit occurs at least partially through the mTOR pathway. Treatment with rapamycin at 600 days also extended mean and maximum lifespans of male and female mice (Harrison et al., 2009). For a more elaborate discussion of possible calorie restriction effects on the epigenome, we refer the readers to a specialized review (Vaquero and Reinberg, 2009).",Epigenetic Mechanisms of Longevity and Aging,Calorie Restriction,2016
Sirtuins and Lifespan Regulation in Mammals,"The lifespan effect of altering mammalian sirtuins is complex. Mammals have seven Sirtuins (SIRT1-7) with SIRT1 being the closest mammalian homolog of yeast Sir2. SIRT1 localizes to the nucleus and deacetylates H1K26, H4K16, H3K9, and a number of non-histone targets including p53, p300, SUV39H1, FOXO transcription factors, and PGC1a (Chen et al., 2012b). Unfortunately, altering systemic SIRT1 levels has not yet shown any lifespan changes in mice, although there are health benefits linked to SIRT1 expression (Libert and Guarente, 2013). Of note, there is one report of lifespan extension in mice by overexpression of neuronal Sirt1 (Satoh et al., 2013).",Epigenetic Mechanisms of Longevity and Aging,Sirtuins,2016
"Resveratrol, Sirtuin Activation, and Mitochondrial Function","In 1997, resveratrol was found to be a potential chemotherapeutic agent in mice (Jang et al., 1997). Resveratrol reduced the Km for binding to both NAD and the acetylated substrate (Howitz et al., 2003; Mitchell et al., 2014). Later, it was shown that resveratrol had beneficial effects on health and survival of mice on a high-calorie diet (Baur et al., 2006). SRT1720, another STAC, was capable of modestly extending mouse lifespan (Mitchell et al., 2014). One of the beneficial effects of SIRT1 activation is improved mitochondrial function. With age and declining NAD+ levels, mitochondrial (but not nuclear) OXPHOS subunits are downregulated, resulting in a pseudohypoxic state. The effect is exaggerated by deleting Sirt1 and attenuated by increasing NAD+ levels, in this case by feeding mice NMN, a precursor of NAD+ (Gomes et al., 2013).",Epigenetic Mechanisms of Longevity and Aging,Resveratrol,2016
SIRT6 and Lifespan Extension,"Surprisingly, the less studied Sirt6 was recently shown to promote longevity in male (but not female) mice by 16% when overexpressed (Kanfi et al., 2012). SIRT6 is nuclear, deacetylates H3K9 and H3K56, and acts as a PARP1 ADP-ribosylase under oxidative conditions to promote DNA repair. These observations support the model whereby histone acetylation and regulation via sirtuins and other metabolic enzymes regulates lifespan. Overall, the epigenetic mechanisms of sirtuin action and their precise targets in aging are still unclear; however, the connection to histone acetylation is strong.",Epigenetic Mechanisms of Longevity and Aging,SIRT6,2016
Metabolism and Sirtuin Activation,"The diverse environmental stimuli (including nutrients) alter the metabolic activity and supply of cofactors and substrates within cells. Cells can sense this altered energy status by, for example, assessing NAD+ levels that are reduced with age. Sirtuins are NAD-dependent deacetylases implicated in aging in multiple model systems (Longo and Kennedy, 2006). Calorie restriction induces an oxidative metabolic state manifested in high NAD+ levels and, consequently, high Sirtuin activity. Overexpression of yeast Sir2 and Sir2 protein homologs (SIR-2.1 in worms, SIRT2 in flies, and SIRT1 in mammals) extend lifespan (Herranz et al., 2010; Kaeberlein et al., 1999; Kim et al., 1999; Rogina and Helfand, 2004; Tissenbaum and Guarente, 2001); however, only yeast Sir2 has been strongly connected to calorie restriction pathways.",Epigenetic Mechanisms of Longevity and Aging,Sirtuins,2016
Sir2 and Sirtuin Activating Compounds,"Importantly, Sir2 is a histone deacetylase and regulates lifespan in yeast through multiple epigenetic mechanisms impacting heterochromatin, ERCs, H4K16ac, and subtelomeric gene expression as discussed above (Dang et al., 2009). Worms grown in presence of sirtuin activating compounds (STACs) like resveratrol (a plant polyphenol) show a slight (10%), but consistent, increase in lifespan (Wood et al., 2004). Boosting NAD+ levels by genetic or pharmacological means also extends worm and mouse lifespan (Mouchiroud et al., 2013; Zhang et al., 2016). Two models for Sir2 action have been found (1) in yeast, in which Sir2 may stimulate heterochromatin formation or reduce H4K16 acetylation at subtelomeric loci to increase lifespan, and (2) in mammals, in which it may directly deacetylate FOXO transcription factors and regulate its target genes (Brunet et al., 2004; Daitoku et al., 2004; Frescas et al., 2005).",Epigenetic Mechanisms of Longevity and Aging,Sirtuins,2016
Histone Deacetylases and Longevity in Flies,"The effect of H4K16 deacetylation on fly lifespan is more complicated as different HDACs have disparate effects on lifespan. As in yeast and worms, Sir2 overexpression and STACs such as resveratrol and fisetin extend lifespan in flies acting via the calorie restriction pathway (Wood et al., 2004). However, haploinsufficiency of Rpd3, another HDAC targeting H4K16, also results in lifespan extension (Rogina et al., 2002). Although the RPD3 findings are similar to yeast, Drosophila longevity is not mediated through heterochromatic gene silencing. Together, these data suggest that, in flies, different deacetylases (SIR2 and RPD3) function in opposite ways at euchromatin to affect longevity by influencing specific gene expression patterns (Frankel and Rogina, 2005). The disparity may stem from the multiple substrates targeted by these proteins but nevertheless underscores the importance of specific gene regulation in longevity, which as discussed above, may be a more dominant mechanism regulating lifespan in some models.",Epigenetic Mechanisms of Longevity and Aging,Histone Deacetylases,2016
Acetyl-CoA and Epigenetic Control of Lifespan,"Another metabolite, acetyl-CoA, increases in aged flies, contributing to increased acetylation of H4K12 and key metabolic enzymes. Targeted reduction of ATP citrate lyase, an acetyl-CoA-producing enzyme and the acetyltransferase for H4K12, Chameau, promotes longevity (Peleg et al., 2016). However, the exact role of acetylation in limiting lifespan in flies is still unclear. For one, different deacetylases, SIR2 and RPD3, have different effects on lifespan. Second, increased H3K9ac achieved by mutation of Su(var) 2-1 has no effect on lifespan (Frankel and Rogina, 2005). Third, administration of different HDAC inhibitors like sodium butyrate, TSA, PBA, and SAHA results in different changes in mean lifespan. The dosage, time of administration, and genetic background of flies used are critical to observing lifespan changes (McDonald et al., 2013). Thus, more work will be required to determine the key epigenetic changes underlying fly lifespan modulation.",Epigenetic Mechanisms of Longevity and Aging,Acetyl-CoA,2016
Calorie Restriction in Vertebrate Models,"The hallmark study that first reported mean and maximum lifespan extension by calorie restriction in a vertebrate model was conducted in rats (McCay et al., 1989). In mice, calorie restriction intervention can be initiated at weaning (3–6 weeks), 1 year, or even up to 19 months to see health benefits. Adult-initiated calorie restriction (1 year) increases mean but not maximum lifespan (Weindruch and Walford, 1982). Calorie restriction initiated in older mice (19 months) has significant effects on mean and maximum lifespans in addition to reducing cancer incidence (Dhahbi et al., 2004). Late-stage benefit occurs at least partially through the mTOR pathway. Treatment with rapamycin at 600 days also extended mean and maximum lifespans of male and female mice (Harrison et al., 2009). For a more elaborate discussion of possible calorie restriction effects on the epigenome, we refer the readers to a specialized review (Vaquero and Reinberg, 2009).",Epigenetic Mechanisms of Longevity and Aging,Calorie Restriction,2016
Mammalian Sirtuins and Lifespan Effects,"The lifespan effect of altering mammalian sirtuins is complex. Mammals have seven Sirtuins (SIRT1-7) with SIRT1 being the closest mammalian homolog of yeast Sir2. SIRT1 localizes to the nucleus and deacetylates H1K26, H4K16, H3K9, and a number of non-histone targets including p53, p300, SUV39H1, FOXO transcription factors, and PGC1a (Chen et al., 2012b). Unfortunately, altering systemic SIRT1 levels has not yet shown any lifespan changes in mice, although there are health benefits linked to SIRT1 expression (Libert and Guarente, 2013). Of note, there is one report of lifespan extension in mice by overexpression of neuronal Sirt1 (Satoh et al., 2013).",Epigenetic Mechanisms of Longevity and Aging,Sirtuins,2016
Resveratrol and Mitochondrial Function,"In 1997, resveratrol was found to be a potential chemotherapeutic agent in mice (Jang et al., 1997). Resveratrol reduced the Km for binding to both NAD and the acetylated substrate (Howitz et al., 2003; Mitchell et al., 2014). Later, it was shown that resveratrol had beneficial effects on health and survival of mice on a high-calorie diet (Baur et al., 2006). SRT1720, another STAC, was capable of modestly extending mouse lifespan (Mitchell et al., 2014). One of the beneficial effects of SIRT1 activation is improved mitochondrial function. With age and declining NAD+ levels, mitochondrial (but not nuclear) OXPHOS subunits are downregulated, resulting in a pseudohypoxic state. The effect is exaggerated by deleting Sirt1 and attenuated by increasing NAD+ levels, in this case by feeding mice NMN, a precursor of NAD+ (Gomes et al., 2013).",Epigenetic Mechanisms of Longevity and Aging,Resveratrol,2016
SIRT6 and Longevity Regulation,"Surprisingly, the less studied Sirt6 was recently shown to promote longevity in male (but not female) mice by 16% when overexpressed (Kanfi et al., 2012). SIRT6 is nuclear, deacetylates H3K9 and H3K56, and acts as a PARP1 ADP-ribosylase under oxidative conditions to promote DNA repair. These observations support the model whereby histone acetylation and regulation via sirtuins and other metabolic enzymes regulates lifespan. Overall, the epigenetic mechanisms of sirtuin action and their precise targets in aging are still unclear; however, the connection to histone acetylation is strong.",Epigenetic Mechanisms of Longevity and Aging,SIRT6,2016
Overview of Laminopathies and Progeria,"Laminopathies are heterogeneous diseases characterized by deleterious changes to nuclear organization, resulting from mutations in genes encoding nuclear lamina proteins (Dechat et al., 2008). Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging laminopathy disease that has been the subject of intense study. HGPS is caused by mutations in lamin A, with most cases involving the expression of progerin, a truncated dominant-negative lamin A protein (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003). HGPS is a complex disease with multiple phenotypes indicating possible defects in normal aging and underlying epigenetic regulation of aging. Of interest to this review, HGPS cells exhibit severe abnormalities in nuclear morphology (Goldman et al., 2004), compromised DNA damage repair, alterations in chromosome organization, abnormal heterochromatin, and accelerated rates of cellular senescence (Burtner and Kennedy, 2010; Kudlow et al., 2007; Merideth et al., 2008; Norton et al., 2011; Prokocimer et al., 2013).",Epigenetic Mechanisms of Longevity and Aging,Laminopathies,2016
Epigenetic Alterations in HGPS,"Specifically, HGPS exhibits many alterations in heterochromatin factors, namely decreased levels of HP1, H3K9me3, and H3K27me3 and increased levels of H4K20me3 (McCord et al., 2013; Scaffidi and Misteli, 2005; Shumaker et al., 2006). Late passage HGPS cells exhibit loss of peripheral heterochromatin (Goldman et al., 2004), reduction in H3K9me3 and H3K27me3 levels (Scaffidi and Misteli, 2006; Shumaker et al., 2006), and the reduction of H3K27me3 methylase EZH2 (Shumaker et al., 2006). A recent genome-wide analysis of H3K27me3 in HGPS cells suggested a redistribution of the remaining amount of this modification across the genome (McCord et al., 2013). In addition, pre-senescent H3K4me3 mesas have been observed in proliferating HGPS cells, indicative of major chromatin reorganization in progeroid cells prior to senescence (Shah et al., 2013).",Epigenetic Mechanisms of Longevity and Aging,HGPS,2016
Mouse Models of Laminopathies,"Laminopathies are well modeled in mice, including in the Zmpste24 and BubR1 models. Zmpste24 encodes a metalloproteinase involved in maturation of lamin A, one of the major components of the lamin network (Bergo et al., 2002; Corrigan et al., 2005; Pendás et al., 2002). BubR1 encodes a key protein involved in the mitotic checkpoint response, ensuring accurate mitotic chromosome segregation: levels of BUBR1 are reduced in normally aged animals (Baker et al., 2004; Hartman et al., 2007; Matsumoto et al., 2007). Mutations in lamin A, Zmpste24, or BubR1 result in premature aging, including the accelerated onset of various age-related pathologies. BubR1-deficient mice also accumulate senescent cells, and in the BubR1 hypomorphic mouse, targeted clearance of senescent cells results in delay of onset for age-related phenotypes and BubR1 overexpression results in increased healthspan and lifespan (Baker et al., 2011), providing a strong link between onset of senescence and aging.",Epigenetic Mechanisms of Longevity and Aging,Laminopathies,2016
Epigenetic and Metabolic Links in Laminopathy Models,"These models suggest other interesting links between epigenetic regulation and aging. Zmpste24 mice have been shown to have hypermethylation at rDNA (Osorio et al., 2010), a phenotype associated with normal rodent aging (Oakes et al., 2003). Zmpste24 mice have also been shown to have global hypoacetylation on core histones H4 and H2B, which has been attributed to changes in expression of key cell-cycle and metabolic genes in pathways associated with age-related pathologies (Zhang et al., 2015). In addition, a recent study demonstrated that BUBR1 is directly acetylated by the histone acetyltransferase CBP, and this modification targets the protein for degradation; however, acetylated BUBR1 is a novel deacetylation target of SIRT2 (North et al., 2014). Overexpression of Sirt2 or treatment with a NAD+ precursor in BubR1 hypomorphic mice results in increased BUBR1 levels and increased median lifespan (Corrigan et al., 2005). However, SIRT2 does not restore BUBR1 levels to WT and cannot reverse many of the age-related phenotypes.",Epigenetic Mechanisms of Longevity and Aging,Epigenetic Regulation,2016
Complexity of Aging Causality in Laminopathies,"We note that laminopathies are complex diseases and determining causality of aging is particularly challenging in these models. Specifically, it remains unclear whether reduction in lifespan and presence of age-related pathology directly results from the lamin or related mutations or whether the phenotypes are secondary to significant metabolic changes. Despite these challenges, it is of great interest to understand how the lifespan is extended in these premature aging models and uncover other epigenetic mechanisms that may underlie maintenance of normal levels of BubR1 or other key checkpoint factors whose reduction contributes to premature aging phenotypes or genetic instability.",Epigenetic Mechanisms of Longevity and Aging,Laminopathies,2016
Protein Folding Requirements and Vulnerabilities,"Introduction Human cells express more than ∼10,000 different proteins at any given time (Kulak et al., 2017), the majority of which must fold (and often assemble) to well-defined, three-dimensional structures to allow a myriad of cellular functions. Although the native conformation of a given protein is encoded by its amino acid sequence (Anfinsen, 1973), in the cell many proteins require assistance by molecular chaperones and other factors to fold efficiently and at a biologically relevant time scale (Balchin et al., 2016). Moreover, proteins often need to retain structural flexibility or contain significant unstructured regions to function, leaving them at risk of misfolding and aggregation (Chiti and Dobson, 2017). Even otherwise stably folded proteins may unfold and possibly aggregate under stress conditions, such as elevated temperatures. Finally, as proteins become terminally misfolded, or are no longer functionally required, they must be degraded to avoid damaging effects of their continued presence. Maintaining an intact proteome (proteostasis) thus requires not only strict control of the initial production and folding of a protein but also its conformational maintenance, control of abundance and subcellular localization, and finally, disposal by degradation.",J Cell Biol,Proteostasis,2018
Proteostasis Network Functions and Aging Challenges,"A complex proteostasis network (PN) acts at each of these steps to maintain a balanced proteome linked by molecular chaperones of different classes as central players. These factors ensure de novo folding in a crowded cellular environment and maintain proteins in a soluble, nonaggregated state. Moreover, in conditions that disfavor folding or solubility, certain chaperones act to target misfolded proteins for degradation or spatial sequestration, thus protecting the rest of the proteome from aberrant interactions (Balchin et al., 2016; Sontag et al., 2017). Here, we describe the major pathways of cellular proteostasis and outline the challenges they face during aging and disease. We exemplify these processes using mainly the proteostasis pathways operating in the cytosol, where most cellular proteins are produced. The major exceptions are the proteins associated with the endomembrane system and secretory proteins. These polypeptides generally fold and assemble in the ER. Although the environment of the ER is oxidizing and differs in several aspects from the reducing cytosol, the core principles governing overall proteostatic balance apply (Skach, 2009; Gidalevitz et al., 2013). Rather than focusing on specific disease states, we discuss common themes that have been shown to be relevant across multiple systems, suggesting a conserved and intimate linkage of proteostasis with the aging process and associated pathologies.",J Cell Biol,Proteostasis,2018
Error-Prone Folding and Proteostasis Burden,"Organization of the PN Because of the astronomically large number of possible conformations a polypeptide chain can adopt, the folding process is inherently error prone (Dobson et al., 1998; Bartlett and Radford, 2009). Production of misfolded proteins is further increased by stochastic errors of protein biogenesis occurring at the level of transcription and mRNA maturation and translation (Sachidanandam et al., 2001; Ng and Henikoff, 2006; Drummond and Wilke, 2008). Such failed protein products must be recognized and degraded to avoid aberrant interactions, making it a challenge to maintain a healthy proteome even under normal conditions. This challenge is exacerbated in the case of disease-associated mutations, environmental stress, and aging and if left unresolved can lead to the formation of toxic aggregate species. Thus, to maintain proteostasis, cells have evolved a wide variety of molecular chaperones and protein quality-control factors that are functionally linked with protein degradation machineries. This system is referred to as the PN (Balch et al., 2008; Fig. 1 A). Defining the exact composition of the PN has proved difficult given the complexity of the human proteome.",J Cell Biol,Proteostasis Network,2018
Composition and Branches of the Proteostasis Network,"The PN has previously been proposed to consist of ∼1,000–1,400 components on the basis of our initial understanding of its organization (Balch et al., 2008; Powers et al., 2009; Kim et al., 2013; Hipp et al., 2014). Based on current annotations in databases and several large-scale genomic studies, we estimate that the PN comprises ∼2,000 factors that act in concert to maintain cellular proteostasis (Fig. 1 B). With increasing availability of functional annotations for the biological pathways in the human genome, these numbers will be further refined. Operationally, the PN can be divided into three branches composed of factors belonging to major processes: (1) protein synthesis, (2) folding and conformational maintenance (often coupled to transport and/or assembly), and (3) protein degradation (the ubiquitin–proteasome system [UPS] and autophagy–lysosome system; Fig. 1). Molecular chaperones and their regulatory cofactors act as liaisons connecting all these processes.",J Cell Biol,Proteostasis Network,2018
Protein Synthesis and Folding Machinery,"A set of ∼280 components participate directly in nascent polypeptide chain synthesis (Wolff et al., 2014; Rouillard et al., 2016; Fig. 1 B). Apart from the core constituents of the translational machinery, several chaperones act on the ribosome to prevent premature misfolding of the nascent chain and assist in cotranslational folding. Quality-control factors of the UPS interface with protein synthesis to remove defective and stalled nascent chains as part of ribosomal quality-control pathways (Brandman and Hegde, 2016). Newly synthesized proteins may fold cotranslationally or may rapidly complete their folding on release from the ribosome. Folding, and in some cases assembly to oligomeric complexes, is mediated by molecular chaperones, often involving sequential interactions with members of different chaperone classes (Langer et al., 1992; Frydman et al., 1994; Balchin et al., 2016). The repertoire of human chaperones (the “chaperome”) contains ∼330 members of several functionally distinct gene families, which cater to diverse substrate clients (Brehme et al., 2014; Sala et al., 2017; Fig. 1 B).",J Cell Biol,Proteostasis Network,2018
Protein Degradation Pathways and PN Adaptation,"Misfolded and aggregated proteins must be removed from the system by proteolytic degradation to prevent the accumulation of toxic species. Eukaryotic cells invest extensively in protein degradation machineries, with the two major pathways of the UPS and autophagy comprising ∼850 and ∼500 different components, respectively (Nijman et al., 2005; Li et al., 2008; Sowa et al., 2009; Varshavsky, 2012; García-Prat et al., 2016; Fig. 1 B). The UPS mainly serves to target individual proteins to the proteasome, whereas the autophagy system clears larger aggregates or membrane-associated proteins (Menzies et al., 2015; Ciechanover and Kwon, 2017). These branches of the PN are functionally coordinated by various signaling cascades, which sense and respond to imbalances in proteostasis (Fig. 1 A). In this way, cells constantly monitor and adjust their proteome status in response to internal and external changes. The PN not only enables this adjustment, but is itself adaptive to the needs of specific cell types.",J Cell Biol,Proteostasis Network,2018
Tissue-Specific PN Variability and Aging-Related Decline,"In “simpler” organisms such as yeast, the basic organization of the PN may be rather constant, only tuning itself to fluctuations in environmental conditions. However, in metazoans (Guisbert et al., 2013), especially in complex mammalian systems (Uhlén et al., 2015), tissue-specific proteomes and regulatory programs imply that there must be a marked heterogeneity in aspects of proteostasis across diverse cell types, suggesting the existence of tissue-specific PNs (Sala et al., 2017) with differing contributions of the three branches. Many diseases, including type II diabetes and the major neurodegenerative pathologies, are associated with a reduced function of the PN, which may be caused by mutations in PN components (Kakkar et al., 4) or by interference of toxic aggregate species with PN function (Hipp et al., 2014). Importantly, as shown in model organisms, aging is also associated with a general decrease in PN capacity and a corresponding increase in protein aggregation, which manifests as a functional decline in many cellular pathways (Taylor and Dillin, 2011; Labbadia and Morimoto, 2015a). In the following sections, we discuss the roles of the major pathways within the PN.",J Cell Biol,Proteostasis Network,2018
Protein Synthesis and Translation Control,"Protein synthesis. Although the production of individual proteins is regulated by specific factors and pathways, the levels of bulk protein synthesis must be adjusted to the protein folding capacity of the cell to avoid the accumulation of misfolded proteins. Indeed, in key lifespan extension pathways such as caloric restriction, increased proteostasis capacity is conferred, at least in part, by a general decrease in protein translation (Hansen et al., 2007; Taylor and Dillin, 2011). Translation attenuation is also critical in relieving PN overload in conditions of conformational stress. This is typically mediated by inhibition of translation initiation factor 2α (eIF2α). For example, on activation of the unfolded protein response (UPR) to the accumulation of misfolded proteins in the ER, protein kinase RNA-like ER kinase (PERK) in the ER membrane phosphorylates eIF2α, thereby attenuating its function in translation (Harding et al., 2001).",J Cell Biol,Proteostasis Network,2018
Protein Folding Dynamics and Misfolding Risks,"Protein folding and aggregation. Polypeptide chains fold by sequestering hydrophobic residues and forming stabilizing intramolecular interactions to achieve a low free-energy state (Fig. 2). Rather than sampling all potential folding states, a process that would take an insurmountable amount of time, polypeptides proceed toward their native conformation by increasingly forming local and long-range contacts between amino acid residues, thereby limiting the conformational space that must be explored (Dinner et al., 2000; Bartlett and Radford, 2009). In this way, many small proteins can achieve their proper fold quickly and efficiently in vitro. However, once placed in the highly crowded cellular environment, proteins often face significant challenges during folding, because partially folded states with exposed hydrophobic amino acids residues are in danger of misfolding and aggregating. Aberrant folding may occur during de novo synthesis or in conditions of conformational stress, where preexisting proteins may fail to maintain their folded states. Destabilizing mutations or the presence of intrinsically unstructured regions can also predispose polypeptides to misfolding (Dunker et al., 2008; Gershenson et al., 2014).",J Cell Biol,Proteostasis Network,2018
Cotranslational Folding and Ribosome-Associated Constraints,"Unlike in vitro folding studies (Anfinsen, 1973; Bartlett and Radford, 2009), which start from complete proteins, in the cell proteins are synthesized vectorially on the ribosome, which means that the structural information necessary for folding becomes available gradually and not all at once. Translation is slow relative to rates of folding, allowing for the possibility of partial structure formation, both native or misfolded, before the completion of protein synthesis. Cotranslational folding limits the amount of time nascent chains populate potentially vulnerable, nonnative states (Balchin et al., 2016). Some very small proteins (∼50 amino acids in length) even fold to completion within the widening exit portal of the ribosome (Holtkamp et al., 2015; Marino et al., 2016). However, the major part of the ribosomal exit channel is too narrow to allow structure formation (Wilson and Beckmann, 2011), and thus the nascent chains of larger proteins must first emerge from the ribosome before they can fold, which puts them at risk of misfolding and aberrant interactions. The ribosomal surface may influence the folding process (Kaiser et al., 2011; Cabrita et al., 2016). Moreover, the topology of ribosomes in the context of polysomes, where translating ribosomes may approach each other closely, is optimized to reduce the risk of interactions between nascent chains. The ribosomes adopt a staggered “pseudohelical” arrangement, in which their polypeptide exit sites are at maximal distance from each other (Brandt et al., 2009). Multidomain proteins often fold their domains sequentially during translation, thereby avoiding nonnative interactions between concomitantly folding domains (Netzer and Hartl, 1997; Frydman et al., 1999).",J Cell Biol,Proteostasis Network,2018
Evolutionary Expansion and Aggregation Tendencies,"Average proteome and protein sizes have increased dramatically during evolution from bacteria to eukarya (Balchin et al., 2016; Fig. 3 A). Proteins of more than ∼100 amino acids in length constitute the vast majority of proteins in all domains of life and typically fold via intermediate states with incompletely buried hydrophobic residues (Dinner et al., 2000; Brockwell and Radford, 2007; Fig. 2). Such intermediates may be kinetically stable and may be highly aggregation prone (Gershenson et al., 2014), particularly in the crowded environment of the cell (∼300 g of protein per liter), where macromolecular interactions are enhanced compared with dilute solution (Ellis and Minton, 2006). Although the majority of resulting aggregates are amorphous (i.e., lacking long-range structural order), a subset of mostly smaller proteins, often containing unstructured regions (Dunker et al., 2008), can form ordered fibrillar aggregates, referred to as amyloid or amyloid-like, and which are characterized by β-strands running perpendicular to the fibril axis (cross-β-structure; Chiti and Dobson, 2017; Fig. 2). Such amyloid aggregates form insoluble deposits and are the hallmark of several age-dependent proteinopathies, including Alzheimer’s, Parkinson’s, and Huntington’s diseases (Ross and Poirier, 2004). More proteins transition to an insoluble, aggregated state when they exceed their normal cellular abundance or when imbalances occur between subunits of oligomeric complexes (Vendruscolo et al., 2011; Ciryam et al., 2013; Chiti and Dobson, 2017), a phenomenon that becomes more prevalent during aging, as shown in the nematode Caenorhabditis elegans (Walther et al., 2015).",J Cell Biol,Proteostasis Network,2018
Molecular Chaperones as Central PN Organizers,"Molecular chaperones—central organizers of the PN. To overcome the challenges to protein folding and solubility, cells have evolved molecular chaperones (Fig. 3 A). We define a molecular chaperone as a protein that assists in the folding, assembly, conformational maintenance, or regulation of another protein without becoming part of its final structure (Hartl, 1996). The chaperones that participate broadly in de novo folding recognize generic structural features of nonnative proteins, primarily exposed hydrophobic amino acid residues, and promote folding by kinetic partitioning of nonnative states (Kim et al., 2013; Fig. 2). Many chaperones are induced under conditions of stress, such as heat shock, and in addition to their functions in de novo folding are also involved in protein refolding, disaggregation, oligomeric assembly, trafficking, and degradation (Balchin et al., 2016; Fig. 1 A). Although the core chaperone machineries (heat-shock protein [Hsp] 70s, Hsp90s, chaperonins, and small Hsps [sHsps]) are already present in prokaryotes, a strong expansion in the number of their regulatory cofactors (Hsp40s, tetratricopeptide repeat proteins) is notable as eukaryotic proteomes increase in complexity (Genevaux et al., 2007; Vos et al., 2008; Brehme et al., 2014; Rizzolo et al., 2017; Fig. 3 B).",J Cell Biol,Molecular Chaperones,2018
Chaperone Systems Across Organisms,"A first tier of chaperones interacts directly with the ribosome close to the polypeptide exit site. These components are typically not stress-inducible (Albanèse et al., 2006) and include Trigger factor in bacteria and specialized chaperone complexes, such as nascent chain-associated complex and ribosome-associated complex, in eukaryotes (Fig. 3, C and D). They interact with exposed hydrophobic sequences of the emerging nascent chain and act to prevent premature (mis)folding, maintaining the polypeptide in a nonaggregated, folding competent state until sufficient structural elements for productive folding are available (Agashe et al., 2004; Kaiser et al., 2006; Preissler and Deuerling, 2012; Nilsson et al., 2016). Although most proteins may only require these chaperones to achieve their native fold, proteins with complex domain topologies and multidomain proteins need assistance by additional chaperone classes that act downstream.",J Cell Biol,Molecular Chaperones,2018
Hsp70 System and Cochaperones,"Such proteins may next interact cotranslationally or posttranslationally with a member of the Hsp70 chaperone family (DnaK in prokaryotes; Fig. 3, C and D), a ubiquitous class of ATP-dependent chaperones of ∼70 kD with a hub position in the PN. The Hsp70 C-terminal domain binds short hydrophobic peptide sequences of about seven residues that are exposed by nascent and nonnative protein substrates (Rüdiger et al., 1997; Mayer et al., 2000; Clerico et al., 2015). The affinity of the C-terminal domain for protein substrate is allosterically regulated by ATP binding and hydrolysis in the N-terminal ATPase domain. Hsp70s rely on regulatory chaperone cofactors of the Hsp40 class (also known as J-proteins), which typically bind first to exposed hydrophobic patches on nonnative proteins and recruit Hsp70 (Kampinga and Craig, 2010; Nillegoda et al., 2017). These factors then stimulate the hydrolysis of Hsp70-bound ATP, thereby catalyzing the closing of the Hsp70 peptide binding cleft (Clerico et al., 2015). There are ∼50 different Hsp40 proteins in human cells (Fig. 3 B), which fall into three structural subtypes and have different subcellular localizations (Kampinga and Craig, 2010). They confer broad functionality to the Hsp70 system, allowing these chaperones not only to participate in the initial folding of nascent chains but also in conformational maintenance, disaggregation, and the targeting of terminally misfolded proteins for degradation.",J Cell Biol,Molecular Chaperones,2018
Chaperonins and Hsp90 Functions,"Proteins that are unable to fold through such Hsp70 cycles may be transferred to the chaperonin class of chaperones (Hsp60s), which includes GroEL/GroES in bacteria, Hsp60 in mitochondria, and TRiC/CCT in the eukaryotic cytosol (Fig. 3, C and D). These chaperonin proteins form multimeric, cylindrical complexes that function by transiently encapsulating individual nonnative proteins so they can fold, unimpaired by aggregation (Lopez et al., 2015; Hayer-Hartl et al., 2016). Although only ∼10% of the proteome requires a chaperonin to fold, substrates include essential and highly abundant proteins, such as actin and tubulin. The highly conserved Hsp90 chaperone system also functions downstream of Hsp70 in maintaining a variety of signaling pathways via the folding and conformational regulation of their signal-transduction molecules (Sharma et al., 2012; Taipale et al., 2012). Hsp90 is active as a homodimer and mediates protein folding via ATP-dependent structural changes in cooperation with a multitude of cofactors (Wandinger et al., 2008).",J Cell Biol,Molecular Chaperones,2018
Maintaining the Metastable Proteome and Aging Decline,"Maintaining the metastable proteome. After initial folding, many proteins continue to require chaperone surveillance to maintain their functional form. Proteins are often active under conditions just at the cusp of stability, and their functional conformational states may be challenged under stress conditions. Many proteins contain intrinsically unstructured regions or sequences of low amino acid complexity important for their function. It has become clear in recent years that a hallmark of cellular aging is a gradual loss of proteome balance and accumulation of protein aggregates. This is thought to be due at least in part to an increase in errors in translation, splicing, or molecular misreading and to an increased production of oxidized and carbonylated proteins. Aged organisms show a markedly reduced ability to maintain metastable proteins in their soluble states. A healthy chaperone network is required to prevent accumulation of toxic aggregate species. The Hsp70 system and sHsps are particularly important. When the system is overburdened, misfolded species may aggregate. Association of sHsps and chaperone cofactors with aggregates enables downstream processing and eventual disaggregation by Hsp70/Hsp40/Hsp110 machineries. In yeast, this capacity is further enhanced by Hsp104, a specialized AAA+ ATPase capable of disaggregating many amyloid aggregates.",J Cell Biol,Molecular Chaperones,2018
Protein Disposal via UPS and Lysosomal Pathways,"Disposal by degradation. Proteins that are unable to fold or refold, despite intervention by chaperones, must be disposed of to prevent the accumulation of potentially toxic aggregate species. Such terminally misfolded proteins undergo proteolytic degradation mainly by the UPS (Varshavsky, 2012; Ciechanover and Kwon, 2017) or by chaperone-mediated lysosomal degradation (Kettern et al., 2010; Cuervo and Wong, 2014). The Hsp70 and Hsp90 chaperone systems are intimately involved in these processes, because the E3 ubiquitin ligase Chip binds the C terminus of these chaperones and ubiquitylates misfolded chaperone-bound proteins (Esser et al., 2004; Fig. 1 A). As shown recently, surplus proteins that fail to assemble with their partner molecules are recognized by a specific E3 ligase (UBE20; Yanagitani et al., 2017).",J Cell Biol,Protein Degradation,2018
Nuclear and ER-Associated Protein Degradation,"A subset of proteins that misfolds in the cytosol undergoes chaperone-mediated transport into the nucleus to be degraded by nuclear proteasomes (Heck et al., 0; Prasad et al., 2010; Park et al., 2013; Shibata and Morimoto, 2014; Fig. 1 A). Most of the proteins known to use this pathway are ectopically expressed secretory proteins. The extent to which endogenous, misfolded proteins are degraded in the nucleus remains to be established. Given that an abundance of proteasomes is found in the nucleus (Russell et al., 1999; von Mikecz, 2006), it is tempting to speculate that compartmentalizing synthesis/folding and degradation provides an evolutionary advantage by preventing premature degradation. The same principle would apply to the process of ER-mediated degradation, wherein misfolded proteins are retrotranslocated from the ER to the cytosol for disposal by the proteasome (Vembar and Brodsky, 2008). Aggregates may be resolved by the Hsp70/Hsp40/Hsp110 machinery before transfer into the proteasome (Hjerpe et al., 2016). Certain aggregate species resistant to disaggregation may be cleared directly by selective autophagy and lysosomal degradation (Lamark and Johansen, 2012), processes that also target a variety of additional substrates including membrane bound organelles (Mizushima, 2007).",J Cell Biol,Protein Degradation,2018
Age-Related Decline of Clearance Systems,"Many cell types show a decline in UPS activity and autophagy during aging (Rubinsztein et al., 2011; Cuervo and Wong, 2014), contributing to the widespread protein aggregation that is observed in postmitotic cells, such as muscle and neurons, and prediposing the latter for certain neurodegenerative diseases (David et al., 2010; Hamer et al., 2010; Walther et al., 2015). Because disease-associated proteins tend to be metastable, a slight increase in their abundance as clearance systems decline can have dramatic effects on their aggregation propensity (Ciryam et al., 2013; Kundra et al., 2017). Aging cells are also less able to cope with and dispose of amyloid-like aggregates (Morley et al., 2002), as exemplified by the fact that cellular aggregate deposits persist although they are typically associated with ubiquitin (Lowe et al., 1988; Bence et al., 2001; Waelter et al., 2001). These aggregates often sequester important components of the PN, which leads to further proteostatic impairment with buildup of damaged protein species and increased risk of aggregation (Bennett et al., 2005; Hipp et al., 2014; Itakura et al., 2016).",J Cell Biol,Protein Degradation,2018
Compartmentalization of Damaged Proteins,"Compartmentalization of damaged proteins. If attempts to prevent, refold, or degrade aberrant protein species fail, a final line of defense against their interference with cellular processes is their controlled sequestration into more benign aggregate deposits or inclusion bodies (Sontag et al., 2017). Depending on the properties of the misfolded proteins and their ability for eventual resolubilization, such deposits can be directed to several different localizations within the cytosol or nucleus and are referred to as an IPOD (for insoluble protein deposit), JUNQ (for juxtanuclear quality-control compartment), or INQ (for intranuclear quality-control compartment) in yeast and as an aggresome in mammalian cells (Johnston et al., 1998; Kaganovich et al., 2008; Miller et al., 2015; Fig. 1 A). Their formation is itself dependent on several quality-control components including chaperones (Malinovska et al., 2012; Escusa-Toret et al., 2013; Wolfe et al., 2013).",J Cell Biol,Proteostasis Compartments,2018
Age-Related Decline of Spatial Quality Control,"In addition to providing an environment in which aggregates may be shielded and thus prevented from engaging in potentially toxic interactions, in dividing cells the inclusions also serve as a way to minimize the amount of aberrant proteins that are passed on to daughter cells (Hill et al., 2017). Like other proteostasis pathways, the ability of a cell to maintain spatial quality control also declines with age (Escusa-Toret et al., 2013), and cells that lack this ability show accelerated aging (Erjavec et al., 2007).",J Cell Biol,Proteostasis Compartments,2018
Aggregation Propensity and Metastable Proteins,"Toxicity caused by aggregation Proteins have an intrinsic capacity to convert from their native state to intractable fibrillar aggregates, but under normal physiological conditions this tendency is resisted by cellular proteostasis mechanisms (Chiti and Dobson, 2017). However, the propensity to form amyloid-like aggregates is more pronounced for certain metastable proteins, including those associated with disease, especially when exceeding the cellular concentrations at which they are soluble (Ciryam et al., 2013). Dysregulation of protein abundance and protein stoichiometries may occur in an age-dependent manner, as observed in nematodes and other model organisms (Walther et al., 2015). Recent research shows that the formation of insoluble protein deposits in neurodegenerative syndromes such as Alzheimer’s disease occurs concomitantly with the aggregation of a large set of highly expressed and aggregation-prone proteins that constitute a metastable subproteome (Kundra et al., 2017).",J Cell Biol,Aggregation Toxicity,2018
Phase Transitions and Aberrant Condensation,"The metastable subproteome includes many RNA-binding proteins that contain unstructured or low-complexity sequences. As shown recently, such proteins often have the ability to undergo liquid–liquid phase transitions (Feric et al., 2016), forming dynamic droplet-like compartments in the nucleus and cytosol that participate in RNA metabolism, ribosome biogenesis, cell signaling, and other processes (Banani et al., 2017). However, the normally dynamic behavior of these condensates is highly sensitive to changes in the physicochemical environment of cells, and aberrant phase transition behavior, leading to fibril formation, has been linked with aging and diseases such as amyotrophic lateral sclerosis (Alberti and Hyman, 2016). These observations help explain how the age-dependent decline in protein homeostasis favors the stochastic manifestation of neurodegenerative aggregation. Even in dominantly inherited neurodegenerative disorders, such as those caused by expanded polyglutamine sequences, manifestation is age dependent and triggered by PN decline.",J Cell Biol,Aggregation Toxicity,2018
Toxic Oligomers and Gain-of-Function Effects,"Aggregation in disease typically causes gain-of-function toxicity, meaning that cytotoxic effects are largely unrelated to the normal function of the disease protein. The appearance of large fibrillar aggregate deposits does not always correlate with disease onset or severity. Work over the past years revealed that the most toxic aggregate species may be soluble oligomers and small insoluble species with little or no fibrillar content. Such oligomers expose hydrophobic residues and unpaired polypeptide backbone structures, making them highly interactive with other proteins, including those enriched in low-complexity sequences and critical factors of the PN, and with membranes. Although the exact nature of the most-toxic species remains under debate, larger amyloid aggregates may exert a relative protective effect by sequestering the more-toxic oligomers and by having a reduced surface-to-volume ratio.",J Cell Biol,Aggregation Toxicity,2018
"Aggregate Sequestration, Cellular Damage, and Chaperone Protection","However, key cellular factors, including PN components, that interact aberrantly with soluble oligomers may also be sequestered in insoluble deposits, contributing to cellular dysfunction. Moreover, large intracellular aggregate deposits sterically displace membrane structures and may cause their fragmentation, as recently shown for inclusions of polyglutamine expansion proteins by cryo-electron tomography. In many model systems, exogenous expression of individual chaperone components, such as Hsp70, or up-regulation of multiple chaperones by pharmacologic induction of the stress response has been shown to either prevent toxic aggregation or direct the formation of less-toxic but still aggregated species.",J Cell Biol,Aggregation Toxicity,2018
Activation of Cellular Stress Responses,"Stress response pathways Although the protein quality-control networks ensure proteostasis under basal conditions, on conformational stress, such as increases in temperature or exposure to oxidative agents, many additional proteins become prone to misfolding, with proteins comprising the metastable subproteome being particularly vulnerable. Cells adapt to such conditions by activating stress-response pathways to increase PN components, decrease substrate load, and resolve misfolded or aggregated species (Fig. 4). In metazoans the stress-response pathways additionally underlie cell nonautonomous regulation, allowing coordination within and between tissues and organs (Taylor et al., 2014; Sala et al., 2017).",J Cell Biol,Stress Response,2018
Cytosolic Heat-Shock Response via Hsf1,"The cytosolic stress response is regulated primarily by heat-shock transcription factor 1 (Hsf1), which is maintained in an inactive state by association with chaperones including Hsp90 and Hsp70. On heat stress, these chaperones are titrated away from Hsf1 by binding to denatured proteins. Hsf1 is then free to induce the transcription of a wide range of proteostasis components, although general protein translation is decreased, reducing the load on the chaperone machinery. Concurrently, expression of chaperones and other quality-control elements, such as proteasomal components, is increased to prevent and resolve misfolded proteins and aggregation. Once the stressor is removed, a negative-feedback loop on Hsf1 activity restores homeostasis.",J Cell Biol,Stress Response,2018
ER and Mitochondrial Unfolded Protein Responses,"Similar stress-response pathways include the UPR in the ER and mitochondria. The UPR of the ER has been studied extensively and is highly conserved from fungi to mammals. The accumulation of misfolded proteins in the ER is sensed by three transmembrane signaling proteins, IRE-1, PERK, and ATF6, which constitute distinct arms of the UPR. These arms activate transcription factors that increase production of proteostasis components. PERK activation also leads to phosphorylation of eIF2α and attenuation of general translation. Concomitantly, proteasome biogenesis is up-regulated by a signaling pathway adjusting degradation capacity to demand. Despite mechanistic distinctions, the goals of the ER UPR mirror those of the cytosolic heat-shock response: increasing quality-control components and decreasing potentially misfolded substrates through transient translation attenuation.",J Cell Biol,Stress Response,2018
Crosstalk Between Stress-Response Pathways,"Increasing evidence supports significant crosstalk between cellular stress-response pathways. Protein misfolding in the ER can lead to the aggregation of metastable proteins in the cytosol, and ER stress can trigger a partially protective cytosolic stress response when components of the UPR are defective. Additionally, mitochondrial stress has been shown to activate cytosolic stress-response pathways capable of protecting cells from disease-associated aggregates. This interconnectedness highlights a coordinated, system-wide attempt to restore proteostasis under stress.",J Cell Biol,Stress Response,2018
Chronic Misfolding and Maladaptive Stress Responses,"Chronic stress response Although the up-regulation of protein quality-control components allows cells to resolve stress-induced misfolded proteins and aggregates that formed as the result of acute environmental stress, the amyloid aggregates associated with age-dependent diseases appear to be largely resistant to these rescue mechanisms. The resulting chronic exposure of cells to misfolded species can have detrimental effects on the PN. Expression of model polyglutamine aggregates interferes with ER-associated protein degradation and leads to prolonged activation of the ER stress response. On chronic production of certain misfolded or aggregated proteins, as may occur in disease or during aging, the stress response becomes activated but unable to clear the offending species. This maladaptive stress response leaves cells vulnerable not only because the aggregates persist but also because cells become refractive to additional stressors, consistent with aged cells being less responsive to stress insults.",J Cell Biol,Chronic Stress Response,2018
"Aging, PN Capacity, and Insulin Signaling","A recent study in nematodes and mammalian cells revealed a relationship among aging, chronic protein folding stress, and PN capacity. Normal turnover of the insulin-like growth factor receptor (Daf2 in C. elegans) involves the E3 ubiquitin ligase Chip. Both aging and the accumulation of protein aggregates were found to interfere with degradation of the insulin receptor, because Chip becomes increasingly engaged by misfolded proteins. The resulting increase in Daf2 levels inhibits the Daf16 transcription factor (FOXO in mammals), causing a down-regulation of PN components and reduced lifespan.",J Cell Biol,Chronic Stress Response,2018
Modulating the PN to Counter Aggregation,"Modulation of the PN The persistence of disease-associated protein aggregates suggests that the cellular PN is generally unable to cope with such substrates. However, cells may be able to adequately handle aberrant protein species for long periods, even decades, as indicated by the late onset of inherited neurodegenerative diseases such as Huntington’s disease. Specific modulation of PN components can impact both aggregate morphology and lifespan in model systems. Expression of chaperones and cochaperones of different classes consistently results in decreased aggregate toxicity and increased lifespan. Therapeutic strategies in aggregate-associated neurodegenerative diseases have focused on preventing further production of misfolded species, stabilizing properly folded species, and clearing existing aggregates.",J Cell Biol,PN Modulation,2018
"Therapeutic Modulation of Translation, Folding, and Clearance","Toward these ends, small molecules have been identified that prolong translation attenuation on stress; stabilize mutant proteins, such as transthyretin, against aggregation; target folding and trafficking defects in specific disease-associated proteins, such as mutant cystic fibrosis transmembrane conductance regulator; and increase the clearance of toxic protein species through activation of the UPS or autophagy. Because many PN components are sequestered by aggregates, enhancement of endogenous stress-response pathways has been particularly effective in extending lifespan and improving proteostatic health.",J Cell Biol,PN Modulation,2018
Proteostasis Decline and Aging,"Concluding remarks Work over the past decades has uncovered a remarkable ability of cells to maintain proteostasis under a variety of challenging conditions. The importance of this ability is underlined by our increasing understanding that many neurodegenerative and aging-associated diseases are caused by a breakdown in this process. In recent years we have gained considerable insight as to why PN capacity may decline with age. Although in some cases the buildup of stochastic mutations and damage can certainly contribute to disease onset, this seems insufficient to explain the universal age-dependent decline in PN health observed across species.",J Cell Biol,Proteostasis and Aging,2018
Disposable Soma Theory and Proteostasis,"Work in metazoans such as worms suggests that this decline may instead be a regulated process. Consistent with the basic tenet of the “disposable soma” theory, organisms may sacrifice their own proteostatic fitness to divert resources toward reproduction. This is a plausible explanation, especially for short-lived species such as C. elegans, in which proteostasis decline occurs abruptly in early adulthood, and the lifespan extension gained by activation of stress-response factors comes at the cost of reduced fecundity. However, the mechanisms underlying the gradual deterioration of proteostasis in long-lived mammals are clearly more complex.",J Cell Biol,Proteostasis and Aging,2018
Future Directions for Enhancing Health Span,"Hopefully, a better understanding of the network connectivity in healthy cells and tissues and its changes during aging and disease will allow us to harness aspects of the PN to combat aggregation disorders and increase health span.",J Cell Biol,Proteostasis and Aging,2018
Proteostasis Networks and Homeostasis,"Protein homeostasis, or proteostasis, is assured through the coordinated action of intricate cellular systems—the proteostasis networks. Under normal conditions, these systems rapidly sense and rectify disturbances in the proteome to restore basal homeostasis. During stress, similar systems preserve proteome solubility and functionality by bringing it to an altered point of proteostasis balance that takes into consideration the stress-induced cellular changes. Although the robustness and adaptability of the proteostasis networks is remarkable, if stressors are chronic, the proteostasis balance becomes difficult to maintain, and proteotoxicity develops.",Nature America,Proteostasis and Aging,2015
Age-Related Loss of Proteostasis and Disease,"With age, the ability of many cells and organs to preserve proteostasis under resting and stress conditions is gradually compromised. Loss of proteostasis is part of the pathogenesis of many human pathologies, including neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease. Many of these diseases—known as proteinopathies or protein conformational diseases—are regarded as age-related disorders, given that the physiological deterioration of proteostasis networks with age is an important aggravating factor. Numerous lines of evidence support a tight relationship between proteostasis and healthy aging. Although a gradual loss of proteostasis can be detected in most organisms as they age, the longest-living species have more stable proteomes, and in long-lived species such as the naked mole rat, proteome stability correlates with enhanced activity in the proteostasis systems.",Nature America,Proteostasis and Aging,2015
Proteostasis Modulation and Integrated Networks,"Interventions that modulate the activity of the proteostasis networks in invertebrates and mammals extend lifespan and healthspan. Although a detailed description of each proteostasis system is beyond the scope of this Perspective, the recognized importance of proteostasis loss in aging is emphasized. Vulnerabilities in the proteostasis networks make them susceptible to chronic stress associated with aging. Newly identified dimensions of proteostasis, beyond the cytoplasm, include organelle proteostasis, tissue proteostasis, and even organism-level proteostasis networks that coordinate a proteostatic response throughout the body. This integrated view of proteostasis is crucial for understanding the basis and consequences of proteostasis loss in aging and has become a promising therapeutic target for age-related diseases.",Nature America,Proteostasis and Aging,2015
Core Components of Proteostasis Systems,"Aging of the components of proteostasis systems The main players in proteostasis maintenance are chaperones and two proteolytic systems, the ubiquitin-proteasome and the lysosome-autophagy systems. These components decide the fate of unfolded proteins: whether they will refold into their original stable conformation or whether they will instead be eliminated from the cell through proteolysis. Chaperones assist proteins through de novo folding, assembly and disassembly, membrane transport, and targeting for degradation. The need for chaperones originates from the crowded environment in the cytoplasm and within organelles. A key aspect of chaperone function is the ability to integrate multiple cellular cues to determine whether a misfolded protein will refold or degrade, depending on ATP levels and chaperone availability.",Nature America,Proteostasis and Aging,2015
Proteasomal and Autophagic Protein Degradation,"Once a commitment to degradation is made, chaperones often also decide the proteolytic pathway. HSC70, a constitutive cellular chaperone, can target proteins for degradation. The two major systems used for protein breakdown are the proteasome and autophagy. The proteasome is a multisubunit protease most abundant in the cytosol, though also present in the nucleus, and degrades proteins often tagged with ubiquitin. Proteins can also be degraded in lysosomes by autophagy, which occurs in distinct forms: macroautophagy, microautophagy, and chaperone-mediated autophagy. Chaperones and cochaperones such as CHIP or BAG3 influence whether HSC70 directs proteins to the proteasome or macroautophagy. Once proteins form oligomers or aggregates, they can only be degraded by selective macroautophagy (aggrephagy) or chaperone-assisted selective autophagy.",Nature America,Proteostasis and Aging,2015
Age-Related Changes that Impair Chaperone Function,"Many age-related cellular changes can influence chaperoning activities. Poor cellular energetics—common in old organisms due to reduced mitochondrial function and dysregulated lipid and glucose metabolism—limit ATP availability, reducing ATP-dependent chaperone function and inducing ATP-independent chaperones observed in aging brains. Chronic stressors, such as persistent metastable proteins, act as a ‘sink’ for chaperones, reducing availability for other substrates. Age-related protein modifications, such as the accumulation of advanced glycation end-products on long-lived proteins, interfere with chaperone recognition. Although enzymes like methionine sulfoxide reductase can repair such modifications, these enzymes decrease in abundance with age, further contributing to accumulation of altered proteins unrecognizable to chaperones.",Nature America,Proteostasis and Aging,2015
Adaptive Shifts in Chaperone Pools with Aging,"Cells may compensate for age-related changes by modifying the chaperone pool involved in proteostasis. Although HSP70 and HSP90 families have well-known roles in maintaining proteome balance under normal conditions, recent studies in nematodes highlight an important function of small heat-shock proteins (sHSPs) in aged organisms. These sHSPs trap excess cytosolic proteins into protective aggregates, thereby preserving proteostasis in the context of aging.",Nature America,Proteostasis and Aging,2015
Proteostasis Systems and Their Core Components,"Aging of the components of proteostasis systems The main players in proteostasis maintenance are chaperones and two proteolytic systems, the ubiquitin-proteasome and the lysosome-autophagy systems. These components decide the fate of unfolded proteins: whether they will refold into their original stable conformation or whether they will instead be eliminated from the cell through proteolysis. Chaperones assist proteins through each of the different conformational changes that they undergo during their lifetime, including de novo folding, assembly and disassembly, membrane transport, and targeting for degradation. An important aspect of chaperone functioning is the ability to integrate multiple cellular cues to determine whether a misfolded protein should refold or be degraded, depending on ATP availability and chaperone load.",Nature America,Proteostasis and Aging,2015
Proteasome and Autophagy Pathways in Degradation,"Once a commitment to degradation is made, chaperones often also determine the proteolytic pathway. HSC70 can target proteins for degradation. The proteasome, a multisubunit protease abundant in the cytosol and present in the nucleus, rapidly degrades proteins often tagged with ubiquitin. Proteins can also be degraded in lysosomes through autophagy. Different types of autophagy exist—macroautophagy, microautophagy and chaperone-mediated autophagy—and the choice depends on how proteins are recognized and delivered to lysosomes. Chaperones and co-chaperones such as CHIP and BAG3 influence whether proteins are targeted toward proteasomal degradation or macroautophagy. Single unfolded proteins may use almost any degradation pathway, but once proteins form oligomers or aggregates, they can only be removed by selective macroautophagy (aggrephagy) or chaperone-assisted selective autophagy. Posttranslational modifications such as ubiquitination or acetylation also influence pathway choice.",Nature America,Proteostasis and Aging,2015
Age-Related Impairments in Chaperone Function,"Many age-related cellular changes influence chaperone activity. Poor cellular energetics due to reduced mitochondrial function and dysregulated lipid and glucose metabolism limits ATP availability, repressing ATP-dependent chaperones and inducing ATP-independent ones found in aging brains. Sustained chronic stressors, such as persistent metastable proteins, act as chaperone ‘sinks,’ reducing their availability for other tasks. Age-associated modifications of substrate proteins, such as advanced glycation end-products on long-lived proteins, interfere with chaperone recognition. Although repair enzymes like methionine sulfoxide reductase can reverse some modifications, these enzymes decline with age, contributing to an accumulation of altered, unrecognizable proteins.",Nature America,Proteostasis and Aging,2015
Adaptive Shifts in Chaperone Pools During Aging,"Cells may partially compensate for age-related disruptions by altering the chaperone pool. Although HSP70 and HSP90 families are central under normal conditions, studies in nematodes highlight the important function of small heat-shock proteins (sHSPs) in preserving proteostasis in aging. These sHSPs trap excess cytosolic proteins into protective aggregates, helping to maintain proteostasis despite compromised chaperone systems.",Nature America,Proteostasis and Aging,2015
Age-Related Decline of Autophagy and Proteasome Activity,"Autophagy and proteasome activity. Age-related changes in proteostasis are not restricted to chaperones. Autophagy and proteasome activity both decrease with aging, but they do so to a lesser degree in association with healthy aging and longevity, such as in centenarians and long-lived species like the naked mole rat. Multiple interventions support the idea that diminishing the proteotoxic load during aging can improve lifespan or healthspan. Promoting proteasome or autophagy activity via overexpression of proteasome subunits or essential autophagy genes increases lifespan and confers stress resistance in Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. Early mammalian evidence shows that whole-body overexpression of the essential autophagy gene Atg5 in mice induces anti-aging phenotypes and extends lifespan by approximately 20%.",Nature America,Proteostasis and Aging,2015
Autophagy-Activating Longevity Interventions,"Most interventions that slow aging in experimental models are associated with improved proteostasis and often show autophagy-activating properties. Calorie restriction, rapamycin, metformin, resveratrol, and spermidine—well-known lifespan- or healthspan-extending compounds—have all been shown to activate autophagy, though likely through distinct mechanisms. As some interventions also modulate chaperone levels and protein synthesis, the relative contribution of autophagy activation to overall lifespan benefits remains under investigation. Protein degradation and proteostasis more broadly can also be positively influenced by non-pharmacological inducers. Physical exercise activates chaperones, the proteasome, and various forms of autophagy. Dietary interventions, including starvation and consumption of common components like olive oil, vitamins, and coffee, stimulate protein quality-control systems.",Nature America,Proteostasis and Aging,2015
Protein Repair and Anti-Aging Potential,"Compared with improving protein degradation, the therapeutic potential of enhancing protein repair has been poorly explored. Limited knowledge about repair enzymes and their age-dependent changes restricts progress. A recent study showed lifespan extension in flies that overexpressed methionine sulfoxide reductase, the enzyme that reduces oxidized methionine, supporting exploration of the anti-aging value of manipulating repair enzyme levels.",Nature America,Proteostasis and Aging,2015
Early-Life Proteostasis Decline and Future Directions,"Recent studies suggest that functional decline of proteostasis networks may occur earlier in the lifespan than previously thought. Work in C. elegans challenges the idea that proteostasis failure results from gradual accumulation of cellular anomalies, instead implicating programmed early-life events in later proteostasis collapse. While current evidence is limited to invertebrates, if similar principles apply to mammals, identifying this early window—and whether it is organism-wide or organ-specific—will be crucial for designing interventions to ameliorate age-related loss of proteostasis or prevent proteinopathy progression.",Nature America,Proteostasis and Aging,2015
Cytosolic Proteostasis and Age-Dependent Protein Imbalance,"Intracellular proteostasis: cytosolic and organelle protein quality control Cytosolic proteostasis. Most advances in our understanding of cellular proteostasis and its changes with age arose from the study of the cytosolic proteostasis system. In these studies, the cytosolic proteome has often been considered to be a uniform entity in which most proteins undergo similar changes. But recent work challenges this assumption. Studies in C. elegans tracking over 5,000 proteins revealed that proteome aging is not subtle: instead, a pronounced increase in protein abundance drives proteome imbalance, loss of stoichiometry and protein aggregation. Similar analyses in aging mammals are needed to determine the universality of abundance-driven proteostasis decline.",Nature America,Proteostasis and Aging,2015
Cross Talk Between Proteasome and Autophagy Pathways,"One major advancement in understanding cytosolic proteostasis is recognition that proteolytic systems function in a coordinated manner. The proteasome and autophagy share substrates, effectors and regulators, enabling continuous cross talk and mutual compensation. This compensation is beneficial when one system becomes impaired, such as during disease or aging. For example, proteasome blockage induces upregulation of macroautophagy, and inhibition of one form of autophagy triggers activation of another. Although these pathways are not redundant, under basal conditions their compensatory interactions preserve homeostasis.",Nature America,Proteostasis and Aging,2015
Organ-Dependent Compensation in Proteolytic Systems,"Compensation between proteolytic pathways should be considered when interpreting experimental inhibition results. While most compensation has been documented in cultured cells, in vivo evidence is increasing. Autophagy is activated in the rat hippocampus following proteasome inhibition; mice lacking chaperone-mediated autophagy in the liver upregulate macroautophagy and the proteasome system. However, compensation is organ-dependent: for example, macroautophagy does not compensate for chaperone-mediated autophagy loss in the retina. Aging negatively impacts this cross talk, with aged mice failing to show compensatory upregulation of proteolytic pathways. Failures in IGF1 signaling and TFEB activity have been implicated in the decline of compensatory mechanisms during aging.",Nature America,Proteostasis and Aging,2015
Proteasome Blockage and Stress Resilience,"Interestingly, chronic moderate blockage of some proteasome subunits improves the ability of this system to respond to acute proteotoxic stress. This rebalancing of proteostasis in response to mild inhibition is conserved from yeast to humans, although the mechanisms underlying proteolytic cross talk and their contributions require further study.",Nature America,Proteostasis and Aging,2015
ER Proteostasis and the Unfolded Protein Response,"Organelle proteostasis. Despite the emphasis of a vast part of the literature on cytosolic proteostasis, the importance of protein homeostasis inside organelles and the existence of organelle-specific proteostasis mechanisms are now well accepted. The high volume of proteins handled by the endoplasmic reticulum (ER) creates a need for robust quality-control systems. A three-arm transcriptional program known as the unfolded protein response (UPR)—activated when ER proteostasis is disturbed—and a retrotranslocation system that reroutes unfolded proteins to the cytosol for degradation together ensure ER homeostasis. The UPR is initiated by the ER transmembrane proteins IRE1, PERK and ATF6, which induce expression of genes involved in protein processing and maturation. If the unfolded-protein load exceeds UPR capacity, retrotranslocation is activated. In addition to proteasome-mediated degradation, a selective autophagy process termed ER-phagy also contributes to ER homeostasis, mediated in part by the ER-resident receptor FAM134B.",Nature America,Proteostasis and Aging,2015
"ER Stress, Aging, and Emerging Therapeutic Targets","The tight connections between ER homeostasis and cytosolic proteolytic systems make the ER vulnerable to age-related declines in proteasome and autophagy activity. Sustained ER stress and inappropriate stress responses are common features of aging and chronic disorders. Accordingly, many small molecules under development for neurodegeneration and metabolic diseases target UPR components. Remarkably, UPR activation can also be triggered by lipid disturbances alone, suggesting dietary modulation as a possible therapeutic strategy. Although global autophagy activation has been observed in most pharmacologic interventions, selective enhancement of ER-phagy is desirable. Overexpression of FAM134B causes ER fragmentation and lysosomal degradation, highlighting its potential as an ER-phagy activator. Additional ER proteostasis mechanisms such as RESET, which exports misfolded proteins to the cell surface for lysosomal degradation, remain insufficiently explored in the context of aging.",Nature America,Proteostasis and Aging,2015
"Mitochondrial Proteostasis, Mitophagy, and the mtUPR","Remarkable progress has also been made in understanding mitochondrial proteostasis. Maintaining mitochondrial proteostasis is challenging due to the high burden of reactive oxygen species (ROS). Severely damaged mitochondrial regions are segregated and eliminated via mitophagy. Multiple types of mitophagy and alternative lysosome-driven mitochondrial clearance pathways highlight the complexity of mitochondrial quality control. Malfunctioning mitophagy is reported in aging and age-related disorders, especially neurodegeneration. Mitochondria contain their own chaperones and proteases, and the mitochondrial unfolded protein response (mtUPR) is activated when unfolded proteins accumulate. The mtUPR triggers nuclear expression of mitochondrial protein genes and has been linked to lifespan extension in multiple organisms. Components of the mtUPR, including SIRT3 and SIRT7, are also associated with age-related disease risk; SIRT7 expression declines in aged hematopoietic stem cells, and its restoration improves regenerative capacity.",Nature America,Proteostasis and Aging,2015
Nuclear Proteostasis and Stress-Induced Nuclear Import,"Quality control in the nuclear compartment is less understood. Although the nuclear envelope was once thought to shield nuclear proteins from cytosolic aggregation, it is now clear that chaperones, ubiquitin ligases and proteasomes in the nucleoplasm contribute to nuclear proteostasis. Some protein-quality components reside permanently in the nucleus, whereas others are transported from the cytosol or ER during proteotoxic stress. These shuttled factors co-localize with nuclear aggregates of proteins such as mutant huntingtin, ataxin-1 and TDP43. This suggests the existence of nuclear proteotoxicity sensors that induce chaperone entry. Certain ubiquitin ligases, including UHRF2 and PML4, recognize mutant nuclear proteins. Although proteasomes degrade damaged histones and surplus ribosomal proteins, it is unclear whether this occurs within the nucleus or after cytosolic export. The extent of nuclear proteostasis decline with age, the vulnerability of specific nuclear protein subsets and potential spillover of altered nuclear proteins into the cytosol remain open questions.",Nature America,Proteostasis and Aging,2015
Intercompartmental Communication and Whole-Organelle Turnover,"Little is known about the principles governing communication between cytoplasmic and organelle proteostasis networks or how they change with age. A recent example of intercompartmental regulation involves the mitochondrial protein SSBP1, which translocates to the nucleus upon heat shock to modulate HSF1-dependent expression of cytosolic, nuclear and mitochondrial chaperones. Whether certain compartments are prioritized for proteostasis maintenance or whether poorly managed proteotoxicity is redistributed among compartments remains unclear. Some organelles, such as the Golgi, endosomes and lysosomes, may undergo full-organelle turnover through lysophagy rather than selective degradation of resident proteins. However, these processes depend on autophagic effectors, making them susceptible to the age-related autophagy decline observed in multiple tissues.",Nature America,Proteostasis and Aging,2015
Intercellular Proteostasis and Non–Cell-Autonomous Stress Signaling,"Intercellular proteostasis: integrating organ and tissue responses Most studies have analyzed age-related proteostasis decline as a cell-autonomous problem, but growing evidence supports the existence of regulatory, intercellular proteostasis networks that coordinate tissue- and organ-wide responses to proteotoxic insults. The effectors of intercellular proteostasis—chaperones and proteolytic systems—mirror those of intracellular proteostasis, but the key question is how stress signals are transmitted between cells. Some communication channels appear to use existing intercellular systems. For example, the coupling of intercellular gap junctions to autophagy activation has been demonstrated by showing that connexins, the components of gap junctions, also function as endogenous inhibitors of autophagy.",Nature America,Proteostasis and Aging,2015
Non–Cell-Autonomous Maintenance of Proteostasis,"Intercellular proteostasis is not limited to transmitting stress signals; components of the proteostasis network, and even toxic proteins, can be transferred between cells to preserve whole-organ proteostasis. In Drosophila, increasing chaperones such as Hsp70 and Hsp40 in one cell group improved proteostasis in other cells of the same tissue, demonstrating non–cell-autonomous regulation. Exosomes play a significant role in this communication. Derived from invaginated endosomal membranes, they release cytosolic material into the extracellular space. Exosomes can transfer chaperones, and their abundance increases under proteotoxic conditions. Adding chaperone-containing exosomes to cultured cells reduces inclusion body formation, showing efficient intercellular chaperone transfer.",Nature America,Proteostasis and Aging,2015
"Exosomes, Aging, and Disease Propagation","The decline of multivesicular endosomal proteostasis with age could reduce exosome transfer efficiency. Studies examining neuronally derived exosomes from individuals with Alzheimer’s disease—before onset and years after diagnosis—showed higher levels of ubiquitinated and lysosomal proteins but reduced HSC70 compared with healthy controls. These findings support exosomal proteins as biomarkers of disease spreading and emphasize the need to understand exosome biology in aged tissues. Another intercellular transfer mechanism involves tunneling nanotubes, which allow direct exchange of materials over distances greater than 100 μm. Transferred cargo includes miRNAs, lysosomes, mitochondria, endosomes and lipid droplets. Nanotube-mediated transfer between neural stem cells and endothelial cells provides neuroprotection, suggesting that lysosome transfer could sustain autophagy in stressed cells without requiring new lysosome biogenesis.",Nature America,Proteostasis and Aging,2015
Prion-Like Propagation of Protein Aggregates,"Pathogenic proteins can also propagate between cells in a prion-like manner, serving as seeds for aggregate formation in healthy cells. Aggregation-prone proteins implicated in neurodegenerative diseases—α-synuclein, tau, β-amyloid and superoxide dismutase 1—have been detected in exosomes. Consequently, exosomes are promising biomarkers for diagnosing and tracking disease progression. It remains unclear whether exosomal propagation is a primary disease mechanism or a result of impaired intracellular proteostasis. This prion-like spreading is increasingly accepted as a mechanism underlying Alzheimer’s disease, tauopathies, Parkinson’s disease, Creutzfeldt–Jakob disease and amyotrophic lateral sclerosis. Nanotubes may also contribute to aggregate propagation, as shown in prion transfer between neuronal cells and from dendritic cells to neurons.",Nature America,Proteostasis and Aging,2015
Regional Proteostasis Differences and Organ Weakness,"Intercellular transfer of chaperones, proteases and protein aggregates may result from heterogeneity in proteostasis robustness across organs. Regional differences in proteasome and autophagy-lysosomal activities correlate with differences in proteostasis capacity and disease susceptibility. For example, chaperone-mediated autophagy activity is lower in aggregation-prone brain regions, even in healthy animals. Intercellular proteostasis may initially sustain weaker regions by exporting aggregates to stronger regions while importing proteostasis effectors. Advances in fluorescent and radiometric proteostasis probes and single-cell tracking technologies are expected to help identify regions of proteostasis weakness and their age-related changes.",Nature America,Proteostasis and Aging,2015
Open Questions in Age-Related Decline of Intercellular Proteostasis,"Many questions remain regarding how aging alters intercellular proteostasis. Do transfer mechanisms, their kinetics or the amount of transferred chaperones and proteases change with age? Do aging cells increasingly offload protein aggregates externally due to overload of their intrinsic proteostasis machinery? Could exosome-based transfer replenish proteostasis capacity in aged tissues? Understanding these mechanisms is essential for determining how tissues respond to proteotoxic insults during aging and how intercellular proteostasis might be harnessed therapeutically.",Nature America,Proteostasis and Aging,2015
Tele-Proteostasis and Systemic Coordination of Proteostasis Networks,"Tele-proteostasis: integrating distant proteostasis networks The realization that proteostasis networks in different organs coordinate with each other and that changes in one organ affect others has emerged as one of the most exciting developments in the field. This 'tele-proteostasis'—proteostasis coordinated from a distance through cell-autonomous and non-autonomous mechanisms—enables systemic responses. For example, activating the heat-shock protein response in neurons alone is sufficient to induce a systemic heat-shock response. Early studies in worms expressing misfolded proteins showed that induction of HSP90 occurred not only in affected cells but also in distant cells, demonstrating a distal proteostasis response. Expression of HSP90 solely in neurons or intestinal cells resolved aggregation problems in muscle cells, supporting non-cell-autonomous modulation of proteostasis.",Nature America,Proteostasis and Aging,2015
UPR-Mediated Systemic Proteostasis and Longevity,"Tele-proteostasis applies to both cytosolic and organelle proteostasis. Its robustness is an important determinant of longevity. Worms with genetically impaired systemic UPR exhibit shortened lifespan, but restoring the missing UPR component solely in neurons and intestinal cells extends lifespan. Neuronal restoration activates the UPR in distal intestinal cells, enhancing ER stress resistance in aged worms. Evidence of similar processes in mammals is emerging: constitutive expression of a UPR component in hypothalamic neurons activates the same component in the liver, demonstrating coupling between neuronal ER stress and hepatocyte proteostasis. This coupling not only stabilizes the liver proteome but also modulates hepatic insulin sensitivity and glucose production, linking tele-proteostasis to whole-body metabolism.",Nature America,Proteostasis and Aging,2015
Systemic Mediators of Tele-Proteostasis,"Following the discovery of systemic activation of chaperone responses and the UPR, research has focused on identifying circulating mediators. UNC-13, a factor involved in neurotransmitter release, is required for distant UPR activation in worms. UNC-13 mutants show reduced longevity, highlighting its physiological relevance. Secretion of the ER chaperone ERDJ3 also contributes to extracellular proteostasis, though its role in distant tissue proteostasis remains unclear. The search for hormone-like systemic proteostasis regulators coincides with renewed interest in circulating anti-aging factors. Early parabiosis studies showed that serum from young mice rejuvenates aged stem cells, a phenomenon recently expanded to include protection against neurodegeneration. Whether these circulating factors exert their beneficial effects through proteostasis regulation remains an open and intriguing possibility.",Nature America,Proteostasis and Aging,2015
"Circulating Factors, Caloric Restriction, and Interorgan Proteostasis","Caloric restriction—which improves proteostasis—has provided additional clues about circulating mediators. Serum from caloric-restricted animals delays senescence and boosts stress resistance of cultured cells, suggesting that systemic factors regulate proteostasis across organs. Open questions include whether circulating mediators of tele-proteostasis are universal or context-specific, whether inflammatory molecules produced during chronic age-related inflammation influence proteostasis coordination, and how metabolic changes in aging affect interorgan proteostasis communication. Understanding these factors is essential for determining whether manipulating proteostasis in one organ can therapeutically benefit distant organs—a possibility with profound implications for treating neurodegenerative diseases through peripheral interventions.",Nature America,Proteostasis and Aging,2015
Overview of Cellular Senescence,"Cellular senescence: the good, the bad and the unknown
Weijun Huang 1,2, LaTonya J. Hickson3, Alfonso Eirin 1, James L. Kirkland 4 and Lilach O. Lerman 1 ✉
Abstract | Cellular senescence is a ubiquitous process with roles in tissue remodelling, including wound repair and embryogenesis. However, prolonged senescence can be maladaptive, leading to cancer development and age-related diseases. Cellular senescence involves cell-cycle arrest and the release of inflammatory cytokines with autocrine, paracrine and endocrine activities. Senescent cells also exhibit morphological alterations, including flattened cell bodies, vacuolization and granularity in the cytoplasm and abnormal organelles. Several biomarkers of cellular senescence have been identified, including SA-βgal, p16 and p21; however, few markers have high sensitivity and specificity. In addition to driving ageing, senescence of immune and parenchymal cells contributes to the development of a variety of diseases and metabolic disorders. In the kidney, senescence might have beneficial roles during development and recovery from injury, but can also contribute to the progression of acute kidney injury and chronic kidney disease. Therapies that target senescence, including senolytic and senomorphic drugs, stem cell therapies and other interventions, have been shown to extend lifespan and reduce tissue injury in various animal models. Early clinical trials confirm that senotherapeutic approaches could be beneficial in human disease. However, larger clinical trials are needed to translate these approaches to patient care.",Nature Reviews Nephrology,Cellular Senescence,2022
Causes and Pathways of Cellular Senescence,"The phenomenon of cellular senescence was discovered in the 1960s in human diploid cell strains that had exhausted their replicative potential. Senescence is characterized by cell-cycle arrest in the G1 or possibly G2 phase, which prevents the proliferation of damaged cells. By contrast, cellular quiescence, a reversible growth arrest state secondary to scarce nutrition and growth factors, takes place in the G0 phase. Cellular senescence occurs during embryonic development and can be induced by cellular impairment, including DNA damage, telomere shortening or dysfunction, oncogene activation or loss of tumour suppressor functions, epigenetic changes and organelle damage.
The principal cause of senescent stress is DNA damage, which activates the DNA damage response (DDR) and the canonical p53–p21 pathway. P21 (also known as cyclin-dependent kinase inhibitor 1) inhibits cyclin–cyclin-dependent kinase complexes that block the formation of the DREAM complex, which represses cell-cycle genes by binding their homology region. Unlike DDR-induced senescence, epigenetic alterations cause senescence mainly via the p16–RB pathway. p16 (also known as cyclin-dependent kinase inhibitor 2A) can inhibit the formation of cyclin D–CDK4/6 complexes and thereby prevent phosphorylation of RB and promote formation of the RB–E2F complex, which inhibits the transcription of cell-cycle genes. Evidence suggests that p21 is mainly activated early during the evolution of senescence, whereas p16 maintains cellular senescence.",Nature Reviews Nephrology,Cellular Senescence,2022
Senescence-Associated Secretory Phenotype and Biological Roles,"The senescence-associated secretory phenotype (SASP) is an important feature of senescent cells that comprises the release of numerous cytokines, chemokines, growth factors and proteases, which are sometimes enclosed within microparticles, into the extracellular environment. Many cell types also release extracellular vesicles, which contain cellular contents, including proteins, lipids and nucleic acids. Extracellular vesicles have a role in inter-cellular communication, and altered extracellular vesicle cargoes are important components of the SASP. Through the release of SASP factors, senescence can modulate pathways in neighbouring cells and tissues as well as at remote sites. Notably, senescent cells that are induced by different stress stimuli may manifest distinctive SASP components.
Cellular senescence has beneficial biological functions in the regulation of embryonic development, wound healing, resolution of fibrosis and tumour suppression. However, prolonged senescence can result in deleterious sequelae, including tumour development, chronic inflammation, immune deficit and stem cell exhaustion. Interest in cellular senescence and in senescence-modulating interventions is increasing owing to observations that in addition to driving ageing, cellular senescence has important roles in the pathogenesis of chronic diseases, including osteoporosis, metabolic syndrome, type 2 diabetes mellitus, cancer, reproductive ageing, atherosclerosis, neurodegeneration, glaucoma and chronic kidney disease (CKD). In this Review, we describe the mechanisms, hallmarks and consequences of cellular senescence, as well as the therapeutic potential of senescence-targeting interventions.",Nature Reviews Nephrology,Cellular Senescence,2022
Types and Triggers of Senescence,"Senescence in physiology and pathology
Diverse types of stimuli trigger senescence, reflecting its spectrum of roles under different conditions. Developmental senescence and replicative senescence (which occurs secondary to telomere shortening) occur under physiological conditions during embryogenesis and ageing, respectively, whereas other types of senescence are often induced by pathological stressors, including tumorigenesis, diabetes mellitus, chemotherapy or radiation.",Nature Reviews Nephrology,Cellular Senescence,2022
Senescence in Embryogenesis and Development,"Embryogenesis and development. In 2006, a transcript of INK4b, which encodes a cyclin-dependent kinase (CDK) inhibitor that blocks progression of the cell cycle beyond the G1 phase, was detected in the roof plate of the developing chicken hindbrain, implicating senescence in the regulation of embryonic development. Subsequently, p66Shc, which regulates oxidative stress-induced senescence, was found to mediate early cleavage arrest in failed bovine embryonic development, suggesting that cellular senescence might fine-tune embryogenesis to prevent the continued development of poor-quality embryos. In the mammalian embryo, senescence occurs at multiple locations, including the limbs, nervous system and gut endoderm. In the developing kidney, accumulation of senescent cells signals immune cells to facilitate mesonephros regression through macrophage-mediated phagocytosis of these senescent cells. Markers of senescence, including senescence-associated β-galactosidase (SABG), p21, p27 (encoded by CDKN1B) and p15 (encoded by CDKN2B), were detected in mouse mesonephros at embryonic day 12.5 to 14.5. Knocking down p21 in mouse embryos results in developmental abnormalities. Senescence has also been shown to regulate the development of multiple tissues in zebrafish embryos.",Nature Reviews Nephrology,Cellular Senescence,2022
Senescence in Endometrium and Embryo Implantation,"Importantly, senescent decidual cells in the mammalian endometrium can secrete multiple canonical implantation factors and form a suitable environment for embryonic implantation. Cellular senescence may therefore have roles in pruning and remodelling developing systems and modulating their microenvironment, and is thus required to ensure fetal integrity.",Nature Reviews Nephrology,Cellular Senescence,2022
Overview of Immunosenescence,"Immunosenescence. In healthy individuals, immunosenescence begins at around the age of 50 years and results in impaired vaccine responses and increased susceptibility to infection, autoimmunity and cancer, as well as chronic inflammation. A reduction in autophagy is an important mechanism that contributes to immunosenescence. Impairment of autophagy hinders the degradation of damaged mitochondria, resulting in the accumulation of ROS and DNA damage. Patients with CKD show accelerated immunosenescence that mimics that of elderly individuals. The resulting chronic inflammation is associated with loss of kidney function and complications such as cardiovascular disease and infections. However, immunosenescence can be beneficial in settings where immune cell activation is undesirable. For example, immunosenescence of CD4+ T cells in transplant recipients facilitates acceptance of kidney allografts.",Nature Reviews Nephrology,Immunosenescence,2022
Age-Related Changes in T Cells,"In people aged >60 years, naive T cells decline, whereas memory T cells increase and show loss of CD28 and elevated expression of the senescence markers CD57 and KLRG1. Although age-related changes are more common in CD8+ T cells, an increased frequency of highly differentiated CD4+ T cells that express NKG2D has been reported in elderly people. Senescent T cells express pro-inflammatory cytokines, exhibit shortened telomeres and might negatively impact human longevity. The offspring of centenarians, who presumably carry longevity-favouring genes, have fewer senescent T cells than age-matched controls. Increased expression of T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain is associated with T cell exhaustion and senescence.",Nature Reviews Nephrology,Immunosenescence,2022
Senescent B Cells and Immune Dysfunction,"Senescent B cells are also prevalent in the elderly. Memory B cells fill the immunological space, whereas naive B cells markedly decrease, resulting in a reduced ability to respond to new pathogens. Senescent B cells also have a decreased capacity for somatic hypermutation and thus show blunted antibody responses to infectious agents. The decrease in naive B cells is caused mainly by a decline of B cell lymphopoiesis, secondary to increased apoptosis of B cell precursors, impaired responsiveness to IL-7 and decreased potency of haematopoietic stem cells. Moreover, senescent B cells, characterized by reduced expression of CD23, CD21 and CD35, accumulate in the bone marrow and suppress B cell lymphopoiesis in aged mice.",Nature Reviews Nephrology,Immunosenescence,2022
"NK Cells, Macrophages, and Senescence-Like States","Although the number of natural killer (NK) cells does not necessarily decline with ageing, their function deteriorates owing to the altered expression of various cytokines, which reduces their ability to recognize infected and malignant target cells. Senescent macrophages express high levels of the senescence-related markers p16INK4a and SABG. They release pro-inflammatory cytokines that promote chronic inflammation and have an overabundance of ROS, which reduces their propensity for efferocytosis. Expression of activating transcription factor 3 and bromodomain-containing protein 4 may contribute to macrophage senescence. Intriguingly, mature macrophages share inherent phenotypic similarities with senescent cells in terms of their signalling pathways, gene expression, metabolism and levels of organelles such as lysosomes. The suitability of p16INK4a and SABG as markers for macrophage senescence has been questioned because these markers are also upregulated in response to stimuli that induce macrophage polarization to a M2 phenotype, such as IL-4 and IL-13. Furthermore, activated macrophages in atherosclerotic lesions resemble senescent cells and show lipid accumulation, SASP and a persistent DDR. Hence, a senescence-like phenotype in macrophages might constitute a physiological activation state adopted in response to challenge rather than true senescence. Notably, cancer cells with chemotherapy-induced senescence undergo transcriptional changes linked to phagocytosis and can engulf adjacent cells. Clearly, the interplay between truly senescent cells and senescent-like macrophages warrants additional studies.",Nature Reviews Nephrology,Immunosenescence,2022
Overview of Stem Cell Senescence,"Stem cell senescence. Stem cells can release paracrine factors to repair injured tissues and organs and have an important role in regeneration. However, risk factors such as ageing, irradiation, and metabolic disorders can induce stem cell senescence (SCS) and dysfunction, which contributes to age-related diseases. For example, senescent p16INK4a-positive MSCs contribute to osteoarthritis by secreting DKK1, which triggers premature bone remodelling and cartilage degradation. Senescent MSCs also lose their cartilage regenerative properties. The mechanisms that underlie SCS are complex. Stressors including ROS can promote MSC senescence induced by ageing, expansion and hypoxia, and transforming growth factor β1 (TGFβ1) can induce SCS by increasing ROS production and upregulating p16INK4A and p21.",Nature Reviews Nephrology,Stem Cell Senescence,2022
Non-coding RNAs and Molecular Regulators of SCS,"In addition, non-coding RNAs regulate SCS. For example, miR-152 induces human dental pulp SCS, whereas the long non-coding RNA Hnscr attenuates hypothalamic SCS. Haematopoietic SCS is regulated by the endogenous Ewing sarcoma gene. Low mitochondrial NAD+ levels contribute to bone-marrow MSC senescence, which may be associated with reduced expression of sirtuin 1 and sirtuin 3. Notably, ageing is linked to compensatory overexpression of sirtuin 6 in bone marrow MSCs, which decreases SCS and confers oxidative stress resistance. Several transcription factors also regulate SCS of different origins. NANOG delays senescence in hair follicle-derived MSCs, GATA6 and SOX11 regulate bone-marrow SCS, MDM2–p53 signalling has a vital role in epidermal SCS and the ROR2–STK4–FOXO1–SMS1 pathway regulates dental pulp SCS. Endometrial SCS, which is associated with recurrent pregnancy loss, may be mediated by SERPINB2–sonic hedgehog signalling.",Nature Reviews Nephrology,Stem Cell Senescence,2022
"Autophagy, Metabolism, and Context-Dependent SCS","The role of autophagy in hypoxia and d-galactose-induced SCS is controversial. Some studies have shown that autophagy inhibits MSC senescence; however, high glucose-induced autophagy increases bone-marrow SCS. Thus, the role of autophagy in SCS likely depends on the context. We found that fat-tissue-derived MSC senescence was elevated in patients with obesity but not in patients with diabetic kidney disease compared with age-matched healthy individuals. Hence, SCS is not necessarily a uniform finding in human disease.",Nature Reviews Nephrology,Stem Cell Senescence,2022
Redox Imbalance and Induction of Senescence,"Redox control of senescence. Oxidant stress caused by overproduction of ROS and/or deficiency of antioxidant defences can induce cellular senescence. Many factors can interrupt redox homeostasis and hence induce DNA damage, including high glucose, radiation and expression of oncogenes. For example, autophagy-deficient melanocytes and keratinocytes with an overabundance of ROS become senescent, and lactose and fumarate can induce fibroblast and renal cancer cell senescence by inducing oxidative stress. Downregulation of cathepsin-D not only increases ROS generation, but also inhibits the activity of nuclear factor erythroid 2-related factor 2 (NRF2), which has an important role in antioxidant defence and prevention of senescence. The integral membrane protein caveolin 1 inhibits the activity of the antioxidant enzyme thioredoxin reductase 1, inducing ROS accumulation and cellular senescence.",Nature Reviews Nephrology,Redox Control of Senescence,2022
"Feedback Loops, Antioxidant Pathways, and ROS Effects","In turn, oxidative stress has a positive feedback effect on caveolin 1 by activation of p38MAPK signalling, forming a vicious cycle. Loss of the anti-apoptotic protein BCL-2 also disrupts redox homeostasis and impairs angiogenic function in senescent endothelial cells. Interestingly, H2 produced by gut bacteria can scavenge the hydroxyl radical and relieve oxidative stress, resulting in suppression of cellular senescence. Many other factors also modulate the senescence process by regulating cellular redox status. In addition to directly inducing DDR-mediated senescence signalling, ROS can regulate Akt signalling, an important pathway to cellular senescence, by modulating the expression of several microRNAs including miR-181a and miR-182. Redox homeostasis is also key to maintaining the integrity of telomeres. Ultimately, telomere shortening and loss hinder DNA copying and lead to replicative cellular senescence.",Nature Reviews Nephrology,Redox Control of Senescence,2022
"ROS Impact on Nuclear Structure, SASP, and Fibrosis","Elevated oxidative stress can also induce senescence accompanied by nuclear shape alteration (and thereby DDR) through overexpression of lamin B1 and can modulate the SASP by regulating the homeostasis of the SASP initiators, IL-1α and Ca2+. In patients with systemic sclerosis, oxidative stress induces fibroblast senescence and contributes to pro-fibrotic and pro-inflammatory phenotypes. By contrast, senescent fibroblasts exhibit antifibrotic phenotypes during normal wound healing. This discrepancy might be the result of the intrinsic characteristics of dermal fibroblasts in patients with systemic sclerosis, which have high intracellular ROS levels, reduced proliferation rate, impaired bioenergetic functions and increased DNA damage and DDR compared with healthy fibroblasts. Conceivably, differing features such as ROS levels may determine the propensity of fibroblast senescence to be linked to a pro-fibrogenic or anti-fibrogenic phenotype.",Nature Reviews Nephrology,Redox Control of Senescence,2022
Antioxidants and Future Directions,"Clearly, increased oxidative stress has a key role in the development of cellular senescence. The ability of exogenous antioxidant strategies to attenuate senescence in selected cells without impairing fibroblast function remains to be explored.",Nature Reviews Nephrology,Redox Control of Senescence,2022
Accumulation and Impact of Senescent Cells,"Functional alterations. Senescent cells have abnormal function and are unable to proliferate owing to their stable cell-cycle arrest in the G1/G2 phase. Senescence thereby reduces the number of cells that are available to execute normal tissue functions. An increased rate of senescent cell production (due to increased exposure to stressors) and a reduced rate of senescent cell removal (due to deregulated immunosurveillance together with pro-survival signals and resistance to apoptosis) lead to the accumulation of these cells in organs with ageing. In turn, senescent cells promote degeneration, ageing, systemic chronic inflammation — termed inflammaging — and age-related diseases due to the release of SASP factors and the resulting spread of senescence to previously non-senescent cells. Congruently, senescent cells accumulate at the sites of age-related degenerative or preneoplastic pathology. In addition to the SASP, the complement system contributes to inflammaging by recruiting macrophages and increasing the release of ROS, TNF and IL-6, which are important inducers of senescence.",Nature Reviews Nephrology,Functional Alterations in Senescence,2022
Beneficial and Deleterious Effects of SASP,"SASP factors can exert both beneficial and deleterious functions. Beneficial effects of SASP factors include the roles of CXCL5 in embryonic development, PDGF-AA in tissue repair, IL-6 in cellular reprogramming, MMP1 and MMP33 in fibrosis resolution and the anti-tumorigenic properties of CCL2. Notably, SASP factors also recruit immune cells to clear pre-malignant senescent cells. This process of senescence surveillance is an important contributor to the anti-tumorigenic properties of senescent cells. Deleterious effects of SASP factors include the roles of IL-8, IL-10 and TNF in inflammation, PDGF-BB in fibrogenesis and MMP1 and MMP3 in tumorigenesis. Some SASP cytokines such as IL-6, IL-8 and CCL2 also promote vascular smooth muscle cell calcification and impair insulin sensitivity.",Nature Reviews Nephrology,Functional Alterations in Senescence,2022
Regulation of SASP and External Triggers,"The SASP is regulated via complex DDR-dependent and DDR-independent mechanisms. Activated NF-κB and the transcription factor CCAAT/enhancer-binding protein-β (C/EBPβ) co-activate promoters of SASP genes. However, NF-κB-independent SASP regulation mechanisms also exist. For example, cytoplasmic DNA accumulation can cause aberrant activation of cGAS-STING cytoplasmic DNA sensors and promote SASP by induction of interferon-β. Downregulation of the lamin B receptor, which regulates heterochromatin organization, is necessary for the generation of cytoplasmic DNA. Factors that are linked to infections, such as lipopolysaccharide and SARS-CoV-2 S antigen, can increase the release of pro-inflammatory, pro-fibrotic and pro-apoptotic SASP factors by senescent cells.",Nature Reviews Nephrology,Functional Alterations in Senescence,2022
Overview of Hallmarks and Limitations of SABG,"Hallmarks and markers of senescence. SABG activity is a widely used marker of senescence in tissues and cell cultures. The activity of SABG can be detected at pH 6 (the intra-lysosomal pH of senescent cells), whereas the activity of acidic β-galactosidase can be detected at pH 4 (the intralysosomal pH of non-senescent cells). However, SABG has some limitations as a marker of senescence. For example, many apoptotic non-senescent cells in developing limbs and healthy neurons during early embryonic development are SABG positive and cell density might influence SABG staining regardless of cell proliferation status. These findings suggest that SABG is not a sufficiently robust and specific marker of senescent cells.",Nature Reviews Nephrology,Senescence Markers,2022
Canonical and Emerging Molecular Markers,"Gene and protein components of senescence-related signalling pathways such as p16 and p21 are canonical markers of senescence. Expression of Met is also considered to be a marker of cellular senescence and the accumulation of nuclear globular actin accumulation has been reported to be a more sensitive marker of cellular senescence than SABG activity. Other markers of senescence include telomere shortening, the DNA double-strand-break marker H2AX, typical chromatin changes such as senescence-associated heterochromatin foci, cytosolic double-stranded DNA and miR-146a. Decreased cellular proliferation potential (which can be measured using BrdU or EdU incorporation assays) and increased apoptosis resistance (for example, as a result of upregulation of BCL proteins) also occur in some types of senescent cells.",Nature Reviews Nephrology,Senescence Markers,2022
Blood and Urine Biomarkers; Extracellular Vesicles,"Potential blood or urine biomarkers of senescence include plasma angiopoietin-like 2, growth/differentiation factor 15, stanniocalcin 1 and serine protease inhibitors, serum T-kininogen and urinary 8-oxoguanosine. Activin A is normally only expressed during embryonic development, but is copiously expressed in injured and senescent kidneys and can be detected in the plasma and urine of patients with kidney disease. SASP signatures enable the detection and characterization of cellular senescence in vivo but need to be defined in specific biological contexts as they might overlap with inflammatory profiles that are not associated with senescence. Extracellular vesicles can be isolated from urine or peripheral blood and analysed as an index of parent cell senescence; for example, increased levels of urinary URAT1+p16+ extracellular vesicles reflect increased proximal tubular cell senescence.",Nature Reviews Nephrology,Senescence Markers,2022
Tissue-Specific Markers and Imaging Challenges,"Several markers characterize specific senescent tissues or cells. For example, loss of EPC1 expression is a marker of senescence in human dermal fibroblasts. In T cells, expression of NKG2D, KLRG1, CD57 and CD28 might reflect senescence. NKG2D and activin A are potential markers of senescence in kidney tissue. Spectrin–phaemoglobin crosslinking is an important feature of senescence in red blood cells, SRSF1 has been postulated to be a key marker of endothelial senescence and maspin is a marker of senescence in oral premalignant lesions. Endogenous autofluorescence of MSCs is considered to be a useful marker to rapidly determine their senescent status in vitro. However, the small number of senescent cells relative to quiescent cells, robust tissue autofluorescence, and low penetration of fluorescence signals hamper the detection of senescent cells in vivo using fluorescence-based imaging.",Nature Reviews Nephrology,Senescence Markers,2022
Advanced Imaging and Future Directions,Near-infrared imaging methods are not clinically applicable because of the limited availability of high-performance second near-infrared region (NIR-II) fluorophores with high brightness and biocompatibility as well as the long-term health risks of using non-biodegradable quantum dots and lanthanide-doped nanoparticles. Positron emission tomography-based methods of detecting senescent cells are being investigated in animal studies but will require the development of sensitive clinical probes. Advances in non-invasive imaging methods may enable the detection and spatial allocation of senescence-prone regions that warrant intervention.,Nature Reviews Nephrology,Senescence Markers,2022
Senescence in Kidney Diseases,"Senescence in kidney diseases. Kidney cell senescence was first described in 1992. In addition to its role in physiological kidney ageing, senescence has important roles in the development of CKD and acute kidney injury (AKI). Interventions that clear senescent cells, including senolytic drugs, are therefore promising novel treatments for kidney diseases.",Nature Reviews Nephrology,Kidney Senescence,2022
Senescence in Acute Kidney Injury (AKI),"Acute kidney injury. Emerging evidence indicates that senescence contributes to the progression of AKI and that senescence inhibition can promote kidney recovery. For example, inhibition of tubular cell senescence using lipoxin A4 restored renal function in a rat model of septic shock-induced AKI. Similarly, in a rat model of contrast-induced AKI, pre-treatment with paricalcitol before contrast medium administration reduced cellular senescence (that is, expression of SABG and p16INK4A) and tissue damage and prevented kidney dysfunction. Notably, patients aged over 70 years have a 3.5-fold higher incidence of AKI than younger individuals. This increased incidence has been linked to immunosenescence, amplification of the SASP, and/or downregulation of the geroprotective protein α-Klotho. Urine and plasma levels of p21 correlate with renal cortical expression of this protein and could be a useful non-invasive biomarker of AKI and kidney ageing.",Nature Reviews Nephrology,Kidney Senescence,2022
Molecular Drivers of Senescence in AKI,"The complement system (C5a), DNA methylation, Wnt4–β-catenin signalling and ROS have been implicated in the development of cellular senescence in AKI. Senescence has been shown to reduce the regenerative capacity of tubular, glomerular and interstitial cells and to delay recovery from AKI induced by ischaemia–reperfusion injury (IRI) in mice. Following IRI, markers of kidney cell senescence (Bax and p16 mRNA) peaked at day 12, suggesting an increase in senescent cells in the chronic phase after AKI that might contribute to maladaptive repair and progression to CKD. Treatment with nicotinamide mononucleotide reduced tubular cell DNA damage and senescence and attenuated renal fibrosis in mouse models of ischaemic AKI.",Nature Reviews Nephrology,Kidney Senescence,2022
Protective and Context-Dependent Roles of Senescence in AKI,"Increasing evidence suggests that cellular senescence might also have beneficial effects in AKI. A small-molecule inhibitor of CDK4 and CDK6 induced proximal tubule cell-cycle arrest and ameliorated kidney injury in a mouse IRI model. Moreover, we found that inhibition of senescence within the first week after induction of renal ischaemia in a mouse model impeded functional recovery (measured using renal perfusion and plasma cystatin-c levels), suggesting a protective role early in the process of AKI. Delineating the time course of kidney injury and the role of cellular senescence during each phase might identify a therapeutic time window to target senescence and interrupt the development of AKI.",Nature Reviews Nephrology,Kidney Senescence,2022
Overview of CKD-Associated Senescence,"Chronic kidney disease. CKD is increasingly recognized to mimic age-related diseases and senescence and the SASP are important drivers of CKD progression. CKD can accelerate the senescence of immune, endothelial and vascular smooth muscle cells via a process known as uraemia-associated ageing, potentially constituting a feed-forward mechanism of cellular damage. Immunosenescence in CKD manifests as an increased proportion of terminally differentiated T cells, telomere shortening of mononuclear cells, low thymic output and reduced immune-mediated clearance of senescent kidney cells, which promotes CKD progression.",Nature Reviews Nephrology,CKD Senescence,2022
Molecular Drivers of Senescence in CKD,"The mechanisms that underlie CKD-induced senescence include hyperphosphataemia, a common complication of CKD that elicits senescence in myoblasts, endothelial cells and aorta smooth muscle cells and contributes to sarcopenia and vascular calcification. Furthermore, uraemic toxins have been implicated in the senescence of proximal tubular cells. Tubular epithelial cell senescence can be induced by inhibition of AMPK–mTOR signalling, activation of the Wnt–β-catenin pathway or overexpression of Wnt9a, and promotes epithelial to mesenchymal transition and consequent fibrosis. α-Klotho, an endogenous antagonist of Wnt–β-catenin signalling, was downregulated in unilateral ureteral obstruction, adriamycin nephropathy, and IRI models of fibrotic kidney disease. Senolytic combination therapy with dasatinib and quercetin alleviated kidney fibrosis in mouse models of chronic renal ischaemia and abrogated the progression of AKI to CKD in mouse models of IRI and cisplatin-induced injury.",Nature Reviews Nephrology,CKD Senescence,2022
"High Glucose, ROS, and Pathway Activation","Podocyte senescence induces glomerulosclerosis in the ageing kidney via AMPK–mTOR signalling, whereas DKD or overfeeding can induce renal cellular senescence by decreasing sirtuin 1 expression. In patients with DKD, the circulating senescence marker activin A is elevated and p16 is upregulated in tubular epithelial cells. In mice with streptozotocin-induced diabetes, glomerular endothelial senescence is driven by M1 macrophages and largely dependent on intracellular ROS. High blood glucose also induces mesangial cell senescence by activating RAGE–STAT5 signalling, which inhibits autophagy and therefore leads to accumulation of injured mitochondria and ROS, which are important inducers of senescence.",Nature Reviews Nephrology,CKD Senescence,2022
Senescence Across Kidney Cell Types and Diseases,"We detected cellular senescence (upregulation of p16, p19 and p21) in the kidneys of mice, pigs and patients with renal artery stenosis and observed that ischaemic renovascular disease induces senescence of renal scattered tubular-like cells, which impairs their reparative capacity. Senescence of renal tubular epithelial cells promotes progression of immunoglobulin A (IgA) nephropathy and the presence of p16INK4a-positive cells in kidney biopsy samples from patients with lupus nephritis is associated with renal injury and a worse prognosis. Elevated gene expression of the senescence markers Tp16, Tp19 and Tp53 was also observed in mice with obesity-induced kidney injury and p16 expression was strikingly increased in biopsy samples from patients with glomerular disease.",Nature Reviews Nephrology,CKD Senescence,2022
"Triggers, Allograft Senescence, and Future Directions","Exposure of kidney cells to nephrotoxic factors including radiation, TNF, hypoxia or glucose oxidase can induce cell-cycle arrest in vitro. Replicative senescence is also involved in the development of chronic allograft nephropathy. The complement factor H-related genes CFHR1 and CFHR3 have been implicated in tubular cell senescence in allografts in transplant recipients with IgA nephropathy. Extensive basic and clinical studies are required to elucidate the complex mechanisms of cellular senescence in CKD. Although these mechanisms are shared across many forms of kidney disease, the extent and unique features of cellular senescence likely vary with the severity and underlying aetiology of CKD.",Nature Reviews Nephrology,CKD Senescence,2022
Overview of Senotherapeutic Interventions,"Interventions targeting senescence. As cellular senescence has key roles in many age-related diseases, interventions targeting senescence, known as senotherapeutics, are potential therapies for these diseases. The promise of such approaches is underscored by the observation that genetic animal models of senescent cell deficiency show improved recovery from kidney injury and extended lifespan. Existing senotherapeutic approaches include drugs that selectively eliminate senescent cells, known as senolytics, drugs that inhibit the SASP, known as senomorphics, exogenous cell-based products and non-pharmacological therapies.",Nature Reviews Nephrology,Senolytics and Senotherapeutics,2022
Senolytic Drugs: Flavonoids and Natural Compounds,"Senolytic interventions. Senolytic drugs were developed to overcome the characteristic resistance to apoptosis of senescent cells by inducing pre-programmed cell death. Quercetin, a natural flavonoid found in some fruit and vegetables, has been shown to eliminate senescent vascular smooth muscle and endothelial cells in animal models by inducing apoptosis through activation of AMPK, sirtuin 1–PINK1-mediated mitophagy and NRF2–NF-κB signalling. We found that quercetin blunted the expression of senescence markers in the kidneys of obese mice and had beneficial, senescence-independent effects on cardiac function in mice fed a high-fat diet owing to pro-angiogenic activity. The plant flavonoid fisetin and procyanidin C1, a flavonoid found in grape seeds, are also senolytics. Herbal extracts can also have senolytic activity; for example, ginsenoside, an extract of ginseng, prevents senescence of bone marrow MSCs by activating NRF2 and PI3K–Akt signalling and can modulate the SASP, reduce inflammation, balance redox status and attenuate organ ageing.",Nature Reviews Nephrology,Senolytics and Senotherapeutics,2022
Pharmacological Senolytics: Targeted Apoptosis Inducers,"AP20187, an FK1012 analogue, selectively induced apoptosis of p16Ink4a-expressing senescent cells in various mouse models, resulting in improvements in age-related brain inflammation, cognitive function and stenotic kidney function. Navitoclax (ABT-263) is an inhibitor of anti-apoptotic BCL-2 family proteins that induces apoptosis and exerts potent senolytic effects in ageing animal models and in some types of senescent cells in vitro. Other BCL-2 family inhibitors, including A-1331852, A-1155463, EF24 and venetoclax, are also effective senolytics. In addition, heat shock protein 90 inhibitors have senolytic activity and radio-electric asymmetric conveyer technology was shown to be effective in reducing the senescence of cultured stem cells. Notably, these interventions do not selectively target senescent cells, and thus can be associated with off-target adverse effects such as abnormal embryo development, dysregulated wound healing and tumorigenesis.",Nature Reviews Nephrology,Senolytics and Senotherapeutics,2022
Targeted Delivery and Immune-Based Senolytics,"Organ-targeted or cell-targeted approaches using protein-based or peptide-based carriers, nanoparticles, extracellular vesicles or other vehicles could increase the specificity, decrease the off-target effects and facilitate the clinical translation of senolytic interventions. Current strategies to selectively target senescent cells include conjugating toxic drugs to antibodies against the senescent membrane marker β2-microglobulin, which is upregulated via a p53-dependent mechanism and is a marker of stress-induced senescence. Another strategy involves activation of invariant NK T cells to improve immunosurveillance and removal of senescent cells. Moreover, chimeric antigen receptor T cells engineered to specifically recognize the urokinase-type plasminogen activator receptor on the surface of senescent cells had senolytic effects in vitro and in vivo. Finally, anti-ageing vaccines have been developed to target CD153+ senescent T cells or GPNMB+ senescent endothelial cells with promising results in obese mouse models.",Nature Reviews Nephrology,Senolytics and Senotherapeutics,2022
Overview of Senomorphic Drugs,"Senomorphic drugs. One of the best-studied senomorphic drugs is metformin, which reduces the incidence of age-related diseases and expands the lifespan of Caenorhabditis elegans, mice and patients with type 2 diabetes mellitus. Metformin also enhances the anticancer efficacy of CDK4 and CDK6 inhibitors by modulating the SASP, inhibits endothelial senescence caused by high glucose-induced metabolic memory by regulating the sirtuin 1–p300–p53–p21 pathway, triggers immune-mediated clearance of senescent cells and restores tumour immunosurveillance.",Nature Reviews Nephrology,Senomorphic Drugs,2022
JAK and mTOR Inhibitors; Autophagy-Inducing Senomorphics,"Other senomorphic drugs include ruxolitinib, a JAK inhibitor that reduces inflammation and alleviates frailty in aged mice by suppressing inflammatory SASP factors. mTOR inhibitors, such as rapamycin, inhibit senescence and suppress the SASP in endothelial cells and fibroblasts by inducing autophagy, thereby reducing the accumulation of damaged organelles. Activation of mTOR leads to peroxisome proliferator-activated receptor-γ coactivator 1β-dependent mitochondrial biogenesis, ROS production and persistent activation of the DDR. Thus, inhibition of mTOR ameliorates cellular senescence.",Nature Reviews Nephrology,Senomorphic Drugs,2022
Herbal Extracts and Epigenetic Senomorphics,"Several herb extracts, such as resveratrol, also show anti-SASP activity. Furthermore, the small molecule ML324 that inhibits KDM4, which is involved in the epigenetic regulation of SASP genes, was shown to decrease the SASP in senescent tumour stromal cells. However, this approach needs to be used cautiously to avoid adverse effects given the sometimes incongruent behaviours of tumour and stromal microenvironments and possibly other cellular niches.",Nature Reviews Nephrology,Senomorphic Drugs,2022
Hormonal Senomorphics and Cautionary Notes,"Some hormones also have senomorphic effects. For example, melatonin suppresses SASP gene expression by interrupting the recruitment of CREB-binding protein (CBP) by poly-(ADP-ribose) polymerase 1 (PARP1), which is a sensor of DNA damage. Melatonin improves senescent T cell activity, alleviated cardiac mitochondrial dysfunction in a mouse model of accelerated senescence, and rescued MSCs from uraemic toxin-induced senescence in CKD. Other hormones, including androgens, oestrogens, oestradiol and glucocorticoids, can also modulate the release of inflammatory cytokines. However, glucocorticoids need to be used with caution as they can induce senescence in primary human tenocytes.",Nature Reviews Nephrology,Senomorphic Drugs,2022
Stem Cells and Extracellular Vesicles as Anti-Senescence Therapies,"Stem cells and extracellular vesicles. In addition to many other salutary effects, stem cells and their extracellular vesicles can exert senolytic activity. For example, bone-marrow-derived MSCs decreased senescence and improved cardiac function in aged mice, pluripotent stem cells prevented stress-induced senescence in cardiomyocyte-derived cells and human umbilical cord-derived MSCs protected rat kidneys from AKI-induced senescence. We observed relatively modest senolytic efficacy of adipose-derived MSCs in post-stenotic mouse and human kidneys. In contrast to these senolytic effects, human umbilical cord-derived MSCs increased splenic CD4+ T cell senescence and alleviated symptoms of lupus in mice. These differing findings suggest that the effects of MSCs are likely cell type and context dependent.",Nature Reviews Nephrology,Stem Cells and EVs,2022
Extracellular Vesicles: Mechanisms and Therapeutic Potential,"Stem cell extracellular vesicles have robust anti-senescence activity and might be less prone to rejection or tumour formation than their parent stem cells. MSC-derived extracellular vesicles inhibited oxidative stress-induced senescence in endothelial cells, promoted wound closure in ageing diabetic mice and decreased myocardial senescence, possibly by improving the systemic inflammatory profile, in a pig model of metabolic renovascular disease. Dental pulp stem cell-derived extracellular vesicles prevented irradiation-induced senescence in submandibular cells by reducing inflammation and oxidative stress and extracellular vesicles from antler progenitor cells alleviated MSC senescence in aged mice. The anti-senescence functions of stem cells and extracellular vesicles have been mainly attributed to their contents, which can regulate the SASP, repair damaged organelles and rescue the function of senescent cells.",Nature Reviews Nephrology,Stem Cells and EVs,2022
Lifestyle Interventions and Senescence,"Lifestyle interventions. Several lifestyle factors might accelerate senescence. For example, sleep deprivation activates the DDR and promotes the SASP in humans, whereas a healthy lifestyle, such as habitual exercise and caloric restriction, retards ageing. Animal and human studies have shown that lifelong exercise or habitual moderate exercise in aged individuals have beneficial effects on immunosenescence and age-related diseases by modulating mitochondrial function, inflammation, the SASP and lipolysis. Overfeeding accelerates senescence in mice, whereas caloric restriction alleviates senescence in dogs and in murine white adipose tissue. These findings might constitute a key mechanism by which caloric restriction extends lifespan and retards age-related chronic diseases.",Nature Reviews Nephrology,Lifestyle and Senescence,2022
"Diet, Microbiome, and Environmental Stressors","Dietary interventions can also influence age-related health through epigenetic alterations and modification of the gut microbiota. For example, nutrients such as betaine, choline and folate promote epigenetic changes that blunt age-related changes and CKD by targeting methylome or chromatin, whereas excessive intake of sugar promotes progression of age-related diseases by decreasing microbial diversity in animal models. Cumulative lifetime exposure to external stressors, such as temperature, oxygen levels and inadequate nutrition, elicit adaptive homeostatic mechanisms, including antioxidant and anti-inflammatory responses, by activating the NRF2–KEAP1 signalling pathway. Modifying these exposures might offer new strategies for improving the health span and combatting CKD and could potentially have beneficial effects on cellular senescence.",Nature Reviews Nephrology,Lifestyle and Senescence,2022
Clinical Trials of Senotherapeutics,"Clinical trials. Clinical studies of senolytic therapies are relatively scarce compared with the numerous animal and in vitro studies, but have shown some success. For example, fractional micro-needling radiofrequency treatment effectively removed senescent keratinocytes and improved hyperpigmentation of aged skin and treatment with dasatinib and quercetin reduced circulating SASP factors and the abundance of senescent cells in adipose tissue and skin in patients with diabetic kidney disease. Raltegravir decreased senescent T lymphocytes in treatment-suppressed people with HIV. A psychosocial intervention with horticultural therapy over a 6-month period alleviated immunosenescence and chronic inflammation in older adults. Hence, non-invasive and cost-effective strategies for improving well-being should be vigorously pursued.",Nature Reviews Nephrology,Senotherapeutics Clinical,2022
"Risks, Timing, and Future Considerations in Senotherapy","Importantly, senotherapeutic approaches can have adverse effects. Some senolytic drugs, such as navitoclax, can induce neutropenia, trabecular bone loss and osteoprogenitor dysfunction. Moreover, as SASP factors have many physiological roles, the potential adverse effects of senomorphic drugs might outweigh their benefits and reduce the success of clinical trials. Furthermore, genes may dictate traits that provide survival benefits in one stage of life but become maladaptive in another phase, a phenomenon known as antagonistic pleiotropy. The effect of senolytic drugs on tumorigenesis might therefore be age-dependent, and given the role of cellular senescence in development, the effects of senolytic drugs might differ between early and later stages of life. Notably, we found that premature delivery reduced the efficacy of senolytic drugs in a renal artery stenosis model, consistent with temporal rather than trait-dependent antagonistic pleiotropy.",Nature Reviews Nephrology,Senotherapeutics Clinical,2022
"Dosing Strategies, Combination Therapies, and Screening","Given that chronic conditions continually generate new senescent cells, the frequency of senotherapy also requires further exploration. A mouse study suggested that intermittent administration of senolytics could be particularly effective at alleviating physical dysfunction and increasing survival. Combination approaches that use senolytics and anti-SASP interventions to target a broad range of cell types might be particularly powerful. Finally, the success of clinical trials of senotherapeutics could potentially be maximized by screening participants prior to enrolment to enable selection of those showing evidence of cellular senescence. However, sensitive screening tools need to be developed and validated to enable selection of those participants who are most likely to benefit from the intervention.",Nature Reviews Nephrology,Senotherapeutics Clinical,2022
Introduction to Cellular Senescence,"Mechanisms and functions of cellular senescence Introduction. Cellular senescence was originally identified as a stable exit from the cell cycle caused by the finite proliferative capacity of cultured human fibroblasts. Currently, senescence is considered a stress response that can be induced by a wide range of intrinsic and extrinsic insults, including oncogenic activation, oxidative and genotoxic stress, mitochondrial dysfunction, irradiation, or chemotherapeutic agents. While the defining characteristic of senescence is the establishment of a stable growth arrest that limits the replication of damaged and old cells, many other phenotypic alterations associated with the senescent program are relevant to understanding the pathophysiological functions of senescent cells. For example, senescent cells undergo morphology changes, chromatin remodeling, and metabolic reprogramming, and secrete a complex mix of mostly proinflammatory factors termed the senescence-associated secretory phenotype (SASP).",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
Physiological Roles of Senescence,"Physiological roles of senescence. Cellular senescence was initially dismissed as a tissue culture artifact. However, a wealth of data has demonstrated that senescent cells can influence disease and aging, as well as normal tissue homeostasis. Indeed, senescence can be engaged during development and is also necessary for tissue remodeling. For instance, transient induction of senescent cells is observed during wound healing and contributes to wound resolution. Senescence can also be a protective stress response. In fact, senescence is best known as a potent anticancer mechanism that prevents malignancies by limiting the replication of preneoplastic cells.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
Senescence in Aging and Disease,"However, the accumulation of senescent cells also drives aging and age-related diseases. The connection between senescence and aging was initially grounded on observations of the accumulation of senescent cells in aged tissues. It was suggested that, during aging, senescence of stem and progenitor cells could hinder tissue homeostasis by interfering with the capacity of tissues to repair and regenerate. In the last 10 years, our understanding of senescence’s detrimental consequences in aging and age-related pathologies has significantly expanded. Two lines of research have facilitated this awareness. First, the use of transgenic models that allow for the detection of senescent cells has enabled a systematic identification of these cells in many age-related pathologies. Second, the development of genetic and drug strategies to selectively eliminate senescent cells, spearheaded by the van Deursen laboratory, has demonstrated that senescent cells can indeed play a causal role in aging and related pathologies.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
"Senolytics, Challenges, and Future Directions","The confirmation that selectively killing senescent cells significantly improves the health span of mice in the context of normal aging and ameliorates the consequences of age-related disease or cancer therapy has ignited interest in the identification of compounds that can clear senescent cells. These so-called senolytic therapies, however, still face important caveats. In addition to their potential side effects, the evaluation of senolytic compounds is compromised by limitations such as the lack of universal senescence biomarkers and the heterogeneity of senescent phenotypes in vivo. Ongoing research into the pathways that initiate and maintain senescence will provide insights to identify biomarkers and potential therapies to target senescent cells.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
Senescence as a Dynamic Multistep Program,"Senescence as a dynamic program. Senescence has been traditionally considered as a defined, static cell fate. However, it is now recognized that senescence is a dynamic multistep process. A simplified model suggests that although the initial senescence-inducing signals are sufficient to initiate cell cycle exit, this merely constitutes an early step in the senescence process. Senescent cells progressively remodel their chromatin and start to sequentially implement other aspects of the senescence program, such as the SASP, to enter into a second step of “full senescence.” If these senescent cells persist for extended periods of time, they continue evolving and can be categorized as entering into a third step of “late senescence,” which can involve adaptation and diversification of the senescent phenotype. Acute senescence is triggered in response to discrete stressors and contributes to tissue homeostasis, whereas chronic senescence results from long-term unscheduled damage and is associated with detrimental processes such as aging.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
SASP Heterogeneity and Immune Clearance,"The senescent secretome is a heterogeneous mix of proteins whose composition depends not only on the stage of senescence progression but also on the affected cell type and the nature of the inducing stressor(s). The efficiency and kinetics of the clearance of senescent cells may vary depending on the organ in which they accumulate or the general ability to mount an effective immune response. For example, while cells undergoing oncogene-induced senescence (OIS) in the liver are efficiently cleared by the immune system, senescent cells in melanocytic nevi often manage to evade immune clearance and persist. Overall, therapeutic approaches aiming to modulate senescent phenotypes will benefit from a better understanding of the steps driving and defining the evolution of senescent cells in vitro.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
Cell Cycle Arrest in Senescence,"Cell cycle arrest. One of the defining features of senescent cells is their stable cell cycle arrest. This cell cycle exit is controlled by activation of the p53/p21CIP1 and p16INK4a/Rb tumor suppressor pathways. Unlike quiescent cells, senescent cells are nonresponsive to mitogenic or growth factor stimuli; thus, they are unable to reenter the cell cycle even in advantageous growth conditions. Senescent cells are also distinct from terminally differentiated cells, which are also irreversibly withdrawn from the cell cycle. While terminal differentiation is the result of a defined developmental program, senescence is mainly implemented as a cellular stress response. However, terminally differentiated cells such as neurons, adipocytes, and hepatocytes can also undergo senescence, or at least show senescence-like features, during aging or in response to oncogenic activation or DNA damage. This indicates that the onset of senescence can occur independently of an active cell cycle arrest.",Journal of Clinical Investigation,Cell Cycle Arrest,2018
DNA Damage Response and Senescence Initiation,"DNA damage response. The senescence growth arrest is often triggered by a persistent DNA damage response (DDR) caused by either intrinsic (oxidative damage, telomere attrition, hyperproliferation) or external insults (ultraviolet, γ-irradiation, chemotherapeutic drugs). During replicative senescence of human fibroblasts, progressive telomere shortening ultimately exposes an uncapped, double-stranded chromosome free end, which is sensed as a double-strand break by the DDR machinery. In cells undergoing OIS, the initial hyperproliferative phase that follows oncogene activation induces this DDR. An increase in usage of DNA replication origins leads to accumulation of genomic damage and activation of a DDR because of stalled replication forks. Senescence is associated with a persistent DDR that results in irreparable DNA damage.",Journal of Clinical Investigation,DNA Damage Response,2018
Limitations of DDR Markers for Senescence Identification,"The DDR associated with replicative senescence is telomere-dependent: it correlates with telomere uncapping and overall loss of telomeric length. During OIS, DDR occurs independently of telomeric length, but it is still associated with telomeric dysfunction. Regardless of which mechanism drives the damage, the DDR is characterized by increased deposition of γ-H2Ax and 53BP1 in the chromatin as well as activation of a kinase cascade involving ATM, ATR, CHK1, and CHK2, which eventually leads to activation of the p53/p21CIP1 axis. Despite DDR’s role in initiating senescence, DDR markers have limited utility for identifying senescence in vivo. Activation of p53 and/or p21CIP1 during senescence can occur in a DDR-independent manner. Moreover, the majority of cells expressing markers of DNA damage in vivo, especially in a nonpathological setting, are not senescent but are responding to transient reparable damage.",Journal of Clinical Investigation,DNA Damage Response,2018
Non–Cell-Autonomous Effects and SASP Overview,"Non–cell-autonomous effects of senescence. Cellular senescence was initially considered to be a cell-intrinsic program. Increasing evidence, however, has shown that senescent cells have the ability to signal and influence their surrounding environment. Senescent cells produce a complex mixture of soluble and insoluble factors that are collectively termed senescence-associated secretory phenotype (SASP) or senescence-messaging secretome. SASP is the general term given to the combination of cytokines, chemokines, extracellular matrix proteases, growth factors, and other signaling molecules secreted by senescent cells. Importantly, its specific composition varies depending on the cell type and the senescence inducer. Likewise, the functions attributed to the SASP depend not only on the nature of the SASP but on the surrounding environment and the genetic context of the cells being exposed to the senescent secretome. Senescent cells can also signal and influence adjacent cells through juxtacrine NOTCH/JAG1 signaling, ROS production, or by cargo transfer via cytoplasmic bridges or exosomes.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
Beneficial and Detrimental Functions of the SASP,"Functions of the SASP. The SASP can have beneficial or detrimental effects. The SASP reinforces the senescence growth arrest in vitro by implementing an autocrine positive-feedback loop. Knockdown of IL-6R, IGFBP7, or CXCR2 prevents senescence. This autocrine loop contributes to the tumor-suppressive function of senescence. Interestingly, the SASP can also induce nonmalignant proliferating neighbor cells to undergo senescence (paracrine senescence). Conversely, the senescent secretome can also promote tumorigenesis. Early work showed that the SASP of senescent fibroblasts can promote proliferation and metastatic features in premalignant epithelial cells or increase tumor vascularization in xenograft transplants. More recently, the SASP of senescent hepatic stellate cells promotes the proliferation and malignancy of surrounding hepatocytes in obese mice treated with carcinogens. The SASP also mediates harmful effects of senescent cells that accumulate upon chemotherapy treatment in vivo, and elimination of senescent cells prevents tumor relapse.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
"SASP, Immunity, and Aging","The interplay between the SASP and the immune response is complex. SASP-dependent recruitment of Th1 cells, NK cells, and macrophages is essential to clear incipient preneoplastic cells and prevent progression of hepatocellular carcinoma. On the other hand, the SASP can have immunosuppressive properties. For instance, when premalignant senescent hepatocytes coexist with liver cancer cells, SASP-dependent recruitment of immature myeloid cells may promote HCC progression by impairing NK cell function. The SASP has also been strongly linked to aging and age-related diseases. Low-level chronic inflammation (“inflammaging”) underlies many age-related pathologies. Elimination of senescent cells reduces levels of proinflammatory cytokines such as IL-6, IL-1α, and TNF-α in aged mice. Although only a small percentage of cells in aged tissues are senescent, removing them greatly improves health span, suggesting SASP suppression may underlie many benefits of senolysis.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
"SASP in Repair, Regeneration, and Fibrosis","Physiological SASP functions also exist. Senescent fibroblasts induced during cutaneous wound repair accelerate wound closure through secretion of factors such as CCN1 and PDGF-AA, reinforcing the idea that acute, nonpersistent senescence can be beneficial. Senescence also limits fibrotic responses. For example, senescence acts as a break in the progression of liver fibrosis induced by acute liver damage: proliferating hepatic stellate cells eventually enter senescence, and the SASP contributes to fibrotic scar degradation, clearance of senescent cells, and restoration of homeostasis. Factors secreted by senescent cells in response to tissue damage can also promote stemness. However, SASP components have limited utility as biomarkers because they are not specific to senescence. Only reliable costaining or single-cell profiling can pinpoint SASP drivers in vivo.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
Regulation of the SASP,"Regulation of the SASP. The SASP is regulated at multiple levels, including transcription, translation, mRNA stability, and secretion. SASP activation depends on autocrine and paracrine positive-feedback loops that enhance robust signal amplification. Multiple signaling pathways, including the DDR, p38 MAP kinase, and cGAS–STING, regulate the SASP. Most converge on NF-κB and C/EBPβ, which are activated and enriched in the chromatin fraction in senescent cells and regulate SASP components such as IL-8 and IL-6. IL-6 and IL-8 act in an autocrine loop to enhance C/EBPβ and NF-κB activity. IL-1α is a master regulator of the SASP. Inhibition of the NLRP3 inflammasome can blunt the SASP. Epigenetic regulation includes MLL1-mediated γ-H2Ax at SASP genes, macroH2A1 involvement in positive-feedback loops, and BRD4 recruitment to senescence-activated super-enhancers adjacent to SASP genes.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
"mTOR, Autophagy, and Alternative SASP Programs","The mTOR pathway is an important node in SASP regulation. mTOR-mediated phosphorylation of 4EBP regulates translation of IL-1α and MAPKAPK2, indirectly controlling SASP mRNA stability by inhibiting ZFP36L1, an mRNA-binding protein that targets proinflammatory SASP components. Cells undergoing OIS boost SASP production by coordinating protein synthesis and autophagy in the TOR–autophagy spatial coupling compartment (TASCC). However, SASP regulation by autophagy is complex, as GATA4 controls the SASP by inhibiting autophagy. Distinct SASP subsets exist, such as mitochondrial dysfunction–associated SASP, and NOTCH signaling can enable switches between TGF-β and inflammatory secretomes.",Journal of Clinical Investigation,Cellular Senescence Mechanisms,2018
SA β-Galactosidase and Lysosomal Markers,"SA β-galactosidase activity. The most widely used senescence marker is SA β-gal activity. This enzymatic activity, which is found in many normal cells under physiological conditions (pH 4.0–4.5), is significantly amplified in senescent cells due to increased lysosomal content. Histochemical detection of β-gal activity at pH 6.0 allows specific identification of senescent cells. Since SA β-gal is detected in most senescent settings, it is considered a hallmark of senescence. However, cells deficient in GLB1 do not exhibit impairments in senescence. α-Fucosidase has been proposed as an alternative biomarker. Increased lysosomal content also occurs in active macrophages, Kupffer cells, and osteoclasts, highlighting the need for additional markers. Lipofuscin colocalizes with SA β-gal in senescent cells and can be detected in paraffin-embedded tissues.",Journal of Clinical Investigation,Senescence Biomarkers,2018
Chromatin Reorganization and SAHF Formation,"Chromatin reorganization. Senescence is associated with large-scale chromatin rearrangements. Beyond DDR and PML body formation, the most striking chromatin change is the formation of senescence-associated heterochromatic foci (SAHFs), prominent in human cells undergoing OIS. These foci can be identified by DAPI staining and are enriched for repressive marks such as H3K9me3 and HP1, accumulation of HMGA proteins, and loss of histone H1. SAHFs represent spatial repositioning of preexisting repressive marks. Upon senescence induction and loss of lamin B1, lamina-associated domains detach and cluster within nuclei. Hi-C analysis suggests decompaction of SAHF-enriched DNA, challenging their classification as purely silencing compartments. LMNB1 degradation compromises nuclear envelope integrity, leading to cytoplasmic chromatin fragments that activate cGAS–STING to initiate SASP and inflammation. However, chromatin changes vary across cell types and inducers, limiting their utility as universal senescence markers.",Journal of Clinical Investigation,Senescence Biomarkers,2018
Metabolic Reprogramming and SASP Energy Demand,"Metabolism, autophagy, and mitochondrial function in senescent cells. Senescent cells display metabolic changes such as increases in glycolysis, mitochondrial metabolism, and autophagy. A clear illustration of this came when Dörr and colleagues showed that the high production of components of the SASP relies on enhanced ATP production mediated by mitochondrial metabolism (i.e., the TCA cycle) and glycolysis. The authors suggested that the increased SASP production and secretion lead to a proteotoxic stress that can be attenuated by activation of autophagy. Previous evidence highlighting the role of autophagy in the sustainability of the SASP had reported that the amino acid supply required to maintain the rapid protein turnover that the SASP demands relies on coupling autophagy (autolysosomes) with protein synthesis.",Journal of Clinical Investigation,Cellular Senescence Metabolism,2018
Controversies in Autophagy and Mitophagy During Senescence,"The role of mitochondrial metabolism and autophagy in senescence, however, remains controversial. Some studies have shown that inhibition of autophagy facilitates senescence. Also, senescent cells display decreased levels of mitophagy (mitochondrial autophagy), which results in a defective mitochondrial network that may contribute to metabolic dysfunction in aging. In agreement with this, Garcia-Prat et al. showed that mitophagy contributes to the maintenance of muscle function during aging by preventing senescence in satellite cells. Defective mitophagy leads to accumulation of dysfunctional mitochondria and ROS-induced senescence. Similar results were observed in a cell model of parkin-mediated mitophagy.",Journal of Clinical Investigation,Cellular Senescence Metabolism,2018
"Mitochondrial Status, ROS, and Senescence Execution","Interestingly, depletion of mitochondria impaired senescence by disrupting a positive-feedback loop involving ROS production and a DDR rather than as a result of insufficient energy levels. Senescent cells lacking mitochondria displayed higher ATP levels due to increased glycolysis. Therefore, it seems that at least in some contexts, the execution of the senescence program is compromised not by insufficient energy levels, but rather by the status of mitochondrial oxidative metabolism. In work related to this concept, Kaplon and colleagues argued that senescent cells must reprogram metabolism in order to support their metabolic demands. They showed that an increase of mitochondrial oxidative respiration through activation of pyruvate dehydrogenase was required for the execution of OIS.",Journal of Clinical Investigation,Cellular Senescence Metabolism,2018
Morphological Alterations in Senescent Cells,"Morphological changes associated with senescence. In cell culture, senescence is normally accompanied by significant morphological changes. Senescent cells become flat, enlarged, and vacuolized, and sometimes appear with multiple or enlarged nuclei. Changes in shape rely on the status of the scaffolding protein caveolin 1 and the Rho GTPases Rac1 and CDC42, and vacuolation has been associated with ER stress caused by the unfolded protein response. Senescent cells also form cytoplasmic bridges that allow them to signal to neighboring cells via direct intercellular protein transfer. Beyond these examples, the functional significance of most morphological changes associated with senescence is unclear. In vivo, senescent cells appear to preserve the morphology dictated by the architecture of the tissue. However, recent studies have discovered that SA β-gal+ cells in aged mice increase in size.",Journal of Clinical Investigation,Senescence Morphology,2018
Resistance to Apoptosis in Senescence,"Resistance to apoptosis. Senescence and apoptosis are alternative cell fates that often can be triggered by the same stressors. While we do not have a full understanding of what makes the cell decide between one and another program, mechanisms must be in place to lock those decisions. In this regard, senescent cells are resistant to extrinsic and intrinsic apoptosis. Recent studies have suggested that this is a result of the upregulation of BCL-2 family proteins such as BCL-W and BCL-XL. This is of extraordinary practical relevance since inhibiting BCL-2 family proteins induces apoptosis on senescent cells.",Journal of Clinical Investigation,Senescence Apoptosis Resistance,2018
Overview of Cellular Senescence,"1. Introduction. Cellular senescence is generally an irreversible proliferative arrest in damaged normal cells that have exited the cell cycle. These cells display high metabolic activities, remain viable, and actively suppress apoptosis. Senescent cells present unique morphological and molecular characteristics and functions that distinguish them from other nondividing cell populations, such as quiescent cells and terminally differentiated cells. The hallmarks of cellular senescence include: prolonged cell cycle arrest, transcriptional changes, acquisition of a bioactive secretome known as the senescence-associated secretory phenotype (SASP), macromolecular damage, and deregulated metabolism.",International Journal of Molecular Sciences,Cellular Senescence Introduction,2021
Triggers and Types of Senescence,"Replicative senescence was the first cellular senescence subtype to be described. It is induced after serial propagation of normal human cells in culture and is caused by telomere erosion and the consequent increase in DNA lesions. The limited lifespan of most cultured primary cells is influenced by the species and tissue type from which they were derived. Senescence can also be triggered by many other intrinsic and extrinsic factors, particularly replicative stress, oxidative damage, metabolism dysfunctions, cytokines, oncogene activation, and chemotherapy agents. All these factors can induce DNA damage and senescence in normal and cancer cells in some contexts. Cellular senescence occurs not only in vitro but also in various tissues in vivo.",International Journal of Molecular Sciences,Cellular Senescence Introduction,2021
Senescence in Cancer and Aging,"Senescence is an important contributor to cancer and aging, two processes characterized by a time-dependent accumulation of cell damage and dysfunction. Senescence markers are detected in premalignant tumor lesions but not at later stages of tumor development. The proliferative arrest imposed by cellular senescence represents an early barrier against cancer initiation by preventing the propagation of damaged DNA to the next generation of cells. Therefore, it has been proposed that senescence escape is required for tumor progression to overt malignancy. On the other hand, senescent fibroblasts can influence their local environment by turning into proinflammatory cells that can promote the growth of transformed or preneoplastic neighboring epithelial cells in culture and in vivo.",International Journal of Molecular Sciences,Cellular Senescence Introduction,2021
"Senescence in Aging, Inflammation, and Regeneration","During aging, senescent cell accumulation in various tissues promotes chronic inflammation that accelerates age-related dysfunctions. Moreover, stem cell senescence caused by telomere shortening can negatively affect tissue homeostasis and regeneration. Importantly, elimination of senescent cells can promote stem cell proliferation and delay the appearance of aging features. However, senescent cells are not effectively removed in aging tissues and this might reflect the age-related decline in immune functions. In addition, senescent cells have been observed in many different physiological contexts and at all stages of life. Unlike chronic senescence, the acute induction of cellular senescence represents a transient physiological response during embryonic development and adult tissue homeostasis, such as facilitating tissue repair after liver damage, in skin fibrosis, and during wound healing.",International Journal of Molecular Sciences,Cellular Senescence Introduction,2021
Heterogeneity and Biological Roles of Senescent Cells,"Senescent cells are heterogeneous and perform various biological functions. Therefore, entry into senescence and the long-term cell cycle exit are regulated through different changes in gene expression, metabolism, and cell organization. Here, we describe some examples of this diversity of senescent cell types, focusing on mammalian systems, and highlight the contribution of cellular senescence to aging and cancer development. We also discuss the functions, regulation, and features of this complex and fascinating cell state.",International Journal of Molecular Sciences,Cellular Senescence Introduction,2021
Control of Cell Cycle Arrest in Senescence,"Senescence is defined by a stable exit from the cell cycle. Although senescence can be triggered by diverse stresses, inhibition of the cell cycle machinery is the central and defining event that establishes and maintains all senescence phenotypes. Senescent cells usually arrest in the G1 phase to prevent DNA replication in damaged cells, but they may also arrest in G2 to prevent mitosis in the presence of DNA damage. Because this arrest prevents propagation of mutations, it must be tightly controlled by key cell-cycle regulators that respond to senescence-inducing signals and exhibit stimulus-specific expression patterns.",International Journal of Molecular Sciences,Cell Cycle Arrest in Senescence,2021
G1 Phase Arrest Mechanisms,"Cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and the retinoblastoma protein (RB) regulate the G1/S transition. Cyclin–CDK complexes promote S-phase entry by phosphorylating RB. Senescence signaling typically inhibits these complexes by decreasing cyclin–CDK activity or increasing CDK inhibitors such as p21, p15, and p16. High levels of p21 or p16 are sufficient to induce cell cycle arrest. p21 rises rapidly upon telomere damage via p53 activation, enforcing arrest by inhibiting cyclin E–CDK2 and cyclin A–CDK2, keeping RB in its active hypophosphorylated state. p16 accumulates more slowly but reinforces irreversible RB-mediated arrest. The loss or reduction of CDK inhibitors can lead to senescence escape and promote abnormal cell cycle reentry.",International Journal of Molecular Sciences,Cell Cycle Arrest Mechanisms,2021
Roles of p21 and p16 in Senescence Initiation,"p21 is crucial for the early, reversible arrest triggered by telomere shortening, allowing time for DNA repair. Loss of p21 permits cells to bypass senescence and continue dividing. Fully senescent cells with low p16 expression can re-enter the cell cycle if p53 is inactivated, indicating early p21-dependent arrest is reversible. Persistent telomere damage gradually increases p16 levels, driving cells toward irreversible, full senescence. Although p21 is key in early arrest, p16 becomes the dominant factor maintaining long-term irreversible growth arrest.",International Journal of Molecular Sciences,p21 and p16 in Senescence,2021
Oncogene-Induced Senescence (OIS),"Telomere damage and oncogenic stress induce p21 and p16 through distinct mechanisms. Upon oncogene activation (e.g., RASV12), both p21 and p16 accumulate rapidly, triggering oncogene-induced senescence. Cells with defective p16 fail to undergo OIS and may instead acquire traits of transformation. Clinical evidence shows high p16 expression in benign melanocytic nevi, representing an in vivo model of OIS.",International Journal of Molecular Sciences,Oncogene-Induced Senescence,2021
Developmental Senescence,"Senescence also occurs physiologically during embryonic development. In this context, p21—but not p16—enforces cell cycle arrest. p21 induction is independent of p53 and DNA damage; instead, it is regulated by TGF-β/SMAD and PI3K/FOXO signaling. Surprisingly, embryos lacking p21 develop normally, suggesting other mechanisms such as apoptosis compensate for the loss of developmental senescence.",International Journal of Molecular Sciences,Developmental Senescence,2021
G2-Mediated Cell Cycle Exit in Senescence,"Although senescence is classically associated with irreversible G1 arrest, cells can also enter senescence during the G2 phase in response to telomere damage, DNA damage, or oncogene activation. After DNA replication, healthy cells accumulate mitotic regulators such as cyclin B1 to ensure proper mitosis progression. These factors are normally degraded by the APC/C ubiquitin ligase after mitosis. In senescence, unscheduled degradation of these regulators causes early exit from the cell cycle at G2. This process occurs rapidly, within hours after damage.",International Journal of Molecular Sciences,G2 Cell Cycle Exit in Senescence,2021
p53–p21 Pathway and Inhibition of Mitosis,"DNA damage in G2 activates p53, which upregulates p21. p21 then sequesters cyclin B1–CDK1 complexes in the nucleus. Although cyclin B1 nuclear localization normally triggers mitosis, the cyclin B1–CDK1 complex bound to p21 is inactive and cannot be reactivated even after CDK1 dephosphorylation. As a result, cells lose their ability to enter mitosis, establishing an irreversible G2 arrest. Without mitotic CDK activity, APC/C-CDH1 becomes prematurely active and degrades cyclin B1 along with other mitotic regulators, reinforcing the G2 block.",International Journal of Molecular Sciences,p53-p21 Regulation of G2 Arrest,2021
Mitotic Bypass and Tetraploidy,"When G2 arrest persists, some cells undergo mitotic bypass – they skip mitosis entirely and enter a permanent G1-like state with 4N DNA content, resulting in tetraploid senescent cells. Although tetraploid cells can be dangerous if they proliferate, senescence prevents this by maintaining strong CDK inhibition and RB activation. Upregulation of p16 in G1 reinforces this block and prevents S-phase re-entry. Mitotic bypass is both necessary and sufficient for oncogene-induced senescence.",International Journal of Molecular Sciences,Mitotic Bypass and Tetraploidy,2021
G2 Arrest in Replicative Senescence,"Unlike acute senescence, replicative senescence arises from chronic, low-grade damage such as telomere shortening and elevated ROS. Senescing fibroblasts often experience prolonged G2 delays due to telomere erosion. With age, mitotic duration increases, expression of mitotic genes decreases, and chromosome segregation errors become more frequent. These defects generate aneuploid daughter cells and micronuclei, eventually causing permanent G1 arrest and full senescence phenotypes.",International Journal of Molecular Sciences,Replicative Senescence and G2,2021
Key Regulators and Senescence Markers,"Different senescence triggers activate distinct pathways, but all converge on increasing levels of core cell cycle inhibitors. Early markers of senescence include p53 and p21, whereas p16 and ARF (from the CDKN2A locus) typically mark late-stage senescence. However, p53 and p16 are not exclusive markers of senescence since they can participate in other cellular processes depending on context. The combination of markers reflects the stage and type of senescence rather than a single universal signature.",International Journal of Molecular Sciences,Senescence Markers and Stages,2021
Overview of Senescence-Inducing Signals,"Senescence can be triggered by diverse intrinsic and extrinsic stressors, including DNA damage (from telomere shortening, oncogene activation, oxidative stress, radiation), metabolic dysfunction (mitochondrial defects, redox imbalance), inflammatory cytokines, and proteotoxic stress from misfolded proteins or autophagy disruption. Because different stressors activate different pathways, senescent cells exhibit heterogeneous phenotypes that vary based on the inducer, cell type, duration of senescence, and tissue environment. Despite this heterogeneity, most senescence programs rely on two conserved tumor suppressor pathways: the p53/ARF axis and the RB/p16 axis. These pathways work cooperatively to block the cell cycle and sustain senescence.",International Journal of Molecular Sciences,Senescence Signaling Overview,2021
Cooperation of p53/ARF and RB/p16 Pathways,"The p53/ARF and RB/p16 pathways form the core machinery enforcing senescence. Their activation depends on stress type, tissue context, and species. For example, loss of RB can activate p53 through ARF or DNA damage signaling, acting as a compensatory safeguard against senescence escape and malignant transformation. During senescence, unresolved DNA damage caused by telomere erosion or oncogene-driven hyperproliferation strongly activates p53 and its transcriptional program. DDR signals can also elevate p16, although p16 frequently acts downstream of telomere dysfunction as a secondary reinforcement mechanism. In oncogene-induced senescence (OIS), RB activation downstream of p16 is essential to establish proliferative arrest.",International Journal of Molecular Sciences,p53–RB Cooperation,2021
Species and Cell-Type Differences in OIS Regulation,"The relative roles of p53 and p16 differ between species and cell types. In mice, p53 is often essential: mouse embryonic fibroblasts lacking p53 escape Ras-induced senescence, whereas loss of p16 or RB does not prevent senescence in these cells. In contrast, human fibroblasts rely more heavily on p16: loss of p16 bypasses Ras-induced senescence, while p53 loss does not. Interestingly, some human cell types, such as mammary epithelial cells and melanocytes, undergo Ras-induced senescence independent of both p53 and p16. These variations emphasize that OIS is not governed by a universal pathway, but rather reflects context-specific regulatory logic.",International Journal of Molecular Sciences,Species Differences in Senescence Signaling,2021
Epigenetic Regulation of the CDKN2A (p16) Locus,"In proliferating cells, expression of p16 is actively repressed by polycomb repressive complexes PRC1 and PRC2, which are recruited to the CDKN2A locus by the long noncoding RNA ANRIL. EZH2, a PRC2 methyltransferase, deposits H3K27me3 marks that silence the locus. PRC1 components such as BMI1, CBX7, and CBX8 reinforce this repression. Overexpression of these factors delays senescence by maintaining p16 repression. During senescence, several changes occur: polycomb complexes dissociate from the locus, EZH2 expression declines, and the demethylase JMJD3 removes H3K27me3 marks. This epigenetic remodeling derepresses the CDKN2A locus and enables strong p16 transcription, stabilizing the irreversible cell cycle arrest.",International Journal of Molecular Sciences,Epigenetic Control of p16,2021
Telomere Shortening as a Trigger of Replicative Senescence,"Telomere erosion is the primary cause of replicative senescence in human fibroblasts. Telomeres consist of repetitive DNA sequences protected by the shelterin complex, which forms t-loops that hide chromosome ends from DNA damage sensors. Because most somatic cells lack telomerase, telomeres shorten with each cell division due to the end-replication problem and end-processing events. Additional mechanisms—oxidative damage, nuclease activity, and loss of telomeric stretches—accelerate this erosion. When telomeres become critically short (often a few extremely short telomeres despite an average length of 6–8 kbp), they fail to maintain protection, exposing chromosome ends and triggering a persistent DNA damage response (DDR) that enforces permanent cell-cycle arrest.",International Journal of Molecular Sciences,Replicative Senescence and Telomeres,2021
DNA Damage Response Activation at Short Telomeres,"Critically short telomeres accumulate γH2AX foci, which recruit DNA repair proteins such as 53BP1, MDC1, and NBS1. This leads to activation of ATM, CHK1, and CHK2, driving strong DDR signaling. Importantly, complete loss of telomeric repeats is not required—shortened telomeres with reduced shelterin occupancy are sufficient to activate DDR. Senescent cells show displacement of shelterin components, especially TRF2, which normally represses ATM activation and prevents non-homologous end joining (NHEJ). Loss or dysfunction of TRF2 (experimentally or due to telomere erosion) produces robust ATM signaling, telomere fusions, and rapid cell-cycle arrest.",International Journal of Molecular Sciences,DNA Damage Response at Telomeres,2021
Short Telomeres Do Not Fully Mimic Acute Shelterin Loss,"Although experimental TRF2 inactivation causes strong ATM activation and telomere–telomere fusions, naturally occurring short telomeres in senescent cells behave differently. They often retain enough TRF2 to prevent NHEJ-mediated chromosome fusions, but not enough to suppress ATM activation. This creates a state where telomeres signal damage without undergoing inter-chromosomal fusions. This differs from the proposed 'intermediate state' model of TRF2 depletion because senescent cells show robust CHK2 and p53 activation, indicating a stronger DDR than predicted by intermediate-state hypotheses.",International Journal of Molecular Sciences,Telomere Dysfunction vs. Shelterin Loss,2021
G2/M Checkpoint Activation by Critically Short Telomeres,"Senescent human fibroblasts frequently activate G2/M checkpoint markers, suggesting telomere dysfunction during or after DNA replication. Severely shortened telomeres may fail to reform t-loops after replication because they lack the minimal length or G-overhang required. Reduced TRF2 occupancy may also impair t-loop formation. Overexpression of TRF2 delays senescence, supporting the idea that t-loop stabilization prevents DDR activation. As telomeres become too short, cells stall in G2 while attempting t-loop reformation. If only a few dysfunctional telomeres remain, cells may still enter mitosis, generating micronuclei or DNA fragments inherited by daughter cells, which then arrest in G1.",International Journal of Molecular Sciences,G2/M Checkpoint and Telomere Dysfunction,2021
Threshold Concept for Enforcing Senescence,"A threshold number of dysfunctional telomeres is necessary to enforce stable senescence. The G2/M checkpoint responds only when ~10–20 DNA breaks are present, meaning cells with a small number of damaged telomeres can still divide. After mitosis, daughter cells inherit micronuclei, fused chromosomes, or residual DNA breaks, which then trigger G1 arrest and full senescence. Thus, senescence often results from cumulative telomere dysfunction and unresolved replication-associated damage.",International Journal of Molecular Sciences,Threshold for Telomere-Driven Senescence,2021
Oxidative Stress as a Driver of Telomeric Damage,"In senescent cells from culture and aged tissues, γH2AX foci often mark persistent DNA lesions that are not due to critically short telomeres. Telomeric DNA is highly vulnerable to oxidative stress because guanine-rich telomeric repeats are prone to oxidation, forming lesions like 8-oxoguanine. This susceptibility increases with age. Studies show that telomere-associated DDR foci accumulate with aging in baboons, mice, and human fibroblasts despite long telomeres and active telomerase, demonstrating that telomere dysfunction can occur independently of telomere length. High oxygen culture (20%) induces senescence in mouse embryonic fibroblasts (MEFs) even though these cells have long telomeres and telomerase activity. Conversely, reducing oxygen to physiological levels prevents senescence during extended passaging or Ras-induced stress.",International Journal of Molecular Sciences,Oxidative Damage and Telomeres,2021
Senescence Triggered by Oxidative Damage Is Not Prevented by Telomerase,"Although telomerase can delay replicative senescence by maintaining telomere length, it does not protect cells from senescence induced by oxidative stress or oncogene-induced senescence (OIS). Oxidative damage leads to DNA lesions at telomeres or replication stress that activates DDR independent of telomere length. Interestingly, very high telomerase activity can prevent DDR activation caused by stalled replication forks inside telomeres during OIS, enabling partial bypass of senescence. However, the mechanism by which telomerase counteracts replication-associated telomeric stress remains unclear.",International Journal of Molecular Sciences,Telomerase and Oxidative Stress,2021
Why Telomeric DNA Damage Persists: Repair Resistance,"Persistent DDR signaling in senescent cells often stems from the cell’s inability to repair telomeric DNA breaks efficiently. TRF2, a key shelterin component, binds throughout telomeric repeats and blocks the completion of double-strand break repair by preventing recruitment of DNA ligase IV to breaks located at or near telomeres. As a result, telomere-associated DNA lesions remain unrepaired and sustain DDR activation, stabilizing the senescence growth arrest. In contrast, non-telomeric DNA breaks are efficiently repaired, and their associated DDR signals are transient.",International Journal of Molecular Sciences,Telomere Repair Resistance,2021
TRF2-Mediated Inhibition of Repair After Exogenous Damage,"When cells experience oxidative damage or DNA-damaging agents, lesions occurring within telomeric regions remain unrepaired because TRF2 inhibits repair machinery access. These unresolved lesions continuously activate the DDR, leading to stable, irreversible cell-cycle arrest. This mechanism explains why even in cells with long telomeres or active telomerase, telomere dysfunction can occur if oxidative stress targets telomeres specifically.",International Journal of Molecular Sciences,TRF2 and Unrepaired Telomeric Lesions,2021
Telomeric Lesions Are Not the Sole Cause of Damage-Induced Senescence,"Although defective repair at telomeres is a major mechanism that stabilizes senescence, it is unlikely to be the only contributor after exposure to DNA-damaging agents. Many senescence-inducing pathways, including widespread genotoxic stress, metabolic dysfunction, and chronic inflammatory signaling, can cooperate with telomeric damage to enforce a stable senescent phenotype.",International Journal of Molecular Sciences,Multiple Pathways to Senescence,2021
Oncogene Activity and Replication Stress,"Oncogene-induced senescence (OIS) is triggered when acute mitogenic signals, exemplified by oncogenic Ras, drive excessive proliferation and deregulate cell-cycle entry in normal fibroblasts. Because endogenous oncogene expression is insufficient for OIS, mutation-driven hyperactivation is generally required. Activated oncogenes increase CDK activity and deregulate replication origin firing, generating replication errors, DNA breaks, and robust DNA damage response (DDR) activation. Oncogenic stress can also reduce RRM2 expression, lowering dNTP pools and contributing to replication stress. High ROS levels, frequently detected in oncogene-overexpressing cells, further intensify DDR through direct DNA damage or oxidative lesions that collapse replication forks. Blocking ATM or CHK2 suppresses this arrest, confirming DDR’s causative role in OIS. Telomeric dysfunction contributes to DDR in OIS independently of telomere length; melanocytic nevi with BRAF mutations show telomeres that are not visibly shorter yet are dysfunctional. A DDR-independent pathway also participates, centered on ARF (p19ARF in mice, p14ARF in humans), which stabilizes p53 by inhibiting MDM2. ARF and p16 arise from the CDKN2A locus but are differently regulated in mice and humans. Mouse p19ARF is strongly induced by oncogenes, leading to p53 activation and senescence, whereas human melanocytes respond to Ras mainly by upregulating p16, not ARF. Benign human nevi typically show high p16 without significant ARF or p53 expression. ARF upregulation correlates more with advanced cancer stages than with early DDR-driven senescence, reflecting the slower transcription-based activation of ARF under persistent oncogenic signaling. Thus, oncogenic stimuli engage DDR rapidly but activate ARF more slowly through distinct promoter regulatory mechanisms.",International Journal of Molecular Sciences,Oncogene Activity and Replication Stress,2021
Transcriptional and Post-Transcriptional Control of Senescence,"Senescent cells undergo extensive transcriptional and post-transcriptional rewiring that reflects their heterogeneity, cell of origin, inducing stimulus, and time since induction. Global transcriptome analyses and single-cell RNA-sequencing show that senescent populations contain distinct subgroups with variable mRNA profiles. A universal early feature is transcriptional repression of cell cycle machinery: genes controlling growth factor signaling, DNA replication, G1/S and G2/M progression, and mitosis are downregulated. Although cycling genes are broadly repressed in non-dividing states, RB is uniquely required during senescence to repress replication gene transcription and block DNA synthesis, whereas RB-family proteins act redundantly during quiescence. About half of the genes differentially expressed in senescent fibroblasts are not shared with quiescent cells, underscoring the specificity of the senescence program. CDK inhibitor genes are consistently upregulated, while DNA repair and chromatin organization programs are repressed. Genes encoding mitotic fidelity regulators, including spindle assembly and segregation components such as CENP-E, are downregulated, potentially contributing to aneuploidy and genomic instability. Senescence also alters metabolic and biosynthetic pathways: genes required for DNA, RNA, and protein synthesis, glucose and lipid metabolism, and nuclear-encoded mitochondrial electron transport chain components are downregulated, consistent with decreased mitochondrial activity and elevated ROS. These changes may reinforce proliferation arrest by limiting available energy and biosynthetic capacity. Conversely, genes involved in membrane trafficking, cell–cell adhesion, and receptor signaling are upregulated, aligning with the characteristic enlarged morphology and increased ECM adhesion. Senescent cells also upregulate anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, BCL-W), downregulate apoptotic mediators such as caspase-3, and exhibit altered p53 signaling, collectively contributing to apoptosis resistance.",International Journal of Molecular Sciences,Transcriptional Regulation in Senescence,2021
Post-Transcriptional Regulation Overview,"Post-transcriptional regulatory pathways also contribute to control senescence through the action of mRNA-binding proteins (RBPs) and noncoding RNAs, particularly specific microRNAs (miRNAs), the levels of which help to mediate the senescence state [179,180]. RBPs, such as human antigen R (HuR), AU-binding factor 1 (AUF1), and tristetraprolin (TTP), can directly or indirectly control the turnover and translation of mRNAs that encode senescence proteins [181–183]. For example, p16 mRNA stability is reduced by the RBPs hnRNP A1, hnRNP A2, and AUF1 [184,185]. Senescent cells express reduced levels of nuclear factor (NF90), an RNA-binding protein that suppresses the translation of SASP factors, such as MCP1, GROα, and IL-6 [186]. Consequently, the reduction in NF90 levels amplifies the production of several SASP factors. RBPs also are major regulators of genes involved in DDR and in the prevention of genome instability [187]; however, only few of these RBPs are functionally involved in promoting or suppressing cellular senescence [188].",International Journal of Molecular Sciences,Cellular Senescence,2021
Role of microRNAs in Senescence,"Gene expression is robustly regulated at the post-transcriptional level also by miRNAs. These short noncoding RNAs (18–25 nucleotides long) repress gene expression by binding to complementary sequences on the 3ʹUTR of the target mRNAs and by blocking their translation and thus promoting their degradation [189,190]. A single miRNA can simultaneously regulate multiple target mRNAs, and different miRNAs might co-regulate the same mRNA, consistent with their role as regulatory molecules that fine-tune gene expression [191,192]. Importantly, miRNAs are differentially expressed during senescence and regulate key nodes of the senescence signaling pathways through direct binding to the mRNAs of p53, p16, SASP factors, and other senescence-regulatory proteins [193]. Overall, the reliance on regulatory miRNAs to regulate SASP and senescence activation allows rapid changes in mRNA stability and translation to ensure a tight control of gene expression.",International Journal of Molecular Sciences,Cellular Senescence,2021
Alternative Splicing in Senescence,"Alternative splicing also plays a role in the post-transcriptional regulation of gene expression during cell senescence: the same pre-mRNA can generate multiple transcripts, and therefore different protein isoforms. Alternative splicing greatly enhances transcriptome diversity and complexity, leading to different protein variants with possible different or modified functionality [194]. The relative abundance of splice isoforms produced from one gene tends to change in cells undergoing senescence in vitro and with aging [195]. Deregulated splicing with age is largely tissue- and species-specific, and many of the affected genes are implicated in mRNA regulatory processes, splicing machinery, inflammation, metabolism, and tissue regeneration [193,196]. Deregulation of the normal splicing patterns can be partially attributed to the use of alternative splice sites and to changes in exon exclusion or intron retention that can alter the protein structure, localization, regulation, and function [197]. Some of these changes, for example, in the genes encoding nuclear lamin A (LMNA), S-endoglin (ENG), p53 (TP53), and the EAAT2 glutamate transporter (EAAT2), contribute to aging-related phenotypes [198–201].",International Journal of Molecular Sciences,Cellular Senescence,2021
Splicing Factor Changes and SASP Regulation,"In addition to the direct changes due to alternative splicing of age-related genes, altered expression of splicing factors has also been associated with cell senescence and age-related phenotypes. For example, the level of the splicing factor SRSF3 decreases in replicatively senescent human fibroblasts, and its knockdown promotes p53-mediated senescence by directly upregulating p53β, an alternatively spliced p53 isoform [201]. The DDR might be implicated in the increased production of p53β by regulating alternative splicing and splicing factor activity [202]. Functionally, p53β is required for senescence induction, possibly through transcriptional repression [202]. Other splicing regulators such as the RNA-binding protein polypyrimidine tract binding protein 1 (PTBP1), regulate the alternative splicing of genes involved in intracellular trafficking and are required for the pro-inflammatory SASP [203]. Consequently, the imbalance in the expression of protein isoforms caused by senescence-related splicing alterations could reflect the inability of senescent cells to properly respond to cellular stress, highlighting the decline in cell adaptability and plasticity during aging.",International Journal of Molecular Sciences,Cellular Senescence,2021
Cell State Changes During Senescence,"Senescence development involves substantial changes in cell metabolism, morphology, and structures (Figure 2). Functional and molecular alterations of cell structures are associated with senescence establishment and are essential for regulating other senescence features, such as the increase in metabolic activity and protein synthesis.",International Journal of Molecular Sciences,Cellular Senescence,2021
Altered Metabolism in Senescent Cells,"Cellular metabolism changes are important for the function and fate of senescent cells [204]. Although senescent cells do not divide, they display a very active but altered metabolism, with increased glycolysis and mTOR activity [204]. The increased metabolic demands are related to their increased size, elevated production of secreted proteins (SASP), and increased oxidative stress and endoplasmic reticulum (ER) stress after cell cycle exit [205]. This results in different metabolic needs compared with proliferating cells and requires changes to support these demands. Senescent cells exhibit a shift toward elevated glycolysis with an imbalanced activity of glycolytic enzymes that results in a reduced energetic state when cell enter replicative senescence [206,207]. Increased aerobic glycolysis compensates for the reduced adenosine triphosphate (ATP) production caused by mitochondrial respiration decline during senescence [1,208–210]. In the early stage of senescence, mitochondria do not function properly and display impaired oxidative phosphorylation capacity and reduced inner membrane potential, resulting in ROS overproduction [120,121,208,211]. Due to their functional defects, the mass and number of mitochondria are increased in senescent fibroblasts [212]. Increased mitochondrial biogenesis is dependent on ATM-mediated activation of the Akt/mTORC1 phosphorylation cascade, leading to stimulation of the mitochondrial biogenesis regulator peroxisome proliferator-activated receptor gamma coactivator α (PGC1-α) [213].",International Journal of Molecular Sciences,Cellular Metabolism,2021
Mitochondrial Dysfunction and SASP Regulation,"Moreover, damaged mitochondria are insensitive to mitophagy (i.e., selective autophagy of mitochondria), and consequently, mitochondrial number and size are not properly regulated in senescent cells [214]. Removal of mitochondria in senescent cells disrupts the feedforward cycle that involves ROS production and persistent DDR activation, while preserving their cell cycle arrest [215]. In these cells, SASP gene expression alteration is not caused by insufficient energy levels because ATP levels are high due to increased glycolysis. Therefore, it seems that at least in some contexts, the execution of the senescence program is compromised not by insufficient energy levels but rather by mitochondrial oxidative metabolism status. Accordingly, a metabolic shift from glycolysis towards mitochondrial oxidative respiration through activation of mitochondrial pyruvate dehydrogenase is required to establish and stabilize the OIS-associated cell growth arrest [209]. Moreover, during OIS, fatty acid metabolism is altered, glucose consumption is enhanced, and the utilization of pyruvate in the tricarboxylic acid cycle and nucleotide deficiency are increased [119,216–219]. Senescence induced by nucleotide deficiency causes aberrant DNA replication but can be overcome by ATM inactivation through restoration of glucose and glutamine consumption [220]. This supports the causative role of metabolic changes in senescence induction (Figure 3).",International Journal of Molecular Sciences,Mitochondrial Dysfunction,2021
Feedback Loop Between DNA Damage and Metabolic Stress,"Despite the increased number of mitochondria, mitochondrial dysfunction in senescent cells impairs metabolism by compromising ATP production, and also through the loss of the biosynthetic precursor pools and the inability to maintain the redox balance. This metabolic stress due to the imbalance in metabolic intermediates can be relayed through metabolic signaling that also contributes to senescence via multiple signaling pathways. Reduced ATP production increases the AMP-to-ATP ratio, a measure of the cell energy charge. This leads to activation of the energy sensor AMPK to coordinate activities for adapting to the metabolic stress. AMPK increases ATP levels through the activation of mitochondrial biogenesis and the stimulation of catabolic pathways, such as autophagy, induction of fatty acid oxidation, and glucose uptake [221,222]. Additionally, chronic AMPK activation promotes senescence via multiple mechanisms. Indeed, AMPK regulates cell cycle arrest and senescence by activating p53 that upregulates p21 transcription [223]. AMPK also prevents cytoplasmic translocation of the mRNA-stabilization factor HuR, thereby increasing p21 and p16 mRNA stability and enhancing RB activity [224].",International Journal of Molecular Sciences,DNA Damage Response,2021
NAD+/NADH Imbalance and SASP Modulation,"AMPK signaling is also induced by the reduced cytosolic NAD+/NADH ratio as a result of mitochondrial defects [225]. In this study, mitochondrial dysfunction, induced by depletion of mitochondrial sirtuins or mitochondrial DNA or by inhibition of the electron transport chain, caused senescence without ROS hyperproduction and DDR activation. Growth arrest due to these mitochondrial perturbations was rescued by the exogenous supply of the electron acceptors pyruvate and potassium ferricyanide that artificially restore NAD+ levels. This identifies imbalanced NAD+/NADH levels as a stress signal secondary to mitochondrial dysfunction. Notably, such mitochondrial dysfunction may engage cellular senescence with a specific secretory profile that lacks the IL-1α/NF-κB-inflammatory component and that is regulated through AMPK-mediated p53 activation [225]. Therefore, mitochondrial dysfunction, initiated by pathways different from DNA damage, can determine the SASP quality because local extracellular factors, such as pyruvate, can modify the SASP in senescent cells [225].",International Journal of Molecular Sciences,SASP Regulation,2021
"p53, RB, mTORC1 and Metabolic Control","In addition to stopping senescent cell proliferation, p53 and RB mediate senescence-related metabolic changes by balancing their effects on glycolysis: p53 restricts the glycolysis activity of senescent cells through several mechanisms [226], while RB upregulates glycolytic genes, resulting in high glycolytic activity [227]. In fully senescent cells, RB also stimulates mitochondrial oxidative phosphorylation [227]. The resulting activated metabolic flow efficiently produces metabolites and the energy molecule ATP that regulate the SASP. As the p53 response efficiency declines during aging [228], aerobic glycolysis is increased as well as the proinflammatory SASP via NF-κB signaling [155]. Similarly, during senescence, mTORC1 activation due to sustained DDR does not favor growth but promotes mitochondrial biogenesis, thus contributing to the ROS-dependent DDR persistence and to SASP regulation [215]. The increase in mTORC1 activity is essential for implementing the SASP by modulating the translation of IL-1α and MAP kinase-activated protein kinase 2 (MAPKAPK2) [162,163]. In turn, IL-1α activates NF-κB to trigger SASP amplification [163]. MAPKAPK2 phosphorylates the RNA-binding protein ZFP36L1, thus preventing its binding to and its ability to degrade SASP RNAs [162].",International Journal of Molecular Sciences,Metabolic Signaling,2021
Phagocytosis by Senescent Cells,"Although still poorly understood, chemotherapy-resistant senescent cells can engulf neighboring normal and tumor cells by phagocytosis for survival advantage [229]. Indeed, upon engulfment and processing through lysosomes of these cells, biosynthetic material and energy are released to sustain the high metabolic needs of senescent cells [229].",International Journal of Molecular Sciences,Senescent Cell Behavior,2021
Morphological and Organelle Alterations,"Senescent cells display characteristic morphological alterations, including flattened, enlarged cell shape and an increase in focal adhesions due to CDK5-dependent activation of the cytoskeleton protein ezrin [230]. These changes reflect the increased alterations in abundance and activity of membranous organelles, particularly mitochondria, lysosomes, and ER. Although in senescent cells, the number and mass of organelles increase, these senescent organelles display functional defects and modified communication through the release of metabolites [215,231]. Consequently, to maintain homeostasis, senescent cells may produce more organelles to compensate for their declined function upon damage by mitochondrial oxidative stress. However, the newly generated organelles also may be exposed to oxidative stress and accumulate alterations, and this aggravates the senescence phenotype. The defective autophagy also can potentiate this effect through loss of its quality control capacity.",International Journal of Molecular Sciences,Cellular Senescence,2021
Lysosomal Expansion and Senescence Biomarkers,"Senescent cells are characterized by an expanded lysosomal compartment and vacuoles [231]. Lysosomes are acidic organelles that contain hydrolytic enzymes required for protein degradation in autophagy. In senescent cells, the activity of lysosomal β-galactosidase is significantly increased as a consequence of the increased lysosomal mass and becomes detectable experimentally at pH 6.0 [232]. Senescence-associated β-galactosidase (SA-β Gal) activity is one of the first and most used biomarkers of senescence, although it has limitations because it can be detected in non-senescent cells that are confluent or serum-starved [233]. Lipofuscin are lysosomal aggregates of non-degradable oxidized protein, lipid, and metal that also accumulate in senescent cells. Lipofuscin accumulation reflects reduced lysosomal activity, altered metabolism, and autophagy dysfunction. Lipofuscin aggregates can be assayed by staining with the Sudan Black B dye, and they are a sensitive biomarker of senescent cells in vitro and even in in situ samples [234,235].",International Journal of Molecular Sciences,Lysosomal Function,2021
"ER Expansion, Stress, and UPR Activation","Senescent cells also display ER expansion and biogenesis to adapt their capacity to the high amount of synthesis, maturation, and secretion of factors involved in the SASP [1,78,236,237]. ER capacity to properly synthetize proteins could be overwhelmed, thus leading to accumulation of misfolded proteins that induces a stress response in the ER. This leads to increased ROS levels that can further impair protein folding and the formation of correct disulfide bonds in many SASP proteins (this requires a controlled oxidant state and glutathione content). Oxidation or decreased expression of some chaperones and folding enzymes with age correlates with the reduction in their enzymatic activity that impairs the ER folding capacity [238]. As a consequence, progressive protein misfolding/aggregation or massive SASP protein synthesis in senescent cells cause the loss of the protein quality control homeostasis (or proteostasis) and activation of the unfolded protein response (UPR). The UPR signaling seeks to limit ER abnormal protein load and to reduce ER stress by inhibiting protein translation [239], by upregulating various ER chaperones that contribute to the correct organization of misfolded proteins [240], and by degrading misfolded proteins via the proteasome through an ER-associated degradation process [241]. For example, in OIS, the increased ROS production caused by ER stress leads to DNA damage that activates ATM. Active ATM triggers the removal from SASP genes of the histone variant macroH2A1, leading to inhibition of their transcription and reduction in ER stress [168].",International Journal of Molecular Sciences,ER Stress and UPR,2021
Autophagy and Senescence Overview,"Senescence involves cellular remodeling, enlarged size, higher organelle content, and increased metabolism. These changes lead to the senescent phenotype, partly supported by mTOR activity that promotes cellular anabolism and inhibits the catabolic autophagy pathway. Macro-autophagy (hereafter autophagy) describes the bulk or selective degradation by lysosomes of damaged macromolecules (e.g., proteins) and organelles, and the recycling of degradation products to sustain cell biosynthetic and bioenergetic demands [242]. Under normal conditions, basal autophagy contributes to maintaining the metabolic homeostasis and controls the quality of cell components [243]. Stress can cause adaptive autophagy responses. Indeed, autophagy is also activated in senescing cells to limit damage by removing defective organelles that may be a source of oxidative stress and consequently DNA damage. Autophagy is also required for the execution of the senescence program because its inhibition delays the senescence-related cell cycle arrest and SASP factor accumulation, at least in OIS [244]. Functionally, autophagy is activated at a later stage than DDR signaling to sustain the bioenergetic needs and to supply metabolites for SASP factor synthesis [245]. Overall, autophagy can both promote and inhibit senescence in different contexts because it modulates several effectors with opposite functions in senescence regulation.",International Journal of Molecular Sciences,Autophagy and Senescence,2021
Spatial Coupling of mTOR and Autophagy,"In senescence induced by oncogenic Ras, protein synthesis and autophagic degradation are simultaneous activated, and the same cell displays high autophagic activity and activated mTOR, the autophagy inhibitor [244]. This paradoxical outcome is possible through the spatiotemporal sub-compartmentalization of mTOR and autophagy that allows their simultaneous activation [246]. The mTOR–autophagy spatial coupling compartment (TASCC), which is close to the nucleus, is highly enriched in mTOR and keeps mTOR away from the autophagy machinery, located away from the TASCC, to avoid its inactivation. In the TASCC, mTOR is closely associated with lysosomes/autolysosomes that generate a high flux of recycled amino acids and other metabolites. These amino acids are then used by mTOR to support the increased biosynthetic demand during the acquisition of the OIS phenotype [246].",International Journal of Molecular Sciences,mTOR-Autophagy Coupling,2021
Selective Autophagy and GATA4 Regulation,"Senescence can be promoted by general autophagy through the TASCC. Conversely, selective autophagy, for instance of GATA4 that accumulates during senescence induction [160], can prevent senescence [160,246]. Under basal conditions, the autophagic adaptor SQSTM1/p62 interacts with GATA4 and mediates its degradation by selective autophagy. Conversely, upon irradiation or oncogene hyperactivation, activation of ATM and ATR suppresses selective autophagy by promoting GATA4 dissociation from SQSTM1/p62, resulting in GATA4 stabilization. Then, GATA4 initiates the synthesis of SASP factors via NF-κB activation, partly mediated by IL-1 α production [160].",International Journal of Molecular Sciences,Selective Autophagy,2021
Autophagic Degradation of Lamin B1 and CCF Formation,"OIS relies not only on the autophagic turnover of cytoplasmic material but also on the specific autophagic degradation of nuclear lamin B1 in lysosomes, where it is delivered by nucleus-to-cytoplasm transport [247]. The lipidated form of the autophagic protein LC3B, which is involved in autophagy, membrane trafficking, and substrate delivery [248], interacts with lamin B1 at the nuclear lamina. However, this interaction does not result in lamin B1 degradation by autophagy under basal conditions. Conversely, upon oncogene activation, lamin B1 and LC3 interact with lamina-associated domains (i.e., transcriptionally inactive heterochromatin) and are extruded through nuclear blebbing into the cytoplasm, forming CCFs that are then degraded by cytoplasmic autophagy. Importantly, autophagy inhibition maintains the nuclear envelope integrity in senescent cells by suppressing lamin B1 degradation and CCFs formation [247]. Therefore, CCFs formation relies on the constitutive interaction between lamin B1 and LC3, and CCFs formation is required to reinforce OIS by initiating the SASP program [171,172].",International Journal of Molecular Sciences,Lamin B1 and CCFs,2021
"Autophagy, CCF Clearance, and SASP Modulation","Accordingly, CCFs clearance by autophagy represses senescence by preventing CCFs-induced cGAS/STING activation and SASP production [249]. Therefore, autophagy might be a protective pathway against micronucleus formation and CCFs accumulation.",International Journal of Molecular Sciences,CCFs and SASP,2021
Persistence of Senescence and DDR Signaling,"Cell viability and a generally irreversible growth arrest are two key features of cellular senescence. These features require protection against apoptosis and stability of the cell cycle exit. In most senescence models, activation of p53 and of DDR proteins initially facilitates cell cycle arrest (as previously discussed). During senescence, the DDR continues to signal through the p53 pathway [47], due to the persistent DNA damage at telomeric sites that cannot be efficiently repaired [109,115] or through the induction of positive-feedback loops that promote constant generation of short-lived repairable non-telomeric lesions [250,251]. These factors remain the main driving force in the establishment of the senescence program. Continuous DNA damage signaling is essential for cell cycle arrest maintenance because inactivation of checkpoint kinases, such as ATM [10,12], CHK2 [11], and p53 [47,48], results in escape from this arrest. The switch from transient to irreversible growth arrest involves positive feedback loops in which mitochondrial dysfunction via ROS [215,250], pro-inflammatory SASP factors [147,154,252], extensive chromatin remodeling [253], and/or nuclear lamina and chromatin degradation [105,171,247] reinforce DDR signaling.",International Journal of Molecular Sciences,Senescence Maintenance,2021
Resistance to Apoptosis in Senescent Cells,"Senescent cells do not undergo apoptosis [143] despite the presence of high and irreparable levels of DNA damage. Indeed, induction of senescence appears protective against p53-dependent apoptosis [146] and high oxidative stress [254,255]. For this, senescent cells rely on the upregulation of pro-survival pathways (e.g., BCL-2 and Ephrins) that actively inhibit apoptosis [3,14,15]. For example, inhibition of the anti-apoptotic BCL2, BCL-W, and BCL-XL proteins induces apoptosis of senescent cells and their elimination in mice [3,14]. Upregulation of the cell cycle inhibitor p21 contributes to apoptosis resistance, for example, by inhibiting p53-mediated apoptosis after DNA damage [256,257] and by preventing the cleavage of the apoptosis effector caspase-3 and the activation of the JNK pathway (both required for apoptosis) [144]. IL-6 also facilitates the senescent phenotype maintenance by inhibiting mitochondrial-mediated apoptosis and by stimulating the pro-survival activity of NF-κB in senescent cells [258]. Senescence-associated heterochromatic focus (SAHF) formation also promotes cell survival by protecting senescent cells against excessive DNA damage signaling during oncogenic stress [259]. Additionally, increased autophagic flux promotes senescent cell survival by facilitating the degradation of damaged proteins and dysfunctional organelles [260].",International Journal of Molecular Sciences,Apoptosis Resistance,2021
Immune-Mediated Clearance of Senescent Cells,"Senescent cells can be cleared by the immune system [27,263,264] rather than directly through apoptosis. Indeed, senescent cells formed following tissue damage can promote their own clearance by secreting chemo-attractants that recruit and activate immune cells [265]. For example, upregulation of secretory factors in senescent hepatic stellate cells during liver damage facilitated their elimination by macrophages. Conversely, cells that could not become senescent due to p53 deletion were not targeted by macrophages [266]. Therefore, if senescent cells are not efficiently eliminated by the immune system, their pro-survival phenotype promotes their persistence within tissues.",International Journal of Molecular Sciences,Immune Clearance,2021
ROS-Driven Feedback Loops,"The escalating ROS production by dysfunctional mitochondria aggravates nuclear DNA damage and stabilizes the chronic DDR activation, contributing to the maintenance of the pro-inflammatory and pro-oxidant senescent phenotype upon DNA damage [215,250]. Conversely, culturing cells in low oxygen or in the presence of antioxidants delays senescence onset [111,212,267,268]. Mitochondrial dysfunction is the main cause of elevated ROS production in senescence. Indeed, the selective and specific removal of mitochondria in senescent cells normalizes ROS levels [215]. Mitochondrial DNA is highly prone to oxidative damage because it is located close to the ROS generation site and is devoid of histones. Damaged mitochondrial DNA alters oxidative phosphorylation reactions and can further enhance ROS production [269]. Downstream of DDR effectors, p16-Protein Kinase C delta (PKC delta) and p21-p38MAPK-TGF-β signaling also contribute to ROS production [250,268]. ROS can also be transferred between cells through gap junctions or released into the environment as hydrogen peroxide, inducing senescence in surrounding cells [270].",International Journal of Molecular Sciences,Mitochondrial ROS,2021
SASP-Driven Feedback Loops,"SASP factor production requires continuous DNA damage signaling [148]. In turn, some SASP factors that strongly accumulate in the secreting senescent cells reinforce the senescence signaling in an autocrine fashion [154,252]. The chemokine IL-8 and other chemokine receptor 2 (CXCR2) ligands strengthen growth arrest in senescent cells by activating a self-amplifying secretory loop through their ability to boost DDR signaling. CXCR2 engagement drives senescence via p53 and RB activation, and CXCR2 depletion allows senescence bypass [153]. Cell surface-bound IL-1α and its receptors maintain high IL-6 and IL-8 secretion [158]. Other proteins, including IGFBP7 that modulates MAPK signaling [271], and PAI-1 that regulates the PI3K pathway [272], inhibit proliferative pathways and stabilize senescence. Type I interferons produced by senescent cells in a cGAS-dependent manner further promote ROS production, DNA damage, and p53 activation [273,274]. Together, these autocrine loops lock the cell into the senescent state.",International Journal of Molecular Sciences,SASP Feedback,2021
Chromatin Remodeling and Epigenetic Alterations,"Senescent cells undergo extensive epigenome and chromatin organization changes that contribute to the persistent proliferative arrest and progression to full senescence. Chromatin remodeling increases the accessibility of normally compacted DNA while restricting other regions, altering transcriptional programs. SAHF formation is a hallmark of oncogene-induced senescence and consists of compact heterochromatic regions enriched in H3K9me3 and HP1 [253,276]. SAHF formation involves components of the p16/RB pathway, HMGA proteins [277], and histone chaperones HIRA and ASF1A [167,276]. SAHF formation is strongly associated with lamin B1 loss [279–281], yet SAHF is not universally present in all senescent cells. Another feature is SADS, the distention of peri/centromeric satellite sequences, which occurs early during senescence and is conserved across models [282]. Senescent cells also exhibit global DNA hypomethylation in heterochromatic regions with local hypermethylation at CpG islands [283], and increased chromatin accessibility in normally condensed repetitive regions, promoting transcription of transposable elements that may contribute to genomic instability [286,287].",International Journal of Molecular Sciences,Chromatin Remodeling,2021
Beneficial and Detrimental Roles of Senescence,"Cellular senescence contributes to tissue homeostasis in many different biological processes. Senescence can be beneficial or detrimental for the organism, in function of the physiological context (Figure 4). Senescent cells play beneficial roles during embryo development, tissue repair/regeneration, and in the protection against cancer [288]. Developmental senescence is induced by developmental cues to regulate cell proliferation in embryonic structures and to induce tissue remodeling signals for proper embryo formation [27,28]. Cellular senescence optimizes tissue remodeling by promoting ECM deposition [30,31] and by inducing the plasticity of neighboring cell populations [289] that are important for mediating the balance between healing and fibrosis in wound closure. In addition, the permanent exit from the cell cycle of senescent cells prevents the propagation of premalignant cells in the context of tumorigenesis. These biological functions of senescent cells largely rely on their ability to communicate with the environment through various intercellular communication processes, including but not limited to the SASP factors, and to stimulate immune surveillance [149,290]. Timely clearance of senescent cells is essential to eliminate SASP factors and for the successful restoration of tissue function [289]. When senescent cells are not efficiently cleared and accumulate in tissues, the continued production of SASP factors can contribute to local inflammation and to the chronic inflammatory milieu, via paracrine and systemic SASP that aggravate tissue dysfunction [7,149].",International Journal of Molecular Sciences,Senescence Functions,2021
Senescent Cells in Aging,"The aberrant accumulation of senescent cells, possibly due to decreased clearance and/or chronic induction, can lead to pathology, as exemplified in aging. Aging is a progressive degenerative state accompanied by loss of tissue homeostasis, decreased regenerative capacity, deterioration of the overall organs function, and increased risk of developing age-associated diseases, such as Alzheimer’s disease, cardiovascular diseases, and cancer [291]. The connection between cellular senescence and aging is supported by the observation that senescent cells accumulate in various tissues during aging, particularly in association with age-related dysfunctions [141,292]. For example, senescent cell burden is higher in adipose tissue of elderly women with frailty and physical dysfunction than in healthier elderly women [293]. Senescent cells also accumulate at sites of age-related chronic diseases, even in younger individuals, and transplantation of a relatively small number of senescent cells accelerates aging in healthy younger mice [294,295]. Importantly, reducing senescent cell burden in mice alleviates features of aging, reduces frailty, brings health benefits, and increases the lifespan of old animals [13,16,295–297]. However, some senescent cells have important structural and functional roles, and the removal of non-replaceable senescent cells in the liver actually shortens the lifespan of mice [298].",International Journal of Molecular Sciences,Senescence and Aging,2021
Cellular Mechanisms Linking Senescence and Aging,"Cellular senescence drives tissue aging and associated disorders by limiting the regenerative potential of stem cell pools and undifferentiated progenitor cells and by increasing chronic inflammation, ECM degradation, and metabolic dysfunction [26,288,299]. As previously discussed, these cell and tissue changes reflect the decline in mitochondrial function, loss of proteostasis, altered intercellular communication, deregulated nutrient sensing, epigenetic profile changes, and defects in DNA repair that lead to genomic instability and damage, including telomere dysfunction [299]. Historically, the discovery that telomere erosion acts as a “mitotic clock” at the cellular level [9] led to the hypothesis that telomere erosion and replicative senescence could concomitantly contribute to organismal aging. Transgenic mouse models without or with inducible telomerase [300,301] allowed establishing a link between short telomeres and the onset of aging phenotypes [300,302,303], progeroid syndromes [304], and chronic inflammatory and degenerative conditions [305,306]. Reactivation of endogenous telomerase reverses tissue degeneration in mice with telomere dysfunction [301].",International Journal of Molecular Sciences,Mechanisms of Aging,2021
Telomere Shortening and Age-Related Senescence,"To unveil the link between progressive telomere shortening and the onset of the human aging phenotype, telomere length in function of age has been extensively studied in blood cells and in various tissues. A consistent mean telomere length shortening and changes in the abundance of short telomeres over time correlate with aging in humans [307,308]. Most tissues, both highly and slowly proliferative, show a decrease in telomere length associated with age [309,310]. However, it is not clear whether this erosion leads to a telomere length short enough to trigger senescence. In addition to telomere erosion, increased oxidative damage, DNA damaging agents, and metabolic changes also may induce cellular senescence in proliferative tissues during normal aging. This DNA damage that occurs independently of telomere shortening may be localized at telomeres [109,115], and it is based on sporadic damage that might contribute to tissue dysfunction and the aging process [13,296]. Indeed, stress-induced senescent cells increase with age [108], and the rate of senescent cell accumulation in some tissues quantitatively predicts lifespan in mouse strains [251].",International Journal of Molecular Sciences,Telomere Dynamics,2021
Senescence Markers Across Tissues and Cell Types,"Biomarkers of senescent cells have been identified in a wide range of tissues in vivo, from tissues with high proliferative index to non-replicating tissues. Senescence markers have been detected in stem cells and somatic cells of aged mice [311] as well as in skin of old baboons and humans [107,232]. Non-dividing cell types, such as neurons and cardiomyocytes, also can display senescence features. Age-dependent accumulation of neurons expressing senescence markers (high ROS, IL-6 production, chromatin reorganization, SA-β-Gal activity) occurs in normally aging mice [312]. Senescent post-mitotic neurons may contribute to neurodegeneration, cognitive decline, and dementia or Alzheimer’s disease [313]. Similarly, senescence markers are enriched in post-mitotic cardiomyocytes in aging mice [314] and may be associated with diminished cardiac function. Although replicative senescence is rare in post-mitotic cells, random DNA damage and persistent DDR signaling may promote senescence-like states [312]. Identification of senescent cells in vivo is limited by the lack of specific markers, requiring multiple complementary markers for proper assessment [315].",International Journal of Molecular Sciences,Senescence Markers,2021
Causal Role of Senescent Cells in Aging,"Direct evidences that senescent cells contribute to age-related tissue dysfunction started to emerge from a transgenic mouse model known as INK-ATTAC in which p16-expressing senescent cells can be specifically eliminated by apoptosis [296]. When this transgenic model is bred in a progeroid mouse genetic background, clearance of p16-positive senescent cells decreases the age-associated dysfunctions and attenuates progression of aged-related disorders [296]. In addition, elimination of senescent cells from naturally aged INK-ATTAC mice extends their healthy lifespan and delays age-associated deterioration of several organs [13]. The physiological accumulation of senescent cells at sites of age-associated pathologies could contribute to the pathogenesis of these diseases. Other studies in mice have demonstrated the contribution of senescent cells to the pathogenesis of age-associated diseases, such as atherosclerosis, cardiovascular diseases, frailty, and osteoarthritis [3,14,15,295,316,317]. For example, in advanced atherosclerosis, plaques contain cells harboring senescence markers, and their clearance leads to a reduction in plaque number and size [316].",International Journal of Molecular Sciences,Senescence and Disease,2021
Senolytics and Functional Rejuvenation,"The elimination of naturally occurring p16-expressing cells in the joints of naturally aged INK-ATTAC transgenic mice decreases age-associated cartilage degradation [317]. Clearance of senescent cells with senolytic drugs (i.e., pharmacological agents that selectively kill senescent cells) improves cardiovascular function in old mice and extends the healthy lifespan of Ercc1−/Δ mice that display progeroid features [3]. Similarly, depletion of senescent cells in normally aged mice after treatment with a senolytic drug rejuvenates hematopoietic stem cells and muscle stem cells [14]. Clearance of senescent cells, using senolytic drugs or in INK-ATTAC transgenic mice, reduces physical dysfunction and extends the lifespan of old mice [295]. Such studies confirm that senescent cells are a typical feature and a contributor to aging and age-related disorders, but the underlying mechanisms are incompletely understood. These effects might be mediated through reduction in the regenerative capacity of progenitor and stem cells or through the SASP detrimental effects on the microenvironment.",International Journal of Molecular Sciences,Senolytic Therapies,2021
Mechanisms of Senescence-Driven Pathology,"Permanent cell cycle arrest of senescent stem cells and progenitor cells leads to their exhaustion, and this can directly impair tissue maintenance, function, and regeneration. Age-related increased p16 expression in hematopoietic stem cells, central nervous system, and pancreatic islets is associated with decreased self-renewal capacity, partly improved by p16 inhibition [292,318,319]. Regeneration of skeletal muscle relies on stem cells that remain quiescent until needed for tissue repair. In old mice, these cells switch to a senescence state caused by p16 expression and lose their self-renewal capacity. Inhibition of p16 restores their regenerative potential [320]. The SASP also compromises cell renewal and organ regeneration/function [321,322]. Chronic exposure to IL-1α reduces hematopoietic stem cell renewal [322], while activin A secretion by senescent fat progenitors inhibits adipogenesis [321]. Senescent fibroblasts accumulate in lungs of idiopathic pulmonary fibrosis patients [323], and senolytic treatments show therapeutic benefit [324]. In mouse fibrosis models, depletion of senescent cells improves lung function even if fibrosis persists [325].",International Journal of Molecular Sciences,SASP and Tissue Dysfunction,2021
Inflammaging and Systemic Effects of SASP,"Systemic chronic low-level inflammation, termed inflammaging, may underlie aging and most age-related pathologies [326]. Inflammatory cytokines such as IL-1α, IL-6, and TNF-α are SASP components associated with inflammaging [327,328]. Mouse models where SASP is specifically altered show that senescent cells contribute to inflammation, regeneration defects, and aging. For example, Nfkb1−/− mice accumulate senescent cells and display chronic inflammation and reduced tissue regeneration, reversible with anti-inflammatory treatment [251]. Conditioned medium from senescent adipocytes induces inflammation in healthy adipose tissue, and SASP inhibition reduces systemic inflammation and enhances physical function in old mice [329]. Interferon-response downregulation reduces inflammaging and improves aging phenotypes [177]. JAK inhibition reduces SASP, increasing bone mass and strength in old mice [294]. Senescent cells also spread senescence to neighboring cells via SASP factors [252,270,330]. IL-1 and TGF-β induce paracrine senescence by generating ROS and DDR signaling in adjacent cells [252,331].",International Journal of Molecular Sciences,Inflammaging,2021
Senescence and COVID-19 Severity,"Accumulation of senescent cells appears to increase the risk of severe outcomes in COVID-19. Mortality is highest in older individuals and those with chronic diseases [332]. Disease severity correlates with increased senescence markers in airway epithelial cells and elevated SASP factors in serum [333–335]. SARS-CoV-2 infection induces cellular senescence with a strong pro-inflammatory phenotype [333,334]. In older patients, infection can amplify SASP activity, exacerbating systemic inflammation and promoting paracrine senescence. Inefficient clearance of infected senescent cells may contribute to hyperinflammation and multi-organ damage [336]. Reducing senescent cells in aged mice lowers mortality after infection with a related β-coronavirus [337], suggesting that senescence is an important driver of severe COVID-19. Persistent senescent cells after recovery may contribute to long COVID symptoms in older patients [338].",International Journal of Molecular Sciences,Senescence and COVID-19,2021
Immune Clearance Failure with Aging,"It is not clear why senescent cells accumulate in many tissues during aging. During wound healing or tissue repair, senescent cells attract immune cells that later clear them [290]. Senescent cells also express immunogenic markers enabling immune-mediated elimination [339]. With aging, immune dysfunction may reduce the capacity to recognize and clear senescent cells, or immune clearance may become overwhelmed. Chronic inflammation may further impair immune function [290]. In mice lacking immune cytotoxicity, senescent cells accumulate with age alongside increased inflammation and accelerated aging [340]. However, senescent cells can persist even in young individuals, such as young mice [341,342], chemotherapy-treated young women [343], and children with benign nevi [19]. Persistent senescent cells may reduce SASP inflammatory signals over time or alter SASP composition [133,150], or evade immunity by upregulating HLA-E, inhibiting CD8+ T cell and NK cell activity [344]. Understanding immune evasion by senescent cells may guide therapies that enhance senescent cell clearance.",International Journal of Molecular Sciences,Immune Clearance Failure,2021
Senescence in Cancer Initiation and Progression,"Cellular senescence plays important but contrasting roles in different steps of tumorigenesis, such as tumor initiation, establishment, and escape. Under some conditions, it represents a potent tumor-suppressive barrier by blocking the proliferation of damaged cells. In other settings, senescent cells may facilitate cancer progression.",International Journal of Molecular Sciences,Senescence and Cancer,2021
Senescence as a Tumorigenesis Barrier,"The senescence program can be activated in normal, pre-neoplastic, and malignant cells in response to a wide variety of stimuli. Critically short, uncapped telomeres [9], oncogene stresses [51], and mitochondrial dysfunction [225] result in proliferative stress and senescence induction, thereby halting the proliferation of cells harboring mutations or genomic instability. The proliferative arrest of preneoplastic cells represents a protective barrier because cancer cells must replicate to produce a macroscopic tumor. Unlimited proliferation is the main means of acquiring oncogenic mutations and fixing the successive genomic alterations that drive clonal expansion and cancer progression [345]. Critically shortened telomeres are frequently observed in early neoplastic lesions [86,346,347], and too short telomeres can induce senescence. Loss of p53 and RB bypassed senescence in cultured human fibroblasts and allowed entry into crisis, which is considered as a second barrier to cancer formation [348].",International Journal of Molecular Sciences,Senescence as Cancer Barrier,2021
Telomere Crisis and Early Tumor Evolution,"This extended proliferative period exacerbates telomere shortening and leads to chromosomal fusions and anaphase bridges. These fused chromosomes can initiate bridge–breakage–fusion cycles, causing more chromosome rearrangements and genomic instability that promote mitotic crisis [349,350]. This mitotic crisis in culture leads to the death of most cells but also produces the very rare immortal cell that has acquired telomerase activity and extensive gene copy number alterations [351,352]. Almost all cancer cells show defects in senescence-controlling signaling pathways downstream of telomere erosion (i.e., the p53 and RB pathways) that allow them to proliferate to the point of telomere crisis. Short telomeres and a sharp increase in genome instability are observed in the early stages of breast cancer before telomerase activation and genome stabilization [353]. Studies in human tumors revealed telomeric fusions indicative of telomere crisis [347,352,354,355], suggesting malignant progression requires crisis-associated genome rearrangements [349,351].",International Journal of Molecular Sciences,Telomere Crisis,2021
Oncogene-Induced Senescence as Tumor Suppression,"Replication stress induced by some oncogenes is a second stringent tumor suppressive mechanism in which the persistent DDR signal could cause senescence. DDR activation and senescence induced by unscheduled DNA replication are frequently observed in early stages of cancer lesions [69,70,356,357]. For example, senescence occurs during the formation of benign cutaneous melanocytic nevi expressing an oncogenic form of BRAF [19]. Malignant progression requires bypass of OIS, typically via additional mutations disabling senescence pathways. In mouse models, inactivating senescence machinery accelerates tumor development, whereas restoration of senescence in growing tumors causes regression [266,358].",International Journal of Molecular Sciences,Oncogene-Induced Senescence,2021
SASP: Tumor Suppression via Immune Recruitment,"Senescent cells communicate with and influence the behavior of neighboring cells partly through SASP factors. SASP-driven paracrine signaling has both tumor-suppressive and tumor-promoting roles. Many SASP factors exert tumor-suppressive activities through autocrine and paracrine signaling that enforce senescence in neighboring cells [153,154,263,271,272,358]. SASP can also mediate non-cell-autonomous tumor suppression by attracting and activating immune cells. Recruitment of T cells and natural killer cells promotes removal of senescent or damaged cells [29,358–360]. In a mouse hepatocellular carcinoma model, premalignant hepatocytes expressing oncogenic Ras were cleared via CD4+ T cell–mediated immune surveillance driven by SASP cytokine secretion [263]. Loss of immune clearance in immunocompromised mice promoted liver cancer development.",International Journal of Molecular Sciences,SASP Tumor Suppression,2021
SASP-Mediated Tumor Promotion,"Paradoxically, SASP can also facilitate cancer progression by promoting growth of pre-neoplastic cells and altering the tumor microenvironment. Conditioned medium from senescent fibroblasts promotes growth of pre-malignant and malignant epithelial cells but not normal cells [22,147]. ECM remodeling and matrix-degrading proteases secreted by senescent cells enhance tumor cell motility, invasion, and metastasis. Senescent stromal cells can induce epithelial-to-mesenchymal transition, a major tumor progression mechanism [23]. Matrix metalloproteinases from senescent fibroblasts enhance tumor growth in xenografts [361]. In vivo, SASP from senescent hepatocytes accelerates liver tumor growth, and peritumoral senescence correlates with poor survival in hepatocellular carcinoma patients [362]. SASP inhibition via mTOR inhibitor rapamycin suppresses senescent fibroblast–stimulated prostate tumor growth [163].",International Journal of Molecular Sciences,SASP Tumor Promotion,2021
Therapy-Induced Senescence (TIS),"Senescent cells can arise within tumors following anticancer treatments. Although chemotherapy is designed to induce apoptosis, some tumor cells enter senescence after DNA damage [363,364]. Senescence markers have been detected in breast cancer samples from patients treated with chemotherapy [343]. Senescent tumor cells stop dividing, possibly blocking further cancer growth. Their secretome, or that of neighboring senescent stromal fibroblasts, can induce paracrine senescence of adjacent tumor cells [270] and recruit immune cells that contribute to cancer cell removal [263,266]. However, senescent tumor cells can also promote inflammation, enhancing tumor growth and invasiveness [148,154,163], and impair immune clearance. Persistent senescent tumor cells may drive chronic inflammation and chemotherapy resistance [363].",International Journal of Molecular Sciences,Therapy-Induced Senescence,2021
Senescence Escape and Tumor Relapse,"A major issue of therapy-induced senescence is that senescence in tumor cells can be incomplete because cell cycle exit may not be irreversible [365]. Tumor cells that escape senescence can resume proliferation, contributing to relapse [342]. Notably, tumor cells escaping senescence after chemotherapy display stem cell–like characteristics and show higher tumor initiation potential in vivo [366].",International Journal of Molecular Sciences,Senescence Escape,2021
Overview of Mitochondrial Biogenesis Regulation,"Although it is well established that physical activity increases mitochondrial content in muscle, the molecular mechanisms underlying this process have only recently been elucidated. Mitochondrial dysfunction is an important component of different diseases associated with aging, such as Type 2 diabetes and Alzheimer’s disease. PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) is a co-transcriptional regulation factor that induces mitochondrial biogenesis by activating different transcription factors, including nuclear respiratory factor 1 and nuclear respiratory factor 2, which activate mitochondrial transcription factor A. The latter drives transcription and replication of mitochondrial DNA. PGC-1α itself is regulated by several different key factors involved in mitochondrial biogenesis, which will be reviewed in this chapter. Of those, AMPK (AMP-activated protein kinase) is of major importance. AMPK acts as an energy sensor of the cell and works as a key regulator of mitochondrial biogenesis. AMPK activity has been shown to decrease with age, which may contribute to decreased mitochondrial biogenesis and function with aging. Given the potentially important role of mitochondrial dysfunction in the pathogenesis of numerous diseases and in the process of aging, understanding the molecular mechanisms regulating mitochondrial biogenesis and function may provide potentially important novel therapeutic targets.",Essays in Biochemistry,Mitochondrial Biogenesis,2010
Definition and Genetic Basis of Mitochondrial Biogenesis,"Mitochondrial biogenesis can be defined as the growth and division of pre-existing mitochondria. Mitochondria are direct descendants of an α-protobacteria endosymbiont that became established in a host cell. Owing to their bacterial origin, mitochondria have their own genome and can autoreplicate. Mitochondrial proteins are encoded by the nuclear and the mitochondrial genomes. The mtDNA (mitochondrial DNA) is a double-stranded circular molecule of approx. 16.5 kb that contains 37 genes encoding 13 subunits of the electron transport chain complexes I, III, IV and V. The only other mitochondrial gene products are the 22 tRNAs and two rRNAs necessary for translation of the respiratory subunit mRNAs within the mitochondrial matrix. Correct mitochondrial biogenesis requires the co-ordinated synthesis and import of approx. 1000–1500 proteins encoded by the nuclear genome and synthesized on cytosolic ribosomes [1]. mtDNA replication, as well as mitochondrial fusion and fission, must also be co-ordinated.",Essays in Biochemistry,Mitochondrial Structure and Genetics,2010
Environmental Regulation of Mitochondrial Biogenesis,"Mitochondrial biogenesis is influenced by environmental stress such as exercise, caloric restriction, low temperature, oxidative stress, cell division and renewal and differentiation. Mitochondrial biogenesis is accompanied not only by variations in number, but also in size and mass. As the majority of proteins are encoded in the nucleus, a mechanism for targeting, import and correct assembly exists to ensure correct mitochondrial function and shape. mRNAs are translated in the cytosol after activation of the nuclear genome to precursor proteins. These precursor proteins are synthesized with an N-terminal positively charged presequence capable of forming a basic, amphipathic α-helix. They traverse inner and outer mitochondrial membranes in an unfolded conformation by crossing through protein translocases. The mitochondrial membrane potential and the action of matrix Hsp70 (heat-shock protein 70) are used to drive translocation. The presequence is then cleaved by a matrix protease and, often with the aid of molecular chaperones, the imported protein is folded.",Essays in Biochemistry,Environmental Regulation,2010
Protein Import Machinery in Mitochondrial Biogenesis,"A majority of mitochondrial protein precursors use different protein-import pathways to the other mitochondrial compartments. Precursor proteins that follow these routes do not typically contain the N-terminal targeting signals but harbour targeting information within their mature sequence. Currently, four major membrane protein translocase complexes are known. The TOM (translocase of the outer membrane) is the universal entry gate for all proteins that are imported into mitochondria. The different protein pathways then diverge at this point. TIM (translocase of the inner membrane) sorts matrix-targeted precursors. The PAM (presequence translocase-associated motor) regulates matrix Hsp70 action to drive precursors into the matrix. Finally, the outer membrane SAM (sorting and assembly machinery) inserts β-barrel proteins into the outer membrane. All of these processes are an integral part of mitochondrial biogenesis.",Essays in Biochemistry,Mitochondrial Protein Import,2010
Mitochondrial Biogenesis and Endurance Exercise,"It is well established that physical activity increases mitochondrial content. The first observations to establish this association were done by comparing the mitochondrial content in different muscle groups in animals. For example, it has been found that the breast muscle of chickens, which fly infrequently, contain less mitochondria than the breast muscle of pigeons, which are able to fly for long periods [3]. Other studies demonstrated that continuously working muscles, such as the heart, had more mitochondrial activity and content than sporadically functioning muscles, such as back muscles [3]. These early studies in animals suggested that muscles involved in regular and sustained physical activity can increase their mitochondrial activity and content. In one study, 6-week-old rats subjected to exercise 5 days per week for 3 months showed an increase in skeletal muscle cytochrome c concentration, as well as increased activities of key mitochondrial enzymes and OXPHOS [4]. Similar findings were later confirmed in humans [5,6]. Trained individuals and elite runners had higher proportions of oxidative slow-twitch fibers and more succinate dehydrogenase activity compared with sedentary controls [7]. Despite this clear link to exercise, the molecular regulators of mitochondrial biogenesis remained elusive until recently.",Essays in Biochemistry,Exercise-Induced Mitochondrial Biogenesis,2010
PGC-1α as Master Regulator of Biogenesis,"PGC-1α (PPAR-γ coactivator-1α) is a major regulator of mitochondrial biogenesis [8] discovered after NRF-1, NRF-2, and Tfam (mitochondrial transcription factor A). In the experiment leading to its discovery, mice exposed to cold (4°C) showed increased PGC-1α mRNA in brown fat and skeletal muscle, along with increased ATP synthase β-subunit and COX (cytochrome c oxidase) subunits. Experiments in rats confirmed a connection between PGC-1α and mitochondrial biogenesis, as electrical stimulation increased COX activity [9]. PGC-1α induces mitochondrial biogenesis by activating NRF-1 and NRF-2, which activate Tfam. NRF-1/2 interact with Tfam to drive mtDNA transcription and replication [10]. In mouse C2C12 myotubes, PGC-1α expression increased mtDNA content, respiration, UCP2 expression, and NRF-1/2 gene expression. PGC-1α also directly co-activates NRF-1 on the Tfam promoter [11]. Acute exercise (e.g., swimming) increases PGC-1α protein levels and enhances NRF-1 and NRF-2 promoter binding (δ-ALAS, COX IV) [12].",Essays in Biochemistry,PGC-1α Regulation,2010
PGC-1α Interaction with Additional Transcription Factors,"In addition to NRF-1 and NRF-2, PGC-1α co-activates several other transcription factors, including PPARs, thyroid hormone receptors, glucocorticoid receptors, estrogen receptors, and ERRα/ERRγ (orphan nuclear receptors). ERRs target extensive gene networks involved in energy homeostasis, fat and glucose metabolism, and mitochondrial biogenesis and function [13,14]. Together, these findings show that PGC-1α links external stimuli such as cold or exercise to internal metabolic adaptation through the NRF transcription cascade and related networks.",Essays in Biochemistry,PGC-1α Transcriptional Network,2010
Role of PGC-1β in Mitochondrial Biogenesis,"PGC-1β shares molecular structure and function with PGC-1α, including nuclear receptor binding and transcriptional activation, and also regulates mitochondrial biogenesis. Ectopic expression of PGC-1β (e.g., in L6 myoblasts) induces mitochondrial biogenesis and increases basal oxygen consumption. However, unlike PGC-1α, PGC-1β is not upregulated in brown adipose tissue after cold exposure, nor in skeletal muscle in response to exercise [15]. This suggests that PGC-1α and PGC-1β are activated independently, but both regulate mitochondrial biogenesis through NRF-1 to support cellular energetic demands.",Essays in Biochemistry,PGC-1β Function,2010
CaMKIV and Mitochondrial Biogenesis,"CaMKIV is another major regulator of mitochondrial biogenesis. It was first noticed that CaMKIV influences gene expression in oxidative fibres of myocytes [22]. To assess its role, a transgenic mouse model muscle-specifically expressing an active form of CaMKIV was created. The skeletal muscle of these mice contained more copies of mtDNA and possessed more mitochondria as a percentage of myocyte volume [23]. mRNA expression of cytochrome b and carnitine palmitoyltransferase-1 was also increased. However, studies in CaMKIV-null mice found similar protein levels of PGC-1α and COX IV compared with wild-type mice, and running increased PGC-1α and COX IV to the same extent in both groups [24]. CaMKIV protein could not be detected in murine skeletal muscle and appears absent in human skeletal muscle, unlike CaMKII, which has an important exercise role [25]. Thus, the role of CaMKIV in exercise-induced mitochondrial biogenesis remains unclear.",Essays in Biochemistry,CaMKIV and Biogenesis,2010
Nitric Oxide (NO) and Mitochondrial Biogenesis,"NO has recently been shown to be involved in mitochondrial biogenesis. HeLa cells expressing eNOS (endothelial NO synthase) display increases in mtDNA content, cytochrome c and COX IV protein expression, as well as PGC-1α, NRF-1 and Tfam mRNA expression [26]. NO produced by eNOS activates guanylate cyclase, increasing cGMP levels, which transmits a signal to the nucleus through an unknown mechanism, leading to PGC-1α transcription and mitochondrial biogenesis.",Essays in Biochemistry,Nitric Oxide and Biogenesis,2010
SIRT1 Activation and Mitochondrial Biogenesis,"SIRT1 (silent mating type information regulation 2 homologue 1) is another activator of PGC-1α. SIRT1 protein is induced in liver during fasting through a nutrient-signalling response mediated by pyruvate kinase. SIRT1 activates PGC-1α by deacetylation [27]. Resveratrol, a phytoalexin produced by some plants, activates SIRT1 and enhances mitochondrial biogenesis in skeletal muscle while improving exercise tolerance [28]. However, recent work indicates resveratrol may not be a direct activator of SIRT1 [28a].",Essays in Biochemistry,SIRT1 and Biogenesis,2010
TORC Proteins and PGC-1α Transcription,"PGC-1α gene transcription is strongly activated by TORC1, a co-activator of CREB. TORC2 and TORC3 also strongly activate PGC-1α transcription. Forced expression of TORCs in primary muscle cells induces endogenous PGC-1α mRNA and its downstream target genes in the mitochondrial respiratory chain and TCA cycle, providing further evidence for the key role of PGC-1α in mitochondrial biogenesis [29].",Essays in Biochemistry,TORC and PGC-1α,2010
Calcineurin and Mitochondrial Gene Regulation,"Calcineurin is a calcium/calmodulin-dependent protein phosphatase and a master regulator of fast-to-slow muscle fibre-type changes [30]. Transgenic mice overexpressing calcineurin show increased slow-twitch fibres and increased skeletal muscle PGC-1α expression, leading to improved insulin action [31]. Inhibition of calcineurin by cyclosporin promotes slow-to-fast fibre transformation [30]. Calcineurin upregulates many genes involved in mitochondrial energy metabolism in cultured cardiomyocytes [32]. However, cyclosporin did not prevent up-regulation of mitochondrial markers in training rats [33]. Thus, calcineurin contributes to mitochondrial biogenesis but does not fully account for exercise-induced adaptations.",Essays in Biochemistry,Calcineurin Signaling,2010
p38 MAPK and Exercise-Induced Biogenesis,"p38 MAPK has an important role in myogenic differentiation [34]. Its activation increases following muscle contraction and running exercise in rodents [35] and humans [36]. Transgenic mice overexpressing p38 MAPK in skeletal muscle show enhanced PGC-1α expression and increased mitochondrial proteins [37]. Acute exercise in mice or rats increases p38 MAPK, leading to PGC-1α activation [37,38]. These findings indicate p38 MAPK plays a key role in exercise-induced mitochondrial biogenesis.",Essays in Biochemistry,p38 MAPK and Biogenesis,2010
RIP140 as a Negative Regulator of Mitochondrial Biogenesis,"RIP140 is a transcriptional co-repressor implicated in mitochondrial biogenesis regulation [39]. It is highly expressed in glycolytic (type II) skeletal muscles and lower in oxidative (type I) muscles [40]. RIP140-null mice show increased mitochondrial gene expression, oxidative capacity, and resistance to diet-induced obesity due to elevated energy expenditure [41]. These mice have more oxidative fibres in extensor digitorum longus. Conversely, muscle-specific overexpression of RIP140 produces more glycolytic fibres and reduces mitochondrial gene expression [39]. RIP140-null adipocytes also show increased mitochondrial biogenesis, fatty acid oxidation, and increased UCP1 expression [42]. These findings reveal opposing roles of RIP140 and PGC-1α in metabolic regulation.",Essays in Biochemistry,RIP140 Regulation,2010
Sin3A and Co-Repressor Control of Metabolism,"Sin3A is a core component of a multiprotein co-repressor complex and its deficiency increases expression of genes involved in oxidative metabolism [43]. It is unclear how the Sin3A complex regulates mitochondrial gene expression, but it may modulate nuclear receptor function through interactions with nuclear receptor co-repressors and silencing mediator complexes [44]. These data suggest that co-activators and co-repressors, including Sin3A, play essential roles in maintaining oxidative metabolism by modulating mitochondrial gene expression.",Essays in Biochemistry,Sin3A and Metabolic Regulation,2010
Clinical Assessment of Mitochondrial Function,"Reduction in mitochondrial function has been found to be associated with many pathologies associated with aging, such as Type 2 diabetes and Alzheimer’s disease [45]. Hepatic and intramyocellular lipid content can be measured non-invasively in vivo with 1H-MRS (magnetic resonance spectroscopy). The rate of ATP synthesis can be assessed by direct observation of 31P-magnetization transfer between Pi and ATP using phosphorus 31P-MRS. Finally, 13C spectroscopy can be used to trace the incorporation of [2-13C]-labelled acetate into glutamate in skeletal muscle during a constant infusion of [2-13C]acetate. The rate of incorporation reflects TCA cycle (Krebs cycle) activity providing an index of mitochondrial function [46,47]. Using these methods, Petersen et al. [46] found reductions in mitochondrial oxidative and phosphorylation activity, as well as increased intramyocellular and intrahepatocellular lipid content and skeletal muscle insulin resistance in aging. Recent studies using similar 13C-MRS methods have also found reductions in neuronal mitochondrial TCA metabolism associated with healthy aging [48].",Essays in Biochemistry,Mitochondrial Dysfunction in Aging,2010
Inherited Mitochondrial Defects and Insulin Resistance,"It is not clear why mitochondrial dysfunction occurs with aging, but age-associated accumulation of mutations in mtDNA may play a role. In further studies, Petersen and co-workers [47,49] measured basal rates of mitochondrial function in human skeletal muscle of young, lean and sedentary insulin-resistant offspring of parents with Type 2 diabetes. They found an approx. 30% reduction in rates of ATP synthesis in muscle using 31P-MRS, and a similar reduction in muscle mitochondrial TCA flux using 13C-MRS [47]. These young, lean and sedentary insulin-resistant subjects also exhibited increased intramyocellular lipid content, which has previously been associated with insulin resistance [50]. Muscle biopsy studies performed in similar subjects showed that mitochondrial density assessed by electron microscopy was reduced by 38% compared with control subjects [51]. These findings suggest that reduced mitochondrial activity may be due to reduced mitochondrial content, providing evidence that hereditary mitochondrial dysfunction contributes to the development of insulin resistance and Type 2 diabetes.",Essays in Biochemistry,Inherited Mitochondrial Dysfunction,2010
Altered Gene Expression in Type 2 Diabetes,"These data further suggest that alterations in nuclear-encoded genes regulating mitochondrial biogenesis may form a genetic basis for inheritance of some forms of Type 2 diabetes. Two studies found reduced NRF-1-dependent gene expression and reduced PGC-1α and PGC-1β expression in skeletal muscle of Type 2 diabetic subjects and overweight first-degree relatives of Type 2 diabetic subjects [52,53]. However, these results could not be replicated in young lean insulin-resistant offspring of parents with Type 2 diabetes [51], suggesting that other factors are responsible for reduced mitochondrial content in these individuals. More recently, Petersen et al. [54] found profound reductions in insulin-stimulated rates of muscle mitochondrial ATP production in insulin-resistant offspring, demonstrating alterations in both basal and insulin-stimulated mitochondrial metabolism.",Essays in Biochemistry,Mitochondrial Gene Expression in Type 2 Diabetes,2010
Physical Inactivity and Mitochondrial Biogenesis,"Physical inactivity can be a confounding factor in studies of mitochondrial biogenesis, especially in Type 2 diabetes. The above-mentioned studies describe skeletal muscle mitochondrial biogenesis in physical-activity-matched subjects, all of whom were sedentary. Taken together, these data demonstrate decreased mitochondrial activity, most likely attributable to reduced mitochondrial content, in healthy young lean individuals prone to develop Type 2 diabetes. A key unanswered question is whether reduced mitochondrial function causes increased intramyocellular lipid content and insulin resistance, or whether mitochondrial dysfunction is secondary to insulin resistance or another factor. Given the key role of intracellular lipid accumulation in the pathogenesis of insulin resistance, reduced mitochondrial activity would be expected to exacerbate metabolic dysfunction.",Essays in Biochemistry,Inactivity and Mitochondrial Deficits,2010
TZDs and Mitochondrial Biogenesis,"TZDs (thiazolidinediones) are activators of PPARγ. TZDs include pioglitazone and rosiglitazone, which are frequently used in the treatment of Type 2 diabetes. Pioglitazone has been shown to induce mitochondrial biogenesis by the activation of the PGC-1α pathway in human subcutaneous adipose tissue [55], but also in HUVECs (human umbilical vein endothelial cells), along with a reduction of mitochondrial ROS (reactive oxygen species) [56]. Rosiglitazone treatment for 8 weeks also increased expression of PGC-1α and activity of oxidative enzymes in skeletal muscle of patients with Type 2 diabetes [57]. These effects of TZDs on skeletal muscle mitochondria are secondary to PPARγ activation in adipocytes, leading to a subsequent increase in adiponectin and mtDNA copy number in human muscles [58]. A potential direct effect of TZDs on muscle cannot be excluded. Indeed, a study showed that rosiglitazone increased AMPK activity in muscle in Type 2 diabetes and obesity [59]. Metformin, another frequently used drug in the treatment of Type 2 diabetes, has also been shown to increase the skeletal muscle content of PGC-1α in rats, suggesting increased mitochondrial biogenesis. These effects seem to be mediated, at least in part, by an increase in AMPK phosphorylation [60].",Essays in Biochemistry,Diabetes Drugs and Mitochondria,2010
Caloric Restriction and Oxidative Stress,"Caloric restriction without malnutrition is a well-known dietary intervention that consistently increases median and maximal life expectancy by delaying the aging process in a wide variety of species, including flies and mice [61]. Oxidative stress theory suggests that the aging process involves the accumulation of oxidative damage to mitochondria and other cellular components. Oxidative damage is induced by ROS, which are a by-product of mitochondrial OXPHOS, a process that is responsible for approx. 85–90% of cellular oxygen consumption [62]. However, this theory remains controversial, as some studies involving mouse models with respiratory chain deficiency induced by tissue-specific mtDNA depletion or by a massive increase of point mutations in mtDNA have very minor or no increase of oxidative stress [63]. Therefore future studies are needed to address the relative importance of mitochondrial dysfunction and ROS in aging.",Essays in Biochemistry,Caloric Restriction and ROS,2010
Effects of Caloric Restriction on Mitochondrial Function,"Several studies have demonstrated that caloric restriction modulates cellular ROS production and damage to cellular macromolecules in various tissues. Studies in mammals have shown that caloric restriction reduces the generation of free radicals by mitochondria, as well as whole-body energy expenditure, and paradoxically induces mitochondrial proliferation [64]. In humans, a study examined the effect of caloric restriction on skeletal muscle mitochondrial function in non-obese humans and found that the expression of genes involved in the regulation of mitochondrial biogenesis, including PGC-1α, Tfam, eNOS and SIRT1, was increased in the caloric restriction group. In parallel, mitochondrial content was significantly increased in the caloric restriction group compared with the control group [65].",Essays in Biochemistry,Caloric Restriction Effects,2010
SIRT1 and AMPK in Caloric Restriction,"SIRT1-null mice are hypermetabolic, utilize ingested food inefficiently, have inefficient liver mitochondria and have elevated rates of lipid oxidation [66]. Their absence of lifespan extension has been interpreted as evidence that SIRT1 is required for the caloric restriction response [67]. AMPK may also play a role linking caloric restriction and mitochondrial biogenesis. AMPK regulates food intake by responding to hormonal and nutrient signalling in the hypothalamus [68]. Fasting induces AMPK in skeletal muscle [69], although caloric restriction seems less efficient. Long-term caloric restriction in mice does not change AMPK activity in skeletal muscle or heart, but increases it in liver [70]. Others have reported decreased AMPK activity in rat liver [71]. Short-term caloric restriction increases AMPK activity in the myocardium of young and old rats [72]. Thus caloric restriction may increase AMPK in rat heart, but not in skeletal muscle or liver.",Essays in Biochemistry,SIRT1 and AMPK,2010
Mechanistic Uncertainty in Caloric Restriction,"These data suggest that caloric restriction increases whole-body energy efficiency by inducing the biogenesis of mitochondria that utilize less oxygen and produce less ROS. However, the exact mechanisms linking caloric restriction and up-regulation of mitochondrial mass and function remain poorly understood.",Essays in Biochemistry,Caloric Restriction Mechanisms,2010
Overview of Mitochondrial Quality Control,"Mitochondria are dynamic organelles essential for energy production, metabolic homeostasis, calcium buffering, innate immunity, and programmed cell death. Due to constant exposure to reactive oxygen species (ROS), mitochondrial DNA mutations, and protein misfolding, mitochondria rely on multilayered quality control systems. These systems coordinate the removal of damaged proteins or mitochondrial fragments via mitophagy and the renewal of components through mitochondrial biogenesis. Because mitochondria contain proteins encoded by both nuclear and mitochondrial genomes, proper maintenance also depends on coordinated protein synthesis, import, and assembly. Mitochondrial quality control therefore operates at protein, organelle, and whole-cell levels, ensuring balanced mitochondrial turnover and sustained cellular function.",Current Biology,Mitochondrial Quality Control,2018
Mitophagy as a Selective Autophagy Pathway,"Mitophagy is a selective form of autophagy responsible for removing damaged or unnecessary mitochondria. While autophagy generally engulfs cytoplasmic components non-selectively, mitophagy specifically targets mitochondrial cargo using unique receptor and signalling proteins activated under defined conditions. The process still relies on core autophagy machinery: the ULK1 complex, Atg12–Atg5–Atg16 conjugation system, LC3/GABARAP lipidation, and SNARE-mediated autophagosome–lysosome fusion. Under physiological conditions, mitophagy does not eliminate the entire mitochondrial population, preserving a basal mitochondrial volume set by energy demands. Instead, it maintains equilibrium with mitochondrial biogenesis to ensure adequate mitochondrial function.",Current Biology,Mitophagy Mechanisms,2018
Mitochondrial Vulnerability and Damage Sources,"Mitochondria generate reactive oxygen species as by-products of oxidative phosphorylation, making their proteins, membranes, and mitochondrial DNA particularly susceptible to damage. Aberrant mtDNA copies can accumulate within a cell, creating heteroplasmy — the coexistence of mutant and wild-type genomes. The ratio of damaged to healthy mitochondrial DNA influences mitochondrial dysfunction and determines whether pathological phenotypes emerge. Beyond energy production, mitochondria are crucial for fatty acid synthesis, amino acid metabolism, heme synthesis, and the biogenesis of iron–sulfur clusters. They also serve as signaling platforms: MAVS on the outer mitochondrial membrane drives antiviral immune responses, while Bax and Bak activation during apoptosis triggers cytochrome c release and caspase activation.",Current Biology,Mitochondrial Damage and Stress,2018
Turnover: Balance Between Mitophagy and Biogenesis,"Mitochondrial turnover results from two opposing processes: degradation of defective organelles through mitophagy and replenishment through mitochondrial biogenesis. Cells must constantly replace mitochondrial components that have variable half-lives and are influenced by developmental cues and stress responses. The mitochondrial network is never fully removed, except in specialized contexts such as vertebrate lens fiber cell maturation. External factors, including nutrient availability, hormonal stimulation, and activity level, shape the steady-state mitochondrial volume. Some organisms exhibit extreme, reversible mitophagy responses — for example, ground squirrel cone photoreceptors lose mitochondria during hibernation but rapidly restore them upon arousal, demonstrating the plasticity of mitochondrial maintenance mechanisms.",Current Biology,Mitochondrial Turnover,2018
Integration with Other Quality Control Pathways,"Mitophagy is interconnected with additional mitochondrial quality control mechanisms, including the mitochondrial unfolded protein response (UPRmt), mitochondrial proteases, ubiquitin–proteasome-mediated degradation of outer membrane proteins, and the production of mitochondrial-derived vesicles. These systems act at different biological scales — from individual protein repair to organelle-level removal — to maintain mitochondrial homeostasis. Dysregulation of these pathways has been implicated in diverse diseases, including diabetes, neurodegeneration, and aging-related decline. Understanding how these quality control layers interact provides opportunities for developing targeted therapeutic strategies to restore mitochondrial function when these systems fail.",Current Biology,Mitochondrial Quality Control Pathways,2018
Mitophagy in Metazoans: Overview,"In mammals, mitophagy is activated in several physiological and stress-related contexts, including cellular differentiation, hypoxia, mitochondrial damage, and post-fertilization removal of paternal mitochondria. Multiple proteins function as mitophagy receptors in these contexts. These receptors typically reside on the outer mitochondrial membrane (OMM) and recruit autophagy machinery to initiate selective mitochondrial degradation. The specific receptor used depends on the biological scenario, highlighting that mitophagy is not a single, universal pathway but a context-dependent process crucial for maintaining mitochondrial quality and cellular homeostasis.",Current Biology,Mitophagy in Metazoans,2018
Mitophagy During Reticulocyte Maturation,"A key physiological example of mitophagy occurs during the maturation of reticulocytes into erythrocytes. As red blood cells mature, they must eliminate mitochondria and other organelles. The protein Nix (also called BNIP3L), an OMM-anchored receptor, is transcriptionally upregulated during this process. Nix-deficient mice display impaired mitochondrial clearance, leading to accumulation of undegraded mitochondria adjacent to autophagosomes and resulting in anemia due to the persistence of immature erythroid cells. Nix interacts with LC3 and GABARAP proteins via an LC3-interacting region (LIR), although regions adjacent to the LIR appear more critical for mitophagy. Importantly, mitochondrial clearance in reticulocytes occurs even when Atg5 or Atg7 are absent, indicating a non-canonical, Atg5/7-independent autophagy mechanism.",Current Biology,Reticulocyte Mitophagy,2018
Nix/BNIP3L as a Mitophagy Receptor,"Nix is a transmembrane outer mitochondrial membrane protein with a cytosolic N-terminus that contains an LC3-interacting region (LIR). Although the LIR interaction contributes to autophagosome recruitment, full mitochondrial clearance depends on additional residues surrounding amino acids 70–86. Nix expression is driven by differentiation cues, and its essential role in mitophagy is demonstrated by the failure of mitochondria to be eliminated in Nix-deficient red blood cells. This identifies Nix as a specialized mitophagy receptor required for erythrocyte maturation and illustrates how specific developmental programs rely on receptor-mediated selective autophagy.",Current Biology,Nix/BNIP3L Function,2018
Non-Canonical Mitophagy in Reticulocytes,"Unlike classical autophagy, which relies heavily on Atg5 and Atg7 for autophagosome formation, reticulocyte mitophagy proceeds even in the absence of these canonical factors. While deletion of ULK1 delays mitochondrial removal, the process still occurs, indicating that ULK1 participates but is not absolutely required. Nix-dependent mitophagy thus represents a non-canonical pathway that bypasses the standard Atg5–Atg7 machinery. This alternative route may allow reticulocytes to efficiently eliminate mitochondria even when canonical autophagy components are downregulated during terminal differentiation.",Current Biology,Non-Canonical Mitophagy,2018
ULK1 and Broader Roles in Mitophagy,"Beyond reticulocytes, ULK1 has a general role in mitophagy across tissues. ULK1-deficient mouse hepatocytes accumulate structurally abnormal mitochondria, and ULK1-deficient fibroblasts show increased mitochondrial content. These findings suggest that ULK1 contributes to mitochondrial turnover in basal and stress-induced contexts, complementing receptor-specific pathways such as Nix-mediated mitophagy. ULK1 therefore acts as part of the upstream autophagy initiation machinery necessary for efficient mitochondrial degradation in diverse cellular settings.",Current Biology,ULK1 in Mitophagy,2018
Hypoxia-Triggered Mitophagy: BNIP3 and Nix,"Under hypoxic conditions, the mitophagy receptors BNIP3 and Nix (also BNIP3L) are transcriptionally upregulated through HIF-1α activation. BNIP3 shares ~50% homology with Nix and contains a single-pass C-terminal transmembrane domain for OMM localization, as well as an LC3-interacting region (LIR). BNIP3 dimerization via its transmembrane domain is essential, as loss of this domain impairs autophagosome formation despite proper targeting. BNIP3 interacts with LC3/GABARAP, and phosphorylation of Ser17 and Ser24—flanking the LIR—regulates this interaction. Although originally thought to contain BH3 domains, BNIP3 and Nix possess atypical motifs that likely do not represent bona fide BH3 domains. Together, BNIP3 and Nix act as hypoxia-induced mitophagy receptors whose expression is tightly controlled by oxygen-sensing pathways.",Current Biology,Hypoxia-Induced Mitophagy,2018
PINK1–Parkin-Dependent Mitophagy: Pathway Overview,"The most extensively studied metazoan mitophagy pathway is governed by PINK1, a mitochondria-targeted kinase, and Parkin, a cytosolic E3 ubiquitin ligase. Under healthy conditions, PINK1 is imported via TOM/TIM complexes and cleaved by MPP and PARL, producing a fragment degraded through the N-end rule pathway. When mitochondrial import is blocked—e.g., upon membrane depolarization—PINK1 accumulates on the OMM. There, PINK1 autophosphorylates and phosphorylates both Parkin (Ser65) and ubiquitin, triggering a feed-forward amplification loop that activates Parkin's ubiquitin ligase activity. Activated Parkin ubiquitinates numerous OMM substrates, generating ubiquitin chains that serve as binding sites for autophagy receptors. This mechanism explains the strong enrichment of ubiquitinated OMM proteins during mitophagy and highlights how PINK1 acts as both a sensor and initiator of mitochondrial elimination.",Current Biology,PINK1–Parkin Pathway,2018
Molecular Activation of Parkin and Ubiquitin Phosphorylation,"Parkin is normally autoinhibited by its UPD/RING0 domain. PINK1-mediated phosphorylation of Parkin’s ubiquitin-like domain (Ser65) induces conformational changes that unlock its catalytic activity. PINK1 also phosphorylates ubiquitin itself at the same serine residue. Phosphorylated ubiquitin (phospho-Ub) binds Parkin, partially activating it, and enhances recruitment of autophagy adaptors. Phospho-Ub is structurally distinct, resistant to deubiquitinases, and accumulates on mitochondria, amplifying mitophagy initiation. This dual phosphorylation of ubiquitin and Parkin is sufficient to recruit, activate, and localize Parkin to damaged mitochondria, establishing an auto-amplifying signaling loop essential for robust mitophagy.",Current Biology,Parkin Activation,2018
Recruitment of Autophagy Receptors: NDP52 and OPTN,"Selective autophagy receptors bind ubiquitinated mitochondrial substrates and recruit autophagosomes. Although earlier models emphasized p62, experiments using receptor penta-knockout (p62, NBR1, NDP52, OPTN, TAX1BP1) cells show that NDP52 and OPTN are the essential (but redundant) receptors for PINK1–Parkin-dependent mitophagy. Their recruitment requires PINK1 kinase activity and intact ubiquitin-binding domains. TBK1, a kinase activated downstream of PINK1 and Parkin, phosphorylates OPTN and NDP52 to increase their affinity for ubiquitin/phospho-Ub and enhances their retention on damaged mitochondria. OPTN Ser177, Ser473, and Ser513 phosphorylation strengthens binding to LC3 and ubiquitin. Tissue-dependent expression patterns determine the relative importance of OPTN versus NDP52.",Current Biology,Mitophagy Receptor Activation,2018
ULK1 Recruitment and Autophagy Machinery Engagement,"Recruitment of autophagy initiation components such as ULK1 depends on receptor engagement. ULK1 fails to localize to damaged mitochondria in pentaKO cells or in NDP52–OPTN double knockouts. Conditional PINK1 expression is sufficient to recruit ULK1 when either receptor is present, demonstrating that receptor engagement upstream is necessary for autophagy machinery assembly. Surprisingly, LC3 proteins are dispensable for PINK1–Parkin mitophagy, indicating that LC3’s classical role in autophagosome formation is not essential here and may instead be involved in later steps such as autophagosome–lysosome fusion.",Current Biology,ULK1 and Autophagy Machinery,2018
Mitophagy Amplification: A Dual-Loop Model,"The PINK1–Parkin system operates via two amplification loops: (1) PINK1 phosphorylates ubiquitin, increasing phospho-Ub density on the OMM and recruiting more Parkin and receptors; (2) TBK1 phosphorylation of OPTN and NDP52 enhances their affinity for ubiquitin, leading to increased receptor accumulation. This dual-loop mechanism explains how a few damaged mitochondria rapidly trigger a robust and irreversible mitophagy response. Parkin ubiquitinates hundreds of OMM proteins, creating a dense ubiquitin landscape that accelerates mitophagosome formation. This revised view positions PINK1 as both sensor and effector, while Parkin serves primarily as an amplifier of the damage signal.",Current Biology,Mitophagy Signaling Amplification,2018
Maternal Inheritance of mtDNA and Rationale for Paternal mtDNA Elimination,"In nearly all metazoans, including humans, mice, Drosophila, and C. elegans, mitochondrial DNA is inherited exclusively from the mother. Deep sequencing across multiple human families reveals no evidence of paternal mtDNA leakage, supporting an active mechanism that eliminates paternal mitochondria after fertilization. This selective removal likely prevents harmful heteroplasmy, as mouse models with mixed mtDNA genomes show metabolic dysfunction, cognitive impairment, and elevated stress responses. Evolutionary theories propose that strict maternal inheritance ensures coordination between nuclear and mitochondrial genomes and prevents propagation of selfish mitochondrial mutations. Although autophagy has been implicated in paternal mtDNA elimination, the exact mechanistic pathways remain incompletely defined.",Current Biology,Maternal mtDNA Inheritance,2018
Mitophagy Machinery Involved in Removal of Paternal Mitochondria,"During early embryogenesis in mammals, paternal mitochondria are rapidly recognized and degraded. In pig and rhesus monkey embryos, p62, GABARAP, and the AAA-ATPase VCP/p97 localize to sperm-derived mitochondria. Blocking p62 or inhibiting VCP/p97 delays paternal mitochondrial removal, suggesting their involvement. VCP/p97 normally extracts ubiquitinated OMM proteins for proteasomal degradation and is also required for mitophagy initiation. Using mitochondrially targeted fluorescent Dendra2 reporters, knockdown of p62, PINK1, Fis1, or the Rab-GAP TBC1D15 inhibited paternal mitochondrial degradation. TBC1D15 binds to Fis1 and modulates Rab7 activity, facilitating autophagosome capture of mitochondria. Loss of Parkin or the mitochondrial E3 ligase Mul1 partially impairs paternal mitochondria removal; double knockdown results in strong retention, highlighting redundancy between these ubiquitin ligases.",Current Biology,Mitophagy in Fertilization,2018
Autophagy–Proteasome Crosstalk in Paternal Mitochondria Clearance,"Evidence suggests that paternal mitochondrial elimination relies on interplay between mitophagy and proteasomal degradation. VCP/p97, a key factor in outer mitochondrial membrane–associated degradation (OMMAD), is necessary for degrading OMM proteins before autophagic engulfment. PINK1–Parkin pathway components localize to paternal mitochondria, indicating that ubiquitin-dependent tagging may help recruit autophagy receptors or promote proteasomal priming. However, the exact degradative signal varies across species. Mammalian paternal mitochondria appear ubiquitinated, while C. elegans sperm mitochondria are not, suggesting diverse recognition strategies. Autophagy receptors such as p62 may bridge ubiquitinated substrates to LC3-positive membranes, supporting selective engulfment of paternal mitochondria.",Current Biology,Ubiquitin–Proteasome in Mitophagy,2018
Mitophagy-Related Pathways Across Metazoans,"Genetic screens in C. elegans show that disrupting autophagy, lysosomal function, or mitochondrial fission delays removal of paternal mtDNA. In flies, autophagy defects similarly impair clearance. Worm sperm mitochondria are not ubiquitinated, but fly paternal mitochondria may be, demonstrating a divergence in mitophagy signaling cues. Endonuclease G, a mitochondrial nuclease, contributes to paternal mtDNA degradation in both worms and flies, but through species-specific mechanisms. Although Parkin contributes partially to paternal mitochondria removal in mammals, Drosophila studies show Parkin-independent pathways also operate. Despite these mechanistic differences, paternal mtDNA is eventually eliminated in all species studied, except for transient persistence in certain mutants.",Current Biology,Cross-Species Mitophagy Mechanisms,2018
Redundancy and Species-Specific Mechanisms Ensuring Maternal mtDNA Transmission,"Even when key mitophagy genes such as p62, LC3, or proteins controlling mitochondrial fission are disrupted, paternal mtDNA is ultimately not inherited, indicating multiple redundant pathways protect maternal inheritance. Mutations in autophagy genes in worms (e.g., LC3 homologs) delay but do not prevent paternal mitochondrial clearance. In mammals, combined loss of Parkin and Mul1 has a more pronounced effect than either alone, suggesting overlapping E3 ligase activities targeting paternal mitochondria. Species-specific adaptations — such as differences in ubiquitination, nuclease involvement, and receptor usage — indicate that paternal mtDNA elimination is executed by diverse mechanisms that converge on a single evolutionary outcome: strict maternal inheritance.",Current Biology,Redundancy in mtDNA Inheritance Systems,2018
Core Mechanism of Yeast Mitophagy: Atg32 as the Selective Receptor,"In Saccharomyces cerevisiae, mitophagy is driven primarily by the mitochondrial outer membrane protein Atg32, which is essential for selective mitochondrial degradation under respiratory growth, starvation, or specific mitochondrial damage. Atg32 contains a transmembrane domain anchoring it to the outer membrane and a cytosolic N-terminal domain that binds directly to Atg11. Atg11 serves as a central adaptor responsible for recruiting the autophagosome machinery and mitochondrial fission components, enabling selective sequestration of mitochondrial segments. Atg32 levels increase significantly (5–10-fold) during respiratory conditions, and its overexpression enhances mitophagy. Activation of mitophagy requires Atg32 phosphorylation at Ser114 and Ser119, which promotes Atg11–Atg32 interaction. Blocking these phosphorylation events disrupts mitophagy. Thus, Atg32 functions as a phosphorylation-regulated mitophagy receptor acting independently of ubiquitin.",Current Biology,Yeast Mitophagy,2018
Regulation and Activation of Atg32,"Although Atg32 is constitutively expressed and mitochondrially localized, its abundance and phosphorylation state are dynamically regulated. During respiratory growth or metabolic transitions, Atg32 expression rises sharply, suggesting transcriptional or post-transcriptional control in response to metabolic demand. Mitophagy-inducing conditions promote phosphorylation by yet-unidentified kinases, enabling Atg32 to bind Atg11 and recruit the autophagy machinery. The upstream sensors that detect mitochondrial fitness and regulate Atg32 activation remain unknown. Further research is needed to identify the kinases/phosphatases responsible for Atg32 modification and to elucidate how mitochondrial stress is translated into mitophagic signals in yeast.",Current Biology,Atg32 Regulation,2018
Comparison of Yeast Mitophagy with Metazoan Pathways,"Yeast mitophagy differs mechanistically from the PINK1–Parkin system in metazoans. Atg32, like NIX and BNIP3 in mammals, is an OMM-anchored receptor that binds autophagy components directly, acting independently of ubiquitin signaling. In contrast, mammalian PINK1–Parkin-dependent mitophagy relies on depolarization-induced PINK1 stabilization and phosphorylation of ubiquitin to trigger Parkin-mediated ubiquitination of OMM substrates. Yeast mitophagy does not require mitochondrial depolarization and does not rely on ubiquitin nor E3 ligase cascade. Instead, it centers on Atg32's phosphorylation state and interaction with Atg11. These distinctions highlight an evolutionarily earlier ubiquitin-independent pathway, while the PINK1–Parkin system may have evolved later to provide higher specificity and speed.",Current Biology,Mitophagy Pathway Comparison,2018
Mitochondrial Biogenesis and Proteome Quality Control in Yeast,"Mitophagy represents only half of the mitochondrial turnover process, with mitochondrial biogenesis acting as the complementary arm. Biogenesis requires coordination between nuclear and mitochondrial genomes to assemble OXPHOS complexes and synthesize hundreds of proteins. NRF1 and NRF2 regulate transcription of nuclear-encoded mitochondrial genes and TFAM, which drives transcription and replication of mtDNA. PGC-1α serves as a co-activator that stimulates NRF1/2 and promotes mitochondrial biogenesis. Loss of PGC-1α decreases mitochondrial transcripts and impairs mitochondrial function, despite minimal changes in total mitochondrial volume. AMPK activation, triggered by low energy states, induces biogenesis and modulates mitochondrial dynamics and autophagy. The phosphorylation of PGC-1α by AMPK activates an autoregulatory loop increasing its own expression. AMPK also phosphorylates mitochondrial fission factor Mff, promoting Drp1 recruitment and mitochondrial fragmentation to facilitate mitophagy.",Current Biology,Mitochondrial Biogenesis,2018
"Interplay Between AMPK, ULK1, TFEB, and Mitophagy","AMPK coordinates mitochondrial quality by modulating biogenesis, mitophagy, and fission. ULK1, required for autophagy initiation, is activated by AMPK and necessary for removal of damaged mitochondria; loss of AMPK or ULK1 causes accumulation of dysfunctional mitochondria. TFEB, a transcription factor regulating autophagy and lysosomal biogenesis, enhances PGC-1α expression during nutrient deprivation, linking mitochondrial biogenesis to lysosomal capacity. TFEB translocates to the nucleus under mitochondrial stress in a PINK1–Parkin-dependent manner, suggesting crosstalk between mitophagy machinery and lysosomal transcription programs. Evidence suggests PGC-1α and TFEB mutually regulate each other, integrating signals from metabolism, nutrient availability, and mitochondrial stress to maintain mitochondrial homeostasis.",Current Biology,AMPK–TFEB–PGC1α Crosstalk,2018
Mitochondrial Proteases in Quality Control,"Over 20 proteases are located in different mitochondrial compartments (matrix, inner membrane, and intermembrane space), where they maintain proteostasis by processing newly imported proteins, degrading misfolded or damaged polypeptides, maintaining stoichiometry within OXPHOS complexes, and regulating mitochondrial dynamics. These proteases prevent accumulation of dysfunctional proteins that could compromise electron transport chain function. Defects in mitochondrial proteases are directly linked to human diseases, demonstrating their essential role in mitochondrial homeostasis.",Current Biology,Mitochondrial Quality Control,2018
OMM-Associated Degradation (OMMAD),"Outer mitochondrial membrane-associated degradation (OMMAD) functions analogously to ERAD. Damaged or misfolded OMM proteins are ubiquitinated and extracted into the cytosol for proteasomal degradation. This retrotranslocation process requires the AAA-ATPase VCP/p97. During mitochondrial stress, Parkin amplifies OMM ubiquitination, triggering p97 recruitment and degradation of several mitochondrial fusion regulators including Mfn1 and Mfn2. p97 also controls Mfn1 and Mcl1 turnover under basal conditions, and ubiquitin ligases such as MITOL/MARCHV and Mul1 influence the fission–fusion machinery. These pathways show that both autophagy and proteasomal degradation contribute to mitochondrial QC.",Current Biology,OMMAD,2018
Mitochondrial Protein Turnover Rates,"Proteomic analyses reveal that mitochondrial proteins display wide variability in half-lives, ranging from several hours to many days. Turnover rates differ across tissues—liver mitochondria turnover faster than those in the heart, reflecting distinct metabolic demands and tissue-specific transcriptional programs. In Drosophila, loss of Parkin or PINK1 slows turnover of OXPHOS components, indicating their role in selective degradation of damaged ETC proteins. Interestingly, Atg7 loss does not specifically affect OXPHOS turnover, suggesting that selective inner-membrane or matrix QC may proceed through mechanisms independent of bulk autophagy, such as MDVs or piecemeal mitophagy.",Current Biology,Protein Turnover,2018
Piecemeal (Bit-by-Bit) Mitophagy,"Piecemeal mitophagy removes only damaged subregions of the mitochondrial network while sparing healthy portions. Laser-induced oxidative damage using MitoKiller Red shows focal Parkin recruitment, localized ubiquitin and LC3 deposition, and budding of LC3- and Parkin-positive mitochondrial fragments. This demonstrates selective subdomain clearance. Experiments with the aggregation-prone mutant DOTC show that damaged matrix proteins can selectively trigger PINK1 stabilization and Parkin recruitment without global depolarization. Damaged regions containing DOTC detach in a Drp1-dependent manner. However, in Drp1 knockout cells, selectivity is lost: both damaged and healthy matrix proteins are removed, and mitophagy becomes global rather than focal. Thus, Drp1-mediated fission helps segregate damaged areas to maintain mitochondrial integrity.",Current Biology,Piecemeal Mitophagy,2018
Mitochondrial-Derived Vesicles (MDVs),"MDVs selectively package subsets of oxidized or damaged mitochondrial proteins from various compartments (matrix, IMM, OMM) into small vesicles that fuse with lysosomes independently of bulk mitophagy. MDV formation requires PINK1 and Parkin and is Drp1-independent. MDVs exclude TOM20 but carry other mitochondrial cargo, indicating selective sorting mechanisms. Syntaxin-17 regulates their fusion with lysosomes. MDVs represent a rapid QC pathway that operates before global mitochondrial dysfunction emerges.",Current Biology,Mitochondrial-Derived Vesicles,2018
Sub-organelle Quality Control in Yeast,"In yeast, selective removal of matrix or imported proteins can occur independently of Atg32. Dnm1 (the yeast homolog of Drp1) mediates mitochondrial fission upstream of selective matrix protein degradation. Damaged or aging mitochondria can form specialized compartments containing specific proteins such as Tom70. These compartments are targeted for autophagy, and disruption of this pathway impairs mitochondrial function. This highlights an evolutionarily conserved mechanism of segregating dysfunctional components for removal without destroying the entire organelle.",Current Biology,Yeast Mitochondrial QC,2018
UPRmt Overview: Organelle-Level Quality Control,"The mitochondrial unfolded protein response (UPRmt) is a stress response pathway that upregulates mitochondrial chaperones and proteases to restore proteostasis when misfolded or damaged proteins accumulate. In C. elegans, UPRmt activation requires ATFS-1, a transcription factor with both mitochondrial and nuclear targeting sequences. Under healthy conditions, ATFS-1 is imported into mitochondria and degraded by the LonP protease. During mitochondrial stress, impaired import prevents ATFS-1 degradation, allowing it to accumulate in the cytosol and translocate to the nucleus. Together with UBL5 and DVE-1, ATFS-1 induces expression of mitochondrial chaperones, proteases, glycolytic enzymes, components of the import machinery, and regulators of mitochondrial biogenesis. ATFS-1 also surprisingly accumulates inside mitochondria during stress and inhibits transcription of mtDNA-encoded OXPHOS proteins, reducing proteotoxic burden.",Current Biology,UPRmt Mechanisms,2018
UPRmt in Mammals: ATF5 and ATF4,"The mammalian UPRmt is more complex than the C. elegans pathway. Early studies detected upregulation of Hsp10, Hsp60, ClpP, and CHOP in response to mitochondrial matrix proteotoxicity or mtDNA depletion. CHOP is necessary but insufficient for UPRmt activation. ATF5 was identified as the mammalian ortholog of ATFS-1: human ATF5 rescues UPRmt signalling in ATFS-1–null worms and binds identical promoter motifs. ATF5 localizes to mitochondria under basal conditions, though nuclear translocation during stress remains technically unconfirmed. Other studies identified ATF4, a stress-induced transcription factor, as a major regulator of transcriptional responses to mitochondrial stress. Complex I inhibition activates ATF4 but not classical UPRmt genes. These findings indicate that the mammalian UPRmt likely involves multiple transcription factors, including ATF4, ATF5, and CHOP.",Current Biology,Mammalian UPRmt,2018
Reduced Import as a Shared Trigger for UPRmt and Mitophagy,"Loss of mitochondrial import efficiency is central to both UPRmt activation and PINK1–Parkin–mediated mitophagy. Reduced import causes ATFS-1/ATF5 to escape degradation and initiate UPRmt, while also stabilizing PINK1 on the outer membrane to trigger mitophagy. In yeast, import defects produce cytosolic stress responses called UPRam or mPOS, which inhibit cytosolic translation and increase proteasome activity. Mitochondria with severe import failure are unlikely to benefit from UPRmt-induced chaperones and proteases, but healthier mitochondria within the network can import and utilize these factors. Thus, UPRmt enhances network-wide fitness while mitophagy selectively removes severely compromised mitochondria.",Current Biology,Import Defects and Stress Responses,2018
Interplay Between UPRmt and Mitophagy: Opposing Effects,"Studies in C. elegans carrying large mtDNA deletions reveal opposing roles for UPRmt and mitophagy. Loss of ATFS-1 decreases mutant mtDNA levels, indicating that UPRmt maintains defective mitochondria and prevents their removal. In contrast, loss of PINK1 or Parkin increases mutant mtDNA accumulation, suggesting that mitophagy normally eliminates mutant genomes. Removing ATFS-1 partially reverses the accumulation seen in mitophagy-deficient backgrounds. ATFS-1 activates genes involved in mitochondrial biogenesis and dynamics, enhancing propagation of mutant mtDNA. Thus, UPRmt preserves defective mitochondria, whereas mitophagy eliminates them, creating an antagonistic relationship in mtDNA quality control.",Current Biology,UPRmt–Mitophagy Crosstalk,2018
Evidence from Mammalian Models: Mutator and Twinkle Mice,"Parkin-knockout mice crossed with mutator mice (POLG-deficient animals that accumulate mtDNA mutations and age prematurely) exhibit dopaminergic neuron loss, elevated phospho-ubiquitin signaling, and reduced mitochondrial function. However, the overall number of mtDNA mutations does not differ, although their pathogenicity increases. In Twinkle 'deletor' mice, which accumulate mtDNA deletions and develop mitochondrial myopathy, UPRmt components such as Hsp60, mtHsp70, and ClpP are elevated. Dopaminergic neuron–specific expression of mutant Twinkle causes neuronal loss and mtDNA mutation accumulation that worsens in the absence of Parkin. These findings illustrate conserved interplay between UPRmt pathways and mitophagy in mammals, though the specific involvement of ATF5 and ATF4 in vivo remains to be fully elucidated.",Current Biology,Mammalian UPRmt–Mitophagy Interactions,2018
Parkin/PINK1 Knockout Models and Lack of Neurodegeneration,"Most mouse models lacking Parkin or PINK1 fail to show hallmark features of human Parkinson’s disease, such as dopaminergic neuron loss or motor dysfunction. Only two exceptions exist: (1) a conditional Parkin knockout restricted to the ventral midbrain, which resulted in progressive loss of dopaminergic neurons, and (2) a PINK1-knockout rat model showing clear dopaminergic degeneration and mobility deficits. These findings suggest that compensatory pathways, redundant mitophagy mechanisms, or robust basal mitochondrial turnover act to protect neurons in many models. Crosses between Parkin-null animals and mitochondrial stress models have produced mixed results: loss of Parkin worsens phenotypes in Twinkle and POLG mutator models (which accumulate mtDNA mutations) but not in MitoPARK mice (which lack TFAM in dopaminergic neurons). The severity and nature of mitochondrial defects may determine whether Parkin/PINK1 loss becomes detrimental.",Current Biology,Mitophagy in Animal Models,2018
Modelling Mitophagy Contributions to Protein Turnover,"Total mitochondrial protein turnover results from both individual protein degradation (via proteases, OMMAD, MDVs) and whole-organelle removal through mitophagy. A conceptual model using three proteins with slow, intermediate, and fast intrinsic turnover rates (5, 10, and 50 molecules/hour) demonstrates how increasing mitophagy rates (from 1 to 50 mitochondria/hour) alters their total degradation. Proteins with slow intrinsic turnover are disproportionately affected by increased mitophagy, whereas fast-turnover proteins remain relatively stable. Even with extremely high mitophagy rates, distinct protein turnover profiles persist. This modeling suggests that mitophagy mainly accelerates removal of long-lived or stable mitochondrial proteins while preserving relative turnover differences across the proteome.",Current Biology,Protein Turnover Modelling,2018
In Vivo Tools for Studying Mitochondrial Turnover,"Several fluorescent reporter systems have been developed to track mitochondrial 'age' and mitophagy in live tissues. MitoTimer is a mitochondrially targeted fluorescent protein that shifts from green (newly synthesized) to red (oxidized/older), allowing discrimination between young, import-competent mitochondria and older, import-deficient regions. Mito-Keima uses a pH-dependent excitation shift to identify mitochondria that have entered acidic lysosomes, providing a direct measurement of mitophagy. The Mito-QC system uses a dual mCherry–GFP mitochondrial tag where GFP is quenched in lysosomes, enabling visualization of mitophagy in fixed tissues. These systems reveal strong heterogeneity of basal mitophagy across tissues, demonstrate developmental mitophagy in organs such as liver and kidney, and show age- and diet-dependent changes in mitochondrial clearance.",Current Biology,Mitophagy Reporters,2018
Physiological and Pathological Modulation of Mitophagy,"Reporter models reveal that mitophagy decreases with age—e.g., a 70% reduction in the dentate gyrus of older mice—and is sensitive to metabolic conditions, as high-fat diets reduce mitophagy signals in the liver. Conversely, in Mito-Keima mice carrying POLG mutations that induce mtDNA instability, mitophagy is increased, indicating that mitochondrial damage stimulates organelle turnover. These insights highlight mitophagy as a dynamic process influenced by environmental factors (diet, exercise), stress, and developmental cues.",Current Biology,Regulation of Mitophagy In Vivo,2018
Future Directions and Open Questions,"Despite extensive knowledge of the PINK1–Parkin pathway in cultured cells, its role in basal and induced mitophagy in vivo remains unclear. Notably, mitophagy reporter models have not yet been crossed into Parkin- or PINK1-null backgrounds, leaving unanswered whether these proteins are essential for physiological mitochondrial turnover in intact animals. These tools may clarify whether mitophagy contributes to neurodegenerative diseases and could reveal tissue-specific reliance on distinct quality control pathways. They also offer opportunities to study how environmental variables—such as nutrient status, exercise, or injury—shape mitochondrial turnover across tissues.",Current Biology,Open Questions in Mitophagy,2018
Overview of mTOR Signaling and Cellular Functions,"The mammalian target of rapamycin (mTOR) is a protein kinase that controls cellular metabolism, catabolism, immune responses, autophagy, survival, proliferation, and migration, to maintain cellular homeostasis. The mTOR signaling cascade consists of two distinct multi-subunit complexes named mTOR complex 1/2 (mTORC1/2). mTOR catalyzes the phosphorylation of several critical proteins like AKT, protein kinase C, insulin growth factor receptor (IGF-1R), 4E binding protein 1 (4E-BP1), ribosomal protein S6 kinase (S6K), transcription factor EB (TFEB), sterol-responsive element-binding proteins (SREBPs), Lipin-1, and Unc-51-like autophagy-activating kinases. mTOR signaling plays a central role in regulating translation, lipid synthesis, nucleotide synthesis, biogenesis of lysosomes, nutrient sensing, and growth factor signaling.",Signal Transduction and Targeted Therapy,mTOR,2023
Pathological Activation and Biological Roles of mTOR,"The emerging pieces of evidence have revealed that the constitutive activation of the mTOR pathway due to mutations/amplification/deletion in either mTOR and its complexes (mTORC1 and mTORC2) or upstream targets is responsible for aging, neurological diseases, and human malignancies. Here, we provide the detailed structure of mTOR, its complexes, and the comprehensive role of upstream regulators, as well as downstream effectors of mTOR signaling cascades in the metabolism, biogenesis of biomolecules, immune responses, and autophagy. Additionally, we summarize the potential of long noncoding RNAs (lncRNAs) as an important modulator of mTOR signaling. Importantly, we have highlighted the potential of mTOR signaling in aging, neurological disorders, human cancers, cancer stem cells, and drug resistance. Here, we discuss the developments for the therapeutic targeting of mTOR signaling with improved anticancer efficacy for the benefit of cancer patients in clinics.",Signal Transduction and Targeted Therapy,mTOR,2023
Introduction to mTOR and Its Biological Functions,"The mTOR belongs to the class of evolutionarily conserved threonine and serine kinases which recognize and incorporate a variety of extracellular and intracellular signals to maintain cellular homeostasis and metabolism.1–4 The name mTOR was obtained from rapamycin isolated from a soil bacterium in 1970 on Rapa Nui.1–4 Further, the structural elucidation of the rapamycin revealed 14–16 membered lactone rings and reduced saccharide substituents. Interestingly, the physiological characterizations have uncovered immunosuppressive properties, curtailed organ rejection, kidney transplantation, and inhibition of T-cell mitogenesis.2,5 Mechanistically, the mTOR has dual kinase activity and can phosphorylate serine/threonine or tyrosine residue. The mTOR has been considered a part of the phosphoinositide 3-kinase (PI3K) family due to the presence of the catalytic domain within the mTOR structure which has a similarity with lipid kinases like PI3K. mTOR has been reported to be crucial for many biological processes, including cell growth, cell survival, immunity, autophagy, and metabolism.1,2",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR Complexes and Their Components,"This has been reported that mTOR can generate two different functional complexes named mTORC1 and mTORC2.6 The mTORC1 was discovered as a complex of several proteins that consist of mTOR, Raptor (regulatory associated protein of mTOR), GβL (G protein β subunit-like protein)/mLST8 (mammalian lethal with SEC13 protein 8), DEPTOR (DEP-domain-containing mTOR-interacting protein), and PRAS40 (the 40 kDa proline-rich Akt substrate).7,8 The mTORC2 is composed of mTOR, GβL/mLST8, Rictor (rapamycin-insensitive companion of mTOR), Protor/PRR5 (Proline-rich protein 5), DEPTOR, and mSIN1 (mammalian stress-activated protein kinase-interacting protein 1).7–14 The mTORC1 amalgamate signals from a variety of growth factors, and nutrients to enhance cellular proliferation especially when there is adequate energy and/or catabolism when the body is hungry.5,15 The mTORC1 is well known for its function in cell growth and metabolism, whereas the mTORC2 regulates proliferation and survival.1 Several groups reported that mTOR is crucial for several signaling cascades like AKT, PI3K, TSC1/TSC2 (tuberous sclerosis complex subunit 1 and 2), Rheb, LKBL/AMP-activated protein kinase (AMPK), VAM6/Rag GTPases.16 The mTOR signaling was demonstrated to enhance gene transcription and translation to control cellular growth, autophagy, and apoptosis.2,5,17",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR Dysregulation and Disease Associations,"Dysregulation of mTOR has been found to be strongly linked with several diseases like aging, arthritis, insulin resistance, osteoporosis, cancers, and neurological disorders.18 Cancer development is a complex, and multifactorial process, including genetic aberration, epigenetic modifications, dysregulated expression of hormones, tumor suppressors, and conversion of proto-oncogenes to oncogenes.19–21 The frequent alteration of mTOR was noticed to play an important role during tumorigenesis, distant metastasis, and drug resistance in human malignancies, such as lung, breast, liver, renal, pancreatic, and prostate.22–25 The stimulation of the mTOR cascade has been displayed to increase tumor growth through the regulation of glycolysis, angiogenesis, growth factor receptor pathway, lipid metabolism, and autophagy.5,9 Therefore, mTOR represents an important and promising target for therapeutic intervention against human malignancies.26,27 In this current review, we have discussed the structure of mTOR complexes along with their molecular functions, upstream regulators, as well as downstream effectors of mTOR signaling, the association of mTOR signaling to modulate cellular metabolism and autophagy. Also, how the dysregulated mTOR signaling is associated with aging, neurological disorders, and cancers (Fig. 1). Importantly, we have highlighted the opportunities and challenges for pharmacological targeting of mTOR signaling for therapeutic intervention and management of human malignancies.",Signal Transduction and Targeted Therapy,mTOR,2023
Structural Architecture of mTOR,"Mechanobiology of mTOR unfolds it as a complex protein kinase intricated with multielement complexes via its communication network with other proteins. The cryo-electron microscopic structure of the mTOR has revealed a hollow rhomboid architecture with dimensions of ~280 × 210 × 130 (Å3).28 This has been noticed that the C-terminus of mTOR was comprised of the FAT domain (FKBP12 rapamycin-associated protein, ataxia telangiectasia, and transformation or transactivation domain associated protein), FRB (FKBP12 rapamycin binding) domain which is responsible for the interaction of FKBP protein bound to rapamycin with mTOR, kinase domain that is an important site for phosphorylation to control the activity of mTOR, and FATC (FAT carboxyterminal domain). Also, the N-terminal part of the mTOR consists of 20 HEAT repeats. The HEAT repeats were found to be essential for interaction with Raptor and Rictor.22,29",Signal Transduction and Targeted Therapy,mTOR,2023
Structural Organization of mTORC1,"The structural architect of mTORC1 defines the complex and symmetric organization of Raptor, PRAS40, DEPTOR, and mLST8 (GβL) as its major components along with centrally located mTOR protein.30 Interlocking interactions allying the two mTOR and two Raptor subunits configure dimeric interfaces. Distal foot-like perturbances of mLST8 (GβL) subunit interpose mTOR inside complex 1. PRAS40 circumscribes itself to the adjoining adjacency of Raptor subunits i.e., the middle section of the central core of complex 1.31,32 Raptor is critical for the assembly, proper localization, and stability of the mTORC1. Raptor was reported to be important for the recruitment of the substrate on mTORC1. PRAS40 has been shown to inhibit the activation of mTORC1 unless it is phosphorylated through growth factor receptor signaling by growth factors/other stimuli. PRAS40 has an essential role in human cancers and metabolic disorders. The mLST8 was found to be associated with the kinase domain of the mTORC1 and can help in the stabilization of kinase activity. DEPTOR acts as an inhibitory subunit in the mTORC1. The crystal structure and functional analysis revealed that rapamycin-FKBP12 can efficiently bind with the FRB domain of mTOR to obstruct the substrates from active sites.1,29,31–33 The mTORC1 has been found to control cellular growth by increasing the biogenesis of ribosomes, mRNA translation, and autophagy.34",Signal Transduction and Targeted Therapy,mTOR,2023
Structural Organization of mTORC2 and Discovery of mTORC3,"The mTORC2 complex is a hollow rhombohedral fold with dimensions of 220 Å × 200 Å × 130Å.28 The complex embraces binary symmetry, and each promoter incorporates one copy of mTOR, GβL/mLST8, mSin1, and Rictor. mTOR-mLST8 (GβL) heterodimer embraces overall architecture alike to complex 1 with a root mean square deviation of 6.7 Å for 3550 α-carbon atoms.30,35 The two monomers of mTOR pack against each other to form a central scaffold yielding a binding surface for the other three components. Two copies of mLST8 (GβL), mSin1, or Rictor bind symmetrically to the mTOR dimer.31,32 Rictor is important for the assembly, substrate recognition, and stability of the mTORC2. This has been observed that mSIN1 acts as a scaffold protein that helps in the mTORC2 interaction with serum and glucocorticoid-activated kinase 1 (SGK1) and negatively controls the kinase activity of mTORC2.12 Protor-1, a Rictor-binding protein was found to regulate mTORC2-dependent phosphorylation of SGK1.36 The mTORC2 has been displayed to be associated with the cytoskeleton, cell proliferation, cell survival, and migration. Harwood et al. have identified another rapamycin-insensitive complex known as mTORC3. The E26 transformation-specific transcription factor ETV7 was found to interact with mTOR in the cytoplasmic compartment. The mTORC3 displayed bimodal mTORC1/2 activity that was independent of the components of mTORC1/2. This was noticed that mTORC3 is robustly activated in several cancers. The loss of mTORC3 expression in cancer cells displayed marked sensitivity to rapamycin. Interestingly, this study also demonstrated that mTORC3 induced tumorigenesis in a murine model of rhabdomyosarcoma. Interestingly, the transgenic ETV7 expression further enhanced tumor onset and penetrance.37 The detailed structure of the mTOR and its complexes, along with their function, has been described in Fig. 2.",Signal Transduction and Targeted Therapy,mTOR,2023
Overview of Upstream Regulation of mTORC1 and mTORC2,"The mTOR signaling cascades control the cellular growth and mitotic divisions by generating prominent metabolic energy from glucose, lipid, protein, and nucleotides while inhibiting catabolic processes like autophagy. Hence, the mTORC1 plays a central role in maintaining the equilibrium between anabolism and catabolism, especially in response to environmental stress. mTOR signaling augments energy depots and consumption. mTORC1 can enhance cellular growth by integrating stimuli from growth factors, DNA damage, oxygen, nutrients, amino acids, and energy, whereas augmented mTORC1 prompts insulin resistance by halting insulin receptor signaling and fat accumulation. This has been observed that dysregulated oncogenes, enhanced anabolism, angiogenesis, and suppression of autophagy underlie the tumorigenic behavior of mTORC1. mTORC2 is activated by growth factors which in turn activates AKT and AGC family leading to increased cellular proliferation and survival. Growth factors are known to regulate several signaling cascades that intersect on TSC, including receptor tyrosine kinases, IGF-1, Wnt, TNFα, inflammatory cytokines, and Ras signaling cascade.",Signal Transduction and Targeted Therapy,mTOR,2023
"Growth Factors, TSC Regulation, and Energy Stress Sensing","These growth factors stimulate the phosphorylation of TSC2 through AKT. The phosphorylation was associated with the inhibition of TSC through its dissociation from the lysosomal membrane. The RTK-mediated Ras signaling was found to activate the mTORC1 pathway through MAPK/ERK and its effector p90RSK leading phosphorylation of TSC2. Moreover, the other growth factors like Wnt and TNFα were noticed with the activation of mTORC1 through repression of TSC1. Importantly, the environmental/extracellular and intracellular stresses are sensed by the mTORC1 and modulate cellular growth and survival under hypoxia, lower levels of ATP, and DNA damage conditions. Under glucose deprivation conditions, there is a marked reduction in cellular energy levels that stimulates energy stress-sensing metabolic regulator AMPK. The stimulation of AMPK was reported to repress the mTORC1 either through direct phosphorylation of Raptor or indirectly by phosphorylating TSC2. Furthermore, low glucose levels were found to suppress mTORC1 signaling by inhibiting the Rag GTPases activity, especially in cells lacking AMPK. AMPK-dependent phosphorylation of WDR24 can modulate the glucose-dependent activation of mTORC1, indicating that mTORC1 can efficiently sense glucose or energy stress through multiple molecular mechanisms.",Signal Transduction and Targeted Therapy,mTOR,2023
"Amino Acid Sensing via Rag GTPases, GATOR Complexes, and CASTOR1","Amino acids are not only required as the building blocks of proteins but also a great source of carbon and energy for a variety of metabolic signaling cascades. The activation of the mTORC1 pathway is coupled with diet-mediated changes in the concentration of amino acids. The mechanism of sensing amino acids through mTORC1 with the help of Rag GTPases as essential components of mTORC1 signaling was one of the groundbreaking discoveries. Rags were discovered as heterodimers of RagA/RagB with RagC/RagD. These are mostly bounded by the membrane of the lysosome through their close association with a pentameric complex composed of p14, HBXIP, p18, MP1, and c7ORF59. Amino acid stimulation converts the Rags to an active nucleotide-bound state which allows Rags to bind with Raptor and recruit mTORC1 to the lysosomal surface where Rheb is localized. SLC38A9, a lysosomal amino acid transporter, interacts with the Rag–v-ATPase complex and is responsible for arginine transport and activation of mTORC1. Cytosolic arginine and leucine signal through GATOR1 and GATOR2 complexes. CASTOR1 interacts and suppresses GATOR2 in the absence of arginine and dissociates upon arginine binding, resulting in mTORC1 activation, confirming CASTOR1 as an arginine sensor.",Signal Transduction and Targeted Therapy,mTOR,2023
Regulators of Amino Acid Sensing and PI3K–mTORC2 Feedback,"Other molecular mechanisms that control amino-acid-mediated mTORC1 signaling include Folliculin–FNIP2 complex recruitment on the lysosome acting as a GAP for RagC/D in the presence of amino acids. Glutamine can stimulate mTORC1 independent of Rag GTPases via Arf family GTPases. Long noncoding RNA SPAR cooperates with the v-ATPase–Ragulator complex to hamper mTORC1 recruitment to lysosomes. Genome-wide CRISPR-Cas9 screening identified ILF3 as a regulator of amino acid sensing in a mTORC1-dependent manner, tethering to GATOR complexes. Additionally, WDR24 or Ring domains in GATOR2 disseminate amino acid availability to mTORC1 during embryonic development. The mSin1 of mTORC2 was found to have a phosphoinositide-binding domain important for insulin-regulated activity of mTORC2. In the absence of insulin, the PH domain of mSin1 hampered mTORC2 catalytic activity. This autoinhibition was rescued upon binding to PI3K-generated PIP3, and mSin1 was phosphorylated by AKT, indicating a positive-feedback loop. S6K1 inhibited mTORC2 signaling through degradation of IRS1. The negative feedback loop between insulin-dependent PI3K and mTORC1 is another mTORC2 regulatory mechanism.",Signal Transduction and Targeted Therapy,mTOR,2023
mTORC1 in Glucose Metabolism and the Warburg Effect,"The energy requirement of the cell is regulated by mTORC1 via AMPK, the sensor of intracellular energy levels. Augmented glucose metabolism promotes mitochondrial activity by prompting AMP levels that disturb the ATP:AMP ratio, resulting in activation of AMPK and phosphorylation of TSC2 to repress mTORC1 activity. AMPK can also directly phosphorylate Raptor to reduce mTORC1 activity under energy-deprived conditions. mTORC1 can favor cellular proliferation through a prominent shift in glucose metabolism from oxidative phosphorylation to glycolysis, known as the Warburg effect. This is characterized by increased glucose uptake and lactate production even in the presence of oxygen. mTORC1 enhances key glycolytic enzymes such as pyruvate kinase muscle isozyme 2, hexokinase 2, and lactate dehydrogenase A. Hypoxic stress promotes reduction of pyruvate to lactate by NADH, increasing lactic acidosis, which favors oncogenesis. HIF-1α increases expression of glycolytic enzymes and glucose transporters. mTORC1 further enhances HIF-1α translation and SREBP-mediated pentose phosphate pathway activation to provide NADPH and intermediates for proliferation.",Signal Transduction and Targeted Therapy,mTOR,2023
mTORC1 in Lipid and Nucleotide Biosynthesis,"mTOR plays a significant role in regulating lipid biosynthesis required for cell growth by maintaining cellular membrane integrity. mTORC1 enhances lipid synthesis through SREBP regulation. Activation occurs via S6K1-mediated SREBP cleavage-activating protein (SCAP) activation or through Lipin-1 phosphorylation. Lipin-1 normally represses SREBP activity but is inactivated by mTORC1, leading to derepression of SREBP and increased lipid biosynthesis. mTORC1 also promotes nucleotide biosynthesis in proliferative cells to support DNA replication and ribosome biogenesis. mTORC1 enhances expression of MTHFD2 in an ATF4-dependent manner to provide carbon units for purine synthesis. S6K1 phosphorylates and stimulates CAD to support pyrimidine synthesis. Phosphoproteomics identified mTOR regulation of over 300 proteins, including CAD phosphorylation at serine 1859, promoting de novo pyrimidine synthesis and S-phase progression. Thus, mTORC1 adjusts RNA and DNA synthesis to support cell growth.",Signal Transduction and Targeted Therapy,mTOR,2023
mTORC1 Regulation of Protein Synthesis via S6K1 and 4EBP,"mTOR signaling cascade is well known for protein synthesis through the phosphorylation of eIF4E binding protein (4EBP), and S6K1. mTORC1 can phosphorylate S6K1 at Thr389 residue which leads to its phosphorylation and activation via PDK1 (3-phosphoinositide-dependent protein kinase 1). S6K1 can lead to the phosphorylation and activation of a variety of substrates that promote mRNA translation initiation, particularly the eIF4B which is crucial for the 5′ cap binding eIF4F complex. Dorrello and colleagues have revealed that programmed cell death protein 4 (PDCD4) repressed the translation initiation factor eIF4A. During mitogen response, the phosphorylation of PDCD4 at Ser67 by the S6K1 leading to its degradation through the ubiquitin ligase SCF (β-TRCP) enables the synthesis of protein synthesis and cellular proliferation. Exon junction complex has been displayed to regulate mRNA synthesis. The SKAR-dependent recruitment of S6K1 to the newly generated mRNPs acts as a bridge between mTOR signaling and translation.",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR-Driven Translational Control in Cancer,"The mTOR kinase has been recognized as one of the master regulators of translation to meet the demand for cancer cells. The global ribosome profiling was performed to unravel the mechanism of translation that regulates gene expression through mTOR in cancers. This study showed the enrichment of the specific genes associated with cellular growth, invasion, and metabolism. These genes were downstream targets of mTOR signaling in prostate cancer. Moreover, the potent and ATP-competitive mTOR inhibitor repressed mRNA translation and suppressed cellular proliferation. Another study has revealed that mRNAs that are controlled by mTORC1 are 5’ terminal oligopyrimidine (TOP) motifs. Moreover, the 4EBPs suppressed the initiation of translation by hampering the interaction between eIF4E and eIF4G1. This diminished the ability of eIF4E to interact with TOP and TOP-like mRNAs which explains why mTOR inhibition selectively repressed their translation. The mTORC1 phosphorylates its substrate 4EBP that triggers its dissociation from eIF4E allowing 5′cap-dependent mRNA translation.",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR Alterations and Oncogenic Mutations in Human Cancers,"The deregulation of mTOR signaling has been noticed in human malignancies. The emerging data has suggested that the mTOR signal is frequently altered in approximately 30% of cancers. The activation of the mTOR pathway is dependent in three different ways (1) the activating mutations in the mTOR, and mTORC1/2 or mutations in upstream genes lead to hyperactivation of the mTOR signaling (2) overexpression/amplification of the components of mTORC1 and mTORC2 (3) loss of function of negative regulators in the mTOR signaling cascade. The gain of the function mutations in the kinase domain of mTOR can directly activate the mTOR pathway. The genome sequencing of human tumors has reported approximately 33 mutations in the mTOR gene. These mutations were associated with the activation of the mTOR pathway in colorectal cancer, endometroid carcinoma, stomach cancer, lung carcinoma, renal cell carcinoma (RCC), and melanoma. This study displayed that mTOR mutations were clustered in six distinct regions in the C-terminal part of mTOR in human tumors. These mutations did not affect the mTOR complex assembly but suppressed the binding of DEPTOR. Interestingly, the cell lines with mTOR mutations displayed marked sensitivity to mTOR inhibitors in both in vitro and murine models.",Signal Transduction and Targeted Therapy,mTOR,2023
"RICTOR Amplification, Upstream Mutations, and Tumorigenesis","Moreover, mutations in the components of mTOR complexes have been observed in several cancers. For instance, RICTOR was reported to be highly amplified in patients with lung and breast carcinoma. The RICTOR amplification in squamous cell lung carcinoma was linked with a bad prognosis and short survival. This study showed the sensitivity of mTORC1/2 inhibitors against RICTOR-amplified lung cancer cells. Interestingly, the patient was treated with mTORC1/2 inhibitors and displayed stabilization of the tumor for at least 18 months. Joly et al. also confirmed that RICTOR was robustly expressed in HER2-amplified breast carcinoma specimens which in turn enhanced phosphorylation of AKT at S473 residue. A case study by Shamieh and colleagues reported that amplification in the RICTOR gene was associated with metastasis and drug resistance in TNBC. Upregulation of RICTOR was associated with increased mTORC2 activity which promoted cellular motility and proliferation of glioma cells. In addition, the hyperactivation of mTOR signaling can be the result of mutations in upstream genes including oncogenes and tumor suppressor genes. Gao et al. reported the overexpression of Rheb1 in acute myeloid leukemia associated with worse survival, and depletion of Rheb1 suppressed mTOR signaling.",Signal Transduction and Targeted Therapy,mTOR,2023
Tumor Suppressor Loss and mTOR Hyperactivation,"Ghosh et al. reported mutations in the FAT domain of the mTOR gene in RCC. This study also identified Rheb mutations in patients with RCC leading to increased mTORC1 activity. The mutations, amplification, and overexpression of PIK3CA, KRAS, AKT, IGFR, and EGFR are more common in cancer and activate the PI3K/AKT/mTOR cascade. Similarly, mutations in KRAS, a key mediator of cell growth and differentiation, can lead to marked activation of the mTOR signaling and contribute to carcinogenesis. Also, the inactivation of p53, PTEN, STK11, and TSC1/2 was noticed to enhance the activation of the mTOR signaling cascade in human tumors. The loss of p53 function contributes to oncogenesis by promoting cell proliferation and survival, reducing apoptosis, and increasing genomic instability. Data indicate that p53 negatively regulates mTOR signaling by inducing REDD1 and inhibiting S6K1. Without functional p53, these negative regulatory mechanisms are lost, leading to mTOR hyperactivation. PTEN alterations are reported in breast, multiple myeloma, and endometrial cancers, where mTOR inhibitors displayed strong antitumor activity.",Signal Transduction and Targeted Therapy,mTOR,2023
lncRNAs Modulating mTOR Signaling in Cancer,"LncRNAs are >200 nucleotides long RNAs similar to mRNA structure but do not code for protein. They perform functions as signal molecules, decoys, guides, and scaffolds. Studies have revealed that dysregulated lncRNAs can modulate mTOR signaling and vice versa. lncRNAs regulate mTOR activity by direct binding to mTOR complex components or by regulating upstream and downstream targets. DLEU1 and HAGLROS were reported as direct mTOR complex targets. DLEU1 was overexpressed in endometrial carcinoma and promoted proliferation, clonogenicity, and migration while suppressing apoptosis through the mTOR pathway. Overexpression of DLEU1 resulted in phosphorylation of mTOR and downstream PI3K, AKT, and pS70K. HAGLROS overexpression was associated with worse outcomes in gastric cancer; silencing HAGLROS suppressed mTOR expression and increased ATG9A and ATG9B. HAGLROS sponged miR-100-5p to activate mTOR and interacted with mTORC1 components to stimulate the mTORC1 pathway.",Signal Transduction and Targeted Therapy,mTOR,2023
Additional lncRNAs Regulating Upstream and Downstream mTOR Pathways,"Several lncRNAs regulate upstream and downstream molecules of the mTOR complexes. NBR2 functions through LKB1-AMPK and maintains an NBR2-AMPK feedback loop; its interaction with AMPK-α increases during glucose starvation. NBR2 regulates cell growth, autophagy, and apoptosis through mTOR signaling. MALAT1 acts as an oncogenic lncRNA in HCC via SRSF1/mTOR/S6K1 axis. LINC00152 increased HCC tumorigenesis through EpCAM expression regulated by mTOR. H19 was downregulated in pituitary adenomas; forced expression suppressed proliferation by interacting with 4E-BP1, hampering its interaction with Raptor. LINC00963 overexpression in NSCLC promoted invasion and metastasis by interacting with PGK1, activating AKT/mTOR signaling. GAS5 lncRNA inhibited gastric carcinoma tumorigenesis through miR-106a-5p/AKT/mTOR. CASC9 depletion suppressed OSCC xenograft tumors via AKT/mTOR autophagy-dependent apoptosis. TUG1 silencing induced apoptosis of HCC cells through mTOR/S6K. CRNDE promoted glioma growth via P70S6K expression.",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR in Cancer Stem Cells,"CSCs are a unique tumor subpopulation with self-renewal, metastasis, and drug resistance abilities. CSCs have been observed in ovarian, pancreatic, breast, liver, and lung cancers. Pathways including Wnt/β-catenin, Hedgehog, STAT3, TGF-β, and PI3K/AKT/mTOR regulate CSCs. mTOR signaling is critical for CSC self-renewal and tumorigenicity. mTOR increased clonogenic ability and tumor formation in breast cancer stem cells. In HCC, branched-chain amino acids activated mTORC1; activation or mTORC2 silencing inhibited CSC population and tumorigenicity by suppressing EpCAM. PI3K/AKT activation was observed in GBM neurospheres; combining alpelisib with mTOR inhibitors reduced glioma stem cell growth. Rapamycin repressed NSC/progenitor markers in GSCs. NVP-BEZ235 effectively suppressed GSC growth in xenografts. PTEN/PI3K pathway was essential for CSC maintenance in prostate carcinoma; PI3K/mTOR inhibitors suppressed CSCs and tumor growth. Hyperactivation of PI3K/Akt/mTOR was linked with CXCR4+ stem-like cells in gefitinib-resistant lung cancer. Rapamycin reduced breast cancer stem cell population in vitro and in vivo.",Signal Transduction and Targeted Therapy,mTOR,2023
Resistance Mechanisms to mTOR Inhibitors,"Drug resistance is a major clinical problem. Reasons include tumor heterogeneity, clonal selection, emergence of new clones, intrinsic resistance to cell death, and pathway crosstalk. Although mTOR inhibitors show strong preclinical activity, resistance emerges frequently. Overexpression of ABC transporters contributes significantly. mTOR inhibitors such as AZD8055 and rapamycin are substrates of ABCB1; NVP-BEZ235 and AZD8055 are substrates of ABCG2. Abcb1/Abcg2 knockout mice showed enhanced brain penetration of these drugs. ABCB1 overexpression caused everolimus resistance in luminal breast cancer. NVP-BEZ235 combined with sunitinib showed synergistic effects in mCRPC. Resistance to PF-4989216 PI3K/mTOR inhibitor in lung carcinoma was driven by ABCG2 upregulation; inhibition of ABCG2 reversed resistance. LY3023414 acted as a substrate for both ABCG2 and ABCB1, and overexpression reduced intracellular uptake. Somatic mutations also confer resistance: A2034V and F2108L in the FRB-FKBP12 domain caused rapamycin resistance; M2327I in the kinase domain conferred AZD8055 resistance. Clinical evidence shows F2108L mutation in a patient who relapsed after everolimus treatment. Somatic kinase-domain mutations including M2327I are also detected in drug-naive patients.",Signal Transduction and Targeted Therapy,mTOR,2023
mTORC1 as a Negative Regulator of Autophagy,"Autophagy is one of the important processes which are critical for cellular digestion to remove damaged organelles and macromolecules. Apart from this, autophagy is critical in maintaining cellular equilibrium by providing energy and building blocks under stress conditions. Autophagy was reported when the electron microscope revealed the structure of vesicles has amorphous materials and cytoplasmic organelles in the kidneys of newborn murine. Later, studies have demonstrated that the deprivation of amino acid can robustly enhance the process of autophagy perfused livers of rats and mammalian cells. Also, several groups have reported that amino acids are one of the important regulators of the mTORC1 cascade. Under nutrient and growth factor-deprived conditions, mTORC1 activity was reported to be suppressed, indicating that there is an inverse relation between autophagy and activation of mTORC1. The induction of autophagy through inhibition of mTORC1 has been well studied in yeast and drosophila models. Interestingly, the molecular basis of mTORC1 to regulate autophagy in mammalian cells is quite recent and emerging.",Signal Transduction and Targeted Therapy,mTOR,2023
"ULK1 Complex, VPS34 Regulation, and Autophagy Suppression","mTOR controls autophagy through the regulation of a protein complex composed of UNC-5-like autophagy-activating kinase 1 (ULK1), autophagy-related gene 13 (ATG13), and focal adhesion kinase family-interacting protein of 200 kDa (FIP200). Studies have revealed that mTORC1 can inhibit the ULK complex through phosphorylation of ATG13 and ULK1/2. Silencing of mTORC1 was found to be associated with enhanced activity of ULK1/2 kinase leading to phosphorylation of ATG13 and FIP200, the important components of the ULK1/2 complex. mTORC1 phosphorylates ULK1 at Ser-758 which in turn blocks the interaction with AMPK, halting ULK1 activation. Moreover, mTORC1 decreases the stability of ULK1 through phosphorylation of autophagy/beclin 1 regulator 1 (AMBRA1). mTORC1 and AMPK control the activity of the VPS34 complex needed for autophagosome generation. The ATG14L-associated VPS34 complex plays a crucial role in autophagy regulation. Under nutrient stress, AMPK stimulates autophagy by phosphorylating Beclin 1 while suppressing non-autophagy VPS34 complexes. In contrast, mTORC1 phosphorylates ATG14L to suppress VPS34 lipid kinase activity, providing another mechanism for autophagy inhibition through mTORC1.",Signal Transduction and Targeted Therapy,mTOR,2023
Dual Role of Autophagy in Cancer Progression,"The precise role of autophagy in human malignancies is still not clear because activation or suppression of autophagy was found to be tumorigenic or anti-tumorigenic. Emerging pieces of evidence have shown that activated autophagy can suppress the process of cancer progression, especially in precancerous lesions. However, several studies indicated that autophagy acts to promote tumor survival and growth in advanced cancers. Therefore, the inhibition of autophagy can be employed as a therapeutic approach. Under stress conditions, autophagy supports the growth and survival of tumors, especially in poorly vascularized tumors. Dysregulated autophagy has emerged as an adaptive mechanism for the initiation and progression of human cancers through the accumulation of DNA damage, macromolecules, organelles like mitochondria, oxidative stress, chromatin instability. In addition, stress-induced autophagy has been noticed with enhanced stemness and drug resistance in human cancers.",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR Signaling and Lifespan Extension Across Model Organisms,"Several studies have observed that mTOR signaling is involved in the key processes associated with aging in a variety of living organisms, such as worms, yeast, flies, and mammals. Initial studies on the Caenorhabditis elegans (C. elegans) have noticed that suppressed expression of the mTOR homolog known as ceTOR or let-363 as well as Raptor known as daf-15 was associated with an increased life span of almost more than double. The mutations in the CeTOR and raptor were reported with dauer-like larval arrest and suggested that CeTOR is important for the regulation of dauer diapause. The let-363 and daf-15 mutants displayed a marked shift in the metabolism that resulted in fat accumulation and an extended adult life span. Later on, other genetic screening studies also noticed that reduced mTOR signaling enhanced the life span of the drosophila, yeast, and murine models. Altogether, these studies have proved that extended life span was highly dependent on nutritional condition (glucose, fat, and protein metabolism) and a close association with the mTOR pathway.",Signal Transduction and Targeted Therapy,mTOR,2023
"Rapamycin, Calorie Restriction, and mTORC1 in Aging","Interestingly, rapamycin, a pharmacological mTOR inhibitor has been proven to increase the life span in different model organisms. This is well-established that mTORC1 has a major role in nutrients and insulin sensing. Therefore, the benefits of a calorie-restricted diet on life span are because of the reduced mTORC1 signaling. This was confirmed when a calorie-restricted diet did not extend the life span upon inhibition of mTOR signaling in C. elegans, Drosophila, and yeast. There are several thoughts regarding the role of mTOR signaling in aging processes in mammalian systems. The repression of the mRNA translation during mTORC1 inhibition was correlated with slower aging by reducing oxidative and proteotoxic stress. This observation was the consistent loss of S6K1 that extends the life span, and resistance to age-related diseases like compromised immunity, motor dysfunction, and loss of insulin sensitivity in mammals. Also, the loss of S6K1-induced gene expression was similar to a calorie-restricted diet or pharmacological activation of AMPK. RNA polymerase III (POL III) was reported to play a critical role in nutrient signaling, and anabolic activities to accelerate aging by mTORC1.",Signal Transduction and Targeted Therapy,mTOR,2023
"Stem Cell Regulation, Clinical Trials, and mTOR Inhibition in Aging","There is another possibility that the depletion of mTORC1 could modulate aging via autophagy. Moreover, this was thought that the attenuation of adult stem cells could be central in maintaining aging processes. Rapamycin treatment was found to increase the self-renewal ability of hematopoietic stem cells and the life span of old age mice. Other studies revealed that mTORC1 and SLC7A5 regulate the self-renewal of intestinal stem cell self-renewal and Paneth cell function in maintaining intestinal niche and physiology. The depletion of foxo was found to restore stem cell aging in germline stem cells of drosophila. Interestingly, the phase IIa clinical trials were conducted on 264 volunteers with an age of ≥65 years at 12 clinical sites to evaluate the safety, and efficacy of mTOR inhibitors to boost immune responses. The low dosages of everolimus markedly decreased the rate of infections and enhanced the vaccination response against influenza with increased antiviral immunity. Based on these data, alternative rapamycin dosage regimens were proposed for better longevity with minimal side effects. Altogether, mTOR inhibition can increase life expectancy and help delay the onset of age-associated diseases. However, the duration of the treatment should be decided carefully as it can produce severe side effects like immunosuppression and glucose intolerance.",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR Signaling in Neural Development and Synaptic Function,"The emerging research studies displayed that mTOR signaling is one of the important regulators of neurological processes like neural stem cells, neural development, synaptic plasticity, circuit formation, learning, and memory. Ablation of Rictor or Raptor in neurons was found to decrease the neuron size and early death. The depletion of Rictor or Raptor was shown to have a differential impact on the differentiation of the oligodendrocyte and myelination of the central nervous system. These data revealed the importance of mTORC1 and mTORC2 during brain development. On the contrary, the hyperactivation of the mTORC1 pathway was observed in the brain and well-documented in human patients with TSC. These patients have been shown with several neurological disorders such as autism, intellectual disability, epilepsy, anxiety, sleep disturbances, and brain tumors. TSC has been characterized as an autosomal disorder caused by the loss of either TSC1 or TSC2. Moreover, the hyperactivation of mTORC1 because of the Tsc1 or Tsc2 loss in the neural cells in the murine models displayed severe epileptic seizures. The rapamycin treatment was effective in reducing epileptic seizures in these mice.",Signal Transduction and Targeted Therapy,mTOR,2023
"mTORC1 Hyperactivation, Epilepsy, and Therapeutic mTOR Inhibition","The mutations in components of the KICSTOR and GATOR1 complexes were associated with epilepsy in humans. Recently, the retrospective analysis of TSC patients treated with everolimus or sirolimus under the age of 2 years has reported promising benefits on epilepsy. This study forms the basis for the testing of mTOR inhibition in a large cohort of patients with TSC. The mTORC1 activation in tissue stems promoted mRNA translation near synapses which required neuronal circuit formation. Further, inhibition of mTOR signaling was found to block the ketamine-induced synaptogenesis and behavioral responses. The mTORC1-mediated autophagy was found to be strongly correlated with the pathogenesis of neurodegenerative disorders like Alzheimer’s disease (AD), and Parkinson’s disease (PD). The rapamycin treatment was found to inhibit the progression of AD and increase the life span in the diseased model of AD. Rapamycin is used in clinical settings and has shown promising effectiveness against AD and emerged as a potential therapeutics. Dactolisib treatment was shown to protect the AD in a transgenic murine Alzheimer model. In the future, next-generation mTOR inhibitors can be designed and tested in AD and PD mice models for better drugs.",Signal Transduction and Targeted Therapy,mTOR,2023
"mTOR Signaling in T-Cell Activation, Differentiation, and Immune Homeostasis","Recent reports have uncovered a significant regulatory function of the mTOR pathway not only in cancer but also in the differentiation, activation, and functional characteristics of immune cells. Tumors can evade the immune system by dampening its ability to detect and eliminate cancer cells. Recent research has focused on tumor immunotherapy as a promising approach to tackle this challenge. Multiple studies indicate that the mTOR pathway, which is frequently overactive in tumors, plays a role in controlling the development and effectiveness of immune cells. Moreover, mTOR signaling plays a vital role in enabling T cells to perceive and merge immune signals originating from dendritic cells (DCs). These signals have been found to involve cytokines, co-stimulatory molecules, and antigenic signals as well as environmental cues derived nutrients, growth, and immunoregulatory factors. T cells rely on mTOR signaling to sense and integrate this comprehensive array of immune and environmental inputs. Studies have identified that TSC1 acts as most crucial for maintaining the naive T-cell quiescence and survival to maintain immune homeostasis. The T cells that were deficient for PTEN were reported to upregulate mTORC1 activity to maintain their quiescence before tumorigenesis indicating the importance of mTORC1 on T-cell homeostasis.",Signal Transduction and Targeted Therapy,mTOR,2023
"mTOR Regulation of T-Cell Proliferation, Effector Fate, and Memory Formation","Other studies have displayed that phosphorylation of liver kinase B1 or STK11 phosphorylates stimulates AMPK under energy deprivation. Further, deletion of Lkb1 in T cells caused massive T-cell apoptosis while compromising thymic selection, altered metabolism, and proliferation of T cells. The stimulation of T- and B-cell receptors as well as other cytokine receptors, such as the IL-2 receptor, causes mTOR to become active in the adaptive immune system. In cytokine-stimulated T cells, mTOR regulates the transition from the G1 to the S phase of the cell cycle. In addition, once T cells are activated by IL-12, mTOR may drive the development of Th1 cells by stimulating the production of IFN-γ. Further, studies have reported that the antigen recognition by naive T cells leads to the mTOR activation, which guides the differentiation of CD4+ T cells into the T-helper cell effector lineages. Also, mTOR was shown to regulate the effector fate of CD8+ T cells during tumor immunity and infections. Interestingly, several studies have primarily focused on mTOR inhibitor rapamycin’s ability to hinder T-cell proliferation and IL-2 production and induce anergy, even in adequately stimulated T cells. Upon T-cell receptor stimulation, both mTORC1 and mTORC2 are activated. The mTORC1 influences effector responses of CD8+ T cells whereas mTORC2 activity is associated with metabolic reprogramming to generate memory CD8+ T cells.",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR Signaling in B-Cell Development and Antibody Production,"The extent of mTOR activation is directly linked to the duration of the interaction between T cells and DCs, as well as the amount of the corresponding antigen. Co-stimulatory signals exert a significant influence on mTOR activity. CD28-mediated co-stimulation enhances the mTOR activity induced by TCR stimulation. The role of mTOR signaling in B-cell development, differentiation, and function is less studied than in T-cell development. The mTOR hypomorph mouse model displayed a partial block of large pre-B to small pre-B stages of B-cell development and compromised proliferation in response to B-cell mitogenic signals. BCR and CD40 signaling were more highly compromised than TLR signaling. This resulted in the alteration in the splenic populations, production of antibodies, and migration towards chemokines. The deficiency of SIN1 increased il7r, rag1, and rag2 expression, enhancing V(D)J recombinase activity and survival of pro-B cells; Akt2 mediated Sin1–mTORC2 effects. Rictor-deficient mice showed increased pro-B, pre-B, and immature B cells with decreased mature B cells. Rapamycin treatment recapitulated early blocks of B-cell development. Transcriptomic analysis revealed that follicular B cells upregulate unfolded protein response genes before antibody secretion, requiring Raptor and mTORC1 kinase adapter.",Signal Transduction and Targeted Therapy,mTOR,2023
mTORC1–mTORC2 Regulation of Innate Immunity: DCs and NK Cells,"Growth factors, TLR ligands, cytokines, and other extracellular signals can all activate the mTORC1–mTORC2 network in innate immune cells. Studies have demonstrated that mTOR signaling plays a crucial role in the differentiation and function of dendritic cells (DCs) and natural killer (NK) cells. DCs possess strong antigen presentation abilities, and NK cells are important immune cells involved in tumor surveillance. Previous studies have reported that both mTORC1 and mTORC2 exert distinct effects on NK cell function. mTORC2 negatively regulates NK cell function by inhibiting the STAT5/SLC7A5 axis, while mTORC1 positively regulates mTORC2 activity, thereby promoting CD122-mediated interleukin-15 signaling. IL-15 induces mTOR activity in NK cells in both humans and mice. Inhibition of mTOR in DCs has been shown to improve their antigen presentation capabilities and enhance the activation of cytotoxic CD8+ T lymphocytes, resulting in increased antitumor activity. Therefore, mTOR inhibitors hold promise for enhancing the efficacy of tumor immunotherapy and autologous DC-based vaccination by extending DC lifespan and improving antigen-processing abilities.",Signal Transduction and Targeted Therapy,mTOR,2023
mTOR in Macrophage Polarization and Immune Modulation in Tumors,"Macrophages, specifically the M1 and M2 subtypes, play critical roles in tumor development and progression. M1 macrophages can kill tumor cells, while M2 macrophages promote tumor growth, invasion, and metastasis. Dysregulation of the mTOR pathway has been implicated in macrophage polarization and function. Decreased expression of miR-30c, which inhibits mTOR activity, leads to reduced M1 differentiation and function, promoting tumor growth and metastasis. Enhanced PI3K and mTOR signaling in mouse macrophages increases M2 macrophage markers and STAT6 pathway activation. Conversely, inhibiting mTORC1 in human macrophages enhances M1 polarization. The role of mTORC1 and mTORC2 is complex: deletion of Tsc1 can promote both M1 and M2 polarization depending on the pathway. AKT isoforms also have specific effects on macrophage polarization. The PI3K–AKT–mTOR pathway senses environmental cues and affects macrophage behavior, although detailed mechanisms remain unresolved. Taken together, mTOR inhibition has enormous potential for vaccine development to boost immunity against human malignancies and pathogens.",Signal Transduction and Targeted Therapy,mTOR,2023
"Rapamycin, Sirolimus, and Early mTOR-Targeted Therapeutics","Rapamycin is a natural macrocyclic lactone that is obtained from the bacterium Streptomyces hygroscopicus. After the discovery of rapamycin, researchers continued to explore the related targets and signaling involved at the molecular and cellular levels. The FKBP12 and FKBP51 expression was found to be the rate-limiting factor that decides the rapamycin drug response in cell lines and tissues. This led to the discovery and development of various moieties including synthetic/semisynthetic which were active against mTOR-induced oncogenesis. Sirolimus is another approved biochemical functional form of rapamycin that displayed target-specific inhibition. Sirolimus has been found to impair the molecular interaction between mTOR and Raptor by targeting mTORC1. Sirolimus is an oral drug that has high protein binding, an elimination half-life of 57–63 h, high drug distribution to the circulation, and is metabolized by the hepatic enzymes like CYP3A4, CYP3A5, and excretion through p-glycoprotein. Sirolimus was approved by FDA for organ transplantation as an immunosuppressive agent in the year 2009. Therapeutic drug monitoring was found to be a critical aspect during sirolimus treatment. Sirolimus received FDA approval in the year 2015 for lymphangioleiomyomatosis. Nab-sirolimus displayed a strong antitumor effect in cases with PEComa and was approved by FDA in 2021. Recently, Nab-sirolimus is going through a clinical trial in solid tumors with genetic mutations in TSC1 and TSC2.",Signal Transduction and Targeted Therapy,mTOR,2023
Rapalogs: Temsirolimus and Everolimus Development and Clinical Use,"The poor bioavailability of the drug led researchers to focus mainly on improving its pharmacokinetics and stability without disturbing its pharmacodynamic profile. Two sides are obligatory to interact with FKBP12 and mTOR providing few options for structural modifications. Synthetic analogs or derivatives thus designed, synthesized, and biologically evaluated by replacing the hydrogen of –OH (C-40) with different moieties led to the development of several newer analogs. These analogs have been named Rapalogs with improved pharmacokinetics and stability without disturbing their pharmacodynamic profile. Temsirolimus (a rapalog) was designed by replacing H of the hydroxyl group (C-40) with di-hydroxyl methyl propionic acid ester and was authorized by the FDA for patients with metastatic RCC in 2007. Temsirolimus interacts with FKBP12 resulting in the formation of a strong trimolecular complex with mTOR and is equivalent to sirolimus in terms of activity. Temsirolimus displayed antitumor potential as monotherapy or in combination with other agents in leukemia, cervix, endometrial, and ovarian cancers. Everolimus was designed as an immunosuppressive drug by replacing H of the hydroxyl group (C-40) with a hydroxylethyl group. Everolimus possesses high protein binding, good half-life, blood–brain penetration, and is metabolized by CYP3A.",Signal Transduction and Targeted Therapy,mTOR,2023
Clinical Efficacy of Everolimus and Ridaforolimus,"Both temsirolimus and everolimus demonstrated anticancer activity in murine models and are used for treating advanced-stage RCC in the clinic. Everolimus is used for patients with advanced breast carcinoma or neuroendocrine pancreatic carcinoma. The combination of exemestane with everolimus enhanced progression-free survival in HR(+) breast carcinoma. Combinations of everolimus with sunitinib/AZD2014 were not tolerated in metastatic RCC, suggesting careful selection of combined pathways. Everolimus showed efficacy in phase-II trials in advanced thyroid carcinoma. Recently, everolimus combined with T-DM1 resulted in antitumor efficacy in HER2-positive breast cancer. Everolimus and letrozole showed promising progression-free survival in ER+ relapsed high-grade ovarian carcinoma. Everolimus combined with rituximab produced complete responses in diffuse large B-cell lymphoma. Everolimus reduced the size of subependymal giant-cell astrocytomas and seizure frequency. Everolimus received FDA approval for pancreatic neuroendocrine tumors and later for gastrointestinal or lung-origin neuroendocrine tumors (2016). Ridaforolimus is an FDA-approved oral/IV mTOR inhibitor for soft tissue and bone sarcoma in phase-II trials and showed strong efficacy and safety in phase-III trials.",Signal Transduction and Targeted Therapy,mTOR,2023
Next-Generation mTOR Inhibitors: TOR-KIs and Dual PI3K/mTOR Agents,"Vistusertib (AZD2014) and AZD8055 are highly potent dual mTORC1/2 inhibitors with superior anticancer effects. AZD8055 enhanced survival in AML transplanted mice and suppressed leukemic cell growth. Rapalogs block mTORC1 selectively, but feedback activation of PI3K, Ras/MAPK, mTORC2, and RTKs compromises long-term effectiveness. Rapamycin therapy was shown to augment AKT phosphorylation at Ser473 despite S6K–IRS1 feedback disruption. mTORC2 can directly phosphorylate AKT, and rapamycin can inhibit AKT by disrupting mTORC2 assembly in some cells. Dual PI3K/mTOR inhibitors were developed to overcome this resistance, but PI3K isoform diversity often caused toxicity. TOR-KIs halt the cell cycle in G1, suppress cyclin D1 transcription, inhibit AKT/SGK1, and prevent RTK accumulation by reducing HIF-2α. However, TOR-KIs still show feedback activation of PI3K and PDK1-dependent AKT T308 phosphorylation and are ineffective against certain mTOR mutations.",Signal Transduction and Targeted Therapy,mTOR,2023
Resistance Mutations and the Development of Rapalink Inhibitors,"Mutations that increase catalytic activity of mTORC1/2 reduce effectiveness of rapalogs, dual inhibitors, and TOR-KIs. Rodrik-Outmezguine and colleagues generated rapamycin-resistant breast carcinoma cell lines with mutations in the mTOR FRB domain (A2034V, F2108L) and AZD8055-resistant clones with kinase domain mutations. Molecular modeling revealed the juxtaposition of rapamycin and AZD8055 binding sites. This enabled the design of bivalent inhibitors linking rapalogs and TOR-KIs with optimized crosslinkers. Rapalink compounds inhibit both mTORC1 and mTORC2, overcoming resistance where rapamycin and TOR-KIs fail. Rapalink-treated xenograft models showed greater sensitivity than previous generations of inhibitors. Rapalinks integrate properties of first- and second-generation inhibitors, simultaneously targeting the FRB domain via FKBP12 and the kinase domain via ATP-competitive inhibition.",Signal Transduction and Targeted Therapy,mTOR,2023
"Sapanisertib (TAK-228), CC-223, and OSI-027: Advanced mTORC1/2 Inhibitors","Sapanisertib (MLN0128 or TAK-228), a selective mTOR inhibitor, showed promising anticancer effects and entered phase-I/II clinical trials for mCRPC, breast carcinoma, lung cancer, endometrial carcinoma, bladder carcinoma, sarcoma, and non-Hodgkin B-cell lymphoma. MLN0128 suppressed tumorigenesis in sarcoma models and sensitized chemoresistant primary effusion lymphoma. MLN0128 suppressed HCC growth and sensitized tumors to sorafenib and cabozantinib. MLN0128 sensitized everolimus-resistant PIK3CA mutant colorectal and pancreatic neuroendocrine tumors. The DICE trial showed TAK-228 + paclitaxel improved outcomes in ovarian cancer. CC-223 suppressed tumorigenesis of HNSCC and HCC through mTORC1/2 inhibition. OSI-027, another potent mTORC1/2 inhibitor, displayed antitumor activity in several cancers and synergized with gemcitabine in pancreatic cancer.",Signal Transduction and Targeted Therapy,mTOR,2023
Dual PI3K/mTOR Inhibitors and Their Applications in Resistant Tumors,"The dual PI3K/mTOR inhibition strategy emerged to address feedback and resistance mechanisms. NVP-BEZ235 (dactolisib) suppresses PI3K isoforms, mTOR, and ATR, with strong activity in solid and blood cancers. NVP-BEZ235 crosses the blood–brain barrier and sensitizes temozolomide-resistant brain tumors. Combination of dactolisib with immune checkpoint inhibition showed robust antitumor responses in CRPC by blocking MDSCs. Synergistic activity was observed with 17AAG in melanoma. Other potent dual inhibitors include paxalisib, apitolisib, voxtalisib, PQR309, omipalisib, and gedatolisib. Paxalisib is in phase-II trials for diffuse midline glioma. Apitolisib (GDC-0980) was tested broadly in solid tumors. Voxtalisib showed activity in follicular lymphoma, DLBCL, CML, and mantle cell lymphoma. Combined voxtalisib with low-intensity pulsed ultrasound inhibited GBM stem cells. PQR309 showed efficacy in lymphomas and solid tumors. LY3023414 (PI3K/mTOR and DNA-PK inhibitor) demonstrated activity alone or in drug combinations.",Signal Transduction and Targeted Therapy,mTOR,2023
Metformin and AMPK-Mediated Suppression of mTOR,Metformin activates AMPK by repressing mitochondrial complex I and increasing the AMP/ATP ratio. AMPK negatively regulates mTORC1. Metformin suppressed mTORC1/2 in myeloma cells through AMPK activation and inhibited tumor formation in mouse models. Metformin can silence mTORC1 through Rag GTPases/REDD1 independent of AMPK. Metformin enhances autophagy via AMPK stimulation and mTORC1 inhibition. Metformin demonstrated anticancer efficacy through repression of mTORC1-dependent protein synthesis in human malignancies including breast cancer. Recent studies highlight the role of metformin-induced autophagy in cancer therapy. Pharmacological targeting of AKT/PI3K/mTOR axis has been summarized in Fig. 7 and Table 1.,Signal Transduction and Targeted Therapy,mTOR,2023
Overview of Exosome Biology and Composition,"The study of extracellular vesicles (EVs) has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and in organ homeostasis and disease. Exosomes, with an average diameter of ~100 nanometers, are a subset of EVs. The biogenesis of exosomes involves their origin in endosomes, and subsequent interactions with other intracellular vesicles and organelles generate the final content of the exosomes. Their diverse constituents include nucleic acids, proteins, lipids, amino acids, and metabolites, which can reflect their cell of origin. In various diseases, exosomes offer a window into altered cellular or tissue states, and their detection in biological fluids potentially offers a multicomponent diagnostic readout. The efficient exchange of cellular components through exosomes can inform their applied use in designing exosome-based therapeutics.",Science,Exosomes,2020
Background on EV Types and Exosome Biogenesis,"All cells, prokaryotes and eu-karyotes, release extracellular vesicles (EVs) as part of their normal physiology and during acquired abnormalities. EVs can be broadly divided into two categories, ectosomes and exosomes. Ectosomes are vesicles that pinch off the surface of the plasma membrane via outward budding, and include microvesicles, microparticles, and large vesicles in the size range of ~50 nm to 1 μm in diameter. Exosomes are EVs with a size range of ~40 to 160 nm (average ~100 nm) in diameter with an endosomal origin. Sequential invagination of the plasma membrane ultimately results in the formation of multivesicular bodies, which can intersect with other intracellular vesicles and organelles, contributing to diversity in the constituents of exosomes. Depending on the cell of origin, EVs, including exosomes, can contain many constituents of a cell, including DNA, RNA, lipids, metabolites, and cytosolic and cell-surface proteins. The physiological purpose of generating exosomes remains largely unknown and needs investigation.",Science,Exosomes,2020
Exosome Function and Intercellular Communication,"One speculated role is that exosomes likely remove excess and/or unnecessary constituents from cells to maintain cellular homeostasis. Recent studies reviewed here also indicate a functional, targeted, mechanism-driven accumulation of specific cellular components in exosomes, suggesting that they have a role in regulating intercellular communication. Exosomes are associated with immune responses, viral pathogenicity, pregnancy, cardiovascular diseases, central nervous system-related diseases, and cancer progression. Proteins, metabolites, and nucleic acids delivered by exosomes into recipient cells effectively alter their biological response. Such exosome-mediated responses can be disease promoting or restraining. The intrinsic properties of exosomes in regulating complex intracellular pathways has advanced their potential utility in the therapeutic control of many diseases, including neurodegenerative conditions and cancer. Exosomes can be engineered to deliver diverse therapeutic payloads, including short interfering RNAs, antisense oligonucleotides, chemotherapeutic agents, and immune modulators, with an ability to direct their delivery to a desired target.",Science,Exosomes,2020
Diagnostic and Therapeutic Potential of Exosomes,"The lipid and protein composition of exosomes can affect their pharmacokinetic properties, and their natural constituents may play a role in enhanced bioavailability and in minimizing adverse reactions. In addition to their therapeutic potential, exosomes also have the potential to aid in disease diagnosis. They have been reported in all biological fluids, and the composition of the complex cargo of exosomes is readily accessible via sampling of biological fluids (liquid biopsies). Exosome-based liquid biopsy highlights their potential utility in diagnosis and determining the prognosis of patients with cancer and other diseases. Disease progression and response to therapy may also be ascertained by a multicomponent analysis of exosomes. The study of exosomes is an active area of research. Ongoing technological and experimental advances are likely to yield valuable information regarding their heterogeneity and biological function(s), as well as enhance our ability to harness their therapeutic and diagnostic potential.",Science,Exosomes,2020
Future Directions in Exosome Research and Applications,"As we develop more standardized purification and analytical procedures for the study of exosomes, this will likely reveal their functional heterogeneity. Functional readouts using EVs enriched for exosomes have already provided new insights into their contribution to various diseases. New genetic mouse models with the ability for de novo or induced generation of cell-specific exosomes in health and disease will likely show the causal role of exosomes in cell-to-cell communication locally and between organs. Whether exosome generation and content change with age needs investigation, and such information could offer new insights into tissue senescence, organ deterioration, and programmed or premature aging. Whether EVs and/or exosomes preceded the first emergence of the single-cell organism on the planet is tempting to speculate, and focused bioelectric and biochemical experiments in the future could reveal their cell-independent biological functions. Single-exosome identification and isolation and cryoelectron microscopy analyses have the potential to substantially improve our understanding of the basic biology of exosomes and their use in applied science and technology.",Science,Exosomes,2020
Graphical Abstract – Overview of Exosome Function,"Exosomes function as a cell-to-cell transit system in the human body with pleiotropic biological effects. These extracellular vesicles (EVs) are generated by all cell types and carry nucleic acids, proteins, lipids, and metabolites. They mediate both near- and long-distance intercellular communication in physiological and pathological contexts. Research on EVs spans diverse areas of biology, including tissue homeostasis, immune regulation, pathogenic injury, and organ remodeling. Because exosomes circulate in all biological fluids, they provide a powerful avenue for diagnostic applications (liquid biopsy) and are being explored for therapeutic delivery. EV biology remains experimentally challenging due to limitations in single-particle detection, isolation heterogeneity, and insufficient in vivo tracking technologies. Despite these caveats, major discoveries have shown that EVs influence disease progression (cancer, neurodegeneration, cardiovascular disease) and may serve as multicomponent biomarkers.",Science,Exosomes,2020
Classification of EVs and Distinction Between Exosomes and Ectosomes,"EVs are broadly separated into two categories: ectosomes and exosomes. Ectosomes arise through outward budding of the plasma membrane and include microvesicles, microparticles, and larger vesicles (~50 nm to 1 μm). Exosomes differ fundamentally in their intracellular origin; they form within endosomes and range from ~40 to 160 nm (~100 nm average). The classification of EVs remains dynamic, and isolation methods vary widely, often producing heterogeneous mixtures containing exosomes and other EVs. This complicates interpretation of biological studies, since current markers likely identify only subpopulations of exosomes. Exosomes are especially important because their formation involves regulated intracellular pathways that determine their molecular contents and possibly their biological functions once secreted. The continuous refinement of EV purification and analytical methods will be necessary to achieve accurate functional characterization of exosomes in vivo.",Science,EV Classification,2020
Biogenesis of Exosomes – Endosomal Pathway and MVB Formation,"Exosome biogenesis begins with double invagination of the plasma membrane. The first inward budding event captures cell-surface proteins and extracellular soluble components, forming early-sorting endosomes (ESEs). These can mature into late-sorting endosomes (LSEs), which subsequently generate multivesicular bodies (MVBs). MVBs contain intraluminal vesicles (ILVs), the precursors of exosomes. MVBs face two possible fates: degradation through fusion with lysosomes/autophagosomes or secretion through fusion with the plasma membrane, releasing ILVs as exosomes. The trans-Golgi network and the endoplasmic reticulum contribute additional components to the endosomal system. Exosome biogenesis relies on multiple regulators, including Rab GTPases, ALIX, TSG101, ESCRT complexes, tetraspanins, ceramides, phospholipids, syndecans, and SNARE proteins. However, interpreting these regulators' roles is difficult because many also control other intracellular trafficking pathways. Cell type, culture conditions, and genomic status further influence exosome formation.",Science,Exosome Biogenesis,2020
Challenges in Studying Exosome Biogenesis and Production Rates,"Functional studies of exosome biogenesis are confounded by overlapping pathways involved in autophagy, lysosomal activity, and Golgi-derived vesicle trafficking. Loss- or gain-of-function experiments targeting Rab proteins or ESCRT machinery often produce indirect effects unrelated to exosome formation. Variations in isolation methods contribute to inconsistent interpretations and may lead to heterogeneous vesicle populations being studied as exosomes. Computing exosome production rates is difficult due to simultaneous production, secretion, and uptake of exosomes by cells. A study using live single-cell imaging with tetraspanin-capture surfaces revealed that cancerous and non-cancerous breast epithelial cells produce exosomes at different rates. Noncancerous cells released ~60–65 exosomes per cell per hour, whereas some cancer cells secreted fewer. Other studies report higher vesicle secretion by cancer cells, but these relied on isolation platforms that may detect both exosomes and ectosomes.",Science,Exosome Production,2020
"Exosome Heterogeneity – Size, Content, and Cellular Origin","The heterogeneity of exosomes is likely reflective of their size, content, functional impact on recipient cells, and cellular origin. Size inequality could be due to uneven invagination of the limiting membrane of the MVB, resulting in distinct total content of fluid and solids, or isolation methods that include other EVs. Refined fractionation methods involving EVs revealed that exosomes may contain subpopulations defined by a distinct size range. Size heterogeneity can also result in different amounts of exosomal content. The microenvironment and the inherent biology of the cells may influence the content of the exosomes and their biological markers. Exosomes can contain membrane proteins, cytosolic and nuclear proteins, extracellular matrix proteins, metabolites, and nucleic acids, namely mRNA, noncoding RNA species, and DNA. Although exosomal cargo analyses require large pools of purified exosomes, not all exosomes contain a similar abundance of a given cargo, as observed, for example, with miRNA. Proteomic analyses of EVs have revealed marker heterogeneity of exosomes, cautioning their utility in experimental design using marker-determined purification. Distinct proteins and nucleic acids can be enriched in exosomes compared with their cell of origin, suggesting a specific protein-sorting mechanism associated with exosome biogenesis or content loading.",Science,Exosome Heterogeneity,2020
Functional Heterogeneity and Organ Tropism of Exosomes,"The effects of exosomes on recipient cells can be different because of their varied expression of cell surface receptors, and such functional heterogeneity can result in one set of exosomes inducing cell survival, another set inducing apoptosis, and a different set inducing immunomodulation in different target cell types. Heterogeneity can also be based on the organ and tissue of origin of the exosomes, including whether they are from cancer cells, giving them distinct properties such as tropism to certain organs and uptake by specific cell types. A combination of size, content, cell of origin, and surface receptor patterns can give rise to a higher order of complexity and heterogeneity of exosomes. The cellular microenvironment and metabolic state also influence exosome composition and functional output.",Science,Exosome Functional Diversity,2020
Intercellular Communication – Exosome Uptake and Secretion,"Questions surrounding the function of exosomes focus on understanding the fate of their constituents and the phenotypic and molecular alterations they induce in recipient cells. Exosome uptake and secretion pathways may intersect, creating mixed pools of endogenously produced and recycled exosomes. Mechanisms of exosome uptake differ across cell types, adding complexity to their communication roles. Oncogenic KRAS expression promotes exosome uptake by macropinocytosis in human pancreatic cancer cells. Human melanoma cells uptake exosomal cargo through fusion with the plasma membrane, whereas neurosecretory PC12 cells rely on clathrin-dependent endocytosis. It remains unknown whether each uptake mode results in distinct localization, degradation, or downstream signaling. The turnover rate of internalized exosomal cargo may depend on cargo type and the metabolic state of the recipient cell. It is also unclear how exogenously administered exosomes compare with physiologically produced exosomes in tropism and retention.",Science,Exosome Uptake Mechanisms,2020
In Vivo Tracking and Functional Cargo Transfer,"To track intercellular exchange of exosomes under physiological conditions, in vivo experiments using genetic mouse models have been explored. These studies demonstrate that exosomes can deliver mRNA to a recipient cell on rare occasions. Such rare events increase during activation and expansion of exosome-producing immune cells in acute inflammation or chronic inflammation. Therapeutic interventions such as chemotherapy may influence exosome uptake and downstream biology; for example, inhibition of a proton pump or altered cellular pH in melanoma cells limits exosome uptake. Although exosomal proteomes reflect their originating cells, cancer cells may alter their exosomal protein cargo. Proteomic studies have revealed that glioblastoma cells expressing EGFRvIII release exosomes enriched in proinvasive molecules. Neural stem cells exposed to inflammatory cytokines produce exosomes carrying IFNγ-bound IFNGR1 that activate STAT1 signaling in recipient cells. These studies support that proteins are selectively sorted into exosomes to modulate development, immune responses, and disease processes.",Science,Exosome Cargo Signaling,2020
Exosomes in Mammalian Reproduction – Semen and Sperm Maturation,"Human reproduction, pregnancy, and embryonic development require precise and dynamic intercellular communication. Semen, amniotic fluid, blood, and breast milk all contain exosomes with putative functions. Seminal plasma exosomes have been implicated in sperm maturation. Molecular profiling indicates that let-7a, let-7b, miR-148a, miR-375, and miR-99a are enriched in seminal plasma-derived exosomes from multiple donors. These miRNAs modulate interleukin expression (IL-10, IL-13), suggesting a role in genital tract immunity. Seminal plasma-derived exosomes also inhibit HIV-1 infection, possibly by blocking the recruitment of the HIV Tat protein and suppressing early viral transcription. Exosomes may therefore contribute to both reproductive physiology and antiviral defense in the male reproductive tract.",Science,Exosomes in Reproduction,2020
Placental and Pregnancy-Associated Exosomes,"Exosomes help protect the placenta from infection by transferring C19MC miRNAs from placental trophoblasts to nonplacental cells. These miRNAs induce autophagy and antiviral defense against poliovirus, cytomegalovirus, and HSV-1. In pregnant women, exosomal miRNA and protein cargo in blood plasma varies by gestational age and differs in cases of preterm birth. In mice, plasma-derived exosomes evolve dynamically throughout pregnancy; late-gestation exosomes can induce preterm birth in near-term pregnancies but have no effect earlier in gestation. These findings indicate that exosomes act as gestational stage–specific mediators influencing labor, immune defense, and maternal–fetal communication.",Science,Exosomes in Pregnancy,2020
Breast Milk Exosomes and Postnatal Development,"Breast milk contains exosomes enriched in immune-related miRNAs and promotes neonatal health. These exosomes enhance peripheral blood–derived T-regulatory cell numbers ex vivo, suggesting a role in immune tolerance. They also promote proliferation of intestinal epithelial cells in porcine models and increase epithelial growth in the mouse intestinal tract in vivo. However, it remains unclear how well nucleic acids and other exosomal cargos survive digestion and are absorbed by the intestinal epithelium. Differences in uptake routes will influence the therapeutic potential of orally delivered exosomes, as the gastrointestinal environment imposes enzymatic and physical barriers.",Science,Breast Milk Exosomes,2020
Exosomes in Immune Responses – General Overview,"The role of exosomes in immune responses is well documented. Interestingly, repeated administration of mouse or human cell-derived exosomes in mice does not trigger severe immune reactions even without matching HLA types, despite transfusions delivering trillions of EVs. This suggests that exosome immune reactivity depends on dose and context. Engineered exosomes have been shown to elicit adaptive and innate immune reactions, supporting therapeutic development. Exosomal functions in immune regulation arise from antigen presentation, delivery of DNA that activates cGAS-STING signaling, gene expression manipulation via miRNAs, and engagement of surface ligands that modulate immune pathways.",Science,Exosomes and Immunity,2020
Exosomes in Antigen Presentation and Adaptive Immunity,"Exosomes from antigen-presenting cells (APCs) carry p-MHC-II and costimulatory signals, enabling direct presentation of peptide antigen to T cells. However, exosomes stimulate T cells less effectively than intact APCs. In mice, intradermal injection of APC-derived exosomes loaded with tumor peptides can delay tumor growth via CD8+ T cell–mediated responses. Exosome-delivered antigens can also be transferred to APCs for indirect antigen presentation. Immature dendritic cells activated by exosomal peptides can indirectly activate APCs and induce CD4+ T cell proliferation. Exosomes from OVA-pulsed dendritic cells activate CD8+ T cells more efficiently than microvesicles, suggesting specificity in exosome-mediated antigen presentation.",Science,Exosomes in Antigen Presentation,2020
"Exosomes in Bacterial, Fungal, and Parasitic Infection","Exosomes influence immune responses to bacterial infection. Macrophage-derived exosomes presenting bacterial antigens promote adaptive immune activation, cytokine production, dendritic cell maturation, and T cell activation. Bacteria-derived EVs also contribute to inflammation and immunity. Exosomal DNA from intracellular bacteria can activate cGAS-STING signaling, but in some cases suppresses T cell responses, weakening host defense. Conversely, exosomal RNA from M. tuberculosis enhances host immunity by promoting phagosome maturation. Parasite-derived exosomes can contribute to virulence; for example, Plasmodium falciparum releases DNA and small RNAs into exosomes that act as decoy signals to manipulate host immune responses.",Science,Exosomes in Infection,2020
Exosomal DNA and Immune Modulation in Cancer,"Exosomal DNA influences both innate and adaptive immune responses. Breast cancer–derived exosomal genomic DNA activates cGAS-STING signaling in dendritic cells, inducing antitumor immunity. T cell–derived exosomal DNA also primes dendritic cells to produce type I interferons. Exosome biogenesis may help cells clear cytoplasmic DNA to prevent aberrant activation of DNA sensing pathways. EGFR inhibition increases exosomal DNA release from cancer cells, strengthening cGAS-STING activation in dendritic cells and suppressing tumor growth. However, tumor-derived exosomal DNA can also promote inflammation by triggering neutrophils to produce IL-8 and tissue factor, contributing to thrombosis and tumor-associated inflammation.",Science,Exosomal DNA in Cancer Immunity,2020
Exosomal miRNA and Immune Signaling Pathways,"Exosomal miRNAs regulate gene expression and immune signaling. miRNA exchange between dendritic cells modulates dendritic cell maturation. Tumor-derived exosomal miR-212-3p suppresses the MHC-II transcription factor RFXAP, potentially enabling immune evasion. Exosomal miR-222-3p down-regulates SOCS3 in monocytes, activating STAT3 and promoting M2 macrophage polarization, which supports immunosuppression. Exosomal PD-L1 suppresses CD8+ T cell function and dendritic cell maturation, while FasL on tumor-derived exosomes induces T cell apoptosis. Many cancer-derived exosomes expressing CD39/CD73 generate adenosine, inhibiting T cell activation and promoting an immunosuppressive microenvironment.",Science,Exosomal miRNA in Immune Modulation,2020
Exosomes in Viral Infection – Dual Roles,"Exosomes participate in both viral spread and antiviral defense. Many viruses exploit exosome biogenesis as a 'Trojan horse' strategy to enhance infectivity, evade immune detection, or promote viral cooperation. HAV and HEV can exist in pseudoenveloped exosome-associated forms. Tetraspanins CD81 and CD9, as well as PtdSer-binding receptor TIM-4 on exosomes, can promote viral entry. Tumor-derived exosomes carrying EGFR suppress macrophage antiviral response via MEKK2–IRF3 signaling. Conversely, exosomes from IFNα-stimulated macrophages deliver APOBEC3G, protecting hepatocytes from HBV infection and impairing HIV-1 infection. CD4-bearing exosomes reduce HIV-1 infectivity, although viral Nef reduces exosomal CD4 to enhance infection.",Science,Exosomes in Viral Infection,2020
Exosomes in Metabolic Disease and Insulin Resistance,"Exosomes contribute to metabolic disease progression by mediating metabolite transfer and intercellular communication among pancreatic β-cells, adipose tissue, skeletal muscle, and liver in mice and humans. In leptin-deficient obese mouse models, reciprocal exosome-mediated signaling between adipocytes and macrophages highlights the role of RBP4 (retinol binding protein 4) in macrophage activation and insulin resistance. High-fat diet–induced obese mice exhibit distinct circulating exosomal miRNAs that are sufficient to promote insulin resistance in lean mice, possibly via down-regulation of Ppara expression in white adipose tissue. These findings indicate that exosomal miRNAs can transmit metabolic dysfunction between tissues and may serve as endocrine-like messengers driving systemic insulin resistance.",Science,Exosomes in Metabolic Disease,2020
Exosomes in Cancer-Associated Cachexia and Paraneoplastic Syndromes,"Cancer cell–derived exosomes can profoundly influence systemic metabolism, contributing to cachexia and metabolic paraneoplastic syndromes such as cancer-associated diabetes. In pancreatic cancer, exosomes carry adrenomedullin, a lipolytic peptide hormone that induces fat breakdown in mouse and human adipocytes and simultaneously inhibits insulin secretion in rat and human islet cells. Additionally, cancer exosomes enriched in heat shock proteins (HSP70 and HSP90) promote muscle wasting in mice, supporting a role in skeletal muscle catabolism. These exosome-driven metabolic effects demonstrate that cancers can remodel distant tissues by exporting bioactive factors through exosomes, thereby driving weight loss, impaired insulin secretion, and multi-organ metabolic dysfunction.",Science,Exosomes and Cancer Cachexia,2020
Exosomes in Cardiovascular Disease and Atherosclerosis,"Exosomes from endothelial cells, cardiac fibroblasts, cardiomyocytes, and cardiac progenitor cells have been implicated in metabolic disorders, atherosclerosis, and cardiovascular disease. In mouse models, platelet-derived exosomes prevent atherosclerosis by reducing macrophage scavenger receptor CD36 expression, thereby limiting the uptake of oxidized LDL and decreasing foam-cell formation. By contrast, human smooth muscle cell–derived exosomes promote thrombogenesis in vitro, highlighting context-dependent effects of vascular exosomes. These observations suggest that exosomes within the cardiovascular system may either protect against or contribute to vascular disease depending on their cellular origin and cargo.",Science,Exosomes in Cardiovascular Disease,2020
Stem Cell–Derived Exosomes in Cardiovascular Protection,"Stem cell–derived exosomes (from bone marrow–derived stem cells, embryonic stem cells, and mesenchymal stromal cells) exhibit cardioprotective properties in mouse and rat models. These effects are mediated largely through exosomal miRNAs. miR-19a and miR-21 from murine cardiac progenitor cells target PDCD4 in rat myoblasts, reducing apoptosis. miR-22 from mouse bone marrow MSCs targets MECP2 in ischemic mouse heart, improving mitochondrial function and preserving cardiac tissue. Human MSC-derived exosomes containing miR-21-5p regulate SERCA2a and L-type calcium channels in human cardiac myocytes derived from pluripotent stem cells, enhancing calcium handling and contractility. Collectively, these cardioprotective exosomal miRNAs promote survival, maintain mitochondrial energetics, and help preserve cardiac performance.",Science,Stem Cell Exosomes and Heart Protection,2020
Exosomes in Neurodegeneration: Bidirectional Roles,"Exosomes intersect with neuronal secretory vesicle pathways and play both protective and harmful roles in neurodegenerative diseases. They can facilitate clearance of unfolded or misfolded proteins, providing detoxifying, neuroprotective functions, or they may propagate misfolded proteins, contributing to their 'infectivity' and spread across neural circuits. Pharmacological manipulation of exosome biogenesis demonstrates these dual effects: GW4869, which inhibits MVB inward budding, reduces transmission of prion protein PrPSc, whereas monensin, which elevates intracellular calcium and boosts MVB formation, increases PrPSc transfer. This duality suggests that exosome production level directly regulates pathogenic protein dissemination. Pathogenic proteins such as Tau and β-amyloid (Aβ) have been identified in exosomes derived from cerebrospinal fluid of patients, microglial cultures, and neuronal cell lines. Exosome-mediated Tau transfer promotes aggregation and may exploit endosomal trafficking pathways. Although Aβ accumulates in MVBs after APP cleavage, the extent to which exosomes drive toxic Aβ oligomer formation in vivo remains unresolved.",Science,Exosomes and Neurodegeneration,2020
"Exosomal Pathways in Alzheimer’s, Parkinson’s, and ALS","Exosome biogenesis can be neuroprotective by assisting in the removal of neurotoxic oligomers or harmful proteins from cells. Conversely, dysregulation of exosome machinery can promote disease. In APP-overexpressing mouse models, increased exosomal Aβ secretion correlates with elevated oligomerized Aβ caused by deregulation of ECE1/2, linking exosome cargo changes to early amyloid pathology. In Parkinson’s disease, α-synuclein appears in cerebrospinal fluid exosomes, and exosome machinery impairment supports intracellular accumulation, partly because α-synuclein suppresses ESCRT activity. ALS-associated proteins such as mutant SOD1 and TDP-43 are also detected in exosomes from astrocytes or neuroblast cell lines. Astrocyte-derived exosomal SOD1 can kill motor neurons in vitro. However, in vivo inhibition of exosome secretion using GW4869 worsened pathology in TDP-43 transgenic mice, indicating that exosome release may be needed for clearing toxic proteins. Although transfer of neurotoxic proteins via exosomes occurs in vitro, the extent and direction of their influence on in vivo disease progression remain unclear.",Science,Exosomal Proteinopathies,2020
Exosomal Nucleic Acids and Neuroinflammation,"Beyond misfolded proteins, exosomal nucleic acids contribute to neurological disorders. Serum-derived exosomes from children with autism spectrum disorder contain mitochondrial DNA capable of inducing IL-1β secretion from microglia, suggesting a role in neuroinflammatory processes associated with ASD. Although causality remains unconfirmed, these findings highlight how exosome cargo beyond proteins—such as mtDNA or other damage-associated molecular patterns—may trigger neuroimmune activation. Exosomes efficiently cross the blood–brain barrier, making them strong candidates for therapeutic delivery vehicles in neurological disease. Their ability to transport RNA, proteins, and small molecules directly into the CNS has encouraged development of exosome-based therapies for neurodegeneration, despite incomplete understanding of their natural physiological roles in these disorders.",Science,Exosomal Nucleic Acids in Neurology,2020
Diagnostic Potential of Exosomes,"Exosomes are present in all biological fluids, making them attractive as minimally invasive liquid biopsies capable of longitudinal monitoring. Their biogenesis captures complex extracellular and intracellular cargo—including proteins, lipids, metabolites, RNA, miRNA, and potentially DNA—enabling multiparameter diagnostic testing. Diseases with emerging exosome-based diagnostics include cardiovascular disease, CNS disorders, cancer, and pathologies of the liver, kidney, and lung. The presence of DNA in exosomes remains contentious: some studies report detectable DNA useful for identifying cancer mutations such as KRAS or TP53, while others report negligible DNA content. Diagnostic miRNAs in exosomes are more consistently observed. Oncogenic miRNAs like miR-21, miR-155, the miR-17-92 cluster, and miR-1246 are elevated across numerous cancers, including glioblastoma, pancreatic, colorectal, liver, breast, prostate, and esophageal cancers. Tumor suppressor miRNAs such as miR-146a and miR-34a also show diagnostic relevance. Additionally, exosomal surface markers—such as GPC1-positive exosomes for pancreatic, breast, and colon cancer, CD147 for colorectal cancer, and phosphatidylserine composition—offer promising detection strategies. Multi-marker diagnostic panels combining RNA, protein, lipid, and metabolite cargo may enhance specificity and sensitivity.",Science,Clinical Applications of Exosomes,2020
Therapeutic Potential of Exosomes,"Exosomes are being actively explored as therapeutic agents or drug-delivery vehicles because of their ability to enter cells efficiently, deliver functional cargo, and evade immune clearance. Repeated injections of mesenchymal or epithelial cell-derived exosomes show minimal toxicity in mice, supporting their safety profile. Natural exosome properties such as CD47-mediated 'don’t eat me' signaling enhance their circulation time, while ligand engineering enables targeted tissue delivery. Engineered exosomes loaded with chemotherapeutic agents show enhanced antitumor efficacy compared with free drugs. Examples include dendritic cell-derived exosomes with RGD peptides delivering doxorubicin in breast cancer models, and macrophage-derived exosomes carrying paclitaxel inducing lung tumor regression. Exosomes engineered to deliver RNA therapeutics represent a major translational focus. Delivery of siRNA or miRNA payloads has shown success in rodent models of mammary carcinoma, glioma, and pancreatic cancer. Clinical-grade MSC-derived exosomes carrying KRasG12D siRNA (iExosomes) increased survival and target engagement in pancreatic cancer mouse models and have advanced to phase I clinical trials. Exosomes can cross the blood-brain barrier, enabling neurological applications. Intranasal or intravenous administration of MSC-, macrophage-, or dendritic cell-derived exosomes has shown neuroprotective effects in models of autism-like behavior, traumatic brain injury, Parkinson’s disease, and Alzheimer’s disease through delivery of BACE1- or α-synuclein–targeting siRNA, catalase, or dopamine. These findings highlight the potential of exosomes to address diseases with limited pharmacological options.",Science,Exosome-Based Therapeutics,2020
Exosomes and Cancer Immunotherapy,"Owing to their role in antigen presentation and modulation of immune responses, exosomes are being explored for immunotherapy. Dendritic-cell-derived exosomes, known as 'dexosomes,' can carry antigenic peptides and costimulatory signals. In early clinical testing, dexosomes generated from IFN-γ–matured dendritic cells and loaded with the melanoma antigen MART1 were administered to patients with advanced non–small-cell lung cancer. Although a strong antigen-specific T cell response was not observed, natural killer cell activity increased, disease stabilization was noted in some patients, and only one case of notable toxicity occurred. Exosomes can also polarize the tumor immune microenvironment. Their engineered forms may enhance antitumor immunity through improved antigen presentation or delivery of immunomodulatory molecules. Preclinical studies indicate that exosome-based immunotherapies can synergize with existing treatments by altering dendritic cell activation, enhancing cytotoxic T cell responses, or modulating macrophage polarization. These early findings, together with substantial preclinical evidence, have positioned exosome engineering as a promising platform for future cancer immunotherapies.",Science,Exosomes in Immunotherapy,2020
Abstract and Overview of Circadian Metabolism,"A majority of mammalian genes exhibit daily fluctuations in expression levels, making circadian expression rhythms the largest known regulatory network in normal physiology. Cell-autonomous circadian clocks interact with daily light-dark and feeding-fasting cycles to generate approximately 24-hour oscillations in the function of thousands of genes. Circadian expression of secreted molecules and signaling components transmits timing information between cells and tissues. Such intra- and intercellular daily rhythms optimize physiology both by managing energy use and by temporally segregating incompatible processes. Experimental animal models and epidemiological data indicate that chronic circadian rhythm disruption increases the risk of metabolic diseases. Conversely, time-restricted feeding, which imposes daily cycles of feeding and fasting without caloric reduction, sustains robust diurnal rhythms and can alleviate metabolic diseases. These findings highlight an integrative role of circadian rhythms in physiology and offer a new perspective for treating chronic diseases in which metabolic disruption is a hallmark.",Science,Circadian Metabolism,2016
Meal Timing and Postprandial Responses,"A transient rise in blood sugar after a meal indicates metabolic health. A larger meal produces a larger spike, whereas a fat or protein-rich meal produces a muted spike (compared with a normal meal of equivalent caloric content). Physiological responses to what and how much we eat represent the foundation for basic and translational science aimed at preventing and treating obesity, diabetes, and metabolic diseases, which together afflict close to a billion people worldwide. However, the timing of food consumption independent of total caloric intake and macronutrient quality has emerged as a critical factor in maintaining metabolic health. For instance, when healthy adults eat identical meals at breakfast, lunch, or dinner, the postprandial glucose rise is lowest after breakfast and highest after dinner (1), as if the dinner were twice the size of the breakfast. In addition, when healthy adults are given a constant glucose infusion over 24 hours, glycemia rises at night and falls around dawn (1), indicating that in addition to what and how much we eat, when we eat helps determine the physiological response to nutrient availability.",Science,Circadian Metabolism,2016
SCN Glucose Uptake and Diurnal Rhythms,"Daily rhythms in nutrient use were first documented almost 40 years ago in cells of the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Experiments in rats fed 14C-labeled deoxyglucose during their habitual (nighttime) feeding period showed that entry of glucose into the SCN was almost negligible, whereas during the day, radio-labeled glucose was readily detected (2). Such glucose uptake rhythms were sustained even in the absence of light cues. In all, this elegant experiment proved the existence of a circadian rhythm in nutrient demand and/or uptake in tissues. Over the next decades, research into circadian rhythms has shown that daily rhythms in the function of numerous genes prime the organism to assimilate nutrients, to mobilize these nutrients for various functions, and to discard metabolic waste at specific times of the 24-hour day (3, 4). Whereas circadian rhythms generally refer to ~24-hour oscillations that occur in the absence of external timing cues, daily or diurnal rhythms apparent during normal living conditions emerge from interactions between the internal circadian clock and timing cues, which include light and food.",Science,Circadian Metabolism,2016
Disruptions in Daily Rhythms and Metabolic Disease,"Accordingly, a consistent daily pattern of eating and fasting maintains normal circadian physiology, whereas frequent disruptions in daily activity-rest and eating-fasting rhythms (as occurs in shiftwork) (5) or genetic disruption of circadian clock in rodents predisposes to metabolic diseases (6). Certain diet regimens (e.g., the frequent eating of energy-dense food) and aging can dampen these daily oscillations and predispose one to metabolic diseases. Therefore, understanding the diurnal physiology of metabolism at a mechanistic level could potentially reveal lifestyle and therapeutic interventions for preventing and treating metabolic diseases.",Science,Circadian Metabolism,2016
Transcriptional-Translational Feedback Loop (TTFL),"In animals, the core mechanism that gives rise to circadian oscillations is a cell-autonomous transcriptional-translational feedback loop (TTFL) present in most cells. The transcription factors CLOCK (or NPAS2) and BMAL1 bind as heterodimers to cis-acting E boxes in the promoters of their own repressors—Cryptochrome (Cry1 and Cry2) and Period (Per−1, −2, and −3)—and of the nuclear hormone receptors Rev-erb (−α and −β), and Ror (−α, −β, and −γ). ROR and REVERB drive rhythmic Bmal1 gene expression by respectively acting so as to activate and repress its expression through RRE elements present in its promoter (Fig. 1) (7). REV and ROR proteins also affect the expression of Cry1, delaying its expression several hours relative to Cry2. Regulated transcriptional and posttranscriptional events involving a growing list of nuclear and cytoplasmic proteins generate endogenous ~24-hour rhythms in the mRNA and protein levels of most of these 13 transcriptional regulators (7). In addition to controlling each other’s expression, these regulators also drive rhythmic expression of thousands of target genes by binding cis-regulatory sites or through downstream transcriptional regulators.",Science,Circadian Oscillator,2016
Coordinated Gene Expression and Phase Regulation,"The transcriptional basis of circadian rhythms enables a set of transcriptional regulators to temporally couple their activity with the synchronous rhythmic expression of hundreds or even thousands of genes, with peak expression at distinct times of the day (phase). Such extensive and coordinated gene expression and function would be difficult to achieve with a timing mechanism based entirely on protein-protein interactions. Circadian transcription factors also interact with a number of coactivators, corepressors, and chromatin-associated factors that read, write, or erase chromatin histone modification marks to activate or repress transcription (Fig. 1). CLOCK/BMAL1 complexes are often associated with the histone acetyl transferase p300 and CREB-binding protein (CBP) (8). CRY/PER repressors are found in complexes with histone deacetylase (HDAC) (9). Additionally, MLL1, MLL3, WDR5, and EZH2 form complexes with circadian transcriptional factors (10). Interactions between REV-ERB and the N-CoR/HDAC3 corepressor are essential for repressive function of REV-ERB (11).",Science,Circadian Oscillator,2016
Chromatin Modifiers and Additional Regulatory Elements,"A histone lysine demethylase, JARID1a (12), and a bHLHPAS protein, USF1 (13), help transition between daily cycles of activation and repression by interacting with CLOCK/BMAL1 and PER/CRY complexes. In addition to rhythms in histone modifications, some circadian clock proteins also undergo acetylation and deacetylation. Additional cis-acting promoter elements (e.g., CRE and HSE) mediate rapid adjustment of circadian clock components in response to sudden changes in cellular state. Altogether, circadian clock–mediated transcriptional regulation involves a large number of proteins and functional interactions.",Science,Circadian Oscillator,2016
Metabolic and Extracellular Inputs to the Circadian Clock,"The elaborate circadian transcriptional mechanism has many advantages, given that each component of the circadian system can serve as a node for integrating cellular physiology with the circadian function or to transmit circadian timing information to nonclock proteins. Cellular concentration of certain metabolites–including but not limited to heme, nicotinamide adenine dinucleotide/reduced form of nicotinamide adenine dinucleotide (NAD/NADH), nicotinamide adenine dinucleotide phosphate/reduced form of NADP (NADP/NADPH), adenosine monophosphate/adenosine triphosphate (AMP/ATP), acetyl coenzyme A (AcCoA), alpha keto glutarate (α-KG), S-adenosyl methionine (SAM), CO, and polyamine—can affect the function of several circadian transcriptional regulators by modulating histone modifications, protein modifications, protein-protein interactions, protein-DNA interactions, or protein turnover (14). Extracellular factors such as temperature, hormones, and metabolites can also affect the clock and thereby constitute mechanisms for local synchrony of cellular clocks or for adjusting the phase of an organ’s clock in response to systemic signals. Hence, the circadian physiology of any given cell emerges from integrating the cell-autonomous TTFL, cellular metabolism, and extracellular systemic signals (Fig. 1). Thus, clock proteins can sense daily changes in cellular metabolism and systemic signals and make predictive changes to the circadian transcriptome.",Science,Circadian Plasticity,2016
Clock Protein Interactions with Nonclock Pathways,"Many circadian clock proteins interact with and modify the function of proteins that are not part of the core clock TTFL. Coupled with their own cycling levels, these clock proteins can “impose” rhythmic functions to nonclock proteins. For example, CRY and REV-ERB proteins interact with the glucocorticoid receptor (GR) to inhibit its transcriptional activity (15, 16). CRY proteins also inhibit signaling downstream of the glucagon receptor, thereby imposing a time-of-day-specific effect of glucagon on gluconeogenesis (17). Similarly, REV-ERB and HNF6 interact to regulate lipid metabolism in adult mouse liver (18). Additionally, several homologous proteins within the core TTFL (or their interacting partners) are not fully redundant but rather have specific functions. For example, the CLOCK/BMAL complex interacts with SIRT1 or SIRT6 in a locus-specific manner to target different subsets of the circadian transcriptome in the liver (19).",Science,Circadian Plasticity,2016
Tissue-Specific Circadian Regulation,"In many cases, these homologs are not expressed in all tissues. Ror-α is expressed in neural tissue, whereas Ror-γ expression dominates in peripheral tissues (20). In summary, the molecular constituents of the clock, partial redundancy among clock components, interactions between clock and nonclock components, and the cyclic expression of downstream tissue-specific components all contribute to tissue-specific molecular circadian physiology, which functions to integrate cell type–specific intra- and extracellular signals to regulate tissue function.",Science,Circadian Plasticity,2016
SCN Control of Systemic Rhythms,"The SCN plays a central, high-order role in the circadian regulation of metabolism by sustaining ~24-hour rhythms in activity-rest and feeding-fasting, even under constant darkness. This is achieved through both synaptic and diffusible factors that couple the SCN oscillator with cell type–specific circadian clocks in different brain regions and endocrine cells (7). Through a poly-synaptic connection, the SCN ensures that the pineal gland produces melatonin in a rhythmic fashion (peak levels at night) to promote sleep in diurnal animals. Similarly, through the paraventricular nucleus (PVN) and the pituitary gland, the SCN drives a circadian rhythm in adrenocorticotropic hormone (ACTH) release, which in turn drives a morning rise in corticosterone release from the adrenal gland. Under natural light-dark (LD) conditions, bright light strongly suppresses the production of melatonin (21) and promotes corticosterone production in the adrenal gland through an ACTH-independent sympathetic pathway (22). Corticosteroids promote arousal and alertness and drive catabolic metabolism in adipose tissue and muscle. Both melatonin and cortisol rhythms are detectable in the blood, and both hormones have pleiotropic effects on multiple tissues.",Science,Tissue-Level Circadian Clocks,2016
Integration of SCN Outputs with Hypothalamic Centers,"Local SCN outputs are intimately integrated with centers in the hypothalamus involved in hunger-satiety, sleep-arousal, thermoregulation, and osmolarity, as well as a forebrain oscillator that mediates an anticipatory drive for food (23). Mechanisms underlying these synaptic interactions are unclear, and both cellular and genetic phenotypes paint a complicated picture. Npas2−/− mice show normal overall diurnal rhythms in activity and rest under a LD cycle, yet they lack a siesta-type rest period in the middle of the active period and cannot adjust normally when meal timing is abruptly changed (24). On the other hand, hypomorphic Clock mutant mice show normal circadian behavior under a LD cycle, yet, owing to low amplitude of the SCN clock, rapidly adjust activity-rest rhythms in response to abrupt changes in the LD cycle, which mimic jet lag or rotating shift work (25). Similarly, mutation of a casein kinase 1 (CK1) phosphorylation site in Per2 advances the sleep onset time (26) but does not affect feeding time. A paralogous mutation in Per1 advances the daily onset of feeding without affecting the timing of activity-rest (27). It is unclear whether the different phenotypes seen in these paralogous mutants arise from shared neural mechanisms.",Science,Tissue-Level Circadian Clocks,2016
Peripheral Clocks in Gut and Pancreas,"The SCN also communicates with peripheral tissues, including the gut and pancreas, which have their own autonomous circadian clocks. These local clocks mediate responses to nutrient intake and control the release of systemic factors. For example, a circadian clock in secretory cells of the gut drives rhythmic expression of SGLT1, accounting for increased glucose uptake at times of anticipated food intake (28). Glucose influx triggers the release of GLP1 incretin that, along with the direct effect of glucose on pancreatic islets, promotes insulin release. In pancreatic islet cells, like in many other cell types, exo-cytosis is modulated in a circadian manner (29). Accordingly, insulin release has a circadian component (Fig. 2A). Overall, the central circadian clock, through direct or indirect effects, generates systemic rhythms in several signaling molecules, including melatonin, glucocorticoids, growth hormones, insulin, glucagon, and GLP1, whose rhythms are further accentuated by LD or feeding-fasting cycles.",Science,Tissue-Level Circadian Clocks,2016
Large-Scale Omics Studies of Liver Circadian Rhythms,"Because the liver plays a central role in nutrient metabolism, is composed of relatively homogeneous cell types, and is easy to access, a number of ≥24-hour time-course “-omics” studies have been conducted using mouse liver tissue. These have included chromatin immunoprecipitation sequencing (ChIP-seq), RNA-seq (and comparable microarray hybridization), ribo-seq, nascentseq, proteomics, and metabolomics. The majority of these studies used young male mice (<20 weeks old) entrained to a 12-hour light/12-hour dark cycle for several days and fed a standard diet (calories from fat <18%, protein ~25%, carb ~60%) ad libitum. Tissue samples have usually been collected under circadian conditions (constant darkness). Such an approach allows the removal of confounding effects derived from light cues. Normally, the majority of a mouse’s daily food intake occurs during the subjective night, but ≥15% of food intake occurs during the subjective day in the form of frequent small snacks. Thus, under the ad libitum–fed condition, mice rarely fast a few contiguous hours.",Science,Liver Circadian Regulation,2016
Extent and Purpose of Rhythmic Gene Expression in the Liver,"Under such conditions, at least 20% of expressed protein coding genes in the liver show circadian rhythms in transcription, mature mRNA, active translation, or protein levels. Rhythmic gene expression has several advantages. First, bioenergetic modeling of gene expression in yeast demonstrates that rhythmic gene expression is more energy efficient than maintaining constant levels of expression. This form of energy conservation is observed in a range of mammalian and insect tissues (30). Conversely, when a mouse is subjected to continuous 24-hour fasting, the number of rhythmic transcripts is reduced by almost 80%. This largely results from reduced peak levels of expression, rather than elevated trough level of rhythmic expression (30). Second, rhythmic expression helps to temporally separate incompatible biochemical processes, thereby preventing futile cycles (e.g., the simultaneous biosynthesis and degradation of a given molecule). Because almost 20% of liver transcripts show daily rhythms, it is logical that at least some of the enzymes and regulators in every metabolic pathway are likely to display circadian rhythmicity.",Science,Liver Circadian Regulation,2016
Metabolic Integration with Circadian Regulation,"More important, these rhythmically regulated pathway components often mediate rate-limiting steps, with peak levels of expression coinciding with substrate availability or metabolic need. Several key rhythmic metabolites exert effects on circadian clock components, thereby integrating the metabolic state with the regulatory mechanism. Sometimes, posttranslational modifiers (e.g., kinases and phosphatases) that regulate key enzymes of metabolic pathways also act on clock components, thus coupling metabolic and circadian regulatory processes. Some of these examples will be highlighted in the following section.",Science,Liver Circadian Regulation,2016
Integration of Metabolism with the Circadian Clock,"Functional annotation of the liver circadian cistrome, transcriptome, and proteome show that these are enriched with metabolic regulators. To maintain energy homeostasis, the liver stores nutrients during feeding periods and taps into this stored energy reserve during fasting periods. Intermediates from these cycles of anabolism and catabolism are used for cellular components and signaling. As feeding and fasting naturally alternate between day and night, interactions among feeding-fasting–driven regulation, metabolism, and circadian clocks have evolved to maintain normal physiology.",Science,Circadian Metabolism,2016
Circadian Regulation of Glucose Metabolism,"Glucose enters hepatocytes where it is phosphorylated. Phosphoglucose is then (i) used for energy production via glycolysis, (ii) stored as glycogen for further use (glucogenesis), or (iii) used in the pentose phosphate pathway (PPP) (Fig. 2B). Expression of the hepatic glucose transporter GLUT2 and glucokinase (GCK) show daily rhythms with peak levels coinciding with periods of feeding (31). In the fed state, insulin activates glycogenesis through a signaling cascade that leads to the inhibition of glycogen synthase kinase (GSK3), thereby releasing the activity of glycogen synthase (GS). GSK3 has daily rhythms of phosphorylation and activity and acts on some circadian clock components (e.g., affecting the stability of REV-ERB) (32). β-linked N-acetylglucosamine (O-β-GlcNAc), the attachment of UDP-GlcNAc to Ser/Thr residues of proteins, is yet another link between the circadian clock and glucose metabolism. The activity of the enzyme O-GlcNac transferase (OGT) is regulated by GSK3 (33) and, accordingly, a number of hepatic proteins show circadian rhythms in O-GlcNAcylation (34), including PER2, CLOCK, and BMAL1.",Science,Circadian Metabolism,2016
"PPP, NADPH, and Glucose Regulation in Fasting","The balance between O-GlcNacylation, glycosylation, CK1 phosphorylation, and protein phosphatase 1 (PP1)–mediated dephosphorylation of PER2 determines its stability (33). In parallel, O-GlcNacylation of CLOCK and BMAL1 interferes with their ubiquitination and degradation (35). Glucose use through the PPP is also connected to the circadian clock. The PPP is essential for nucleotide and amino acid biosynthesis, as well for replenishing the pool of cytoplasmic NADPH. Low NADPH levels activate the transcription factor NRF2, which can drive transcription of Rev-erb, thereby affecting the molecular clock (36). In the fasted state, the circadian clock also influences glucose metabolism by interacting with glucagon signaling (Fig. 2C). Glucagon signals through its G-protein–coupled receptor and adenylate cyclase to activate protein kinase A (PKA), which in turn promotes glycogenolysis and gluconeogenesis to supply glucose (37).",Science,Circadian Metabolism,2016
"CREB, CRY1, AMPK, and Fasting Responses","PKA phosphorylates and thereby activates the bZIP transcription factor cyclic AMP response element–binding protein (CREB) so that it binds to cis-acting CRE sites at the Per1 and several gluconeogenic promoters, thereby stimulating their transcription (38). Additionally, CRY1 inhibits the activation of PKA by negatively regulating the G protein or adenylate cyclase (17, 39). On the other hand, prolonged fasting also increases the ratio of AMP/ATP and activates AMP-activated kinase (AMPK), which phosphorylates CRYs and targets them for degradation (40). Together, this suggests a mechanism by which CRY1 acts as a balance point between the short- and long-term responses to nutrient deficit.",Science,Circadian Metabolism,2016
Circadian Control of Lipid Metabolism,"Fatty acid synthesis and β oxidation are tightly controlled in the liver (Fig. 2D). Mitochondrial acetyl CoA is exported to the cytoplasm via a citrate/palmitate shuttle, where ATP citrate lyase (ACLY) is a rate-limiting enzyme. The circadian peak of ACLY expression coincides with feeding (31). The first committed step of fatty acid synthesis is the carboxylation of acetyl CoA by ACACA (acetyl CoA carboxylase) to produce malonyl CoA. ACACA is inactivated via phosphorylation by fasting-induced AMPK. The rate of mitochondrial β oxidation is limited by the entry of fatty acyl groups into the mitochondria by carnitine palmitoyl transferase 1 (CPT1) and CPT2. The levels of L-carnitine, CPT1, and CPT2 show daily rhythms (41). High levels of malonyl CoA, produced during fatty acid synthesis and peaking during feeding, inhibit CPT activity. Such circadian and product-mediated regulation generates a daily rhythm in fatty acid synthesis and oxidation, peaking during feeding and fasting, respectively. REV-ERBα-dependent repression produces daily rhythms in transcripts of the synthesis pathway, and Rev-erbα−/− mice exhibit fatty liver disease (43, 44).",Science,Circadian Metabolism,2016
Protein Metabolism and Circadian Regulation,"Ingested proteins are degraded to amino acids in the small intestine and transported to the liver. Amino acids rarely remain free in cells, because they are used for protein synthesis during feeding, for gluconeogenesis during fasting, for conversion into bioactive molecules (e.g., methionine → SAM), or for degradation into ammonia for the urea cycle. During feeding, AKT activates the mTOR-S6K pathway to promote translation. AKT or S6K1 also phosphorylates BMAL1 and recruits it to translation complexes, promoting activity (Fig. 2E) (45, 46). Along with circadian rhythms in ribosome biogenesis (47) and preferential translation of specific mRNAs (48), this rhythm in protein synthesis is crucial for liver function because it produces major secreted proteins such as albumin, retinol-binding protein, transthyretin, and complement components.",Science,Circadian Metabolism,2016
"Amino Acid Mobilization, Urea Cycle, and Ion Rhythms","During overnight fasting, circadian regulation of the transcription factor KLF15 in muscle and liver drives rhythmic expression of downstream enzymes involved in mobilizing amino acids from muscle and reusing them in the liver for gluconeogenesis and ammonia production for the urea cycle (49). Plasma levels of total amino acids, branched-chain amino acids, and urea show circadian rhythms in humans, peaking at night (49). In the urea cycle, mitochondrial L-ornithine use by ornithine carbamoyl transferase (OCT) is a critical step in clearing CO2 (Fig. 2F). Circadian regulation of Oct is imposed by KLF15. Klf15−/− mice show acute metabolic disruption when fed a protein-rich diet, featuring hypoglycemia, hyperammonemia, and impaired ureagenesis (49). Additionally, circadian rhythms in Ca2+ (at least in the SCN) (50) and Mg2+ can influence the activity of Ca2+-activated kinases and Mg2+-ATP or Mg2+-UTP dependent enzymes (51).",Science,Circadian Metabolism,2016
"Polyamines, One-Carbon Metabolism, and Circadian Regulation","Intermediate products of nutrient metabolism give rise to several small molecules that can affect clock function in the cytoplasm or nucleus. Cytoplasmic ornithine is decarboxylated by ornithine decarboxylase (ODC) as the initial step in polyamine biosynthesis. Several genes involved in polyamine production are transcriptionally regulated by the circadian clock components, giving rise to a daily rhythm in polyamine (Fig. 2F) (52, 53). Circadian expression of the Odc gene is directly driven by CLOCK/BMAL (53). A derivative of methionine plays an important role in this pathway. mRNAs encoding regulatory proteins in methionine production and its adenylation to SAM (Bhmt, Mtrr, Mat1, and Mat2) show daily rhythms (31). SAM is a reactive methyl carrier used in methyl-group transfer reactions, including histone methylation, as well as the biosynthesis of polyamine, phosphatidylcholine, and phosphocreatine.",Science,Circadian Metabolism,2016
"SAM, One-Carbon Metabolism, and Polyamine Feedback","SAM also inhibits the activity of methyl tetrahydrofolate reductase (MTHFR) and thereby is tightly linked to tetrahydrofolate or one-carbon metabolism, which is required for purine metabolism and RNA synthesis in non-dividing cells (54). Several genes involved in tetrahydrofolate metabolism exhibit a circadian rhythm (31, 55). Both reactive methyl-group metabolism and one-carbon metabolism likely signal to the circadian clock through polyamines. Polyamines modulate many protein-protein and protein-DNA interactions, regulating the circadian clock by promoting the interaction between PER2 and CRY1. Age-related dampening of the circadian clock may lead to reductions in polyamine, because supplementing mouse food with polyamine improves circadian rhythms in older mice (53).",Science,Circadian Metabolism,2016
NAD+ Rhythms and SIRT/PARP Regulation of Clock Proteins,"NAD+ (oxidized form of NAD) is emerging as a key regulator of metabolism because it (i) functions as an electron carrier, (ii) modulates protein function, and (iii) serves as a substrate for ADP ribosylation. In animal tissues, NAD+ is either synthesized de novo, or the nicotinic moiety is salvaged from nicotinamide for the synthesis of NAD+ in a reaction catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). Owing to the short half-life of NAD+, this salvage pathway is essential for sustained availability of NAD+. The circadian clock drives daily rhythms in NAMPT, as well as the resulting rhythm in cellular NAD+ levels (56, 57). Direct binding of NAD+ to SIRT proteins inhibits their ability to deacetylate target proteins, including clock components (58, 59) and several metabolic enzymes. NAD+ is also used by PARP1 to add poly(ADP) ribose moieties to CLOCK, which increases CLOCK/BMAL1 affinity to DNA and delays repression by CRY/PER complex (Fig. 2G). This makes the hepatic clock less susceptible to abrupt changes in eating time (60).",Science,Circadian Metabolism,2016
Acetyl-CoA Production and Chromatin Regulation,"Acetyl CoA is a major energy metabolite generated by glycolysis, β-oxidation, and amino acid metabolism in the mitochondria, then transported out of mitochondria by ACLY. Acetyl CoA is also produced in the cytoplasm or nucleus from acetate and CoA by AceCS1. The circadian oscillator exerts strong control over pathways that generate acetyl CoA: key rate-limiting metabolic steps are circadian-regulated, ACLY expression is rhythmic, and AceCS1 activity is regulated by the NAD-dependent deacetylase SIRT1 (61, 62). Cytoplasmic acetyl CoA can diffuse into the nucleus and modulate lysine acetyltransferases such as p300 and CBP (63), both associated with CLOCK/BMAL1 and capable of activating histone acetylation (55). Acetyl CoA is required for synthesis of fatty acids, cholesterol, bile acids, steroid hormones, and ketone bodies, many of which show circadian regulation at the mRNA and protein levels.",Science,Circadian Metabolism,2016
"TCA Cycle Intermediates, α-KG, and Transcriptional Control","In mitochondria, acetyl CoA from pyruvate and fatty acid oxidation is fed into the TCA cycle. Two intermediates, α-KG and succinyl CoA, can have gene-regulatory effects. Alpha-KG is the cofactor for several histone demethylases, and the ratio of succinyl CoA to α-KG affects histone methylation state (Fig. 2H) (64).",Science,Circadian Metabolism,2016
"Heme, Tetrapyrroles, and Xenobiotic Metabolism","Succinyl CoA is the starting material for the synthesis of aminolevulinic acid, catalyzed by ALAS1, whose expression is circadian (65). Aminolevulinate is then used to synthesize tetrapyrroles such as heme and cytochromes. Heme acts as a ligand for REV-ERB and is degraded by heme oxygenase (HO), which also shows circadian expression (66, 67). These rhythms likely cause oscillations in heme and CO (a product of heme degradation), which can diffuse into the nucleus and influence NPAS2 DNA binding (Fig. 2H) (68). Cytochromes, also tetrapyrroles, function as cofactors for mitochondrial electron transport and for cytochrome p450 (Cyp) involved in xenobiotic metabolism. The PAR bZIP transcription factors DBP, TEF, and HLF, which are clock-controlled genes, regulate expression of numerous Cyp genes, generating rhythmic xenobiotic metabolism (69).",Science,Circadian Metabolism,2016
"Cholesterol, Bile Acids, BAT Thermogenesis, and Temperature Feedback","Both fasting and circadian clock regulate cholesterol biosynthesis from acetyl CoA (Fig. 2I). Metabolism of cholesterol into ligands for nuclear hormone receptors shows daily rhythms. The Cyp7 enzymes (Cyp7a and Cyp7b) mediate the first step of cholesterol conversion to bile acids. Hepatic Cyp7 expression is strongly circadian, with peak levels correlating with reduced cholesterol and increased bile acids (70, 71). Excess bile acids can circulate and activate UCP expression in brown adipose tissue (BAT), contributing to thermoregulation (72). In BAT, REV-ERBα imposes circadian rhythms on UCP expression and daily thermogenic rhythms (73). BAT-specific Rev-erbα−/− mice lose rhythmic repression of UCPs and body temperature oscillations (73). Temperature changes also affect circadian rhythms: heating activates HSF1, which activates Per2 transcription via an HSE site (75), while cooling induces CIRBP, which binds CLOCK pre-mRNA and modulates its processing (76).",Science,Circadian Metabolism,2016
Melatonin Synthesis and Light Suppression,"In the pineal gland, acetyl CoA is used to synthesize melatonin via AANAT (77). Circadian expression of AANAT produces the daily melatonin rhythm, peaking at night. Blue-cyan light strongly suppresses AANAT expression and melatonin production (Fig. 2A) (78).",Science,Circadian Metabolism,2016
Peripheral Signals and Central Clock Modulation,"The mechanisms described above illustrate the extensive, reciprocal regulation between the circadian clock and cellular metabolism. Notably, these connections extend to the systemic level. For example, metabolic signals from the periphery also affect brain-specific circadian clocks. Although peripheral signals provide feedback to the SCN, these signals are often insufficient to override the robust influence of light. Forcing nocturnal rodents to eat during the day changes the peak phase of expression for nearly all rhythmically expressed genes in the liver without affecting phases of gene expression in the SCN, which remains tied to the LD cycle (31, 79, 80). However, the phase of circadian gene expression in the pituitary, dorsomedial hypothalamus (DMH), and PVN are affected by daytime feeding (81). Daytime access to a limited amount of food also elicits a survival strategy in nocturnal rodents by suppressing the natural circadian drive for daytime sleep and increasing food-seeking activity before the arrival of the daytime meal (82). This food anticipatory activity (FAA) seems to be mediated by ketone bodies produced by the liver that act on the dorsal striatum in a Per2-dependent manner (83). Such FAA is independent of the circadian clock in the SCN, as normal FAA can be triggered in mice lacking the SCN.",Science,Circadian Integration,2016
Nocturnal–Diurnal Behavior and Environmental Influences,"The effect of eating patterns on central nervous system clocks (e.g., the emergence of FAA, which seems to override SCN control of the activity-rest cycle) raises a larger question about the origin of nocturnal or diurnal (N-D) behavior in different species. The phase of the SCN circadian clock in both nocturnal and diurnal species is similar (84), implying that the N-D switch may not involve the SCN. The effect of food access time on extra-SCN brain clocks raises the provocative hypothesis that the N-D switch may be driven by complex interactions between the light-dark cycle, the feeding-fasting cycle, and overall energy balance. In mice, reducing the ambient temperature and restricting nutrition quantity can trigger daytime activity, suggesting that diurnality is a strategy for conserving energy (85). Understanding the mechanisms by which food, light, and ambient temperature affect the daily sleep-wake cycle and metabolism has increasing importance for humans who are living under diverse work schedules, lifestyles, and food preferences.",Science,Circadian Integration,2016
Impact of Eating Patterns on Rhythmic Physiology in Model Organisms,"Reciprocal interactions between metabolism and the circadian clock imply that nutrition quality, quantity, and daily eating pattern can affect diurnal rhythms, which in turn determines whole-body physiology. When mice are fed a standard diet ad libitum, they typically consume a majority of their daily food intake during the night. The vast majority of circadian -omics studies have been performed using mice fed a standard diet ad libitum. However, relative to these ad libitum–fed animals, the number and amplitude of rhythmic transcripts is substantially reduced in animals deprived of food or in circadian mutant mice, whereas it is increased in mice fed the same calories within an 8- to 12-hour interval [time-restricted feeding (TRF)]. TRF alone cannot sustain rhythmic expression of a vast majority of hepatic rhythmic transcripts, proteins, or metabolites in mice lacking a functional circadian clock (31, 41, 42, 48). When mice are fed a high-fat diet ad libitum, which is widely used for diet-induced obesity (DIO), mice spread their caloric intake evenly throughout the 24-hour day (86). This eating pattern reprograms the hepatic diurnal transcriptome by dampening the oscillation of numerous circadian clock targets (compared to the diurnal transcriptome of mice fed a standard diet) (87). However, as seen in mice fed a standard diet, TRF of a high-fat diet improves molecular oscillations (70, 71, 88).",Science,Circadian Integration,2016
"TRF, Diet Quality, and Metabolic Outcomes","Subjecting genetically identical animals to caloric restriction or a high-fat diet (beneficial and adverse effects, respectively) has served as a powerful experimental system for understanding the roles of nutrition quantity and quality on health. Similarly, feeding isogenic animals identical, isocaloric diets ad libitum or via TRF has offered a foundation for understanding how the daily eating pattern affects diurnal rhythms and health (89). TRF can attenuate the adverse metabolic consequences (i.e., diet-induced pathologies) of high-fat, high-sucrose, or high-fructose diets in rodents and insects (Fig. 3) (70, 71, 88, 90). Comparing mice fed a normal or high-fat diet—either ad libitum or via TRF—is yielding new insights into how circadian regulation of metabolism is an integral part of physiology. DIO disrupts the temporal regulation of metabolism by tonic activation, by tonic suppression, or by mistiming the activation of several liver metabolic pathways (e.g., gluconeogenesis, fatty acid synthesis, cholesterol synthesis, bile acid production, and the pentose phosphate pathways). TRF reverses the adverse effects of a high-fat diet in the liver and other metabolic organs.",Science,Circadian Integration,2016
Physiological Diversity of TRF Benefits,"Although the benefit of TRF on body weight is comparable for 8-, 9-, or 12-hour feeding intervals, several metabolic and physiological health indicators are differentially affected by these regimens, suggesting that the duration of fasting is also important (91). Similarly, TRF does not reduce body weight in mice fed a standard diet but increases lean mass at the expense of fat mass. TRF benefits are also seen in insects, where it has been shown to (i) support body-weight homeostasis, (ii) reduce age-dependent or high-fat diet-dependent deterioration of cardiac function, (iii) maintain sleep patterns, and (iv) promote flight-muscle function (90). Because subjecting model organisms to caloric restriction or DIO has revealed molecular mechanisms of metabolic health, the TRF model will likely yield new insights into how metabolism is temporally regulated. Preliminary studies in flies have shown that TRF and caloric restriction elicit different gene expression signatures and that TRF benefits on cardiac function depend on a functional circadian clock (90).",Science,Circadian Integration,2016
"TRF, Fasting, and Clock-Dependent Health Effects","However, it is still possible that fasting-induced molecular changes could contribute to TRF benefits. Similarly, it is not known whether TRF has beneficial effects in the absence of a functional clock in rodents. It is also worth mentioning that for many caloric-restriction experiments (both in rodents and in higher animals), the restricted group is often given food at a fixed time of the day and the animals consume the daily ration within a few hours, similar to TRF. In contrast, the control animals are given ad libitum access to food. Because of this experimental design, some of the health benefits seen with caloric restriction may have resulted from TRF. Altogether, these experiments stress the importance of eating patterns in metabolic regulation and have begun to inspire researchers to examine the contribution of daily eating patterns on metabolic outcomes in experimental animals and humans. For example, efforts to improve metabolic homeostasis in mice (or hepatocytes) have revealed two promising strategies: (i) behavioral intervention to improve circadian rhythm and (ii) pharmacological agents that target CRY, REV-ERB, or CLOCK (92–94). Targeting the interface between circadian rhythms and metabolism may therefore prove effective in alleviating the effect of metabolic disorders.",Science,Circadian Integration,2016
Unresolved Questions and Energy Balance under TRF,"Although TRF results in health benefits irrespective of nutrition quality and quantity, numerous questions remain. How is “energy balance” explained in TRF? How do different macronutrients, micronutrients, supplements, and medications affect the clock? If timing of food intake can determine metabolic outcomes, can timing of medication be optimized for efficacy? Does TRF during the day versus night have different effects? How can we translate these findings to clinical practice or standard of care? Close examination of TRF reveals that diurnal rhythms may affect components of energy balance (energy intake = absorption + storage + expenditure). In addition to circadian rhythms in the gut epithelium that affect nutrient absorption (95, 96), the composition of the gut microbiome, with respect to nutrient metabolism, also shows diurnal rhythms (97). Compared with ad libitum feeding, TRF does not affect the composition of the predominant cecal microbiome, but TRF mice excrete more simple sugars, which are microbially derived from complex carbohydrates in food (98).",Science,Circadian Physiology,2016
Effects of TRF on Energy Storage and Expenditure,"This implies that complex sugars are digested in the lower intestine (where absorption is relatively low) or that TRF somehow reduces overall sugar absorption. TRF also changes energy storage by increasing metabolically active lean mass and preventing the accumulation of fat mass. Even fat mass in TRF mice has a higher mitochondrial content (70, 71). TRF increases the peak level of Cyp7a/b expression, an effect that correlates with reduced cholesterol and increases in bile acids. Bile acids can act through TGR5 and DIO2 to increase BAT thermogenesis (72) and contribute to higher energy expenditure and increased O2 consumption in TRF mice. However, it is unclear why a considerable amount of bile acids are excreted in the feces of TRF mice. Nevertheless, inhibition of bile acid reabsorption in the gut protects against fatty liver disease (99).",Science,Circadian Physiology,2016
"Food Components, Microbiome, and Circadian Modulation","Beyond the simple effects of nutrition on energy balance, some food components may affect the circadian clock even when consumed in moderation (i.e., at small caloric levels typically ignored for energy balance). For example, caffeine itself can change the phase of the circadian clock (100). As the mere presence of the gut microbiome is necessary for a robust liver circadian rhythm (101), noncaloric artificial sweeteners (102), as well as antibiotics known to change the gut microbiome composition, are likely to affect gut or hepatic circadian rhythms. Similarly, the absorption, target function, and clearance of many drugs are likely circadian (103). Therefore, systematic analyses of the timing of drug activities (specifically for those with a short half-life or those provided in small doses), as well the resulting prognosis, are warranted.",Science,Circadian Physiology,2016
Translating Circadian–Metabolic Insights to Human Health,"How might we translate these results into the standard of care? Epidemiological studies have repeatedly shown that sleep deprivation and shift work correlate with higher incidence of metabolic diseases in humans (104, 105). Conversely, overnight fasting (≥13 hours after controlling for sleep and activity) both prevents breast cancer and improves the prognosis of breast cancer patients (106, 107). These observations suggest that daily patterns of activity, sleep, and food intake may dramatically affect human heath and that these patterns should be systematically dissected to determine their roles in health. Preliminary studies using a smartphone app have shown that nearly 50% of nonshift workers distribute their food intake over greater than 15 hours and, therefore, implementing a 10-hour TRF may promote weight loss and improve sleep (108). Retrospective analyses of a weight-loss study showed that eating earlier may lead to increased weight loss (109), suggesting that the relationship between the eating interval and the day-night cycle may affect metabolism.",Science,Circadian Physiology,2016
"Circadian Regulation of Glucose, Melatonin, and Environmental Factors","As stated above, hyperglycemia is sustained for a longer period of time after the evening meal than after the morning meal. This is likely because melatonin inhibits insulin release from pancreatic islets through the melatonin receptor 1B (Fig. 2A). Therefore, the evening rise in melatonin likely causes hyperglycemia (110). Because light in the blue spectrum strongly suppresses plasma melatonin level, it raises the interesting possibility that adjusting spectral quality and quantity in the indoor environment may affect metabolism. [However, melatonin action on metabolism extends beyond the pancreas (111), and its rise during nighttime feeding in nocturnal rodents adds further complexity to generalization of melatonin effects in both diurnal and nocturnal animals.] The rise of consumer markets for wearable sensors, ubiquity of smartphones, and their increasing use in research offer an unprecedented opportunity to longitudinally measure human feeding behavior, sleep patterns, activity levels, ambient light, body temperature, heart rate, and blood glucose to understand how these factors interact in free-living conditions. These data may reveal how the environment and diet may be manipulated to optimize the circadian physiology of metabolism.",Science,Circadian Physiology,2016
Overview of Circadian Genomics and Health,"Circadian clocks are endogenous oscillators that control 24-h physiological and behavioral processes. The central circadian clock exerts control over myriad aspects of mammalian physiology, including the regulation of sleep, metabolism, and the immune system. Here, we review advances in understanding the genetic regulation of sleep through the circadian system, as well as the impact of dysregulated gene expression on metabolic function. We also review recent studies that have begun to unravel the circadian clock’s role in controlling the cardiovascular and nervous systems, gut microbiota, cancer, and aging. Such circadian control of these systems relies, in part, on transcriptional regulation, with recent evidence for genome-wide regulation of the clock through circadian chromosome organization. These novel insights into the genomic regulation of human physiology provide opportunities for the discovery of improved treatment strategies and new understanding of the biological underpinnings of human disease.",Genome Medicine,Circadian Genomics,2019
Genetic Basis and Discovery of Circadian Clock Genes,"Circadian rhythms are driven by an internal timing system regulated at the transcriptional level that gives rise to gene networks that oscillate with a 24-h cycle. Within these networks are clock genes that control rhythms in physiology and behavior. The circadian clock genes were among the first genes identified as controlling behavior. Following studies by Konopka and Benzer [1], who identified the first circadian mutant—period—in fruit flies, a forward-genetic behavioral screen was implemented in mice. Through this screen, the first circadian mutant mouse was identified [2], followed by the cloning of the first mammalian circadian gene, Clock [3]. Research into the mechanisms of mammalian circadian rhythms then expanded rapidly, with many additional genes added to the core loop [4–11]. Since then, it has become clear that the circadian system plays an overarching role in regulating human physiology. Disruption of circadian rhythms is associated with sleep disorders, cancer, susceptibility to infections, metabolic syndrome, Alzheimer’s disease, and aging. There is also evidence that clock genes can influence cellular functions in a non-circadian manner.",Genome Medicine,Circadian Genomics,2019
Modern Genomics Approaches and Chronopharmacology,"This review highlights recent advances in mammalian circadian rhythms research, emphasizing novel techniques and their implications for human disease, translational research, and medicine. Modern genomics approaches to study circadian rhythms include evaluating chromatin dynamics and gene regulation. Because circadian dysfunction contributes to numerous diseases, there is growing interest in timed drug administration (chronopharmacology) or targeting clock components directly. Considering circadian timing in treating metabolic disorders, cardiovascular disease, and cancer may offer significant benefits.",Genome Medicine,Circadian Genomics,2019
Core Mechanism of the Mammalian Circadian Clock,"The circadian clock in mammals is cell-autonomous and depends on transcriptional autoregulatory feedback loops. Circadian rhythms are also tuned at the post-transcriptional and post-translational levels, although gene transcription remains central for generating rhythmicity. Genome-wide approaches have shown that rhythmic transcription is accompanied by rhythmic transcription factor binding and histone modifications in enhancers, as well as circadian recruitment of RNA polymerase II (Pol II) to DNA. Another regulatory layer involves chromosome organization, with oscillations in interactions between active and repressive chromosomal domains.",Genome Medicine,Circadian Genomics,2019
Chromatin Accessibility and 3D Genome Organization,"Studies in mouse tissues have greatly enhanced understanding of circadian regulatory mechanisms for rhythmic transcription. Sobel et al. mapped DNase I hypersensitive sites (DHSs) in mouse liver over 24 h, identifying that 8% of 65,000 DHSs cycled with a 24-h period, coinciding with Pol II binding and H3K27ac marks. Two additional studies advanced understanding of chromatin interactions. Mermet et al. used 4C-seq to explore chromatin interactions for the Cry1 promoter and the liver-specific clock-controlled gene Gys2. These genes show opposite-phase rhythmic transcription, and their chromatin interactions peak at times corresponding to maximal expression. Disruption of an enhancer rhythmically recruited to Cry1 shortened locomotor period, indicating chromatin looping is essential for rhythmic behavior.",Genome Medicine,Circadian Genomics,2019
Circadian Proteome and Phosphoproteome Insights,"Despite advances in genomics, circadian regulation at the protein level remains less understood due to technical challenges in quantitative proteomics. Recent progress has enabled quantification of the circadian proteome, nuclear proteome, and phosphoproteome. These studies revealed ~500 proteins (~10%) in the nucleus with rhythmic abundance, many involved in transcriptional regulation, ribosome biogenesis, DNA repair, and cell cycle pathways. Notably, more than 5000 (~25%) phosphorylation sites are rhythmic, greatly exceeding rhythms in protein abundance. Overall, these studies highlight the extensive genome-wide regulatory reach of the molecular circadian clock.",Genome Medicine,Circadian Genomics,2019
Genetic Causes of Circadian Sleep Disorders,"In humans, mutations in circadian clocks have been associated with circadian rhythm sleep disorders. Familial advanced sleep phase disorder (FASPD) is a circadian rhythm sleep disorder with habitual sleep times that are earlier than the societal norm. The first identified cause of FASPD was a missense mutation (S662G) in the PER2 gene [28]. Casein kinases Iδ and Iε (CKIδ/ε) regulate levels of PER2 by phosphorylation-mediated degradation and cellular localization. The S662G mutation appears to be in the CKIε binding site, which causes hypophosphorylation by CKIε in vitro. Deficient phosphorylation of PER2 in the cytoplasm may impair its degradation and lead to nuclear accumulation [28, 74]. FASPD has also been linked to a missense mutation (T44A) in the human CKIδ gene, which leads to reduced kinase activity in vitro and a shorter circadian period in mice [75]. Hirano et al. [48] described another mutation in human CRY2 associated with FASPD. The alanine-to-threonine mutation (A260T) is located in the FAD binding domain and increases FAD affinity for FBXL3, promoting CRY2 degradation.",Genome Medicine,Circadian Sleep Genetics,2019
Delayed Sleep Phase Disorder and Chronotype Genetics,"A more common circadian rhythm sleep disorder, affecting nearly 10% of the population, is delayed sleep phase disorder (DSPD). It is characterized by delayed sleep onset and offset times relative to societal norms. Familial DSPD cases suggest Mendelian inheritance, with polymorphisms in CLOCK or PER3 implicated (reviewed in [87]). Patke et al. [47] reported a hereditary form of DSPD associated with a CRY1 mutation at the 5′ splice site of exon 11, resulting in exon skipping and deletion of 24 residues in CRY1’s C-terminal region. This enhances CRY1 affinity for CLOCK/BMAL1, lengthening the circadian period. Biobank-scale studies have associated multiple loci with chronotype (morningness/eveningness), including PER1, CRY1, and BMAL1 [88].",Genome Medicine,Circadian Sleep Genetics,2019
Metabolic Link to Sleep: The Role of SIK3,"In mice, a recent study suggested a novel link between metabolism and sleep regulation. Salt-inducible kinase 3 (SIK3), a serine-threonine kinase in the AMPK family, acts as an energy sensor. Sik3−/− mice exhibit severe metabolic symptoms such as hypolipidemia and hypoglycemia, with many dying shortly after birth [89]. SIK3 influences PER2 stability, but unlike Per2 mutants, Sik3−/− mice have a longer circadian period and a 6-hour phase delay in oxygen consumption rhythms. A forward-genetics screen identified a SIK3 mutation that increases sleep time profoundly. Whole-exome sequencing revealed exon 13 skipping, which contains a PKA recognition site. Unlike Sik3−/− mice, these mutants show no effect on circadian period under constant darkness [90]. Together, these findings suggest SIK3 plays a critical role in the regulation of sleep and circadian rhythms.",Genome Medicine,Circadian Sleep Genetics,2019
Circadian Regulation of Metabolism,"Driven by the circadian clock, a regular daily pattern of eating and fasting maintains normal circadian physiology. However, recurrent disruption of daily activity–rest rhythms, and thus feeding patterns (as occurs in shift workers), is associated with metabolic syndrome [91]. Genetic disruption of the circadian clock also predisposes rodents to metabolic disease [32, 33]. The clock controls metabolism directly by driving transcriptional programs for certain metabolic pathways. For example, CRY1 suppresses hepatic gluconeogenesis during fasting through the regulation of cAMP/CREB signaling, the rhythmic repression of the glucocorticoid receptor gene, and the suppression of nuclear FOXO1 that, in turn, downregulates gluconeogenesis [92–94]. Another clock repressor, PER2, controls lipid metabolism by direct regulation of peroxisome proliferator-activated receptor gamma (PPARγ) and mitochondrial rate-limiting enzymes [95, 96]. The nuclear hormone receptors, REV-ERBs, also directly regulate the transcription of several key rate-limiting enzymes for fatty acid and cholesterol metabolism [97]. Disruption of CLOCK and BMAL1 has also been associated with obesity, hyperinsulinemia, and diabetes [32, 33, 99, 100]. The circadian posttranscriptional regulator Nocturnin also controls lipid and cholesterol metabolism [101].",Genome Medicine,Circadian Metabolism,2019
Temporal Coordination of Metabolism Across Tissues,"Recently, an atlas of circadian metabolic profiles across eight tissues revealed temporal cohesion among tissues, whereas nutritional challenge (a high-fat diet) impacted each tissue differentially [102]. In addition to direct modulation of mammalian metabolism, indirect control by the clock occurs through its regulation of behavior, food intake, and the oscillation of hormones such as insulin, glucagon, peptide YY, glucagon-like peptide 1, corticosterone, leptin, and ghrelin (reviewed in [103]). Although much is known about circadian control of metabolism, the mechanisms behind this control are still far from understood [104]. How nutritional challenges dysregulate the clock and how clock disruption increases adipogenesis remain open questions in the field.",Genome Medicine,Circadian Metabolism,2019
Dietary Influences on Circadian Enhancers,"In recent years, time-restricted feeding has revolutionized dietary restriction protocols. Body weight increases are kept to a minimum even when animals are placed on high-fat and/or high-fructose diets by simply restricting food ingestion to an 8–12-h window [105, 106]. The time during which food is consumed should be in sync with the animals’ circadian rhythms, as misalignment leads to metabolic dysfunction [108–111]. Nutrient-sensing neurons (AgRP) also experience daily rhythms in response to leptin [112]. The nutritive environment itself impacts feeding behavior and imposes dramatic changes in circadian gene expression in diet-induced obesity (DIO) models [113, 114]. Guan et al. [53] showed that one of these DIO-related changes is the emergence of newly rhythmic oscillations of SREBP, which regulates fatty acid synthesis and oxidation, and of PPARα, a major regulator of fatty acid oxidation. This likely results from circadian rhythms evoked at enhancers of genes that are not normally rhythmic [53]. A PPARα agonist (WY-14,643) is more effective at lowering lipids when administered at the circadian peak of PPARα expression, supporting chrono-pharmacological approaches.",Genome Medicine,Circadian Metabolism,2019
Clock-Modulating Compounds and Metabolic Effects,"A high-throughput screen of 200,000 synthetic small molecules using circadian reporter assays identified compounds that lengthen or shorten circadian period in central and peripheral clocks [115]. More recently, nobiletin (NOB), a polymethoxylated flavone, was discovered as a clock amplitude–enhancing molecule. In mice with metabolic syndrome caused by DIO or genetic disruption (db/db obese mice), NOB augments energy expenditure and locomotor activity in a Clock gene–dependent manner while reducing body-weight gain, lowering fasting glucose, and improving glucose tolerance and insulin sensitivity. These benefits are absent in DIO Clock mutants [117], suggesting potential for pharmacological modulation of metabolism through enhancement of circadian rhythms.",Genome Medicine,Circadian Metabolism,2019
Glucocorticoid Rhythms and Adipocyte Differentiation,"Glucocorticoids and other adipogenic hormones are secreted in mammals in a circadian manner. High-resolution automated sampling has revealed ultradian glucocorticoid cycles of ~1-hour period, with higher amplitude coinciding with the onset of circadian activity [118]. Loss of glucocorticoid oscillations correlates with obesity, but how hormone dynamics affect adipocyte differentiation has not been clear. A quantitative study by Bahrami-Nejad et al. [81] showed that adipocyte differentiation does not progress under normal circadian hormonal cycles. Differentiation is induced only when hormonal pulses shorten or when glucocorticoid signals are flat or continuously elevated. Flattened glucocorticoid profiles increase subcutaneous and visceral fat pad mass in mice. This differentiation appears linked to PPARγ, which acts as a filter for circadian hormonal stimuli.",Genome Medicine,Circadian Metabolism,2019
"Autophagy, CRY1 Degradation, and Clock-Metabolism Integration","A recently described link between circadian clocks and autophagy reveals the role of this degradation pathway in recycling circadian proteins. Autophagy degrades the repressor CRY1, which suppresses hepatic gluconeogenesis. Toledo et al. [120] found that timely degradation of CRY1 by autophagic pathways enables glucose production. Obesity increases autophagic degradation of CRY1, leading to elevated glucose production and blood-sugar levels. Conversely, loss of autophagy leads to CRY1 accumulation and clock disruption. These results highlight that regulation of clock rhythmicity is highly intertwined with central metabolic processes. The mechanisms defining the diurnal window of autophagy and its timing remain unclear, but likely many additional circadian-regulated cellular functions remain to be uncovered.",Genome Medicine,Circadian Metabolism,2019
Circadian Control of Immune Function,"Dramatic temporal variation in sensitivity to endotoxins between morning and evening was first discovered in the 1960s [13], but only in the past decade have major inroads been made in our understanding of clock control over the immune system. Circadian clock control impinges upon many aspects of the immune response, from the trafficking of immune cells, to the activation of innate and adaptive immunity, to host–pathogen interactions. Immune cells respond to circadian cues to maximize protection, with rhythmic behaviors observed across both innate and adaptive branches.",Genome Medicine,Circadian Immunity,2019
Circadian Regulation of Immune Cell Trafficking,"Cells of the innate immune system, such as neutrophils and monocytes, exhibit circadian patterns of migration from the blood to tissues [122]. T and B lymphocytes of the adaptive immune system also show strong circadian oscillations in the blood, with numbers peaking during the resting phase. This rhythmicity continues as lymphocytes traffic to the lymph nodes, with homing efficiency peaking at activity onset and cells leaving lymph nodes during the resting period [123–125]. Lineage-specific ablation of circadian clock function showed that rhythmic lymph node trafficking depends on periodic expression of promigratory factors. Trafficking rhythmicity is linked to CXCR4 expression and glucocorticoid signaling [126]. IL-7R, containing a GRE in its enhancer, is induced rhythmically by glucocorticoids, promoting T cell survival, CXCR4 expression, and tissue recruitment. Diurnal variation in T cell distribution enhances immune responses to antigens and systemic bacterial infection at night [124].",Genome Medicine,Circadian Immunity,2019
Clock Proteins in Innate and Adaptive Immunity,"Clock proteins play key roles in regulating immune responses. BMAL1 and REV-ERBα exert anti-inflammatory effects [39]. Loss of Bmal1 in macrophages eliminates rhythmic cytokine storm responses to endotoxins and abolishes normal daily protection against sepsis during the early rest phase [122]. Endotoxins repress BMAL1 via miR-155, which targets the 3′ UTR of Bmal1; BMAL1 normally inhibits miR-155 induction and protects against LPS-induced sepsis [129]. Circadian disruption also impacts autoimmune disease: loss of myeloid BMAL1 increases IL-1β–secreting monocytes that infiltrate the CNS, promoting pathogenic T lymphocytes and worsening neuroinflammation in a multiple sclerosis model [130]. These findings highlight coordinated circadian regulation of innate and adaptive immune cells.",Genome Medicine,Circadian Immunity,2019
"Oxidative Stress, NRF2, and Circadian Immune Regulation","An additional mechanism through which the clock regulates immunity involves antioxidant control. BMAL1 directly binds to an E-box in the promoter of Nrf2, regulating NRF2 expression in myeloid cells [131]. In macrophages, ROS promote IL-1β production by stabilizing HIF-1α, which induces downstream proinflammatory molecules [132, 133]. NRF2 protects against oxidative damage. Early et al. [131] showed that activating NRF2—genetically or pharmacologically—rescues the proinflammatory phenotype of Bmal1−/− macrophages. Thus, the molecular clock regulates NRF2 to modulate inflammation. Despite increasing insights, further studies are needed to fully understand circadian influences on immune surveillance and activity.",Genome Medicine,Circadian Immunity,2019
Time-of-Day Effects on Host–Pathogen Interactions,"Many studies have shown that the outcome of an infection (bacterial, viral, or parasitic) depends on the time of day at which the infection is initiated [40, 51, 52, 134]. For example, Salmonella enterica serovar Typhimurium levels are higher following infection during the rest phase compared to infection in the active phase in mice, and this difference depends on a functional copy of CLOCK [40]. Viral infections—including herpes, influenza A, and respiratory Paramyxoviridae viruses—are enhanced when host circadian rhythms are abolished by disrupting Bmal1 [52, 135]. Bmal1−/− mice infected intranasally with respiratory syncytial virus (RSV) have higher viral loads than wild-type mice [135]. Misalignment of circadian rhythms through chronic jet lag exacerbates acute viral bronchiolitis caused by Sendai virus (SeV) or influenza A virus in mice [136]. In humans, respiratory tract expression of many clock genes (BMAL1, NPAS2, PER2, DBP, NR1D1) is reduced in adult asthma patients.",Genome Medicine,Circadian Immunity,2019
Circadian Regulation of Parasitic Infection,"Parasite infection is also shaped by host circadian timing. Leishmania parasite burden is circadian, and Bmal1 in monocytes modulates the magnitude of infection [51]. Similar findings appear for the intestinal helminth Trichuris muris, with mice infected at the start of the active phase showing delayed resistance. This response pattern shifts with daytime-restricted feeding. Lineage-specific genetic ablation of Bmal1 in antigen-presenting dendritic cells (DCs) eliminates time-of-day dependency for helminth expulsion [134]. Thus, BMAL1 plays a central role in regulating immunity against bacteria, viruses, and parasites.",Genome Medicine,Circadian Immunity,2019
Infection-Induced Disruption of the Circadian Clock,"Infections or resulting inflammation can disrupt circadian rhythms by decreasing amplitude. This has been observed in infections with Trypanosoma cruzi (Chagas disease) [137], Trypanosoma brucei (sleeping sickness) [138], and Plasmodium chabaudi (malaria) [138]. Downregulation of clock genes is likely driven by the massive immune response, as proinflammatory cytokines can decrease circadian amplitude in vitro. These immune responses also drive 'sickness-like behavior' [139]. A study of sleeping sickness showed that T. brucei may disrupt patient sleep by modulating their circadian clocks, possibly through a systemic signal produced by the parasite or host response, shortening the circadian period [138]. These results highlight that host–pathogen interactions are subject to circadian modulation, and pathogen rhythms may also play a role.",Genome Medicine,Circadian Immunity,2019
Microbiota–Circadian Interactions,"Host circadian rhythms strongly influence commensal microbiota, and vice versa. Disruption of host clock genes abolishes rhythms in certain microbial populations [141], which can be restored with time-restricted feeding [141, 142]. Conversely, absence of gut microbes perturbs circadian gene expression in the mouse liver [143]. Thaiss et al. [142] showed that intestinal microbiota undergo rhythmic fluctuations in biogeography and metabolome. Wang et al. [85] discovered that the gut microbiota regulates body composition via NFIL3, a circadian basic leucine zipper transcription factor. NFIL3 shows diurnal oscillation in intestinal epithelial cells, enhanced by microbiota, and its expression is reduced in germ-free animals. Epithelial-specific Nfil3 knockout mice are resistant to diet-induced obesity. NFIL3 regulates a circadian lipid metabolic program and lipid absorption. Additionally, circadian clocks in intestinal cells, particularly ILC3s, regulate susceptibility to bowel infection and lipid metabolism [144]. These findings reveal a complex interplay between commensal microbiota, metabolism, immunity, and circadian rhythms.",Genome Medicine,Circadian Immunity,2019
Circadian Influence on Cardiovascular Events,"Cardiovascular complications have higher incidence in the morning. Many different studies have connected the clock with cardiovascular function, including daily variation in blood pressure, and even response to aspirin [82, 145, 146]. Some studies suggest that pharmacological targeting of REV-ERB decreases atherosclerotic plaque burden in mice [147]. On the other hand, other studies suggest that deletion of Bmal1 in myeloid cells increased monocyte recruitment and atherosclerosis lesion size [148].",Genome Medicine,Circadian Cardiovascular Physiology,2019
Daily Rhythms in Leukocyte Recruitment and Atherosclerosis,"A recent study has shed light on a mechanism that may contribute to this phenomenon. The adherence of myeloid cells to microvascular beds peaks during the early active phase, which appears to be a consequence of peak cell recruitment to atherosclerotic lesions 12 h earlier [57]. Winter et al. [57] showed that both the upregulation of cell adhesion molecules during the active phase by endothelial cells and the presence of immobilized chemokines (emitted by either endothelial cells or myeloid cells) on arterial vessels attract leukocytes into atherosclerotic lesions. Thus, the chemokine CCL2 and its receptor CCR2 are at the core of this daily pattern of leukocyte migration and adhesion. Timed pharmacological CCR2 neutralization inhibited atherosclerosis without disturbing microvascular recruitment, providing a proof-of-principle treatment schedule for chrono-pharmacological intervention in atherosclerosis.",Genome Medicine,Circadian Cardiovascular Physiology,2019
"BMAL1, Aging, and Cardiovascular Dysfunction","Loss of Bmal1 results in an acceleration of aging and a shortened life span in mice [84]. The cardiovascular system is among the systems affected by aging, with Bmal1−/− mice being predisposed to developing atherosclerosis. Using an inducible knockout (iKO), Yang et al. [149] tested whether these age-related phenotypes remained if mice lost BMAL1 as adults. They found that both Bmal1−/− and iKO models exhibit markers consistent with accelerated aging (ocular abnormalities and brain astrogliosis), behavioral disruption, and transcriptional dysregulation. Conditional ablation of the pancreatic clock still causes diabetes mellitus [99]. However, some other biomarkers of aging, including premature death in Bmal1−/− mice, were not replicated in the iKOs. The predisposition for atherosclerosis appears to be reversed in iKOs [149].",Genome Medicine,Circadian Cardiovascular Physiology,2019
Developmental vs. Adult BMAL1 Function in Cardiovascular Health,"These findings suggest that some cardiovascular phenotypes associated with Bmal1 depletion—such as predisposition to atherosclerosis—may result from BMAL1 function during development rather than its function alone in adulthood. Although a link between the circadian clock and atherosclerosis is clear, further investigation into the contributions of BMAL1 and other clock proteins is necessary to fully understand their roles in cardiovascular disease.",Genome Medicine,Circadian Cardiovascular Physiology,2019
Circadian Rhythms in the Nervous System,"Circadian rhythms in the suprachiasmatic nucleus (SCN) have been the focus of many years of research; but how the SCN imposes rhythmicity throughout the body (or even locally in the brain) is not fully understood. Recent studies have broadened the focus from neurons to astrocytes, demonstrating the important role of these glial cells in maintaining circadian rhythmicity [150–152]. A recent circadian atlas of non-human primates includes 64 tissues, including 22 brain regions [153]. Genes were found cycling across the day in all these regions, providing a comprehensive view of the widespread influence of the circadian clock throughout the CNS. While further work is needed to fully understand the impact of rhythmicity in the nervous system, the following findings highlight emerging directions.",Genome Medicine,Circadian Neuroscience,2019
Circadian Regulation of the Blood–Brain Barrier,"The blood–brain barrier (BBB) is highly selective in what it allows into the brain, and its permeability is regulated in part by the circadian clock. Mice lacking Bmal1 in both the CNS and peripheral nervous system exhibit BBB hyperpermeability with age-dependent loss of pericyte coverage of brain blood vessels [154], suggesting that the clock regulates BBB homeostasis. Similarly, the Drosophila BBB is more permeable at night [155]. In flies, the BBB consists of subperineurial and perineurial glia. Zhang et al. [155] showed that at night, the perineurial glia clock increases gap junctions and lowers Mg2+ levels, reducing transporter efflux activity and increasing xenobiotic entry into the brain. These findings have practical implications for CNS drug delivery. For example, an anti-seizure drug was more effective when administered at night [155]. Given structural and functional similarities between insect and mammalian BBBs, these discoveries may be highly relevant to human physiology.",Genome Medicine,Circadian Neuroscience,2019
"Light, Mood, and Learning","Light is a strong external cue for the circadian system [156]. Its detection involves three classes of retinal photoreceptors: rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs), which express melanopsin (Opn4) [25–27]. When ipRGCs are lost, the SCN no longer receives light information. Surprisingly, ablation of these photoreceptors affects mood and learning in mice, indicating that ipRGCs also drive non-SCN behavioral effects [156]. Fernandez et al. [157] found that a subset of ipRGCs transmits light information to the perihabenular nucleus of the thalamus, influencing cognitive functions in an SCN-independent manner. These findings reveal two distinct retina–brain pathways that integrate light, underscoring the influence of light on mood and learning and pointing toward future strategies for light-based treatments of mood disorders.",Genome Medicine,Circadian Neuroscience,2019
Circadian Disruption and Cancer Susceptibility,"Epidemiological studies have linked circadian disruption to increased cancer susceptibility in all key organ systems [158–160]. Polymorphisms in the core circadian genes Per1, Per2, and Per3 are frequently found in human cancers and often result in decreased expression of these genes [158]. Oncogenic MYC has also been shown to suppress the clock [161]. Genetic loss of Per2 or Bmal1 promotes lung tumorigenesis in mice, leading to increased c-Myc expression, enhanced proliferation, and metabolic dysregulation [50]. Hepatocellular carcinoma (HCC) can be induced by chronic jet lag in mice in a manner similar to obese humans, progressing from NAFLD to steatohepatitis, fibrosis, and ultimately HCC [49], providing strong evidence for mechanistic links between clock disruption and cancer development.",Genome Medicine,Circadian Cancer Biology,2019
"Clock Genes, microRNAs, and Oncogenesis","MicroRNAs also contribute to circadian disruption in cancer. miR-211 suppresses Clock and Bmal1 and promotes tumor progression [162]. Pharmacological targeting of REV-ERBs has emerged as a potential therapeutic strategy that selectively disrupts cancer cell viability. Using anticancer REV-ERB agonists SR9009 and SR9011, Sulli et al. [58] interfered with at least two metabolic hallmarks of cancer—de novo lipogenesis and autophagy—processes essential for meeting the metabolic demands of cancer cells.",Genome Medicine,Circadian Cancer Biology,2019
"Hypoxia, Tumor Microenvironment, and Clock Disruption","Low oxygen levels in solid tumors stabilize hypoxia-inducible factors (HIFs), transcription factors that acidify the tumor microenvironment. Recent research shows that HIFs can influence various clock transcripts [163–165]. Walton et al. [166] demonstrated that acidification of the tumor microenvironment disrupts the circadian clock and rhythmic transcriptome. Low pH suppresses mTORC1 signaling, inhibiting translation. Restoring mTORC1 signaling—either by buffering against acidification or inhibiting lactic acid production—fully rescues translation and circadian oscillations [166].",Genome Medicine,Circadian Cancer Biology,2019
Implications for Circadian-Informed Cancer Therapies,"Overall, recent research on circadian rhythms and cancer has provided major insights into how clock disruption contributes to tumor initiation, metabolic reprogramming, and microenvironmental dysregulation. These mechanistic findings suggest opportunities for improved treatments that leverage circadian timing or target clock components directly. Chronotherapy and circadian-informed pharmacology may enhance treatment efficacy while minimizing adverse effects on normal tissues.",Genome Medicine,Circadian Cancer Biology,2019
Age-Related Decline of Circadian Rhythms,"Circadian rhythms decline with age in both amplitude and stability. Neuronal activity rhythms weaken in the suprachiasmatic nucleus (SCN), the master circadian clock [169]. Genetic ablation of Bmal1—an essential core clock gene—leads to premature aging in mice, including early onset of age-related pathologies [84]. Recent transcriptomic studies show that aging does not simply dampen circadian rhythms; instead, aging rewires transcriptional programs. Rather than losing rhythmicity, aged tissues begin cycling entirely new sets of genes [83, 170]. Aged epidermal and skeletal muscle stem cells retain rhythmic core clock gene expression but redirect circadian control toward stress response, inflammation, and DNA-damage pathways [83]. This reprogramming may reflect changes in DNA methylation patterns that occur with aging [171].",Genome Medicine,Circadian Rhythms and Aging,2019
Polyamines and Circadian Period Regulation in Aging,"Polyamines regulate multiple cellular processes, and altered polyamine metabolism is associated with aging. Zwighaft et al. [55] demonstrated that the circadian clock controls polyamine levels and, conversely, that polyamines regulate circadian period length. Polyamines modulate the interaction between the repressors PER2 and CRY1. Aging reduces polyamine levels, contributing to a lengthened circadian period. Remarkably, supplementation of polyamines in aged mice shortens this prolonged circadian period, partially restoring youthful clock function.",Genome Medicine,Circadian Rhythms and Aging,2019
DNA Methylation and Epigenetic Oscillations Across Aging,"DNA methylation is a key epigenetic regulator that changes with age. De novo DNA methylation is mediated by DNMT3A/3B, while TET enzymes contribute to active demethylation. Oh et al. [172] found that many cytosines show circadian oscillations in methylation and that these oscillations correlate with rhythmic mRNA expression in liver and lung. In aged animals, these oscillatory cytosine modifications decline, mirroring the reduced rhythmicity seen in some transcripts. This suggests that epigenetic rhythmicity is an additional layer of circadian regulation that deteriorates with age.",Genome Medicine,Circadian Rhythms and Aging,2019
Circadian Rhythms and Alzheimer’s Disease,"Alzheimer’s disease (AD) is associated with disrupted sleep–wake cycles, including increased daytime sleep and nighttime wakefulness [54]. β-amyloid (Aβ) levels exhibit robust daily oscillations in mouse hippocampal interstitial fluid [78, 173]. Tau levels also fluctuate diurnally in both mice and human cerebrospinal fluid. Sleep deprivation markedly increases tau levels and abolishes normal rhythmicity [54]. Kress et al. [175] showed that deleting Bmal1 in the entire brain eliminates Aβ rhythmicity and increases amyloid plaque burden, whereas preserving Bmal1 in the SCN alone maintains normal Aβ rhythms. Cross-sectional studies in humans show that preclinical AD—marked by higher phosphorylated-tau/Aβ-42 ratios—is associated with fragmented activity–rest rhythms [176]. These findings suggest that circadian dysfunction may contribute to early AD pathogenesis and may serve as a biomarker.",Genome Medicine,Circadian Rhythms and Aging,2019
Implications for Healthy Aging and Disease Prevention,"Collectively, these findings highlight that aging affects the circadian system at multiple biological layers, from transcriptional rewiring and epigenetic regulation to metabolic and neurodegenerative processes. Maintaining robust sleep–wake cycles and circadian alignment may be protective against age-related diseases such as Alzheimer’s. Future research may reveal interventions—behavioral, nutritional, or pharmacological—that can stabilize circadian rhythms to slow aging-associated decline and neurodegeneration.",Genome Medicine,Circadian Rhythms and Aging,2019
Chronopharmacology and Therapeutic Timing,"Circadian research has increasingly demonstrated that the timing of medical interventions can significantly influence therapeutic efficacy. Chronopharmacology—aligning treatment with the body's internal rhythmic cycles—has reshaped approaches to immunity, metabolism, inflammation, and host–pathogen interactions. Many genes that cycle across the day encode proteins considered druggable targets by the FDA, highlighting the potential for circadian-informed therapies. Circadian transcriptome atlases from mice, non-human primates, and humans reveal that rhythmic gene expression is widespread across tissues, suggesting that synchronizing drug administration with the peaks of target gene activity may improve outcomes. This alignment of treatment timing with internal biological phases provides an emerging framework for optimizing intervention effectiveness across diverse diseases.",Genome Medicine,Circadian Rhythms and Therapeutic Translation,2019
Implications for Infectious Disease Treatment and Vaccination,"Circadian control of immunity suggests that susceptibility to infection and responsiveness to treatment vary across the day. Studies show that administering vaccines or anti-infective interventions at specific times can enhance efficacy. Notably, influenza vaccination in older adults produces stronger antibody responses when administered in the morning rather than the afternoon. This reflects alignment of vaccination timing with the circadian peak of immune responsiveness. Additionally, pathogens may themselves possess intrinsic circadian rhythms; for example, the parasite Trypanosoma brucei exhibits endogenous oscillations that affect its sensitivity to the drug suramin. Such findings imply that both host and pathogen clocks may influence therapeutic outcomes and that future anti-infective strategies may benefit from time-based optimization.",Genome Medicine,Circadian Rhythms and Therapeutic Translation,2019
Targeting the Circadian Machinery for Disease Treatment,"Modulating core clock components has emerged as a promising therapeutic strategy. In cancer, targeting the circadian nuclear receptors REV-ERBs with pharmacological agonists can suppress cancer hallmarks such as de novo lipogenesis and autophagy without harming normal cells. Clock-modulating compounds may also have potential for treating sleep disorders, anxiety, and circadian misalignment conditions. For example, molecules capable of altering the circadian period may help mitigate the effects of jet lag or assist patients with delayed sleep phase disorder (DSPD). Since physiological processes are tightly intertwined with circadian rhythms, adjusting clock protein activity could improve metabolic regulation, immune function, and neurological health.",Genome Medicine,Circadian Rhythms and Therapeutic Translation,2019
Timing as a Universal Variable in Human Disease Intervention,"The intimate relationship between circadian rhythmicity and physiology raises a fundamental clinical question: for any given disease, is there an optimal time of day to intervene? Circadian phases influence drug absorption, metabolism, immune activation, hormone release, and even cellular susceptibility to stress or injury. Understanding these rhythms could allow clinicians to schedule treatments—medications, vaccinations, behavioral therapies—at times of maximal benefit and minimal side effects. Furthermore, synchronizing or stabilizing circadian rhythms through lifestyle, pharmacological, or molecular interventions may represent a broad strategy for improving health across metabolic, infectious, neurological, and oncological diseases.",Genome Medicine,Circadian Rhythms and Therapeutic Translation,2019
Emerging Layers of Circadian Regulation,"Recent years have revealed that circadian biology lies at the core of animal physiology. New regulatory layers of the circadian clock continue to be discovered, expanding beyond the classic transcription–translation feedback loops. These include dynamic chromatin conformation changes, polyamine-mediated modulation of clock protein interactions, fluctuations in the NADP+:NADPH redox ratio, cytosine modifications, and autophagy-based degradation of clock components. Together, these mechanisms highlight that circadian regulation extends deeply into genome organization, metabolic state, and cellular quality-control pathways. Genomic studies have significantly advanced our understanding of daily physiological rhythms in both health and disease by identifying rhythmic regulatory elements, enhancer activity, chromatin looping, and oscillatory transcriptional programs.",Genome Medicine,Circadian Rhythms – Mechanisms,2019
Ultradian Rhythms and 12-Hour Cycles,"Beyond the familiar 24-hour circadian cycle, shorter biological rhythms—ultradian rhythms—have gained increased attention. A distinct set of genes oscillates with a 12-hour rhythm in several peripheral tissues, many of which respond to feeding cues. Emerging evidence suggests that these 12-hour rhythms arise from a cell-autonomous pacemaker that is independent from the 24-hour clock and plays an important role in metabolic homeostasis. The presence of these ultradian cycles raises the possibility that multiple internal clocks operate simultaneously to coordinate physiology. Future work will determine how these 12-hour oscillators interact with circadian rhythms and which physiological systems depend on their precise timing.",Genome Medicine,Ultradian Rhythms,2019
"Circadian Rhythms, Health, and Lifespan","A rapidly growing body of research reveals a strong link between circadian rhythms and human health. Disruptions to circadian biology influence disease susceptibility, metabolic regulation, immune function, and aging. However, the mechanisms underlying this broad influence remain incompletely understood. New findings continue to emerge regarding dietary modulation of circadian physiology, sex-specific circadian differences, and the impact of circadian disruption on lifespan. These data emphasize that circadian medicine is inherently interdisciplinary, requiring integration of molecular biology, neuroscience, immunology, metabolism research, and clinical science. Understanding these interactions will be essential for translating circadian knowledge into practical health strategies.",Genome Medicine,Circadian Rhythms – Health and Aging,2019
Future Directions and Opportunities in Circadian Medicine,"Technological advances—including improved sequencing, proteomics, chromatin mapping, and high-resolution temporal sampling—have transformed circadian research and will continue to propel the field. Incorporating the temporal dimension into physiology and medicine may allow alignment of internal rhythms with external environmental cues, offering opportunities for lifestyle-based and pharmacological interventions. Future circadian medicine aims to optimize treatment timing, improve metabolic and immune outcomes, enhance response to disease, and promote healthy aging. Fully understanding the interplay between circadian, ultradian, environmental, and behavioral rhythms will be central to developing time-aligned strategies that improve human health and longevity.",Genome Medicine,Circadian Medicine – Future Directions,2019
Overview of Aging and the Gut Microbiome,"Aging results from complex interactions between genetic and environmental factors, and accumulating evidence identifies the gut microbiome as a central regulator of age-associated biological changes. Alterations in microbial communities influence immune function, metabolic pathways, inflammation, and susceptibility to age-related diseases. Across the lifespan, the gut microbiota undergoes substantial shifts in diversity, taxonomic composition, and functional capacity. This systematic review synthesizes findings from 27 empirical human studies examining normal and successful aging, focusing on how microbial diversity and downstream metabolic pathways differ across developmental stages and age groups.",Nutrients,Gut Microbiome and Aging,2020
Microbial Diversity in Aging,"Alpha diversity of microbial taxa, metabolic pathways, and microbially derived metabolites was generally higher in older adults, especially among the oldest-old. Beta diversity analyses demonstrated significant differences in microbial community composition across age groups, including distinctions between young-old and oldest-old individuals. These findings suggest that aging is associated with unique diversification and restructuring of the gut microbiome, with the oldest-old exhibiting microbiome characteristics that differ not only from young adults but also from younger elderly adults.",Nutrients,Microbial Diversity in Aging,2020
Taxonomic and Functional Shifts,"Although taxonomic findings varied across studies, some genera showed consistent age-related trends. Akkermansia was more abundant with aging, whereas Faecalibacterium, Bacteroidaceae, and Lachnospiraceae were often reduced. Functional analyses indicated that older adults exhibit decreased pathways associated with carbohydrate metabolism and amino acid synthesis. However, oldest-old adults demonstrated functional distinctions that may reflect healthy or successful aging, including increased short-chain fatty acid (SCFA) production potential, elevated butyrate derivatives, and enrichment of metabolic pathways linked to gut epithelial health and reduced inflammation.",Nutrients,Gut Microbiome Functional Shifts,2020
Interpretation and Implications,"Most available studies employ cross-sectional designs, limiting causal interpretations. Nevertheless, integrating compositional and functional findings suggests that age-associated microbiome signatures may influence inflammation, immunosenescence, metabolic health, and cognitive function. Differences between normal and successful agers imply that specific microbial taxa and metabolite profiles may support longevity or resilience against age-related decline. These insights highlight the potential of targeting microbial pathways to promote healthy aging, though longitudinal studies are required to clarify mechanistic relationships.",Nutrients,Healthy Aging and Microbiome,2020
Introduction — Aging Biology Overview,"Aging is a genetically driven and environmentally modulated process involving progressive changes across biological, behavioral, environmental, and social domains. Core cellular and molecular hallmarks include genomic instability, telomere attrition, epigenetic alterations, and loss of proteostasis, which induce compensatory mechanisms such as deregulated nutrient sensing, mitochondrial dysfunction, and cellular senescence. These processes ultimately contribute to stem cell exhaustion and altered intercellular communication, leading to functional decline. Advances in next-generation sequencing have accelerated the discovery of biological and genetic mechanisms underlying aging and age-related diseases. Growing evidence positions the gut microbiome as a central regulator of many age-associated changes, influencing longevity across species. Host physiology and the gut microbiota affect each other reciprocally, and disruptions in this relationship can alter immune responses and systemic homeostasis.",Nutrients,Aging Biology and Microbiome,2020
"Introduction — Immunity, Inflammaging, and Gut–Brain Axis","The gut microbiome is a major determinant of immune function, and its dysregulation can maintain chronic low-grade inflammation. Aging is characterized by weakening immune competence, creating an imbalance between pro-inflammatory and anti-inflammatory activity. This age-associated pro-inflammatory state, termed inflammaging, contributes to heightened vulnerability to chronic diseases such as cardiovascular disorders, cognitive decline, metabolic disease, frailty, and increased mortality risk. In addition, gut microbes communicate with the central nervous system through the gut–brain axis using neural, immune, and hormonal pathways, thereby influencing mood, behavior, and higher-order cognitive functions. These interactions highlight the microbiome as a key entry point for understanding both physical and cognitive aspects of aging.",Nutrients,Inflammaging and Gut–Brain Axis,2020
"Introduction — Healthy, Normal, and Successful Aging","Healthy aging lacks a universally accepted definition due to the inherent heterogeneity of aging trajectories. Individuals diverge more biologically and functionally as they get older, and even different tissues within the same individual age at different rates. Aging can be categorized into normal aging, pathological aging associated with disease, and successful aging characterized by preserved function and resilience. Centenarians exemplify successful aging, having avoided or survived major age-related diseases while still exhibiting certain biological signs of aging. In these individuals, inflammaging persists but is counterbalanced by enhanced anti-inflammatory responses. Gut microbiota composition across the lifespan may therefore influence health outcomes and modulate aging trajectories.",Nutrients,Healthy vs Successful Aging,2020
Introduction — Knowledge Gaps and Scope of Review,"Despite numerous reviews exploring microbiome changes in aging-related diseases, few have focused specifically on aging itself independent of pathology. No prior work has systematically evaluated gut microbiome and metabolome changes across normal and successful aging in humans. Longitudinal microbiome studies in aging are notably absent, creating difficulty distinguishing cohort effects from true within-individual aging processes. This systematic review synthesizes current evidence on gut microbiota composition, functional capacity, and metabolic products across adulthood, including the oldest-old. It further integrates associations between microbiome features and clinical variables such as cognition, living environment, and results from interventions targeting the microbiome in aging populations.",Nutrients,Microbiome and Aging Research Gaps,2020
Overview of Vascular Stiffness in Aging,"The goal of this review is to provide further understanding of increased vascular stiffness with aging, and how it contributes to the adverse effects of major human diseases. Differences in stiffness down the aortic tree are discussed, a topic requiring further research, because most prior work only examined one location in the aorta. It is also important to understand the divergent effects of increased aortic stiffness between males and females, principally due to the protective role of female sex hormones prior to menopause.",Frontiers in Cardiovascular Medicine,Vascular Stiffness,2021
Sex Differences and Model Limitations,"Another goal is to review human and non-human primate data and contrast them with data in rodents. This is particularly important for understanding sex differences in vascular stiffness with aging as well as the changes in vascular stiffness before and after menopause in females, as this is controversial. This area of research necessitates studies in humans and non-human primates, since rodents do not go through menopause.",Frontiers in Cardiovascular Medicine,Vascular Stiffness,2021
Mechanisms Underlying Increased Vascular Stiffness,"The most important mechanism studied as a cause of age-related increases in vascular stiffness is an alteration in the vascular extracellular matrix resulting from an increase in collagen and decrease in elastin. However, there are other mechanisms mediating increased vascular stiffness, such as collagen and elastin disarray, calcium deposition, endothelial dysfunction, and the number of vascular smooth muscle cells (VSMCs).",Frontiers in Cardiovascular Medicine,Vascular Stiffness,2021
Longevity Populations and Disease Connections,"Populations with increased longevity, who live in areas called “Blue Zones,” are also discussed as they provide additional insights into mechanisms that protect against age-related increases in vascular stiffness. Such increases in vascular stiffness are important in mediating the adverse effects of major cardiovascular diseases, including atherosclerosis, hypertension and diabetes, but require further research into their mechanisms and treatment. Keywords: aortic stiffness, aging, cardiovascular diseases, human, non-human primate",Frontiers in Cardiovascular Medicine,Vascular Stiffness,2021
Introduction to Vascular Stiffness in Aging,"The goal of this article is to review what is known about changes in vascular stiffness with aging and disease. It is widely accepted that aortic stiffness increases with advancing age. However, most existing research employs measures of aortic stiffness at a single aortic location as an estimate of overall aortic stiffness. This makes it a challenge to understand age-related stiffness along the length of the aortic tree, from the aortic root to its bifurcation into the iliac arteries, and regional vessels. Another goal is to review human and non-human primate data, which is particularly important for understanding sex differences in vascular stiffness with aging and the changes in vascular stiffness before and after menopause in females. Several mechanisms that mediate the increases in vascular stiffness will be reviewed.",Frontiers in Physiology,Vascular Stiffness,2021
Extracellular Matrix and Additional Mechanisms,"The most well-studied mechanism involves the extracellular matrix, with increases in vascular collagen and decreases in vascular elastin. There are other mechanisms, less well studied, that also contribute to the increased vascular stiffness, e.g., collagen and elastin disarray and increased vascular smooth muscle cell stiffness and numbers. Further insight can also be gained from populations with an extended lifespan, living in areas called 'Blue Zones,' where a healthy diet and exercise ameliorate the increases in vascular stiffness observed with age.",Frontiers in Physiology,Vascular Stiffness,2021
Aorta Anatomy Overview,"The aorta is divided into sections by location; the ascending aorta, aortic arch, and the descending aorta. The descending aorta can be divided into thoracic and abdominal sections. Branches of interest include the left and right coronary arteries, which branch from the aortic root, and the brachiocephalic, left carotid, and left subclavian, which branch from the aortic arch. As the aorta descends there are numerous branches which supply the surrounding muscles and organs including intercostal, celiac, hepatic, gastric, splenic, renal, mesenteric, and gonadal arteries. The abdominal aorta bifurcates into the iliac arteries which extend inferiorly, turning into the femoral arteries, which supply blood flow to each leg.",Frontiers in Physiology,Aorta Anatomy,2021
Aorta Morphometry Across Regions,"The morphometric properties of the aorta differ along its length. The aorta tapers, with the average systolic diameter decreasing from the proximal to the distal aortic tree (Hickson et al., 2010). In healthy humans, helical computed tomography showed that maximum aortic diameter is in the ascending aorta, distal to the aortic valve sinus and proximal to the innominate artery (Hager et al., 2002). The aortic diameter then decreases progressively along the thoracic aorta and continues to decrease from the infrarenal abdominal aorta to the lower abdominal aorta (Hager et al., 2002; Rogers et al., 2013). Overall thickness of the aortic wall also decreases down the thoracic aorta, but then remains constant in the abdominal aorta (Sokolis, 2007).",Frontiers in Physiology,Aorta Morphometry,2021
Age-Related Morphometric Changes,"In the pig, the tunica media decreases in thickness distally along the thoracic and abdominal aorta, while the tunica adventitia thickness is negligible in the thoracic aorta, but increases down the length of the abdominal aorta (Sokolis, 2007). Aging results in morphometric changes to the diameter, length, and thickness of the aorta. Overall, the diameter and length increase progressively with age (Komutrattananont et al., 2019), with the greatest change in diameter occurring at the level of the ascending aorta (+0.96 mm/decade) (Hickson et al., 2010). The tunica intima and media of the aortic wall thicken with age (Komutrattananont et al., 2019).",Frontiers in Physiology,Aorta Morphometry,2021
PWV and Regional Differences in Aging,"One measure of aortic stiffness, carotid-femoral pulse wave velocity (PWV), is an estimate of the pulse transit-time between the carotid and femoral arteries (O’Rourke et al., 2002; Pannier et al., 2002; Laurent and Boutouyrie, 2020). This is an approximation that averages the many branches of the aortic tree and does not consider the influence of regional differences in stiffness and diameter (Millasseau et al., 2005). With aging, large elastic arteries, such as the aorta, show increases in arterial stiffness, which correlate with histological and biochemical changes within the arterial wall. Several studies have examined both thoracic and abdominal aortic stiffness with aging, in vivo (Farrar et al., 1984; Rogers et al., 2001; Nelson et al., 2009; Hickson et al., 2010; Taviani et al., 2011; Westenberg et al., 2011; Devos et al., 2015). In humans, where PWV was measured by cine phase contrast magnetic resonance imaging (PCMRI) in four segments of the aorta, it was found that the greatest age-related increase in aortic stiffness occurred in the abdominal aorta (+0.9 m/s per decade) followed by the thoracic-descending region (+0.7 m/s), the mid-descending region (+0.6 m/s), and aortic arch (+0.4 m/s) (Hickson et al., 2010).",Frontiers in Physiology,Aortic Stiffness,2021
Variation in Stiffness Down the Aortic Tree,"Another study, focusing on the ascending, descending, and infrarenal aorta showed increases in stiffness down the aortic tree in humans aged 40, 60, and 75 years (Cuomo et al., 2017). Variation in stiffness down the aortic tree has also been addressed via computational modeling of the human aortic tree using several metrics for stiffness and geometric and hemodynamic data from the literature. In silico examination of the effect of aging showed that pulse pressure and stiffness increase down the aortic tree and are most marked with advanced age. PWV may deviate from this pattern when it comes to the most distal sections of the aorta, as these are likely influenced by arterial tapering and branching (Cuomo et al., 2017). Isolated in vitro studies have also found that abdominal aortic stiffness is increased more with aging (Haskett et al., 2010).",Frontiers in Physiology,Aortic Stiffness,2021
Conflicting Human Study Results,"Although many studies have reported that stiffness increases down the aortic tree, there still is some controversy. Some in vivo studies reported that increases in thoracic aortic stiffness with aging were greater than, or similar to those observed in the abdominal aorta (Farrar et al., 1984; Rogers et al., 2001; Nelson et al., 2009; Hickson et al., 2010; Taviani et al., 2011; Westenberg et al., 2011; Devos et al., 2015). Using PCMRI in a single para-sagittal plane to measure PWV in different regions, one study in humans found that participants below 55 years of age had similar PWVs at different aortic locations, but those older than 55 experienced the reverse of what is generally thought, i.e., stiffness decreased down the aortic tree (Rogers et al., 2001). In this study the investigators also suggested that the most significant mechanisms for increasing aortic stiffness with age are fragmentation of elastin, which would primarily affect the proximal aorta due to its higher elastin content, and diminished nitric oxide activity (Rogers et al., 2001). However, it’s possible that the changes noted are not statistically significant, since the population studied had a low probability of having atherosclerosis and PWV variability increased markedly with age. Another study in humans found no significant difference in aortic PWV in pre- and post-menopausal women, but significantly lower brachial and femoral PWV values in pre-menopausal women (London et al., 1995).",Frontiers in Physiology,Aortic Stiffness,2021
Non-Human Primate Evidence,"By comparison with these studies in humans, we found significantly greater increases in stiffness in the abdominal aorta compared to the thoracic aorta in studies of non-human primates (Zhang et al., 2016; Babici et al., 2020; Figures 1, 2). Recording of aortic dimensions using implanted ultrasonic crystals in monkeys (Figure 3) showed that abdominal aortic stiffness was greater than thoracic aortic stiffness in both young and old monkeys (Figures 1, 2; Zhang et al., 2016). These measurements of arterial stiffness using direct and continuous measurements of arterial pressure and diameter are more precise than measurements of stiffness using PWV, since they permit assessment of stiffness at distinct locations in the aorta. Examination of old premenopausal female monkeys also showed that the aortic stiffness index (β) was significantly higher in the abdominal vs. the thoracic aorta, both in older (20 ± 1.8) and younger (8 ± 1.1) monkeys (Babici et al., 2020). In addition, histological correlates of vessel stiffness were greatest in the iliac artery, suggesting iliac artery stiffness was even greater than abdominal aortic stiffness (Babici et al., 2020).",Frontiers in Physiology,Aortic Stiffness,2021
Implications and Challenges of Primate Research,"Our in vivo studies of monkeys clearly indicate that age-related increases in aortic stiffness are greater in the abdominal compared to the thoracic aorta (Figures 1, 2). Our previous studies also found significantly greater aortic stiffness in the abdominal compared to the thoracic aorta, in young monkeys (Zhang et al., 2016; Babici et al., 2020). Similarly, in normal rabbits PWV increased more with age in the abdominal than the thoracic aorta (Katsuda et al., 2014). As noted above there are several reasons why studies in non-human primates are ideal for understanding vascular stiffness. Although it would be best to conduct these studies in humans there are ethical limitations to those studies and it is challenging to study changes in vascular stiffness in the absence of other disease states that normally evolve in older patients. The non-human primate is closest to humans on the evolutionary tree and therefore has the closest changes in genomics to humans among animal models, which occur with age. Moreover, sex-specific changes with aging, particularly the similarity between menopause in humans and non-human primates, is another important feature. On the other hand, there are features that make it considerably more difficult to study non-human primates than other animal models. First of all cost: there is a difference of thousands of dollars in purchasing non-human primates compared to other laboratory animals. Secondly, their supply is limited. Thirdly, care for these animals is more complex and most vivariums do not have the appropriate veterinary staff and facilities to house primates. Finally, there is increasing criticism for the use on non-human primates on an ethical basis.",Frontiers in Physiology,Aortic Stiffness,2021
Developmental Changes from Fetal to Adult Life,"Most interest in age-related vascular stiffness has focused on the changes observed between midlife and older age in adults. However, it would also be of interest to know if changes in vascular stiffness occur between birth and midlife. To this end, we examined fetal, newborn and adult sheep, chronically instrumented for measurements of aortic diameter and pressure. At baseline levels of arterial pressure the elastic modulus of the aorta of young adult sheep was higher than that of the newborn lamb or the fetus, associated with a higher stress level (Pagani et al., 1979). However, when data were evaluated at common levels of stress, the aorta of the adult had a lower elastic modulus, than either the newborn or fetal animals (Figure 4). Furthermore, in the adult, a marked shift in the pressure-diameter and stress-radius relationships were observed in response to alpha-adrenergic mediated vasoconstriction. In contrast, no shift was observed in the newborn or fetal lambs (Pagani et al., 1979). The mechanism could either be at the level of either alpha adrenergic receptor signaling development or the inability of the aortic smooth muscle to constrict.",Frontiers in Physiology,Aortic Stiffness Development,2021
Peripheral vs. Central Artery Characteristics,"In comparison to the aorta, peripheral arteries are less elastic, more muscular, and inherently stiffer (Yu and Mceniery, 2020). In humans, the femoral artery has a more rigid wall, a greater diastolic diameter, and a twofold lower distensibility coefficient than the carotid artery (Benetos et al., 1993). The diastolic diameter of the carotid artery increases with age, while in the femoral artery, arterial diameter only increases slightly with age (Benetos et al., 1993). Carotid artery distensibility decreases linearly with aging, and cross-sectional compliance also decreases (Benetos et al., 1993). Although stiffness of peripheral arteries prior to the age of 50 is higher than that of central arteries, the increases in stiffness with aging is less in peripheral arteries than in central arteries (Mitchell et al., 2004).",Frontiers in Physiology,Peripheral Vessels,2021
Age Effects on Major Peripheral Arteries,"A study of static mechanical properties using an ultrasonic phase locked echo tracking system showed that the common carotid, femoral, and brachial arteries all increase in diameter with age (Kawasaki et al., 1987). Stiffness increases in all arteries as well; however, this was only significant in the common carotid and changes in stiffness of the brachial and femoral arteries varies greatly among individuals (Kawasaki et al., 1987). As with the aging aorta, it is believed that collagen content in the pulmonary and carotid arteries increases, while elastin content and the number of vascular smooth muscle cells (VSMCs) decrease with age (Greenwald, 2007). However, aging likely impacts large conduit arteries, such as the aorta and carotid, differently than small resistance vessels, which have fewer layers of VSMC and less matrix (Trache et al., 2020).",Frontiers in Physiology,Peripheral Vessels,2021
Regional Stiffness in Non-Human Primates,"In non-human primates, as noted above, stiffness increases from the thoracic to the abdominal aorta and even distally to the iliac artery in both male and female monkeys (Zhang et al., 2016; Babici et al., 2020). However, most other studies have not measured stiffness directly but have examined the histological properties at different vessel levels. For example, the canine femoral artery has a higher content of collagen and lower content of elastin when compared to the ascending aorta, which would correlate to increases in stiffness physiologically (Fischer and Llaurado, 1966). However, stiffness was not directly measured in this study at any of the sites analyzed (Fischer and Llaurado, 1966).",Frontiers in Physiology,Peripheral Vessels,2021
Overview of Mechanisms Increasing Vascular Stiffness,"The mechanisms of increased stiffness in aging are both extracellular and cellular (Figure 5). The three main aortic wall components, elastin, collagen, and smooth muscle cells, vary along the length of the aortic tree. With aging, these components of the aortic wall are altered. The number of elastic fibers and smooth muscle cells in the tunica media decrease, while collagen fibers increase with advancing age (Maurel et al., 1987). The number of smooth muscle cells in the tunica media decreases with age and vascular smooth muscle cell migration from the tunica media thickens the intima (Collins et al., 2014).",Frontiers in Physiology,Mechanisms of Vascular Stiffness,2021
ECM Remodeling as a Driver of Stiffness,"The most important mechanism studied as a cause of age-related increases in vascular stiffness is alteration in the extracellular matrix (ECM), resulting from an increase in collagen and decrease in elastin. The ECM is composed of a complex network of different matrix proteins, metalloproteases, and glycosaminoglycans, which are also responsible for the structural integrity of the vasculature, and therefore contribute to its stiffness (Ma et al., 2020). Collagen is a very stiff protein with the function of limiting vessel elasticity and distension (Briones et al., 2010), and is therefore fundamental to defining the stiffness of the arterial wall. Collagen deposition throughout the vasculature increases with age, which alters the normal ECM network (Kohn et al., 2015). This has been shown to occur in the intima, media, and adventitia of the vessel wall leading to substantial changes in its morphology and function (Greenberg, 1986; Fleenor et al., 2010, 2012). In addition to increased collagen deposition, there is also increased non-enzymatic glycation. This is also responsible for age-related increases in arterial stiffness (Schleicher et al., 1997), as it induces collagen cross-linking, which increases stiffness (Reddy, 2004).",Frontiers in Physiology,Extracellular Matrix Remodeling,2021
"Elastin Structure, Degradation, and Aortic Gradients","Unlike collagen, elastin, the other major ECM protein, provides flexibility and extensibility of the vessel wall (Wagenseil and Mecham, 2012). Elastin fibers are mainly found in the medial layer of large elastic arteries and are oriented around smooth muscle cells and collagen. Elastin content decreases, while collagen content increases from the proximal to distal aorta (Fischer and Llaurado, 1966; Sokolis, 2007). The elastin/collagen ratio is highest in the thoracic aorta and decreases distally (Hager et al., 2002), whereas the reverse is found in the collagen/elastin ratio (Figures 1, 2). Smooth muscle cell content remains similar throughout the length of the aorta, but increases with aging and is another mechanism for increased aortic stiffness. Degradation of elastin fibers with aging is mediated by the increases of proteolytic enzymes, e.g., matrix metalloproteases (MMP), which degrade elastin fibers, resulting in an increase in collagen/elastin ratio, which in turn increase vessel wall stiffness (Wolinsky, 1970). Nevertheless, the extent to which increases in collagen and decreases in elastin contribute to increased vascular stiffness with aging remains controversial.",Frontiers in Physiology,Extracellular Matrix Remodeling,2021
Regional ECM Changes in Monkeys,"Using old world monkeys, we previously found that collagen density in the thoracic aorta did not change with age, whereas that in the abdominal aorta increased. In contrast, we found that elastin in both the thoracic and abdominal aorta decreased with age (Zhang et al., 2016; Babici et al., 2020; Figures 1, 2, 6). Relatively few studies have examined changes in both thoracic and abdominal aortic stiffness with age, in vivo (Farrar et al., 1984; Rogers et al., 2001; Nelson et al., 2009; Hickson et al., 2010; Taviani et al., 2011; Westenberg et al., 2011; Devos et al., 2015). Some studies measured collagen and elastin with aging, but did not measure aortic stiffness, in vivo. Interestingly, the results of these studies are controversial, with some studies finding an increase (Faber and Oller-Hou, 1952; Chamiot-Clerc et al., 2001; Aronson, 2003; Nosaka et al., 2003; Astrand et al., 2011; Taviani et al., 2011; Wheeler et al., 2015) and others finding no change in collagen (Hosoda et al., 1984) or a decrease in collagen (Nosaka et al., 2003; Qiu et al., 2007a; Astrand et al., 2011; Taviani et al., 2011; Atanasova et al., 2012) or no change in elastin (Wheeler et al., 2015).",Frontiers in Physiology,Extracellular Matrix Remodeling,2021
Abdominal vs. Thoracic ECM Differences,"In contrast to the thoracic aorta, collagen content rose by 34% in the abdominal aorta with aging, which was a significantly greater increase than that observed in the thoracic aorta (Zhang et al., 2016). Elastin was decreased in the abdominal compared to the thoracic aorta in young animals, and decreased to even lower levels with aging (Zhang et al., 2016). Consistent with our finding that stiffness in the abdominal aorta of young monkeys equaled or was greater than that of the thoracic aorta in old monkeys, the collagen and elastin levels in the abdominal aortas of young monkeys equaled the values observed in the thoracic aorta for old monkeys (Zhang et al., 2016; Babici et al., 2020), emphasizing the importance for studying regional aortic stiffness changes with aging. However, basal levels of collagen and elastin are not the only mechanism accounting for greater stiffness observed in the abdominal compared to the thoracic aorta both in young and old monkeys.",Frontiers in Physiology,Extracellular Matrix Remodeling,2021
Collagen/Elastin Disarray and Additional Mechanisms,"From our previous studies, we also observed marked disarray of both collagen and elastin; a finding that was more prominent with aging and in the abdominal vs. thoracic aorta. In fact, the marked disarray of elastin and collagen in the young abdominal aorta is likely responsible for the unexpected more severe stiffness than even in the old thoracic aorta (Zhang et al., 2016; Babici et al., 2020). The elastin and collagen disarray correlated better with stiffness than did elastin and collagen content (Figure 6). Another study also found elastic tissue in the abdominal aorta is most affected by aging (Maurel et al., 1987). Elastic fibers become damaged and thicken the tunica intima. Within the tunica media, elastic lamellae become damaged and elastic fibers become fragmented and disarrayed (Maurel et al., 1987). It is surprising that this marked architectural disarray we observed in the aging aorta with increased stiffness has not been noted extensively in the past, even though isolated observations have previously found disarray in aortae related to aneurysms (Hofmann Bowman et al., 2010; Pezzini et al., 2012; Lee et al., 2014), hypertension (Sans and Moragas, 1993), and aging (Fornieri et al., 1992). Other important mechanisms mediating increased vascular stiffness, include increases in calcium deposition, endothelial dysfunction, and increases in the stiffness of vascular smooth muscle cells.",Frontiers in Physiology,Extracellular Matrix Remodeling,2021
Calcium Deposition and Vascular Stiffness,"Calcification of the vessel wall occurs with normal aging, reducing the vessel wall’s distensibility (London et al., 2003). In humans there is a direct correlation between aortic calcification and arterial stiffness (Guo et al., 2017). Calcinosis of arterial walls with aging has been associated with increased cholesterol content in the elderly, suggesting a relationship between these processes (Kanabrocki et al., 1960). However, it is unknown which process occurs first, although some have speculated that calcinosis increases interaction with cholesterol molecules in the arterial wall (Hornebeck and Partridge, 1975). Another explanation for the increase in calcium deposition within the arterial wall is via an increase in inflammation and oxidative stress, both of which occur with normal aging. Increases in oxidative stress that occur with aging, mainly due to decreases in mitophagy and autophagy (Pescatore et al., 2019), stimulate vascular calcification by activating several signaling cascades (Byon et al., 2008). One of the best studied signaling pathways involves the upregulation of bone morphogenetic proteins due to increases in oxidative stress, which results in increased vascular calcification (Sorescu et al., 2004; Johnson et al., 2006).",Frontiers in Physiology,Calcium Deposition,2021
Regional Variability in Calcification Effects,"The relationship between calcification and aortic stiffness differs depending on the location of the calcification. Carotid artery stiffness was more strongly associated with thoracic aorta calcification than calcification of the coronary arteries (Blaha et al., 2009). This may be because calcification of the coronary arteries usually involves only the intimal layer, while in large arteries calcification involves both the intima and media (Blaha et al., 2009); with medial calcification being more strongly associated with aging, diabetes, and severe renal disease (Iribarren et al., 2000). In addition, structural features may be involved: carotid arteries, being more elastic, are more similar to the aorta than to coronary arteries, which are non-elastic, predominantly conduit vessels.",Frontiers in Physiology,Calcium Deposition,2021
Endothelial Dysfunction and NO Loss,"The vascular endothelium is the innermost, monolayer of cells in blood vessels. When the endothelium is healthy, vascular tone is regulated by a balance of vasoconstriction and vasodilation; the latter controlled by nitric oxide (NO) release (Furchgott and Zawadzki, 1980). Reduced bioavailability of nitric oxide leads to endothelial dysfunction, resulting in impaired vasodilation, which increases arterial stiffness (Mceniery et al., 2006). Endothelium impairment and decreased NO bioavailability occur with normal aging, ultimately leading to a proinflammatory, vasoconstrictive state, resulting in increased vascular fibrosis and arterial stiffness (Santhanam et al., 2010; Santos-Parker et al., 2014). Furthermore, endothelial dysfunction leads to an increase in oxidative stress through an increase in the production of superoxide causing damage to the vessels leading to changes in hemodynamics (Donato et al., 2018). Recently, it has also been proposed that autophagy, the cellular housekeeping mechanism that maintains cellular homeostasis, decreases in the aging endothelium, further leading to increases in oxidative stress (Larocca et al., 2012). This was further confirmed with the use of a pro-autophagy treatment, which reduced arterial stiffness and oxidative stress in aged mice (Larocca et al., 2013).",Frontiers in Physiology,Endothelial Dysfunction,2021
Location-Dependent Endothelial Impairment,"Age-related endothelial dysfunction may affect the arterial network differently based on location and vessel type. Aging results in endothelial dysfunction in the aorta but not in the femoral artery (Barton et al., 1997). The anatomic variation in the influence of age on endothelial function may be related to increased pulse pressure and reduced eNOS mRNA expression in the aorta (Barton et al., 1997). In rats, acetylcholine-induced vasorelaxation is impaired in large conduit arteries (abdominal aorta and iliac arteries) but not in smaller conduit (femoral arteries) or resistance arteries (Luttrell et al., 2020).",Frontiers in Physiology,Endothelial Dysfunction,2021
VSMC Stiffness and Aging,"Vascular smooth muscle cells have recently been discovered as important contributors to age-related increases in arterial stiffness (Trache et al., 2020). This mechanism, which we named “Vascular Smooth Muscle Cell Stiffness Syndrome” (Sehgel et al., 2015b), was elucidated in aged non-human primates that displayed an increase in VSMC stiffness in large arterial vessels (Qiu et al., 2010; Sehgel et al., 2015a; Figure 7). This increased VSMC stiffness is due to the direct relationship between VSMCs and endothelial cells. Endothelial cells regulate vascular tone mainly through the release of nitric oxide. This reduces active tone of VSMCs (Furchgott and Zawadzki, 1980; Van Bussel et al., 2015), which counteracts the increase in wall shear stress that occurs with both aging and high blood pressure (Boutouyrie et al., 1995; Van Bussel et al., 2015; Jaminon et al., 2019; Figure 8). However, aging also leads to a decrease in the number of cells within the vascular wall due to a decrease in cell proliferation with age (Greenwald, 2007; Chi et al., 2019). Multiple mechanisms mediate the decrease of VSMCs with age, but most notably inflammation and calcification, which increase VSMC apoptosis (Lacolley et al., 2012). In humans, the VSMCs lost with aging are replaced by collagen fibers in the media of the arterial wall, resulting in increased vascular stiffness (Schlatmann and Becker, 1977).",Frontiers in Physiology,Vascular Smooth Muscle Cells,2021
Regional vs. Cellular Contributions to Stiffness,"Interestingly, when the stiffness of isolated VSMCs was measured with atomic force microscopy, it did not differ with age between the thoracic and abdominal aorta (Zhang et al., 2016). This suggests that VSMC stiffness is not the mechanism by which stiffness varies regionally within the aorta, unless it is related to differences in VSMC function as a result of altered endothelial cell function, inflammation or calcium mechanisms that occur variably along the aortic tree with aging.",Frontiers in Physiology,Vascular Smooth Muscle Cells,2021
Premature Aging and Progeria Models,"As emphasized in this review, increased aortic stiffness is mainly associated with aging. Interestingly, children with Hutchinson-Gilford progeria syndrome, a premature aging syndrome with a mean lifespan of 13 years, experience severe arterial stiffening from a young age. Patients aged 7 years have the aortic stiffness of a 60–69-year old without additional diseases (Gordon et al., 2005). This is thought to be related to abnormal elastin production (Sloop et al., 2015). These patients die prematurely, primarily of atherosclerotic disease and its complications. Since increased vascular stiffness, particularly in coronary arteries is linked to atherosclerosis, this provides further support for the concept that vascular stiffness is an important determinant of longevity.",Frontiers in Physiology,Lifespan Models,2021
Extended Lifespan Populations and Blue Zones,"Studying populations and animal models that experience longevity without cardiovascular decline and consequent increases in vascular stiffness could provide new insights into delaying vascular aging and promoting vascular health. Populations with extended lifespan live in areas known as Blue Zones (Vatner et al., 2020). They are geographically widespread, and include locations in the United States, Costa Rica, the Mediterranean, and East-Asia, Loma Linda, United States; the Nicoya peninsula in Costa Rica; Sardinia, Italy; Ikaria, Greece; Okinawa, Japan (Buettner and Skemp, 2016; Huang and Mark Jacquez, 2017). In these areas the number of centenarians, i.e., those reaching the age of 100, is 10 times greater than the average in the United States. The inhabitants share general features leading to healthful aging, e.g., frequent ambulation, healthy social relationships and psychological wellbeing, and diets that prevent weight gain, as opposed to Western diets, which are associated with increased vascular stiffness (Dupont et al., 2019).",Frontiers in Physiology,Lifespan Models,2021
Blue Zone Physiology and Aortic Protection,"As noted above, protection against vascular stiffness plays a role in extended lifespan. Data supporting this can be found in a study in one of these Blue Zone populations, people living on the island of Ikaria, where aortic stiffness increases gradually with age, but then begins to decelerate at 50 years of age, with PWV being significantly lower than that in normal lifespan populations (Pietri et al., 2015). In the Ikaria population, habitual physical exercise, which is known to ameliorate the effects of vascular aging (Siasos et al., 2013), is associated with increased endothelial function, also known to protect against vascular stiffness (Anderson, 2006). It has also been shown that body fat-percentage is directly associated with arterial stiffness in long-lived populations, consistent with individuals with more lean muscle having more elastic arteries (Melo et al., 2021). Exercise also preserves muscle mass and a healthy diet can promote anti-inflammatory and anti-oxidative pathways (Redei and Mehta, 2015). Another interesting population are the Yanomami Indians of South America, whose diets are low in fat and salt, and high in fiber, plantains, cassavas (a root vegetable), and fruit. Vascular stiffness has not been studied in Yanomami Indians, but their blood pressure remains largely unchanged from age 1 to 60 years (Mueller et al., 2018).",Frontiers in Physiology,Lifespan Models,2021
"Obesity, Caloric Restriction, and Stiffness","Obesity, one of the most common worldwide problems, is associated with almost all cardiovascular diseases, including increased vascular stiffness (Dupont et al., 2019). Conversely, caloric restriction has also been reported to increase lifespan, both in humans and animal models, and protects against obesity, diabetes, hypertension, cancer, and cardiovascular disease (Trepanowski et al., 2011; Yan et al., 2012, 2013; Ravussin et al., 2015; Redman et al., 2018; Vatner et al., 2020). Caloric restriction also protects against arterial stiffness. In rats, this is evidenced by increased aortic distensibility and decreased PWV (Ahmet et al., 2011). Less collagen builds up, more elastin remains, and vascular smooth muscle is preserved in the aorta (Fornieri et al., 1999). A major mechanism by which caloric restriction is protective and prolongs longevity is through increased eNOS levels, which increase nitric oxide bioavailability, and protect against oxidative stress (Wilkinson et al., 2002).",Frontiers in Physiology,Lifespan Models,2021
Longevity in Unique Animal Species,"However, vascular stiffness has not been evaluated in most studies of animal models with an extended lifespan. Whales, for example, are some of the longest living mammals but not much is known about the aging of their vasculature. They have a vastly different structure to their aortic tree with an anatomy representing arterial adaptation by diving mammals (Shadwick and Gosline, 1994). Of interest, Japanese women, who are lifelong pearl divers, demonstrate significantly lower arterial stiffness in proximal and elastic arteries and lower carotid artery impedance modulus compared with non-diving residents in the same village (Sugawara et al., 2018). Vascular stiffness, however, has not been studied in other long-lived animal models, such as, bats (Podlutsky et al., 2005), and tortoises (living over 100 years) (Quesada et al., 2019).",Frontiers in Physiology,Lifespan Models,2021
Naked Mole-Rats and Resistance to Vascular Aging,"An exception to this is the naked mole-rat, which is the longest-lived rodent known (Dammann and Burda, 2006; Grimes et al., 2014). This rodent does not display the age-related pathology seen in other mammalian species, including shorter living rodents. In naked mole-rats, systolic, mean, and pulse pressure as well as PWV remain unchanged with age (Grimes et al., 2014). Additionally, they maintain normal cardiovascular structure and function at 24 years of age, which is 8 times the lifespan of normal rats, an age physiologically equivalent to a 92-year-old human (Grimes et al., 2014). Studies suggest that their youthful vasculature may be attributed to sustained nitric oxide availability and protection against oxidative stress (Csiszar et al., 2007). Further studies of vascular stiffness in animal models with extended or abbreviated lifespan are warranted as they might provide novel therapeutic targets for the prevention of age-related increases in vascular stiffness.",Frontiers in Physiology,Lifespan Models,2021
Sex Differences in Age-Related Stiffening,"Aging-related vascular stiffening is sexually dimorphic, a topic that has not been studied extensively. Although arterial stiffness increases from young adulthood to older ages in both men and women, the increases in stiffness in women before menopause are less than those in age-matched men (Ogola et al., 2018). This pattern reverses after menopause (Nethononda et al., 2015; Babici et al., 2020). This may partly explain why women tend to live longer than men but are in worse health at older ages when compared to men (Hagg and Jylhava, 2021). In humans, postmenopausal women have a higher carotid-femoral PWV than premenopausal women; a step-up in PWV that is not observed in age-adjusted men (Staessen et al., 2001). This finding suggests that investigations into sex-specific gene regulation and sex hormones, may provide new insights into the mechanisms mediating these patterns.",Frontiers in Physiology,Sex Differences,2021
Limitations of Rodent Models,"Much of the previous experimental work on vascular stiffness has been performed in rodent models, which have a short lifespan and do not experience menopause. Because of this, the most relevant studies are those in non-human primates and humans. Non-human primates have a longer lifespan (>30 years), and undergo menopause like humans but are exempt from diseases, such as atherosclerosis, hypertension and diabetes, which are confounding factors when studying age-related changes in vascular stiffness in humans (Qiu et al., 2007a).",Frontiers in Physiology,Sex Differences,2021
Sex-Specific Patterns in PWV and Distensibility,"The studies considered here, on sex differences in arterial stiffness in humans, will focus predominantly on primary measures of arterial stiffness, namely, PWV, pulse pressure, and aortic distensibility; PWV and pulse pressure increasing and aortic distensibility decreasing as age and stiffness increases. In a study involving 777 people aged 21–85 years, aortic distensibility and aortic PWV were assessed using cardiovascular MRI, with age-related differences examined in successive deciles in each sex (Nethononda et al., 2015). In the first age group (20–29 years), aortic distensibility was significantly higher in women than men. This pattern was reversed in older aged groups, the sex differences being most marked in those aged around 60 years. In both sex groups, aortic distensibility decreased with increasing age in all regions studied, namely, the ascending, proximal aorta, and abdominal aorta. In both men and women, the greatest decreases in aortic distensibility occurred between those aged 50–59 and 60–69 years, although the decrease between these age groups was larger in women (47–61%) than in men (31–45%). These findings indicate that the decline in aortic distensibility is sex-independent, although the steepest decline occurs in women between the pre- to the postmenopausal periods.",Frontiers in Physiology,Sex Differences,2021
Distensibility–PWV Discrepancies and Menopause,"Surprisingly, however, these aortic distensibility changes were not mirrored by commensurate steep increases in PWV in the peri-menopausal period (Nethononda et al., 2015). One mechanism that may contribute to the rapid decline in aortic distensibility in females between these age groups is body weight changes that accompany menopause. Greater weight gain and an increase in the waist:hip ratio, suggesting increased abdominal fat, are seen in women compared to the increases in correspondingly aged men (Nethononda et al., 2015). Interestingly, other studies have also noted similar age-related but sex-independent PWV increases (Smulyan et al., 2001; Hickson et al., 2010). It is possible that no sex differences are observed in aortic PWV because despite distensibility decreasing rapidly after menopause, blood viscosity increases with menopause (Schillaci et al., 1998). The mathematical relationship between PWV and distensibility is demonstrated by the Bramwell-Hill equation; PWV (ρ × Distensibility)1/2, with ρ being blood density (Nethononda et al., 2015). Given that blood viscosity increases greatly with menopause, the equation aids us in understanding why a large decrease in distensibility does not correlate with an increase in PWV of the same magnitude. However, in contrast, the Baltimore Longitudinal Study of Aging identified a steeper longitudinal increase of PWV in men compared to age-matched women (Alghatrif et al., 2013).",Frontiers in Physiology,Sex Differences,2021
Premenopausal Female Primate Studies,"It is important to note that, although aortic stiffness is more severe in males than females prior to menopause, premenopausal women also exhibit age-dependent increases in aortic stiffness, as observed in primates (Babici et al., 2020). An aortic pressure catheter and ultrasonic diameter transducers implanted in young (7 ± 0.7 years old) and aging premenopausal female monkeys (24 ± 0.7 years old) found that the aortic pulse pressure was increased in old premenopausal monkeys (48 ± 2.7 mmHg) compared to young monkeys (33 ± 2.5 mmHg) (Babici et al., 2020). The aortic stiffness index, a function of aortic pressure and aortic strain, was increased in the old vs. young subjects in both the thoracic and abdominal aortas. Furthermore, the collagen/elastin ratio increased down the aortic tree and was consistently higher in the old premenopausal monkeys (Babici et al., 2020). Elastin and collagen showed progressively more disarray down the aortic tree and were more marked in premenopausal monkeys.",Frontiers in Physiology,Sex Differences,2021
Sex-Specific ECM and Structural Protein Changes,"Twice as much disarray was noted in the older group compared to the younger group in the thoracic aorta, abdominal aorta, and iliac artery (Babici et al., 2020). Elastin- and collagen-fiber disarray and breaks also increased down the aortic tree and were more marked in premenopausal monkeys. In a previous study, elastin and collagen disarray correlated better with stiffness than elastin and collagen content (Zhang et al., 2016). Studies on these structural proteins also identified sex differences in the specific characteristics of elastin and collagen. In the human abdominal aorta, elastin content decreased but the stiffness of elastin and collagen increased with age in men (Astrand et al., 2011). There was a much lesser age-related change in aortic elastin- and collagen-stiffness between young, middle-aged, and elderly women (Astrand et al., 2011). Collagen and elastin seem less affected in the female aortic wall due to the influence of sex hormones.",Frontiers in Physiology,Sex Differences,2021
Sex-Dependent Gene Regulation Mechanisms,"The mechanisms underlying age-related and regional differences in aortic stiffness are also sexually dimorphic. In non-human primate models, aging-related changes in gene expression have been shown to be sex-dependent and to involve key contributors of vascular stiffness, such as ECM composition, VSMC phenotype, cell signaling pathways, resistance to apoptosis, metabolism, protein synthesis, and transcription factors. Aging male, but not female monkeys, show downregulation of collagen type III protein expression and upregulation of collagen type VIII transcript levels. Collagen type III decreases collagen bundle size and increases vascular elasticity while collagen type VIII promotes VSMC migration into the intima (Qiu et al., 2007b). This may explain the finding that elastin stiffness increases with age in men, but not in women (Astrand et al., 2011). Similar protein and gene expression changes have also been observed in another study of a non-human primate model, in which female premenopausal animals were compared with their aged-matched male counterparts (Qiu et al., 2007a). In addition to changes in gene regulation, sex hormones are also potential contributors to sex-specific differences in age-related increases in vascular stiffness.",Frontriers in Physiology,Sex Differences,2021
Hypertension as Cause and Consequence of Stiffness,"Aortic stiffness and arterial pressure are strongly correlated in hypertension with vascular stiffness being both a cause and a consequence of hypertension (Humphrey et al., 2016). High blood pressure may cause vascular damage and elastin fragmentation, leading to increased stiffness. On the other hand, aortic stiffness widens pulse pressure which affects systolic blood pressure. Hypertension and aging may have an additive effect evidenced by elderly hypertensive patients having stiffer arteries than age-matched normotensive patients (Verwoert et al., 2014). However, these differences may be attributed to hypertension, rather than intrinsic vascular changes associated with increased stiffness (Bavishi et al., 2016). Vascular stiffness is linearly related to age both in normotensive and severely hypertensive subjects (Figure 9; Safar et al., 2018). Interestingly the slope of these linear relationships is not that different; arterial stiffness rising in normotensive people almost as much as in those who are hypertensive. Aortic stiffness is also increased in spontaneously hypertensive rats, even at a young age, but much more in older rats (Sehgel et al., 2013, 2015a,b).",Frontiers in Physiology,Hypertension,2021
"MMPs, Elastin Breakdown, and Isolated Systolic Hypertension","As discussed above, elastin breakdown, due to matrix metallopeptidases and serum elastase, is a major mediator of increased vascular stiffness. Serum MMP-9 and MMP-2 levels, and serum elastase activity, which degrade elastin and increase aortic and brachial PWV, are increased in subjects with isolated systolic hypertension (Yasmin et al., 2005). Isolated systolic hypertension, defined as systolic blood pressure > 140 mmHg and diastolic blood pressure < 90 mmHg (Mancia et al., 2013), is the predominant form of hypertension in the elderly, and is associated with increased arterial stiffness (Franklin et al., 1997; Yasmin et al., 2005; Bavishi et al., 2016). Interestingly, in older patients, systolic arterial pressure continues to increase along with aortic stiffness, but diastolic hypertension declines, further demonstrating the important relationship between systolic arterial hypertension and aortic stiffness (Franklin et al., 1997). Systolic-diastolic hypertension due to elevation of both systolic and diastolic arterial pressures is less common in older adults (Tsimploulis et al., 2017), but is associated with an increased incidence of heart failure and cardiovascular mortality. Pre-eclampsia, which induces hypertension during pregnancy, is also associated with increased vascular stiffness (Hausvater et al., 2012).",Frontiers in Physiology,Hypertension,2021
"Salt Intake, Calcification, and Treatment Resistance","Salt intake, a key mechanism mediating hypertension, is positively correlated with carotid-femoral PWV; the slope of the linear regression line linking these two parameters was steeper in women than in men (0.0199 ± 0.0045 vs. 0.0326 ± 0.0052 m/s per gram of salt, respectively, P < 0.05). However, after adjustment in the data of outliers, the association remained significant only in men (Baldo et al., 2019). Calcium deposition in the aorta is another mechanism mediating the increase in arterial stiffness in hypertension (Guo et al., 2017). Interestingly, it was most pronounced in subjects who were resistant to anti-hypertensive therapy, suggesting that arterial stiffness not only contributes to isolated systolic hypertension development, but may also be involved in resistance to hypertension treatment (Mceniery et al., 2009).",Frontiers in Physiology,Hypertension,2021
VSMC Stiffness in Hypertension,"Another important mechanism of aortic stiffness in hypertension is vascular smooth muscle stiffness (Sehgel et al., 2013, 2015a,b). Changes to the intrinsic stiffness of VSMCs and to their adhesion properties are observed in hypertension. Using atomic force microscopy and a reconstituted aortic tissue model, it was found that spontaneously hypertensive rats had increased aortic VSMC stiffness as well as different temporal oscillations in VSMC stiffness compared to normotensive control rats (Sehgel et al., 2013). In a later study in which VSMC stiffness and adhesions to the ECM were also found to be increased in hypertensive rats (Sehgel et al., 2015a), hypertension did not increase the amount of collagen in the thoracic aorta, suggesting that increased vascular stiffness associated with hypertension is likely not exclusively mediated by altered collagen and elastin content, but by increased VSMC stiffness and adhesion. VSMC changes with aging are augmented when hypertension is superimposed on aging.",Frontiers in Physiology,Hypertension,2021
"Atherosclerosis, Aging, and Early-Onset Forms","Atherosclerosis, which is more common with aging, is a chronic inflammatory disease in which atheromatous plaques form, resulting in arterial narrowing. Many studies, such as the Rotterdam study (Van Popele et al., 2001), show that stiffness of the aorta increases with plaque burden and conclude that arterial stiffness is strongly associated with atherosclerosis. However, these conclusions must be tempered by the fact that increased vascular stiffness is also a feature of aging in the absence of atherosclerosis (Sun, 2015). Atherosclerosis can occur at an earlier age in a condition known as Pediatric atherosclerosis (Wilson, 2000). Also, as discussed above the autosomal recessive premature aging disorder, Hutchison-Gilford Progeria syndrome, is characterized by precocious atherosclerosis and stiffening of the arteries, which cause early death in affected individuals (Keay et al., 1955). Consistent with the arterial stiffness found in these children, their carotid-femoral PWV is also markedly increased (Gerhard-Herman et al., 2012; Gordon et al., 2012).",Frontiers in Physiology,Atherosclerosis,2021
Young Animal Models of Atherosclerosis,"One way to address the question of mechanisms of atherosclerosis in a younger population is to examine atherosclerosis mechanisms in animal models that develop disease at a young age. Our laboratory has begun to study aortic stiffness in Watanabe rabbits, an animal model of atherosclerosis (Aliev and Burnstock, 1998). Our preliminary data suggest that aortic stiffness is increased even in relatively young Watanabe rabbits, as compared to aged-matched New Zealand White rabbit controls. In hypercholesterolemia Kurosawa and Kusanagi rabbits, PWV reflecting the atherosclerotic regions found that vascular stiffness was increased more in these regions and more in the abdominal vs. the thoracic aorta (Katsuda et al., 2014).",Frontiers in Physiology,Atherosclerosis,2021
ECM Remodeling and Hypercholesterolemia Mechanisms,"Many different mechanisms have been implicated in the stiffening of arteries associated with atherosclerosis. Hypercholesterolemia is most commonly implicated in the pathogenesis of atherosclerosis in humans and animal models, ranging from rodents to rabbits, pigs and monkeys (Linton et al., 2000; Getz and Reardon, 2012). Extracellular matrix proteins are also involved in the development of increased vascular stiffness in atherosclerosis. Elastin degradation is increased by the build-up of atheromatous plaques. Non-atherosclerotic arteries contain MMP-2 as well as inhibitors of MMP, such as TIMP 1 and 2 (Galis et al., 1994). In contrast, atheromatous plaques also contain macrophages that secrete MMP-1, MMP-9, and MMP-3, smooth muscle cells, lymphocytes, and endothelium (Galis et al., 1994). Based on SDS-PAGE zymography, plaques have been found to contain activated forms of MMP-2 and MMP-9 (Galis et al., 1994). In addition, patients with hypercholesterolemia exhibit more circulating CD31+/CD42− microparticles, less endothelial progenitors (EPCs), and have stiffer aortae than controls. The ratio of CD31+/CD42− microparticles to EPCs was found to be directly associated with arterial PWV (aPWV) (Pirro et al., 2006). This suggests that hypercholesterolemia contributes to large artery stiffness by increasing microparticle release and by reducing the number of circulating EPCs. In addition, the extracellular matrix protein, fibrillin-1, has been found to modulate large-artery stiffness and pulse pressure (Medley et al., 2002).",Frontiers in Physiology,Atherosclerosis,2021
Oxidative Stress and Calcification in Atherosclerosis,"Elevated oxidative stress also plays a role in increased arterial stiffening in patients with atherosclerosis. A study examining patients with peripheral arterial disease found an independent association of aPWV with serum levels of osteopontin and oxidized low-density lipoprotein, which are involved in oxidative stress, thus supporting the role for oxidative stress in mediating arterial stiffness in patients with atherosclerosis (Zagura et al., 2012). Intimal arterial calcification within atherosclerotic plaques may also be responsible for increased vascular stiffness (Mackey et al., 2007). A study in the Twins United Kingdom population suggests that it is the propensity of plaques to calcify rather than the amount of plaque that determines arterial stiffness (Cecelja et al., 2013). Aortic stiffness was correlated with calcified plaques in the carotid and femoral arteries detected by ultrasound and with total aortic calcification measured by computed tomography (Cecelja et al., 2013).",Frontiers in Physiology,Atherosclerosis,2021
Diabetes and Accelerated Arterial Aging,"Diabetes predisposes to cardiovascular disease and accelerated arterial stiffness. The magnitude of the effect of diabetes on central stiffness has been compared to the equivalent of 6–15 years of chronological aging on vessels (Cameron and Cruickshank, 2007; Loehr et al., 2016). It has been suggested that vascular stiffness in diabetic patients may be attributed more to the role of diabetes and metabolism, than to aging, per se (Cameron et al., 2003). One major metabolic mechanism is the non-enzymatic advanced glycation of proteins observed in diabetes. The accelerated production of advanced glycation end-products (AGEs) is implicated in diabetes-associated increasing stiffness. AGEs form in hyperglycemic environments, accumulate in the vessel wall, and form cross-links with collagen and elastin fibers, decreasing arterial wall distensibility (Goldin et al., 2006). The incidence of atherosclerosis is also increased in diabetic patients (Poznyak et al., 2020) and, thus, increased vascular stiffening due to accelerated atherosclerosis also contributes to the increase vascular stiffness in diabetic patients (Stehouwer et al., 2008; Prenner and Chirinos, 2015). Patients with type 2 diabetes also displayed endothelial dysfunction and a reduced contractile response to endothelin-1, suggesting these mechanisms factor into the development of vascular stiffness, due to the role of vasoconstriction in mediating vascular stiffness (Rizzoni et al., 2001).",Frontiers in Physiology,Diabetes,2021
"Glucose Tolerance, Central vs Peripheral Stiffness","It has been shown that vascular stiffness increases as glucose tolerance deteriorates. Impaired glucose metabolism and type 2 diabetes (DM-2) are associated with decreased total systemic arterial compliance and increased aortic augmentation index, indicating increased central artery stiffness (Schram et al., 2004). Central artery stiffness is greater and carotid-femoral transit time is decreased in patients with DM-2 (Schram et al., 2004). It has also been shown that stiffness of the peripheral arteries increases with deteriorating glucose tolerance (Henry et al., 2003). Together, these studies suggest that stiffness due to impaired glucose metabolism and DM-2 are worse in peripheral than central arteries (Henry et al., 2003; Schram et al., 2004). Interestingly, in children with type 1 diabetes, especially males, stiffness of peripheral arteries is more common than of central arteries (Urbina et al., 2010).",Frontiers in Physiology,Diabetes,2021
Oxidative Stress Mechanisms in Diabetes,"As noted throughout this review, an important mechanism mediating increased vascular stiffness is increased oxidative stress (Giacco and Brownlee, 2010; Pasupuleti et al., 2020). It is well known that diabetes leads to increased oxidative stress, involving mitochondrial superoxide overproduction in the vasculature and in the myocardium (Giacco and Brownlee, 2010; Pasupuleti et al., 2020). It is also recognized that increased intracellular reactive oxygen species cause defective angiogenesis in response to ischemia, and activate a number of proinflammatory pathways in diabetes and mediate the atherosclerosis and cardiomyopathy associated with diabetes (Giacco and Brownlee, 2010; Pasupuleti et al., 2020). Even patients with prediabetes experience increased arterial stiffness. In one study, diabetes was associated with higher aortic PWV and prediabetes was associated with higher brachial-ankle PWV, a measure of composite stiffness (Loehr et al., 2016). Similarly, it has been shown that higher baPWV is associated with an increased risk of developing diabetes and that arterial stiffness may precede the increase in fasting blood glucose (Zheng et al., 2020).",Frontiers in Physiology,Diabetes,2021
Impaired Fasting Glucose and Early Stiffness Indicators,"Arterial stiffness is also increased in patients with impaired fasting glucose but no other cardiovascular complications (Rerkpattanapipat et al., 2009). That study found that total vascular stiffness, but not thoracic aortic stiffness, is increased in patients with impaired fasting glucose compared to control subjects (Rerkpattanapipat et al., 2009).",Frontiers in Physiology,Diabetes,2021
Summary of Age-Related Vascular Stiffness,"One of the most important effects of aging on the cardiovascular system is a progressive increase in vascular stiffness. Understanding the extent to which vascular stiffness increases with aging and the mechanisms involved are important, since vascular stiffness is a critical factor in mediating the adverse effects of most cardiovascular diseases, including atherosclerosis, hypertension and diabetes. Many prior studies are limited in defining changes in vascular stiffness down the aortic tree, because only one section of the aorta was studied. We found that abdominal aortic stiffness is greater than thoracic aortic stiffness. However, this topic warrants further investigation as there are major sex differences. In men vascular stiffness increases progressively from young adulthood to old age. In women vascular stiffness increases, but to a lesser extent up to menopause, and then increases at a rate exceeding that for males after menopause.",Frontiers in Physiology,Vascular Stiffness,2021
"Sex Differences, Mechanisms, and Key Contributors","Less data are available on sex differences in animals, since the most commonly studied species are rodents, where females do not go through menopause. Their relevance for understanding human disease, therefore, is limited. Studies of sex differences in the changes in vascular stiffness associated with age are best carried out in humans without associated cardiovascular diseases, and in non-human primates that live over 30 years and, like human females, go through menopause. Several mechanisms mediate the protection in females, with the most significant one being the female hormone, estrogen, which is present up to menopause and then declines. Other important mechanisms of increased vascular stiffness include changes in the extracellular matrix, with increases in vascular collagen and decreases in vascular elastin. It is also known that calcium deposition and endothelial dysfunction in the vessels contribute to increased vascular stiffness. Less well studied mechanisms may also contribute, such as collagen and elastin disarray, and increased vascular smooth muscle cell stiffness and numbers.",Frontiers in Physiology,Vascular Stiffness,2021
"Lifestyle, Longevity, and Environmental Factors","Additional insights come from studies in populations with an extended lifespan that live in areas known as “Blue Zones.” People in these areas maintain a healthy diet and daily exercise and have lesser increases in vascular stiffness with age. Understanding how these environmental factors influence the progression of vascular stiffness may provide critical insights into retarding its progression and, thereby reducing cardiovascular disease.",Frontiers in Physiology,Vascular Stiffness,2021
Overview of Endothelial Aging and Cardiovascular Risk,"Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Oxidative Stress Mechanisms in Aged Endothelium,"Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
"Inflammation, Senescence, and Genomic Instability","This review discusses the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage have been shown to trigger cell senescence via the p53/p21 pathway that results in increased inflammatory signaling in arteries from older adults.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Energy-Sensing Pathways Altered with Age,"Energy sensitive/stress resistance pathways (SIRT-1, AMPK, mTOR) are altered in endothelial cells and/or arteries with aging and these pathways may modulate endothelial function via key oxidative stress and inflammation-related transcription factors. This review also discusses what is known about the role of 'energy sensing' longevity pathways in modulating endothelial function with advancing age. With the growing population of older adults, elucidating the cellular and molecular mechanisms of endothelial dysfunction with age is critical to establishing appropriate and measured strategies to utilize pharmacological and lifestyle interventions aimed at alleviating CVD risk.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Aging as a Major Driver of Cardiovascular Disease,"Cardiovascular diseases (CVD), largely defined as stroke, coronary artery disease, heart failure, and cardiac arrest, are the predominant killers of Americans, accounting for ~752,000 deaths per year according to current statistics from the Centers for Disease Control. CVDs cause ~35% of all deaths for Americans 65 years of age or older, making them the leading causes of death in this age group. Furthermore, with advancing age, the prevalence of CVDs among Americans increases progressively, from ~5.5% in 25–44 year olds to ~41% in people 65 years of age or older. Thus, CVDs can be considered true diseases of aging. Heart disease, stroke, and hypertension are all diseases currently recognized to be caused, in part, by arterial dysfunction.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Arterial Dysfunction as a Precursor to CVD,"Age-related alterations to arteries are thought to lead to a dysfunctional phenotype that precedes CVDs. Importantly, the dysfunctional phenotype that develops in arteries with advancing age can occur in the absence of overt CVD and conventional CVD risk factors, supporting the idea that these changes are a primary effect of aging that may be a precursor to the development of CVD. Large landmark studies, like the Baltimore Longitudinal Study on Aging (BLSA) and the Framingham Heart Study, demonstrated that the age-associated phenotype of arteries involves, among other changes, the development of a dysfunctional arterial endothelium. This dysfunctional endothelial phenotype is common to humans and non-human primates as well as rodents and contributes to hemodynamic changes, including augmented large and resistance arterial tone, induction of greater oscillatory shear stress and elevated large artery stiffness, that contribute to increases in arterial blood pressure and atherosclerosis seen with advancing age.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Endothelial Function and Systemic Vascular Regulation,"The arterial endothelium is extremely dynamic and performs many vital functions that vary from one segment of the arterial tree to another as well as from one organ system to another. The vascular endothelium releases molecules that act in an autocrine and paracrine manner to regulate the function and health of the vascular network. These include the maintenance of blood in a fluid state; exchange of fluid and molecules between the blood and surrounding tissues; creation of new vascular networks; participation and facilitation of the immune response; and the control of vascular resistance in response to changes in blood flow by regulating arterial tone in resistance arteries. A healthy vascular endothelium is in a tightly regulated state of balance between pro- and anti-oxidants, vasodilators and vasoconstrictors, pro- and anti-inflammatory molecules, and pro- and anti-thrombotic signals.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Endothelial Dysfunction and Clinical Importance,"A diseased or dysfunctional endothelium has lost its tightly regulated balance and displays pro-oxidant, vasoconstrictor, pro-inflammatory and pro-thrombotic properties. One hallmark of vascular endothelial dysfunction is impaired endothelial dependent dilation (EDD), which is predictive of future CVD events. Indeed, the Framingham Heart Study has recently demonstrated that increased age is the strongest independent correlate of EDD. Therefore, it is of great clinical significance that we obtain a better understanding of the mechanisms underlying age-related decreases in endothelial function and to test the efficacy of interventions that may restore endothelial function in middle-aged and older adults.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Scope and Focus of the Review,"The first goal of this review will be to introduce the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction in healthy older adults and rodent models. Next, we will discuss the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Then, we will discuss the emerging concepts of senescence and genomic instability as it relates to the aforementioned processes. Lastly, we explore how “energy sensing” longevity pathways that appear to modulate endothelial function with advancing age.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Methodological Considerations for Studying Endothelial Aging,"In this review, we will focus first on in vivo or ex vivo studies that directly examined endothelial cells. However, a major obstacle to our understanding of the events that lead to endothelial dysfunction with advancing age is access to pure primary endothelial cells from humans or rodent models. Next, we will consider studies utilizing whole artery homogenates with the understanding that protein expression in whole arteries is strongly biased toward the smooth muscle cell component rather than the endothelium.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Focus on Endothelial-Dependent Dilation (EDD),"Lastly, this review will explore the mechanisms of endothelium dysfunction defined as reductions in EDD assessed by the response to pharmacological or physiological stimuli. Our focus on EDD is because a majority of studies that perform mechanistic studies utilize this marker. It is not to say measures of angiogenesis, permeability, fibrinolysis or other markers of endothelial function are not as important; indeed these are critical functions which are vastly understudied, but at the present time, the mechanisms leading to impairments in these functions in aged endothelial cells or their direct relation to CVD development are not clearly understood.",Journal of Molecular and Cellular Cardiology,Endothelial Aging,2015
Age-Associated Vascular Endothelial Dysfunction,"Aging is associated with endothelial dysfunction in both men and women, even without overt clinical disease. Most studies quantify this dysfunction using endothelial-dependent dilation (EDD), but aging also impairs other endothelial functions such as fibrinolysis, permeability, and angiogenesis. Animal models replicate these human findings, demonstrating age-related reductions in EDD, fibrinolytic capacity, endothelial barrier integrity, and angiogenic responses. These conserved impairments suggest that endothelial dysfunction is a fundamental biological feature of vascular aging.",Journal of Molecular and Cellular Cardiology,Endothelial Aging Mechanisms,2015
Modulators of Endothelial Dysfunction in Older Adults,"In older adults, traditional cardiovascular risk factors—including elevated blood pressure, LDL cholesterol, blood glucose, and sodium intake—exacerbate endothelial dysfunction as measured by EDD. Additionally, non-traditional markers such as increased white blood cell count and elevated plasma norepinephrine also worsen endothelial function. Many of these modulators act by increasing oxidative stress and inflammation within the endothelium, two central processes that drive age-related endothelial decline.",Journal of Molecular and Cellular Cardiology,Risk Factors in Endothelial Aging,2015
Role of Postprandial Metabolic Stress,"With advancing age, the clearance of postprandial glucose and lipids becomes significantly impaired, even in otherwise healthy adults. This delayed clearance leads to repeated, prolonged metabolic exposures after meals, which are known to acutely impair endothelial function in younger adults. Endothelial cell culture studies show that physiological elevations in glucose and fatty acids trigger oxidative stress, inflammation, and alterations in energy-sensitive signaling pathways. Chronic exposure to these postprandial insults may create a persistent pro-oxidant, pro-inflammatory endothelial environment, contributing to sustained vascular aging. Although this model is strongly supported by associative evidence, it remains a hypothesis requiring direct experimental validation.",Journal of Molecular and Cellular Cardiology,Postprandial Stress and Endothelial Aging,2015
"Aging, Oxidative Stress and Endothelial Dysfunction","Aging—particularly when accompanied by low physical activity—is considered a state of chronic systemic oxidative stress, largely due to increased production of reactive oxygen species (ROS) relative to antioxidant defenses. A major mechanism driving age-related reductions in endothelial-dependent dilation (EDD) and nitric oxide (NO) bioavailability is the accumulation of vascular oxidative stress. Superoxide (O2−), produced by enzymes such as NADPH oxidase, xanthine oxidase, cytochrome P450, and uncoupled eNOS, rapidly reacts with NO to form peroxynitrite (ONOO−), which nitrates proteins and reduces NO signaling. Aging endothelial cells show elevated nitrotyrosine, lipid peroxidation markers (4-HNE, MDA), and glutathionylation, all reflecting oxidative stress-driven damage.

Acute antioxidant administration (vitamin C, SOD mimetics) improves NO-mediated dilation in older adults, demonstrating the direct role of O2− in suppressing endothelial function. NADPH oxidase activation and eNOS uncoupling—often due to BH4 deficiency—are major ROS sources in aging; restoring BH4 improves endothelial function. Mitochondrial ROS also contribute indirectly, mainly as hydrogen peroxide (H2O2), and interventions targeting mitochondrial oxidative stress (e.g., MitoQ or deletion of p66Shc) improve vascular function. Evidence for reduced antioxidant defenses with age is mixed: while some antioxidant enzymes decline in aged arteries, others remain unchanged. Importantly, normal physiological ROS levels are required for signaling and vasodilation, but chronic excess ROS disrupts the balance, leading to impaired NO signaling and endothelial dysfunction.",Journal of Molecular and Cellular Cardiology,Oxidative Stress and Endothelial Aging,2015
Structural Changes in Aging Arteries,"Arterial walls stiffen with age. The most consistent and well-reported changes are luminal enlargement with wall thickening (remodeling) and a reduction of elastic properties (stiffening) at the level of large elastic arteries, namely arteriosclerosis. Interestingly, this aging process in the arterial tree is heterogeneous, with distal muscular arteries not exhibiting these stiffening changes, which is different from the atherosclerotic process.",Circulation Journal,Arterial Stiffness,2010
Medial Degeneration and Elastin Fatigue,"The media of large arteries is mainly composed of an integrated assembly of vascular smooth muscle cells and elastic and collagen fibers, comprising functional musculoelastic sheets. Cross-links between extracellular matrix components and smooth muscle cell–matrix interactions confer adequate mechanical properties. The principal structural change with aging is medial degeneration, which leads to progressive stiffening of the large elastic arteries. Longstanding arterial pulsation in the central artery has a direct effect on the structural matrix proteins, collagen and elastin in the arterial wall, disrupting muscular attachments and causing elastin fibers to fatigue and fracture.",Circulation Journal,Arterial Stiffness,2010
AGE Accumulation and Calcium Deposition,"Accumulation of advanced glycation endproducts (AGE) on the proteins alters their physical properties and causes stiffness of the fibers. Another major change in the arterial wall is calcium deposition. The calcium content of the arterial wall increases with age, particularly after the 5th decade, which might also contribute to the loss of arterial distensibility. Though the structure of the peripheral muscular arteries/ arterioles is only minimally affected by aging, impaired vasomotor function associated with endothelial dysfunction leads to thickening of the intima–media layer, and can contribute to peripheral resistance.",Circulation Journal,Arterial Stiffness,2010
Endothelial Dysfunction and Smooth Muscle Cell Stiffness,"Endothelial dysfunction is characteristic of arterial aging, triggered by a decrease in antioxidative capacity and an increase in oxidative stress. Moreover, Qiu et al recently suggested that, as well as changes in the extracellular matrix, increased stiffness of the vascular smooth muscle cells themselves also mediates aging-associated vascular stiffness by increasing adhesion molecule expression. Finally, various comorbid risk factors, which are invariably highly prevalent among the elderly, accelerate the atherosclerotic process. Age-related changes in the arterial structure may create a vicious cycle of arterial stiffness. Wide pulse pressure is transmitted to other arteries, such as the carotid, which further aggravates the process of large artery remodeling to reduce wall stress, leading to intima–media thickening. The causes of vascular aging are summarized in Figure 1.",Circulation Journal,Arterial Stiffness,2010
Non-Invasive Measures of Arterial Stiffness,"Several non-invasive methods are currently used to assess vascular stiffness. Pulse wave velocity (PWV) and the augmentation index (AI) are the 2 major non-invasive methods of assessing arterial stiffness. PWV reflects the elasticity of the segmental artery. Cardiac contraction generates a pulse wave, which is propongated distally to the extremities. PWV is calculated as the distance traveled by the pulse wave divided by the time taken to travel the distance. Increased arterial stiffness results in increased speed of the pulse wave in the artery. PWV can be measured in any arterial segment between 2 regions. Carotid–femoral PWV is considered as the gold standard for assessing central arterial stiffness, and is an independent predictor of cardiovascular mortality and morbidity in elderly subjects, as well as in the general population.",Circulation Journal,Arterial Stiffness,2010
Carotid–Femoral and Brachial–Ankle PWV,"However, a relatively high level of skill combined with the need to expose the inguinal region are barriers to its wide clinical use. In contrast, the brachial–ankle PWV is easy to measure and has potential for screening purposes. In small studies, brachial–ankle PWV was an independent predictor of cardiovascular death and events in elderly community dwelling people and in patients with coronary artery disease. Though brachial–ankle PWV reflects not only elastic central arterial stiffness but also muscular peripheral arterial stiffness, it shows a close correlation with aortic or carotid–femoral PWV. The main limitation of PWV interpretation is that the PWV is significantly influenced by blood pressure (BP). Because increased BP increases the arterial wall tension, thus adding functional stiffening of the arteries, BP becomes a confounding variable when comparing the degree of structural arterial stiffening.",Circulation Journal,Arterial Stiffness,2010
Stiffness Index β and CAVI,"The stiffness index, β, can be derived with minimal influence of BP; however, β is a marker of regional and not segmental arterial stiffness. Because β is estimated at the same site of PWV measurement, but with an adjustment for BP, it cannot be used to obtain segmental arterial stiffness when the BP differs between the proximal and distal parts of the measured segment. Recently, a new arterial stiffness index, the cardio-ankle vascular index (CAVI), has been suggested as a marker of arterial stiffness independent of BP. CAVI is derived by measuring PWV and then adjusted by the stiffness parameter, β, thus enabling BP-independent measurement of arterial stiffness. Early results show good reproducibility, BP-independence, and risk predictability; however, further studies are warranted for wide acceptance.",Circulation Journal,Arterial Stiffness,2010
Augmentation Index and Wave Reflection,"AI reflects stiffness of the systemic arterial tree. The arterial pressure wave is a composition of the forward pressure wave arising from left ventricular ejection and the backward (reflected) pressure wave. Backward pressure occurs mainly as a consequence of wave reflection and wave reflection is mainly created by impedance mismatch at the branch point of the arterial system and very small resistance arterioles. The interaction between the forward and reflected pulse waves is assessed by pulse wave analysis and expressed as the AI. The pressure waveform from the radial artery is recorded non-invasively with applanation tonometry, and then the aortic pressure waveform is derived by a generalized transfer function. Therefore, central arterial stiffness and peripheral reflectance are important determinants of the AI.",Circulation Journal,Arterial Stiffness,2010
Physiological Consequences of Increased AI,"Normally, the reflected wave arrives at the aortic root during diastole and augments the coronary circulation. Increased stiffness of the aorta increases the aortic PWV and the reflected wave returns earlier to the aortic root during late systole when the ventricle is still ejecting blood, adding the reflected wave to the forward wave and augmenting the central systolic pressure. Increased central systolic pressure and pulse pressure result in an increase in arterial wall stress, progression of atherosclerosis and the development of left ventricular hypertrophy because of increased left ventricular afterload. Early return of the reflected wave also causes a decrease in central diastolic pressure, resulting in reduced coronary artery perfusion pressure. AI is also influenced by BP and, importantly, by heart rate. Though brachial systolic BP is not influenced by heart rate, central BP is significantly increased by heart rate reduction, which can be a confounding factor.",Circulation Journal,Arterial Stiffness,2010
Age-Related Changes in PWV and AI,"A number of studies have investigated the effects of age on aortic PWV and AI. Most studies suggest a linear, age-related increase in both PWV and AI; however, central AI and aortic PWV do not always show a linear correlation, but rather they are differentially affected by aging. Data from a large cohort of healthy individuals in the Anglo-Cardiff Collaborative Trial (ACCT) showed these different patterns in age-related changes between central AI and aortic PWV. Changes in AI were more prominent in younger individuals (<50 years), whereas the changes in aortic PWV were more marked in those older than 50 years. Therefore, central AI might be a more sensitive marker of arterial aging in younger individuals, and aortic PWV is more sensitive in those over 50 years of age.",Circulation Journal,Arterial Stiffness,2010
Differential Wave Reflection Mechanisms,"Similarly, Mitchell et al reported that central AI changes less with age in older individuals and actually declines after the age of 60 years. McEniery et al suggested a hypothesis for those discrepant responses. In younger individuals, the rise in augmented pressure is because of an increase in the magnitude of wave reflection rather than increased wave velocity, whereas in older individuals, the rise in augmented pressure is driven by an earlier return of the reflected wave and a less compliant aorta rather than predominant changes in the magnitude of wave reflection. Age-related dilatation usually occurs in large arteriosclerotic arteries, thus increasing PWV, but minimally affecting impedance.",Circulation Journal,Arterial Stiffness,2010
Sex Differences and Menopause Effects,"Though it is still controversial, there is a reported greater increase in aortic stiffness with age among women, particularly with the menopause. In particular, AI is higher in women than in men by approximately 7% after menopause, in part because of women’s shorter height and therefore closer physical proximity between the heart and reflecting sites. Another report found a steeper age-related rise in pulse pressure among women after menopause. Central PWV also increases more rapidly with age in women than in men, crossing over around the age of 45 years, whereas there is no significant difference across the sexes in femoral PWV.",Circulation Journal,Arterial Stiffness,2010
Korean Arterial Aging Study (KAAS) Findings,"Recently, a Korean vascular research working group performed the Korean Arterial Aging Study (KAAS) to determine the effect of aging on the stiffness parameters such as AI and PWV in apparently healthy individuals with or without CV risk factors. That study enrolled 1,750 subjects aged 17–87 years (mean, 46.5 years) who were apparently healthy and not taking any medication for hypertension, diabetes or dyslipidemia. We observed the same tendency in the aging and sex effects. In younger ages (age quartile 1 and 2), both central and peripheral pulse pressures were lower in women than in men, but around 45 years, the pulse pressures of the women sharply rose, so that in their 50s and over, women had higher pulse pressures than men. The same tendency of a rapid increase was observed in central pulse wave velocity.",Circulation Journal,Arterial Stiffness,2010
Accelerated Arterial Aging with Comorbidities,"Once, the aging-associated changes in arterial structural and functional changes were thought to be part of normative aging, but this concept changed when data emerged showing that these changes are accelerated with coexistent cardiovascular disease. For example, patients with hypertension, metabolic syndrome, or diabetes exhibit increased carotid wall thickness and stiffness even after adjusting for age. And this ‘accelerated’ arterial aging is well confirmed to be a risk.",Circulation Journal,Arterial Stiffness,2010
Hemodynamic Consequences of Arterial Stiffness,"Stiffening of large arteries results in various adverse hemodynamic consequences. Aging-associated arterial stiffness leads to a rise in pulse pressure and isolated systolic hypertension. Following aging, systolic BP increases linearly, while diastolic BP increases until approximately age 50 then declines. Mean arterial pressure increases until approximately age 50 then reaches a plateau, while pulse pressure is constant until approximately age 50 and then increases. In individuals over 60 years of age, isolated systolic hypertension affects more than 50%, and results in excess morbidity and mortality.",Circulation Journal,Arterial Stiffness,2010
Microvascular Damage and Stroke Risk,"Increased pulsatile pressure and flow stresses extend to the vulnerable microcirculation of vasodilated organs, such as the brain and kidneys, and can predispose to cerebral lacunar infarction and albuminuria. Indeed, isolated systolic hypertension caused by widening of pulse pressure is the most common form of hypertension among the elderly, and carries a 3-fold increase in the risk of stroke. Moreover, arterial stiffness correlates with cognitive function in the very elderly over 80s.",Circulation Journal,Arterial Stiffness,2010
Cardiac Effects: Hypertrophy and Ischemia,"Elevated systolic BP because of large arterial stiffening also promotes left ventricular hypertrophy and ventricular stiffening, thus leading to diastolic dysfunction and heart failure, the incidence of which doubles among isolated systolic hypertensive patients. In addition, low diastolic pressure reduces coronary blood flow, aggravating the situation and predisposing to ischemia.",Circulation Journal,Arterial Stiffness,2010
Non-Pharmacological and Pharmacological Interventions,"There are a large number of studies reporting changes in arterial stiffness and wave reflection after various interventions, either non-pharmacological or pharmacological. Non-pharmacological treatments that are able to reduce arterial stiffness include exercise training, weight loss, and various dietary modifications, including low-salt diet, moderate alcohol consumption, garlic powder, α-linoleic acid, dark chocolate, and fish oil. Pharmacological treatments include (1) antihypertensive treatment such as diuretics, α-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin-receptor blockers (ARB), and calcium-channel antagonists; (2) treatment of congestive heart failure, such as with ACE inhibitors, nitrates, and aldosterone antagonists; (3) lipid-lowering agents such as statins; (4) antidiabetic agents, such as thiazolidinediones; and (5) AGE breakers.",Circulation Journal,Arterial Aging Management,2010
RAAS Inhibition and Limitations of Beta-Blockers,"It is still debated whether the reduction in arterial stiffness after antihypertensive treatment is only attributable to BP lowering, or if additional BP-independent effects are involved. However, renin–angiotensin–aldosterone system (RAAS) inhibitors, such as the ACE inhibitors and ARBs, have been widely suggested to have a BP-independent effect on arterial stiffness. Those observations are relevant to the widely reported fibrosis-inducing effect of the RAAS. In contrast, β-blockers have been suggested to be inferior to other classes of drugs in reducing vascular stiffness because β-blockers devoid of vasodilating properties are less effective for reducing central pulse pressure and AI than other antihypertensive drugs. Non-vasodilating β-blockers may increase vasoconstriction and facilitate the return of wave reflection in late systole rather than in diastole, thereby increasing the AI.",Circulation Journal,Arterial Aging Management,2010
ASCOT and CAFE Trials: Central vs Brachial BP,"The roles of arterial stiffness and wave reflections in the mechanisms of central BP reduction in subjects under antihypertensive drug therapy might be best demonstrated in the ASCOT and CAFE studies. In both trials, an ACE inhibitor, perindopril, was used with a calcium-channel blocker, amlodipine, and compared with an association of diuretic and β-blockers. In the ASCOT study, amlodipine-based treatments proved to be more effective than atenolol-based treatments for reducing cardiovascular events. The CAFE study showed that the reduction in central systolic BP and pulse pressure was higher in the amlodipine- than in the atenolol-based treatment group, despite similar reductions in both pressures at the brachial level.",Circulation Journal,Arterial Aging Management,2010
REASON Study Findings,"The results of the REASON study (Preterax in Regression of Arterial Stiffness in a Controlled Double-Blind Study), which compared the antihypertensive effects of the very-low-dose combination of indapamide (0.625 mg) and perindopril (2 mg) with the β-blocking agent atenolol (50 mg), showed a superior effect of the ACE inhibitor, perindopril, to atenolol in reducing both aortic PWV and wave reflections. In the primary results of 1-year follow-up for the same diastolic BP reduction, perindopril/indapamide lowered brachial and carotid systolic BP and thus pulse pressure more than atenolol. This difference was significantly more pronounced for the carotid artery than for the brachial artery. Also, the wave-reflection changes resulting from the reduction of peripheral reflection coefficients were the principal factor to consider in explaining the difference between atenolol and perindopril/indapamide with regard to systolic BP reduction.",Circulation Journal,Arterial Aging Management,2010
Olmesartan Combination Therapy,"Finally, the effect on central BP reduction by the ARB, olmesartan, in combination with either a calcium-channel blocker or a diuretic was compared in hypertensive patients (mean age, 68.4 years). Though brachial systolic BP was similar between the 2 groups, the reduction in central systolic BP in the olmesartan/calcium-channel blocker group was significantly greater than in the olmesartan/diuretic group. In addition, aortic PWV was significantly more reduced in the olmesartan/calcium-channel blocker group. Again, these results suggest that the regulating capacity of arterial stiffness and wave reflections might differ among antihypertensive patients.",Circulation Journal,Arterial Aging Management,2010
Okinawan Longevity and Cardiovascular Health,"Okinawans are the longest-living population out of any country or state in the world, according to reports of WHO and Japan Ministry of Health (Koseishou). Okinawans seem to have delayed the aging process and minimized debilitating diseases that accompany the elder years, especially coronary heart disease (CHD). The three leading killers in the west, CHD, stroke and cancer, occur in Okinawa with the lowest frequency in the world. For example, out of 100 000 inhabitants, an average of only 18 die from CHD in a typical year, compared to 20 in Japan and 100 people in the USA. The Okinawan lifestyle provides real, scientifically verifiable reasons why these people are so incredibly robust and healthy so far into their senior years.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
"Homocysteine, Cholesterol, and the Okinawan Paradox","In 1995, when medical researchers were just beginning to take note of homocysteine, it was conservatively estimated to cause 10% of all coronary heart disease deaths in the west. Okinawans have among the lowest homocysteine levels in the world. Serum cholesterol levels between centenarians and septuagenarians in Okinawa have been calculated. The centenarian level is 166.2 mg/dL of total cholesterol and is lower than the septuagenarian level. The centenarian level of LDL cholesterol is 102.4 mg/dL and is also lower than the septuagenarian level. However, the diet has recently changed greatly, to include fast fatty foods and processed foods, so that the young Okinawan’s homocysteine and cholesterol levels have risen. Compared to the Japanese norm, young Okinawans now have higher than average risk for CHD, while older Okinawans have lower than average risk. We think of this reversal as the Okinawan paradox.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
Arterial Aging Patterns in Okinawan Centenarians,"According to physical and biochemical evaluations for arteriosclerotic conditions for healthy Okinawan centenarians, pulse wave velocity data are generally in the lower range, below 10 m/s, which is considered to be a relatively young level. However, arteriosclerotic index shows generally high levels. This discrepancy may be because of the time lag of the development of arteriosclerosis. In centenarians, the development of peripheral arteriosclerosis is slower than that of arteriosclerosis of the aorta. This phenomenon suggests that the arterial changes in healthy centenarians are not as a result of atheromatous change, but from physiological aging mainly based on fibrosis of arteries, possibly because of higher age-related shear pressure.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
"Blood Pressure, Salt Intake, and Cardiac Pathology","High blood pressure was recognized in only 1.5% of apparently healthy centenarian subjects. Most never developed a taste for salt and, partly as a result, their heart disease and stroke rates have traditionally been much lower than the Japanese average. Most subjects who have segmental deviation on electrocardiogram have rarely experienced obvious episodes of chest pain or discomfort. This suggests that centenarians might not have developed coronary ischemia, but instead there are degenerative damages of the myocardium. Pathological changes include amyloid accumulation and deposition in the regional myocardium in more than 50% of centenarians.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
"Cardiac Function, Aging, and Longevity Factors","Cardiac functional disturbances as a result of valvular regurgitation and arrhythmias, secondary to diseased myocardium and valvular calcification, occur with increasing frequency with age. Generally, ventricular pump function is well compensated, but heart failure can be triggered more easily with extreme age. Much of centenarians’ long life expectancy can be attributed to a minimization of cardiovascular diseases, especially through low cholesterol levels and minimal hypertension. Indeed, it would be difficult to age successfully without some degree of protection against cardiovascular diseases.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
Low Risk of Hormone-Dependent Cancers,"Okinawans have an extremely low-risk for hormone-dependent cancers, including cancer of the breast, ovary, prostate and colon. The profound differences in hormone-dependent cancer death rates can be observed between several long-lived countries. Compared to the USA, they have 90% less chance of breast cancer, 80% less chance of prostate cancer and less than 50% chance of ovarian and colon cancer. We believe this is likely due to several factors including lifelong low caloric intake, low BMI, high intake of plant foods especially green-yellow vegetables and soy products. According to our research, caloric intakes in Okinawan centenarians are 1407 calories for men and 1096 calories for women. The most common cooking oil used by Okinawans’ stir-fry cooking at home is a canola-soy oil blend that contains mainly monounsaturated fat with a significant portion of omega-3 fatty acids. Low-calorie diets and the high intake of monounsaturated fat result in less production of cell-damaging free radicals and less exposure to mitogenic hormones.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
Antioxidant Protection and Free Radical Reduction,"We measured the amount of one of the main free radical byproducts, called lipid peroxide. The centenarians’ level of 1.67 nmol/mL was significantly lower than the septuagenarians’ level of 3.40. This is compelling evidence that they suffer less free radical-induced damage. According to our study, Okinawan centenarians’ superoxide dismutase (SOD) activity is 1.41 U/mL, which is almost the same level as octogenarians. Other anti-oxidant defenses come from dietary anti-oxidants, such as vitamins C and E, carotenoids and flavonoids. Contrary to expectations, plasma and intracellular α-tocopherol levels of centenarians are not significantly higher compared to septuagenarian levels, possibly reflecting lower caloric intake or lower intake of vitamin E-rich foods. Okinawan consumption of flavonoids is six times higher than Japanese–Canadians. Plant sterols of flavonoids taken from soy and legumes may offer protection against arteriosclerosis and hormone effects. The carotenoid lycopene is thought to be a more powerful anti-oxidant than vitamin E. Okinawan diets have been found to have the highest lycopene content in Japan, likely from the reddish-purple Okinawan sweet potato.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
Bone Health and Collagen Metabolism,"Okinawans have strong bone density compared with mainland Japanese. They have a 25% reduced risk of hip fracture versus mainland Japanese and about 50% less hip fractures than Americans. Bone mineral density measured in 50- to 60-year-olds showed values of 485 mg/cm2 for men and 375 mg/cm2 for women, higher than age-matched mainland Japanese. Several factors likely play a role including high calcium intake from foods and natural drinking water, high flavonoid intake, high vitamin D from sunlight exposure, and increased physical activity at older ages. Serum hydroxyproline levels in centenarians were 67.1 nmol/mL for men and 72.1 for women, remarkably high compared to septuagenarians. Hydroxyproline is a key component of collagen fibers in arterial walls and bone matrix, and is abundant in soy products.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
Sex Hormones and Healthy Aging,"We studied several major hormones including DHEA, thyroid hormones, cortisol and sex steroids in Okinawan elders. The sex hormones in Okinawans may decline more slowly with age. Higher levels of DHEA, testosterone and oestrogen may indicate a physiologically younger sex hormone axis. High oestrogen may help maintain vascular and bone health, while the deleterious effects of endogenous oestrogen on hormone-dependent cancers may be offset by very high flavonoid intake, which can block oestrogen activity in vitro. This interaction requires further study. Psycho-spiritual practices may also contribute to reduced stress-induced radical oxidant damage, although this topic requires additional investigation.",Asia Pacific Journal of Clinical Nutrition,Okinawan Longevity,2001
Overview of Structural and Functional Changes in Arterial Ageing,"Complex structural and functional changes occur in the arterial system with advancing age. The aged artery is characterized by changes in microRNA expression patterns, autophagy, smooth muscle cell migration and proliferation, and arterial calcification with progressively increased mechanical vessel rigidity and stiffness. With age the vascular smooth muscle cells modify their phenotype from contractile to ‘synthetic’ determining the development of intimal thickening as early as the second decade of life as an adaptive response to forces acting on the arterial wall.",Journal of Internal Medicine,Arterial Ageing,2017
Intimal Thickening and Atherosclerosis Progression,The increased permeability observed in intimal thickening could represent the substrate on which low-level atherosclerotic stimuli can promote the development of advanced atherosclerotic lesions. In elderly patients the atherosclerotic plaques tend to be larger with increased vascular stenosis. In these plaques there is a progressive accumulation of both lipids and collagen and a decrease of inflammation. Similarly the plaques from elderly patients show more calcification as compared with those from younger patients. The coronary artery calcium score is a well-established marker of adverse cardiovascular outcomes.,Journal of Internal Medicine,Arterial Ageing,2017
Plaque Calcification and Mechanical Instability,The presence of diffuse calcification in a severely stenotic segment probably induces changes in mechanical properties and shear stress of the arterial wall favouring the rupture of a vulnerable lesion in a less stenotic adjacent segment. Oxidative stress and inflammation appear to be the two primary pathological mechanisms of ageing-related endothelial dysfunction even in the absence of clinical disease.,Journal of Internal Medicine,Arterial Ageing,2017
Towards Prevention and Reversal of Vascular Ageing,Arterial ageing is no longer considered an inexorable process. Only a better understanding of the link between ageing and vascular dysfunction can lead to significant advances in both preventative and therapeutic treatments with the aim that in the future vascular ageing may be halted or even reversed.,Journal of Internal Medicine,Arterial Ageing,2017
Atherosclerosis and Age-Related Metabolic Changes,"Atherosclerosis, with its clinical complications, is the leading cause of death in Western nations. It is a chronic silent inflammatory disease, the clinical symptoms of which become manifest in adulthood and usually involve plaque rupture and thrombosis. The histological composition of the plaque and the biological behaviour of the cells involved vary in relation to the presence of parietal and extramural factors, which are affected by the main atherosclerosis risk factors. Ageing determines irreversible changes at the metabolic and physiological levels. These changes are strongly affected by energy metabolism, that is, by caloric restriction, and present in arterial wall components (smooth muscle cells and inflammatory cells) and persist as independent risk factors for atherosclerosis even in the absence of all other known risk factors.",Journal of Internal Medicine,Arterial Ageing,2017
Molecular and Structural Alterations in Ageing Arteries,"Modifications of microRNAs, signalling pathways or autophagy correlated with ageing determine various structural and physiological changes of the arterial wall that predispose it to atherosclerosis. In particular, inversion of the elastin/collagen ratio and the nonenzymatic glycosylation of some proteins present in the arterial wall, such as collagen, capable of crosslinking adjacent proteins, increase mechanical vessel rigidity and stiffness. The reduction in elasticity and distensibility of the arterial wall determines a reduction in compliance leading to increased systolic and decreased diastolic blood pressure. Ageing is also accompanied by an increase in thickness and a decrease in the cellularity of the tunica media. These structural changes are associated with an increase in collagen and a corresponding decrease in elastin content.",Journal of Internal Medicine,Arterial Ageing,2017
Extracellular Matrix Changes and Elastin Degradation,"Collagen tends to accumulate in ‘gaps’ interposed between two adjacent lamellar units. Ultrastructural studies have demonstrated that with age aortic elastin changes its structure and appears as ‘encrusted’ with an insoluble, collagenase-resistant, fibrillar protein. Advancing age, independent of other risk factors, leads to important biochemical, histological and structural vascular alterations that determine a proatherogenic environment upon which major cardiovascular risk factors act to favour the progression and destabilization of an atherosclerotic stable plaque.",Journal of Internal Medicine,Arterial Ageing,2017
"Plaque Instability, Immune Activation and Inflammation","Plaque stability is influenced not only by systemic factors, that is, the ‘major’ atherosclerotic risk factors (cigarette smoking, hypertension, high serum cholesterol and its fractions, low levels of HDL and diabetes mellitus), but also by local factors such as modifications of the arterial wall inducing progression from stable to unstable plaque. Ageing influences the local inflammatory response and the histological plaque composition; the unstable plaque is characterized by an active chronic inflammation different from that observed in stable lesions. Macrophages and T cells become activated in vulnerable plaques, producing inflammatory cytokines and lytic enzymes, such as metalloproteases, which predispose towards plaque rupture by digesting and reducing the thickness of the fibrous cap. The Th1 subpopulation of T cells is detected in the plaque and affects its stability through macrophage activation, smooth muscle cell growth and extracellular matrix synthesis.",Journal of Internal Medicine,Arterial Ageing,2017
Inflammatory Regulation and Hemodynamic Stress,"The balance between inflammatory and anti-inflammatory activity is influenced by the recruitment of inflammatory cells as well as the haemodynamic stress that favours the expression of adhesion molecules by cytokines produced in the plaque. Moreover, several molecules produced during lipid peroxidation are able to stimulate both the inflammatory processes and the defensive reactions, binding to nuclear receptors regulating genes involved in inflammation.",Journal of Internal Medicine,Arterial Ageing,2017
Endothelial Function and Nitric Oxide Homeostasis,"The endothelium is important in maintaining vascular homoeostasis and is involved in many physiological functions including regulation of blood pressure, promotion of angiogenesis and control of the coagulation process. The function and health of the vascular network are regulated by the vascular endothelium releasing molecules that act in an autocrine and paracrine manner. In addition to the redox balance, nitric oxide (NO) is the most important mediator of normal endothelial function, through its powerful vasodilator action. NO is synthesized by the endothelial NO synthase (eNOS or NOS III) via the action of different neurohumoral mediators such as acetylcholine and circulating hormones. Reduction in the bioavailability or increased degradation of NO is a marker of endothelial dysfunction associated with impaired endothelium-dependent dilatation (EDD), which predicts future cardiovascular events.",Journal of Internal Medicine,Endothelial Dysfunction,2017
Balance of Vasoactive and Inflammatory Signals,"A healthy vascular endothelium depends on the balance between pro and antioxidants, vasodilators and vasoconstrictors, pro and anti-inflammatory molecules and pro and antithrombotic signals. Endothelial dysfunction occurs when this balance is perturbed, inducing pro-oxidant, vasoconstrictor, proinflammatory and prothrombotic effects. Ageing represents the strongest independent correlate of EDD, as demonstrated by the Framingham Heart Study. Elderly men and women also showed endothelial dysfunction in the absence of clinical disease. Several studies have demonstrated that oxidative stress and inflammation are the most important pathological processes that determine endothelial dysfunction in healthy older adults and in rodent models.",Journal of Internal Medicine,Endothelial Dysfunction,2017
"NO Decline, eNOS Regulation, and Reactive Nitrogen Species","In experimental animals and humans, the age-related reduction in EDD is due to the reduced vascular NO bioavailability caused by reduced expression of eNOS and its downstream effects. However, the precise mechanism underlying the age-associated decrease in NO remains unclear. Prostanoid vasodilators such as prostacyclin are also reduced with age, whilst the reduction of NO induces vasoconstriction mediated by endothelin 1 (ET-1). NO reacts with tyrosine, resulting in the formation of nitrotyrosine, which is increased in abundance in aged arteries. During oxidative stress, elevated reactive oxygen species (ROS) and particularly superoxide anion react with NO resulting in the production of peroxynitrite (ONOO-), a very reactive and toxic product. Peroxynitrite further inactivates nearby proteins irreversibly in ageing endothelial cells.",Journal of Internal Medicine,Endothelial Dysfunction,2017
"Redox Signaling, Apoptosis Regulation, and eNOS Suppression","At the molecular level, NO can induce the S-nitrosylation of reactive cysteines, resulting in regulation or inactivation of clotting factor XII, AP-1 and CD95L, transglutaminases or mitochondrial adenine nucleotide translocator. This redox-based signalling mechanism regulates the switch between apoptosis and necrosis, which plays a relevant role in endothelial dysfunction. Changes in eNOS regulatory proteins such as caveolin-1, pAkt and heat shock protein 90 contribute to decreased eNOS activity in aged endothelial cells. Oxidative stress-associated suppression of NO and EDD in old arteries is supported by antioxidant supplementation such as superoxide dismutase and vitamin C, which improve NO bioavailability and endothelial function. Decreased eNOS expression is also partly due to L-arginine degradation by arginase II, whose expression increases with advanced age.",Journal of Internal Medicine,Endothelial Dysfunction,2017
"Chronic Inflammation, NF-κB Activation and Senescence","Plasma concentrations of inflammatory proteins increase with age in healthy individuals, including interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). Their upregulation in advanced ageing underlies a low-grade, chronic, systemic proinflammatory state correlated with age-associated diseases such as diabetes and obesity. This proinflammatory condition is tightly linked to nuclear factor-kappa B (NF-κB) signalling in endothelial dysfunction, which both reduces cytokine levels and enhances EDD. IκB kinase beta is activated in response to inflammatory stimuli or ROS, phosphorylating IκB-α and permitting NF-κB nuclear translocation and activation of proinflammatory cytokine genes. Endothelial cell senescence is thus associated with increased oxidative stress and inflammation, defined as the senescence-associated secretory phenotype.",Journal of Internal Medicine,Endothelial Dysfunction,2017
Intimal Thickening and Early Structural Changes,"With age, vascular SMCs undergo functional changes that alter the normal structure of the vessel wall predisposing it to the development of atherosclerosis. From the second decade of life, intimal thickening (IT) develops as an adaptive response to forces acting on the arterial wall. Such thickening comprises various layers of SMCs separated by thin elastic fibres and collagen. Typically IT has a widespread and concentric development in the aorta, whilst appearing eccentric in the coronary arteries. Small plasma-like deposits of alcianophilic material, corresponding to glycosaminoglycans, can be observed within the thickened intima. Electron microscopy has shown that such material consists of granules with an irregular shape associated with thin filaments that appear in connection with the cell membranes of endothelial cells and SMCs. IT represents a preatherosclerotic lesion because there is a topographic correspondence between the localisation of IT and the presence of atherosclerotic plaques.",Journal of Internal Medicine,Vascular Smooth Muscle Cells,2017
Increased Permeability and Atherosclerosis Progression,"The increased permeability observed during IT, evidenced by the presence in the subendothelial space of cholesterol and phospholipids as early as the third decade of life, could be the substrate on which low-level atherosclerotic stimuli (mild hypercholesterolaemia and/or mild hypertension) promote the development of advanced atherosclerotic lesions. Aged rabbits, compared to young animals, showed more severe atherosclerotic lesions when fed a long-term hyperlipaemic diet containing a low dose of cholesterol. The increased susceptibility to atherosclerosis with age is also related to changes in the mechanisms that regulate vascular SMC proliferation, including the synthesis and release of various growth factors.",Journal of Internal Medicine,Vascular Smooth Muscle Cells,2017
"SMC Proliferation, Growth Factors, and Loss of Inhibition","Stemerman et al. demonstrated that after removal of aortic endothelium vascular SMCs incorporate more 3H-thymidine in older rats compared to younger animals. SMCs cultured from aged rats showed a higher proliferative rate than cells from young rats, associated with a higher increase in α-SM actin protein levels and mRNA expression. This correlates with increased platelet-derived growth factor-like activity and reduced heparin-like activity. By contrast, Orlandi et al. and McCaffrey and Falcone demonstrated an age-related modification of the antiproliferative response to transforming growth factor beta 1 (TGFβ-1) in vascular SMCs, which failed to respond to the autocrine growth inhibitory effects of this factor, leading to increased proliferation.",Journal of Internal Medicine,Vascular Smooth Muscle Cells,2017
Phenotypic Switching and Inflammatory Activation,"Local environmental factors determine in adult vascular SMCs reversible changes in their phenotype, from ‘contractile’ to ‘synthetic’. The phenotypic modulation often includes all mesenchymal lineages, that is, osteoblasts, chondrocytes and adipocytes. Deregulated signalling of the TGF-β superfamily has been involved in these changes. With ageing, SMCs also show modification of the production of NO under specific stimulation. SMCs from old rats present markedly enhanced inducible NOS (iNOS) activity compared to newborn rats. Aged SMCs show greater expression of ICAM-1 in response to cytokines and higher production of angiotensinogen, which likely contributes to the regulation of vascular tone via increased local renin–angiotensin system activity.",Journal of Internal Medicine,Vascular Smooth Muscle Cells,2017
"Shear Stress, Aging, and Atherosclerotic Plaque Development","The development of the atherosclerotic plaque is influenced by the presence of mechanical forces exerted on the vessel wall or on the endothelial surface, such as shear stress. Mechanical forces regulate the cellular and molecular composition of plaques as well as plaque growth. Ageing is associated with a reduction in endothelial shear stress, with subsequent development of atherosclerotic plaques. High shear stress favours the maintenance of the physiological properties of the endothelial barrier, including anticoagulant, anti-inflammatory and antioxidant properties. By contrast, low shear causes endothelial cell death and increased platelet and macrophage adhesion to the arterial wall, favouring the formation of atherosclerotic plaques through mechanisms targeting the MAPK and NFκB pathways. These effects are likely modulated by the expression of specific noncoding RNAs as well as mRNAs in endothelial cells. Shear stress can also influence endothelial cell injury by inducing signalling pathways regulating apoptosis.",Journal of Internal Medicine,Vascular Smooth Muscle Cells,2017
Age-Related Changes in Carotid Plaque Morphology,"Relatively little is known about changes in carotid plaque morphology during ageing and the possible impact on cardiovascular events. Few studies have addressed age-related modifications within atherosclerotic lesions. Comparison between studies is difficult because patient populations are not uniform, distribution of age groups differs, and various cut-off values have been used to define the elderly population. Histological changes in the atherosclerotic plaque with age have been studied mainly in the carotid arteries, because coronary arteries can be evaluated only on autopsy series.",Journal of Internal Medicine,Atherosclerotic Plaque Morphology,2017
Histological Studies of Plaques Across Age Groups,"Our research group performed the first histological study evaluating the changes in histological composition of carotid plaques with ageing in humans. Carotid plaques from 180 patients with neurological symptoms were divided into three subgroups: patients aged <50 years, 51–70 years and >70 years. Multivariate discriminant analysis demonstrated that plaques from older groups were larger, more stenotic, and composed of a large amount of connective tissue associated with newly formed vessels and few or no giant cells. The presence of inflammatory cells was not evaluated in this study. van Oostrom et al. observed a decrease of fibrous plaques and an increase of atheromatous plaques with ageing, accompanied by decreased SMC content and slightly increased macrophage content and MMP-9 in carotid plaques.",Journal of Internal Medicine,Atherosclerotic Plaque Morphology,2017
Plaque Instability and Calcification in Older Adults,"van Lammeren et al. investigated whether the increased risk of stroke in elderly subjects correlated with plaque morphological changes and found a decrease in plaque stability with age. Older patients with carotid stenosis showed more unstable plaques characterized by low SMC content, larger lipid cores and more calcification compared with younger patients. Similarly, Redgrave et al., in a large series of symptomatic carotid plaques, found an increase in plaque calcification and lipid core size with age, paralleled by a decrease in fibrous tissue. These morphological changes were associated with reduced overall plaque inflammation.",Journal of Internal Medicine,Atherosclerotic Plaque Morphology,2017
Conflicting Evidence and Inflammation in the Elderly,"Partially conflicting results were reported by Grufman et al. in a study of 200 plaques from symptomatic and asymptomatic patients divided into two age groups (<70 and ≥70 years). Macrophage and lipid content were significantly higher in plaques from symptomatic elderly patients but not in younger patients. Some inflammatory markers, such as IFN-γ, TNF-α, soluble CD40 ligand and fractalkine, were significantly decreased in atherosclerotic lesions from older individuals. The authors concluded that increased plaque vulnerability in symptomatic elderly patients was associated with lipid accumulation and impaired tissue repair, rather than inflammatory processes.",Journal of Internal Medicine,Atherosclerotic Plaque Morphology,2017
Inflamm-Ageing and Mechanisms of Plaque Vulnerability,"These morphological observations are partially inconsistent with experimental data in healthy elderly persons showing that ageing is related to an increased proinflammatory status, originally termed 'inflamm-ageing'. Elevated circulating levels of proinflammatory cytokines including IL-1, IL-6, TNF-α and PGE2 contribute to vascular pathology. Increased plaque vulnerability in elderly patients is probably correlated with several mechanisms linked to the release of inflammatory cytokines, such as endothelial adhesion of inflammatory cells, increased absorption of atherogenic lipoproteins, enlargement of the necrotic lipid core and plaque calcification.",Journal of Internal Medicine,Atherosclerotic Plaque Morphology,2017
Sarcopenia Overview and Clinical Impact,"Sarcopenia is a geriatric syndrome currently defined as pathological loss of muscle mass and function. Sarcopenia is not only a major contributor to loss of physical function in older adults but is also associated with increased risk of morbidity, mortality, and increased healthcare costs. As a complex and multifactorial syndrome, sarcopenia has been associated with numerous degenerative changes during the aging process, but there is building evidence for significant contributions to the development of sarcopenia from neurodegenerative changes in the peripheral nervous system. A variety of interventions have been investigated for the treatment of sarcopenia, but current management is primarily focused on nutrition and therapeutic exercise interventions. Great strides have been made to improve screening procedures and diagnostic criteria for sarcopenia, but continued optimization of diagnostic and screening strategies is needed to better identify individuals with sarcopenia or at risk of developing sarcopenia.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia,2020
Introduction and Definition of Sarcopenia,"Maintaining and optimizing physical function and performance in older adults is becoming an increasingly important task. The geriatric syndrome, sarcopenia, which is currently defined as pathological loss of muscle mass and function, is a major contributor to impaired physical function and loss of independence in older adults. Sarcopenia results not only in physical impairments but also increased risk of mortality and morbidity. At best, some decline of physical function is an inevitable consequence of aging, but the rates at which individuals lose physical function vary remarkably due to a combination of intrinsic and extrinsic factors. When the term sarcopenia was first coined by Rosenberg in 1989 it was defined simply as loss of muscle mass, and until recently, the majority of research efforts have focused largely on understanding the mechanisms of muscle wasting and the development of treatments to increase mass.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia,2020
Distinguishing Sarcopenia and Dynapenia,"Dynapenia, a term coined by Manini and Clark, is defined as age-related loss of muscle strength. Dynapenia is a multifactorial phenomenon that can be related to deficits in muscle size or quality (muscle function per unit of muscle) as well as neuromuscular control. Longitudinal studies clearly demonstrate that loss of muscle function in older adults is more tightly associated with physical function and adverse outcomes as compared with muscle mass. As such, the European Working Group on Sarcopenia in Older People (EWGOS) developed diagnostic criteria that encompass both loss of muscle mass (sarcopenia) and either the loss of muscle strength or physical function (dynapenia). The worldwide prevalence of sarcopenia in individuals aged 60 or older has been estimated at about 10%, but figures vary widely depending on the diagnostic cut points used and the population being assessed.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia vs Dynapenia,2020
Intrinsic and Extrinsic Modifiers of Functional Decline,"Understanding, modifying, and exploiting the intrinsic and extrinsic factors that modify individual risk can help facilitate development of strategies to mitigate loss of physical function and lessen the burden of healthcare costs on society. In this review, the authors aim to provide an overview of the current understanding of age-related muscle dysfunction and sarcopenia, current clinical practices and recommendations for managing sarcopenia, and insight into future needs for maintaining and improving muscle function in older adults. The major goal is to provide an overview of the current understanding of muscle dysfunction in older adults from a systems-based perspective, outside of the muscle-centric view that has historically dominated the study of sarcopenia. Age-related degeneration and decline results from a variety of disruptions in normal cellular and molecular pathways or “hallmarks” of aging, all of which at some point have been implicated in loss of physical function in older adults.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia Pathogenesis,2020
Neuromuscular Requirements for Muscle Function,"Muscle function is dependent on intact excitation of muscle fibers via sufficient neuromuscular junction transmission, endplate potential generation, and intact coupling of excitation and contraction at the subsarcolemmal level. Not only is neuromuscular integrity required for muscle contraction and force production but also maintenance of trophic support for retained muscle bulk. There has long been interest in the impact of age on the form and function of the neuromuscular system. Historically, contrasts have primarily been made between young adults and older individuals (or between young and old preclinical models), but more recently the emphasis has shifted to investigating changes that contribute to geriatric syndromes such as sarcopenia or physical frailty by comparing sarcopenic adults to older adults with healthy or successful aging.",American Journal of Physical Medicine & Rehabilitation,Age-Related Neuromuscular Decline,2020
Muscle Mass vs Muscle Function Decline with Age,"Historically, decline of muscle performance has been simplistically attributed to muscle mass, but longitudinal studies have shown that losses of muscle performance outpace losses of muscle mass at rates that are two fold or greater than that of muscle wasting. The discordance between changes of muscle mass and muscle function highlights the importance of muscle 'quality' as a measure of muscle function per unit of muscle. An array of changes in muscle are associated with advancing age including losses of muscle size, mass, as well as muscle fiber numbers. The effects of aging appear to affect type II fibers to a greater extent as compared with type I fibers. There is building evidence for neurological origins of sarcopenia, and neurological factors have increasingly been considered an important factor in determining muscle quality.",American Journal of Physical Medicine & Rehabilitation,Muscle Quality and Aging,2020
Motor Neuron and Motor Unit Changes with Aging,"Over four decades ago, Tomlinson and colleagues demonstrated 25% losses of motor neuron counts in post-mortem tissues from the 2nd to the 9th decade with some individuals losing up to 50%. Electrophysiological recordings of motor unit numbers have consistently shown significant reductions in older individuals and aged preclinical models, and similarly motor unit firing rates are also reduced. Preclinical studies have consistently shown alterations of neuromuscular junction (NMJ) morphology in aged animals. There is much less known about morphological changes at the NMJ in older adults due to challenges in obtaining suitable muscle tissue for analyses.",American Journal of Physical Medicine & Rehabilitation,Motor Neuron Aging,2020
NMJ Morphology and Transmission in Aging,"In two older studies investigating intercostal muscles post-mortem, using light and electron microscopy, aging was associated with fragmentation and reduced post-synaptic folding. A more recent study suggested human NMJ morphology is much less complex compared to rodents, and also suggested that no overt changes of NMJ morphology occur across the lifespan in humans. A major caveat was that muscles were obtained during limb amputations related to peripheral vascular disease and diabetes, both associated with peripheral nerve pathology. Prior work in aged mice suggested that physical function is related to NMJ transmission defects on single fiber electromyography (SFEMG), and more recent studies showed features of NMJ transmission failure likely related to hypoexcitability of muscle fibers rather than synaptic dysfunction.",American Journal of Physical Medicine & Rehabilitation,Neuromuscular Junction Aging,2020
"Sarcopenia, Motor Units, and CNS Contributions","There is less electrophysiological evidence supporting abnormal NMJ transmission in older adults, but in one clinical study, Piasecki and colleagues showed that aging was associated with motor unit losses that were indistinguishable between older men with and without sarcopenia. Yet, in contrast to non-sarcopenic older men, the sarcopenic men showed small motor unit potential sizes and increased interpotential variability suggesting less reinnervation capacity and NMJ dysfunction. Sarcopenia is independently associated with cognitive decline. Therefore, although outside the scope of this article, it is also important to consider loss of integrity and function of the central nervous system in the context of muscle function. Considering muscle function in the context of deficits within both peripheral and central nervous systems will be critical for improving diagnostic and therapeutic developments.",American Journal of Physical Medicine & Rehabilitation,Neurological Factors in Sarcopenia,2020
Evolution of Sarcopenia Diagnostic Criteria,"Sarcopenia is a complex, multifactorial process which presents significant challenges for designing and implementing simple methods for patient identification, diagnosis, and prognosis. Several diagnostic criteria were originally developed based only on muscle mass, and more recent criteria have encompassed measures of both muscle mass and muscle function. In 2010, the European Working Group developed and published cutpoints and criteria based on the presence of reduced muscle mass and either reduced muscle strength or physical function. Other groups including the Asian Working Group on Sarcopenia (AWGS) and the Foundation for the National Institute of Health have defined cutpoints from different populations.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia Diagnostics,2020
EWGSOP2 Diagnostic Pathway (F-A-C-S),"The EWGSOP reconvened in 2018 (EWGSOP2) and provided updated recommendations regarding the use of a pathway of 'Find-Assess-Confirm-Severity' (F-A-C-S) for use across clinical practice and research studies. In clinical practice, EWGSOP2 advises use of the simple SARC-F questionnaire to screen ('Find') individuals with probable sarcopenia. Although the SARC-F has low sensitivity (~30%), it has good specificity, and adding calf circumference improves sensitivity to ~61%. Grip strength and the chair stand test are recommended for identifying reduced muscle strength. Following screening, the diagnosis of sarcopenia is established by findings of muscle weakness and loss of muscle mass. DXA and BIA are recommended in clinical care to identify reduced muscle mass, while DXA, MRI, and CT are recommended in research studies. Additional physical performance measures include SPPB, TUG, and the 400 m walk test.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia Diagnostics,2020
Subcategories of Sarcopenia,"The EWGSOP suggested subdividing sarcopenia based on chronicity as well as association with other medical conditions. Acute sarcopenia is defined as an acute condition of less than 6 months duration, whereas chronic sarcopenia has a duration of at least 6 months. Individuals with co-morbid medical conditions including organ failure, inflammatory conditions, degenerative joint diseases, and neurological disorders such as Alzheimer's disease are at increased risk of developing sarcopenia. Secondary sarcopenia refers to cases associated with identifiable causes or risk factors, while primary sarcopenia refers to age-related sarcopenia alone. Sarcopenic obesity describes sarcopenia in the context of excess adiposity and is associated with worsened physical function and increased mortality.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia Subtypes,2020
AWGS 2019 Recommendations,"The AWGS reconvened in 2019 to update recommendations regarding the diagnosis and treatment of sarcopenia. Updated recommendations included a case finding, assessment, and diagnostic testing strategy. Methods for case finding include calf circumference (<34 cm for men, <33 cm for women), SARC-F ≥4, or SARC-CalF ≥11. A diagnosis of 'Possible sarcopenia' is based on reduced muscle strength (handgrip strength) or reduced physical performance (5-time chair stand test). Diagnostic testing includes measures of muscle strength, physical performance, and appendicular skeletal muscle mass. 'Sarcopenia' is diagnosed when low appendicular muscle mass is combined with reduced strength or physical performance. 'Severe sarcopenia' requires findings of low muscle mass, low strength, and low physical performance.",American Journal of Physical Medicine & Rehabilitation,AWGS Sarcopenia Criteria,2020
"Distinguishing Sarcopenia, Cachexia, and Disuse Atrophy","Cachexia and disuse atrophy are other muscle wasting conditions that share similarities and differences with sarcopenia. Sarcopenia is a degenerative condition resulting in loss of muscle mass and strength and is associated with increased intramuscular fat. Cachexia, in contrast, is a catabolic disorder that results in loss of both muscle and fat mass. Disuse atrophy results in muscle atrophy without loss of muscle fibers. Importantly, all three—cachexia, sarcopenia, and disuse atrophy—may co-exist within the same patient, compounding muscle dysfunction.",American Journal of Physical Medicine & Rehabilitation,Muscle Wasting Disorders,2020
Current Management Strategies for Sarcopenia,"There are currently no pharmacological interventions available that are specifically designed for the treatment of sarcopenia. Management of sarcopenia currently includes reducing risk factors, minimizing the impact of contributory co-morbid medical conditions, and initiation of interventions such as nutrition and exercise. The primary intervention for prevention and management of sarcopenia is physical exercise. While resistance exercise, alone or combined with other training modes, demonstrates positive effects in older adults, evidence in sarcopenic older adults is more limited and based on short-term studies.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia Management,2020
Exercise Interventions and Considerations,"Physical exercise interventions should address all attributes of physical fitness including strength, stamina, endurance, coordination, balance, and body composition. Consideration must be given to an individual’s current abilities and fitness, available resources, and needs during daily activities. Challenges may include inadequate patient engagement, lack of resources, or limitations imposed by co-morbid medical conditions such as degenerative or inflammatory joint disease. Although muscle mass and strength are typically emphasized, aerobic, range of motion, and balance training are also important. Aerobic exercise interventions have been less frequently implemented in sarcopenic populations, likely due to expectations of limited improvements in muscle mass and strength, yet combining strengthening and aerobic exercise may offer protective effects against aging-associated muscle loss. Functional movements depend not only on contractile function but also adequate passive and active range of motion and balance.",American Journal of Physical Medicine & Rehabilitation,Exercise for Sarcopenia,2020
Role of Nutrition in Sarcopenia,"Nutrition is an important consideration in the management of sarcopenia. Adequate nutritional intake is a prerequisite for maintaining quality of life and preventing deficiencies and malnutrition in older adults. Meta-analyses have shown variable results regarding nutritional interventions, alone or combined with exercise, on improving lean mass and muscle strength. Generally, protein intake of 1.2–1.5 g/kg/day is recommended. A recent interventional trial in community-dwelling sarcopenic older adults demonstrated no benefit with protein supplementation when combined with a low-intensity exercise program, suggesting nutritional support may only benefit populations with deficiency or subclinical malnutrition. Another study demonstrated a synergistic effect when amino acid supplementation was combined with moderate intensity comprehensive exercise.",American Journal of Physical Medicine & Rehabilitation,Nutrition and Sarcopenia,2020
"Vitamin D, Amino Acids, and Supplements","Adequate vitamin D is essential for muscle contraction and induces protein synthesis, improves muscle performance, strength, and muscle fiber composition. Assessment and repletion of vitamin D levels is important, particularly because older adults are at increased risk for deficiency due to decreased sun exposure. Beta-hydroxy beta-methylbutyrate (HMB), a metabolite of the essential amino acid leucine, has shown possible positive effects on muscle mass, but effects on muscle function have been less consistent. Overall, nutritional strategies must be individualized and combined with exercise to optimize outcomes in sarcopenic patients.",American Journal of Physical Medicine & Rehabilitation,Micronutrients and Supplements in Sarcopenia,2020
Lifestyle Factors Influencing Sarcopenia,"The influences of lifestyle factors such as alcohol consumption and smoking may also contribute to loss of muscle mass and function. Chronic alcoholic myopathy is characterized by selective atrophy of type II muscle fibers, leading to reduction of muscle mass by up to 30%. Alcohol abuse appears to affect skeletal muscle severely, promoting its damage and wasting. Although alcohol consumption is not known as a direct cause of sarcopenia, the adverse effects of alcohol on skeletal muscle suggest that chronic alcohol consumption may promote loss of muscle mass and strength in old age. Some in vivo studies indicate that alcohol-induced muscle damage may be the result of impaired synthesis of muscle protein rather than increased muscle catabolism. Therefore, high alcohol intake is a lifestyle habit that may contribute to the development of sarcopenia.",American Journal of Physical Medicine & Rehabilitation,Lifestyle Risk Factors in Sarcopenia,2020
Impact of Smoking on Muscle Biology,"Montes de Oca et al. explored the effects of smoking on skeletal muscle by studying biopsies of the vastus lateralis muscle and found structural and metabolic damage in skeletal muscle of smokers, including decreased cross-sectional area of type I muscle fibers, and a similar trend in type IIa fibers. Petersen et al. studied the effect of smoking on protein metabolism in skeletal muscle of smokers and non-smokers about the age of 60 and found that the fractional synthesis rate of muscle was significantly lower in smokers compared with non-smokers. Petersen et al. concluded that smoking may increase the risk of sarcopenia by impairing muscle protein synthesis and up-regulating genes associated with impaired muscle maintenance. Thus, lifestyle habits regarding alcohol and tobacco use have a substantial impact on the progression of sarcopenia and the ability to prevent and treat the loss of muscle mass and function in old age.",American Journal of Physical Medicine & Rehabilitation,Smoking and Muscle Decline,2020
Sarcopenia as a Multifactorial Syndrome,"Sarcopenia is a complex geriatric syndrome, and the major factors that drive the phenotype of muscle weakness and wasting in older adults remain to be fully defined. It is unclear whether sarcopenia should be considered primarily a myopathic or neurogenic condition (or a combination). There is strong evidence for degenerative changes in both the neurological and muscular components of the neuromuscular system, and it remains possible that different pathological processes may occur between different populations affected by sarcopenia, potentially resulting in neurogenic and myopathic variants. Determining the main drivers of a sarcopenia phenotype may be important for prognosis or for the design and implementation of effective therapeutic strategies.",American Journal of Physical Medicine & Rehabilitation,Sarcopenia Pathogenesis,2020
Future Directions and Phenotype-Specific Approaches,"In the future, different aspects or stages of neuromuscular degeneration may need to be addressed with differing potential therapeutic strategies and mechanisms. Table 3 presents a forward-looking view on how different sarcopenia phenotypes could require specifically designed therapeutics. A phenotype-specific approach to therapeutic design and testing may be critical to achieve satisfactory patient stratification and positive clinical outcomes. Understanding whether sarcopenia is driven predominantly by muscular degeneration, neuromuscular failure, or combined mechanisms may guide precision interventions.",American Journal of Physical Medicine & Rehabilitation,Precision Therapeutics for Sarcopenia,2020
Exerkines and Myokines in Aging Sarcopenia – Overview,"Aging sarcopenia is an unavoidable condition affecting most older adults and is characterized by progressive declines in skeletal muscle mass, strength, and function. Exercise has been extensively researched as an effective intervention for sarcopenia. During physical activity, skeletal muscle releases exerkines and myokines, small molecules such as peptides, metabolites, and nucleic acids that mediate crosstalk between organs. These factors exert beneficial effects on neurological, metabolic, cardiovascular, and immune processes. Certain molecules, including IL-6, irisin, FGF21, and BDNF, function as both myokines and exerkines. These substances are key mediators linking exercise, metabolism, and inflammation, and even small exercise-induced changes can affect whole-body physiology.",Frontiers in Endocrinology,Exerkines and Myokines in Sarcopenia,2025
Biological Mechanisms Underlying Myokine Protection,"Exerkines exert protective effects on aging skeletal muscle through multiple mechanisms. First, they mediate energy redistribution toward skeletal muscle, ensuring adequate energy supply. Second, they enhance skeletal muscle satellite cell activation, promoting muscle repair and regeneration. Third, exerkines upregulate genes responsible for muscle regeneration while suppressing genes linked to muscle atrophy, thereby improving anabolic–catabolic balance. Finally, they improve neuromuscular junction function, strengthening neural control of muscle contraction. These combined actions constitute a core protective mechanism through which exercise-derived myokines help counteract age-associated muscle decline.",Frontiers in Endocrinology,Myokine Protective Mechanisms,2025
"Aging, Muscle Homeostasis, and Selective Fiber Loss","Aging involves a progressive decline in physiological function accompanied by increased susceptibility to disease. Sarcopenia specifically involves reductions in muscle mass and contractile function together with metabolic, inflammatory, and endocrine abnormalities. Muscle atrophy occurs when protein breakdown exceeds synthesis, making maintenance of proteostasis crucial. With age, disturbances in muscle homeostasis combined with neuronal degeneration lead to preferential loss of type II fast-twitch fibers. This selective loss is accompanied by reduced motor units, contributing to muscle weakness, slowed movement, and impaired physical function.",Frontiers in Endocrinology,Aging-Related Muscle Degeneration,2025
Exercise as a Therapeutic Strategy,"Exercise represents a powerful non-pharmacological intervention for age-related diseases, including sarcopenia. Resistance training in particular activates the nervous system and accelerates muscle protein synthesis, enhancing muscle mass and strength. Exercise-induced secretion of exerkines and myokines supports systemic health, influencing neurological, metabolic, cardiovascular, and immune functions. Because myokines are closely linked to skeletal muscle biology, understanding their regulation offers insights into muscular homeostasis, aging, and potential rejuvenation therapies.",Frontiers in Endocrinology,Exercise-Induced Myokines,2025
Future Directions and Research Aims,The study of exerkines offers a promising therapeutic avenue for aging sarcopenia. Current research aims to clarify the causal relationship between sarcopenia severity and circulating myokine levels. Understanding how exerkines regulate muscle homeostasis may lead to novel strategies for rejuvenating aging skeletal muscle. The overarching goal is to leverage exercise-induced molecular signals to improve skeletal muscle health and slow or reverse age-related functional decline.,Frontiers in Endocrinology,Future Research in Exerkines and Sarcopenia,2025
Overview of Aging Sarcopenia,"Sarcopenia was originally recognized as age-related loss of lean body mass and was formally defined as a distinct condition in 2010. According to the European Working Group on Sarcopenia in Older People (EWGSOP), sarcopenia is a progressive skeletal muscle disease characterized by reduced muscle mass, decreased muscle strength, and impaired physical function, with prevalence rising sharply with age. Sarcopenia is classified as primary when caused mainly by aging, or secondary when driven by chronic diseases, malnutrition, or other contributing factors. The condition typically progresses from muscle atrophy to impaired muscle function and ultimately to decline in muscle strength. Sarcopenia must be distinguished from skeletal muscle atrophy: although both involve reduced muscle mass and fiber size, sarcopenia additionally includes functional decline and is influenced by both environmental and genetic factors.",Frontiers in Endocrinology,Sarcopenia Definition and Classification,2025
Mechanistic Basis of Muscle Atrophy and Sarcopenia,"Skeletal muscle atrophy involves decreased muscle mass and fiber size resulting from an imbalance between protein synthesis and degradation. Major pathways contributing to muscle atrophy include the ubiquitin–proteasome system, autophagy–lysosome pathways, the IGF1–AKT–FoxO signaling axis, inflammatory cytokines, and NF-κB signaling. The mechanistic drivers of sarcopenia overlap with those of general muscle atrophy but also include unique age-related contributors such as reduced physical activity, hormonal dysregulation, impaired nutrient absorption, and chronic inflammation. These changes influence myokine secretion and disrupt skeletal muscle homeostasis.",Frontiers in Endocrinology,Mechanisms of Muscle Atrophy and Sarcopenia,2025
Multifactorial Pathogenesis of Aging-Related Sarcopenia,"The development of aging-related sarcopenia involves several interconnected biological processes. First, aging disrupts protein homeostasis, and when muscle protein breakdown exceeds synthesis, muscle atrophy ensues. Second, aging-related degeneration of the neuromuscular system—including motor neuron loss, neuromuscular junction deterioration, and satellite cell senescence—reduces regenerative capacity and contributes to loss of muscle fibers and fiber size. Third, mitochondrial dysfunction plays a key role by reducing cellular energy production and promoting oxidative stress; this dysfunction is recognized as a major driver of sarcopenia. Hormonal declines and abnormal production of muscle-derived factors further contribute to disease progression.",Frontiers in Endocrinology,Pathogenesis of Aging Sarcopenia,2025
Role of Physical Activity and Myokines in Counteracting Sarcopenia,"Physical activity and exercise are effective interventions for delaying or mitigating skeletal muscle aging. Exercise enhances muscle contraction, stimulates protein synthesis, and promotes the release of various myokines that regulate muscle mass, metabolic homeostasis, and regeneration. Altered myokine signaling in individuals with sarcopenia contributes to muscle atrophy and functional decline, while reduced muscle mass itself further diminishes myokine expression, creating a negative feedback loop. Exercise interrupts this cycle by restoring myokine signaling, improving muscle metabolism, and enhancing regeneration and repair.",Frontiers in Endocrinology,"Exercise, Myokines, and Muscle Homeostasis",2025
Overview of Exerkines,"Exerkines are peptides, lipids, metabolites, and nucleic acids released during or after physical exercise. They are secreted by multiple tissues, and their classification is based on the organ of origin: myokines from skeletal muscle, cardiokines from the heart, hepatokines from the liver, adipokines from white adipose tissue, batokines from brown adipose tissue, and neurokines from neurons. Exerkines enter circulation either directly or via extracellular vesicles, enabling systemic communication between organs. Their receptors and target pathways are expressed widely across the body, including in the brain, heart, pancreas, bones, adipose tissue, immune cells, and skeletal muscle. Animal and human studies demonstrate that exerkines mediate inter-organ crosstalk, contributing to metabolic, neurological, cardiovascular, and musculoskeletal adaptations to exercise.",Frontiers in Endocrinology,Exerkine Classification and Systemic Crosstalk,2025
Systemic Actions of Exerkines,"Exerkines influence nearly all major physiological systems. In the cardiovascular system, they improve cardiac metabolic health and contribute to myocardial protection. Cathepsin B and BDNF released during exercise enhance neuronal maturation and cognition. In the brain, exerkines improve mitochondrial function, reduce oxidative stress, maintain proteostasis, and enhance synaptic plasticity. Bone metabolism is modulated through exerkines such as irisin and BAIBA, which promote bone formation, while myostatin negatively regulates osteoblast and osteoclast activity. Exerkines also play key roles in skeletal myogenesis by regulating myoblast proliferation, migration, and differentiation. Individual exerkines can act on multiple tissues to support coordinated metabolic homeostasis.",Frontiers in Endocrinology,Systemic Biological Effects of Exerkines,2025
Exerkines in Skeletal Muscle Regulation,"Exerkines are essential modulators of skeletal muscle homeostasis. They regulate energy allocation, mitochondrial activity, inflammation, satellite cell activation, and muscle regeneration. Some exerkines, such as irisin, influence pancreatic and adipose tissue activity, thereby indirectly enhancing energy supply to skeletal muscle. Others work synergistically within muscle tissue to maintain internal metabolic balance. Despite increasing recognition of their importance, the detailed mechanistic actions of many exerkines remain insufficiently understood. This gap limits current ability to fully leverage exerkines as targeted therapeutic interventions for muscle aging or sarcopenia.",Frontiers in Endocrinology,Exerkines and Skeletal Muscle Homeostasis,2025
Classification and Research Overview,"Due to the diversity of exerkines, they are categorized based on their tissue of origin. This classification includes myokines, cardiokines, hepatokines, adipokines, batokines, and neurokines. Current research uses large-scale database searches (PubMed, Web of Science, Sci-Hub) to map the molecular relationships between exercise and muscle physiology. In particular, several myokines with strong therapeutic potential for aging-related sarcopenia have been identified and will be discussed in detail in subsequent sections. Understanding these exerkines may enable the development of precision exercise-based therapies for muscle aging.",Frontiers in Endocrinology,Exerkine Classification and Research Landscape,2025
Overview of Myokines,"Skeletal muscle functions as an endocrine organ capable of synthesizing and secreting hundreds of cytokines and peptides termed myokines. These molecules are released during muscle contraction and exert autocrine, paracrine, and endocrine effects. Targeted skeletal muscle biopsy analyses from both acute and chronic exercise studies have identified numerous myokines whose abundance rises in interstitial muscle fluid following exercise. Different types of exercise stimulate distinct myokine profiles: concentric contractions preferentially induce IL-6 and IL-8; resistance training prominently increases IL-15; a single exercise bout releases IL-6, IL-1ra, and IL-8; whereas repeated strenuous exercise triggers TNF-α release. Muscle fiber type also influences myokine expression, with glycolytic fibers producing actin, angiopoietin, and muscarinic acetylcholine receptors, while oxidative fibers preferentially release myosin and irisin.",Frontiers in Endocrinology,Myokine Secretion and Exercise-Dependent Regulation,2025
Functional Diversity of Myokines,"Non-quantitative proteomic approaches have identified over 600 myokines, many of which exert systemic effects through circulation. These molecules participate in metabolic regulation by promoting glucose uptake, improving insulin sensitivity, modulating cognitive functions, stimulating osteoblast differentiation, influencing blood pressure, and regulating cardiac contractility. Some myokines contribute to immune modulation by combating acute inflammatory responses or attenuating chronic low-grade inflammation associated with aging. Their most fundamental role, however, is the protection of skeletal muscle function through enhancement of contractility, support of regenerative processes, and maintenance of muscle homeostasis.",Frontiers in Endocrinology,Physiological Roles of Myokines,2025
Myokines in Myogenesis and Tissue Crosstalk,"During skeletal muscle cell proliferation, myoblast-derived myokines typically inhibit neurogenesis and adipogenesis, whereas during differentiation, myoblasts release factors that promote myotube formation, vascular development, and neurogenesis. Myokines form a complex communication network among tissues: adipose tissue, pancreas, brain, vasculature, and bone are all responsive to myokine signaling. This network enables coordinated metabolic, inflammatory, and regenerative responses across the organism, positioning myokines as key mediators of exercise-induced benefits and essential regulators of muscle integrity during aging.",Frontiers in Endocrinology,Myokines in Myogenesis and Inter-Organ Communication,2025
Exerkines in Aging Sarcopenia: Overview,"Current literature indicates that numerous exerkines are associated with skeletal muscle physiology, yet only a subset has been directly investigated in the context of aging sarcopenia. Most studies focus on exerkines involved in muscle regeneration or contractile function, whereas fewer have examined their therapeutic potential in reversing age-related muscle loss. Among the known exerkines, myonectin, meteorin-like protein (Metrnl), adiponectin, and leptin have emerged as particularly relevant to sarcopenia. These molecules influence systemic and local pathways related to muscle homeostasis, regeneration, inflammation, and metabolism. Understanding the mechanistic contribution of these exerkines may enable the development of new systemic therapeutic strategies for aging sarcopenia.",Frontiers in Endocrinology,Exerkines and Aging Sarcopenia,2025
Myonectin and Aging Sarcopenia,"Myonectin, also referred to as CTRP15, plays an important role in maintaining metabolic homeostasis. In experimental models, myonectin deficiency accelerates age-related muscle atrophy and reduces muscle strength through suppression of the AMPK–PGC-1α pathway. Supplementation of exogenous myonectin prevents skeletal muscle atrophy via an AMPKα2/PGC-1α4/IGF-1–dependent mechanism. Despite its clear physiological relevance in murine models, human studies involving 142 older adults reported no significant association between serum myonectin levels and sarcopenia. These findings suggest that while myonectin is required for muscle maintenance in vivo, serum myonectin concentration may not reliably reflect sarcopenic status in elderly individuals.",Frontiers in Endocrinology,Myonectin Function in Muscle Aging,2025
Metrnl and Aging Sarcopenia,"Meteorin-like protein (Metrnl) is expressed in multiple tissues including skeletal muscle, liver, macrophages, spleen, and the central nervous system. Although muscle fiber–derived Metrnl is not essential for regeneration, macrophage-derived Metrnl is critical for muscle repair, particularly through induction of IL-10, IL-6, and IGF-1 expression to activate satellite cell proliferation. Aging alters macrophage function, leading to impaired innate immune signaling and a decline in Metrnl expression during muscle injury, which contributes to reduced regenerative capacity. In aged murine models, Metrnl administration enhances muscle regeneration and satellite cell proliferation. Clinically, serum Metrnl correlates positively with weight loss and cardiac insufficiency severity in elderly heart failure patients. Moreover, recombinant Metrnl activates cAMP/PKA/SIRT1 and AMPK/PAK2 pathways, reducing cardiomyocyte apoptosis after ischemia–reperfusion injury. These findings indicate that Metrnl possesses both regenerative and cardioprotective functions that may be relevant to sarcopenia.",Frontiers in Endocrinology,Metrnl and Muscle Regeneration,2025
Adiponectin and Aging Sarcopenia,"Adiponectin (ApN) is primarily secreted by white adipose tissue, but also by skeletal muscle cells, cardiomyocytes, hepatocytes, and osteoblasts. Structurally, it exists as full-length adiponectin (fApN) and globular adiponectin (gApN). During skeletal muscle injury, elastase released from infiltrating immune cells cleaves fApN into gApN, the latter exerting strong pro-regenerative effects. ApN is considered a protective factor against aging sarcopenia, although its circulating levels correlate negatively with skeletal muscle density, physical function, and bone mineral density. gApN promotes myoblast proliferation and differentiation by inducing MyoD expression and activating myogenin and MRF4, thereby facilitating myotube formation. Additionally, the ApN/AdipoR1–AMPK signaling axis mediates exercise-induced satellite cell activation and improves locomotor performance in aged mice. Notably, these effects show muscle-type specificity: treatment of 25-month-old mice with the adiponectin receptor agonist AdipoRon preferentially enhances function and metabolism of fast-twitch fibers without significant influence on slow-twitch fibers. Collectively, ApN improves mitochondrial metabolism, supports muscle fiber regeneration, and enhances autophagy, thereby contributing to the protection of aged skeletal muscle.",Frontiers in Endocrinology,Adiponectin and Sarcopenia,2025
Leptin and Aging Sarcopenia,"Leptin is predominantly produced by adipose tissue, and its circulating concentration reflects total body fat mass. As a key metabolic hormone, leptin regulates appetite, energy balance, insulin sensitivity, and glucose metabolism. However, its relationship with aging sarcopenia is complex and inconsistent across studies. Some large population analyses, including a cross-sectional cohort of 4,062 adults aged ≥69 years, show that elevated leptin levels are associated with a higher risk of obesity-related sarcopenia. Conversely, other studies report that higher leptin levels are linked to a reduced risk of sarcopenia, a discrepancy likely influenced by body fat content, leptin resistance, and variances in receptor sensitivity. Mechanistically, chronically high leptin levels impair skeletal muscle fatty acid oxidation and lipid handling, leading to metabolic dysfunction within muscle fibers. Genetic models provide additional insight: fat-free (FF) mice generated by crossing ApN-Cre lines—resulting in total congenital absence of adipose tissue—exhibit severely reduced leptin levels and display profound muscle mass deficits due to pronounced fast-twitch fiber atrophy. Despite these observations, the precise mechanisms through which leptin regulates skeletal muscle homeostasis remain poorly defined and require further investigation.",Frontiers in Endocrinology,Leptin and Muscle Aging,2025
Myokines in Aging Sarcopenia,"Exercise is one of the most effective interventions for mitigating sarcopenia, and myokines have emerged as key molecular mediators of this benefit. Myokines exert bidirectional effects on skeletal muscle mass, with some promoting muscle hypertrophy and others contributing to atrophy. Their protective influence in aging skeletal muscle involves several coordinated mechanisms: enhancing skeletal muscle protein synthesis, regulating glucose and fatty-acid uptake, modulating mitochondrial metabolism, and improving neuromuscular junction (NMJ) signaling. Collectively, these actions help maintain muscle mass, strength, and function during aging and represent a promising direction for understanding how exercise counteracts sarcopenia.",Frontiers in Endocrinology,Myokines and Sarcopenia,2025
Myostatin and Aging Sarcopenia,"Myostatin (MSTN), a member of the TGF-β superfamily, is expressed not only in skeletal muscle but also in cardiac tissue, adipose tissue, the brain, kidneys, and leukocytes. Initially synthesized as an inactive precursor, MSTN undergoes sequential processing: furin-mediated removal of signal peptides, followed by BMP1/Tolloid metalloproteinase cleavage to expose the active ligand. MSTN is one of the most potent negative regulators of skeletal muscle mass. Older men (~70 years) exhibit approximately double the MSTN gene expression observed in younger men (~20 years), together with a ~40% reduction in type II fiber cross-sectional area. Elevated serum MSTN is strongly associated with reduced muscle strength—an increase of 1 ng/mL corresponds to ~11% higher odds of sarcopenia. Mechanistically, active MSTN binds the ActRIIB receptor, inducing phosphorylation of Smad2/3 and downstream activation of ALK4/ALK5 signaling. This suppresses key myogenic transcription factors such as MEF2 and MyoD1, inhibiting myoblast proliferation and differentiation. MSTN also inhibits hypertrophy by suppressing mTOR signaling and promotes muscle catabolism via FoxO1-dependent pathways. Given its central role as a negative regulator of muscle mass, MSTN inhibition or gene-level modulation represents a promising therapeutic strategy for age-related sarcopenia.",Frontiers in Endocrinology,Myostatin and Muscle Aging,2025
Interleukin-6 and Aging Sarcopenia,"Interleukin-6 (IL-6) is a multifunctional cytokine involved in immune regulation, inflammation, and the balance between anabolic and catabolic processes. It signals through three pathways—classical, trans-signaling, and cluster signaling—each influencing satellite-cell proliferation and migration, which supports physiological muscle hypertrophy. Skeletal muscle contractions induce acute IL-6 release, leading to transient rises in serum IL-6 during exercise. However, circulating IL-6 levels are chronically elevated in elderly individuals with sarcopenia, and this persistent elevation is associated with increased intramuscular fat infiltration and impaired energy metabolism. Experimental IL-6 overexpression in NSE/IL-6 mice leads to reduced early-life muscle growth, severe adult muscle atrophy, and decreased muscle fiber number and cross-sectional area. These results highlight a dual effect of IL-6: acute, exercise-induced IL-6 promotes muscle regeneration and satellite-cell activation, while chronic, low-grade elevation suppresses myogenesis, reduces protein synthesis, and accelerates muscle degeneration. Whether sustained IL-6 elevation in the elderly directly drives fiber-type transitions or metabolic dysregulation contributing to whole-muscle atrophy remains an open question.",Frontiers in Endocrinology,Interleukin-6 and Sarcopenia,2025
Irisin and Aging Sarcopenia,"Irisin is a peptide hormone produced when FNDC5 is cleaved following PGC-1α activation during exercise. It is expressed in skeletal muscle and the brain and plays a central role in muscle regeneration and metabolic adaptation. Animal studies indicate that irisin enhances satellite-cell activity, reduces protein degradation, protects against muscle fibrosis, and improves sarcolemmal stability across multiple muscle-loss models, including denervation, hindlimb unloading, and muscular dystrophy. Irisin promotes protein synthesis through p-Erk, Akt, and mTOR signaling, and suppresses atrophy-related genes such as Atrogin-1 and MuRF-1. In aged mice, irisin levels and FNDC5 expression decrease; irisin knockout accelerates sarcopenic phenotypes whereas recombinant irisin administration restores muscle mass and grip strength. These protective effects are mediated via the AMPK–PGC-1α–FNDC5 axis and IGF-1/Akt/mTOR signaling. Human studies, however, show inconsistent results. Some report no differences in circulating irisin between healthy and sarcopenic elderly adults and no association with muscle mass or function. These discrepancies may reflect differences in patient populations, metabolic status, or methodological limitations. Overall, irisin shows strong therapeutic potential in preclinical aging models, but translation to humans requires further investigation.",Frontiers in Endocrinology,Irisin and Muscle Aging,2025
BDNF and Aging Sarcopenia,"The mammalian neurotrophic factor family includes nerve growth factor (NGF), neurotrophin-3, neurotrophin-4/5, and BDNF. BDNF is secreted out of the cell as Pro-BDNF after being translated and cut into mature BDNF. BDNF and Pro-BDNF act on specific receptors in the nucleus and cell membrane. Different skeletal muscles secrete BDNF in response to different exercise durations and intensities. Post-exercise plasma BDNF levels depend on exercise duration, its intensity, type of exercise, the level of previous training, and the functional status of the body. In skeletal muscle, BDNF regulates glycolytic fiber-type recognition, fatty acid oxidation, and satellite cell differentiation, and strengthens the neuromuscular junction. BDNF takes part in the generation of regenerating muscle fibers after injury and is necessary for the formation of regenerating muscle fibers after injury or damage. In contrast, plasma BDNF levels are significantly lower in patients with aging sarcopenia and in debilitated patients with diminished muscle strength and physical activity.",Frontiers in Endocrinology,BDNF,2025
"BDNF, Neuromuscular Junctions, and Muscle Innervation","In animal models, intramuscular injection of BDNF promotes functional repair after nerve injury. BDNF plays an important role in protecting the neuromuscular junction. The ameliorative effect of BDNF on aging sarcopenia involves muscle repair signal cascades and neuromuscular signaling connections. Activation of TrkB receptors by BDNF enhances presynaptic protein kinase C family (cPKCa, cPKCbI, and cPKCe), and activation of PKCs enhances synaptic vesicle fusion and neurotransmitter release, which enhances the functional innervation of the muscles. Muscle wasting during aging is partly due to the retraction and death of motor neurons, resulting in the detachment of muscle fibers from neuronal innervation. Muscle fiber degeneration and muscle atrophy can only be avoided if these muscle fibers are re-innervated by neighboring neurons. Perhaps we may speculate that BDNF delays skeletal muscle atrophy during aging by enhancing neuromuscular connections in patients with aging sarcopenia.",Frontiers in Endocrinology,BDNF,2025
"Apelin Distribution, Regulation, and Exercise Effects","Apelin belong to cardiac factor, myokine and adipokine, and is extensively distributed across various organs and tissues, including skeletal muscle, adipose tissue, the central nervous system, the gastrointestinal tract, lungs, liver, and heart. Apelin-13 and apelin-17 are the predominant isomers of apelin found in human plasma, with apelin-13 exhibiting greater biological activity and receiving more extensive research attention. Exercise and aging are important factors influencing apelin secretion. Exercise induces apelin, while overall levels decrease with aging. However, the effect of exercise on apelin levels is controversial. One study demonstrated that prolonged aerobic exercise significantly elevated plasma apelin levels, whereas another study indicated that it did not influence apelin expression.",Frontiers in Endocrinology,Apelin,2025
"Apelin Signaling, Muscle Regeneration, and Aging","Apelin enhances myocyte metabolism and stem cell function to stimulate skeletal muscle formation during aging. The Apelin/Apelin receptor system stimulates skeletal muscle stem cells through the Forkhead box 03–MuRF-1–Atrogin axis and simultaneously activates the AMP-activated protein kinase (AMPK) and P7050K pathways to promote protein production in myofibers, which together promote skeletal muscle regeneration. Knockdown of the apelin gene in the skeletal muscle of aged mice led to muscle mass reduction, muscle weakness, and motor dysfunction, whereas aged mice that are administered apelin or subjected to adenovirus-mediated enhancement of the apelin gene expression exhibit improved muscle function and hypertrophy of muscle fibers. Apelin-administered mice had increased expression of the markers Pax7, Myf5, and Myogenin in satellite cells and target muscle stem cells to promote muscle regeneration.",Frontiers in Endocrinology,Apelin,2025
"Apelin Deficiency, Fiber-Type Shifts, and Therapeutic Potential","Apelin deficiency resulted in changes in skeletal muscle fiber types. Compared to wild-type mice, apelin knockout induces a shift from fast type II to slow-oxidizing type I fiber in mice and increases the proportion of MHC-1 type fibers. Additionally, apelin knockout mice exhibited a decreased number of mitochondria in myogenic fibers, a significant reduction in mitochondria-related enzyme activities, and diminished muscle tonic contractility and grip strength compared to wild-type mice. Exercise-induced apelin enhances skeletal muscle function and alleviates sarcopenia, which makes apelin a potential target for the treatment of myofibrillar atrophy, muscle weakness, and oxidative stress in aging mice. However, with age, both systemic and local apelin levels show a decreasing trend. Exogenous administration of apelin can lead to significant improvements in age-related pathologies. Apelin may serve not only as a novel tool for the early diagnosis of sarcopenia but also as a prognostic marker for evaluating the benefits of exercise in older adults.",Frontiers in Endocrinology,Apelin,2025
IGF-1 Physiology and Age-Related Decline,"Insulin-like growth factor-1 (IGF-1) is an anabolic growth factor that facilitates tissue development, maturation, cellular adaptation, and regeneration during growth and development. In skeletal muscle, IGF-1 is secreted by muscle fibers into the extracellular matrix, subsequently binding to insulin-like growth factor binding proteins. Age and exercise are key factors influencing circulating IGF-1 concentrations. Serum IGF-1 levels decrease with age, but exercise promotes IGF-1 secretion. Aerobic exercise, resistance exercise, whole-body vibration, and electrical stimulation all activate the IGF-1 pathway, increase protein synthesis and skeletal muscle mass, inhibit protein degradation and apoptosis, and enhance the exercise capacity of skeletal muscle in early aging mice.",Frontiers in Endocrinology,IGF-1,2025
"IGF-1 Signaling, Muscle Regeneration, and Protective Effects","In animal experiments, IGF-1 knockout in mouse monocytes/macrophages resulted in impaired muscle regeneration after injury, with reduced size of regenerating muscle fibers, enlarged interstitial gaps, and deposition of lipid tissue. In contrast, IGF-1-overexpressing mice maintain high IGF-1 levels even in old age, thereby maintaining skeletal muscle function. IGF-1 achieves its protective effects on skeletal muscle by activating muscle signaling responses and skeletal myogenesis. Elevated plasma levels of IGF-1 lead to IGF-1 Akt/Protein Kinase B-mTOR pathway stress, which promotes ribosomal biosynthesis and facilitates the formation of new myofibrillar proteins to provide a condition for skeletal muscle remodeling. Additionally, a high level of IGF-1 inhibits skeletal muscle via the ubiquitin ligases MuRF1 and MAFbx. Skeletal muscle secretion of IGF-1 decreases during aging, which results in a decline in skeletal muscle mass and function. However, high plasma IGF-1 levels can increase muscle mass to decrease the incidence of aging sarcopenia.",Frontiers in Endocrinology,IGF-1,2025
Follistatin Function and Muscle Regulation,"Follistatin (FST) is a multifunctional protein whose main function is to antagonize the TGB-b superfamily, such as the muscle growth inhibitors, activin, and BMPs. FST, as a cytokine expressed systemically, is particularly abundant in skeletal muscle, the heart, adipose tissue, the kidneys, and the lungs. FST overexpression through gene transfer or a transgene induces skeletal muscle hypertrophy, myofiber regeneration, and satellite cell proliferation. Circulating MSTN and FST are negatively correlated with muscle function in older women. In patients with severe muscle atrophy, the MSTN pathway was found to be significantly downregulated with a progressive increase in FST, which may partially delay muscle atrophy. Although FST overexpression increases muscle mass and excitability, it does not prevent the age-related decline in motor unit function.",Frontiers in Endocrinology,Myokines,2025
Decorin and Muscle Regeneration,"Decorin is an exercise-induced muscle factor expressed in various tissues, including intestinal tissue, heart, adipose tissue, and skeletal muscle. It plays a role in regulating autophagy, inflammation, and glucose homeostasis, and has been shown to effectively prevent muscle atrophy by inhibiting MSTN. Decorin is an anti-fibrotic and pro-myogenic generating agent. When it is injected into damaged skeletal muscle directly, it can promote the process of complete skeletal muscle regeneration and reduce the formation of fibrotic scar tissue. Decorin reduces MSTN-induced phosphorylation of Smad2 and inhibits the activation of the Smad signaling pathway in a dose-dependent manner. Intramuscular injection of recombinant Decorin may significantly enhance muscle mass in dystrophic mice by activating skeletal muscle cell differentiation.",Frontiers in Endocrinology,Myokines,2025
LIF and Exercise-Induced Muscle Adaptation,"Leukemia inhibitory factor (LIF), which belongs to the IL-16 family, regulates skeletal muscle growth and regeneration and is associated with skeletal muscle after prolonged exercise. Aerobic exercise upregulates LIF expression in human skeletal muscle to inhibit myasthenia gravis and improve muscle performance. Exogenous LIF intervention may enable human myoblast proliferation by inducing the cell proliferation factors c-Myc and JunB.",Frontiers in Endocrinology,Myokines,2025
"IL-15, Metabolism, and Muscle Protein Synthesis","IL-15 is a contraction-induced myokine that improves energy metabolism in skeletal muscle locally. High IL-15 levels protect against high-fat diet-induced obesity, glucose intolerance, and insulin resistance. A comparison of wild-type, IL-15 knockout, and IL-15 transgenic mice reveals that IL-15 promotes muscle protein synthesis and myofiber regeneration by activating critical regulators of skeletal muscle autophagy. IL-15 and its cognate receptor a (IL-15 receptor a) are involved in the regulation of anabolic and catabolic homeostasis in skeletal muscle. IL-15Ra may play a role in the increased synthesis of myofibrillar proteins in skeletal muscle after a single bout of resistance exercise. However, few studies have reported an association between the above muscle factors and sarcopenia in aging, but they all have the function of maintaining muscle mass and enhancing muscle strength.",Frontiers in Endocrinology,Myokines,2025
Interleukin-6 and Aging Sarcopenia,"Interleukin-6 (IL-6) is a multifunctional cytokine involved in immune regulation, inflammation, and the balance between anabolic and catabolic processes. It signals through three pathways—classical, trans-signaling, and cluster signaling—each influencing satellite-cell proliferation and migration, which supports physiological muscle hypertrophy. Skeletal muscle contractions induce acute IL-6 release, leading to transient rises in serum IL-6 during exercise. However, circulating IL-6 levels are chronically elevated in elderly individuals with sarcopenia, and this persistent elevation is associated with increased intramuscular fat infiltration and impaired energy metabolism. Experimental IL-6 overexpression in NSE/IL-6 mice leads to reduced early-life muscle growth, severe adult muscle atrophy, and decreased muscle fiber number and cross-sectional area. These results highlight a dual effect of IL-6: acute, exercise-induced IL-6 promotes muscle regeneration and satellite-cell activation, while chronic, low-grade elevation suppresses myogenesis, reduces protein synthesis, and accelerates muscle degeneration. Whether sustained IL-6 elevation in the elderly directly drives fiber-type transitions or metabolic dysregulation contributing to whole-muscle atrophy remains an open question.",Frontiers in Endocrinology,Interleukin-6 and Sarcopenia,2025
Irisin and Aging Sarcopenia,"Irisin is a peptide hormone produced when FNDC5 is cleaved following PGC-1α activation during exercise. It is expressed in skeletal muscle and the brain and plays a central role in muscle regeneration and metabolic adaptation. Animal studies indicate that irisin enhances satellite-cell activity, reduces protein degradation, protects against muscle fibrosis, and improves sarcolemmal stability across multiple muscle-loss models, including denervation, hindlimb unloading, and muscular dystrophy. Irisin promotes protein synthesis through p-Erk, Akt, and mTOR signaling, and suppresses atrophy-related genes such as Atrogin-1 and MuRF-1. In aged mice, irisin levels and FNDC5 expression decrease; irisin knockout accelerates sarcopenic phenotypes whereas recombinant irisin administration restores muscle mass and grip strength. These protective effects are mediated via the AMPK–PGC-1α–FNDC5 axis and IGF-1/Akt/mTOR signaling. Human studies, however, show inconsistent results. Some report no differences in circulating irisin between healthy and sarcopenic elderly adults and no association with muscle mass or function. These discrepancies may reflect differences in patient populations, metabolic status, or methodological limitations. Overall, irisin shows strong therapeutic potential in preclinical aging models, but translation to humans requires further investigation.",Frontiers in Endocrinology,Irisin and Muscle Aging,2025
Introduction to Satellite Cell Aging,"Skeletal muscle regeneration relies on satellite cells—quiescent muscle stem cells that activate, proliferate, and differentiate upon injury to form new myofibers. Aging disrupts this process through a substantial decline in satellite cell numbers and function. Age-related impairments arise from both cell-intrinsic defects (such as genomic instability, metabolic dysfunction, and entry into pre-senescent states) and extrinsic alterations in the niche and systemic environment. Although the contribution of satellite cells to sarcopenia remains debated, maintaining youthful satellite cell functionality or introducing rejuvenated progenitors holds strong therapeutic potential for combating age-related muscle wasting. Skeletal muscle therefore provides a powerful model to study stem cell aging and explore regenerative interventions aimed at restoring youthful tissue repair capacity.",FEBS Journal,Satellite Cell Aging Overview,2019
Decline of the Satellite Cell Pool with Age,"Aging progressively reduces the size of the satellite cell pool due to disruptions in quiescence regulation, increased spontaneous activation, and apoptosis. One major driver is elevated myofiber-derived FGF2 in old muscle, which forces satellite cells out of quiescence and contributes to premature stem cell exhaustion. FGF2 simultaneously suppresses Sprouty1—a key inhibitor of the ERK/MAPK pathway—preventing proper return to quiescence and promoting apoptosis. Additional niche defects intensify this decline: aging reduces Notch-activating signals, causing mitotic catastrophe during satellite cell proliferation. Human studies confirm the presence of apoptosis-prone satellite cells in aged muscle. While some satellite cells persist into advanced age, they exhibit diminished activation, limited proliferative expansion, decreased differentiation capacity, and increased lineage diversion into fibrotic or adipogenic fates. These intrinsic and extrinsic defects together drive reduced muscle regeneration and heightened fibrosis.",FEBS Journal,Satellite Cell Pool Decline,2019
Intrinsic and Extrinsic Drivers of Satellite Cell Dysfunction,"Aged muscle stem cells are impaired due to combined changes in their environment and their internal regulatory systems. Extrinsically, the aged niche exhibits increased TGFβ and FGF signaling, reduced Notch activity, and decreased fibronectin deposition, all of which distort activation–differentiation balance and impair self-renewal. Fibro-adipogenic progenitors (FAPs) also lose their supportive role, failing to induce the matricellular protein WISP1 necessary for satellite cell expansion. Systemically, circulating factors such as complement C1q (promoting fibrosis), and decreased levels of rejuvenating hormones like oxytocin and potentially GDF11, influence stem cell behavior. Exercise-induced apelin declines with age but enhances regeneration when restored. Immune dysregulation further contributes: aging macrophages and bone-marrow–derived cells impose TNFα–NFκB–driven inflammatory stress on satellite cells, while excessive STAT3 activation limits symmetric expansion. Reduced IL-33–dependent recruitment of regulatory T cells (Tregs) hampers the transition from pro- to anti-inflammatory phases, restricting muscle repair.",FEBS Journal,Drivers of Satellite Cell Dysfunction,2019
Environmental Influences on Stem Cell Aging,"Aging of the systemic environment plays a decisive role in satellite cell decline. Classic heterochronic transplantation and parabiosis experiments show that young blood rejuvenates old stem cells, while aged systemic factors suppress regeneration even in youthful tissue. Elevated circulating Wnt-like signals promote aberrant fibrogenic differentiation in aged satellite cells, contributing to maladaptive tissue remodeling. Conversely, rejuvenating systemic molecules—such as oxytocin and the exercise-inducible myokine apelin—decline with age and demonstrate the capacity to restore regenerative potential when replenished. Additional niche-level alterations — including chronic inflammation, overactive TNFα/NFκB and JAK/STAT signaling, and impaired Treg-mediated immune transitions — collectively generate a non-permissive environment for effective stem cell-mediated repair. These findings underscore that satellite cell aging is not solely a cell-autonomous process but is strongly dictated by age-associated environmental dysregulation.",FEBS Journal,Environmental Regulation of Satellite Cell Aging,2019
Epigenetic Dysregulation in Aged Satellite Cells,"Aging causes extensive epigenetic reprogramming in satellite cells, translating chronic environmental stress and accumulated molecular damage into dysfunctional gene expression patterns. Comparative studies show that aged quiescent satellite cells acquire increased repressive chromatin marks, altering transcription of genes regulating self-renewal and lineage commitment. In contrast, aged activated satellite cells exhibit abnormally permissive chromatin states that inappropriately activate developmental pathways, impairing proper myogenic progression. These genome-wide alterations contribute to the transition of satellite cells into a pre-senescent state, especially in geriatric muscles. A key locus affected by aging is p16INK4a: aged satellite cells display loss of ubiquitinated H2A—a Polycomb-associated repressive mark—leading to derepression of p16INK4a. The aging process also reduces expression of the transcriptional repressor Slug, further enabling p16INK4a activation and promoting senescence. Beyond intrinsic effects, epigenetic alterations within the muscle niche—such as increased enhancer activation of ECM genes marked by H3K27ac—promote fibrosis and reduce myogenic potential. These modifications demonstrate that epigenetic dysregulation is both a driver and amplifier of satellite cell aging.",FEBS Journal,Epigenetic Regulation in Satellite Cell Aging,2019
Genomic Instability and Accumulated DNA Damage,"Despite their predominantly quiescent state—which lowers replication-induced damage—satellite cells accumulate significant DNA lesions with age. Aged satellite cells show elevated γH2AX foci, indicating persistent DNA double-strand breaks. Whole-genome sequencing reveals a progressive increase in somatic mutations across decades of human life, confirming the accumulation of genomic instability as a major contributor to stem cell aging. DNA damage not only compromises genomic integrity but also perturbs the epigenetic landscape, as many DNA repair pathways intersect with chromatin-regulatory mechanisms. However, the precise environmental factors and signaling pathways that converge to induce these changes in aged satellite cells remain incompletely understood. Clarifying these mechanisms would provide important insight into strategies for preserving or restoring genomic health in aging stem cells.",FEBS Journal,Genomic Instability in Muscle Stem Cell Aging,2019
Proteostasis Collapse and Autophagic Decline,"Long-lived quiescent satellite cells are highly vulnerable to proteostatic stress because they cannot dilute damaged proteins or organelles through cell division. Healthy quiescent satellite cells rely on robust autophagy to remove dysfunctional mitochondria and aggregated proteins. With aging, a marked decline in autophagic flux leads to the accumulation of defective organelles and elevated levels of reactive oxygen species (ROS). These metabolic and oxidative stresses reinforce epigenetic derepression of p16INK4a, directly driving satellite cells into senescence. Evidence suggests that age-related autophagy decline may stem from oscillatory dysregulation of autophagy gene expression. Because proteostasis is a central determinant of stem cell quiescence and survival, impaired autophagy represents a major mechanism underlying satellite cell aging.",FEBS Journal,Autophagy and Proteostasis in Satellite Cell Aging,2019
Metabolic Rewiring and Mitochondrial Dysfunction,"Autophagy plays additional roles during satellite cell activation by supplying the rapid energy required for proliferation. When autophagy is insufficient, AMPK/p27Kip1 signaling becomes critical in determining satellite cell fate. Artificial activation of this pathway improves autophagy, reduces senescence markers, and enhances transplantation potential of aged satellite cells. Mitochondrial oxidative respiration is also essential for satellite cell function. Aging reduces NAD+ levels, impairing the mitochondrial unfolded protein response and promoting senescence. Mitochondrial DNA damage further disrupts bioenergetics and structural integrity. The anti-aging protein α-Klotho, diminished in aged satellite cells, is required to maintain mitochondrial function; its loss recapitulates mitochondrial defects seen in old cells. Additionally, senescent human satellite cells show oxidation of glycolytic enzymes, leading to impaired glucose metabolism and altered energy substrate usage. This metabolic reprogramming represents a hallmark of stem cell aging and highlights the tight coupling between cellular energetics, proteostasis, and epigenome stability.",FEBS Journal,Metabolic and Mitochondrial Regulation of Satellite Cells,2019
Ex Vivo Rejuvenation of Aged Satellite Cells,"Multiple interventions can restore intrinsic functionality of aged satellite cells when applied ex vivo. One strategy is the genetic repression of p16INK4a, which restores quiescence, enhances proliferation, and reinstates regenerative potential in aged satellite cells. Similarly, pharmacological inhibition of p38 MAPK reduces expression of cell cycle inhibitors such as p16INK4a and enables the re-establishment of asymmetric division—a hallmark of functional stem cell behavior. Satellite cells treated with p38 inhibitors show significantly improved engraftment capacity in transplantation experiments. These ex vivo manipulations highlight the reversibility of several intrinsic aging phenotypes. Additional approaches include modulating JAK/STAT signaling ex vivo, which improves transplantation efficiency by reducing aberrant differentiation commitment and restoring balanced expansion. Together, these strategies demonstrate that many cell-intrinsic defects associated with aged satellite cells—including senescence, impaired self-renewal, and reduced proliferative capacity—can be reversed through targeted molecular interventions before transplantation.",FEBS Journal,Satellite Cell Rejuvenation Strategies,2019
Local and Systemic In Vivo Rejuvenation Strategies,"Several in vivo strategies can restore regenerative capacity in aged skeletal muscle by targeting both the niche and systemic environment. Systemic restoration of autophagy enhances satellite cell quiescence and regenerative potential, while systemic oxytocin supplementation improves activation and proliferation of aged satellite cells. The matricellular protein WISP1, reduced in aged fibro-adipogenic progenitors, can be systemically delivered to enhance myogenic commitment. Systemic delivery of α-Klotho improves mitochondrial bioenergetics and reduces senescence in aged muscle stem cells. Local interventions also show strong effects: intramuscular delivery of fibronectin or β1-integrin activators restores integrin-dependent signaling required for satellite cell responsiveness; Wnt inhibitors locally reverse aberrant fibrogenic conversion; and IL-33 restores age-impaired Treg recruitment essential for proper regeneration. Additionally, JAK/STAT inhibitors delivered intramuscularly during regeneration improve satellite cell expansion and muscle repair in aged mice. These interventions collectively demonstrate that targeting extracellular signaling, immune modulation, and ECM integrity can rejuvenate stem cell function in aged muscle.",FEBS Journal,Niche and Systemic Rejuvenation,2019
Whole-Body Rejuvenation Approaches Targeting Common Hallmarks of Aging,"Interventions that act on the entire organism can also improve satellite cell function by addressing broad hallmarks of aging. Caloric restriction, rapamycin treatment, and senescent cell ablation alleviate chronic inflammation and proteostatic decline, enabling more effective satellite cell responses. Supplementation with nicotinamide riboside restores NAD+ levels, improving mitochondrial function and reducing senescence in aged satellite cells. Targeting chronic inflammation through systemic NFκB inhibition enhances myogenic function in old muscles. IL-33 supplementation, delivered locally or systemically, restores Treg recruitment required for proper macrophage transitions and regenerative outcomes. Inhibition of JAK/STAT signaling enhances regenerative capacity, but these interventions must be applied with caution: inflammatory pathways are essential for early phases of regeneration, as excessive suppression may impair repair in young muscle. These systemic strategies highlight conserved molecular pathways—autophagy decline, NAD+ depletion, chronic inflammation—as actionable targets for improving aged muscle regeneration.",FEBS Journal,Systemic Anti-Aging Interventions,2019
Stem Cell-Based Therapies and Limitations in Aged Muscle,"Satellite cells and myogenic progenitors hold therapeutic potential for reversing muscle aging, although their contribution to sarcopenia remains debated. While lifelong satellite cell ablation does not induce premature sarcopenia in sedentary mice, satellite cells contribute to exercise-induced hypertrophy and maintenance of muscle mass. Preserving functional satellite cells in old muscle prevents sarcopenia, as shown by spry1 overexpression. Proof-of-concept transplantation studies reveal that satellite cells delivered to young muscle can increase muscle mass and force throughout life. However, translating these findings to aged or sarcopenic muscle is challenging: aged environments exhibit chronic inflammation, impaired niche signaling, and reduced pro-regenerative cues, all of which hinder engraftment and myogenic differentiation. Successful engraftment requires an injury signal, which is not clinically feasible for sarcopenia. Exercise-induced injury can enhance engraftment, but aged muscle fails to upregulate key exerkines like apelin, reducing this effect. Thus, effective cell therapy will require co-adjuvant strategies that simulate pro-regenerative signals, modulate inflammation, or introduce exercise-mimicking molecules to support donor cell survival, expansion, and differentiation in aged environments.",FEBS Journal,Stem Cell Therapy for Sarcopenia,2019
Overview of Muscle Regenerative Decline with Aging,"Aging is characterized by the progressive dysfunction of most tissues and organs, which has been linked to the regenerative decline of their resident stem cells over time. Skeletal muscle provides a stark example of this decline. Its stem cells, also called satellite cells, sustain muscle regeneration throughout life, but at advanced age they fail for largely undefined reasons. Here, we discuss current understanding of the molecular processes regulating satellite cell maintenance throughout life and how age-related failure of these processes contributes to muscle aging. We also highlight the emerging field of rejuvenating biology to restore features of youthfulness in satellite cells, with the ultimate goal of slowing down or reversing the age-related decline in muscle regeneration.",FEBS Journal,Satellite Cells and Aging,2019
"Stem Cells, Tissue Maintenance, and Regenerative Requirements","Stem cells are rare and specialized cells within a fully differentiated tissue that are essential for host-organ repair and renewal. In an adult organism, stem cell populations have the unique capacity both to self-renew and to generate progeny that differentiates to replace lost or damaged cells. In most instances, stem cell properties and behavior are determined by the regenerative requirements of the host tissue: High-turnover tissues like the intestine or the hematopoietic system maintain active populations of stem- or progenitor-cell populations, whereas tissues like the skeletal muscle maintain most of their stem cell pool in a quiescent state, activating it only upon injury. The central role played by stem cells in tissue replacement throughout the human lifespan means that their functional decline often results in compromised organ maintenance and regeneration. At the same time, stem cell-based therapies hold immense potential for regenerative medicine to restore or rejuvenate tissues.",FEBS Journal,Stem Cell Biology,2019
"Aging, Systemic Dysregulation, and Satellite Cell Function","Aging is accompanied by a progressive decline in tissue function and increased vulnerability to disease. Current understanding of the aging process centers on the interplay of cell-intrinsic, intercellular communication and systemic dysregulations. In this context, the adult stem cell is simultaneously an important integrator of multiple age-related alterations and a key contributor to the progression of age-related tissue dysfunction. Skeletal muscle harbors a population of quiescent stem cells, called satellite cells, that are essential for its extraordinary regenerative capacity. Upon injury, satellite cells exit quiescence and proliferate, giving rise to a population of committed progenitors capable of engaging the myogenic program to generate new myofibers and replace damaged tissue. This remarkable regenerative capacity is greatly affected by the aging process and is associated with an age-related decline in satellite cell numbers and functionality.",FEBS Journal,Satellite Cells and Aging,2019
"Satellite Cells, Sarcopenia, and Rejuvenation Potential","Skeletal muscle aging is characterized by a loss of mass and decline in muscle strength, a phenotype that broadly defines sarcopenia. Although the involvement of stem cells in the etiology of sarcopenia is debated, the maintenance of a healthy population of satellite cells or the exogenous delivery of a rejuvenated progenitor population to the aging muscle has the potential to correct acquired defects that give rise to age-related muscle wasting. Skeletal muscle is thus an attractive model for the study of stem cell and tissue aging, as well as a potential future target tissue for stem cell-based rejuvenating interventions. In this review, we examine and discuss our current understanding of the determinants of satellite cell aging and their contribution to age-related loss of regenerative capacity in skeletal muscle. We also highlight recent advances in the field pointing to promising rejuvenating interventions that could help restore skeletal muscle regenerative capacity in the elderly. Finally, we discuss the potential of stem cell-based interventions as rejuvenating strategies to prevent or delay age-related loss of muscle mass and force.",FEBS Journal,Satellite Cells and Sarcopenia,2019
Satellite Cell Pool Decline and Quiescence Disruption,"Skeletal muscle aging is accompanied by a considerable reduction in the size of the satellite cell pool. The decline in satellite cell numbers occurs in the early stages of muscle aging and is likely due to changes affecting the niche and to cell-autonomous alterations that together disturb the proper balance between cell quiescence, proliferation, and apoptosis. Increased expression of myofiber-derived Fibroblast growth factor (FGF)2 in old muscles has been shown to bring satellite cells out of quiescence, causing spontaneous mitogenic activity that can contribute to the exhaustion of the satellite cell pool. Additionally, FGF2 inhibits the expression of sprouty1, an intracellular inhibitor of the Extracellular signal-regulated kinase (ERK)/Mitogen-activated protein kinase (MAPK) signaling pathway, and mouse models with satellite cell-specific deletion of Sprouty1 have defects in the muscle stem cell pool. Sprouty1-null cycling satellite cells are impaired in their capacity to return to quiescence, succumbing instead to apoptosis.",FEBS Journal,Satellite Cell Aging,2019
Niche Deficiencies and Early Aging-Related Satellite Cell Loss,"Depletion of resident satellite cells after regenerative events in aged muscles also involves deficiencies in the aged niche microenvironment. A recent study revealed that an age-related decline in Notch activators in the niche provokes satellite cell death by mitotic catastrophe, impairing proliferative expansion of muscle stem cell in aged mice. The idea that stem cell survival is an important factor in the maintenance of the satellite cell pool is further supported by the observations that old human satellite cells are susceptible to nuclear apoptosis and that the promotion of anti-apoptotic pathways in aged satellite cells improves muscle regenerative capacity.",FEBS Journal,Satellite Cell Aging,2019
Functional Defects and Pre-Senescence in Aged Satellite Cells,"Although the satellite cell pool diminishes with age, a fraction of the satellite cells survives within the skeletal muscle until very old age. However, even when they are present, these satellite cells have functional defects that undermine their ability to sustain muscle regenerative capacity. The defects in satellite cell proliferative activity in response to regenerative pressure seen in the early stages of the aging process are exacerbated in very old (geriatric) mice. Whereas the early defects are driven by alterations in the environment, at advanced ages quiescent satellite cells are intrinsically changed and become pre-senescent, a state that leads to full senescence in response to regenerative pressure. As a consequence of these defects, old satellite cells show a reduced capacity for activation and expansion after injury and produce insufficient progeny to sustain muscle regeneration.",FEBS Journal,Satellite Cell Function,2019
Differentiation Defects and Fibrogenic Conversion in Aged Muscle,"Moreover, the progeny of muscle stem cells that overcome these limitations have a limited differentiation capacity, with poor myogenic potential and a tendency to commit to alternative lineages. The consequence of these defects in old skeletal muscle is an enhanced fibrotic response to injury. These functional alterations have multiple underlying molecular causes, including altered signaling cues from the aging environment and intrinsic alterations in genomic integrity and metabolic regulation within the satellite cell.",FEBS Journal,Satellite Cell Function,2019
Extrinsic and Intrinsic Drivers of Satellite Cell Aging,"Changes in niche-derived and systemic signaling molecules, along with intrinsic changes in the satellite cell, contribute to the functional impairments of aged muscle stem cells and the consequent defects in regenerative capacity of the aged skeletal muscle. Changes in the niche and systemic environment include alterations in the inflammatory signaling and changes in secreted and local growth factors that synergize with alterations in ECM signaling to impair satellite cell function. Intrinsic changes to the satellite cell include epigenetic changes and defects in autophagy, leading to increased senescence and apoptosis.",FEBS Journal,Satellite Cell Aging,2019
Aging Niche and Systemic Effects on Satellite Cells,"In a young organism, satellite cell function is tightly regulated by signals originating from the niche and the systemic environment. Alterations to the surrounding milieu have profound effects on satellite cell quiescence, differentiation, and self-renewal, and deterioration of the extracellular environment is one of the main factors determining age-related stem cell dysfunction. Heterochronic tissue transplant studies revealed that the age of the host animal was a key determinant factor of the regenerative success. Studies employing heterochronic parabiosis demonstrated that the regenerative capacity of the muscle is modulated by exposure to blood from an animal of a different age. A recent study obtained similar results through direct blood exchange, supporting the idea that circulatory factors directly affect satellite cell aging.",FEBS Journal,Satellite Cell Niche,2019
Myofiber Signaling Changes and Their Impact on Satellite Cells,"Several signaling molecules, including niche-derived signaling cues and systemic factors, have been identified as mediators of the extrinsic effects of the aging environment on satellite cell function. Changes in the myofiber, the most abundant source of niche signaling, have a significant impact on satellite cell activity: an age-related increase in Transforming growth factor (TGF)β and FGF signaling from the myofiber synergize with a decline in Dll-driven Notch signaling and decreased deposition of the Extracellular matrix (ECM) protein fibronectin to disrupt satellite cell activity. Changes in TGFβ and Notch activity contribute to an imbalance in satellite cell activation and differentiation cues, while increased FGF signaling breaks satellite cell quiescence leading to stem cell loss.",FEBS Journal,Niche Signaling and Aging,2019
"Fibronectin, FAPs, and Impaired Regeneration","The remaining satellite cells become unresponsive to FGF under regenerative pressure and fail to expand or self-renew, a defect exacerbated by defective fibronectin deposition in old muscles undergoing regeneration, and consequent impairment in integrin signaling. Aging also impairs the supportive function of fibro-adipogenic progenitors (FAPs), which fail to induce the matricellular protein Wnt1-inducible signaling pathway protein 1 (WISP1), required for satellite cell expansion and commitment.",FEBS Journal,Satellite Cell Niche,2019
Systemic Regulators Identified by Parabiosis,"Heterochronic parabiosis has identified systemic factors as key regulators of muscle stem cell function. These include the unconventional Wnt-activating ligand complement component 1q, the TGFβ family member growth differentiation factor 11 (GDF11), and the hormone oxytocin. Increased Wnt signaling driven by systemic factors promotes aberrant fibrogenic commitment in aged satellite cells, leading to fibrotic response upon regenerative pressure. Conversely, oxytocin and GDF11 have been identified as rejuvenating factors decreased in the circulation of old animals. However, the effects of GDF11 on muscle and age-related changes remain debated, with contradictory reports highlighting the need for further investigation.",FEBS Journal,Systemic Aging Factors,2019
Immune Dysregulation and Inflammatory Drivers of Aging,Age-related alterations in the immune environment have emerged as important contributors to satellite cell impairments in old muscles. Regenerative success depends on a regulated immune response involving multiple immune cell types and coordinated pro- and anti-inflammatory signaling. Bone marrow transplant from old donors is sufficient to impair satellite cell function in young mice. An age-related increase in TNFα signaling and over-activation of the NFκB pathway disrupt satellite cell function. Increased STAT3 activation in old satellite cells compromises symmetric expansion by promoting direct myogenic commitment and limits regenerative capacity. Impaired Regulatory T cell (Treg) signaling and reduced Interleukin 33 (IL-33)-dependent recruitment of Tregs further compromise regeneration.,FEBS Journal,Inflammation and Satellite Cells,2019
Epigenetic Remodeling and Chromatin Changes in Aged Satellite Cells,"Satellite cell aging is accompanied by genome-wide epigenetic changes that translate abnormal signaling from the aged environment and a lifelong accumulation of molecular damage into altered gene expression programs. Comparative epigenomic analysis of young and old satellite cells has revealed complex changes with distinct consequences for quiescent and activated stem cells: While in quiescent satellite cells, an age-related increase in repressive chromatin marks can contribute to changes in the expression of genes involved in stem cell self-renewal and lineage commitment, in old activated satellite cells permissive chromatin states cause the aberrant induction of developmental pathways that impair stem cell function. Epigenetic changes are known to play an important role in the conversion of quiescent satellite cells to a pre-senescent state in geriatric mice. Specific analysis of the p16INK4a locus in satellite cells isolated from geriatric muscles revealed the loss of ubiquitinated H2A, a chromatin repressive mark associated with the polycomb repressor complex 1. The transcriptional repressor Slug was also found to be downregulated in aged satellite cells, contributing to the derepression of the p16INK4a gene and the conversion of aged muscle stem cells to a pre-senescent state.",FEBS Journal,Epigenetics and Aging,2019
"Chromatin Accessibility, ECM Signaling, and Fibrogenic Conversion","Future studies of global chromatin accessibility will likely reveal other changes to specific genomic loci that trigger age-related perturbations in satellite cell function. Epigenetic alterations can also impact the health of neighboring cells in the satellite cell niche, indirectly contributing to muscle stem cell loss of function. Analysis of histone modifications in whole human skeletal muscle tissues found an age-associated increase in the active enhancer marker H3K27ac. In mouse models, this enhancer activation is associated with the upregulation of ECM genes during aging, contributing to a decline in myogenic capacity and increased fibrogenic conversion of aged satellite cells.",FEBS Journal,Epigenetics and ECM,2019
"DNA Damage, Mutational Burden, and Genomic Instability","The accumulation of DNA damage is a potential source of genomic instability and an important contributor to global changes in the epigenome with aging. The quiescent satellite cell combines a low risk of replication-induced DNA damage with highly efficient mechanisms of DNA repair. However, satellite cells isolated from aged mice still have an elevated number of foci containing the DNA damage marker γH2AX. Whole-genome sequencing of human satellite cells isolated from individuals of different ages revealed an age-related increase in the somatic mutation burden, reinforcing the notion that loss of genomic integrity is an important factor in satellite cell aging. Knowledge remains limited about how age-related environmental changes converge to drive alterations in the satellite cell epigenetic landscape, but understanding these processes could provide insight into expanding the muscle stem cell healthspan.",FEBS Journal,Genomic Stability,2019
"Autophagy Decline, Proteostatic Stress, and ROS Accumulation","The lifelong accumulation of altered or damaged proteins is an important factor in age-related stem cell dysfunction, affecting multiple intracellular signaling pathways and ultimately driving genomic instability, senescence, and apoptosis. Satellite cells, mostly quiescent throughout life, cannot eliminate toxic organelles and protein aggregates through cell division and are therefore particularly susceptible to proteostatic stress. Mechanisms of intracellular macromolecular clearance, such as autophagy, play a fundamental role in the maintenance of satellite cell quiescence. Impairment of autophagy causes the accumulation of damaged mitochondria and the generation of high reactive oxygen species (ROS) levels, propagating protein and DNA damage. During aging, a decline in autophagic flux and the consequent increase in ROS levels are directly linked to the epigenetic derepression of the p16INK4a locus in old satellite cells and their entry into senescence.",FEBS Journal,Autophagy and Aging,2019
"Autophagy Oscillation, AMPK Signaling, and Energy Stress","The decline in defective organelle clearance in aged quiescent satellite cells may be related to an oscillatory rewiring that affects the expression of autophagy genes. Autophagy is also required for rapid energy mobilization to meet the metabolic demands of satellite cell activation. The 5' AMP-activated protein kinase (AMPK) signaling pathway plays an important role in satellite cell fate decisions when autophagy is unable to meet the energy needs of activated cells. Ectopic activation of the AMPK/p27Kip1 pathway enhances autophagy and reduces markers of cell senescence in aged satellite cells, improving their transplantation potential.",FEBS Journal,Metabolism and Stem Cells,2019
"Mitochondrial Dysfunction, NAD+ Decline, and α-Klotho","The interconnection between proteostatic and metabolic pathways is illustrated by studies showing that mitochondrial oxidative respiration is fundamental for the functional maintenance of satellite cells. A reduction in the levels of oxidized nicotinamide adenine dinucleotide (NAD+) affects the mitochondrial protein response, ultimately leading to satellite cell senescence. Mitochondrial DNA damage can also affect mitochondrial ultrastructure and bioenergetics. Levels of α-Klotho, a membrane-bound and circulating hormonal protein, are decreased in satellite cells from aged mice, and genetic knockdown of α-Klotho in young muscle stem cells provokes an aged phenotype with decreased mitochondrial bioenergetics, mitochondrial DNA damage, and increased senescence.",FEBS Journal,Mitochondria and Aging,2019
Replicative Senescence and Metabolic Shifts in Human Satellite Cells,"Replicative senescence of human satellite cells is associated with the oxidation of glycolytic enzymes, resulting in impaired glucose metabolism and a metabolic shift of energy substrates in senescent muscle stem cells. These metabolic impairments further contribute to the reduced regenerative potential of aged satellite cells.",FEBS Journal,Senescence and Metabolism,2019
Overview of Satellite Cell Rejuvenation Strategies,"Interventions for satellite cell rejuvenation
Considering the combined role of extrinsic and intrinsic determinants on satellite cell aging, interventions that aim to restore the functionality of an aged muscle stem cell are likely to require combinatorial strategies that target age-dependent deregulations in niche signaling and cell-intrinsic alterations simultaneously. The studies described in the previous chapter have contributed promising new approaches to the restoration of regenerative capacity in sarcopenic muscles.

Interventions for satellite cell rejuvenation. Rejuvenation of the regenerative capacity of aged skeletal muscle can be achieved through multiple interventions, including ex vivo rejuvenation of the satellite cell pool prior to transplantation, intramuscular delivery of agents that regulate niche signaling to improve satellite cell function, and systemic treatments that target satellite cell and niche-specific age-related alterations. Ex vivo interventions include inhibition of STAT and p38 or genetic repression of the p16INK4a locus. Niche-specific interventions, delivered through intramuscular injections, include inhibition of STAT and Wnt signaling, or supplementation of fibronectin, integrin signaling activators, or Il-33. Systemic interventions include modulators of inflammation, hormones, growth factors, and metabolic regulators targeting specific pathways affected in the aged skeletal muscle and/or the aged satellite cell.",FEBS Journal,Satellite Cell Rejuvenation,2019
Ex Vivo Manipulations and Restoration of Intrinsic Defects,"Ex vivo manipulations of aged satellite cells have proven to be effective strategies to reverse some of the intrinsic alterations limiting their regenerative potential. These manipulations include genetic interventions to silence p16INK4a expression, thereby restoring quiescence and regenerative capacity to the aged satellite cell. Similarly, ex vivo pharmacological inhibition of p38 MAPK signaling decreases the expression of cell-cycle inhibitors, such as p16INK4a, and restores asymmetric division in satellite cells, contributing to enhanced regenerative potential of aged satellite cells in muscle transplantation experiments.",FEBS Journal,Satellite Cell Rejuvenation,2019
Local and Systemic Interventions Targeting Aged Satellite Cells,"In vivo, local and systemic interventions have also shown promise in reversing age-related satellite cell defects. For example, systemic pharmacological treatments to restore basal autophagy flux preserved quiescence and muscle stem cell regenerative capacity in old muscles. Similarly, systemic delivery of oxytocin restores age-related regenerative capacity in old muscles, promoting satellite cell activation and proliferation, while systemic delivery of WISP1 during a regenerative event improves myogenic commitment and regenerative success. Moreover, systemic delivery of exogenous α-Klotho improves muscle stem cell bioenergetics and improves regenerative capacity in aged animals. Local delivery of fibronectin or β1-integrin activators also restores satellite cell responsiveness in old mice, enhancing regenerative capacity, while intramuscular injection of Wnt inhibitors restore the myogenic potential of old muscle stem cells.",FEBS Journal,Satellite Cell Rejuvenation,2019
Whole-Organism Rejuvenation and Inflammatory Pathways,"Rejuvenating interventions able to target the whole organism have also a positive impact on satellite cell function during aging. Successful interventions include caloric restriction, rapamycin treatment, supplementation with the NAD+ precursor nicotinamide riboside, senescent cell ablation, and in vivo reprogramming. These studies anticipate the existence of common hallmarks of aging associated with satellite cell loss of function in old animals, which can be considered common targets for intervention. Consistently, targeting chronic inflammation (a shared feature of several age-related pathologies) through systemic treatment with an inhibitor of NFκB activation improves myogenic function in aged satellite cells. The regenerative capacity of old skeletal muscle is also improved by intramuscular or systemic supplementation with IL-33, which reestablishes the recruitment of Tregs into injured muscles. Another related approach is the inhibition of JAK/STAT pathway: Isolated satellite cells treated ex vivo with JAK-STAT inhibitors show improved engraftment in transplantation experiments, and intramuscular delivery of STAT inhibitors during regeneration in old mice also significantly improved muscle regenerative capacity.",FEBS Journal,Systemic Rejuvenation Interventions,2019
Caution with Modulating Inflammatory and STAT Signaling,"However, inflammatory pathways are essential regulators of the muscle regenerative response, and pro-inflammatory signaling is essential for the activation phase of satellite cell function; therefore, caution is advised when using these interventions. For example, inhibition of pro-inflammatory mediators impairs the regenerative response in young animals, and Interleukin 6-driven STAT activation may be essential for myogenic commitment during regenerative events and satellite cell-dependent muscle growth during hypertrophy.",FEBS Journal,Inflammation and Regeneration,2019
Role of Satellite Cells in Muscle Aging and Sarcopenia,"Stem cell-based interventions in muscle aging
The use of stem cells as sources for tissue repair and renewal introduces their potential as rejuvenating interventions. In addition to their primary role as agents of tissue regeneration after injury, satellite cells also contribute to the homeostatic maintenance of muscle fibers. However, debate continues about the contribution of satellite cells to the pathogenesis of sarcopenia. Experiments involving lifelong ablation of satellite cells did not produce precocious or exacerbated signs of sarcopenia in sedentary mice, leading the authors to conclude that the loss of satellite cells that happens during aging does not contribute the development of sarcopenia. However, in young animals, exercise increases muscle mass through the combined effect on new fiber production in response to exercise-induced fiber damage and the hypertrophy of existing fibers, with both processes depending, at least in part, on satellite cell function.",FEBS Journal,Stem Cells and Sarcopenia,2019
"Satellite Cells, Daily Activity, and Preservation of Muscle Mass","It is thus possible that the wear and tear associated with daily activity in humans provides a signal for satellite cell dependent maintenance of muscle mass that was not captured in the ablation study in mice. Moreover, given the functional impairment of satellite cells that persist in the old skeletal muscle, these may be unable to contribute to fiber maintenance in old age. Supporting this view, a recent study showed that preserving functional satellite cells in the old skeletal muscle (through the overexpression of spry1) preserves muscle mass and force and attenuates sarcopenia development.",FEBS Journal,Stem Cells and Sarcopenia,2019
Therapeutic Potential of Muscle Stem Cells in Sarcopenia,"Nevertheless, the possible causal relationship of satellite cell loss of function to sarcopenia does not negate the potential of satellite cells as an intervention to restore sarcopenic muscle. The use of healthy muscle stem cells to generate new muscle fibers, and/or to contribute new myonuclei to existing fibers, can be envisioned as a viable strategy to correct the defects that drive age-related muscle wasting, restoring muscle mass and force, and rejuvenating the sarcopenic muscle. In a proof-of-principle study supporting this idea, Hall et al. showed that satellite cells transplanted into injured muscle of young mice (along with the associated muscle fiber) contribute to an increase in muscle mass and force that persists as the mice age. In this model, the lifelong persistence of the enhanced muscle mass results from an increase in myofiber numbers and a progressive myofiber hypertrophy, attributed to myonuclear accretion.",FEBS Journal,Stem Cell Therapy for Sarcopenia,2019
Limitations of Progenitor Cell Delivery in Aged Muscle,"Satellite cells and other myogenic progenitor-cell types have been successfully used in young animals to ameliorate the effects of degenerative muscle diseases caused by genetic conditions. However, there are no published reports of direct delivery of muscle progenitors into the muscles of old sarcopenic mice. Moreover, while these studies are promising, they have also brought to light several limitations with important implications for the use of cell therapy applied in aged skeletal muscle. Successful engraftment with the current methodology used in preclinical settings to deliver progenitor cells to the muscle requires a concurrent injury, which is not a viable strategy for the treatment of sarcopenic muscles. Moreover, the systemic and local environment of chronic inflammation in old organisms presents a further obstacle to the success of these interventions and would likely worsen survival, engraftment, and the myogenic potential of transplanted progenitors.",FEBS Journal,Stem Cell Therapy for Sarcopenia,2019
"Inflammation, Immune Environment, and In Situ Modulation","The age-related inflammation and dysregulated immune environment have been consistently demonstrated to have a negative impact on satellite cell function, limiting their myogenic potential and regenerative capacity, contributing significantly to the development of sarcopenia. In a model of Duchene muscular dystrophy (DMD), macrophages have been used to deliver pro-activating signals that enable expansion of myoblasts in situ before differentiation is initiated. These experiments, together with the use of pharmacological interventions that promote myoblast migration, showed that it is possible to modulate muscle progenitor activity in situ to promote muscle repair. Successful cell therapies for sarcopenia will likely require the development of concurrent interventions to provide the pro-repair signals elicited by the injury and to promote myogenesis and the subsequent steps of muscle regeneration that are inhibited by the pro-inflammatory environment.",FEBS Journal,Stem Cell Therapy for Sarcopenia,2019
"Exercise, Exerkines, and Limits of Pro-Repair Signaling in Aging","One possible source of such signals is exercise. In DMD models, the injury signal produced by exercise increases the success of muscle progenitor engraftment, removing the need for a concurrent injury induced by toxins or chemicals. However, analysis of muscles subjected to overload induced by synergistic ablation suggests that physical exercise is insufficient to induce myofiber hypertrophy in the old skeletal muscle, but can improve satellite cell responses in injury models. Thus, it is likely that in aged animals, the ability of exercise to induce pro-growth and pro-repair signals is not fully functional. Recent work suggests that this is indeed the case, at least for the exerkine apelin. The authors showed that young but not aged mice or humans increase apelin levels in response to exercise. Since low apelin levels are associated with sarcopenia and apelin signaling promotes fiber hypertrophy and satellite cell-mediated muscle repair, it is likely that the lack of such signals in aging would blunt the beneficial effects of exercise in promoting myoblast engraftment. It is therefore critical to identify other factors that mediate the beneficial effects of exercise and that could be used as co-adjuvants in skeletal muscle cell therapy in sarcopenia.",FEBS Journal,"Exercise, Exerkines, and Therapy",2019
Neuroinflammaging and Immune Shifts in Ageing,"Although the central nervous system (CNS) is generally considered an immune-privileged organ, it maintains distinguished immune niches with unique immune cells, such as microglia and glia. Constant communication is maintained between the CNS and peripheral immune cells through the blood–brain barrier (BBB), which tightly regulates immune responses. The term ""inflammaging"" describes chronic low-grade systemic inflammation as a hallmark of the ageing process. Normal ageing is also associated with neuroimmune shifts from a resting to a hyperactive and inflammatory state, termed 'Neuroinflammageing'. Microglial activation, hyperactive astrocytes, cytokine and chemokine release, BBB leakage, infiltration of peripheral immune cells into the CNS, and a dysregulated glymphatic system are key features. Age-associated escalation of neuroinflammation enhances immune surveillance and helps eliminate altered or infected cells in the ageing brain but also increases vulnerability to CNS tumors and infections.",Biogerontology,Neuroinflammaging,2025
"Systemic Inflammation, Senescence, and Neuroimmune Dysfunction","The molecular mechanisms of cellular senescence and ageing—including DNA damage, epigenetic changes, metabolic shifts, mitochondrial dysfunction, and protein misfolding—have inflammation as their root cause. These profound changes in the ageing immune system, along with escalating baseline systemic inflammation, result in neuropathology and diminished responses to foreign pathogens in older adults. Since age-related alterations in neuroimmune responses are potential drivers of brain health decline, identifying mechanistic relationships and molecular targets is essential for designing interventions to promote healthy brain ageing. Maintaining immune resilience may help prevent progression of immune-mediated neuroinflammation. Interventions such as dietary or caloric restriction, caloric restriction mimetics, medications like metformin, mTOR inhibitors, and senolytics may greatly contribute to immune and brain health.",Biogerontology,Neuroimmune Aging,2025
"Reviews on Autophagy, cSVD, Viral Neuroinflammation, and VEGF","The review by Tamatta et al. discusses the interplay of autophagy and cellular senescence with brain immune cells and their role in moderating neuroinflammation in ageing. Defective autophagy and dysregulated neuroimmune systems lead to cytotoxic protein accumulation, damaged organelles, and disrupted senescence, aggravating neuroinflammation and neurological disease. The review by Van der Taelen et al. examines circulating immune cells in cerebral small vessel disease (cSVD), a major cause of stroke and vascular cognitive impairment. Pro-inflammatory immune cell phenotypes contribute to endothelial activation, dysfunctional neurovascular units, BBB disruption, and cognitive decline. Another review by Ghosh and Das Sarma highlights peripheral-CNS cross-talk in coronavirus-induced neuroinflammation in older adults, including mechanisms of viral neuroinvasion and persistent inflammatory responses contributing to long-COVID neurological complications. Agrawal et al. discuss roles of VEGF in vascular development, neuroprotection, and cognitive preservation, and how ageing-related endothelial dysfunction and chronic inflammation disrupt VEGF regulation.",Biogerontology,Neuroimmune Mechanisms,2025
"Ageing, BBB Dysfunction, Myelin Loss, and Gut–Brain Axis","Alaqel et al. review the progressive decline in neurovascular units and BBB integrity with ageing, identifying oxidative stress, endothelial senescence, and tight-junction degradation as key mechanisms underlying BBB dysfunction. Jana and Das Sarma examine age-related myelin loss and axonal degeneration, emphasizing alterations in glial and peripheral immune cell function affecting myelin formation. Ahmadi et al. discuss gut dysbiosis as a contributor to age-related neuroinflammation and cognitive decline, highlighting how fecal microbial transplantation (FMT) may restore microbial balance and support healthy ageing. Conflicting reports exist regarding inflammaging in centenarians. Biscetti et al. analyzed pro- and anti-inflammatory gene expression in brain regions of adult, old, and oldest-old rats, showing significant upregulation of pro-inflammatory cytokines in the oldest-old, with females exhibiting more pronounced changes.",Biogerontology,Ageing and Neuroinflammation,2025
"nAChRs, Cognitive Decline, and Sulforaphane Interventions","Nicotinic acetylcholine receptors (nAChRs), expressed on neurons and glia, play essential roles in CNS and immune functions. Clinical trials targeting the α7 subtype (α7nAChR) show promise for cognitive enhancement in Alzheimer's-related dementias. Kohn et al. provide evidence linking inflammation, peripheral monocyte nAChRs, and cognitive function in older adults, demonstrating subset-specific deficits in anti-inflammatory nAChR activity correlated with cognitive impairment. Santín-Márquez et al. evaluated sulforaphane (SFN), an isothiocyanate, for its ability to modulate SASP factors in ageing. Long-term SFN treatment attenuated pro-inflammatory cytokines, senescence markers, and senescent cell numbers in cortex and hippocampus of adult rats and improved memory, but these effects were absent in old rats. SFN is known for hormetic and polypharmacological activity.",Biogerontology,nAChRs and Anti-Inflammatory Interventions,2025
Importance of Brain Health in Healthy Longevity,"Ageing is characterized by a gradual decline in the efficiency of physiological functions and increased vulnerability to diseases. Ageing affects the entire body, including physical, mental, and social well-being, but its impact on the brain and cognition can have a particularly significant effect on an individual’s overall quality of life. Therefore, enhancing lifespan and physical health in longevity studies will be incomplete if cognitive ageing is overlooked. Promoting successful cognitive ageing encompasses the objectives of mitigating cognitive decline, as well as simultaneously enhancing brain function and cognitive reserve.",Frontiers in Aging Neuroscience,Cognitive Ageing,
Synaptic Changes as the Basis of Age-Related Cognitive Decline,"Studies in both humans and animal models indicate that cognitive decline related to normal ageing and age-associated brain disorders are more likely linked to changes in synaptic connections that form the basis of learning and memory. This activity-dependent synaptic plasticity reorganizes the structure and function of neurons not only to adapt to new environments, but also to remain robust and stable over time. Therefore, understanding the neural mechanisms that are responsible for age-related cognitive decline becomes increasingly important.",Frontiers in Aging Neuroscience,Synaptic Plasticity,
Focus of the Review: Synaptic Plasticity and Modifiable Factors,"In this review, we explore the multifaceted aspects of healthy brain ageing with emphasis on synaptic plasticity, its adaptive mechanisms and the various factors affecting the decline in cognitive functions during ageing. We will also explore the dynamic brain and neuroplasticity, and the role of lifestyle in shaping neuronal plasticity.",Frontiers in Aging Neuroscience,Neural Ageing,
Cognitive Decline and Age-Associated Brain Changes,"The brain, like any other biological system in the body, experiences physiological ageing, which is reflected by a decline in cognitive, social and motor abilities. It includes a decline in information processing, executive functions, planning and working memory. Along with the decline in mental abilities, ageing also predisposes the brain to various diseases. The term “benign forgetfulness of senescence” or “age-associated memory decline” was used to differentiate individuals undergoing memory decline due to ageing from those whose memory impairment is linked to neurological damage or disease.",Frontiers in Aging Neuroscience,Cognitive Ageing,
Evolutionary Conservation of Cognitive Ageing Pathways,"Analysis of various model systems suggests that the genetic pathways regulating cognitive ageing are highly conserved in organisms ranging from yeast, worms, flies to mammals. These models indicate a dynamic association between cognitive functions and ageing. Therefore, it is important to identify key regulators of both cognitive impairment and pathways related to longevity. Interventions directed at longevity pathways could potentially offer benefits in addressing cognitive decline and neurodegenerative conditions, and conversely, targeting cognitive health may also impact longevity pathways.",Frontiers in Aging Neuroscience,Longevity Pathways,
Synaptic Plasticity and Its Age-Related Impairment,"Unlike other systems in the body, a remarkable characteristic of the brain is its intrinsic capacity to adapt to a rapidly changing environment, known as neuronal or synaptic plasticity. This activity-dependent plasticity allows the nervous system to change its connections according to experiences. With ageing, the brain undergoes a gradual decline in its capacity to both physically and functionally adapt to changing or novel environments. This decline in plasticity is implicated in the functional decline of many cognitive functions and increases vulnerability to neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Therefore, mechanisms governing how neural structure and function can be altered also undergo changes throughout the lifespan.",Frontiers in Aging Neuroscience,Synaptic Plasticity,
Knowledge Gaps and Review Objectives,"Although significant advancements have been made in understanding the mechanisms involved in synaptic plasticity, its influence on healthy brain ageing, lifespan extension and adaptive plasticity remains poorly understood. This review details the mechanisms of cognitive decline, including adaptive mechanisms and dysfunction in synaptic plasticity that contribute to the ageing process, with an emphasis on the hippocampus and the factors that make it vulnerable to neurodegenerative disorders. The review also discusses the dynamic brain, the role of lifestyle changes that affect synaptic plasticity, and provides a comparative account of ageing in humans, rodents, and non-human primates as models for ageing research.",Frontiers in Aging Neuroscience,Neural Ageing,
Adaptive Capacity and Resilience of the Ageing Brain,"Evidence from neuroimaging, cognitive psychology, neuropathology, metabolic, cellular and molecular studies points to the adaptability and resilience of the ageing brain. These include metabolic, physiological and neural network adaptations that improve cognitive, mental and physical well-being. Enhancing adaptive mechanisms within neural cells could potentially protect or delay the ageing process. A range of physiological and pharmacological interventions can activate these adaptive mechanisms, including education, social interactions, balanced diet, physical exercise, anti-inflammatory drugs, senolytics, antihypertensive drugs, reducing stress, and mental training such as mindfulness. Collectively, this highlights that the ageing brain exhibits adaptive changes that can delay the onset of functional decline.",Frontiers in Aging Neuroscience,Adaptive Neural Mechanisms,2024
"Neural Excitation, REST Signaling, and Longevity","A study by Zullo et al. showed that reduced neural activity and downregulation of genes that mediate excitatory neurotransmission is an adaptive mechanism that delays cognitive decline and increases lifespan. Targeting excess neural excitation may improve cognition and attenuate Alzheimer's pathology in late-onset AD. The restrictive element-1 silencing transcription factor (REST)/NRSF represses excitation-related genes and is upregulated in humans with extended longevity. Mice lacking REST show increased cortical activity and neuronal excitability with ageing. Similarly, mutations reducing function of C. elegans REST orthologs increase neural excitation and shorten lifespan. REST is activated by stressors including oxidative stress, DNA damage, and amyloid beta, reflecting adaptive brain responses. It also promotes neuroprotection by repressing genes involved in oxidative stress and amyloid toxicity. Exploring targets to activate REST or drugs that reduce excitation (e.g., nemadipine, ivermectin) may offer novel strategies to slow ageing and reduce neurodegenerative risk.",Frontiers in Aging Neuroscience,REST and Neural Excitation,2024
Metabolic Adaptations and Conserved Longevity Pathways,"Metabolic adaptations within the body and brain have played significant roles in the evolution of the primate brain. Loss of metabolic homeostasis is a hallmark of ageing and is characterized by altered regulatory processes controlling cellular functions. Age-related homeostatic disruption is influenced by damage to biological macromolecules, changes in energy metabolism, deregulated tissue integrity, and impaired physiological coordination. Conserved genetic mechanisms have evolved to counteract these processes. Key metabolic pathways with adaptive roles during ageing include insulin/insulin-like growth factor signaling (IIS), mTOR, AMPK and sirtuins. These adaptive metabolic changes provide neuroprotection by decreasing inflammation, activating stress resistance factors, promoting autophagy, or modulating other molecular pathways that influence ageing. These mechanisms can be enhanced through lifestyle changes such as reduced stress, meditation, yoga, physical exercise, dietary approaches, non-invasive brain stimulation, or pharmacological agents including rapamycin, alpha-ketoglutarate, metformin, and NAD+ precursors.",Frontiers in Aging Neuroscience,Metabolic Regulation in Ageing,2024
"Obesity, Metabolic Dysregulation, and Cognitive Ageing","Metabolic dysregulation linked with obesity resembles patterns observed in normal ageing, and obesity can accelerate ageing processes. Obesity in ageing is associated with blood–brain barrier disruption, oxidative stress, neuroinflammation, microglial activation, and impaired hippocampus-dependent learning and memory. From an evolutionary perspective, neural pathways adapted to food scarcity are disrupted by chronic overconsumption and sedentary lifestyles, accelerating brain ageing. Nutritional regulation and gene-targeting therapies focusing on Sirtuin 1, Klotho, p66Shc, and FOXO proteins hold promise in mitigating obesity-driven cognitive decline. Behavioral interventions such as physical exercise, healthy diet, stress management, social engagement, and sleep hygiene offer potential to lessen obesity’s impact on brain health. Physical exercise improves cognitive ageing by influencing cognition directly and indirectly through reduced obesity. Optimization of macro- and micronutrient intake forms a strategy to improve cognitive functioning in the ageing brain.",Frontiers in Aging Neuroscience,Obesity and Cognitive Ageing,2024
Lifestyle Medicine and Brain Health in Ageing,"Applying the six pillars of lifestyle medicine—plant-based nutrition, regular physical activity, stress management, avoidance of harmful substances, quality sleep, and fostering social connections—can enhance neurocognitive functions and support healthy weight and wellbeing. Prioritizing lifestyle changes is therefore crucial in promoting brain health and preventing cognitive decline. Interventions designed to optimize diet, physical activity and stress reduction may form the basis for strategies aimed at improving cognitive functioning and promoting healthy ageing.",Frontiers in Aging Neuroscience,Lifestyle Interventions,2024
General Characteristics of Cognitive Decline in Ageing,"Normal ageing is characterized by significant declines in cognitive performance, especially on tasks necessitating rapid information processing, decision-making, speed of processing, working memory, and executive cognitive function. Decline or loss of brain functions, including learning and memory, and loss of the ability to live independently is one of the greatest fears of ageing. According to the World Health Organisation (WHO), cognitive decline has become a major health concern in old age. As the average age of the population rises, understanding the basis of cognitive decline during ageing is critical for healthy ageing.",Frontiers in Aging Neuroscience,Cognitive Decline,2024
Differential Vulnerability of Memory Systems,"Memory or cognition is not a unitary concept but a complex, multifactorial process. Various memory components are influenced by the ageing process in different ways. Some mental functions, such as verbal ability, certain numerical skills, and general knowledge experience minimal age-related decline. Language skills and vocabulary developed during youth are mostly retained. Ageing primarily affects episodic memory rather than semantic memory. Episodic memory is the recollection of day-to-day events, while semantic memory refers to factual knowledge. While both semantic and episodic memory decline with age, episodic memory shows progressive decline from midlife, whereas semantic memory is affected mostly in late life.",Frontiers in Aging Neuroscience,Memory Systems,2024
"Executive Function, Processing Speed, and Attention","Executive dysfunction is characterized by difficulties in planning, mental flexibility, inhibiting inappropriate actions, distinguishing relevant sensory information, and problem solving. Another hallmark of ageing is the slowdown of information processing or decreased speed in decision-making tasks. Attentive efficiency, which is the ability to sustain focused attention and cognitive processing, is reduced with increasing age. The ability to encode new information also declines, while retention of previously learned information is preserved in older adults. Visual construction skills, such as assembling parts into a whole, also decline with age. Age-related changes exhibit significant variability, with some individuals showing high resilience while others experience pronounced decline.",Frontiers in Aging Neuroscience,Executive Function and Attention,2024
Synaptic Dysfunction and Hippocampal Vulnerability,"The decline in cognitive functions is not always accompanied by neuronal loss; instead, it is often associated with synaptic changes, including loss of synapses and connections or dysfunctions in synaptic plasticity. Animal models of ageing, including rodents and non-human primates, show functional declines in memory processes similar to humans. This decline in memory and plasticity is particularly evident in the hippocampus, which is especially vulnerable to ageing and age-associated diseases.",Frontiers in Aging Neuroscience,Hippocampal Ageing,2024
Hippocampus as a Key Region in Age-Related Cognitive Decline,"One brain structure of significant relevance in ageing and cognitive decline is the hippocampus, a region known for its crucial involvement in the processes of learning and memory that is found deep within the medial temporal lobe of the brain. Although functional MRI studies have shed light on the functional role of the hippocampus in various cognitive functions, the impact of subtle changes in hippocampal structure and function during normal healthy ageing remains poorly understood.",Frontiers in Aging Neuroscience,Hippocampal Ageing,2024
Hippocampal Circuitry and Vulnerable Subfields,"In the hippocampus, information processing occurs through the trisynaptic pathway, involving connections from the entorhinal cortex (EC) to the dentate gyrus (DG) via the perforant path. Mossy fibers from the DG terminate in the DG and CA3. From CA3, Schaffer collaterals project to CA1, which then sends outputs to the subiculum and EC. Among the various subfields of the hippocampus, the CA1 is more susceptible to age-associated changes.",Frontiers in Aging Neuroscience,Hippocampal Circuitry,2024
"Molecular, Cellular, and Structural Changes in the Ageing Hippocampus","Age-related changes in the hippocampus include alterations in gene expression, intracellular signaling, increased oxidative stress, neuroinflammation, and impaired neurogenesis. A correlation has been observed in rats between age-related declines in hippocampal-dependent learning and memory and reduced hippocampal volume. Hyperexcitability in hippocampal neurons is another pathological change that increases vulnerability to neurodegenerative diseases. Expression of the DNA methyltransferase Dnmt3a2 declines in the ageing hippocampus, and restoring its expression mitigates age-associated cognitive decline. Cellular changes include impaired glucose uptake, decreased abundance of enzymes associated with glycolysis and gluconeogenesis, and increased mitochondrial dysfunction leading to reduced ATP production and higher ROS generation.",Frontiers in Aging Neuroscience,Molecular Changes in Hippocampus,2024
Synaptic Alterations and Decline in Plasticity,"Structural alterations in the ageing hippocampus include decreased neuronal count and reduced synaptic connections. Subtle synaptic and functional changes underlie the majority of age-related memory impairment. Age-related changes within the hippocampus contribute to a gradual loss of synaptic plasticity, which serves as a major driver of functional and cognitive decline.",Frontiers in Aging Neuroscience,Synaptic Aging,2024
Foundations of Synaptic Plasticity and Age-Related Changes,"Donald Hebb proposed that increases in synaptic efficacy arise from a presynaptic cell’s repeated and persistent stimulation of a postsynaptic cell, summarized as “cells that fire together will wire together.” Later, Bliss and Lomo discovered long-term potentiation (LTP), the strengthening of synaptic connections after high-frequency stimulation, and long-term depression (LTD), the weakening of synapses after low-frequency stimulation. LTP and LTD are the most widely studied cellular correlates of learning and memory. Alterations in hippocampal LTP are involved in age-related learning deficits. Reductions in the induction and maintenance of LTP, along with increased LTD propensity, have been observed in aged rodents, providing a neural basis for memory decline associated with ageing.",Frontiers in Aging Neuroscience,Synaptic Plasticity,2024
NMDA- and VGCC-Dependent LTP Alterations in Ageing,"Two forms of LTP coexist at Schaffer collateral–CA1 synapses: NMDA receptor-dependent LTP and L-type voltage-gated calcium channel (VGCC)-dependent LTP. NMDA-receptor-LTP tends to diminish with age, while VGCC-LTP increases. This shift toward NMDA-independent plasticity is most evident in high-performing aged rats, suggesting an adaptive mechanism for maintaining cognitive function. Ageing alters NMDA receptor function, increasing postsynaptic calcium levels and disrupting the LTP/LTD balance. Decreases in the NMDA receptor subunit GluN1 and reductions in proteins like reelin impair NMDA-receptor signaling. Additional changes, such as altered neural cell adhesion molecule (NCAM) function, contribute to impaired LTP and LTD, which can be partly rescued by NMDA-modulating drugs like d-cycloserine.",Frontiers in Aging Neuroscience,NMDA Receptor Aging,2024
Structural Synaptic Alterations and Dendritic Spine Loss,"Structural changes in the ageing hippocampus, including synaptic loss, are key contributors to cognitive decline. A 20–40% reduction in dendritic spines in the cortex and hippocampus has been observed in ageing humans and animal models. Aged rats with spatial learning deficits exhibit reduced postsynaptic density size in perforated synapses within CA1. In aged monkeys, reductions in synaptic contacts per axonal bouton and increases in non-synaptic boutons in the molecular layer correlate with cognitive impairment. Age-related decreases in NMDA and AMPA receptor expression and downregulation of genes associated with synaptic transmission further impair hippocampal plasticity.",Frontiers in Aging Neuroscience,Synaptic Structure Aging,2024
Functional Impairments and Altered Neural Dynamics,"Comparisons of spatial firing patterns reveal that aged CA3 neurons fail to rapidly encode new spatial information, while CA1 neurons show relatively preserved firing characteristics. Altered synaptic plasticity in hippocampal neurons contributes to changes in neural dynamics during ageing. These functional deficits underscore the need to identify causal cellular and molecular factors driving synaptic aging to inform therapeutic interventions.",Frontiers in Aging Neuroscience,Neural Dynamics in Aging,2024
The Role of Inflammation in Cognitive Decline in Ageing,"Ageing is characterized by systemic inflammation, immunosenescence and inflammageing, where chronic inflammation occurs without overt infection. The ageing brain shifts from competent immune function to heightened immune activation, contributing to cognitive decline. Age-associated immune alterations increase vulnerability to diseases and inflammatory markers correlate with AD, PD, and mild cognitive impairment. Postmortem AD brains show beta-amyloid plaques co-localized with inflammatory factors, including activated microglia and proinflammatory cytokines, though it remains unclear whether neuroinflammation precedes disease or results from pathology. Systemic inflammation impairs multiple cognitive domains: mice overexpressing IL-1 and rats receiving chronic LPS display spatial working-memory deficits. LPS-induced microglial activation enhances immune factor expression and disrupts hippocampal LTP via NMDA-receptor-mediated metaplasticity. Ageing exacerbates the cognitive impact of neuroinflammation, increasing susceptibility to learning and memory impairments. Therapeutic targets include NSAIDs (COX-1/COX-2 inhibitors), which correlate with reduced AD risk. Estrogen, particularly ERβ agonists, exerts anti-inflammatory effects by reducing IL-1β and TNF-α and improves cognition in aged primates. Endocannabinoids modulate CB1/CB2 receptors: CB2 activation decreases proinflammatory cytokines and enhances anti-inflammatory signaling, and chronic CB2 stimulation improves excitatory synaptic transmission and reverses amyloid-induced cognitive deficits.",Frontiers in Aging Neuroscience,Neuroinflammation and Cognition,2024
"Caloric Restriction, Autophagy, and Neuroinflammation","Caloric restriction (CR), one of the most robust longevity interventions, reduces inflammation in both peripheral and central nervous systems and can reverse age-associated impairments in LTP and NMDA receptor expression. Enhancing autophagy reduces inflammation by clearing inflammasomes or inhibiting transcriptional modulators of cytokines, positioning autophagy as a promising therapeutic target for regulating neuroinflammation. Autophagy is a conserved lysosomal degradation pathway that removes misfolded proteins and damaged organelles to maintain homeostasis. Ageing is associated with reduced autophagy, contributing to functional decline of biological systems. Many autophagy-regulating genes are linked to lifespan regulation. Declines in autophagy contribute to age-related cognitive impairment, and enhancing autophagy facilitates degradation of Aβ, tau, and α-synuclein. Autophagy activation improves longevity across mice, flies, and worms, while repression shortens lifespan. Enhancing autophagy improves hippocampus-dependent memory in aged animals; young plasma administration restores autophagy, reversing age-related memory deficits. Memory stimuli themselves upregulate autophagy, promoting dendritic spine formation and adaptive responses to novel experiences. Spermidine-induced autophagy reduces protein aggregation and cognitive decline in animal models and improves memory in elderly humans with MCI.",Frontiers in Aging Neuroscience,Autophagy and Brain Aging,2024
Autophagy as a Convergent Mechanism of Longevity Pathways,"Recent studies in C. elegans and mammals suggest autophagy is a convergent downstream mechanism across diverse longevity paradigms. Longevity-extending interventions—reduced insulin/IGF-1 signaling, reduced mTOR signaling, caloric restriction, and pharmacological activation with spermidine, resveratrol, rapamycin, or tomatidine—all enhance autophagy. Learning induces hippocampal autophagy; drugs that inhibit autophagy impair long-term memory, while autophagy activators facilitate memory formation. Acute autophagy inhibition disrupts hippocampal LTP and reduces paired-pulse facilitation, indicating a presynaptic role. Spermidine, an autophagy activator, significantly improves hippocampal-dependent memory in elderly individuals with mild cognitive impairment. Fly models show that cognitive decline correlates with autophagy reduction, and spermidine-induced autophagy reduces ubiquitinated protein aggregation, improving cognition. Autophagy enhancers correlate with memory improvements, but the cellular mechanisms underlying these benefits remain unclear, providing an important direction for future research into autophagy-inducing drugs aimed at preventing memory decline in aged brains.",Frontiers in Aging Neuroscience,Longevity Pathways and Autophagy,2024
Calcium Homeostasis and Ageing Synapses,"Calcium is a central regulator of neuronal processes including autophagy, synaptic structural and functional plasticity, neurotransmitter release, activation of kinases and phosphatases, and overall neuronal excitability. Because Ca2+ influences multiple neuronal pathways, even subtle alterations in its regulation produce major functional consequences. Brain ageing is strongly associated with dysregulation of calcium homeostasis, and changes in Ca2+ handling mechanisms function as biomarkers of ageing as well as contributors to cognitive decline. Age-related plasticity deficits are tightly linked to disrupted calcium regulation. At synapses, intracellular Ca2+ level and duration determine the form of synaptic plasticity: brief, high-amplitude Ca2+ rises induce LTP, while modest, prolonged Ca2+ elevations induce LTD. Earlier hypotheses suggested that ageing causes elevated resting Ca2+ levels; however, current evidence indicates that ageing causes a shift in the sources of intracellular Ca2+ during synaptic activity. Specifically, ageing decreases the contribution of NMDA receptors and increases the contribution of L-type voltage-dependent Ca2+ channels (LTCCs) and intracellular calcium stores (ICS). Calcineurin (CaN) increases with ageing and dephosphorylates NMDA receptors, reducing their function. Increased oxidative stress elevates LTCC and ICS activity while reducing NMDA-receptor signaling. Ca2+ influx through LTCCs and ICS further activates CaN, perpetuating the decline in NMDA-receptor activation and raising the threshold for LTP induction.",Frontiers in Aging Neuroscience,Calcium Regulation and Synaptic Aging,2024
Age-Dependent Shifts in Calcium Sources and Plasticity,"The age-related reduction in NMDA-receptor signaling explains why high-frequency stimulation can still induce LTP, yet weaker stimulation paradigms fail, reflecting elevated thresholds for LTP induction. Ageing also increases susceptibility to LTD, which is associated with forgetting of hippocampal-dependent memory. Although the shift from NMDA-receptor-driven Ca2+ to LTCC/ICS-driven Ca2+ appears subtle, its functional effects are profound. Synapse specificity—central to Hebb's postulate—is impaired in ageing: high-frequency stimulation produces non–input-specific LTP in aged mice, which can be restored by blocking LTCCs or Ca2+-induced Ca2+ release (CICR). Increased afterhyperpolarization (AHP) accompanies elevated intracellular Ca2+ and inhibits NMDA-receptor activation, forming a feedback loop. LTCC blockade reduces AHP and facilitates LTP induction. In addition to NMDA- and LTCC-driven effects, calcium-permeable AMPA receptors (CP-AMPARs) may contribute to pathological Ca2+ influx. Defects in the GluA2 subunit of AMPA receptors are linked to AD and certain neurodevelopmental disorders; CP-AMPAR involvement offers new perspectives on AD pathophysiology, particularly regarding amyloid-beta–mediated effects. CICR from endoplasmic reticulum stores through inositol trisphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs) is elevated in aged neurons, and ryanodine release may serve as a biomarker of functional neuronal ageing.",Frontiers in Aging Neuroscience,Age-Related Plasticity Mechanisms,2024
"Calcium-Induced Calcium Release, Oxidative Stress, and Therapeutic Targets","Ca2+-induced Ca2+ release (CICR) from ER stores via RyRs is triggered by Ca2+ influx through LTCCs and amplified by oxidative stress. This mechanism becomes increasingly dominant in aged neurons, compounding dysregulated calcium dynamics. CICR contributes to altered LTP/LTD thresholds, impaired synapse specificity, and vulnerability to cognitive decline. The combined increase in LTCC activity, enhanced RyR sensitivity, reduced Ca2+-buffering capacity, and weakened NMDA-receptor function drive Ca2+ dyshomeostasis. Early interventions targeting these pathways show promise: nootropic drugs that enhance synaptic transmission, steroid-based therapies that influence Ca2+-dependent processes, and hormonal strategies that reduce oxidative damage may alleviate age-associated calcium dysregulation. Understanding the complex alterations in Ca2+ signaling—including NMDA receptor dephosphorylation, LTCC upregulation, CP-AMPAR contributions, oxidative-stress–driven CICR, and expanded AHP—is essential for developing effective therapeutics for age-related cognitive impairment and neurodegenerative diseases. The intricate interplay between calcium sources, intracellular stores, and regulatory enzymes suggests multiple therapeutic entry points for restoring synaptic plasticity and cognitive resilience in the ageing brain.",Frontiers in Aging Neuroscience,Calcium Dysregulation and Therapeutics,2024
The Brain as a Modulator of Longevity,"The view that the CNS regulates systemic ageing originated from studies in model organisms such as C. elegans, D. melanogaster, and mice. Restoring insulin signaling in neurons alone was sufficient to increase lifespan in C. elegans, illustrating the centrality of neural circuits in longevity control. Insulin and insulin-like growth factor (IGF) signaling (IIS) is evolutionarily conserved and strongly influences lifespan. Regulation of IRS2 signaling in the brain links metabolic control with lifespan extension; reducing insulin signaling either systemically or neuronally can extend lifespan by up to 18% by promoting metabolic health. Ageing is associated with reduced cortical insulin concentrations and impaired insulin receptor binding. Overexpression of Klotho inhibits IIS and extends lifespan, whereas Klotho deficiency causes premature ageing through disrupted insulin and IGF1 signaling. Insulin receptor null mice (Irs1−/−) have extended lifespan and show resistance to age-sensitive biomarkers. In humans, reduced IIS is linked to diabetes, insulin resistance, and higher cardiovascular risk, yet preserved insulin sensitivity is characteristic of centenarians, who maintain glucose tolerance and insulin action. Manipulating insulin/IGF1 pathways increases longevity despite causing insulin resistance and hyperglycemia, highlighting the need to identify neural circuits that mediate lifespan extension while preserving brain energy balance and peripheral glucose homeostasis.",Frontiers in Aging Neuroscience,Neural Regulation of Longevity,2024
"Neuronal Longevity Pathways (FOXO, REST, AMPK, Sirtuins, NRF2)","DAF-16/FOXO transcription factors are major downstream targets of IIS and key regulators of lifespan. JNK signaling operates in parallel with insulin-like pathways and converges on DAF-16 to promote longevity. In flies, neuronal activation of JNK extends lifespan through FOXO-mediated transcription. In humans, exceptional longevity is associated with downregulation of genes linked to neural excitation; the transcriptional repressor REST/NRSF is upregulated in long-lived individuals. REST represses excitation-related genes, promotes neuroprotection, preserves memory, and modulates activity-dependent synaptic plasticity during ageing. AMPK activation also prolongs lifespan and delays ageing by regulating metabolism, stress resistance, autophagy, and cellular homeostasis. Age-related reduction of AMPK responsiveness contributes to impaired metabolic regulation, increased oxidative stress, and reduced autophagy. While AMPK extends lifespan, it is unclear whether neuronal AMPK alone is sufficient. Resveratrol activates AMPK and increases lifespan in mammalian neurons and fish, delaying age-dependent cognitive decline. AMPK inhibits mTOR, a conserved longevity pathway. Sirtuins (NAD+-dependent deacetylases) regulate DNA repair, inflammation, and metabolism. Brain-specific Sirt1 overexpression delays ageing and extends lifespan via increased neural activity in hypothalamic nuclei through enhanced orexin receptor expression. Sirtuins interact with IIS, AMPK, PKA, mTOR, and FOXO pathways. NRF2 signaling also influences longevity; its decline with age contributes to oxidative stress and inflammation. Transient NRF2 activation protects aged cells, whereas chronic activation induces senescence, highlighting its tight regulation. NRF2 activation improves cognition in AD mouse models, indicating therapeutic potential.",Frontiers in Aging Neuroscience,FOXO–REST–AMPK–Sirtuin Longevity Pathways,2024
"BDNF, Lifestyle Factors, and Neural Circuits of Longevity","FOXO transcription factors integrate signals from IIS, AMPK, JNK, and TOR pathways to regulate ageing and longevity. DAF-16/FOXO shuttles between cytoplasm and nucleus to orchestrate gene expression related to autophagy, proteostasis, and antioxidant defense. BDNF (Brain-Derived Neurotrophic Factor), a central regulator of synaptic plasticity, learning, and memory, is emerging as an important player in longevity. Lifestyle interventions known to extend lifespan—calorie restriction and physical exercise—increase BDNF levels and improve cognitive function. In older adults, short sessions of physical exercise, cognitive training, or mindfulness elevate BDNF levels. BDNF also modulates autophagy, which underlies plasticity mechanisms; autophagy induction through BDNF signaling enhances synaptic remodeling. Metformin alleviates age-induced neurocognitive deficits via AMPK/BDNF/PI3K pathways. CREB and CREB-regulated transcriptional coactivators influence lifespan downstream of AMPK and calcineurin, further linking BDNF regulation with longevity. Alterations in BDNF expression affect normal and pathological ageing, especially in hippocampal and parahippocampal regions. Modulating BDNF through dietary, lifestyle, or pharmacological interventions may serve as a route toward promoting cognitive resilience and longevity. These findings support a broader paradigm in which the brain acts as a master regulator of lifespan by integrating metabolic, synaptic, inflammatory, and neurotrophic signals.",Frontiers in Aging Neuroscience,BDNF and Neural Longevity Modulation,2024
Lifestyle Interventions and Synaptic Plasticity in Ageing,"Interventions in ageing by lifestyle changes or therapeutic strategies that promote hippocampal rejuvenation to restore synaptic plasticity and cognitive functions hold promise in preventing age associated memory decline. Exercise can preserve hippocampal function with age. In rodents, improved acquisition and retention of memory in Morris water maze, inhibitory avoidance, contextual fear, and object recognition tasks were observed after long-term voluntary running in aged animals. In humans, cardiovascular exercise correlates with more accurate spatial short-term memory and reduced hippocampal atrophy. Exercise increases long-term potentiation (LTP) and lowers the threshold for LTP induction. In rats with cerebral infarction, exercise training enhanced LTP and synaptic efficacy in CA3 compared to non-exercised controls. These benefits are mediated by enhanced neurogenesis, reduced inflammation, and improved plasticity. Running reverses age-associated LTP impairments in dentate gyrus by increasing neurogenesis. In elderly humans, running enhances hippocampal blood flow. Exercise induces release of cathepsin B from muscle, which increases BDNF expression, neurogenesis, and memory performance. Parabiosis and young blood studies demonstrate systemic factors can rescue age-related decline in LTP, increase dendritic spine numbers, and enhance neuronal activity in aged hippocampus. Young plasma administration reverses hippocampus-dependent memory impairments, highlighting systemic rejuvenation as a promising clinical avenue.",Frontiers in Aging Neuroscience,Lifestyle Plasticity Interventions,2024
"Neuroplasticity, Learning, Nutrition, and Sleep in Ageing","Neuroplastic alterations in healthy individuals arise from routine processes such as learning. London taxi drivers, who acquire extensive spatial knowledge, exhibit increased gray matter volume in posterior hippocampus, demonstrating structural plasticity driven by cognitive experiences. Mental rehearsal alone can induce neuroplastic changes, and sensory deprivation reorganizes sensory cortices: blind or deaf individuals frequently develop enhanced abilities in remaining senses, with brain regions supporting lost modalities being repurposed. Nutrition profoundly shapes neuroplasticity. Long Evans rats on high saturated fat and cholesterol diets exhibit increased working memory errors, especially under high cognitive load. High-fat diets impair hippocampus-dependent memory by elevating glutamate uptake, decreasing synaptic efficacy, and inhibiting LTP and NMDA-receptor dependent LTD. Intermittent fasting alters metaplastic properties of CA1 neurons and enhances synaptic tagging/capture via increased neurogenesis and upregulation of BDNF and Prkcz. Periodic fasting-like diets improve cognitive performance and healthspan in mice. Sleep deprivation impairs plasticity and memory; REM sleep deprivation disrupts LTP and learning. Brief sleep deprivation impairs hippocampal LTP by interfering with cAMP signaling through enhanced PDE4 activity, suggesting cAMP-targeted interventions. Sleep deprivation also disrupts synaptic tagging and capture. Longer-lived individuals typically exhibit early sleep times, early waking, and afternoon napping, highlighting sleep quality as a determinant of longevity.",Frontiers in Aging Neuroscience,Neuroplasticity–Lifestyle–Sleep–Nutrition,2024
"Stress, Dynamic Brain Plasticity, and Comparative Ageing Models","Quality of life strongly influences longevity. Mild stress may enhance memory and lifespan, while chronic stress accelerates ageing, disrupting endocrine, autonomic, and immune systems. Elevated corticosteroids reduce hippocampal LTP. Brain ageing remains dynamic, allowing restoration of degraded cognitive functions through lifestyle-driven interventions. Re-establishing synaptic connections through behavioral changes holds importance for cognitive ageing. Comparative ageing across humans, rodents, and non-human primates is critical for translational research. Lifespan varies dramatically across species, and biological age must be assessed through physiological change rather than simple lifespan proportion. Mice are primary ageing models but differ significantly from humans in maturation rates: 3–6 month-old mice approximate 20–30-year-old humans; 10-month-old mice correspond to middle age; 18–24 months map to 56–69-year-old humans; over 24 months represents very old age. Rodents share physiological ageing characteristics with humans but differ in disease pathogenesis. Non-human primates more accurately replicate human ageing and disease mechanisms. Monkeys aged 20+ equate to human 60+, while those aged 30+ correspond to human 90+. Marmosets, despite shorter lifespan, serve as valuable models due to similarity in disease biomarkers and ageing trajectories. These animal models enable investigation of molecular, cellular, biochemical, and behavioral mechanisms underlying human ageing.",Frontiers in Aging Neuroscience,"Stress, Dynamic Brain, Comparative Ageing Models",2024
Citation and Article Information,"Nutrition, Physical Activity, and Other Lifestyle Factors in the Prevention of Cognitive Decline and Dementia
Ligia J. Dominguez 1,2,*, Nicola Veronese 1, Laura Vernuccio 1, Giuseppina Catanese 1, Flora Inzerillo 1, Giuseppe Salemi 3,4 and Mario Barbagallo 1. Citation: Dominguez, L.J.; Veronese, N.; Vernuccio, L.; Catanese, G.; Inzerillo, F.; Salemi, G.; Barbagallo, M. Nutrition, Physical Activity, and Other Lifestyle Factors in the Prevention of Cognitive Decline and Dementia. Nutrients 2021, 13, 4080. https://doi.org/10.3390/nu13114080. Academic Editor: Panteleimon Giannakopoulos. Received: 17 September 2021. Accepted: 27 October 2021. Published: 15 November 2021. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.",Nutrients,Cognitive Decline Prevention,2021
Author Affiliations,"1 Geriatric Unit, Department of Medicine, University of Palermo, 90100 Palermo, Italy; nicola.veronese@unipa.it (N.V.); lvernuccio@virgilio.it (L.V.); giusi.catanese@libero.it (G.C.); florainzerillo@gmail.com (F.I.); mario.barbagallo@unipa.it (M.B.)
2 Faculty of Medicine and Surgery, University of Enna “Kore”, 94100 Enna, Italy
3 Department of Biomedicine, Neuroscience, and Advanced Diagnostics, University of Palermo, 90100 Palermo, Italy; giuseppe.salemi@unipa.it
4 UOC of Neurology, University Hospital “Paolo Giaccone”, 90100 Palermo, Italy
* Correspondence: ligia.dominguez@unipa.it; +39-0916554828",Nutrients,Cognitive Decline Prevention,2021
Abstract,"Abstract: Multiple factors combined are currently recognized as contributors to cognitive decline. The main independent risk factor for cognitive impairment and dementia is advanced age followed by other determinants such as genetic, socioeconomic, and environmental factors, including nutrition and physical activity. In the next decades, a rise in dementia cases is expected due largely to the aging of the world population. There are no hitherto effective pharmaceutical therapies to treat age-associated cognitive impairment and dementia, which underscores the crucial role of prevention. A relationship among diet, physical activity, and other lifestyle factors with cognitive function has been intensively studied with mounting evidence supporting the role of these determinants in the development of cognitive decline and dementia, which is a chief cause of disability globally. Several dietary patterns, foods, and nutrients have been investigated in this regard, with some encouraging and other disappointing results. This review presents the current evidence for the effects of dietary patterns, dietary components, some supplements, physical activity, sleep patterns, and social engagement on the prevention or delay of the onset of age-related cognitive decline and dementia.",Nutrients,Cognitive Decline Prevention,2021
Keywords and Context,"Keywords: cognitive decline; Alzheimer; dementia; aging; nutrition; physical activity; exercise; diet; sleep; socialization; oxidative stress; inflammation.
1. Introduction. We are living in an aging world. Currently, most humans worldwide may expect to live beyond 60 years, while demographic data foresee that half of children alive in 2010 in nations with the highest life expectancies would be centenarians. However, this optimistic scenario can be shadowed by the numerous people that would be affected with age-associated cognitive decline, which is a chief cause of disability worldwide. About forty-seven million people have dementia worldwide with an estimated eight million new diagnoses every year. The most common causes of dementia are Alzheimer’s disease (AD), vascular dementia, and mixed forms. Currently, there is no effective treatment that may significantly modify the course of dementia. Brain pathological changes seem to initiate long before clinical manifestations, which mostly occur in old age. This provides a large period of time to implement prevention strategies effectively delaying age-related cognitive decline and dementia, which is a major public health concern.",Nutrients,Cognitive Decline Prevention,2021
Limitations of Pharmacological Interventions,"However, there is no solid evidence supporting a role for pharmacological therapies (i.e., anti-inflammatory medications, estrogen/progestin supplementation, antihypertensives, antidiabetics, dementia medications) in the prevention of cognitive decline in persons with preserved cognitive function or with mild cognitive impairment (MCI) according to a systematic review of fifty-one trials. The evidence is not yet solid for cognitive training examined in a systematic review of eleven trials and on exercise in a systematic review including sixteen trials. Conversely, a multidomain intervention including diet, cognitive training, and physical activity reported significant improvements in a range of cognitive outcomes. The interest on dietary and nutritional components as potential modifiable factors for postponing the onset and severity of age-related cognitive function deterioration has grown in recent decades. In fact, unhealthy diet seems to be a key risk factor. This is exemplified by the fact that during Japan’s nutrition transition from the traditional Japanese diet to the Western diet, AD rates rose from 1% in 1985 to 7% in 2008.",Nutrients,Cognitive Decline Prevention,2021
Dietary Components and Supplements,"The antioxidant and anti-inflammatory actions of various minerals, micronutrients, and vitamins in regard to cognitive function have been studied as well as components of neuronal membranes, e.g., dietary essential fatty acids. Several studies on single or multi-component supplements suggest biological plausibility for the cognitive effects of these preparations, but they do not show solid evidence of these effects. Lately, nutrition research has moved from focusing attention only on single nutrients and foods to investigating dietary patterns based on the notion that combinations of foods and nutrients can have synergistic and/or antagonistic effects beyond single components. It has been calculated that about 3% of all dementia cases could be prevented by increasing levels of physical activity. Likewise, an increasing number of studies have emerged indicating the importance of physical activity and exercise for the prevention of the pathological process and complications of dementia.",Nutrients,Cognitive Decline Prevention,2021
Scope of the Review,"In this narrative review, we examine updated evidence of the effects of dietary patterns, dietary components, and supplements on cognitive function decline and dementia. We also review some non-dietary lifestyle factors (i.e., physical activity/exercise, sleep quality, and socialization), which can contribute in association with dietary factors. We included systematic reviews with or without formal meta-analysis of intervention or observational studies dealing with dietary and non-dietary factors as exposure and cognitive and other related parameters as outcomes. We will also briefly review possible mechanisms that may help explain potential benefits.",Nutrients,Cognitive Decline Prevention,2021
Overview of Dietary Patterns,"2. Dietary Patterns
Several food combinations in specific dietary patterns have been studied in regard to cognitive decline from MCI to dementia in various contexts with some exhibiting favorable results and others showing no evidence of such effects. Indeed, a multinutrient approach seems to better support outcomes than single nutrient intervention. Although there is no single dietary intervention that has been definitely proven in randomized control trials (RCTs) to effectively prevent cognitive deterioration and dementia, data from epidemiological studies suggest that following a healthy, balanced diet and lifestyle, which has been confirmed to reduce cardiovascular (CV) risk, may also help with preventing or delaying the onset of AD.",Nutrients,Cognitive Decline Prevention,2021
Mediterranean Diet – Evidence Base,"2.1. Mediterranean Diet (MedDiet)
This dietary pattern that has been traditionally followed for centuries by populations from the Mediterranean countries has been intensely studied in prospective observational studies and trials conducted in Mediterranean and non-Mediterranean countries with different health outcomes. This extensive evidence supports its beneficial action on the prevention of several non-communicable diseases (NCDs), including cognitive decline and dementia. A systematic review of prospective cohort studies reported that participants with the highest adherence to the MedDiet vs. those with lowest adherence had a 33% lower risk of developing MCI or AD.",Nutrients,Mediterranean Diet,2021
PREDIMED Trial and Cognitive Outcomes,"In the PREDIMED (Prevención con Dieta Mediterranea) trial, among adults (aged 55 to 80 years) at high CV risk, those following a MedDiet supplemented with extra virgin olive oil (EVOO) or nuts had a reduced incidence of CV events, which are a known risk factor for cognitive decline, compared to participants following a low-fat diet over 5 years of follow-up. In sub-analyses of this trial, an improvement in cognitive function with the MedDiet supplemented with either EVOO or nuts vs. a low-fat diet has been reported. In prospective longitudinal studies conducted in the USA and France, a higher adherence to a MedDiet was associated with slower cognitive decline and incident AD.",Nutrients,Mediterranean Diet,2021
Mixed Evidence Across Populations,"Some reviews suggested that there is some evidence showing that following a MedDiet is associated with a reduced risk of developing AD, but that still extensive confirmation in different populations and ethnicities is necessary. There are also some conflicting results. For example, an Australian cohort of healthy adults in The Personality and Total Health (PATH) through Life longitudinal study did not find any protection of the MedDiet for cognitive decline, while another Australian study, the Australian Imaging, Biomarkers and Lifestyle (AIBL) study, reported that patients with MCI or AD had lower adherence to the MedDiet when compared to healthy controls.",Nutrients,Mediterranean Diet,2021
Comparison With Other Diet Quality Scores,"Tangney et al. evaluated adherence to the Healthy Eating Index (HEI)-2005 or to the MedDiet in regard to cognitive function modifications in 3790 over-65 adults followed for seven years from the Chicago Health and Aging Project longitudinal study. They observed that higher MedDiet adherence scores were associated with slower rates of cognitive decline, after adjusting for confounders, but no association was observed for HEI-2005 scores. Table 1 shows the summary of systematic reviews and meta-analyses exploring the association of adherence to the MedDiet with cognitive decline and/or incident dementia. As mentioned, the MedDiet is the dietary pattern with the highest number of studies published hitherto in the medical literature.",Nutrients,Mediterranean Diet,2021
Lifestyle Factors Within the MedDiet,"Noteworthy, the MedDiet comprises several other lifestyle parameters, such as physical activity, social engagement, culinary activities, and adequate rest, which have shown positive effects on delaying cognitive function deterioration. Therefore, it is crucial to include these other lifestyle factors when evaluating the MedDiet and cognitive performance decline.",Nutrients,Mediterranean Diet,2021
Ketogenic Diet – Overview and Rationale,"2.4. Ketogenic Diet
Ketone bodies, products of fat metabolism, are a source of energy for the brain and are available even when glucose supplies are insufficient (e.g., during severe carbohydrate restriction) or when its metabolism is defective as in AD. Based on evidence that this dietary pattern has been associated with positive results in some forms of drug-resistant epilepsy proposed to be mediated by the action of medium-chain triglycerides (MCT) and generation of ketones as an alternative metabolic substrate for the brain, enhancing mitochondrial function and reducing the expression of inflammatory and apoptotic mediators, this diet has been proposed as neuroprotective. In the last few years, some investigations have suggested that the ketogenic diet, which is characterized by a high consumption of fat, moderate consumption of protein, and very low carbohydrate composition, may be a useful instrument for the prevention of age-associated cognitive decline. However, most available studies are experimental, and there are not yet enough data from clinical trials to draw definitive conclusions on the prevention and treatment of cognitive decline and AD.",Nutrients,Ketogenic Diet,2021
Ketogenic Diet – Experimental Evidence,"In experimental aged animals, the ketogenic diet improved cognitive performance even under hypoxic conditions without changes in motor performance. Another two experimental studies found that ketogenic diet started at a young age improved cognition and extended longevity. Late-life ketogenic diet intervention improved behavior and cognitive tasks that required working memory in rats. A preliminary double-blind placebo-controlled study in twenty patients with MCI or AD reported improvement in AD Assessment Scale-Cognitive subscale (ADAS-cog) scores in APOE ε4-negative patients receiving medium-chain triglycerides, which was not observed in APOE ε4-positive patients; higher ketone concentrations were associated with greater improvement in paragraph recall with MCT treatment compared to placebo among all participants. Another study involving 152 participants reported similar results.",Nutrients,Ketogenic Diet,2021
Modified Atkins Diet and Early Human Trials,"A recent preliminary human phase I/II RCT examined the feasibility of using a modified Atkins diet (MAD) to induce ketogenesis in persons with MCI or early AD, and it tested as a primary outcome the effects of this diet on a Memory Composite Score (MCS), the sum of the delayed recall trials for the Hopkins Verbal Learning Test—Revised (HVLT-R) and Brief Visuospatial Memory Test—Revised (BVMT-R). Secondary outcomes for efficacy were changes in the MMSE-2—Expanded Version, Profile of Mood States, bipolar form, and instrumental activities of daily living scores and other clinical outcomes. Twenty-seven participants were eligible. After extensive assessment and education, participants were randomly assigned to either the MAD or the National Institute on Aging recommended diet for older people for twelve weeks. Preliminary results showed that nine patients in the MAD arm and five in the NIA arm had completed the trial. Despite extensive training, adherence to both diets was only fair. Among those in the MAD arm who generated at least trace amounts of urinary ketones, there was a large and statistically significant increase in MCS between the baseline and week-6 assessment.",Nutrients,Ketogenic Diet,2021
Ketogenic Diet – Cognitive Effects and RCT Findings,"MAD participants also reported increased energy between baseline and week-6 assessment. These preliminary results suggest that the generation of even trace ketones might enhance episodic memory and vitality in very early AD. Another RCT involving twenty-one patients with clinically confirmed AD were randomly assigned to a modified ketogenic diet or usual diet supplemented with low-fat healthy-eating guidelines in a crossover trial (two 12-week treatment periods, separated by a 10-week washout period). Participants on the ketogenic diet achieved sustained physiological ketosis measured with blood beta-hydroxybutyrate concentrations. Compared with the usual diet, patients on the ketogenic diet had better scores in the AD Cooperative Study—Activities of Daily Living and Quality of Life in AD testing; Addenbrookes Cognitive Examination-III also improved but not significantly. Changes in CV risk factors were mostly favorable, while adverse effects were mild. These results encourage the design of larger RCTs in AD patients to confirm the positive effects on daily function and quality of life.",Nutrients,Ketogenic Diet,2021
Nordic Diet – Definition and Cognitive Effects,"2.5. Nordic Diet
The Nordic diet attempts to reflect the diet consumed in Nordic countries, where the usual eating habits include high consumption of fish, cabbages, apples, and pears, whole grains from oat, berries, root vegetables, barley and rye, low-fat dairy products, potatoes, and rapeseed oil. A study including participants aged fifty-seven to seventy-eight years with normal cognition at baseline reported that better adherence to the Nordic diet was associated with higher scores in global cognition over a 4-year study period after adjustment for demographic and lifestyle factors. A large cohort study involving 2223 dementia-free adults aged over sixty years followed for six years found that moderate to high adherence to the Nordic diet was more closely associated with less cognitive decline than moderate to high adherence to other healthy dietary patterns, i.e., the MedDiet, DASH, and MIND.",Nutrients,Nordic Diet,2021
Nordic Diet – Interaction With Lifestyle and Summary,"Subsequent analyses of this cohort study aiming to verify whether an active lifestyle may reinforce the positive effects of the Nordic Prudent Dietary Pattern (NPDP) on cognitive function showed that the association of NPDP with a reduced decline in MMSE scoring became stronger when combined with moderate-to-intense physical, mental, or social activities. Thus, the combination of NPDP and an active lifestyle may result in even better preservation of cognitive function and further decreased risk of cognitive decline. In summary, the results of studies with the Nordic diet are still scarce. Although they do appear promising for people with preserved cognitive function, whether such diet might improve cognition in a population with cognitive decline and in populations different from that of Nordic countries remains to be established.",Nutrients,Nordic Diet,2021
Vegetarian Diet – Overview and Epidemiology,"2.6. Vegetarian Diet
It is accepted today that dietary patterns that emphasize plant foods, which are plenty of bioactive compounds but not strictly vegetarian/vegan can exert neuroprotective effects. Likewise, it is recognized that an increased consumption of fruits and vegetables can be favorable to brain health. A recent prospective study involving 27,842 men with a mean age of 51 years in 1986 examined the relation of vegetable and fruit consumption, which was measured with five repeated food frequency questionnaire (FFQ)s collected every 4 years, to future incident subjective cognitive function. Higher intakes of total vegetables, total fruits, and fruit juice 18 to 22 years before were each significantly associated with lower odds of moderate or poor subjective cognitive function. Nevertheless, there is no direct evidence yet to support the benefits of a strict vegetarian or vegan diet in preventing cognitive decline despite evidence for brain-health-promoting effects, mainly in experimental and observational investigations, of several plant foods rich in polyphenols and anti-inflammatory components that may impact neuroinflammation.",Nutrients,Vegetarian Diet,2021
Vegetarian Diet – Mixed Findings and Meta-analyses,"In 1993, Giem et al. published preliminary findings of two cohort sub-studies of the Adventist Health Study examining the association of following a vegetarian diet or a meat-eating diet with the incidence of dementia. One of the studies (n = 272 participants) showed that persons who frequently consumed meat had an over two-fold increased risk of becoming demented than vegetarians. The second study (n = 2984 participants) reported no significant difference in incident dementia in meat-eating participants vs. vegetarians. No explanation for these contrasting results were proposed, and there was no evidence of standardized cognitive assessments during the studies. Although vegetarian/vegan diets may provide beneficial health effects, they may also lead to nutritional deficiencies. A meta-analysis examining the relationship between vegan or vegetarian diets and cognitive and mental health (n = 13 articles; 17,809 participants) found no significant association between diet and the continuous depression score, stress, well-being, or cognitive impairment. Vegans/vegetarians were at increased risk for depression and had lower anxiety scores, but no differences for other outcomes, including cognition, were found. Heterogeneity was large, impeding subgroup analyses.",Nutrients,Vegetarian Diet,2021
Healthy Eating Index–2005 (HEI-2005),"2.7. Healthy Eating Index-2005 (HEI-2005)
The HEI was developed based on foods aligning the key recommendations of the Dietary Guidelines for Americans in order to measure diet quality. There are conflicting results on the effects of this dietary pattern on cognitive function. Former cross-sectional studies showed associations of HEI-2005 with improvements in cognitive tests. Conversely, other longitudinal studies involving participants followed for up to 7.6 years reported no association of HEI-2005 with any modification of cognitive performance. Wengreen et al. conducted a longer study with a follow-up of eleven years showing an association between increased HEI scores with lower cognitive decline, suggesting that a longer period of time is needed in order to reveal the effects of diet on cognitive function.",Nutrients,Healthy Eating Index,2021
Okinawa Diet – Longevity and Cognitive Outcomes,"2.8. Okinawa Diet
This type of diet describes the eating habits followed by people originally from the Ryukyu Island in Japan, which counts the largest number of centenarians in the world. Although this dietary pattern has been associated with a long and healthy life, there is no direct evidence of its effects on the prevention of cognitive deterioration and AD. A not fully reliable study reported a higher incidence of dementia in 157 migrants from Okinawa to Brazil compared to 2217 residents of Okinawa. However, there was a period of nine years between the evaluation of the two groups and a remarkably different sample size, which hindered definitive conclusions.",Nutrients,Okinawa Diet,2021
Low-Copper Diet – Cognitive Associations,"2.9. Low-Copper Diet
A former longitudinal study involving 3718 participants reported that elevated intakes of copper supplements in combination with a diet rich in saturated and trans-fatty acids were associated with faster cognitive decline after a follow-up of five and a half years. Conversely, a small study in thirty-two patients with AD at mild to moderate stages showed that those participants with low plasma concentrations of copper had higher (worse) ADAS-cog scores. It has been suggested that the promotion of a low-copper diet may potentially reduce the risk of AD, but there is no evidence that this can modify the incidence or pathological features of AD.",Nutrients,Low-Copper Diet,2021
Paleolithic Diet – Evidence and Cognitive Outcomes,"2.10. Paleolithic Diet
Although there is no evidence on the prevention of cognitive decline or AD with the Paleolithic diet, some small studies have been conducted in overweight and diabetic patients. One of these studies compared the effects of Paleolithic diet vs. the Nordic Nutrition Recommendations in a sample of twenty overweight postmenopausal women followed for six months. The outcomes included parameters of functional magnetic resonance imaging (fMRI), episodic memory fMRI tasks (a face-name paradigm was used to examine brain responses related to episodic memory), and weight loss. The authors reported significant improvements in fMRI episodic memory tests, which were associated with increased hippocampal activity, decreased waist circumference, and reduced plasma free fatty acid with similar results for both dietary patterns. Another study was conducted in twelve sedentary patients with type 2 diabetes (with or without metformin treatment) randomized to follow a Paleolithic diet with or without high-intensity exercise. After twelve weeks, both interventions induced similar significant weight loss, improved insulin sensitivity, and increased oxygen uptake.",Nutrients,Paleolithic Diet,2021
Paleolithic Diet – Brain Responses and Need for Long-Term Studies,"Both interventions were associated with increased functional brain responses within the right anterior hippocampus and right inferior occipital gyrus as well as increased volume of the right posterior hippocampus, which are areas that are linked to cognitive functions. No changes in episodic memory function and associated functional brain responses were observed in this short period. Further studies are still needed to test the long-term effects of this dietary pattern.
3. Dietary Components and Supplements
3.1. Vegetables
There is growing evidence that plant-based diets rich in vegetables can be protective, together with other dietary and lifestyle factors, against age-related cognitive decline and can help prevent dementia. In the preceding sections, we have discussed dietary patterns with evidence of cognitive decline and dementia protection. Although those that are most protective, namely the MedDiet, DASH, and MIND, are not strictly vegetarian, these diets are mainly composed of foods with plant origin, including abundant amounts of vegetables. This means that they are rich in phytonutrients, such as polyphenols, carotenoids, antioxidant vitamins, healthy fats, and other phytochemicals linked to lower chronic inflammation and oxidative stress and to better scores of the Dietary Inflammatory Index.",Nutrients,Vegetables,2021
Vegetables – Mechanisms and Studies,"The Dietary Inflammatory Index has been shown to be inversely associated with memory and cognitive functioning. In animal models, these nutrients and non-nutrients have been found to enhance neurogenesis, synaptic plasticity, and neuronal survival by reducing oxidative stress and neuroinflammation. In particular, a variety of vegetables have attracted the attention of the lay public and also the scientific community for their content, although in reduced amounts, of numerous molecules increasingly investigated for their healthy properties. Among them, plant polyphenols have raised notable interest. Various clinical trials have indicated that many health benefits of the Mediterranean and Asian diets can be related to the presence of significant amounts of polyphenols, even if in some cases, inconsistent results have been reported as well, which highlights the need for further investigation.",Nutrients,Vegetables,2021
Vegetables – Cohort Evidence and Meta-analyses,"A recent analysis of data from the Survey of Health, Ageing, and Retirement in Europe (SHARE) including 22,635 participants from eleven European countries showed that frequent consumption of fruits and vegetables was associated with improved health outcomes, including cognitive and mental health. In a biracial cohort of 3231 men and women aged 18–30 years at baseline in 1985–1986, followed for up to 25 years in the Coronary Artery Risk Development in Young Adults Study in the US, intake of whole vegetables (excluding potatoes) was significantly associated with a better cognitive performance after adjustment for potential confounders in all three cognitive tests used, supporting the long-term benefits of vegetables and fruits consumption on cognitive performance. Among 27,842 men from the Health Professionals Follow-up Study with a mean age of 51 years in 1986 and followed for 18 to 22 years, higher intakes of total vegetables, total fruits, and fruit juice were each significantly associated with lower odds of moderate or poor subjective cognitive function.",Nutrients,Vegetables,2021
Vegetables – Dementia Risk Reduction,"A systematic review and meta-analysis identified six cohorts involving 21,175 participants. The pooled analysis found that the consumption of fruit and vegetables was inversely associated with the incident risk of cognitive disorders, with evidence of significant heterogeneity attributed to ethnic differences. An observational study examined the diet of 17,700 community-living dementia-free Chinese older adults who attended the Elderly Health Centers in Hong Kong at baseline and followed their cognitive status for six years, evaluating whether the minimal daily requirement of vegetables and fruits recommended by the World Health Organization would independently lower dementia risk. Multivariable logistic regression analysis showed that having at least three servings of vegetables and two servings of fruits daily might help prevent dementia in older adults. Another meta-analysis including nine studies with 31,104 participants and 4583 incident cases showed that increased fruit and vegetable consumption was associated with a significant reduction in cognitive impairment and dementia risk. Dose–response analysis showed that an increment of 100 g per day was related to a near 13% reduction in risk.",Nutrients,Vegetables,2021
EVOO – Composition and Types of Olive Oil,"3.2. Extra-Virgin Olive Oil (EVOO)
Olive oil, a staple of the MedDiet, is a natural product rich in oleic acid, which is a MUFA. Olive oil is the typical culinary fat used in Mediterranean countries where it is the main source of fat in this dietary pattern. There are various types of olive oil (i.e., EVOO, virgin olive oil, refined olive oil, and pomace oil), which exhibit diverse chemical characteristics and potential impacts on human health. Unfortunately, olive oil subtypes are often not discriminated in research investigation with possible heterogeneous results due to the diverse effects of various subtypes. In particular, EVOO arises from the first pressing of fresh olives, usually within 24 h after their harvest. It is extracted by non-chemically induced mechanical means at temperatures below 28 °C. Its free fatty acid or acidity level is lower than 0.8%, having optimal taste and odor.",Nutrients,Extra-Virgin Olive Oil,2021
EVOO – Phenolic Content and Comparison With Other Oils,"Olives contain hydrophilic phenol compounds, including simple phenolic compounds (e.g., gallic, vanillic, caffeic and coumaric acids, tyrosol, and hydroxytyrosol), complex compounds (e.g., secoiridoids such as oleuropein and ligstroside), and lignans. Total polyphenol content is highest in EVOO as opposed to concentrations in the other subtypes of olive oil. EVOO has been associated with beneficial effects on human health, including the prevention of CV diseases, which is attributable to its composition that gives it antioxidant and anti-inflammatory properties. Virgin olive oil also arises from the first pressing of olives, albeit with acidity levels below 2%. Virgin olive oil contains hydrophilic phenols—including phenolic acids and alcohols, flavonoids, lignans, and secoiridoids—in lower concentrations compared to EVOO and is apparently not as beneficial as EVOO. Even so, perhaps because it is more easily available, virgin olive oil is highly recommended in the literature.",Nutrients,Extra-Virgin Olive Oil,2021
Refined and Pomace Oils – Processing and Nutrient Loss,"The refinement of olive oil is produced using acids, alkalis, and heat to extract as much oil as possible from the olive pulp that remains after the first pressing. This processing renders refined oil higher in fat and acidic content than EVOO and virgin olive oil. Therefore, refined olive oil is deprived of optimal taste, aroma, and natural antioxidants and anti-inflammatory compounds. Pomace oil is a by-product of EVOO. Olive skins, pulp, and seeds are heated, and the oil that remains is extracted using hexane as a solvent. Pomace oil has few bioactive compounds and hence scarce antioxidant actions. Other types of oil produced through poor practices, such as lampante oil, are not recommended for human use unless they are refined. Olive oil is composed mainly of triacylglycerols and contains small quantities of free fatty acids, glycerol, pigments, phosphatides, sterols, and flavor compounds.",Nutrients,Extra-Virgin Olive Oil,2021
EVOO – Fatty Acid Profile and Phenolic Antioxidants,"Olive oil has a high proportion of unsaturated and a low proportion of saturated fats. It consists of about 15% saturated fats including palmitic and stearic acid and about 85% unsaturated fats (70% oleic acid, a monounsaturated omega-9 fatty acid; 15% linoleic acid, an omega-6 PUFA; and 3.5% palmitoleic acid). Olive oil contains more oleic acid and less linolenic acids (i.e., more MUFA than PUFA) than other vegetable oils; it is free of trans fatty acids. Several epidemiological observational studies have indicated that a higher proportion of MUFA in the diet may be linked with reduced CV disease risk. The flavonoid polyphenols in olive oil are natural antioxidants that contribute to a bitter taste, astringency, and resistance to oxidation. The major phenolic compounds—simple phenols, secoiridoids, and lignans—have notable antioxidant properties.",Nutrients,Extra-Virgin Olive Oil,2021
EVOO – Health Effects and Cognitive Study Evidence,"A high consumption of EVOO, particularly rich in these compounds, is associated with diminished risk in colon, breast, and skin cancer and has beneficial effects on coronary heart disease incidence as well as healthy aging. Nevertheless, polyphenol content is determined by many factors (e.g., olive variety, environmental factors, harvest time, extraction, and storage). Regarding consumption of EVOO, a study including 6947 participants in the Three Cities Study cohort reported that an intensive use of EVOO slowed cognitive decline during the four years of follow-up. Compared with those participants who never used EVOO, participants with moderate or intensive EVOO use showed an improvement in visual memory and verbal fluency. In the same cohort, analysis of 1329 older participants with high consumption of EVOO and other plant-derived polyphenols found that the risk of dementia was reduced by 50% in multivariable-adjusted models.",Nutrients,Extra-Virgin Olive Oil,2021
EVOO – PREDIMED Results and Experimental Models,"Vals-Predet et al. in a sample of 334 older participants of the PREDIMED trial at high risk of CV disease reported that after four years of a MedDiet supplemented with EVOO, memory scores, frontal cognition, and global cognition scores declined less compared to the control diet. In the PREDIMED-Navarra trial involving 522 participants, those receiving supplemented EVOO or mixed nuts showed higher cognitive scores and improved cognition vs. the control group. Participants in the EVOO group had fewer cases of MCI than those following a low-fat diet. In animal models of AD (senile SAMP8), mice fed with EVOO had improved learning and memory associated with better profiles of oxidative stress markers. Aged rats fed with EVOO also showed improved biochemical parameters, memory, and motor coordination by reducing oxidative stress and enhancing antioxidant enzymes.",Nutrients,Extra-Virgin Olive Oil,2021
EVOO – Summary of Evidence and Research Gaps,"In summary, EVOO seems to have numerous health benefits shown in experimental and clinical trials. EVOO consumption appears neuroprotective as a crucial component of the MedDiet. However, there is not yet strong evidence for the effects of EVOO in isolation on cognitive decline and dementia because very few clinical studies are available. EVOO should be tested in RCTs for the prevention of cognitive deterioration.",Nutrients,Extra-Virgin Olive Oil,2021
Nuts – Bioactive Components and Mechanisms,"3.3. Nuts
Tree nuts provide macronutrients, micronutrients, and phytochemicals that may affect several pathways in AD pathogenesis such as amyloidogenesis, tau phosphorylation, oxidative stress, cholinergic pathways, and other non-target mechanisms including cholesterol lowering and anti-inflammatory properties, as well as effects on neurogenesis. Among nuts, walnuts contain the largest amount of free and total polyphenols, which are followed by Brazil nuts and almonds. Phytochemicals contained in tree nuts comprise carotenoids, phenolic acids, phytosterols and polyphenolic compounds such as flavonoids, proanthocyanidins, and stilbenes, as well as phytates, sphingolipids, alkylphenols, and lignans. The phytochemical content of tree nuts can vary considerably by nut type, genotype, pre- and post-harvest conditions, as well as storage conditions. Although the beneficial effects of nuts on cardiometabolic diseases have been well established, less is known about the effects of nuts on cognitive well-being. Many of the biological pathways are common; hence, it is plausible that diets rich in nuts might be beneficial in ameliorating other age-related conditions as well.",Nutrients,Nuts,2021
Nuts – Preclinical and Human Evidence Overview,"Most studies on the effects of tree nuts on cognition or AD have been performed in preclinical studies in experimental animal models or in cell cultures. While tree nut phytochemicals are bioaccessible and bioavailable in humans, the number of intervention trials conducted in humans to date is limited, while most of the evidence in humans comes from observational studies. Considering the available human studies together—those designed specifically for nuts and those considering them within a dietary pattern—there are nineteen studies conducted in countries from different world regions (Europe, USA, Asia), of which there are thirteen observational and six interventional studies, reporting nut consumption effects and cognitive function in older adults. From the seven observational studies (three cross-sectional and four prospective) specifically investigating nuts consumption and cognitive function, only one study did not find significant differences in the cognitive test scores for participants with high vs. low nuts consumption after adjusting for confounders. The remaining six studies reported a significant positive association of various cognitive test and nuts consumption.",Nutrients,Nuts,2021
Nuts – Dietary Pattern Studies and Cognitive Outcomes,"The other six studies (three cross-sectional and three prospective) investigated the associations of dietary patterns including nuts and cognitive function of older adults. It is worth clarifying that in these studies, it is not possible to attribute the findings to the intake of nuts specifically. Nevertheless, only one long-term study (nine-year follow-up) including 6000 older women did not find significant associations between nut consumption and global cognition or verbal memory. The remaining five studies reported a lower risk of incident cognitive impairment or better overall cognition and verbal memory with a higher consumption of nuts. As regards intervention studies investigating the effects of nut supplementation on the cognitive functioning of older adults, there are three studies available. A former pilot study involving participants with MCI with a diet supplemented with one Brazil nut daily for six months found improvements in verbal fluency and constructional praxis scores but no difference in global cognition (two of six subsets of neuropsychological battery tests were improved).",Nutrients,Nuts,2021
Nuts – Intervention Trials and Summary of Evidence,"Two more recent and larger studies supplementing diets with almonds or walnuts as 15% of total daily energy intake did not observe any significant differences in cognitive performance or mood after the intervention. Three more studies including nuts as part of a MedDiet intervention reported no benefit in a battery of cognitive tests, significant improvements in MMSE scores and clock drawing test, and improvements in a composite memory score but not in frontal and global cognitive function, all after adjustment for confounders. As it happens with other dietary components, in the interventional studies that used an overall dietary pattern approach, nuts consumption was only one component of the overall interventions, making it difficult to assess their independent effects. In summary, most observational studies reported positive associations between nut consumption and cognitive function in older adults, while almost all interventional studies failed to demonstrate the benefits of nut supplementation (alone or within an overall dietary pattern) on cognitive function measurements. Larger and longer RCTs are warranted.",Nutrients,Nuts,2021
Berries – Flavonoids and Neuroprotective Mechanisms,"3.4. Berries
Berries generally are rich in flavonoids including the flavan-3-ols catechin and epicatechin, the flavanols kaempferol and myricetin, and the anthocyanins delphinidin and petunidin. In studies conducted in experimental animals and cellular cultures, it has been shown that flavonoids are neuroprotective and can slow brain aging and cognitive decline through a number of potential mechanisms, including the suppression of microglia-mediated inflammation as well as blood pressure and oxidative stress decrease with a consequent reduction of vascular risk, which is facilitated in part through the production of neuronal and inducible nitric oxide. Specific studies assessing the possible cognitive effects of berries have been conducted experimentally, supporting the role of berry-rich diets in motor function, working memory, and increased neurogenesis. Analyses of data from 16,010 participants from the observational Nurses’ Health Study aged ≥70 years found that a greater consumption of blueberries and strawberries in the long-term were significantly associated with slower rates of cognitive decline comparing extreme categories of consumption after adjusting for multiple potential confounders.",Nutrients,Berries,2021
Berries – Anthocyanin Studies and Systematic Reviews,"There are some small clinical studies on the effects of the consumption of berries or berry juices on cognitive parameters. A systematic review aiming to identify studies on food-based anthocyanin consumption (i.e., berry juices) and cognitive outcomes in human intervention trials included seven studies, comprising four acute trials and three longer-term interventions (twelve to sixteen weeks). Six of seven studies reported improvements in either a single or multiple cognitive outcomes, including verbal learning and memory, after anthocyanin-rich food consumption. However, the authors found important methodological limitations due to the diversity of the studies, most of them very small trials, that prevented the pooling of data for quantitative analysis. Therefore, they concluded that even if food-based anthocyanin consumption seems promising, adequately powered studies are still needed. Another systematic review evaluated RCTs investigating the effects of blueberries and blueberry products on cognition. Eleven articles (that included twelve studies) were identified; nine studies used freeze-dried blueberries, two studies used whole blueberries, and one study used blueberry concentrate.",Nutrients,Berries,2021
Berries – Blueberry Interventions and Cognitive Effects,"Eight studies reported an improvement in cognitive performance, particularly short- and long-term memory and spatial memory, after blueberry consumption or supplementation at various doses and time lengths. However, considerable differences in the study design, dosages, and anthocyanin content hinder between-study comparison. A double blinded, chronic intervention RCT investigated the effect of two blueberry formulations (whole wild blueberry powder at 500 mg (WBP500) and 1000 mg (WBP1000) and a purified extract at 100 mg (WBE111)) for six months vs. placebo on cognitive performance in 120 older adults (aged 65 to 80 years). The study results indicated that a three-month intervention with WBE111 facilitated better episodic memory performance and reduced CV risk factors over 6 months. Another small RCT involving thirteen men and twenty-four women, aged 60 to 75 years, randomized participants to consume either freeze-dried blueberry (24 g/d, equivalent to 1 cup of fresh blueberries) or a blueberry placebo for ninety days. Participants in the blueberry group showed significantly fewer repetition errors in the California Verbal Learning test and reduced switch cost on a task-switching test across study visits compared to controls.",Nutrients,Berries,2021
Berries – fMRI Studies and Mixed Cognitive Results,"Another small trial included healthy older adults randomized to consume either 30 mL of blueberry concentrate providing 387 mg anthocyanidins or isoenergetic placebo for twelve weeks undertaking a battery of cognitive function tests and fMRI pre- and post-supplementation. Significant increases in brain activity and in working memory were observed in response to blueberry supplementation relative to the placebo group in brain areas associated with cognitive function. In another study, 40 healthy 50 to 70-year-old participants were provided a berry beverage based on a mixture of berries (blueberries, blackcurrant, elderberry, lingonberries, strawberry, and tomatoes) or a control water-based beverage daily during five weeks in a randomized crossover design. The berry intervention significantly reduced total- and low-density lipoprotein (LDL) cholesterol vs. baseline and vs. the control beverage. Participants performed better in the working memory test after the berry beverage when compared to after the control beverage. No changes in the cognitive tests were observed.",Nutrients,Berries,2021
Berries – Additional Trials and Overall Summary,"Krokorian et al. conducted another small study in a sample of nine older adults with early memory changes randomized to daily consumption of wild blueberry juice. After twelve weeks of intervention, the authors observed improved paired associate learning and word list recall vs. baseline values. They also compared the memory performances of participants receiving the intervention with a demographically matched sample who consumed a berry placebo beverage and observed comparable results for paired associate learning. In a 24-week double-blind RCT, older men and women received daily fish oil or blueberry or both and neuropsychological assessment. Both fish oil and blueberry groups reported fewer cognitive symptoms, and the blueberry group showed improved memory discrimination. Another double-blind RCT among 215 healthy older adults receiving 600 mg/d of a polyphenol-rich extract from grape and blueberry for six months found no significant effect on the primary visuospatial memory outcome, but a subgroup with advanced cognitive decline responded positively. In summary, available data indicate possible neuroprotective effects of berries or their products, but results come from small studies, and large long-term RCTs are still lacking.",Nutrients,Berries,2021
Coffee – Mechanisms and Early Evidence,"3.5. Coffee
There is evidence from in vitro studies indicating that caffeine has antioxidant properties and from experiments in AD animal models showing amyloid-beta suppression. The decrease in amyloid-plaques was associated with greater levels of phosphorcyclic amp-response element binding protein (CREB), protein kinase A activity stimulation, and a reduction in the expression of phosphor-c-Jun N-terminal kinase (JNK) and phosphorextracellular receptor kinase (ERK) in mouse models of AD, which may activate brain survival cascades. In the short-term, coffee and caffeine are recognized stimulants to memory and cognition. Yet, the evidence on long-term effects is scarce. A case-control study reported that elevated serum levels of caffeine were associated with lack of progression to dementia in a sample of 124 older adults with MCI. In the Cardiovascular Risk Factors, Aging and Dementia (CAIDE) study, coffee drinkers had lower risk of developing AD and dementia; the lowest risk (65% reduction) was reported for participants consuming three to five cups of coffee daily after a 20-year follow-up.",Nutrients,Coffee,2021
Coffee – Observational Findings and Inconsistent Results,"Analysis of data from the Honolulu-Asia Study reported that the highest caffeine intake was associated with a lower risk of post-mortem neuropathological dementia lesions versus the lowest caffeine intake. Nevertheless, coffee and caffeine intake during midlife were not associated with AD, vascular dementia, cognitive decline, or neuropathological lesions. A study conducted in Portugal reported an association between caffeine intake with slower cognitive decline, while another study conducted in France did not report any association. A longitudinal study by Arab et al. showed a lower risk of cognitive decline among participants with higher coffee intake, but there was no dose response. A Finnish study showed no relationship. A meta-analysis of studies evaluating the association of caffeine intake and cognitive deterioration did not find significant effects; the studies included were highly heterogeneous.",Nutrients,Coffee,2021
Coffee – Dose–Response Studies and Brain Imaging Results,"Recent investigations continue to provide inconsistent results. A meta-analysis aiming to investigate the dose–response relationship between alcohol, coffee, or tea consumption and cognitive deficits including prospective cohort studies or nested case-control studies (n = 29) found that a low consumption of coffee reduced the risk of any cognitive deficit (<2.8 cups/day) or dementia (<2.3 cups/day). Conversely, a study exploring the effect of lifetime coffee consumption on the volume of white matter hyperintensities (VWMH) in late life among 492 cognitively normal adults found that higher cumulative lifetime coffee consumption was associated with significantly higher log VWMH in both sexes. Participants consuming >2 cups of coffee per day showed higher log VWMH in late life than those who consumed less. These findings suggest that prolonged high coffee consumption may be associated with the risk of WMH in late life.",Nutrients,Coffee,2021
Coffee – Cohort Studies and Cognitive Associations,"A prospective cohort study from Japan examining the association of green tea and coffee consumption with repeated MMSE scores in 620 men and 685 women from the NILS-LSA did not observe any association between coffee consumption and incident cognitive decline (MMSE < 27) after a mean follow-up of 5.3 years, while tea showed a significant association. Other contrasting results include a study investigating AD pathology (beta-amyloid deposition and WMH). Among 411 non-demented older adults, lifetime coffee intake of ≥2 cups/day was significantly associated with lower beta-amyloid positivity versus <2 cups/day after adjusting for confounders. However, lifetime or current coffee intake was not related to hypometabolism, atrophy of AD-signature regions, or WMH volume.",Nutrients,Coffee,2021
Coffee – PREDIMED-plus Findings and Summary,"Since coffee is rich in polyphenols (caffeine, diterpenes, melanoidins, trigonelline), which can stimulate brain activity, a cross-sectional analysis of participants in the PREDIMED-plus study explored the association of coffee consumption and dietary caffeine intake with poor cognitive functioning. Total coffee consumers and caffeinated coffee consumers had better cognitive functioning than non-consumers after adjusting for potential confounders (OR 0.63; 95% CI 0.44–0.90 and OR 0.56; 95% CI 0.38–0.83). No associations were observed for decaffeinated coffee. Participants in the highest tertile of dietary caffeine intake had significantly lower odds of poor cognitive functioning than those in the lowest tertile. There is no specific evidence on the effects of coffee on cognitive decline in RCTs. In conclusion, there is no consistency in the literature on the association of positive effects of coffee on long-term cognition. More research is still needed on this important subject.",Nutrients,Coffee,2021
Garlic – Experimental Neuroprotective Findings,"3.7. Garlic
In experimental animals, extracts of garlic have exhibited antioxidant properties and protective actions against amyloid-beta-induced neurotoxic effects. In vitro studies have reported that allicin, an organosulfur compound contained in garlic, inhibited cholinesterase enzymes and upregulated brain acetylcholine concentrations. In experimental animals, S-allyl cysteine, the active compound of aged garlic extract, has shown a mitigation of LPS-induced cognitive deficits via the attenuation of oxidative stress, neuroinflammation, astrogliosis, and acetylcholinesterase activity. However, no clinical trials for these compounds are available. There are some negative reports, such as those from the Doetinchem Cohort Study comprising 2613 participants aged 43–70 years, in which a higher consumption of allium (onion, garlic, and leek) was associated with worse scores on speed of cognitive processes and cognitive flexibility in cross-sectional analyses, and there was no association with cognitive decline in longitudinal analyses. More recent experimental studies confirm that garlic extract may be effective in alleviating cognitive impairment and neuropathology in AD animal models, but there is no evidence of possible effects on cognition in human studies.",Nutrients,Garlic,2021
Tea – Traditional Use and Mechanistic Evidence,"3.8. Tea
In Asian cultures where tea is largely consumed, it is traditionally considered a cognitive enhancer. Acute effects of tea consumption on mood and cognitive performance have been reported in some studies and have been linked to antioxidants contained in tea, such as epigallocatechin-3-gallate (EGCG), L-theanine, and caffeine. In addition, it has been suggested that the neuroprotective actions of tea consumption may be mediated by inhibition of acetylcholinesterase and regulation of stress hormones. Nevertheless, there is no definitive evidence on tea consumption neuroprotective actions due to inconsistent results of available studies. Tea bioactive components, i.e., L-theanine and EGCG, may have shown anti-amyloidogenic and antioxidant properties in vitro, but the evidence for their use in humans as nutraceuticals is limited; hence, their use in the clinical practice is not currently recommended.",Nutrients,Tea,2021
Tea – Cohort Evidence From Japan,"Recently, a prospective cohort study analyzing data from 1305 cognitively competent participants at baseline of the NILS-LSA in Japan, aged 60–85 years, examined the mean consumption of green tea and coffee in the previous year in relation to cognitive decline assessed with repeated measurements of MMSE score (up to six times biennially). During follow-up (mean of 5.3 ± 2.9 years), 432 participants had incident cognitive decline (MMSE < 27). In multivariable-adjusted Cox proportional hazard regression, participants who consumed green tea once/day, 2–3 times/day, and ≥4 times/day had a progressively significant lower risk of incident cognitive decline compared to participants who consumed green tea < once/day. No significant association was found between coffee intake and cognitive decline.",Nutrients,Tea,2021
Tea – Meta-Analysis and Summary,"Since the results of studies in humans on alcohol, coffee, and tea consumption in relation to cognitive decline have been incongruous, a recent meta-analysis of 29 prospective cohort studies or nested case-control studies in a cohort from different countries worldwide aimed to find the dose–response relationship between alcohol, coffee, or tea consumption and cognitive deficits. The dose–response relationships showed that compared to non-drinkers, green tea consumption was a significant protective factor for cognitive health; one cup of tea per day was associated with a 6% reduction in the risk of cognitive deficits. Nevertheless, the available studies are all observational, and there is no solid evidence of protective effects of tea consumption on cognitive decline.",Nutrients,Tea,2021
Alcohol – Cardiovascular Links and Cognitive Risk,"3.9. Alcohol
We have mentioned how both risk and protective factors for CV disease are also risk/protective factors for cognitive impairment and dementia. The MedDiet dietary pattern includes the possibility of consuming light to moderate amounts of alcohol (5–25 g/day for women and 10–50 g/day for men), which has been found to be associated with a lower risk of total mortality, type 2 diabetes, coronary heart disease, stroke, and heart failure. The evidence of wine’s cardioprotective effects emerged in earlier studies. However, the proposed CV protective actions of alcohol have been lately a matter of debate with evidence showing an inverse association of alcohol consumption with coronary heart disease but also with an increased risk of different types of stroke, which is very relevant for the development of cognitive decline and dementia. It is clear that heavy drinking (over 4 drinks/day) represents a risk factor for CV and other chronic diseases and is a leading cause of premature deaths in the USA.",Nutrients,Alcohol,2021
Alcohol – APOE ε4 Interactions and Observational Evidence,"Supporting the message that there is no safe level of alcohol consumption, studies based on Mendelian randomization analyses in mega-cohorts have questioned the CV benefits of alcohol consumption. However, the effects of alcohol consumption on the risk of cognitive deterioration appear to be strongly modified by the presence of APOE ε4 allele. Analyses of data from the Epidemiology of Vascular Aging (EVA) prospective study involving 1,389 participants aged 59–71 years followed for 4 years found that alcohol drinking was associated with a decreased risk of cognitive deterioration in non-APOE ε4 carriers, whereas an opposite association was observed in APOE ε4 carriers. Other data have accumulated confirming that moderate alcohol consumption may be beneficial for cognition.",Nutrients,Alcohol,2021
Alcohol – Meta-analyses and Epidemiological Patterns,"A recent meta-analysis of observational studies concluded that light to moderate alcohol consumption is associated with a reduced risk of dementia, whereas both abstinence and heavy drinking are associated with a higher risk of dementia. Similar results were obtained in the Whitehall II cohort study. Furthermore, it is well known that both abstinence and excess alcohol are associated with increased risk of CV disease and diabetes, which are established links with dementia. Concerning cognition, there is abundant epidemiological evidence that low to moderate alcohol consumption is associated with better cognitive function than abstinence or excess drinking.",Nutrients,Alcohol,2021
Alcohol – Wine Polyphenols and Uncertain Benefits,"For red wine, it has been suggested that its composition comprising not only alcohol but also bioactive compounds (polyphenols) can impact oxidative stress and chronic inflammation. Nevertheless, research on resveratrol, the most intensely studied wine polyphenol, has not demonstrated that the neuroprotective effects obtained in experimental animals are replicable in humans. A meta-analysis of twenty-nine prospective studies aiming to explore possible dose–response relationship between alcohol, coffee, and tea consumption and cognitive deficits found that compared to non-drinkers, low consumption (<11 g/d) of alcohol could reduce the risk of cognitive deficits or dementia, but there was no significant effect of heavier drinking (>11 g/d).",Nutrients,Alcohol,2021
Alcohol – Inconsistent Findings and Dose–Response Studies,"A recent review summarizing relevant studies concluded that no definitive results can clarify if light to moderate alcohol drinking is detrimental to cognition and dementia, or if alcohol intake could reduce the risk of developing AD. A study using data from the Baltimore Longitudinal Study of Aging including ten cognitive scores found mixed effects of alcohol on letter fluency, attention, and working memory depending on sex and baseline age. A recent dose–response meta-analysis including six prospective studies (n = 4244) with data on at least three levels of alcohol exposure found that heavy alcohol intake (>14 drinks/week) was significantly associated with higher risk of progression to dementia in people with MCI, with a nonlinear relationship: drinking over 16 drinks/week significantly increased progression risk.",Nutrients,Alcohol,2021
Alcohol – Summary and Risks,"In summary, there are still contrasting results regarding the protective effects of low/moderate alcohol consumption on the risk of cognitive impairment and dementia. Certainly, there is accumulated evidence of benefit, but there are also studies that fail to reach definitive conclusions. The point on which there is general agreement is that excessive alcohol consumption can be detrimental for cognitive functions, and besides, in some cases, even moderate consumption can engender increased risk of drowning, violence, and injuries from car accidents and falls, and with a higher risk of breast cancer.",Nutrients,Alcohol,2021
Curcumin – Background and Epidemiological Observations,"3.10. Curcumin
The rhizome of Curcuma longa, part of the curry spice widely used traditionally in Asian cuisine, contains the polyphenolic compound turmeric curcumin, which is used as therapy for various conditions in traditional Indian medicine and a potent antioxidant. It has been reported that older healthy people who consumed curry frequently had better cognitive performance vs. non-consumers of curry, while the Indian population that widely used curry seem to have a lower prevalence of AD compared to the US population. A number of experimental studies have shown the potent anti-oxidant and anti-inflammatory properties of curcumin and its protective effects against AD in animal models, while few RCTs and case reports are available.",Nutrients,Curcumin,2021
Curcumin – Clinical Trials in Alzheimer's Disease,"A study involving thirty-four AD patients receiving either 1 or 4 g/d of curcumin or placebo for six months found no significant effects in MMSE. Another RCT included thirty-six patients with dementia receiving 2 or 4 g/d of curcumin C3 complex (95% curcuminoids) or placebo for twenty-four weeks, followed by a 48-week open-label trial with curcumin C3 complex for the placebo arm. There were no significant differences between treatment groups and placebo in cognitive scores (ADAS-Cog, NPI, ADCS-ADL, MMSE) or cerebrospinal fluid markers. A case report article described three patients with dementia and severe behavioral and psychological symptoms who had a remarkable improvement of these symptoms after treatment with 100 mg/day of curcumin and donepezil for twelve weeks with significantly decreased NPI scoring.",Nutrients,Curcumin,2021
Curcumin – RCTs in Cognitively Healthy Older Adults,"A double-blind RCT examined the acute (1 and 3 h after a single dose), chronic (4 weeks), and acute-on-chronic (1 and 3 h after single dose following chronic treatment) effects of solid lipid curcumin formulation (400 mg as Longvida®) on cognitive function, mood, and biomarkers in sixty healthy adults (age 60–85). Curcumin significantly improved performance on sustained attention and working memory tasks after one hour of administration vs. placebo. Following chronic treatment, working memory and mood (general fatigue and stress-induced calmness/contentedness) were significantly better vs. placebo. Another double-blind RCT investigated curcumin to prevent cognitive decline in community-dwelling older adults. Ninety-six participants received placebo or 1500 mg/d Biocurcumax® for 12 months. An interaction was observed for the Montreal Cognitive Assessment, driven by decline in the placebo group at 6 months, not seen in the curcumin group. No differences were observed for other cognitive measures.",Nutrients,Curcumin,2021
Curcumin – Review of Translational Evidence,"A recent review of preclinical and clinical studies evaluated the efficacy of curcumin in ameliorating and preventing age-associated cognitive decline. Results from preclinical studies consistently demonstrated that curcumin and its analogues were efficacious for various aspects of cognitive impairment and processes contributing to age-associated decline. Results of the few published clinical studies were not univocal, but some continue to show promising results for curcumin’s use against cognitive decline yet overall remain inconclusive. Both in vitro and in vivo studies reported that curcumin can significantly decrease oxidative stress and systemic inflammation and hinder pathways that activate transcription factors associated with these processes. Further clinical studies are needed including biomarker evaluation (amyloid, tau) and appropriate population targeting.",Nutrients,Curcumin,2021
Curcumin – Bioavailability and New Formulations,"Turmeric supplements generally have low bioavailability, which is why new formulations have been proposed, such as polymeric micelles, polymer nanoparticles, nanogels, dendrimers, nanoemulsions, inclusion complexes, phytosomes, solid–lipid nanoparticles, curcumin nanoparticles, liposomes, micelles, nanogel, dendrimers, nanoemulsions, inclusion complexes, and phytosomes with potential to reduce intestinal degradation and increase curcumin bioavailability, ultimately enhancing its efficacy throughout the body and the brain.",Nutrients,Curcumin,2021
Omega-3 Fatty Acids – Physiological Role and Early Evidence,"3.11. Omega-3 Fatty Acids
The PUFAs are crucial components of the neuronal cell membranes, which preserve membrane fluidity for synaptic vesicle fusion and neurotransmitter communication. PUFAs may be lipid messengers and precursors for signaling processes to promote protection or prevent neuronal damage. A deficit of PUFAs in the hippocampus, cortex, and cerebellum has been reported in the aged brain, which may be worse in AD. The most extensively studied PUFAs regarding cognitive deterioration are omega-3 long chain (LC) PUFAs with conflicting results. A systematic review concluded that omega-3 long chain (LC)PUFAs play a relevant role in the reduction of cognitive decline, while other studies have shown negative results.",Nutrients,Omega-3 Fatty Acids,2021
Omega-3 Fatty Acids – Large Clinical Trials and Meta-Analyses,"For example, the “Supplementation with Folate, vitamin B6 and B12 and/or Omega-3 fatty acids” trial, involving 1748 participants with a history of CV disease, did not find any significant effect of vitamin B and omega-3 PUFA supplementation on cognitive function. A double-blind RCT including 302 cognitively intact persons older than 65 years receiving 1.8 g/d EPA-DHA, 0.4 g/d EPA-DHA, or placebo for 26 weeks found no significant effects. A meta-analysis of three trials comprising data from 3536 participants aged over sixty years with preserved cognitive performance at baseline supplemented with omega-3 PUFAs reported no significant effects on cognitive function. A study conducted in China found that participants older than 65 years who consumed ≥100 g/week of fish had a reduction of near 65% in the mean annual rate of global cognitive decline with no associations among participants aged 55–64 years.",Nutrients,Omega-3 Fatty Acids,2021
Omega-3 Fatty Acids – MAPT Trial and Intrinsic Capacity,"Likewise, the Multidomain Alzheimer Preventive Trial (MAPT) including 1525 participants found that a multidomain intervention plus omega-3 PUFAs supplementation, either alone or in combination, had no significant effects on cognitive deterioration over 3 years. More recent analyses of data from 1445 MAPT participants explored the effects of PUFAs and the mentioned multidomain intervention on levels of intrinsic capacity (IC), which is a construct proposed by the WHO including the Geriatric Depression Scale, Short Physical Performance Battery, Z-score combining four tests (cognitive function), and handgrip strength. After three years, the IC Z-score decreased significantly among all groups with no significant differences between groups, confirming no effect of the intervention.",Nutrients,Omega-3 Fatty Acids,2021
Omega-3 Fatty Acids – Observational Studies in Older Adults,"A recent cross-sectional study conducted in Australia investigated the relationship between erythrocyte omega-3 LCPUFA, DHA and EPA levels and their corresponding dietary intakes with cognition and physical function in a cohort of 142 community-dwelling older adults (60–85 years) at risk of dementia. Higher dietary DHA and EPA were associated with better global cognitive function, better attention/psychomotor composite scores, mobility, and gait speed. No associations were found between erythrocyte omega-3 LCPUFA and cognitive or functional performance. A recent narrative review examined the available evidence on the association between DHA/EPA and brain volume in non-demented older adults. Most studies reviewed provided mixed findings and were brain-region dependent.",Nutrients,Omega-3 Fatty Acids,2021
Omega-3 Fatty Acids – Additional Trials and Meta-Analyses,"Another recent trial and a meta-analysis reported negative results. The small RCT (n = 33) evaluated the effect of supplementation with omega-3 fatty PUFAs (2.3 g/d) on biomarkers in CSF of AD patients after six months. There were no significant differences between groups for CSF biomarkers, with only a small increase in two biomarkers that did not correlate with MMSE. A systematic review and meta-analysis of thirty-eight RCTs (49,757 participants) assessed the effects of increasing omega-3, omega-6, or total PUFA on incident neurocognitive illness and cognition. The meta-analysis suggested no or very little effect of omega-3 LCPUFA on incident neurocognitive illness, new cognitive impairment, or global cognition. Effects did not change with sensitivity analyses, doses, durations, or intervention types.",Nutrients,Omega-3 Fatty Acids,2021
Omega-3 Fatty Acids – Sporadic Positive Results and Summary,"There are also some sporadic positive results. Among 285 participants with stable coronary artery disease on statin treatment randomized to 3.36 g/d EPA and DHA or none for 30 months, participants on EPA/DHA treatment had significantly better scores for verbal fluency, language, recall memory, and visual–motor coordination. A post-doc analysis of the placebo-controlled FACIT trial found that the efficacy of folic acid treatment on cognitive functioning depended on omega-3 PUFA status: participants with lower omega-3 PUFA at baseline benefited, while those with higher levels did not. In summary, findings of studies on the effects of omega-3 PUFAs on cognition are mixed. At present, there is insufficient evidence to recommend this supplementation to improve cognitive performance or prevent cognitive decline.",Nutrients,Omega-3 Fatty Acids,2021
Magnesium – Biological Roles and Early Evidence,"3.12. Magnesium
There is substantial evidence showing that a deficit of magnesium (Mg) leads to increased free radicals production in various tissues, increased oxidative tissue damage, greater superoxide anion production by inflammatory cells, reduced antioxidant enzyme expression and activity, lessened cellular and tissue antioxidant concentrations, and augmented oxygen peroxide production. Mg is crucial for synaptic conduction, N-methyl-D-aspartate (NMDA) receptor response to excitatory amino acids, inhibition of calcium channels, calcium influx, and glutamate release, and for the stability and viscosity of cell membranes. Vasospasm has been reported in conditions of Mg deficiency; contrariwise, high Mg levels induce tone relaxation in cerebral arteries. The previous results have aroused interest in studying the role of Mg in cognitive decline and dementia in recent years.",Nutrients,Magnesium,2021
Magnesium – Associations With Alzheimer’s Disease,"Earlier investigations had shown that AD patients exhibited low serum Mg levels and decreased Mg brain tissue concentrations in autoptic studies. We found reduced plasma concentrations of ionized free Mg in AD patients that were associated with the severity of the cognitive dysfunction. A study reported a negative significant association of serum Mg concentrations with two scales of AD clinical severity (Global Deterioration Scale and Clinical Dementia Rating), supporting the likely protective role of Mg on cognitive performance. More recent analyses of observational studies have confirmed the association of Mg deficit with an increased possibility of incident cognitive decline.",Nutrients,Magnesium,2021
Magnesium – Systematic Reviews and Nutrient Interactions,"A systematic review comparing Mg status in AD with healthy controls (HCs) or medical controls (MCs) included 13 studies (AD: 559; HCs: 381; MCs: 126). Compared to HCs, patients with AD had significantly lower Mg in cerebrospinal fluid (two studies) and hair (two studies). No differences were evident for serum Mg. Mg plays a critical role in vitamin D biosynthesis and metabolism. Deficiencies in both nutrients, common in old age, have been associated with poor cognition. A study using NHANES 2011–2014 data (n = 2466, ≥60 years) found that higher total Mg intake was independently associated with higher DSST cognitive scores, especially among women, non-Hispanic whites, physically active participants, and those with sufficient serum 25(OH)D.",Nutrients,Magnesium,2021
Magnesium – Prospective Cohort Findings,"In the REGARDS US cohort (n = 2063), baseline serum Mg concentration showed an inverse threshold association with incident cognitive impairment. Compared to those with hypomagnesemia (<0.75 mmol/L), intermediate serum Mg (0.75–<0.81) was associated with 41% reduced odds of cognitive impairment; higher Mg levels did not provide further benefit. Another study examined plasma Mg in 102,648 participants from the Copenhagen General Population Study. Hazard ratios for non-AD dementia were 1.50 for the lowest and 1.34 for the highest quintile vs. the fourth quintile (reference). No associations were observed for AD.",Nutrients,Magnesium,2021
Magnesium – Dementia Risk and Cognitive Decline,"In mediation analyses, diabetes explained part of the association between low Mg and high risk of non-AD dementia, while smoking, stroke, and systolic blood pressure played minor roles. Data from 12,040 cognitively intact participants in the ARIC study showed 2519 dementia cases over a median 24.2 years. Participants in the lowest quintile of serum Mg had a 24% higher rate of incident dementia than those in the highest quintile. No relationship was found between serum Mg and cognitive decline across domains. Thus, low midlife serum Mg was associated with increased risk of incident dementia but did not appear to impact cognitive decline rate.",Nutrients,Magnesium,2021
Magnesium – Summary and Research Gaps,"In summary, at present, there is accumulated evidence supporting a role for Mg in cognitive decline, both as an antioxidant and neuroprotective agent as well as in association with the incidence of dementia in observational studies. However, so far, there are no trials testing whether Mg supplementation can directly be preventive or therapeutic in cognitive disorders; thus, the protection of Mg supplementation for the development of AD remains to be further elucidated in well-designed RCTs.",Nutrients,Magnesium,2021
Ginkgo biloba – Composition and Experimental Evidence,"3.13. Ginkgo biloba
The leaves of the tree Ginkgo biloba (Gb) native to China are a popular herbal medicine in Traditional Chinese Medicine, whose extracts have been used for various disorders including cognitive disorders, hypertension, coronary heart disease, and cerebral ischemia. In animal models and in vitro, the extract of Gb leaves has been reported to attenuate neuronal damage, in particular that induced by amyloid-beta and even to have beneficial effects on cognition in combination with donepezil in animal models of AD. The chemical constituents of Gb comprise flavonoids, terpene lactones, and ginkgolic acids that may explain its favorable effects in experimental studies possibly mediated by antioxidant activities, improvements in microcirculation, modulation of neurotransmitters, enhancement of neuroplasticity, prevention of brain edema, and other neuroprotective actions.",Nutrients,Ginkgo biloba,2021
Ginkgo biloba – Clinical Studies and Mixed Outcomes,"Ginkgo biloba extracts have been used empirically in the treatment of dementia in humans for several decades. However, whether Gb can improve cognitive function and prevent cognitive decline and dementia in clinical studies is as yet a controversial question. Even if few available large-scale clinical trials suggest that Gb extracts are relatively efficacious in delaying the progress of dementia, other trials showed negative results. Some systematic reviews and meta-analyses have been conducted to assess Gb extracts effects on the prevention and treatment of MCI and dementia with mixed results. The available studies are very heterogeneous, but in general, those carried out in healthy people are more frequently negative than those involving patients with MCI or dementia. The effects seem to be dose-dependent with better results for doses higher than 240 mg/d and longer duration. However, the results are variable, and no definitive conclusions can be made for the use of Gb in cognitive disorders or in the prevention of dementia.",Nutrients,Ginkgo biloba,2021
Resveratrol – Sources and Experimental Findings,"3.14. Resveratrol
This is a phytoalexin from the group of polyphenols that is contained in berries. Most of the resveratrol that is consumed through the diet in humans derives from grapes and red wine. Resveratrol as other polyphenols has shown antioxidant and anti-inflammatory actions. Studies conducted in experimental models of AD reporting reduced hippocampal neurodegeneration and improved cognitive memory performance with resveratrol administration provided some optimism to the possibility that this polyphenol could contribute to the prevention of cognitive decline and dementia. Clinical trials in humans are small, fewer, and have shown mixed results.",Nutrients,Resveratrol,2021
Resveratrol – Human Trials and Meta-analysis Findings,"Some small trials exploring intermediate outcomes have reported seemingly beneficial results. A placebo-controlled trial involving twenty-two healthy adults reported a dose-dependent (250–500 mg/d) increase in cerebral blood flow in the prefrontal cortex during cognitive tasks. Another small study of twenty-three overweight participants aged 50–75 receiving 200 mg/d of resveratrol or placebo for 26 weeks reported improved memory performance and higher functional connectivity of the hippocampus. However, a recent systematic review and meta-analysis of human clinical trials and animal studies showed that while most animal studies reported positive effects, eleven meta-analyses of human placebo vs. resveratrol, grape, or wine treatment trials did not find significant effects on cognitive performance, mood, gray matter volume, or blood pressure.",Nutrients,Resveratrol,2021
Resveratrol – Summary and Clinical Implications,"In summary, the promising effects of resveratrol on cognition and dementia prevention in experimental models are not replicated in human clinical trials based on currently available data. Likewise, there is no evidence on the effects of light wine consumption in this regard, while heavy alcohol consumption can be detrimental for brain health. Therefore, there is no validated evidence for prescribing this supplement or advice recommending wine consumption to improve cognitive function or prevent AD and other types of dementia.",Nutrients,Resveratrol,2021
Phytoestrogens – Definition and Experimental Findings,"3.15. Phytoestrogens
These are plant-derived naturally occurring polyphenolic non-esteroidal xenoestrogens that, because of its structural similarity with estradiol, have the ability to cause estrogenic and/or anti-estrogenic effects by binding to the estrogen receptors. Phytoestrogens have been described in over 300 plants. Isoflavones are the most extensively investigated phytoestrogens, and the neuroprotective effects of soy isoflavones have been demonstrated in experimental animal research and cell cultures studies, which are attributable to both their antioxidant properties and their interaction with the estrogen receptor. However, the studies include mixtures of them, such as genistein and daidzein, which prevents the identification of which component the cognitive effects depend on. The studies carried out on soy consumption and phytoestrogen supplements on cognitive function in rodent models and in vitro have shown variable and inconclusive results, most reporting positive neuronal effects, but high doses may have negative brain and cellular effects with an overall absence of adverse events.",Nutrients,Phytoestrogens,2021
"Phytoestrogens – Human Studies, Equol Production, and WISH Trial","Some clinical studies reported cognitive positive effects in adult women that were reversed in older women, while in men, the results were even more ambivalent. The inconsistencies found have been explained by the use of diverse isoflavone supplements at various doses in short-term clinical trials. Most studies reporting no effects have been conducted in European cohorts with low soy intake, while Asian populations with higher soy consumption show a lower incidence of cognitive decline. The phytoestrogen daidzein is transformed into S-equol by the gut microbiota, and about 70% of Japanese individuals are equol producers, helping explain cross-population differences. In the WISH trial (n = 313 postmenopausal women), 25 g/d of isoflavone-rich soy protein for 2.5 years showed no overall cognitive differences vs. placebo, although women within 5–10 years of menopause exhibited a non-significant trend toward improvement and significant enhancement of verbal episodic memory. S-equol producers also showed a non-significant trend toward improvement. In conclusion, data on cognitive benefits are inconclusive and no RCTs exist for AD prevention.",Nutrients,Phytoestrogens,2021
"Vitamins – Overview and Mixed Findings (A, B Vitamins)","3.16. Vitamins
There are numerous studies investigating effects of vitamin supplements on cognitive function and dementia, with no definitive conclusions. Data from the Physicians’ Health Study showed no short-term cognitive effects of beta-carotene but beneficial effects with long-term (18-year) use. Studies of vitamin B on cognition are mixed: an RCT of 299 men over 75 reported no effects after 2 years of folic acid, B12, and B6; a meta-analysis of nine RCTs found no significant effects; whereas another RCT of 900 participants aged 60–74 found significant improvements with folic acid and B12. A large Danish cohort study found no association between low plasma B12 and dementia risk, nor effects of B12 treatment.",Nutrients,Vitamins,2021
"Vitamins – Antioxidant Vitamins (C, E) and Mixed Evidence","Studies of vitamins C and E have inconsistent results. The Women’s Health Study found no cognitive effects of vitamin E supplementation in 6377 women over 65. Data from the Duke EPESE cohort showed no effect of vitamins C/E on AD incidence. Conversely, the Canadian Study of Health and Aging reported that combined vitamins C and E decreased cognitive decline. Brain tissue studies found gamma-tocopherol levels associated with lower amyloid and tangle burden, but alpha-tocopherol was not associated. The AREDS trial and the Women’s Antioxidant Cardiovascular Study found no cognitive benefits from antioxidant mixtures. Overall, evidence remains inconsistent.",Nutrients,Vitamins,2021
"Vitamin D – Brain Physiology, Observational Data, and Clinical Trials","Vitamin D plays roles in brain physiology via VDR expressed in hippocampus, orbitofrontal cortex, cingulate, amygdala, and thalamus. Low vitamin D levels are common in older adults and have been associated with dementia in cross-sectional and some longitudinal studies, although other studies report no associations. Reverse causality remains possible. Intervention trials have failed to show prevention of cognitive decline or AD with vitamin D supplementation. A recent Cochrane review concluded that vitamin or mineral supplementation strategies in healthy adults show no meaningful effect on cognitive decline, although evidence remains insufficient for definitive conclusions.",Nutrients,Vitamins,2021
Multinutrient Combination – LipiDiDiet Trial Overview,"3.17. Multinutrient Combination
LipiDiDiet is the first double-blind, multicentre, international RCT of a non-pharmacological intervention in prodromal AD. The intervention is the multinutrient combination (Fortasyn Connect) containing DHA, EPA, uridine monophosphate, choline, vitamins B12, B6, C, E, and folic acid, phospholipids, and selenium. These nutrients were selected based on their biological and neuroprotective properties, shown in experimental models and in 3 previous clinical trials of 12–24 weeks treatment, in which improved memory was reported in mild, but not mild-to-moderate AD. LipiDiDiet focused on prodromal AD with a longer duration of the intervention. A first report involving 153 participants treated with the intervention vs. 158 controls found no significant effect on the neuropsychological test battery (NTB) score over two years of intervention, attributable to inadequate statistical power.",Nutrients,Multinutrient Combination,2021
Multinutrient Combination – Secondary Outcomes and 36-Month Follow-up,"Group differences on secondary endpoints of disease progression measuring cognition and function and hippocampal atrophy were observed. A second report of data from 162 participants (85 from the active group and 77 from the control group) who completed 36 months of intervention found reduction in NTB decline, Clinical Dementia Rating-Sum of Boxes, memory, and brain atrophy measures. Future studies may assess further benefits by integrating the multinutrient intervention with multidomain interventions such as those discussed below (Section 5).",Nutrients,Multinutrient Combination,2021
Physical Activity – Protective Role and Risk Reduction,"4. Other Non-Dietary Factors That Together with Diet May Influence Cognitive Decline
4.1. Physical Activity
Whilst dementia is a common condition in older people, no definitive treatments for this condition are available. Recent works have pointed out that about 3% of all dementia cases could be prevented by increasing levels of physical activity [18,19]. At the same time, increasing literature shows the importance of physical activity and exercise for preventing/slowing down the pathological process and dementia-related problems [20]. It is widely known that older people who are more physically active can maintain cognition for a longer time than sedentary people [18]. In a large meta-analysis, including several cohort studies and 33,816 individuals, higher physical activity levels were associated with a significant reduction in the onset of dementia in a linear dose–response manner [349]. At the same time, the effect of physical activity/exercise in MCI or in early phases of dementia should be better explored. Some studies, in fact, have reported that physical activity/exercise can delay the transition from MCI to dementia [350], but other recent investigations have reported that a moderate-to-high intensity multi-component exercise program is not beneficial in people with early dementia [351]. In our opinion, this topic is important, since MCI is one of the most diffused risk factors for dementia, with 10% to 15% of people with MCI becoming demented during one year of observation [352,353].",Nutrients,Physical Activity and Cognitive Decline,2021
"Physical Activity – MCI, Mechanisms, and Neurobiology","Again, whilst several risk factors associated with a faster conversion from MCI to dementia are already known, the research regarding sedentary behavior and physical activity is still limited to a few investigations [354]. In one umbrella review of our group, we have reported that physical activity/exercise was able to significantly improve global cognition and specific cognitive tests in MCI, even if supported by some biases, suggesting a positive role of these interventions, but studies exploring the conversion to dementia are still not present [354]. In people already affected by dementia, it seems that physical activity and exercise can improve global cognition. This finding is probably due to several pathways. First, physical activity/exercise improves the management of cardiovascular risk factors (e.g., diabetes, hypertension, dyslipidemia, and obesity), which are traditionally associated to poor cognitive performance [355]. Furthermore, it is reported that physical activity/exercise may increase neurogenesis and synaptic plasticity in animal models [350,356]. Aerobic exercise is also associated with an increase in brain-derived neurotrophic factor (BDNF), which is a factor that can stimulate neuronal cell growth and can maintain neurons in an optimal status [357]. Finally, using neuroimaging techniques, additional evidence for the impact of physical activity on brain function and structure is reported in human beings [358–360]. Physical activity/exercise might be a good predictor of long-term changes in brain structure, such as brain volume [361].",Nutrients,Physical Activity and Cognitive Decline,2021
"Physical Activity – Falls, Neuropsychiatric Symptoms, and Conclusion","Moreover, in people affected by dementia, physical activity/exercise interventions may have other effects such as decreasing the risk of disability, falls, and neuropsychiatric symptoms [354]. Again, we believe that all these outcomes are important. For example, in one study, it was reported that the incidence of falls in dementia is extremely higher and associated more frequently than controls to fractures and hypokinetic syndrome [362]. Physical activity/exercise may decrease the risk of falls by approximately 31% (204 falls every 1000 people affected by dementia treated with this intervention). Since falls are among the most important contributors of disability, we may hypothesize that the beneficial effect of physical activity/exercise in decreasing the risk of falls may consequently improve activities of daily living [363]. Moreover, another important consequence of physical activity/exercise is to decrease the presence and the severity of neuropsychiatric symptoms [364], in particular depression [365]. Again, it seems that physical activity/exercise may increase the production of neurotransmitters, neurotrophins, and BDNF as well as lead to a reduction of oxidative stress and inflammatory levels, increase cerebral blood flow, regulate the hypothalamic–pituitary–adrenal axis, and support neurogenesis and synaptogenesis [366]. Table 2 shows the summary of systematic reviews and meta-analyses exploring the association of physical activity with cognitive decline and/or incident dementia.
In conclusion, physical activity and exercise are good options for improving not only cognitive aspects but also non-cognitive outcomes in people with MCI and dementia. However, the literature is still sparse, and often, the combination with other nonpharmacological interventions, such as diet, is still limited. In this sense, we warmly suggest future intervention studies that better account for healthy lifestyle in the treatment and prevention of MCI and dementia.",Nutrients,Physical Activity and Cognitive Decline,2021
Sleep Pattern – Bidirectional Relationship and Epidemiology,"4.2. Sleep Pattern
There appears to be a bidirectional relationship between sleep pattern and dementia, with disturbed sleeping representing both a risk factor for, and symptom of, the neurocognitive syndrome [394–396]. Indeed, the notion that sleep quality and duration is critical for cognitive processing has gained increasing attention. However, it is not simple to establish causal relations, because there are vicious circles among different aspects of the disease. It is currently accepted that sleep is essential to consolidate memory and to remove the excess of beta-amyloid and hyperphosphorylated tau accumulated in AD patients’ brains [397]. Sleep disturbances frequently precede AD pathological traits and cognitive decline [398]. It has been proposed that sleep alterations (i.e., specific oscillatory patterns) may be biomarkers to predict the risk of developing AD [394]. A systematic review and meta-analysis found that sleep disturbances, including both short and long sleep duration, insomnia, obstructive sleep apnea (OSA), impaired circadian rhythm, and sleep quality, were all associated with an increased relative risk of preclinical AD, cognitive impairment, and AD [399]. Former studies found that sleep disturbances occur frequently in AD and other forms of dementia [400,401]. Recent epidemiological investigations have shown that sleep disorders in AD patients go far beyond the physiological changes that occur in normal aging [399]. As pointed out in a meta-analysis including 5634 AD patients, the prevalence was widely variable, estimated in 14–69% [402].",Nutrients,Sleep Patterns and Cognitive Decline,2021
"Sleep Pattern – Prevalence, Phenotypes, and Caregiver Impact","A retrospective study conducted in Japan involving 684 AD patients found a prevalence of 21.3% [403]. Other studies reported that over 60% of patients with MCI and AD had at least one clinical sleep disorder, of which insomnia and OSA were the most common [404,405]. The inconsistent prevalence reported may be related to the use of sleep questionnaires in many studies; understandably, cognitive impairment in patients with AD renders the information on sleep disturbances provided by a questionnaire not fully reliable. It has been reported that patients with AD can have increased sleep onset latency and reduced time spent in restorative slow wave sleep and rapid eye movement sleep [406]. In dementia with Lewy bodies, sleep disturbance may be even more common than in AD, and they have been associated with rapid eye movement sleep behavior disorder [407]. A relevant point is that sleep disturbances may significantly impact caregiver burden, caregiver sleep, and quality of life, and it is associated with a higher risk of institutionalization [408,409]. Various medications are commonly used for sleep disturbance in dementia, but evidence is lacking for most of them [410]. Melatonin may be beneficial in some cases, and it may help for nocturnal behavioral and psychological symptoms of dementia (BPSD), together with non-pharmacological interventions, which should be first-line therapy [411,412]. Trazadone may be effective for sleep disturbance and BPSD, in case the pharmacological intervention is considered necessary [413].",Nutrients,Sleep Patterns and Cognitive Decline,2021
Sleep Pattern – Non-Pharmacological Interventions and Evidence,"Indeed, non-pharmacological management has an important role due to the risk of adverse effects associated with the use of hypnotics [414]. A systematic review and meta-analysis [415] explored non-pharmacological treatments for sleep disturbance in MCI and dementia including 48 articles with participants aged 67.3 to 89.4 years. Most studies (79%) had small samples (less than 50 participants), and many were conducted in long-term care settings (62%). Most recruited participants had moderate–severe dementia (85%) with a wide range in MMSE scores (0 to 28.3/30), while only four studies examined MCI. Light therapy for one to ten weeks was the most frequently studied intervention, with most studies (81.5%) showing improvements on objective or subjective sleep measures, although with significant clinical and methodological heterogeneity that prevented a definitive conclusion. There were seven multi-modal intervention studies, all incorporating light exposure, and six of them reported improved sleep. Other less frequently used interventions included electrotherapy stimulation, physical exercises/activities, acupressure/acupuncture, and mindfulness/cognitive behavioral therapy. The authors performed a meta-analysis of data from RCTs reporting a statistically significant improvement in sleep efficiency between multi-modal interventions and controls, favoring the combined interventions (bright light, multidomain, and other therapies) [415]. Therefore, non-pharmacological sleep interventions seem useful, particularly multidomain approaches, but the available studies are heterogeneous and small. More research evaluating the efficacy of multimodal interventions in community-dwellers and those with MCI is still needed.",Nutrients,Sleep Patterns and Cognitive Decline,2021
Multidomain Lifestyle-Based Interventions – Rationale and Major Trials,"5. Multidomain lifestyle-based interventions
Due to the complex, multifactorial, and heterogeneous nature of age-related cognitive decline and of late-onset AD and dementia, it is unlikely that a single intervention can have a significant impact. A crucial consideration regarding preventive interventions is the fact that multiple risk and protective factors for cognitive decline and dementia usually coexist and interact across the lifespan to determine the overall risk of dementia. Therefore, interventions targeting several risk factors and mechanisms simultaneously may be required for optimal preventive effects. Up to now, the results of three large multidomain lifestyle-based prevention trials have been published: the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) [10], the French Multidomain Alzheimer Preventive Trial (MAPT) [243], and the Dutch Prevention of Dementia by Intensive Vascular Care (PreDIVA) [434].
The Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) is the first large, long-term RCT to demonstrate that a two-year multidomain lifestyle-based intervention by improving vascular and lifestyle-related risk factors can preserve cognitive performance and reduce the risk of cognitive decline among older adults at increased risk of dementia [10]. The FINGER RCT comprised 1260 older adults aged 60-77 years; the multidomain intervention delivered by trained professionals through individual and group sessions consisted of dietary counseling, exercise, cognitive training, social activities, and monitoring and management of vascular and metabolic risk factors. The control group was offered regular health advice.",Nutrients,Multidomain Lifestyle Interventions,2021
Multidomain Interventions – FINGER Outcomes and MAPT Trial,"After two years, the intervention showed significant beneficial effects on the neuropsychological test battery composite score (25% more improvement vs. control), executive functioning (83% more improvement), processing speed (150% more improvement), and complex memory tasks (40% more improvement) [10]. There were also favorable effects on BMI reduction, improved adherence to dietary guidelines and recommendations [435], and increase in physical activity [10].
The other two RCTs, MAPT [243] and PreDIVA [434], reported negative results for their primary outcomes although subsequent subgroup analyses showed some benefit. The MAPT RCT enrolled 1680 community dwellers aged 70 years or older who had either subjective memory complaints, limitation in one IADL, or slow gait speed. In two arms, participants received a multidomain lifestyle intervention consisting in cognitive training and counseling on nutrition and physical activity, either alone or in combination with omega-3 fatty acid supplementation. In other two arms, participants received only the omega-3 fatty acid supplementation or placebo. The primary outcome was change in a cognitive composite score. Although the trial failed to meet its primary outcome, beneficial intervention effects were observed when both groups receiving the multidomain lifestyle intervention were combined. Beneficial effects were also reported for specific subgroups: those with brain amyloid pathology or severe cognitive impairment (CAIDE risk score ≥6 points) [243,436].",Nutrients,Multidomain Lifestyle Interventions,2021
Multidomain Interventions – PreDIVA Trial and Methodological Considerations,"The PreDIVA trial enrolled 3526 unselected older adults aged 70-78 years from general practices [434]. The multidomain intervention consisted of advice concerning healthy lifestyle and intensive vascular care and risk factor management, including initiation or optimization of antithrombotics and pharmacological treatments for hypertension, diabetes, or dyslipidemia, when necessary vs. regular care for the control group. After six years of intervention, there was no difference in the primary outcome (i.e., dementia incidence) between the intervention and control groups. However, in the exploratory analyses, a reduction in the incidence of dementia was observed among participants with untreated hypertension who adhered to the treatment during the trial.
Several issues may help to explain the dissimilar results of the last two trials, including the difference in the selection of the studied populations; also the fact that the intervention should start early enough to reduce the long-term exposure to the risk factors and should have a long-term follow-up to make evident any benefit for a robust outcome such as dementia.",Nutrients,Multidomain Lifestyle Interventions,2021
Multidomain Interventions – eHealth Models and WW-FINGERS,"Innovative proposals of multidomain prevention trials have started to use novel technologies, such as eHealth and mHealth, to optimize the delivery of multidomain interventions. For example, the Healthy Aging Through Internet Counselling in the Elderly (HATICE), a European open-label 18-month RCT testing the efficacy of an Internet platform in improving self-management of CV risk factors for prevention of CV disease and cognitive decline in over-65 participants reported a modest but significant improvement in CV risk profiles with the interactive Internet intervention after 18 months [437]. If preventive interventions through Internet or via mobile applications prove to be feasible and effective and induce sustained behavioral changes on a large scale, it could support self-management and be a cost-effective way to reach and involve a large population across the world.
Following the encouraging results of the FINGER trial, the World-Wide (WW)-FINGERS network was launched in July 2017 [438]. By collectively convoking international research teams, WW-FINGERS aims to facilitate data sharing and joint analyses of studies across country borders, and strengthen the potential evidence-base for multidomain lifestyle interventions worldwide in at-risk populations from diverse geographical and cultural settings, by means of local and cultural adaptations of content and delivery method of interventions. Several countries worldwide have joined the WW-FINGERS network and are currently at different stages of planning and conducting their FINGER-type prevention trials [438].",Nutrients,Multidomain Lifestyle Interventions,2021
Oxidative Stress and Chronic Inflammation – Mechanistic Basis,"6. Proposed Mechanisms Mediating the Effects of Nutrition and Other Lifestyle Factors on Cognitive Decline
6.1. Oxidative Stress and Chronic Inflammation
Aging and age-related degenerative diseases have been linked to a pro-oxidant and pro-inflammatory state, which leads to the damage of cellular components. The brain tissue is highly susceptible to oxidative damage because cerebral metabolism necessitates large quantities of energy, is dependent on aerobic conditions, and is rich in oxidizable compounds such as PUFAs and in transition metals that facilitate free radicals generation. Furthermore, compared to other body structures, the brain has low concentrations of antioxidant systems. This may help to explain why the brain tissue is so susceptible to damage due to an accumulation of neurotoxic peptides such as amyloid-beta [439]. In studies performed in autopsies from AD patients, brains exhibited increased protein oxidative damage, glycol-oxidation, lipid peroxidation, and reduced antioxidant enzyme systems [440].
There is growing evidence linking neuroinflammation to AD pathogenesis. Misfolded and aggregated proteins, such as amyloid, bind to toll-like receptors (TLRs) and CD4 in the microglia, starting innate immune responses with the production and release of inflammatory mediators [441,442]. Alois Alzheimer already had described the microglia surrounding amyloid plaques and tangles describing the disease for the first time, but the key role of microglia inflammation has been investigated only recently.",Nutrients,Oxidative Stress and Neuroinflammation,2021
Oxidative Stress and Chronic Inflammation – Neuroinflammatory Cascade,"In normal conditions, neuroinflammation is a crucial protective mechanism. Conversely, when it becomes excessive, chronic, and uncontrolled, it may become damaging by the continuous release of cytokines, proteolytic enzymes, free radicals, complementary factors, nitric oxide, or excitatory amino acids [442,443]. Amyloid-beta accumulation further induces neuroinflammation, which produces more amyloid-beta aggregation, leading to a vicious cycle of propagating injury [444]. Increased neuroinflammation has been shown in MCI and AD patients vs. healthy controls using positron emission tomography and imaging with radioligand C-11-DAA1106 [445]. Amyloid-beta may promote a preponderance of macrophage (M)1 pro-inflammatory cells vs. M2 (anti-inflammatory cells) and decrease the switching of cellular phenotypes to lessen destruction [442,443]. Excess of nitric oxide may also contribute to induce inflammatory signals, which are key players in neurodegenerative diseases with resulting neuronal death [446].",Nutrients,Oxidative Stress and Neuroinflammation,2021
Overweight and Obesity – Epidemiology and Dementia Risk,"6.2. Overweight and Obesity
The well-known CV and metabolic risk factors, both in isolation and combined, have been recognized as risk factors for cognitive decline and AD as well [18,447]. The metabolic syndrome—namely, the simultaneous occurrence of diverse risk factors (i.e., central obesity, hypertension, hyperglycemia, dyslipidemia, and prothrombotic state)—is directly related to increased visceral adiposity in midlife as a consequence of overeating and sedentary lifestyle with harmful consequences in late life [448]. Obesity is currently a pandemic at all ages [449–451]. Currently, there is a large availability of inexpensive processed food, full of calories and poor in nutrients, which together with little physical activity have paradoxically contributed to the increased life expectancy in the last century. Overeating and a sedentary lifestyle are considered powerful risk factors for the genesis of chronic non-communicable diseases, which are becoming unsustainable [452]. A systematic review of twenty-eight cohort studies conducted from 2003 to 2013 with a follow-up range between five and forty years reported up to a 2.44-fold increased risk of late-onset dementia in participants who were overweight and obese in midlife [453].",Nutrients,Overweight and Obesity,2021
Overweight and Obesity – Mechanisms Linking Adiposity to Cognitive Decline,"Although the precise mechanism behind this association is not yet completely clear, various possible mechanisms have been suggested. Overweight and obesity are strong risk factors for cardiometabolic disease (i.e., hypertension, diabetes, and dyslipidemia), which are also recognized risk factors for dementia [18,447]. However, these factors have been taken into account in most studies as potential confounders. Obese persons with diabetes had higher concentrations of plasma amyloid proteins [454], and those with history of being overweight or obese in midlife had lower blood–brain barrier integrity after twenty-five years of follow-up [455]. Another source of inflammation that may trigger systemic and neuroinflammation lies in the accumulation and activation of macrophages around the excess fat cells that accompany obesity. These immune cells present in adipose tissue can produce pro-inflammatory cytokines (e.g., interleukin (IL)-1b, IL-6, tumor necrosis factor –TNF-) as well as less anti-inflammatory cytokines (e.g., adiponectin and IL-10), which sustain the state of low-grade chronic inflammation [456]. Obesity has been also associated with a switch from M2 macrophages to the pro-inflammatory M1 phenotype [442,457].",Nutrients,Overweight and Obesity,2021
Overweight and Obesity – Neuroinflammatory and Metabolic Consequences,"Lipids contained in the diet, and more so saturated fatty acids, induce inflammatory responses on microglia, with local cytokine secretion, i.e., hypothalamic nuclear factor kappa B (NF-kB), which may promote the apoptosis of neurons linked to body weight control, glucose homeostasis, central regulation of energy balance, and blood pressure [458,459]. Obesity may induce alterations in the developing brain of children and adolescents [460], with long-term adverse consequences. Contrariwise, weight reduction, low-calorie diets, and frequent consumption of food rich in anti-oxidant/anti-inflammatory properties, or food patterns with combinations of them, have been associated with reduced levels of systemic and adipose tissue inflammation markers [461]. Clinical studies have shown a link between insulin resistance and cognition [462] as well as between glucose regulation abnormalities in type 2 diabetes and cognitive function [463]. Accordingly, diet interventions that improved insulin resistance have been associated with decreased inflammatory cytokines concentrations and improved cognition [464,465].",Nutrients,Overweight and Obesity,2021
Microbiota – Composition and Role in Health and Disease,"6.3. Microbiota
The human gut microbiota comprises trillions of symbiotic microorganisms, which are crucial for immune and brain health and disease [466]. The main component of gut microbiota is bacteria, of which most are strict anaerobes, but it also comprises fungi and viruses. The main reported phyla in gut microbiota from adults are Bacteroidetes and Firmicutes, while Proteobacteria and Actinobacteria are found in relatively low amounts [467]. The number of gut microbes within the gastrointestinal tract is estimated to be approximately 100 trillion, which is 10 times the number of cells found in the human body. The gut microbiome is about 150 times greater than that of the human genome and encodes 100-fold more unique genes than our genome, which suggests their vast possibilities in influencing human health. This microbiome shows vast diversity amongst different population groups and has a significant influence on human health, disease state, and overall well-being [468]. Several factors may alter the composition of gut microbiota, such as diet, antibiotic exposure, and infection, which may promote the loss of homeostasis and have been implicated in the development of various diseases, including obesity, metabolic syndrome, colorectal cancer, type 2 diabetes, allergies, inflammatory bowel disease, heart failure, and neurodegenerative disorders [469–472]. Recent investigations suggests that the amyloid cascade model does not fully explain AD pathogenesis and that alterations in the gut microbiome may play a significant role in the genesis and progression of the disease [473].",Nutrients,Microbiota,2021
Microbiota – Gut–Brain Axis and Communication Pathways,"The bilateral interaction of the gastrointestinal tract and the brain was recognized for the first time in the 1880s by William James and Carl Lange, proposing that this gut–brain communication could play a role in emotional regulation. At present, the microbiota has been added to this connection (microbiota–gut–brain axis), and it is believed to constitute a two-way homeostatic pathway, through which the gastrointestinal tract exerts modifications on brain function and vice versa [474–476]. This microbiota–gut–brain axis bidirectional communication occurs through various pathways. (1) First, over thirty hormones and neurotransmitters are produced in the gastrointestinal tract. These hormones affect the brain centers that regulate metabolic control, appetite, and behavioral pathways linked to reward, anxiety, mood, memory, stress, etc. They can also act locally and activate afferent vagal terminals in the gut, thus generating afferent signals [474,475,477]. Through entero-endocrine cells, enteric neurons, and gut microbiota, the gastrointestinal tract can as well synthesize neurotransmitters that influence the functioning of both the gastrointestinal tract and the central nervous system (CNS) [475]. Neurotransmitters that impact several brain functions, including cognition (i.e., serotonin, dopamine, acetylcholine, gamma-aminobutyric acid, epinephrine, and norepinephrine), can all be synthesized in the gastrointestinal tract [478].",Nutrients,Microbiota,2021
"Microbiota – Vagus Nerve, HPA Axis, SCFAs, Immune Signaling, and BDNF","(2) In neuroanatomic communication, the main neuroanatomical communication between the enteric nervous system and the CNS is provided by the vagus nerve (parasympathetic input) and spinal nerves (sympathetic input). Interestingly, 90% of vagal fibers are afferent, suggesting that the brain is primarily a receiver of information, rather than a transmitter, with regard to gut–brain communication [479]. The vagus nerve has receptors for some hormones and neurotransmitters produced in the gastrointestinal tract, such as serotonin, ghrelin, cholecystokinin, and YY peptide, as well as receptors for bacterial fragments, such as lipopolisaccaride (LPS), whose activation can signal the brain regarding gut events. Furthermore, short-chain fatty acids (SCFAs) produced by the gut microbiota can also activate afferent fibers of the vagus nerve [480]. In support of this bidirectional communication, vagotomy has recently been shown to prevent the beneficial neurobehavioral effects induced by probiotics [481]. (3) Activation of the hypothalamic–pituitary–adrenal (HPA) axis leads to the release of cortisol by the adrenal gland during acute stress. In normal conditions, the neuroendocrine stress response is counter-regulated by a negative feedback mechanism. However, if stress is exaggerated and/or persistent, excess cortisol can be deleterious [482]. Stress can cause increased gastrointestinal motility, secretions, permeability, and also adverse effects on the gut microbiota [483]. Contrariwise, changes in microbiota can also modify the stress response. A study showed that germ-free animals had an exaggerated HPA stress response compared to animals colonized with beneficial bacteria [484]. Another preclinical study demonstrated that microbiota can modulate the stress-dependent activation of pituitary and adrenal glands [485].",Nutrients,Microbiota,2021
"Microbiota – SCFAs, Immune Pathways, BDNF, and Human Evidence","It is well recognized that chronic stress can affect the brain (and in particular hippocampal) functioning, including learning and memory processes, as well as mood regulation [482,486]. (4) Metabolites produced by bacteria, such as SCFAs, including acetate, propionate, and butyrate, can also influence the gut microbiota–gut–brain crosstalk [487]. SCFAs are produced by bacterial fermentation of substrate (mostly dietary fiber) in the large intestine. The functions of SCFAs are not yet totally clarified, but it has been suggested that they may have local effects in the colon (e.g., decrease inflammation, improve mucus production), affect gene expression by inhibiting histone deacetylases, impact hormone regulation (e.g., glucagon-like peptide 1 and peptide YY), and interact with vagal afferents. Furthermore, SCFAs can also regulate blood–brain barrier integrity and function, acting directly on the brain [488]. (5) The gastrointestinal tract contains about 70% to 80% of the body’s immune cells [489]. Components of Gram-negative bacteria such as LPS can interact with TLRs, influencing the immune response and the production of inflammatory cytokines [490], which can reach or send signals to the CNS through several mechanisms and result in neuroinflammation [471,491]. Moreover, the CNS regulates innate immune responses through neuronal and hormonal routes [492]. (6) BDNF is involved in synaptic and structural plasticity, learning, and memory, and it has been associated with the pathophysiology and treatment of several neurological diseases [493]. A preclinical study showed that germ-free mice had reduced BDNF expression in the cerebral cortex and hippocampus compared to controls [484]. Germ-free mice had lower BDNF mRNA expression in the hippocampus, cingulate cortex, and amygdala [494], while animals supplemented with probiotics or prebiotics showed increased BDNF levels in various brain regions [495]. (7) A number of intrinsic and extrinsic factors can influence the microbiota–gut–brain axis. These include genetic and epigenetic factors, as well as environmental factors, such as exercise, medications, and consumption of probiotics [476]. Diet composition and nutritional status have repeatedly been shown to be crucial modifiable factors regulating the gut microbiota across the lifespan and under various health conditions [496].",Nutrients,Microbiota,2021
Microbiota – Limitations of Animal Research and Human Evidence,"Most investigations on host–microbiota interactions come from animal models, which represent crucial tools for studying the various pathways linking the gut and the brain. Nevertheless, there are complexities and marked limitations in translating complex human disease to reductionist animal models. Studies in humans are few and small. For example, several bacteria taxa (i.e., Bacteroides Actinobacteria, Ruminococcus, Lachnospiraceae, and Selenomonadales) were found to be different in forty-three AD patients compared to those in healthy controls using RNA sequencing [497]. In summary, despite the preclinical evidence, well-designed clinical studies are still lacking to elucidate the role of the microbiota or the microbiota–gut–brain axis on the prevention and treatment of cognitive decline.",Nutrients,Microbiota,2021
Autophagy – Age-Related Decline and Link to AD Pathology,"6.4. Autophagy
The efficiency of autophagy declines with age. This crucial mechanism mediates the degradation and recycling of cellular proteins and clearance of misfolded proteins and aggregates such as amyloid, which is one of the most studied pathophysiological mechanisms in AD [498]. The mammalian target of rapamycin (mTOR) signaling, a fundamental player of cellular senescence, affects glucose metabolism, mitochondrial function, energy production, and autophagy in the brain. These events are crucial in age-associated cognitive decline and AD [499].",Nutrients,Autophagy,2021
Prothrombotic State – Vascular Dysfunction and Platelet Activation in AD,"6.5. Prothrombotic State
Vascular disorders are other features characteristic of AD (e.g., cerebrovascular dysfunction, blood–brain barrier disruption, and decreased cerebral blood flow) as well as a prothrombotic state (e.g., activated platelets, clot formation, decreased fibrinolysis). Fibrinogen accumulates with amyloid-beta, which triggers amyloid-beta fibrillization and the generation of fibrin resistant to degradation. A study showed higher platelet activating factor acetylhydrolase activity and higher oxidized-LDL concentrations in AD patients compared to healthy controls [500].",Nutrients,Prothrombotic State,2021
Summary and Conclusions,"7. Summary/Conclusions
The aging of world populations is an undeniable reality that leads to an inexorable increase in aging-related cognitive decline and its worst manifestation, dementia. The accumulation of negative results in the last decades together with the heavy financial burden while searching for effective pharmacological therapies suggest that this is the wrong path. Compelling evidence has accumulated on the critical role that adequate nutrition together with other lifestyle factors can play in the maintenance of cognitive health and in the prevention of cognitive decline and its progression to dementia. It is credible that this must be the path to travel.
However, the illusion that one or few nutritional components can be effective in this complex battle is short-lived. Nutrition research has shifted its focus in recent years from examining the effects of foods or nutrients in isolation to focusing the attention on the effects of foods/nutrients combinations in dietary patterns [17], which the results presented in this review once more corroborate as a wiser choice. In this review, more focused on nutrition but without neglecting other crucial lifestyle factors, it is clear that no single food or nutrient is the magic bullet to prevent dementia. Most components have been studied in vitro or in animals, but evidence in humans is limited by small samples, short follow-up, or unsuitable designs.
On the contrary, combinations of foods and nutrients, as shown by dietary patterns such as MedDiet, DASH, and MIND, comprising mostly plant-based unprocessed or minimally processed foods and neuroprotective nutrients, appear ideal. Essential non-dietary factors such as physical activity, sleep quality, and socialization must also be added. A recent review and Bayesian network meta-analysis identified factors associated with lower dementia risk: no sleep disturbances, high education, no diabetes, non-obesity, no smoking, living with family, physical exercise, no alcohol, and no hypertension [19].
It is reasonable to anticipate that complex diseases arise from combinations of factors, and preventive measures should be similarly combined. Though mechanisms are not fully clear, evidence on anti-inflammatory and antioxidant effects, microbiota, vascular changes, prothrombotic states, and protein clearance may explain benefits. Dementia is feared due to loss of autonomy, and often perceived as unavoidable. Increasing awareness that dementia can be largely prevented with lifelong healthy nutrition and lifestyle may encourage adherence to healthier habits for active aging.",Nutrients,Summary and Conclusions,2021
Thyroid Hormones and Aging,"Thyroid hormones have vital roles in development, growth and energy metabolism. Within the past two decades, disturbances in thyroid hormone action have been implicated in ageing and the development of age-related diseases. This Review considers results from biomedical studies identifying the importance of precise temporospatial regulation of thyroid hormone action for local tissue maintenance and repair. Age-related disturbances in tissue homeostasis are thought to be important drivers of age-related disease. In most iodine-proficient human populations without thyroid disease, the mean, median and 97.5 centile for circulating concentrations of thyroid-stimulating hormone (TSH) are progressively higher in adults over 80 years of age compared with middle-aged (50–59 years) and younger (20–29 years) adults. This trend extends into advanced ages (over 100 years). Potential causes and consequences of altered thyroid status in old age, and its association with longevity, are discussed. In about 5–20% of adults at least 65 years of age, TSH concentrations are elevated but circulating thyroid hormones remain within the population reference range, a condition referred to as subclinical hypothyroidism. Results from randomized clinical trials testing the clinical benefit of thyroid hormone replacement therapy in older adults with mild subclinical hypothyroidism are reviewed, along with implications for screening and treatment of subclinical hypothyroidism in older populations.",Thyroid Hormones and Aging Review,Endocrine Aging and Longevity,2023
Overview of Immunosenescence,"Immunosenescence, a systematic reduction in the immune system connected with age, profoundly affects the health and well-being of elderly individuals. This review outlines the hallmark features of immunosenescence, including thymic involution, inflammaging, cellular metabolic adaptations, and hematopoietic changes, and their impact on immune cells such as macrophages, neutrophils, T cells, dendritic cells, B cells, and natural killer (NK) cells.",Vaccines (Basel),Immunosenescence,2024
Thymic Involution and Inflammaging,"Thymic involution impairs the immune system’s capacity to react to novel antigens by reducing thymopoiesis and shifting toward memory T cells. Inflammaging, characterized by chronic systemic inflammation, further impairs immune function. Cellular metabolic adaptations and hematopoietic changes alter immune cell function, contributing to a diminished immune response.",Vaccines (Basel),Immunosenescence,2024
Implications and Need for Intervention,Developing ways to reduce immunosenescence and enhance immunological function in the elderly population requires an understanding of these mechanisms. Vaccines (Basel). 2024 Nov 23;12(12):1314. doi: 10.3390/vaccines12121314 Immunosenescence: Aging and Immune System Decline,Vaccines (Basel),Immunosenescence,2024
Inflammaging and Chronic Inflammation,"Inflammaging is a gradual increase in chronic inflammation that occurs with aging, characterized by higher concentrations of inflammatory markers in the body, including tumor necrosis factor-alpha (TNF-α), C-reactive proteins (CRP), and interleukins such as IL-6, IL-1 receptor antagonist (IL-1ra), IL-18, and IL-1b [13,14,15,16,17]. Older adults with elevated IL-6 levels and TNF-α in their blood are more likely to have less muscle mass and weaker muscles [18]. Age-related chronic inflammation is not directly caused by infection. This is driven by cell damage from free radicals, an imbalance in inflammatory cytokines, and cellular senescence. Aging leads to increased production of p16 (a tumor suppressor protein) and sterile α-motif domain- and HD domain-containing protein 1 (SAMHD1) in mouse models [19]. Additionally, aging alters the gut microbiota, resulting in increased lipopolysaccharide (LPS) production, which activates nuclear factor-kappa B (NF-κB), suggesting that gut bacteria’s LPS contributes to age related inflammation. NF-κB activation increases the expression of IL-6, which may lead to an increase in inflammation [20]. It is believed that these inflammatory markers affect an adult’s capacity to fight off infection, which leads to a decrease in overall immune function. Chronic inflammation plays a significant role in the development and progression of cardiovascular diseases (CVDs). Elevated levels of inflammatory markers, such as high-sensitivity C-reactive protein (hsCRP), have been associated with an increased risk of cardiovascular events [21].",Vaccines (Basel),Inflammaging,2024
Metabolic Adaptations in Senescent T Cells,"Senescent T cells exhibit metabolic reprogramming characterized by a transition from oxidative phosphorylation to glycolysis [22]. This age-related adaptation results in less efficient ATP production, compromising cellular processes essential for immune function, including proliferation and cytokine production. Consequently, T cells exhibit a reduced capacity for proliferation and cytokine production, crucial for a robust immune response. Aging significantly impacts mitochondrial DNA (mtDNA) quality and quantity [23,24]. Specifically, mtDNA number decreases with age, leading to reduced mitochondrial biogenesis and impaired energy production capacity. This decrease in mtDNA number compromises cell function, as mitochondria play a crucial role in powering cellular processes. Senescent T cells shift their energy production towards glycolysis for metabolic needs. Inhibiting p38 MAPK signaling can promote mitochondrial biogenesis and autophagy [25]. This could suggest a potential therapeutic strategy to rejuvenate aged immune cells, thereby enhancing overall immune capabilities.",Vaccines (Basel),Cellular Metabolic Adaptations,2024
Hematopoietic Changes in Aging,"The aging process profoundly impacts the hematopoietic system, particularly hematopoietic stem cells (HSCs) [26]. Hematopoietic stem cells (HSCs) undergo a significant shift towards myeloid differentiation, altering the composition and functionality of immune cells [27,28,29,30]. This shift is driven by increased expression of hypoxia-inducible factor 1-alpha, leading to inflammatory responses. Additionally, aging impairs HSCs’ diversity and differentiation potential, resulting in reduced polymorphism and a decreased number of CD62L+ HSCs, which are crucial for lymphocyte homing in secondary lymphatic organs. Aged HSCs show general hypermethylation [31]. Specific transcription factors (TFs) contribute to myeloid bias associated with aging [32]. Knocking down Klf5 in aged long-term HSCs enhances lymphoid production and reduces myeloid output. In contrast, the knocking down of Ikzf1 in young LT-HSCs diminishes lymphoid production. These hematopoietic alterations contribute to the general decline in immune function seen in aging.",Vaccines (Basel),Hematopoietic Changes,2024
Macrophage Aging and Functional Decline,"Aging imparts a variety of changes to the various immune cells (see Figure 2). Older mice macrophages produced less pro-IL-1β than macrophages from young mice [33]. The macrophages’ capacity to control inflammation and fight infection is hampered by this lack of cytokine synthesis. Age-related dysregulation of genes linked to interferon-gamma (IFN-γ) responses has been observed in old mice [34]. IFN-γ is a crucial cytokine for immune control and tissue repair. A study by Zhang et al. [34] showed decreased inflammatory responses during muscle regeneration after acute injury, hindering efficient healing. Telomere loss contributes to oxidative stress, abnormal macrophage mitochondria, and hyperactivation of the NLRP3 inflammasome [35]. Research by Dube et al. demonstrated that older animals’ macrophages express more pro-inflammatory genes and have reduced capabilities for cellular proliferation and DNA repair [36]. These alterations impair the wound healing mechanism, which causes older mice to experience increased tissue damage and a delayed resolution of inflammation. Changes in macrophages as a result of aging, emphasize a broader damage to the immune system. and its contribution to age-related consequences from acute injuries, which, in older people, can develop into chronic ones.",Vaccines (Basel),Macrophages,2024
T Cell Aging and Functional Shifts,"As people age, CD8+ T cells and CD45RA+ naïve T cells decrease, but CD4+ T cells do not change [37]. This alteration reduces the immune system’s response to novel antigens, as opposed to it being highly responsive. Aged CD4+ and CD8+ naïve T cells exhibit elevated immune suppression and checkpoint regulation gene expression [38]. Additionally, CD28 expression decreases on CD8+ T cells, which is vital for the survival of T cells and their activation [39]. T cell receptor repertoire attrition reduces diversity, especially in CD8+ T cells [40]. This suggests that T cells have changed functional capacities, which increase susceptibility to infections and lower the effectiveness of vaccinations in older persons. Furthermore, a history of multiple cell division, short telomeres, and a general lack of ability to proliferate are all indicated by high expression of CD57 on CD8+ T cells [41]. In peripheral blood, CD8+ T cells from healthy donors exhibit higher p53β expression and lower Δ133p53, indicating senescent cell accumulation (CD28− CD57+) that is age-dependent [42]. Within human T cells, the endogenous p53 isoforms p53β and Δ133p53 function as physiological regulators of senescence and proliferation. Elevated p53β expression may increase autoimmune diseases risk, contributing to chronic inflammation.",Vaccines (Basel),T Cells,2024
"T Cell Senescence, Telomere Erosion, and Oxidative Stress","Telomere erosion is caused by prolonged stimulation throughout the course of a person’s life due to T cells, with high proliferation propensity upon activation [43]. Upon activation, highly differentiated CD8+CD28−CD27− T cells progress toward a replicative end stage, as indicated by telomerase down-regulation [44,45]. A strategy to improve the function of T cells in the elderly could involve increasing telomerase activity, as telomerase expression gradually decreases during T cell differentiation [45]. Oxidative stress and significant protein oxidative modification result from a rise in reactive oxygen species and a decrease in proteasome activity, respectively [46]. These events set the stage for cellular senescence and inflammation.",Vaccines (Basel),T Cell Senescence,2024
B Cell Aging and Repertoire Decline,"Aging reduces the diversity of the B cell repertoire by influencing the selection process during B cell affinity maturation [47]. Decreases in naïve B cells and increases in memory B cells are two age-related changes in B cell populations. The immune system’s capacity to react to novel antigens is compromised by this change. Certain aged B cell subsets show elevated mitochondrial mass and mitochondrial reactive oxygen species because of age-related changes to B cell mitochondrial functioning [48]. Reduced mitochondrial energy production and abnormalities in one-carbon metabolism, which are necessary for amino acid and nucleotide synthesis and the antibody production and activation of B cells, are the outcomes of these alterations. Lee et al. reported that compared to B cells from younger individuals, older individuals’ B cells do not exhibit fundamental abnormalities in their ability to proliferate and develop into antibody-secreting cells in vitro [49]. The GC response is delayed by age-related intrinsic B cell alterations, but this is not the cause of the compromised antibody-secreting response.",Vaccines (Basel),B Cells,2024
B-1 Cell Function Decline with Age,"Rodriguez-Zhurbenko et al. found that as people aged, their B-1 cell percentage and their capacity to secrete IgM spontaneously declined [50]. Additionally, healthy donors over 65 years of age had significantly lower levels of XBP-1 and Blimp-1 transcription factor expression than did healthy young donors. However, PAX-5, a hallmark of non-secreting B cells, was much greater. Rodriguez-Zhurbenko et al. also found variation in the utilization of VH- and DH-specific genes and a decreased IgM antibody repertoire diversity in B-1 cells derived from elderly donors compared to younger ones. When combined, these modifications may result in decreased antibody production and a diminished capacity to react to immunization and novel antigens.",Vaccines (Basel),B Cell Function,2024
Neutrophil Structural and Functional Changes,"Age-related increases in infection rates may result from structural alterations in neutrophils, such as lower plasma membrane viscosity and diminished adhesion capabilities [51]. When compared to neutrophils that are not from elderly people, senescent neutrophils have increased phagocytic activity [52,53]. Although this may appear to be a good thing, if neutrophils are few where they are needed, the immune system may be affected. According to a Study by Weisel et al., the expression of the chemokine receptor CXCR2, which is essential for directing neutrophils to the inflammatory location, is reduced [54]. Because of their decreased expression of CXCR2, elderly neutrophils are less likely to be drawn to the site of inflammation, which may influence the inflammatory response.",Vaccines (Basel),Neutrophils,2024
Neutrophil Trafficking and NET Formation Defects,"Neutrophils’ capacity to travel to and infiltrate infection sites is impacted by changes in neutrophil trafficking [55]. A reduced ability to eliminate pathogenic E. coli during septic peritonitis was noted in elderly mice, with increased neutrophil recruitment during LPS-induced peritonitis but not aseptic peritonitis [56]. Older mice’s neutrophils showed decreased E. coli killing and decreased reactive oxygen species (ROS) production after LPS priming. An important loss of neutrophil activity that leads to a weakened immune response is shown by the elderly population’s impaired ability to generate neutrophil extracellular traps (NETs) [57].",Vaccines (Basel),Neutrophil Dysfunction,2024
NK Cell Aging and Receptor Changes,"As people age, killer immunoglobulin-like receptor (KIR) expression increases, and NKG2A expression decreases [58,59]. NKG2A serves as an inhibitory receptor; inhibitory signaling may be diminished because of decreased NKG2A expression. This could result in increased lysis of healthy cells, which triggers autoimmunity. Isolated NK cells from elderly people produce more IL-8 responses to cytokine stimulation [60]. However, compared to younger NK cells, the amount of IL-8 produced is noticeably smaller. Elderly patients exhibit alterations in the distribution of NK cell subsets instead of a simple drop in cell numbers, in contrast to adult patients, who show a decline in the overall NK cell count [61]. It has been demonstrated that as people age, the CD56dim subset increases, while the CD56bright subset decreases [62,63]. Aging impairs NK cells’ proliferative capacity [64]. In response to immunological challenges, younger individuals (<41 years) demonstrate robust NK cell proliferation, whereas elderly individuals (41–80 years) show reduced proliferation capacity.",Vaccines (Basel),NK Cells,2024
Dendritic Cell Aging and Immune Dysfunction,"Dendritic cell (DC) development, cytokine production, and antigen presentation are all hampered by aging and are essential for monitoring and controlling immune responses [65]. Blood samples from aged patients showed lower myeloid DC (mDCs) counts than those from younger subjects [66]. When the antigen-presenting ability of elderly and young plasmacytoid DCs (pDCs) was evaluated, it was shown that the older patients’ pDCs were less able to stimulate IFN-γ secretion and CD4+ and CD8+ T cell proliferation [67]. Jing et al. reported a noteworthy decline in pDC counts with aging [68]. Older adults’ weakened immune response to viral infections is a result of age-related alterations in pDCs. Research by Bashir et al. has illuminated how aging affects gut dysbiosis and how this affects the loss of DC tolerance [69]. For instance, DCs produced in young mice with gut dysbiosis or in elderly mice show a notable decrease in immunological tolerance. Compared to DCs from young, healthy mice, they are unable to moderate the overactivation of CD4+ T cells and to successfully stimulate the production of Tregs. The general immunosenescence that older adults experience is influenced by the reported loss in these processes, which results in a weakened immune system.",Vaccines (Basel),Dendritic Cells,2024
Strategies for Managing Age-Related Immune Decline: Vaccines and Microbiome,"To change the immune landscape caused by aging in seniors, a variety of approaches seem promising (see Figure 3). New vaccine strategies are one of several strategies that warrant further exploration. The immune system’s response is impacted by modifications to hematopoietic stem cells, especially a move towards a myeloid lineage [27]. Elderly people who receive high-dose influenza vaccines produce more antibodies and have better protection against the influenza virus [70]. Pneumococcal 23-valent and 13-valent offer protection to elderly people against usual Streptococcus pneumoniae serotypes containing several antigen subtypes, which demonstrates that the use of multivalent vaccines in elderly individuals promotes robust immune responses and protects against multiple disease-causing pathogens [71,72]. Modulating immune function is significantly dependent on the gut microbiome [73]. Poor health outcomes have been associated with changes in microbiomes, such as a decrease in microbiota diversity. Moreover, the pathophysiology of autoimmune diseases is greatly influenced by the gut microbiota [74]. Different bacterial communities were discovered through various mouse models. Gut microbiota dysbiosis leads to increased colonic oxidative stress, permeability alterations, and inflammatory reactions, interfering with barrier function and resulting in tissue damage and elevated markers of autoimmune disease.",Vaccines (Basel),Immune Decline Management,2024
"Probiotics, Diet, and Microbiome-Based Immune Support","Bacteroides fragilis polarizes macrophages to an M1 phenotype, which improves their phagocytic capabilities [75]. According to the study by Schulthess et al., butyrate, a short-chain fatty acid (a metabolite produced by bacteria), differentiates macrophages with stronger antimicrobial activity that produce more antimicrobial peptides [76]. Compared to the younger group, the elderly group had lower levels of Bifidobacterium and Lactobacillus [77]. Elderly subjects who consumed Bifidobacterium lactis HN019 for three weeks had a high number of natural killer cells, Th cells, activated T cells, and total T lymphocytes [78]. Additionally, the probiotic supplementation increased the natural killer cells’ ability to kill tumors and the ex vivo phagocytic ability of polymorphonuclear and mononuclear phagocytes. By supplementing Bacillus subtilis CU1, elderly subjects experienced a notable decline in the frequency of respiratory infections and a rise in IgA levels in their saliva and feces [79]. Although the mean number of days with common infectious disease symptoms did not decrease statistically, this result does point to the possibility that Bacillus subtilis CU1 may be a safe and efficient method of improving immune responses in the older population. The Mediterranean diet has been associated with favorable gut microbiota traits, such as elevated levels of Candida albicans, a higher ratio of Bifidobacteria to Escherichia coli, and lower counts of Escherichia coli [80]. Reintroducing Lactobacillus plantarum into the gut of aged mice can restore dendritic cell tolerance, which is diminished due to aging-associated gut microbiota dysbiosis. By supporting a balanced gut microbiota and optimal gastrointestinal function, this diet may be a helpful strategy for managing immunosenescence.",Vaccines (Basel),Microbiome and Diet,2024
"Molecular Targets, Exercise, and Supplements for Immune Support","A study by Mondal et al. reveals that Δ133p53 and p53β play a role in cellular growth and aging, potentially opening a new therapeutic avenue for immunosenescence disorders [42]. Future research should focus on targeting p53β and Δ133p53 treatment, maintaining optimal p53 isoform balance, and exploring therapeutic interventions to modulate expression. Long-lived B cell depletion may aid in immunological competence enhancement and B-lineage rejuvenation, indicating that in the B-lineage, immunosenescence is not permanent [81]. There is evidence to support the idea that medications like metformin may reduce oxidative stress and enhance mitochondrial function, two things that are essential for preserving the health of immune cells in the elderly [82]. Regular physical activity can improve immune responses in the elderly population. In contrast to sedentary women, those engaging in physical activities have increased NK and T cell function, according to a study by Nieman et al. [83]. A study by Bartlett et al. suggests that decreased physical activity in older adults may contribute to impaired neutrophil migration, while regular physical activity may be beneficial for neutrophil-mediated immunity [84]. One approach to combat the age-related decrease in cellular immune response is to take vitamin E supplements [85]. Elderly participants, especially those with lower baseline immune responsiveness or less physical activity, showed improvements in delayed-type hypersensitivity and interleukin-2 production after taking a 100 mg vitamin E supplement for six months.",Vaccines (Basel),Interventions,2024
Conclusions on Immunosenescence and Healthy Aging,"Immunosenescence negatively affects the health and well-being of older populations by affecting various immune cells (see Figure 4 for comparison of young vs. aged immune systems). The complex interplay between genetic, molecular, and cellular processes plays a crucial role in the aging immune system. Key phenomena such as thymic involution, inflammaging, alterations in metabolic processes, and hematopoiesis underscore the multifaceted nature of immune decline. These changes result in diminished immune surveillance, reduced vaccine efficacy, and heightened vulnerability to infections and age-related diseases, such as cardiovascular diseases, autoimmune diseases, neurodegenerative diseases, cancers, and COVID-19. Comprehensive approaches, including dietary supplementation, lifestyle interventions, pharmacological treatments, and physical activity, may help to manage age-related immune decline and promote healthy aging. By combining these approaches, it may be possible to develop effective strategies for enhancing gastrointestinal health, promoting a balanced gut microbiota, and improving immune responses in older adults.",Vaccines (Basel),Conclusions,2024
Overview of Immunosenescence and Inflammaging,"Thomas et al. Immunity & Ageing (2020) 17:2. Immune system aging is characterized by the paradox of immunosenescence (insufficiency) and inflammaging (over-reaction), which incorporate two sides of the same coin, resulting in immune disorder. Immunosenescence refers to disruption in the structural architecture of immune organs and dysfunction in immune responses, resulting from both aged innate and adaptive immunity. Inflammaging, described as a chronic, sterile, systemic inflammatory condition associated with advanced age, is mainly attributed to somatic cellular senescence-associated secretory phenotype (SASP) and age-related autoimmune predisposition. However, the inability to reduce senescent somatic cells (SSCs), because of immunosenescence, exacerbates inflammaging. Age-related adaptive immune system deviations, particularly altered T cell function, are derived from age-related thymic atrophy or involution, a hallmark of thymic aging.",Immunity & Ageing,Thymic Involution,2020
Thymic Involution and Its Role in Immune Dysfunction,"Recently, there have been major developments in understanding how age-related thymic involution contributes to inflammaging and immunosenescence at the cellular and molecular levels, including genetic and epigenetic regulation, as well as developments of many potential rejuvenation strategies. Herein, we discuss the research progress uncovering how age-related thymic involution contributes to immunosenescence and inflammaging, as well as their intersection. We also describe how T cell adaptive immunity mediates inflammaging and plays a crucial role in the progression of age-related neurological and cardiovascular diseases, as well as cancer. We then briefly outline the underlying cellular and molecular mechanisms of age-related thymic involution, and finally summarize potential rejuvenation strategies to restore aged thymic function.",Immunity & Ageing,Thymic Involution,2020
Characteristics of Immunosenescence,"The aged immune system has various characteristics. One of which is immunosenescence, which describes the vast and varied changes in the structure and function of the immune system as a result of age. Many early observations, such as reduced ability to fight new infections, diminished vaccine immunity, and reduced tumor clearance, are generally categorized as immune insufficiencies. Immunosenescence is not due to the lack of immune cells, but due to reduced immune repertoire diversity, attributed to insufficient production of naïve immune cells and amplified oligo-clonal expansion of memory immune cells. Immunosenescence is therefore linked to the thymus. Natural aging causes the thymus to progressively atrophy, a process called thymic involution.",Immunity & Ageing,Immunosenescence,2020
Impact of Thymic Atrophy on T Cell Diversity,"Thymic involution is readily observed in most vertebrates and results in structural alterations, as well as functional decline, ultimately resulting in significantly decreased thymic output of naïve T cells that reduces the diversity of the T cell antigen receptor (TCR) repertoire, culminating in disrupted T cell homeostasis. The second characteristic of aged immunity is termed inflammaging. Inflammaging describes the elevated self-reactivity in the elderly, resulting in the typical chronic, low-grade, but above baseline, systemic inflammatory phenotype observed in the absence of acute infection. Inflammaging was originally attributed to somatic cell senescence-associated secretory phenotype (SASP) and chronic innate immune activation.",Immunity & Ageing,T Cell Aging,2020
Interplay Between Adaptive Immunity and Inflammaging,"In recent years, however, the contribution of aged adaptive immune components and specifically self-reactive T lymphocytes, has been realized as a probable primary contributor to the age-related development of subclinical autoimmune predisposition. Although immunosenescence and inflammaging appear to be opposing phenotypes, they comprise two sides of the same coin when attempting to holistically understand age-related immune dysfunction. It has been proposed that the basal inflammatory state in the elderly, defined by inflammaging, greatly contributes to many age-related degenerative diseases, including Type-II Diabetes, Alzheimer’s disease, and atherosclerosis.",Immunity & Ageing,Inflammaging,2020
Central T Cell Tolerance and Aging,"T lymphocyte development and selection occurs in the thymus. Included in this process is central tolerance establishment, which occurs via two mechanisms. First is thymocyte negative selection, during which the majority of self-reactive T cells are depleted from the repertoire via apoptosis. Second is the generation of CD4 single positive FoxP3+ regulatory T (Treg) cells, whose primary function is to suppress T cell-mediated self-reactivity and preserve immune homeostasis in the periphery. These arms of central tolerance work in tandem, and Treg cells most likely compensate for imperfections of negative selection, as some self-reactive T cells escape negative selection. With age, however, the atrophied thymus declines in its capacity to establish central tolerance.",Immunity & Ageing,Central Tolerance,2020
Theories of Decreased Thymopoiesis,"Historically, there have been two schools of thought regarding theoretical causes of age-related decreased thymopoiesis. First is the idea of defective hematopoietic stem cells, since there are reduced numbers of HSC progenitors produced by aged bone marrow. It therefore follows that there are fewer early T-cell progenitors entering the thymus from the bone marrow, resulting in shrinkage of the thymus. Second is the idea of a defect in stromal niches of the bone marrow or thymus. Therefore, age-related hallmarks of thymic involution primarily occur within the thymic niche and then extend to impact the development of early T-cell progenitors.",Immunity & Ageing,Thymopoiesis Decline,2020
Thymic Epithelial Cell Dysfunction and FOXN1,"It is our belief that the latter theory is more substantiated in light of recent advances, and that the extensive age-related alterations in thymic structure and microenvironment contribute most to the diminished thymopoiesis observed in the elderly. Thymic epithelial cells (TECs) are the primary thymic stromal cells and include medullary TECs (mTECs) and cortical TECs (cTECs). These two populations are distinct in their localization, function, and molecular expression. Evidence shows that age-related thymic atrophy is tightly associated with postnatal TEC homeostasis, regulated by TEC-autonomous transcription factors such as FOXN1. To this end, rejuvenation of age-related thymic involution by developing FOXN1-TEC–based therapeutics is reasonable, although other strategies are under investigation.",Immunity & Ageing,FOXN1 and TECs,2020
Scope of the Review and Future Directions,"This review discusses recent research progress exploring how age-related thymic involution contributes to inflammaging progression in conjunction with immune insufficiency, resulting in reduced clearance of senescent somatic cells, coupled with increased T cell–mediated self-reactivity and inflammation. We outline the differences in general senescence and immunosenescence as it pertains to inflammaging and age-related immune dysregulation. We describe how the involvement of T cell adaptive immunity in mediating inflammaging plays a crucial role in the progression of age-related neurological and cardiovascular diseases, as well as cancer. Finally, we outline underlying cellular and molecular mechanisms of age-related thymic involution and summarize potential rejuvenation strategies to restore aged thymic function.",Immunity & Ageing,Thymic Rejuvenation Strategies,2020
Thymic Involution in T Cell Immune Aging,"Contributions of Thymic Involution to T Cell Immune System Aging. Since T cell immune system aging mainly includes two aspects: immunosenescence and inflammaging, in this section we discuss recently published papers about how they intersect, how they are induced, and how age-related thymic involution participates in these processes. We outline this intricate relationship between immunosenescence and inflammaging associated with age-related thymic involution in Fig. 1. Intersection of Immunosenescence and Inflammaging. When discussing hallmarks of biological aging, seven overarching pillars are thought to collapse, namely: decreased adaptation to stress, loss of proteostasis, exhaustion of stem cells, derangement of metabolism, macromolecular damage, epigenetic dysregulation, and intercellular communication disorder. These changes are intricately linked through the crossroads of immunosenescence and inflammaging, which characterize immunology of aging.",Immunity & Ageing,Thymic Involution,2020
Somatic Cell Senescence and SASP,"Conventional senescence is a general term usually denoting somatic cellular senescence, referring to permanent or durable cell-cycle arrest first observed in cultured fibroblasts. The original observations leading to the discovery of senescence were not fully acknowledged by the scientific community because the initial observations were described in in vitro cultured cells, although this group believed there to be cell intrinsic factors leading to the observed “degeneration” of the cells. It was later demonstrated that senescence occurs in vivo and has since been more adequately defined as cells exhibiting permanent cell cycle arrest, lack of proliferation, expression of corresponding anti-proliferation markers, such as p16INK4a and senescence-associated β-galactosidase (SA-β-gal), shortened telomeres, and activation of DNA-damage signaling cascades. Somatic cellular senescence is believed to be advantageous as an evolutionary protection against cancer development.",Immunity & Ageing,Somatic Senescence,2020
"SASP, Inflammaging, and Resistance to Apoptosis","However, senescence of somatic cells during aging is thought to significantly contribute not only to degeneration of aged tissue function if SSCs are accumulated in certain organs, but also to the systemic inflammatory milieu via induction of SASP. This largely pro-inflammatory cellular secretion pattern induces increased basal levels of serum IL-6 and IL-1, as well as matrix metalloproteinases (MMPs). SASP has therefore been cited as a major contributor to inflammaging. Some of the mechanisms suggested to trigger cellular senescence are prolonged or chronic insults that accumulate over time, such as oxidative stress, gradual telomere shortening, and chronic infections. One additional characteristic of senescent cells is that they actively resist apoptosis. The anti-apoptotic pathways involve many factors including downregulation of Capsase-3 and increased Cyclin-dependent kinase inhibitors, p16 and p21. More recently, histone modification studies have implicated altered expression ratios of Bcl-2 and Bax family genes in mediating the anti-apoptotic phenotype of senescent fibroblasts.",Immunity & Ageing,SASP and Inflammaging,2020
Immunosenescence and T Cell Exhaustion,"Immunosenescence is a much broader term that encompasses all age-related changes to the immune system, both innate and adaptive. The primary hallmarks of immunosenescence are dampened immune responses to new infection or vaccination, and diminished anti-tumor immunosurveillance, including altered immune response phenotypes in activated T cells, increased memory T cell accumulation, and an inverted T lymphocyte subset ratio. Immunosenescence in T cells is commonly termed “cellular exhaustion”. This is usually characterized as loss of co-stimulatory surface molecule CD28 and expression of Tim-3, in addition to the other features of cellular senescence. T cell exhaustion differs from conventional senescence because of upregulation of surface markers such as PD-1 and Tim-3. Additionally, this type of growth arrest is not permanent, as blocking PD-1 can reverse T cell exhaustion, as demonstrated by recent clinical trials.",Immunity & Ageing,T Cell Exhaustion,2020
Links Between Immunosenescence and Somatic Senescence,"Recently, a link between immunosenescence and somatic cellular senescence has been established, in which the SSCs are no longer homeostatically reduced by the immune response. This results when natural killer (NK) cells, macrophages, astrocytes, and T cells undergo diminished chemotaxis toward accumulated SSCs for targeted depletion. The mechanisms by which T cells deplete accumulated SSCs could include CD8+ cytotoxic T lymphocytes (CTLs), CD4+ Th1-like cells producing cytotoxic inflammatory cytokines (such as IFN-γ), and Th2-like cells producing IL-4 and TGF-β. In addition to diminished chemotaxis, there is also dampened phagocytosis by neutrophils and macrophages associated with age that facilitates SSC accumulation. This ultimately results in increased production of SASP, which significantly contributes to inflammaging and subsequent development of age-related diseases.",Immunity & Ageing,Senescent Cell Clearance,2020
Etiology of Immunosenescence and Inflammaging,"Underlying Etiology of Immunosenescence and Inflammaging. There are several proposed components underlying immunosenescence and inflammaging etiology. In addition to cellular SASP secretions which contribute to inflammaging as discussed above, chronic innate immune activation due to long-term latent or persistent viral infection, for example, with members of the Herpesviridae family, have been proposed to contribute to low level pro-inflammatory cytokine production. Most notably, cytomegalovirus (CMV) infection has been explored as a potential biomarker in aging human patients. For example, several longitudinal studies of aging adults saw correlations with CMV sero-positivity and increased morbidity. Importantly, the role of the aged adaptive immune responses to self-tissues (in the absence of acute infection), primarily induced by the T cell compartment, has been found to be a major player in the onset and progression of inflammaging and associated with immunosenscence.",Immunity & Ageing,Etiology of Immunosenescence,2020
Thymic Dysfunction and TCR Repertoire Contraction,"The aged, atrophied thymus, continues to select T cells throughout the lifetime of the individual. However, the atrophied thymus is less able to negatively select self-reactive T cells, releasing these harmful, self-reactive T cells to the periphery, thereby increasing subclinical autoimmune predisposition in the elderly. Additionally, age-related thymic atrophy results in reduced output of functional naïve T cells, or recent thymic emigrant cells (RTEs), over time. Since peripheral T cell numbers remain unchanged or relatively elevated in aged individuals, the reduced thymic output in combination with peripheral oligo-clonal expansion of memory T cells, which occupy immunological space in the periphery, results in an overall contracted TCR repertoire diversity thereby inducing immune insufficiency (immunosenescence).",Immunity & Ageing,Thymic Dysfunction,2020
Thymic Involution Drives Immunosenescence and Inflammaging,"Thymic involution directs Immunosenescence and Inflammaging. Given both the altered output of naïve T cells and disruption of central tolerance establishment, it follows that thymic involution contributes to T cell-associated immunosenescence and inflammaging. Herein, we review recently determined evidence in this field. As indicated above, an age-related subclinical autoimmune predisposition induced by adaptive immune reaction to self-tissues by self-reactive T cells has recently been recognized as a potential factor underlying inflammaging. This results mainly from the increased output of self-reactive T cells by the atrophied thymus, which should be depleted through negative selection as the first boundary for preventing self-reactivity.",Immunity & Ageing,Thymic Involution,2020
Treg Decline and Broken Central Tolerance,"Treg cells suppress self-reactivity as the second frontier to prevent self-inflicted tissue damage. However, aged Treg cells usually are unable to do so, potentially due to the lack of Treg TCR diversity, as seen in an autoimmune diabetes model. These changes are attributed to defects in central tolerance establishment during the thymocyte development process, encompassing negative selection and thymic Treg (tTreg) cell generation.",Immunity & Ageing,Treg Dysfunction,2020
Overview of Defective Negative Selection,"Defective Negative Selection. Under the current paradigm, negative selection is the systematic removal of thymocytes expressing a TCR that exhibits high affinity for self-peptides presented by major histocompatibility complex class II (MHC-II) on mTECs. In support of this paradigm, it has been shown that when these high affinity TCRs receive strong signaling, negative selection follows via apoptosis of the thymocyte. However, overall TCR signaling strength is a culmination of TCR affinity for the self-peptide and avidity, or the combination of the affinity of TCR for self-peptide/MHC-II (self-pMHC-II) complexes and the number of TCR/self-pMHC-II interactions that occur (Fig. 2). Therefore, if thymocyte-dependent factors (i.e. TCR affinity and number) of self-reactive thymocytes are unchanged, then TCR signaling strength depends on the efficiency of self-pMHC-II expression by mTECs.",Immunity & Ageing,Negative Selection,2020
Age-Related mTEC Defects and Altered TCR Signaling Strength,"Since aging induces mTEC defects, such as decreased expression of autoimmune regulator (AIRE) and MHC-II, there is reduced capacity for self-pMHC-II ligand expression. Therefore, we suggest that a strong signaling strength shifts either to an intermediate strength, which favors CD4SPFoxP3+ tTreg cell generation (Fig. 2, arrow-a), or to an even lower (weak) strength, resulting in the release of self-reactive thymocytes (Fig. 2, arrow-b) with the potential to initiate self-reactivity and auto-inflammation. The FOXN1 cKO mouse model has proven to be a beneficial model for studying the capacity for efficient self-pMHC-II ligand expression, because it maintains young hematopoietic precursor cells and a young periphery in order to isolate the effects of TEC defects associated with thymic involution.",Immunity & Ageing,mTEC Dysfunction,2020
Experimental Evidence for Disrupted Negative Selection,"We showed that thymic involution disrupts negative selection, as revealed by the enhanced release of self-reactive T cells recognizing interphotoreceptor retinoid-binding protein (IRBP) from the atrophied thymus of FOXN1 cKO mice compared to control. This result was confirmed using a mock self-antigen model in which expression of ovalbumin (under control of the AIRE-regulated rat insulin promoter) was decreased in the involuted thymus compared to control. These findings support the hypothesis that age-related thymic involution weakens the strength of TCR signaling during thymocyte selection, increasing the escape of antigen-specific self-reactive T cells and contributing to inflammaging and autoimmune predisposition.",Immunity & Ageing,Thymic Involution,2020
Thymic-Derived Treg Cell Generation and Central Tolerance,"Thymic-Derived Regulatory T Cell Generation. The second arm of central tolerance induction is the generation of tTreg cells, which function to suppress autoimmune or chronic immune reactions in the periphery as peripheral Treg (pTreg) cells. It is proposed that Treg cells compensate for imperfections in negative selection that allow some self-reactive T cells to enter the periphery. It is currently accepted that 80 - 95% of pTreg cells are directly generated in the thymus, as opposed to Treg cells that are induced in the periphery. Under the current paradigm, the processes of both negative selection and tTreg generation in the thymus utilize the same set of agonist self-peptides. In this setting, TCR signaling strength determines whether developing thymocytes are deleted via negative selection or enter the tTreg cell lineage. Moreover, strong signaling induces apoptosis of highly self-reactive thymocytes, while intermediately high signaling leads to tTreg generation. Weak signaling results in the survival of thymocytes that differentiate into conventional T (Tcon) cells.",Immunity & Ageing,Treg Generation,2020
Impact of Aging on mTEC Antigen Presentation and Treg/Tcon Balance,"As mentioned earlier, the mTECs of the aged, involuted thymus are less apt to express self-antigens, and this could definitively dampen the overall avidity of TCR signaling received by developing thymocytes. We have observed a relatively increased tTreg generation by the atrophied thymus, which showed no change in overall tTreg numbers, but an increased %tTreg:%tTcon cell ratio in the aged, atrophied thymus compared to controls with the normal thymus. This phenotype was also observed in a mouse model with partial defects in MHC-II through microRNA inhibition. We believe this to be a demonstration of the atrophied thymus attempting to compensate for defective negative selection in order to maintain central T cell tolerance in the elderly. Additionally, several studies investigating the effects of diminished ability for thymic self-antigen presentation in mTECs, such as AIRE gene knock, have shown similar results of no change in overall Treg production. In combination with age-related accumulation of pTreg cells in the periphery of mice and humans, the relative proportion of pTreg cells is increased in the elderly, though these aged pTregs exhibit unimpaired functionality.",Immunity & Ageing,Aging mTECs and Treg Ratios,2020
Defects in Antigen-Specific Treg Repertoires,"Therefore, why is the increased or unchanged proportion of Treg cells unable to successfully manage self-reactivity in the elderly? We hypothesize that despite increased polyclonal Treg cells, certain self-tissue-specific Treg cells are reduced or imbalanced with self-reactive T effector (Teff) cells due to thymic atrophy, creating holes in the Treg repertoire. There are several mouse models using AIRE gene alterations that result in similar defects in self-antigen presentation seen in the aged thymus that support our hypothesis. One study assessed the effects of AIRE knock-out thymus on generation of a particular self-antigen-specific tTreg population, namely for the TCAF3 epitope of a prostate antigen, and saw a significant redirection of this TCR-bearing thymocyte from the tTreg to the Tcon lineage. These redirected TCAF3-specific Tcon cells were able to infiltrate the prostate tissue and cause visible lesions, but few TCAF3-specific Treg cells were observed.",Immunity & Ageing,Antigen-Specific Treg Defects,2020
Age-Related Impairment of Monoclonal Treg Generation,"Other studies investigating defects in mTEC self-peptide expression due to specific self-protein knock-out are beginning to indicate that similar impairments exhibited by the aged thymus may negatively impact antigen-specific (monoclonal) tTreg generation despite an unchanged or increased total (polyclonal) tTreg population. In contrast, these age-related thymic impairments seem to increase output of Tcon cells recognizing the same self-antigens and may contribute to increased peripheral self-tissue damage and inflammation. Further investigation will hopefully shed more light on how these subtle deficits in central tolerance establishment by the aged thymus impact the aged Treg TCR repertoire, in spite of a relatively increased aged polyclonal Treg population.",Immunity & Ageing,Central Tolerance Decline,2020
"Immunosenescence, Inflammaging, and Age-Related Diseases","Participation of Immunosenescence and Inflammaging in Age-Related Diseases. Immunosenescence and inflammaging begin as largely subclinical conditions, which eventually underlie age-related diseases. High-risk diseases in the elderly including neurodegenerative diseases, cardiovascular diseases, and late-life cancers are associated with a persistent, chronic pro-inflammatory status and impaired regulation of aberrant pro-inflammatory cells due to immunosenescence in aged individuals. Age-related neurodegenerative diseases. Most age-related neurodegenerative diseases, such as Alzheimer’s disease (AD), are associated with immunosenescence and/or inflammaging, which cause structural and functional disturbances of the blood-brain barriers (BBB), thereby leading to the infiltration of immune cells into the central nervous system (CNS). However, whether these disease outcomes are a cause or an effect of the imbalanced pro-inflammatory and anti-inflammatory immune cells is under investigation.",Immunity & Ageing,Immunosenescence and Disease,2020
Pro-Inflammatory T Cells and the CNS in Alzheimer’s Disease,"Among these immune cells are IFN-γ-producing Th1 cells and IL-17A-producing Th17 cells that are pro-inflammatory. They interact with the CNS resident microglia and exacerbate AD. Treg cells in AD play dual roles, either pathogenic or protective in various animal studies. Conversely, Th2 cells specific for amyloid beta (Aβ), which is a peptide that is accumulated in the AD brain, ameliorate AD in mice, showing improved cognition and reduced burden of Aβ depositions. In addition to the BBB, the choroid plexus (CP) is also an important barrier that maintains CNS homeostasis. The CP harbors CD4+ Th1 cells producing IFN-γ that stimulate CP epithelium to express leukocyte trafficking factors and recruit small numbers of leukocytes, including T cells and monocyte-derived macrophages. In contrast to the pro-inflammatory Th1 phenotype associated with disease exacerbation when in the CNS parenchyma, the IFN-γ producing Th1 cells in the CP support CNS tissue repair and maintain normal cognitive conditions.",Immunity & Ageing,Neuroinflammation,2020
Age-Related Decline in CP Immunity and Cognitive Impairment,"However, insufficient Th1 function occurs in the aged CP, leading to reduced IFN-γ and premature cognitive impairment in several mouse models. This decreased Th1 immune response might represent one of the profiles of immunosenescence, but the definite mechanisms remain to be explored. The role of infiltrating T cells in the CNS, including effector T (Teff) and Treg cells, is another critical element in AD, which could be associated with age-related thymic involution. Teff cells, especially CNS-infiltrating Th1 cells, are recognized as pathogenic by multiple studies. For example, Th1 cells specific for Aβ-antigen in the AD brain was verified to promote the disease in mice. Based on this fact, it remains an interesting question whether the altered negative selection of antigen-specific T cells in the aged thymus is more favorable for the development of Aβ-specific Teff cells, and thereby could potentially predispose aged individuals to AD.",Immunity & Ageing,Th1 and Treg Roles in AD,2020
Dichotomous Roles of Treg Cells in Alzheimer’s Disease,"Additionally, Treg cells could play dichotomous roles in AD, either protective or pathogenic likely depending on their location in the brain. The fundamental function of Treg cells in immune response is to suppress the activity of other immune cells including Teff and myeloid leukocytes. Thus, they are generally believed to inhibit neuroinflammation inside the CNS. However, Treg cells which are residing at the CP, but not infiltrating into the CNS, can be detrimental for AD, because the CP is an important gateway for leukocyte trafficking into the CNS to maintain its homeostasis. CP leukocyte trafficking can be suppressed by Treg cell-produced IL-10. Additionally, Treg cells can directly inhibit the expression of leukocyte trafficking molecules by the CP, which was verified to aggravate AD in an animal model.",Immunity & Ageing,Treg Function in CNS,2020
Age-Related Treg Accumulation and AD Susceptibility,"Treg cells are accumulated in the periphery with advancing age in both mice and humans, partially due to the relatively enhanced Treg generation in the aged, atrophied thymus, and Treg cells also have an increased suppressive function in aged mice. Therefore, the negative effects of Treg cells in the CP and increased Treg proportion and function in the aged periphery could be an important aspect for AD susceptibility and provide a potential therapeutic target.",Immunity & Ageing,Aging Tregs and AD Risk,2020
Age as a Risk Factor for Cardiovascular Disease,"Age-related cardiovascular diseases. Age is also a predominant risk factor for cardiovascular diseases, the principal pathological consequences of which involves vascular endothelial dysfunction and arterial stiffness. These basic pathologies are associated with immunosenescence and inflammaging, particularly on the cardiovascular wall, and lead to hypertension, atherosclerosis, and ultimately heart failure. In recent years, accumulating evidence has implicated the senescent T cell immune system in the pathogenesis of cardiovascular diseases, such as atherosclerosis, which is proposed to be related to thymic involution, as well as links to clonal expansion of senescent T cells and accumulation of effector memory T cells in the elderly.",Immunity & Ageing,Cardiovascular Aging,2020
Role of ApoB-Specific T Cells in Atherosclerosis,"Apolipoprotein B (ApoB) is the major apolipoprotein constituent of low-density lipoprotein (LDL), which is a causal agent for atherosclerosis. Although T cells are not the primary pathogenic cells in atherosclerotic lesions, ApoB-100-specific T cells were reported in an atherogenic mouse model and ApoB p18-specific Treg cells were found in healthy individuals. This indicates that age-related thymic involution might predispose elderly individuals to atherosclerosis by biasing the balance of ApoB-specific Teff versus Treg cells.",Immunity & Ageing,ApoB-Specific Immunity,2020
Senescent T Cells and Macrophage Activation in Atherosclerosis,"It is known that macrophages are the primary pathogenic cells in atherosclerotic lesion onset. The accumulating CD8+ CD28null CD27- senescent T cells on the inflammatory cardiovascular wall constantly produce IFN-γ, which activates macrophages to release MMPs for extracellular matrix degradation. This is an important underlying mechanism of atherosclerosis etiology related to T cells. In addition, CD4+ CD28null senescent T cells are relevant to the recurrence of acute coronary events. Circulating CD4+ effector memory T cells were reported to be associated with atherosclerosis in humans and in mouse models, providing further indication for the role of immunosenescence in cardiovascular disease.",Immunity & Ageing,T Cell Senescence in CVD,2020
"Immunosenescence, Inflammaging, and Declining Cancer Immunosurveillance","Late-Life Cancers. There is substantial knowledge regarding aged immune function and cancer. Immunosenescence with advanced age is known to decrease cancer cell immunosurveillance, and inflammaging creates a favorable cytokine microenvironment for tumorigenesis. However, knowledge about how age-related thymic involution directly contributes to tumor development is insufficient. Declined immunosurveillance of cancer cells is related to reduced thymopoiesis leading to an altered or contracted TCR repertoire diversity. If the range of tumor antigen recognition is narrowed by thymic involution, the aged T cell immune system will be less apt to clear cancerous cells. Likewise, if the proportion of pro-tumorigenic TCRs is biased, the risk for cancer development is increased. For example, a pro-tumorigenic γδ-T cell subset bearing Vγ6 and Vδ1 TCR chains, which is related to a higher risk of cancer development, was reported to be accumulated in aged mice, but it remains undetermined whether this pro-tumorigenic γδ-T cell population is increased by the altered negative selection in the aged thymus or by clonal expansion in the aged periphery.",Immunity & Ageing,Cancer Immunosurveillance,2020
Age-Related Treg Accumulation and Tumor Immunosuppression,"Treg cells, on the other hand, contribute substantially to the suppression of anti-tumor T cell responses, and they frequently accumulate in the tumor microenvironment, dampening anti-tumor immunity. Numerous studies have shown that cancer patients have increased Treg cells in peripheral blood and tumor microenvironment. For example, elderly lung cancer patients have more Treg cells in peripheral blood than age-matched controls. This corresponds to the peripheral accumulation of Treg cells and the potentially enhanced tTreg generation by the aged thymus, which could be an important factor predisposing elderly individuals to late-life cancer. An important aspect for cancer prognosis is metastatic relapse, which typically occurs several years after removal of the primary tumor and treatment with adjuvant therapy. The question is where the residual tumor cells hide during chemo- and/or radiotherapy. It has been shown that lymphoid cancers can hide in the thymus in mice. Also, we recently reported that in mice the atrophied thymus can be a pre-metastatic cancer reservoir to protect non-lymphoid, solid cancer cells from chemotherapy because the thymus provides an inflammatory microenvironment favorable for solid tumor cell dormancy during chemotherapy.",Immunity & Ageing,Treg Cells and Tumor Microenvironment,2020
Inflammation-Driven Tumorigenesis and Cytokine Pathways,"Inflammation is a double-edged sword that is necessary for anti-tumor responses, but it can also induce drug resistance in the tumor cells. Particularly, chronic inflammation is associated with increased risk of cancer, as supported by many studies. Inflammation-driven cancers are induced by inflammatory cytokines, initiating or promoting multiple processes in tumorigenesis including cellular mutations, metastasis, tumor growth, and angiogenesis. For example, macrophages and T cells release TNFα which can exacerbate DNA damage and tumor-associated macrophages secrete macrophage migration inhibitory factor that dampens p53-dependent protection. TNFα was also found to increase cancer metastasis to the lung and the liver in animal models. Additionally, tumor growth is promoted by IL-6 via the IL-6/JAK2/STAT3 pathway in kidney, lung and breast cancer, and angiogenesis in prostate cancer patients was found to be associated with TGFβ. These examples demonstrate the mutagenic potential of several classic cytokines.",Immunity & Ageing,Cytokine-Driven Tumorigenesis,2020
"Myeloid Bias, MDSCs, and Cancer Progression in Aging","One additional component contributing to age-related increased cancer incidence is the skew toward myelopoesis compared to lymphopoesis that is readily observed in both animal models and in humans when studying BM progenitor hematopoiesis. A subset of these myeloid cells termed myeloid-derived suppressor cells (MDSCs) are increased in aged individuals and are highly associated with cancer development and progression. For example, in a study of colorectal cancer patients, a positive correlation was observed for circulating MDSCs and overall tumor burden. These cells suppress anti-tumor responses through mechanisms that differ from Treg cell immunosuppression, but nonetheless are correlated with age-related cancer incidence. MDSC induction has been attributed to pro-inflammatory cytokines, such as IL-6, which we know to be increased during inflammaging. Therefore, perhaps if the thymic niche was rejuvenated for enhanced lymphopoesis and the inflammatory environment during inflammaging was dampened, rebalance of myeloid-to-lymphoid hematopoiesis could reduce MDSC induction and alleviate their role in cancer progression. Taken together, the axis connecting age-related thymic involution, T cell immunosenescence and chronic inflammatory environment, to tumorigenesis and tumor metastasis is intriguing, but the current knowledge is insufficient, and more evidence is necessary.",Immunity & Ageing,MDSCs and Aging,2020
Overview of Age-Related Thymic Involution,"Key Triggers Associated with Induction of Age-Related Thymic Involution. Age-related thymic involution is characterized by a reduction in thymic size and thymocyte numbers as well as overt remodeling of the thymic microstructure. The thymus is a meshwork structure, in which thymocytes of hematopoietic origin undergo development and selection within various compartments containing TECs of non-hematopoietic origin. The aged, involuted thymus declines in both TECs and thymocytes. The initial question was which cellular compartment contained the primary defect that triggered thymic involution. It has been noted that BM hematopoietic stem cells (HSCs) are decreased with age and exhibit a skewed developmental pathway resulting in a decreased ratio of lymphoid-to-myeloid cells. Since the thymocyte progenitor cells immigrate to the thymus from the BM, this raised a natural question of whether aged BM-derived HSC lymphoid progenitors are sufficiently able to seed the thymus.",Immunity & Ageing,Thymic Involution Mechanisms,2020
HSC Aging vs Microenvironmental Aging,"Therefore, many studies have investigated this aspect. The outcome was that aged HSCs contain defects that could contribute to insufficient thymic seeding by early T-cell progenitors (ETPs), culminating in decreased thymic output with age. The conclusion was largely based on BM transplantation experiments in mice or in vitro fetal thymic organ culture experiments to assess ETP proliferation. Therefore, aged HSCs and ETPs were regarded as having an intrinsic defect. This conclusion was confirmed using BM aspirate samples from young and elderly patients in which gene expression profiling of the HSCs showed differential gene expression associated with skewed myeloid lineage determination, however, it is possible that circulating factors in the aged periphery, such as cytokines could be initiating such lineage shifts. Importantly, the role of non-hematopoietic origin TECs and BM stromal cells in age-related thymic involution was neglected by these studies.",Immunity & Ageing,HSC vs TEC Aging,2020
Evidence for Microenvironment-Driven Thymic Aging,"We focused on the role of HSC/thymocyte niche cells by several experimental designs. For BM transplantation, we avoided the usual whole body irradiation and reduced artifacts of in vitro HSC manipulation by instead utilizing young or aged IL-7R knockout mice as recipients, since these mice have a BM niche that is accessible to seeding exogenous BM cells without irradiation. After BM cell engraftment, the young BM cells exhibited a young phenotype in young recipients, but the young BM cells exhibited an old phenotype in aged recipients. This suggests that the microenvironmental cells, rather than the HSCs, directs BM cell aging. We also performed transplantation of “microenvironmental niche”, i.e. fetal mouse thymi, into young or aged mice under the kidney capsule, in which BM progenitors from the host mice directly seed the engrafted fetal thymus in vivo. After engraftment, BM progenitors from young and aged mice developed equally well in the young engrafted thymus.",Immunity & Ageing,Microenvironment and Thymus Aging,2020
TEC Compartment as the Initiator of Thymic Involution,"These comprehensive experiments provide substantial evidence demonstrating that the aged non-hematopoietic microenvironment, rather than aged HSCs or ETPs, mediates age-related thymic involution. The result can be explained by the “seed and soil” theory, which describes how stem niches (soil) direct progenitor cell (seed) fate, and how thymocytes and the stromal microenvironment (TECs) cross-talk in the thymus, leading us to conclude that age-related thymic involution begins with defects in the TEC compartment. Therefore, it is possible that diminished thymic factors, such as IL-7, in the aged, involuted thymus could provide signals to HSCs that facilitates the shift in lymphoid-to-myeloid lineage observed in aged HSCs.",Immunity & Ageing,TEC Homeostasis,2020
FOXN1 as the Central Regulator of TEC Aging,"To identify which specific factors mediate cellular and molecular TEC aging, many groups have performed substantial work. They found many age-related TEC influencing factors, including sex steroids, cytokines, transcription factors, and microRNAs, but the single most predominant mechanistic factor currently accepted as causal to thymic involution is the TEC autonomous transcription factor FOXN1, which is uniquely expressed in epithelial cells of the thymus and skin to help regulate epithelial cell differentiation. It is required for thymic organogenesis and responsible for thymocyte development, as well as hair follicle development in the skin. Many past and current studies utilize nude mice as a model, which exhibit a null mutation in FOXN1 resulting in the lack of hair and thymus, and therefore lack of T cells.",Immunity & Ageing,FOXN1 and Thymic Involution,2020
FOXN1 Decline and Experimental Models,"FOXN1 expression is reduced in the aged thymus and has even been described as one of the first markers of the onset of thymic involution. The question of the cause-and-effect relationship of FOXN1 decline and thymic involution had been largely under debate until the advent of a FOXN1 cKO mouse model. In this model, the murine FOXN1 gene is loxP-floxed and the ubiquitous Cre-recombinase with tamoxifen (TM)-inducible fused estrogen receptor blocker (uCreERT) is introduced through crossbreeding, in which a low level of spontaneous activation takes place over time, even without tamoxifen induction. This causes a gradual excision of the FOXN1flox/flox gene over time and results in a progressive loss of FOXN1 with age. The thymic involution that results is positively correlated with reduced FOXN1 levels.",Immunity & Ageing,FOXN1 Decline,2020
FOXN1 Restoration and Rejuvenation,"Furthermore, supplying exogenous FOXN1, such as via plasmid or transgene, into the aged thymus greatly reduces thymic atrophy and improves thymic function. Additionally, use of FOXN1 reporter mice have enabled further elucidation of the timeline and kinetics of thymic atrophy with age. It is now largely accepted that progressively decreased FOXN1 expression resulting from age introduces defects in TEC homeostasis, resulting in age-related thymic involution.",Immunity & Ageing,Thymus Rejuvenation,2020
FOXN1 Gene Therapy Approaches,"Gene therapy. Similar to the TEC-based cellular therapy, some groups have utilized genetically-based methods to enhance exogenous FOXN1 expression, either with FOXN1 cDNA plasmid or FOXN1 transgenes. One group intrathymically injected plasmid vectors carrying FOXN1-cDNA into middle-aged and aged mice and observed partial rescue of thymic size and thymocyte numbers compared to empty vector controls. Another group, utilizing an inducible FOXN1 overexpression reporter gene system, showed in vivo upregulation of FOXN1 expression in middle-aged and aged mice resulted in increased thymic size and thymocyte numbers. They also observed enhanced ETP cell numbers, and the mTECs:cTECs ratio was restored to normal levels. Moreover, these targeted FOXN1 gene therapies also show great promise for rejuvenation of aged thymic structure and function.",Immunity & Ageing,FOXN1 Gene Therapy,2020
Growth Hormones and Thymic Rejuvenation,"Periphery–thymus axis. Growth hormones. Decline in growth hormone during aging has been suggested to contribute to age-related thymic involution and animal studies using growth hormone supplementation show rescue of thymic atrophy, increased T cell progenitor recruitment into the thymus, as well as enhanced thymic microenvironmental cytokine production. Studies of growth hormone date back to the early 1999s after the observations that TECs express growth hormone receptors and that insulin-like growth factor is expressed in the thymus. Studies of insulin-like growth factor 1 (IGF-1), which is closely related to growth hormone, show similar thymic functional and structural improvements upon increased IGF1 levels in aged mice. Although the effects of crosstalk between growth hormones and many other neuroendocrine hormones with thymocytes and TECs are under investigation, these systemic pathways are extremely interwoven and thus difficult to compartmentally delineate.",Immunity & Ageing,Growth Hormone and Thymus,2020
Sex Hormones and Thymic Involution,"Sex hormones. The effects of sex hormones on the thymus have long been characterized, with the earliest reports of thymic atrophy correlating with adolescence and reproductive hormones dating back to a 1904 study in cattle. Early studies using castration and sex steroid antagonists in both male mice and male patients receiving androgen blockade for prostate cancer therapy demonstrated phenotypes varying from delayed onset of thymic involution to complete thymic regeneration. Most of these early studies, however, focused primarily on phenotypic data, such as an increase of thymopoiesis, with insufficient mechanistic results. Generally, the rejuvenation is thought to occur in the TEC compartment because androgen receptors are expressed by TECs. One of the potential mechanisms reported was that sex steroids inhibit cTEC expression of Notch ligand Delta-like 4 (DLL4), shown in one study utilizing a luteinizing hormone-releasing hormone blockade that saw enhanced thymopoesis after blockade in mice. DLL4 is an important factor for promoting T cell differentiation and development.",Immunity & Ageing,Sex Hormones,2020
Effects of Hormone Ablation and Autoimmune Risks,"It remains unclear whether Notch ligands (there are four types) are decreased in the aged thymus and how this might play a role in decreased thymopoiesis with age. In contrast, other studies of thymic rejuvenation through sex steroid ablation exhibited in the least, only a short-lived rejuvenation, and at most no influence whatsoever on thymic involution in mice. Others suggest that the observable thymic restoration can be transient (only 2 weeks) but harmful, asserting that the “rejuvenated” thymus potentially produces more harmful T cells and increasing self-reactivity. In support of the opinion that sex hormone ablation may cause detrimental autoimmune implications, a human study, which used medical castration resulted in a declined %CD4+CD25+ Treg cells and increased NK cells, which may compromise immune tolerance. Recently, studies on sex hormones and their impact on thymocyte selection of the TCR repertoire via AIRE gene expression by TECs in the thymus demonstrate that there are differences in males and females in both mouse and human samples.",Immunity & Ageing,Hormone Ablation and Autoimmunity,2020
Sex Differences in AIRE Regulation and Autoimmunity,"Androgens from males promote AIRE expression in mTECs to enhance thymocyte negative selection, while estrogens reduce AIRE expression, dampening thymocyte negative selection and potentially increasing autoimmunity. Therefore, these hormones may mediate thymic functionality to a greater extent than simply structural atrophy. In light of this, sex steroid antagonists or castration-based rejuvenation of thymic aging may have more disadvantages (inducing autoimmune predisposition in the elderly) than advantages. Blood-borne factors. Of note, there are likely circulating factors that impact age-related thymic involution, including proteins, mRNAs, microRNAs and other signaling molecules. One method to test this is a heterochronic parabiosis model, in which young and aged mice are surgically conjoined resulting in mutual influence of blood-borne factors. These experiments, however, have not demonstrated rejuvenation of the aged thymus.",Immunity & Ageing,AIRE and Sex Hormones,2020
Blood-Borne Factors and Extracellular Vesicle Rejuvenation,"Conversely, when serum-derived extracellular vesicles, which carry cellular factors throughout the body, were taken from young mice and given to aged hosts, partial thymic rejuvenation with increased negative selection signaling was observed. Interestingly, we also observed decreased levels of circulating pro-inflammatory IL-6, suggesting rescue from inflammaging following treatment with these young serum-derived extracellular vesicles. Further work to elucidate the mechanism of ameliorated inflammaging phenotype is necessary, as it could be due to increased targeted deletion of senescent cells in the periphery causing less SASP secretion, enhanced Treg production, or other unknown mechanisms.",Immunity & Ageing,Blood-Borne Rejuvenation Factors,2020
Lifestyle Factors and Thymic Health,"Life-Style/Physical Exercise. Finally, life-style habits should not be overlooked pertaining to immune health and healthy aging. Indeed, CT scans of patient thymus tissue demonstrate that advanced fatty degeneration of the thymus is positively correlated with increased BMI and with smoking. Additionally, physical exercise has demonstrated countless benefits for immune health, some of which have recently been reported. One such study has documented an intriguing correlation between physical exercise and improved thymic function in elderly patients. This in-depth study compared numerous aspects of immunosenescence and thymic output in aged adults who participated in high levels of regular exercise for much of their adult lives and aged adults who had been inactive. This study found that the aged individuals who maintained physical exercise regimens exhibited reduction in typical decline in thymic output, decreased markers of inflammaging, such as reduced serum IL-6, and increased serum IL-7 and IL-15, which may foster thymic health and function.",Immunity & Ageing,Lifestyle and Thymus,2020
Exercise Effects on Immune Phenotypes,"The age-associated increase in Th17 phenotype was also significantly lessened in the aged cohort with physical exercise and lower peripheral Treg cell numbers were observed in these individuals compared to the inactive aged cohort. Though not all aspects of immunosenescence were lessened in the exercising cohort, as both groups maintained the age-related accumulation of senescent T cells, this study does present some compelling findings. This group published a recent review and discussed the direct cross-talk between skeletal muscles during exercise and the immune compartment, even describing exercise as a potential adjuvant to immunizations, as some studies have also shown enhanced T cell priming and increased naïve T cell frequency. Therefore, it is significant to mention the effects of physical exercise and overall healthy life-style habits on immune health and directly on thymic health over the lifespan.",Immunity & Ageing,Exercise and Immunity,2020
Limits and Considerations in Thymic Rejuvenation Strategies,"In sum, there are many varied avenues for restoration of aged thymic structure and function as well as its influences on inflammaging. Many of these rejuvenation strategies focus on the TEC compartment, since decline in TECs and TEC-associated factors are implicated in thymic involution onset and progression, however, the role of other systemic players are still under investigation. Additionally, each strategy has disadvantages. For example, intrathymic injection of newborn TECs can rejuvenate middle-aged thymus, but the source of newborn TECs is limited and may not be ideal as a translational therapy. Additionally, generation of an ectopic de novo thymus under the kidney capsule can generate naïve T cells, but this does not remedy the increased self-reactive T cells released by the original atrophied thymus remaining in the host. Also, the use of thymus-targeted cytokines may be beneficial, but caution is needed, as systemic cytokine therapies usually encompass adverse effects.",Immunity & Ageing,Thymus Rejuvenation Strategies,2020
Conclusion: Importance of Targeting Thymic Aging,"Moreover, continued investigation is required for future development of practical and effective interventions for age-related thymic involution and inflammaging. Conclusion. Age-related thymic involution is a dynamic process that impacts overall T cell development and central T cell tolerance establishment throughout life. Immunosenscence and inflammaging describe two opposing arms of the aged immune system: immune insufficiency, with regard to infection, vaccination, and tumor surveillance, coupled with increased self-reactivity and chronic, systemic inflammation. The contributions of the aged thymus to the manifestations of immunosenscence and inflammaging have recently come to be appreciated. However, continued investigation into their synergy in the aged immune system is needed. Additionally, as we shift our focus towards improving quality of life with age, research into potential avenues for reversing the adverse effects of age-related thymic involution on the aged T cell immune system is of paramount importance. Moreover, there are numerous areas still to explore in this field with far-reaching applications.",Immunity & Ageing,Thymic Aging Conclusion,2020
Introduction to Immunosenescence,"Immunosenescence and Its Hallmarks: How to Oppose Aging Strategically? A Review of Potential Options for Therapeutic Intervention. Aging is accompanied by remodeling of the immune system. With time, this leads to a decline in immune efficacy, resulting in increased vulnerability to infectious diseases, diminished responses to vaccination, and a susceptibility to age-related inflammatory diseases. An age-associated immune alteration, extensively reported in previous studies, is the reduction in the number of peripheral blood naïve cells, with a relative increase in the frequency of memory cells. These two alterations, together with inflamm-aging, are considered the hallmarks of immunosenescence.",Frontiers in Immunology,Immunosenescence Review,2019
Nutrition and Immunosenescence,"Because aging is a plastic process, it is influenced by both nutritional and pharmacological interventions. Therefore, the role of nutrition and of immunomodulation in immunosenescence is discussed, due to the multifactorial influence on these hallmarks. The close connection between nutrition, intake of bioactive nutrients and supplements, immune function, and inflammation demonstrate the key role of dietary strategies as regulators of immune response and inflammatory status, hence as possible modulators of the rate of immunosenescence.",Frontiers in Immunology,Nutrition and Immunity,2019
Therapeutic Options,"In addition, potential options for therapeutic intervention are clarified. In particular, the use of interleukin-7 as growth factor for naïve T cells, the function of checkpoint inhibitors in improving T cell responses during aging, and the potential of drugs that inhibit mitogen-activated protein kinases and their interaction with nutrient signaling pathways are discussed. Finally, it is suggested that the inclusion of appropriate combinations of toll-like receptor agonists may enhance the efficacy of vaccination in older adults.",Frontiers in Immunology,Therapeutic Interventions,2019
Introduction Part 1,"INTRODUCTION. People worldwide are living longer. In 2025, there will be about 1.2 billion people over the age of 60, increasing to 2 billion by 2050. However, the increase in lifespan does not coincide with the increase in healthspan, i.e., the period of life free from serious chronic diseases and disability. The influence of aging on humans is responsible for physiological dysfunctions in different tissues, organs, and systems, including the immune system. The age-related involvement of the immune system leads to a progressive reduction in the ability to trigger effective antibody and cellular responses against infections and vaccinations. This phenomenon, called immunosenescence, a term coined by Roy Walford, is multifactorial and affects both natural and acquired immunity, although T lymphocytes are dramatically affected. Aging more extensively affects acquired immunity than innate immunity. Several factors, such as genetics, nutrition, exercise, previous exposure to microorganisms, biological and cultural sex, and human cytomegalovirus (HCMV) status can influence immunosenescence.",Frontiers in Immunology,Immunosenescence Introduction,2019
Introduction Part 2,"Concerning sex, steroid hormones linking to specific receptors differentially modulate the immune system. In general, estrogens increase the immune response, whereas progesterone and androgens have immunosuppressive actions. Few studies have analyzed the postmenopausal immune system. Therefore, it is unclear whether age-related changes are different between men and women, although some data show that immunosenescence develops earlier in men than in women, related to the longer life expectancy of women. In addition, no evidence exists that males and females respond differently to therapeutic interventions against immunosenescence. Many studies have emphasized the importance of viruses such as herpes viruses, responsible for both latent and chronic infections, in shaping T cell compartments during aging. In particular, HCMV seropositivity seems related to many functional T cell changes. HCMV status has a greater impact than age on the immune system, because the virus contributes to shaping immune profile and function during normal human aging.",Frontiers in Immunology,Immunosenescence Introduction,2019
Introduction Part 3,"Understanding mechanisms of age-related disorders in immune regulation is important to identify more efficient strategies for immune rejuvenation and for effective induction of vaccination-mediated immunity in older individuals. Aging is a malleable process, affected by both nutritional and pharmacological interventions. Therefore, the immune system might also be prone to intervention. However, therapies aimed at nonspecifically 'rejuvenating' the immune system might be counterproductive. Differences observed between young and older people could also be a product of adaptation to the exposome, meaning all the stimuli the immune system has undergone during life. Therefore, a targeted intervention for safe rejuvenation of the immune status in older people is necessary. Numerous studies of underlying mechanisms of age-related immune decline have laid the groundwork for identification of targeted approaches. The authors discuss nutritional strategies, growth factors such as IL-7, monoclonal antibodies affecting immune checkpoints, MAPK-inhibiting drugs interacting with nutrient signaling pathways, and combinations of TLR agonists to enhance vaccine efficacy. Future approaches are outlined in the conclusion.",Frontiers in Immunology,Immunosenescence Introduction,2019
Micronutrients – Overview,"Micronutrients. Nutritional status is crucial for the health of older adults. Changes in phenotypic features, mainly loss of teeth and alterations in taste receptors, along with gut disorders, determine variations in both quality and quantity of food intake, contributing to general alterations in metabolism. Many studies have examined the influence of micronutrients and their impact on enhancing immune function in older adults. Micronutrients such as vitamins and minerals are essential for efficient immune performance. They are needed only in trace quantities because their homeodynamic range is small, but maintaining correct levels is very rare in older people (and often even in young adults), due to both deficiency and excessive use from unnecessary supplementation. Zinc is one of the main micronutrients associated with immune physiology and is one of the most studied factors. It is involved in molecular processes including signal transduction, apoptosis, proliferation, and differentiation of immune cells. Even slight zinc deficiencies can have significant consequences.",Frontiers in Immunology,Micronutrients & Immunosenescence,2019
Micronutrients – Zinc and Thymulin,"Zinc deficiency reduces serum thymulin, a zinc-dependent peptide hormone produced by thymic epithelial cells whose activity declines with age, peaking in pre-adolescence. Active thymulin induces expression of T lymphocyte activation markers and promotes T-cell–mediated functions, acting on both early and late lymphocyte differentiation phases. In a randomized, double-blind, placebo-controlled trial, zinc supplementation (45 mg elemental zinc gluconate/day for 12 months) significantly reduced infection incidence, increased plasma zinc, and lowered TNF-α and oxidative stress markers in participants aged 55–87. Another double-blind, randomized trial with zinc supplementation (25 mg zinc sulfate once daily for 3 months; placebo mean age 80.6 ± 7.8, supplemented 79.5 ± 6.8) demonstrated increased levels of activated T helper and cytotoxic T lymphocytes, with a higher relative percentage of T cells among total circulating lymphocytes. Given zinc’s dose-dependent effects as both pro- and antioxidant, maintaining normal physiological levels is essential for regulating reactive oxygen species.",Frontiers in Immunology,Micronutrients & Immunosenescence,2019
Micronutrients – Vitamins E and C,"Vitamin supplementation studies in older adults show that vitamin E supports IL-2 production and activation-induced T cell proliferation in naïve but not memory T cells. However, responses vary depending on genetics and immune functionality. Age-related oxidative stress and inflamm-aging may be counteracted by vitamin C supplementation. Vitamin C appears to enhance antibody generation and support differentiation and maturation of immature T cells and NK cells. Because vitamin C is water-soluble and humans have low storage capacity, regular intake is required at levels up to 100-fold higher than many other vitamins. These findings demonstrate essential roles for vitamins E and C in modulating immune function in older individuals, though results remain inconsistent due to heterogeneity of participants, baseline nutritional status, and methodological limitations.",Frontiers in Immunology,Micronutrients & Immunosenescence,2019
"Probiotics, Prebiotics, and Symbiotics – Overview","The use of probiotics, prebiotics, and symbiotics (the combination of pro- and prebiotics) as immunomodulators acting on the microbiota is very common. However, strong cause–effect relationships are often lacking between their use and specific clinical end-points. Gut microbiota, which plays an active role in maintaining health status, is compromised in older adults due to malnutrition, medication use, and immunosenescence itself. Therefore, administering specific strains of Lactobacilli and Bifidobacteria as probiotics, as well as fructooligosaccharides, galactooligosaccharides, and other prebiotics, or their combination, may benefit immunocompromised individuals.",Frontiers in Immunology,Dietary Immunomodulation & Immunosenescence,2019
Probiotics and Prebiotics – Immune Effects and Limitations,"Supplementation studies with pro- or prebiotics in older adults show modulation of inflammatory status, with reduced production of TNF-α, IL-1β, and IL-6, as well as increased anti-inflammatory cytokine IL-10 by PBMCs. These biotics also improve innate immune responses through enhanced phagocytosis and cytotoxicity against bacteria such as Staphylococcus aureus, increased NK cell activity, and reduced CD25 expression on resting T lymphocytes. Despite this, the complexity of randomized controlled trials and lack of specific biomarkers in humans make reproducibility difficult. Moreover, healthy status appears crucial for their action, as shown by null immunomodulatory results after prebiotic administration in older adults vaccinated with influenza or pneumococcal vaccines. Reviews, such as that of Suez et al., highlight the weakness and inconsistency of existing data. A major limitation is the lack of mechanistic studies to reveal molecular pathways underlying their effects, which would enable targeted use and reduce variability and conflicting findings.",Frontiers in Immunology,Dietary Immunomodulation & Immunosenescence,2019
Nutraceuticals – Overview,"Various bioactive food components with health-related effects have been termed nutraceuticals. These compounds, mostly from plant-based foods and fatty fish, offer physiological benefits beyond basic nutrition. There is growing interest in optimizing immune responses and reducing inflammation in older adults by increasing intake of certain bioactive agents. Many studies have shown that increasing nutraceutical intake above habitual levels can enhance aspects of immune function and reduce inflammatory status, improving cellular resistance to aging. Three main classes are examined: carotenoids, polyphenols, and polyunsaturated fatty acids (PUFAs), with emphasis on their interactions with immunosenescence.",Frontiers in Immunology,Dietary Immunomodulation & Immunosenescence,2019
Carotenoids – Immune Modulation,"Carotenoids, naturally occurring pigments found in most fruits and vegetables, primarily exert antioxidant and anti-inflammatory effects, though individual carotenoids may also act through other mechanisms including immune enhancement. Lutein and astaxanthin increased ex vivo antibody responses of mouse splenocytes to T-cell antigens. Older adults supplemented with carotenoids (30 mg β-carotene, 15 mg lycopene, 9 mg lutein) showed a shift to T cells with a mature phenotype, increased serum IgA levels, and increased NK cell numbers. Higher doses of β-carotene (30–60 mg/day) increased T helper and NK cells. Enhanced NK cell cytotoxicity was also observed after oral β-carotene treatment, and long-term supplementation increased NK cell activity in older adults.",Frontiers in Immunology,Dietary Immunomodulation & Immunosenescence,2019
Polyphenols – Anti-inflammatory and Immune Effects,"Dietary polyphenols are bioactive non-nutrient plant compounds found in fruits, vegetables, grains, and other plant foods, and their consumption is linked to reduced risk of major age-related diseases. Their primary action is inflammation control. In participants at high cardiovascular risk, consumption of cocoa polyphenols rich in flavonoids (40 g/day with 500 ml skimmed milk) significantly reduced expression of adhesion molecules VLA-4, CD40, and CD36 on monocytes, and lowered circulating P-selectin and ICAM-1. Olive oil polyphenols (caffeic acid and oleuropein glycoside) reduced IL-1β levels in LPS-stimulated human whole blood cultures. A pilot study found that daily consumption of 12 green olives reduced serum IL-6 and malondialdehyde. Polyphenols also increase IL-2 and IFN-γ release; resveratrol increased T helper cells and delayed-type hypersensitivity responses in aged rats.",Frontiers in Immunology,Dietary Immunomodulation & Immunosenescence,2019
Polyunsaturated Fatty Acids (PUFAs),"Dietary lipids also modulate immune responses. Relevant fatty acids include long-chain PUFAs of the omega-3 (n-3) and omega-6 (n-6) classes. n-6 PUFAs, derived from plants and land animals, have minimal immune effects. n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), found in fish and some plants, exert anti-inflammatory properties by inhibiting formation of eicosanoids and synthesis of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6), chemokines (IL-8, MCP-1), and adhesion molecules (ICAM-1, VCAM-1, selectins). Dose, timing, and participant age strongly influence outcomes, leading to contrasting results. Only a few studies specifically evaluate PUFA effects in older adults.",Frontiers in Immunology,Dietary Immunomodulation & Immunosenescence,2019
IL-7 Physiology and Rationale for Therapy,"The various aspects of IL-7 physiology raise the possibility that reduction of this pleiotropic cytokine level contributes to the age-related decrease in thymocytes and naïve T cells. Thus IL-7 might be used therapeutically to enhance thymopoiesis in lymphopenic patients or older individuals, counteracting the first hallmark of immunosenescence. Thymic involution remodels thymic epithelial cells, decreasing intrathymic IL-7 production. IL-7 from thymic epithelial cells provides survival and proliferative signals to double negative CD4−CD8− thymocytes and promotes V(D)J recombination of the TCR γ-c locus. Mutations in IL-7Rα or γc lead to severe combined immunodeficiency, underscoring its importance in T cell development. At later stages IL-7 signaling supports homeostatic proliferation of naïve T cells, especially cytotoxic T cell subsets. High IL-7Rα expression on naïve T cells maintains this pool, but IL-7 availability is limited. Upon antigen encounter, naïve T cells lose IL-7Rα and differentiate, ensuring IL-7 is efficiently used by naïve cells. IL-7Rα is re-expressed at the memory stage to support survival in memory pools.",Frontiers in Immunology,Clinical Immunosenescence Interventions,2019
IL-7 Clinical Trials and Limitations,"IL-7Rα is also part of the receptor for thymic stromal lymphopoietin (TSLP), which has weaker co-mitogenic activity than IL-7, though no studies have examined its role in immunosenescence. In the first human trial, metastatic cancer patients (age 20–59) treated with IL-7 showed dose-dependent increases in circulating CD4+ and CD8+ lymphocytes and decreases in Tregs. Subsequent trials in malignancies and chronic viral infections demonstrated increases in naïve and memory CD4+ and naïve CD8+ T cells, including in HIV patients with low CD4 counts despite viral suppression. These data suggest IL-7 could improve immune responses when enhancement is clinically necessary. However, in aging, IL-7’s immunorestorative effects may be limited by deterioration of thymic architecture, as functional cortical and medullary organization and intact epithelial cells are required for thymopoiesis. IL-7 therapy likely requires prior restoration of thymic structure for optimal effects on T cell development.",Frontiers in Immunology,Clinical Immunosenescence Interventions,2019
Checkpoint Inhibitors – PD-1 and CTLA-4,"Immune checkpoint inhibitors—monoclonal antibodies that block inhibitory receptors or their activation—are pivotal in cancer management in both young and older patients. They promote immune control of cancer cells by inhibiting pathways that normally suppress T cell responses after antigen clearance. Data on their role in older adults are limited and often statistically underpowered. PD-1 and CTLA-4 are key inhibitory receptors: PD-1 fine-tunes T cell responses, while CTLA-4 contributes to Treg suppressive function and interacts with CD28. CD28 activation induces PD-1 expression; PD-1 engagement with PD-L1 inhibits CD28-mediated costimulation, reducing proliferation and cytokine secretion. Notably, PD-1 expression increases on T cells of older adults, and its blockade partially restores T cell functionality. Immunosenescence influences checkpoint inhibitor efficacy, with reduced therapy benefit in patients ≥75 years, likely due to greater immune aging. Still, several studies show encouraging responses in older individuals.",Frontiers in Immunology,Clinical Immunosenescence Interventions,2019
Checkpoint Inhibitor Clinical Evidence,"In metastatic melanoma, Nivolumab (PD-1 inhibitor), alone or with other antagonists, confers survival benefits independent of age. PD-L1 antibody Atezolizumab shows positive results across age groups. In older patients with NSCLC, anti-PD-1/PD-L1 therapy provides benefits similar to younger patients. CTLA-4 blockade with Ipilimumab can establish anti-leukemic activity post-transplant and restore anti-tumor reactivity in relapsed patients, though efficacy varies. Meta-analysis of tremelimumab, ipilimumab, nivolumab, and pembrolizumab across melanoma, prostate cancer, renal carcinoma, and NSCLC showed a 37% reduction in mortality risk over control treatments. Microbiota composition influences therapy effectiveness: responders show enrichment in Akkermansia muciniphila, Ruminococcaceae, or Bacteroides species. Antibiotic-treated or germ-free mice fail to respond to CTLA-4 blockade. Patients with Firmicutes-rich baseline gut microbiota respond better to Ipilimumab. These findings highlight the interplay between microbiota, aging, and immune checkpoint therapy responsiveness.",Frontiers in Immunology,Clinical Immunosenescence Interventions,2019
Mechanistic Insights and Implications for Aging,"Checkpoint inhibitor efficacy declines in patients ≥75 years. Metcalf et al. demonstrated that CD28 costimulation is required for expansion of PD-1+ CD8 T cells and for PD-1 therapy effectiveness in chronic viral infection and cancer models. In lung cancer patients, PD-1+ CD8 T cells proliferating after blockade express CD28, suggesting preferential expansion of CD28+ subsets and explaining weaker responses in older individuals who accumulate CD28− senescent T cells. Understanding checkpoint biology is essential for designing immunomodulatory strategies for aging. PD-1 blockade enhances effector functions in HIV, hepatitis B, and hepatitis C infections. Future studies should examine combinatorial blockade of PD-1, other inhibitory receptors, and immunosuppressive cytokines to improve T effector responses, vaccine efficacy, and therapeutic immunity in older adults.",Frontiers in Immunology,Clinical Immunosenescence Interventions,2019
Nutrient Signaling Pathways: AMPK–mTOR Interactions,"The mechanisms exposed above are distinct from another sestrin-inhibitory complex containing GATOR and RAG A/B GTPase that involves the mTOR pathway. In particular, sestrins stimulate the activation of AMPK by an unknown mechanism, inhibiting mTORC1 signaling. This suggests that the anti/pro-aging dichotomy of sestrin action in T cells vs. other cell types may depend on different sestrin-protein interactions. Senescent human CD27−/CD28−/CD4+ T cells trigger AMPK to stimulate p38 recruitment, causing p38 autophosphorylation mediated by the protein scaffold TAB1. This pathway can inhibit telomerase activity, T cell proliferation, and expression of key components of the TCR signalosome. Under low-nutrient conditions and DNA-damage signaling, the proliferative defect of senescent T cells is reversed by blocking AMPK–TAB1–dependent p38 activation. Moreover, in senescent CD8+ T cells, p38-MAPK induces an increase in autophagy through interactions between a p38 interacting protein and autophagy protein 9, in a mTOR-independent manner, suggesting that p38-MAPK blockade reverses senescence via a pathway independent of mTOR.",Frontiers in Immunology,Clinical Immunosenescence Interventions,2019
mTOR Function in T Cells and Aging,"mTOR plays an important role in T cell activation and differentiation, especially in naïve CD4+ T cells transitioning toward Th1 or Th17 phenotypes. The activation of mTOR signaling is under the control of TCR/CD28 stimulation. A growing body of research has highlighted mTOR inhibitors, such as rapamycin and everolimus, as treatments for several age-related pathologies, including immunosenescence, prolonging lifespan in yeast, worms, flies, and mice. Partial inhibition of mTOR may be beneficial for immune function in older individuals, although mTOR activity normally inhibits autophagy. At high doses, rapamycin is immunosuppressive, blocking protein synthesis and cell division. In a clinical trial of over 200 older participants, daily low-dose everolimus regimens for six weeks followed by a two-week drug-free interval and seasonal influenza vaccination improved immune function without serious side effects. Participants showed improved hematopoietic stem cell function and decreased PD-1+ lymphocyte proportions. A follow-up study showed that combined BEZ235 (a dual PI3K/mTOR inhibitor) and everolimus treatment for six weeks resulted in better infection control in older adults for a year after treatment ended.",Frontiers in Immunology,Clinical Immunosenescence Interventions,2019
"mTOR Inhibition: Conflicts, Immune Modulation, and Future Directions","Rapamycin and Torin, another mTOR inhibitor, are also reported to suppress anti-inflammatory effects of circulating glucocorticoids. These findings conflict with earlier studies showing the central role of mTOR in innate immunity, specifically production of anti-inflammatory IL-10 and suppression of pro-inflammatory cytokines IL-21 and IL-1β. The improved immune response observed after rapamycin treatment, which may involve a decrease in PD-1+ T cells, requires more detailed study. Data suggesting that nutrient signaling pathways may negatively influence lymphocyte function in aging indicate that inhibition of these pathways may enhance lymphocyte activity in older adults. Broad ranges of pharmacological agents with anti-immunosenescence properties have been identified, and additional trials with rapamycin analogs are underway. This represents a promising therapeutic approach for improving the healthspan of older individuals. See Figure 2 for the main clinical approaches in immunomodulatory interventions.",Frontiers in Immunology,Clinical Immunosenescence Interventions,2019
Novel Vaccine Approaches for Aging Immune Systems,"Other approaches focus on development of novel vaccines especially suited to raise protective immunity in older adults by overcoming the decrease in naïve cells. This approach includes high-dose vaccines, booster vaccinations, different immunization routes, and use of new adjuvants. The most used adjuvants are based on aluminum salts. These adjuvants induce the activation of APCs and strengthen the antigen immunogenicity by their slower release and higher persistence at the vaccination site. Another interesting compound is MF59, a squalene-based adjuvant, which increases the chemokine-dependent recruitment of APCs. However, adjuvants have shown only modest success. The most effective is generally considered complete Freund adjuvant, which can only be used in animals because it can cause damaging skin inflammation. Therefore, there is an unmet need for new vaccine strategies for older people. The development and identification of appropriate adjuvants and cytokines might effectively remedy defects in T cell functions from older adults, both directly and by better activation of DCs.",Frontiers in Immunology,Immunosenescence Interventions – Vaccine/Adjuvant Strategies,2019
TLR Agonist-Based Solutions and CASAC Strategy,"Stimulation of TLRs by agonists seems to be a promising strategy to enhance vaccine efficacy, because TLR triggering can induce cytokine production by APCs and promote germinal center antibody production. Age-related variations in cytokine production are seen in APCs isolated from older donors, and efficient TLR stimulation may overcome TLR signaling dysfunction. Triggering multiple TLRs using a combined adjuvant for synergistic activation of cellular immunity (CASAC) is an intriguing strategy. CASAC incorporates CpG, a potent inducer of IFN-α by pDCs, together with poly I:C, a synthetic analog of viral dsRNA that targets TLR3 inducing type I IFNs. CASAC also contains IFN-γ and MHC class I and II peptides. This formulation results in potent cytotoxic T cell–mediated immunity in young mice. Immunization with multiple TLR agonists, an activator anti-CD40 antibody, IFN-γ, and surfactants drives unprecedented levels of CD8 responses and highly polarized Th1 CD4 responses. CASAC stimulation activates both mDCs and pDCs with IL-12 secretion and provides a technological platform for rapid vaccine development.",Frontiers in Immunology,Immunosenescence Interventions – Vaccine/Adjuvant Strategies,2019
CASAC Performance in Aged Models and TLR Combinations,"In aged mice, antigen-specific CD8+ T cell responses were stimulated after serial vaccinations with CASAC and a class I epitope derived from ovalbumin or the melanoma-associated self-antigen tyrosinase-related protein-2. Pentamer analysis showed that aged CASAC-vaccinated animals had substantially higher levels of antigen-specific CD8+ T cells than those vaccinated with complete or incomplete Freund adjuvant. CASAC promoted significantly better functional CD8+ T cell activity. An approach that overcomes defects in CD4 responses in vivo comes from the activation of peripheral blood DCs isolated from older donors with combined TLR ligands. Preliminary in vitro screens suggest that the most effective activator is the combination of TLR7/TLR8 agonist with TLR4 agonist. This combination induces significantly greater cytokine production than individual ligands due to activation of both MyD88 and TRIF-dependent pathways. The specific combination of R848 (TLR7/8 agonist) and monophosphoryl lipid A (TLR4 agonist) induces significantly higher cytokine secretion by mDCs and pDCs from older adults.",Frontiers in Immunology,Immunosenescence Interventions – Vaccine/Adjuvant Strategies,2019
Implications for Elderly Vaccination and Cytokine Restoration,"The reduced production of cytokines by pDCs from older people, caused by defects in TLR signaling pathways, is associated with an ineffective antibody response to influenza vaccination. The findings demonstrating enhanced cytokine secretion via combined TLR agonists highlight the efficient effect of adjuvants in stimulating cytokine production. These results point toward the potential use of appropriately selected combinations of TLR agonists in future vaccination approaches for older adults, to overcome the CD4 inability to respond effectively. The section concludes by emphasizing that these strategies may provide routes to restore immunity in the elderly through enhancement of both innate signaling and adaptive T cell priming.",Frontiers in Immunology,Immunosenescence Interventions – Vaccine/Adjuvant Strategies,2019
Impact of Age on Thymic Stromal Cells – Structural Decline,"Aged-related thymic involution reduces thymic cellularity in mice by 50% at 16 weeks in comparison with its adult peak at 4 weeks, eventually leading to less than 5% thymic cellularity. In humans, thymus size reduction begins as early as 1 year of age, and it continues to decline at a rate of approximately 3% per year until middle age before slowing down to less than 1% per year. Morphological analysis has shown that cortical and medullary thymic epithelial region structure becomes increasingly less reticular and less globular with age in mice, and the definite cortical-medullary junction is also gradually lost with age. Furthermore, aging is concomitant with thymic epithelial space contraction and perivascular space augmentation in humans. The aged thymus displays obvious TEC reduction, fibroblast and adipocyte expansion, and senescent cell accumulation. Compared with 4 week old mice, TEC cellularity is reduced by about 50% at 16 weeks and over 80% at 50 weeks. The mTEC population decreases gradually with age, leading to a decline in mTEC/cTEC ratio. Although TEC number reduction is widely accepted, a recent study suggested that aging contracts cTEC projections but may not reduce TEC number, with earlier studies potentially underestimating TECs due to isolation methods.",Aging Cell,Age-related Thymic Involution – Stromal Cells,2022
"Impact of Age on TEC Function – Proliferation, Markers, and Antigen Presentation","Proliferation of both CD45− non-TECs stromal cells and TECs decreases dramatically with thymic aging in mice. Impaired TEC proliferation in aged mice was demonstrated using transcriptome analysis. Thymic aging is accompanied by a decline in TEC marker expression, including EpCAM, keratin, CD205, and Ulex Europaeus Agglutinin 1. During thymic aging, the ratio of MHCIIhi TECs to MHCIIlo TECs clearly decreases, reflecting a reduced TEC antigen presentation ability in aged mice. However, some recent studies have shown that emerging MHCIIlo TEC subsets during thymic development have other specific roles such as supporting invariant NKT cell development. Tissue-restricted antigen expression also diminishes with age, representing a potential mechanism for age-related increase in autoimmune diseases. Aging also impairs TEC secretion ability, as demonstrated by diminished production of the thymopoietic cytokine IL-7 in mice. IL-7 administration in older mice and in the rhesus macaque increases thymic output.",Aging Cell,Age-related Thymic Involution – Stromal Cells,2022
Impact of Age on TEC Subsets – scRNA-seq Insights,"Advances in bulk RNA-seq and scRNA-seq technology have allowed more comprehensive investigation of TEC subpopulation and transcriptional profile changes during thymic aging. A scRNA-seq study comparing TEC subsets in young and old mice found that most mTECs considerably diminished and most cTECs dramatically increased in percentage upon aging. mTEC progenitor subsets also reduced with age; however, there was a much higher frequency of bipotent TEC progenitors in the aged thymus. Another study subdivided TECs from 1-, 4-, 16-, 32-, and 52-week-old mice into 9 subtypes using scRNA-seq. The proportion of perinatal cTECs and mature mTECs were significantly reduced with aging, in contrast to mature cTECs and intertypical TECs, which increased. Intertypical TECs represent a TEC progenitor state, and aging compromises their differentiation into mature mTECs. Inflamm-aging transcriptional signatures were restricted to mature cTECs and mature mTECs. Transcriptome comparisons from 1-, 3-, and 6-month-old mice showed that E2F3 and cell cycle-associated gene expression decreased early in cTECs and mTECs. E2F3 downregulation impairs TEC cell-cycle progression. Decline in myc targets and ribosomal genes was also reported, and a FoxN1MycTg mouse model demonstrated that myc promotes ribosomal gene expression in TECs.",Aging Cell,Age-related Thymic Involution – Stromal Cells,2022
Impact of Age on Non-TEC Stromal Cells,"In addition to TECs, aging affects other stromal cells in the thymus. Thymic aging coincides with adipocyte accumulation around the thymus, and the increase in adipose tissue may inhibit thymic function through adipocytokine secretion. Fibroblast percentage also increases in the aging thymus in species including mice, humans, and fish, suggesting a conserved feature. A thymic stromal cell transcriptome analysis revealed that proinflammatory gene expression increased with aging in mouse thymic dendritic cells, which in turn may accelerate thymic aging. Another study showed that thymic B cell function is impaired with aging in mice; the authors demonstrated that Aire and Aire-dependent tissue-restricted antigen expression decline in aging thymic B cells. Thus, aging impairs many cell subsets in the thymic microenvironment.",Aging Cell,Age-related Thymic Involution – Stromal Cells,2022
"Impact of Aging on HSCs, ETPs, and Early Thymocyte Seeding","In addition to thymic stromal cells, thymocyte development is also drastically disturbed during thymic aging. Some studies have shown that hematopoietic stem cells (HSCs) of aged mice display an increased bias toward myeloid differentiation concomitant with a diminished lymphoid lineage differentiation ability. HSC abnormalities in aged mice may affect the seeding of early T-lineage progenitors (ETPs) within the thymus. Indeed, ETP frequency declines with aging, and their potential ability to reconstitute the thymus is also reduced. ETPs from young mice are able to differentiate into all stages of thymocytes when seeded into thymic lobes; in contrast, this differentiation ability is impaired in ETPs from aged mice. However, the effect of aging on HSCs and ETPs is controversial. Zhu et al. established a mouse model in which they transplanted a fetal thymus into the kidney capsule of aged mice, providing a young thymic microenvironment for aged lymphohematopoietic progenitors. They demonstrated that progenitors from aged and young mice have similar abilities to differentiate into ETPs and subsequent thymocyte subpopulations when placed in a young thymic environment, indicating LPCs do not have intrinsic defects synchronized with age-related thymic involution. Another study showed that ETP defects in aged thymi are mainly due to disrupted thymic epithelial architecture—including poorly defined cortico-medullary junction and reduced medulla cellularity—rather than intrinsic ETP defects.",Aging Cell,Age-related Thymic Involution – Thymocyte Development,2022
"Impact of Aging on DN, DP, and SP Thymocyte Differentiation","ETPs subsequently differentiate into double negative (DN) CD4−CD8− subpopulations including DN1, DN2, DN3, and DN4. These subsets then become double positive (DP) CD4+CD8+ cells that further differentiate into CD4 or CD8 single positive T cells through positive and negative selection. Although both DN and DP thymocyte population numbers significantly reduce with aging, the DN subset frequency increases 2–3 times in aged (24–27 months old) mice compared with young (2–3 months old) mice, whereas the percentage of DP subpopulations significantly diminishes. Among DN subsets, DN2 and DN3 subset numbers show considerable reduction with thymic aging. Additionally, thymic aging is concomitant with abnormal accumulation of CD3+ DN cells within the thymus. Aging also interferes with later stages of thymocyte development. DP and SP thymocytes in aged mice display deregulated CD3 expression, which may lead to attenuated TCR-dependent stimulation. Thymocytes from older mice exhibit impaired mitogen response ability, manifested by failure to upregulate the activation marker CD69 and failure to proliferate. Consistent with impaired thymocyte differentiation in the aged thymus, T-cell receptor excision circles (TRECs) significantly decline with aging in both mice and humans. Thus, aging impairs multiple thymocyte developmental stages, affecting early progenitors, DN transitions, DP survival, SP maturation, and the generation of newly formed naïve T cells.",Aging Cell,Age-related Thymic Involution – Thymocyte Development,2022
Thymic Output Decline and Peripheral Naïve T-Cell Maintenance,"Mature CD4 SP and CD8 SP thymocytes are exported to the periphery where they play a role in immunological surveillance. Age-related thymic involution causes an obvious reduction in the thymic output of naïve T cells and subsequently decreases peripheral T-cell diversity. Diminished thymic production of naïve T cells leads to homeostatic expansion of existing T cells, resulting in memory T-cell augmentation. Although it is well accepted that the thymic output of peripheral naïve T cells progressively declines with aging in mice, in humans the relationship of thymic involution to peripheral naïve T-cell maintenance is debated. Many studies using T-cell receptor excision circles (TRECs) as a measurement of thymic output demonstrate that peripheral naïve T-cell thymic output declines with aging in humans. However, den Braber et al. showed that adult human peripheral naïve T-cell pool maintenance occurs almost exclusively through cell proliferation rather than thymic output. Therefore, the contribution of thymic export to naïve T-cell maintenance in adults requires further investigation. Declining thymic output combined with increased peripheral proliferation alters the balance between naïve and memory populations, contributing to immunosenescence.",Aging Cell,Age-related Thymic Involution – Thymic Output,2022
Aging Effects on Naïve T-Cell Function and Immunological Surveillance,"Aging also interferes with naïve T-cell properties and functions. Naïve T cells from aged mice express elevated levels of senescence markers and display reduced proliferation ability upon antigen stimulation. Chemokine receptor expression is altered in CD4+ T cells of aged mice, exhibiting deregulation of CCR1, CCR7, CCR8, and CXCR2, CXCR4, and CXCR5, which may impair their migration ability. These molecular and phenotypic changes compromise the ability of naïve T cells to properly respond to antigenic challenge. The reduced number of naïve T cells together with the disrupted function of remaining naïve T cells during aging leads to impaired immunological surveillance ability in aged organisms. The combined impact of reduced thymic export, altered homeostatic proliferation, diminished TCR repertoire diversity, and functional impairment of naïve T cells contributes significantly to the increased susceptibility of aged individuals to infections, reduced vaccine responsiveness, and greater risk of malignancy.",Aging Cell,Age-related Thymic Involution – Naïve T-Cell Function,2022
Thymic Stromal Cell Degeneration as the Primary Driver of Involution,"Although the T-lineage differentiation potential of HSCs and ETPs is partially compromised in aged mice compared with young mice, increasing evidence suggests that thymic involution is mainly caused by age-related thymic stromal cell degeneration, particularly TEC degeneration. A global transcriptome analysis of thymic stromal cells and lymphocytes revealed that mouse thymic stromal cells, in contrast to lymphocytes, are deficient in catalase. This deficiency leads to elevated H2O2 levels and stromal cell oxidative damage, which subsequently results in thymic atrophy. Restoration of antioxidant activity, both genetically and biochemically, ameliorates thymic atrophy in mouse models. Similar findings have been reported in humans: the thymi of Down syndrome patients exhibit premature senescence, and TECs show increased oxidative stress. Using a genome-wide computational approach, another study showed that age-associated thymic degeneration is primarily a stromal cell functional decline. These results strongly support the idea that alterations in thymic stromal cells are central to the process of age-related thymic involution.",Aging Cell,Thymic Involution Mechanisms – Stromal Degeneration,2022
Evidence from Transplant and Reconstitution Studies,"Many studies support the pivotal role of the thymic stroma in thymic aging. Mackall et al. demonstrated that lethally irradiated older mice exhibit impaired thymopoiesis compared with lethally irradiated young mice after both were injected with young bone marrow, suggesting that the aged thymic stromal microenvironment cannot support normal thymocyte development. Another experiment showed that intrathymic injection of young ETPs failed to restore normal thymopoiesis in older mice but did restore thymopoiesis in young mice. In contrast, fetal thymus transplants into the kidney capsules of young or old mice supported similar levels of thymopoiesis regardless of the age of the host. These findings indicate that defects intrinsic to the aged thymic stroma—rather than defects in hematopoietic progenitors—are primarily responsible for thymic involution. The durable identity and functional capacity of the thymus are therefore established by its stromal components, whereas developing thymocytes are only transient inhabitants of the organ.",Aging Cell,Thymic Involution Mechanisms – Stromal Microenvironment,2022
Overview of Menopause and Reproductive Ageing,"The post-reproductive phase or menopause in females is triggered by a physiological timer that depends on a threshold of follicle number in the ovary. Curiously, reproductive senescence appears to be decoupled from chronological age and is instead thought to be a function of physiological ageing. Ovarian ageing is associated with a decrease in oocyte developmental competence, attributed to a concomitant increase in meiotic errors. Although many biological hallmarks of general ageing are well characterized, the precise mechanisms underlying the programmed ageing of the female reproductive system remain elusive. In particular, the molecular pathways linking the external menopause trigger to the internal oocyte chromosome segregation machinery that controls fertility outcomes is unclear.",Human Reproduction,Reproductive Aging,2022
Genomics Studies and Molecular Pathways in Menopause,"However, recent large scale genomics studies have begun to provide insights into this process. Next-generation sequencing integrated with systems biology offers the advantage of sampling large datasets to uncover molecular pathways associated with a phenotype such as ageing. In this mini-review, we discuss findings from these studies that are crucial for advancing female reproductive senescence research. Targets identified in these studies can inform future animal models for menopause. We present three potential hypotheses for how external pathways governing ovarian ageing can influence meiotic chromosome segregation, with evidence from both animal models and molecular targets revealed from genomics studies.",Human Reproduction,Reproductive Aging,2022
Future Directions and Biomarker Development,"Although still in incipient stages, we discuss the potential of genomics studies combined with epigenetic age acceleration models for providing a predictive toolkit of biomarkers controlling menopause onset in women. We also speculate on future research directions to investigate extending female reproductive lifespan, such as comparative genomics in model systems that lack menopause. Novel genomics insights from such organisms are predicted to provide clues to preserving female fertility.",Human Reproduction,Reproductive Aging,2022
Hallmarks of General and Female Reproductive Ageing,"Universally, ageing is a progressive decline of physiological integrity resulting in functional deterioration. Many biological processes are established hallmarks of ageing across mammalian species including genomic instability, epigenetic changes, telomere shortening, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication (Lopez-Otin et al., 2013). Female reproductive ageing is unique in terms of its apparent disconnection with the onset of ageing processes in the other organs. The most intriguing aspect is that the ovary operates a physiological timer or clock which depends on the number of non-growing follicles that comprise the ovarian reserve after birth, enforcing reproductive senescence when a threshold of 1000 follicles remain in the ovary (te Velde and Pearson, 2002; Broekmans et al., 2009; Wallace and Kelsey, 2010). Numerous outstanding questions exist regarding what determines the follicular threshold that controls the female reproductive lifespan.",Human Reproduction,Reproductive Aging,2022
The Ovarian Physiological Clock and Knowledge Gaps,"Undeniably, female reproductive ageing research will benefit from defining the biological hallmarks specific to the female reproductive system, which remain elusive to date. Developments in genomics are beginning to elucidate the cell type and developmental stage-specific molecular players that constitute the biological clock of female fertility. However, our understanding of how it works to time the reproductive life span, and how its molecular components interact with environmental factors to irreversibly shut down the reproductive system before other systems, is far from complete. Understanding this clock and its constituents will spark the development of strategies to mitigate the impact of female reproductive ageing on women’s reproductive choices.",Human Reproduction,Reproductive Aging,2022
"Genomics, Comparative Biology, and Future Directions","In this mini-review, we not only address new findings from genomics but also discuss the importance of building a strong bridge with comparative and molecular biology to accelerate discovery and development of predictive or anti-ageing strategies.",Human Reproduction,Reproductive Aging,2022
Ovarian Biological Clock and Meiotic Error Hypotheses,"Genomics identifies pathways that operate the ovarian biological clock: insights into the irreversible end of a woman’s reproductive life
The relationship between natural fertility and age follows an inverse U-shaped curve with reduced rates at both age extremes (Hawkes and Smith, 2010). Chromosome segregation errors during meiosis were shown to follow the same U-shaped trend, with increased frequencies in teenage and advanced maternal age (AMA) women (Gruhn et al., 2019) and were proposed to be the main determinant of female fecundity. Based on these observations, the hypothesis of a molecular meiotic chromosome driven ‘timing’ mechanism of ovarian ageing, such as cohesion weakening (discussed below), was brought forward (Gruhn et al., 2019). The critical role of oocyte quality as a determinant in reproductive ageing is best demonstrated in IVF cycles where women of AMA who use oocytes from reproductively young donors increase their birth rate per embryo transferred from 4% in autologous transfers to ∼25% (HFEA report, 2020). But is oocyte genome integrity the only driver of fecundity decline as females age? The very elegant hemi-ovariectomy surgical experiments in rodents (discussed in the next section) show that the size of the initial oocyte pool determines the time of onset of both ovarian senescence and oocyte aneuploidy (Brook et al., 1984; Nelson and Felicio, 1986).",Human Reproduction,Reproductive Aging,2022
GWAS Insights Into Menopause Timing and DDR Pathways,"Which mechanisms associate with variation of the age at menopause onset in humans? Analytical dissection of the genetic associations from genome-wide association studies (GWAS) of age at natural menopause (ANM) (Day et al., 2015; Ruth et al., 2021) yielded novel insights into these themes. Genetics contribute to >50% of the variation in the ANM onset (Snieder et al., 1998; Murabito et al., 2005) and significantly associated common variants explain up to 32.4% of the UK biobank genotype array heritability and 10% of ANM variance in an independent cohort (Ruth et al., 2021). Further elegant integrative analyses including an expression quantitative trait locus (QTL) survey for 48 tissues, network analysis, pathway enrichment and gene expression quantitation, fine mapped the GWA loci to genes mediating molecular pathways including DNA damage response (DDR), apoptosis and metabolic signaling (Ruth et al., 2021). Integrative genomics and systems concepts such as gene network inference from single-cell expression studies in foetal primordial germ cells (PGCs), oocytes and granulosa cells (GCs) from primordial, primary, secondary antral follicle and mature metaphase II (MII) oocytes yielded novel insights into the temporal activation of each mechanism from foetal to adult life (Ruth et al., 2021).",Human Reproduction,Reproductive Aging,2022
DNA Repair Mechanisms Across Ovarian Development,"Importantly, we now have more accurate information on the varied insults which occur in different developmental stages, follicular events and cell types (oocytes versus GCs), and which invoke specific DDR mechanisms (Day et al., 2015; Ruth and Murray, 2016; Ruth et al., 2021). For example, genetic variation in genes mediating single- and double-strand break (DSB) repair, mismatch repair (MSH5, MSH6), base excision repair, DNA damage checkpoint kinase genes (CHEK1, CHEK2) and repair of replication stress-induced inter-strand crosslinks (BRCA1, FANCA, FANCB, FANCM, FAAP24) is expected to impact the development of foetal oocytes and follicular growth at different stages (Ruth et al., 2021). Similarly, variation in apoptotic genes (TP63, BCL2, BCL2L11, PPARG, PPP5C) located within 300 kb of the single-nucleotide polymorphisms with the strongest statistical association for ANM, suggests varying activation downstream of DDR, probably regulating attrition and depletion of the ovarian reserve.

Can the size of the follicular endowment be maintained at optimal levels at birth and can we delay depletion of the ovarian reserve over time? Experiments in mice show that manipulation of CHEK1 and CHEK2 increases the reproductive lifespan, and women carrying loss-of-function variants in CHEK2 reported an ANM almost 3.5 years later than non-carriers (Ruth et al., 2021).",Human Reproduction,Reproductive Aging,2022
"DDR in Foetal Oocytes, PGC Clearance, and Metabolic DNA Damage","Through these experiments, we gained deeper knowledge on the activity of specific DDR mechanisms in the establishment and the depletion of the ovarian reserve (discussed in the next section). Several DDR ANM-associated genes (MSH5, MSH6, BRCA1, BRCA2, FANCA, FANCB, RAD51) are important for genomic integrity surveillance in the foetal germline where diverse DDR mechanisms engage various pathways to repair different lesions across ovarian development. Meiotic DDR pathways target errors during homologous recombination and play a pivotal role in the surveillance of genome integrity during meiosis I where foetal oocytes appear to escape the ‘metaphase I check point’ (Lenzi et al., 2005). The size of the follicular reserve is determined at birth by ‘clearing’ defective oocytes during the perinatal attrition period (Martinez-Marchal et al., 2020). Importantly, pre-meiotic DDR-mediated pathways also clears defective PGCs (Hill and Crossan, 2019). When unrepaired DNA lesions accumulate in PGCs, because of a defective or absent inter-strand cross-link repair pathway, the affected cells are cleared by apoptosis during a very narrow developmental window during epigenetic reprogramming and proliferation. Metabolic insults, such as the impaired catabolism of aldehydes, cause DNA damage to PGCs (Hill and Crossan, 2019). In contrast, the surrounding gonadal somatic cells do not differ in the frequency of DNA DSBs (Hill and Crossan, 2019). This observation indicated that perhaps mutagenic insults in somatic cells are less frequent than in PGCs.",Human Reproduction,Reproductive Aging,2022
"Somatic Mutation Rates, NAD+ Pathways, and Environmental Impacts","Hence there is a decreased requirement for repair in the soma compared to the germline, which is in line with purifying selection preventing the development of germline cells passing mutations to the next generation.

DNA repair has emerged as a universal functional cog of the ageing clock, by quantifying the mutation rate—defined as the number of mutations per year—across 16 mammalian species (Cagan et al., 2022). The somatic mutational rate is inversely correlated with lifespan (Cagan et al., 2022), suggesting that the DNA repair mechanisms are more efficient in longer-lived species, which in turn indicates that a conserved somatic tissue biological clock also relies on DNA repair.

What do we know about DNA damage in the adult ovary? Temporal expression of the genes implicated in the NAD+ salvage pathway (NAMPT, PPARG, FOXO1, SIRT1) and in caloric restriction (IGF1) through the IGF1-axis across the consecutive follicular developmental stages suggests a link between environmental inducers of age-related DNA damage which then triggers the DDR and apoptosis. SIRT1 sensing of NAD+ is a central mediator of the anti-ageing benefits of caloric restriction.",Human Reproduction,Reproductive Aging,2022
"Caloric Restriction, Diet Effects, and Single-Cell Aging Signatures","The association between SIRT1 sensing NAD+ and defective DNA repair has been shown in accelerated neurodegeneration in Cockayne syndrome (Scheibye-Knudsen et al., 2014). Caloric restriction preserves female fertility in aged mice by reducing meiotic spindles defects and by maintaining mitochondrial distribution and ATP production in mature MII oocytes (Selesniemi et al., 2011). Importantly, exposures to an obesogenic diet, both in utero and after weaning, affects Dmc1 recombinase and Brsk1 DNA damage sensor expression (Ruth et al., 2021).

Single-cell RNA-seq changes in non-human primates modelling human reproductive ageing (pre- and peri-menopausal stages) investigated the impact of age (in a cell-type distinctive manner) on the expression of genes that regulate protection from oxidative stress (GPX1, GSR), preserve mitochondrial function in oocytes from the early follicular stages, and mediate the protective response to oxidative damage and apoptosis in GCs (IDH1, NDUFB10) (Wang et al., 2020). Furthermore, DNA oxidation, DNA damage and apoptosis in GCs increased in ageing oocytes compared to those from young animals (Wang et al., 2020).",Human Reproduction,Reproductive Aging,2022
Central Role of DDR in Ovarian Senescence Timing,"Collectively, these seminal genomics studies provide critical links between the central ageing molecular mechanism, the DDR, in timing the onset of ovarian senescence in animal models. The link predicts that a disturbance in the balance between DNA damage and repair leads to increased follicular attrition rates either in utero or during adult life. In the following sections, we address recent and propose future experiments in models of reproductive ageing that aim at further delineating the mechanistic link between the size of ovarian reserve and the pathways highlighted by genomics studies.",Human Reproduction,Reproductive Aging,2022
Ovarian Biological Clock and Meiotic Error Hypotheses,"Genomics identifies pathways that operate the ovarian biological clock: insights into the irreversible end of a woman’s reproductive life
The relationship between natural fertility and age follows an inverse U-shaped curve with reduced rates at both age extremes (Hawkes and Smith, 2010). Chromosome segregation errors during meiosis were shown to follow the same U-shaped trend, with increased frequencies in teenage and advanced maternal age (AMA) women (Gruhn et al., 2019) and were proposed to be the main determinant of female fecundity. Based on these observations, the hypothesis of a molecular meiotic chromosome driven ‘timing’ mechanism of ovarian ageing, such as cohesion weakening (discussed below), was brought forward (Gruhn et al., 2019). The critical role of oocyte quality as a determinant in reproductive ageing is best demonstrated in IVF cycles where women of AMA who use oocytes from reproductively young donors increase their birth rate per embryo transferred from 4% in autologous transfers to ∼25% (HFEA report, 2020). But is oocyte genome integrity the only driver of fecundity decline as females age? The very elegant hemi-ovariectomy surgical experiments in rodents (discussed in the next section) show that the size of the initial oocyte pool determines the time of onset of both ovarian senescence and oocyte aneuploidy (Brook et al., 1984; Nelson and Felicio, 1986).",Human Reproduction,Reproductive Aging,2022
GWAS Insights Into Menopause Timing and DDR Pathways,"Which mechanisms associate with variation of the age at menopause onset in humans? Analytical dissection of the genetic associations from genome-wide association studies (GWAS) of age at natural menopause (ANM) (Day et al., 2015; Ruth et al., 2021) yielded novel insights into these themes. Genetics contribute to >50% of the variation in the ANM onset (Snieder et al., 1998; Murabito et al., 2005) and significantly associated common variants explain up to 32.4% of the UK biobank genotype array heritability and 10% of ANM variance in an independent cohort (Ruth et al., 2021). Further elegant integrative analyses including an expression quantitative trait locus (QTL) survey for 48 tissues, network analysis, pathway enrichment and gene expression quantitation, fine mapped the GWA loci to genes mediating molecular pathways including DNA damage response (DDR), apoptosis and metabolic signaling (Ruth et al., 2021). Integrative genomics and systems concepts such as gene network inference from single-cell expression studies in foetal primordial germ cells (PGCs), oocytes and granulosa cells (GCs) from primordial, primary, secondary antral follicle and mature metaphase II (MII) oocytes yielded novel insights into the temporal activation of each mechanism from foetal to adult life (Ruth et al., 2021).",Human Reproduction,Reproductive Aging,2022
DNA Repair Mechanisms Across Ovarian Development,"Importantly, we now have more accurate information on the varied insults which occur in different developmental stages, follicular events and cell types (oocytes versus GCs), and which invoke specific DDR mechanisms (Day et al., 2015; Ruth and Murray, 2016; Ruth et al., 2021). For example, genetic variation in genes mediating single- and double-strand break (DSB) repair, mismatch repair (MSH5, MSH6), base excision repair, DNA damage checkpoint kinase genes (CHEK1, CHEK2) and repair of replication stress-induced inter-strand crosslinks (BRCA1, FANCA, FANCB, FANCM, FAAP24) is expected to impact the development of foetal oocytes and follicular growth at different stages (Ruth et al., 2021). Similarly, variation in apoptotic genes (TP63, BCL2, BCL2L11, PPARG, PPP5C) located within 300 kb of the single-nucleotide polymorphisms with the strongest statistical association for ANM, suggests varying activation downstream of DDR, probably regulating attrition and depletion of the ovarian reserve.

Can the size of the follicular endowment be maintained at optimal levels at birth and can we delay depletion of the ovarian reserve over time? Experiments in mice show that manipulation of CHEK1 and CHEK2 increases the reproductive lifespan, and women carrying loss-of-function variants in CHEK2 reported an ANM almost 3.5 years later than non-carriers (Ruth et al., 2021).",Human Reproduction,Reproductive Aging,2022
"DDR in Foetal Oocytes, PGC Clearance, and Metabolic DNA Damage","Through these experiments, we gained deeper knowledge on the activity of specific DDR mechanisms in the establishment and the depletion of the ovarian reserve (discussed in the next section). Several DDR ANM-associated genes (MSH5, MSH6, BRCA1, BRCA2, FANCA, FANCB, RAD51) are important for genomic integrity surveillance in the foetal germline where diverse DDR mechanisms engage various pathways to repair different lesions across ovarian development. Meiotic DDR pathways target errors during homologous recombination and play a pivotal role in the surveillance of genome integrity during meiosis I where foetal oocytes appear to escape the ‘metaphase I check point’ (Lenzi et al., 2005). The size of the follicular reserve is determined at birth by ‘clearing’ defective oocytes during the perinatal attrition period (Martinez-Marchal et al., 2020). Importantly, pre-meiotic DDR-mediated pathways also clears defective PGCs (Hill and Crossan, 2019). When unrepaired DNA lesions accumulate in PGCs, because of a defective or absent inter-strand cross-link repair pathway, the affected cells are cleared by apoptosis during a very narrow developmental window during epigenetic reprogramming and proliferation. Metabolic insults, such as the impaired catabolism of aldehydes, cause DNA damage to PGCs (Hill and Crossan, 2019). In contrast, the surrounding gonadal somatic cells do not differ in the frequency of DNA DSBs (Hill and Crossan, 2019). This observation indicated that perhaps mutagenic insults in somatic cells are less frequent than in PGCs.",Human Reproduction,Reproductive Aging,2022
"Somatic Mutation Rates, NAD+ Pathways, and Environmental Impacts","Hence there is a decreased requirement for repair in the soma compared to the germline, which is in line with purifying selection preventing the development of germline cells passing mutations to the next generation.

DNA repair has emerged as a universal functional cog of the ageing clock, by quantifying the mutation rate—defined as the number of mutations per year—across 16 mammalian species (Cagan et al., 2022). The somatic mutational rate is inversely correlated with lifespan (Cagan et al., 2022), suggesting that the DNA repair mechanisms are more efficient in longer-lived species, which in turn indicates that a conserved somatic tissue biological clock also relies on DNA repair.

What do we know about DNA damage in the adult ovary? Temporal expression of the genes implicated in the NAD+ salvage pathway (NAMPT, PPARG, FOXO1, SIRT1) and in caloric restriction (IGF1) through the IGF1-axis across the consecutive follicular developmental stages suggests a link between environmental inducers of age-related DNA damage which then triggers the DDR and apoptosis. SIRT1 sensing of NAD+ is a central mediator of the anti-ageing benefits of caloric restriction.",Human Reproduction,Reproductive Aging,2022
"Caloric Restriction, Diet Effects, and Single-Cell Aging Signatures","The association between SIRT1 sensing NAD+ and defective DNA repair has been shown in accelerated neurodegeneration in Cockayne syndrome (Scheibye-Knudsen et al., 2014). Caloric restriction preserves female fertility in aged mice by reducing meiotic spindles defects and by maintaining mitochondrial distribution and ATP production in mature MII oocytes (Selesniemi et al., 2011). Importantly, exposures to an obesogenic diet, both in utero and after weaning, affects Dmc1 recombinase and Brsk1 DNA damage sensor expression (Ruth et al., 2021).

Single-cell RNA-seq changes in non-human primates modelling human reproductive ageing (pre- and peri-menopausal stages) investigated the impact of age (in a cell-type distinctive manner) on the expression of genes that regulate protection from oxidative stress (GPX1, GSR), preserve mitochondrial function in oocytes from the early follicular stages, and mediate the protective response to oxidative damage and apoptosis in GCs (IDH1, NDUFB10) (Wang et al., 2020). Furthermore, DNA oxidation, DNA damage and apoptosis in GCs increased in ageing oocytes compared to those from young animals (Wang et al., 2020).",Human Reproduction,Reproductive Aging,2022
Central Role of DDR in Ovarian Senescence Timing,"Collectively, these seminal genomics studies provide critical links between the central ageing molecular mechanism, the DDR, in timing the onset of ovarian senescence in animal models. The link predicts that a disturbance in the balance between DNA damage and repair leads to increased follicular attrition rates either in utero or during adult life. In the following sections, we address recent and propose future experiments in models of reproductive ageing that aim at further delineating the mechanistic link between the size of ovarian reserve and the pathways highlighted by genomics studies.",Human Reproduction,Reproductive Aging,2022
External Physiological Factors and Meiotic Aneuploidy,"Genomics and chromosome biology of reproductive senescence: how strong is the bridge?
Increased meiotic aneuploidy has also been reported to be a function of reduced ovarian reserve count from both mouse models of menopause and in humans, although other factors confound the data obtained from humans (Brook et al., 1984; Finch, 2014). This suggests that external physiological factors such as ovulation frequency, follicular atresia and follicle count also underpin the quality of ovulated oocytes. Indeed, an intriguing aspect of reproductive senescence is that it is thought to be a function of physiological rather than chronological ageing since mice oocytes age over months whereas human oocytes age over decades. At the level of chromosome segregation, it is well-established that one of the major causes of the maternal age effect is declining levels of chromosome-associated proteins that are not replenished with age (Chiang et al., 2010). This includes but is not limited to cohesins, like Rec8, Shugoshin-like 2 (SGO2), that ensure centromeric cohesion (Liu and Keefe, 2008; Duncan et al., 2012), and certain actin cytoskeleton proteins that support the oocyte spindle (Dunkley and Mogessie, 2022).",Human Reproduction,Reproductive Aging,2022
"Spindle Proteins, DNA Repair, and Oocyte Aneuploidy","A recent exome sequencing study found that genes functioning in centriole formation like Cep120, and DNA damage repair were enriched in blastocysts with higher incidence of maternally derived aneuploidy (Tyc et al., 2020; Wartosch et al., 2021). Likewise, the oocyte-specific spindle protein TUBB8, was also found to cause female infertility (Feng et al., 2016). Altogether, the aetiology of human oocyte aneuploidy has been attributed to inherent meiotic spindle instability, increased merotelic attachments, and age-related changes in kinetochore and chromosome architecture (Fellmeth et al., 2015; Zielinska et al., 2015, 2019; Thomas et al., 2021). However, how the external ovarian changes communicate to the internal segregation machinery remains unclear. Identifying and defining the molecular networks that control these ovarian processes will be key to delaying maternal ageing.",Human Reproduction,Reproductive Aging,2022
Genomics Pathways and Need for Functional In Vivo Validation,"Genomics studies have revealed several pathways that are implicated in determining the ANM and the next steps for the field will be testing and validating the role of these target pathways functionally in vivo. In this section, we will discuss some models based on functional studies that provide a link between external physiological changes in an ageing ovary and fidelity of the oocyte chromosome segregation. Indeed, some of these studies support findings from the GWAS.",Human Reproduction,Reproductive Aging,2022
"Ovulation Frequency, Inflammation, and Aneuploidy","The ovulation suppression model
A recent study tested the hypothesis that cohesion dysfunction is linked to ovulation frequency. Ovulation is an intensely inflammatory process that generates reactive oxygen species and leads to oxidative damage (Duffy et al., 2019). Under this hypothesis, physiological ageing associated with repeated ovulation can cause protein turnover leading to increased segregation errors. Using three mouse models that converge on reducing ovulation frequency, Chatzidaki and colleagues (Chatzidaki et al., 2021) showed that ovulation suppression does indeed reduce aneuploidy by preserving centromeric cohesion in aged oocytes. Thus, a direct molecular consequence of repetitive ovulation cycles is that it negatively impacts oocyte segregation and hence reproductive outcomes. Methods that can reduce ovulation frequency are thus predicted to have a protective effect against reproductive senescence, e.g. usage of oral hormonal contraception. The study by Chatzidaki and colleagues, along with a few others in humans, supports oral contraceptives as a potential clinical method to preserve fertility in women (Janerich et al., 1976; Farrow et al., 2002; Chatzidaki et al., 2021). However, the risks associated with long-term hormonal treatments must be weighed against the benefits of extending reproductive lifespan, especially for younger mothers.",Human Reproduction,Reproductive Aging,2022
"Inflammation, Fibrosis, and Protective Interventions","The hypothesis that inflammation associated with incessant ovulation exacerbates ageing is further supported by the fact that ablation of pro-inflammatory cytokines such as interleukin 1 results in increased fertility in mice at 2.5 months of age that persists until 12 months of age (Uri-Belapolsky et al., 2014), suggesting that inflammation is one of the molecular determinants of the ANM (Foley et al., 2021). Indeed, a link was experimentally established between the accumulating tissue damage from repeated ovulation in nulliparous 12-month-old virgin mice with gradually increasing ovarian fibrosis and ovulation rate decline compared to 12-month-old repeat breeders (Umehara et al., 2022). The same group showed that administration of pirfenidone (a TGFb1 suppressor, anti-oxidant and anti-inflammatory drug) used to treat pulmonary fibrosis resulted in reproductively aged mice being able to ovulate as opposed to the untreated controls. Moreover, the ovulated oocytes were reproductively competent resulting in blastocyst formation following IVF (Umehara et al., 2022).",Human Reproduction,Reproductive Aging,2022
CENP-A Preservation and Long-Lived Oocyte Proteins,"Although this study outlines the deleterious effects of repeated ovulation on internal chromosome-associated proteins, there are some proteins like the centromeric histone CENP-A, that are impervious to it. CENP-A, is a histone H3 variant that marks the location of the centromere where a kinetochore is built for spindle attachment and segregation. Therefore, preserving and transmitting CENP-A nucleosomes through the female germline to progeny is essential for chromosome inheritance (Das et al., 2017). Remarkably, CENP-A persists in mouse oocyte chromatin throughout their reproductive lifespan and shows little decline with age (Smoak et al., 2016). It is not known how the centromeric nucleosome is retained through reproductive senescence, unlike Rec8 and SGO2. Since the centromeres, designated by CENP-A, are crucial for meiotic divisions and beyond, perhaps the oocytes have evolved mechanisms to preserve this information long term, and protect against loss of centromere identity in progeny.",Human Reproduction,Reproductive Aging,2022
Recovery of Centromere Strength and Oocyte Protein Longevity,"Indeed, any reduction in centromere strength in one generation is completely recovered in the female germline of progeny possibly through increased CENP-A assembly during oogenesis to compensate for age-related loss (Das et al., 2022). Investigating mechanisms that retain long lived oocyte proteins through prophase arrest will also provide insights into what maintains oocyte quality over time (Das et al., 2017).",Human Reproduction,Reproductive Aging,2022
Reduced Follicle Count as a Driver of Aneuploidy,"Reduced ovarian reserve or follicle counter model
The above model cannot explain all age-related oocyte aneuploidy. Therefore, there must be other drivers of the female fecundity decline. It has been proposed that the reduced follicle count with age can have consequences on the remaining oocytes in terms of aneuploidy (Fu et al., 2014), but the evidence for this in humans is conflicting (Kline et al., 2004, 2011; Honorato et al., 2015). Under the follicle counter model, reduced ovarian reserve even in young females would be sufficient to yield high rates of oocyte aneuploidy. The hemi-ovariectomy surgical experiments in rodents have shown that the initial oocyte pool determines both ANM and oocyte aneuploidy (Brook et al., 1984; Nelson and Felicio, 1986). Surgical depletion of primordial follicles is associated with progressively earlier times of ovarian senescence and accelerated aneuploidy onset relative to the size of the ovarian reserve. These experiments suggest that factors such as DNA damage (discussed below), ovulation frequency and pathways controlling follicular attrition, which determine the size of the ovarian reserve, can directly influence oocyte chromosome segregation and reproductive outcomes. However, the molecular links between reduced follicle count and chromosome aneuploidy, if any, are still unknown. It may be worthwhile to revisit such mouse models with earlier onset of ovarian senescence in order to ascertain whether chromosome aneuploidy is directly related to a decreased ovarian reserve size, independent of natural ageing.",Human Reproduction,Reproductive Aging,2022
"DOR, POI, and Genetic Determinants of Ovarian Reserve","Women with either diminished ovarian reserve (DOR) or premature ovarian insufficiency (POI) have reduced follicle count in common, despite showing disparate clinical symptoms (Studer et al., 1984; Greene et al., 2014; Practice Committee of the American Society for Reproductive Medicine, 2020). Both conditions are associated with multiple genetic factors that affect the quantity of oocytes, identified through whole-exome sequencing (Jaillard et al., 2020; Tang and Yu, 2020), but whether the quality of the remaining oocytes is affected in terms of preserving segregation fidelity, is unknown. An attractive option to parse out the molecular links between the follicle counter mechanism and oocyte quality is to leverage the myriad genetic determinants of DOR and/or POI identified from GWA studies (Chon et al., 2021). These studies identified SNPs in genes associated with DNA repair (BRCA1, BRCA2, CHEK2), meiosis (SYCE1, STAG3) and the different pathways controlling oocyte growth and folliculogenesis (GDF9, FIGLA; Qin et al., 2015). As of now, the field uses aged mouse models to study the links between reduced ovarian reserve and oocyte chromosome segregation fidelity. Studying aged animals has the disadvantages of having limited material resulting in reduced statistical power, other detrimental effects from chronological ageing that confound physiological ageing defects, expense and labour intensity.",Human Reproduction,Reproductive Aging,2022
Mouse Models of Premature Ovarian Senescence,"Utilizing mouse models of candidate genes known to induce premature ovarian senescence, provides the advantage of assessing the detrimental effects of physiological ageing on oocyte segregation fidelity in an otherwise young female. Such models can decouple physiological ageing defects from chronological ones, do not require long experimental times, and provide valuable insight into molecular pathways governing premature ovarian senescence.

Harnessing the power of genomics in organisms that lack reproductive senescence also represents an exciting future direction for the field that could uncover the molecular components of the follicle counter mechanism. One such organism is the mammal, the naked mole rat, whose social colonies contain both reproductive (queen) and non-reproductive females. The females suppress ovulation until they are designated to become the queen after which they are fertile for their entire lifespan (Buffenstein, 2005). They also carry a large ovarian reserve (Place et al., 2021), which may contribute to their extended fertility, suggesting that multiple pathways involving both ovulation suppression and large follicle reserves may be required to extend their reproductive potential.",Human Reproduction,Reproductive Aging,2022
Comparative Genomics and the Naked Mole Rat,Future efforts on comparative genomics between reproductive (queen) and non-reproductive females can provide novel genetic targets specific to the queen that are crucial for extending the reproductive lifespan.,Human Reproduction,Reproductive Aging,2022
DDR Pathways and the Oocyte Quality Control Checkpoint,"Oocyte quality control checkpoint model
The total number of follicles in human females peaks at 20 weeks of gestation, after which the number dramatically reduces at birth to 300000–400000 follicles, which are then cyclically recruited upon menarche (Block, 1953). Menopause is initiated when this number declines over time to ∼1000 (te Velde and Pearson, 2002). Recent genomics studies have revealed an essential role for DNA damage repair (DDR) proteins such as BRCA1 and HELB, in regulating the ANM (Day et al., 2015; Ruth et al., 2021). Notably, ANM-associated mutant variants were also found in seven binding partners of BRCA1 suggesting that homologous recombination repair is pivotal for ovarian ageing. However, variants in mismatch repair (MSH5, MSH6) and base excision repair (APEX1 and PARP2) proteins, were also represented in the data set as being linked with ANM (Day et al., 2015). These proteins constitute a canonical surveillance mechanism that detects genome instability and activates the DNA damage checkpoint at the G2/M boundary of the cell cycle (Gould and Nurse, 1989). It is not surprising that oocytes, which are arrested in G2, have co-opted DDR proteins to monitor genome integrity. Multiple lines of evidence support a critical role of the DDR pathway in oocyte quality control and reproductive ageing.",Human Reproduction,Reproductive Aging,2022
Checkpoint-Controlled Atresia and CHEK1/CHEK2 Mechanisms,"First, DNA damage checkpoint proteins control the process of follicular atresia thus regulating both follicle count and oocyte quality. Indeed, Chek2 knock-out mice had lower rates of ovarian reserve depletion and responded with higher numbers of oocytes following hormonal stimulation in comparison to the wild-type aged controls (Ruth et al., 2021), consistent with the role of Chek2 in culling mouse oocytes with damage (Bolcun-Filas et al., 2014; Tuppi et al., 2018). However, Chek2 knock-out females were fertile with equivalent litter sizes to controls, suggesting that the DNA damage that CHEK2 responds to while inducing atresia does not irreversibly damage the genome. This suggests that oocytes that are normally culled are not necessarily poor quality oocytes. It remains a mystery as to why the DDR checkpoint eliminates such oocytes and what the selection criteria is. On the other hand, Chek1 overexpression led to an increased ovarian reserve at birth which delayed the time of onset of ovarian senescence in mice (Ruth et al., 2021).",Human Reproduction,Reproductive Aging,2022
"DDR Gene Dosage, POI, and Progeroid Syndromes","Second, reduced dosage of DDR proteins (e.g. FANCA and BRCA1) is linked to POI, indicating that proteins in this pathway impact physiological ageing. Indeed, FANCA hypomorphs and BRCA1 deficient mice show reduced follicle count and decreased reproductive potential (Lin et al., 2017; Pan et al., 2021). Women carrying BRCA1 mutations also show an accelerated decline in primordial follicles compared to a control group with no mutations, consistent with a depleted ovarian reserve. How DNA damage repair proteins regulate follicle count is unknown, but as stated above, utilizing these models represent an excellent method of studying ovarian senescence and its relation to biological events inside the oocyte.

Third, other rare genetic progeroid (premature-ageing like) syndromes are all caused by mutations in DDR proteins, including but not limited to the RecQ helicases, required for DNA replication, repair and recombination, transcription coupled repair (ERCC6, ERCC8) and inter-crosslink repair (FANCA, FANCC, FANCG) (Schumacher et al., 2021). Hence, DNA damage is a central theme in the process of ageing, and oocytes are particularly susceptible to it due to long periods in a dormant state during which genome instability can accumulate. Altogether, the DDR pathway, as the acting sentinels of the genome, may represent the unifying link between oocyte quality and age.",Human Reproduction,Reproductive Aging,2022
Evolutionary Explanations for Oocyte Attrition,"It is still mysterious why mammals have evolved to generate large numbers of oocytes during oogenesis just to have massive amounts of attrition early in life. One possibility is that somehow the process of oogenesis itself creates oocytes that may contain DNA damage requiring attrition and quality control. Otherwise, it is hard to understand how widespread apoptosis in the ovary could be beneficial. This is especially perplexing given that oocyte chromosome segregation is error prone as well. The only way to reconcile the damaging and error prone processes of oogenesis and oocyte chromosome segregation with their essential functions in ensuring species continuity, is to assume it confers an evolutionary advantage. One speculation is that these inherently vulnerable processes are an evolutionary adaptation that makes them susceptible to physiological ageing. This would either allow females a post-reproductive phase where they can care for the next generation’s progeny (i.e. the ‘grandmother’ hypothesis) or offset the morbidity risks of late-life reproduction (i.e. the ‘mother’ hypothesis); although both are debatable (Croft et al., 2015).",Human Reproduction,Reproductive Aging,2022
Epigenetic Clocks and Biomarkers for Reproductive Aging,"Epigenetic age acceleration: opportunities and challenges for developing predictive tools for reproductive and post-reproductive ageing
Establishing a non-invasive tool that can measure the effect of anti-ageing interventions on female reproduction (reviewed in Tesarik et al. (2021)) or monitor the rate of ovarian reserve exhaustion under the influence of external insults and environmental toxicants would be of great value given the inaccessibility of ovarian tissue. In mice, an epigenetic clock based on 90 CpG methylation profiles from blood (Petkovich et al., 2017) can predict biological age and the effects of environmental factors on longevity (e.g. caloric restriction). In humans, pan-tissue and blood-based epigenetic clocks have been developed as a biomarker of chronological age based on DNA cytosine-5′ methylation level measurements at specific CpGs (Hannum et al., 2013; Horvath, 2013). Expanding on these DNA methylation clocks, estimators were developed to predict lifespan (time to death for all-cause mortality) after adjusting the regression model for risk factors and chronological age (Levine et al., 2018; Lu et al., 2019; McCrory et al., 2021).",Human Reproduction,Reproductive Aging,2022
Epigenetic Age Acceleration and Menopause,"How do the epigenetic clocks perform in predicting ovarian age? The Horvath pan tissue epigenetic clock concept, which is based on estimating the relative methylation levels of 353 CpG islands, was implemented in whole blood cells to interrogate possible correlations between menopause and biological ageing as predicted by epigenetic age (Levine et al., 2016). Meta-analysis on the blood methylation levels from three cohorts (WHI, InCHIANTI and PEG) uncovered a strong association between epigenetic age acceleration and earlier onset of menopause (Levine et al., 2016). Menopause was also found to accelerate epigenetic ageing in blood, indicating that the associated endocrine changes during the post-reproductive period could affect epigenetic age of other tissues, thus inducing ageing (Levine et al., 2016). The same concept was implemented to construct epigenetic clocks on human white blood cell and cumulus cell (CC) methylation profiles from a cohort of poor and good responders to ovarian stimulation (Hanson et al., 2020). Blood epigenetic age acceleration estimates were statistically significant between poor and good responders when participants <38 years old were analysed but not when all samples were included in the analysis. There was no association between poor and good responders with the epigenetic age acceleration estimates on CC methylomes.",Human Reproduction,Reproductive Aging,2022
Cross-Species Epigenetic Clocks and Oocyte Aging,"Importantly, the CC methylome-based epigenetic clock did not associate with chronological age, which most likely can be attributed to the different mechanisms defining age in the follicular cells (oocytes and CCs). Further work from the Horvath group, exploited bovine oocytes to test whether epigenetic age estimates from the blood methylome parallel those measured by the epigenetic clock in oocytes and whether bovine oocyte epigenetic age can be used to model human oocytes (Kordowitzki et al., 2021). However, the correlation between DNA methylation ageing was low between oocytes and blood and could not be captured by the targeted array-based CpG methyl profiling (Kordowitzki et al., 2021). It is also worth noting that single-cell sequencing in mouse oocytes, revealed that although global CpG methylation was modestly reduced in old compared to young oocytes, the characteristic oocyte methylation patterns and imprinted regions were unaffected by age (Castillo-Fernandez et al., 2020). Therefore, a relationship between earlier age at menopause and accelerated epigenetic ageing, and whether the epigenetic clocks could predict the time to reproductive senescence is unresolved. It remains to be seen whether an epigenetic clock developed in one species can effectively be translated to another. A longitudinal study of epigenetic ageing before and after menopause would provide insights into the association of epigenetic age acceleration/deceleration with the age at the onset of menopause.",Human Reproduction,Reproductive Aging,2022
Cell-Type Specific Methylation and Granulosa Cell Clocks,"Given the differences between somatic and germline cells comprising a follicle, prior to implementing the statistical and mathematical concepts of the epigenetic clocks, comprehensive methylation studies to identify the differentially methylated regions (DMRs) that capture the effect of age in cell type-specific methylomes are needed. In this direction, Olsen and colleagues developed a mural granulosa cell (MGC) specific epigenetic clock (Granulosa Cell Clock), using 296 CpGs defined for this somatic ovarian cell type (Olsen et al., 2020). The granulosa clock performed well in predicting MGC chronological age, in contrast to the 353 CpG pan-tissue clock. In line with the prediction of different methylation patterns between ovarian and non-ovarian somatic tissues, the age-associated MGC DMRs were not reproducible in leucocytes. Genes with strong age-associated MGC DMRs and a lack of association with non-ovarian somatic tissues, include VTRNA2-1, AMH, ZFP57, PGF, GHSR, GHR and GAPDH, all of which have important roles in folliculogenesis (summarized in Olsen et al. (2020)), suggesting that MGCs age differently to non-ovarian somatic cells in the human body. MGCs play a pivotal role in folliculogenesis and in interactions with the oocyte, therefore further knowledge about ageing of the MGC may provide important insights into ageing of the oocyte itself (Olsen et al., 2020). Taken together, the findings of that study, strengthen the hypothesis that MGCs age differently to non-ovarian somatic cells in humans.",Human Reproduction,Reproductive Aging,2022
Single-Cell Transcriptomics and Predictive Ovarian Aging Tools,"Single-cell transcriptomic studies emphasize the role of understanding tissue heterogeneity in regulating functional heterogeneity through diverse cellular programs in the ovary (Fan et al., 2019; Wagner et al., 2020). Further dissection of these programs at temporal and spatial resolution across developmental and follicular stages and their association with methylome changes will (i) advance our understanding of ovarian remodelling in the context of ageing and (ii) create an opportunity for the development of a predictive tool based on ovarian DMRs. It would still have to be associated with peripheral blood signatures to render it non-invasive.",Human Reproduction,Reproductive Aging,2022
Overview of Skin Aging Mechanisms,"Skin aging is a multifaceted process that involves intrinsic and extrinsic mechanisms that lead to various structural and physiological changes in the skin. Intrinsic aging is associated with programmed aging and cellular senescence, which are caused by endogenous oxidative stress and cellular damage. Extrinsic aging is the result of environmental factors, such as ultraviolet (UV) radiation and pollution, and leads to the production of reactive oxygen species, ultimately causing DNA damage and cellular dysfunction. In aged skin, senescent cells accumulate and contribute to the degradation of the extracellular matrix, which further contributes to the aging process. To combat the symptoms of aging, various topical agents and clinical procedures such as chemical peels, injectables, and energy-based devices have been developed. These procedures address different symptoms of aging, but to devise an effective anti-aging treatment protocol, it is essential to thoroughly understand the mechanisms of skin aging. This review provides an overview of the mechanisms of skin aging and their significance in the development of anti-aging treatments.",Frontiers in Physiology,Skin Aging,2023
Introduction to Dermal Aging and Clinical Approaches,"1 Introduction
Skin aging is a complex process that involves numerous biological and biochemical changes as well as secondary structural changes of the skin, underlying muscles, subcutaneous fat tissue, and bony structures. Common aesthetic procedures performed in clinical practice, such as chemical peels, energy-based treatments, injectable treatments, and threads, may share similar mechanisms; however, they often address the symptoms and signs of skin aging in distinct ways. Furthermore, as research on the mechanism of skin aging continues to expand, existing theories are replaced with new concepts, such as cellular senescence of dermal fibroblasts (Fang et al., 2022; Shvedova et al., 2022). Thus, clinicians must possess a thorough comprehension of skin aging physiology to devise a treatment plan that entails selecting anti-aging procedures that target specific mechanisms of skin aging while simultaneously reducing side effects. It is hoped that this narrative review will provide new avenues to comprehensively describe the complex skin aging process and help clinicians to establish anti-aging treatment protocols.",Frontiers in Physiology,Skin Aging,2023
Publication Details,"OPEN ACCESS
EDITED BY
Dong Hun Lee, Seoul National University Hospital, Republic of Korea
REVIEWED BY
Si-Hyung Lee, Seoul National University, Republic of Korea
Abigail Langton, The University of Manchester, United Kingdom
Jung Won Shin, Seoul National University Bundang Hospital, Republic of Korea
*CORRESPONDENCE
Kui Young Park, kyky@cauhs.or.kr
RECEIVED 28 March 2023
ACCEPTED 02 May 2023
PUBLISHED 10 May 2023
CITATION
Shin SH, Lee YH, Rho N-K and Park KY (2023), Skin aging from mechanisms to interventions: focusing on dermal aging. Front. Physiol. 14:1195272. doi: 10.3389/fphys.2023.1195272
COPYRIGHT © 2023 Shin, Lee, Rho and Park. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.",Frontiers in Physiology,Skin Aging,2023
Intrinsic and Extrinsic Drivers of Skin Aging,"2 Mechanisms of skin aging
2.1 Molecular mechanisms of skin aging
The skin is the body’s largest organ and is continuously exposed to various environmental factors, including ultraviolet (UV) rays, smoking, heat, and air pollution. Therefore, the skin undergoes extrinsic aging as well as intrinsic aging, which is also referred to as chronological aging. The process of intrinsic aging can be considered alongside programmed aging, and it results from continuous chromatic damage by various factors, of which the most representative is oxidative stress caused by reactive oxygen species (ROS). The cells have an endogenous defense system against oxidative stress including superoxide dismutase (SOD), tripeptide glutathione, and catalase (Steenvoorden and van Henegouwen, 1997). Age-related impairment in its redox capacity results in the accumulation of ROS, thereby causing a detrimental effect on cellular components including proteins, lipids, and DNA, consequently leading to cellular dysfunction (Gniadecka et al., 1998; Gu et al., 2020). ROS generated by exogenous factors such as UV rays and air pollution also play a significant role in extrinsic aging.",Frontiers in Physiology,Skin Aging,2023
Cellular Senescence and SASP in Skin Aging,"In response to stress factors including DNA damage, cells enter a state of irreversible growth arrest, which is called cellular senescence (Hayflick, 1965). Recent research has uncovered that cellular senescence plays a major role in the skin aging process (Fitsiou et al., 2021; Wlaschek et al., 2021; Kim et al., 2022a; Kim et al., 2022b; Gerasymchuk et al., 2022; Papaccio et al., 2022). Senescent cells exhibit several biomarkers: 1) increased activity of the cell cycle arrest proteins p21WAF1 and p16INK4A, 2) lysosomal enzyme senescence-associated β galactosidase (SA-β-gal), and 3) decreased expression of nuclear high mobility group box-1 (HMGB1) and lamin B1, a structural component of the nuclear lamina (Ho and Dreesen, 2021). They also release humoral factors known as senescence-associated secretory phenotype (SASP), which includes various inflammatory cytokines, chemokines, matrix proteases, and microRNAs (Coppe et al., 2010; Kim et al., 2016). The temporary cellular senescence signals that physiologically occur during wound healing promote the formation of granulation tissue and skin regeneration while inhibiting excessive cell growth that can progress to precancerous or cancerous lesions (Demaria et al., 2014; Wang and Dreesen, 2018). As age increases, the accumulation of senescent keratinocytes, melanocytes, and, most importantly, fibroblasts can cause various age-related diseases and disrupt the homeostasis of the skin (Wlaschek et al., 2021).",Frontiers in Physiology,Skin Aging,2023
Extracellular Matrix Degradation and Inflammatory Drivers,"Moreover, the degradation of the extracellular matrix (ECM) is observed as a result of altered senescent cells and excessive ROS production. Excessive ROS activate the mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) pathway, which consequently induces the expression of matrix metalloproteinase (MMP), resulting in collagen breakdown (Chung et al., 2000). It also downregulates the collagen production via the TGF-β/Smad signaling pathway (Quan et al., 2004; Quan et al., 2010; He et al., 2014). In addition, tissue inhibitors of metalloproteinases (TIMPs) are downregulated during the aging process. Furthermore, the presence of senescent cells contributes to ECM degradation by promoting chronic inflammatory responses and collagen breakdown. In particular, the senescent fibroblasts express SASP containing MMP-2, MMP-9, and proinflammatory cytokines such as interleukin (IL)-6 and IL-8 (Kuilman et al., 2008; Wang and Dreesen, 2018; Wlaschek et al., 2021). The migration of neutrophils after inflammation or UV exposure further accelerates the collagen and elastin fragmentation via production of neutrophil-derived proteolytic enzymes (Li et al., 2013; Sharma et al., 2020).",Frontiers in Physiology,Skin Aging,2023
Clinical Features and Shared Mechanisms of Dermal Aging,"2.2 Dermal aging
These two types of skin aging have several overlapping molecular mechanisms including ROS generation, DNA damage, and structural deterioration of ECM components. Therefore, the clinical phenotypes of skin aging are similar in some respects; however, some facets differ based on the aging process (Park, 2022). Intrinsic aging results in overall thinning of the skin, dry and pale skin, fine wrinkles, and skin sagging with decreased elasticity (Walker, 2022). The functions of the sweat and sebaceous glands also decrease, with less sebum secretion caused by decreased peroxisome proliferator-activated receptor gamma (PPAR-γ) expression ultimately leading to dry skin, while sebaceous gland hyperplasia can occur due to increased gland size (Zouboulis and Boschnakow, 2001; Kim et al., 2014). Furthermore, extrinsic aging manifests as relatively coarse wrinkles, severe loss of elasticity, and dyspigmentation (Walker, 2022).
Despite these differences, biochemical and biophysical changes in the dermis are common to both aging processes and are major contributing factors to the aging phenotypes such as wrinkles and loss of skin elasticity. The dermis of the skin consists of connective tissue that is rich in collagen, which provides mechanical support and structure. Recently, the changes in the dermal components in skin aging and treatments to reverse or combat them to reduce the signs of aging have become the focus of many dermatologists. Therefore, in this part, we will discuss these dermal aging processes in more detail.",Frontiers in Physiology,Skin Aging,2023
"Fibroblast Senescence, CCN1, and ECM Remodeling","The fibroblasts within the dermis are responsible for the synthesis, organization, and remodeling of collagen and thus play a major role in maintaining the integrity of the ECM. As aforementioned, aging causes the accumulation of senescent fibroblasts in the dermis, which causes gradual degradation and dysfunction of the ECM via release of proteolytic, matrix-degrading SASPs (Ressler et al., 2006).
In particular, the matricellular protein CCN1, also known as cysteine-rich protein 61, has been suggested to be a contributor to the age-associated dermal microenvironment. CCN1 is markedly elevated in the human dermal fibroblasts in aged skin, and Quan et al. demonstrated that elevated expression of CCN1 accelerates dermal aging by dysregulating the production and homeostasis of collagen using a transgenic mouse model (Quan et al., 2011; Quan et al., 2021). Their results showed that the fibroblasts of COL1A2-CCN1 mice had increased MMP expression and impaired TGF-β/Smad signaling, resulting in reduced COL-1 expression and fragmentation of ECM. Furthermore, CCN1 induces increased expression of proinflammatory cytokines, thus further promoting dermal aging (Quan et al., 2011).",Frontiers in Physiology,Skin Aging,2023
Senescent Fibroblast Paracrine Effects and Dermal Structure Changes,"In addition, Ezure et al. found that complement factor D secreted from senescent dermal fibroblasts induces increased MMP-1 expression and negatively impacts matrix production in surrounding young dermal fibroblasts in vitro (Ezure et al., 2019). Collectively, these changes disrupt the complex interaction of dermal fibroblasts with the ECM, including by reducing the mechanical forces exerted on the fibroblasts, which negatively affects their morphology and function (Qin et al., 2014; Fisher et al., 2016).
In addition, Solé-Boldo et al. (2020) found that there is decrease in the number and heterogeneity of dermal fibroblasts with age. These skin aging-associated changes were mainly observed in the papillary dermis rather than in the reticular dermis with decreased papillary dermal fibroblasts (Mine et al., 2008). Collectively, these changes impair the structure and function of the skin and create a microenvironment that is conducive to age-related skin pathologies, including delayed wound healing and skin cancer (Woodley, 2017; Blair et al., 2020; Fane and Weeraratna, 2020; Xue et al., 2022).",Frontiers in Physiology,Skin Aging,2023
Fibroblast–Keratinocyte–Melanocyte Interactions and Aging,"Furthermore, senescent fibroblasts contribute to skin aging by interacting with other neighboring cells, including keratinocytes and melanocytes, through paracrine signaling. Insulin-like growth factor (IGF)-1, which is mainly released by dermal fibroblasts, is known to be necessary for mesenchymal stem cell niches and the modulation of epidermal cell proliferation and differentiation (Hodak et al., 1996; Youssef et al., 2017; Muraguchi et al., 2019). In addition, IGF-1 signaling is essential for the appropriate protective responses (DNA damage response, DDR) of keratinocytes to UV-induced DNA damage by inducing favorable cellular senescence or DNA damage repair (Lewis et al., 2009; Loesch et al., 2016; Alkawar et al., 2020). Aged skin exhibits decreased synthesis of IGF-1, which results in the epidermal atrophy and proliferation of keratinocytes with unrepaired DNA, leading to the development of age-related non-melanoma skin cancers (Stachelscheid et al., 2008; Lewis et al., 2009; Alkawar et al., 2020; Wlaschek et al., 2021).",Frontiers in Physiology,Skin Aging,2023
"Senescent Fibroblast EVs, Pigmentation, and Photoaging Changes","More recently, Terlecki-Zaniewicz et al. (2019) showed that extracellular vesicles derived from the senescent fibroblasts affect the terminal differentiation of keratinocytes with decreased expression levels of involucrin in a 2D cell culture model, which is reported to be a major initiator of cornification. Senescent fibroblasts have also been suggested to contribute to age-related pigmentation by inducing activation of melanocytes through several factors such as secreted frizzled-related protein 2, growth differentiation factor 15, and stromal-derived factor 1 (Kim et al., 2016; Yoon et al., 2018; Kim Y. et al., 2020; Kim J. C. et al., 2022). This was also supported by reduced epidermal pigmentation after radiofrequency treatment with reduced p16INK4A-positive senescent fibroblasts in a pilot study by (Kim et al., 2019).
There are also age-related structural changes in the dermal elastic fibers. Elastic fiber networks are composed of elastin and fibrillin, forming a unique arrangement within the dermis. In the upper papillary dermis, oxytalan fibers, which are microfibrillar bundles abundant in fibrillin, play a role in preventing the epidermis from easily detaching from the dermo-epidermal junction (DEJ) by forming a candlestick-shaped organic bond with the DEJ (Heinz, 2021). In photoaged skin, the oxytalan fibers undergo degeneration, and the elastic fibers of upper dermis are degraded by elastolytic enzymes including MMPs and neutrophil elastases (Bernstein et al., 1996; Naylor et al., 2011; Bonta et al., 2013). In addition, the altered, disorganized elastic fibers gradually accumulate in the reticular dermis, appearing as solar elastosis.",Frontiers in Physiology,Skin Aging,2023
Intrinsic Elastic Fiber Depletion and Basement Membrane Aging,"In contrast, intrinsic skin aging is characterized by overall depletion of the elastic fiber network (El-Domyati et al., 2002).
Recently, it has been recognized that the basement membrane not only provides physical support for keratinocytes but also plays a major role in the regulation of signaling and communication between epidermal and dermal cells (Tsutsui et al., 2021). With age, the protein components of the basement membrane zone, including collagen 7 and 17, nidogen, integrins, and laminin 332, decrease, and the papillary pattern of the DEJ flattens (Iriyama et al., 2011a; Amano, 2016; Roig-Rosello and Rousselle, 2020). It has been postulated that disrupted basement membranes allow soluble melanogenic regulators from senescent fibroblasts to more easily stimulate melanocyte activity and accelerate age-related pigmentation (Goyarts et al., 2007; Amano, 2009; Iriyama et al., 2011b; Bastonini et al., 2016). Iriyama et al. (2022) have shown that the inhibition of basement membrane degradation with MMP inhibitors and heparinase inhibitors promotes the deposition of laminin-511 at the DEJ, which in turn promotes the secretion of platelet-derived growth factor consisting of 2 B subunits (PDGF-BB). Expression of COL5A1 and COL1A1 genes was increased in the fibroblasts stimulated with PDGF-BB, suggesting increased collagen expression in the papillary dermis (Iriyama et al., 2022).",Frontiers in Physiology,Skin Aging,2023
"Proteoglycans, GAGs, and Combined Intrinsic–Extrinsic Aging","Therefore, strengthening the damaged basement membrane and restoring epidermal-dermal integrity have been proposed as new anti-ageing targets, but the actual clinical significance of the DEJ and its role in aging requires much further research.
Age-related changes in proteoglycans (PGs) and glycosaminoglycans (GAGs) are very complex, and there are still many unknown aspects (Oh et al., 2011b; Lee et al., 2016). Although previous research has often reported conflicting results in the changes of PGs and GAGs, they have received attention as promising targets for skin rejuvenation (Oh et al., 2011a; Oh et al., 2011b; Lee et al., 2016; Wang et al., 2021). Unlike collagen, which has a relatively long half-life, GAGs, such as hyaluronic acid (HA), have a much shorter half-life ranging from 24 to 36 h in human skin (Jiang et al., 2007; Fallacara et al., 2018). While the regulation of collagen metabolism takes a long time to show any visible changes, GAGs have the advantage that a treatment effect can be observed within a short period of time. However, further follow-up studies are needed to understand the role of PGs and GAGs in skin aging.
Table 1 summarizes the differences between intrinsic and extrinsic aging of the skin that have been generally recognized to date. However, recent research suggests that this distinction is not as clear-cut as textbooks describe and can often be confusing. It would be more clinically appropriate to understand that middle-aged adults visiting dermatologic clinics for skin rejuvenation undergo concomitant intrinsic and extrinsic aging. Thus, the histological and molecular changes related to skin aging that have been acknowledged to date should be organized to establish an appropriate treatment plan.",Frontiers in Physiology,Skin Aging,2023
UV Protection and Fundamentals of Anti-Aging Care,"3 Management of skin aging: focus on dermal aging
3.1 UV protection
As mentioned above, UV rays play a critical role in cellular aging and skin aging; thus, Sun protection—using sunscreen or protective clothing and staying in the shade—is the most basic and essential option for preventing skin aging and slowing the rate of aging-related changes.",Frontiers in Physiology,Skin Aging,2023
Energy-Based Devices for Dermal Rejuvenation,"3.2 Energy-based devices
Various energy-based devices, such as lasers, high-intensity focused ultrasound (HFU), and radiofrequency (RF) devices, have grown increasingly common to address aging phenotypes. These devices deliver thermal energy to the reticular dermis and subcutaneous tissue, which subsequently causes tissue contraction and stimulates neocollagenesis, leading to improvement in skin laxity and rhytides (Orringer et al., 2012; Majidian et al., 2021; Chen et al., 2022).
An ablative laser, such as a CO2 laser or an Erbium:YAG laser, which requires re-epithelialization, has been used in the past, but recently, a non-ablative fractional laser has been used mainly to reduce the downtime and risk of adverse events including postinflammatory hyperpigmentation or scarring (Nanni and Alster, 1998; Chen et al., 2022). In contrast, fractional picosecond lasers produce nonthermal, photomechanical stress in the dermis and promote fibroblast proliferation (Tanghetti, 2016; K et al., 2021).",Frontiers in Physiology,Skin Aging,2023
"Laser Modalities, HFU, RF, and LED Therapies","Recent ex vivo animal and clinical studies also support that 532-nm and 1,064-nm picosecond Nd:YAG lasers may improve photoaged skin (Yim et al., 2020; Connor et al., 2021; Han et al., 2023). In addition, various lasers including low fluence Q-switched Nd:YAG lasers, Q-switched ruby lasers, and Q-switched alexandrite lasers are effective for treating aging-related pigmentation through selective photothermolysis of melanosomes (Anderson and Parrish, 1983; Sadighha et al., 2008; Vachiramon et al., 2016).
HFU is a noninvasive and safe treatment that focuses ultrasound waves on a localized area, much like a magnifying glass focuses light, causing thermal coagulation of the subcutaneous tissue and rearranging the collagen and elastic fibers of the subcutaneous tissue without affecting the skin surface. In contrast, RF devices deliver relatively diffuse thermal energy throughout the dermis (Suh et al., 2015). Kwon et al. (2021) showed that bipolar RF device treatment reduces the number of p16INK4A-positive senescent fibroblasts and increases the expression of HSP70 and HSP90 in melasma skin. More recently, fractional RF microneedling devices that deliver targeted bipolar RF energy directly to the reticular dermis via microneedles have been developed. Fractional RF microneedling devices have also shown to be effective in treating UV-induced hyperpigmentation by upregulating the antisenescence pathways (Rangarajan et al., 2013; Yoon et al., 2018; Lee et al., 2021).
Furthermore, recent evidence suggests that a light emitting diode (LED) can also ameliorate UV-induced changes in dermal fibroblasts and promote collagen synthesis by photobiomodulation (Baez and Reilly, 2007; Kim et al., 2015; Mamalis and Jagdeo, 2018; Hong et al., 2022). The mechanisms underlying the effects of LEDs aren’t fully understood, and clinical data are insufficient; therefore, further studies are needed.",Frontiers in Physiology,Skin Aging,2023
"Topical Retinoids, Antioxidants, Peels, and Emerging Therapies","3.3 Topical agents
A variety of topical agents have been used to improve the signs of skin aging, but retinoids are currently considered the most effective option (Samuel et al., 2005). Retinoids have been shown to increase types Ⅰ, III, and VII collagen and GAG deposition and to normalize elastic tissue organization (Woodley et al., 1990). In addition, topical tretinoin treatment also induces thickening of the granular layer and compaction of the stratum corneum, resulting in smooth skin (Berardesca et al., 1990). Clinical evidence also supports the role of topical retinoids in the reversal of skin aging phenotypes including fine wrinkling, dyschromia, and skin elasticity (Weinstein et al., 1991; Olsen et al., 1992; Darlenski et al., 2010; Milosheska and Roskar, 2022).
Topical antioxidants, such as ascorbic acid (vitamin C), have also been shown to be effective in reducing skin aging. Ascorbic acid reduces ROS and is required for collagen synthesis in human skin fibroblasts. However, its poor skin penetration and chemical instability can reduce its clinical efficacy.
In addition, chemical peeling using topical alpha-hydroxy acids, such as glycolic or lactic acid, have been shown to improve the quality of elastic fibers, stimulate GAG and collagen production in the dermis, and increase the epidermal thickness (Bernstein et al., 2001; Hussein et al., 2008). Tricholoroacetic acid peels also have been shown to promote neocollagenesis and improve benign pigmented lesions (Kitzmiller et al., 2003; Chun et al., 2004).
The development of new formulations through advances in nanotechnology and drug delivery systems is expected to further increase the use of topical agents as well as cosmeceuticals. Microneedling with a dermaroller has also been used to enhance drug delivery by creating pores in the stratum corneum, promoting neocollagenesis through release of various growth factors during the micro-wound healing process (Hou et al., 2017). Similarly, microdermabrasion using aluminum oxide crystals has been shown to be effective in improving the drug delivery and promoting dermal collagen synthesis. Recently, there is increasing evidence that stem cell-derived exosomes can ameliorate aging-related changes including UV-induced DNA damage and ROS generation and MMP-1 expression in senescent fibroblasts, and promote the expression of ECM proteins (Oh et al., 2018; Gao et al., 2021). The autologous stromal vascular fraction extracted from adipose tissue-derived stem cells has also shown to be effective in dermal rejuvenation due to its regenerative capacity (Charles-de-Sa et al., 2015; Rigotti et al., 2016). Still, clinical data are still insufficient, and further studies are needed to elucidate the anti-aging effects of exosomes and the stromal vascular fraction.",Frontiers in Physiology,Skin Aging,2023
Hyaluronic Acid and Biostimulatory Injectables,"3.4 Injectables
The use of injectables in the dermatologic field has been increasing to improve rhytides and restore the soft tissue volume in aged skin. HA is one of the most commonly used injectables available due to its biocompatibility, ease of use, and reversibility. The injection of HA causes the dermis to stretch mechanically and enhances the structural support of the ECM, which activates dermal fibroblasts and leads to the production of type I collagen by activating the TGF-β signaling pathway (Wang et al., 2007; Turlier et al., 2013; Landau and Fagien, 2015). In addition, HA directly activates fibroblasts through its hyaluronan receptors, CD44 and CD168, causing them to migrate and proliferate (Mast et al., 1993; Turley et al., 2002). HA injection is also effective in improving skin hydration and texture (Ayatollahi et al., 2020).
Recently, a novel EGF-containing HA filler was shown to induce types I and III collagen production and downregulate the expression of MMP-9 (Shin et al., 2022). In addition to HA, biocompatible polymers such as poly-L-lactic acid, polycaprolactone, and polynucleotide have also been found to stimulate fibroblasts and induce neocollagenesis and are thus increasingly used as injectables (Park et al., 2016; Kim J. H. et al., 2020; Oh et al., 2021). Furthermore, botulinum toxin injection not only overcomes hyperkinetic rhytides but also improves skin elasticity, skin hydration level and decrease skin erythema via suppression of neurogenic inflammation (Gazerani et al., 2009; Zhu et al., 2017). Table 2 summarizes the mechanisms of skin aging and corresponding dermatological interventions.",Frontiers in Physiology,Skin Aging,2023
Senotherapeutics and Future Anti-Aging Strategies,"3.5 Future perspectives
In very recent decades, researchers have attempted to counteract aging using senotherapeutics that selectively target senescent cells. Senotherapeutics are categorized into two groups. Senolytic drugs selectively eliminate senescent cells, and senomorphic drugs inhibit the negative effects of their SASPs. Since the combination of dasatinib and quercetin was proposed as the first senolytic drug to suppress genes that are increased in senescent cells, many studies have shown that various substances such as ABT-737, ABT-263, A1155463, and fiestin have anti-aging properties (Zhu et al., 2015; Thompson et al., 2022).
In particular, ABT-263 and ABT-737, which are Bcl-2 inhibitors, have been found to selectively eliminate SA β-gal-positive senescent cells in skin both in vitro and ex vivo (Victorelli et al., 2019; Kim et al., 2022a; Kim et al., 2022b; Park et al., 2022). Kim and his colleagues demonstrated that either ABT-263 or ABT-737 treatment selectively eliminated dermal fibroblasts in an intrinsic skin aging mouse model (Kim et al., 2022a). They also showed that the treatment increased the collagen density, epidermal thickness, and keratinocyte proliferation while reducing SASPs including MMP-1 and IL-6.
After, this team revealed that treatment with ABT-263 and ABT-737 also attenuated the induction of MMPs and decreased collagen density in the photoaging mouse model (Kim et al., 2022b). In addition, ABT-263 showed potential in reducing pigmentation caused by photoaging in human skin inducing apoptosis of p16INK4A-positive fibroblasts with its senolytic activity, resulting in decreased levels of melanin and tyrosinase activity (Park et al., 2022).",Frontiers in Physiology,Skin Aging,2023
mTOR Pathway Inhibition and Rapamycin in Skin Aging,"One of the most notable targets of senomorphic agents is the mechanistic/mammalian target of rapamycin (mTOR) pathway, which regulates cellular metabolism and is linked to cellular growth, proliferation, and autophagy (Papadopoli et al., 2019). The mTOR pathway is also involved in the synthesis of SASPs (Cayo et al., 2021). Rapamycin, an mTOR inhibitor, exhibited significant reduction in senescence markers and SASPs as well as oxidative cellular stress in UV-induced photoaged human dermal fibroblasts (Bai et al., 2021).
Moreover, Chung et al. revealed the potential anti-aging effect of topical application of rapamycin (an mTOR inhibitor) (Chung et al., 2019). A total of 17 subjects over the age of 40 years with age-related photoaging of the skin applied a rapamycin-containing hand cream to the dorsum of one hand and a placebo hand cream to the other hand daily for 8 months and found that the rapamycin-treated hand had a decrease in p16 and an increase in collagen VII protein.
Taken together, these promising results suggest that senotherapeutics may be a novel therapeutic option for skin aging; however, the limitations of these drugs, such as their specificity, selectivity, and efficiency, still need to be addressed, and their mechanisms of action and side effects must be better understood.",Frontiers in Physiology,Skin Aging,2023
Introduction to Skin Aging and UV Radiation,"Skin aging involves internal and external processes. Changes occurring as a result of genetic conditions (internal, chronological aging) overlap with aging symptoms stimulated by environmental conditions (extrinsic aging). The most harmful external factor threatening the skin is ultraviolet (UV) radiation. UV radiation consists of three components: UVA (λ = 320−400 nm), UVB (λ = 280−320 nm) and UVC (λ = 100−280 nm). UVC radiation, unlike UVA and UVB radiation, is almost completely absorbed by the ozone layer. UVA and UVB rays reach the earth in sufficient quantities to damage skin structures. Nevertheless, the negative impact has been also observed during skin exposure to infrared radiation (IR; λ = 760 nm−1 mm). IR penetrates deeper layers of the skin than the rest of optical radiation. Even up to 17% of the incident infrared light can directly penetrate into the subcutaneous tissue. IR is absorbed by tissue chromophores (e.g., water) and converted into heat so that deep tissues can be heated. The heat is then transferred deeper by conduction, and pathological changes such as skin and corneal burns can occur.",Journal of Cosmetic Dermatology,UV Radiation and Photoaging,2021
Skin Defense Mechanisms Against UV Exposure,"The human skin, an important part of the innate immune system, has various molecular mechanisms that protect this organ from UV exposure. The first of these is the layered structure of the epidermis, which provides the first line of defense against harmful external agents. Additionally, immune cells such as Langerhans cells and T lymphocytes are located within the skin. Another line of protection for the skin is the melanocytes. Melanin, a pigment synthesized by these cells, impedes the penetration of UV radiation into the living layers of the epidermis by absorbing it. Furthermore, to maintain homeostasis, UV-induced DNA damage can be repaired at the molecular level by nucleotide repair and base excision mechanisms or apoptotic mechanisms as well as cell cycle checkpoints are activated.",Journal of Cosmetic Dermatology,UV Radiation and Photoaging,2021
Clinical Consequences of UVA and UVB Exposure,"UV radiation increases the risk of long-term damages such as photoaging, photoimmunosuppresion, and photocarcinogenesis. UVA radiation has its negative effect on the epidermal keratinocytes and dermal fibroblast and induces long-term changes. Changes arising as a result of UVB radiation are visible mainly within the epidermis but it also penetrates the upper part of dermis. The harmful effects of ultraviolet exposure mainly include skin side effects such as sunburn, photodermatoses, hyperpigmentation, photoaging of the skin and precancerous lesions and cancers. The mechanisms discussed in this paper are involved in the formation of these clinical changes in the skin.",Journal of Cosmetic Dermatology,UV Radiation and Photoaging,2021
"Oxidative Stress, ROS, and ECM Damage in Photoaging","Common point of photoaging is less dermal fibers expression. Overexposure to UV radiation increases the formation of reactive oxygen species (ROS), which at higher concentrations can damage the main proteins that make up the skin, collagen and elastin. A characteristic feature of the skin affected by photoaging is the presence of solar elastosis in the dermis. Solar elastose is a dystrophic elastic material that is formed as a result of a cycle of processes leading to the degradation of elastic fibers, followed by the formation of the extracellular matrix (ECM) and its reconnection into a structure other than its original one.",Journal of Cosmetic Dermatology,UV Radiation and Photoaging,2021
Historical Context and Aim of Photoaging Research,"The first report about the modulating influence of UV radiation on the progress and formation of some dermatoses is dated on 1910. Since then, there has been a lot of research to find out the relationship between solar radiation and skin lesions, both disease and esthetic. This review focuses on gathering information about photoaging, especially the impact of UV radiation on skin cells metabolism, formation of oxidative stress and modulation of skin enzymes. The information collected in this review about the mechanisms that are occurring in skin cells under the influence of UVA and UVB radiation may be helpful in the search for new, effective compounds that show protective activity on the skin and reduce the effects of photoaging.",Journal of Cosmetic Dermatology,UV Radiation and Photoaging,2021
UV Effects on Cellular Metabolism and DNA Damage,"UV radiation can penetrate into the skin and interact with skin cells, both fibroblasts and keratinocytes. Senescent cells secrete a number of factors such as cytokines, chemokines, growth factors and matrix metalloproteinases (MMPs), that is known as senescence-associated secretory phenotype. The formation of premutagenic photoproducts is dependent on the type and dose of UV radiation. Cyclobutane dimers Py (CPD) are mainly induced by UVB, while 8-hydroxy-2-deoxyguanine (8-OHdG) is one of the most common markers for the estimate of DNA damage from UVA. DNA damage is one of the most serious effect of excessive skin exposure to UV radiation and plays a major role in inducing photocarcinogenesis and is also directly involved in photoaging.",Journal of Cosmetic Dermatology,UV Radiation and Cellular Metabolism,2021
Mechanisms of UVA and UVB-Induced DNA Alterations,"The destructive mechanism of UVB and UVA action on DNA molecules differs, which is related to the amount of energy absorbed by base pairs in the DNA chain. The direct action of UVB radiation on cellular DNA results in characteristic mutations in the structure of the nucleic chain, such as the formation of CPD and pyrimidine base transverssions. DNA damage due to UVA radiation, like UVB, includes the formation of CPD, pyrimidine (6-4) pyrimidone photoproducts, as well as damage to and transition of DNA bases. Exposure to UVA causes direct damage of skin cells through an inflammatory reaction and indirectly through the induced oxidative stress. This initiates peroxidation of polyunsaturated fatty acids (PUFA) in the skin membrane and the formation of DNA adduct, 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is the most numerous and highly mutagenic factor, considered as a reliable marker for oxidative DNA damage.",Journal of Cosmetic Dermatology,UV Radiation and DNA Damage,2021
Inflammatory Pathways and nc886 Response to UV Exposure,"Under exposure to UVB radiation on the skin, an inflammatory response is triggered in keratinocytes, resulting in the activation of the protein kinase R signal transduction pathway, which blocks this signal transduction pathway. A long non-coding RNA, nc886, suppresses the signal transduction pathway with protein kinase R to protect the cell from UV radiation. Autophagy, which is an intracellular cleansing system, is essential for maintaining homeostasis in skin structures. In the case of skin aging, the basic level of autophagy increases during the replicable aging of human facial fibroblasts. Furthermore, UVA induce autophagy in fibroblasts and UVB in human keratinocytes. However, the process of autophagy is not capable of completely cancelling out the aging reaction of these cell types and only delaying it.",Journal of Cosmetic Dermatology,UV-Induced Inflammatory Signaling,2021
Autophagy Regulation Under UVA and UVB Stress,"ROS production induced by UV stimulates autophagy, which regulates the reaction to oxidative stress due to solar radiation. Exposure to UVA radiation results in an increase in the quantity of oxidized phospholipids, oxysterols and cholesterols in epidermal cells, which is a signal to induce autophagy in keratinocytes. Autophagy plays a multiple role in response to oxidative stress caused by UVA radiation by removing oxidized molecules while minimizing the antioxidative reaction in various cell types. It has been shown that UVA regulates the transcription of a certain of genes which take part in autophagy, for example adaptive protein p62, as well as autophagic activators p53 and Sestrin2 (SESN2) that can induce autophagy through 5’ adenosine monophosphate-activated protein kinase (AMPK) signalling.",Journal of Cosmetic Dermatology,Autophagy and UV Response,2021
UVA-Induced Autophagy Impairment and Lysosomal Dysfunction,"The results of an experiment conducted by Endo et al. (2020) showed that repeated UVA radiation negatively affects the autophagy process in fibroblasts due to modifications in lysosomal functioning. The impairment of intracellular degradation in UVA-treated fibroblasts occurs through molecular mechanisms underlying impaired autophagy such as decreased lysosomal acidity and reduced expression of cathepsins B, L and D. That suggests that anomalies in the process of autophagy are the leading agent in the process of photoaging of the skin. However, the basic dysfunctional mechanism of lysosomes in repeated UVA radiation fibroblasts is still not clear.",Journal of Cosmetic Dermatology,Lysosomal Dysfunction Under UVA,2021
UVB-Induced Sunburn Cells and Autophagy Activation,"Multiple exposition to UVB radiation causes an fail-safe process in the epidermis, which results in the forming of sunburn cells (SBC), that is, keratinocytes which undergo apoptosis. Damages of keratinocytes DNA caused by UVB leads to the release of signals which initiate the release of inflammatory response mediators, for example, cytokines IL-1α, IL-6, and TNF-α. UVB directly induces the AMPK autophagy activator, a gene associated with UV resistance (UVRAG) and p53. Stabilized by UVB p53, initiates transcription of AMPK, SESN2, tuberous sclerosis complex 2 (TSC2), and UVRAG for activation of autophagy.",Journal of Cosmetic Dermatology,UVB Response and Apoptosis,2021
ROS Formation and Molecular Damage Under UV Exposure,"One of the main effects of UV radiation on skin molecules (e.g., urocanic acid, nicotinamide-adenine dinucleotide (NADH) or melanin) is sensitization to ROS formation from absorbed energy. ROS (e.g., singlet oxygen, hydrogen peroxide, peroxide) are able to react and damage most of the molecules in their pathways, such as lipid membranes of cells, proteins or DNA. Moreover, ROS stimulate cell surface receptors, especially those for the epidermal growth factor (EGF), keratinocyte growth factor (KGF), interleukin (IL)-1, and tumor necrosis factor (TNF)-α. In addition, ROS causes damage to the membrane lipids, which leads to the release of ceramides and then the activation of AP-1.",Journal of Cosmetic Dermatology,UV-Induced Oxidative Stress,2021
UVA-Induced Inflammation and Lipid Metabolism Changes,"UVA radiation causes the release of prostaglandin-F2α (PGF2α) and 12-HETE from arachidonic acid (AA) by causing an inflammatory response in skin and upregulating cyclooxygenase and lipoxygenase enzymes. The formation of these metabolites has been found to be associated with immunological reactions, inflammatory disorders, skin pigmentation and the wounds healing. Short-term exposure of keratinocytes to sunlight may modify the composition of PUFA and its metabolism in skin cells. An in vitro study on epidermal cells carried out by Leung et al. (2017) has shown that keratinocytes may have defensive mechanisms at exposure to UVA, for example raised docosahexaenoic acid (DHA) levels, which prove to be helpful in regeneration after potential lesions.",Journal of Cosmetic Dermatology,UVA and Lipid Oxidation,2021
UVB-Induced ROS Generation and NF-κB Activation,"The effects of UVB radiation on the generation and release of ROS in human keratinocytes have been studied by Beak et al. (2004). The results showed an increase in intrinsic cellular production and release of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase and cyclooxygenase (COX), that might play an essential role in UVB-induced ROS production and nuclear factor B (NF-κB) activation in keratinocytes in a dose-dependent mode. Cavinato et al. (2017) have studied the role of protein quality control systems and their functional interaction in mediating the cellular aging of UVB-treated fibroblasts in vitro. The results suggest that early events in the process of senescence of fibroblast after UVB exposure include increased production of ROS and an inhibition of proteasome, followed by initiation of autophagy.",Journal of Cosmetic Dermatology,UVB-Induced Oxidative Stress,2021
"Proteasome Inhibition, Autophagy, and Fibroblast Senescence","The obtained results suggest that increased ROS generation and autophagy and reduced proteasome activity contribute to the aging of fibroblasts treated with UVB. Autophagy is necessary to establish the phenotype of aging of UVB-induced fibroblasts, and suppression of autophagy is needed to modify the path of cells from aging to apoptosis death. Deactivation of proteasomal system in fibroblast cells under the influence of UV radiation is associated with the generation of singlet oxygen, oxidation of proteins and activation of transcription agents known from the regulation of MMP-1 expression.",Journal of Cosmetic Dermatology,Oxidative Stress and Proteostasis,2021
MMP Activation and ECM Degradation by UV,"One of the main effects of UV radiation on the skin is an increase in expression of MMPs, which are responsible for the degradation of ECM proteins such as collagen, fibronectin, elastin, and proteoglycans. Excessive degradation of these proteins caused by excessive production of MMP-1, MMP-3, and MMP-9 contributes to the photoaging of the skin and thus to the formation of thick wrinkles and sagging of the skin through photodestruction, phototransformation and photooxidation of collagen and elastin. MMPs play an important role in the development of solar radiation-related cancer, as they regulate various processes associated with the cancer process, including tumor location, growth, angiogenesis and metastasis.",Journal of Cosmetic Dermatology,UV-Induced Enzyme Changes,2021
UVB-Induced MMP-1 Expression and Protein Regulation Pathways,"The data presented by Dong et al. (2008), as the main initiator of processes leading to the induction of MMP-1 production, indicate DNA damage caused by UVB radiation. Their studies revealed a more than fourfold increase of the expression of the MMP-1 gene in keratinocytes mRNA induced by UVB treatment of cells. Changes in protein expression play an important role in the process of skin photoaging, including the transforming growth factor-β (TGF-β), Smad2, MMP-1, MMP-3, MMP-9. Moreover, UV radiation, the increase in the amount of ROS and the expression of MMPs, which are one of the major agents involved in the process of skin changes associated with photoaging, may also interact with catepsins.",Journal of Cosmetic Dermatology,UVB and MMP Regulation,2021
Cathepsins as Biomarkers and Effectors of Photoaging,"Citing the results of a Zheng et al. (2011) study, the modified gene expression of cathepsins by repetitive exposures to UVA radiation is a prospective clarification of the alteration of cathepsin activity and content in photoaging skin. Their results suggest that the level and activity of cathepsins B, D, K and G may be considered as potential biomarkers in photoaging of human skin. Cathepsin K is one of the factors involved in the degradation of elastin, which leads to the formation of solar elastosis. It plays a dominant role in this process due to its elastolytic activity. Cathepsin D is a factor that induces cellular mitosis, which leads to weakening of the immune response and inhibition of dendritic cell function. It is assumed that down-regulation of cathepsin D, as a growth regulator of keratinocytes, may contribute to photoaging-related disorders of epidermal cell proliferation, such as disorders of the keratinization process. Also cathepsin B is responsible for matrix degradation and cell invasion.",Journal of Cosmetic Dermatology,Cathepsins and ECM Breakdown,2021
Opsin 3 (OPN3) Regulation of MMP Activity,"Another factor responsible for regulating MMP-1, MMP-2, MMP-3, and MMP-9 in human UVA-treated fibroblasts is opsin 3 (OPN3), as first demonstrated by Lan et al. (2020). Through a calcium-dependent signalling pathway coupled with G-proteins, OPN3 initiates the process of transduction in UVA-exposed fibroblast cells. OPN3 influences the phosphorylation of activator 1 protein and regulates the action of MMPs by activating protein kinase II. The process of activation of protein kinase II is dependent on many factors, including Ca2+/calmodulin, cyclic adenosine monophosphate protein binding response elements, extracellular signal kinase, N-terminal c-JUN and p38.",Journal of Cosmetic Dermatology,OPN3 and MMP Regulation,2021
"Role of AP-1, MAPK, and NF-κB in Collagen Degradation","The ROS produced as a result of UV radiation stimulates the mitogen-activated protein kinase family (MAPK), which is responsible for the formation of activator-1 protein (AP-1). AP-1 plays an instrumental role in regulating the transcription of MMP-1, MMP-3, and MMP-9, which results in progressive degradation of collagen. In addition, AP-1 inhibits the signalling of TGF-β, which is responsible for regulating the synthesis of pro-collagen type I in human skin, resulting in a reduction in the synthesis of this collagen precursor. One other important transcription factor that is activated in response to UV radiation is NF-κB. The main role of NF-κB is to regulate gene expression of growth factors, chemokines, cytokines and cell adhesion molecules, both in physiological state and in many diseases. NF-κB activity is responsible for regulating the expression of MMPs such as MMP-1 and MMP-3 in human skin fibroblasts. Therefore, both transcription factors, both AP-1 and NF-κB, are responsible for the formation of changes characteristic for photoaging.",Journal of Cosmetic Dermatology,Transcriptional Control of Photoaging,2021
Overview of Skin Aging and Collagen Decline,"Aging leads to a decline in skin function due to intrinsic factors (genetics, hormones) and extrinsic factors (sun exposure, pollutants). Type I collagen plays a vital role in maintaining skin integrity and elasticity. As aging progresses, collagen synthesis diminishes, resulting in weakened skin structure and wrinkle formation. This systematic review explores the role of type I collagen in skin aging by summarizing key clinical findings.",Cosmetics,Type I Collagen and Skin Aging,2025
Systematic Review Design and Study Selection,"A systematic search was conducted using PubMed and ScienceDirect as the primary databases, including studies published between 2014 and 2025 that addressed type I collagen and skin aging. Eleven clinical studies were selected following PRISMA guidelines. The results consistently show the decline of type I collagen as a central contributor to dermal thinning, loss of elasticity, and the appearance of wrinkles and sagging.",Cosmetics,Type I Collagen and Skin Aging,2025
Effects of Collagen Supplementation in Clinical Trials,"Clinical trials demonstrate that collagen supplementation, particularly from hydrolyzed fish cartilage and low-molecular-weight peptides, enhances collagen production, improves skin hydration and texture, and reduces signs of photoaging. Overall, the evidence emphasizes the critical role of type I collagen in skin aging and suggests that targeted collagen supplementation may serve as an effective strategy to maintain skin structure and combat visible signs of aging.",Cosmetics,Collagen Supplementation,2025
Introduction to Skin Aging and Biological Decline,"Aging is an inexorable time-dependent deterioration of biological function and regenerative capacity of higher organisms, resulting in a progressive increase in the risk of disease and death. Being the largest organ in the body, skin undergoes structural changes over time due to the combination of complex biological processes caused by intrinsic as well as extrinsic factors. Signs of aging skin include wrinkles, loss of elasticity, sagging, laxity, dullness, rough texture, and pigmentation changes. Of these, the occurrence of deep wrinkles merits special mention. Aging is an interdependent process that involves integrated molecular, cellular, and tissue-level changes. At the cellular level, mitotically active cells enter into senescence, a state characterized as irreversible G1 phase cell cycle arrest yet remain metabolically active. Cellular senescence acts not only as a model for human tissue aging research but plays a critical role itself in systemic aging.",Cosmetics,Type I Collagen and Skin Aging,2025
Intrinsic and Extrinsic Aging Mechanisms,"In skin, aging is associated with an insidious loss of regenerative potential in almost all components of its structural and functional integrity. As the first line of defense of the body, the skin is especially vulnerable to intrinsic biological processes and extrinsic physical damage. Intrinsic aging, a genetic process including hormonal behavior and natural physiological changes, has been associated with decreased collagen production, specifically with the decrease in type I collagen leading to a loss of skin elasticity and the presence of wrinkles. Extrinsic aging is driven by chronic exposure to environmental agents such as ultraviolet light (UV) radiation, pollution, and microorganisms. These stressors contribute to the formation of reactive oxygen species (ROS), which increase expression of matrix metalloproteinases (MMPs), leading to degradation of type I collagen in the dermal extracellular matrix. UV radiation induces oxidative stress and upregulates MMPs while suppressing new collagen synthesis. Air pollution sustains inflammation and damages fibroblasts, while microbial imbalance activates immune responses that promote inflammatory mediators.",Cosmetics,Intrinsic and Extrinsic Aging,2025
Environmental Stressors and Collagen Breakdown,"Altogether, these extrinsic factors contribute to visible signs of skin aging such as wrinkles, dryness, and loss of elasticity through cumulative detrimental effects on collagen homeostasis. These outside stressors also speed up DNA damage, leading to continued structural and functional decline of skin, compounding with intrinsic aging. These mechanisms work together to drive visible and biological aspects of skin aging by impacting rates of collagen breakdown and degradation while inhibiting tissue regeneration, as shown by numerous studies that have examined the role of UV radiation on collagen breakdown. These changes are responsible for visible aging: wrinkles, dryness, decreased barrier function, and epidermal thinning. The skin serves as an essential protective barrier against threats such as temperature extremes, UV, infections, mechanical damage, and water loss.",Cosmetics,Environmental Stressors and Collagen,2025
"ECM Structure, Collagen Types, and Age-Related Changes","Collagen, the most abundant protein in the extracellular matrix (ECM) and connective tissue, is most abundant in mammals. The ECM is a large and complex structure formed by proteins and glycosaminoglycans within the dermis, providing necessary structural support to fibroblasts, keratinocytes, and endothelial cells that maintain tissue architecture and skin structure. Keratinocytes utilize the ECM for structural stability and barrier function. Previous studies confirm that collagen decreases with aging and is one of the important molecules in the aging process of skin. Twenty-eight different forms of collagen have been characterized, distinguished by structural and functional traits. Many forms, such as type I, are abundant across tissues and serve as fundamental structural building blocks.",Cosmetics,ECM and Collagen Types,2025
"Type I Collagen Structure, Function, and Gene Components","Type I collagen is the most abundant collagen in the skin, accounting for 80–85% of the dermal ECM. Other subtypes, like type III and type V, are also present, but type I supplies tensile strength and determines dermal durability. As skin ages, there is progressive loss and fragmentation of dermal collagen fibrils, leading to reduced skin thickness and biomechanical strength. Type I collagen, a major fibrillar component of connective tissues, has a triple-helix structure composed of two α1 chains encoded by COL1A1 and one α2 chain encoded by COL1A2. COL1A1 expression is a biomarker of skin aging, declining with age. This organization supports mechanical strength, elasticity, thermal stability, and interactions with biomolecules. Collagen fragmentation, a hallmark of aging, results from altered interactions between fibroblasts and ECM.",Cosmetics,Type I Collagen Structure,2025
"Fibroblasts, ECM Interactions, and Collagen Homeostasis","Fibroblasts are the main collagen-producing cells and synthesize connective tissue matrix. They use integrins as surface receptors to bind ECM proteins such as type I collagen. Integrin binding leads to focal adhesion formation, linking ECM to intracellular signaling pathways. With advancing age, collagen homeostasis is perturbed, resulting in decreased collagen production, structural weakening, and diminished dermal strength. Due to collagen degradation, hallmark features such as loss of elasticity and wrinkle formation emerge. MMPs digest endogenous type I collagen. In young skin, MMP expression is low, regulated by tissue inhibitors of metalloproteinases (TIMPs). Collagen turnover is slow, with replacement every ~30 years, allowing accumulation of cross-links that disturb ECM architecture. Fragmented collagen reduces fibroblast attachment sites, decreasing mechanical resistance and causing fibroblast collapse. This leads to reduced collagen synthesis and increased MMP activity, forming a self-reinforcing aging loop.",Cosmetics,Fibroblasts and ECM Aging,2025
TGF-β Signaling Decline and Inflammaging,"Transforming growth factor-beta (TGF-β) is a chief modulator of ECM components such as collagen and elastin. With age, expression of the TGF-β type II receptor (TβRII) decreases in dermal fibroblasts, reducing downstream signaling. Impairment of collagen synthesis is accompanied by increased activity of MMPs responsible for collagen degradation. Impaired TGF-β signaling promotes inflammaging, associated with decreased skin elasticity, firmness, and resilience, leading to sagging, wrinkles, and general skin aging. Specific interventions targeting these impairments may increase collagen synthesis, stabilize the ECM, and decrease inflammation, representing promising anti-aging strategies. The aim of this systematic review is to evaluate the role of type I collagen in skin aging, synthesizing findings from clinical studies.",Cosmetics,TGF-β Signaling and Inflammaging,2025
Study Design and Objectives,"This comprehensive systematic review was conducted to evaluate the role of type I collagen in skin aging based on clinical studies. The review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to ensure methodological transparency and reproducibility. The search focused on identifying high-quality, peer-reviewed clinical studies published between 2014 and 2025.",Cosmetics,Type I Collagen Review Methods,2025
Search Strategy and Databases,"A comprehensive literature search was performed using PubMed and ScienceDirect which were selected as primary databases due to their extensive coverage of biomedical and dermatological research. The following keywords and Boolean operators were applied to refine the search: “Type I collagen AND skin aging”, “Collagen AND anti-aging”, and “Collagen type I AND clinical study”. The search was limited to clinical studies published in English, ensuring relevance to human applications in dermatology.",Cosmetics,Literature Search Strategy,2025
Inclusion and Exclusion Criteria,"Studies were included if they met the following criteria: clinical studies assessing the impact of type I collagen on skin aging; published between 2014 and 2025; full-text availability in English; investigated collagen-related changes in skin elasticity, hydration, or wrinkle formation in human participants. Studies were excluded if they were not clinical studies (e.g., in vitro, ex vivo, or animal studies); did not specifically evaluate type I collagen’s role in skin aging; were literature reviews, meta-analyses, or opinion pieces; or were duplicates or superseded by more recent research.",Cosmetics,Screening Criteria,2025
Study Selection and PRISMA Framework,"The PRISMA framework was applied to systematically screen and select eligible studies. Initially, all retrieved articles were screened based on titles and abstracts for relevance. Full-text reviews were conducted for studies meeting the inclusion criteria. The PRISMA flowchart provides an overview of the selection process, ensuring a structured and reproducible approach.",Cosmetics,PRISMA Screening,2025
Data Extraction and Screening Process,"A systematic and transparent approach was used to identify, screen, and select clinical studies evaluating the role of type I collagen in skin aging. The search was conducted across PubMed and ScienceDirect using predefined keywords, yielding a total of 9243 records. Duplicate records (n = 2019) were removed; articles published before 2014 (n = 1230) were excluded; and studies irrelevant to the clinical investigation of type I collagen in the context of skin aging, including those investigating collagen type I in non-cutaneous tissues or lacking specific focus on the COL1A1 gene (n = 3027), were excluded, leaving 2967 records for further screening.",Cosmetics,Data Extraction Process,2025
Screening Results and Final Study Selection,"The remaining 2967 records underwent title and abstract screening, resulting in the exclusion of 2200 articles that did not directly examine the role of type I collagen in skin aging. This left 767 reports sought for retrieval, of which 123 full-text articles were assessed for eligibility. Reports excluded due to insufficient methodological quality (n = 110). Ultimately, 11 studies met all inclusion criteria and were included in the final systematic review. These selected studies served as the foundation for evaluating the impact of type I collagen on skin aging and were analyzed in detail.",Cosmetics,Study Selection Outcome,2025
Synthesis and Data Integration,"The structured methodology ensured that only high-quality, clinically relevant studies were incorporated, strengthening the validity of the findings. A total of 11 studies were included in the final analysis. These articles were systematically examined to extract key information related to the effect of type I collagen on skin aging parameters including elasticity, hydration, and wrinkle formation. The extracted data were synthesized into a comprehensive table to facilitate comparison and interpretation, forming the basis for the Results and Discussion sections.",Cosmetics,Data Synthesis,2025
Ethical Considerations,"This study is a systematic review based on previously published studies and does not involve new studies with human or animal participants. Therefore, ethical approval was not required.",Cosmetics,Ethical Considerations,2025
Overview of Evidence on Type I Collagen in Skin Aging,"To ensure a structured and comprehensive evaluation of type I collagen in skin aging, a summary of evidence obtained from 11 selected studies (Table 1). Due to the scope of this systematic review, clinical studies were selected that investigated the influence of type I collagen on skin elasticity, hydration, and wrinkle generation. The table summarizes core components of each study, such as design, methods, intervention characteristics, key findings, and clinical implications. Yoon et al. evaluated the potential benefit of topical estrogen treatment in photoaged skin by measuring wrinkle severity and collagen levels. What the investigators noted was that compared to placebo, the topical estrone significantly enhanced skin elasticity and decreased wrinkle severity but there were no statistically significant differences between the groups observed on physical examination, with the exception that topical estrone resulted in a marked increase in matrix metalloproteinase-1 (MMP-1), a collagen-degrading enzyme.",Cosmetics,Type I Collagen Clinical Evidence,2025
"Topical Estrogen, MMP-1 Activation, and Collagen Homeostasis","Collectively, the data indicate that, against the expectation, estrogen treatment adversely impacts photoaged skin by negatively modulating collagen homeostasis. The results of this study may not be widely applicable because the duration of the study was short and the target population was limited to a specific age group, post-menopausal women. Studies on other doses, formulations, or concomitant use with other drugs could expand upon our findings by preventing the negative effects of MMP-1 activation and promoting the effects on collagen synthesis. This study highlights the equilibrium that exists between collagen synthesis and degradation in skin aging and that pharmaceutical interventions should be approached with caution. This study employed both objective skin measurements (Visiometer and Cutometer) and molecular analyses (qPCR and immunohistochemistry), revealing that while type I procollagen mRNA levels increased, protein expression did not, likely due to a significant 10.3-fold upregulation of MMP-1, which underscores the complex role of topical estrogen in collagen homeostasis under UV exposure.",Cosmetics,MMP-1 and Estrogen Effects,2025
Mechanical Stimulation (Mécano-Stimulation) and Collagen Synthesis,"Humbert et al. investigated the impacts of Mécano-Stimulation, a method of mechanical stimulation, on age-related skin deterioration and collagen synthesis. The trial concluded that skin firmness significantly increases and skin sagging decreases with 24 sessions of Mécano-Stimulation, and the activity of dermal fibroblasts also increases, leading to an increase in collagen deposition. Though the study did not identify significant improvements in elasticity, the authors note that mechanical stimulation effects may be more apparent in firmness and collagen synthesis than elasticity. Another limitation of this study is that there was no longer follow-up to determine the lasting effects of changes. Moreover, while the effect was stronger at the inferior and lateral region of the cheek, there was limited change for the superior region and nasolabial fold—suggesting possible pitfalls of a one-size-fits-all approach.",Cosmetics,Mechanical Stimulation and Collagen,2025
"LMW Collagen Peptides and Hydration, Elasticity, and Wrinkle Outcomes","Kim et al. investigated the effect of oral supplementation with low-molecular-weight collagen peptides (LMWCP) on skin hydration, elasticity, and wrinkling. The researchers concluded that LMWCP supplementation significantly improved skin hydration and was shown to reduce wrinkle severity after 12 weeks in the study population, particularly those with lower baseline skin hydration. One strength of this study is that multiple skin parameters have been evaluated in the skin and time with each compound, giving an overall assessment of the LMWCP and skin effects on these parameters. The study found no noteworthy improvement in skin elasticity, which implied that LMWCP has limited efficacy with regard to skin elasticity and works better with regard to hydration and wrinkling. Although the presence of a placebo group and the randomized nature of the study boost the reliability of the findings, the absence of histological findings limits deeper evaluation.",Cosmetics,Collagen Peptides Clinical Effects,2025
10-Hydroxystearic Acid (HSA) and Collagen Modulation,"Schütz et al. studied the impact of 10-hydroxystearic acid (HSA) on age spots and pores on the human face. The results revealed that HSA significantly decreased facial pore area and pigmentation of age spots after 8 weeks of application. The strength of the study lies in the use of a multi-level approach to address the effect of HSA on skin aging, including in vitro, ex vivo, and clinical evaluations. Significant biochemical changes, including modulation of MMP-1 and collagen expression, supported the clinical findings and indicated that HSA improved skin texture via modulation of collagen metabolism. The relatively short follow-up time of the study makes the long-term effect of HSA difficult to discern, and longer follow-up studies are needed to validate whether these improvements were maintained.",Cosmetics,HSA and Collagen Regulation,2025
Angelica gigas Nakai Extract and Collagen Synthesis,"Kang et al. examined the anti-wrinkle effect of Angelica gigas Nakai root extract (ARE) in vitro and in clinical trials. This study showed that ARE upregulated type I collagen synthesis in fibroblast cells and significantly ameliorated the appearance of crow’s feet wrinkles in 8-week treatment participants. Strength of the study: both in vitro and clinical trials, and the overall effects of ARE look good. ARE appears to have potential as a protective agent to reduce the development of crow’s feet wrinkles. Nonetheless, the mechanism of how ARE stimulates collagen synthesis remains unclear. Generalization is limited due to the shorter treatment period and small number of participants; further studies of larger sizes and longer durations will be helpful to support ARE as an anti-aging agent.",Cosmetics,ARE and Collagen Synthesis,2025
Marine Collagen Supplementation (VWC) and Anti-Aging Outcomes,"Evans et al. investigated the effects of supplementation of hydrolyzed marine collagen (Vinh Wellness Collagen, VWC) on skin health in women aged 45 to 60. Following 12-week VWC supplementation, the study noted clinically meaningful increases in skin hydration and skin elasticity and decreases in wrinkle severity. The randomized, triple-blind, placebo-controlled design strengthens reliability. Reduction in wrinkles and improvements in skin texture and radiance indicate that VWC supplementation can target multiple aspects of skin aging. However, reliance on self-reported measures introduces potential bias. Further studies should evaluate long-term effects and combinations with other nutrients.",Cosmetics,Marine Collagen Clinical Effects,2025
Collagen Peptide Supplementation and Dermal Structural Changes,"Laing et al. conducted a randomized, placebo-controlled trial to demonstrate the effects of a food supplement containing collagen peptides, vitamins, and other nutrients on skin quality. Remarkable enhancement of collagen structure in facial skin was found with confocal laser scanning microscopy at 12 weeks, suggesting improved skin smoothness and elasticity. The triple-blind, expert-assessed design supports the validity of the results. However, the study focused specifically on facial cheek skin, limiting generalizability to other body regions. Although the supplement was well tolerated, long-term effects were not assessed, highlighting the need for extended follow-up.",Cosmetics,Collagen Peptides and Skin Structure,2025
Hydrolyzed Fish Cartilage and Collagen Remodeling,"Campos et al. examined the impact of oral supplementation with hydrolyzed fish cartilage on skin aging variables such as wrinkle formation, dermal thickness, and collagen morphology. Supplementation for 90 days led to significant improvement in skin microrelief, dermis echogenicity, and collagen structure. Findings indicate that supplementation can increase skin density and decrease wrinkles. The specific collagen source and low dosage raise questions regarding optimal dosing. Larger sample sizes and longer follow-up periods are needed to confirm longer-term effects on skin health. In this study, dermal collagen was evaluated using in vivo reflectance confocal microscopy, providing insight into collagen remodeling.",Cosmetics,Fish Cartilage Collagen Effects,2025
Hyaluronan (HA2k) Oligosaccharides and Collagen Metabolism,"Abe et al. explored the role of 2 kDa hyaluronan (HA2K) oligosaccharides in collagen remodeling and wrinkle reduction. The study showed that HA2k penetrated the stratum corneum, activated collagen metabolism, and improved dermal collagen density. The randomized controlled trial showed visible wrinkle reduction in nasolabial folds and crow’s feet areas after 8 weeks. However, HA2k simultaneously upregulated collagen synthesis (COL1A1) and degradation (MMP-1), suggesting complex regulatory effects. The 0.1% HA2k solution appeared promising but requires further dose optimization. Short study duration and small sample size necessitate larger, longer trials.",Cosmetics,HA2k and Collagen Remodeling,2025
Collagen and Hyaluronic Acid Supplementation Combined,"Žmitek et al. investigated supplementation of hydrolyzed collagen (HC) alone or in combination with hyaluronic acid (HA). Results showed improvements in dermal density, wrinkle severity, and skin roughness in both groups, with no significant differences between collagen alone and collagen with HA. Thus, combining HA with collagen may not enhance efficacy. Strengths include the rigorous randomized, placebo-controlled design and objective imaging techniques. However, the focus on a single dose makes it difficult to determine optimal formulations. Larger sample sizes and extended treatment times are warranted.",Cosmetics,Collagen + HA Clinical Effects,2025
Endo180 Stimulation by Lemon Balm Extract,"Iwahashi et al. focused on screening materials that enhance Endo180, a collagen receptor implicated in collagen degradation and synthesis. Lemon balm (Melissa officinalis) leaf extract (MOLE) enhanced Endo180 production and thereby type I collagen synthesis in dermal fibroblasts. In a clinical trial, a MOLE-containing cream decreased eye-corner wrinkles at 8 weeks. Rosmarinic acid was identified as a potential active compound. However, precise molecular events mediating the effects remain to be fully elucidated. The clinical sample size was small, and larger studies are needed to verify results and explore long-term effects.",Cosmetics,Endo180 and Collagen Modulation,2025
Mechanisms of Type I Collagen Degradation and Therapeutic Targets,"A more detailed overview of the specific processes involved in type I collagen degradation in aging and potential targets for therapeutic interventions are illustrated in a summary table (Table 2). Collagen breakdown is induced by different factors, which consist of MMP activities, collagen fiber fragmentation, and perturbation of signaling pathways like TGF-β signaling, which is crucial in collagen synthesis and maintenance. Here, we summarize the former mechanisms, together with therapeutic strategies such as oral supplementation, topical treatments, and mechanical stimulation, which counteract the degradation of collagen and favor its regeneration.",Cosmetics,Collagen Degradation Mechanisms,2025
Limitations of Current Clinical Studies,"Although the reviewed studies offer important insights into understanding the role of type I collagen in skin aging, as well as additional potential benefits of collagen supplementation as humans age, there were weaknesses that must be noted. Some of the studies had smaller sample sizes, for example, the ones by Kim et al. (n = 64) and Iwahashi et al. (n = 20), which may restrict the generalizability of the results to larger and more heterogeneous populations. Finally, most interventions are between 8 and 12 weeks in duration, which is relatively short and may not allow for the long-term evaluation of collagen supplementation and its effects on skin aging. Some studies, like the one by Yoon et al., do not have long-term follow-up, making it impossible to determine if the treatment effects would be sustained. Additionally, differences in populations studied, such as age or other cutaneous conditions, may render findings less relevant to the general population.",Cosmetics,Study Limitations,2025
"Variability, Methodological Weaknesses, and Areas for Improvement","Even those studies had some limitations—for example, Humbert et al. based their findings largely on visual assessments that may be subjective. Fifthly, some of the studies reviewed, such as those performed by Žmitek et al., only focused on the effect of collagen supplementation, without exploring potential interactions with other skin treatments or environmental factors that could have influenced outcomes. Further shortcomings in the studies that may be addressed in future research include smaller sample sizes, lack of long-term follow-up, or just vague definitions for skin aging. All these disadvantages aside, however, the strengths of the present review are that it encompasses a variety of studies, those regarding topical versus oral collagen supplementation, and as such provides a broader perspective on the therapeutic potential of collagen when it comes to skin wellness and rejuvenation.",Cosmetics,Methodological Weaknesses,2025
Future Directions and Research Opportunities,"The effectiveness of collagen supplementation on skin hydration, elasticity, and wrinkle reduction has been repeatedly demonstrated in studies evaluating both topical collagen preparations and oral collagen peptides, emphasizing the key role collagen plays in the quest for retaining youthful skin. Additional studies should investigate the long-term effects of collagen supplementation, especially in diverse populations, to determine its optimal doses and treatment durations. Moreover, potential interactions and synergistic effects between collagen peptides and other anti-aging treatments such as antioxidants or hyaluronic acid could also be explored. In addition, gaining more knowledge about the molecular mechanisms controlling collagen synthesis and degradation in various skin types and environmental settings may shed further light on how to more effectively tailor therapies to target skin aging in a more precise manner.",Cosmetics,Future Research on Collagen,2025
Introduction to Calorie Restriction and Fasting,"Genetic and pharmacological interventions have successfully extended healthspan and lifespan in animals, but their genetic interventions are not appropriate options for human applications and pharmacological intervention needs more solid clinical evidence. Consequently, dietary manipulations are the only practical and probable strategies to promote health and longevity in humans. Caloric restriction (CR), reduction of calorie intake to a level that does not compromise overall health, has been considered as being one of the most promising dietary interventions to extend lifespan in humans. Although it is straightforward, continuous reduction of calorie or food intake is not easy to practice in real lives of humans.",Mechanisms of Lifespan Regulation by Calorie Restriction and Intermittent Fasting,Calorie Restriction,2020
Fasting Strategies and Their Emergence,"Recently, fasting-related interventions such as intermittent fasting (IF) and time-restricted feeding (TRF) have emerged as alternatives of CR. Here, we review the history of CR and fasting-related strategies in animal models, discuss the molecular mechanisms underlying these interventions, and propose future directions that can fill the missing gaps in the current understanding of these dietary interventions. CR and fasting appear to extend lifespan by both partially overlapping common mechanisms such as the target of rapamycin (TOR) pathway and circadian clock, and distinct independent mechanisms that remain to be discovered.",Mechanisms of Lifespan Regulation by Calorie Restriction and Intermittent Fasting,Intermittent Fasting,2020
Systems Approaches for Understanding Longevity Mechanisms,"We propose that a systems approach combining global transcriptomic, metabolomic, and proteomic analyses followed by genetic perturbation studies targeting multiple candidate pathways will allow us to better understand how CR and fasting interact with each other to promote longevity.",Mechanisms of Lifespan Regulation by Calorie Restriction and Intermittent Fasting,Longevity Mechanisms,2020
Determinants of Lifespan Regulation Through Diet,"CR regards the daily caloric intake per se as a key determinant in lifespan regulation. For example, a reduction of calorie intake without a reduction of protein intake increased the lifespan of rats, and lifespan was not altered in rats fed isocaloric diets in which either fat or mineral components had been reduced. These studies indicated that the total calories are a key determinant in regulating the lifespan of rats. However, recent evidence had indicated that the amount of calorie intake might not be a key determinant of lifespan regulation by CR. The lifespans of rats and fruit flies have been increased by nutritional changes or protein reduction while providing the same calorie intake.",Nutrients,Calorie Restriction and Lifespan Determinants,2020
Role of Amino Acids and Protein Restriction,"Moreover, the results of several studies have suggested that amino acids are key modulators of lifespan in organisms. Furthermore, reducing only one type of amino acid, methionine, is sufficient enough to increase the lifespan of yeast, nematodes, fruit flies, and rodents. Beneficial effects of TRF on health and longevity indicated that there might be a third determinant in lifespan extension, other than total calories or nutrient composition, since TRF exerts its effect without exhibiting notable changes in total calories or nutrient composition.",Nutrients,Amino Acid Restriction and Methionine,2020
Need for Identifying Key Nutrient Determinants,"A more thorough investigation into the key determinant(s) of nutrient restriction effect is necessary. This includes identifying how nutrient composition, timing of feeding, and specific amino acids interact to modulate lifespan independently or synergistically with caloric restriction.",Nutrients,Nutrient Determinants of Longevity,2020
Yeast as a Model for Dietary Manipulation,"Yeast aging is classified into two different types as replicative and chronological aging. Replicative aging is defined by the number of daughter cells produced by a mother cell, while chronological aging is defined by the time in which a nondividing cell can maintain viability. Although two yeast aging paradigms have been used in aging studies, replicative aging is more widely used in CR-related aging studies. Generally, CR in yeast is performed by reducing the glucose level in growth medium, which commonly contains 2% peptone, 1% yeast extract and 2% glucose. The concentrations of glucose are reduced to ~0.5–0.005% for CR. In these settings, replicative lifespan of budding yeast was extended by about 10 times in the low-dose glucose medium compared to control. Yeast is also cultured in water in order to undergo fasting.",Nutrients,Dietary Restriction Models,2020
Dietary Restriction and Fasting in C. elegans,"C. elegans has several advantages in aging studies—a relatively short lifespan/reproductive cycle, a translucent body, it is easy to culture, has a small genome, and there are many available mutants. DR is mainly performed in nematodes by controlling the concentration of bacteria such as Escherichia coli in the media that they feed. In the worms, genetic perturbations that mimic DR were also introduced by inhibiting specific nutrient transporters and reducing pharyngeal pumping. For IF, worms are placed every other day in medium with and without bacteria. This IF regimen (alternate 2 days eating / 2 days fasting) successfully extended lifespan in the worms. Furthermore, chronic fasting also increased the lifespan of worms compared to normal diet-fed worms.",Nutrients,C. elegans Dietary Restriction,2020
Dietary Manipulation in Drosophila melanogaster,"The fruit fly, D. melanogaster, is another invertebrate model organism widely used for aging and dietary intervention studies. Similar to C. elegans, the fruit fly also has many advantages such as a relatively short lifespan and high productivity. However, compared to C. elegans, the fruit fly has more complicated and diverse tissues such as the heart and kidney that are functionally homologous to mammals. Gene manipulation and editing tools are also readily available. Although food composition varies, the most general method for DR is dilution of food ingredients including yeast as a protein source, sugar, or fat. Limiting amino acids such as methionine or limiting protein sources were sufficient to increase lifespan. IF and TRF protocols have also been implemented, including 2-day feeding/5-day fasting regimens and TRF with daily food access by day and water by night.",Nutrients,Drosophila Dietary Restriction,2020
"Rodent Models of CR, DR, IF, and TRF","Rodents can fill gaps between invertebrate models and humans due to their closer phylogenetic relationship and physiological similarities. Many studies have shown beneficial effects of CR/DR on aging in rodents. CR/DR reduced the incidence of age-related diseases such as cancer, neurodegenerative diseases, and cardiovascular diseases, and prolonged lifespan by 30% in rats and 15% in mice. Generally, CR in rodents is conducted by reducing 20–50% of calories compared to ad libitum food intake. Trials modulating macromolecule composition such as proteins or carbohydrates were also attempted. Reducing specific amino acids such as methionine or tryptophan also extended lifespan. IF regimens include providing water or minimal nutrients for less than 24 h followed by normal feeding, whereas PF involves ~48 h fasting cycles. TRF is performed by regulating daily cage transfers between food-available and water-only environments. IF effects on lifespan in rodents are mixed, but multiple studies show that TRF inhibits chronic diseases, tumor progression, and increases lifespan.",Nutrients,Rodent CR and Fasting Models,2020
CR Studies in Non-Human Primates,"The use of NHP in dietary studies provides unique evidence that cannot be obtained by studying a lower-order model animal. Although the results of NHP studies have high reliability in human applications, NHP studies can encounter several technical, financial and ethical difficulties. Three independent groups, the NIA, the WNPRC, and the University of Maryland have investigated, or are currently investigating, the beneficial effects of CR on NHP by using the rhesus monkey model. A research group at the University of Maryland have focused on the effects of short-term CR on obesity and diabetes, while the NIA and WNPRC have been investigating the effects of CR in rhesus monkeys throughout their entire lifetime. Although the rhesus monkeys in the CR groups were provided with about 70% food compared to ad libitum groups in both the NIA and WNPRC studies, there is a key difference between them in terms of dietary composition. The NIA provided unpurified natural ingredient-based food, while the WNPRC provided a purified diet to monkeys. Natural ingredient-based food contains phytochemicals and minerals which might have beneficial effects on health and lifespan, whereas a purified diet allows the precise manipulation of nutrient composition. In addition, the NIA provided approximate ad libitum intake based on age and bodyweight without overfeeding, but WNPRC established individualized ad libitum references and applied CR based on these standards.",Nutrients,Calorie Restriction in Primates,2020
Comparative Outcomes of NIA and WNPRC CR Studies,"Lifelong CR in rhesus monkeys led to lifespan extension at the WNPRC, but there was no lifespan extension effect by CR at NIA. The NIA used food lower in calories and fat, and higher in protein and fiber compared to the WNPRC diet. These dietary differences led to a longer lifespan of the control old-onset group at the NIA compared to what is known as the median lifespan of rhesus monkey (~35 years). Juvenile and adult males without CR at NIA showed lifespans similar to monkeys with CR at WNPRC, suggesting that the difference in diet between control and CR groups at NIA may have been insufficient to affect lifespan. The NIA also used rhesus macaques of various ages, sexes, and different genetic backgrounds (Indian and Chinese), enabling comparisons of CR effects across demographic variables.",Nutrients,Diet Composition and Longevity,2020
Health Benefits of CR in Rhesus Monkeys,"Although the lifespan outcomes differed between NIA and WNPRC studies, both groups reported significant health benefits of CR, including reduced body weight and fat mass, lower risk of cancer, and decreased incidence of cardiovascular disorders. These findings suggest that if all variables such as diet composition, genetic background, and feeding regimen were controlled consistently, CR could robustly increase lifespan in monkeys and potentially provide translational insight for human aging interventions.",Nutrients,Health Effects of Calorie Restriction,2020
Overview of Dietary Manipulations in Humans,"Many studies have shown that dietary manipulation can retard the aging process through some well conserved mechanisms in diverse organisms from yeast to NHP. The determination of conserved mechanisms that produce beneficial effects of dietary manipulation in humans would require additional investigation, due to the limited number of studies examining the effects of CR/IF in humans. However, several epidemiological and cross-sectional studies using centenarians and individuals who volunteered CR practice indicate the beneficial effect of CR in humans. Epidemiological data can be gathered from people who follow food restrictions due to religious guidelines. For example, Muslims ingest no food or water for approximately 15 h between sunrise and sunset for a month during Ramadan every year. Thus, this long-term food restriction during Ramadan could be considered a human IF model.",Nutrients,Human Dietary Manipulations,2020
Human Health Effects of Calorie Restriction (CR),"Some studies have shown that Ramadan fasting has the effect of promoting human health. The Comprehensive Assessment of the Long-term Effects of Reducing Intake of Energy (CALERIE) research program was designed to systematically investigate sustained CR effects in healthy volunteer humans over a two-year period. The CALERIE program produced several results that demonstrate the beneficial effect of CR on aging and health in humans, including observation of an increase in metabolism and a decrease in oxidative stress; however, the study did not indicate the presence of beneficial effects of CR on age-related bone and muscle impairment.",Nutrients,Calorie Restriction in Humans,2020
Intermittent Fasting (IF) Outcomes in Humans,"Additionally, some studies have shown that IF can improve metabolic health and physiological function in humans. IF reduced fat mass, lean mass, and body weight in healthy humans and obese patients. Similarly, IF improved lipid and glucose metabolism, reduced inflammatory response, lowered blood pressure, and improved cardiovascular health. Several studies have shown that IF is an effective intervention, especially for people who are overweight or diabetic. IF reduced overall fat mass and decreased insulin resistance.",Nutrients,Intermittent Fasting Effects,2020
Time-Restricted Feeding (TRF) in Human Health,"Some researchers also conducted the studies to evaluate the effects of TRF on human health, and demonstrated that TRF improved insulin sensitivity, blood pressure, oxidative stress, and quality of life in overweight or diabetic adults. Results of the studies also showed that TRF improved cardiovascular function and other indicators of healthspan (e.g., walking distance and heart rate) in healthy middle-aged and older adults although weight loss observed with other IF methods were not accompanied by TRF. These results suggest that IF including TRF may be a promising manipulation to extend the healthspan of humans.",Nutrients,TRF and Healthspan,2020
Healthspan vs. Lifespan and Importance of Mechanistic Studies,"The ultimate goal for animal studies on CR/IF is to identify the conserved molecular mechanisms that can extend the healthspan of humans. Healthspan, the period of life that is free from disease, is measured by examining declines of functional health parameters and disease states. Because healthspan is a multifactorial complex phenotype affected by genotypes (G), environmental factors (E), and interactions between them (G × E), measuring healthspan often gets complicated. Delayed functional aging in one parameter is not always linked to extension of healthspan in other parameters. Unlike healthspan, lifespan is unequivocally recorded by following mortality. Lifespan extension in animal models is strongly correlated with decreased morbidity and improved health. Although health-related findings from CR/IF may translate to humans, this manuscript focuses on mechanisms of lifespan extension in animal models.",Nutrients,Healthspan and Lifespan Mechanisms,2020
Core Molecular Pathways in CR-Mediated Lifespan Extension,"Studies over the last two decades have provided extensive details on CR mechanisms. Advances in OMICs and bioinformatics combined with genetic perturbation analyses expanded understanding of molecular pathways mediating lifespan extension by CR. CR works through key nutrient and stress-responsive pathways including IIS/FOXO, TOR, AMPK, Sirtuins, NRF2, and autophagy. While these pathways can act independently, cross-talk exists among them, and upstream networks such as the circadian clock also contribute. Although fewer studies exist for IF, evidence shows that IF extends lifespan in both vertebrates and invertebrates. Increased survival by nutrient deprivation in E. coli further emphasizes evolutionary conservation. However, mechanisms responsible for IF-mediated lifespan extension remain less understood.",Nutrients,CR Molecular Pathways,2020
Shared and Distinct Effects of CR and IF,"CR and IF share a common strategy: reduction of caloric intake or timing-based nutrient limitations. Both CR and IF induce common metabolic and physiological changes across tissues, including increased ketone bodies, insulin sensitivity, and adiponectin, along with reduced insulin, IGF-1, leptin, inflammation, and oxidative stress. Behavioral changes such as increased hunger response and improved cognitive performance also occur. These similarities support the idea that overlapping molecular mechanisms mediate lifespan extension by CR and IF. A proposed model suggests both interventions alter TOR, IIS, and sirtuin pathway activity. However, independent mechanisms must also exist because IF aims to extend lifespan without reducing total caloric intake by leveraging fasting-responsive pathways.",Nutrients,CR vs IF Mechanisms,2020
Body Weight Considerations in CR and IF,"Chronic CR that extends healthspan and lifespan typically involves body weight loss in animal models. Body weight reduction is also common under IF. Although modest weight loss may benefit health, excessive loss could counteract positive outcomes. Importantly, fasting-based interventions can decouple weight loss from health benefits, suggesting that body weight reduction is a side effect rather than a mechanistic determinant. In the CALERIE trial, weight loss was mild and accompanied by improvements in metabolic and physiological parameters. Thus, body weight changes are not considered the primary driver of CR/IF benefits.",Nutrients,Weight Loss and Longevity,2020
Need for Genetic Validation of CR and IF Pathways,"CR and IF significantly reorganize genomic, metabolomic, and proteomic landscapes at tissue and organism levels in age-, sex-, and strain-dependent ways. However, these molecular changes do not inherently demonstrate causality for lifespan extension. Genetic perturbation studies must accompany OMICs findings to validate mechanistic pathways. Due to fewer genetic studies available for IF compared to CR, the molecular mechanisms of CR are better defined, and future sections focus on CR pathways first before examining potential overlap with IF.",Nutrients,Genetic Mechanisms of CR and IF,2020
TOR Pathway in Nutrient and Growth Regulation,"In eukaryotes, the target of the Rapamycin (TOR) pathway plays a central role in nutrient and energy sensing to control cellular and organismal growth. The TOR pathway regulates growth and metabolism by promoting protein synthesis in response to nutritional availability including dietary amino acids. A number of genetic studies showed that suppression or downregulation of the TOR pathway extends lifespan in multiple model organisms including the yeast S. cerevisiae, the worm C. elegans, the fly D. melanogaster, and the mouse M. musculus. As CR downregulates the TOR signaling cascade, it has long been suggested that CR may extend lifespan by at least partially suppressing the TOR pathway at the cost of reduced growth. Mutant animals for components of the TOR pathway often fail to show lifespan extension by CR, indicating that TOR antagonizes the full benefit of CR-mediated longevity.",Nutrients,TOR Signaling and Longevity,2020
AMPK as an Energy Sensor and Longevity Regulator,"In addition to amino acids, the TOR pathway is also regulated by cell energy status through AMP-dependent protein kinase (AMPK), a conserved energy sensor in eukaryotes. Increased AMP:ATP ratio by energy depletion such as CR activates AMPK, which in turn inhibits the TOR pathway. Thus, CR activates AMPK while suppressing the TOR cascade. Unlike the TOR pathway where suppression extends lifespan, AMPK extends lifespan in model organisms when activated. Genetic perturbation studies showed that AMPK mediates lifespan extension by CR. For example, lifespan extension by CR in worms was suppressed in mutant worms lacking aak-2, a catalytic subunit of AMPK. However, another type of CR in worms (feeding diluted bacteria in liquid culture) did not require AMPK signaling to extend lifespan, suggesting distinct CR subtypes may recruit different mechanisms.",Nutrients,AMPK Activation and CR,2020
Fasting-Related AMPK-TOR Interactions,"Mild nutritional stress through feeding 2-deoxy-D-glucose (2-DG) or food deprivation, which mimic fasting, extended lifespan in worms through AMPK signaling. In mammals, acute starvation readily activates AMPK, but activation depends on the duration and type of CR, and extended CR can fail to activate AMPK. This suggests that AMPK-TOR dependent lifespan extension may partially result from fasting-induced mechanisms independent of CR. Supporting this, Honjoh et al. showed that lifespan extension by IF (every-other-day feeding) was dependent on RHEB, a small GTPase that activates the TOR pathway, at least in worms. Their findings also indicated that RHEB-dependent IF-mediated lifespan extension was partially due to IIS/FOXO signaling, supporting the idea that tightly regulated networks between IIS/FOXO and TOR signaling mediate both DR- and IF-dependent longevity.",Nutrients,AMPK-TOR in Fasting and IF,2020
Overview of IIS-FOXO in Growth and Longevity,"In mammals, growth hormone (GH) secreted from the pituitary gland promotes somatic growth by activating downstream Insulin/Insulin-like growth factor-1 signaling (IIS). Activated IIS drives FOXO transcription factors out of the nucleus into the cytoplasm, suppressing their longevity-associated transcriptional programs. When GH/IIS signals are reduced, FOXO translocates into the nucleus and activates target genes involved in stress resistance, DNA repair, detoxification, and cell cycle arrest. These responses promote longevity by shifting the organism from growth toward maintenance. Although lower organisms such as yeast, worms, and flies lack GH, strong evidence indicates that reduced IIS and activation of FOXO orthologs extend lifespan across these species. Roughly one third of over 40 known longevity-extending mutations in rodents involve GH/IIS pathways, supporting the centrality of this axis in aging.",Nutrients,IIS-FOXO Longevity Pathway,2020
"CR, GH/IIS Downregulation, and Lifespan Extension","Because caloric restriction (CR) reduces GH and IIS, the prevailing view is that CR extends lifespan partly by limiting IIS, thereby activating FOXO transcription factors. However, studies testing whether IIS-FOXO is required for CR longevity yield mixed results. Bonkowski et al. reported that dwarf mice lacking the GH receptor did not show lifespan extension under 30% CR, suggesting CR acts by suppressing IIS. Conversely, another study showed that CR further increased lifespan in GH-deficient dwarf mice with tissue-specific reductions in GH production, implying that GH suppression may act independently of CR or require broader tissue-level suppression to fully synergize with CR. FOXO3 mutant mice, both heterozygous and homozygous, also failed to benefit from CR, highlighting the requirement of FOXO for full CR-mediated longevity. These findings collectively indicate that IIS-FOXO contributes significantly to CR-driven lifespan extension, but the interaction is tissue-specific and context-dependent.",Nutrients,CR and IIS-FOXO Mechanistic Interaction,2020
IIS-FOXO in Lower Organisms and Interactions with IF,"In lower organisms, whether IIS-FOXO mediates CR longevity remains unresolved. In Drosophila, different studies suggest IIS-FOXO modulates but is not the primary driver of CR-induced lifespan extension. In C. elegans, the requirement of IIS-FOXO (DAF-16) for CR longevity depends strongly on the specific CR protocol; some CR regimens extend lifespan independently of DAF-16. No direct genetic tests have yet established whether IIS-FOXO is required for IF-mediated lifespan extension. However, IF produces physiological and metabolic changes similar to CR—improved insulin sensitivity, reduced inflammation, and enhanced stress resistance. Because these benefits correspond to FOXO-driven transcriptional programs, current evidence suggests IIS-FOXO likely contributes at least partially to IF-mediated longevity, even though the pathway may not be the sole determinant.",Nutrients,IIS-FOXO and Intermittent Fasting,2020
Sirtuins as NAD⁺-Dependent Metabolic Sensors in Longevity,"Sirtuins (Sir2 family proteins) are NAD⁺-dependent deacetylases that function as metabolic sensors, linking cellular energy status to chromatin regulation and stress responses. Because NAD⁺ levels rise under nutritional stress, including caloric restriction (CR) and fasting, sirtuins become activated and can modulate pathways associated with longevity. Genetic overexpression of sirtuins has been shown to extend lifespan in yeast, worms, flies, and mice, while pharmacological activation using agents such as resveratrol produces similar effects. Multiple studies demonstrate that sirtuins are required for CR-mediated lifespan extension: deletion of SIR2 in yeast eliminates the longevity benefit of glucose-dilution CR, and sirtuin loss-of-function in flies and worms impairs CR's lifespan-extending effects depending on the CR protocol used. Collectively, these observations support the idea that sirtuins represent a conserved pro-longevity mechanism responsive to nutrient availability.",Nutrients,Sirtuin Function and Longevity,2020
CR Protocol Dependence and Complexity of Sirtuin-Mediated Longevity,"The requirement for sirtuins in CR-mediated longevity varies strongly depending on the organism and the specific CR regimen. In yeast, mild CR (0.5% glucose) requires SIR2 for lifespan extension, whereas severe CR (0.05% glucose) extends lifespan independently of SIR2, suggesting that extreme nutrient depletion may trigger fasting-like mechanisms distinct from classical CR. Similar protocol-dependent effects appear in worms: the necessity of sir-2.1 for CR longevity depends on the method of dietary reduction. In flies, sir2 overexpression extends lifespan but is not additive with CR, and CR does not increase lifespan in sir2 null mutants, implying that sirtuin activation is an obligatory component of fly CR response. Importantly, sirtuin dosage is critical—excessive overexpression (>45-fold) shortens lifespan, whereas modest overexpression (≤11-fold) increases it. This dosage sensitivity likely contributes to conflicting claims in earlier literature regarding the reproducibility of sirtuin-mediated longevity.",Nutrients,CR Protocol Dependence of Sirtuin Effects,2020
Sirtuins in Mammalian CR and the Open Question of IF,"In mammals, sirtuins (SIRT1–SIRT7) similarly regulate metabolic, stress-responsive, and chromatin-associated processes. Mouse knockout studies of SIRT1 demonstrate that CR fails to extend lifespan in the absence of SIRT1, indicating that sirtuin-dependent mechanisms are conserved in vertebrates. Several studies show that activating sirtuins genetically or pharmacologically extends lifespan in mice. However, despite strong evidence that NAD⁺ rises during fasting and that sirtuins contribute to beneficial fasting-induced physiological responses, the role of sirtuins in lifespan extension by intermittent fasting (IF) remains poorly defined. No animal studies have yet directly tested whether sirtuin loss abrogates IF-mediated longevity. A recent discovery showing that fasting induces the mitochondrial sirtuin dSirt4 in flies—and that overexpressing dSirt4 extends lifespan—suggests a promising link between fasting-specific pathways and sirtuin regulation. Because sirtuin levels themselves can have opposite effects on lifespan depending on dosage, profiling sirtuin expression across different CR and IF protocols will be essential for disentangling their mechanistic roles.",Nutrients,Sirtuins in CR vs IF Longevity Pathways,2020
Circadian Clock as a Regulator of CR-Mediated Longevity,"Circadian (~24 h) clocks regulate rhythmic metabolic, physiological, and behavioral processes through oscillatory transcriptional control of output genes. Misalignment between internal clocks and the external light–dark cycle is linked to metabolic dysfunction, accelerated aging, and increased risk of age-related disease. Because circadian rhythms coordinate nutrient processing and energy utilization, they were proposed to mediate beneficial effects of caloric restriction (CR). Recent evidence shows that CR enhances the amplitude of core circadian genes in the mouse liver, restoring rhythmicity that typically declines with age. This suggests that CR may promote longevity by reinforcing metabolic rhythmicity. Genetic studies support this idea: CR fails to extend lifespan in Bmal1 knockout mice, demonstrating that a functional circadian clock is required for CR-mediated longevity. In flies, CR similarly enhances the amplitude of core clock gene oscillations, and mutations in key clock components such as timeless impair the lifespan extension normally induced by CR. These findings highlight the circadian clock as a conserved determinant of CR-driven lifespan extension across species.",Nutrients,Circadian Clock and CR Longevity Mechanisms,2020
Clock Control of Metabolic Pathways Linked to CR and IF,"Many major nutrient- and stress-responsive pathways implicated in CR-mediated longevity—including GH/IGF-1, FOXO, TOR, AMPK, sirtuins, and NRF2—are under circadian regulation. This positions the circadian clock as a potential master coordinator of longevity pathways. During the fasting phase (night in nocturnal rodents), NAD⁺ levels rise and activate sirtuins, while reductions in ATP:AMP ratio suppress IIS/TOR signaling and activate AMPK and FOXO, thereby promoting catabolic, pro-longevity processes. During feeding, elevated nutrient availability increases IIS/TOR activity and reduces AMPK signaling, fostering anabolic processes that may accelerate aging. These temporal oscillations suggest that both CR and intermittent fasting (IF) rely on circadian coordination to optimize metabolic transitions between anabolic and catabolic states. Thus, the circadian system may relay anti-aging signals by regulating the timing at which CR- and IF-associated metabolic events occur.",Nutrients,Circadian Regulation of CR/IF Signaling Pathways,2020
Fasting Duration vs Caloric Intake: Evidence for Circadian Timing as a Key Factor,"A major unresolved question concerns whether the benefits of CR and IF arise primarily from reduced calories, nutrient composition, rhythmicity of feeding–fasting cycles, or fasting duration itself. Emerging evidence points to fasting time and circadian alignment as dominant drivers of longevity. Mice on CR naturally consume all food rapidly, then undergo extended fasting periods, effectively self-imposing time-restricted feeding (TRF). Experimental studies show that TRF extends lifespan even in mice fed an ad libitum caloric load, demonstrating that feeding schedule alone can override the negative effects of calorie excess. Conversely, reducing calories without imposing fasting fails to extend lifespan. These findings imply that eating patterns—especially circadian fasting duration—may be more critical for longevity than caloric intake itself. Nutrient-specific interventions such as methionine restriction may also extend lifespan partially by altering feeding rhythms in ways similar to TRF. Importantly, invertebrate models like flies and worms do not undergo enforced fasting during CR, suggesting that CR and IF operate through overlapping but distinct mechanisms across species.",Nutrients,Fasting Duration and Circadian Feeding Patterns in Longevity,2020
"Distinct Transcriptional Signatures of CR, TRF, and Starvation","Genome-wide expression analyses reveal that CR, TRF, and starvation produce distinct transcriptional signatures. In flies, TRF-induced changes differ markedly from both CR and prolonged starvation, indicating TRF engages unique circadian-driven pathways rather than simply mimicking nutrient deprivation. TRF ameliorates age-related cardiac dysfunction independently of CR and starvation, and its benefits rely on circadian clock function and the TCP-1 ring complex chaperonin. These findings reinforce the idea that circadian timing—not caloric deficit—drives many TRF-specific longevity effects. Although yeast and worms lack canonical circadian clock genes, they exhibit oscillatory metabolic behaviors whose interactions with CR remain to be fully investigated. These oscillations may represent evolutionarily ancient timing mechanisms that modulate nutrient-response pathways, raising the possibility that circadian-like regulation contributes to CR benefits even in organisms without a classical clock system.",Nutrients,Distinct Molecular Signatures of CR vs TRF vs Starvation,2020
"Diet, Meal Timing, and Longevity Overview","Reduced food intake, avoiding malnutrition, can ameliorate aging and aging-associated diseases in invertebrate model organisms, rodents, primates, and humans. Recent findings indicate that meal timing is crucial, with both intermittent fasting and adjusted diurnal rhythm of feeding improving health and function, in the absence of changes in overall intake. Lowered intake of particular nutrients rather than of overall calories is also key, with protein and specific amino acids playing prominent roles. Nutritional modulation of the microbiome can also be important, and there are long-term, including inter-generational, effects of diet. The metabolic, molecular, and cellular mechanisms that mediate both improvement in health during aging to diet and genetic variation in the response to diet are being identified. These new findings are opening the way to specific dietary and pharmacological interventions to recapture the full potential benefits of dietary restriction, which humans can find difficult to maintain voluntarily.",Cell,"Diet, Meal Timing, Nutrient Intake",2015
Introduction to Dietary and Genetic Interventions,"The discovery that aging can be ameliorated by dietary, genetic, and pharmacological interventions has opened up the prospect of a broad-spectrum, preventive medicine for aging-related diseases. Single-gene mutations that extend animal lifespan can ameliorate natural, age-dependent loss of function and the pathology of aging-related diseases, including neurodegeneration. Furthermore, laboratory animal models of slowed aging, naturally long-lived species such as the naked mole rat, and some humans that achieve the age of 100 have all demonstrated that a long life is not inevitably associated with late-life disability and disease. Recent work has shown that specific dietary interventions can also promote long life and healthy old age.",Cell,Dietary and Genetic Longevity Interventions,2015
Effects of Dietary Restriction in Mammals,"Dietary restriction (DR), implemented as chronic and coordinate reduced intake of all dietary constituents except vitamins and minerals, was first shown 80 years ago to extend lifespan in rats. DR in both rats and mice improves most aspects of health during aging. Exceptions include resistance to infection and wound healing. However, these conditions rapidly improve with re-feeding, and DR animals can then outperform controls. DR can produce substantial benefits with, for instance, ~30% of DR animals dying at old ages without gross pathological lesions, compared with only 6% of ad-libitum-fed controls. DR started in young, adult Rhesus monkeys greatly improves metabolic health; prevents obesity; delays the onset of sarcopenia, presbycusis, and brain atrophy; and reduces the risk of developing and dying of type 2 diabetes, cancer, and cardiovascular disease.",Cell,Dietary Restriction in Mammals,2015
Dietary Restriction in Humans,"In humans, severe food restriction without malnutrition results in many of the same physiological, metabolic, and molecular changes associated with DR in animals, including less age-associated myocardial stiffness and autonomic dysfunction, lower core body temperature, and downregulation of the pi3k/akt/foxo and inflammatory pathways in skeletal muscle. Furthermore, humans voluntarily undertaking long-term DR score lower than controls on multiple risk factors for cardiovascular disease and cancer. In short-term, randomized clinical trials in aging humans, DR improves several markers of health. However, severe DR with adequate nutrition is not an option for most people because it is difficult to practice and sustain and, with inadequate nutrition, can increase the risk of impaired menstrual and reproductive function, osteoporotic bone fractures, anemia, and cardiac arrhythmias. Dietary interventions that avoid unrealistic levels of self-deprivation, and pharmacological interventions that recapture beneficial effects of DR, are therefore important goals to improve human health during aging.",Cell,Dietary Restriction in Humans,2015
Intermittent Fasting in Evolution and Model Organisms,"Meal Frequency and Timing
Only recently have humans and domesticated animals had constant access to food. During their evolution, many animals and humans ate only intermittently. For many microorganisms and invertebrates, long periods of starvation are normal and, correspondingly, many of them (including C. elegans) have evolved forms of quiescence in response to the onset of food shortage. Many of the genes that control quiescence are also important in the control of lifespan (Baugh, 2013). Interestingly, intermittent fasting (IF), with alternation of 2 days of ad libitum feeding with 2 day fasting, also extends worm lifespan, through a mechanism involving the small GTP-ase RHEB-1 and insulin/Igf signaling (Honjoh et al., 2009). Even chronic starvation extends lifespan in C. elegans (Kaeberlein et al., 2006; Lee et al., 2006), through mechanisms that overlap with those mediating the response to IF and that include the combined activity of FOXO, FOXA, and AP-1 transcription factors in two parallel starvation-responsive pathways (Uno et al., 2013).",Cell,Intermittent Fasting in Evolution and C. elegans,2015
Intermittent Fasting Lifespan Effects in Rodents,"In rodents, both fasting for 24 hr every other day or twice weekly extends lifespan up to 30%, independent of both total food intake and weight loss (Mattson et al., 2014). As for chronic DR, the magnitude of the life extension induced by IF can be influenced by the age of initiation and mouse genotype. In A/J mice, for example, IF started at 6 months did not increase lifespan and, when started at 10 months of age, reduced mean lifespan by 15% (Goodrick et al., 1990), although it is not clear whether a more or less severe form of IF could extend lifespan in these mice. IF can also protect against obesity, cardiovascular disease, hypertension, diabetes, neurodegeneration, and the clinical progression of several neurodegenerative diseases (Mattson et al., 2014). In contrast, although many studies report a protective effect against cancer progression (Berrigan et al., 2002; Lee et al., 2012), others suggest detrimental cancer initiation and promotion (Tessitore and Bollito, 2006). In rodents, multiple changes mediate benefits of fasting, including increased production of the neurotrophic factors BDNF and FGF2, reduced inflammation and oxidative stress, and enhanced cellular and molecular adaptive stress responses.",Cell,Intermittent Fasting in Rodents,2015
Molecular and Cellular Effects of Fasting,"Treatment of cells and mice with bOHB, an endogenous histone deacetylase inhibitor powerfully induced by fasting, protects against oxidative stress (Shimazu et al., 2013). Fasting improves mitochondrial function, stimulates the production of chaperones such as HSP-70 and GRP-78, and, by inhibiting the AKT/mTOR pathway, triggers autophagy and DNA repair pathways in multiple cell types (Brown-Borg and Rakoczy, 2013; Mattson et al., 2014). Moreover, in mice, multiple cycles of fasting modulate hematopoietic stem cell protection, self-renewal, and regeneration via IGF-1 or PKA inhibition (Cheng et al., 2014). Finally, short-term fasting (1–3 days) has been shown to protect rodents against the damage induced by ischemia-reperfusion of the liver and kidney, by improving insulin sensitivity, reducing expression of markers of inflammation and insulin/igf-1 signaling, and increasing cytoprotective gene expression (Hine et al., 2015).",Cell,Fasting-Induced Molecular Mechanisms,2015
Human Clinical Trials of Intermittent Fasting,"Many trials on the effects of IF in humans are underway. A 6 month, randomized, clinical trial in overweight or obese premenopausal women showed that fasting for 2 non-consecutive days per week results in reduced body weight, fat mass, and waist circumference and also reduced serum concentrations of total and LDL cholesterol, triglycerides, C-reactive protein, and arterial blood pressure. Several serum biomarkers of cancer risk also improved, but total and free IGF-1 did not change (Harvie et al., 2011). Similarly, in three small, short-term (8–12 weeks), randomized clinical trials in non-obese and obese individuals, alternate day fasting lowered body weight, fat mass, and risk factors for cardiovascular disease (Kroeger et al., 2014). Preliminary evidence from dogs and humans suggests that short-term fasting (24–48 hr) prior to chemotherapy reduces some chemotherapy-associated side effects by protecting normal cells, but not cancer cells, from toxicity (Safdie et al., 2009; Withers et al., 2014).",Cell,Intermittent Fasting in Humans,2015
Time-Restricted Feeding and Circadian Effects,"Patterns of eating over the day can also have substantial effects. Limiting daily food intake of an isocaloric diet to a 5 to 7 hr time window in humans can induce health benefits compared with a standard three to five meals per day (Mattson et al., 2014). Delaying feeding until the evening in diurnally active fruit flies, which normally eat predominantly in the morning, causes an uncoupling of their metabolic cycle from the central circadian rhythm and reduces egg laying (Gill and Panda, 2011). Time-restricted feeding of mice during 8 hr of the dark phase of the daily cycle does not affect overall calorie intake of mice on a high-fat diet but restores normal circadian rhythms of activity in metabolic pathways and protects the mice against weight gain, fat accumulation, inflammation, glucose intolerance, insulin resistance, and loss of endurance and motor coordination (Chaix et al., 2014). Humans who eat and sleep ~12 hr out of phase from their habitual patterns experience increased blood pressure, worsening of glucose tolerance, a reduction of the satiety hormone leptin, and a complete inverse pattern of the cortisol rhythm (Scheer et al., 2009).",Cell,Time-Restricted Feeding and Circadian Rhythm,2015
"Circadian Disruption, Meal Timing, and Longevity","Although randomized clinical studies on effects of chronic disruption of meal patterns and circadian rhythms, as in shift workers, have yet to be performed, epidemiological data suggest an increased risk of obesity, type 2 diabetes, cardiovascular disease, cancer, and neurodegenerative diseases (Wang et al., 2011). Furthermore, 30% calorie restriction by dietary dilution, in which mice ate all day to compensate for the low energy density of the diet, had no beneficial effects on lifespan (Solon-Biet et al., 2014), possibly because of the disrupted meal pattern. Long-lived, hungry DR mice and rats consume their restricted portion of food rapidly, with an extended period of fasting (22–23 hr) between meals. Chronic DR may hence improve health at least in part through IF. Similarly, in the Wisconsin Rhesus monkey DR trial, the animals fed mainly once a day—in the National Institute on Aging (NIA) trial twice daily—which may have contributed to the Wisconsin DR monkeys having a 1.83 lowered rate of death from any cause in contrast with the absence of a difference in death rate in the NIA study. Interestingly, both in overweight/obese and lean women with polycystic ovary syndrome, subjects randomized to earlier meal timing lost more weight, displayed higher insulin sensitivity, lower serum testosterone concentration, and increased ovulation rate than controls eating isocaloric diets with a later meal pattern.",Cell,Circadian Disruption and Meal Timing,2015
Energy Sensing Pathways in Meal Timing,"The molecular mechanisms responsible for the effects of altered meal patterns on metabolic health are not fully understood. There may be compensatory changes in energy sensing pathways, such as AMPK, AKT/mTOR, and cyclic AMP response element binding protein (CREB), which are all implicated in cellular homeostasis and rhythmic oscillations of circadian clock targets (Mattson et al., 2014).",Cell,Energy Sensing Pathways and Meal Timing,2015
Calorie Intake vs Specific Nutrients,"Calories or Specific Nutrients?
Determining the optimal overall intake and dietary ratios of carbohydrate, fat, and protein is challenging. The effects of reduced consumption of a specific macronutrient will depend partly upon how much of it is consumed by the controls and also upon the composition of the rest of the diet. Even for just three macronutrients, the possible combinations are vast. Combinatorial effects can be explored using a geometric framework (see the Essay by Simpson et al. (2015) on page 18 of this issue), but this is often not feasible in practice. Dietary composition can also affect overall food intake and its timing through effects on hedonistic value and satiety. Although such mechanisms are important to understand, they can also complicate analysis of the effects of nutrient intake per se. The effects of diet on health may also be age specific, a possibility that is starting to be explored experimentally in humans (Wolfe et al., 2008).",Cell,Calorie vs Nutrient Contribution,2015
Macronutrients and Lifespan,"Macronutrients
Until recently, reduced intake of calories, rather than of specific macronutrients, was considered important for health benefits of DR. This assumption was primarily based on a flawed interpretation of experimental data showing that 40% calorie restriction, but not 40% protein restriction, increased lifespan in rats (Maeda et al., 1985). However, the protein-restricted rats were not food restricted, because their growth rate was normal, a point overlooked by the authors of the study. A subsequent series of studies in yeast, invertebrate model organisms and rodents has instead clearly demonstrated that a reduction in specific nutrients in the diet, rather than reduced calorie intake, is primarily responsible for improvements in health and extended lifespan, which is why we use the term DR rather than CR.",Cell,Macronutrients in Dietary Restriction,2015
Protein Intake and Healthspan,"Protein and Amino Acids
Dietary guidelines from the medical literature and popular press often promulgate high protein intake, especially from animal sources rich in essential amino acids, including sulfur-containing and branched chain, to combat obesity, sarcopenia, osteoporosis, frailty, surgical stress, and mortality. However, accumulating evidence points instead to a restriction of protein or specific amino acids in the diet as promoting healthspan (Grandison et al., 2009; Solon-Biet et al., 2014; Ables et al., 2014; Nakagawa et al., 2012; Pamplona and Barja, 2006; Mirzaei et al., 2014). This conclusion initially emerged from investigation of the nutrients mediating the effects of DR and has since been amplified in broader studies of the effects of dietary composition and intake on health and lifespan.",Cell,Protein Restriction and Healthspan,2015
Amino Acid Restriction in Model Organisms,"In Drosophila, restriction of protein-containing yeast, but not carbohydrate or energy, extends lifespan (Mair et al., 2005). Adding back essential amino acids to the diet of DR flies decreases lifespan, with the addition of non-essential amino acids, lipid, or carbohydrate exerting little or no effect (Grandison et al., 2009). In mice, also, health and lifespan are strongly affected by the protein component of the diet, with median lifespan progressively increasing up to 30% as the dietary protein-to-carbohydrate ratio is decreased, despite a parallel increase in overall food intake and body fat and reduction in lean mass (Solon-Biet et al., 2014).
Dietary protein intake is an important regulator of the IGF-1/mTOR network (Efeyan et al., 2012). In humans, unlike rodents, chronic severe calorie restriction does not reduce serum IGF-1 concentration unless protein intake is also reduced (Fontana et al., 2008), suggesting that dietary protein or specific amino acid intake may be as or more important than calorie intake in modulating IGF-related biological processes and disease risk in men and women.",Cell,Protein and Amino Acid Restriction Effects,2015
"Protein, IGF-1, and Cancer Risk","In rodents, over-stimulation of the GH/IGF pathway accelerates aging and increases mortality, whereas downregulation slows aging, prevents cancer, and increases lifespan (Junnila et al., 2013). Moreover, serum IGF-1 concentration is inversely correlated with median lifespan in 31 genetically diverse, inbred mouse strains (Yuan et al., 2009) and with the risk of developing some common cancers in humans (Pollak, 2012). Interestingly, isocaloric restriction of protein and substitution of plant for animal proteins markedly inhibit prostate and breast cancer growth in human xenograft animal models of neoplasia, with reduced serum IGF-1 levels and downregulation of intratumor mTOR activity, histone methyltransferase EZH2, and the associated histone mark H3K27me3, two epigenetic markers of prostate cancer progression (Fontana et al., 2013).",Cell,"Protein Intake, IGF-1, and Cancer",2015
Specific Amino Acids and Longevity Mechanisms,"Within the protein component of the diet, specific amino acids or their ratio can be important for health and lifespan. Selective restriction of asparagine, glutamate, or methionine in the medium has been shown to extend chronological lifespan in yeast (Dilova et al., 2007; Wu et al., 2013a, 2013b). In Drosophila and rodents, restriction of methionine and tryptophan, respectively, extends average and maximal lifespan (Ables et al., 2014; Miller et al., 2005; Zimmerman et al., 2003). In the DR Rhesus monkey trials, the Wisconsin diet contained higher concentrations of methionine and branched-chain amino acids derived from lactalbumin than did the NIA diet (Table 2), which could explain some of the differences in effects on cancer and mortality.
Amino acid availability is sensed by multiple evolutionarily conserved molecular pathways, the most important being mTOR and GCN2. Activation of mTOR is modulated by different essential amino acids in a tissue-specific manner, with branched-chain amino acids playing a key role (Efeyan et al., 2012). In contrast, absence of any individual amino acid activates GCN2, which is required for the protective effect of short-term protein or tryptophan deprivation on surgical stress in mice (Peng et al., 2012).",Cell,Amino Acid Modulation of Longevity Pathways,2015
Methionine Restriction and Trans-Sulfuration Pathway,"The protein/amino acid restriction-induced protective mechanisms against stress, damage accumulation, and aging downstream of TOR inhibition and GCN2 activation are unknown. Nonetheless, GCN2 activation stabilizes ATF4, a transcription factor essential for the Integrated Stress Response, which is elevated in several dietary, genetic, and pharmacological animal models of longevity (Li et al., 2014; Hine et al., 2015).
Reduced dietary methionine, in particular, induces specific, protective, molecular mechanisms. The sulfur-containing amino acids methionine and cysteine are metabolized through the trans-sulfuration pathway, lesions in which are associated with increased incidence of age-related pathologies in humans. DR in Drosophila results in increased activity of the rate-limiting enzyme in the trans-sulfuration pathway, and inhibition of the pathway blocks the response of fly lifespan to DR. Furthermore, transgene-mediated increase in activity of the pathway increases fly lifespan (Kabil et al., 2011). Interestingly, the trans-sulfuration pathway is the primary source of hydrogen sulfide in cells, and hydrogen sulfide can increase lifespan in C. elegans (Miller and Roth, 2007).",Cell,Methionine Restriction and Trans-Sulfuration,2015
"Hydrogen Sulfide, GH Pathway, and Longevity","Furthermore, a trans-sulfuration-pathway-dependent increase in production of hydrogen sulfide is seen in yeast, worm, fruit fly, and rodent models of DR, and in mice DR-induced resistance against ischemia reperfusion injury requires this increase (Hine et al., 2015). Interestingly, in the long-lived Ames dwarf mouse, which lacks growth hormone, expression of genes in the trans-sulfuration pathway and the flux of methionine to the pathway are increased, associated with higher levels of GSH (Uthus and Brown-Borg, 2006). The lifespan of Ames dwarf mice and of mice lacking the growth hormone receptor is extended relative to controls on normal diet but does not respond to methionine restriction and then becomes similar to that of controls (Brown-Borg et al., 2014). Thus, the increased health during aging of these somatotropic mutant mice may be at least in part attributable to increased trans-sulfuration activity.",Cell,Hydrogen Sulfide and Somatotropic Longevity,2015
Protein Restriction in Humans,"In humans, little is known on the effects of dietary modifications of protein quantity and quality in modulating molecular pathways that control aging, stress resistance, and age-associated diseases. Nonetheless, ongoing clinical trials should soon begin to reveal the metabolic and molecular adaptations induced by protein restriction and alterations of amino acid intake in relativity healthy overweight human subjects and in cancer patients.",Cell,Protein Restriction in Human Aging,2015
Gut Microbiota and Nutrient-Responsive Signaling,"Microbiota-Derived Factors and Healthy Aging
In humans, only ~10% of cells and less than 1% of genes are human, and the rest come from trillions of microbes in the gastrointestinal tract. Rapidly accumulating metagenomic data indicate that altered food intake, especially protein and insoluble fiber, have rapid and profound effects on gut microbiota structure, function, and secretion of factors that modulate multiple inflammatory and metabolic pathways (Muegge et al., 2011; Clemente et al., 2012; David et al., 2014; Thorburn et al., 2014). G-protein-coupled receptors expressed by enteroendocrine and immune cells may be important mediators of the effects of the microbiome (Thorburn et al., 2014). For example, oral administration to mice of 17 non-pathogenic Clostridia species isolated from healthy human fecal samples results in gut microbiota that provide short-chain fatty acids, bacterial antigens, and a TGF-b-rich environment, which help differentiation, expansion, and colonic homing of Treg cells and reduce disease severity in multiple models of colitis and allergic diarrhea (Atarashi et al., 2013). In contrast, diet-induced microbiota dysbiosis is associated with increased risk of developing cardiovascular disease, obesity-associated metabolic abnormalities, cancer, and autoimmune and allergic disease (Clemente et al., 2012).",Cell,Gut Microbiota and Dietary Modulation,2015
Microbiome Influences on C. elegans Longevity,"In nature, C. elegans feeds on a variety of bacterial species that grow on rotting vegetation, which also constitute the gut microbiome of the worm. Feeding C. elegans with soil bacteria, Bacillus mycoides, and Bacillus soli instead of the standard laboratory E. coli OP50 strain, significantly extended lifespan and stress resistance, suggesting that microbial-derived factors may modulate pro-longevity pathways (Abada et al., 2009). Moreover, wild-type C. elegans fed respiratory-incompetent E. coli show increased lifespan (Saiki et al., 2008). A comparison of the effects of E. coli and Comamonas aquatica on the C. elegans host identified vitamin B12 as a major diffusable factor from Comamonas that influenced patterns of gene expression and the rate of development and fertility of the worm (Watson et al., 2014). Interestingly, effects of chemicals on the worm can also be mediated by the gut microbiome. Metformin, the drug used as the first line of defense against type 2 diabetes, extends lifespan of C. elegans fed on live, but not killed, E. coli, and it does so by disrupting the folate cycle and methionine metabolism of the E. coli. In consequence, the supply of bacterial methionine to the worm is reduced, inducing a type of methionine restriction, which is consistent with the action of metformin as a DR mimetic (Cabreiro et al., 2013).",Cell,Microbiome Effects in C. elegans,2015
Microbiome Effects in Drosophila and Aging,"The relatively simple gut microbiome of Drosophila is also derived from its food intake, and bacterial density and composition have a substantial effect upon the fly host (reviewed in Erkosar and Leulier, 2014). Bacterial density increases during the aging of the fly and can compromise gut integrity (Guo et al., 2014). The normal complement of gut bacteria enables the flies to use low-nutrient or unbalanced diets by providing them with B vitamins, particularly riboflavin, and by promoting protein nutrition (Wong et al., 2014). Alterations in the gut microbiome may contribute to the improvement in health from DR and time-restricted feeding. Long-term dietary restriction of mice, with either a normal or a high-fat diet, leads to alterations in composition of the gut microbiome, although any contribution of these changes to the health of the DR mice remains to be elucidated (Zhang et al., 2013). In mice on a high-fat diet, time-restricted feeding during 8 hr of the dark phase decreased representation of Lactobacillus species, which are associated with obesity, and increased Ruminococcaceae species, which protect against metabolic disease associated with obesity (Zarrinpar et al., 2014). Experimental transplantations of microbiota associated with healthy eating could be revealing of their causal role.",Cell,Microbiome and Aging in Drosophila and Mice,2015
Timescales of Dietary Restriction Effects,"Health, Disease, and Longevity on Various Timescales, Including Inter-generational
The effects of nutrition, including DR, can be exerted on timescales ranging from more or less instantaneous to inter-generational. For instance, in Drosophila, DR acts acutely, with flies switched between DR and ad libitum feeding almost immediately adopting the mortality pattern of a control group kept permanently in the feeding regime that the switched flies join (Mair et al., 2003). At least as reflected in their mortality rate, these flies thus have no memory of their nutritional history, and their patterns of gene expression also change rapidly in response to DR (Whitaker et al., 2014).",Cell,Short-Term Responses to Dietary Restriction,2015
Dietary Memory in C. elegans and Rodents,"In contrast, the mortality rates of C. elegans subjected to one form of DR retain a permanent memory of the previous feeding regime after a dietary switch (Wu et al., 2009). The mortality rates of mice and rats subjected to switches between DR and AL feeding later in life have shown mixed responses, but a meta-analysis suggests that there is a permanent memory of diet in these animals (Simons et al., 2013). Similarly, even short episodes of DR early in adulthood in male mice can induce a glycemic memory apparent as increased glucose tolerance (Cameron et al., 2012; Selman and Hempenstall, 2012).",Cell,Dietary Memory and Metabolic Programming,2015
Acute Physiological Responses to Dietary Restriction,"However, in mice and humans, acute responses to DR also occur, including improved insulin sensitivity, reduced inflammation, and protection against ischemia reperfusion injury and other surgical stressors (Hine et al., 2015).",Cell,Acute Physiological Responses to DR,2015
Developmental Programming and Early-Life Nutrition,"Developmental Programming
In contrast to immediate effects of diet, in mammals (including humans), nutrition in early life (including in utero) can have lasting effects on health during aging, often referred to as developmental programming. For instance, undernourished rat and mouse mothers produce offspring with low birth weight and multiple metabolic defects, including early-life adiposity, altered pancreatic function, and progressive glucose intolerance (Tarry-Adkins and Ozanne, 2014; Vickers, 2014). Maternal effects on offspring can include changes to the composition of the egg, alterations to the environment in utero, and peri-natal effects such as transmission of the microbiome and alterations to lactation, and can be manifest in the offspring as changes in gene expression and epigenetic modifications, including DNA methylation, histone modification, and expression of microRNAs, as well as evidence of increased cellular aging (Aiken and Ozanne, 2014; Colaneri et al., 2013; Radford et al., 2012).",Cell,Developmental Programming and Early Nutrition,2015
Human Evidence and Thrifty Phenotype Hypothesis,"Epidemiological data from humans also show a consistent effect of developmental programming by early—including in utero—nutrition, although the evidence on the mechanisms involved is necessarily correlational rather than experimental. The thrifty phenotype hypothesis (Hales and Barker, 2001) postulates that many of the changes in organ structure and metabolism seen in humans in response to restricted nutrition—particularly of protein—in utero can be understood as the consequences of immediate responses of the fetus to ensure survival and spare vital organs such as the brain. Viewed in this way, an under-nourished fetus makes the best of a bad job with adverse consequences for health in later life, including reduced glucose tolerance and a higher incidence of ischemic heart disease, problems that are greatly exacerbated by subsequent adequate or over-nutrition.",Cell,Thrifty Phenotype and Long-Term Health,2015
Adaptive vs Maladaptive Responses to Low Nutrition,"However, a poor functional capacity for insulin secretion would not be detrimental to individuals who continued to be poorly nourished and remained thin and, therefore, insulin sensitive, and it remains possible that some fetal and post-natal responses to low nutrition are advantageous in conditions of continuing poor nutrition.",Cell,Adaptive Responses to Early-Life Undernutrition,2015
Inter-generational Signaling and Predictive Adaptive Responses,"Inter-generational Effects of Diet
Current nutrition may act as a predictor of future nutritional conditions if food availability shows local variation or if timing of natural cycles of food scarcity and abundance occurs on an appropriate timescale. Under these circumstances, information gained early in life or even in earlier generations could be profitably used to anticipate future nutritional prospects and adjust physiology accordingly (Rando, 2012). Such considerations may partly explain why inter-generational effects of diet can also be transmitted through males. The evidence for a role of epigenetic inheritance in these cases is largely correlational, and a direct experimental testing of the hypothesis is challenging (Heard and Martienssen, 2014; Rando, 2012). Cellular mechanisms by which metabolic changes can be communicated to chromatin are being increasingly discussed (Gut and Verdin, 2013; Katada et al., 2012; Lu and Thompson, 2012).",Cell,Inter-generational Dietary Signaling,2015
"Paternal Diet, Chromatin Reprogramming, and Offspring Metabolism","In Drosophila, the sugar content of the paternal diet, even over a 2 day period during which the offspring are sired, can elicit increased lipid content in offspring. Sugar in the diet de-silences chromatin-state-defined domains both in mature sperm and in offspring embryos, and H3K9/K27me3-dependent reprogramming of metabolic genes in two time windows in the germline and the zygote is required for increased lipid content of offspring. Furthermore, data from mice and humans, including discordant human monozygotic twins, show a similar signature of chromatin de-repression associated with obesity (Öst et al., 2014).
Livers of offspring of male mice fed a low-protein diet show elevated expression of genes involved in lipid and cholesterol biosynthesis. These alterations are accompanied by subtle (in the region of 20%) changes in DNA cytosine methylation in specific gene regions, including a putative enhancer for the lipid regulator Ppara (Carone et al., 2010).",Cell,Paternal Diet and Epigenetic Reprogramming,2015
Epigenetic Transmission of Undernutrition Effects,"Mice subjected to in utero undernourishment are glucose intolerant, and they can transmit the glucose intolerance even though they themselves are not undernourished. They experience the effects of maternal undernourishment during a period that includes the time when their germ cell DNA reacquires methylation. The sperm DNA of these males is hypomethylated at multiple sites, especially ones enriched in regulatory elements and regulators of chromatin. Although these altered methylation patterns are not apparent in the tissues of their offspring, there are perturbations to gene expression, possibly attributable to other types of epigenetic alteration (Radford et al., 2014).",Cell,Transgenerational Epigenetic Effects of Undernutrition,2015
Starvation-Induced Small RNAs and Multigenerational Longevity,"Evidence is also starting to point to truly inter-generational effects of diet, where information about dietary history is epigenetically transmitted in the germline in the absence of any further input from the organism or its environment. For instance, in the nematode worm Caenorhabditis elegans, starvation-induced developmental arrest has effects that persist for at least three generations, with the third generation offspring of the starved great-grandparents showing increased adult lifespan. The starvation event leads to the generation of small RNAs that are also inherited for at least three generations, that target the mRNAs of genes involved in nutrient reservoir activity (Rechavi et al., 2014), and that are possibly also causal in the increased lifespan of the third generation descendants. It is at present not clear whether these more persistent effects of diet represent non-adaptive perturbations to physiology, anticipatory programming of an adaptive response to nutritional circumstances, or both.",Cell,Small RNAs and Multigenerational Longevity,2015
Human Inter-generational Dietary Effects and Parent-of-Origin Influence,"Effects of nutrition of the paternal grandfather on grandchildren have been reported in humans, but the mechanisms responsible are unknown (Pembrey et al., 2014). Recent work with mice has suggested that sex-of-parent-of-origin effects may be much more pervasive and influential than previously supposed. Even though the number of imprinted genes in the mammalian genome is predicted to be small, non-imprinted genes can regulate the tissue-specific expression of many other genes differently when transmitted by females or males, possibly by physical interaction with imprinted loci (Mott et al., 2014). These findings could have profound implications for human aging and disease.",Cell,Human Inter-generational Diet Effects,2015
Genetic Differences in Dietary Responses,"Genetic Variation in Response to Diet
Individuals of different genotypes can respond differently to diet. Although little studied outside the context of inborn errors of metabolism, such genetic effects in humans are potentially important for identifying sub-groups that would benefit from dietary modulation. Females and males often respond very differently to dietary and pharmacological interventions, and evidence is mounting for the importance of other types of genetic variation.
DR has proved to extend lifespan in most species examined, including many non-model organisms, although it has been suggested that increased lifespan in response to DR may have evolved in part as an artifact of laboratory culture (Nakagawa et al., 2012). However, strains of C. elegans and Drosophila collected directly from nature respond normally to DR (Metaxakis et al., 2014). Wild-derived mice, on the other hand, can show little or no response (Harper et al., 2006). The stresses of captivity in non-domesticated animals could be part of the explanation for this finding.",Cell,Genetic Variation in Dietary Responses,2015
Strain-Specific Variation in Lifespan Response to DR,"In addition, wild mice may respond to milder or stronger DR than the single level used in the investigation (Gems et al., 2002). A similar experimental approach applied to recombinant inbred mouse strains, using a single, 40% reduction in overall intake showed a range of responses from a 98% extension of lifespan to a 68% reduction (Liao et al., 2010). A wider range of reductions intake would have revealed whether these strains differ only in the intake level at which their lifespan peaks under DR or whether DR indeed does not extend lifespan in some strains.
In general, the lifespans of model organisms show a tent-shaped response to the level of food intake, with peak lifespan at intermediate food intake and a decline through starvation to the left and through increased levels of food intake to the right (Partridge et al., 2005). In order to determine whether a strain or species responds to DR, a range of degrees of food intake should therefore be explored (Gems et al., 2002). The apparent lack of response of lifespan to DR trials in the NIA rhesus monkey trial may have been at least in part attributable to the 5%–10% DR applied to the control animals, which may have been sufficient to bring them close to peak lifespan.",Cell,Strain-Specific DR Lifespan Responses,2015
Genetic Risk Factors Affecting Dietary Interventions,"Experimental analysis of genetic effects on the response to DR has tended to focus on major genetic variation, caused by either specific gene mutants or inbreeding (Schleit et al., 2013). Although this approach can be informative about mechanisms of the normal response to DR and hence candidates for disease prevention in humans, it may not reveal much about the effects of natural genetic variation in outbred human populations. It is becoming clear that such variation is important. For instance, interventions to reduce weight often have beneficial effects on blood lipid profiles, type 2 diabetes, and risk of cardiovascular disease, but some individuals respond poorly or not at all, thus limiting the effectiveness and increasing the cost of intervention programs.
A study of the effects on blood lipid profiles and diabetes of increased exercise and lowered intake of fat revealed that a higher genetic risk score for dyslipidemia based upon SNP genotyping was associated with a substantially diminished response to intervention (Pollin et al., 2012). Studies of this kind could also throw light on underlying mechanisms and, hence, individually targeted dietary and other types of intervention that could be effective in preventing disease.",Cell,Genetic Modifiers of Diet and Disease Risk,2015
Metabolic Clock and Aging Overview,"Several metabolic alterations accumulate over time along with a reduction in biological fitness, suggesting the existence of a ""metabolic clock"" that controls aging. Multiple inborn defects in metabolic circuitries accelerate aging, whereas genetic loci linked to exceptional longevity influence metabolism. Each of the nine hallmarks of aging is connected to undesirable metabolic alterations. The main features of the ""westernized"" lifestyle, including hypercaloric nutrition and sedentariness, can accelerate aging as they have detrimental metabolic consequences. Conversely, lifespan-extending maneuvers including caloric restriction impose beneficial pleiotropic effects on metabolism. The introduction of strategies that promote metabolic fitness may extend healthspan in humans.

Introduction
The human superorganism (i.e., the host and its microbiome) is a complex metabolic system in which nutrient intake, physical activity, and elimination of waste orchestrate anabolic and catabolic reactions that ultimately determine development, maturation, and aging.",Cell,Metabolic Control of Longevity,2016
Metabolites and Longevity Correlations,"After many years of being subordinate to the surge in cellular and molecular biology, the study of metabolism is now experiencing its own Renaissance. A clear understanding is emerging of the key roles that metabolites play in all biological processes, including physiological and pathological aging. Recent trans-species comparisons have tried to link longevity with metabolic parameters from different organs (Ma et al., 2015a). These studies have revealed a positive correlation between longevity and sphingomyelin levels. Conversely, the levels of triacylglycerols containing polyunsaturated fatty acid (PUFA) side chains and by-products of inflammatory processes correlate negatively with longevity. Consistently, female familial longevity in humans has been associated with high levels of plasma sphingomyelin and low levels of PUFA-containing triacylglycerols (Gonzalez-Covarrubias et al., 2013). Longevity across mammalian species also correlates negatively with the hepatic levels of enzymatic cofactors involved in amino acid metabolism and with the hepatic concentrations of tryptophan degradation products.",Cell,Metabolic Control of Longevity,2016
"Amino Acids, Energy Expenditure, and Metabolic Aging","Accordingly, reduction of dietary amino acids—notably, tryptophan and methionine—can extend lifespan in animal models (Fontana and Partridge, 2015). Hence, a reduced energy expenditure per body mass per day (mass-specific basal metabolism) may characterize long-lived mammals. Multiple studies have tried to determine the biological age of humans by measuring proxies including telomere length, gene methylation (that would reflect an ""epigenetic clock""), and transcriptional signatures (that would mirror ""transcriptomic aging"") (Peters et al., 2015). Similarly, it has been attempted to determine aging-related metabolic features. Thus, NMR spectroscopy has been used to measure age-associated changes in the urine metabolome and to determine a ""metabolic age score,"" which was shown to predict survival independent of chronological age and other risk factors (Hertel et al., 2016). Consistently, age-associated alterations and some premature manifestations of age-related diseases in humans can be detected in the serum metabolome and lipidome (Gonzalez-Covarrubias et al., 2013).",Cell,Metabolic Control of Longevity,2016
Hallmarks of Aging and Metabolic Links,"Moreover, two of the nine hallmarks of aging that we have previously defined, namely, ""deregulated nutrient sensing"" and ""mitochondrial dysfunction,"" are tightly linked to metabolic alterations. Similarly, the hallmark ""altered intercellular communication,"" which gathers any age-related change that trespasses the boundaries of single cells, encompasses major biochemical and neuroendocrine alterations affecting whole-body metabolism (Lopez-Otin et al., 2013). In line with this consideration, many human gene variants that increase the likelihood of becoming a centenarian—such as those in forkhead box O3 (FOXO3) and other genes involved in PI3K/AKT1 signaling—are linked to metabolism (Broer and van Duijn, 2015). Moreover, the male offspring of two parents reaching a nonagenarian age exhibits reduced abdominal visceral fat, suggesting that familial longevity is linked to a healthy metabolic profile (Sala et al., 2015). Conversely, many genetic syndromes that cause premature aging in humans are directly linked to metabolic defects.",Cell,Metabolic Control of Longevity,2016
Metabolic Deficiencies and Progeroid Syndromes,"This applies to cutis laxa (defects in proline biosynthesis), Ehlers-Danlos syndrome (deficient proteoglycan synthesis), the Lenz-Majewski hyperostotic dwarfism (defects in phosphatidylserine synthesis), SHORT syndrome (hypomorphic mutations in PIK3R1), and progressive external ophtalmoplegia (mitochondrial DNA instability) (Vermeij et al., 2016). Although most other genetically determined progeroid syndromes stem from alterations in genes that maintain genome integrity, and hence affect metabolism indirectly, these examples underscore a potential key role for metabolic deficiencies in aging. Here, we describe the links between each hallmark of aging and metabolic perturbations, discuss current strategies to manipulate metabolism for increasing healthspan and lifespan, and elaborate on the major threat posed to public health in the developed world, i.e., the incipient ""westernization"" of lifestyle.",Cell,Metabolic Control of Longevity,2016
Hallmarks of Aging and Metabolic Impact,"Metabolic Repercussions of the Hallmarks of Aging
We have previously classified the nine candidate hallmarks of aging into three categories (López-Otín et al., 2013). The primary hallmarks (genomic instability, telomere attrition, epigenetic alterations, and loss of proteostasis) are the main causes of molecular damage underlying aging. The antagonistic hallmarks (deregulated nutrient sensing, mitochondrial dysfunction, and cellular senescence) mediate beneficial effects at low levels and protect the organism from damage and nutrient scarcity but become deleterious at high levels. Finally, the integrative hallmarks (stem cell exhaustion and altered intercellular communication) are the culprits of aging and arise when the accumulating damage cannot be compensated by homeostatic mechanisms. All these denominators of aging have important repercussions on cellular metabolism (Figure 1).",Cell,Metabolic Control of Longevity,2016
Genomic Instability and Metabolic Consequences,"Genomic Instability
Nuclear DNA damage and the consequent activation of repair mechanisms have multiple effects on cellular metabolism. Patients with age-accelerating diseases such as Hutchinson-Gilford progeria syndrome and Werner syndrome frequently develop type 2 diabetes, suggesting that chronic DNA damage can promote metabolic disorders (Shimizu et al., 2014). Mouse models with defective transcription-coupled repair (for instance upon knockout of Ercc1) exhibit multiple biochemical aberrations including altered nutrient sensing, energy metabolism, and redox balance. This complex response to persistent DNA damage not only affects all circuitries underlying bioenergetic metabolism (mitochondrial oxidative phosphorylation, glycolysis, and the pentose phosphate shunt required for antioxidant defense) but also blocks the anabolic reactions driven by trophic signals such as insulin (INS), insulin-like growth factor 1 (IGF1), and growth hormone 1 (GH1), which are required for cell growth and proliferation (Garinis et al., 2008).",Cell,Metabolic Control of Longevity,2016
"DNA Damage Signaling, Inflammation, and Systemic Metabolic Effects","This situation may reflect a strategy whereby cells experiencing DNA damage and other forms of stress favor adaptive processes over anabolic reactions. Indeed, several components of the DNA-repair machinery, including Chek1 and p53, are phosphorylated and inhibited by the growth factor-responsive kinase Akt1 (Vermeij et al., 2016). The systemic or tissue-specific knockout of Ercc1 is sufficient to cause the dissociation of a multiprotein complex containing nuclear receptor corepressor 1 (Ncor1), Ncor2, and histone deacetylase 3 (Hdac3) from the promoters of interleukin 6 (Il-6), Il-8, and tumor necrosis factor (Tnf) in mouse adipocytes. The consequent secretion of pro-inflammatory cytokines promotes chronic inflammation and lipodystrophy. Importantly, the adipocyte-specific knockout of one Ercc1 allele causes type 2 diabetes, confirming that DNA-damage responses in selected cell types can provoke a systemic perturbation of metabolism (Karakasilioti et al., 2013). Cells experiencing DNA damage also secrete type I interferon, which amplifies the response to damage, induces cellular senescence, and inhibits stem cell function. Consistently, suppression of type I interferon signaling abrogates the establishment of various progeroid phenotypes in mice with erosion-prone telomeres (Yu et al., 2015).",Cell,Metabolic Control of Longevity,2016
"PARP1 Hyperactivation, NAD+ Depletion, and Mitochondrial Dysfunction","Some of the best-studied human progerias, ataxia telangiectasia, Cockayne syndrome, and xeroderma pigmentosum, are disorders of nucleotide excision repair characterized by severe neurodegeneration. These diseases are accompanied by the hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1), a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme involved in DNA repair. PARP1 hyperactivation leads to NAD+ depletion, hence inhibiting the NAD+-dependent deacetylase sirtuin 1 (SIRT1) (Fang et al., 2014). These events lead to mitochondrial abnormalities, including reactive oxygen species (ROS) generation, increased transmembrane potential, and limited mitochondrion-selective autophagy (mitophagy), all of which can be rescued in mice by PARP1 inhibition or external supply of the NAD+ precursor nicotinamide riboside (Fang et al., 2014; Scheibye-Knudsen et al., 2014). Mechanistically, these effects (which result in the accumulation of dysfunctional mitochondria) stem from reduced peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PPARGC1A; best known as PGC1a) activity and consequent uncoupling protein 2 (UCP2) downregulation (Fang et al., 2014). Thus, genomic instability may trigger different metabolic alterations that favor cellular senescence and organismal aging.",Cell,Metabolic Control of Longevity,2016
Telomere Attrition and Metabolic Dysfunction in Mice,"Telomere Attrition
Telomere shortening caused by telomerase inactivation can precipitate aging in mice. However, telomerase also has telomere-independent functions that counteract premature aging, a finding that should stimulate further assessments of the relative role of telomerase and telomere erosion in the aging process. Telomere attrition ensuing the knockout of telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) accelerates aging as it causes insulin resistance, β cell failure, and glucose intolerance (Shimizu et al., 2014). Telomere attrition also promotes p53 activation, hence causing the repression of Pgc1a and Pgc1b, the consequent suppression of the transcription factors nuclear respiratory factor 1 (Nrf1), estrogen-related receptor alpha (Esrra), and Ppara, and hence the inhibition of mitochondrial biogenesis and function (Sahin et al., 2011). Thus, telomere erosion may limit mitochondrial turnover and favor age-related metabolic perturbations, at least in mice.",Cell,Metabolic Control of Longevity,2016
Glucose Dependence and Lifespan Effects in Telomerase-Deficient Models,"Telomere dysfunction also enhances the requirement of glucose for the maintenance of energy homeostasis, as well as for mechanistic target of rapamycin (Mtor)- and Igf1-dependent mitochondrial biogenesis, in mouse aging tissues. Accordingly, a glucose-enriched diet significantly extends the lifespan of Terc−/− mice by stimulating glycolysis, mitochondrial biogenesis, and oxidative glucose metabolism (Missios et al., 2014). Several metabolic alterations linked to telomere attrition are detectable in humans. In a cohort of 3,511 women, leukocyte telomere length (LTL) negatively correlated with two markers of oxidative stress, γ-glutamyltyrosine and γ-glutamylphenylalanine, as well as with two lysolipids that are positively associated with phospholipase A2 expression and may reflect poor membrane fluidity (Zierer et al., 2016). These results, which have been confirmed in an independent cohort of 904 women, suggest that telomere length might influence metabolism (or vice versa) in humans. The existence of such a cause-effect relationship remains to be explored in suitable animal models.",Cell,Metabolic Control of Longevity,2016
Human Evidence and Interactions with Other Hallmarks,"Of note, dietary interventions performed in humans suggest that a Mediterranean diet with a strong “anti-inflammatory” profile may slow LTL shortening (García-Calzón et al., 2015), although these findings are mostly correlative. Beyond these pending questions, current evidence indicates that telomere attrition triggers metabolic changes that also impinge on other hallmarks of aging, including mitochondrial dysfunction, stem cell exhaustion, and altered intercellular communication.",Cell,Metabolic Control of Longevity,2016
Epigenetic Shifts and Metabolic Linkages in Aging,"Epigenetic Alterations
Aging is accompanied by multiple epigenetic alterations, and many of the enzymes that catalyze these changes utilize cofactors and substrates generated by intermediate metabolism, suggesting the existence of a strong link between the epigenetic control of aging and metabolism (Benayoun et al., 2015). Accordingly, the enhancer and insulator regions of genes whose expression levels in peripheral blood change with age tend to be enriched in functional CpG methylation sites. Moreover, the premature upregulation of the so-called ""transcriptomic age"" (an age-associated metagene) of an individual has been associated with signs of metabolic syndrome, such as enhanced blood pressure, increased levels of circulating glucose and cholesterol, as well as high body-mass index (Peters et al., 2015). Indeed, accumulating data indicate that major metabolic shifts, such as starvation-induced reduction of acetyl-coenzyme A and methionine restriction-associated depletion of S-adenosylmethionine (SAM), affect chromatin acetylation and methylation, respectively, leaving a durable epigenetic signature of past metabolic experiences that may condition the organismal response to future challenges (Su et al., 2016).",Cell,Metabolic Control of Longevity,2016
Intergenerational Transmission of Metabolic Epigenetic Marks,"Such epigenetic mechanisms, as well as alterations in small non-coding RNA levels, may also explain how parental obesity or other metabolic alterations experienced during gestation and lactation may favor the transmission of various aspects of metabolic syndrome to the next generation. Irrespective of the underlying mechanisms, the ""transmission"" of metabolic signatures from one generation to the next may be difficult to explain by a simple pattern of epigenetic alterations affecting all cell types. For instance, feeding mouse dams a high-fat diet during lactation strongly inhibits the formation of projections of anorexigenic proopiomelanocortin (POMC)- and orexigenic agouti-related neuropeptide (AGRP)-producing neurons in the hypothalami of newborns, most likely as a result of hyperinsulinemia, which altogether predisposes the offspring to hyperphagia, obesity, and diabetes (Vogt et al., 2014).",Cell,Metabolic Control of Longevity,2016
Epigenetic Rewiring from Early-Life Diet and Potential for Intervention,"Interestingly, such a protocol of transgenerational metabolic derangement decreases the expression levels of DNA methyltransferase 1 (Dnmt1) in the hypothalamus of the newborn and also affects the levels of Sirt1 and Hdac1 (another histone deacetylase) into adulthood (Desai et al., 2016). Altogether, these findings suggest that epigenetic traits are profoundly affected by past metabolic experiences, both within an individual’s aging trajectory and across generations. It is therefore likely that manipulating the epigenome by metabolic interventions or by enhancing the activity of relevant enzymes, such as SIRT6, may improve various manifestations of age-related diseases and extend healthspan.",Cell,Metabolic Control of Longevity,2016
Proteostasis Decline and Metabolic Connections,"Loss of Proteostasis
Aging and various aging-associated diseases are associated with impaired proteostasis (protein homeostasis) (Labbadia and Morimoto, 2015). The integrity of the proteome is preserved by folding mechanisms involving a complex network of molecular chaperones, as well as by degradation processes mediated by the ubiquitin-proteasome and the autophagy-lysosome systems. Signaling pathways that impinge on intermediate metabolism and regulate proteostasis influence aging as well as the onset and progression of age-related diseases (Vilchez et al., 2014). For example, metabolites from the hexosamine pathway enhance protein quality control, improve resistance to proteotoxic stress, and increase lifespan in animal models (Denzel et al., 2014). Conversely, proteotoxic stress associated with aging causes the loss of redox homeostasis, triggering adaptive changes in multiple subcellular compartments.",Cell,Metabolic Control of Longevity,2016
"Proteasome Aging, AMPK Signaling, and Metabolic Impact","Moreover, age-related metabolic and bioenergetic changes reduce the activity and availability of ATP-dependent chaperones, further hampering the preservation of cellular and organismal proteostasis (Brehme et al., 2014). Proteasome activity also declines with time, a process that markedly alters the proteome of aging cells and tissues. In contrast, healthy centenarians maintain remarkable proteasomal activity (Chondrogianni et al., 2015). The age-related decline in proteasomal efficiency may have detrimental metabolic outcomes. Indeed, the beneficial effect of elevated proteasomal activity on yeast lifespan has been attributed to its impact on AMP-activated protein kinase (AMPK) signaling (Yao et al., 2015). Robust proteasomal functions also correlate with an elevated respiratory activity and increased oxidative stress response. Consistently, genomic studies have revealed that polymorphisms affecting different proteasomal subunits are associated with increased susceptibility to metabolic disorders including diabetes in humans.",Cell,Metabolic Control of Longevity,2016
Autophagy Decline and Metabolic Dysregulation,"Moreover, one of the most important alterations that accompanies normal aging is a decline in autophagic proficiency (Madeo et al., 2015). Although most work on this topic has focused on macroautophagy, defects in chaperone-mediated autophagy (CMA) have also been documented in the aging mouse liver. Thus, the liver-specific knockout of lysosomal-associated membrane protein 2 (Lamp2) induces the accumulation of key enzymes of carbohydrate and lipid metabolism in hepatocytes, which is paralleled by a reduction in gluconeogenesis, an increase in glycolysis, and hepatic steatosis (Schneider et al., 2014). Reduced mitophagy has also been associated with aging, at least in specific brain areas (Sun et al., 2015). Genetic manipulations designed to promote autophagy, such as systemic overexpression of the essential component of the autophagic machinery Atg5, increase longevity and improve insulin sensitivity, leanness, and motor function in mice (Pyo et al., 2013).",Cell,Metabolic Control of Longevity,2016
"TFEB, Klotho, and Autophagy-Dependent Lifespan Effects","Similarly, transgene-driven expression of transcription factor EB (TFEB; a pro-autophagic transcriptional regulator) increases lifespan in Caenorhabditis elegans and antagonizes the lipotoxicity of high-fat diet in mice (Settembre et al., 2013). Accumulating evidence suggests that any kind of manipulation that extends lifespan loses its beneficial effect when autophagy is inhibited (Madeo et al., 2015). Importantly, deficient autophagy also plays an unexpected key role in animal models of accelerated aging, such as mice deficient in klotho (Kl), which develop a progeria-like syndrome accompanied by arteriosclerosis and reduced levels of autophagy at baseline (Kurosu et al., 2005; Shi et al., 2015). Conversely, transgene-driven Kl overexpression extends lifespan (Kurosu et al., 2005), and recombinant Kl injection into mice promotes cytoprotective autophagy (Shi et al., 2015). These results suggest that differences in Kl expression might affect the aging process secondary to alterations in autophagic flux.",Cell,Metabolic Control of Longevity,2016
Autophagy in Progeria Models and Metabolic Compensation,"Paradoxically, mice lacking zinc metallopeptidase STE24 (Zmpste24)—which are a model of Hutchinson-Gilford progeria syndrome—exhibit increased autophagic flux associated with metabolic changes that normally accompany lifespan extension (Mariño et al., 2008). These metabolic alterations are linked to changes in circulating leptin, glucose, insulin, and adiponectin levels, which altogether lead to peripheral AMPK activation and MTOR inhibition. It has therefore been proposed that the nuclear damage causing premature aging in Zmpste24−/− mice triggers a metabolic response involving the compensatory activation of autophagy. However, the chronic activation of this pathway turns a pro-survival mechanism into an etiological determinant of the systemic degeneration found in Zmpste24−/− progeroid mice (Mariño et al., 2008).",Cell,Metabolic Control of Longevity,2016
Proteostasis Collapse and Systemic Metabolic Aging,"In summary, metabolic changes associated with aging can impinge on the collapse of the proteostasis network and vice versa. Recent findings suggesting that the integrity of the cellular proteome is also preserved systemically, via non-cell-autonomous mechanisms, emphasize the need to identify the metabolic factors that may extend longevity by preventing age-associated proteome degeneration.",Cell,Metabolic Control of Longevity,2016
"IIS Pathway, Caloric Restriction, and Aging","Deregulated Nutrient Sensing
Different signaling pathways that sense and respond to fluctuations in nutrient levels are commonly deregulated during aging and in the presence of metabolic disorders (Efeyan et al., 2015). Among them, the ""insulin and IGF1 signaling"" (IIS) pathway has prominent aging-modulating effects. Consistent evidence indicates that the most efficient measure to extend lifespan across species, namely, caloric restriction (CR), relies on the suppression of the IIS pathway, coupled to the activation of various members of the FOXO protein family, and MTOR inhibition. Accordingly, nematodes overexpressing the FOXO-like transcription factor DAF-16 or lacking the worm ortholog of MTOR (i.e., LET-363) exhibit considerable lifespan extension as compared to control worms (Lapierre and Hansen, 2012). Paradoxically, normal aging as well as the aging-accelerating effects of obesity (see below) can be linked to the progressive inactivation of the IIS pathway, perhaps as a response aimed at minimizing cell growth in the context of systemic damage (Garinis et al., 2008).",Cell,Metabolic Control of Longevity,2016
"AMPK, Sirtuins, and NAD+ Loss in Aging","Other nutrient sensors, in particular AMPK and sirtuins, tend to be downregulated with aging, and their pharmacological activation is reputed to increase longevity. In C. elegans, the α subunit of AMPK (i.e., AAK-2) is required for the lifespan-extending effects of several genetic interventions, and AAK-2 overexpression per se increases lifespan (Lapierre and Hansen, 2012). Among age-regulating sirtuins, SIRT1 is shut off secondary to the age-associated depletion of its main cofactor, NAD+. The mechanisms accounting for NAD+ loss are presumably multifactorial, encompassing downregulation of the biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT), hyperactivation of the NAD+-consuming enzyme PARP1, disruption of circadian rhythms, and chronic inflammation (Verdin, 2015). Sestrins—a family of stress-inducible proteins that modulate nutrient-sensing pathways such as those orchestrated by AMPK and MTOR—are also emerging as regulators of metabolic homeostasis and appear to attenuate aging in various model organisms (Lee et al., 2013).",Cell,Metabolic Control of Longevity,2016
"Sestrins, MTORC Signaling, and Metabolic Homeostasis","The lack of sestrin 3 (Sesn3) in mice results in diverse metabolic disorders that generally accompany accelerated aging, including fat accumulation, diabetes, and muscle degeneration. Conversely, transgenic Sesn3 expression protects mice against insulin resistance caused by high-fat diet, as a direct consequence of MTOR complex 2 (MTORC2)-Akt1 signaling (Tao et al., 2015). Interestingly, Sesn2 is a leucine sensor for the MTORC1 pathway, connecting the availability of this amino acid to the control of organismal growth (Wolfson et al., 2016). Thus, proficient nutrient-sensing pathways are required to preserve metabolic fitness at the organismal level and hence suppress the development of aging-associated diseases.",Cell,Metabolic Control of Longevity,2016
"NAD+ Loss, SIRT1 Impairment, and Mitonuclear Communication","Mitochondrial Dysfunction
Human aging is generally linked to a progressive mitochondrial dysfunction (Wang and Hekimi, 2015). Part of this deterioration is caused by the abovementioned decrease in NAD+ availability and consequent functional impairment of the deacetylase SIRT1 (Gomes et al., 2013). Indeed, low SIRT1 activity results in the acetylation-dependent inactivation of PGC1a, MYC, and HIF1A, which limits the PGC1a-dependent expression of nuclear genes encoding mitochondrial proteins, as well as the MYC- and HIF1A-dependent expression of the mitochondrial transcription factor TFAM (Gomes et al., 2013). This example illustrates how alterations in the intracellular levels of one single metabolite can contribute to mitochondrial aging by impairing mitonuclear communication.",Cell,Metabolic Control of Longevity,2016
Longevity from Mild Mitochondrial Stress in Model Organisms,"Paradoxically, mild perturbations of mitochondrial function can extend longevity in various model organisms (Palikaras et al., 2015). The mechanisms accounting for this counterintuitive finding have been elucidated in C. elegans, where partial mitochondrial dysfunction extends longevity upon epigenetic changes linked to histone demethylation (Merkwirth et al., 2016) and coupled to the activation of multiple transcription factors including the worm orthologs of mammalian p53, NRF2, and HIF1A (Ventura et al., 2009). Upon stabilization, HIF1A activates the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2), which contributes to promoting longevity in worms (Leiser et al., 2015). Moreover, mitophagy induction by the worm ortholog of NRF2 (SKN-1) is required for the longevity-extending effects of mild mitochondrial dysfunction (Palikaras et al., 2015). Intriguingly, adaptive responses to mild mitochondrial perturbations can activate a longevity-promoting mechanism that depends on proteins usually involved in cell death signaling (Yee et al., 2014).",Cell,Metabolic Control of Longevity,2016
Mitochondrial Peptides Humanin and MOTS-c in Longevity,"Mouse mitochondria can release peptides such as humanin and MOTS-c (encoded within mt-Rnr2 and mt-Rnr1, respectively) with potential longevity-extending activity. Humanin has cytoprotective effects in vitro and ameliorates cardiovascular and neurodegenerative disorders in rodents (Yen et al., 2013). MOTS-c promotes the biosynthesis of an endogenous AMP analog, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which stimulates AMPK and hence counteracts diabetes, obesity, and aging (Lee et al., 2015). Intriguingly, a polymorphism in the MOTS-c-coding sequence of mt-Rnr1 is frequent among Northeast Asians, and this may partially contribute to the exceptional longevity of the Japanese population (Fuku et al., 2015).",Cell,Metabolic Control of Longevity,2016
"Mitochondrial Turnover, ROS, and Aging Pathways","The predominant link between mitochondria and longevity may consist in the acceleration of mitochondrial turnover, implying a combination of increased synthesis (Finley et al., 2012) and mitophagic degradation (Palikaras et al., 2015), globally assuring optimal quality control. Preserving mitochondrial fitness is expected to have a beneficial impact on several aspects of the aging process, including not only primary mitochondrial function but also (1) genomic instability (dysfunctional mitochondria are major sources of genotoxic ROS; see above) and (2) altered intercellular communication (ROS overgeneration is connected to the secretion of inflammatory mediators, see below). Further substantiating this notion, both PGC1a levels and mitophagy decline with age in mice (Chabi et al., 2008). It has been speculated that the mitochondrial unfolded protein response (UPRmt)—an adaptive reaction initiated by the accumulation of aberrant proteins in the mitochondrial matrix or by an unbalanced mitonuclear communication—may underlie the ability of mild perturbations of mitochondrial homeostasis to extend longevity in C. elegans (Wang and Hekimi, 2015).",Cell,Metabolic Control of Longevity,2016
UPRmt Activation and Nicotinamide Riboside,"Recently, the lifespan-extending effects of nicotinamide riboside have been linked to the activation of the UPRmt in various stem cell compartments in mice (Zhang et al., 2016), suggesting that the UPRmt may be important for longevity in several organisms beyond worms.",Cell,Metabolic Control of Longevity,2016
"Senescence, Proliferative Arrest, and Metabolic Links","Cellular Senescence
Accumulating in vitro and in vivo evidence indicates that cellular senescence—a process that imposes a permanent proliferative arrest on cells responding to different stressors—is intimately associated with several metabolic alterations. The available data linking cellular senescence with aging and aging-associated diseases, including metabolic disorders, are mainly circumstantial. However, targeted clearance of senescent cells from progeroid mice reduces circulating levels of activin A, enhances adipogenesis, and prevents lipodystrophy, a common feature of advanced age (Xu et al., 2015). Furthermore, the elimination of naturally occurring senescent cells has recently been shown to attenuate the age-related deterioration of several organs and tissues and to extend lifespan in mice (Baker et al., 2016).",Cell,Metabolic Control of Longevity,2016
"SASP, GATA4, Autophagy, and Inflammatory Signaling","The so-called ""senescence-associated secretory phenotype"" (SASP), which is a pathognomonic shift in protein secretion, stems from the dissociation of the transcription factor GATA4 and the autophagic adaptor p62 (Kang et al., 2015). Such dissociation abolishes the autophagic degradation of GATA4 and hence allows it to support the SASP and consequent inflammatory response, which is a driver of aging. This mechanism links cellular senescence to another hallmark of aging, altered intercellular communication, via a metabolic effect on autophagy. Autophagy may also contribute to senescence triggered by DNA damage through a specific mechanism in which a nuclear pool of LC3 (a core component of the autophagic machinery) interacts with lamin B1 and damaged heterochromatin, hence favoring their export to the cytoplasm and lysosomal degradation (Dou et al., 2015). Autophagy inhibition limits oncogene-induced senescence in human cells, but nutrient deprivation (which potently activates autophagy) per se does not cause the degradation of lamin B1 and senescence (Dou et al., 2015).",Cell,Metabolic Control of Longevity,2016
MiDAS: Mitochondrial Dysfunction–Associated Senescence,"Interestingly, mitochondrial dysfunction can precipitate a peculiar type of cellular senescence that has been dubbed ""mitochondrial dysfunction-associated senescence"" (MiDAS). At odds with other forms of cellular senescence, MiDAS does not involve IL-1β secretion but promotes the release of other conventional components of the SASP, including IL-10, TNF, CCL27, and HMGB1 (Wiley et al., 2016). MiDAS is characterized by reduced NAD+/NADH ratio, which causes growth arrest and prevents IL-1β secretion as a result of AMPK-driven p53 activation. Although MiDAS appears to be rather frequent in homozygous PolgD257A/D257A mice, which have a progeroid phenotype linked to an increased rate of mitochondrial DNA mutations (Wiley et al., 2016), the exact contribution of this phenomenon to the aging process remains to be determined. Nonetheless, MiDAS provides additional support to the existence of strong connections between cellular senescence and metabolic dysfunction.",Cell,Metabolic Control of Longevity,2016
"Senolytics, SASP Inhibitors, and Metabolic Therapies","Furthermore, the fact that some senolytic agents (i.e., compounds that selectively remove senescent cells from tissues) and SASP inhibitors target central metabolic pathways, alleviate adipose tissue dysfunction, and improve metabolism in aged mice suggests that targeting metabolism may be part of future approaches aimed at extending lifespan in humans.",Cell,Metabolic Control of Longevity,2016
"Stem Cell Aging, Metabolism, and Systemic Influences","Stem Cell Exhaustion
The progressive decline in stem cell function that generally accompanies aging may result from many of the aforementioned hallmarks of aging, alone or in combination, and hence is intimately linked to metabolic alterations. Heterochronic transplantation experiments (in which tissues from an aged mouse are transplanted into a young mouse or vice versa) and parabiosis studies (in which the circulatory system of an aged mouse is shared with a young mouse) have delineated some of the metabolic alterations that affect stem cell aging (Goodell and Rando, 2015). Metabolic assessments have also revealed that, as they age, stem cells experience important changes in the balance between glycolysis, oxidative phosphorylation, and response to oxidative stress (Shyh-Chang et al., 2013). Stem cells in general, including hematopoietic stem cells (HSCs), are particularly sensitive to ROS, and ROS increase with age in this cellular compartment. Accordingly, treatment with the antioxidant N-acetyl-L-cysteine enhances the replicative potential of mouse HSCs in serial transplantation experiments (Ito et al., 2006).",Cell,Metabolic Control of Longevity,2016
Nutrient Sensing Pathways Regulating Stem Cell Fate,"Nutrient sensors including the insulin receptor (INSR), MTORC1, and AMPK, as well as downstream signal transducers like PI3K, AKT1, and FOXO transcription factors, also modulate the balance between stem cell quiescence and proliferation in the course of aging, at least in mice. For example, excessive MTOR signaling causes epidermal stem cell exhaustion and hair loss (Castilho et al., 2009), defects in the FOXO system dampen the oxidative stress response and promote HSC depletion, and alterations in AMPK signaling contract the size of long-term HSC pools and impair hematopoiesis (Shyh-Chang et al., 2013). Of note, SIRT7 is downregulated in HSCs from old individuals, which causes unwarranted NRF1-dependent mitochondrial biogenesis, incapability to maintain the quiescent state, and myeloid-biased differentiation (Mohrin et al., 2015). Thus, a reduced number of mitochondria paradoxically appears to be an inalienable property of stem cells.",Cell,Metabolic Control of Longevity,2016
Metabolic Control of iPSC Reprogramming,"Intermediate metabolites from glycolytic and oxidative reactions also influence epigenetic changes that accompany the reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) (Ryall et al., 2015). iPSCs rely on glycolysis to generate ATP and have reduced mitochondrial mass as compared to somatic cells. The inhibition of autophagy-related proteins like Ulk1 and Rab9, as well as that of their upstream activator AMPK, can prevent the wave of mitochondrial clearance that accompanies the reprogramming of somatic cells into iPSCs, whereas multiple autophagy inducers favor the iPSC generation (Ma et al., 2015b). These findings suggest that non-canonical autophagy may contribute to the generation of iPSCs. It has not been determined yet whether this atypical autophagic pathway crosstalks with NF-kB signaling, which has a major inhibitory effect on iPSC generation as it activates the repressor DOT1L (Soria-Valles et al., 2015).",Cell,Metabolic Control of Longevity,2016
Future Directions: Metabolic Modulation of Stem Cell Aging,"Further studies of the impact of metabolism on the age-related exhaustion of stem cells, as well as on the metabolic aspects of stem cell reprogramming, will likely result in new approaches for modulating aging at the cellular level or even prolonging organismal longevity.",Cell,Metabolic Control of Longevity,2016
"Intercellular Communication, Microbiota, and Metabolism","Altered Intercellular Communication
Multiple age-related alterations of metabolism are intertwined with major perturbations in intercellular communication. Such an intersection between metabolism and the orchestration of multicellular life concerns a variety of complex processes including neuroendocrine signaling, inflammation, and circadian rhythms. Even the gut microbiota of the elderly differs from that of the young, in particular as it contains a reduced abundance of Ruminococcus spp. and Prevotella spp., correlating with signs of frailty, co-morbidity, and inflammation (Claesson et al., 2012). Experiments in young mice demonstrate that depleting the gut microbiota favors browning of white adipose tissue and reduces obesity (Suárez-Zamorano et al., 2015), and aging is well-known to involve a re-organization of whole-body metabolism that causes a reduction in brown and beige fat. However, the actual relationship between age-associated changes in the gut microbiota and brown fat reduction has not been investigated thus far.",Cell,Metabolic Control of Longevity,2016
Neuroendocrine Circuits and Longevity Extension,"Neuroendocrine circuits affecting metabolic phenotypes also change over time, and some of them constitute targets for the experimental extension of longevity. As an example, the knockout of Trpv1, which encodes a specific pain receptor, improves metabolic fitness and extends survival in mice by limiting the production of the neuropeptide Cgrp from sensory terminals innervating pancreatic islets (Riera et al., 2014). This situation favors insulin secretion, which would otherwise decline with age due to the enhanced local secretion of Cgrp. Age-associated inflammation and consequent insulin resistance, bone loss, cognitive decline, and frailty can be reduced by genetic ablation of the NLRP3 inflammasome, a multicomponent platform responsible for the secretion of mature IL-1b and IL-18 (Goldberg and Dixit, 2015). In the course of aging, NLRP3 (which normally responds to microbial or neoplastic threats) is activated by the accumulation of endogenous danger signals including ATP, cholesterol crystals, excess glucose, and urate in the extracellular space, a process that is further favored by intracellular ROS overgeneration secondary to mitochondrial dysfunction.",Cell,Metabolic Control of Longevity,2016
"Inflammasomes, Fasting, fTregs, and Metabolic Aging","Interestingly, ketone bodies that rise upon fasting (i.e., β-hydroxybutyrate) potently inhibit the NLRP3 inflammasome (Youm et al., 2015), proving another link between CR and lifespan extension. In the visceral adipose tissue (VAT), fat-resident regulatory T (fTreg) cells accumulate with age. Depletion of fTreg cells by conditional knockout of Pparg abolishes virtually all age-related metabolic changes, as it ameliorates insulin sensitivity and glucose uptake by VAT (Bapat et al., 2015). Such improvements can be partially recapitulated by acute fTreg depletion with an antibody that neutralizes the IL-33 receptor interleukin-1 receptor-like 1 (Il1rl1, also known as St2) and are specific for age-associated insulin resistance, but not for obesity-driven metabolic syndrome. Conversely, both aging and obesity are associated with an exacerbated secretion of transforming growth factor beta 1 (TGF-b1) by hypothalamic astrocytes, constituting a common determinant of glucose intolerance and insulin resistance (Yan et al., 2014). Thus, obesity and aging cause insulin resistance through inflammatory pathways that overlap only to partial extents.",Cell,Metabolic Control of Longevity,2016
"Circadian Rhythms, NAD+ Decline, and Metabolic Flexibility","Aging correlates with a decrease in the circadian oscillation of the so-called ""respiratory exchange ratio"" (which reflects the relative consumption of carbohydrate or lipid for energy metabolism), and old mice develop a substrate preference toward lipids, losing the capacity to switch between distinct fuel sources, which is commonly referred to as ""metabolic flexibility."" Although this phenotype might stem from declining mitochondrial proficiency and the development of insulin resistance, it may also be associated with changes in circadian control. Aging has been linked to an attenuated circadian oscillation in transcriptional processes of the suprachiasmatic nucleus of the hypothalamus (and perhaps of other cell-autonomous clocks in the periphery as well), which in turn has been attributed to declining NAD+ levels (Chang and Guarente, 2013). Such a decline reflects a decrease in NAMPT expression levels and inhibits SIRT1 deacetylase activity. SIRT1 contributes to the regulation of circadian rhythms by a transcriptional mechanism impinging on the deacetylation of histone H3, aryl hydrocarbon receptor nuclear translocator-like (Arntl, also known as Bmal1), and period circadian clock 2 (Per2) (Asher et al., 2008).",Cell,Metabolic Control of Longevity,2016
"Polyamines, Circadian Clock Control, and Aging","In addition, SIRT1 deacetylates acyl-CoA synthetase long-chain family member 1 (Acsl1), thereby causing circadian oscillations of acetyl-CoA levels (Sahar et al., 2014), with wide-ranging consequences for several metabolic circuitries, including autophagy. The complex crosstalk between circadian clocks and metabolic pathways through mechanisms that decay with aging also involves polyamines (e.g., putrescine and spermidine). Indeed, the availability of polyamines oscillates with circadian periodicity, mostly reflecting feeding behavior (Zwighaft et al., 2015). Reciprocally, putrescine and spermidine control the circadian period in cultured cells and mice by modulating the interaction between the core clock proteins Per2 and cryptochrome 1 (Cry1). In mice, the age-related decline in polyamine levels is linked to an increased circadian periodicity, which can be reversed upon dietary polyamine supplementation (Zwighaft et al., 2015). Collectively, these studies offer novel targets for metabolic and nutritional interventions aimed at preventing the functional decay of the circadian clock over time.",Cell,Metabolic Control of Longevity,2016
Introduction to Calorie Restriction and Sirtuins,"Calorie or dietary restriction (CR) has attracted attention because it is the oldest and most robust way to extend rodent life span. The idea that the nutrient sensors, termed sirtuins, might mediate effects of CR was proposed 13 years ago and has been challenged in the intervening years. This review addresses these challenges and draws from a great body of new data in the sirtuin field that shows a systematic redirection of mammalian physiology in response to diet by sirtuins. The prospects for drugs that can deliver at least a subset of the benefits of CR seems very real. Toward the resolution of discordances. Sirtuins and aging. Perhaps the greatest challenge to the idea that sirtuins mediate effects of CR was a study describing the failure to observe extension of life span in worms or flies transgenic for the corresponding SIR2 orthologs after controlling for genetic background (Burnett et al. 2011). These findings contradicted other, earlier studies that showed extension of life span in transgenic worms (Tissenbaum and Guarente 2001; Viswanathan et al. 2005; Berdichevsky et al. 2006) and flies (Rogina and Helfand 2004; Wood et al. 2004; Bauer et al. 2009). In fact, a subset of these earlier studies did control for genetic background (e.g., Bauer et al. 2009), creating conflicting claims about the role of sirtuins in aging.",Genes & Development,Calorie Restriction & Sirtuins,2013
Re-examining Sirtuins in Yeast and Flies,"Recently, a group of papers re-examined the effects of sirtuins on life spans in different organisms and reinforced the idea that they are of central importance. In budding yeast, one study underscored the established importance of SIR2 in replicative aging (Stumpferl et al. 2012). Two highly divergent yeast strains (a laboratory strain and a clinical isolate) were crossed, and a genome-wide quantitative trait locus (QTL) analysis pinpointed those genetic differences responsible for the difference in replicative life span. Indeed, the most important locus was SIR2, explaining more than half of the difference in the replicative life span between the strains (due to five codon differences between the SIR2 alleles). In flies, the critical role of Drosophila Sir2 (dSir2) was revealed by tissue-specific manipulation of the gene under an inducible promoter (Banerjee et al. 2012), a more specific system compared with the constitutive whole-body overexpression at a single dose in the prior study (Burnett et al. 2011). Overexpression of dSir2 in the fat body extended the life span of flies on the normal diet. This fits well with earlier findings that the fat body is a critical determinant of Drosophila life span (Giannakou et al. 2004; Hwangbo et al. 2004). In a second experiment, Banerjee et al. (2012) found that deletion of dSir2 in the fat body abolished the extension of life span by a CR-like protocol.",Genes & Development,Calorie Restriction & Sirtuins,2013
Sirtuins in Worm Longevity,"A second recent study confirms that dSir2 overexpression increases the life span in flies (Hoffmann et al. 2013). In worms, two studies concurrent with Burnett et al. (2011) showed that increasing the dose of Caenorhabditis elegans sir-2.1 did indeed extend the life span (Rizki et al. 2011; Viswanathan and Guarente 2011), although not to the extreme level previously reported (Tissenbaum and Guarente 2001). The 2001 study was complicated by an unrecognized, unlinked mutation that made the extension appear larger. In the past year, three additional studies implicate sir-2.1 in worm longevity. First, the dauer-inducing natural products of the worm, ascarosides, were shown to extend worm life span in a pathway requiring SIR-2.1 (Ludewig et al. 2013). Second, a recent study confirmed that SIR-2.1 overexpression extends life span (Mouchiroud et al. 2013). In addition, boosting internal NAD levels could also extend the life span via sir-2.1. NAD/SIR-2.1 induced two distinct longevity pathways; one involved insulin-like signaling and the FOXO homolog DAF-16, and the other involved the mitochondrial unfolded protein response resulting from an imbalance between nuclear and mitochondrial gene expression. Importantly, this study also pinpoints a possible mechanism of normal aging—NAD depletion by chronic DNA damage-induced PARP activation.",Genes & Development,Calorie Restriction & Sirtuins,2013
Sirtuin Overexpression Studies in Worms and Mammals,"In a third study, life span extension in SIR-2.1-overexpressing strains was again confirmed by Ristow and colleagues (Schmeisser et al. 2013) using strains from Burnett et al. (2011) as well as Viswanathan and Guarente (2011). In mammals, Kanfi et al. (2012) showed that two transgenic mouse lines overexpressing the mammalian SIRT6 had significantly extended male life spans. Mammals have seven sirtuins, SIRT1–7. SIRT1, SIRT6, and SIRT7 are nuclear; SIRT3, SIRT4, and SIRT5 are mitochondrial; and SIRT2 is cytoplasmic. In a very recent study, brain-specific overexpression of SIRT1 was also shown to extend the life span (Satoh et al. 2013). The failure of whole-body SIRT1 overexpression to extend the life span may be due to untoward effects on cancer, which counteract the observed slowing of aging in these mice (Herranz et al. 2010). Finally, in humans, a link between SNPs at the SIRT3 locus and longevity has also been described (Albani et al. 2013). All told, the above body of work strongly supports the proposal that sirtuins are conserved mediators of longevity.",Genes & Development,Calorie Restriction & Sirtuins,2013
Dietary Restriction and Sirtuins in Yeast,"CR in rodents was first shown to extend life span in the 1930s (McCay et al. 1935) and is a very specific regimen—a 30%–40% reduction in the ad libitum levels of chow intake. We originally attempted to apply the concept of CR to yeast by mutating nutrient-sensing pathways or providing 0.5% glucose instead of the usual 2% and found that the replicative life span was extended (Fig. 1; Lin et al. 2000, 2002). Importantly, extension was abolished when SIR2 was knocked out. However, other studies, which generally (but not always) used a more severe limitation of the glucose, found life span extension that was not SIR2-dependent (Kaeberlein et al. 2004). Consistent with a role of SIR2 in CR, the NAD salvage pathway enzyme Pnc1p was shown to be up-regulated by 0.5% CR and required for longevity (Anderson et al. 2003). Furthermore, the SIR2 paralog HST2 was implicated in CR at the extreme concentrations of glucose (Lamming et al. 2005). As discussed below, subtle differences in media or other laboratory conditions may account for observed experimental differences.",Genes & Development,Calorie Restriction & Sirtuins,2013
CR and Sirtuin Dependence in Worms and Flies,"In C. elegans, both genetic and physiological regimens of CR were contrived, and life span extension by one required sir-2.1 (Narasimhan et al. 2009), but extension by others did not (e.g., Bishop and Guarente 2007). Similarly, in flies, a limitation of the yeast extract in the diet was shown to extend the life span. In two studies, dSir2 was essential (Rogina and Helfand 2004; Banerjee et al. 2012), but in another, it was not (Burnett et al. 2011). How can we interpret these disparate findings? We now know that metazoans have multiple nutrient-sensing pathways that can affect the life span (e.g., insulin signaling) (Kenyon 2010), TOR (Johnson et al. 2013), AMP kinase (Kahn et al. 2005), and sirtuins (Guarente 2000). Any variability in laboratory conditions might favor signaling through different subsets of these pathways, explaining the reported differences in the genetic requirements for CR in lower organisms (Speakman and Mitchell 2011).",Genes & Development,Calorie Restriction & Sirtuins,2013
Challenges and Evidence for SIRT1 Activators,"Finally, putative SIRT1-activating compounds (resveratrol and newer, synthetic STACs) were reported to activate the enzyme in vitro by lowering its Km for substrate (Howitz et al. 2003) and also elicit health benefits in mice, especially in animals exposed to the high-fat diet (Baur et al. 2006; Lagouge et al. 2006; Milne et al. 2007). However, the proposed mechanism of both resveratrol and newer STACs was challenged because activation appeared to require the presence of a fluorescent tag on the substrate peptide used in vitro (Kaeberlein et al. 2005). In addition, resveratrol was suggested to activate SIRT1 in vivo indirectly by binding to phosphodiesterases and triggering cAMP signaling to activate SIRT1 (Park et al. 2012). Three subsequent studies provide strong evidence that these compounds really work by directly activating SIRT1. First, activation can, in fact, be demonstrated using peptide substrates without any fluorescent conjugate (Dai et al. 2010). Importantly, the presence of aromatic amino acid side chains at residues positioned near the deacetylated lysine was required, suggesting a substrate specificity for activation foreshadowed by the earlier apparent requirement for the fluorescent tags.",Genes & Development,SIRT1 Activators,2013
Mechanistic Insights into Direct SIRT1 Activation,"Second, acute deletion of SIRT1 in adult mice prevented many of the physiological effects of resveratrol and other STACs (Price et al. 2012). Third, a single mutation in SIRT1 abolished the ability of resveratrol and all 117 other STACs tested to activate the enzyme in vitro or promote the canonical physiological changes in cells (Hubbard et al. 2013). This mutation lies adjacent to but outside of the catalytic domain and is thought to define an allosteric site in the enzyme for activation by small molecules. At present, it is difficult to interpret all of these findings in any model other than direct activation of SIRT1 by resveratrol and other STACs. Of course, the dose of resveratrol or other STACs may affect whether additional targets are also engaged in vivo.",Genes & Development,SIRT1 Activators,2013
General Effects of CR and Sirtuins in Mice,"CR studies in mice have the advantage of being more standardized than dietary studies in lower organisms; e.g., many of these experiments use C57BL/6 mice under a 30% limitation of the ad libitum consumption of chow food. The lines of evidence that sirtuins mediate effects of CR in mammals are many. First, the substrates targeted by sirtuins SIRT1, SIRT3, SIRT4, SIRT5, and SIRT6 (detailed in the next section) closely define those pathways at the heart of the metabolic shift induced by CR. Second, CR induces the expression levels of at least a subset of the sirtuins—SIRT1 (Cohen et al. 2004), SIRT3 (Lombard et al. 2007), and SIRT5 (Nakagawa et al. 2009). SIRT1 is also induced by CR in humans (Civitarese et al. 2007). Reciprocally, a high-fat diet leads to the loss of SIRT1 in mice (Chalkiadaki and Guarente 2012), and obesity has the same effect in humans (Pedersen et al. 2008; Costa Cdos et al. 2010). Third, loss-of-function mutation of specific sirtuins ablates specific outputs of CR. For example, knocking out SIRT1 abolishes changes in physical activity (Chen et al. 2005), and brain-specific ablation has a similar effect on the somatotrophic axis (growth hormone/IGF-1) (Cohen et al. 2009). In addition, SIRT1 knockout mice do not live longer on a CR diet (Boily et al. 2008; Mercken et al. 2013). Knocking out the mitochondrial SIRT3 prevents the protective effect of CR against hearing loss (Someya et al. 2010). In this case, SIRT3 is required for CR to mitigate oxidative damage in crucial neurons of the inner ear. Deletion of SIRT5 prevents the up-regulation of the urea cycle, which is required to reduce blood ammonia when amino acids serve as energy sources (Nakagawa et al. 2009).",Genes & Development,Sirtuins and Calorie Restriction,2013
Sirtuin Overexpression and Disease Mitigation,"Fourth, transgenic overexpression of SIRT1 or STACs mitigates disease syndromes much like CR; these include diabetes, neurodegenerative diseases, liver steatosis, bone loss, and inflammation (Baur et al. 2006; Lagouge et al. 2006; Bordone et al. 2007; Pfluger et al. 2008; Herranz et al. 2010; Guarente 2011). Tissues likely responsible for these effects are discussed in another section below, but one aspect may involve a positive effect of SIRT1 on insulin secretion by pancreatic b cells (Moynihan et al. 2005; Bordone et al. 2006). Conversely, compromised sirtuin activity contributes to metabolic syndrome and diabetes in mice and humans (Hirschey et al. 2011; Chalkiadaki and Guarente 2012; Biason-Lauber et al. 2013). Fifth, SIRT1 activators like resveratrol exert effects that overlap those of CR at the level of whole-animal physiology (Lam et al. 2013) or transcriptional profiling (Barger et al. 2008). All told, one may conclude that this is the most complete set of evidence for the involvement of any genetic pathway in CR.",Genes & Development,Sirtuins and Calorie Restriction,2013
Cellular Roles of Sirtuins Under CR,"Perhaps the most direct indication that sirtuins play an important role in the physiological adaptation to CR comes from a more detailed analysis of their substrates and physiological effects. Two hallmarks of CR are metabolic reprogramming to oxidative metabolism (to gain the most possible energy from fuel sources) and resistance to stress, particularly oxidative stress. This section considers general cellular effects of sirtuins on these hallmarks, and the subsequent section examines tissue-specific effects. SIRT1 plays a central role in inducing mitochondrial biogenesis, stress tolerance, and fat metabolism. This sirtuin deacetylates PGC-1a (Rodgers et al. 2005; Gerhart-Hines et al. 2007), FOXO1 (Brunet et al. 2004; Motta et al. 2004), and PPARa (Purushotham et al. 2009). In this regard, SIRT1 activity is tightly linked to AMP kinase (AMPK) (Feige et al. 2008; Fulco et al. 2008), since AMPK drives expression of the NAD synthetic enzyme NAMPT (Canto et al. 2009, 2010), and SIRT1 deacetylates and activates the AMPK activator kinase LKB1 (Hou et al. 2008; Lan et al. 2008). At the same time, SIRT1 turns down glycolytic metabolism by deacetylating glycolytic enzymes (Hallows et al. 2012) and one of their key transcriptional inducers, HIF-1a (Lim et al. 2010). One must assume that repressing HIF-1a is especially important, since SIRT3 and SIRT6 also target this pathway.",Genes & Development,Sirtuins and Calorie Restriction,2013
Mitochondrial Sirtuins and Cancer-Related Metabolism,"SIRT3 reduces reactive oxygen species (ROS) production by mitochondria, thus blunting HIF-1a induction as well as generally reducing the ROS burden to cells (Bell et al. 2011; Finley et al. 2011). SIRT6 corepresses HIF-1a target genes by deacetylating histones at those loci (Zhong et al. 2010). The metabolic shift away from glycolysis and toward mitochondria along with the accompanying stress resistance are reinforced by the deacetylation and desuccinylation of mitochondrial proteins by SIRT3 (Lombard et al. 2007) and SIRT5 (Du et al. 2011; Peng et al. 2011), respectively. Mitochondrial targets thus activated by these sirtuins include superoxide dismutase 2 (Qiu et al. 2010; Tao et al. 2010) and metabolic enzymes for fatty acid oxidation (Hirschey et al. 2010), the urea cycle (Nakagawa et al. 2009; Hallows et al. 2011), and acetate metabolism (Hallows et al. 2006; Schwer et al. 2006). Interestingly, the third mitochondrial sirtuin, SIRT4, seems to be wired oppositely to SIRT3 and SIRT5; i.e., its expression goes down in CR. This also makes sense because SIRT4 ADP-ribosylates and represses glutamate dehydrogenase (Haigis et al. 2006), the gateway for glutamine and glutamate to enter the TCA cycle and central metabolism to provide energy during CR.",Genes & Development,Sirtuins and Calorie Restriction,2013
Sirtuins in Cancer Suppression and Inflammation,"These actions of SIRT1, SIRT3, SIRT4, and SIRT6 are relevant to the Warburg effect, in which cancer cells show a massive up-regulation of glycolysis and glutaminolysis, thereby suggesting tumor suppressor functions. Indeed, loss of SIRT3 (Finley et al. 2011) or SIRT6 (Sebastian et al. 2012), which would induce glycolysis, or loss of SIRT4, which would induce glutaminolysis, has been found in many tumors (Jeong et al. 2013). Interestingly, SIRT4 is the sirtuin most highly induced by DNA damage and impedes glutamine entry into metabolism, raising the possibility that a “glutamine checkpoint” may play a tumor suppressor role by limiting growth of precancerous cells to allow repair of damage (Jeong et al. 2013). A second study also implicates SIRT4 in cancer suppression and shows that levels of this sirtuin are regulated by mTORC1 (Csibi et al. 2013). One should note for SIRT1, however, that evidence for both a tumor prevention function (e.g., Firestein et al. 2008) and tumor enhancement function in established tumors (e.g., Li et al. 2013) has been reported. Besides HIF-1a, the only known target of three or more sirtuins is NF-kB, involved in a proinflammatory arm of the immune response. p65 of this transcription factor is deacetylated and repressed by SIRT1 (Yeung et al. 2004) and SIRT2 (Rothgiesser et al. 2010), and histones at NFkB-regulated genes are deacetylated by SIRT6 (Kawahara et al. 2009) to reinforce repression. These sirtuin functions may help explain the global anti-inflammatory effect of CR, which may be an important mechanism by which this diet slows aging. Indeed, NF-kB activation has been linked to aging (Adler et al. 2007), a topic explored further below.",Genes & Development,Sirtuins and Calorie Restriction,2013
Overview of Tissue-Specific Roles of SIRT1,"Effects in specific tissues. It is clear that the effects of CR must be a coordinated, systemic response, prompting the question of what tissues are most important and how they interact. The next section reviews roles of SIRT1 in various tissues, as outlined in Figure 3, which may help explain the organismal protection conferred by this diet. Figure 3. Sirtuin targets in different tissues relevant to CR. The figure shows validated sirtuin targets in seven tissues. The color coding of sirtuins is shown in the key. Calorie restriction and sirtuins revisited.",Genes & Development,Sirtuins and Calorie Restriction,2013
SIRT1 in the Hypothalamus: Metabolic and Behavioral Control,"The hypothalamus contains different groups of neurons that control much of mammalian physiology, including feeding behavior, energy expenditure, physical activity, body temperature, and central circadian control (Coppari 2012). It was originally inferred that this brain region may play a key signaling role in CR from studies in worms, in which ablation of two neurons prevented the increased energy expenditure and longevity conferred by a CR-like protocol (Bishop and Guarente 2007). Many hypothalamic functions of SIRT1 have been reported (Fig. 4). A plausible role of hypothalamic SIRT1 in mammals was first suggested by the observation that levels of this sirtuin in this brain region change with the diet (Ramadori et al. 2008). Further evidence is the finding that brain depletion of SIRT1 prevents CR regulation of the somatatrophic axis (Cohen et al. 2009). Genetic manipulation of SIRT1 protein levels in the hypothalamus affects feeding behavior, although the direction of the changes and the effects of the diet on these changes are not fully consistent from study to study (Cakir et al. 2009; Sasaki et al. 2010). Nonetheless, evidence indicates that SIRT1 in the agouti-related peptide-producing neurons (AgRPs) controls the response to the gut hormone ghrelin and feeding behavior (Dietrich et al. 2010, 2012; Sasaki et al. 2010).",Genes & Development,Sirtuins and Calorie Restriction,2013
"SIRT1 Regulation of VMH, DMH, LH, and POMC Neurons","SIRT1 in the ventromedial hypothalamic (VMH) neurons determines physiological outputs to ghrelin signaling (Velasquez et al. 2011; Porteiro et al. 2013). SIRT1 in the dorsomedial hypothalamus (DMH) and lateral hypothalamus (LH) is induced by CR and mediates outputs such as physical activity and body temperature by determining the levels of the orexin receptor 2 (Satoh et al. 2010). Correspondingly, SIRT1 in the pro-opiomelanocortin (POMC) neurons (Ramadori et al. 2010) protects against metabolic decline induced by high-fat diets. Administering resveratrol directly to the CNS of rodents mediates insulin sensitivity, and this effect is abolished by knocking down SIRT1 expression in the hypothalamus (Knight et al. 2011). Another hypothalamic region of importance is the suprachiasmatic nucleus (SCN), which determines central circadian control of metabolism and other physiological functions in addition to the sleep–wake cycle. Negative health consequences of circadian disruptions are well documented, and mice with intrinsic circadian periods closest to 24 hours are the longest lived in a 12 h light/12 h dark cycle (Wyse et al. 2010; Libert et al. 2012).",Genes & Development,Sirtuins and Calorie Restriction,2013
SIRT1 and Circadian Regulation in SCN and Aging,"SIRT1 was first linked to circadian control via the peripheral and autonomous clock in the liver. In this tissue, SIRT1 deacetylates circadian clock proteins BMAL1 and PER2 (Asher et al. 2008; Nakahata et al. 2008). The clock also regulates NAMPT, rendering sirtuin activity circadian and linking the clock to metabolism (Nakahata et al. 2009; Ramsey et al. 2009). Recently, SIRT1 was shown to control central circadian function in the brain by amplifying expression of BMAL1 (Chang and Guarente 2013). SIRT1 levels decline in the SCN of aged mice, concomitant with reduced levels of circadian components, triggering degradation of central circadian function with aging (Valentinuzzi et al. 1997). Overexpressing SIRT1 in the brain blunts aging effects, while deletion compromises function in young animals. SIRT1 deacetylates PGC-1a in neurons to enhance BMAL activation. A loop of SIRT1, PGC-1a, and NAMPT amplifies circadian clock protein expression and maintains SCN function. Given that SIRT1 appears to be a key vulnerability in aging, brain-targeted sirtuin drugs may help sustain circadian function.",Genes & Development,Sirtuins and Calorie Restriction,2013
"Hypothalamic SIRT1, NF-κB, and Aging","A recent study directly illustrates the importance of the hypothalamus (and NF-kB) in mammalian aging. Tissue-specific deletion of the inhibitor of the NF-kB inhibitor I-kB results in reduced NF-kB activity in hypothalamic neurons and significantly extends mouse lifespan (Zhang et al. 2013). Satoh et al. (2013) also conclude that SIRT1 in the hypothalamus is key to lifespan extension in transgenic mice through its activation of the orexin type 2 receptor in the LH and DMH. Multiple lines of evidence now indicate that the hypothalamus plays a dominant role in mammalian aging and that SIRT1 is a critical regulatory factor in this compartment. Many additional studies show that SIRT1 functions throughout the brain. For example, SIRT1 protects against neurodegenerative diseases in the cortex and striatum (Kim et al. 2007; Donmez et al. 2010; Min et al. 2010; Jeong et al. 2011; Jiang et al. 2011) and enhances learning and memory in the hippocampus (Gao et al. 2010; Michan et al. 2010). Since physiological functions of these regions deteriorate with aging, sustaining SIRT1 activity may be generally critical for brain maintenance.",Genes & Development,Sirtuins and Calorie Restriction,2013
SIRT1 and Sirtuin Regulation in White Adipose Tissue,"White adipose tissue (WAT). SIRT1 and NAD+ levels are induced in WAT of CR mice (Chen et al. 2008). Conversely, SIRT1 levels are reduced in white fat in obese mice (Chalkiadaki and Guarente 2012) and obese humans (Pedersen et al. 2008; Costa Cdos et al. 2010). One possible mechanism for loss of SIRT1 in obese animals is suggested by the finding that a high-fat diet in mice triggers cleavage of SIRT1 in WAT by caspase 1 of the inflammasome (Chalkiadaki and Guarente 2012). Mice genetically knocked out for SIRT1 in WAT are predisposed to diabetes, likely because the first “hit” toward diabetes (i.e., SIRT1 loss in WAT) has already taken place. Interestingly, SIRT2 also plays an important functional role in adipocyte biology by regulating differentiation of preadipocytes (Jing et al. 2007). Other functional data are consistent with the hypothesis that SIRT1 in WAT promotes metabolic health by causing fat reduction in these depots. SIRT1 promotes fat mobilization from WAT to blood, for example, during fasting (Picard et al. 2004), to facilitate its oxidation in the liver and muscle. SIRT1 also drives the browning of white fat cells (Qiang et al. 2012), which would trigger fat oxidation in situ. Consistent with these functions, inhibition of SIRT1 in WAT is associated with macrophage recruitment and inflammation (Gillum et al. 2011).",Genes & Development,Sirtuins in Adipose Tissue,2013
"PPARγ, Adiponectin, and Endocrine Crosstalk in WAT","One target for deacetylation by SIRT1 in WAT is the nuclear receptor PPARγ. SIRT1 alters gene expression of a subset of PPARγ target genes (Wang et al. 2008) and restrains differentiation of preadipocyte precursor cells (Picard et al. 2004). Last, SIRT1 transgenic mice show higher levels of the insulin-sensitizing adipokine adiponectin (Banks et al. 2008). The expanded role of sirtuins in adipokine production and endocrine cross-talk between WAT and the hypothalamus remains a fascinating area to explore.",Genes & Development,Sirtuins in Adipose Tissue,2013
SIRT1-Mediated Control of Gluconeogenesis in Liver,"In the liver, SIRT1 has numerous deacetylation targets that affect gluconeogenesis and fat homeostasis. Many of these deacetylation events have opposing effects, revealing the complexity of SIRT1 function in this tissue. For example, SIRT1 deacetylates two coactivators that drive gluconeogenesis: PGC-1a to activate it (Rodgers et al. 2005) and CRTC2 to trigger its ubiquitination and degradation (Liu et al. 2008). It is thought that SIRT1 thus stages a temporal shift during fasting by switching gluconeogenesis from an early CRTC2-driven mechanism to a later mechanism driven by the other SIRT1 target, PGC-1a. SIRT1 expression declines at the time of the shift due to the fact that the SIRT1 promoter itself is regulated by CRTC2 (via the transcription factor CREB) (Noriega et al. 2011). The decline in CRTC2 (and correspondingly SIRT1) may set a proper, lower activity level of the SIRT1 substrate PGC-1a during the later stage of fasting.",Genes & Development,Sirtuins in Liver Metabolism,2013
SIRT1 Regulation of Hepatic Fat Metabolism,"With regard to fat homeostasis, SIRT1 deacetylates the nuclear receptor LXR (Li et al. 2007) to activate the SREBP1 gene for synthesis. However, SIRT1 also deacetylates SREBP1 itself to repress its activity (Ponugoti et al. 2010; Walker et al. 2010). Other substrates of SIRT1 include the nuclear receptor FXR for bile synthesis (Kemper et al. 2009) and LKB1 linking SIRT1 and AMPK (Hou et al. 2008; Lan et al. 2008). What is the net effect of summing the various SIRT1 hepatic activities? Numerous studies have examined the role of hepatic SIRT1 under different dietary conditions. Most studies show that liver-specific deletion of SIRT1 triggers physiological hypersensitivity to a high-fat diet (You et al. 2008; Erion et al. 2009; Purushotham et al. 2009; Wang et al. 2010, 2011; Xu et al. 2010; but an exception is Chen et al. 2008), while overexpression gives protection against steatosis (Li et al. 2011b). Consistent with this idea, high-fat diet-induced inflammation triggers phosphorylation of SIRT1 by JNK1 kinase, leading to its degradation (Gao et al. 2011), reminiscent of what is observed in WAT.",Genes & Development,Sirtuins in Liver Metabolism,2013
Roles of SIRT4 and SIRT6 in Hepatic Lipid Homeostasis,"Liver SIRT4 again appears to oppose SIRT1, since its deletion protects against high-fat diet-induced steatosis (Nasrin et al. 0). This may be because SIRT4 deacetylates and inhibits malonyl CoA decarboxylase 1 (MCD1), which converts malonyl CoA to acetyl CoA (Laurent et al. 2013). Thus, SIRT4 deletion would prevent the accumulation of malonyl CoA, the key precursor for fat synthesis, as would the reduction in SIRT4 levels in CR. Finally, hepatic SIRT6, like SIRT1, appears to be protective, since two studies show that its deletion sensitizes animals to steatosis (Kim et al. 2010; Dominy et al. 2012).",Genes & Development,Sirtuins in Liver Metabolism,2013
"SIRT1, Exercise, and Mitochondrial Biogenesis in Skeletal Muscle","Many studies suggest that CR, exercise, and resveratrol induce mitochondrial biogenesis and increased stress tolerance in the skeletal muscle of mice and humans to improve physiology systemically (Baur et al. 2006; Lagouge et al. 2006; Feige et al. 2008; Koltai et al. 2010). At least three pathways have been implicated in mediating these effects. First, the activity of the nitric oxide synthase expressed in skeletal muscle (eNOS) is induced by CR in mice and humans (Nisoli et al. 2005; Civitarese et al. 2007). Since SIRT1 is known to deacetylate and activate eNOS (Mattagajasingh et al. 2007), the induction of this sirtuin in CR muscle can explain this chain of events. Importantly, eNOS−/− mice do not show the induction of mitochondria by CR (Nisoli et al. 2005). Second, the deacetylation of PGC-1a is also induced by CR or resveratrol and also drives mitochondrial biogenesis (Rodgers et al. 2005; Gerhart-Hines et al. 2007). Third, the CR-induced adipokine adiponectin binds to its receptor in muscle and stimulates the SIRT1/AMPK axis by causing Ca++ release and activation of the AMPK kinase calmodulin-dependent protein kinase (Iwabu et al. 2010). This pathway may also be triggered by exercise (Iwabu et al. 2010). The effects of glucose on cultured skeletal muscle cells depend on the interaction of SIRT1 with AMPK (Fulco et al. 2008; Canto et al. 2010) and FOXO1 (Hariharan et al. 2010). Not surprisingly, tissue-specific knockout of SIRT1 in skeletal muscle blunts physiological changes induced by CR (Schenk et al. 2011).",Genes & Development,Sirtuins in Muscle Physiology,2013
Sirtuins in Cardiac Stress Resistance and Hypertrophy,"With respect to the heart, studies with gain-of-function or loss-of-function mice indicate a protective role of SIRT1 against oxidative stress; for example, in ischemia/reperfusion challenges (Hsu et al. 2010; Tanno et al. 2010). SIRT1 can also protect against hypertrophy at moderate levels of cardiac-specific overexpression but appears to be deleterious at higher levels (Alcendor et al. 2007). Protection against hypertrophy may involve a pathway including PPARa and fat oxidation (Planavila et al. 2011), although, surprisingly, one study showed that haploinsufficiency of SIRT1 or PPARa protected against hypertrophy induced by pressure overload (Oka et al. 2011). CR induces nuclear localization of SIRT1 in a mechanism requiring eNOS, and this is associated with increased ischemic tolerance (Shinmura et al. 2008). SIRT1 and eNOS may comprise a mutually reinforcing activity loop, as for SIRT1 and AMPK. With regard to other sirtuins, SIRT6 has been found to attenuate AKT signaling in the heart to protect against hypertrophy (Sundaresan et al. 2012). A new study shows that mice knocked out for one subunit of complex I in the heart show a buildup of NADH, inactivation of SIRT3, and hyperacetylation of cardiac mitochondrial proteins (Karamanlidis et al. 2013). These mice are highly susceptible to heart failure, reinforcing the importance of SIRT3 in cardiac function (Pillai et al. 2010).",Genes & Development,Sirtuins in Cardiac Function,2013
Sirtuins in Renal Protection and Kidney Injury,"Numerous studies have indicated protection of renal function by SIRT1, initiating with the report that SIRT2 mitigates oxidative stress in HK-2 cells (Hasegawa et al. 2008). In mice, SIRT1 genetic activation in transgenic mice or the application of STACs protects against various models of kidney injury (Funk et al. 2010; Hasegawa et al. 2010; He et al. 2010; Fan et al. 2013; Kim et al. 2013). SIRT1 also mitigates fibrosis following acute kidney injury by deacetylating Smad4 and suppressing TGF-β signaling (Simic et al. 2013). In addition, SIRT1 is required for CR-induced renal protection against hypoxia, in this case by deacetylating FOXO3 and activating autophagy (Kume et al. 2010). CR is also renal-protective in a diabetes model in rats, associated with activation of SIRT1 and deacetylation of NF-kB (Kitada et al. 2011).",Genes & Development,Sirtuins in Kidney Physiology,2013
Inter-compartment Kidney Signaling via NMN and SIRT1,"A recent study points to a novel kind of communication between two different kidney compartments mediated by SIRT1. In one compartment, consisting of proximal tubule cells, SIRT1 determines the levels of the NAD precursor nicotinamide mononucleotide (NMN) that are secreted. The secreted NMN then activates SIRT1 in a different kidney compartment, consisting of podocytes, to trigger stress resistance. The net effect of this circuit is the protection of the kidney and suppression of diabetic albuminuria (Hasegawa et al. 2013). Opposite to these protective effects, SIRT1 may contribute to the pathophysiology of autosomal dominant polycystic kidney disease (ADPKD). This heritable disease, due to genetic defects in PKD1 or PKD2, affects up to one in 400 individuals and leads to kidney failure in midlife. A recent study shows that up-regulation of SIRT1 by c-MYC occurs in a murine model of ADPKD (Zhou et al. 2013). Genetic or pharmacological inhibition of SIRT1 suppresses cyst growth in this model, suggesting that SIRT1 may abet cyst formation by promoting cell survival mechanisms such as deacetylation and inhibition of p53. Interestingly, activation of SIRT1 is renal-protective in normal settings, but inhibition of SIRT1 may be efficacious in certain disease contexts.",Genes & Development,Sirtuins in Kidney Physiology,2013
SIRT1 Regulation of Endothelial Cell Function,"Endothelium and smooth muscle. Many studies have revealed a deep connection between SIRT1 and the vasculature. Among the important substrates deacetylated by SIRT1 in endothelial cells (ECs) are eNOS (Mattagajasingh et al. 2007), LKB1 (Zu et al. 2010), Notch (Guarani et al. 2011), and p66shc (Zhou et al. 2011). The net effect of SIRT1 in ECs appears to be control of vessel growth (Potente et al. 2007) and protection against EC senescence (Ota et al. 2007) and, more generally, atherosclerosis. For example, sheer stress induces SIRT1, AMPK, eNOS, and mitochondrial biogenesis (Chen et al. 2010), and this is associated with phosphorylation and stabilization of SIRT1 by calmodulin-dependent protein kinase kinase (Wen et al. 2013). Inhibition of this SIRT1 induction sensitizes ECs to damage inflicted by oxidized LDL (Guo et al. 2013) and sensitizes apoE−/− mice to atherosclerosis (Zhang et al. 2008). The SIRT1/AMPK axis in ECs has been shown to be inducible by exercise or resveratrol in mice (Cacicedo et al. 2011; Takizawa et al. 2013).",Genes & Development,Sirtuins in Vascular Biology,2013
SIRT1 in Smooth Muscle Cells and Atheroprotection,"In a second critical cell type of the vasculature, smooth muscle cells (SMCs), SIRT1 is also athero-protective, since selective depletion of this sirtuin from SMCs predisposes mice to atherosclerosis (Gorenne et al. 2013). In addition, SIRT1 restrains the induction of hypertrophy in SMCs by angiotensin II by reducing levels of its receptor (Li et al. 2011a). The effects of SIRT1 in ECs and SMCs may help explain the protective role of CR against atherosclerosis (Fontana et al. 2004) and (along with effects in other tissues) protection by this diet against cardiovascular disease in general.",Genes & Development,Sirtuins in Vascular Biology,2013
Introduction to mTOR and Aging,"NIH Public Access Author Manuscript Nature. Author manuscript; available in PMC 2013 July 17. Published in final edited form as: Nature. 2013 January 17; 493(7432): 338–345. doi:10.1038/nature11861. mTOR is a key modulator of ageing and age-related disease Simon C. Johnson1, Peter S. Rabinovitch1, and Matt Kaeberlein1,2 1Department of Pathology, University of Washington, Seattle, Washington 98195, USA 2 Institute of Aging Research, Guangdong Medical College, Dongguan 523808, China Abstract Many experts in the biology of ageing believe that pharmacological interventions to slow ageing are a matter of ‘when’ rather than ‘if’. A leading target for such interventions is the nutrient response pathway defined by the mechanistic target of rapamycin (mTOR). Inhibition of this pathway extends lifespan in model organisms and confers protection against a growing list of age-related pathologies. Characterized inhibitors of this pathway are already clinically approved, and others are under development. Although adverse side effects currently preclude use in otherwise healthy individuals, drugs that target the mTOR pathway could one day become widely used to slow ageing and reduce age-related pathologies in humans.",Nature,mTOR,2013
Discovery of Rapamycin and Early Mechanistic Insights,"The mechanistic target of rapamycin (mTOR) story began in the 1970s when new antifungal activity was discovered in soil samples from the Polynesian island of Rapa Nui (Box 1). Thus, the compound, which was isolated from Streptomyces hygroscopicus, was named rapamycin. Also known as sirolimus, rapamycin was widely studied as an immunosuppressant before its mechanism of action was well understood, and in 1999 it was approved for use in post-transplantation therapy. Since then, rapamycin and several derivative compounds (including everolimus, temsirolimus, ridaforolimus, umirolimus and zotarolimus, collectively referred to as ‘rapamycins’ in this Review) have been approved for a variety of uses, including prevention of restenosis following angioplasty and as a treatment for certain forms of cancer. Studies in the budding yeast Saccharomyces cerevisiae first identified the target of rapamycin genes TOR1 and TOR2 as genetic mediators of rapamycin’s growth inhibitory effects, and soon afterwards the mTOR protein was purified from mammalian cells and demonstrated to be the physical target of rapamycin.",Nature,mTOR,2013
mTOR Complexes and Rapamycin Effects,"mTOR is a serine/threonine protein kinase of the phosphatidylinositol-3-OH kinase (PI(3)K)-related family that functions as a master regulator of cellular growth and metabolism in response to nutrient and hormonal cues. mTOR functions in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Fig. 1). Rapamycins inhibit mTORC1 by binding the FK506-binding protein FKBP12, which then interacts physically with the complex and decreases activity. Although mTORC2 is not directly affected by rapamycin, chronic exposure can sequester mTOR from mTORC2, inhibiting mTORC2 assembly. This effect on mTORC2 is thought to contribute to metabolic complications associated with chronic rapamycin treatment, including glucose intolerance and abnormal lipid profiles. Much more is known about both the upstream regulation and downstream outputs of mTORC1 compared with mTORC2.",Nature,mTOR,2013
Upstream Regulation and Downstream Outputs of mTORC1,"mTORC1 is activated by insulin and other growth factors through PI(3)K and AKT kinase signalling. mTORC1 is also activated by environmental nutrients (for example, amino acids) and repressed by AMP-activated protein kinase (AMPK), a key sensor of cellular energy status. In response to these growth signals, mTORC1 is thought to promote messenger RNA translation and protein synthesis through at least two mTORC1 substrates, ribosomal protein S6 kinases (S6Ks) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). mTORC1 also promotes lipid biosynthesis, represses degradation through the autophagy pathway, and regulates glucose metabolism and mitochondrial function through the hypoxic response transcription factor HIF-1α as well as the peroxisome-proliferator-activated receptor coactivator PGC-1α. These multiple inputs and outputs place mTORC1 as a key regulatory nexus, modulating anabolic processes versus catabolic processes in response to nutrients, growth cues and cellular energy status.",Nature,mTOR,2013
mTORC1 and Longevity Across Species,"The first indication that mTOR regulates ageing came from studies in S. cerevisiae, in which it was found that deletion of the gene encoding the yeast orthologue of S6K — SCH9 — resulted in a doubling of yeast chronological lifespan. Soon after, mTORC1 was shown to affect longevity with the finding that mutation or RNA interference (RNAi) knockdown of mTOR (let-363) or the mTORC1 component raptor (daf-15) could extend lifespan in the nematode Caenorhabditis elegans. This was followed by studies in the fruitfly Drosophila melanogaster and using the yeast replicative ageing model, showing that mutations in mTOR and several other components of the mTORC1 pathway also increase lifespan in these systems. In addition, a series of studies showed that rapamycin extended lifespan in yeast, nematodes, fruitflies and mice, firmly establishing mTORC1 as a central, evolutionarily conserved regulator of longevity.",Nature,mTOR,2013
Dietary Restriction and mTORC1,"Reduction in nutrient intake in the absence of malnutrition, or dietary restriction, extends lifespan in many different species. In fact, other than mTORC1 inhibition, dietary restriction is currently the only intervention known to extend lifespan in yeast ageing models and in worms, flies and mice. mTORC1 is thought to play a part in mediating longevity and health benefits as a result of dietary restriction, which is intuitive given its function in responding to nutrient and growth cues. Dietary restriction reduces mTORC1 activity in invertebrate organisms and in some mammalian tissues, and pharmacological or genetic disruption of mTORC1 is sufficient to extend lifespan in both invertebrates and mice under non-dietary restriction conditions. Genetic evidence that suggests mTORC1 acts downstream of dietary restriction comes mainly from epistasis experiments in which dietary restriction is combined with mutations in the mTORC1 pathway.",Nature,mTOR,2013
Interactions Between Dietary Restriction and mTOR Pathway,"In yeast, dietary restriction fails to further extend replicative lifespan when combined with deletion of the genes encoding the mTOR and S6K homologues. A similar relationship is observed when RNAi knockdown of mTOR is combined with dietary restriction in C. elegans. In fruitflies, 4E-BP1 is required for maximal lifespan extension from at least one method of dietary restriction, and lifespan extension from dominant-negative alleles of mTOR and S6K extend lifespan in fruitflies in a nutrient-dependent manner. However, the interaction between dietary restriction and mTORC1 signalling is complex, as evidenced in reports that RNAi knockdown of S6K and translation initiation factors yield an additive lifespan extension when combined with dietary restriction in C. elegans. Despite these challenges, numerous studies have linked mTORC1-regulated processes, including both reduced mRNA translation and induction of autophagy, to lifespan extension from dietary restriction in different organisms.",Nature,mTOR,2013
mTOR and Insulin/IGF-1 Signalling,"Reduced insulin/IGF-1-like signalling increases longevity in nematodes, fruitflies and mice. Lifespan extension from reduced IIS is mediated by the FOXO family of transcription factors, and several studies have associated polymorphisms in FOXO3 with longevity in humans. mTOR activity is linked to IIS through multiple connections. mTORC1 is activated by IIS through AKT, and mTORC1 can negatively regulate IIS through S6K, which inhibits insulin receptor substrate 1 (IRS-1). Additional studies have indicated that FOXO3A can transcriptionally regulate tuberous sclerosis protein 1 (TSC1), an upstream inhibitor of mTORC1, in mammalian cells and that the mTORC1 target 4E-BP1 is also a transcriptional target of FOXO in fruitflies. mTOR and IIS also interact through mTORC2, which activates AKT to repress FOXO1 and FOXO3 in mammalian cells.",Nature,mTOR,2013
Genetic Interactions Between mTORC1 and IIS in Longevity,"Genetic studies that examine the relationship between mTORC1 and IIS with respect to longevity reflect this complexity. In nematodes, the FOXO orthologue daf-16 is proposed to repress raptor expression, and lifespan extension from mutation of raptor requires DAF-16 — a defining characteristic of components of the IIS pathway. Paradoxically, mutation of mTOR itself or treatment with rapamycin does not require DAF-16 for lifespan extension. Similarly, lifespan extension that results from mutations or RNAi knockdown of mTOR-pathway downstream targets such as S6K and the translation initiation factors eIF4E and eIF4G does not require DAF-16. Thus, current evidence supports the idea that mTORC1 modulates ageing by mechanisms that overlap but are distinct from IIS. This is consistent with the model that mTORC1 acts mainly downstream of dietary restriction, which, based on similar epistasis experiments, also seems to increase lifespan by mechanisms that are overlapping but distinct from IIS.",Nature,mTOR,2013
AMPK Regulation of mTORC1,"AMP-activated protein kinase AMPK is a conserved sensor of energy status that is activated in response to low ATP levels and negatively regulates mTORC1 (Fig. 2). Overexpression of AMPK is sufficient to extend lifespan in C. elegans, and the AMPK activator metformin (a widely used antidiabetic drug) is reported to extend lifespan in C. elegans and in short-lived, cancer-prone strains of mice25–27. AMPK inhibits mTORC1 through at least two distinct mechanisms: AMPK phosphorylates TSC2 on conserved serine sites, resulting in activation of TSC2 and downregulation of mTORC1 activity28, and AMPK directly phosphorylates raptor to impair mTORC1 signalling29. These data suggest that dietary restriction could reduce mTORC1 signalling partly through activation of AMPK. The actual situation is likely to be more complex, however, as both AMPK and mTOR interact with multiple additional factors, and it has been reported that activation of AMPK extends the lifespan of C. elegans through the CREB-regulated transcriptional coactivator 1 (CRTC-1) rather than through mTORC1 (ref. 30). Importantly, so far, there is no direct evidence for regulation of mTORC1 by AMPK in C. elegans, which does not have a TSC2 orthologue.",Nature,mTOR,2013
Hypoxic Response and mTOR,"The hypoxic response HIF-1α is a highly conserved transcription factor that is activated in response to low oxygen availability (hypoxia) and regulates the expression of a suite of genes involved in maintaining cellular homeostasis under these conditions31. Under normoxic conditions, HIF-1α is targeted for proteasomal degradation, and ectopic stabilization of HIF-1α is a hallmark of many cancers. A major feature of the hypoxic response is to drive ATP production under oxygen-limiting conditions by promoting a metabolic shift from oxidative phosphorylation towards glycolysis and lactic acid fermentation. mTORC1 activates the early hypoxic response in mammals by enhancing translation and stabilization of HIF-1 itself, as well as by enhancing translation of mRNAs encoding a subset of HIF-1 target genes that include vascular endothelial growth factor (VEGF)32. Prolonged hypoxia, however, results in downregulation of mTORC1. The hypoxic response has been implicated in ageing by studies in C. elegans in which HIF-1 acts as both a positive and negative regulator of longevity31.",Nature,mTOR,2013
"HIF-1, Longevity, and mTORC1 Interactions","Stabilization of HIF-1 results in lifespan extension by a mechanism that is distinct from both IIS and dietary restriction33,34. Interestingly, deletion of hif-1 also extends lifespan of animals grown at a high temperature (25 °C), but not at a low temperature (15 °C)35. In this context, HIF-1 has been proposed to act downstream of dietary restriction and mTORC1 (ref. 36) (Fig. 2). However, direct evidence for regulation of HIF-1 by mTORC1 in C. elegans has yet to be obtained. mTOR in mammalian ageing and healthspan The first indication that mTOR modulates mammalian ageing came from longevity studies as part of the National Institute on Aging’s Interventions Testing Program (ITP). Rapamycin was found to significantly extend lifespan in a genetically heterogeneous strain background at three independent test locations13. A particularly interesting aspect of this study was that the animals did not begin receiving the drug until they had reached 600 days of age, roughly equivalent to 60 years of age in a person. Follow-up studies that began rapamycin treatment at an earlier age replicated the initial result but failed to show substantially larger effects on longevity14.",Nature,mTOR,2013
"mTORC1, Healthspan, and Sex-Specific Longevity Effects","In mice treated with rapamycin from 6 months of age, mean lifespan extension was about 18% in females, but only 10% in males14. Both higher and lower doses of rapamycin are currently being tested, and it will be of particular interest to see whether a greater longevity benefit can be achieved. Soon after the ITP rapamycin study was published, knockout of the mouse gene encoding S6K1 (Rps6kb1) was reported to increase lifespan in females37. In this case, the lifespan extension was sex-specific, with males receiving no longevity benefit. A key prediction of the hypothesis that ageing is caused by specific molecular changes is that it should be possible to slow the ageing process and, thereby, delay the onset and progression of multiple age-related diseases. Thus, slowing ageing should increase both lifespan and healthspan — the period of life spent in relatively good health, free from chronic disease or disability. This seems to be the case for dietary restriction, which not only extends lifespan but also delays the incidence of age-related decline and disease in rodents, including cancer, cognitive decline and neurodegeneration16.",Nature,mTOR,2013
"mTORC1 Inhibition, Healthspan, and Compression of Morbidity","Similarly, dietary restriction reduces the incidence of cancer, cardiovascular disease, brain atrophy and diabetes in rhesus monkeys38. Emerging evidence suggests that, like dietary restriction, mTORC1 inhibition may have similar positive effects on multiple age-related pathologies in rodents and, in some cases, humans (Box 2). From a public-health perspective, an intervention that results in compression of morbidity, in which most of a lifetime’s illness is compressed into a shorter period of time near the end of life39, is particularly desirable. Although it is too early to know whether rapamycins could represent such an intervention, end-of-life analysis of the long-lived rapamycin-treated mice in the ITP studies indicates that the spectrum of causes of death was not significantly altered14. However, age-related alterations in heart, liver, adrenal glands, endometrium and tendon, as well as the decline in spontaneous activity, all occur more slowly in rapamycin-treated mice40.",Nature,mTOR,2013
"Rapamycin, Tumorigenesis, and Delayed Age-Related Pathologies","Another study found that, in addition to lifespan extension, spontaneous tumorigenesis and deaths owing to cancer in the inbred C57BL/6 mouse strain were significantly reduced by rapamycin15. These data suggest that rapamycin slows the ageing process in mice such that many normal causes of morbidity and death are delayed. The evidence that rapamycin can protect against multiple age-related diseases — including reduced cognitive function41,42 — is consistent with a compression of morbidity, although further studies will be needed to definitively test this. As previously alluded to, there has not yet been a careful analysis of different rapamycin regimens, and the observed effects on longevity and healthspan may be quite different at higher or lower doses of the drug.",Nature,mTOR,2013
Mechanisms of Longevity Regulation by mTOR,"Mechanisms of longevity regulation by mTOR In recent years, much effort has been applied to defining the mechanisms by which inhibition of mTORC1 enhances longevity. Not surprisingly, the picture that has emerged is complex. Multiple mTORC1-regulated processes seem to contribute to the pro-longevity effects of mTORC1 inhibition in a coordinated and overlapping manner. mRNA translation Among the most crucial of mTORC1 functions is the promotion of mRNA translation and protein synthesis under conditions that are favourable for growth, and there is evidence that regulation of mRNA translation can modulate longevity in yeast, nematodes, fruitflies and mice43. For example, mutations in S6K have been found to extend lifespan in all four of these model organisms (Table 1). Likewise, mutation or knockdown of multiple translation initiation factors and ribosomal proteins has also been shown to extend lifespan in yeast and nematodes44, and overexpression of an activated allele of 4E-BP extends lifespan in flies18.",Nature,mTOR,2013
"mTORC1, Translation, and Lifespan Mechanisms","It has been proposed that a global reduction in mRNA translation could be beneficial during ageing by allowing endogenous protein repair and degradation machinery to better maintain protein homeostasis in the face of protein aggregation and oxidative damage45. Alternatively, a general reduction in mRNA translation could attenuate age-associated pathologies that result from ‘hyperfunctional’ overactive biosynthetic and proliferative processes that are important during development but detrimental during adulthood46. Instead of a global reduction in mRNA translation, however, molecular evidence suggests that differential translation of specific mRNAs is more important for effects on lifespan. The evidence for this came from yeast, in which lifespan extension in ribosomal protein mutants is associated with increased translation of the transcriptional activator Gcn4, which regulates the expression of many target genes, including several that are involved in enhanced stress resistance and response to amino acid starvation47. This regulation of Gcn4 is mediated by structural features of the 5′ region of the GCN4 mRNA that cause it to be preferentially translated when mTORC1 is inhibited.",Nature,mTOR,2013
Differential mRNA Translation Under mTORC1 Inhibition,"Mechanistically similar observations have been made in both nematodes and fruitflies, in which a subset of mRNAs are translated more efficiently when global mRNA translation is reduced through inhibition of mTORC1-pathway components. In worms, these differentially translated mRNAs are enriched for stress response genes, and in flies they are enriched for mRNAs encoding components of the mitochondrial electron transport chain18,48. Although the specific mRNAs seem to differ across species, a common theme of enhanced translation efficiency of metabolic and stress genes in response to mTORC1 inhibition has emerged. Johnson et al. Page 5 Nature. Author manuscript; available in PMC 2013 July 17. The mechanism by which mTORC1 regulates mRNA translation in mammals continues to be an active area of research. Although S6k1 knockout mice are long-lived and have a small body size37, there is no direct evidence that these animals have reduced rates of global mRNA translation. In both yeast and worms, a deficiency of S6K causes a substantial reduction in global mRNA translation and protein synthesis24,47, so it would be somewhat surprising if mice that lack S6K1 do not.",Nature,mTOR,2013
mTORC1 Translation Control in Mammals,"However, one recent study has questioned whether S6K1 is a major component of mTORC1 regulation of mRNA translation in mammalian cells49. In experiments using the mTOR catalytic inhibitor Torin 1, acute control of translation by mTORC1 in p53−/− mouse embryonic fibroblasts was found to be mediated mainly by 4E-BPs, and was largely limited to mRNAs with 5′ terminal oligopyrimidine (TOP) motifs49. This study also found no evidence that complexity of the 5′ untranslated region is a determinant of mTORC1-dependent differential mRNA translation49, which is in contrast with the yeast and invertebrate models already described. It may be that the precise mechanisms by which mTORC1 regulates mRNA translation, and the impact of this regulation on longevity, is different in mammals and invertebrate species. Alternatively, these differences could arise from effects of the catalytic inhibition of mTOR, as opposed to genetic inhibition or inhibition with rapamycin, or from differences in mTOR function in cells grown between culture and in vivo.",Nature,mTOR,2013
Autophagy and Longevity,"Autophagy Activation of autophagy is another key mTORC1-regulated process that probably has a central role in promoting longevity. Autophagy is a major degradation pathway in eukaryotic cells that is essential for removing damaged organelles and macromolecules from the cytoplasm and recycling amino acids during periods of starvation50. Evidence suggests that autophagic degradation declines with age, and it has been proposed that this leads to an accumulation of damage, such as protein aggregates and degenerate mitochondria, that contribute to age-related cellular dysfunction51. Activation of autophagy by inhibition of mTORC1 presumably maintains cellular function during ageing by allowing enhanced degradation of aged cellular components. However, this hypothesis has been difficult to test directly, partly because the tools for quantifying autophagic flux are inadequate and rely mainly on secondary assays — such as the abundance of proteins involved in various steps of autophagy.",Nature,mTOR,2013
Autophagy Requirements for Lifespan Extension,"Evidence from studies in yeast and invertebrates supports the model that mTORC1-mediated induction of autophagy is required for lifespan extension from dietary restriction or from rapamycin12,52,53, although it remains to be determined whether induction of autophagy is sufficient to promote longevity in the absence of mTORC1 inhibition. In addition to longevity, aberrant regulation of autophagy has been linked to several diseases of ageing, including cancer, diabetes, cardiovascular disease and neurodegenerative diseases50, and it seems likely that enhanced autophagy underlies many of the beneficial effects of mTORC1 inhibition in these disease models. Stress resistance and xenobiotic metabolism Inhibition of mTORC1 signalling has been linked most clearly to enhanced stress resistance in yeast and nematodes. In yeast, for example, reduced activity of Tor1 and Sch9 activates the protein kinase Rim15, which in turn activates the stress response transcription factors Msn2, Msn4 and Gis1 (ref. 54). Induction of these stress response pathways is required for yeast chronological lifespan extension from dietary restriction, or in response to reduced mTORC1 signalling through deletion of SCH9 (ref. 54).",Nature,mTOR,2013
Stress Response Pathways and Xenobiotic Defence,"In nematodes, treatment with rapamycin has been shown to activate the stress response transcription factor SKN-1, which is encoded by the orthologue of mammalian NRF2 (ref. 11). SKN-1 is required for lifespan extension from rapamycin11, and has also been implicated in lifespan extension from dietary restriction55. Interestingly, NRF2 is activated in mice treated with rapamycin and in those subjected to dietary restriction56. In both cases, NRF2 activation results in the induction of several enzymes involved in xenobiotic defence, suggesting that enhanced resistance to such environmental insults may be important for lifespan extension. Mitochondrial function Regulation of mitochondrial function by mTORC1 is complex and seems to involve multiple mechanisms. As mentioned previously, mTORC1 promotes activation of HIF-1, which in turn enhances glycolytic flux while simultaneously downregulating mitochondrial oxygen consumption32. Another report suggests that mTORC1 actively promotes mitochondrial biogenesis and metabolism through PGC-1α and the transcription factor YY-1 (ref. 57).",Nature,mTOR,2013
Mitochondrial Respiration and mTORC1,"The best evidence for a direct mitochondrial role in longevity downstream of mTOR signalling comes from yeast, in which inhibition of mTORC1 results in a metabolic shift towards greater mitochondrial respiration, thereby increasing chronological lifespan58. This effect has been proposed to involve an adaptive signalling response owing to elevated levels of mitochondrial super-oxide59. Mice that lack mTORC1 activity in adipose tissue also show enhanced mitochondrial respiration60, suggesting a similar relationship between mTORC1 and mitochondrial function in mammals, at least in some tissues. Inflammation Inflammation is associated with several age-related disorders61, and a reduction in inflammation is proposed to be a primary mechanism by which dietary restriction promotes longevity and healthspan62. Hyper-activation of mTOR is often associated with inflammation, and rapamycin has been shown to have anti-inflammatory effects in multiple settings, including chronic kidney disease63, vascular inflammation after angioplasty64, atherosclerotic plaques65 and lung infection66.",Nature,mTOR,2013
mTORC1 Inhibition and Reduced Inflammation,"Thus, a reduction in chronic, age-associated inflammation is another attractive mechanism by which mTORC1 inhibition could slow multiple age-related pathologies in mammals.",Nature,mTOR,2013
Stem Cell Function and mTORC1,"Stem cells In mammals, a decline in stem-cell function is likely to be an important cause of age-related pathology67. There is increasing evidence that mTORC1 has a central role in this process, and that inhibition of mTORC1 can preserve, and perhaps even rejuvenate, stem-cell function in a variety of tissues. For example, rapamycin protects old mice from an immune challenge with influenza virus, which has been attributed to a rejuvenation of haematopoietic stem-cell function in the treated animals68. Furthermore, treating old mice with rapamycin has been found to enhance intestinal stem-cell function69. Interestingly, the mechanism accounting for this improvement is linked to inhibition of mTORC1 in the surrounding Paneth cells, resulting in a more favourable niche, rather than a direct effect of mTORC1 inhibition in the intestinal stem cells. In another study, dietary restriction was shown to enhance the function of muscle stem cells in both young and old animals through both cell-intrinsic and cell-extrinsic mechanisms70.",Nature,mTOR,2013
Stem Cells and Future Directions,"Although the authors did not directly examine a role for mTORC1, these effects have been proposed to be partly mediated by reduced mTORC1 activity71. Future directions and perspectives mTOR is a key modulator of ageing in evolutionarily divergent organisms, ranging from yeast to rodents, and it is likely that this function has been conserved to some extent in humans. The complexity of the mTOR network presents a hurdle in defining the mechanistic details of how mTOR influences longevity and healthspan. Several mTOR-regulated processes are likely to contribute to the effects of mTORC1 inhibition on ageing and disease, and it will be challenging to untangle the context-specific and tissue-specific relationships. Nonetheless, impressive progress has been made in this area over the past few years, and we anticipate that this trend will continue. As the list of beneficial effects of rapamycin in invertebrate and mouse models continues to grow longer (Box 2), it becomes increasingly tempting to speculate on similar benefits in people.",Nature,mTOR,2013
Rapamycin Clinical Potential and Cognitive Aging,"As mentioned previously, rapamycins have been approved clinically for a variety of uses but have yet to be tested against a broad spectrum of age-related diseases. At the time of writing, the search term ‘rapamycin’ generates 1,343 results in the National Institutes of Health clinical trials database (http://clinicaltrials.gov). We therefore anticipate a bounty of additional data on the effects of rapamycin in humans over the next few years. All of this then raises the question, at what point should we consider giving these drugs to otherwise healthy people? Perhaps the greatest upside of mTORC1 inhibitors is their potential to delay cognitive decline during ageing. The evidence for improved cognitive function in old mice treated with rapamyicin is striking41. Current estimates suggest cognitive decline can be detected as early as 45 years of age in otherwise healthy people72. Loss of cognitive function is a leading concern among geriatricians and their elderly patients and is a significant and growing public-health burden.",Nature,mTOR,2013
Potential Health Benefits and Risks of mTOR Inhibition,"If mTORC1 inhibition has even a modest positive effect on cognitive function, it could improve the quality of life for millions of middle-aged and older adults. Add to this the likelihood that risks of developing some forms of cancer, cardiovascular disease, and neurodegenerative disease would be reduced, then mTOR inhibitors may offer an attractive opportunity to have a significant impact on preventive health care. Before this can happen, however, important questions must be answered. Rapamycin is not without side effects, including hyperlipidaemia and hyperglycaemia, anaemia and stomatitis, in patients. A recent study of long-term rapamycin treatment in mice reported increased incidence of cataracts and testicular degeneration40. The effects of mTORC1 inhibition on immune function and wound healing are also of particular concern. Although it is unclear whether rapamycin alone has substantial immunosuppressive effects in healthy individuals, it undoubtedly is immunomodulatory.",Nature,mTOR,2013
"Concerns About Immune Effects, Dosage, and Side Effects","It would be unfortunate to take a drug that slows the rate of ageing, only to succumb to infection from an otherwise innocuous bacterium or virus at an early age. Optimal dosage and duration of treatment are also unknown factors. Almost no information exists on the most effective dose of rapamycin for longevity or healthspan in mice, and the current data suggest that rapamycin therapy that begins late in life is nearly as effective as therapy that begins early in life, at least for longevity. Understanding whether this is also true for a variety of age-related pathologies will be important. Assessing risks and side effects from studies of patients with disorders such as the rare lung disease lymphangioleiomyomatosis, for which rapamycin is indicated for chronic use, may be informative in determining the clinical potential of this drug and other mTOR inhibitors in the setting of age-related disease.",Nature,mTOR,2013
New mTOR Pathway Inhibitors and Longevity,"The development of chemical inhibitors of mTOR, as well as drugs that target other components of the mTOR pathway, promises to greatly aid research, while also potentially providing drugs of therapeutic value. Until recently, pharmacological inhibition of mTOR has been largely limited to rapamycins, but newer ATP-competitive inhibitors of mTOR kinase activity have been developed that block the phosphorylation of all known substrates of both mTORC1 and mTORC2 (ref. 73). These drugs show early promise as anticancer therapies, but as yet there are no data regarding their effects on longevity and other age-related diseases. S6K inhibitors, 4E-BP1 activators, mTORC1-specific inhibitors and mTOR-independent activators of autophagy are also attractive approaches for potentially modulating longevity and healthspan without incurring the side effects that are associated with rapamycins.",Nature,mTOR,2013
Prospects for Delaying Human Aging,"The search for a way to delay human ageing has proven long and elusive. Although still far from certain, there is reason to be optimistic that mTOR inhibitors may accomplish this goal. mTORC1 inhibition slows ageing in yeast and invertebrates, extends lifespan in mice, and has an impact on a diverse array of age-related diseases. Are we finally on the threshold of being able to fundamentally alter human ageing? Only time will tell, but if the pace and direction of recent progress are any indication, the next few chapters in the mTOR story should prove very interesting indeed.",Nature,mTOR,2013
SIRT1 Regulation of Endothelial Cell Function,"Endothelium and smooth muscle. Many studies have revealed a deep connection between SIRT1 and the vasculature. Among the important substrates deacetylated by SIRT1 in endothelial cells (ECs) are eNOS (Mattagajasingh et al. 2007), LKB1 (Zu et al. 2010), Notch (Guarani et al. 2011), and p66shc (Zhou et al. 2011). The net effect of SIRT1 in ECs appears to be control of vessel growth (Potente et al. 2007) and protection against EC senescence (Ota et al. 2007) and, more generally, atherosclerosis. For example, sheer stress induces SIRT1, AMPK, eNOS, and mitochondrial biogenesis (Chen et al. 2010), and this is associated with phosphorylation and stabilization of SIRT1 by calmodulin-dependent protein kinase kinase (Wen et al. 2013). Inhibition of this SIRT1 induction sensitizes ECs to damage inflicted by oxidized LDL (Guo et al. 2013) and sensitizes apoE−/− mice to atherosclerosis (Zhang et al. 2008). The SIRT1/AMPK axis in ECs has been shown to be inducible by exercise or resveratrol in mice (Cacicedo et al. 2011; Takizawa et al. 2013).",Genes & Development,Sirtuins in Vascular Biology,2013
SIRT1 in Smooth Muscle Cells and Atheroprotection,"In a second critical cell type of the vasculature, smooth muscle cells (SMCs), SIRT1 is also athero-protective, since selective depletion of this sirtuin from SMCs predisposes mice to atherosclerosis (Gorenne et al. 2013). In addition, SIRT1 restrains the induction of hypertrophy in SMCs by angiotensin II by reducing levels of its receptor (Li et al. 2011a). The effects of SIRT1 in ECs and SMCs may help explain the protective role of CR against atherosclerosis (Fontana et al. 2004) and (along with effects in other tissues) protection by this diet against cardiovascular disease in general.",Genes & Development,Sirtuins in Vascular Biology,2013
"Overview of Diet, Health, and Longevity","Promoting Health and Longevity through Diet: From Model Organisms to Humans. Luigi Fontana1,2,3,* and Linda Partridge4,5,* 1Division of Geriatrics and Nutritional Science, Washington University School of Medicine, St. Louis, MO 63110, USA 2Department of Clinical and Experimental Science, Brescia University, 25123 Brescia, Italy 3CEINGE Biotecnologie Avanzate, 80145 Napoli, Italy 4Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany 5Institute of Healthy Ageing and Department of Genetics, Environment, and Evolution, University College London, London WC1E 6BT, UK *Correspondence: lfontana@dom.wustl.edu (L.F.), linda.partridge@age.mpg.de (L.P.) Reduced food intake, avoiding malnutrition, can ameliorate aging and aging-associated diseases in invertebrate model organisms, rodents, primates, and humans. Recent findings indicate that meal timing is crucial, with both intermittent fasting and adjusted diurnal rhythm of feeding improving health and function, in the absence of changes in overall intake. Lowered intake of particular nutrients rather than of overall calories is also key, with protein and specific amino acids playing prominent roles. Nutritional modulation of the microbiome can also be important, and there are long-term, including inter-generational, effects of diet.",Cell,Dietary Restriction and Longevity,2015
"Mechanisms Linking Diet, Aging, and Health","The metabolic, molecular, and cellular mechanisms that mediate both improvement in health during aging to diet and genetic variation in the response to diet are being identified. These new findings are opening the way to specific dietary and pharmacological interventions to recapture the full potential benefits of dietary restriction, which humans can find difficult to maintain voluntarily. Introduction. The discovery that aging can be ameliorated by dietary, genetic, and pharmacological interventions has opened up the prospect of a broad-spectrum, preventive medicine for aging-related diseases (Table 1) (Fontana et al., 2014; Goldman et al., 2013; Partridge, 2010). Single-gene mutations that extend animal lifespan can ameliorate natural, age-dependent loss of function (Metaxakis et al., 2014; Stein and Murphy, 2012) and the pathology of aging-related diseases, including neurodegeneration (Cohen et al., 2009; Killick et al., 2009; Menzies and Rubinsztein, 2010; Pinkston-Gosse and Kenyon, 2007; Stohr et al., 2013). Laboratory animal models of slowed aging, naturally long-lived species such as the naked mole rat, and some humans that achieve the age of 100 have demonstrated that a long life is not inevitably associated with late-life disability and disease (Ikeno et al., 2006; Edrey et al., 2011; Ailshire et al., 2014).",Cell,Dietary Restriction and Longevity,2015
Dietary Restriction Across Species,"Recent work has shown that specific dietary interventions can also promote long life and healthy old age. Dietary restriction (DR), implemented as chronic and coordinate reduced intake of all dietary constituents except vitamins and minerals, was first shown 80 years ago to extend lifespan in rats. DR in both rats and mice improves most aspects of health during aging (Fontana et al., 2010a; Ikeno et al., 2006; Maeda et al., 1985). Exceptions include resistance to infection and wound healing. However, these conditions rapidly improve with re-feeding, and DR animals can then outperform controls (Kristan, 2008; Hunt et al., 2012). DR can produce substantial benefits with, for instance, ∼30% of DR animals dying at old ages without gross pathological lesions, compared with only 6% of ad-libitum-fed controls (Ikeno et al., 2006). DR started in young, adult Rhesus monkeys greatly improves metabolic health; prevents obesity; delays the onset of sarcopenia, presbycusis, and brain atrophy; and reduces the risk of type 2 diabetes, cancer, and cardiovascular disease (Colman et al., 2014; Mattison et al., 2012).",Cell,Dietary Restriction and Longevity,2015
Effects of DR in Humans,"In humans, severe food restriction without malnutrition results in many of the same physiological, metabolic, and molecular changes associated with DR in animals, including less age-associated myocardial stiffness and autonomic dysfunction, lower core body temperature, and downregulation of the pi3k/akt/foxo and inflammatory pathways in skeletal muscle (Cava and Fontana, 2013; Mercken et al., 2013). Humans voluntarily undertaking long-term DR score lower than controls on multiple risk factors for cardiovascular disease and cancer (Fontana et al., 2010b). In short-term, randomized clinical trials in aging humans, DR improves several markers of health (Heilbronn et al., 2006; Fontana et al., 2010b). However, severe DR with adequate nutrition (i.e., consuming at least 100% of the RDI for each essential nutrient) is not an option for most people because it is difficult to practice and sustain and, with inadequate nutrition, can increase the risk of impaired menstrual and reproductive function, osteoporotic bone fractures, anemia, and cardiac arrhythmias (Fairburn and Harrison, 2003). Dietary interventions that avoid unrealistic levels of self-deprivation, and pharmacological interventions that recapture beneficial effects of DR, are important goals to improve human health during aging.",Cell,Dietary Restriction and Longevity,2015
DR Effects in Short-Lived Model Organisms,"DR increases healthy lifespan in many shorter-lived organisms, including budding yeast Saccharomyces cerevisiae, the nematode worm Caenorhabditis elegans, and the fruit fly Drosophila melanogaster (Figure 1). The experimental tractability of yeast and invertebrates facilitates discovery of the—often evolutionarily conserved—mechanisms through which genetic and environmental intervention improve health during aging. The mechanisms mediating the health benefits of DR are not fully understood in any organism. A wide range of interventions has been used to impose DR, even within single species, and the mechanisms through which they extend lifespan can differ (Bass et al., 2007; Cypser et al., 2013; Mair and Dillin, 2008; Tatar et al., 2014). Multiple neural, systemic, tissue-specific, and cell-autonomous mechanisms are involved (Figure 2).",Cell,Dietary Restriction Mechanisms,2015
Sensory and Neural Mechanisms in DR Longevity,"In both C. elegans and Drosophila, altered sensory perception alone may play a role, with specific subsets of sensory, including olfactory and gustatory, neurons influencing lifespan (Alcedo and Kenyon, 2004; Allen et al., 2014; Apfeld and Kenyon, 1999; Ostojic et al., 2014; Waterson et al., 2014). In C. elegans, Drosophila, and mice, neural circuits both detect nutrient status and control the responses to it, in mice mediated mainly by the hypothalamus (Dacks et al., 2013). Alterations in the following also play important systemic roles: the levels of metabolites (Chin et al., 2014), the activity of the nutrient-sensing insulin/igf signaling network (Johnson et al., 2013; Kenyon, 2010) in C. elegans, steroid hormone signaling (Thondamal et al., 2014), and growth hormone in the mouse (Johnson et al., 2013; Kenyon, 2010). Cellular effector processes can include enhanced genomic stability and chromatin remodeling (Dang et al., 2014); improved chaperone-mediated protein homeostasis and cellular turnover processes, including autophagy (Singh and Cuervo, 2011); and increases in various forms of stress resistance (Hine et al., 2015).",Cell,Dietary Restriction Mechanisms,2015
Molecular Effectors of DR Longevity,"Molecular effectors that have been shown to mediate the effects of DR on health and longevity include FOXO (Tullet et al., 2008; Webb and Brunet, 2014), TOR (Kapahi et al., 2010; Johnson et al., 2013), AMPK (Greer et al., 2007; Burkewitz et al., 2014), sirtuins (Mouchiroud et al., 2013; Guarente, 2013), HSF, and NRF2 (Akerfelt et al., 2010; Martín-Montalvo et al., 2011). Inhibition of AKT activates FOXO, a transcription factor that upregulates several “longevity pathways” controlling DNA repair, autophagy, antioxidant activity, stress resistance, and cell proliferation (Webb and Brunet, 2014; Wang et al., 2014). Inhibition of mTORC1 improves proteostasis, increases autophagy, and enhances stem cell function (Kapahi et al., 2010; Efeyan et al., 2012; Johnson et al., 2013). Systemic or tissue-specific overexpression of some sirtuins (i.e., SIRT1, SIRT3, and SIRT6) also increases genomic stability, reduces NF-kB signaling, and improves metabolic homeostasis through histone deacetylation (Mouchiroud et al., 2013; Guarente, 2013).",Cell,Dietary Restriction Mechanisms,2015
Parallel Pathways and Timing Effects in DR,"Activation of SIRT1 and AMPK activates PGC-1a, a transcriptional regulator of mitochondrial function, antioxidant defenses, and fatty acid oxidation (Wu et al., 1999). Activation of transcription factors heat shock factor 1 and Nrf2, by upregulating HSP70, p62, and the transcription factor ATF3, induces several antioxidant and drug-metabolizing enzymes, prevents the age-dependent impairment of proteostasis, and promotes the maintenance of cell structure, redox, and intermediary metabolism (Akerfelt et al., 2010; Martín-Montalvo et al., 2011). Multiple, parallel processes thus contribute to the increase in health during aging from DR, and the relative contribution of these may vary between DR regimes and organisms. Interestingly, recent work has revealed the importance of timing of food intake, the role of specific nutrients, the nature of the effector mechanisms, the longer-term—including transgenerational—consequences of diet, and the key role played by the gut microbiota (Figure 3). These findings point to less drastic dietary manipulations that could be combined with pharmacological interventions to improve health and prevent disease during aging.",Cell,Dietary Restriction Mechanisms,2015
Intermittent Fasting and Evolutionary Eating Patterns,"Meal Frequency and Timing. Only recently have humans and domesticated animals had constant access to food. During their evolution, many animals and humans ate only intermittently. For many microorganisms and invertebrates, long periods of starvation are normal and, correspondingly, many of them (including C. elegans) have evolved forms of quiescence in response to the onset of food shortage. Many of the genes that control quiescence are also important in the control of lifespan (Baugh, 2013). Intermittent fasting (IF), with alternation of 2 days of ad libitum feeding with 2 day fasting, also extends worm lifespan, through a mechanism involving the small GTP-ase RHEB-1 and insulin/Igf signaling (Honjoh et al., 2009). Even chronic starvation extends lifespan in C. elegans (Kaeberlein et al., 2006; Lee et al., 2006), through mechanisms that overlap with those mediating the response to IF and that include the combined activity of FOXO, FOXA, and AP-1 transcription factors in two parallel starvation-responsive pathways (Uno et al., 2013).",Cell,Intermittent Fasting,2015
Intermittent Fasting in Rodents: Longevity and Disease Protection,"In rodents, both fasting for 24 hr every other day or twice weekly extends lifespan up to 30%, independent of both total food intake and weight loss (Mattson et al., 2014). As for chronic DR, the magnitude of IF-induced life extension can be influenced by the age of initiation and mouse genotype. In A/J mice, IF started at 6 months did not increase lifespan and, when started at 10 months of age, reduced mean lifespan by 15% (Goodrick et al., 1990). IF can also protect against obesity, cardiovascular disease, hypertension, diabetes, neurodegeneration, and progression of several neurodegenerative diseases (Mattson et al., 2014). Some studies show protection against cancer progression (Berrigan et al., 2002; Lee et al., 2012), whereas others suggest detrimental effects on cancer initiation and promotion (Tessitore and Bollito, 2006). Mechanisms include increased production of neurotrophic factors BDNF and FGF2, reduced inflammation and oxidative stress, and enhanced cellular adaptive stress responses. Treatment of cells and mice with bOHB, a fasting-induced histone deacetylase inhibitor, protects against oxidative stress (Shimazu et al., 2013).",Cell,Intermittent Fasting,2015
Fasting-Induced Stress Resistance and Autophagy,"Fasting improves mitochondrial function, stimulates production of chaperones such as HSP-70 and GRP-78, and, by inhibiting the AKT/mTOR pathway, triggers autophagy and DNA repair (Brown-Borg and Rakoczy, 2013; Mattson et al., 2014). In mice, multiple fasting cycles modulate hematopoietic stem cell protection, self-renewal, and regeneration via IGF-1 or PKA inhibition (Cheng et al., 2014). Short-term fasting (1–3 days) protects rodents against ischemia-reperfusion injury of the liver and kidney, by improving insulin sensitivity, reducing inflammatory markers, and increasing cytoprotective gene expression (Hine et al., 2015).",Cell,Intermittent Fasting,2015
Human Trials of Intermittent Fasting,"Many trials on the effects of IF in humans are underway. A 6-month randomized clinical trial in overweight or obese premenopausal women showed that fasting for 2 non-consecutive days per week reduced body weight, fat mass, waist circumference, total and LDL cholesterol, triglycerides, CRP, and arterial blood pressure; cancer risk biomarkers improved, but IGF-1 did not change (Harvie et al., 2011). Similarly, in three small, short-term (8–12 weeks) randomized trials, alternate-day fasting lowered body weight, fat mass, and cardiovascular risk factors (Kroeger et al., 2014). Preliminary evidence from dogs and humans suggests that short-term fasting prior to chemotherapy reduces certain toxicities by protecting normal but not cancer cells (Safdie et al., 2009; Withers et al., 2014).",Cell,Intermittent F Fasting,2015
Circadian Timing and Meal Scheduling,"Patterns of eating over the day can have substantial effects. Limiting food intake of an isocaloric diet to a 5–7 hr window in humans induces health benefits compared to three to five meals per day (Mattson et al., 2014). Delaying feeding until evening in diurnally active fruit flies uncouples metabolic cycles from circadian rhythms and reduces egg laying (Gill and Panda, 2011). In mice, time-restricted feeding during 8 hr of the dark phase restores normal circadian metabolic rhythms and protects against weight gain, fat accumulation, inflammation, glucose intolerance, insulin resistance, and motor decline without reducing calorie intake (Chaix et al., 2014). Humans who eat and sleep 12 hr out of phase from habit show increased blood pressure, reduced glucose tolerance, lower leptin, and inverted cortisol rhythms (Scheer et al., 2009).",Cell,Meal Timing,2015
Consequences of Disrupted Meal Patterns and DR Timing,"30% calorie restriction by dietary dilution, in which mice ate all day to compensate for lower energy density, had no beneficial effect on lifespan (Solon-Biet et al., 2014), possibly due to disrupted meal pattern. Long-lived DR mice and rats consume restricted food rapidly, producing an extended fasting window. DR may improve health partly through IF. In the Wisconsin Rhesus monkey DR trial, monkeys were fed mainly once per day (versus twice daily in the NIA trial), which may have contributed to differing mortality outcomes (Colman et al., 2014; Mattison et al., 2012). In women with PCOS, earlier meal timing (large breakfast, medium lunch, small dinner) improved weight loss, insulin sensitivity, testosterone levels, and ovulation compared with isocaloric late-eating patterns (Jakubowicz et al., 2013).",Cell,Meal Timing,2015
Mechanisms Linking Meal Timing and Metabolic Health,"The molecular mechanisms responsible for effects of altered meal patterns are not fully understood. Compensatory changes may occur in AMPK, AKT/mTOR, and CREB, all central to cellular homeostasis and circadian oscillation. These pathways interact with circadian clock targets and nutrient-sensing signals (Mattson et al., 2014).",Cell,Meal Timing,2015
Challenges in Determining Optimal Macronutrient Intake,"Calories or Specific Nutrients? Determining the optimal overall intake and dietary ratios of carbohydrate, fat, and protein is challenging. The effects of reduced consumption of a specific macronutrient will depend partly upon how much of it is consumed by the controls and also upon the composition of the rest of the diet. Even for just three macronutrients, the possible combinations are vast. Combinatorial effects can be explored using a geometric framework (see the Essay by Simpson et al. (2015) on page 18 of this issue), but this is often not feasible in practice. Dietary composition can also affect overall food intake and its timing through effects on hedonistic value and satiety. Although such mechanisms are important to understand, they can also complicate analysis of the effects of nutrient intake per se. The effects of diet on health may also be age specific, a possibility that is starting to be explored experimentally in humans (Wolfe et al., 2008).",Cell,Macronutrients and Longevity,2015
Reevaluating Macronutrient Restriction vs. Calorie Restriction,"Until recently, reduced intake of calories, rather than of specific macronutrients, was considered important for health benefits of DR. This assumption was primarily based on a flawed interpretation of experimental data showing that 40% calorie restriction, but not 40% protein restriction, increased lifespan in rats (Maeda et al., 1985). However, the protein-restricted rats were not food restricted, because their growth rate was normal, a point overlooked by the authors of the study. A subsequent series of studies in yeast, invertebrate model organisms and rodents has instead clearly demonstrated that a reduction in specific nutrients in the diet, rather than reduced calorie intake, is primarily responsible for improvements in health and extended lifespan, which is why we use the term DR rather than CR.",Cell,Macronutrients and Longevity,2015
Protein Intake and Healthspan,"Dietary guidelines from the medical literature and popular press often promulgate high protein intake, especially from animal sources rich in essential amino acids, including sulfur-containing and branched chain, to combat obesity, sarcopenia, osteoporosis, frailty, surgical stress, and mortality. However, accumulating evidence points instead to a restriction of protein or specific amino acids in the diet as promoting healthspan (Grandison et al., 2009; Solon-Biet et al., 2014; Ables et al., 2014; Nakagawa et al., 2012; Pamplona and Barja, 2006; Mirzaei et al., 2014). This conclusion initially emerged from investigation of the nutrients mediating the effects of DR and has since been amplified in broader studies of the effects of dietary composition and intake on health and lifespan. In Drosophila, restriction of protein-containing yeast, but not carbohydrate or energy, extends lifespan (Mair et al., 2005). Adding back essential amino acids to the diet of DR flies decreases lifespan, with non-essential amino acids, lipid, or carbohydrate exerting little or no effect (Grandison et al., 2009).",Cell,Protein Restriction,2015
Protein-to-Carbohydrate Ratio and Longevity,"In mice, health and lifespan are strongly affected by the protein component of the diet, with median lifespan progressively increasing up to 30% as the dietary protein-to-carbohydrate ratio is decreased, despite a parallel increase in overall food intake and body fat and reduction in lean mass (Solon-Biet et al., 2014). Dietary protein intake is an important regulator of the IGF-1/mTOR network (Efeyan et al., 2012). In humans, unlike rodents, chronic severe calorie restriction does not reduce serum IGF-1 concentration unless protein intake is also reduced (Fontana et al., 2008), suggesting that dietary protein or specific amino acid intake may be as or more important than calorie intake in modulating IGF-related biological processes and disease risk in men and women.",Cell,Protein Restriction,2015
"Protein Restriction, IGF-1, and Cancer","Over-stimulation of the GH/IGF pathway accelerates aging and increases mortality in rodents, whereas downregulation slows aging, prevents cancer, and increases lifespan (Junnila et al., 2013). Serum IGF-1 concentration is inversely correlated with median lifespan in 31 genetically diverse mouse strains (Yuan et al., 2009) and with the risk of several cancers in humans (Pollak, 2012). Isocaloric restriction of protein and substitution of plant for animal proteins markedly inhibit prostate and breast cancer growth in human xenograft models, reducing serum IGF-1 and downregulating intratumor mTOR activity, EZH2, and H3K27me3, markers of cancer progression (Fontana et al., 2013).",Cell,Protein Restriction,2015
Amino Acid Restriction and Longevity Mechanisms,"Within the protein component of the diet, specific amino acids or their ratio can be important for health and lifespan. Selective restriction of asparagine, glutamate, or methionine extends chronological lifespan in yeast (Dilova et al., 2007; Wu et al., 2013a, 2013b). In Drosophila and rodents, restriction of methionine and tryptophan extends average and maximal lifespan (Ables et al., 2014; Miller et al., 2005; Zimmerman et al., 2003). In Rhesus monkey DR trials, the Wisconsin diet contained higher methionine and branched-chain amino acids than the NIA diet, possibly explaining differing outcomes in cancer and mortality.",Cell,Amino Acid Restriction,2015
"mTOR, GCN2, and Amino Acid Sensing","Amino acid availability is sensed by conserved pathways, especially mTOR and GCN2. Activation of mTOR is modulated by essential amino acids, with branched-chain amino acids playing a key role. Absence of any individual amino acid activates GCN2, required for the protective effect of short-term protein or tryptophan deprivation on surgical stress in mice (Peng et al., 2012). Protein/amino acid restriction-induced protective mechanisms downstream of TOR inhibition and GCN2 activation are still unknown. GCN2 activation stabilizes ATF4, essential for the Integrated Stress Response, elevated in multiple longevity models.",Cell,Amino Acid Restriction,2015
Methionine Restriction and Trans-Sulfuration Pathway,"Reduced dietary methionine induces specific protective mechanisms. Methionine and cysteine are metabolized via the trans-sulfuration pathway, defects in which are linked to age-related diseases. DR in Drosophila increases activity of the rate-limiting enzyme in this pathway, and blocking the pathway prevents lifespan extension. Transgene-mediated activation of the pathway increases lifespan. Hydrogen sulfide, produced via this pathway, also increases lifespan in C. elegans. DR-induced hydrogen sulfide production is observed in yeast, worms, flies, and rodents, and DR-induced resistance to ischemia reperfusion injury in mice requires this increase.",Cell,Methionine Restriction,2015
Methionine Metabolism in Long-Lived Mutant Mice,"In the long-lived Ames dwarf mouse, which lacks growth hormone, expression of trans-sulfuration pathway genes and methionine flux are increased, associated with higher GSH levels (Uthus and Brown-Borg, 2006). These mice and mice lacking the GH receptor have extended lifespan on normal diet but do not respond to methionine restriction, resulting in a lifespan similar to controls (Brown-Borg et al., 2014). Thus, increased trans-sulfuration activity may partly underlie increased healthspan in somatotropic mutant mice.",Cell,Methionine Restriction,2015
Human Evidence and Ongoing Trials,"In humans, little is known about the effects of altering protein quantity and quality on pathways controlling aging, stress resistance, and age-associated diseases. Ongoing clinical trials aim to reveal metabolic and molecular adaptations induced by protein restriction and amino acid modification in overweight subjects and cancer patients.",Cell,Protein Restriction,2015
Microbiota-Derived Factors in Healthy Aging,"In humans, only ~10% of cells and less than 1% of genes are human, and the rest come from trillions of microbes in the gastrointestinal tract. Rapidly accumulating metagenomic data indicate that altered food intake, especially protein and insoluble fiber, have rapid and profound effects on gut microbiota structure, function, and secretion of factors that modulate multiple inflammatory and metabolic pathways (Muegge et al., 2011; Clemente et al., 2012; David et al., 2014; Thorburn et al., 2014). G-protein-coupled receptors expressed by enteroendocrine and immune cells may be important mediators of the effects of the microbiome (Thorburn et al., 2014). For example, oral administration to mice of 17 non-pathogenic Clostridia species isolated from healthy human fecal samples results in gut microbiota that provide short-chain fatty acids, bacterial antigens, and a TGF-β-rich environment, which help differentiation, expansion, and colonic homing of Treg cells and reduce disease severity in multiple models of colitis and allergic diarrhea (Atarashi et al., 2013). In contrast, diet-induced microbiota dysbiosis is associated with increased risk of cardiovascular disease, obesity-associated metabolic abnormalities, cancer, and autoimmune and allergic disease (Clemente et al., 2012).",Cell,Gut Microbiome and Aging,2015
Microbial Effects on Longevity in C. elegans,"In nature, C. elegans feeds on a variety of bacterial species that grow on rotting vegetation, which also constitute the gut microbiome of the worm. Feeding C. elegans with soil bacteria, Bacillus mycoides, and Bacillus soli instead of the standard laboratory E. coli OP50 strain significantly extended lifespan and stress resistance, suggesting that microbial-derived factors may modulate pro-longevity pathways (Abada et al., 2009). Moreover, wild-type C. elegans fed respiratory-incompetent E. coli show increased lifespan (Saiki et al., 2008). A comparison of the effects of E. coli and Comamonas aquatica on the C. elegans host identified vitamin B12 as a major diffusable factor from Comamonas that influenced patterns of gene expression and the rate of development and fertility of the worm (Watson et al., 2014). Interestingly, effects of chemicals on the worm can also be mediated by the gut microbiome. Metformin, the drug used as the first line of defense against type 2 diabetes, extends lifespan of C. elegans fed on live, but not killed, E. coli, and it does so by disrupting the folate cycle and methionine metabolism of the E. coli. In consequence, the supply of bacterial methionine to the worm is reduced, inducing a type of methionine restriction, consistent with the action of metformin as a DR mimetic (Cabreiro et al., 2013).",Cell,Gut Microbiome and Aging,2015
Microbiome Effects in Drosophila and Aging,"The relatively simple gut microbiome of Drosophila is also derived from its food intake, and bacterial density and composition have a substantial effect upon the fly host (reviewed in Erkosar and Leulier, 2014). Bacterial density increases during the aging of the fly and can compromise gut integrity (Guo et al., 2014). The normal complement of gut bacteria enables the flies to use low-nutrient or unbalanced diets by providing them with B vitamins, particularly riboflavin, and by promoting protein nutrition (Wong et al., 2014). Alterations in the gut microbiome may contribute to the improvement in health from DR and time-restricted feeding. Long-term dietary restriction of mice, with either a normal or a high-fat diet, leads to alterations in composition of the gut microbiome, although any contribution of these changes to the health of the DR mice remains to be elucidated (Zhang et al., 2013).",Cell,Gut Microbiome and Aging,2015
Microbiome Changes with Time-Restricted Feeding,"In mice on a high-fat diet, time-restricted feeding during 8 hr of the dark phase decreased representation of Lactobacillus species, which are associated with obesity, and increased Ruminococcaceae species, which protect against metabolic disease associated with obesity (Zarrinpar et al., 2014). Experimental transplantations of microbiota associated with healthy eating could be revealing of their causal role.",Cell,Gut Microbiome and Aging,2015
Immediate vs. Long-Term Effects of Dietary Restriction,"Health, Disease, and Longevity on Various Timescales, Including Inter-generational. The effects of nutrition, including DR, can be exerted on timescales ranging from more or less instantaneous to inter-generational. For instance, in Drosophila, DR acts acutely, with flies switched between DR and ad libitum feeding almost immediately adopting the mortality pattern of a control group kept permanently in the feeding regime that the switched flies join (Mair et al., 2003). At least as reflected in their mortality rate, these flies thus have no memory of their nutritional history, and their patterns of gene expression also change rapidly in response to DR (Whitaker et al., 2014). In contrast, the mortality rates of C. elegans subjected to one form of DR retain a permanent memory of the previous feeding regime after a dietary switch (Wu et al., 2009). The mortality rates of mice and rats subjected to switches between DR and AL feeding later in life have shown mixed responses, but a meta-analysis suggests that there is a permanent memory of diet in these animals (Simons et al., 2013).",Cell,Nutritional Timescales,2015
Acute Nutritional Responses and Glycemic Memory,"Similarly, even short episodes of DR early in adulthood in male mice can induce a glycemic memory apparent as increased glucose tolerance (Cameron et al., 2012; Selman and Hempenstall, 2012). However, in mice and humans, acute responses to DR also occur, including improved insulin sensitivity, reduced inflammation, and protection against ischemia reperfusion injury and other surgical stressors (Hine et al., 2015).",Cell,Nutritional Timescales,2015
Developmental Programming and Early-Life Nutrition,"In contrast to immediate effects of diet, in mammals (including humans), nutrition in early life (including in utero) can have lasting effects on health during aging, often referred to as developmental programming. Undernourished rat and mouse mothers produce offspring with low birth weight and multiple metabolic defects, including early-life adiposity, altered pancreatic function, and progressive glucose intolerance (Tarry-Adkins and Ozanne, 2014; Vickers, 2014). Maternal effects on offspring can include changes to the composition of the egg, alterations to the environment in utero, and peri-natal effects such as transmission of the microbiome and alterations to lactation, and can be manifest in the offspring as changes in gene expression and epigenetic modifications, including DNA methylation, histone modification, and expression of microRNAs, as well as evidence of increased cellular aging (Aiken and Ozanne, 2014; Colaneri et al., 2013; Radford et al., 2012).",Cell,Developmental Programming,2015
Human Evidence and the Thrifty Phenotype Hypothesis,"Epidemiological data from humans also show a consistent effect of developmental programming by early—including in utero—nutrition, although the evidence on the mechanisms involved is necessarily correlational rather than experimental. The thrifty phenotype hypothesis (Hales and Barker, 2001) postulates that many of the changes in organ structure and metabolism seen in humans in response to restricted nutrition—particularly of protein—in utero can be understood as the consequences of immediate responses of the fetus to ensure survival and spare vital organs such as the brain. Viewed in this way, an under-nourished fetus makes the best of a bad job with adverse consequences for health in later life, including reduced glucose tolerance and a higher incidence of ischemic heart disease, problems that are greatly exacerbated by subsequent adequate or over-nutrition. However, a poor functional capacity for insulin secretion would not be detrimental to individuals who continued to be poorly nourished and remained thin and therefore insulin sensitive, and it remains possible that some fetal and post-natal responses to low nutrition are advantageous in conditions of continuing poor nutrition.",Cell,Developmental Programming,2015
Predictive Value of Nutrition Across Generations,"Inter-generational Effects of Diet. Current nutrition may act as a predictor of future nutritional conditions if food availability shows local variation or if timing of natural cycles of food scarcity and abundance occurs on an appropriate timescale. Under these circumstances, information gained early in life or even in earlier generations could be profitably used to anticipate future nutritional prospects and adjust physiology accordingly (Rando, 2012). Such considerations may partly explain why inter-generational effects of diet can also be transmitted through males. The evidence for a role of epigenetic inheritance in these cases is largely correlational, and a direct experimental testing of the hypothesis is challenging (Heard and Martienssen, 2014; Rando, 2012). Cellular mechanisms by which metabolic changes can be communicated to chromatin are being increasingly discussed (Gut and Verdin, 2013; Katada et al., 2012; Lu and Thompson, 2012).",Cell,Intergenerational Nutrition,2015
Paternal Diet Effects via Epigenetic Reprogramming,"In Drosophila, the sugar content of the paternal diet, even over a 2 day period during which the offspring are sired, can elicit increased lipid content in offspring. Sugar in the diet de-silences chromatin-state-defined domains both in mature sperm and in offspring embryos, and H3K9/K27me3-dependent reprogramming of metabolic genes in two time windows in the germline and the zygote is required for increased lipid content of offspring. Furthermore, data from mice and humans, including discordant human monozygotic twins, show a similar signature of chromatin de-repression associated with obesity (Öst et al., 2014).",Cell,Intergenerational Epigenetics,2015
Male-Mediated Epigenetic Transmission in Mammals,"Livers of offspring of male mice fed a low-protein diet show elevated expression of genes involved in lipid and cholesterol biosynthesis. These alterations are accompanied by subtle (in the region of 20%) changes in DNA cytosine methylation in specific gene regions, including a putative enhancer for the lipid regulator Ppara (Carone et al., 2010). Mice subjected to in utero undernourishment are glucose intolerant, and they can transmit the glucose intolerance even though they themselves are not undernourished. They experience the effects of maternal undernourishment during a period that includes the time when their germ cell DNA reacquires methylation. The sperm DNA of these males is hypomethylated at multiple sites, especially ones enriched in regulatory elements and regulators of chromatin. Although these altered methylation patterns are not apparent in the tissues of their offspring, there are perturbations to gene expression, possibly attributable to other types of epigenetic alteration (Radford et al., 2014).",Cell,Intergenerational Epigenetics,2015
Transgenerational Nutritional Memory in C. elegans,"Evidence is also starting to point to truly inter-generational effects of diet, where information about dietary history is epigenetically transmitted in the germline in the absence of any further input from the organism or its environment. In the nematode worm Caenorhabditis elegans, starvation-induced developmental arrest has effects that persist for at least three generations, with the third generation offspring of the starved great-grandparents showing increased adult lifespan. The starvation event leads to the generation of small RNAs that are also inherited for at least three generations, that target the mRNAs of genes involved in nutrient reservoir activity (Rechavi et al., 2014), and that are possibly also causal in the increased lifespan of the third generation descendants. It is not clear whether these persistent effects represent non-adaptive perturbations, anticipatory programming, or both. Effects of nutrition of the paternal grandfather on grandchildren have been reported in humans, but the mechanisms responsible are unknown (Pembrey et al., 2014).",Cell,Transgenerational Epigenetics,2015
Parent-of-Origin Effects and Imprinting,"Recent work with mice has suggested that sex-of-parent-of-origin effects may be much more pervasive and influential than previously supposed. Even though the number of imprinted genes in the mammalian genome is predicted to be small, non-imprinted genes can regulate the tissue-specific expression of many other genes differently when transmitted by females or males, possibly by physical interaction with imprinted loci (Mott et al., 2014). These findings could have profound implications for human aging and disease.",Cell,Genomic Imprinting and Diet,2015
Genetic Variation in Dietary Response,"Individuals of different genotypes can respond differently to diet. Although little studied outside the context of inborn errors of metabolism, such genetic effects in humans are potentially important for identifying sub-groups that would benefit from dietary modulation. Females and males often respond very differently to dietary and pharmacological interventions, and evidence is mounting for the importance of other types of genetic variation. DR has proved to extend lifespan in most species examined, although it has been suggested that increased lifespan in response to DR may have evolved partly as an artifact of laboratory culture (Nakagawa et al., 2012).",Cell,Genetic Variation and Diet,2015
Strain-Specific Variation in DR Lifespan Response,"Strains of C. elegans and Drosophila collected directly from nature respond normally to DR (Metaxakis et al., 2014). Wild-derived mice, on the other hand, can show little or no response (Harper et al., 2006). The stresses of captivity in non-domesticated animals could explain this finding, and wild mice may also respond to milder or stronger DR than the single level tested (Gems et al., 2002). In recombinant inbred mouse strains, a 40% reduction in intake resulted in responses ranging from a 98% extension to a 68% reduction in lifespan (Liao et al., 2010). Lifespan typically shows a tent-shaped response to food intake, peaking at intermediate levels. Thus, determining whether a strain responds to DR requires testing multiple levels of restriction (Gems et al., 2002).",Cell,Genetic Variation and Diet,2015
Human Genetic Variation and Dietary Interventions,"Interventions to reduce weight often have beneficial effects on blood lipid profiles, type 2 diabetes, and cardiovascular disease risk, but some individuals respond poorly or not at all. A study showed that a higher genetic risk score for dyslipidemia based on SNP genotyping was associated with a substantially diminished response to increased exercise and lowered fat intake (Pollin et al., 2012). Studies of this kind could also illuminate underlying mechanisms and enable individualized dietary and lifestyle interventions for disease prevention.",Cell,Genetic Variation and Diet,2015
Study Summary and Key Findings,"Low Protein Intake Is Associated with a Major Reduction in IGF-1, Cancer, and Overall Mortality in the 65 and Younger but Not Older Population. Mice and humans with growth hormone receptor/IGF-1 deficiencies display major reductions in age-related diseases. Because protein restriction reduces GHR-IGF-1 activity, we examined links between protein intake and mortality. Respondents aged 50–65 reporting high protein intake had a 75% increase in overall mortality and a 4-fold increase in cancer death risk during the following 18 years. These associations were either abolished or attenuated if the proteins were plant derived. Conversely, high protein intake was associated with reduced cancer and overall mortality in respondents over 65, but a 5-fold increase in diabetes mortality across all ages. Mouse studies confirmed the effect of high protein intake and GHR-IGF-1 signaling on the incidence and progression of breast and melanoma tumors, but also the detrimental effects of a low protein diet in the very old. These results suggest that low protein intake during middle age followed by moderate to high protein consumption in old adults may optimize healthspan and longevity.",Cell Metabolism,Protein Intake and Longevity,2014
Background on Caloric Restriction and IGF-1 Signaling,"Caloric restriction (CR) without malnutrition has been consistently shown to increase longevity in a number of animal models, including yeast, C. elegans, and mice (Fontana et al., 2010). However, the effect of CR on the lifespan of nonhuman primates remains controversial and may be heavily influenced by dietary composition (Cava and Fontana, 2013; Colman et al., 2009; Fontana and Klein, 2007; Mattison et al., 2012; Mercken et al., 2013; Stein et al., 2012). The lifespan extension associated with CR in model organisms is believed to operate through its effects on growth hormone (GH) and GH receptor (GHR), leading to subsequent deficiencies in IGF-1 and insulin levels and signaling (Bartke et al., 2001; Bellush et al., 2000; Fontana et al., 2010; Hauck et al., 2002; Wei et al., 2009). Mutations in the insulin/IGF-1 receptor or downstream genes such as age-1 cause several-fold lifespan extensions in C. elegans (Johnson, 1990; Kenyon et al., 1993; Kenyon, 2010). Other mutations affecting TOR-S6K and RAS-cAMP-PKA signaling pathways also regulate aging in multiple organisms.",Cell Metabolism,Protein Intake and Longevity,2014
GH/IGF-1 Deficiency and Human Disease Protection,"In mice, growth hormone receptor deficiency (GHRD) or growth hormone deficiency (GHD), both of which display low IGF-1 and insulin levels, cause significant lifespan extension and reduction in age-related pathologies including cancer and insulin resistance/diabetes (Brown-Borg and Bartke, 2012; Brown-Borg et al., 1996; Masternak and Bartke, 2012). Recently, humans with GHRD, who also exhibit major deficiencies in serum IGF-1 and insulin, displayed no cancer mortality or diabetes. Despite higher obesity prevalence, combined deaths from cardiac disease and stroke in this group were similar to those in their relatives (Guevara-Aguirre et al., 2011). Similar protection from cancer was also reported in a survey of 230 GHRDs (Steuerman et al., 2011).",Cell Metabolism,Protein Intake and Longevity,2014
Role of Protein Restriction in Longevity,"Protein restriction or restriction of particular amino acids, such as methionine and tryptophan, may explain part of the effects of calorie restriction and GHRD mutations on longevity and disease risk, since protein restriction is sufficient to reduce IGF-1 levels and can reduce cancer incidence or increase longevity in model organisms, independently of calorie intake (Ayala et al., 2007; Fontana et al., 2008, 2013; Gallinetti et al., 2013; Horáková et al., 1988; Hursting et al., 2007; Leto et al., 1976; Mair et al., 2005; Pamplona and Barja, 2006; Peng et al., 2012; Ross, 1961; Sanz et al., 2006; Smith et al., 1995; Youngman, 1993). Here, we combined an epidemiological study of 6,381 US men and women aged 50 and above from NHANES III with mouse and cellular studies to understand the link between the level and source of proteins and amino acids, aging, diseases, and mortality.",Cell Metabolism,Protein Intake and Longevity,2014
Study Summary and Key Findings,"Ketogenic Diet Reduces Midlife Mortality and Improves Memory in Aging Mice. Ketogenic diets mimic several metabolic features of dietary restriction, including reliance on fatty acid metabolism and ketone body production. This study investigated whether an isoprotein ketogenic diet (KD) might affect longevity and healthspan in C57BL/6 male mice. Cyclic KD (alternating KD weekly with a control diet to prevent obesity) reduced midlife mortality but did not increase maximum lifespan. A non-ketogenic high-fat diet (HF) under the same feeding structure may have an intermediate survival effect. Cyclic KD improved memory performance in old age and modestly improved composite healthspan scores. Gene expression revealed suppression of insulin signaling, protein synthesis, and fatty acid synthesis pathways in both KD and HF groups. However, KD uniquely upregulated PPARα target genes across tissues, and this signature persisted into old age. Overall, a non-obesogenic KD improves survival, memory, and general health in aging mice.",Cell Metabolism,Ketogenic Diet and Aging,2017
Background on Ketone Bodies and Longevity Mechanisms,"Ketogenic diets recapitulate metabolic features of dietary restriction such as increased fatty acid oxidation and production of the ketone body beta-hydroxybutyrate (BHB). BHB functions not only as an energy substrate but also as a signaling molecule. It inhibits the NLRP3 inflammasome, inhibits histone deacetylases, acts as a ligand for GPCRs, and forms a fasting-associated histone modification. These signaling roles position BHB as a potential regulator of inflammation, gene expression, and aging. Elevated ketone bodies occur naturally during intermittent fasting and dietary restriction, which are linked to extended longevity, reduced cancer incidence, improved cognition, and immune rejuvenation.",Cell Metabolism,Ketogenic Diet and Aging,2017
Experimental Design and Dietary Implementation,"The researchers used an isoprotein ketogenic diet to avoid confounding effects of protein deficiency. A cyclic diet approach was used: KD for one week alternating with a standard control diet the next week (Cyclic KD). This approach prevented obesity, a common side effect of continuous KD feeding in mice. Parallel comparisons were made with a non-ketogenic high-fat (HF) diet implemented with the same cyclic schedule. This design allowed investigation of lifespan, survival curves, healthspan metrics, and molecular pathways influenced specifically by ketosis versus high fat intake.",Cell Metabolism,Ketogenic Diet and Aging,2017
Molecular Mechanisms and Gene Expression Effects,"Gene expression analysis showed that both KD and HF diets downregulated insulin signaling, protein synthesis pathways, and fatty acid synthesis. These shared changes resemble the metabolic signatures observed under dietary restriction. However, KD uniquely upregulated PPARα target genes—key regulators of fatty acid oxidation and ketogenesis—across multiple tissues. This PPARα activation appeared robust and remained present even in aged mice. The unique transcriptional footprint of KD may explain its specific health benefits beyond those seen in high-fat feeding.",Cell Metabolism,Ketogenic Diet and Aging,2017
Experimental Design for Ketogenic Diet Survival Study,"The researchers designed AIN-93M–based diets with matched protein, micronutrient, and fiber content, while varying carbohydrate and fat levels to determine which formula maximized ketone body production. Zero-carbohydrate ketogenic diet (KD) produced the highest plasma β-hydroxybutyrate (BHB), with ketogenesis suppressed progressively as carbohydrate content increased, reaching baseline at 15% carbohydrate. Three diets were finalized: (1) zero-carbohydrate KD, (2) a standard Control diet, and (3) a high-fat, non-ketogenic diet (HF) containing 15% carbohydrate. Both KD and HF produced high fat utilization, but only KD elevated BHB. Continuous KD or HF feeding caused obesity; however, alternating these diets weekly with the Control diet (Cyclic KD and Cyclic HF) prevented long-term weight gain and maintained metabolic stability. Cyclic KD produced BHB levels of 1–2 mM, similar to fasting. The study then implemented five regimens: Cyclic KD, Cyclic HF, continuous Control, continuous KD, and continuous HF, beginning in C57BL/6 male mice at 12 months of age.",Cell Metabolism,Ketogenic Diet and Longevity,2017
Metabolic and Physiological Effects of Cyclic KD,"Cyclic KD mice maintained body weights nearly identical to Controls across lifespan, while Cyclic HF mice were slightly heavier. Continuous KD and HF groups were more obese. Although caloric intake was higher during KD or HF weeks, the total lifetime caloric intake of cyclic groups matched Controls. Weekly feeding cycles produced predictable weight oscillations, with larger fluctuations in Cyclic HF than Cyclic KD. Weight loss occurred consistently during early Control-diet days each cycle. BHB levels were higher in Cyclic KD than in continuous KD, especially during daytime, suggesting enhanced reliance on ketone metabolism caused by the cyclic regimen.",Cell Metabolism,Ketogenic Diet and Metabolism,2017
Impact of Cyclic KD on Survival and Lifespan,"A small pilot suggested that Cyclic KD might reduce midlife mortality without extending maximum lifespan. In the main cohort, both Cyclic KD and Cyclic HF reduced early mortality and increased median lifespan relative to Control. Maximum lifespan was unchanged. Day-by-day statistical analysis showed significantly improved survival in Cyclic KD versus Control up to ~30 months of age, after which survival curves converged. Cyclic HF exhibited similar but weaker effects. Continuous KD and HF resulted in shorter lifespan due to obesity, but KD still produced lower early mortality than HF. Monte Carlo label-randomization simulations confirmed the robustness of the Cyclic KD survival benefit (appearing in only 21/1,000 chi-square simulations), while Cyclic HF demonstrated an intermediate and less robust effect.",Cell Metabolism,Survival Effects of Ketogenic Diet,2017
Necropsy Findings and Causes of Death,"Necropsy analysis indicated no major differences in causes of early versus late deaths across groups. Internal tumor prevalence was lowest in the Cyclic KD group, though not statistically significant compared to Controls. A rare tumor type—Harderian gland carcinoma—was more frequent in Cyclic KD and continuous KD groups, though overall incidence remained low. These observations suggest that reduced midlife mortality in Cyclic KD is unlikely explained by a single disease category.",Cell Metabolism,Ketogenic Diet Pathology Findings,2017
Healthspan Study Design and Goals,"To evaluate whether the cyclic ketogenic diet (Cyclic KD) improves healthspan beyond survival, the authors ran a parallel longitudinal functional study. Male C57BL/6 mice were baseline-tested at 12–14 months for cognitive, physical, and behavioral function, then half were randomized to Cyclic KD while the rest remained on Control diet. All mice were fed Control diet during the testing periods to avoid acute metabolic confounding. After 10 months of cyclic dietary intervention, aged testing was performed at 22–24 months; a follow-up limited assessment occurred at 28–30 months. The healthspan battery included place avoidance learning, novel object recognition, rotarod, balance beam, wire hang, frailty scoring, spontaneous activity measures, and echocardiography. To analyze broad aging effects, the authors generated composite scores normalized to young performance using 35 quantitative parameters.",Cell Metabolism,Ketogenic Diet and Healthspan,2017
Cognitive Benefits: Preservation of Memory,"Cyclic KD produced a robust and consistent preservation of memory during aging. In the primary cognitive task—place avoidance—the Cyclic KD group exhibited markedly better memory retention (recall) than Controls at 22–24 months, despite similar age-related slowing during the learning phase. Cyclic KD mice also showed improved maximum escape velocity following shock, indicating partial preservation of motor responsiveness. At 28–30 months, Cyclic KD again demonstrated superior memory in a different paradigm (novel object recognition). These results highlight a reproducible, cross-task cognitive advantage attributable to Cyclic KD.",Cell Metabolism,Cognitive Aging and Ketogenic Diet,2017
"Physical, Behavioral, and Cardiovascular Outcomes","Most physical performance outcomes showed modest or no differences between groups. Frailty scores were only slightly improved in Cyclic KD mice, and many strength- and coordination-based tasks (rotarod, wire hang) did not differ significantly. One spontaneous activity measure—exploration in the elevated plus maze—declined with age in Controls but was preserved in Cyclic KD mice, suggesting maintained exploratory drive. Importantly, echocardiographic analysis revealed that Cyclic KD preserved a more youthful cardiac phenotype, reflected in a composite score combining heart rate, LV mass, valve pressure gradients, and fractional shortening.",Cell Metabolism,Physical and Cardiovascular Aging,2017
Overall Healthspan Effects,"When combining all 35 healthspan parameters into a global composite index, the Cyclic KD group showed a modest but consistent attenuation of age-related functional decline. The improvements were not driven by a single system but were distributed across many physiological domains. This indicates that Cyclic KD has broad, mild protective effects on aging physiology, with especially strong benefits in cognitive preservation.",Cell Metabolism,Global Healthspan Assessment,2017
Fasting-like Transcriptional Signature of Ketogenic Diet,"Gene expression analysis revealed that KD induces a transcriptional program highly reminiscent of fasting. In 9-month-old mice fed KD for 5 months, hepatic protein acetylation increased—both globally and within mitochondria—alongside activation of fasting-responsive transcriptional networks. Lipogenesis genes (Fasn, Scd1) were strongly repressed, and a fasting-like metabolic switch was evident. These observations suggested that BHB and KD feeding activate conserved nutrient-sensing pathways.",Cell Metabolism,Ketogenic Diet Gene Expression,2017
Transcriptomic Comparison of KD vs HF vs Control,"RNA-seq of liver and kidney following 1 week of diet exposure showed that KD and HF share many downregulated genes, especially those involved in glucose metabolism, insulin/IGF signaling, fatty acid synthesis, and mTOR/ribosomal protein expression—mechanisms linked to longevity. However, the upregulated gene sets differed dramatically: most KD-induced genes were not induced by HF. Pathway enrichment demonstrated that KD uniquely activates PPARα target genes across tissues, enhancing fatty acid oxidation and mitochondrial programs. HF failed to activate PPARα despite its high fat content. KD also showed unique activation of p53 and p21 relative to HF, suggesting enhanced tumor-suppressive signaling.",Cell Metabolism,KD vs HF Transcriptomics,2017
Long-Term Persistence of Ketogenic Gene Expression Programs,"To determine whether KD-induced transcriptional changes persist during the long-term cyclic regimen, qPCR was performed on 26-month-old mice after 14 months of Cyclic KD. Tissue was collected mid-Control week to avoid acute KD effects. Downregulation of mTOR pathway genes persisted, while suppression of glucose metabolism and fatty acid synthesis did not. Crucially, upregulation of PPARα targets and fatty-acid oxidation genes remained strong and durable into old age, mirroring the physiological improvements in survival and memory. Thus, the combination of chronic TOR suppression and sustained PPARα activation may underlie the healthspan and early-life survival benefits of Cyclic KD.",Cell Metabolism,Longevity Mechanisms of KD,2017
Study Objective and Design,"Objective: Dietary carbohydrate is the major determinant of postprandial glucose levels, and several clinical studies have shown that low-carbohydrate diets improve glycemic control. In this study, we tested the hypothesis that a diet lower in carbohydrate would lead to greater improvement in glycemic control over a 24-week period in patients with obesity and type 2 diabetes mellitus.
Research design and methods: Eighty-four community volunteers with obesity and type 2 diabetes were randomized to either a low-carbohydrate, ketogenic diet (<20 g of carbohydrate daily; LCKD) or a low-glycemic, reduced-calorie diet (500 kcal/day deficit from weight maintenance diet; LGID). Both groups received group meetings, nutritional supplementation, and an exercise recommendation. The main outcome was glycemic control, measured by hemoglobin A1c.
Results: Forty-nine (58.3%) participants completed the study. Both interventions led to improvements in hemoglobin A1c, fasting glucose, fasting insulin, and weight loss. The LCKD group had greater improvements in hemoglobin A1c (-1.5% vs. -0.5%, p = 0.03), body weight (-11.1 kg vs. -6.9 kg, p = 0.008), and high density lipoprotein cholesterol (+5.6 mg/dL vs. 0 mg/dL, p < 0.001) compared to the LGID group. Diabetes medications were reduced or eliminated in 95.2% of LCKD vs. 62% of LGID participants (p < 0.01). Conclusion: Dietary modification led to improvements in glycemic control and medication reduction/elimination in motivated volunteers with type 2 diabetes. The diet lower in carbohydrate led to greater improvements in glycemic control, and more frequent medication reduction/elimination than the low glycemic index diet. Lifestyle modification using low carbohydrate interventions is effective for improving and reversing type 2 diabetes.",Nutrition & Metabolism,Ketogenic Diet,2008
Background on Carbohydrates and Glycemic Control,"Background: The dietary macronutrient that raises postprandial serum glucose and insulin most potently is carbohydrate. This observation led to the use of diets low in carbohydrate for the treatment of diabetes before insulin or other medication therapies were available. In like fashion, individuals who are insulin-deficient are instructed to estimate the amount of carbohydrate in the meal and then to administer the insulin dosage based upon the amount of dietary carbohydrate. This strong relationship between dietary carbohydrate and postprandial serum glucose led to the development of medications that block carbohydrate absorption for the treatment of type 2 diabetes. Clinical studies that have lowered the percentage of dietary carbohydrate and/or the glycemic index of the carbohydrate have consistently shown improvements in glycemic control among individuals with type 2 diabetes. In randomized studies, low-carbohydrate diets have been found effective for the treatment of obesity for durations up to 24 months. While glycemic control was not a primary outcome, some of these studies additionally demonstrated improvement in glycemic parameters when carbohydrate intake was lowered. In the Nurse's Health Study cohort study, low-glycemic load diets were found to be associated with lower cardiac risk over a 20 year period.",Nutrition & Metabolism,Ketogenic Diet,2008
Rationale for Low-Carbohydrate Ketogenic Diets,"One mechanism to explain these findings is that when patients are instructed to limit carbohydrate intake to low levels without mention of caloric intake, there is an overall reduction in caloric intake. In several recent studies, in the outpatient setting and metabolic ward, low-carbohydrate ketogenic diets led to improvements in glycemic control among patients with diabetes. While it may be intuitive that a low-carbohydrate ketogenic diet with fewer than 20 grams of carbohydrate intake per day would lead to better glycemic control than a low-glycemic diet, we are not aware that this idea has been actually tested. In the present study, our hypothesis was that a diet lower in carbohydrate would lead to greater improvement in glycemic control in patients with obesity and type 2 diabetes mellitus over 24 weeks in the outpatient setting.",Nutrition & Metabolism,Ketogenic Diet,2008
Participant Recruitment and Eligibility,"Participants were recruited from the community by newspaper advertisements. After telephone screening, potential participants were scheduled for a ""screening visit"" which included informed consent approved by the local institutional review board, a medical history, physical examination and laboratory tests. The inclusion criteria were: diagnosis of type 2 diabetes mellitus > 1 year (confirmed by hemoglobin A1c > 6.0%), onset of diabetes after age 15 years, no history of diabetic ketoacidosis, age 18–65 years old, body mass index (BMI) from 27–50 kg/m2, and desire to lose weight. Exclusion criteria were: unstable or serious medical condition; significant co-morbid illnesses such as liver disease (AST or ALT > 100 IU/L), kidney disease (serum creatinine > 1.5 mg/dL), cancer; pregnancy; or nursing mothers. No monetary incentives were given.",Nutrition & Metabolism,Ketogenic Diet,2008
Randomization and General Interventions,"If study criteria were met, participants were randomized to one of two treatment groups stratified upon BMI greater or less than 32 kg/m2 using a computer-generated list, and invited to attend the ""baseline visit."" (Measurements taken at the ""screening visit"" were used as the initial value in comparison testing for laboratory tests; measurements from the ""baseline visit"" were used as the initial value for other outcomes.) The intervention for both groups included group sessions, diet instruction, nutritional supplements, and an exercise recommendation. Group meetings took place at an outpatient research clinic every week for 3 months, then every other week for 3 months. If a participant was taking medication for diabetes or hypertension, a physician reviewed the blood glucose and blood pressure readings and made medication changes according to a pre-specified algorithm. Participants were encouraged to exercise for 30 minutes at least 3 times per week, but no formal exercise program was provided. Both groups received the same nutritional supplements known to have a mild lowering effect on blood glucose levels (vanadyl sulfate 200 mcg/day, chromium dicotinate glycinate 600 mcg/day, alpha-lipoic acid 200 mg/day).",Nutrition & Metabolism,Ketogenic Diet,2008
Low-Carbohydrate Ketogenic Diet Intervention (LCKD),"Using a lay-press diet book and additional handouts, a registered dietitian instructed participants to restrict intake of dietary carbohydrate to fewer than 20 grams per day, without explicitly restricting caloric intake. Allowed foods were unlimited amounts of animal foods (i.e., meat, chicken, turkey, other fowl, fish, shellfish) and eggs; limited amounts of hard cheese (e.g., cheddar or swiss, 4 ounces per day), fresh cheese (e.g., cottage or ricotta, 2 ounces per day), salad vegetables (2 cupfuls per day), and non-starchy vegetables (1 cupful per day). Participants were encouraged to drink at least 6 glasses of permitted fluids daily. Drinking bouillon dissolved in water was recommended 2–3 times a day during the first two weeks to reduce possible side effects.",Nutrition & Metabolism,Ketogenic Diet,2008
Low-Glycemic Index Diet Intervention (LGID),"Using a lay-press diet book and additional handouts, a registered dietitian instructed participants to follow a low-glycemic index, reduced-calorie diet with approximately 55% of daily caloric intake from carbohydrate. The energy intake was individualized to be 2.1 MJ (500 kcal) less than the participant's calculated energy intake for weight maintenance (21.6*lean body mass + 370 kcal + activity factor).",Nutrition & Metabolism,Ketogenic Diet,2008
Primary Outcome Measure: Hemoglobin A1c,"Hemoglobin A1c was measured at baseline, week 12, and week 24. The primary outcome was change in hemoglobin A1c from baseline to week 24, using an immunoassay technique. The hemoglobin A1c provides an estimate of glycemic control for the previous 3-month period and is predictive of clinical outcomes.",Nutrition & Metabolism,Ketogenic Diet,2008
Diet Composition Assessment,"All participants completed food records (5 consecutive days, including a weekend) at baseline, and during the intervention (weeks 4, 12, and 24). Participants were instructed how to document food record information and given handouts with examples of how to complete the records. A sample of completers (n = 8 for low-carbohydrate diet group; n = 7 for low-glycemic diet group) was selected for food record analysis based upon record detail. A registered dietitian analyzed the food records using a nutrition software program (Nutritionist Five, Version 1.6, First DataBank Inc., San Bruno, CA). Food record results were averaged over weeks 4, 12, and 24.",Nutrition & Metabolism,Ketogenic Diet,2008
Vital Signs and Anthropometrics,"Wearing light clothing and no shoes, participants were weighed at each visit on the same calibrated scale. Body mass index was calculated as: (body weight in kilograms)/(height in meters)2. Systolic and diastolic blood pressures were measured in the non-dominant arm using an automated digital cuff (model HEM-725C, Omron Corp., Vernon Hills, IL) after sitting for 3 minutes. Two measurements were taken per visit and averaged for the analysis.",Nutrition & Metabolism,Ketogenic Diet,2008
Metabolic and Laboratory Assessments,"Blood tests were obtained in the morning after at least 8 hours of fasting and processed by a commercial laboratory (Labcorp, Burlington NC). Glomerular filtration rate was estimated by using an equation containing the variables age, gender, race, and serum albumin, creatinine, and blood urea nitrogen (Modification of Diet in Renal Disease (MDRD) Study equation). Twenty-four hour urine collections for protein were collected at baseline and at 24 weeks.",Nutrition & Metabolism,Ketogenic Diet,2008
Adverse Effects and Medication Tracking,"At all return visits, participants completed an open-ended side effects questionnaire. To enhance the description of side effects, participants completed a checklist of side effects commonly mentioned during weight loss studies at both the 20 and 24-week visit. These two measures were combined to report the proportion in each group who experienced an adverse effect at any time during the study. At baseline and at all return visits, participants recorded all of their current medications with dosages and schedules.",Nutrition & Metabolism,Ketogenic Diet,2008
Adherence and Intervention Delivery,"Adherence with the diet and exercise recommendations was measured by self-report, food records, and urinary ketones. The delivery of the intervention and the assessment of outcomes were not blinded to the treatment assignment.",Nutrition & Metabolism,Ketogenic Diet,2008
Statistical Analysis Framework,"For categorical outcomes, comparisons between groups were performed using the chi square test or Fisher's exact test, as appropriate. For all continuous outcomes, comparisons were made using a t-test or Wilcoxon rank-sum test as appropriate, testing the difference between groups for the change from baseline to week 24. For the primary outcome variable, a completer's analysis and last observation carried forward (LOCF) were performed, and a multiple linear regression analysis adjusting for weight change was performed to determine if the change in hemoglobin A1c was independent of weight loss. A p value of ≤ 0.05 was considered statistically significant. Analyses were performed using SAS Statistical Software, Version 8.02 (SAS Institute Inc., Cary, NC). In order to detect a clinically meaningful change in hemoglobin A1c (absolute change of 1%, SD = 1.5) with 80% power (two-sided alpha of .05) in a completers analysis, a total of 60 participants was required. To protect for dropouts, 97 participants were recruited.",Nutrition & Metabolism,Ketogenic Diet,2008
Funding Statement,The investigators conducted the study independently of the funding source. The funding source had no involvement in conduct of the study.,Nutrition & Metabolism,Ketogenic Diet,2008
Participant Flow and Completion Rates,"213 potential participants were screened for eligibility, and 97 were randomized. Ten participants of 48 randomized to the LCKD group, and 3 of 49 participants randomized to the LGID group discontinued the study prior to the Week 0 visit and did not receive instruction, leaving 38 in the LCKD group and 46 in the LGID group for the analyses. For the LCKD group, 21 (55.3%) completed the study; reasons for discontinuation were: 3 refused assigned diet, 2 were unsatisfied with the diet, 2 were lost to follow-up, 2 were too busy, 1 relocated, and 7 cited no reason. For the LGID group 29 (63.0%) completed the study; reasons for discontinuation were: 1 refused assigned diet, 1 was unsatisfied with the diet, 2 were lost to follow-up, 3 were too busy, 1 relocated, 1 had difficulty adhering to the diet and 9 cited no reason. The baseline characteristics of study participants are shown in Table 1. There were no clinically significant differences between the treatment groups.",Nutrition & Metabolism,Ketogenic Diet,2008
Hemoglobin A1c Outcomes,"From baseline to 24 weeks, the reduction of mean ± SD hemoglobin A1c was greater for the LCKD group (8.8 ± 1.8% to 7.3 ± 1.5%, p = 0.009, within group change, n = 21) than for the LGID group (8.3 ± 1.9% to 7.8 ± 2.1% p = NS, within group change, n = 29; between groups comparison p = 0.03). The mean change in hemoglobin A1c for the LCKD group was -1.5% (95% CI: -2.30, -0.71), and for the LGID group was -0.5% (95% CI: -1.04, 0.10). Using a theoretical probability matrix comparing the change in hemoglobin A1c for each individual in one group to each individual in the other group, the probability of having a greater improvement in hemoglobin A1c was 0.683 for being assigned to the LCKD group, compared to 0.300 for being in the LGID group. Fasting blood glucose and insulin improved similarly for both groups over the 24 weeks. In the LOCF analysis, the mean hemoglobin A1c at baseline and week 24 was 8.5% and 7.5% for the LCKD group, and 8.3% and 8.0% for the LGID group (p = 0.02, between groups comparison). In a multivariate linear regression model adjusting for weight change or BMI change, the between group comparison in change in hemoglobin A1c approached statistical significance (p = 0.06). Additionally, there was no correlation between change in hemoglobin A1c and change in weight.",Nutrition & Metabolism,Ketogenic Diet,2008
Medication Changes,"At baseline, 22 (75.9%) of the LGID group were taking hypoglycemic medications (insulin only n = 3, oral agents only n = 19), and 20 (95.2%) of the LCKD group were taking hypoglycemic medications (insulin + oral agents n = 4, insulin only n = 4, oral agents only n = 12). Twenty of 21 (95.2%) LCKD group participants had an elimination or reduction in medication, compared with 18 of 29 (62.1%) LGID group participants (p < 0.01). Five individuals (4 in the LCKD group, 1 in the LGID group) who were taking over 20 units of insulin at baseline were no longer taking insulin at the end of the study.",Nutrition & Metabolism,Ketogenic Diet,2008
Dietary Intake and Exercise Adherence,"Prior to the study intervention, the mean ± SD dietary intake for both groups was 2128 ± 993 kcal, 245 ± 136 g of carbohydrate (46% of daily energy intake), 86 ± 33 g of protein (18% of daily energy intake), 88 ± 57 g of fat (36% of daily energy intake). Over the 24-week duration of the intervention, the LCKD group consumed 1550 ± 440 kcal per day, 49 ± 33 g of carbohydrate (13% of daily energy intake), 108 ± 33 g of protein (28% of daily energy intake), 101 ± 35 g of fat (59% of daily energy intake). In comparison, the LGID group consumed 1335 ± 372 kcal per day, 149 ± 46 g of carbohydrate (44% of daily energy intake), 67 ± 20 g of protein (20% of daily energy intake), 55 ± 23 g of fat (36% of daily energy intake). There was no difference in self-reported exercise between the groups: the mean number of exercise sessions per week increased from 2.0 ± 2.0 to 3.0 ± 2.0 for the LCKD group and from 2.2 ± 2.2 to 3.8 ± 2.9 for the LGID group (p = 0.39).",Nutrition & Metabolism,Ketogenic Diet,2008
Weight Loss and Vital Sign Changes,"There was significantly greater weight loss for the LCKD than the LGID group over the 24 weeks: body weight decreased from 108.4 ± 20.5 kg to 97.3 ± 17.6 kg for the LCKD group, and from 105.2 ± 19.8 to 98.3 ± 20.3 kg for the LGID group. Both groups had reductions in systolic blood pressure and diastolic blood pressure. All 7 parameters associated with the metabolic syndrome showed improvement for the LCKD group; 5 of 7 improved for the LGID group. In terms of renal function, serum creatinine and calculated GFR did not change significantly over the 24 weeks for either group. There was a greater reduction in 24-hour urine protein for the LCKD group (baseline = 445 ± 1175 mg/24 hour, week 24 = 296 ± 750 mg/24 hours, n = 18), as compared with the LGID group (baseline = 276 ± 705 mg/24 hour, week 24 = 223 ± 623 mg/24 hours, n = 24, p = 0.007).",Nutrition & Metabolism,Ketogenic Diet,2008
Adverse Effects,"There were no statistically significant differences between groups in reported symptomatic adverse effects. The most common symptoms experienced at any point during the study were headache (LCKD: 53.1%, LGID: 46.3%), constipation (LCKD: 53.1%, LGID: 39.0%), diarrhea (LCKD: 40.6%, LGID: 36.6%), insomnia (LCKD: 31.2%, LGID: 19.5%), and back pain (LCKD: 34.4%, LGID: 39.0%) (p > 0.05 for all comparisons).",Nutrition & Metabolism,Ketogenic Diet,2008
NR Supplementation Trial Overview,"Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults. Nicotinamide adenine dinucleotide (NAD+) has emerged as a critical co-substrate for enzymes involved in the beneficial effects of regular calorie restriction on healthspan. As such, the use of NAD+ precursors to augment NAD+ bioavailability has been proposed as a strategy for improving cardiovascular and other physiological functions with aging in humans. Here we provide the evidence in a 2 × 6-week randomized, double-blind, placebo-controlled, crossover clinical trial that chronic supplementation with the NAD+ precursor vitamin, nicotinamide riboside (NR), is well tolerated and effectively stimulates NAD+ metabolism in healthy middle-aged and older adults. Our results also provide initial insight into the effects of chronic NR supplementation on physiological function in humans, and suggest that, in particular, future clinical trials should further assess the potential benefits of NR for reducing blood pressure and arterial stiffness in this group.",Nature Communications,Nicotinamide Riboside,2018
"Aging, Cardiovascular Disease, and Need for CR-Mimetics","Advancing age is the primary risk factor for the development of cardiovascular disease (CVD), which remains the leading cause of morbidity and mortality in industrial and post-industrial societies. The increase in CVD risk with aging is driven largely by adverse changes to arteries, including stiffening of the aorta, and by increases in systolic blood pressure. As such, interventions designed to lower blood pressure and/or improve arterial function hold promise for preventing age-related CVD. Chronic calorie restriction (CR) prevents the development of arterial dysfunction and increases in blood pressure with aging in rodents, and lowers arterial stiffness and blood pressure in overweight-obese middle-aged and older adults. Despite numerous health benefits, adherence to chronic CR remains poor and possibly even unsafe in normal weight older adults. As such, there is a critical need to establish safe, practical alternatives to regular CR for enhancing cardiovascular function and health with aging in humans.",Nature Communications,Nicotinamide Riboside,2018
"NAD+, Sirtuins, and Decline of NAD+ With Aging","The recent identification of several key molecular mechanisms responsible for CR-mediated longevity in model organisms has led to an exciting search for “CR-mimetic” interventions to improve cardiovascular and other physiological functions with aging. In this regard, nicotinamide adenine dinucleotide (NAD+) has emerged as a critical signaling molecule and essential substrate for sirtuins, a class of enzymes that mediate several of the beneficial effects of CR in model organisms, including the maintenance of cardiovascular function. Moreover, CR has been shown to increase NAD+ levels in pre-clinical models. The cellular bioavailability of NAD+ and related metabolites declines in animals and in humans during normal aging and may contribute to physiological aging by reducing sirtuin activity. Although NAD+ can be synthesized de novo from the amino acid tryptophan, this process does not occur in all tissues, requiring most cells to rely on a salvage pathway for regenerating NAD+ from other intracellular intermediates.",Nature Communications,Nicotinamide Riboside,2018
Limitations of Niacin and Advantage of NR/NMN,"Vitamin B3 (niacin: i.e., nicotinic acid and nicotinamide) enters this salvage pathway and acts as a NAD+ precursor; however, nicotinic acid is associated with undesirable flushing at therapeutic doses and nicotinamide does not reliably activate (and may even inhibit) sirtuins despite raising concentrations of NAD+. Therefore, administration of nicotinic acid or nicotinamide is unlikely to be widely adopted for maintaining health and function with aging. In contrast, oral supplementation with either of the NAD+ metabolites, nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), increases levels of NAD+ and improves multiple physiological functions in animal models. Indeed, supplementation of NMN in the drinking water improved cardiovascular function in old mice. Moreover, CR increases concentrations of NR and NAD+ and restores normal circadian gene transcription in the liver, further suggesting that NR may act as a CR mimetic.",Nature Communications,Nicotinamide Riboside,2018
Rationale for Human NR Trials and Study Design,"Despite these encouraging results from preclinical studies, the tolerability and effectiveness of chronic supplementation with NMN or NR have not been established in humans. Because NR is readily taken up by cells and acts as a direct vitamin precursor for NAD+ synthesis, its recent development as a dietary ingredient (NIAGEN®, ChromaDex Inc., Irvine, CA) has provided the first opportunity to translate the potential benefits of NAD+ boosting molecules to people. A recent study showed that single doses of NR stimulated blood cellular NAD+ metabolism in healthy humans in a dose-dependent manner. However, the tolerability of chronic NR supplementation and its efficacy for increasing NAD+ bioavailability have not been established in humans, and we lack even initial insight into the potential of NR for improving cardiovascular and other physiological functions with human aging.",Nature Communications,Nicotinamide Riboside,2018
Trial Goals and Key Findings,"To address these important research gaps, we conducted a small randomized, placebo-controlled, crossover clinical trial of NR supplementation (500 mg, 2×/day) to assess its overall tolerability and efficacy vs. placebo for raising levels of NAD+-related metabolites in healthy middle-aged and older men and women. We also took the opportunity to gain preliminary insight into the effects of chronic NR supplementation for improving cardiovascular and other physiological functions associated with risk of clinical diseases and/or disability with aging. Our results demonstrate that 6 weeks of NR supplementation at this dose is well-tolerated in humans and effectively increases blood cellular NAD+ concentrations. Exploratory analyses suggest that the potential for reducing systolic blood pressure and arterial stiffness may be the most promising hypotheses to investigate in future larger-scale clinical trials, particularly in individuals with elevated baseline blood pressure.",Nature Communications,Nicotinamide Riboside,2018
Subject Enrollment and Randomization,"Subject enrollment and baseline characteristics. Information on subject consent, randomization, testing and completion is presented in Fig. 1. Sixty healthy middle-aged and older men and women between the ages of 55 and 79 years were consented for this study, which was registered on clinicaltrials.gov under the identifier NCT02921659 and conducted between March 2015 and September 2016. The individuals recruited for this study were lean (average BMI = 24 ± 4 kg m−2) and healthy, and were representative of the late middle-aged/older adult population within the greater Boulder County Colorado community. Twenty-five participants did not meet inclusion criteria and were excluded without being randomized. Four participants dropped out of the study prior to randomization due to a conflict with time commitment, and one individual was unresponsive to scheduling requests, resulting in a total of 30 subjects remaining for randomization. Of these, 15 subjects were randomized to Group A, which received placebo capsules during the first 6 weeks before crossing over to receive NR capsules for the remaining 6 weeks. The other 15 subjects were randomized to Group B, which received NR capsules first followed by placebo.",Nature Communications,Nicotinamide Riboside,2018
Study Completion and Baseline Matching,"One subject was withdrawn from Group A due to a change in medication status that no longer met inclusion criteria, and two subjects in Group A elected to drop out of the study due to a complaint of side effects. Two subjects were withdrawn from Group B due to a change in health or medication status, and one subject elected to drop out of Group B due to a non-study-related injury, resulting in a total of 24 subjects who completed the trial. Removal of these six subjects did not influence the overall makeup of the group because the characteristics for the 24 subjects who completed the trial were similar to those for all 30 subjects who were initially randomized. The subjects that completed the study were well matched between groups for age, sex and clinical characteristics, and all baseline values were within normal clinical ranges.",Nature Communications,Nicotinamide Riboside,2018
Treatment-Emergent Adverse Events and Safety,"Adherence to the study treatments was excellent, with all subjects consuming greater than 95% of all NR and placebo capsules administered. NR was well tolerated at the dose tested, and no serious adverse events occurred. A total of 14 treatment-emergent adverse events (AEs) were reported by 7 of the 30 participants enrolled in the study, with the other 23 subjects reporting no AEs. All self-reported AEs were mild in severity. The reported symptoms included nausea, flushing, leg cramps and increased bruising during the NR condition, and headache, skin rash, flushing, fainting and drowsiness during the placebo condition. Only 2 out of the 30 enrolled subjects (<10%) dropped out of the study due to a complaint of side effects, both occurring while subjects were in the placebo phase; no subject dropped out during the NR treatment condition.",Nature Communications,Nicotinamide Riboside,2018
Clinical Laboratory Findings,"Clinical laboratory values were obtained from blood samples collected at the end of each treatment phase in 21 of the 24 subjects who completed the study. Complete blood work could not be obtained from the remaining three subjects due to failed catheterization, administrative error, or study nurse error. No meaningful differences were observed between treatment conditions for hematology, blood chemistry including markers of renal function and liver enzymes, or blood lipid profiles. Importantly, all clinical laboratory values remained within the normal reference range during both the placebo and NR conditions. Collectively, these results indicate that oral supplementation with NR for 6 weeks at this dose is well-tolerated in healthy middle-aged and older adults.",Nature Communications,Nicotinamide Riboside,2018
Effect of NR on NAD+ and Related Metabolites,"After demonstrating the tolerability of chronic NR supplementation, our primary objective was to determine if NR raises blood cellular NAD+ metabolism in humans. Because blood NAD+ and related metabolites have recently been shown to be measurable in circulating peripheral blood mononuclear cells (PBMCs), we assessed the NAD+ metabolome in PBMCs. Oral NR supplementation effectively elevated levels of NAD+ in PBMCs by ~60% compared with placebo (mean change = 6.2 pmol per mg protein; one-sided 95% CI (0.074, ∞)). The mean level of NADP+ also increased, but did not reach statistical significance. NR also elevated levels of nicotinic acid adenine dinucleotide (NAAD) nearly fivefold above placebo (mean change = 1.1 pmol per mg protein), confirming NAAD as a sensitive biomarker of increased NAD+ metabolism. NR also elevated the mean concentration of nicotinamide (NaM), though not statistically significant, suggesting increased activity of NAD+-consuming enzymes.",Nature Communications,Nicotinamide Riboside,2018
Metabolite Changes and Energy Pathways,"We also observed an ~1.5-fold increase in NMN, which may indicate conversion of NR to NMN by NRK enzymes or further metabolism of NaM by NAMPT. Consistent with previous findings, we were unable to detect NR concentrations in PBMCs despite optimized recovery methods. The magnitude of NAD+ increase in response to NR was negatively associated with baseline NAD+ levels (R = −0.49), suggesting greater response in individuals with naturally low NAD+. In addition to NAD+-specific metabolites, NR supplementation increased the mean concentration of other metabolites involved in energy production, including adenosine and ATP (mean change = 699 pmol per mg protein). NR also tended to raise levels of ADP and AMP, though these changes did not reach statistical significance. Collectively, these findings indicate that chronic NR supplementation effectively stimulates NAD+ metabolism in healthy middle-aged and older adults.",Nature Communications,Nicotinamide Riboside,2018
NR Effects on Blood Pressure,"Effect of NR on indicators of cardiovascular health. Supplementation with NR tended to lower mean systolic (SBP; mean change = −3.9 mmHg; one-sided 95% CI (−∞, −0.058)) and diastolic (DBP; mean change = −2.0 mmHg; one-sided 95% CI (−∞, −0.26)) blood pressure (BP) in all subjects as a group; however, these comparisons were not statistically significant after correction for multiple comparisons. Because the risk of cardiovascular events is greatly increased in individuals with above-normal baseline BP, we performed a follow-up analysis to compare the effect of NR on BP in the participants with BP in the normal range versus those with BP in the elevated/stage I hypertension range. Of particular note, mean SBP was 9 mmHg lower after NR vs. placebo in individuals with elevated/stage I hypertension, whereas no change was observed in subjects with initial SBP in the normal range. Because this post-hoc subgroup analysis was exploratory, no statistical inferences can be made.",Nature Communications,Nicotinamide Riboside,2018
NR Effects on Arterial Stiffness,"We also observed a trend towards a reduction in the mean carotid-femoral pulse wave velocity (PWV) with NR supplementation, the clinical “gold standard” measure of the stiffness of the aorta and a strong independent risk factor for incident cardiovascular events. However, this reduction was not statistically significant after correction for multiple comparisons. Similar to our exploratory analysis of BP, NR supplementation tended to lower aortic stiffness more in individuals with higher baseline BP, although no statistical inferences were made for this post-hoc comparison. No effect of NR was observed on ultrasound-determined carotid artery compliance or brachial artery flow-mediated dilation, a measure of vascular endothelial function.",Nature Communications,Nicotinamide Riboside,2018
Exploratory Physiological Outcomes,"To gain exploratory insight into potential benefits of NR supplementation on other domains of physiological function, we assessed outcomes indicative of metabolic function, motor function, and exercise capacity/performance. Total energy intake and expenditure, oxidative fuel source (carbohydrate vs. fat), and physical activity patterns were not affected by NR. Likewise, we observed no difference in body mass, body mass index or percent body fat compared with placebo and no differences were observed in measures of glucose or insulin regulation. Finally, there was no effect of the intervention on overall motor function, maximal exercise capacity as assessed by VO2 max and treadmill time to exhaustion, or on markers of submaximal exercise performance.",Nature Communications,Nicotinamide Riboside,2018
Primary Interpretation of Findings,"The primary finding of this study is that chronic oral supplementation with 1000 mg per day of NR is a well-tolerated and effective strategy for stimulating NAD+ metabolism in healthy middle-aged and older humans. Additionally, our exploratory analyses suggest that the ability of NR to reduce SBP and aortic stiffness, two clinically important risk indicators of cardiovascular function and health, are among the most promising hypotheses to test in a future larger-scale clinical trial, particularly in individuals with above-normal baseline SBP. NR stimulated NAD+ metabolism without any difference in treatment-emergent adverse events compared with placebo, supporting previous suggestions that NR may be a more suitable NAD+ precursor than niacin, which is associated with a painful flushing sensation at therapeutic doses.",Nature Communications,Nicotinamide Riboside,2018
Safety Considerations and Mechanistic Insight,"Despite these promising findings, the size of the cohort in the present study is insufficient to establish the broader safety profile of NR at this dose. Larger clinical trials will be necessary to confirm tolerability. Niacin serves as an important dietary precursor to NAD+ and protects against pellagra. Like niacin, NR has been detected in cow’s milk and may theoretically act as another vitamin precursor form of NAD+. It is plausible that the daily requirement for NAD+ precursors may increase with advancing age due to decreasing NAD+ bioavailability. NAD+ is an obligate substrate for sirtuin 1 (SIRT1), which is implicated in maintaining healthy vascular function. Preclinical studies show that SIRT1 activation protects against aortic stiffness and hypertension and that NMN supplementation reverses aortic stiffening in old mice. It is possible that NR may similarly affect BP and aortic stiffness in humans through a mechanism involving SIRT1 activation.",Nature Communications,Nicotinamide Riboside,2018
Implications and Future Directions,"Future studies should explore the role of NR supplementation in groups with impaired mobility, frailty, chronic diseases associated with reduced cardiorespiratory fitness, or metabolic dysfunction. The present study’s findings suggest that NR does not improve aerobic exercise capacity or motor function in healthy adults with good baseline physical status. Larger-scale clinical trials are needed to confirm the effects of NR supplementation on SBP and carotid-femoral PWV. It is important to note that the dose tested exceeds the label-recommended dose for NIAGEN® and should be considered investigational until further safety and efficacy data become available. Future investigations should include studies on groups with cardio-metabolic diseases, motor deficits, impaired NAD+ metabolism, and other disorders to determine the efficacy of NR for enhancing health status in populations with impaired baseline physiological function.",Nature Communications,Nicotinamide Riboside,2018
Overview of NAD and Its Therapeutic Promise,"Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence. Nicotinamide adenine dinucleotide (NAD), the cell’s hydrogen carrier for redox enzymes, is well known for its role in redox reactions. More recently, it has emerged as a signaling molecule. By modulating NAD+-sensing enzymes, NAD+ controls hundreds of key processes from energy metabolism to cell survival, rising and falling depending on food intake, exercise, and the time of day. NAD+ levels steadily decline with age, resulting in altered metabolism and increased disease susceptibility. Restoration of NAD+ levels in old or diseased animals can promote health and extend lifespan, prompting a search for safe and efficacious NAD-boosting molecules that hold the promise of increasing the body’s resilience, not just to one disease, but to many, thereby extending healthy human lifespan.",Cell Metabolism,NAD+,2018
Historical Rise and Rediscovery of NAD+,"The Rise, Fall, and Rise of NAD+. Nicotinamide adenine dinucleotide (NAD) is one of the most important and interesting molecules in the body. It is required for over 500 enzymatic reactions and plays key roles in the regulation of almost all major biological processes. NAD was first described in 1906 by Harden and Young as a cell component that enhanced alcohol fermentation. In 1936, Warburg showed that NAD is required for redox reactions and solidified the nomenclature: 'NAD' refers to the chemical backbone, while 'NAD+' and 'NADH' refer to the oxidized and reduced forms. A breakthrough came in 1963 with the discovery that NAD+ is a co-substrate for the addition of poly-ADP-ribose to proteins. PARylation is carried out by poly-ADP-ribose polymerases (PARPs), which control cellular functions from DNA repair to gene expression. Renewed interest in NAD over the last decade can be attributed to the sirtuins, a family of NAD+-dependent protein deacylases. Mammalian sirtuins metabolize NAD+, and yeast Sir2 is an NAD+-dependent histone deacetylase. Sirtuins influence inflammation, cell growth, circadian rhythm, metabolism, neuronal function, and stress resistance.",Cell Metabolism,NAD+,2018
NAD+ Synthesis and Compartmentalization,"NAD+ Synthesis. NAD+ is one of the most abundant molecules in the human body, required for approximately 500 enzymatic reactions and present at about three grams in the average person. It is in a constant state of synthesis, degradation, and recycling, not only in the cytoplasm but also in major organelles including the nucleus, Golgi, and peroxisomes. High-sensitivity NAD+ metabolite tracing methods such as mitoPARP, PARAPLAY, and Apollo-NADP+ have revealed that the concentration and distribution of NAD+ differ across organelles and change in response to physiological stimuli and cellular stress. NAD+ exists as a free pool and a protein-bound pool, with ratios varying across tissues and with age. With the exception of neurons, mammalian cells cannot import NAD+, so they synthesize it de novo via the kynurenine pathway from tryptophan or from vitamin B3 forms such as nicotinamide (NAM) or nicotinic acid (NA). Most NAD+ is salvaged rather than generated de novo. Precursors such as NAM, NA, nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN) feed into salvage pathways.",Cell Metabolism,NAD+,2018
NAD+ Salvage Pathway and Key Enzymes,"The precursor NR is taken up via equilibrative nucleoside transporters (ENTs) and converted to NMN by NR kinases (NRK1/2). NR generates unexpectedly high levels of nicotinic acid adenine dinucleotide (NAAD) in mouse liver and heart, as well as in human PBMCs, though the mechanism remains unclear. The rate of NAD+ synthesis is largely determined by the first step in the salvage pathway converting NAM to NMN, carried out by NAM phosphoribosyltransferase (NAMPT). NAMPT levels are dynamic and respond to NAD+ demand, DNA damage, starvation, exercise, and nutrient status. NAMPT exists in intracellular (iNAMPT) and extracellular (eNAMPT) forms. eNAMPT is present in cerebrospinal fluid and in serum of both mice and humans. NAMPT expression controls the body’s response to stress, exercise, nutrient status, and circadian rhythms. Obesity and high-calorie diets reduce NAMPT and NAD+ levels in tissues including liver, white adipose, muscle, and brown adipose. Increased eNAMPT is observed in conditions such as NAFLD, obesity, type 2 diabetes, cardiovascular disease, and cancer.",Cell Metabolism,NAD+,2018
NAD+ Metabolism Across Species,"Unlike mammals, invertebrates and yeast convert NAM to NA via Pnc1, a nutrient- and stress-responsive NAM deamidase that serves a similar role to NAMPT. Some microorganisms are incapable of synthesizing NAD+ de novo and therefore rely exclusively on external sources and salvage pathways. Overall, NAD+ metabolism is highly conserved yet exhibits key differences across species, particularly in mechanisms of salvage pathway utilization and regulation. The NAD+ network is tightly regulated by stress signals, metabolic state, and cellular demands, suggesting that therapeutic boosting of NAD+ may influence a wide array of physiological processes.",Cell Metabolism,NAD+,2018
NAD+ Degradation by CD38 and CD157,"CD38 and CD157 (BST1) are glycohydrolases that cleave NAD+ to generate nicotinamide (NAM) and ADP-ribose (ADPR), while CD38 also hydrolyzes cyclic ADPR (cADPR) to ADPR. Both enzymes can also act as ADP-ribosyl cyclases, producing NAM and cADPR—a Ca2+-mobilizing second messenger active in many cell types. CD38 is ubiquitously expressed, whereas CD157 is mainly found in lymphoid tissue and the gut. These enzymes influence energy metabolism, immune responses, and cell adhesion, and have been associated with human diseases such as Parkinson's disease, ovarian cancer, and leukemia. CD38 is a major consumer of NAD+ in mammals, as shown by increased NAD+ levels in CD38-knockout mice and age-related increases in CD38 protein expression that coincide with declining NAD+. Inhibiting CD38 (e.g., with apigenin or 78c) increases NAD+ levels and serves as proof-of-concept for therapies targeting metabolic and age-related diseases.",Cell Metabolism,NAD+,2018
SARM1 as a Neuronal NADase,"SARM1 is a newly discovered NAD+-cleaving enzyme found in neurons and potentially other cell types. It belongs to a new class of NAD-consuming enzymes and is unique because its catalytic activity is dependent on a Toll/interleukin-1 receptor (TIR) domain—previously known only for protein–protein interactions. In response to neuronal injury, the TIR domain converts cytoplasmic NAD+ into ADPR, cADPR, and NAM, rapidly depleting NAD+ and initiating axonal degeneration. Overexpression of NAMPT or NMNAT, as well as supplementation with NR, can block SARM1-induced axon degeneration. SARM1 knockout rescues embryonic lethality and neuronal degeneration in NMNAT2-deficient mice. These findings identify SARM1 as an attractive therapeutic target for acute neuronal injury and neurodegenerative diseases.",Cell Metabolism,NAD+,2018
NAD+-Responsive Signaling Pathways: Sirtuins,"The sirtuins (SIRT1–7) are major NAD+-responsive signaling proteins. First identified in yeast as genes involved in silencing and telomere maintenance, sirtuins regulate mitochondrial metabolism, inflammation, autophagy, circadian rhythms, meiosis, and apoptosis. Sirtuins use NAD+ as a co-substrate during deacetylation reactions, producing NAM. The canonical reaction involves removing an acetyl group from lysine residues, forming a peptidyl-ADP-ribose intermediate and ultimately generating O-acetyl-ADP-ribose. Some sirtuins have specialized functions: SIRT5 desuccinylates proteins, while SIRT6 performs defatty-acylation. SIRT4 and SIRT6 can also mono-ADP-ribosylate target proteins.",Cell Metabolism,NAD+,2018
NAD+-Responsive Signaling Pathways: PARPs and MARTs,"The poly-ADP-ribose polymerases (PARPs) are another major NAD+-responsive protein family. PARP1, PARP2, and PARP5 add long poly-ADP–ribose chains to target proteins, whereas PARP3, PARP4, and PARP6–16 function as mono-ADP-ribose transferases (MARTs). PARPs modify proteins by transferring ADP-ribose to amino acid residues such as asparagine, glutamate, arginine, lysine, or cysteine. PARP1 and PARP2 regulate DNA repair and transcription, and hyperactivation of PARP1 in response to DNA damage depletes NAD+ and induces apoptosis. PARP inhibition increases NAD+ levels, improves mitochondrial function, and protects against high-fat–diet stress in mice. Many PARPs/MARTs have diverse roles, including Wnt signaling (PARP5a/b), unfolded protein response (PARP16), immune signaling (PARP9/10/14), post-transcriptional RNA regulation (PARP7/10/12/13), and nuclear envelope organization (PARP11).",Cell Metabolism,NAD+,2018
Cross-Talk Between Sirtuins and PARPs,"Sirtuin and PARP pathways interact extensively. The Deleted in Breast Cancer 1 (DBC1) protein inhibits both SIRT1 and PARP1, and its regulation depends on NAD+ binding. SIRT6 enhances PARP1-mediated DNA repair by mono-ADP-ribosylation, whereas PARP1 inhibition increases expression of multiple sirtuins (SIRT1, SIRT4, SIRT6). These intertwined pathways illustrate the coordinated role of NAD+ metabolism in stress responses, DNA repair, and aging processes.",Cell Metabolism,NAD+,2018
Inhibition of NAD+ Degradation as a Strategy to Raise NAD+,"An alternative approach to increasing NAD+ levels is to inhibit its degradation through blockade of PARPs or NADases (glycohydrolases). CD38, the major mammalian NADase, can be inhibited at low micromolar concentrations in vitro by flavonoids such as luteolinidin, kuromanin, luteolin, quercetin, and apigenin (IC50 < 10 μM). In vivo, these inhibitors also elevate NAD+ levels: apigenin increases NAD+ across multiple tissues, reduces global proteome acetylation, and improves glucose and lipid metabolism in obese mice, likely through SIRT1 and SIRT3 activation. Luteolinidin similarly preserves NAD+ and protects cardiac endothelial and myocardial function after ischemic injury. More potent small-molecule inhibitors include thiazoloquin(az)olinones like 78c, which raise NAD+ in plasma, liver, and muscle, although their specificity and cellular uptake mechanisms remain unclear. PARP1 inhibitors, widely used in oncology, also raise NAD+ by decreasing PARP-driven NAD+ consumption. Another target, SARM1, drives axonal degeneration by rapidly degrading NAD+ into ADPR, cADPR, and NAM after neuronal injury. XAV939, originally identified as a Wnt-pathway modulator inhibiting PARP5a/5b, may also inhibit SARM1 and is being explored clinically for neurological disorders and axonal injuries.",Cell Metabolism,NAD+,2018
Uptake and Biodistribution of NAD+ Precursors,"Cellular uptake routes for NAD+ precursors remain incompletely understood. Tryptophan uptake occurs via SLC7A5 and SLC36A4, while nicotinic acid (NA) uses SLC5A8 and SLC22A13. Nicotinamide (NAM), being uncharged, passively diffuses across cellular and mitochondrial membranes and synergizes with NA in raising NAD+. Most cells cannot import intact NAD+, with neurons being a possible exception. NR and NAR uptake in yeast occurs through transporters such as Nrt1 and FUN26, and mammalian uptake may involve equilibrative nucleoside transporters, as dipyridamole reduces NAD+ availability. NAD+ and potentially NMN exist in serum and extracellular fluid, where they may act as systemic signals; however, debate persists regarding extracellular NMN stability and the physiological relevance of extracellular NAMPT activity. Extracellular degradation converts NAD+ into NMN and AMP via CD73, or into NAM and ADPR via CD38/CD157, with cADPR contributing to immune and redox signaling. Whether NMN requires a transporter is disputed: some groups propose extracellular degradation to NR/NAM as the true uptake route, while others provide evidence for rapid, cell-type-specific NMN transport. Isotopic tracer studies are required to resolve these controversies.",Cell Metabolism,NAD+,2018
Tissue-Specific Metabolism of NAD+ Precursors,"NAD+ precursor metabolism varies markedly across tissues due to differences in transporter expression, salvage enzymes, and extracellular processing. NAMPT is widely expressed but shows large tissue-to-tissue differences. In most tissues, the NMNAT enzymes responsible for converting NMN to NAD+ are not rate-limiting, except in blood. In contrast, NADS, required for the deamidated pathway (NA → NAAD → NAD+), is rate-limiting in lung and skeletal muscle, leading to NAAD accumulation. NRK-dependent salvage predominates in skeletal muscle, with NRK1 being ubiquitous and NRK2 primarily expressed in muscle. Consistent with this, chronic NR supplementation raises NAD+ in muscle but not in brain or white adipose tissue. CD38 inhibition shows strong tissue specificity: compound 78c increases NAD+ >5-fold in liver but only ~1.2-fold in muscle. Whether these effects stem from intra- or extracellular CD38 inhibition remains unresolved.",Cell Metabolism,NAD+,2018
Physiological Effects of NAD+ Boosters in Mouse Models: Liver Function,"NAD+-linked signaling pathways strongly influence hepatic metabolism, protecting against fat accumulation, fibrosis, and insulin resistance. SIRT1 regulates mitochondrial biogenesis (via PGC-1α), cholesterol metabolism (PSK9), and lipogenesis (SREBP1). SIRT2 modulates gluconeogenesis; SIRT3 regulates oxidative phosphorylation, fatty acid oxidation, ketogenesis, and antioxidant defense; SIRT6 controls glucose production. Aging and obesity decrease NAMPT expression and increase CD38 activity, halving NAD+ levels by mid-life. Restoring hepatic NAD+ to youthful levels using NR, NMN, PARP inhibitors, CD38 inhibitors, or NNMT inhibitors improves mitochondrial function, steatosis, insulin sensitivity, and inflammatory status in mouse models of obesity, alcoholic liver disease, and NASH. NAD+ boosting also enhances liver regeneration following partial hepatectomy and protects against hepatotoxicity.",Cell Metabolism,NAD+,2018
Physiological Effects of NAD+ Boosters in Mouse Models: Kidney Function,"Age-related decline in kidney NAD+ and sirtuin activity contributes to diminished renal resilience. Boosting NAD+ via SIRT1/SIRT3 activation or precursor supplementation protects kidney cells from hypertrophy, oxidative stress, and metabolic dysfunction. NMN protects against cisplatin-induced acute kidney injury (AKI) through SIRT1-dependent mechanisms, while AMPK activation (via AICAR) raises NAD+ and protects against nephrotoxicity in a SIRT3-dependent manner. Nicotinamide supplementation increases renal prostaglandin PGE2, improves recovery from ischemic injury, and reduces cisplatin-induced renal damage by stimulating NAD+ synthesis.",Cell Metabolism,NAD+,2018
Spermidine and Aging Overview,"High incidences of morbidity and mortality associated with age-related diseases among the elderly population are a socio-economic challenge. Aging is an irreversible and inevitable process that is a risk factor for pathological progression of diverse age-related diseases. Spermidine, a natural polyamine, plays a critical role in molecular and cellular interactions involved in various physiological and functional processes. Spermidine has been shown to modulate aging, suppress the occurrence and severity of age-related diseases, and prolong lifespan. However, the precise mechanisms through which spermidine exerts its anti-aging effects have not been established. In this review, we elucidate the mechanisms and roles underlying the beneficial effects of spermidine in aging from a molecular and cellular perspective, and provide new insights into its promising diagnostic and therapeutic applications.",Aging and Disease,Spermidine and Aging,2021
Physiological Roles of Spermidine,"Polyamines are ubiquitous polycations found in all cells, tissues, and organs. They interact with negatively charged molecules such as DNA, RNA, ATP, and proteins. These molecules exert multiple functions in physiological and pathophysiological processes, including cell proliferation, differentiation, growth, tissue regeneration, and gene regulation. Due to its antioxidant functions, anti-inflammatory properties, enhanced proteostasis, and improved mitochondrial metabolic functions, spermidine is involved in apoptosis, transcription, and DNA stability. Concentrations of spermidine decline with age, and exogenous supplementation reverses age-associated adverse changes and prolongs lifespan. Spermidine is associated with longevity and influences aging through diverse mechanisms due to its interactions with various molecular substrates.",Aging and Disease,Spermidine Longevity,2021
Mechanisms of Spermidine: Autophagy and Anti-Inflammatory Action,"Spermidine is an inducer of autophagy, which is considered the main mechanism of its anti-aging effects. Spermidine triggers autophagy by modulating the expressions of Atg genes. It also regulates transcription factor eIF5A to promote synthesis of TFEB. Furthermore, spermidine inhibits EP300, which directly promotes acetylation of Atg genes and indirectly stimulates deacetylation of tubulin via inhibition of aTAT1. Spermidine exerts anti-inflammatory roles by suppressing multiple inflammatory cytokines such as ROS, NF-κB, IL-1β, and IL-18. Additionally, it regulates cell proliferation, differentiation, senescence, apoptosis, and necrosis, thereby promoting cell growth and inhibiting cell death. Spermidine also suppresses histone acetylation as an anti-aging mechanism and regulates lipid metabolism by promoting differentiation of preadipocytes into mature adipocytes, altering lipid profiles, modulating lipogenic gene expressions, and repressing lipid accumulation.",Aging and Disease,Spermidine Mechanisms,2021
Signaling Pathways Regulated by Spermidine,"Spermidine delays aging through specific signaling pathways, including SIRT1/PGC-1α, insulin/IGF, AMPK-FOXO3a, and CK2/MAPK. Through these pathways, spermidine influences metabolism, mitochondrial biogenesis, stress resistance, and cell survival. The compound’s ability to activate autophagy, modulate transcription factors, and regulate inflammatory mediators contributes to its broad protective functions in age-related diseases. These pathways are closely associated with longevity regulation, nutrient sensing, and mitochondrial homeostasis, placing spermidine at the intersection of several hallmark aging mechanisms.",Aging and Disease,Spermidine Signaling Pathways,2021
Mechanisms of Spermidine in Aging,"Even though aging is inevitable, it can be modified by biological and genetic interventions, pharmaceuticals, lifestyle, and the systemic environment. Spermidine has been shown to be important for prolonging survival outcomes, and abnormal changes in spermidine levels are associated with aging as well as disease development. Intracellular concentrations of spermidine are suppressed during aging. Exogenous spermidine supplementation has been shown to extend the lifespans of flies, nematodes and yeast. Moreover, a diet enriched in spermidine was shown to prolong the lifespans of mice. However, studies on the mechanisms of action of spermidine are rare. Autophagy is the main mechanism of spermidine in delaying aging and prolonging the lifespan. In addition, spermidine exerts its effects through other mechanisms, including anti-inflammation, histone acetylation reduction, lipid metabolism, and regulation of cell growth and signaling pathways. This section discusses the updated mechanisms of spermidine in aging.",Aging and Disease,Spermidine Mechanisms,2021
Autophagy Mechanisms,"Autophagy is an intracellular degradation system delivering damaged or unnecessary cytoplasmic components into lysosomes, and can be divided into microautophagy, macroautophagy, and chaperone-mediated autophagy. Macroautophagy is the most common process, through which cytoplasmic material is sequestered within an autophagosome before fusing with a lysosome or vacuole. At basal levels, autophagy maintains cellular function, but can be adjusted by stimuli such as aging, oxidative stress, or inflammation. Aging enhances the accumulation of damaged cellular constituents, including proteins and organelles, and suppresses cellular ability to degrade these components. Induction of autophagy prolongs lifespan, while its deficiency shortens lifespan. Spermidine induces autophagy in liver, heart, and muscle in mice, as well as in aging yeast, worms, flies, and mammalian cells. It induces autophagy by adjusting expression levels of autophagy-related genes (Atg), including upregulation of Atg7, Atg15, and Atg11. Knockout of Atg genes abolishes spermidine-induced lifespan extension.",Aging and Disease,Spermidine Autophagy,2021
Autophagy Mechanisms (Part 2),"Spermidine regulates autophagy by inducing the expression of the translation factor eIF5A to increase synthesis of transcription factor TFEB. It also initiates autophagy by inhibiting protein acetylation. EP300, an acetyltransferase, directly promotes acetylation of multiple autophagy-essential proteins and indirectly stimulates tubulin deacetylation by inhibiting a-tubulin acetyltransferase 1 (aTAT1). Spermidine enhances deacetylation by reducing EP300 expression. In addition, spermidine decreases acetylation by reducing the availability of acetyl-CoA. Spermidine can also induce autophagy through other pathways, including regulation of inflammation and lipid metabolism. Altogether, autophagy is considered the most important mechanism through which spermidine exerts anti-aging effects.",Aging and Disease,Spermidine Autophagy,2021
Anti-inflammatory Mechanisms,"Inflammation plays a crucial role in immunity, but excessive inflammatory responses, referred to as 'inflammatory aging', are a major risk factor for aging. Elevated expression levels of pro-inflammatory biomarkers such as CRP, TNF-α, and IL-6 are associated with the risk of cardiovascular diseases, cerebrovascular diseases, chronic kidney diseases, and metabolic syndrome. Spermidine delays aging through systemic anti-inflammatory processes. Spermidine supplementation reduces chronic inflammation by decreasing TNF-α expression, suppressing cardiovascular dysfunction progression. Spermidine exerts anti-inflammatory effects by inhibiting accumulation of reactive oxygen species (ROS) and the translocation of nuclear factor-kappa B (NF-κB). It also inhibits inflammation-related migration of immune cells. Spermidine supplementation suppresses protein expression levels of IL-1β and IL-18. These findings indicate that anti-inflammation is essential for spermidine-mediated delay of aging.",Aging and Disease,Spermidine Inflammation,2021
Cellular Lifecycle Overview,"Cellular lifecycle involves proliferation, differentiation, senescence and apoptosis, which are structural and functional bases for organism growth, development, aging and death, respectively. Most cells exhibit a normal lifecycle, while a few cells deviate from a normal lifecycle due to interference of certain factors, including damage, necrosis or cancer. Dysregulated cellular lifecycle has been implicated in the pathogenesis of aging and age-related diseases. Spermidine is involved in regulating the lifecycle of cells. With the relevant cumulative findings, studies now discuss the correlation between spermidine and cellular lifecycle.",Aging and Disease,Spermidine Cellular Lifecycle,2021
Spermidine and Cell Proliferation,"Cell proliferation, attributed to cell division, is an important characteristic of living organisms. Cell cycle progression is responsible for cell growth, survival and death. Spermidine plays a causative role in modulating the cell cycle, with small amounts of spermidine shown to sustain normal cell cycles. Landau et al. reported that the absence of spermidine can cause growth cessation at the G1 phase by affecting the expression of cell cycle regulators. Spermidine was also shown to enhance the proportion of S phase cells and maintain mitochondrial membrane potential, thereby improving the senescence of mouse neuroblastoma cells.",Aging and Disease,Spermidine Proliferation,2021
Spermidine and Cell Differentiation,Cell differentiation refers to the process through which cells from the same source gradually produce groups with different morphological structures and functional characteristics. Recent studies have revealed that spermidine is involved in cell differentiation. Emerging evidence indicates a role for spermidine in enhancing differentiation in differentiated chondrocytes and in adult stem cells. Cervelli et al. proved that exogenous supplementation of spermidine impacts D-gal-induced aging-related skeletal muscle atrophy during skeletal muscle differentiation.,Aging and Disease,Spermidine Differentiation,2021
Spermidine and Cell Senescence,"Cell senescence is characterized by cessation of replication, loss of proliferation potential, resistance to apoptosis, and increased protein production. Spermidine prevents cell senescence. Elevated spermidine levels were associated with improved functions of 'old' B cells, which might reverse immune aging. Zhu et al. demonstrated that spermidine inhibits high-glucose and neurotoxicity-induced senescence by upregulating cannabinoid receptor type 1. Suppressed p21 and p16 expression levels and reduced senescence-associated β-gal staining indicated that spermidine improved bleomycin-stimulated premature cell senescence.",Aging and Disease,Spermidine Senescence,2021
Spermidine and Cell Death,"Physiological apoptosis and pathological necrosis are collectively referred to as cell death. Cell apoptosis is a basic biological phenomenon that removes unwanted or abnormal cells and is involved in organismal evolution, internal homeostasis, and development of multiple systems. Spermidine can modulate cell apoptosis. Its interaction with the mitochondrial membrane induces the release of cytochrome C, which is the prelude to apoptosis. In addition to interfering with the cell cycle, spermidine slows down aging by preventing apoptosis. Cell necrosis refers to death induced by extreme physical, chemical or pathological factors. Spermidine's role in cell necrosis has been reported, with elevated spermidine levels shown to suppress cell necrosis, prolong lifespan, and improve health in aging yeast.",Aging and Disease,Spermidine Cell Death,2021
2.6 Signaling pathways,"Multiple signaling pathways are involved in modulation of aging and age-related diseases. Spermidine interacts with various signaling pathways to regulate the aging process. However, the specific mechanisms have not been established. Sirtuin-1/peroxisome proliferator-activated receptor gamma coactivator alpha (SIRT1/PGC-1α) signaling pathway is a major modulator of mitochondrial function and a vital contributor to aging and cardiovascular diseases. Wang et al. confirmed that spermidine stimulates mitochondrial biogenesis through the SIRT1/PGC-1α pathway and could, therefore, be used to prevent cardiac function degradation during aging. In drosophila, dietary spermidine supplementation was associated with extended lifespan by suppressing insulin/insulin-like growth factor (IGF) signaling. FOXO3a, a downstream effector of AMP-activated protein kinase (AMPK), which is involved in the aging process, has been associated with longevity. Fan et al. showed that spermidine protects against aging-related skeletal muscle atrophy by suppressing apoptosis and enhancing autophagy through the mediation of the AMPK-FOXO3a signaling pathway. The ubiquitous kinase, CK2, has been reported to translate information in the mitogen-activated protein kinase (MAPK) pathway by detecting spermidine levels. Moreover, spermidine upregulates the expression of MAPK family genes and to regulate MAPK phosphorylation. In conclusion, spermidine exerts its anti-aging properties by activating or suppressing signaling pathways.",Aging and Disease,Spermidine Signaling Pathways,2021
2.7 Other mechanisms,"In addition to the above mechanisms through which spermidine modulates aging, biological functions of spermidine in protecting replicating DNA from oxidative damage have also been proposed. Oxidative damage by singlet oxygen, 1O2, leads to harmful effects on cells. Spermidine, as a positively charged molecule, can bind and precipitate DNA. Khan et al. documented that spermidine protects DNA against oxidative attack, ensuring the integrity of DNA and RNA, thereby guaranteeing protein synthesis.",Aging and Disease,Spermidine DNA Protection,2021
3. Introduction to Age-Related Diseases,"Aging refers to a gradual deterioration of functionality and physiological integrity processes, which enhances susceptibility to age-related diseases. Since the aging population is rapidly increasing, there is a need to thoroughly understand aging and age-related diseases. Studies have reported on the mechanisms involved in aging, especially vascular aging. Spermidine is a critical factor in aging and age-related diseases, including CVDs, neurodegenerative diseases, metabolic diseases, musculoskeletal diseases, and immune diseases. In this section, we discuss on the role of spermidine in age-related diseases.",Aging and Disease,Spermidine in Age-Related Diseases,2021
3.1 Spermidine and Age-Related Cardiovascular Diseases,"Aging is a major risk factor for the development of CVDs, which is a major cause of disability and death in the elderly population. Spermidine prevents against cardiac aging by improving left ventricular elasticity, diastolic function, and mitochondrial function. It has been reported that CVDs such as coronary artery disease (CAD), essential hypertension (EH), and heart failure (HF) are highly influenced by spermidine levels. CAD, one of the primary CVDs, is caused by the narrowing or blocking of vascular lumen due to atherosclerotic lesions in coronary arteries. Atherosclerosis (AS) is the major cause of CAD. Studies have reported on the role of spermidine in CAD. Han et al. found an association between spermidine and myocardial ischemic reperfusion. A prospective study found an inverse parallel relationship between spermidine and AS.",Aging and Disease,Spermidine and Cardiovascular Aging,2021
3.1.1 Spermidine and CAD – Part 2,"Tyrrell et al. documented that spermidine supplementation in aged mice inhibits AS via decreasing inflammatory cytokines and improving mitochondrial functions. The risk of AS can also be reduced by the role of spermidine in autophagy. Besides, spermidine attenuates AS due to its antagonistic action on platelet aggregation, which is regarded as a causative factor for AS. Plasma hyaluronan-binding protein (PHBP) is a factor VII activating protease involved in the modulation of vascular function, inflammation, and AS. Spermidine promotes the conversion of PHBP from a single-chain to a two-chain form, thereby protecting against AS development. In type 2 diabetes mellitus (T2DM), restoration of endothelial nitric oxide synthase (eNOS) activation by spermidine was found to be blocked by autophagy inhibitors, resulting in AS.",Aging and Disease,Spermidine and CAD,2021
3.1.2 Spermidine and Essential Hypertension,"EH is characterized by increased vascular resistance, due to endothelial dysfunction and vascular remodeling, representing age-related functional and structural alterations, respectively. Spermidine attenuates the development of EH during aging. Maione et al. reported on the beneficial effects of spermidine on N-methyl-D-aspartate (NMDA) induced EH. Eisenberg et al. proved that dietary spermidine reduces high blood pressure by improving age-related diastolic. Ornithine decarboxylase (ODC) is a crucial enzyme in the polyamine biosynthesis. In hypertensive tissues, spermidine concentrations have been shown to increase in tandem with alterations in ODC activity. Ibrahim et al. proved that spermidine regulates blood pressure because it is an essential component of the blood pressure effect of angiotensin II.",Aging and Disease,Spermidine and Hypertension,2021
3.1.3 Spermidine and Heart Failure,"HF is a clinical syndrome of aging-related phenotypes. An association between spermidine and HF has been reported. Appropriate induction of autophagy by spermidine might be involved in resistance to HF. Moreover, spermidine supplementation was shown to prevent cardiac hypertrophy and protect cardiomyocytes, thereby delaying HF progression. Mitochondria are crucial in myocardial maintenance and development. Spermidine attenuates mitochondrial dysfunction during aging, which is the primary cause of HF development. Wirth et al. found that the cardioprotective effect of spermidine at the histological level was associated with reduced telomere attrition in cardiac tissues. Moreover, Tantini et al. indicated the effect of spermidine on the apoptosis of myocardial ischemic cells, which inhibited HF development.",Aging and Disease,Spermidine and Heart Failure,2021
3.2 Spermidine and Neurodegenerative Diseases,"Neurodegenerative diseases are featured by a progressive loss of selective populations of vulnerable neurons, and they can be classified as Alzheimer's disease (AD), Parkinson’s disease (PD), or motor neuron disease according to clinical characteristics. Spermidine protects against neuronal cell damage by inducing autophagy. Therefore, supplementation with spermidine inhibits multiple neurological pathologies including neurodegeneration, memory loss, cognitive decline, and motor impairment in aging.",Aging and Disease,Spermidine and Neurodegeneration,2021
3.2.1 Spermidine and Alzheimer’s Disease,"AD, also referred to as senile dementia, is characterized by progressive cognitive dysfunction and behavioral impairments. Clinically, it manifests as memory impairment, aphasia, agnosia, personality and behavioral alterations, among others. Age-associated memory decline can be attenuated by the autophagic effect of spermidine. Besides, spermidine has been reported to ameliorate age-related dementia. It relieves mitochondrial dysfunction to maintain neuronal energy, prevent nerve cell apoptosis and inflammation as well as improve the expression of neurotrophic factors. Wirth et al. revealed that spermidine exerts a positive influence on memory performance among the elderly, which might be regulated by stimulating the neuromodulators in the memory system.",Aging and Disease,Spermidine and Alzheimer's Disease,2021
3.2.2 Spermidine and Parkinson’s Disease,"PD, namely paralysis agitans, is a common neurodegenerative disease that is manifested by tremors, myotonia and decreased movement abilities. Degeneration and death of dopaminergic neurons in the substantia nigra is the main pathological basis of PD. McCarty et al. proved that spermidine protects against PD by maintaining dopaminergic neurons functions in the mitochondria. Jadiya et al. reported that spermidine protected against PD by inducing the Atg 7 dependent autophagy pathway in C. elegans. Besides, it also protected cells in a PD model of C. elegans against the toxic effects through the PINK1-PDR1-dependent mitophagy pathway. α-synuclein is considered to be the primary toxic trigger of PD. Previous studies found that higher spermidine concentration alleviates the process of PD through inhibition of α-synuclein and promotion of climbing activity. Guerra et al. suggested that spermidine exhibits neuroprotective effects against PD, which are mediated through its anti-inflammatory and antioxidant properties. DENSPM, a polyamine analogue, and Berenil, a pharmacological agent, increases or decreases SAT1 activities, respectively. It has been confirmed that DENSPM attenuates PD histopathology while Berenil aggravates it.",Aging and Disease,Spermidine and Parkinson's Disease,2021
3.3 Spermidine and age-related metabolic diseases,"Metabolic diseases are caused by disorders in substance anabolism and catabolism, which are closely correlated with aging. Spermidine is involved in the development of metabolic diseases, such as T2DM, obesity, and metabolic syndrome. 3.3.1 Spermidine and T2DM. T2DM is characterized by hyperglycemia due to insulin resistance. Its risk factors are complex, including aging, obesity, a strong family history of diabetes, and physical inactivity. Exogenous spermidine supplementation improves insulin sensitivity and maintains glucose homeostasis. Levasseur et al. clarified that spermidine binds deoxyhypusine synthase (DHPS) in β cells to mRNA translation, which promotes facultative cell proliferation and glucose homeostasis maintenance. Méndez et al. revealed that L-arginine and spermidine play an inhibitory role in lipid peroxidation and hemoglobin glycation, which may prevent diabetic complications.",Aging and Disease,Spermidine and T2DM,2021
3.3.1 Spermidine and T2DM – Part 2,"Besides, Wang et al. proved that spermidine enhanced glucose utilization through AMPK activation in myotubes, possessing a potential hypoglycemic activity in vitro. Serum spermidine oxidase activity has been shown to regulate T2DM and its microvascular complications in patients. Furthermore, Marx et al. elaborated that spermidine and agmatine were involved in renal collagen reduction in diabetic mice, thereby reducing the complications associated with diabetic nephropathy.",Aging and Disease,Spermidine and Glucose Homeostasis,2021
3.3.2 Spermidine and obesity,"Obesity is associated with multiple alterations at hormonal, inflammatory and endothelial levels, thereby enhancing morbidity rates from CVDs. Spermidine was found to reduce adiposity and hepatic fat accumulation in diet-induced obese mice. Besides, spermidine dietary can cause a significant weight loss and has the potential for treating obesity due to its beneficial effects in regulating lipid metabolism, inflammatory responses, and thermogenesis. Spermidine intake was negatively correlated with obesity caused by high-calorie diets and was accompanied by the induction of autophagy in white adipose tissues.",Aging and Disease,Spermidine and Obesity,2021
3.3.2 Spermidine and obesity – Part 2,"Notably, Ma et al. demonstrated that spermidine supplementation alleviated obesity in both mice and humans because of its effects in enhancement of intestinal barrier functions and alteration of microbiota composition and functions. Moreover, spermidine suppresses adiposity by inhibiting lipogenic genes expression through an AMPK-mediated mechanism. Up-regulation of spermidine is accompanied by down-regulation of nicotinamide N-methyltransferase (Nnmt), which results in nicotinamide salvage regeneration of NAD+, increased energy expenditure, and resistance against obesity.",Aging and Disease,"Spermidine, Microbiome, and Adiposity",2021
3.3.3 Spermidine and metabolic syndrome,"Metabolic syndrome is characterized by insulin resistance, abdominal obesity, hypertension, and hyperlipidemia. Triethylenetetramine dihydrochloride (TETA), a copper-chelator agent, is a safe pharmaceutical that can reduce obesity associated with excessive sucrose intake, high-fat diet, or leptin deficiency, since it can reduce hepatic steatosis and glucose intolerance. It has been shown that the TETA effects depended on the SAT1 activation, which can correct metabolic syndrome. Moreover, Ma et al. confirmed that spermidine inhibited metabolic syndrome in obese mice by ameliorating hepatic steatosis and adipose tissue inflammation.",Aging and Disease,Spermidine and Metabolic Syndrome,2021
3.4 Spermidine and musculoskeletal diseases,"Musculoskeletal diseases are a range of degenerative and inflammatory disorders, which are a vital cause of disability. Spermidine exhibits protective roles against various musculoskeletal diseases, such as osteoporosis, sarcopenia, and osteoarthritis. 3.4.1 Spermidine and osteoporosis. Osteoporosis is characterized by microarchitectural deterioration and low bone mass. Spermidine concentration is inversely proportional to osteoporosis. Spermidine dietary supplementation enhances bone strength. Increased spermidine biosynthesis in vivo promoted warmth regeneration, which prevented bone loss through gut microbiota.",Aging and Disease,Spermidine and Musculoskeletal Aging,2021
3.4.1 Spermidine and osteoporosis – Part 2,Spermidine was shown to prevent against bone loss by preferentially disturbing osteoclastic activation in ovariectomized mice. Yeon et al. documented that spermidine exerts anti-osteoclastogensis and anti-migration effects by inhibiting RANKLE-mediated signaling pathway and by preventing the expression of transcription factors such as NF-κB.,Aging and Disease,Spermidine and Osteoporosis Mechanisms,2021
3.4.2 Spermidine and sarcopenia,"Skeletal muscles are essential in inhibiting the development of multiple chronic diseases, including CVDs, T2DM, and cancer. Sarcopenia refers to age-associated progressive loss of skeletal muscle mass and function. Spermidine concentrations are associated with sarcopenia. Cervelli et al. hypothesized that spermidine protects against aging-related skeletal muscle atrophy by regulating skeletal muscle differentiation. Chrisam et al. revealed that systemic administration of spermidine induced autophagy in mice, leading to a concurrent amelioration of both ultrastructural and histological muscle defects.",Aging and Disease,Spermidine and Sarcopenia,2021
3.4.3 Spermidine and osteoarthritis,"Osteoarthritis is one of the most prevalent and debilitating chronic joint diseases, associated with decline and loss in life quality. Sacitharan et al. hypothesized that spermidine is a potential therapy for osteoarthritis because it activates autophagy in osteoarthritic cartilage and reverses the reduction in polyamine synthesis. Chen et al. showed that spermidine alleviates synovitis, osteophyte formation, and cartilage degeneration by inhibiting TNF-α induced NF-κB/p65 signaling pathway in osteoarthritis. In addition, spermidine was shown to exhibit antioxidant, anti-inflammatory, and chondroprotective roles in osteoarthritic chondrocytes.",Aging and Disease,Spermidine and Osteoarthritis,2021
Abstract Overview of CoQ10 and Cognition,"Background and Objective: With an increase in the number of older citizens in most Western countries, cognitive decline is becoming an increasingly significant issue. Numerous age-related metabolic and physiological changes, such as increased inflammation and oxidative stress, decreased adenosine triphosphate (ATP) production, poorer cardiovascular function, and reduced cerebral blood flow, have been implicated in cognitive decline, prompting research into interventions. Among these, Coenzyme Q10 (CoQ10), an antioxidant and metabolic stimulant, has shown promise in improving some of the underlying biological mechanisms of cognitive decline. However, not much is known about the efficacy of CoQ10 supplementation on cognition in the elderly. Therefore, the aim of this review is to explore the efficacy of CoQ10 supplementation on cognitive function. Methods: We conducted a review of animal studies and human clinical trials investigating the effect of CoQ10 supplementation on cognition in samples who were healthy or with specific diseases. Overall, twelve studies demonstrated improved cognitive function and two showed a reduction in oxidative stress in response to CoQ10 supplementation, either alone or in combination with other compounds.",Nutrients,Coenzyme Q10,2025
Abstract Results and Conclusions,"Out of eight human clinical trials in healthy subjects (n = 2) and disease states (n = 6), four showed evidence of a beneficial effect of CoQ10 supplementation on cognition, while two demonstrated an increase in cerebral blood flow. Disparity in the results of the clinical trials presented here is likely due to differing testing procedures, inconsistent use of cognitive assessments, and/or varying bioavailability of different preparations of CoQ10. Conclusions: There is some evidence to suggest that cognition and the biological mechanisms that regulate it are positively impacted by CoQ10 therapy. However, it is crucial to note that the literature presents mixed results, with many human clinical trials also reporting no benefit of CoQ10 supplementation on cognitive performance. To fully evaluate the benefits of CoQ10 on cognitive function in ageing and in neurodegenerative diseases, future studies are needed that target possible mechanisms and utilise a wider range of cognitive assessments. Keywords: Coenzyme Q10; CoQ10; cognition; ageing.",Nutrients,Coenzyme Q10,2025
Introduction to Ageing and Cognitive Decline,"It is estimated that by 2050, 22% of the global population will be 60 years or older. Associated with this changing demographic will be an increase in the number of people experiencing cognitive decline due to increasing age and prevalence of neurodegenerative diseases. The causes of cognitive decline and neurodegenerative diseases are multifactorial but inflammation, oxidative stress, mitochondrial dysfunction, poorer cardiovascular health and reduced cerebral blood flow are major contributors. These multiple processes occurring in the brain and body require different targeted therapeutic approaches. A wide range of approaches have been used to counteract age-related cognitive decline, including dietary modification, physical exercise, and use of medication and dietary supplements. While reviews of these approaches have often shown favorable outcomes, a comprehensive evaluation of the literature in areas such as the use of herbal supplements reveals a lack of high-quality evidence of efficacy, possibly due to inconsistencies in testing procedures.",Nutrients,Coenzyme Q10,2025
Introduction to CoQ10 as a Therapeutic Option,"Therefore, exploring new avenues is crucial, and CoQ10 supplementation with its unique biological effect on the body presents a promising option. This review will provide an overview of CoQ10 and its physiological roles. It will summarise research on the beneficial effects of CoQ10 therapy in cardiovascular health, followed by a summary of studies demonstrating additional broader health benefits. Finally, a comprehensive literature review examining the effects of CoQ10 on cognition in both animal and human studies will be provided. Coenzyme Q10 (CoQ10) is a naturally occurring fat-soluble antioxidant found in all cells in the body, mainly within mitochondria. There are two main recognised forms of CoQ10, oxidised CoQ10 (ubiquinone), and the reduced form (ubiquinol). CoQ10 has inherently poor bioavailability, but various innovative formulations have been developed to enhance absorption.",Nutrients,Coenzyme Q10,2025
Article Selection Method,"To conduct this review, the authors searched for clinical trial papers using Scopus (n = 2249), PubMed (n = 288), and Cochrane library (n = 140) to search for clinical research conducted on the effects of CoQ10 on cognition. The complete list of key search terms were: (“CoQ10” or “Coenzyme Q10” or “Ubiquinol” or “coenzyme”) and (“cognition” or “cognitive decline” or “cognitive function” or “neurocognition” or “neuropsych*” or “memory”). All clinical trials were required to have measurable cognitive assessments. Overall, 12 relevant animal and in vitro studies and 8 human randomised controlled trials (RCTs) reporting cognitive outcomes were identified. These studies are outlined separately, with animal and in vitro research presented in one table and human clinical trials in another.",Nutrients,Coenzyme Q10,2025
CoQ10 in Animal and In Vitro Cognitive Studies,"Examining the mechanisms of CoQ10’s actions in animals assists in understanding the effects of CoQ10 on the brain and cognition. As such, Table 1 outlines the cognitive effects reported in the reviewed animal studies involving mice and rats, as well as details pertaining to potential mechanisms of effect (both in vitro and in vivo). Table 1. Animal studies of CoQ10 and cognition. 4.1. Ageing Mice Like humans, mice exhibit a deterioration in cognitive functions as they age. Sumien et al. examined whether high or low dose CoQ10 supplementation, versus a normal diet, slowed age-related cognitive decline in mice. It was found that there was no significant effect of CoQ10 on the performance of mice completing the Morris water maze, measuring spatial memory and learning. CoQ10 levels were examined in the cortex, hippocampus-striatum, midbrain-diencephalon, cerebellum, and brainstem, with CoQ10 supplementation significantly increasing CoQ10 levels within the cortex.",Nutrients,Coenzyme Q10,2025
Ageing Mice Studies and Vitamin E Interactions,"These findings suggest that CoQ10 may have limited effects on cognitive function, despite its increased presence in brain tissue. Building on the prior evidence supporting the benefits of co-administrating alpha-tocopherol (vitamin E) and CoQ10, Shetty et al. investigated their combined effect on cognition across multiple trials. Mice were tested to assess if CoQ10, alpha-tocopherol, or both over three weeks could reduce age-related cognitive deficits. The results from the Morris water maze task showed no significant effect on performance due to any treatment. Contrary to these findings, McDonald et al. demonstrated benefits of the combination of CoQ10 and alpha-tocopherol on learning. When completing a discriminated avoidance testing, the effect of CoQ10 and alpha-tocopherol given independently did not produce significant avoidance responses. However, mice taking the combination had significantly more avoidance responses in the learning phase, suggesting a greater ability to learn. The varied outcomes of these trials indicate that additional research is necessary to fully comprehend the individual effects and interactions between these compounds.",Nutrients,Coenzyme Q10,2025
CoQ10 in Alzheimer’s Disease Models,"Alzheimer’s disease (AD) is characterised by the accumulation of amyloid plaques and neurofibrillary tangles in the brain, with possible contributions from mitochondrial dysfunction and oxidative stress. Ishrat et al. examined the impact of CoQ10 on cognitive deficits in a rat model of AD induced via the administration of streptozotocin, a diabetes-inducing chemical. CoQ10 therapy enhanced memory, learning and retention in rats with AD. Biochemical analysis indicated substantial reductions in oxidative stress and elevations in ATP levels inside the hippocampus and cerebral cortex of rats exhibiting AD. Rats undergoing CoQ10 therapy exhibited considerable enhancement in these metrics. The authors determined that CoQ10 treatment mitigated oxidative stress and enhanced ATP levels, thereby ameliorating learning and memory deficiencies. The neuroprotective effects of CoQ10 were illustrated by Singh et al., including improved memory and learning in rats with cognitive dysfunction induced by amyloid beta injections into the hippocampal region. CoQ10’s beneficial impact was largely attributed to its ability to inhibit microglial activation and restore mitochondrial function.",Nutrients,Coenzyme Q10,2025
Plaque Reduction and Mitochondrial Benefits in AD Models,"The effects of CoQ10 on brain pathology and behaviour in genetically modified mice with AD were demonstrated by Dumont et al. Mice that were fed a diet supplemented with CoQ10 had lower carbonyl density, lower plaque numbers, and smaller plaque areas in their retrosplenial cortex and hippocampus. Amyloid beta levels were also lower in mice administered CoQ10 compared to the control group. CoQ10 administration significantly decreased oxidative stress. CoQ10 treatment significantly enhanced cognitive performance in AD mice, as evidenced by improved learning compared to untreated controls. There were significant reductions in hippocampal plaque area and number that corresponded with improved spatial memory. To better understand the mechanism of action of CoQ10 in improving cognition in AD, Ying et al. studied genetically modified 3xTg-AD mice. They found that administration of CoQ10 altered the expression levels of 12 proteins in the hippocampus, alongside improvement in spatial memory.",Nutrients,Coenzyme Q10,2025
Mechanisms in AD: Protein Expression and Autophagy,"Specifically, Ying et al. determined that there was an over-expression of CPLX-1 and CPLX-2 in AD mice following CoQ10 therapy, proposing that this prevented spatial memory impairment. The therapeutic potential of CoQ10 in AD was also reported by Muthukumaran et al., demonstrating efficacy in halting AD-related behavioural and pathological symptoms in a double transgenic mouse model. CoQ10 treatment significantly enhanced cognitive functions, including memory and learning, and appeared to prevent AD pathology progression by reducing oxidative stress, inflammation, and activating autophagy, particularly in the hippocampus. These findings suggest that if these results were translated to human studies, CoQ10 could improve the quality of life for individuals with neurodegenerative diseases. Further evidence was reported by Sheykhhasan et al. (2002), where CoQ10-loaded exosomes enhanced cognitive function and memory in streptozotocin-induced AD rats. This was achieved by boosting BDNF and SOX2 levels in the hippocampus and promoting neuronal differentiation.",Nutrients,Coenzyme Q10,2025
"CoQ10 with Biotin, Folic Acid, and Omega-3 in AD Models","In the brains of individuals with AD, poor neuronal survival has been associated with increased insulin resistance, contributing to inflammation and pathological changes. Supplements such as biotin (vitamin B7) have shown benefits in insulin resistance in experimental diabetes. Attia et al. investigated neuroprotective effects of biotin and CoQ10 in an AD model induced by aluminium chloride in rats. Findings indicate that both biotin and CoQ10 independently, but more effectively when combined, can protect against AD by reducing neuroinflammation and enhancing insulin signalling within the brain. Vitamins such as folic acid (vitamin B9), essential for DNA synthesis and homocysteine remethylation, were studied alongside CoQ10 by Dolatabadi et al., showing significantly improved learning and memory in AD rats receiving CoQ10. More recently, Fouad et al. studied hypercholesterolemia-induced AD in rats, examining CoQ10 and Omega-3. Treated rats were significantly faster in maze tasks, showed reduced oxidative stress, enhanced cholinergic function, and nearly normal brain histology.",Nutrients,Coenzyme Q10,2025
Epilepsy in Rats,"Seizures resulting from heightened activation of brain neurons due to epilepsy are linked to neurodegeneration induced by reactive oxygen species (ROS). However, anticonvulsant drugs used for treating epilepsy, such as phenytoin, can additionally cause cognitive impairment and oxidative damage. Research by Tawfik et al. aimed to investigate the effects of CoQ10 supplementation on oxidative stress, seizure severity, and cognitive function in epileptic rats. Two weeks of phenytoin impaired memory in rats, while CoQ10 supplementation improved it. Phenytoin administration significantly increased oxidative stress, but CoQ10 supplementation significantly ameliorated this increase.",Nutrients,Coenzyme Q10,2025
Parkinson’s Disease Models,"While many of the symptoms of Parkinson’s disease are motor-related, cognition, behaviour, and mood can also be negatively impacted. In a preclinical investigation of CoQ10’s neuroprotective potential in Parkinson’s disease, Abu-Elfotuh et al. demonstrated that CoQ10 supplementation improved manganese-induced short-term memory impairments in a rat model. These effects were further enhanced when CoQ10 was combined with sesamol, thymol, and wheatgrass. Mechanistically, CoQ10 was associated with reductions in oxidative stress and neuroinflammation, as well as the modulation of apoptotic signalling pathways.",Nutrients,Coenzyme Q10,2025
Introduction to Ageing and Cognitive Decline,"It is estimated that by 2050, 22% of the global population will be 60 years or older. Associated with this changing demographic will be an increase in the number of people experiencing cognitive decline due to increasing age and prevalence of neurodegenerative diseases. The causes of cognitive decline and neurodegenerative diseases are multifactorial but inflammation, oxidative stress, mitochondrial dysfunction, poorer cardiovascular health and reduced cerebral blood flow are major contributors. These multiple processes occurring in the brain and body require different targeted therapeutic approaches.
A wide range of approaches have been used to counteract age-related cognitive decline, including dietary modification, physical exercise, and use of medication and dietary supplements. While reviews of these approaches have often shown favorable outcomes, a comprehensive evaluation of the literature in areas such as the use of herbal supplements reveals a lack of high-quality evidence of efficacy, possibly due to inconsistencies in testing procedures. Therefore, exploring new avenues is crucial, and CoQ10 supplementation with its unique biological effect on the body presents a promising option.",Nutrients,Coenzyme Q10,2025
Purpose and Structure of the Review,"This review will provide an overview of CoQ10 and its physiological roles. It will summarise research on the beneficial effects of CoQ10 therapy in cardiovascular health, followed by a summary of studies demonstrating additional broader health benefits. Finally, a comprehensive literature review examining the effects of CoQ10 on cognition in both animal and human studies will be provided.
Coenzyme Q10 (CoQ10) is a naturally occurring fat-soluble antioxidant found in all cells in the body, mainly within mitochondria. There are two main recognised forms of CoQ10, oxidised CoQ10 (ubiquinone), and the reduced form (ubiquinol). CoQ10 has inherently poor bioavailability, but various innovative formulations have been developed to enhance absorption. Notably, ubiquinol (Ub) supplementation has high bioavailability and has been demonstrated to both improve in vivo CoQ10 absorption and clinical recovery in patients with severe heart failure. Endogenous production of CoQ10 declines with age and is accompanied by an increase in reactive oxygen species (ROS) levels.",Nutrients,Coenzyme Q10,2025
CoQ10 Biological Function and Mitochondrial Role,"CoQ10’s primary function is within the mitochondrial electron transport chain, where it plays a crucial role in the synthesis of adenosine triphosphate (ATP) by transporting electrons from Complex I and Complex II to Complex III. ATP is important for cellular function to maintain the health and energy of all bodily organs, especially those with high energy requirements such as the heart and brain. The evidence presented in this review draws from cardiovascular, metabolic, and neurological research to build a comprehensive understanding of how CoQ10 supplementation may impact cognition indirectly through systemic health improvements and directly through neurobiological mechanisms.",Nutrients,Coenzyme Q10,2025
CoQ10 Clinical Trials in the Cardiovascular System: Surgical and Heart Failure Evidence,"CoQ10 supplementation has been shown to increase cellular energy production. In one clinical trial, patients scheduled for elective heart surgery were randomised to receive either oral CoQ10 (300 mg/d) or a placebo for two weeks prior to surgery. Analysis of myocardial tissue samples indicated that preoperative oral CoQ10 therapy elevated myocardial and cardiac mitochondrial CoQ10 levels and enhanced the efficiency of mitochondrial energy production. A meta-analysis of published randomised placebo-controlled trials of CoQ10 therapy in heart failure (HF) up to 2013 revealed that CoQ10 supplementation markedly increased ejection fraction (EF) and improved the New York Heart Association (NYHA) functional class, a tool used to diagnose heart failure. Specifically, supplementation led to a net enhancement of 3.67% in ejection fraction and a reduction of −0.30 in NYHA functional class.",Nutrients,Coenzyme Q10,2025
Landmark Heart Failure Trial and Blood Pressure Meta-Analyses,"In 2014, Mortensen et al. published a landmark prospective, randomised placebo-controlled multicenter trial of the efficacy of CoQ10 therapy in chronic heart failure (HF). Patients with moderate to severe heart failure (n = 420) were randomised to receive either CoQ10 at a dosage of 100 mg three times daily or a placebo, alongside normal care. The findings indicated that over the two-year follow up period, the CoQ10 group showed significant improvements relative to the placebo group in several key endpoints: cardiovascular mortality (9% vs. 16%, p = 0.026), all-cause mortality (10% vs. 18%, p = 0.018), and a reduced incidence of hospitalisation for heart failure (p = 0.033). Additionally, a notable improvement in NYHA class was observed in the CoQ10 group (p = 0.028). The conclusion was that long-term CoQ10 therapy in individuals with chronic heart failure is safe, alleviates symptoms, and reduces significant adverse cardiovascular events.",Nutrients,Coenzyme Q10,2025
CoQ10 in Hypertension and Systemic Cardiovascular Benefits,"In a review published in 2007 of trials of CoQ10 in the management of hypertension, the overall efficacy and consistency of therapeutic action and incidence of side effects associated with CoQ10 treatment was assessed. Meta-analysis was performed using 12 clinical trials (362 patients) comprising three randomised controlled trials, one crossover study, and eight open label studies. The review concluded that CoQ10 has the potential to lower systolic blood pressure by up to 17 mm Hg and diastolic blood pressure by up to 10 mm Hg in hypertensive patients without significant side effects. Subsequently, a meta-analysis by Zhao et al. containing 26 clinical trials (1831 participants) of CoQ10 in hypertension revealed decreased systolic blood pressure after CoQ10 supplementation in individuals with cardiometabolic diseases compared to control groups. The best effects were observed with CoQ10 dosages of 100–200 mg daily.",Nutrients,Coenzyme Q10,2025
"CoQ10, Vasodilation, Cerebral Blood Flow, and BBB Transport","Research has shown that the bioavailability of CoQ10 within heart tissue and mitochondria has been found to significantly increase with CoQ10 supplementation. In addition to strengthening the heart, CoQ10 also improves vasodilation in the circulatory system, supporting overall cardiovascular function. Similarly, CoQ10 has been shown to improve blood flow in the upper limb in Type II diabetic patients. A study by Kure et al. showed that, in patients with heart failure, patients with a higher bioavailability of CoQ10 displayed higher cerebral blood flow. Moreover, given that reduced cerebral blood flow is one cause of impaired cognition in heart failure and in ageing, CoQ10 might have a beneficial effect on the brain and its function by increasing cerebral blood flow. Whether CoQ10 can cross the blood–brain barrier (BBB) and have a direct effect on the brain is not clear. The BBB is created by the endothelial cells that line the blood vessels of the central nervous system, tightly regulating movement of ions, molecules, and cells.",Nutrients,Coenzyme Q10,2025
BBB Transport Mechanisms and Cardiovascular Predictors of Cognition,"Recent work has identified many molecules required for proper BBB function as well as many of the cellular and molecular signalling events that regulate the formation of the BBB during development, its function in adulthood, and its response to injury and disease. An in vitro study by Wainwright et al. using BBB endothelial cell models of CoQ10 deficiency was the first to identify lipoprotein-associated uptake and efflux mechanisms regulating CoQ10 distribution across the BBB. The results implied that the uptake of exogenous CoQ10 into the brain might be improved by the administration of low-density lipoprotein receptor inhibitors or by interventions that stimulate luminal activity of SR-B1 transporters. Cardiovascular health is a known predictor of cognitive function. A systematic review involving 50 studies and over 100,000 participants determined that elevated blood pressures predicted poorer global cognitive functioning, memory, language, and attention. Elevated arterial stiffness is also a predictor of reduced cognitive performance.",Nutrients,Coenzyme Q10,2025
"Cerebral Blood Flow, Cognitive Domains, and Implications for CoQ10","Cerebral blood flow is also associated with cognitive function, with positive associations reported between cerebral blood flow and performance in different cognitive domains in the SABRE study. Considering the varied benefits of CoQ10 supplementation on cardiovascular health outlined above, it is plausible that any benefits to cognition following CoQ10 supplementation may be, at least in part, due to positive effects upon cardiovascular health.",Nutrients,Coenzyme Q10,2025
Human Clinical Research Overview,"Insights gained from animal and in vitro studies have laid the groundwork for the development of human clinical trials. The final selection of human clinical trials investigating the effects of CoQ10 on cognitive function, identified through the literature search, is presented in Table 2. Although CoQ10 supplements are widely available, research on their cognitive effect in healthy individuals is limited. To date, only two randomised clinical trials have been published. The first of these trials was a randomised double-blind controlled trial by Kennedy et al. This study assessed the impact of a low dose CoQ10 (4.5 mg per day) in conjunction with multivitamin and mineral supplements in 106 females aged between 25 and 49 years. It was found that when completing cognitive tasks, cerebral blood flow significantly increased in the treatment group. However, there was no associated change in task performance. Plasma CoQ10 serum levels exhibited a significant increase, but the low dosage and presence of 22 additional supplement ingredients complicate interpretation of the results.",Nutrients,Coenzyme Q10,2025
Healthy Human Trials and Methodological Limitations,"The second study in healthy humans was conducted by Kinoshita et al., using 50 mg Ub capsules twice daily, and revealed no statistically significant differences between groups in Memory Performance Index (MPI) score, Trail Making Test (TMT), or Digit Symbol Substitution Test. Participants considered cognitively normal at baseline demonstrated significant improvement in memory after 34 weeks of Ub supplementation. Attention and processing speeds also improved on the TMT. However, methodological shortcomings undermine the reliability of these findings. Limitations included unclear MCI classification, exclusion of a participant that shifted p-values into significance, absence of sample size calculation, untested normality assumptions, sex-based sampling imbalance, and inappropriate analytical methods. These issues weaken the credibility of the results and highlight the need for more rigorous methodologies.",Nutrients,Coenzyme Q10,2025
Ongoing Trial in Healthy Elderly,"To further investigate the effects of CoQ10 on cognition in healthy humans, a randomised clinical trial was conducted by the current authors. This ongoing trial investigated the effects of Ub (200 mg/day) on cognitive decline over a 90-day period in healthy elderly participants aged 60 years or older. These participants were assessed and identified as experiencing subjective cognitive decline, increasing the likelihood of observing a benefit. The primary outcome was cognition. Secondary outcomes included cardiovascular health, oxidative stress, liver function, and mood. Results will be published in due course. Given the scarcity of studies in healthy individuals that help determine whether CoQ10 supplementation improves cognition, it is important to examine findings related to CoQ10 supplementation in pathological conditions.",Nutrients,Coenzyme Q10,2025
Alzheimer’s Disease Clinical Trials,"AD is an increasingly common disease especially in the elderly; however, there are few effective treatments. After reviewing studies on CoQ10 and other antioxidants, Galasko et al. identified optimal combinations and dosages, and investigated whether these supplements would affect cognitive function and cerebrospinal fluid biomarkers of oxidative stress or neurodegeneration over 16 weeks. Three treatment groups were formed: vitamin C + E + α-lipoic acid; CoQ10 (400 mg three times daily); and placebo. CoQ10 showed no detectable effect on cerebrospinal fluid biomarkers such as tau, Aβ42, and F2-isoprostanes, suggesting no significant impact on oxidative stress or neurodegeneration. CoQ10 supplementation caused no significant changes in cognition.",Nutrients,Coenzyme Q10,2025
Mild Cognitive Impairment Trials,"Mild cognitive impairment (MCI) involves cognitive deficits with preserved daily function and is often considered a transitional phase between normal ageing and dementia. Amnestic MCI is more strongly associated with progression to AD, while non-amnestic MCI relates more to vascular or frontotemporal dementia. García-Carpintero et al. studied 69 patients with diagnosed MCI using nine cognitive assessments and cerebral Doppler sonography. Inflammatory markers in plasma were also measured. Participants received 200 mg/day of Ub or placebo for one year. Plasma Ub concentration significantly increased after supplementation. However, cerebral vasoreactivity improved and inflammation decreased only in males. No cognitive changes occurred in either sex.",Nutrients,Coenzyme Q10,2025
Parkinson’s Disease Human Trials,"Previous research has shown that Ub significantly improves Parkinson’s symptoms as assessed by the Unified Parkinson’s Disease Rating Scale. Although CoQ10 supplementation improves mitochondrial function in Parkinson’s disease, randomised clinical trials combining CoQ10 with vitamin E found no improvement in self-reported cognitive function. Follow-up research investigated tolerability of CoQ10 up to 3000 mg/day with 1200 IU/day vitamin E. Plasma CoQ10 plateaued beyond 2400 mg/day, indicating that 2400 mg/day is the upper effective limit. Replication studies using these dosages showed no cognitive benefit relative to placebo. However, combinations of CoQ10 and creatine showed neuroprotective mechanisms, including reduced phospholipid indicators and improved cognitive function at 12 and 18 months.",Nutrients,Coenzyme Q10,2025
Progressive Supranuclear Palsy,"Progressive supranuclear palsy is characterised by akinesia, postural instability, bulbar symptoms, speech/language dysfunction, ocular motor dysfunction, and frontal cognitive and behavioural deficits. The condition involves impairment in the respiratory chain beginning at Complex I (NADH dehydrogenase), reducing oxidative phosphorylation. CoQ10 supplementation reduces neurotoxicity of Complex I inhibitors. Stamelou et al. administered CoQ10 (5 mg/kg/day) to 21 patients for 6 weeks, reporting significant improvement in frontal assessment battery (FAB) scores compared to controls. This suggests CoQ10 may restore respiratory chain function and frontal lobe activity.",Nutrients,Coenzyme Q10,2025
Chronic Fatigue Syndrome,"Chronic fatigue syndrome (CFS), or myalgic encephalomyelitis (ME), is a poorly understood syndrome characterised by physical and mental fatigue worsened by exertion. Symptoms include depression, impaired cognition, and neurocognitive dysfunction. A study by Fukuda et al. found that 150 mg of CoQ10 daily for 8 and 12 weeks improved cognitive function in CFS patients. The treatment group showed improved performance in arithmetic tasks, working memory, and fatigue symptoms. Additionally, CoQ10 supplementation reduced oxidative stress levels, suggesting that lowering oxidative stress may improve the pathophysiology of chronic fatigue syndrome.",Nutrients,Coenzyme Q10,2025
Discussion Overview,"In this review, multiple studies with animals have revealed protective effects of CoQ10 on cognition in healthy ageing and neurodegenerative disease models, though null effects have also been reported. While human clinical trials have shown some evidence of benefit in disease states but none in healthy ageing, multiple studies reported null or non-significant results. While these null effects may be attributable, at least in part, to limitations such as small sample sizes and insufficient statistical power, rather than the absence of a true effect, the overall mixed results reported in the current literature highlight the critical need for further high-quality, well-designed randomised clinical trials. There is a lack of studies examining cognitive improvements in non-diseased animal trials. This highlights a potential gap in research that warrants further exploration. When considering healthy ageing, three studies were conducted; however, two of these studies reported no significant improvement in cognitive function. Notably, one study that utilised CoQ10 in combination with vitamin E did yield positive results, suggesting that joint supplementation may hold promise for enhancing spatial memory and learning in healthy ageing.",Nutrients,Coenzyme Q10,2025
Epilepsy and Alzheimer’s Disease in Animal Models,"A single epilepsy study indicated an enhancement in cognitive function and a reduction in oxidative stress when CoQ10 was taken to reverse damage done by antiepileptic drugs such as phenytoin. This suggests potential neuroprotective mechanisms when facing oxidative stress and learning deficits due to chronic antiepileptic use. Given that this was conducted on only one antiepileptic agent, further research is required to determine if similar benefits extend to other therapies. Findings from Alzheimer’s disease (AD) studies present a promising therapeutic potential. Out of nine studies, all demonstrated improvements in cognition, particularly in learning. These improvements were accompanied by reductions in histological markers of AD, lower oxidative stress levels, and increased ATP production. One study demonstrating the protection of 12 hippocampal proteins, including CPLX-1 and CPLX-2, strengthens the hypothesis that targeted interventions like CoQ10 may mitigate the progression of cognitive decline associated with AD.",Nutrients,Coenzyme Q10,2025
Human Studies: Healthy Ageing and Cognitive Disorders,"In human studies, results are mixed. For healthy humans, one CoQ10 study yielded no effect, while another demonstrated borderline cognitive improvement. A recent study of healthy ageing is ongoing, with results expected in 2025. In AD research, one study found no significant impact, reflecting the complexity and variability of cognitive interventions. Mild cognitive impairment showed no improvement in one study, though an enhancement in cerebral vasoreactivity was noted, with unclear clinical relevance given the lack of cognitive effects. For Parkinson’s disease, two studies produced contrasting outcomes: one reported positive effects, while another found no significant impact, underscoring the need for more comprehensive research. Studies on progressive supranuclear palsy and chronic fatigue syndrome yielded favourable outcomes, demonstrating cognitive enhancements and suggesting potential utility across diverse neurological disorders.",Nutrients,Coenzyme Q10,2025
"Mechanisms: Oxidative Stress, Inflammation, and Cardiovascular Factors","Potential mechanistic pathways underpinning cognitive effects—particularly effects upon inflammation, oxidative stress, and histological markers associated with Alzheimer’s disease—were predominantly examined in animal studies. Few human trials explored these mechanisms, though considerable evidence suggests that elevated inflammation, oxidative stress, and poorer cardiovascular and cerebrovascular health predict poorer cognitive performance in humans. Systematic reviews and meta-analyses have reported that CoQ10 supplementation can lower inflammation, reduce oxidative stress, and lower blood pressure. Our review identified improvements in oxidative stress markers in studies involving AD and epilepsy, while reductions in inflammatory markers occurred exclusively in AD studies. Improvements in cardiovascular function were also noted in trials involving individuals with MCI and healthy adults, though results in healthy adults were confounded by multiple co-administered supplements.",Nutrients,Coenzyme Q10,2025
Future Directions and Research Justification,"In animal studies, CoQ10 has demonstrated its potential to improve cognitive function in ageing and neurodegenerative diseases, reduce oxidative stress, and improve mitochondrial function. Very few clinical trials on cognition in ageing and neurodegenerative diseases exist. Earlier diagnosis in conditions such as Parkinson’s disease may increase treatment benefits. Given rising rates of neurodegenerative diseases, there is a need for new clinical research into therapeutic options like CoQ10. Differences in BBB permeability and supplement preparations may explain discrepancies between animal and human findings. High CoQ10 doses used in animal studies are often not feasible in humans, suggesting the need to explore whether similar effects can be achieved with sustainable doses. Future work should target human populations with heightened risk factors such as inflammation, oxidative stress, or cardiovascular dysfunction. Additional research into BBB transport differences between humans and animals is needed, as well as well-powered trials assessing mechanistic pathways of cognitive improvement.",Nutrients,Coenzyme Q10,2025
Methodological Considerations and Safety,"Future research must justify participant quantities and cognitive assessment methodologies to ensure disciplinary uniformity. A diverse set of cognitive tasks is needed to provide a comprehensive overview of cognition. While CoQ10 is generally regarded as safe, some gastrointestinal effects such as abdominal pain and soft stools have been reported. The reported safe level of CoQ10 in humans is 1200 mg daily, below doses used in some clinical trials. Polypharmacy must be considered, as CoQ10 may negatively interact with medications such as warfarin and antihypertensives, potentially confounding cognitive outcomes. A recently completed prospective randomised clinical trial that satisfies most of these methodological criteria will soon be published.",Nutrients,Coenzyme Q10,2025
Conclusions,"CoQ10, a naturally occurring antioxidant essential for mitochondrial energy production, has demonstrated clinical benefits in cardiovascular health and physical performance and a role in reducing oxidative stress and increasing cerebral blood flow. These findings support the therapeutic potential of CoQ10 for improving cognitive function. Studies in animals have demonstrated the potential to improve cognitive function in ageing and neurodegenerative diseases and the ability for CoQ10 to reduce oxidative stress and improve mitochondrial function in the brain. However, clinical trials with humans have produced mixed results as to cognitive benefits in response to CoQ10 supplementation. Despite this, there is good evidence to suggest that the several mechanisms that maintain optimal cognition are positively impacted by CoQ10 therapy. To fully evaluate the benefits of CoQ10 on cognitive function in ageing and in neurodegenerative diseases, additional well designed, high quality randomised clinical trials are required. Such studies could target risk factors associated with cognitive decline that are also amenable to CoQ10 treatment (e.g., oxidative stress, inflammation, poor cardiovascular health) and examine treatment effects in a broader array of cognitive functions. Recruitment of larger study samples is also essential to ensure greater statistical power and robustness of findings.",Nutrients,Coenzyme Q10,2025
Introduction: Cancer Risk in Older Adults,"As the incidence of most cancers increases markedly with age, and cancer is the second leading cause of mortality in older adults, cancer is considered a major age-related disease in the United States and Europe (White et al., 2014; Laconi et al., 2020). Furthermore, accelerated aging and cancer development appear to be promoted by some of the same lifestyle-risk factors, such as low physical activity and an unhealthy diet (Lopez-Otin et al., 2013). However, apart from some preventive recommendations such as smoking cessation for lung cancer (Dragnev et al., 2003), public health efforts that focus on cancer prevention at midlife and older age have been largely focused on vaccination and screening efforts (White et al., 2014; Emmons and Colditz, 2017). This may in part be explained by mixed findings from clinical trials that tested single public health interventions for cancer prevention. Alternatively, combined interventions taking advantage of potentially small additive benefits from several public health strategies are largely lacking.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Rationale for Combined Interventions,"Although novel cancer treatments aim to block multiple pathways for cancer development by combining several agents (Bayat Mokhtari et al., 2017), this concept has not been translated into cancer prevention (Sabia et al., 2012). With regard to vitamin D and cancer prevention, mechanistic studies indicate that vitamin D inhibits the growth of cancer cells by regulating several genes responsible for cell proliferation and differentiation. Observational studies also support an inverse association between vitamin D blood levels and total cancer risk (Han et al., 2019). However, randomized trials testing supplemental vitamin D have produced mixed results, with suggestions that vitamin D has no benefit on cancer prevention overall, but may reduce the risk of advanced or fatal cancer (Haykal et al., 2019; Keum et al., 2019; Chandler et al., 2020).",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Omega-3 Fatty Acids and Cancer Prevention,"With regard to omega-3 and cancer prevention, mechanistic studies show that omega-3s may inhibit carcinogenesis by suppression of inflammation, cell proliferation, and angiogenesis (Fay et al., 1997; Larsson et al., 2004; Calviello et al., 2007). More recent research highlights the role of lipid metabolism in cancer cells, suggesting a mechanism by which omega-3 and omega-6 supplements can induce cancer cell death of acidic cancer cells by ferroptosis (Dierge et al., 2021). Despite these promising mechanistic pathways, cohort studies on omega-3 intake and total cancer risk have been inconclusive (MacLean et al., 2006). A 2020 meta-analysis of 27 trials involving 113,557 participants receiving a mean dose of 1.7 g of omega-3 over an average duration of 32 months suggested little or no benefit on the risk of any cancer diagnosis or cancer-related death (Hanson et al., 2020).",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Exercise and Cancer Risk Reduction,"Exercise may reduce cancer risk through several mechanistic pathways, including decreases in inflammation and improvements in immune function (McTiernan, 2008; Hong and Lee, 2020; Wang and Zhou, 2020). Observational studies support that higher physical activity reduces the risk of several cancers (Rezende et al., 2018) and increases cancer survival (Cormie et al., 2017). However, clinical trials directly testing the effect of exercise on cancer prevention are still lacking (McTiernan et al., 1999). These gaps highlight the need for well-designed intervention trials that combine lifestyle factors with potential additive or synergistic effects.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Aim of the Exploratory Trial,"The aim of the present exploratory trial among 2,157 generally healthy adults aged 70 and older was to address these knowledge gaps by testing the effect of daily high-dose vitamin D3, daily supplemental omega-3s, and a simple home exercise program (SHEP), alone and in combination, on the risk of any invasive cancer among adults aged 70 and older. This trial was designed to evaluate whether multimodal preventive strategies could provide additive benefits beyond those of single interventions. The study investigated whether combining nutritional supplementation with structured physical activity could serve as a feasible and effective public health approach to reducing cancer incidence in an aging population.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Trial Design,"The DO-HEALTH trial is a multicenter, double-blind, and 2 × 2 × 2 factorial design randomized-controlled clinical trial designed to support healthy aging in European adults aged 70 years and older (NCT01745263) addressing six primary endpoints (Bischoff-Ferrari et al., 2020). A verified cancer risk was a pre-defined exploratory outcome including all 2,157 participants. Clinical visits were at baseline, one, two, and three years, and there were phone calls every 3 months. The detailed trial design and protocol are provided elsewhere (Bischoff-Ferrari et al., 2021).",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Participants,"We targeted 2,157 participants who were generally healthy community-dwelling adults and recruited them from seven centers in five European countries, namely, Switzerland, Germany, Austria, France, and Portugal (Bischoff-Ferrari et al., 2021). The inclusion criteria were the absence of major health events in the 5 years prior to enrollment including cancer diagnosis, recurrence, treatment, sufficient mobility to come to the study centers, and good cognitive function with an MMSE score of at least 24 (Bischoff-Ferrari et al., 2020). The participants were required to limit the use of vitamin D from all supplemental sources to the recommended dietary allowance intake for older adults (800 IU per day), and to forego any supplemental omega-3 intake. All participants signed informed consent, and the ethical and regulatory agencies of all five countries approved the study protocol.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Randomization and Masking,"We randomized participants to eight treatment groups using block randomization (block sizes of 16 individuals): 2000 IU/day of vitamin D3 and 1 g/day of omega-3s and SHEP (n = 264); vitamin D3 and marine omega-3s (n = 265); vitamin D3 and SHEP (n = 275); vitamin D3 alone (n = 272); omega-3s and SHEP (n = 275); omega-3s alone (n = 269); SHEP alone (n = 267); or placebo (n = 270). Each participant received two study capsules per day identical in size, appearance, taste, and weight, and all capsules had coatings to prevent unblinding by the aftertaste. Each active omega-3s capsule contained 500 mg of EPA and DHA in a 1:2 ratio; each active vitamin D capsule contained 1000 IU of Vitamin D3 stabilized with dl-α-tocopherol (vitamin E); and each placebo capsule contained high-oleic sunflower oil.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Interventions and Dosing Rationale,"The SHEP and control exercise program are outlined in Supplementary Table S1. The dosing of the interventions was based on main trial outcomes including cardiovascular, bone, muscle, brain health, and immunity. Details on supporting evidence are provided in Bischoff-Ferrari et al., 2021. The consort flow diagram was published elsewhere (Bischoff-Ferrari et al., 2020).",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Cancer Outcomes Assessment,"The risk of verified invasive cancer was a pre-defined exploratory analysis of the DO-HEALTH trial, with time to any verified invasive cancer as the primary outcome. Secondary endpoints included prevention of three site-specific cancers: gastro-intestinal cancer, prostate cancer in men, and breast cancer in women. Cancer events were assessed prospectively at each in-person contact every 3 months throughout the 3-year follow-up in all 2,157 participants. Any reported cancer event triggered a detailed report and a medical record review to verify the diagnosis by an Independent Physician Endpoint Committee. All cancer events including ICD-10 D03:D04 were considered, excluding non-melanoma skin cancer (ICD-10 C44) and benign neoplasms (ICD-10 D13). The earliest verified event date was used for outcome time, with censoring at death (n = 25) or end of follow-up.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Cancer History Prior to Enrollment,"The history of cancer prior to the 5-year study eligibility window was assessed using medical history recorded by the study physician at the baseline visit, documenting cancer onset and ICD-10 codes (Bischoff-Ferrari et al., 2021).",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Statistical Analysis Methods,"We analyzed the trial based on the intent-to-treat. For the primary analysis, we included only the 81 participants whose self-reported invasive cancer could be verified as cases. To assess the effect of treatment on the incidence of any invasive cancer, we fitted Cox-proportional hazard models, adjusted for history of cancer, sex, body mass index (BMI), prior fall, study site, and age (70–84 and 85+). The primary predictors were indicator variables for the three treatments since we did not find significant interactions between the treatments in preliminary testing. We evaluated combination treatments within subgroups of participants, comparing participants who had a particular combination of treatments to participants who had neither/none of the treatments. Because of the factorial design, about half of participants in each of these two groups received the SHEP intervention. Sensitivity analyses excluded participants with any history of invasive cancer or cases from participants who self-reported invasive cancer, even if they were not verified.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Statistical Considerations and Software,"Given the small numbers of site-specific cancer cases and cancer deaths, we used the same statistical approach but presented unadjusted model results. Given that our statistical models report on several comparisons, we caution on interpretation of p-values. Following recent warnings by the American Statistical Association, we do not classify results as statistically significant or not based on a p-value threshold. Instead, we consider p-values < 0.05 as evidence for rejecting the null hypothesis. We also examine the magnitudes of estimates and the absolute number of cancer cases for each comparison. To provide an estimate for clinical relevance, we calculated probabilities of remaining cancer-free at 3 years from Kaplan–Meier curve analysis and from those, the number needed to treat (NNT) to prevent one incident case of cancer at 3 years. Statistical analyses were performed in SAS (v9.4) and R (v4.02) within RStudio (v1.2.1578). The patients and the public were not involved in setting the research question, design, outcome measures, interpretation, or manuscript preparation.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Baseline Characteristics of Participants,"Table 1 shows the baseline characteristics of all 2,157 participants. Overall, characteristics of the treatment and non-treatment groups were balanced. The mean age was 74.9 years, 61.7% were women, BMI 26.3 (SD 4.3), and only 5.2% were active smokers. Participants reported on average 3.3 comorbidities (SD 3.0), had good mobility [median SPPB score 11.0 (IQR 10.0–12.0)], and 82.6% were physically active at baseline with moderate-to-high activity levels. The majority were vitamin D replete (59.3%). At baseline, 24% were taking vitamin D supplements, increasing to 33% taking up to 800 IU/day at year 3, in addition to study medication.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Verified Invasive Cancer Cases,"Among 2,157 participants and 5,562.4 person-years of follow-up (median 2.99 years), 119 invasive cancer events were self-reported during quarterly in-person interviews. Of those, 29 could not be verified, and for three cases, the physician committee could not classify malignancy. Six reported cases were medically verified as noncancerous. This left 81 verified invasive cancer cases for the main intent-to-treat analysis. Because there was no significant effect modification by pre-defined subgroups, no subgroup analyses were performed.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Intent-to-Treat Hazard Ratios,"Including all 2,157 participants and 81 verified invasive cancers, individual and combined benefits of treatments were evaluated. Adjusted hazard ratios (HRs; 95% CI) for individual treatments were: vitamin D3: 0.76 (0.49–1.18; 36 vs. 45 cases), omega-3s: 0.70 (0.44–1.09; 32 vs. 49 cases), and SHEP: 0.74 (0.48–1.15; 35 vs. 46 cases). For combinations of two treatments: omega-3s + vitamin D3: 0.53 (0.28–1.00; 15 vs. 28 cases), vitamin D3 + SHEP: 0.56 (0.30–1.04; 11 vs. 21 cases), omega-3s + SHEP: 0.52 (0.28–0.97; 12 vs. 26 cases). For all three combined: HR = 0.39 (0.18–0.85; 4 vs. 12 cases). The NNT to prevent one cancer case at 3 years with all treatments combined was 35 (95% CI 26–137).",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Sensitivity Analyses and Site-Specific Cancers,"Excluding 185 participants with cancer history (69 verified new cases), all three treatments combined yielded an adjusted HR of 0.35 (0.15–0.80; 2 vs. 11 cases), NNT = 33 (95% CI 25–108). Including all 2,157 participants and 113 reported invasive cancers, HR for all treatments combined was 0.51 (0.27–0.98; 9 vs. 16 cases). For site-specific cancers (very low numbers): no treatment reduced gastrointestinal cancers (n = 22) or breast cancer in women (n = 13). Omega-3 alone [HR 0.17 (0.04–0.75); 2 vs. 12 cases] and omega-3 + SHEP [HR 0.12 (0.02–0.74); 0 vs. 6 cases] suggested possible reduction in prostate cancer risk (n = 14). Cancer mortality was not a predefined endpoint; only eight cases were observed.",Frontiers in Aging,"Vitamin D, Omega-3, Exercise",2022
Longevity Proteins and Triage Theory,"It is proposed that proteins/enzymes be classified into two classes according to their essentiality for immediate survival/reproduction and their function in long-term health: survival proteins versus longevity proteins. As proposed by the triage theory, a modest deficiency of one of the nutrients/cofactors triggers a built-in rationing mechanism that favors the proteins needed for immediate survival and reproduction (survival proteins) while sacrificing those needed to protect against future damage (longevity proteins). Impairment of the function of longevity proteins results in an insidious acceleration of the risk of diseases associated with aging. I also propose that nutrients required for the function of longevity proteins constitute a class of vitamins that are here named “longevity vitamins.” I suggest that many such nutrients play a dual role for both survival and longevity. The evidence for classifying taurine as a conditional vitamin, and the following 10 compounds as putative longevity vitamins, is reviewed: ergothioneine, pyrroloquinoline quinone (PQQ), queuine, lutein, zeaxanthin, lycopene, α- and β-carotene, β-cryptoxanthin, and astaxanthin. Because nutrient deficiencies are highly prevalent in the United States, appropriate supplementation and/or an improved diet could reduce much of the consequent risk of chronic disease and premature aging.",PNAS,Longevity Vitamins,2018
Concept of Longevity Vitamins and Proteins,"I propose that an optimal level of many of the known 30 vitamins and essential minerals/elements (V/M), plus that of 11 new putative vitamins described herein, is necessary for promoting healthy aging. The triage theory had previously introduced the concept that proteins/enzymes sacrificed during V/M shortage are necessary for supporting long-term health. This insight is broadened here to classify many V/M as necessary for maintaining long-term health. I present evidence that deficiency of many V/M specifically increases the risk of future disease and shortens lifespan. Thus, such V/M should be named “longevity vitamins,” and proteins associated with them should be named “longevity proteins.” Prolongation of healthy aging has not been generally understood as being related to V/M levels.",PNAS,Longevity Vitamins,2018
List of Essential Vitamins and Minerals,"Approximately 30 V/M are cofactors necessary for metabolism to function properly and were discovered because severe dietary deficiencies were linked to serious adverse health effects. They include vitamins A, B1, B2, B6, B12, biotin, C, choline, D, E, folic acid, K, niacin, pantothenate; and minerals/elements calcium, chloride, chromium, cobalt, copper, iodine, iron, manganese, magnesium, molybdenum, phosphorus, potassium, selenium, sodium, sulfur, and zinc. Additional important nutrients, the marine omega-3 fatty acids DHA and EPA, are discussed although they are not known as vitamins. Nine essential dietary amino acids are also important for protein and hormone synthesis but are not discussed here.",PNAS,Longevity Vitamins,2018
Prevalence of Vitamin and Mineral Deficiencies,"Most of the world’s population—even in developed countries—consume many V/M at levels below those recommended. Using the estimated average requirement (EAR) as a reference, the following high percentages of the U.S. population ingest quantities below EAR (including fortifications and supplements): vitamin D, 70%; vitamin E, 60%; magnesium, 45%; calcium, 38%; vitamin K, 35%; vitamin A, 34%; vitamin C, 25%; zinc, 8%; vitamin B6, 8%; folate, 8%. Intakes of DHA and EPA are also remarkably low; an EAR has not been set. A varied diet could provide enough V/M for healthier and longer life, whereas a diet high in refined foods and sugar is deficient in V/M and leads to an unhealthy and shorter life.",PNAS,Longevity Vitamins,2018
Linking V/M Deficiencies to Age-Related Disease,"The association or causality between various diseases of aging and numerous V/M deficiencies is analyzed by screening literature using clinical trials, epidemiology, Mendelian randomization studies, and biochemical and medical literature. A sampling of the literature covering links between diseases and deficiencies is provided in the supplemental appendix. Many deficiencies increase risk of long-term damage because longevity proteins lose function under nutrient scarcity, supporting the argument that optimizing V/M intake is essential for healthy aging and reducing chronic disease burden. These ideas reinforce the need to conceptualize V/M not only as survival nutrients but also as regulators of long-term maintenance and repair systems.",PNAS,Longevity Vitamins,2018
Triage Theory Overview,"The triage theory provides a unifying rationale for why modest V/M deficiencies—insufficient to elicit overt symptoms of severe deficiency—might contribute significantly to the aging process and the diseases of aging. Briefly, the triage theory posits that a strategic rationing response has been selected through evolution, which ensures that when a moderate shortage of a V/M is encountered, the scarce V/M is preferentially retained by those V/M-dependent proteins/enzymes that are essential for survival and reproduction, such as proteins essential for early development and immediate survival (i.e., “survival proteins”). At the same time, proteins/enzymes needed for maintaining long-term health by preventing insidious damage are starved for that V/M and become increasingly inactive, thus leading to an increase in diseases of aging. A major aspect of degenerative aging is that the damage is insidious and clinically not obvious because it accumulates slowly over time and is apparent only later in life. The connection to V/M shortages is underappreciated.",PNAS,Longevity Vitamins / Triage Theory,2018
Evidence Supporting Triage Theory,"This concept of triage has been buttressed by analyses of vitamin K and the element selenium. Vitamin K-dependent proteins could be categorized into those required for short-term survival (primarily blood-clotting functions) and those involved in long-term health. A similar triage rationing was found for selenium-dependent proteins. Recent human studies provide additional support for the triage theory with respect to vitamin K and selenium. It is noteworthy that in both of these cases about half of the proteins are affected negatively by a V/M shortage, suggesting that a large price could be paid in terms of accelerated aging by such a shortage. The mechanisms by which triage rationing occurs vary: for vitamin K the proteins are found in physically unconnected tissues. In the case of selenium the rationing is based on the use of two different transfer RNAs controlled by a modified base in the tRNA. Thus, different mechanisms were adopted for the same ultimate outcome in these two examples, suggesting that they evolved independently.",PNAS,Longevity Vitamins / Triage Theory,2018
Broadening the Triage Concept,"Although the triage theory was originally thought of as a cofactor-rationing system, upon further consideration it should be more broadly construed to include a larger variety of components that are not enzyme cofactors, but are still essential elements in triage rationing. The trading-off of metabolic resources to achieve a balance between somatic maintenance and reproductive fitness was proposed in an evolutionary theory of why we age. The triage theory provides a mechanism for achieving such a trade-off. Vitamins and essential minerals are usually thought of as compounds crucial for survival or protection against severe ill health, as shown by their dramatic short-term effect upon removal from the diet. The role of vitamins in healthy aging has been less appreciated. I am introducing two concepts inherent in the triage-rationing mechanism: longevity proteins and longevity vitamins.",PNAS,Longevity Vitamins / Triage Theory,2018
Longevity Proteins vs Survival Proteins,"Among the insights derived from the triage theory are the following concepts: (i) not all proteins/enzymes are affected equally by a V/M deficiency; (ii) not all V/M are exclusively needed for short-term survival; (iii) adequate V/M throughout life plays an important role in healthy aging. I propose that during triage rationing proteins that are sacrificed to allow for survival belong to a category with the specific function of protecting against diseases of aging. I propose to name them longevity proteins. In contrast, those needed for short-term survival and reproduction, and thus preferentially supplied with a V/M necessary for their function (besides being also necessary for later health) are referred to as “survival enzymes/proteins.” A fraction of the cofactor-requiring proteins that are subject to triage rationing are not technically enzymes (e.g., regulatory or structural proteins).",PNAS,Longevity Vitamins / Triage Theory,2018
Definition of Longevity Vitamins,"A redefinition of vitamins emerges from the definition of longevity proteins: the concept that the dietary compounds needed for the function of longevity proteins belong to a special category—that is, longevity vitamins—the shortage of which results in damage that is cumulative and insidious. Thus, vitamins may be divided into two general categories: (i) supporting both survival and longevity proteins, and thus subject to triage rationing; and (ii) supporting health without emphasis on early survival, but with their shortage leading continuously to accelerated aging, which may or may not be rationed. Some of those that are rationed may not involve interaction with a protein (e.g., carotenoids). In addition, I propose the likely existence of compounds needed only for longevity and therefore not essential for short-term survival. Dietary compounds used exclusively for protecting and improving healthy longevity would not yet have been recognized as V/M because the effects of their deficiency would be evident only in the form of insidious late damage. Evidence for 10 putative longevity vitamins is presented below.",PNAS,Longevity Vitamins / Triage Theory,2018
Dual-Role Vitamins and Minerals,"Inherent in the triage theory is the concept that most V/M necessary for the proper function of longevity proteins/enzymes are also survival V/M, having been originally discovered as cofactors for survival proteins. Thus, these V/M have two effects: on short-term survival and on long-term health. Besides the examples of vitamin K and selenium as being both essential and longevity V/M, three additional examples of V/M that have both effects are vitamin D, marine omega-3 fatty acids (DHA/EPA), and magnesium. The levels of each of these are inadequate in a large percentage of the American population, and these deficiencies are a major contributor to unhealthy aging.",PNAS,Longevity Vitamins / Survival and Longevity V/M,2018
Vitamin D as a Longevity Vitamin,"Vitamin D levels are inadequate in 70% of the United States population. Almost all dark-skinned people residing in northern latitudes are particularly deficient. Vitamin D was long considered responsible only for protecting against rickets, but it has now been shown to be involved in a myriad of functions. A cholesterol derivative, 7-dehydro-cholesterol, is converted by UV light to a precursor of vitamin D steroid hormone. Then the final steroid hormone binds to the vitamin D receptor protein; the latter interacts with a 12-base regulatory sequence in vitamin D receptor-dependent genes and regulates them either positively or negatively. About 2,700 such binding sites have been found in the human genome. Extensive evidence shows that vitamin D deficiency causes—or is associated with—many diseases that affect healthy aging, including all-cause mortality, cancer, cardiovascular disease, diabetes, and brain function. It is particularly important to tune up metabolism with respect to vitamin D.",PNAS,Longevity Vitamins / Vitamin D,2018
Vitamin D Trials and Measurement Issues,Supplementation with vitamin D has been discouraged historically because of fear of toxicity and because many clinical trials were inadequate. Newer evidence on all-cause mortality shows no increased risk even when blood levels of 25(OH)D reach 100 ng/mL. Randomized clinical trials for nutrients can be misinterpreted unless the nutrient level is measured both before supplementation and at its end. Many subjects in such trials may not be deficient and supplementation may not raise levels sufficiently. Failure to measure levels has led to many erroneous negative results in vitamin D RCTs. Conclusions that supplemental vitamins are ineffective should be viewed skeptically if the RCT did not measure nutrient levels. Vitamin D performs much more than its originally assigned function of maintaining bone health and thus qualifies as a longevity vitamin.,PNAS,Longevity Vitamins / Vitamin D,2018
Omega-3 Fatty Acids (DHA/EPA),"The intake of DHA and EPA is inadequate in most of the United States population. Low EPA and DHA levels in red blood cells were associated with increased all-cause mortality in 6,501 elderly women followed for nearly 15 years. A meta-analysis reported that each 1% increase in plasma DHA/EPA was linked with a 20% decreased risk of all-cause mortality. DHA/EPA are present in high levels in the central nervous system and are important for brain and retinal structure and function. In an RCT on first-episode schizophrenia patients, omega-3 supplementation prevented cortical loss of gray matter thickness. DHA has been implicated in aging, Alzheimer’s disease, Parkinson’s disease, schizophrenia, bipolar disorder, and depression. Low levels of DHA/EPA were associated with faster telomere shortening, and supplementation slowed telomere loss. DHA supplementation increased amyloid plaque clearance in individuals with mild cognitive impairment. DHA/EPA are also important for vitamin D steroid hormone effectiveness. Evidence also supports their role in reducing heart disease risk.",PNAS,Longevity Vitamins / Omega-3 Fatty Acids,2018
Magnesium as a Longevity V/M,"Magnesium is present in the center of the chlorophyll molecule, with plants, whole grains, nuts, and seeds being major dietary sources. Mg deficiency affects about 45% of the United States population and has been associated with increased all-cause mortality, poor DNA repair capacity, increased risk of lung cancer, several other cancers, heart disease, telomere shortening, and stroke. A recent review on the subclinical effects of Mg deficiency makes the case that this deficiency is a principal driver of cardiovascular disease and an underrecognized global public health crisis. Magnesium is required to convert vitamin D to its active steroid hormone form. Together with vitamin K, selenium, vitamin D, and DHA/EPA, Mg provides considerable evidence for being a longevity V/M as well as essential for survival, and optimizing intake offers a way to lengthen healthy longevity.",PNAS,Longevity Vitamins / Magnesium,2018
Abstract,"With regular practice, resistance exercise can lead to gains in skeletal muscle mass by means of hypertrophy. The process of skeletal muscle fiber hypertrophy comes about as a result of the confluence of positive muscle protein balance and satellite cell addition to muscle fibers. Positive muscle protein balance is achieved when the rate of new muscle protein synthesis (MPS) exceeds that of muscle protein breakdown (MPB). While resistance exercise and postprandial hyperaminoacidemia both stimulate MPS, it is through the synergistic effects of these two stimuli that a net gain in muscle proteins occurs and muscle fiber hypertrophy takes place. Current evidence favors the post-exercise period as a time when rapid hyperaminoacidemia promotes a marked rise in the rate of MPS. Dietary proteins with a full complement of essential amino acids and high leucine contents that are rapidly digested are more likely to be efficacious in this regard. Various other compounds have been added to complete proteins, including carbohydrate, arginine and glutamine, in an attempt to augment the effectiveness of the protein in stimulating MPS (or suppressing MPB), but none has proved particularly effective.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
Abstract (continued),"Evidence points to a higher protein intake in combination with resistance exercise as being efficacious in allowing preservation, and on occasion increases, in skeletal muscle mass with dietary energy restriction aimed at the promotion of weight loss. The goal of this review is to examine practices of protein ingestion in combination with resistance exercise that have some evidence for efficacy and to highlight future areas for investigation.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
Introduction,"The process of skeletal muscle protein turnover is constant and ongoing. Protein turnover within muscle is the sum of the processes of both muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Beyond childhood growth, chronic imbalances between the processes of MPS and MPB lead to a net gain in protein pool size (hypertrophy: MPS > MPB) or a net loss (atrophy: MPB > MPS). Often, athletes seek to maximize a hypertrophic response to exercise with the general acceptance that this may translate into performance gains. Hypertrophy, or the offsetting of atrophy, may also be a goal for athletes in recovery from injury, and so understanding the mechanisms that regulate muscle mass are important. The goal of this review is to provide a brief overview of the factors that regulate hypertrophy and how they can be affected by nutritional factors with a focus on protein.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
Regulation of Muscle Protein Turnover,"Resistance exercise provides a loading stimulus to skeletal muscle that results in increases in skeletal MPS and, if performed in the fasted state, an increase in MPB. The increase in fasted-state MPS with resistance exercise is long-lasting and persists for at least 48 h, and maybe longer with a higher volume of focused contractions. Provision of amino acids intravenously, as isolated proteins, or in foods such as beef and milk that promote hyperaminoacidemia and hyperinsulinemia are all effective in stimulating MPS. In addition, post-exercise hyperaminoacidemia suppresses the rise in MPB that occurs following resistance exercise in the fasted state.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
Regulation of Muscle Protein Turnover (continued),"Post-exercise hyperinsulinemia is not overtly stimulatory for MPS, but will also simultaneously suppress MPB. It thus appears that rather than being strictly anabolic, the hyperinsulinemia that accompanies post-exercise protein consumption is not stimulatory but probably merely permissive for MPS and suppressive for MPB. Therefore, when protein is ingested after resistance exercise it is the amino acids themselves that are driving the rise in post-exercise MPS. It is also now quite clear that it is really only the essential amino acids (EAA) that drive the process of MPS. However, perhaps more important is that the key EAA is leucine, as it alone appears to be the metabolic trigger for MPS. A complete mechanistic explanation of muscle protein turnover and its regulation is beyond the scope of this review; however, several reviews have covered this topic in detail.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
"Feeding, Exercise, and the Anabolic Window","With feeding, we now know that meal-to-meal fluctuations in MPS dictate the fed-state gains, and fasted-state losses, in muscle protein. Resistance exercise amplifies the inherent feeding response, which is actually quite transient, both immediately after exercise and at 24 h post-exercise. An important study by Tipton et al. showed that 24-h net protein balance reflected the acute changes in muscle protein turnover induced by both aminoacidemia and resistance exercise. However, the MPS response to aminoacidemia post-exercise wanes with time and the acute period post-exercise appears to be an optimal time to ingest protein—promoting hyperaminoacidemia and a robust stimulation of MPS. The nascent stimulation of MPS from resistance exercise alone lasts at least 24 h. We recently proposed that enhanced amino acid sensitivity of protein synthesis in this window of 'anabolic potential' probably persists for just as long.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
Mechanistic Considerations,"The mechanisms for enhanced sensitivity to amino acid feeding at each timepoint may be different, with the intriguing hypothesis that at later times (i.e., 24 h and beyond) following resistance exercise amino acid transport may be enhanced. Together, these findings highlight that resistance exercise and protein feeding interact synergistically to elevate MPS, suppress MPB, and promote a net positive muscle protein balance supporting hypertrophy.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
Dose–Response of Dietary Protein and MPS (Part 1),"To date only three true dose–response studies in which MPS has been measured have been published. In those studies, the main message was that MPS is a saturable process in young people at protein ingestion doses of approximately 20–25 g (~8.5–10 g of EAA) regardless of whether subjects exercised or not. Moore et al. also noted that, in parallel with the rise in MPS, the albumin protein synthetic rate showed a strikingly similar saturable dose–response curve, demonstrating that at least one other body protein had similar synthetic kinetics. In an attempt to standardize this protein dose to body mass (BM), and using the subjects’ mass from Moore et al., the dose of protein that was maximally effective (20 g) post-exercise equated to approximately 0.25 g protein/kg BM. While egg was the protein source used in that study, the rationale being that it is the internationally recognized standard protein, similar data would be expected with other high-quality proteins.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
Dose–Response of Dietary Protein and MPS (Part 2),"However, the dose of protein that is maximally stimulatory in older adults is closer to 40 g following resistance exercise and 20 g at rest. Beyond the levels at which MPS is maximally stimulated, it has been noted that the oxidation of an indicator amino acid, leucine, rises quite sharply, indicating that amino acids are not being used for protein synthesis and instead are oxidized, probably leading to urea production. While oxidative amino acid loss has been used as an indication of protein excess, it may well be that oxidative losses would still occur despite the fact that protein synthesis is not maximally stimulated as a result of lower Km values of enzymes involved in amino acid degradation compared with, for example, the Km for the activation of mTOR. The traditional interpretation of amino acid oxidation as being 'wasteful' may not be a true sentiment where optimal stimulation of MPS is concerned.",Sports Medicine,Exercise-Induced Hypertrophy / Protein Metabolism,2014
Protein Quality and Muscle Protein Turnover (Part 1),"Protein quality has traditionally been defined by the protein digestibility-corrected amino acid score (PDCAAS). This estimate of quality is derived from measures of the limiting EAA content in the protein compared with that of a reference protein (egg protein) multiplied by the digestibility of the protein. However, issues with the PDCAAS method of scoring proteins have been raised and relate to the validity of the preschool-age child amino acid requirement values, the use of fecal versus ileal digestibility, and the truncation of values at 1.0. The restriction of a PDCAAS value at 1.0 obscures the fact that the content of particular amino acids, such as leucine, are higher in milk-derived proteins casein and whey compared with soy by 33% and 76%, respectively. This difference in leucine content probably has some functional significance because leucine has been shown to be an important regulatory activator of skeletal MPS.",Sports Medicine,Protein Quality / Leucine Trigger,2014
Protein Quality and Muscle Protein Turnover (Part 2),"It has recently been reported that even small doses of protein, that were only 25% of the maximally effective protein dose for stimulating MPS, could be made to be maximally effective with the addition of leucine. Therefore, despite an equivalent PDCAAS score, it is perhaps not surprising that whey was found to be superior to soy protein in stimulating MPS in both a rested and contracted muscle. Interestingly, the same result was found in older men. While isolated proteins are an interesting model, most athletes consume whole foods. It was previously shown that skimmed milk was superior to a nutrient-matched soy beverage, which was attributed to the high leucine content of milk proteins in the 4:1 ratio of casein:whey in bovine milk.",Sports Medicine,Protein Quality / Food vs Isolated Protein,2014
Protein Quality and Muscle Protein Turnover (Part 3),"Of note, whey was also found to be superior to casein in stimulating MPS in both rested and contracted muscles. This is an interesting observation given that the leucine content of whey is only 20% higher than that of casein. However, casein is digested much more slowly than whey and has even been termed a ‘slow’ protein by comparison to whey, which is an acid-soluble and rapidly digested protein. Pennings et al. recently reported that whey was superior to both casein and casein hydrolysate in stimulating muscle protein accretion. Therefore, even hydrolysis of casein to speed up its digestion did not result in a greater stimulation of MPS. When protein was fed in small pulses, resulting in protracted hyperaminoacidemia with low amplitude, compared with a large bolus with rapid and transient aminoacidemia, a smaller rise in MPS occurred.",Sports Medicine,Protein Digestion Speed / Whey vs Casein,2014
Leucine Trigger Hypothesis,"Much of the evidence reviewed above has led to the proposal of the leucine 'trigger' hypothesis that revolves around the concept that leucine is the key amino acid that triggers a rise in MPS. As such, proteins that are richer in leucine would be more effective than proteins with lower leucine content. In addition, the rapidity of digestion, and thus the peak leucinemia, would be an important consideration as it would dictate the supply of leucine to trigger MPS. Exercise generally increases the sensitivity to leucine and thus lowers the leucine threshold, whereas aging and inactivity increase the threshold and the muscle develops a state of anabolic resistance. Current evidence would thus lead to a guideline stating that to achieve peak rates of MPS, a high leucine-containing protein that is rapidly digested, leading to rapid leucinemia and hyperaminoacidemia, should be consumed post-exercise.",Sports Medicine,Leucine Trigger / Anabolic Resistance,2014
High Protein Intake During Weight Loss (Part 1),"A number of studies have compared higher than normally consumed (i.e. ~15–17% of total dietary energy intake from protein) protein intakes in their effects on weight loss. While there is little doubt that the energy deficit per se will determine weight loss, the focus with weight loss and higher protein diets should be more on what is referred to as the 'quality' of the weight loss. The operational definition of weight loss quality is loss of a high ratio of fat to lean tissue, with an emphasis on the loss of visceral fat. Therefore, while general conclusions regarding weight loss in long-term free-living individuals have suggested that weight loss is no different with higher protein intakes, short-term trials have shown important differences in the weight lost as fat with muscle 'spared'. What is evident is that with respect to weight loss with exercise, higher protein and higher dairy protein in particular provide a protective effect for muscle, even allowing its accrual in certain circumstances. An important observation from an exercise performance standpoint is that in a group of overweight women who consumed a higher protein diet (1.3 g/kg BM/day) during diet and exercise-induced weight loss, they experienced greater gains in strength.",Sports Medicine,High Protein Diets / Weight Loss Composition,2014
High Protein Intake During Weight Loss (Part 2),"However, protein is not able to ablate the loss of skeletal muscle mass completely, especially if the energy deficit is substantial and rapid weight loss occurs, even in exercising athletes. However, when weight loss is more moderate then higher protein intake (1.6 g/kg BM/day) can not only preserve lean mass but allow performance gains. Unfortunately, without continued supervision, the same athletes who lost fat and gained muscle in the 8-week study period returned to their pre-intervention body composition after 12 months.",Sports Medicine,Protein Intake / Lean Mass Preservation,2014
Adjunctive Strategies to Augment MPS (Carbohydrates),"While it is clear that aminoacidemia following protein ingestion drives the rise in MPS, other nutrients have been added to protein in an attempt to augment its impact on MPS. Carbohydrates have been a primary focus in this area, with the rationale that their energy may serve to reverse an exercise-induced suppression of protein synthesis, either by activation of AMP kinase or through a calcium–calmodulin-dependent mechanism. Alternatively, insulin as a result of carbohydrate ingestion could either promote protein synthesis, suppress proteolysis, or both. However, to date several studies combining protein and carbohydrate have shown no augmentation of protein synthesis when protein is provided in adequate amounts. These data do not preclude the hypothesis that carbohydrate is not stimulatory with lower-than-optimal protein doses. In addition, the restoration of muscle glycogen by means of carbohydrate ingestion is also obviously important for athletes and should not be neglected.",Sports Medicine,Carbohydrates / MPS Modulation,2014
Adjunctive Strategies — Glutamine,"Only a few amino acids have been tested in their capacity to augment MPS, but none has proved beneficial in young men. Glutamine (0.3 g/kg BM) was given to young men following 90 min of cycling at 65% of peak oxygen uptake in addition to carbohydrate and balanced EAA, and there was no difference in post-exercise MPS compared with the placebo trial. The lack of an effect of glutamine on MPS following endurance exercise is at odds with data showing that even endurance exercise is anabolic for mitochondrial and myofibrillar protein synthesis. Congruent with the absence of any benefit of glutamine on MPS after endurance exercise are data from young men performing resistance training who received glutamine throughout 6 weeks of training (0.9 g/kg lean tissue/day). Glutamine supplementation has been shown to be useful in certain clinical populations, in whom there is a relative lack of intracellular glutamine. However, it is perhaps not overly surprising that glutamine is ineffective in populations who have adequate levels of the amino acid, because it is hard for even a high dose of glutamine to increase intramuscular glutamine, and conclusions of recent reviews have been that glutamine appears to be far from useful for athletes.",Sports Medicine,Glutamine / MPS Ineffectiveness,2014
Adjunctive Strategies — Arginine / Nitric Oxide,"As a precursor for nitric oxide biosynthesis, the amino acid arginine has received some attention for its potential role to promote blood flow and enhance nutrient or hormonal delivery to muscles allowing enhanced anabolism. The one study in which MPS has been measured in humans following exercise with arginine supplementation showed no effect of a bolus dose (10 g) of arginine on nitrate or nitrite concentration, femoral artery flow, or MPS. An interesting observation was that growth hormone concentrations were enhanced by arginine supplementation but, similar to other studies, the transiently increased growth hormone concentration did not enhance MPS. Other attempts to enhance blood flow after resistance exercise by means of arginine or other nitric oxide-enhancing compounds have proved unsuccessful, at least in healthy young men.",Sports Medicine,Arginine / NO / Blood Flow / MPS,2014
Conclusion,"Changes in MPS are variable throughout the day on a meal-to-meal basis and are augmented immediately and for a prolonged time period after resistive exercise. Endurance exercise also stimulates MPS, but the responses are different to those with resistance exercise, and there is far less clarity on the length of time that they persist. Dietary protein appears to be most effective when consumed after exercise, to take advantage of the 'receptive state' of the muscle for mounting a robust MPS response. This would appear to be a guideline that athletes engaging in resistance and endurance training should follow to allow the synthesis of new proteins specific to their activity, and also to promote adaptive remodeling and repair of any cellular damage. The dose of protein that appears most effective following resistance exercise, and possibly endurance exercise, is approximately 0.25–0.30 g protein/kg BM/meal, at least when consuming isolated proteins. Leucine is a key amino acid in stimulating MPS and its content in, for example, whey protein is probably a primary reason why whey protein is so effective at stimulating MPS as opposed to isolated soy and casein proteins. Therefore, proteins containing a high content of leucine that are digested rapidly are most effectively directed toward MPS; however, ingestion of foods such as milk promote a robust stimulation of MPS and highlight the fact that blends of fast and slow proteins are still effective in stimulating MPS. When protein is sufficient, dietary carbohydrate and the ensuing insulinemia does not augment the response of MPS, but carbohydrate is still a practical macronutrient to consume to promote glycogen resynthesis. Neither arginine nor glutamine have been demonstrated to be effective at promoting resistance exercise-induced anabolism in humans and their inclusion in supplements has, on the basis of current evidence, no grounds.",Sports Medicine,Exercise-Induced Muscular Hypertrophy — Conclusion,2014
Overview of Resistance Training and Mitochondrial Adaptations,"Impact of Resistance Training on Skeletal Muscle Mitochondrial Biogenesis, Content, and Function Thomas Groennebaek and Kristian Vissing* Skeletal muscle metabolic and contractile properties are reliant on muscle mitochondrial and myofibrillar protein turnover. The turnover of these specific protein pools is compromised during disease, aging, and inactivity. Oppositely, exercise can accentuate muscle protein turnover, thereby counteracting decay in muscle function. According to a traditional consensus, endurance exercise is required to drive mitochondrial adaptations, while resistance exercise is required to drive myofibrillar adaptations. However, concurrent practice of traditional endurance exercise and resistance exercise regimens to achieve both types of muscle adaptations is time-consuming, motivationally demanding, and contended to entail practice at intensity levels, that may not comply with clinical settings. It is therefore of principle interest to identify effective, yet feasible, exercise strategies that may positively affect both mitochondrial and myofibrillar protein turnover. Recently, reports indicate that traditional high-load resistance exercise can stimulate muscle mitochondrial biogenesis and mitochondrial respiratory function. Moreover, fatiguing low-load resistance exercise has been shown capable of promoting muscle hypertrophy and expectedly entails greater metabolic stress to potentially enhance mitochondrial adaptations.",Frontiers in Physiology,Resistance Training and Mitochondria,2017
Low-Load Resistance Exercise and Mitochondrial Potential,"Consequently, fatiguing low-load resistance exercise regimens may possess the ability to stimulate muscle mitochondrial adaptations without compromising muscle myofibrillar accretion. However, the exact ability of resistance exercise to drive mitochondrial adaptations is debatable, not least due to some methodological challenges. The current review therefore aims to address the evidence on the effects of resistance exercise on skeletal muscle mitochondrial biogenesis, content and function. In prolongation, a perspective is taken on the specific potential of low-load resistance exercise on promoting mitochondrial adaptations. Keywords: mitochondria, strength training, bioenergetics, mitochondrial protein synthesis, mitochondrial volume density, blood flow restricted exercise INTRODUCTION Mitochondria are intracellular organelles that play a key role in metabolism and cellular homeostasis. Consequently, mitochondrial content and function exert great influence on substrate utilization capacity and skeletal muscle health, with skeletal muscle mitochondrial abnormalities implicated in the pathology of chronic disorders such as diabetes, obesity, and peripheral arterial disease (Kim et al., 2000; Petersen et al., 2004; Rontoyanni et al., 2017).",Frontiers in Physiology,Resistance Training and Mitochondria,2017
Mitochondrial Biogenesis and Exercise-Induced Regulation,"Synthesis of new mitochondrial reticular components (i.e., mitochondrial biogenesis) has profound effect on mitochondrial content and function. Mitochondrial biogenesis is reported to be attenuated with aging, prolonged inactivity, and/or chronic disease (Rooyackers et al., 1996a,b; Abadi et al., 2009; Gram et al., 2014), while metabolic stressors inherent of exercise possess the ability to stimulate mitochondrial biogenesis. In accordance, metabolic stress inherent of endurance exercise has been demonstrated to stimulate mitochondrial biogenesis (Perry et al., 2010; Di Donato et al., 2014), which, when repeated through prolonged training (i.e., endurance training), can accumulate into adaptational changes in mitochondrial content and function (Hoppeler et al., 1985; Jacobs and Lundby, 2013). Employment of animal models and omics-based studies has improved our understanding on the molecular mechanisms underlying endurance exercise-induced mitochondrial biogenesis. Accordingly, alterations in intramuscular homeostasis (e.g., alterations in AMP, calcium, and reactive oxygen species) inferred by endurance exercise are described to exert regulatory action on specific proteins involved in transcriptional regulation of mitochondrial biogenesis, such as calcium/calmodulin-dependent protein kinase (CaMK), AMP-activated protein kinase (AMPK), and p38 mitogen-activated protein kinase (p38-MAPK) (Hawley et al., 1995; Wright et al., 2007; Combes et al., 2015).",Frontiers in Physiology,Resistance Training and Mitochondria,2017
Assessment Methods for Mitochondrial Content,"EFFECT OF PROLONGED RESISTANCE TRAINING ON MUSCLE MITOCHONDRIAL CONTENT Multiple analytical approaches are available for assessment of mitochondrial content. Among these, electron microscopy-based determination of mitochondrial volume density is regarded as the golden standard (Larsen et al., 2012). However, the studies utilizing electron microscopy to deduce resistance training-induced adaptational changes in mitochondrial volume density are few and characterized by methodological constraints, such as low sample size and absence of control experiments. These methodological constraints notwithstanding, two previous studies utilizing electron microscopy surprisingly suggest a dilution of mitochondrial volume density to occur following high-load resistance training in young men (MacDougall et al., 1979; Luthi et al., 1986). Most studies on the effect of resistance training on mitochondrial content rely on utilization of citrate synthase (CS) activity as a biomarker, because CS activity has been shown to exhibit a degree of association with mitochondrial volume density determined by electron microscopy, while simultaneously offering a significantly less time-consuming approach (Larsen et al., 2012; Meinild Lundby et al., 2017).",Frontiers in Physiology,Resistance Training and Mitochondria,2017
Divergent Findings on Mitochondrial Content After Resistance Training,"However, results from studies on high-load resistance training in young untrained individuals that employ measures of CS activity are somewhat equivocal, with most studies reporting unaltered CS activity (Tesch et al., 1990; Wang et al., 1993; Wilkinson et al., 2008; Porter et al., 2015), whereas other studies report either decreased (Kon et al., 2014) or increased (Tang et al., 2006) CS activity. A similar discordance is evident from studies evaluating the effect of high-load resistance training on single fiber mitochondrial content by use of histochemical staining for succinate dehydrogenase (Ploutz et al., 1994; Chilibeck et al., 1999; Green et al., 1999; Bell et al., 2000). In conclusion, while these divergent results may in part adhere to the degree of muscle hypertrophy achieved (Tang et al., 2006), they are also the product of a number of small-scale studies, with only few to include control experiments and with the vast majority of these studies relying on surrogate markers of mitochondrial content.",Frontiers in Physiology,Resistance Training and Mitochondria,2017
Cristae Density and Functional Insight Beyond Volume Measures,"More recently, a study that has used electron microscopy to evaluate mitochondrial cristae density after prolonged endurance training, suggests that mitochondrial cristae density may provide a stronger predictor of an individual’s maximal oxygen uptake compared to mitochondrial volume density (Nielsen et al., 2016). Still, whereas insight on mitochondrial volume density and cristae density can provide relevant information on permanent quantitative changes, such measures are not necessarily telling on adaptations in mitochondrial function.",Frontiers in Physiology,Resistance Training and Mitochondria,2017
High-Resolution Respirometry and Resistance Training,"EFFECT OF PROLONGED RESISTANCE TRAINING ON MUSCLE MITOCHONDRIAL RESPIRATORY FUNCTION Utilization of high resolution respirometry in permeabillized myofibers and/or isolated mitochondria from muscle biopsies may allow for a more physiologically relevant approach to study mitochondrial function (Haller et al., 1994; Kuznetsov et al., 2008). In this regard, a recent study utilizing high resolution respirometry in permeabilized myofibers observed increased maximal coupled respiration supported by electron transfer from complex 1 and 2 after 12 weeks of high-load resistance training in young, untrained subjects, with only a modest (and non-significant) increase in CS activity (Porter et al., 2015). This implies that the maximal ATP-producing capacity can be improved independently of changes in mitochondrial content. Similarly, by a comparative approach also utilizing high resolution respirometry in permeabilized myofibers, Pesta et al. (2011) found identical magnitudes of improvements in intrinsic mitochondrial function following 10 weeks of high-load resistance training and endurance training undertaken under normoxic as well as hypoxic conditions, although it needs to be emphasized that only three participants completed the normoxic high-load resistance training-intervention.",Frontiers in Physiology,Resistance Training and Mitochondria,2017
Qualitative Adaptations and Supercomplex Assembly,"Furthermore, the authors employed measures of mitochondrial DNA as a biomarker, which has elsewhere been contended to constitute a poor predictor of mitochondrial content (Larsen et al., 2012). Further supporting these findings, a cross-sectional study reported higher maximal coupled respiration and tighter coupling of oxidative phosphorylation (suggestive of increased efficiency of the phosphorylation system) in permeabilized myofibers of highly resistance trained subjects compared to untrained controls, despite no differences in CS activity (Salvadego et al., 2013). Such qualitative adaptations may be ascribed simply to increased abundance of electron transport chain complexes following prolonged high-load resistance training (Porter et al., 2015), but may also rely on other contributing mechanisms. Accordingly, recent pioneering studies have provided evidence for increased assemblies of respiratory supercomplexes as well as increased mitochondrial cristae density in response to long term endurance training, thus facilitating qualitative modulation of oxidative capacity (Nielsen et al., 2016; Greggio et al., 2017). Whether similar adaptational changes also adhere to resistance training, remain to be investigated.",Frontiers in Physiology,Resistance Training and Mitochondria,2017
Permeabilized Fibers vs Isolated Mitochondria,"Peculiarly, contrary to studies on permeabilized fibers, employment of high resolution respirometry on isolated mitochondria indicates little effect of high-load resistance training on mitochondrial function. In accordance, two recent randomized controlled studies collectively favor the contention that high-load resistance training is not able to augment mitochondrial respiratory function (Irving et al., 2015; Robinson et al., 2017). It can be speculated that the discordance in results between analyses on permeabilized fibers vs. isolated mitochondria stem from organelle interactions inherent of the normal milieu of the muscle cell. Hence, in the living cell, mitochondria exist in fused networks with preserved interactions with other organelles such as the endoplasmic reticulum and the cytoskeleton (Kirkwood et al., 1986; Rizzuto et al., 1998). From this perspective, analysis on permeabilized myofibers can be considered as more closely resembling genuine physiological conditions. Oppositely, mitochondrial isolation procedures can be contended to disrupt the mitochondrial structure and consequently interfere with respiratory function (Picard et al., 2011), but this needs to be further investigated. For comprehensive considerations on the utilization of permeabilized fibers vs. isolated mitochondria, we refer to a previous report (Kuznetsov et al., 2008).",Frontiers in Physiology,Resistance Training and Mitochondria,2017
Exercise Modalities and Biogenesis Overview,"Skeletal Muscle Ribosome and Mitochondrial Biogenesis in Response to Different Exercise Training Modalities Skeletal muscle adaptations to resistance and endurance training include increased ribosome and mitochondrial biogenesis, respectively. Such adaptations are believed to contribute to the notable increases in hypertrophy and aerobic capacity observed with each exercise mode. Data from multiple studies suggest the existence of a competition between ribosome and mitochondrial biogenesis, in which the first adaptation is prioritized with resistance training while the latter is prioritized with endurance training. In addition, reports have shown an interference effect when both exercise modes are performed concurrently. This prioritization/interference may be due to the interplay between the 5’ AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) signaling cascades and/or the high skeletal muscle energy requirements for the synthesis and maintenance of cellular organelles. Negative associations between ribosomal DNA and mitochondrial DNA copy number in human blood cells also provide evidence of potential competition in skeletal muscle. However, several lines of evidence suggest that ribosome and mitochondrial biogenesis can occur simultaneously in response to different types of exercise and that the AMPK-mTORC1 interaction is more complex than initially thought.",Frontiers in Physiology,Ribosome and Mitochondrial Biogenesis,2021
Introduction and Research Background,"The purpose of this review is to provide in-depth discussions of these topics. We discuss whether a curious competition between mitochondrial and ribosome biogenesis exists and show the available evidence both in favor and against it. Finally, we provide future research avenues in this area of exercise physiology. Keywords: skeletal muscle, ribosomes, mitochondria, AMP-activated protein kinase, mechanistic target of rapamycin, exercise training, concurrent training INTRODUCTION Research interest in the fields of ribosome and mitochondrial biogenesis has been growing considerably over the last decades. While the number of overall publications listed on MEDLINE has been increasing steadily during the last 20 years (~200% increase when comparing 2020–2000), during the same period, there was an even greater increase (over 2,500%) in the number of publications with the search terms “ribosome biogenesis” or “mitochondrial biogenesis.” Much of the research in “ribosome biogenesis” and/or “mitochondrial biogenesis” has dealt with cancer biology (Derenzini et al., 2017; Vanderveen et al., 2017; Pelletier et al., 2018), aging (Tiku and Antebi, 2018; Correll et al., 2019; Roque et al., 2020), and other disciplines unrelated to exercise physiology.",Frontiers in Physiology,Ribosome and Mitochondrial Biogenesis,2021
Definitions of Ribosome and Mitochondrial Biogenesis,"However, in recent years, several exercise physiology laboratories have been utilizing more mechanistic molecular tools to study the adaptations that occur with exercise to discern the well documented health and/or performance benefits following exercise. Ribosome and mitochondrial biogenesis are both complex processes. A detailed description of the molecular underpinnings of each process is beyond the scope of this review and readers are referred to other excellent reviews on the topics [ribosome biogenesis (Henras et al., 2015; Kressler et al., 2017), mitochondrial biogenesis (Jornayvaz and Shulman, 2010; Bouchez and Devin, 2019)]. For the purpose of this review, ribosome biogenesis refers to the de novo synthesis of ribosomes, a process that involves the transcription and processing of rRNA and the assembly of several ribosomal proteins. The rate-limiting step of ribosome biogenesis is thought to be generation of the 45S pre-rRNA by RNA Polymerase I (Kopp et al., 2007). This precursor is then processed, yielding the 18S, 5.8S, and 28S mature rRNA transcripts. These transcripts are exported to the nucleus and associate with 5S rRNA and different ribosomal proteins resulting in the assembly of the mature ribosome.",Frontiers in Physiology,Ribosome and Mitochondrial Biogenesis,2021
Mechanisms of Mitochondrial Biogenesis,"Mitochondrial biogenesis is accomplished through the recruitment of newly synthesized mitochondrial proteins to existing organelles, which can grow and divide (Ryan and Hoogenraad, 2007; Miller and Hamilton, 2012). Mitochondrial biogenesis involves the transcription of proteins encoded by both nuclear and mitochondrial genomes. Considered a major regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) activates nuclear respiratory factors, increasing nuclear transcription of mitochondrial genes (Ryan and Hoogenraad, 2007). These nuclear respiratory factors activate mitochondrial transcription factor A (TFAM), which promotes the transcription and replication of mitochondrial DNA (Wu et al., 1999). Importantly, researchers frequently use activation markers of cell signaling pathways, mRNA expression, protein levels, and/or enzymatic activity as a measure of mitochondrial biogenesis. However, as nicely discussed by Miller and Hamilton (2012), such variables present important limitations and are not direct measures of biogenesis. The only direct measure of mitochondrial biogenesis currently available is the measure of mitochondrial protein synthesis using stable isotopic tracers.",Frontiers in Physiology,Ribosome and Mitochondrial Biogenesis,2021
Indirect and Direct Measurement Challenges,"Similarly, ribosome biogenesis is usually assessed through measurements of cell signaling, mRNA expression, protein levels, total RNA and/or rRNA levels, variables that are at best only indirect measures of biogenesis. Tracer methodologies have also been developed and used to measure de novo ribosomal biogenesis (Brook et al., 2017). However, considering the fact that only few exercise training studies have used direct measures of ribosome and/or mitochondrial biogenesis, studies using indirect measures will be included and discussed in the current review. Readers are strongly encouraged to consider whether direct or indirect measures were used when interpreting the results of the studies presented herein. Resistance and endurance training increase skeletal muscle ribosome biogenesis and mitochondrial biogenesis, respectively. Mitochondrial biogenesis increases aerobic capacity (Costill et al., 1976; Burgomaster et al., 2008; Yeo et al., 2008; Murias et al., 2011; Cochran et al., 2014; Vigelso et al., 2014), and ribosome biogenesis has been associated with skeletal muscle hypertrophy.",Frontiers in Physiology,Ribosome and Mitochondrial Biogenesis,2021
"Specificity, Prioritization, and Interference","It is generally believed that skeletal muscle adaptations to exercise are highly specific. Increased ribosome biogenesis with resistance training is seemingly prioritized over mitochondrial biogenesis (Wilkinson et al., 2008; Figueiredo et al., 2021), while there is evidence to suggest increased mitochondrial biogenesis with endurance training is prioritized over ribosome biogenesis (Morrison et al., 1989; Gibala et al., 2009). In addition, an interference effect may occur if both endurance and resistance exercise are included in the same training session or program (i.e., concurrent training). For example, published reports show that endurance training compromises muscle hypertrophy response to resistance training (Kraemer et al., 1995; Jones et al., 2013) and, although researchers have tried to unveil the mechanisms underlying the interplay between resistance and endurance training, the molecular underpinnings of these observations are still unclear. However, evidence suggests that both processes can occur simultaneously (Tang et al., 2006; Fyfe et al., 2016, 2018). Therefore, the purpose of this review is to discuss whether this curious competition between mitochondrial and ribosome biogenesis exists during different exercise training programs and show the available evidence both in favor and against it.",Frontiers in Physiology,Ribosome and Mitochondrial Biogenesis,2021
AMPK Signaling and Mitochondrial Biogenesis,"AMPK AND mTOR SIGNALING HELP REGULATE MITOCHONDRIAL AND RIBOSOME BIOGENESIS, RESPECTIVELY Two critical signaling proteins that facilitate the adaptive responses to exercise training include the 5' AMP-activated protein kinase (AMPK) and the mechanistic target of rapamycin (mTOR). As an important regulator of cellular energy homeostasis, AMPK is a hetero-trimeric cytosolic enzyme with a catalytic α-subunit and regulatory β and γ subunits. The α-subunit phosphorylates cytoplasmic and nuclear proteins to affect the expression of various mRNAs. High adenosine monophosphate (AMP) concentrations during exercise (as a result of high ATP turnover) lead to increased binding of AMP with AMPK (Richter and Ruderman, 2009), but it has been shown that ADP could also activate AMPK (Oakhill et al., 2011). In addition, glycogen interacts with the β-subunit of AMPK, and muscle glycogen depletion during exercise results in the loss of the interaction between these molecules, which increases AMPK activity (Steinberg et al., 2006). Stress-responsive proteins, such as serine/threonine kinase 11 and calcium/calmodulin-dependent protein kinase 2, can also act to phosphorylate AMPK at the Thr172 residue and increase its activity (Richter and Ruderman, 2009). Evidence in multiple cell lines and tissues suggests that increased AMPK signaling facilitates mitochondrial gene expression to provide for mitochondrial biogenesis (Reznick et al., 2007; Yan et al., 2013; Marin et al., 2017).",Frontiers in Physiology,AMPK–mTOR Signaling,2021
AMPK Activation During Exercise,"In this regard, endurance exercise studies with rodents and humans have shown AMPK signaling and mRNAs involved in mitochondrial biogenesis increase hours following exercise (Fujii et al., 2000; Atherton et al., 2005; Jorgensen et al., 2005). Additionally, researchers have used the muscle-specific double knockout AMPK β1 and β2 mouse model (β1β2M-KO) to demonstrate functional AMPK is critical in maintaining muscle mitochondrial content (O'neill et al., 2011). The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is widely recognized as a regulatory hub for overload-induced skeletal muscle hypertrophy (Goodman, 2019). mTORC1 is a multi-subunit complex that consists of the mTOR protein as well as Raptor and mTOR associated protein LST8 homolog (mLST8; Saxton and Sabatini, 2017). Like AMPK, active mTORC1 complexes possess kinase activity to phosphorylate downstream proteins that facilitate the assembly and initiation of translation-competent ribosomes. Bodine et al. (2001) were the first to demonstrate mTOR signaling was required for muscle hypertrophy. Specifically, the authors administered rapamycin (an mTOR inhibitor) to mice, and observed synergist ablation-induced hypertrophy was completely abrogated in the plantaris muscle.",Frontiers in Physiology,AMPK–mTOR Signaling,2021
mTORC1 Signaling and Ribosome Biogenesis,"Human studies have since shown that phosphorylation of mTOR and its downstream substrates (i.e., p70s6k, 4EBP1) are critically involved in facilitating post-exercise increases in muscle protein synthesis (Drummond et al., 2009; Gundermann et al., 2014). Further, acute increases in mTOR signaling markers following one bout of resistance exercise are associated with muscle hypertrophy following weeks of resistance training (Terzis et al., 2008; Hulmi et al., 2009; Mayhew et al., 2009; Mitchell et al., 2013). Aside from upregulating muscle protein synthesis, more recent evidence suggests mTOR signaling regulates ribosome biogenesis across multiple cell lines [reviewed in (Mayer and Grummt, 2006)]. Notably, Nader et al. (2005) were the first to demonstrate this mechanism occurs in skeletal muscle cells in vitro. Von Walden et al. (2016) later demonstrated that mTOR signaling enhances ribosome biogenesis in skeletal muscle cells in vitro by modifying chromatin at the rDNA promoter. For further information on this topic, readers are encouraged to refer to other excellent reviews (Kim et al., 2019; Von Walden, 2019).",Frontiers in Physiology,AMPK–mTOR Signaling,2021
Evidence for Competition Between Mitochondrial and Ribosome Biogenesis,"WHAT EVIDENCE IS THERE SUGGESTING MITOCHONDRIAL AND RIBOSOME BIOGENESIS MAY COMPETE? Several lines of evidence exist suggesting skeletal muscle mitochondrial and ribosome biogenesis may compete at the molecular level in response to different modes of exercise training. For instance, we have reported that Otsuka Long-Evans Tokushima Fatty rats exposed to 12 weeks of treadmill training demonstrated ~60% lower total RNA per mg wet tissue (a surrogate of skeletal muscle ribosome density) compared to untrained animals (Romero et al., 2017), and data from these same animals showed skeletal muscle citrate synthase activity (a surrogate of mitochondrial volume) was ~16% higher in trained vs. untrained animals (Martin et al., 2012). While we did not assess markers of AMPK activation it is notable that others have shown treadmill running results in acute increases in markers of AMPK activity following exercise (Ruderman et al., 2003). Other rodent studies partially agree with our findings. For instance, Morrison et al. (1989) reported that rats that underwent 2 weeks of treadmill training had greater hindlimb citrate synthase activity (~40%, p<0.05) compared to untrained rats, while 18S rRNA (a surrogate of ribosome density) was similar between groups.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis,2021
Rodent and Human Evidence for Competition,"Hayase and Yokogoshi (1992) reported that rats that underwent 7 days of treadmill exercise had non-significantly lower levels of total RNA/mg protein in the mixed gastrocnemius muscle (−5.6%, p=0.060) and soleus muscle (−4.7%, p=0.111) compared to untrained rats. Regarding human studies, transcriptomic results from the 20-week HERITAGE cardiovascular training study indicated that certain ribosomal mRNAs in the vastus lateralis were downregulated from pre- to post-training (Teran-Garcia et al., 2005). Additionally, Wilkinson et al. (2008) used a 10-week unilateral leg training protocol to demonstrate the differential molecular adaptations to resistance vs. endurance training. Specifically, 10 healthy men with minimal training >8 months prior to the initiation of the study trained one leg using the knee extensor exercise (2–3 days per week) and the other leg using a cycle ergometer (2–3 days per week). The authors reported that basal myofibrillar protein synthesis rates increased from pre- to post-intervention within the resistance-trained leg only (~0.08%/h POST vs. ~0.06%/h PRE). Myofibrillar protein synthesis rates were also greater at the 10-week time point in the resistance vs. endurance-trained leg.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis,2021
Human Studies Supporting Competition Theory,"While markers of ribosome biogenesis were not assessed, these data suggest resistance training may have increased ribosome density via biogenesis given the ~30% increase in basal myofibrillar protein synthesis rates. These data additionally suggest ribosome biogenesis was likely unaffected with endurance training. A recent study conducted by Figueiredo et al. (2021) supports the competition between mitochondrial and ribosome biogenesis theory. The authors investigated the genetic and epigenetic regulation of ribosome biogenesis with either endurance or resistance exercise and found that markers of ribosome biogenesis were increased with resistance exercise but decreased with endurance exercise (30 min post-exercise). In addition, the authors reported that, in general, resistance exercise activated the mTOR pathway while endurance exercise activated the AMPK pathway. Collectively, these studies suggest endurance training does not alter ribosome biogenesis or may interfere with certain aspects of the process. However, more human endurance training studies are needed before definitive conclusions can be drawn.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis,2021
Resistance Training Increasing Ribosome Density,"Despite sparse evidence linking endurance training to unaltered or decreased ribosome biogenesis, several human studies have shown that resistance training increases ribosome density (as measured by total RNA per mg tissue; Kadi et al., 2004; Figueiredo et al., 2015; Stec et al., 2016; Brook et al., 2017; Reidy et al., 2017; Mobley et al., 2018; Hammarstrom et al., 2020). Separate reports have also shown that resistance training does not alter or decreases mitochondrial volume (as measured by citrate synthase activity assays or transmission electron microscopy; Macdougall et al., 1982; Luthi et al., 1986; Tesch et al., 1987; Parise et al., 2005; Porter et al., 2015). It is uncommon for the same study to report both variables. However, two human studies from our laboratory have examined changes in markers of skeletal muscle ribosome density and mitochondrial volume in response to resistance training. In one study, untrained young men participated in 12 weeks (3 days per week) of full-body resistance training (Roberts et al., 2018b), and following training, total RNA per mg tissue increased by 23% (p<0.05), while vastus lateralis citrate synthase activity non-significantly decreased by 11% (p=0.064).",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis,2021
Ribosome Biogenesis Increases While Mitochondria Lag,"Similar to these findings, we reported 6 weeks of unaccustomed high volume resistance training in previously-trained young men increased vastus lateralis total RNA per mg tissue by 28% (p<0.05). In contrast, vastus lateralis citrate synthase activity decreased by 12% (p<0.05; Haun et al., 2019). Critically, both studies suggest ribosome biogenesis occurred with unaccustomed resistance training, whereas mitochondrial biogenesis either did not occur or was delayed relative to increases in myofiber hypertrophy. In addition, Hanson et al. (2019) found that performing a bout of endurance exercise before resistance exercise led to an acute decrease in markers of ribosome biogenesis compared to resistance exercise alone. However, it is important to note that markers of ribosome biogenesis were restored 3 h post-exercise. To summarize, several human studies suggest that unaccustomed resistance training increases ribosome density (likely through increased ribosome biogenesis), whereas mitochondrial density remains constant or decreases. Whether decrements in citrate synthase activity resulted from “mitochondrial dilution” via skeletal muscle hypertrophy rather than a decrease in mitochondrial biogenesis is debatable.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis,2021
Limitations and Need for Further Research,"Notably, most studies only used measures of ribosome and/or mitochondrial content or other indirect measures of biogenesis and their results should be interpreted with caution. Given the overall lack of data in this area, more research is needed to interpret the relevance of these findings.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis,2021
Mechanistic Basis for Competition: AMPK vs mTORC1,"WHY WOULD MITOCHONDRIAL AND RIBOSOME BIOGENESIS COMPETE WITH ONE ANOTHER IN RESPONSE TO EXERCISE TRAINING? Ample molecular evidence exists to explain why mitochondrial and ribosome biogenesis may compete with one another during periods of exercise training. First, AMPK mechanistically blocks mTORC1 signaling through direct phosphorylation of the complex (Shaw, 2009) as well as through the phosphorylation and activation of the hamartin-tuberin (TSC1/2) complex [reviewed in (Shaw, 2009)], which is an upstream inhibitor of mTORC1 signaling. Given the proposed role mTORC1 signaling has on skeletal muscle ribosome biogenesis, it seems plausible that this process is impaired during situations of heightened AMPK signaling. In support of this hypothesis, we have reported that treating C2C12-derived myotubes with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, a stimulator of AMPK activity) for 6 hours reduced 47S pre-rRNA levels by 16% compared to vehicle-treated cells (Mobley et al., 2016). Researchers have also reported similar phenomena in other cell lines. For instance, several AMPK activators (e.g., phenformin, resveratrol, and AICAR) have been shown to disrupt nucleolar organization and inhibit ribosomal RNA synthesis in LLC-PK1 kidney proximal tubule epithelial cells (Kodiha et al., 2014).",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis Competition,2021
AMPK Suppression of Ribosome Biogenesis,"In HEK293T cells, glucose deprivation-induced AMPK activation has been reported to lead to increased phosphorylation of the RNA polymerase I-associated transcription factor TIF-IA at Ser635 (Hoppe et al., 2009). This phosphorylation event reduced the interaction of TIF-IA with other transcription factors and ultimately reduced the assembly of functional transcription initiation complexes at the rDNA promoter. Others have also shown a reduction in ribosome biogenesis in COS7 and HEK293 cells and transgenic mice overexpressing γ2-AMPK (Cao et al., 2017). Thus, it is apparent that a conserved outcome of AMPK activation in several cell types involves inhibition of ribosome biogenesis. Evidence also exists suggesting mTORC1 signaling may reduce certain aspects of mitochondrial biogenesis. For instance, Deepa et al. (2013) reported mRNAs involved with mitochondrial biogenesis (i.e., Ppargc1a, Nrf1, and Esrra) increased in the white adipose tissue of female db/db mice administered rapamycin (an mTOR inhibitor) for 6 months. Chiao et al. (2016) reported that cardiac muscle mitochondrial biogenesis markers (i.e., PPARGC1A and TFAM protein levels) increased during the first 2 weeks of a 10-week rapamycin feeding experiment in mice.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis Competition,2021
mTORC1 Effects on Mitochondria and Autophagy,"There are also data showing mTORC1 signaling may disrupt autophagy, which in turn may affect mitochondrial remodeling (Choi et al., 2012). This is relevant to the competition paradigm given that autophagy is critical for mitochondrial remodeling and function in skeletal muscle cells in vitro (Sin et al., 2016). Furthermore, Johnson et al. (2014) presented evidence of reciprocal regulation of protein synthesis in the cytosol and the mitochondria of human embryonic kidney cells. The authors found that amino acid starvation led to an inhibition of mTORC1 and a decrease in cytosolic protein synthesis, whereas there was an increase in active AMPK, mitochondria density (i.e., increased citrate synthase activity), mitochondrial translation and function. Collectively, several lines of evidence support the notion that AMPK activation impairs ribosome biogenesis, and some evidence suggests that mTORC1 signaling may negatively affect certain aspects of mitochondrial biogenesis. However, the latter data are not as conclusive.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis Competition,2021
Inverse rDNA-mtDNA Relationships,"Aside from the aforementioned AMPK and mTORC1 data, sequencing data from human blood cells show ribosomal DNA (rDNA) and mitochondrial copy number (or “dose”), both of which can vary between individuals, are inversely correlated between one another (Gibbons et al., 2014). In explaining these findings, the authors suggested a tight regulatory relationship exists between rDNA abundance, the mRNA expression of ribosomal proteins, and mitochondrial DNA (mtDNA) abundance. While these data are provocative in making the case for ribosome and mitochondria competition, determining whether this relationship exists in skeletal muscle remains unknown.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis Competition,2021
Energetic Constraints on Dual Biogenesis,"Finally, ribosome biogenesis and mitochondrial biogenesis require cellular energy that is greater than metabolic homeostasis. In general, transcription and translation are ATP-consuming processes (Lynch and Marinov, 2015). The 80S ribosome contains 79 proteins and four rRNAs, and there are ~1,500 mitochondrial proteins (Boengler et al., 2011). Thus, the transcription of these components requires ATP, and the translation of mRNAs into protein requires additional ATP. It has also been suggested that ribosome assembly in eukaryotes is an energy-consuming process given that the nuclear export and assembly of the ribosome subunits involves various nucleotide-hydrolyzing enzymes (Strunk and Karbstein, 2009). Rodent studies have reported that muscle ribosomes and mitochondria exhibit rapid decay rates in response to unloading schemes (Steffen and Musacchia, 1984; Wagatsuma et al., 2011). These findings also support the notion that maintaining ribosome and mitochondrial densities are an energetic burden to muscle cells. Therefore, aside from the aforementioned mechanisms, which may contribute to the competition between ribosome and mitochondrial biogenesis, these latter points call into question as to whether or not muscle cells have the “energy bandwidth” to simultaneously promote both processes.",Frontiers in Physiology,Mitochondrial vs Ribosome Biogenesis Competition,2021
Evidence Against Biogenesis Competition: Simultaneous Adaptations,"WHAT EVIDENCE IS THERE SUGGESTING MITOCHONDRIAL AND RIBOSOME BIOGENESIS DO NOT COMPETE? To this point, we have provided evidence in favor of the biogenesis competition paradigm, in which ribosome biogenesis is prioritized with resistance training while mitochondrial biogenesis is prioritized with endurance training, or an interference effect is observed when both modes of exercise are performed concurrently. However, there is also evidence available suggesting that both processes can occur simultaneously. Tang et al. (2006), for example, reported increased muscle fiber hypertrophy and mitochondrial density (i.e., citrate synthase activity) in young males after 12 weeks of resistance training, although markers of ribosomal or mitochondrial biogenesis were not assessed. Our laboratory has also reported increased citrate synthase activity after resistance training in a cohort of older participants, concomitantly with an increase in hypertrophy (Lamb et al., 2020). The same cohort of participants showed an increase in protein content of the mitochondrial electron transport chain complexes and markers of mitochondrial remodeling (Mesquita et al., 2020). However, there was no change in PGC-1a and TFAM protein content, and the activation of these signaling pathways were not interrogated.",Frontiers in Physiology,Biogenesis Non-Competition,2021
Direct Tracer Evidence for Dual Biogenesis,"Notably, studies using tracer methodology to directly measure ribosome and mitochondrial biogenesis have shown that resistance training is capable of increasing both ribosome (Sieljacks et al., 2019) and mitochondrial biogenesis (Groennebaek et al., 2018). Regarding changes in the signaling pathway involved in mitochondrial adaptations, resistance training increased ACC (Ser79) and p38-MAPK phosphorylation, but AMPK phosphorylation remained unchanged (Groennebaek et al., 2018). Importantly, the authors reported no significant correlation between mitochondrial protein synthesis and changes in citrate synthase activity. Similarly, even though total RNA content also increased in Sieljacks et al. (2019) study, the authors found no significant difference between total RNA content and RNA synthesis rate. The results of both studies suggest that resistance exercise can lead to both ribosome and mitochondrial biogenesis but reinforce the need to be careful when using measures of organelle content (i.e., citrate synthase activity and total RNA content) as an indicative of biogenesis. In addition, concurrent training, which involves simultaneously engaging in resistance and endurance training, is a prime candidate for increasing ribosome and mitochondrial biogenesis.",Frontiers in Physiology,Biogenesis Non-Competition,2021
Concurrent Training Showing Dual Adaptations,"A landmark study by Hickson (1980) showed that concurrent training interfered with strength and hypertrophy adaptations when compared with resistance training alone. However, a comprehensive review by Fyfe et al. (2014) challenges the notion as to whether concurrent training interferes with resistance training adaptations. Moreover, a series of meta-analyses (Denadai et al., 2017; Murlasits et al., 2018; Sabag et al., 2018) suggest the interference effect elicited through endurance training is contextual and depends on factors such as endurance training modality (e.g., run training vs. cycle training) as well as endurance training frequency and duration. A number of other variables can be manipulated and potentially affect the outcome, including the training timing, which mode of training is done first, the time between the two bouts, and whether nutritional support is given between bouts. Furthermore, studies show that concurrent training increases maximal aerobic capacity as well as strength and hypertrophy (Mccarthy et al., 1995; Balabinis et al., 2003; Sillanpaa et al., 2008; Lundberg et al., 2014). These studies did not determine if phenotypic changes coincided with increased mitochondrial and ribosome biogenesis.",Frontiers in Physiology,Biogenesis Non-Competition,2021
Studies Showing Dual Activation of AMPK and mTOR,"However, Fyfe and colleagues have published two reports suggesting concurrent resistance training and high-intensity interval training may increase both processes. The first study (Fyfe et al., 2016) showed that compared with resistance exercise only, high-intensity interval training and resistance exercise enhanced ACC phosphorylation (Ser79; a readout of AMPK activity), PPARGC1A mRNA expression (suggestive of increased mitochondrial biogenesis), and mTOR phosphorylation [Ser2448; which may indicate enhanced mTOR activity, although this has been debated (Figueiredo et al., 2017)]. The second study by Fyfe et al. (2018) involved three groups of participants who undertook resistance training only, high-intensity interval training + resistance training, or moderate-intensity continuous training + resistance training for 8 weeks. Following the training intervention, basal 45S pre-rRNA, 28S rRNA, and 5.8S rRNA expression were greater in the two groups that incorporated high-intensity interval training or moderate-intensity continuous training vs. resistance training alone. Total RNA per mg tissue also increased in the high-intensity interval training + resistance training, or moderate-intensity continuous training + resistance groups by ~20–30%, albeit these increases were not statistically significant.",Frontiers in Physiology,Biogenesis Non-Competition,2021
Low-Load and Blood-Flow Restricted Training,"Lundberg et al. (2014) have also reported that 5 weeks of concurrent training increases quadriceps hypertrophy (+6%), endurance performance (+22%), and muscle citrate synthase activity (+18%). Moreover, the order of exercise (resistance exercise followed by endurance exercise or the opposite) is an important variable in concurrent studies. Wang et al. (2011) showed that performing a bout of resistance exercise after endurance exercise enhanced the signaling cascade for mitochondrial biogenesis. The authors found a concomitant activation of AMPK and mTOR and an increased expression of PGC-1a and PGC-1-related coactivator (PRC). However, markers of ribosome biogenesis were not examined, making it difficult to determine if mitochondrial and ribosome biogenesis coincided. Apro et al. (2013), on the other hand, investigated the effects of performing endurance exercise after resistance exercise on mTORC1 and AMPK signaling pathways. Activation of the mTORC1 by resistance exercise was not impaired by subsequent concurrent endurance exercise. However, the authors found that phosphorylation of AMPK was decreased 3 h after both resistance exercise-only and concurrent exercise, suggesting that prior activation of mTORC may suppress AMPK activation.",Frontiers in Physiology,Biogenesis Non-Competition,2021
Low-Load High-Volume and BFR Training Evidence,"Beyond concurrent training, it is possible that other types of training, such as low-load blood flow restricted or low-load/high-volume resistance training to failure may simultaneously enhance mitochondrial and ribosome biogenesis. The studies of Groennebaek et al. and Sieljacks et al. cited previously found that low-load blood flow restricted resistance training increased both mitochondrial (Groennebaek et al., 2018) and ribosome biogenesis (Sieljacks et al., 2019), with no difference when compared to a high-load resistance training. Furthermore, low-load/high-volume resistance training paradigm can assume several forms, but the most studied paradigm involves participants performing sets at 30% 1RM to failure (30FAIL; Mitchell et al., 2012; Jenkins et al., 2016, 2017; Morton et al., 2016, 2019; Haun et al., 2017). Lim et al. (2019) recently published a study which compared three groups of participants who trained for 10 weeks (3 days/week) with either 80FAIL, 30FAIL, or 30% 1RM loads, which were volume-matched to the 80FAIL group. While the authors did not report significant changes in mitochondrial volume markers (i.e., cytochrome C and COX IV protein levels), robust alterations in these markers occurred in the 30FAIL group. Markers of mitochondrial remodeling (i.e., PARKIN, OPA1, and FIS1 protein levels) also increased only in the 30FAIL group. Indeed, this evidence suggests mitochondrial biogenesis may have increased in the 30FAIL group, albeit markers of ribosome biogenesis were not assessed. Nonetheless, muscle hypertrophy did occur in the 30FAIL group.",Frontiers in Physiology,Biogenesis Non-Competition,2021
Limitations of the Competition Paradigm,"OTHER CONSIDERATIONS TO THE COMPETITION PARADIGM A major limitation to the biogenesis competition paradigm is that AMPK and mTORC1 is primarily responsible for said competition. If this is indeed the case, then the paradigm would likely have to operate through an AMPK-mTORC1 signaling “switch” in response to each form of training. This switch has been proposed to occur in the skeletal muscle of rats following prolonged low-frequency stimulation vs. short bursts of high-frequency stimulation (Atherton et al., 2005). However, the acute post-exercise time course data regarding AMPK and mTORC1 activity in humans are more nuanced. For instance, Dreyer et al. (2006) reported that one bout of unaccustomed resistance exercise concomitantly increases AMPK activity and mTORC1 signaling markers 2 hours post-exercise. Likewise, Mascher et al. (2011) reported that a 60-min cycling bout concomitantly increases AMPK activity and mTORC1 signaling markers 2 hours post-exercise. The study by Wilkinson et al. (2008) similarly demonstrated that a bout of unilateral resistance and endurance training increased the phosphorylation of AMPK (Thr172) immediately following exercise.",Frontiers in Physiology,Biogenesis Competition Paradigm,2021
Training Status and Generic vs Specific Signaling,"According to the competition paradigm, these findings suggest that resistance and endurance exercise should initiate mitochondrial and ribosomal biogenesis. Furthermore, Coffey et al. (2006) explored the effects of training status and accustomization to different exercise training modes (resistance vs. endurance exercise) and their data illustrate the complexity of the signaling AMPK and mTORC pathways response to exercise. The authors had a group of endurance trained and a group of resistance trained individuals perform one bout of endurance exercise and one bout of resistance exercise on different sessions. Their results suggest that untrained individuals might present a more generic response to exercise, with increases in both signaling pathways with either endurance or resistance exercise. However, as one becomes more accustomed to an exercise mode through training, the signaling responses to exercise seem to be attenuated. Moreover, AMPK signaling seems to be less specific, being activated with both endurance and resistance exercises, while mTORC is preferentially activated in response to resistance exercise (Vissing et al., 2013).",Frontiers in Physiology,Biogenesis Competition Paradigm,2021
Timing of Biopsies and Temporal Dynamics,"In addition, considering that the response to exercise in untrained subjects seems to be fairly generic, performing concurrent training instead of resistance-only or endurance-only exercise could have an additive instead of an interference effect. This is supported by the work of Wang et al. (2011), which showed that performing resistance exercise after cycling enhanced markers of mitochondrial adaptations compared to cycling-only. However, this effect is likely dependent on a myriad of other factors, such as interval between exercise bouts and the volume of each differentiated exercise mode. The timing of skeletal muscle biopsies and therefore of the measurements of AMPK/mTORC1 activation is commonly referred as a limitation and a possible source of inconsistencies found between different studies (Gibala et al., 2009; Figueiredo et al., 2015; Stec et al., 2015). There is evidence to suggest that even though both mTORC1 and AMPK can be activated in response to resistance exercise, mTORC1 is activated once AMPK signaling subsides (Vissing et al., 2013). Similar findings have been reported with an ex-vivo endurance exercise model (Jakobsgaard et al., 2021).",Frontiers in Physiology,Biogenesis Competition Paradigm,2021
Temporal Separation of AMPK and mTORC1 Activation,"In addition, Vissing et al. (2013) results showed that mTORC1 peak activation was at 5 h post-exercise and remained upregulated until 22 h post-exercise. Therefore, if studies do not collect muscle tissue in several time-points (e.g., only perform biopsies 1 h and/or 3 h post-exercise), this important information about the signaling response to exercise could be missed. However, it is important to note that as previously mentioned, other studies have shown that AMPK and mTORC1 can be concomitantly activated (Wang et al., 2011; Fyfe et al., 2016). Data from both animal and in vitro models also challenge the AMPK-mTORC1 switch theory. Drake et al. (2013) demonstrated a null effect of mTOR inhibition on mitochondrial biogenesis markers in mice fed a rapamycin-supplemented diet for 12 weeks. There are also in vitro data suggesting mTOR signaling enhances mitochondrial biogenesis (Morita et al., 2013). Likewise, a review by Morita et al. (2015) provides several lines of evidence to suggest mTORC1 enhances mitochondrial function through the increased translation of transcription factors that regulate the expression of nuclear-encoded mitochondrial genes.",Frontiers in Physiology,Biogenesis Competition Paradigm,2021
mTORC1/2 and Mitochondrial Regulation,"An elegant study performed by Cunningham et al. (2007) demonstrated through a series of experiments that mTOR is necessary for proper mitochondrial oxidative function and biogenesis. The authors found that inhibition of mTOR by rapamycin decreased the expression of important mitochondrial transcription factors, gene targets of PGC-1α, and mitochondrial respiration in C2C12 myotubes. In addition, mice exposed to the same treatment also experienced similar effects. The authors proceeded with additional experiments to show that mTOR-dependent regulation of mitochondrial biogenesis and function is achieved through direct modulation of YY1-PGC-1α. Furthermore, it is notable that mTOR complex 2 (mTORC2) is also involved with mitochondrial physiology. The differences between the mTORC1 and mTORC2 complexes are subtle; specifically, mTORC2 contains the mTOR, Rictor, LST8 and SIN1 proteins (Loewith et al., 2002). Whereas mTORC1 functions as a nutrient/amino acid sensing complex, mTORC2 receives intracellular signals from extracellular growth factor binding (Jhanwar-Uniyal et al., 2019).",Frontiers in Physiology,Biogenesis Competition Paradigm,2021
"mTORC2, Exercise, and Mitochondrial Biogenesis","Interestingly, data suggest that mTORC2 stimulates mitochondrial biogenesis in liver (Betz et al., 2013) and myeloid dendritic cells (Watson et al., 2019), although equivocal data exist in macrophages from Rictor-knockout mice (Oh et al., 2017). Studies examining mTORC2 activity responses to exercise bouts or training are sparse relative to studies examining mTORC1 responses. However, evidence suggests that mTORC2 activity increases in response to an endurance bout in rodents (Kleinert et al., 2017). In contrast, skeletal muscle mTORC2 activity seems unresponsive to a bout of resistance training in humans based upon the localization of the complex not being altered following a bout of resistance training (Hodson et al., 2017). These data add to the proposed competition paradigm in that mTOR may be involved in both biogenesis processes depending upon whether mTORC1 or mTORC2 is stimulated. However, again, more studies are needed before definitive conclusions can be drawn.",Frontiers in Physiology,Biogenesis Competition Paradigm,2021
Energetic Demands and Coordination of Biogenesis,"Additionally, we have previously made the case that both ribosome and mitochondrial biogenesis are metabolically demanding processes and that maintaining a high density of both organelles would place an energetic demand on the cells. However, as mitochondria are the main energy-producing organelles in the cell, it could also be argued that it is counterintuitive to decrease its density when the cell is facing an increased energy demand, such as during increased ribosome biogenesis and cytosolic protein synthesis after resistance exercise. Moreover, several proteins needed for mitochondrial biogenesis are encoded in nuclear DNA and synthesized by cytosolic ribosomes before they can be imported into the mitochondria (Jornayvaz and Shulman, 2010; Perry and Hawley, 2018). Again, it would be counterintuitive to decrease ribosome density when there is an increased demand for nuclear-encoded proteins needed for mitochondrial biogenesis. Therefore, ribosome and mitochondrial biogenesis would be expected to be closely related processes.",Frontiers in Physiology,Biogenesis Competition Paradigm,2021
"Aging, Physical Activity, and Disease Prevention","Aging, a universal and inevitable process, is characterized by a progressive accumulation of physiological alterations and functional decline over time, leading to increased vulnerability to diseases and ultimately mortality as age advances. Lifestyle factors, notably physical activity (PA) and exercise, significantly modulate aging phenotypes. Physical activity and exercise can prevent or ameliorate lifestyle-related diseases, extend health span, enhance physical function, and reduce the burden of non-communicable chronic diseases including cardiometabolic disease, cancer, musculoskeletal and neurological conditions, and chronic respiratory diseases as well as premature mortality. Physical activity influences the cellular and molecular drivers of biological aging, slowing aging rates—a foundational aspect of geroscience. Thus, PA serves both as preventive medicine and therapeutic agent in pathological states. Sub-optimal PA levels correlate with increased disease prevalence in aging populations.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
Need for Structured and Personalized Exercise Prescriptions,"Structured exercise prescriptions should therefore be customized and monitored like any other medical treatment, considering the dose-response relationships and specific adaptations necessary for intended outcomes. Current guidelines recommend a multifaceted exercise regimen that includes aerobic, resistance, balance, and flexibility training through structured and incidental (integrated lifestyle) activities. Tailored exercise programs have proven effective in helping older adults maintain their functional capacities, extending their health span, and enhancing their quality of life. Particularly important are anabolic exercises, such as Progressive resistance training (PRT), which are indispensable for maintaining or improving functional capacity in older adults, particularly those with frailty, sarcopenia or osteoporosis, or those hospitalized or in residential aged care.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
Multicomponent Training and Individual Variability,"Multicomponent exercise interventions that include cognitive tasks significantly enhance the hallmarks of frailty (low body mass, strength, mobility, PA level, and energy) and cognitive function, thus preventing falls and optimizing functional capacity during aging. Importantly, PA/exercise displays dose-response characteristics and varies between individuals, necessitating personalized modalities tailored to specific medical conditions. Precision in exercise prescriptions remains a significant area of further research, given the global impact of aging and broad effects of PA. Economic analyses underscore the cost benefits of exercise programs, justifying broader integration into health care for older adults. However, despite these benefits, exercise is far from fully integrated into medical practice for older people.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
Barriers in Medical Integration and Polypharmacy,"Many healthcare professionals, including geriatricians, need more training to incorporate exercise directly into patient care, whether in settings including hospitals, outpatient clinics, or residential care. Education about the use of exercise as isolated or adjunctive treatment for geriatric syndromes and chronic diseases would do much to ease the problems of polypharmacy and widespread prescription of potentially inappropriate medications. This intersection of prescriptive practices and PA/exercise offers a promising approach to enhance the well-being of older adults. An integrated strategy that combines exercise prescriptions with pharmacotherapy would optimize the vitality and functional independence of older people whilst minimizing adverse drug reactions.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
Consensus Purpose and Scope,"This consensus provides the rationale for the integration of PA into health promotion, disease prevention, and management strategies for older adults. Guidelines are included for specific modalities and dosages of exercise with proven efficacy in randomized controlled trials. Descriptions of the beneficial physiological changes, attenuation of aging phenotypes, and role of exercise in chronic disease and disability management in older adults are provided. The use of exercise in cardiometabolic disease, cancer, musculoskeletal conditions, frailty, sarcopenia, and neuropsychological health is emphasized. Recommendations to bridge existing knowledge and implementation gaps and fully integrate PA into the mainstream of geriatric care are provided. Particular attention is paid to the need for personalized medicine as it applies to exercise and geroscience, given the inter-individual variability in adaptation to exercise demonstrated in older adult cohorts.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
Foundational Contribution to Exercise as Medicine,"Overall, this consensus provides a foundation for applying and extending the current knowledge base of exercise as medicine for an aging population to optimize health span and quality of life.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
Global Aging Demographics and Physiological Decline,"1. Optimizing health in an aging population: Leveraging physical activity and exercise The global population is rapidly aging, with projections indicating that the number of adults ≥65 years will double to 1.5 billion by 2050. At the same time, those ≥80 are expected to triple between 2019 and 2050, reaching 426 million [1], with 80% living in low- and middle-income countries. This demographic shift impacts multiple sectors, including healthcare, social quality of life, retirement planning, and caregiving. More critically, it brings an increased burden of non-communicable diseases and disabilities. Aging is an inevitable, universal process marked by a progressive decline in several physiological functions, although the rate and extent of this decline is highly variable. Human physiological systems exhibit varying peak development timelines: bone mass peaks around age 20, muscle mass often remains stable until approximately age 50, while cognitive functions such as crystallized intelligence, wisdom, and emotional intelligence can continue to develop and improve well into advanced age [2]. Biological aging begins at different stages for different systems and progresses over subsequent decades.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
Aging Phenotype and Hallmarks of Aging,"Aging is typically defined as the progressive accumulation of physiological changes and functional decline over time, leading to increased vulnerability to diseases and mortality [3]. This raises an important question: Do health outcomes during the aging process correlate with inherent physiological capabilities? The “aging phenotype” encompasses the diverse array of observable traits, behaviors, and physiological alterations that lead to a gradual decline in organismal functions and an increased susceptibility to age-related diseases [4]. It is characterized by morphological changes such as wrinkling and greying of hair, functional declines in various organ systems, cognitive modifications, and increased susceptibility to chronic diseases. Current research into the aging phenotype focuses on delineating biological mechanisms underlying these alterations and developing interventions to delay detrimental effects of aging. Researchers have identified twelve interconnected aging hallmarks — genomic instability, telomere shortening, epigenetic alterations, mitochondrial dysfunction, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis — that intensify with age [7].","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
"Disuse vs Biological Aging, Resilience, and Human Potential","Experimentally targeting these hallmarks offers possibilities for therapeutic interventions that can slow, halt, or even reverse some aspects of biological aging [7]. However, it is critical to evaluate the practicality of targeting these hallmarks and the extent to which they reflect true biological aging versus lifestyle-driven decline [8,9]. Additionally, maintaining or enhancing functional capacity and increasing years lived without disabilities remain central considerations for extending health span. The overlap of features between disuse and biological aging has long been recognized [10], highlighting the need to differentiate between the two. As Prof. Walter Bortz II stated, “The mission of medicine is the assertion and the assurance of the human potential” [11]. Achieving this involves promoting resilience — the capacity to withstand adversity and maintain stability amidst physical, psychological, and social challenges. Resilience is not synonymous with the absence of frailty, just as happiness is not simply the absence of depression but the attainment of ‘eudaimonia’, the Aristotelian concept of flourishing.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
"Role of Physical Activity, Exercise Definitions, and Sedentary Behavior","Regular exercise is widely acknowledged for mitigating cognitive and physical decline and the psychological and social challenges linked with aging [10]. Research suggests that many declines are more likely due to inactivity rather than inevitable biological deterioration. Lifestyle factors, particularly physical activity (PA) and exercise, substantially influence aging phenotypes [13,14]. Tailored exercise programs effectively help older adults maintain functional capacity, extend health span, and improve quality of life [15]. Physical activity is defined as any bodily movement produced by skeletal muscles that increases energy expenditure [16]. Exercise is a structured, planned subset of PA aimed at improving fitness components such as aerobic capacity, strength, endurance, balance, coordination, or flexibility [17]. The metabolic equivalent (MET) standardizes activity intensity, where one MET represents resting energy expenditure. Sedentary behavior involves ≤1.5 MET activities performed while sitting or reclining. A sedentary lifestyle combines low levels of moderate-to-vigorous PA with prolonged sedentary behaviors.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
"Physical Inactivity, WHO Guidelines, and Need for Implementation","""Insufficient PA"" describes individuals who do not meet the WHO guidelines of 150–300 minutes per week of moderate-to-vigorous intensity PA or country-specific equivalents [20]. Promoting healthy and dignified aging involves supporting healthcare systems in implementing evidence-based exercise programs for older adults across all settings. Given rapid advancements in exercise science, this consensus focuses on changes in functional capacity, fitness, body composition, quality of life, and disease burden rather than the entirety of aging. Understanding PA patterns is essential for optimizing aging, addressing concerns of clinicians and caregivers, and improving outcomes in older individuals. This paper advocates using PA/exercise to enhance health, prevent disease, and manage conditions in older adults. It presents findings from the latest randomized controlled trials demonstrating the benefits of specific PA/exercise modalities on physiological aging, disease prevention, and treatment of chronic conditions. Updated evidence-based recommendations address gaps in prior guidelines and support practical clinical implementation.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
1.1 Distinctive Phenotypes of Aging – Exercise Spectrum and Resilience,"1.1. Distinctive phenotypes of aging Engaging in regular exercise, maintaining social connections, keeping the mind active, and consuming a healthful diet while avoiding harmful substances and toxic environmental exposures are essential for promoting good health and well-being at all life stages [8]. Exercise is a preventive and therapeutic measure, synergistically enhancing the benefits of other lifestyle factors [13,14]. At one end of the PA/exercise spectrum are lifelong exercisers and competitive master athletes. Despite a decline in competitive performance, as evidenced by diminishing world records, the absolute levels of physical performance in these individuals are impressive compared to their physically inactive age-matched peers [21,22]. These athletes exemplify how exercise (and likely genotype) can preserve physiological function and enhance human health and functional ability in old age. Thus, PA is fundamental to achieving and maintaining a state of resilience and ensuring full human potential that is age appropriate, despite the social, physical, and psychological challenges of aging [8]. On the other end of the spectrum, with suboptimal levels of PA, aging is more likely to be accompanied by cardiovascular disease, cancer, type 2 diabetes (T2D), obesity, diminished muscular function, Alzheimer's disease and related disorders, depression, frailty, and increased end-of-life morbidity.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
1.1 Distinctive Phenotypes of Aging – Impact of Lifestyle and Disease Risk,"While PA is generally beneficial, even among the small proportion of individuals who maintain a high level of PA throughout life, some will develop cardiometabolic, musculoskeletal, neurological, or other diseases with age. Overall, the choice, or ability, to engage in PA significantly impacts the trajectory of decline with aging, with PA optimizing physiology and reducing the risk of many chronic diseases, particularly if social determinants of health (economic, cultural, geographic) are favorable. Conversely, a sedentary lifestyle increases susceptibility to these same disorders (Fig. 1).","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
"1.2 Lifestyle, Genetics, and Environment – Determinants of Healthy Aging","1.2. Lifestyle, genetics, and environment: Interacting determinants of healthy aging Biological aging and chronic diseases share a bidirectional relationship. Chronic conditions, including geriatric syndromes, can exacerbate age-related decline. Additionally, certain treatments for these conditions may contribute to further health deterioration, especially in older adults. Notably, conditions such as frailty, sarcopenia, and dementia accelerate the onset of age-related disability, posing significant health risks [23]. The World Health Organization (WHO)'s 2015 World Report on Aging and Health defined healthy aging as developing and maintaining functional abilities [24]. Achieving an optimal aging trajectory involves considering an individual’s intrinsic characteristics, behaviors, and ecological influences. While homeostatic capacities providing resilience to stressors may diminish with age [25], these factors can be optimized to sustain a person's functional ability and intrinsic capacity throughout their life, especially in older age (Fig. 1).","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
"1.2 Determinants of Healthy Aging – Epigenetics, Environment, Intrinsic Capacity","The primary determinants of health and longevity—genetics, environment, and behavior—interact and influence the manifestations of aging, chronic diseases, and multi-morbidities. This interaction is often evidenced by epigenetic changes to the genome, which can result from lifestyle choices or exposure to maternal or fetal stress/toxins/metabolic abnormalities in utero [26,27]. Insufficient PA and a sedentary lifestyle are major public health issues, together with other modifiable risk factors that have significant beneficial effects across the life course. In this context, the WHO has published the World Report on Aging and Health [24], which describes functional autonomy as the interaction between an individual's physical and mental capacity (intrinsic capacity domains) and the context of each individual's life (environment) [28]. Accordingly, healthy aging depends on intrinsic capacity, socio-economic status, physical environment, as well as the interactions between these factors [29]. The WHO has proposed five domains—locomotion, vitality, cognition, psychological, and sensory—that can be used to evaluate an individual's intrinsic capacity.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
"1.2 PA, Intrinsic Capacity, and Exercise Prescription as Medicine","The WHO recommends actively addressing these issues to promote healthy aging [30]. Although the specific mechanisms and optimal modalities of PA's health benefits are not fully understood, evidence to date suggests that engaging in PA can substantially enhance domains of intrinsic capacity [31]. This, in turn, reduces mortality, promotes functional ability, and healthy aging [32,33]. However, not all PA is equally effective, as dose-response relationships and exercise modality-specific adaptations exist. There are associations between the intensity, volume, and modality of exercise and many health outcomes [34]. For example, for over three decades [17,35,36], high-intensity progressive resistance training (PRT) has been recognized as a safe and effective means to combat frailty. In contrast, simple stretching exercises do not offer therapeutic benefits for this condition. Therefore, exercise used to promote healthy aging must be prescribed in the same evidence-based manner as any other medicine.","Journal of Nutrition, Health and Aging",Exercise & Longevity (ICFSR Consensus),2025
1.4 Personalizing exercise – Part 1,"1.4. Personalizing exercise: understanding the sequencing of the prescription, dose-response and interindividual variability. The data reviewed above on PA and mortality are primarily drawn from large epidemiological studies. Implementing these findings into guidelines to improve health outcomes for individuals in clinical practice is the next step. Personalizing exercise prescriptions is critical in clinical care, especially for older adults, in whom adapting exercise modalities to the unique physiological changes that come with aging is important. As people age, their ability to perform vigorous aerobic exercise decreases significantly, while simultaneously they exhibit declines in muscle mass and function, making anabolic or PRT increasingly necessary. This shift in exercise strategy is not just a matter of preference but a vital adaptation to diminished aerobic capacity and the growing need to preserve muscle mass and strength. Clinical observations indicate that in older adults with advanced frailty and multiple comorbidities resulting in mobility and functional impairment, it is often difficult or impossible to implement robust, moderate, or high-intensity aerobic exercise. However, such individuals remain very capable of undertaking high intensity PRT, even in the tenth decade of life [35,84]. This shift highlights the urgent need for healthcare providers to recalibrate exercise recommendations, tailoring them to meet the specific capabilities and needs of the aging population.","Journal of Nutrition, Health and Aging",Exercise Prescription & Longevity,2025
1.4 Personalizing exercise – Part 2,"Adopting a personalized approach and prescribing PRT as a cornerstone of physical activity for older adults, combined with aerobic and balance training where indicated and feasible, holds the potential to maintain functional autonomy and enhance quality of life while mitigating the risk of various age-related conditions. Integrating this personalized strategy into routine practice will likely yield substantial benefits for the aging population (Fig. 2). Exercise prescriptions must consider the relationship between dose and response, volume, intensity, and the specific adaptations necessary for desired outcomes. Resistance, aerobic, balance, and mobility training can each target distinct age-related deficits. Multicomponent exercise programs that integrate cognitive tasks effectively improve frailty characteristics such as low muscle mass, strength, endurance, mobility, PA level, and energy, while also enhancing cognition [85,86]. Older adults should participate in multicomponent programs emphasizing functional balance and moderate-to-high intensity PRT at least three times per week to prevent falls [15]. Power training gains importance with advancing age due to the profound relationship between muscle power output and functional abilities. Age-related declines in muscle power—largely attributed to Type II fiber atrophy and neural recruitment changes—support including explosive PRT with rapid, forceful concentric contractions whenever possible [88].","Journal of Nutrition, Health and Aging",Exercise Prescription & Longevity,2025
1.4 Personalizing exercise – Part 3,"Sarcopenia, involving losses in muscle mass, strength, and function with aging, requires high-intensity PRT for optimal adaptation. This underscores the importance of both modality and intensity in managing this widespread condition [58]. Training techniques that simultaneously optimize strength and power—fast velocity at moderate-to-high loads—are ideal [89]. However, evidence is still scarce regarding the optimal quantity of muscle-strengthening activity for older adults. Dosage, intensity, volume (sets/repetitions), frequency, and rest intervals remain insufficiently defined. The minimum clinically important difference (MCID) for strength, power, muscle mass, bone density, and function after PRT also remains poorly established. Interindividual heterogeneity further complicates optimal prescription design, as individuals vary widely in physiological responses to exercise. Current debates focus on categorizing individuals as responders, non-responders, or adverse responders, but uniform definitions are lacking across exercise modalities [90–92]. Genotypes predictive of aerobic adaptation do not predict anabolic adaptation to PRT [60,93]. The relationship between exercise intensity and strength gains is well-established: higher-intensity PRT yields greater strength improvements. Contrary to the notion of “non-responders,” low strength gains in older adults typically stem from improperly prescribed or inadequately progressed PRT rather than inherent inability to adapt [94].","Journal of Nutrition, Health and Aging",Exercise Prescription & Longevity,2025
1.4 Personalizing exercise – Part 4,"While some individuals may show variable functional performance outcomes despite strength gains, lack of strength improvement is usually linked to suboptimal training intensity or prescription, not a biological failure to respond. Exercise should be considered a medical treatment, with prescriptions tailored to intended outcomes—primary prevention, improved fitness, enhanced function, or disease treatment. It is critical to personalize, adjust, and manage exercise prescriptions just as with pharmacological therapies. External variables (intensity, volume, modality, rest intervals) and internal variables (acute physiological response to exercise) are shaped by personal, genetic, functional, psychosocial, and environmental factors [95]. Thus, achieving precision in exercise prescriptions remains a major research priority. Understanding how to match modality, intensity, and volume to individual phenotypes, intrinsic capacity, disease status, and goals will be essential for optimizing healthy aging outcomes. These principles reflect the broader shift toward precision geroscience, leveraging individual variability to maximize health span benefits of physical activity and structured exercise in older adults.","Journal of Nutrition, Health and Aging",Exercise Prescription & Longevity,2025
1.5 Global Exercise Recommendations – Part 1,"1.5. Physical activity and exercise: global health recommendations. Strategies to increase population-level physical activity increasingly focus on integrating exercise directly into daily life. Simple habitual actions—taking stairs instead of elevators, standing on one leg while washing dishes, or performing sit-to-stand transitions without arm support—allow older adults to incorporate aerobic, balance, and strengthening elements into routine activities. Research is examining whether lifestyle-integrated prescriptions improve adherence and outcomes more effectively than traditional structured exercise, particularly for fall prevention in older adults. The World Health Organization recommends that adults aged 65+ accumulate at least 150 minutes of moderate-intensity or 75 minutes of vigorous-intensity aerobic activity weekly, along with muscle strengthening activities on two or more days per week. The U.S. Department of Health and Human Services further advises older adults to undertake multicomponent exercise training—balance, muscle strengthening, and moderate aerobic activities—three or more times per week for 30–45 minutes per session to enhance functional ability in individuals with frailty. However, global adherence remains low; in U.S. surveillance data, only 17–23% of adults met both aerobic and resistance exercise guidelines between 2015 and 2019. Sedentary lifestyle coupled with insufficient activity is strongly linked to sarcopenia, frailty, obesity, and chronic disease. Worldwide, inadequate PA contributes to an estimated 1.3 million premature deaths annually.","Journal of Nutrition, Health and Aging",Global Exercise Guidelines,2025
1.5 Global Exercise Recommendations – Part 2,"The role of exercise in disease prevention and management—especially for age-related conditions in which medications show limited success—is increasingly recognized. Substantial evidence supports physical activity and structured exercise as preventive and therapeutic tools for cardiovascular disease, type 2 diabetes, obesity, and metabolic dysregulation. Exercise improves muscle function, mental health, quality of life and reduces mortality. Combining balance training with progressive resistance training is the most effective strategy to reduce falls, while PRT alone is the strongest evidence-based treatment for sarcopenia, outperforming currently available pharmacological options. A comparative framework of exercise versus pharmacologic interventions highlights that exercise is universally beneficial in improving aerobic fitness, muscle mass, metabolic health, and psychological well-being. Pharmacological treatments retain importance in conditions requiring specific biochemical modulation, such as dyslipidemia, insulin resistance, systemic inflammation, respiratory disease, and cancer. However, for many aging-related declines—frailty, mobility loss, disability, sarcopenia, cognitive impairment—no effective drugs exist, whereas exercise consistently produces clinically meaningful improvements. Many psychotropic medications used for depression, insomnia, or anxiety increase risks of falls, fractures, delirium, and cognitive decline, while exercise independently improves these conditions with fewer adverse effects. This strengthens the case for exercise as a primary therapy: preventing diseases with existing treatments, supplementing effective medical interventions, substituting unsafe treatments, and providing the only effective therapy where drugs fail.","Journal of Nutrition, Health and Aging",Exercise vs Pharmacology,2025
1.5 Global Exercise Recommendations – Part 3,"Despite robust evidence, exercise prescription remains poorly integrated into geriatric medicine and is not a core competency for many healthcare professionals. A significant gap exists in research on tailored PA guidelines that optimize functional abilities, daily living activities, and cognitive, psychological, sensory, and locomotor domains for older adults. Effective interventions must account for behavioral and social determinants to improve adherence. Enhancing self-efficacy, promoting active lifestyles, and addressing social barriers are essential components. Public health strategies must also tackle environmental inequities that limit older adults’ ability to engage in PA. This includes improving access to exercise facilities, subsidizing transportation for older individuals, and modifying community spaces, workplaces, and residential environments to promote movement and reduce sedentary behavior. Multilevel approaches that target behavioral risk factors—smoking, alcohol use, unhealthy diet, inactivity—combined with structural changes that improve social and physical environments are necessary to raise PA levels in aging populations. Tailored, accessible, and behaviorally informed exercise prescriptions will be essential to meet the needs of older adults and close the gap between evidence and real-world implementation.","Journal of Nutrition, Health and Aging",Exercise Adherence & Public Health,2025
1.6 Preserving Exercise Capacity – Part 1,"1.6. Preserving exercise capacity with age via habitual engagement in physical activity and exercise. Regular physical activity significantly mitigates age-associated reductions in aerobic capacity. One unavoidable change is the decline in maximal heart rate due to reduced sensitivity to beta-adrenergic stimulation in aging cardiac tissue. Although peak workloads decline with age, older adults who engage in long-term aerobic training display improved cardiovascular and musculoskeletal efficiency, allowing them to perform higher submaximal workloads with lower heart rate, blood pressure, dyspnea, and reduced fatigue. Exercise adaptations remain highly modality-specific. Moderate-to-vigorous intensity aerobic exercise produces the greatest improvements in aerobic capacity, with high-intensity interval training (HIIT, 85–95% peak heart rate for 1–4 minute intervals) eliciting the strongest cardiorespiratory gains. High-intensity progressive resistance training (PRT) remains optimal for treating sarcopenia and also improves balance in older individuals. An important exception to strict modality specificity is the finding that high-intensity PRT enhances aerobic capacity nearly as effectively as moderate-intensity aerobic exercise. Therefore, an optimal exercise prescription for aging integrates both aerobic and resistance modalities to counteract key physiological declines in cardiorespiratory fitness and muscle function.","Journal of Nutrition, Health and Aging",Exercise Capacity and Aging,2025
1.6 Preserving Exercise Capacity – Part 2,"Aerobic exercise alone cannot improve muscular strength or balance and is therefore insufficient as a standalone modality for older adults. Isolated balance training improves postural control, reduces falls and fall-related injuries, and enhances functional mobility, with additional support from Tai Chi interventions. However, balance training does not enhance aerobic capacity or counteract sarcopenia. Conversely, multicomponent exercise programs that integrate progressive resistance training, balance challenges, and functional exercises are consistently shown to reduce fall incidence in older adults. The greatest reductions in falls occur when moderate-to-high intensity PRT is paired with sufficiently challenging balance exercises. Despite its frequent inclusion in guidelines, stretching offers limited clinical benefit. Flexibility work is best used during cooldown routines. Acute static stretching before exercise, especially before PRT, is not effective for injury reduction and may transiently decrease muscle performance, lowering potential strength gains. The most effective warm-up involves performing a low-intensity version of the intended activity—e.g., lighter resistance movements or slower walking—aligning with evidence that neuromuscular priming enhances performance more than passive stretching.","Journal of Nutrition, Health and Aging",Exercise Modalities in Aging,2025
1.6 Preserving Exercise Capacity – Part 3,"Although static stretching is commonly promoted, its effects on muscle strength are modest and based largely on studies of younger adults (median age ~22). In these studies, observed strength increases often reflected improved isometric maximum voluntary contraction (MVC), likely due to neuromuscular familiarization rather than true dynamic strength gains. Evidence for meaningful increases in dynamic strength, such as 1RM performance, remains limited. Flexibility improvements depend on a higher number of repetitions, longer time under tension per session, and greater cumulative stretching exposure. However, a recent meta-analysis of 38 studies involving more than 1,000 participants found no significant differences in range of motion or stiffness when comparing stretching or foam rolling to interventions such as walking, vibration, cycling, calisthenics, strength training, electrical stimulation, heat exposure, or cryotherapy. These findings highlight the need for higher-quality research to determine the specific benefits of stretching and foam rolling. Collectively, the evidence underscores that preserving exercise capacity with age requires prioritizing resistance training, balance work, and aerobic conditioning, whereas stretching should be viewed as supplemental rather than central to functional preservation.","Journal of Nutrition, Health and Aging",Flexibility and Functional Aging,2025
1.7 Bridging Research and Clinical Practice – Part 1,"1.7. Bridging the gap: from research to clinical implementation in exercise prescription for older persons. Despite the extensive evidence supporting exercise as one of the most powerful interventions for aging, its integration into mainstream clinical practice remains limited. Many healthcare professionals, including geriatricians, lack adequate training to incorporate structured exercise into routine patient care. Although some progress has been made in introducing exercise counseling within healthcare settings, it is frequently reserved for older adults who do not exhibit significant physical or cognitive impairments. Even then, recommendations often promote only mild activities such as walking, typically at intensities or volumes that do not align with evidence-based guidelines. This overly cautious approach is commonly rooted in an exaggerated fear of exercise-related injuries or concerns about the safety of vigorous exercise in older individuals. In reality, sedentariness—not exercise—is the true risk factor linked to disease, functional decline, and mortality. Current prescribing patterns often fail to match the established science supporting moderate-to-high intensity progressive resistance training as essential for preventing frailty and sarcopenia.","Journal of Nutrition, Health and Aging",Exercise Implementation in Geriatrics,2025
1.7 Bridging Research and Clinical Practice – Part 2,"Prescribing exercise at suboptimal intensities or volumes should be regarded similarly to prescribing inadequate doses of medication. In pharmacology, the goal is to achieve a therapeutic dose—yet in exercise prescriptions for older adults, especially those involving resistance training, recommendations frequently resemble a placebo. Low-load, non-progressive, or insufficiently dosed exercise programs are unlikely to generate meaningful improvements in strength, function, or physiological resilience, yet these are often the default in clinical environments. More intensive and progressive models of exercise have repeatedly been shown to be safe, even for individuals in advanced age or with multiple comorbidities. Thus, the gap between research evidence and typical clinical practice reflects a fundamental inconsistency in applying scientific standards. Given the substantial benefits and minimal risks associated with properly prescribed exercise, it should be embraced as a core therapeutic intervention rather than an optional adjunct. The effectiveness of PRT, aerobic conditioning, and multicomponent training is well established, yet insufficiently implemented across healthcare systems.","Journal of Nutrition, Health and Aging",Therapeutic Exercise Standards,2025
1.7 Bridging Research and Clinical Practice – Part 3,"Integrating evidence-based exercise prescriptions into a standardized component of care requires implementation across hospitals, outpatient clinics, rehabilitation settings, and long-term aged care facilities. Exercise should be included in care plans just as routinely as pharmacotherapy, dietary interventions, or psychological support. Community initiatives, public health policies, and targeted programs in residential care can amplify the reach of such interventions. Individualized prescriptions—adjusted to functional level, frailty status, and comorbidities—ensure safety while maximizing therapeutic effect. Promoting active lifestyles through societal support, accessible exercise environments, and education for healthcare providers is essential for successful implementation. Finally, deepening scientific understanding of how exercise influences the biological hallmarks of aging can further validate its role as a frontline therapeutic strategy. The subsequent sections of the document elaborate on how exercise modulates aging biology and why it should be central to geriatric care.","Journal of Nutrition, Health and Aging",Implementation Science for Exercise in Aging,2025
2. Evidence for Exercise Recommendations – Part 1,"An extensive body of epidemiological and experimental evidence supports integrating exercise prescription training into routine education for physicians, including geriatricians. While physicians are not expected to serve as fitness trainers, they must understand the physiological benefits of exercise, be able to explain them to patients, and make appropriate referrals to exercise professionals. Exercise is an effective intervention for managing chronic diseases common in older adults, including cognitive impairment, frailty, sarcopenia, falls, and mobility limitations. Promoting physical activity requires a broad, multisectoral approach involving clinicians, physiotherapists, public health agencies, insurers, fitness professionals, community planners, and policymakers. Screening for sedentary behavior and insufficient activity is recommended at all healthcare encounters, as these factors increase risk for mortality, obesity, hypertension, insulin resistance, chronic kidney disease progression, cardiovascular disease, diabetes, cancer, dementia, osteoarthritis, osteoporosis, recurrent falls, disability, and frailty. Exercise prescriptions should be incorporated whenever medically feasible, with programs adjusted to individual health status and goals.","Journal of Nutrition, Health and Aging",Exercise Recommendations for Older Adults,2025
2. Evidence for Exercise Recommendations – Part 2,"Exercise prescriptions must be personalized according to the type of activity, frequency, duration, and intensity required to meet specific health goals. Support systems for monitoring progress and providing feedback enhance adherence. Although counseling on physical activity during routine healthcare has increased, it is still disproportionately focused on individuals without significant frailty or cognitive impairment, despite strong evidence showing exercise benefits across the full functional spectrum. Excluding tailored exercise programs from clinical practice constitutes sub-optimal care. A key challenge is integrating structured exercise interventions into hospitals, outpatient clinics, and long-term care settings. Current guidelines recommend a multicomponent regimen combining aerobic training, progressive resistance training, balance work, and mobility exercises delivered through structured or lifestyle-integrated formats. Sedentary older adults may begin with a single exercise modality to facilitate adaptation before progressing toward comprehensive programs. Even small increases in activity provide measurable health benefits among highly inactive individuals, but exercise prescriptions should aim beyond minimal thresholds to achieve clinically meaningful improvements.","Journal of Nutrition, Health and Aging",Personalized Exercise Guidelines,2025
2. Evidence for Exercise Recommendations – Part 3,"Before initiating structured aerobic training, clinicians should assess key musculoskeletal factors such as strength, balance, and joint range of motion, as these underpin safe and effective performance. Aerobic training alone does not improve these domains and therefore cannot adequately address sarcopenia or fall risk. A multicomponent program integrating resistance training, gait training, and balance exercises is necessary to optimize musculoskeletal function and reduce frailty-related outcomes such as falls, fractures, and mobility decline. Resistance training improves muscle mass, strength, and power; balance training enhances postural control; and aerobic training improves endurance and cardiorespiratory fitness. Programs must be individualized based on risk factors, comorbidities, medical history, and personal preferences. Properly sequenced exercise prescriptions are especially important for older adults with mobility limitations and should follow foundational physiological principles to ensure safety and maximize functional gains.","Journal of Nutrition, Health and Aging",Multicomponent Training Strategies,2025
2.1 Principles for Older Adults with Mobility Challenges – Part 1,"Exercise prescription for older adults with frailty must follow principles of sequencing, specificity, and safety. A comprehensive geriatric assessment is essential to identify mobility limitations, comorbidities, fall risk, cognitive factors, and functional goals. Physicians initiate the prescription, while ongoing monitoring and progression are managed by a multidisciplinary team including physiotherapists, occupational therapists, and exercise specialists. Patient empowerment and contextual considerations—such as prior activity levels, cultural factors, access to equipment, and preferences—enhance long-term adherence. Sequencing exercises according to the physical requirements of mobility is critical: strength and power are required to stand up and lift body weight; balance is required to remain upright; and endurance is needed for ambulation. Therefore, resistance training to improve foundational strength must precede balance and endurance training. Attempting to initiate walking in individuals lacking basic sit-to-stand strength or standing balance increases fall risk and leads to unsuccessful outcomes.","Journal of Nutrition, Health and Aging",Exercise Sequencing for Frailty,2025
2.1 Principles for Older Adults with Mobility Challenges – Part 2,"Training should begin with low-intensity exercises and gradually increase duration, load, and frequency to allow adaptation and prevent excessive fatigue. Specificity is essential: exercise programs must target the physiological determinants of functional limitations. For example, individuals requiring assistance during wheelchair-to-bed transfers may need focused upper-body strengthening such as triceps and shoulder exercises. Older adults with severe knee osteoarthritis may benefit more from low-impact seated resistance training or aquatic-based exercise rather than weight-bearing aerobic activities. Aquatic resistance training has demonstrated effectiveness in improving strength, muscle mass, and metabolic health in older adults and patients with non-communicable diseases. Programs should incorporate functional movements, postural control, range of motion work, and task-specific practice to enhance real-world transferability. Ultimately, exercise prescription must aim to improve not only physical capability but also psychological well-being, autonomy, and perceived quality of life in frail individuals.","Journal of Nutrition, Health and Aging",Mobility Prescription Principles,2025
2.1 Principles of Exercise Prescription – Safety and Risk Mitigation,"Safety and risk mitigation are essential components of exercise prescription for older adults, particularly those living with chronic diseases, frailty, or mobility limitations. Clinicians must evaluate risks associated with different exercise modalities relative to the individual’s health status. Some conditions respond equally well to resistance or aerobic training, such as depression; in these cases, the modality chosen should reflect safety, tolerance, and comorbidities. For example, individuals with severe knee osteoarthritis, recurrent falls, or low thresholds for peripheral ischemia may tolerate progressive resistance training better than aerobic exercise, making PRT the safer antidepressant approach. Effective prioritization requires weighing risks and benefits of each modality and identifying exercises capable of addressing multiple conditions simultaneously. For instance, in individuals with both osteoporosis and depression, resistance training is superior to aerobic training because it improves bone density while also benefiting mental health. The exercise environment must be secure, with activities performed within the individual’s functional capacity to minimize risk of injury.","Journal of Nutrition, Health and Aging",Exercise Safety and Risk Mitigation,2025
2.1 Principles of Exercise Prescription – Personalization and Holistic Factors,"Personal preference and individualization are central to designing effective and sustainable exercise prescriptions. Older adults vary widely in preferences regarding exercise setting (group vs. individual), modality (structured vs. lifestyle-integrated), supervision needs, and specific likes or dislikes. A comprehensive assessment of physical, cognitive, and functional status ensures that exercise programs align with individual goals and capacities. This enhances enjoyment, motivation, adherence, and long-term outcomes. Holistic health considerations are essential, particularly for frail adults with multiple chronic conditions. Exercise modalities should be selected to target several health issues simultaneously. This approach maximizes physiological benefit and efficiency of training. For example, resistance training can improve bone density, muscle mass, metabolic function, and mood in a single modality. Multicomponent training—including strength, aerobic, balance, and flexibility exercises—should be introduced sequentially based on feasibility and priority of adaptations needed.","Journal of Nutrition, Health and Aging",Personalized and Holistic Exercise Planning,2025
"2.1 Principles of Exercise Prescription – Functional, Balance, and Cognitive Training","Functional training emphasizes exercises that simulate daily tasks such as standing, walking, carrying objects, and transitional movements. These activities improve independence, mobility, and quality of life by strengthening the neuromuscular patterns required for everyday living. Balance and fall-prevention exercises—targeting coordination, proprioception, and postural control—are essential components of training for older adults, as they reduce fall risk and promote safe ambulation. Cognitive engagement can be integrated through dual-tasking, memory components, or problem-solving embedded within physical tasks, which improves cognitive flexibility and adherence. Social support through group sessions, caregiver involvement, or peer interaction enhances motivation and consistency. Ongoing evaluation and adaptation are required to prevent plateaus and ensure progression, while supervision ensures proper technique and safety. Adaptive equipment such as chairs, handrails, water-based environments, or stability tools can accommodate limitations and facilitate safe, effective training.","Journal of Nutrition, Health and Aging","Functional Training, Balance, Cognition",2025
2.2 Gait Training,"Gait training is essential for maintaining mobility, preventing falls, and preserving independence in older adults. Research on exercise effects on gait stability shows mixed results: some interventions improve gait, while others show no change. Notably, the studies demonstrating the most consistent improvements used multicomponent exercise programs combining strength, balance, and functional activities, rather than isolated resistance or aerobic programs. Gait speed is one of the strongest predictors of survival in older adults, and gait variability is a reliable predictor of fall risk across clinical populations. Practical exercises to improve walking stability and mobility include walking with changes in pace and direction, treadmill walking, stair climbing, step-ups, and balance tasks. Weight-bearing aerobic activities mimicking real-life movement are generally preferable. However, for individuals with severe osteoarthritis, pain, or balance deficits, alternatives such as aquatic exercise, seated steppers, or recumbent cycles may be used initially, although these do not directly improve gait. For those unable to support body weight, seated resistance training and power training should precede progression to upright ambulation. Transitioning to walking-based exercise should occur as soon as safely feasible to optimize functional mobility.","Journal of Nutrition, Health and Aging",Gait Training in Older Adults,2025
2.3 Aerobic Training,"Aerobic training focuses on improving cardiovascular endurance through rhythmic, continuous activities such as walking, cycling, and swimming. It relies on thousands of low-resistance muscle contractions using large muscle groups. For those unable to use their legs—due to stroke, neuropathy, amputation, or pain—arm-based aerobic activities can be used. Sessions typically begin with 5–10 minutes of low-intensity work and gradually progress to 20–30 minutes. Exercise intensity is usually prescribed using the heart rate reserve method, beginning around 40–60% HRR and progressing toward 60–85% as fitness improves. For individuals where HR is unreliable (e.g., arrhythmias, beta-blockers, pacemakers), perceived exertion scales such as the Borg Scale are essential tools for adjusting intensity. Monitoring symptoms such as dizziness, dyspnea, and unusual fatigue is essential for safety. Hydration, temperature regulation, and appropriate clothing also contribute to safe training. When properly implemented, aerobic exercise significantly enhances cardiovascular fitness, reduces morbidity, and supports healthy aging.","Journal of Nutrition, Health and Aging",Aerobic Training Guidelines,2025
2.4 High-Intensity Interval Training (HIIT) – Benefits and Protocols,"High-intensity interval training (HIIT) consists of brief periods of high-intensity exercise separated by rest or low-intensity recovery. HIIT is increasingly used in older adults and has been shown to improve cardiovascular health, VO2 peak, submaximal performance, and cardiac function, often with time-efficiency advantages over moderate-intensity continuous training (MICT). Typical HIIT protocols for older adults involve 20-minute sessions, two to three times per week for under 12 weeks, with intervals lasting 30 seconds to 4 minutes at 70–89% of peak HR or higher, interspersed with up to 180 seconds of rest. Evidence indicates HIIT can be performed safely in supervised settings among low-to-moderate risk individuals, including those with cardiovascular disease, and meta-analyses show beneficial effects on VO2 peak (typically 15–20% improvements). A five-year RCT in Norway demonstrated that community-based HIIT can be feasible and safe in generally healthy older adults. Additionally, HIIT shows comparable effects to MICT on lipids, blood pressure, fat mass, and general cardiovascular risk reduction. HIIT may stimulate anabolic signaling and muscle hypertrophy, although whether these benefits translate to clinically relevant strength or mass improvements in older adults remains unclear.","Journal of Nutrition, Health and Aging",HIIT Benefits and Protocols for Older Adults,2025
"2.4 High-Intensity Interval Training (HIIT) – Risks, Psychological Responses, and Limitations","Despite its benefits, HIIT poses slightly higher cardiovascular risk than MICT, especially in patients at elevated cardiovascular risk. Retrospective analyses report one fatal cardiac event per ~23,000 patient-hours of HIIT compared with ~129,000 hours for MICT. Most HIIT studies exclude high-risk cardiac patients and are conducted under clinician supervision, limiting generalizability to frail or high-risk populations. Psychological responses to HIIT vary: some older adults experience discomfort as exercise approaches ventilatory or lactate threshold, while others experience euphoria, reduced pain sensitivity, and improved mood (‘runner’s high’). Genetic differences may influence tolerance to catecholamine-driven sensations. Many older adults find HIIT empowering, reporting improved self-efficacy and satisfaction. However, feasibility for frail individuals remains uncertain—treadmill HIIT may be unsafe for those with gait impairment, and cycling HIIT does not improve balance or bone density. While HIIT can induce metabolic and mitochondrial improvements, its effects on sarcopenia-related outcomes remain insufficiently studied. Overall, HIIT offers time-efficient improvements in aerobic fitness, but careful screening, supervision, and individualized programming are required before widespread adoption in older adults with comorbidities or mobility impairments.","Journal of Nutrition, Health and Aging",HIIT Risks and Older Adult Considerations,2025
Power Training Overview and Age-Related Decline,"Power training is a specific type of muscle training that targets both the force production and velocity components of muscle power. Unlike traditional PRT, which involves overcoming resistance using a high force at slow speed during the concentric (shortening) phase, power training emphasizes overcoming resistance at maximal volitional speed. Muscle power declines significantly with age, leading to an increased risk of physical impairment, falls, disability, and mortality. The ability to perform daily activities is closely linked to muscle power output and the rate of force development, with research showing strong correlations between these metrics and performance in functional capacity tests in healthy older adults. Recent studies have also linked muscle power and explosiveness to enhanced functional capacity and reduced incidence of falls in frail, oldest-old populations. Aging-associated physiological changes, such as the loss of type II fast-twitch fibers and deficits in neural recruitment, can diminish power output in ways that traditional slow-velocity strength training does not address, such as the early onset of muscle force and the maximal rate of force development.","Journal of Nutrition, Health and Aging",2.6 Power Training Recommendation,2025
Neuromuscular Benefits and Implementation in Clinical Settings,"Power training can significantly enhance physical capabilities in older adults by promoting the recruitment of fast-twitch fibers and improving neuromuscular coordination through high-velocity contractions, optimizing motor unit firing rates, and enhancing muscle activation and intermuscular coordination, which are vital for daily tasks. Although evidence indicates benefits for individuals ranging from robust to frail, including hospitalized older persons, power training remains underused in clinical practice. Bodyweight exercises, such as quick standing from a chair, can initially provide resistance as long as it is safe. Individuals may start these exercises slowly with assistance and progress to performing them by themselves and quickly. If an individual’s body weight does not provide a sufficient load, additional weights or machines can be introduced to ensure progression. Both free weights and specialized PRT machines are used for power training, showing similar improvements in neuromuscular, functional, and overall fitness. Studies employing pneumatic resistance machines specifically designed for power training have shown comparable benefits.","Journal of Nutrition, Health and Aging",2.6 Power Training Recommendation,2025
"Exercise Variations, Monitoring, and Safety Considerations","An alternative way to increase intensity without PRT machines is to change from bilateral to unilateral body weight exercises such as lunges, which can also be performed at increasing speeds during the concentric phase. Monitoring movement speed is essential because power training relies on explosiveness, whereas endurance methods focus on repetition. Plyometric training, such as jumping onto platforms or boxes, may be an alternative when power training machines are unavailable. However, lower extremity or spinal arthritis, osteoporosis of the spine, and balance impairment frequently preclude the use of lunges or plyometrics by many older adults with frailty. Power training should include fast concentric (shortening lifting) and controlled eccentric phases for optimal gains, focusing mainly on the lower extremities. Explosive resistance training sessions can be combined with traditional PRT in the same workout, avoiding concentric failure and maximizing effectiveness and safety. The ideal intensity for these sessions might range from 60% of the maximal load for upper-body exercises and 80% for lower-body exercises.","Journal of Nutrition, Health and Aging",2.6 Power Training Recommendation,2025
"Training Intensities, Risks, and Recommendations for Older Adults","A dose-response study indicated that peak muscle power improved similarly across light (20%), moderate (50%), and heavy (80%) resistance levels, but the 80% load provided the best improvements in strength as well. There is a known correlation between training intensity and muscle strength and endurance improvements, with higher intensities yielding better results. However, moderate-intensity power training (60% of 1RM) can lead to superior functional capacity adaptations. Caution is advised with low-load power training, as it may increase injury risk owing to undiagnosed joint issues exacerbated by the very high velocity of the movement. Starting with a minimal dose for the first few 2–3 weeks is recommended, particularly for older participants. Two–three sessions per week are typically sufficient to achieve significant neuromuscular and functional benefits. Screening for musculoskeletal and joint conditions before initiating power-based training is essential, as some conditions may contraindicate such activities or require specific adjustments.","Journal of Nutrition, Health and Aging",2.6 Power Training Recommendation,2025
"Injury Prevention, Adaptations, and Mental Imagery Techniques","If free weights are used, instructions to avoid the use of momentum to achieve high velocity are critical, as this will both minimize muscle adaptation and increase the likelihood of tendon or cartilage tears in open chain exercises with unrestricted end ranges or back strain in standing biceps curls. While the risks associated with power training are generally low and comparable to those associated with traditional PRT, careful screening and exercise selection are essential to minimize injury risk and ensure safety. Two potential adverse events related to muscle power training are injury to tendons or cartilage, particularly of the rotator cuff and knee, and exacerbations of abdominal or inguinal hernias. A systematic review of PRT in frail older adults reported only one case of shoulder pain related to interventions in 20 studies and 2544 subjects. To optimize muscle adaptations, mental imagery can be used to enhance motor performance in both elite athletes and older adults. Practically, this means coaching the person to imagine moving the weight as rapidly and forcefully as possible before each repetition, then using a clap or vocal command to elicit maximal intended velocity.","Journal of Nutrition, Health and Aging",2.6 Power Training Recommendation,2025
Concurrent vs Combined Training and Interference Effect,"A combination of resistance/power and aerobic training, known as ""combined or concurrent training"", is the most effective method for enhancing both neuromuscular and cardiorespiratory functions and is essential for maintaining functional capacity during aging. ‘Concurrent’ resistance and aerobic training involves performing both exercise modalities within the same session, either sequentially or interspersed as in circuit training. In contrast, ‘combined’ training refers to performing both modalities within the same training period but on separate days. Importantly, in the classical work by Hickson it was observed that when resistance and aerobic training are performed concurrently, a phenomenon known as the ""interference effect"" can occur. This effect refers to the potential reduction in strength and hypertrophy adaptations from resistance training when preceded by, or interspersed with, aerobic exercise in the same session in comparison with a resistance-only training regimen. More recent studies showed that concurrent training performed twice weekly resulted in similar adaptations in lower-upper body strength, power, and VO2 peak compared to PRT alone and comparable aerobic fitness to aerobic training alone in moderately trained or untrained healthy adults. Thus, the clinical relevance of the interference effect is not completely clear.","Journal of Nutrition, Health and Aging",2.7 Sequencing Resistance and Aerobic Training,2025
Combined Training in Older Adults and Early Adaptations,"By contrast, a combined training study in healthy older men (65–74 yr) compared the effects of 16 weeks of twice-weekly PRT alone (50–80% of 1RM), or twice weekly cycling aerobic training alone (HR between 70% and 90% of maximal HR) or combined PRT (once weekly) and aerobic (once weekly) training on muscle mass, maximal strength and power, and cardiovascular performance. The main findings were that combined resistance and aerobic training led to similar gains in muscle mass, maximal leg strength, and muscle power output as resistance training alone and to similar gains in maximal peak power output measured in an incremental cycling test as aerobic training alone, despite the lower volume in the combined group. Thus, during the initial 16 weeks of training a minimum weekly frequency of combined training—one session of resistance plus one session of aerobic training—might be sufficient to enhance neuromuscular and cardiovascular functions in previously untrained older adults. Comparable improvements may have occurred due to the lower limbs cycling stimulus, requiring more strength and power output compared with other aerobic modes.","Journal of Nutrition, Health and Aging",2.7 Sequencing Resistance and Aerobic Training,2025
Effects of Scheduling and Sequencing Within the Same Session,"Collectively, these studies demonstrate that given specific training volumes, frequencies, and training schedules, the combined or concurrent performance of resistance and aerobic training does not impede neuromuscular and cardiorespiratory adaptations; it may not result in an interference effect in healthy older adults. However, the sequence of exercises within the same session significantly impacts neuromuscular adaptations. Specifically, performing aerobic exercises after PRT within the same session does not blunt strength gains and neuromuscular adaptations. By contrast, performing aerobic exercise before PRT may blunt strength and hypertrophy gains. This effect is likely due to the fact that resistance training induces specific gene expression changes in muscle fibers, enhancing neuromuscular recruitment and contractile function. This includes upregulation of genes associated with myofiber hypertrophy, mitochondrial biogenesis, and angiogenesis, which are crucial for muscle adaptation and performance improvements. The interference effect between AE and PRT is largely attributed to the activation of AMPK, a key regulator of energy homeostasis and cellular stress responses, by aerobic exercise.","Journal of Nutrition, Health and Aging",2.7 Sequencing Resistance and Aerobic Training,2025
"AMPK-mTOR Interactions, Fatigue, and Training Adaptation","Aerobic exercise activates AMPK, particularly under low energy availability conditions, as it restores energy balance by increasing glucose uptake and fatty acid oxidation. However, this activation also promotes catabolic pathways, such as mitochondrial biogenesis and autophagy, which can counteract the anabolic processes necessary for muscle hypertrophy induced by resistance training. As a result, elevated AMPK activity following aerobic exercise can interfere with signaling pathways like mTOR, which are crucial for muscle protein synthesis and growth in response to PRT. In addition, blunted adaptation to PRT may be due to the onset of neuromuscular fatigue. This fatigue reduces the ability to generate maximal force during resistance exercises, diminishing the effectiveness of PRT in promoting muscle hypertrophy and strength gains. Consequently, performing aerobic exercise before resistance training in the same session may reduce the overall effectiveness of the resistance training in terms of muscle growth and strength development. Combined training should also be progressively structured in volume and intensity to maximize benefits and avoid excessive load.","Journal of Nutrition, Health and Aging",2.7 Sequencing Resistance and Aerobic Training,2025
Optimal Exercise Ordering and Integration of Training Modalities,"Traditional PRT may reach 80–85% of the one-repetition maximum (1RM). For optimal adaptations within a resistance training session, performing large multi-joint exercises (e.g., leg press, chest press) before smaller, single-joint exercises (e.g., biceps curls, ankle dorsiflexion) is recommended, maximizing neuromuscular benefits. Aerobic training can target up to 95% of the anaerobic threshold using continuous training or HIIT. Power training combined with HIIT can enhance maximal strength, muscle mass, and cardiorespiratory fitness, providing benefits comparable to traditional combined training and improving explosive power. However, integrating all modalities—resistance, aerobic, balance, and gait training—into a single session may lead to suboptimal intensity and volume for each modality, even with optimized sequencing. This dilution of exercise quality may blunt adaptations more than the molecular interference effect. In conclusion, combined training—when performed on separate days—does not compromise the effectiveness of either resistance or aerobic exercise for older adults. Concurrent training, however, may reduce resistance training benefits if aerobic exercise precedes it, especially in frail individuals.","Journal of Nutrition, Health and Aging",2.7 Sequencing Resistance and Aerobic Training,2025
Practical Recommendations for Older and Frail Adults,"Special consideration should be given to physically frail individuals, as fatigue is a hallmark of this population. For these individuals, resistance and aerobic training should ideally be scheduled on separate days to guarantee that both modalities can be performed effectively, avoiding reduced strength gains due to neuromuscular fatigue or blunted molecular adaptation. While the potential advantages of a combined training regimen may exceed those of concurrent training, the increased burden—time, transportation, cost—must be considered. A practical compromise may involve performing resistance training under supervision in a clinical setting twice weekly, while prescribing aerobic activities such as community-based walking on alternate days. This minimizes costs, transportation challenges, fatigue, and potential interference effects, while maximizing the likelihood of physiological adaptation. Separating modalities across different days ensures each can be performed with adequate intensity, optimizing health benefits. Ultimately, programs must be tailored to each individual’s needs, preferences, limitations, and practical constraints to maximize outcomes in older adults.","Journal of Nutrition, Health and Aging",2.7 Sequencing Resistance and Aerobic Training,2025
Balance Training Fundamentals and Progression,"Balance training is an essential component of physical conditioning for older adults, especially those with medical conditions that may compromise their postural stability and increase their risk of falls. Before initiating aerobic exercise or gait retraining programs, evaluating and addressing any balance deficits through targeted balance training interventions is critical. The implementation of balance training exercises, however, presents challenges due to the inherent risk of accidental falls. A cautious approach to balance training involves progressively challenging postural control and stability in a secure environment. The initial phase should concentrate on mastering basic postures or movements, such as standing on one leg without hand support, while ensuring adequate safety measures are in place. Once proficiency at this level is achieved, the individual can progress to more complex variations, such as performing the exercise with eyes closed. This progressive overload principle mirrors the well-established concept of PRT, where the load is gradually increased to continue eliciting physical adaptation and improvement. Additionally, various forms of balance training, including virtual reality systems, specific balance strategies, sensory and muscular training, cognitive dual-task training, and programs using tools like Wii Fit or activities conducted in public parks, have been shown to effectively improve balance, boost confidence, enhance function and mobility, and reduce falls in older adults. An extensive discussion of balance training principles is beyond the scope of this article, but the fundamental principles are summarized in Table 1.","Journal of Nutrition, Health and Aging",2.8 Balance Training,2025
Multicomponent Training and Frailty Prevention,"Individualized multicomponent exercise interventions, including resistance and power training, cardiovascular exercises, balance, and gait exercises, have been proven to be more effective in improving the primary indicators of frailty syndrome, such as instability, declining muscle strength, decreased walking abilities, and an increased risk of falls. It is recommended that these exercise programs be adopted to prevent frailty in older individuals and target those in the pre-frail stage. For example, a multicomponent exercise program incorporating cognitive dual-tasking effectively enhances the clinical hallmarks of frailty in those with cognitive impairment (low body mass, strength, endurance, mobility, PA level, energy, and cognition). This exercise training modality may also be prescribed to the most vulnerable populations, including the acutely ill, hospitalized, or institutionalized older adults. There is emerging evidence that an individualized multicomponent exercise training program for older adults can partially reverse the dependence in activities of daily living (i.e., toileting, transfers, mobility, and stair climbing) that frequently occurs during and after hospitalization. Multicomponent training programs should include gradual increases in individual exercise volume, intensity, and complexity. Table 3 presents some critical points for the multicomponent exercise prescriptions.","Journal of Nutrition, Health and Aging",2.9 Multicomponent Training,2025
VIVIFRAIL Program Structure and Functional Assessment,"The VIVIFRAIL multicomponent Physical Exercise Program to Prevent Frailty and the Risk of Falls is an exemplary evidence-based program recommended by the WHO in the Integrated Care Program for Older People guidance for person-centered assessment and pathways in primary care. The VIVIFRAIL physical exercise guide includes lower-limb exercises such as squats from a chair, leg presses, and bilateral knee extensions, alongside upper-body exercises like the seated bench press. It also features balance and gait retraining exercises such as semi-tandem line walking, single-leg standing, stepping practice, walking with small obstacles, and proprioceptive exercises on unstable surfaces such as foam pads. Additionally, the program facilitates weight transfer exercises from one leg to another. It includes individual prescription passports for older adults, which allow for the implementation of unsupervised sessions tailored to the person's functional capacity level, assessed using the SPPB and walking speed test, and their risk of falling. The VIVIFRAIL program is effective for enhancing intrinsic capacity in older adults with conditions ranging from pre-frailty to frailty and mild cognitive impairment or mild dementia, showing more significant benefits than usual care. The program improves IC domains such as locomotion, cognition, and vitality and increases physical performance and HGS in frail individuals while modulating circulatory miRNA expression.","Journal of Nutrition, Health and Aging",2.9 Multicomponent Training,2025
"Cognitive Integration, Exercise Interruptions, and Detraining Effects","Furthermore, integrating the VIVIFRAIL exercise program with executive function-based cognitive training has demonstrated the potential to prevent falls and fall-related negative outcomes in frail older adults. Exercise is medicine, and like most medicines for chronic disease, treatment is recommended without interruption. However, older adults frequently encounter disruptions in their exercise routines due to adverse events, hospitalizations, or periods of travel and relocation, where physical activity may be reduced if social support or healthcare infrastructure is lacking. Despite these interruptions, recent evidence suggests that exercise confers a protective effect even after short and long-term cessations, which is particularly relevant for frail and institutionalized populations. For instance, a study by Courel-Ibáñez examined the impact of a tailored multicomponent exercise program (VIVIFRAIL) on the prevention of weakness and falls in older adults with sarcopenia living in nursing homes. Twenty-four residents participated in either a 24-week or a 4-week intervention, followed by detraining periods of 6 or 14 weeks. The results demonstrated significant improvements in functional capacity and strength, with 36% of participants changing scores to “robust” or “non-frail” and 59% achieving a high level of self-autonomy at 4 weeks.","Journal of Nutrition, Health and Aging",2.9 Multicomponent Training,2025
Detraining Outcomes and Intermittent Training Strategies,"Participants in the long-term training group saw additional benefits, with a further 10%–20% enhancement in performance compared to the short-term group. Although detraining led to a 10%–25% decline in functional capacity, the improvements remained well above baseline levels. These findings suggest the potential of intermittent exercise strategies—such as 4-week training blocks repeated three times per year—to sustain and enhance functional capacity in frail older adults, even in the face of inevitable periods of inactivity. This approach should be tested in future studies. The Otago Exercise Program (OEP) is also a multicomponent, evidence-based program that is effective in reducing fear of falling, falls, and mortality in community-dwelling older adults. The OEP is a comprehensive home-based exercise program that encompasses warm-up exercises, progressive muscle strength training, balance training, and walking aerobic sessions. The program offers four levels of difficulty for strength training and balance training. The OEP is currently customized to suit frail nursing home residents' physical conditions and abilities by adjusting the difficulty level.","Journal of Nutrition, Health and Aging",2.9 Multicomponent Training,2025
OEP Effectiveness and Benefits for Frailty and Fall Prevention,"Research has demonstrated that the OEP can effectively improve physical function in older community groups through balance and strength training, thereby reducing the incidence of falls and fall-related injuries by 35%. Additionally, it can delay or reverse the frailty status, bolster cognitive function, and promote the overall health status of residents. The OEP has also been shown to reduce the self-reported number of falls over 12 months among community-dwelling older adults who have already experienced a fall.","Journal of Nutrition, Health and Aging",2.9 Multicomponent Training,2025
Definition and Causes of Deconditioning in Older Adults,"Deconditioning in older adults refers either not engaging in appropriate levels of PA per se or to the cessation of established exercise routines and their impact on various health indicators. Older individuals often face interruptions in PA and exercise due to illness, injury, or other factors, leading to a reduction in or complete cessation of their usual PA levels. The impact of deconditioning depends on its duration and an individual’s prior training levels. Deconditioning is also relevant when older adults are hospitalized, ill, or temporarily immobilized, thereby disrupting their PA. Stopping training and reducing usual daily PA can negatively affect muscle mass, strength, cognitive performance, and functional capacity. The degree of muscle mass and strength loss varies with the duration of the detraining period, with significant decreases occurring over extended periods. For instance, stopping training for merely 3–4 weeks led to only minor changes in muscle power and strength loss. However, reductions of approximately 5% in maximal strength are seen after six weeks, and more substantial decreases (~15%) occur after 12–20 weeks. Conversely, cognitive performance, executive function, and mobility can continue to improve or retain improvements even after training stops.","Journal of Nutrition, Health and Aging",2.10 Deconditioning,2025
Long-Term Detraining and Partial Preservation of Benefits,"A two-year longitudinal study showed that the benefits of 16 weeks of PRT on functional capacity, maximal muscle strength, and power output partially persisted and were maintained above baseline values in pre-frail and frail older adults aged >70 years with T2D, even after 38 weeks of detraining. This study demonstrated that intermittent multimodal interventions could partially maintain improvements in functional capacity and muscle power in frail older adults with T2D who are at increased risk of adverse events such as falls, hospitalization, disability, and mortality. This maintenance may have occurred due to the increased level of activities of daily living following training-induced improvements. Although there might be a brief period after training cessation, during which some functional reserves are retained, the long-term benefits of exercise are best maintained through ongoing activity. This is particularly true for T2D, where the insulin sensitivity benefits are primarily present in the first 48 h after an exercise bout, apart from any benefits attributable to changes in body composition. Thus, healthcare professionals and policymakers should encourage older adults to exercise regularly to maintain functional independence.","Journal of Nutrition, Health and Aging",2.10 Deconditioning,2025
Retraining Effects and Recovery of Neuromuscular Function,"Following a period of deconditioning, retraining in older adults has been shown to quickly restore neuromuscular function and the cardiometabolic health benefits that were initially achieved. This recovery has been observed in the general older population and frail individuals, highlighting the adaptability of the aging neuromuscular system and its ability to regain strength and overall PA levels after a break in training. This suggests that retraining can be a powerful tool to reinstate and enhance the health benefits initially obtained from exercise regimens. Guidelines from the American College of Sports Medicine and the American Heart Association emphasize the importance of pre-exercise screening to detect cardiovascular and musculoskeletal conditions that could pose risks during exercise. This allows programs to be tailored to match individual capabilities and health status. Before starting an exercise program, comprehensive screening and assessment are essential to identify potential risks requiring specific modifications. This process may involve obtaining a detailed medical history, performing a physical examination, and reviewing relevant diagnostic tests and laboratory findings.","Journal of Nutrition, Health and Aging",2.11 Exercise Safety and Tolerance,2025
"Screening, Preparation, and Monitoring During Exercise","Healthy older adults who plan to increase PA gradually do not need to be seen by a healthcare professional. However, according to ACSM and AHA guidelines, those with CV or renal or metabolic disease or signs or symptoms of them need medical clearance. This ensures that any undiagnosed symptoms are identified, preventive care is in place, and any existing medical conditions are stable before making changes to their physical activity levels. Proper preparation to engage in PA is vital. Older adults should be educated about appropriate exercise attire, including well-fitting, comfortable clothing and adequate footwear, to minimize the risk of trips, falls, or skin irritation. Maintaining adequate hydration before, during, and after exercise sessions is also crucial to prevent dehydration and related complications. Avoiding the Valsalva maneuver and breath-holding can help minimize hemodynamic excursions or abdominal/inguinal hernia risk. Monitoring signs of exercise intolerance is critical. This includes checking vital signs before and during exercise sessions (such as heart rate and orthostatic blood pressure), with individualized target ranges established based on age, frailty status, health status, and exercise capacity.","Journal of Nutrition, Health and Aging",2.11 Exercise Safety and Tolerance,2025
"Exercise Progression, Long-Term Safety, and Cardiovascular Risk","It is essential that older adults and caregivers receive education on recognizing symptoms such as angina, claudication, undue shortness of breath, dizziness, or excessive fatigue that may require modifying or terminating exercise. Exercise programs should begin slowly with low-intensity activities. Exercise can then progressively increase intensity and duration to allow physiological adaptations and minimize injury risk. Incorporating warm-up and cool-down periods can also prevent injuries and support cardiovascular health. Long-term (> one year) physical exercise interventions have been shown to reduce the risk of falls and improve muscle strength, balance, physical function, and cognition without increasing the risk of health-related dropouts, mortality, or fractures compared to usual care. Epidemiological data show that although the risk of myocardial infarction (MI) is more significant during exercise than at rest, the overall risk of MI is 50% lower in those who are regularly active compared to sedentary adults and nearly 50-fold lower following an acute bout of exercise in highly active adults. Therefore, long-term physical exercise is safe and effective in older adults, and its benefits accrue regardless of age, physical function, or cognitive status at baseline.","Journal of Nutrition, Health and Aging",2.11 Exercise Safety and Tolerance,2025
"Training Modalities, Attrition, and High-Intensity Exercise Considerations","The type and frequency of exercise (PRT, aerobic MICT, or HIIT) and the individual's age, as well as cognitive and physical function levels, do not affect attrition rates owing to medical problems or mortality. Despite the proven safety of PRT for older adults, including those who are frail or have multiple chronic conditions, clinicians often hesitate to prescribe this exercise modality. High-intensity interval training is now recommended as a beneficial aerobic exercise option, offering efficiency and better tolerance for some individuals with chronic conditions. However, its efficacy and safety in older adults with frailty and multimorbidity need further study.","Journal of Nutrition, Health and Aging",2.11 Exercise Safety and Tolerance,2025
Limitations of Traditional Exercise Prescription and Emerging Role of Apps,"Traditional exercise prescription methods for older adults have several disadvantages, including a lack of adaptability to changing needs, limited personalization to individual health conditions, reduced accessibility for those with mobility limitations, and lower engagement without interactive features. These limitations can be overcome by using mobile applications. The use of apps makes exercise prescriptions more accessible and enjoyable to older adults, who can access personalized exercise programs directly on their mobile devices. Additionally, many apps are dynamic and can adapt to users’ needs anytime, conduct self-assessments, and promote intergenerational usage. The increasing popularity of health apps can facilitate the scaling up of exercise prescriptions to reach a larger population of frail older adults. Moreover, apps enable the prescription of tailored exercise programs according to individual needs, preferences, and health status. They can also incorporate reminders, progress tracking, and social elements to help motivate and engage older adults in their exercise routines.","Journal of Nutrition, Health and Aging",2.12 Mobile Applications for Exercise Prescription,2025
"Limitations, Inequities, and Evidence Quality in Exercise Apps","Despite certain benefits, mobile applications prescribing physical exercise to older individuals present certain restrictions. A recent review revealed that out of 15 exercise apps analyzed, only one was based on scientific evidence, which underscores a gap in catering to the needs of older adults. This study emphasizes the importance of adapting apps to older users' cognitive and physical requirements, suggesting that involving older individuals in the ‘co-design’ of the app creation process is crucial for effectiveness. In addition, although most older adults have access to smart phones, there are still a substantial proportion who have low computer and/or health literacy, visual impairments, motor coordination difficulties, language limitations, low self-efficacy, or financial limitations which preclude use of such technology. Such social inequity needs to be considered in PA promotion efforts.","Journal of Nutrition, Health and Aging",2.12 Mobile Applications for Exercise Prescription,2025
VIVIFRAIL App and Community-Based Technological Solutions,"Based on research findings, the most widely used mobile application for physical exercise prescription in frail older adults is VIVIFRAIL. This evidence-based app fulfills the needs of frail older adults by providing adaptable, accessible, and progressive exercise programs that provide both written and audiovisual information. In addition, an integrated system based on VIVIFRAIL multicomponent training was developed to monitor frailty within a community-dwelling environment and provide a multimodal tailored intervention. This technological solution enabled older users to engage with the intervention over a six-month period. Older users and their healthcare professionals also perceived it as a usable, user-friendly, and satisfactory solution. In summary, while mobile apps show potential for prescribing exercise for older adults, further development and customization are urgently needed to address this population's specific needs better and to assure access is equitable in lower-income and culturally diverse settings.","Journal of Nutrition, Health and Aging",2.12 Mobile Applications for Exercise Prescription,2025
"Exercise, Aging, and Body Composition Overview","Many studies suggest that habitual engagement in PA/exercise can markedly attenuate most decreases in exercise capacity that would otherwise occur with aging. In the last few years, evidence from well-designed studies has been accumulating, supporting the benefits of PA for bone health, increase in muscle mass and strength/power, and reduction in adipose tissue. Bone mass begins to decrease well before menopause in women (as early as the 20s in the femur of sedentary women). It accelerates in perimenopausal years, with a continuous decline through age. Similar patterns are observed in men, although with no acceleration related to the loss of ovarian function during menopause seen in women. As with the loss of muscle tissue, strength, and function (sarcopenia), many factors related to genetics, lifestyle, nutrition, disease, and medications may predict bone density at a given age. Epidemiological studies suggest that a 10% increase in peak bone mass (PBM) at the population level would be predicted to reduce the risk of fracture later in life by 50%. Thus, the accretion of PBM and bone strength among young people is essential for attenuating bone mass loss and osteoporosis risk later in life. Mechanical loading of the skeleton generally leads to favorable site-specific changes in bone density, morphology, and strength, whereas unloading produces rapid and sometimes dramatic resorption of bone and increased fracture susceptibility.","Journal of Nutrition, Health and Aging","3. Exercise, Bone Health, Adipose, Muscle, Strength, Power",2025
"Bone Density, Activity Levels, and Fracture Risk","A significantly greater bone density has been observed in athletic cohorts, with effects depending on the type, intensity, and duration of exercise training and the characteristics of the athletes. Exceptions include non-weight-bearing activities (e.g., swimming, cycling) and competitive distance runners, whose bone density appears similar to or lower than non-exercising controls. The incidence of hip fractures is 30–50% lower in older adults with a history of higher PA levels than in age-matched, less active individuals. In the prospective Epidemiology of Osteoporosis (EPIDOS) study of 6901 white women aged ≥75 years who were followed for 3.6 years, low PA levels increased the risk of proximal humerus fracture by more than two-fold. Significant changes in bone health of the femur, lumbar spine, and radius have been observed following high-impact aerobic training, PRT, and combined aerobic and resistive exercise programs. The effectiveness of isolated high-impact training (jumping, skipping, heel drops) documented in young women has yet to be replicated in studies of postmenopausal women, possibly due to lower ground-reaction forces generated in older women with lower muscle power attempting to jump, or simply performing heel drops.","Journal of Nutrition, Health and Aging","3. Exercise, Bone Health, Adipose, Muscle, Strength, Power",2025
Exercise Modalities for Body Composition and Bone Health,"Table 4 provides exercise recommendations for optimal body composition for older adults, including decreased adipose tissue mass, increased muscle mass and strength, and increased bone mass and density. Aerobic or resistance training reduces adiposity, while resistance training increases muscle mass. For bone health, resistance training and high-impact activities (e.g., jumping using weighted vests during exercise) are recommended when tolerated; these activities are not recommended for individuals with vertebral osteoporosis. Balance training is recommended up to seven days per week. Aerobic training volumes include 30–60 minutes per session; resistance training includes 2–3 sets of 8–10 repetitions of 6–8 muscle groups. Intensities of 70–80% of one-repetition maximum are recommended for both resistance and bone-building modalities, while impact activities may include 5–10% of body weight in weighted vests during jumps. Proven benefits of high-impact training are mostly observed in premenopausal women and adolescents, or when combined with multi-modality exercise in older adults.","Journal of Nutrition, Health and Aging","3. Exercise, Bone Health, Adipose, Muscle, Strength, Power",2025
Optimal Exercise Modality and Intensity for Bone Health,"The predominant exercise training factors that influence bone adaptation are the intensity and novelty of the load. Studies on mechanical loading in animals show that bone is most sensitive to short loading periods characterized by unusual strain distribution, high strain magnitudes, and rapid loading rates. In older women, aerobic activities with high ground-reaction forces (walking, jogging, stair climbing) and exercises with high joint reaction forces (weightlifting, rowing) significantly increased whole-body BMD, lumbar spine, and Ward’s triangle. Only the ground-reaction group showed increased femoral neck BMD. Lean mass and muscle strength increased only in the weight-lifting group. In postmenopausal women, PRT significantly increased total and intertrochanteric BMD after two years. Compared with aerobic exercise, PRT in older adults is more favorable because of its broader benefits for muscle, bone, balance, and fall risk. If aerobic training is used, weight-bearing and high-impact activities have greater efficacy for bone health than non-weight-bearing or low-impact aerobic activities. A multimodal 12-month program combining high-intensity PRT and weight-bearing circuits resulted in significant BMD improvements, with results linearly related to total weight lifted.","Journal of Nutrition, Health and Aging","3. Exercise, Bone Health, Adipose, Muscle, Strength, Power",2025
Efficacy of Exercise Across Age Groups and Key Clinical Trial Findings,"Multicomponent exercise training that included moderate-to-vigorous PRT prevented increases in bone turnover and attenuated decreases in hip BMD in frail older adults with obesity engaged in a weight-loss intervention. Most studies demonstrating efficacy of exercise on BMD have been conducted in women aged 50–70, and it is unknown whether similar benefits occur in women over 80 with multiple comorbidities, who are often excluded from trials. A randomized clinical trial of 90 men and 90 women aged 65–74 comparing Tai Chi, resistance exercise, and control interventions three times per week for 12 months showed modest BMD effects, which may not translate to better clinical outcomes, although adherence was high. More recent studies suggest optimal adaptations continue to accrue with high-intensity resistance and power training in older adults. A meta-analysis of 20 RCTs found exercise was associated with a 26% reduction in fall-related fractures in older adults. However, simple walking increased fall-related fractures in two studies, indicating it is contraindicated in recurrent or high-risk fallers. The DO-HEALTH trial found no benefit of low-intensity, home-based elastic band training on fracture reduction or BMD in highly active adults aged 70+, likely due to lack of overload and progression.","Journal of Nutrition, Health and Aging","3. Exercise, Bone Health, Adipose, Muscle, Strength, Power",2025
"Aging, Adipose Redistribution, and Disease Risk","Aging is associated with changes in body composition, including increased visceral adipose tissue, redistribution of adipose tissue from the subcutaneous to internal organs, appendicular to central deposition, infiltration of skeletal muscle tissue with fat, increases in intracellular adiposity, and deposition of ectopic adipose tissue. All of these are risk factors for diseases, including osteoarthritis, cardiovascular disease, gall bladder disease, T2D, breast, colon, and endometrial cancer, hypertension, peripheral artery disease, stroke, reduction in vascularization and hypoxia, increased fibrosis, and senescent cell accumulation. Reduced visceral fat has been shown to improve glucose tolerance and insulin sensitivity in individuals with and without diabetes. Reductions in trunk fat correlate with improved glycemic control in T2D. Therefore, exercise has the potential to favourably affect the accretion and distribution of adipose tissue. In this section, we review the significance of exercise's effects on reducing the disease burden associated with reducing adipose tissue in older adults.","Journal of Nutrition, Health and Aging",3.2 Adipose Tissue and Physical Exercise,2025
Experimental Studies on Exercise and Abdominal Fat,"Evidence from well-designed studies supports the benefits of PA in reducing total abdominal fat. Most studies have included middle-aged to older populations with higher abdominal and visceral fat accumulations than younger adults. These studies were more likely to demonstrate a greater magnitude of change in these individuals than in those with lower abdominal fat mass at baseline. Furthermore, the potential for PA to attenuate the gain in visceral fat is evident in obese individuals as early as childhood. Decreases in total adipose tissue accumulation and abdominal (visceral) deposition are achievable by both aerobic exercise and PRT. However, reductions in total body weight are more rapid when combined with energy-restricted diets or when performing substantial volumes of exercise (i.e., 7 h per week resulting in high energy expenditure), both of which support a negative energy balance. Preferential visceral fat mobilization is often observed in response to exercise and dietary intervention, meaning a slight reduction in total body weight or fat mass (5%) may be associated with substantial changes in visceral fat (25% or more). These changes have important metabolic implications for preventing and treating insulin resistance syndrome.","Journal of Nutrition, Health and Aging",3.2 Adipose Tissue and Physical Exercise,2025
Exercise plus Diet: Effects on Obesity and Body Composition,"Combining exercise and diet is the most effective nonsurgical treatment for obesity and metabolic health. All international consensus panels advocate this approach. One of the most common undesired effects of hypocaloric diets in older people is impairment in functioning due to loss of skeletal muscle, with the highest impact in those with sarcopenia and frailty. The advantages of adding anabolic exercise to the diet attenuate this side effect, whereas aerobic exercise does not. Other benefits include more significant weight loss, preservation of fat-free mass (both muscle and bone), preservation of resting metabolic rate (when PRT is included), improved fitness levels, correction of metabolic abnormalities associated with visceral obesity, and better long-term adherence to dietary modifications. Therefore, exercise including PRT plus diet appears to be an optimal evidence-based treatment for obesity in individuals of all ages. In general, weight loss parallels energy expenditure via exercise, whether achieved by greater volume, intensity, or duration of the exercise prescription. There is no evidence from well-designed studies that low-intensity exercise effectively reduces abdominal fat. Most robust studies used moderate- to high-intensity aerobic interventions, and higher-intensity stimuli can be delivered via intermittent intensities with resistance or interval training.","Journal of Nutrition, Health and Aging",3.2 Adipose Tissue and Physical Exercise,2025
"Exercise Intensity, HIIT vs MICT, and Role of Energy Restriction","A recent meta-analysis comparing body composition changes between HIIT and MICT suggested that exercise intensity minimally influences longitudinal changes in fat mass and lean mass. These findings underscore the importance of exercise volume (and resulting energy expenditure) in facilitating fat mass loss. However, the amount of exercise required to achieve practically meaningful fat loss (~100 min/day) is not feasible for most of the general public and therefore has limited practical relevance. Dietary energy restriction plays a vital role in creating an energy deficit and facilitating fat mass loss. Exercise may help preserve lean mass and functional performance during periods of energy restriction and should be considered an essential complement to nutritional strategies for those seeking to alter body composition.","Journal of Nutrition, Health and Aging",3.2 Adipose Tissue and Physical Exercise,2025
Aerobic vs Resistance Training for Fat Reduction,"There is some evidence that aerobic training may be better than PRT at reducing abdominal fat. However, at doses resulting in a sustained negative energy balance for several months, resistance and aerobic exercises generally result in significant reductions in fat mass when sensitive measurement techniques (not anthropometrics) are used. Resistance exercise may be more suitable as a fat-reduction strategy for obese older individuals with cardiovascular disease, osteoarthritis, osteoporosis, or mobility limitations, who may not tolerate moderate- to high-intensity aerobic training or may need the added benefits of PRT for maintaining muscle and bone mass. Importantly, energy restriction results in significant loss of muscle and bone. The addition of PRT to hypocaloric dieting has been shown to prevent such adverse changes in body composition, which are not attained with aerobic exercise alone. The combination of aerobic and PRT has demonstrated superiority in reducing trunk fat in older men compared to aerobic training alone. Further well-designed studies are needed, particularly in overweight older adults, to explore the relative benefits of these exercise modes in optimizing body composition.","Journal of Nutrition, Health and Aging",3.2 Adipose Tissue and Physical Exercise,2025
Exercise and Muscle Mass Preservation With Aging,"In contrast to the changes in fat and bone, a significant increase in muscle mass is achievable only with progressive PRT or weight gain from extra energy and protein consumption. The accretion of lean tissue with exercise has a potentially beneficial effect in preventing diabetes and metabolic syndrome, functional dependency, falls, and fractures as well as in the treatment of chronic diseases and disabilities that are often accompanied by disuse, catabolism, and sarcopenia. For persons with T2D, there are potential advantages to minimizing fat and maximizing muscle tissue because these compartments have opposite and likely independent effects on insulin resistance. Resistance exercise coupled with leucine-enriched essential amino acid supplements (when the diet is inadequate in energy and protein provision) is recommended to treat sarcopenia. Various epidemiological and experimental studies have shown that muscle weakness, decreased muscle mass, reduced activation of glycogen synthase, and alterations in the numbers of glycolytic skeletal muscle fibers are related to, and may precede, insulin resistance, glucose intolerance, and T2D.","Journal of Nutrition, Health and Aging","3.3 Muscle Mass, Strength, Energetics & Exercise",2025
Resistance Training to Maintain or Increase Muscle Mass,"A properly designed PRT program can counteract age-related changes in contractile function, atrophy, and morphology of the aging human skeletal muscles. Appropriate progressive PRT programs of 3–6 months duration can increase muscle strength by an average of 40–150%, depending on the person’s characteristics and intensity of the program, and increase total body lean mass by 1–3 kg or muscle fiber area by 10–30%. Exercise training reduces frailty in older adults by suppressing muscle inflammation and promoting anabolism, thereby increasing muscle protein synthesis rate. Thus, even if some of the neural control of muscles and the absolute number of motor units are not affected by exercise, adaptation to muscle loading causes neural, metabolic, and structural changes in muscles, which can compensate for strength losses and, in some cases, age-related atrophy. Strength gains after exercise often exceed changes in muscle size due to neural adaptation, particularly in early training phases. High-load PRT is more beneficial than low-intensity training for maximizing muscle and bone mass/strength and treating gait disorders, functional impairments, and disability. It is ideal as a multiple-risk factor intervention strategy for injurious fall prevention in osteopenic adults.","Journal of Nutrition, Health and Aging","3.3 Muscle Mass, Strength, Energetics & Exercise",2025
Predictors of Muscle Hypertrophy After Exercise,"There is mixed evidence regarding whether there are significant sex differences in the functional or hypertrophic response to PRT in older adults, heavily influenced by whether results are presented in absolute or relative terms. Some studies have found that women have smaller gains in muscle strength, power, or hypertrophic response, whereas others have shown no differences or even more significant gains. Differences in training regimens (mainly intensity) and measurement techniques used to assess muscle mass, cross-sectional area, or volume may explain discrepant findings. Malnutrition, impaired protein synthesis rates, inflammatory cytokines, and depression are other factors detrimental to robust anabolic and functional adaptations to PRT. The roles of genetic and epigenetic factors remain under investigation.","Journal of Nutrition, Health and Aging","3.3 Muscle Mass, Strength, Energetics & Exercise",2025
"Skeletal Muscle Energetics, Mitochondrial Function, and Exercise","Mitochondria play a crucial role in cellular redox balance, calcium handling, metabolism, and ATP production. Declining mitochondrial function is frequently described as a characteristic of skeletal muscle aging. However, debate continues regarding the nature of age-associated mitochondrial dysfunction, with several studies finding no effect of age on mitochondrial oxidative capacity. Some older athletes even show healthier mitochondria than young non-exercisers. Studies observing age effects often fail to consider covariates such as PA level, cardiovascular fitness, and adiposity. Regardless, loss of mitochondrial energetics contributes to slower walking speed, fatigability, multimorbidity, frailty, and sarcopenia. Endurance and resistance exercise are the only proven measures to improve muscle health and mitochondrial bioenergetics in older adults. Resistance training remains the most effective way to counteract the progression of sarcopenia. In 1990, Fiatarone et al. showed that an 8-week high-intensity PRT program in frail institutionalized nonagenarians led to a 9% increase in mid-thigh muscle area.","Journal of Nutrition, Health and Aging","3.3 Muscle Mass, Strength, Energetics & Exercise",2025
Resistance and Endurance Training Effects on Mitochondria,"Although aerobic training improves myofiber size and strength, PRT is more effective for increasing muscle size and muscle fiber cross-sectional area in older adults. Studies examine varying intensities and durations of PRT and the effects of nutritional supplements. Whey protein supplementation combined with PRT results in larger muscle mass gains, particularly in low-functioning older adults. Resistance training also reduces muscle mass loss during hypocaloric weight loss, whereas aerobic exercise does not. Resistance training was previously thought not to affect mitochondrial content or function, but strong evidence now shows that PRT increases mitochondrial protein fractional synthesis rates and improves mitochondrial function. Improvements in mitochondrial respiration from PRT are modest compared with endurance training. Yet in older adults, improvements in phosphocreatine recovery rates and oxidative capacity appear comparable between modalities. Resistance exercise increases state III mitochondrial respiration and maximal oxidative phosphorylation capacity in older adults, concurrent with improved ADP sensitivity.","Journal of Nutrition, Health and Aging","3.3 Muscle Mass, Strength, Energetics & Exercise",2025
"Endurance Exercise, Mitochondrial Biogenesis, and Protection Against Sarcopenia","Endurance exercise induces mitochondrial biogenesis, first demonstrated by Holloszy in 1975. Studies consistently show improvements in mitochondrial content and function through increased mitochondrial turnover, including increased protein synthesis and mitophagy. Improved mitochondrial efficiency and antioxidant capacity reduce oxidative stress and proteome damage. Enhanced mitochondrial function sustains greater energetic flux, decreases lipotoxic intermediates, and reduces inflammation and oxidative modification of proteins. Thus, aerobic exercise improves cellular integrity and adaptability to stress, and enhances the ability of muscle to provide energy on demand for mobility in older adults. However, as shown by Klitgaard in 1990, older recreationally strength-trained men (mean age 68) had muscle morphology and function comparable to young controls, whereas older habitual runners and swimmers resembled sedentary older men. This and other evidence convincingly show that aerobic exercise alone cannot prevent sarcopenia.","Journal of Nutrition, Health and Aging","3.3 Muscle Mass, Strength, Energetics & Exercise",2025
"Exercise in Primary, Secondary, and Tertiary Disease Prevention","Physical activity, particularly exercise, can reduce the burden of comorbidity, disability, and premature death caused by incident disease and is valuable for primary, secondary, and tertiary prevention. Exercise patterns may vary with age and genotype, influencing physiological capacity, psychological health, dietary intake, adverse behaviors, and risk factors. These are the potential pathways through which exercise can affect the prevalence of chronic diseases in a population. Although optimal levels of PA can ameliorate risk profiles, the presence of risk factors may lead to reduced PA, thereby increasing disease risk. For example, inactivity can lower muscle mass, followed by muscle weakness and further reduction of activity levels, subsequently contributing to osteoporosis, gait abnormalities, and, ultimately, a high risk of falls and hip fractures. Preventive exercise prescriptions for middle-aged sedentary adults with low fitness markedly reduce cardiovascular mortality, suggesting that exercise in middle age can be as effective as activities started at a younger age to reduce mortality. Empirical data have shown that exercise can prevent some diseases (e.g., secondary cardiovascular events, diabetes mellitus, and osteoporotic fractures).","Journal of Nutrition, Health and Aging",4. Exercise in Disease Prevention,2025
"Exercise for Disease Prevention: Diabetes, Cardiometabolic Disorders, Mortality","Evidence confirms epidemiological risk reduction for other conditions (renal failure, stroke, dementia, and depression). Based on findings from the Finnish Diabetes Study, the Diabetes Prevention Program (DPP), and similar trials, diabetes can be prevented in high-risk obese adults with impaired glucose tolerance through diet and exercise interventions. In the DPP, participants assigned to an intensive lifestyle intervention including diet and exercise reduced their risk of incident T2D by 58% at three years compared to the control group, and this intervention was significantly better than metformin prescription. Notably, those older than 60 years showed the best response, with a 71% reduction in incident diabetes during this timeframe. By contrast, metformin was no more effective than the control condition in older adults. Although metformin is often subsidized by some governments and health insurance plans for diabetes prevention in older adults (despite its lack of efficacy in this cohort), proven long-term lifestyle interventions are not covered. Table 5 lists the major diseases and syndromes for which exercise could serve as a preventive strategy or aid in disease prevention and the specific modality of exercise most relevant for these outcomes.","Journal of Nutrition, Health and Aging",4. Exercise in Disease Prevention,2025
Exercise in Secondary and Tertiary Prevention,"Exercise is particularly effective in targeting syndromes of disuse and decelerating the trajectory of decline, notably in conditions such as Parkinson’s disease, chronic obstructive pulmonary disease, and cardiometabolic disorders. Some disease-related pathophysiological abnormalities are specifically addressed by exercise, making it a valuable adjunct to pharmacological treatment. Muscle-derived myokines are known for their beneficial effects of promoting a healthy anti-inflammatory and anabolic environment. Excess adipose tissue is associated with inflammation. Loss of visceral fat through resistance or aerobic training improves insulin resistance and complements dietary and pharmacological management in older adults with T2D and central obesity. Regular exercise fosters anti-atherogenic changes in vascular function and structure independent of traditional CVD risk factors. Exercises that stimulate skeletal muscle hypertrophy in congestive heart failure counteract the catabolic effects of circulating cytokines, which cannot be achieved by available medications. Lower extremity exercises in individuals with osteoarthritis improve joint stability.","Journal of Nutrition, Health and Aging",4. Exercise in Disease Prevention,2025
"Exercise, Chronic Disease Management, and Geriatric Syndromes","It is not feasible to discuss every disease for which exercise has beneficial effects. Type 2 diabetes, cancer, cognitive impairment, dementia, and mental health are prototypical examples. The role of exercise interventions in geriatric syndromes (frailty, falls, and sarcopenia) is also essential for preventing and treating disability, counteracting iatrogenic diseases, and improving outcomes in acute hospitalized older persons. Table 5 outlines the recommended exercise modalities, considerations for exercise prescription, and disease-specific mechanisms by which exercise influences outcomes across arthritis, cancer, COPD, chronic renal failure, congestive heart failure, coronary artery disease, dementia, depression, osteoporosis, peripheral vascular disease, stroke, type 2 diabetes mellitus, and venous insufficiency. These mechanisms include improvements in body composition, enhanced vascular function, reductions in inflammation, improved glycemic control, increased muscle mass and strength, and reductions in visceral adiposity. Exercise contributes significantly to secondary and tertiary disease management by improving functional independence, reducing falls, enhancing psychological well-being, and counteracting metabolic disturbances.","Journal of Nutrition, Health and Aging",4. Exercise in Disease Prevention,2025
Exercise in Type 2 Diabetes: Limitations of Diet Alone and Need for Combined Approaches,"Targeting glycemic control without simultaneously addressing central obesity and a sedentary lifestyle may hasten the emergence of disease complications and add to the burden of polypharmacy in individuals with insulin resistance. Weight loss due to diet alone in older adults with obesity leads to loss of lean tissue (muscle and bone), exacerbating age-related sarcopenia and osteopenia. Many consensus and position statements recommend moderate- to high-intensity aerobic exercise for 3–4 h per week to improve insulin sensitivity and glucose homeostasis, assist in maintaining lower body weight, reduce visceral fat, modestly improve blood pressure and lipids, and lower the risk of cardiovascular morbidity and mortality in individuals with T2D. However, the clinical management of obese individuals with T2D is often complicated by multiple comorbid conditions, such as cognitive impairment, osteoarthritis, ischemic heart disease, peripheral vascular disease, renal failure, peripheral neuropathy, and hypertension, which may impede adherence to dietary and aerobic exercise recommendations. This clustering makes implementation of guidelines challenging because performing aerobic exercise at the volumes or intensities needed for metabolic benefits is unrealistic for many individuals.","Journal of Nutrition, Health and Aging",4.1 Disease-Specific Exercise Interventions,2025
"Resistance Training in Type 2 Diabetes: Functional, Metabolic, and Clinical Benefits","An alternative approach in adults with diabetes is PRT. The specific benefits for older adults include combating age- and diabetes-related sarcopenia, preventing loss of muscle and bone mass, reduced resting metabolic rate accompanying hypocaloric dieting, increased glucose uptake and storage in skeletal muscle, reduced visceral fat, lowered C-reactive protein, and improved resting blood pressure, functional status, mobility, sleep, and depressive symptoms. The effects on muscle mass are distinct from those of aerobic exercise and are essential for preventing dementia in older adults with diabetes, although more evidence is needed. Current recommendations include aerobic and PRT and dietary modifications for T2D. In older adults with coexisting frailty, diabetes, and functional decline, evidence from a large RCT shows functional benefits with a combined approach of PRT, nutritional education, and readaptation of clinical targets for glycosylated hemoglobin and blood pressure. Benefits were evident early (week 8) and continued long-term (12–24 months). A multicomponent exercise program consisting of resistance, endurance, balance, and gait training is also recommended to increase functional capacity and quality of life and to avoid falls, institutionalization, and disability. Strategies to develop skeletal muscle power in this population are essential to prevent or delay functional limitations and subsequent disability.","Journal of Nutrition, Health and Aging",4.1 Disease-Specific Exercise Interventions,2025
"Cancer: Disease Burden, Treatment Side Effects, and Functional Decline","Cancer is a group of diseases characterized by abnormal growth and spread of cells due to multiple genetic mutations. Cancer cells often accumulate and adhere together to form a tumor at the site of initial carcinogenesis. These tumors can shed cancer cells that metastasize throughout the body and form colonies at distant sites. Once widely disseminated, cancer cells invade and destroy organs and tissues. In 2020, over 19 million people were diagnosed with cancer, projected to reach 28 million by 2040. Cancer fatalities have also followed this trend. Cancer is a disease of older adults, with approximately 80% of all diagnoses in people over 50 and 60% in those over 65 in highly developed countries. Cancer treatments—including surgery, radiation, chemotherapy, endocrine therapy, immunotherapy, and targeted therapy—often involve multiple lines administered sequentially or concurrently. Side effects affect all organ systems and include nausea, vomiting, diarrhea, mucositis, peripheral neuropathy, fatigue, depression, cardiotoxicity, cognitive dysfunction, and lymphedema. Cancer and its treatments can accelerate aging, impair fitness, reduce muscle strength and balance, worsen body composition, and lead to sarcopenia and frailty.","Journal of Nutrition, Health and Aging",4.1 Disease-Specific Exercise Interventions,2025
"Exercise Benefits in Cancer: Treatment Tolerance, Function, and Clinical Outcomes","Exercise assists cancer patients in preparing for treatments, managing treatments, recovering afterward, and improving long-term outcomes. It benefits health-related fitness, physical functioning, some treatment side effects, psychosocial functioning, treatment tolerance, treatment response, and possibly survival. Exercise can prevent and reverse sarcopenia and sarcopenic obesity in cancer patients, with benefits even in frail individuals. Counteracting catabolic effects of steroids or androgen deprivation therapy requires specific attention to anabolic exercise (PRT) to prevent body composition deterioration. Countries and organizations worldwide have issued exercise guidelines for cancer patients. ASCO recommends that oncology providers prescribe both aerobic and resistance exercises during curative treatment to mitigate side effects. ACSM recommends at least 150 min/week of aerobic exercise plus two days per week of strength exercise. ACSM also provides symptom-specific prescriptions. Risks and contraindications must be considered, such as fracture risk in bone metastases, fall risk due to neuropathy or cognitive dysfunction, and worsening of fatigue or hand-foot syndrome. Exercise prescriptions must be tailored carefully, particularly during cumulative toxic treatments, to achieve precision exercise oncology.","Journal of Nutrition, Health and Aging",4.1 Disease-Specific Exercise Interventions,2025
"Exercise, Cognition, and Dementia Risk","Both observational and experimental studies show PA and exercise can positively impact a broad range of cognitive functions in older individuals with and without cognitive impairment. Age-related cognitive dysfunction may be influenced by suboptimal PA participation across the life course. Reduced PA and sedentary behavior are precursors of cardiometabolic diseases and systemic inflammation that contribute to cognitive decline. Evidence also suggests a link between sarcopenia and various neurocognitive disorders. Cross-sectional and longitudinal studies consistently demonstrate that higher lifetime PA levels are associated with a lower likelihood of cognitive impairment and a reduced risk of dementia. For instance, in a prospective cohort study, walking reduced dementia risk in a dose-dependent manner, with walking less than 0.25 miles per day compared to walking more than 2 miles per day. Evidence further supports a dose-dependent inverse relationship between PA levels and the risk of mild cognitive impairment (MCI) and dementia.","Journal of Nutrition, Health and Aging","4.1.3–4.1.5 Exercise, Cognitive Impairment, Dementia",2025
Multidomain Interventions and Cognitive Benefits,"Multidomain interventions that incorporate physical exercise have emerged as promising non-pharmacological strategies for dementia prevention. These interventions integrate exercise with cognitive training, nutritional guidance such as vitamin B, vitamin D, and omega-3 supplementation, socioemotional support, and cardiovascular risk factor management. By addressing multiple modifiable risk factors simultaneously, these programs have shown modest but meaningful benefits in mitigating cognitive decline among older adults at high risk for dementia. The FINGER trial, a two-year lifestyle intervention involving 1,190 older adults, demonstrated significant improvements in global cognitive function, executive function, and processing speed. A meta-analysis evaluating long-term effects of sustained exercise and multidomain interventions found inconclusive results for exercise alone but small significant cognitive benefits for multidomain approaches. A trend toward a protective effect of exercise on dementia risk was observed, though exercise's role in preventing MCI remains uncertain.","Journal of Nutrition, Health and Aging","4.1.3–4.1.5 Exercise, Cognitive Impairment, Dementia",2025
Mechanisms Linking Exercise and Brain Health,"Effects of exercise on cognition are partially mediated by structural and functional adaptations in the brain, including changes in gray matter volumes and white matter microstructural integrity. Exercise attenuates cognitive decline through mechanisms involving increased cerebral blood flow, higher levels of neurotrophic factors such as BDNF and IGF-1, reduced levels of neurotoxic factors including C-reactive protein, cortisol, and IL-6, and modulation of inflammatory cytokines. Exercise also contributes to better control of chronic diseases such as stroke, diabetes, cardiovascular disease, and depression. Progressive resistance training induces long-term structural brain changes in older adults as shown by MRI and fMRI. While aerobic exercise is traditionally recommended to improve cognition, resistance and mind-body exercises such as Tai Chi have demonstrated significant cognitive benefits. High-intensity PRT in the SMART study improved global and cognitive function in older adults with MCI, associated with increases in posterior cingulate cortex thickness and hippocampal preservation.","Journal of Nutrition, Health and Aging","4.1.3–4.1.5 Exercise, Cognitive Impairment, Dementia",2025
Exercise in Alzheimer’s Disease and Established Dementia,"Exercise decelerates cognitive decline in patients with Alzheimer’s disease and reduces the behavioral and psychological symptoms of dementia. Although many studies of older adults with dementia show improvements in physical function, cognitive gains are less consistent. Exercise programs addressing falls, frailty, sarcopenia, depression, osteoporosis, and cardiometabolic diseases are recommended in aged care settings and dementia cohorts. The cognitive frailty construct reflects coexisting physical frailty and potentially reversible cognitive impairment without dementia. Four months of low-intensity high-speed PRT using elastic bands improves cognitive and physical performance in older adults with MCI. Additional beneficial exercises include multicomponent and dual-task training. Multicomponent high-speed PRT combined with walking and balance exercises improves gait, balance, and strength and reduces falls in frail patients with dementia, including those previously restrained.","Journal of Nutrition, Health and Aging","4.1.3–4.1.5 Exercise, Cognitive Impairment, Dementia",2025
Multicomponent Exercise in Dementia and Training Considerations,"Research in older adults with cognitive impairment demonstrates the feasibility and efficacy of multicomponent exercise programs combining cognitive training, nutrition strategies, and social enrichment. However, effectiveness over single-mode exercise remains uncertain. Proper supervision is crucial, as there is no evidence that low-intensity, minimally progressive multimodal exercise improves cognition in mild or moderate dementia. Additional recommendations include addressing dementia-related behavioral issues and communication challenges. Simplified instructions, reassurance, and mirror techniques help promote exercise adherence. Creating a respectful and empathetic training atmosphere improves participation. Evidence-based guidelines that combine mindful caregiving with progressively intense resistance and balance training for caregiver–patient dyads are available at strongmindshomecare.org.","Journal of Nutrition, Health and Aging","4.1.3–4.1.5 Exercise, Cognitive Impairment, Dementia",2025
Exercise and Depression Risk Reduction,"The risk of incident depression is estimated to be 21% lower in older adults who engage in PA, especially at moderate to vigorous intensities. A dose-response relationship has been observed, where an activity volume equivalent to 2.5 h of brisk walking per week is associated with a 25% lower risk of depression, and half that dose yields an 18% lower risk than no activity. Most benefits are achieved when moving from no activity to at least one activity. Similar effects are observed in older adults with other mental health symptoms, such as anxiety. PA is linked to positive psychological attributes and a reduced prevalence and incidence of depressive symptoms, which are most significant in those with comorbid illnesses such as CVD, pulmonary disease, and major depression. Despite numerous trials documenting the benefits of physical exercise for older adults, the integration of PA and exercise programs into routine medical treatment remains ad hoc.","Journal of Nutrition, Health and Aging",4.1.6 Exercise and Mental Health,2025
Exercise as Treatment for Clinical Depression,"Evidence of exercise as an isolated intervention for treating clinical depression across age groups is robust and consistent. Both aerobic and PRT exercises have produced clinically meaningful improvements in depression, with response rates ranging from 25 to 88%. Aerobic, resistance, and mind-body exercises, including yoga, are equivalent in mitigating symptoms of depression in older adults. PRT was comparable to aerobic training in young adults with depression, and yoga was as effective as aerobic exercise. Blumenthal et al. compared high-intensity aerobic exercise with antidepressant medications in older adults with major depression and found the two approaches were equipotent, with no added benefit from combining exercise and medication, and a reduced remission rate in the aerobic exercise-only group. Singh et al. found that high-intensity PRT produced a clinical response in 61%, compared to 29% with low-intensity PRT and 21% in a usual-care control group.","Journal of Nutrition, Health and Aging",4.1.6 Exercise and Mental Health,2025
"Exercise Dose, Anxiety Reduction, and Adherence Challenges","Low-intensity aerobic training in older adults with depression is similar in efficacy to social-contact controls, reducing depression scores by only 30%, equivalent to placebo. A single exposure to exercise can improve mood in depressed individuals and act as short-term symptom relief during exacerbations. Evidence supports the anxiety-reducing effects of exercise in older adults and those with multiple comorbidities. Trials have focused mainly on moderate-intensity aerobic exercise such as walking or cycling. Progressive resistance training has also been shown to reduce anxiety symptoms in older adults. Tsutsumi et al. found that high and moderate-intensity PRT induced benefits in anxiety after 12 weeks in sedentary older adults compared to non-exercising controls. However, non-adherence to exercise prescriptions, particularly non-compliance with exercise dose, remains a challenge. Practitioners should identify risk factors for low adherence, monitor compliance, address barriers, and promote self-efficacy and motivation during exercise interventions.","Journal of Nutrition, Health and Aging",4.1.6 Exercise and Mental Health,2025
Biological Mechanisms and Choosing Exercise Modalities for Depression,"Overall, the literature suggests exercise is effective for depression in younger and older adults and is as effective as antidepressants in clinical cohorts. Aerobic and resistance modalities appear equally beneficial, with optimal responses observed at higher resistance training intensities. The mechanism of the antidepressant effect is likely multifactorial, involving neurotransmitters, inflammatory cytokines, neuroendocrine pathways, dopamine processing, oxidative stress, neuroplasticity, reward processing, body image, self-efficacy, and functional independence. Dose-response characteristics for aerobic exercise and intensity requirements for PRT support exercise as a potent biological agent, where specific parameters such as intensity and volume play crucial roles. Ultimately, choice of exercise modality depends on comorbidities. For example, PRT is particularly suitable for older adults with depression plus frailty, sarcopenia, mobility impairments, osteoporosis, or recurrent falls—as it improves depressive symptoms while also enhancing strength, bone density, cognition, and reducing fall risk.","Journal of Nutrition, Health and Aging",4.1.6 Exercise and Mental Health,2025
Overview of Exercise Effects on Geriatric Syndromes,"Geriatric diseases and syndromes such as frailty, sarcopenia, and falls significantly affect the health and well-being of older adults. Exercise interventions have emerged as effective non-pharmacological strategies for complex, multifactorial conditions that do not fit traditional disease models. Evidence shows that structured exercise can modify risk factors, restore function, and improve outcomes across multiple geriatric domains. Frailty, sarcopenia, mobility impairments, and fall risk all respond to targeted exercise approaches that combine resistance, power training, balance work, and functional tasks. Table 7 outlines the major syndromes for which exercise is beneficial, explaining mechanisms and the most relevant exercise modalities. These include improvements in muscle mass and strength, better neuromuscular coordination, enhanced gait stability, and reductions in fear of falling. Multicomponent programs integrating resistance, balance, and gait training are repeatedly shown to address multiple deficits simultaneously. Exercise also contributes to psychological and cognitive resilience, making it a cornerstone intervention for geriatric syndromes.","Journal of Nutrition, Health and Aging",4.2 Effects of Exercise Interventions on Geriatric Syndromes,2025
Frailty: Mechanisms and Exercise Strategies,"Frailty is a state of reduced physiological reserve resulting in vulnerability to stressors, disability, and mortality. It spans cognitive, social, and physical domains. Resistance training programs and multicomponent interventions with robust PRT have repeatedly shown improvements in muscle strength among older adults with frailty and sarcopenia. Numerous studies demonstrate that exercise, either as a single modality or part of a multicomponent intervention, can prevent or reverse frailty. These programs typically include low-to-moderate intensity aerobic and resistance training, balance work, and gait retraining. Multicomponent interventions improve key frailty components such as poor balance, reduced muscle strength, impaired gait, and increased fall risk. Programs such as VIVIFRAIL provide structured training materials to help clinicians prescribe exercise for older adults. Explosive resistance training (power training) has special relevance in frailty due to its effects on functional capacity, muscle power, balance, and reduction of fall risk. Studies in institutionalized nonagenarians show improvements in muscle CSA, fat infiltration, strength, gait, and sit-to-stand performance after 12 weeks. In contrast, low-load or unsupervised home-based exercises result in minimal strength gains due to inadequate overload.","Journal of Nutrition, Health and Aging",4.2 Effects of Exercise Interventions on Geriatric Syndromes,2025
Sarcopenia: Evidence and Exercise Prescription,"Sarcopenia involves progressive loss of muscle mass, strength, and function and is closely linked to frailty, falls, disability, cognitive decline, and mortality. International clinical guidelines consistently recommend progressive resistance training as the primary treatment for sarcopenia. Evidence shows that PRT improves strength, muscle power, gait speed, functional capacity, and skeletal muscle index across community, hospital, and institutional settings. No pharmacological therapy has yet demonstrated clinically meaningful improvements in physical performance. PRT also prevents or ameliorates sarcopenia in individuals with T2D, sarcopenic obesity, and cancer. Combining PRT with whey protein supplementation yields greater muscle mass gains than PRT alone, especially in low-functioning older adults. Power training recommendations detailed in earlier sections reinforce the need for progressive, high-quality mechanical loading to support hypertrophy and functional improvement. Together, these findings establish PRT as the most effective, evidence-based therapy for managing and preventing sarcopenia in older adults.","Journal of Nutrition, Health and Aging",4.2 Effects of Exercise Interventions on Geriatric Syndromes,2025
"Falls: Evidence, Effective Modalities, and Implementation Challenges","Exercise is the most effective intervention to prevent falls and fall-related injuries in older adults. World guidelines identify robust balance and strength training as essential components. Multicomponent programs combining resistance training, balance exercises, functional training, and Tai Chi can reduce falls by 20–40%. However, programs that rely primarily on resistance exercise without balance work, dance interventions, or gait retraining show uncertain evidence. Walking alone may increase fracture risk in osteoporotic women, underscoring the importance of modality selection. USPSTF reviews show moderate-certainty evidence that exercise yields a moderate net benefit in preventing falls among high-risk older adults. Higher exercise doses (≈3 hours/week) that target balance produce up to a 42% reduction in fall rates. Despite strong trial evidence, many community programs fail due to insufficient intensity and challenge. Large trials like STRIDE and DO-HEALTH showed no benefit because prescribed exercises lacked adequate load or balance challenge. Digital delivery of balance programs, such as the Standing Tall RCT, has shown partial success, reducing falls by 16–20% over 24 months. Sustaining long-term adherence remains the primary barrier, as falls prevention exercise must be maintained lifelong in high-risk individuals. Effective implementation strategies are now a greater challenge than defining successful exercise modalities.","Journal of Nutrition, Health and Aging",4.2 Effects of Exercise Interventions on Geriatric Syndromes,2025
Exercise Needs and Barriers in Nursing Home Residents,"Nursing home residents are typically frail, multimorbid, and often live with dementia and multiple disabilities, creating highly complex exercise needs. Mobility impairment is common, highlighting the large potential benefits of appropriately structured physical activity. Understanding each resident’s motivation, preferences, and individual limitations is essential when promoting physical activity. Frailty and advanced dementia are especially prevalent, yet a systematic review shows that only 60–67% of nursing home residents receive physiotherapy services. Chair-based exercises are widely used due to perceived safety and lower risk of adverse events, and evidence shows they can produce task-specific improvements in physical and cognitive outcomes, contributing to enhanced well-being. Numerous randomized controlled trials have evaluated exercise in long-term care residents. A systematic review of ten RCTs targeting residents with dementia found that nine reported improvements or slower decline in mobility, physical function, or functional limitations, though many studies had methodological weaknesses. A larger meta-analysis of 105 randomized trials (n = 7759) found that exercise interventions significantly improved physical function across cognitive and functional levels. Key benefits included increased independence in activities of daily living, improved muscle strength, gait, balance, flexibility, and physical performance. The most consistent improvements occurred with approximately three hours of weekly exercise, with little difference between types of exercise modalities.","Journal of Nutrition, Health and Aging",4.3 Exercise in Nursing Home Residents,2025
Evidence for Multicomponent Programs and Progressive Resistance Training,"High-quality trials demonstrate meaningful benefits of structured exercise programs in nursing home settings. A rigorous French RCT providing twice-weekly one-hour exercise sessions for a year showed a significantly slower decline in Activities of Daily Living performance versus controls. Another systematic review identified 30 trials studying either multicomponent exercise (19 studies) or progressive resistance training (12 studies). Seventeen of these were included in a meta-analysis, revealing that both multicomponent training and PRT effectively improved physical performance in institutionalized older adults. Improvements were consistently shown in standardized measures such as the Short Physical Performance Battery, the 30-second Chair Stand test, Timed Up and Go, and gait ability. These programs have the potential to ameliorate frailty and reduce fall risk. The Otago Exercise Program (OEP) has been evaluated in nine nursing home studies and has demonstrated improvements in postural control, lower limb strength, and short-distance functional mobility. A meta-analysis of 14 trials emphasized balance training as a key component for reducing falls in nursing home residents. The VIVIFRAIL multicomponent program has also shown promise for improving or maintaining physical frailty in vulnerable institutionalized populations. However, not all trials have found reductions in falls, likely due to differences in program intensity, fidelity, and adherence, underscoring the importance of robust implementation.","Journal of Nutrition, Health and Aging",4.3 Exercise in Nursing Home Residents,2025
"Nutrition, Cognition, and Implementation Challenges","Combining exercise with nutritional supplementation has produced mixed results. The VIVE2 trial found that six months of exercise alone improved gait speed, while adding whey protein and vitamin D did not further enhance physical function, although muscle density increased. Two additional trials similarly reported no further functional gains from adding dietary supplements to physical activity programs. Exercise also supports cognitive health, mental well-being, and perceived quality of life among nursing home residents, demonstrating its broad therapeutic value. The primary barrier to implementing effective, high-intensity exercise programs remains structural: insufficient financial reimbursement, lack of trained exercise professionals, and limited access to proper equipment. The FRIEND trial (Frailty Reduction via the Implementation of Exercise, Nutrition and Deprescribing) addressed these challenges by applying the Asia-Pacific Frailty Management Guidelines within residential aged care. This required establishing an exercise gym with PRT equipment, hiring Accredited Exercise Physiologists, redesigning nutrition services, and training staff in deprescribing and anabolic exercise principles. The program produced improvements in physical function, fall rates, drug burden, and body weight, demonstrating the substantial potential of comprehensive interventions combining exercise, nutrition, and medication optimization for frail nursing home residents.","Journal of Nutrition, Health and Aging",4.3 Exercise in Nursing Home Residents,2025
"Physical Activity, Disability Trajectories, and Risk Factors","Physical activity plays a crucial role in shaping disability trajectories in older adults. Data from large cohorts such as EPESE show that active older individuals are more likely to survive to age 80 or beyond and have nearly half the risk of dying with disability compared to sedentary peers. Longitudinal Study of Aging findings similarly demonstrate that regular physical activity slows the progression of functional limitations and delays the transition to ADL and IADL disability. Many risk factors for disability overlap with correlates of inactivity, including older age, female sex, minority status, low education, and lower income. Psychosocial features such as social isolation, low self-esteem, low self-efficacy, anxiety, depressive symptoms, smoking, and harmful alcohol intake contribute to vulnerability. Body composition changes associated with inactivity—sarcopenia, obesity, visceral fat accumulation, and bone loss—further impair gait stability, balance, mobility, and lower extremity function. Reduced exercise capacity, including declines in strength, endurance, power, balance, and flexibility, is frequently observed in disabled older adults. Although cross-sectional designs limit causal inference, chronic inactivity contributes to disease risk and functional decline across obesity, osteoarthritis, cardiovascular disease, stroke, osteoporosis, and type 2 diabetes. Disability emerges from complex interactions among sensory deficits, impaired glycemic control, psychological factors, musculoskeletal limitations, and comorbid disease expression. Exercise directly addresses many of these pathways and represents a central non-pharmacological strategy for modifying the onset and progression of disability.","Journal of Nutrition, Health and Aging",4.4 Disability Prevention and Treatment,2025
Exercise Interventions to Modify Disability Risk and Progression,"Evidence from large randomized trials demonstrates that exercise alters the trajectory of disability in frail older adults. One of the most comprehensive RCTs randomized 704 nursing home residents to resistance, balance, aerobic training, nursing rehabilitation, or usual care. After 17 months, residents in both exercise and nursing rehabilitation facilities experienced significantly less decline in activities of daily living compared to controls. The LIFE trial (n = 1635) showed a significant reduction in major mobility disability over 2.6 years among older adults randomized to structured physical activity versus health education. Similarly, the SPRINTT trial (n = 1519) showed that among older adults with physical frailty and sarcopenia, a multicomponent program combining resistance, endurance, balance, and nutrition counseling reduced the incidence of mobility disability, with the greatest effect observed in those with low baseline SPPB scores (≤7). Exercise interventions are also effective in disease-specific disability populations, including individuals with osteoarthritis, COPD, CVD, stroke, and depression. In knee osteoarthritis, numerous studies using weight-bearing functional exercises, walking programs, and progressive resistance training consistently demonstrate improvements in pain, function, gait, balance, and quality of life. Land-based exercise is superior to aquatic activity and passive stretching despite misconceptions regarding its tolerability in this population. Disability reduction in osteoarthritis is mediated through enhanced muscle strength, gait efficiency, reduced pain, improved mood and self-efficacy, and better management of comorbid conditions. Overall, exercise serves as a primary and adjunctive therapy across chronic diseases contributing to disability, aligning with WHO recommendations for musculoskeletal and mobility-related conditions.","Journal of Nutrition, Health and Aging",4.4 Disability Prevention and Treatment,2025
Customized Exercise Prescriptions Amidst Pharmacotherapy,"Customized exercise prescriptions should be designed to address specific drug interactions and enhance overall health. Balance and resistance exercises can compensate for orthostatic hypotension, peripheral neuropathy, and gait and balance impairments. Aerobic exercise can alleviate constipation, whereas aerobic and resistance exercises benefit depression, fatigue, and cognitive impairment. Where feasible, aerobic or resistance exercise should substitute for psychotropics or analgesics [8] [537]. Aerobic or resistance exercises can help manage blood pressure and cardiovascular health for patients taking multiple antihypertensives, beta-blockers, and nitrates. For individuals with diabetes, it is critical to adjust for insulin and other glycemic agents in conjunction with PA [591]. Proper exercise timing after meals is crucial for maintaining glycemic balance, enhancing glucose utilization, and preventing hyperglycemia or post-activity hypoglycemia. Exercise can also reduce the need for NSAIDs in the treatment of osteoarthritis and chronic pain syndrome. Addressing drug-nutrient interactions, such as vitamin B12 deficiency from metformin or proton-pump inhibitors and reduced calcium and vitamin D absorption from corticosteroids through balance and power training, can mitigate these adverse effects [559–562].","The Journal of Nutrition, Health and Aging",Exercise-Drug Interactions,2025
Exercise to Offset Cognitive and Mobility Side Effects of Medications,"Importantly, exercise can substitute for medications that impair cognitive function and mobility. Psychotropic medications (antipsychotics, anxiolytics, insomniacs, antidepressants), which can cause confusion, sedation and fatigue, may be replaced with aerobic or PRT, significantly enhancing a patient’s physical and cognitive health. Similarly, for patients who cannot to cease medications that impair mobility or mental function, exercises such as balance, resistance, or aerobic training should be prescribed to offset these side effects [540–543].","The Journal of Nutrition, Health and Aging",Exercise-Drug Interactions,2025
Nutritional Considerations in Exercise–Drug Regimens,"Nutritional considerations are also crucial for managing drug-exercise interactions, yet this three-way framework is not commonly appreciated. For example, to prevent falls and cognitive decline, potential vitamin B12 deficiency due to medications should be screened for in those with peripheral neuropathy taking metformin or proton-pump inhibitors (PPIs), addressed with balance training and supplementation if needed [559–562], and consideration given to alternatives to PPIs. Resistance and power training should be used to counteract anabolic deficits in bones and muscles due to protein-calorie undernutrition and/or catabolic drugs [537]. Drug-induced hypoglycemia in diabetes should be managed by adjusting exercise timing and intensity, adjusting dosages of insulin on exercise days when needed to ensure safe glucose levels, and using snacks and available monitoring tools [553–558]. Exercising about one hour after a meal is ideal for management of post-prandial hyperglycemia with no risk of hypoglycemia.","The Journal of Nutrition, Health and Aging",Nutrition–Exercise–Drug Interactions,2025
"Exercise, Nutrition, and Management of Catabolic Effects","Resistance exercise is essential for preventing and treating chronic conditions such as osteoporosis in COPD or rheumatic diseases, and where catabolic drugs are often used, complicating the anorexia, cachexia and inflammation of the diseases themselves, protein supplementation may be needed as well [592,593]. Balance training, aerobic exercise, and increased hydration and fiber intake can help manage side effects of anticholinergic and other drugs causing constipation. Incorporating nutritional support may further improve the benefits of exercise in malnourished individuals, those with catabolic or inflammatory diseases or those taking medications adversely affecting muscle mass/function, such as androgen deprivation therapy. Ensuring adequate protein intake, vitamin D supplementation when needed, adequate calcium intake, high-intensity resistance and power training can help mitigate catabolic effects on muscle and bone [579,580]. The complexity of age-related nutritional needs, multiple co-morbidities, polypharmacy, and exercise specificity require this holistic oversight, as summarized in Table 8 and Figs 5, 6 and 7.","The Journal of Nutrition, Health and Aging",Nutrition–Exercise–Drug Interactions,2025
Prioritizing Exercise in Treatment Plans,"Exercise as a central component of treatment plans addresses primary health concerns and enhances overall resilience and well-being. For example, aerobic exercise can reduce the severity of depression and anxiety, often managed with psychotropics, while improving cardiovascular health. Resistance training can mitigate the muscle-wasting effects of corticosteroids and enhance bone density while simultaneously addressing primary and secondary health concerns [537]. Fig. 7 examines the diverse effects of medication on physical ability. It highlights particular side effects, such as sarcopenia and osteoporosis, which are prevalent in the older population and presents evidence-based modifications for exercise regimens. This figure illustrates how PRT and other forms of exercise can effectively alleviate these effects, improve physical outcomes, and promote general health and well-being.","The Journal of Nutrition, Health and Aging",Exercise as Treatment,2025
Interindividual Variability and Dose-Response Heterogeneity,"Dose-response heterogeneity is not exclusive to pharmaceutical treatments; it is also applies to the physiological decline associated with aging [594]. Similarly, there is significant variability in the physiological, performance, and health-related adaptations of individuals undergoing the same exercise training program [595]. Various factors, such as intensity, duration, frequency, mode of exercise, functional status, adherence, and age, determine the effectiveness of exercise training interventions. Other responses to exercise training can be influenced by factors such as macronutrient intake and genetics [596]. The concept of heterogeneity in response to exercise training gained attention in the 1980s [597], with studies focusing on the trainability of sedentary adults. Understanding the causes and potential strategies to manage interindividual variability and dose-response heterogeneity during exercise is essential. This knowledge has profound implications for public health.","The Journal of Nutrition, Health and Aging",Exercise Response Variability,2025
Understanding Variability in Exercise Response,"Physiological adaptations to chronic exercise have been shown to enhance several age-related health outcomes, demonstrating a positive impact of exercise on the fundamental biology of aging [598,599]. Interindividual variability and dose-response heterogeneity to exercise can be influenced by various factors, both intrinsic (e.g., sex, race, ethnicity, age, genotype) and extrinsic (e.g., comorbidities, functional capacity, diet, medications, recovery sleep, stochastic factors) [600]. Exercise has benefits for many clinically important outcomes in older adults, such as reducing fall risk, cardiovascular disease, and death. However, despite engaging in exercise at the same relative intensity, not only young but also older populations exhibit significant variations in acute physiological responses to exercise and the time to task failure [601]. Understanding the threshold and optimal levels of activity necessary for health promotion and disease management has become increasingly important in recent years [602].","The Journal of Nutrition, Health and Aging",Exercise Response Variability,2025
Challenges in Classifying Responders and Non-Responders,"A significant hurdle in understanding training response variability is the need for a standardized definition for classifying individuals as responders or non-responders. This challenge is compounded by technical difficulties in accurately measuring the physiological responses to many exercise protocols. To determine the impact of exercise on overall disease risk, disease pathobiology, health status, and outcomes, some studies have classified a fixed proportion of the lowest training response, absolute changes in pre- to post-intervention values, and changes of more than one standard deviation [603,604]. More recently, it has been suggested that technical error, minimal clinically important differences, and the combination of day-to-day biological variability and measurement error should be considered when categorizing response rates [600]. Consensus is yet to be reached on the ideal analytical approach for studying exercise response variation, which is a critical unmet need that must be addressed.","The Journal of Nutrition, Health and Aging",Exercise Response Variability,2025
Intrinsic and Extrinsic Factors Influencing Training Response,"Significant efforts have been made to explain variations in training response variability by assessing intrinsic (non-modifiable) and extrinsic (modifiable) factors [605]. Meyer et al. [606] systematically explored the impact of biological factors on the variability of cardiorespiratory fitness (V̇O2 max) responses, finding that sex and age collectively account for less than 10–16% of the V̇O2 peak response [607]. Furthermore, cardiac autonomic recovery, as measured by heart rate variability, provides a practical assessment of physical recovery, although it does not reflect all physiological systems affected by exercise. Precisely, baseline cardiovascular autonomic function, characterized by high-frequency power (indicating high vagal activity) [608], has emerged as one of the strongest predictors of V̇O2 peak response, accounting for 27% of the response after eight weeks of aerobic training and 34% after 12 weeks of interval training [609]. Another possible reason for non-response is insufficient training stimuli, such as intensity or specificity to intervention, sex-related differences in response to exercise, and baseline physical fitness levels.","The Journal of Nutrition, Health and Aging",Exercise Adaptation Predictors,2025
Outcome-Specific Responses and Research Needs,"The literature has shown age-related declines in physical function, muscle mass, and metabolic efficiency and wide heterogeneity in exercise response among older adults [90]. It is essential to note that a physiological non-response to exercise in one outcome does not equate to a non-response in other outcomes. It is well recognized that physiological and phenotypic responses to exercise are highly variable and depend significantly on the outcome of interest. An individual may benefit from an exercise intervention that differs from the chosen response variable. For instance, a person may experience an improvement in V̇O2 peak, without a corresponding reduction in fasting glucose levels, despite showing significant improvements in HbA1c, waist circumference, and body fat percentage across all subjects [610]. Which outcome is most important depends on an individual’s current health status and health-related goals. However, no study has comprehensively assessed all possible parameters determining exercise response variability in older adults, including extrinsic and intrinsic factors. Therefore, while response heterogeneity poses a challenge for investigators, it also allows one to explore its mechanistic basis, highlighting the importance of continued research in clinical settings when studying non-responsiveness to exercise [611].","The Journal of Nutrition, Health and Aging",Exercise Response Variability,2025
Genetic Predisposition and Variability in Exercise Benefits,"Understanding the benefits of exercise in older adults is mainly based on typical standardized responses from small sample sizes with restricted or prescribed exercise regimens and considering a limited number of outcomes [90]. Furthermore, researchers have examined the individual interactions of physiological and molecular factors (such as genetics, epigenetics, transcriptomics, and metabolic factors) and environmental factors as potential mediators of the lack of response to exercise in certain participants [612]. From a public health viewpoint, a high V̇O2 peak is a proven negative predictor of CVD and overall mortality, and thus a major target of global exercise recommendations, yet V̇O2 peak varies more than twofold among sedentary individuals, supporting the existence of significant genetic or other contributions to V̇O2 peak in addition to the influence of PA patterns. Additionally, studies designed to investigate the response variability in aerobic training adaptation have reported significant differences across cohorts, supporting the heritability of V̇O2 peak. For example, investigating the genetic contributions to changes in cardiorespiratory fitness in response to a 20-week standardized exercise program, Bouchard et al. [93] estimated that 47% of the variation of V̇O2 peak response was genetically determined.","The Journal of Nutrition, Health and Aging",Genetics and Exercise Response,2025
Genetic Architecture of Aerobic Training Response,"In a subsequent report, a genome-wide association study (GWAS) suggested that multiple genes influence V̇O2 peak trainability, each with minor effects; however, much remains to be discovered regarding this variability in the response [613]. Recent data show that the variation in V̇O2 peak changes among individuals ranges from -4.7 to 47.8% [614]. Similarly, other clinical outcomes in older adults exhibit heterogeneity in the magnitude of their exercise response. For instance, in the HEalth, RIsk factors, exercise Training And GEnetics (HERITAGE) Family Study involving 316 women and 280 men (173 black and 423 white) and healthy sedentary individuals, approximately 42% of the participants showed no change in glycemic control indices after completing a 20-week exercise program on cycle ergometers three times a week for 60 sessions [615]. Similarly, a secondary analysis of the Functional Improvement from Aerobic Training in Alzheimer’s Disease (FIT-AD trial), which aimed to evaluate the effects of aerobic exercise on community-dwelling older adults with mild-to-moderate dementia due to Alzheimer’s disease, revealed interindividual differences in aerobic fitness and cognitive responses to aerobic exercise [616].","The Journal of Nutrition, Health and Aging",Genetics and Exercise Response,2025
Variability Across Clinical Populations and Non-Response Rates,"Whipple et al. [617] reported a high prevalence of non-responders among individuals with peripheral artery disease, with or without T2D, who completed at least two-thirds of their prescribed exercise sessions. When non-response was defined as a negative or no change in the 6-minute walk test distance, its prevalence was 35%. However, when defined as a lack of clinically meaningful change (20 meters), non-response prevalence was as high as 56% [618]. Despite these varying responses, all participants improved in at least one outcome, and only one individual improved across all measures. Physiological, performance, and health-related adaptations to PRT also display considerable heterogeneity. Previous studies have shown either no responsiveness or substantial gains in the whole-muscle cross-sectional area (ranging from -2% to +59%) and maximal dynamic strength (ranging from 0% to +250%) [91,92]. Heritability estimates for general muscle strength range from 30% to 60%, with the overall heritability of strength-related phenotypes estimated at approximately 50% [91,92,619]. More than 200 polymorphisms have been linked to strength/power phenotypes, particularly in athletic performance [620].","The Journal of Nutrition, Health and Aging",Genetics and Exercise Response,2025
Gene–Training Interactions and Strength Adaptations,"At baseline, individuals with the CC genotype of the PPARGC1A Gly482Ser (rs8192678) had lower 1-RM values compared to T-allele carriers. However, after eight weeks of maximal strength training, C-allele carriers showed a greater improvement in 1-RM compared to those with the TT genotype [621]. A systematic review of genetic influences on functional adaptations to aerobic or PRT in older adults identified seven studies that measured ten single-nucleotide polymorphisms and nine different functional performance test outcomes. The ACE (D), ACTN3 (RR), UCP2 (GG), IL-6-174 (GG), TNF-a-308 (GG), and IL-10-1082 (GG) genotypes all predicted significantly superior adaptations in at least one functional outcome in older men and women after prescribed exercise or in those with higher levels of PA [622]. Although genetics have been shown to play a significant role in this variability, research utilizing candidate gene or GWAS approaches has yet to conclusively identify genetic predictors that fully explain the intrinsic biological mechanisms driving individual variability in response to exercise. While specific genetic variants and the proteins they code for have been identified, it remains unclear whether these proteins are solely responsible for the variability in exercise response.","The Journal of Nutrition, Health and Aging",Genetics and Exercise Response,2025
Beyond Genetics: Environmental and Epigenetic Influences,"These findings suggest that other factors, such as environmental or epigenetic influences, may counteract any genetic 'handicap'. Therefore, overemphasizing genetics when tailoring exercise prescriptions may be counterproductive [623]. Identifying the genetic predictors of blunted adaptations to exercise may enhance our ability to target individuals at risk of poor outcomes by recommending advanced training techniques, better behavioral strategies, or physiological augmentation via nutritional or pharmacological co-interventions. An important research direction involves the identification of the genetic factors contributing to variations in responsiveness to exercise across clinically relevant endpoints, which could lead to more precise and effective exercise prescriptions to optimize the clinical benefits of exercise training in older adults.","The Journal of Nutrition, Health and Aging",Genetics and Exercise Response,2025
Exercise as a Geroscience Intervention,"Physical activity and exercise are associated with a reduced risk of developing most several chronic diseases, geriatric syndromes, and disabilities during aging [41,284,502,624–628]. Physical activity and exercise benefits apply to conditions characterized by very distinct pathophysiology, such as Alzheimer’s disease [629], cancer [628], heart disease [630], and pneumonia/other infections [631,632]. One possible explanation for such pleiotropic effects is that PA and exercise positively affect resilience mechanisms that oppose aging and exert positive effects by decelerating the pace of aging. This would prevent or delay the onset of different clinical conditions (e.g., several diseases, frailty, and disability) and reduce their severity. Thus, PA and exercise may be considered central geriatric care interventions, that is, interventions that act on the cellular and molecular drivers of biological aging to decelerate the rates of aging, which constitute the basics of the geroscience field [633,634].","The Journal of Nutrition, Health and Aging",Exercise and Geroscience,2025
Biological Mechanisms Through Which Exercise Slows Aging,"Several reviews have discussed the biological mechanisms through which PA and exercise may modify the pace of aging [635–638], including their benefits for immune function [639,640] (in particular, adaptive immune function), low-grade chronic inflammation, their effects on mitochondrial biogenesis and mitophagy [641,642], and as regulators of multiple metabolic cascades important to aging, such as insulin/IGF1, Akt-mTOR, AMPK [638], sirtuins, and PGC1a. A seminal work by Contrepois et al. [643] found that a simple amount of acute exercise (and up to 1-h recovery after the training in individuals aged 40–75 years) induced substantial changes in more than half (n = 9,816 analytes) of the 17,662 analytes evaluated through a multi-omics approach. Many of these pathways are central to aging biology, including mitochondrial stress markers (e.g., GDF-15), immune function pathways, inflammatory cytokines (e.g., IL-6, TNF-a), oxidative stress markers (e.g., myeloperoxidase), and mTOR signaling.","The Journal of Nutrition, Health and Aging",Exercise and Geroscience,2025
Age Dependency and Challenges in Exercise Geroscience Research,"Epidemiological data and biological investigations converge, indicating PA and exercise as major geriatric care interventions. However, most data linking exercise to the main drivers of biological aging have been obtained from animal experiments or human studies on young adults. Only a few investigations employed a sample with a significant age range covering adulthood or recruited older people, challenging the generalizability of findings to the aging process. While the magnitude of the response of the biological mechanisms of aging to PA and exercise depending on age range is unknown, knowledge of human physiology suggests that adaptive and resilience mechanisms tend to decrease over time during aging. The specificity and age dependency of the response to exercise could have important consequences for prevention strategies. The prescription of exercise in gerotherapeutic counseling may need to be different for a specific range of ages, with certain PA programs more suited for different life stages. This underscores the need for continued investment in translational research on exercise interventions for older adults.","The Journal of Nutrition, Health and Aging",Exercise and Geroscience,2025
Limitations of Current Evidence and Need for Long-Term Protocols,"Most investigations used one bout of acute exercise or an observational design, which makes it challenging to establish the best exercise regimen for decelerating the pace of aging, which may depend heavily on how gradually the intensity of exercise increases over time. Non-pharmacological prevention strategies in aging must be long term, especially because exercise in very old individuals may not be without risks. This requires strategies that consider acceptability, adherence, and safety procedures to minimize adverse effects. No previous investigation has gathered evidence on how exercise affects the main determinants of aging using prospective experimental exercise trial designs. Therefore, to provide initial pragmatic guidance on exercise regimen as a gerotherapeutic intervention, trials testing the effects of exercise on biological drivers of aging were examined [7].","The Journal of Nutrition, Health and Aging",Exercise and Geroscience,2025
Effects of Exercise on Inflammation and Other Aging Biomarkers,"Meta-analyses on both modalities [645,646] or either aerobic exercise [647,648] or PRT alone [649,650] found that exercise was able to reduce the levels of traditional biomarkers of chronic inflammation, such as CRP, IL-6, and TNF-a, including in the context of age-related diseases such as T2D [651], heart failure [652], and mild cognitive impairment and dementia [653]. Multicomponent training (aerobic plus strength) appeared to offer the best results. Evidence suggests that exercising 3–5 times a week at moderate-to-vigorous intensity for at least 25 minutes per session over 12 weeks or more can reduce inflammatory biomarkers of aging and disease.","The Journal of Nutrition, Health and Aging",Exercise and Aging Biomarkers,2025
"Exercise Effects on Mitochondria, Senescence, and Muscle Quality","Evidence on other biological determinants of aging is emerging: exercise increases mitochondrial biogenesis (PRT) [654], oxidative metabolism (cycling-aerobic exercise [655]), reduces cellular senescence [72,73], and improves muscle oxidative capacity [656]. A well-designed RCT by Colleluori et al. showed that 6-month multicomponent training in obese older adults was more effective than aerobic or PRT alone for improving muscle protein synthesis and myocellular quality, despite mitochondrial biogenesis being highest in the aerobic group. A recent meta-analysis (n = 164) concluded that exercise improves mitochondrial quality, density, and oxidative capacities [657], although evidence remains limited. PRT and multicomponent training also improved IGF1 levels in older people with sarcopenic obesity [474,658–660].","The Journal of Nutrition, Health and Aging",Exercise Effects on Aging Biology,2025
Exercise and Cellular Aging Pathways,"Improvements in autophagy markers were found in older adults engaging in either aerobic exercise [661] or PRT [662], including increases in phosphorylated ULK-1, beclin-1, Atg12, and Atg16. Resistance training also reduced differentiated CD8 T-cells [663], while aerobic exercise and HIIT reduced p16INK4a levels in young adults [664,665], supporting the senescence-targeting effect of exercise. Aerobic exercise increased telomerase activity [666], and multicomponent training improved gut microbiota composition [667]. Despite heterogeneity in interventions, multicomponent training appears to yield the broadest anti-aging benefits.","The Journal of Nutrition, Health and Aging",Exercise Effects on Cellular Aging,2025
Toward Practical Exercise Recommendations for Healthy Aging,"Although evidence does not allow precise recommendations for all elements of the exercise regimen, it is plausible that exercising 3–5 times per week at moderate-to-vigorous intensity for at least 25 minutes per session and at least 8–12 weeks provides a reasonable balance between feasibility and effectiveness for slowing biological aging. A key message is that any physical activity is better than none, and greater amounts yield greater benefits. Observational evidence shows mortality reductions even at activity levels below recommended thresholds [65,628,668], with benefits following a curvilinear dose-response pattern.","The Journal of Nutrition, Health and Aging",Exercise Guidelines for Aging,2025
Research Gaps and Life-Course Considerations,"Several gaps remain. The optimal exercise protocol across the life course is unknown, and benefits from short-term interventions may dissipate with detraining. Long-term protocols with intermittent supervised training (e.g., 12-weeks/year in a 3-year study) should be examined. Determining whether exercise should be intensified at mid-life is crucial, as many age-related diseases begin their pathogenesis decades before clinical symptoms, making middle-aged adults a key target population for intensive exercise strategies.","The Journal of Nutrition, Health and Aging",Exercise and Geroscience Research Gaps,2025
Economic Burden of Physical Inactivity in Older Adults,"The global healthcare system incurs over $50 billion in costs due to physical inactivity, underscoring the critical importance of PA programs in reducing health-related expenses [669–671]. These economic effects encompass direct healthcare expenditures and indirect financial impacts such as reduced productivity, costs of informal caregiving, and losses in human capital due to early mortality. Aging is associated with increased healthcare costs, which increase with declining health and functional ability [672–674]. Research has shown that very old individuals with lower functional capacities face greater healthcare costs than their more physically capable peers [659], with these expenses being notably higher for those in care facilities [672]. Therefore, improving functional capacity and health in institutionalized older adults could significantly impact healthcare spending. Conditions common in geriatrics, such as sarcopenia, frailty, cognitive impairment, and falls, escalate healthcare costs.","The Journal of Nutrition, Health and Aging",Cost-Effectiveness of Exercise,2025
Economic Burden of Cognitive Impairment and Falls,"Older adults with cognitive deficits are more frequently hospitalized [675] and admitted to care facilities [676], and dementia patients require more complex, prolonged, and expensive care [677,678]. Falls result in a substantial economic burden owing to hospitalization and long-term rehabilitation costs [679,680]. Consequently, the cost-effectiveness of exercise interventions designed to prevent falls in older adults has been extensively studied [681]. It was shown that the OEP, when delivered to individuals over 80 years and targeted at high-risk groups, can prevent the most significant number of falls at the lowest incremental costs [681]. Hewitt et al. [682] found that an exercise program encompassing strength and balance exercises is cost-effective in preventing falls in institutionalized older adults aged 65–100 years. Earlier work reported reduced healthcare costs in community-dwelling older adults participating in outdoor PA interventions aimed at fall prevention, resulting in fewer acute care and rehabilitation admissions and lower expenditures on allied health and community services [683].","The Journal of Nutrition, Health and Aging",Cost-Effectiveness of Exercise,2025
Healthcare Cost Reduction Through Exercise in Institutionalized Elders,"Bays-Moneo et al. [684] evaluated the economic impact of two exercise interventions versus usual care among nonagenarian nursing home residents. A tailored multicomponent exercise program emphasizing power training remarkably reduced monthly healthcare costs for institutionalized oldest-old individuals (mean change = -€330.43), while costs in the usual care group significantly increased (mean change = +€300.00 over 12 months). A daily calisthenics group maintained monthly mean costs during the intervention and had lower healthcare costs after 12 months compared to usual care. Both exercise interventions reduced associated care needs (bathing, dressing, walking, medication assistance, physiotherapy) and reduced hospital stays. The multicomponent program incurred higher implementation costs (€672.00/year) than calisthenics (€134.40/year), mainly due to its personalized nature.","The Journal of Nutrition, Health and Aging",Cost-Effectiveness of Exercise,2025
National-Level Economic Evaluations of Exercise Programs,"A comprehensive German study involving over 1.5 million individuals aged 70 and older compared two exercise interventions targeting fall prevention: a tailored individual program (""LIFE"") and a group-based program (""gLIFE"") [685]. Over five years, both programs prevented 2,692 deaths and 618,060 falls. The tailored intervention incurred substantially higher costs (€510 million) compared to the group-based approach (€186 million), and neither intervention achieved cost savings compared to standard care. However, cost savings were more pronounced among participants with higher care needs, particularly in formal care, outpatient treatment, and inpatient rehabilitation. This emphasizes that while fall prevention initiatives target a broad population, risk varies greatly and more targeted approaches may improve cost-effectiveness.","The Journal of Nutrition, Health and Aging",Cost-Effectiveness of Exercise,2025
Economic Benefits of Diverse and Multimodal PA Programs,"Several studies underscore the economic benefits of diverse PA programs for older adults [682,686,687]. Multicomponent exercise regimens and supervised initiatives often prove more economically viable than single-modality programs. Combining exercise with other interventions, such as PRT within multimodal approaches (exercise, nutrition, education), has demonstrated economic benefits. Rodriguez-Mañas et al. [277] reported a significant reduction in healthcare costs—primarily via reduced hospitalization—amounting to €420 per patient over one year compared to controls. The development and implementation of cost-effective exercise programs are essential public policy objectives, given the increasing economic burden of aging populations.","The Journal of Nutrition, Health and Aging",Cost-Effectiveness of Exercise,2025
Need for More Research and Policy Implications,"Despite promising findings, data on the cost-efficiency of tailored exercise programs remain limited. More extensive research is urgently needed to evaluate long-term financial benefits. Preliminary evidence suggests that tailored programs may be prudent investments to improve health outcomes later in life. To manage expenses and enhance outcomes for older patients, healthcare systems and long-term care facilities must integrate structured PA into their services as an immediate priority.","The Journal of Nutrition, Health and Aging",Cost-Effectiveness of Exercise,2025
Implementation Science and Physical Activity in Older Adults,"Older adults, a diverse group with varying physiological capacities, frequently lead sedentary and inactive lifestyles despite the well-documented advantages of physical activity for health. Understanding the culturally specific factors influencing exercise adherence across different societies and socioeconomic backgrounds is essential to promote regular physical activity. This understanding is particularly critical in the fields of geriatrics and public health. Implementation science, which examines how evidence-based interventions can be integrated into routine practice and policy, is crucial in addressing barriers and enhancing facilitators to exercise adherence among older adults [688]. The Consolidated Framework for Implementation Research (CFIR) presents a comprehensive and structured approach, encompassing intervention characteristics such as the strength of evidence, adaptability, and complexity, as well as the inner setting (e.g., culture, readiness, resources), outer setting (e.g., patient needs, incentives, public health policy), characteristics of individuals involved (e.g., knowledge, attitudes, beliefs, perceptions), and the implementation process (e.g., planning, engagement, evaluation [689]. Researchers and practitioners can employ CFIR principles to systematically identify barriers and facilitators in exercise interventions for older adults, allowing for the development of targeted strategies to enhance the uptake, sustainability, and effectiveness of exercise programs tailored for older populations.","The Journal of Nutrition, Health and Aging",Exercise Implementation Science,2025
Behavior Change Models and Exercise Determinants,"The application of behavior change theory is crucial in encouraging physical activity among older adults. The Capabilities, Opportunities, Motivations, Behavior (COM-B) model, which considers an individual's capability, opportunity, and motivation, helps to understand the determinants that influence exercise behavior in this population [690]. This model emphasizes the interplay between physical ability, social support, and personal motivation in shaping exercise habits among older adults. Both participants and healthcare professionals encounter facilitators and barriers that impact exercise adherence. Older adults' barriers to exercise adherence include low self-confidence, competing priorities, perceived risks, and limited perceived benefits. Healthcare providers, on the other hand, face challenges such as time constraints, uncertainty in the referral process, and reimbursement issues, which can hinder their ability to prescribe effective exercise regimens [691]. Additionally, external factors such as community resources, social support availability, and policy frameworks significantly influence the uptake of exercise recommendations among frail older adults.","The Journal of Nutrition, Health and Aging",Exercise Implementation Science,2025
"Barriers, Facilitators, and Strategies to Improve Adherence","Older adults often face challenges in engaging in regular physical activity due to intervention-specific factors such as the need for tailored interventions based on individual profiles and health conditions, the importance of intensity, dosage (considering factors like pain, cognition, frailty, and falls), and social support. These challenges can be addressed by implementing comprehensive strategies that include tailored interventions, enhancing social support, educating participants, and empowering them to maintain exercise engagement [690]. In summary, promoting exercise adherence in older adults requires a multifaceted approach that considers both individual and systemic factors. By addressing barriers and leveraging facilitators through the integration of implementation science and behavior change theory, healthcare professionals and policymakers can help promote healthier aging through regular physical activity.","The Journal of Nutrition, Health and Aging",Exercise Implementation Science,2025
Importance of Physical Activity for Healthy Aging,"Given the increasing prevalence of older adults worldwide, effective interventions that promote functional capacity and prolong the number of years lived without disabilities are vital when evaluating the impact of life-extending interventions on the health span of older adults. Insufficient PA and sedentary behaviors are potent risk factors for a range of age-related health issues, including all-cause and cardiovascular mortality, obesity, sarcopenia, frailty, and disability, as well as other chronic illnesses. On the other hand, adopting a healthy diet, engaging in regular physical activity, avoiding smoking, consuming alcohol in moderation, and maintaining an appropriate body mass index can significantly contribute to overall health and well-being across all age groups. Regular physical activity and structured exercise can help counteract age-related declines in physical, cognitive, and psychological health, extending health span and improving quality of life.","The Journal of Nutrition, Health and Aging",Exercise and Healthy Aging,2025
Biological Mechanisms and Disease Prevention,"Exercise participation influences various biological mechanisms, such as chronic inflammation, mitochondrial dysfunction, myokine release, autophagy, oxidative damage, and insulin-like growth factor signaling. Engaging in physical activity and exercise can improve physical function and reduce the burden of non-communicable diseases and premature mortality, including cause-specific mortality from cardiovascular disease, cancer, and chronic lower respiratory tract diseases. Exercise should be viewed as a form of treatment, with prescriptions tailored to specific outcomes, such as primary prevention, enhanced fitness or functional status, or disease treatment. Just like any other medical intervention, it is crucial to personalize, adjust, and manage these prescriptions, considering external (exercise variables) and internal (acute response to exercise) factors influenced by personal, genetic, functional, psychosocial, and environmental factors.","The Journal of Nutrition, Health and Aging",Exercise Mechanisms and Disease Prevention,2025
Exercise Modalities and Personalized Prescriptions,"Exercise recommendations for achieving health-related outcomes must consider the relationship between dose and response, volume, intensity, and the specific adaptations necessary for the desired outcomes. For instance, resistance, aerobic, balance, and mobility training can specifically target age-related deficits. Multicomponent exercise programs that integrate cognitive tasks effectively improve frailty characteristics, such as low muscle mass, strength, endurance, mobility, PA level, and energy, while also enhancing cognition to optimize functional capacity during aging. The development of wearable technology has facilitated personalized exercise regimens by monitoring physiological responses in real time. Omic technologies, encompassing phenomics, metagenomics, metabolomics, proteomics, and genomics, enable multi-omic approaches crucial for identifying molecular transducers of exercise, revealing substantial interindividual variability, and suggesting evidence-based doses for treating both healthy and unhealthy populations.","The Journal of Nutrition, Health and Aging",Exercise Prescription Personalization,2025
"Polypharmacy, Medication Interactions, and Exercise","Polypharmacy and potentially inappropriate medications (PIMs) in older adults often overlook the importance of exercise prescription. The connection between medication use and PA/exercise offers a promising approach to enhancing the well-being of older adults. By combining exercise prescriptions with pharmacotherapy, a comprehensive strategy can optimize vitality and functionality while minimizing adverse pharmaceutical reactions. Exercise can serve as a better alternative for managing the side effects of medications, as it often provides benefits beyond the targeted condition and shifts the risk-benefit balance in favor of exercise. Exercise can lead to dose reductions or substitute some potentially hazardous drugs whenever possible, enhancing overall safety and efficacy.","The Journal of Nutrition, Health and Aging",Exercise and Medication Interactions,2025
Barriers to Exercise Integration in Healthcare Settings,"Despite the numerous advantages of exercise, its integration into medical practice for older people remains limited. Many healthcare professionals, including geriatricians, lack adequate training to incorporate exercise directly into patient care. Although progress has been made, exercise counseling is often reserved for those without significant physical or mental limitations and typically includes mild activities at dosages not supported by evidence. This cautious approach stems from unfounded fears of exercise-related injuries or perceived risks of vigorous activities in older adults, even though the true danger lies in sedentariness. Integrating evidence-based, comprehensive, and adaptable exercise programs into all healthcare settings—including community and institutional environments—is crucial for promoting healthy aging and addressing the growing burden of non-communicable diseases linked to inactivity.","The Journal of Nutrition, Health and Aging",Exercise Implementation Challenges,2025
Societal and Policy-Level Strategies for Promoting Physical Activity,"Promoting PA in older adults is a multifaceted societal issue that involves geriatricians, family doctors, fitness professionals, policymakers, health agencies, insurance companies, and urban planners. Effective promotion of physical activity requires a collaborative approach and the development of environments and policies that encourage active lifestyles among older adults. This consensus calls for evidence-based exercise programs tailored to the needs and capabilities of older adults, ensuring that these programs are comprehensive and adaptable to individual health conditions. The ultimate goal is enhanced quality of life, regardless of age or initial state of fitness or frailty.","The Journal of Nutrition, Health and Aging",Exercise Policy and Public Health,2025
Melatonin Decline and Neurodegeneration,"The synthesis and release of melatonin in the brain harmonize various physiological functions. The apparent decline in melatonin levels with advanced aging is an aperture to the neurodegenerative processes. It has been indicated that down regulation of melatonin leads to alterations of circadian rhythm components, which further causes a desynchronization of several genes and results in an increased susceptibility to develop neurodegenerative diseases. Additionally, as circadian rhythms and memory are intertwined, such rhythmic disturbances influence memory formation and recall. Besides, cell cycle events exhibit a remarkable oscillatory system, which is downstream of the circadian phenomena. The linkage between the molecular machinery of the cell cycle and complex fundamental regulatory proteins emphasizes the conjectural regulatory role of cell cycle components in neurodegenerative disorders such as Alzheimer’s disease. Among the mechanisms intervening long before the signs of the disease appear, the disturbances of the circadian cycle, as well as the alteration of the machinery of the cell cycle and impaired neurogenesis, must hold our interest.",Current Neuropharmacology,Melatonin,2023
Introduction to Alzheimer’s Pathogenesis and Circadian Disruption,"Several theories have been postulated with respect to the pathogenesis of Alzheimer’s disease (AD), a neurodegenerative disorder presenting a very peculiar conformity in terms of initiation of pathology and symptomology. The neurodegenerative countenances befall years before the symptoms show up, with the formation of senile plaques and neurofibrillary tangles being contemplated as its characteristic pathological hallmarks. Given the prevalence of rhythmic anomalies during aging and in neurodegenerative pathologies, the molecular circadian network directly influences the course of such states, thereby supporting the hypothesis that circadian disruption is one major symptom in neurodegenerative diseases. Indeed, circadian rhythm upheaval is precisely involved in the etiopathogenesis and progression of AD, and alterations in this rhythm could conceivably presage the onset of dementia.",Current Neuropharmacology,Melatonin,2023
"Circadian System, SCN, and Neurodegenerative Links","The zeitgebers-like light synchronizes the daily rhythms, which are generated in the suprachiasmatic nucleus (SCN) located in the ventral hypothalamus. Circadian rhythms are commanded by this principal pacemaker that controls the coordinated peripheral oscillators and regulates the physiological, biochemical, and behavioural activities. Studies have shown that apart from the hypothalamus, other regions in the brain, such as the hippocampus and the cortex, possess oscillators capable of generating circadian rhythms. The crosstalk between the circadian clock and cellular signalling thereby contributes to the synchronization of circadian oscillations and physiological homeostasis. It is interesting to point out that abnormal circadian rhythms are one of the characteristic features of not only AD but also of several other neurodegenerative diseases, including Parkinson’s disease and Huntington's disease, one extremely important aspect of the fundamental function of circadian clocks being the regulation of proteostasis, where altered daily rhythms could significantly influence and enhance pro-neurodegenerative molecular aggregations.",Current Neuropharmacology,Melatonin,2023
Circadian Disruptions as Early Markers and Therapeutic Targets,"Moreover, the occurrence of circadian rhythm disruptions before the onset of typical clinical symptoms of neurodegeneration suggests that such rhythmic alterations are early events that might also be a potential risk factor for developing AD and PD. The circadian system, therefore, could be a new diagnostic and therapeutic target in neurodegenerative disorders. As such, clinical trials become essential to determine whether circadian interventions could prevent or delay the onset of AD and related dementias and if modulating the circadian state of brain cells susceptible to neurodegeneration could represent a possible anti-AD therapeutic track. One additional important point is that adult neurogenesis, which is altered in AD, is under the control of the circadian system, thereby suggesting that restored neurogenesis could assist future innovative circadian rhythms-synchronizing treatments in alleviating AD-related cognitive symptoms.",Current Neuropharmacology,Melatonin,2023
Cell Cycle Dysregulation in Alzheimer’s Disease,"The cell cycle is a highly regulated process that maintains a homeostatic balance between cell proliferation and cell death and that consists of four phases G1 (cell growth), S (DNA replication), G2 (cell growth) and M (mitosis), the progression of which is under the control of cell cycle regulators that coordinate the transition from one phase to another. Cell cycle oddity is an early feature of AD that has been extensively evidenced in the aetiology of the disease as manifesting abnormal cell cycle re-entry that precedes the pathological hallmarks of AD. Thus, the neuronal cell cycle re-entry in humanized Ab plaque-producing AppNLF knock-in mice results in the development of subsequent AD-related pathological events, including tauopathy, neuroinflammation, brain leucocytes infiltration, DNA damage response and neurodegeneration.",Current Neuropharmacology,Melatonin,2023
Neuronal Cell Cycle Reactivation and Apoptosis,"Neurons generally remain in the G0 phase (quiescent state), but toxic insults and different kinds of stressors force neurons to exit G0 and induce cell cycle re-entry leading to cell death via apoptosis. Cell cycle reactivation in adult neurons is a sign of neurodegeneration and central nervous system lesions, and the cell-cycle hypothesis of AD considers this disease a consequence of deregulation of the cell cycle in neurons. Although studies in rodents and primates have indicated that regulation of the cell cycle plays a crucial role in controlling area-specific neuron production, an aberrant reactivation of the cell cycle in these neurons initiates the complex process of apoptosis, which likely contributes to AD development and is associated with the cardinal hallmarks of AD including tau hyperphosphorylation and amyloid beta (Aβ) accumulation along with consequent degeneration of neurons.",Current Neuropharmacology,Melatonin,2023
Melatonin as a Circadian Regulator and Therapeutic Molecule,"Melatonin is a key output of the circadian clock, a circadian regulator and plays a major role in peripheral clock synchronization due to its strong circadian expression pattern. Under direct control from the SCN, the pineal gland synthesizes and releases melatonin into circulation in response to darkness, where its secretion is known to be suppressed by light. Also, animal studies showed that retinal melatonin plays an important role in the regulation of retinal daily and circadian rhythms. The most direct route by which melatonin can reach the SCN is considered to be via the cerebrospinal fluid of the third ventricle. Melatonin can also reach the pars tuberalis of the pituitary, another melatonin-sensitive tissue, via this route. Melatonin, in turn, acts on the SCN by directly influencing the circadian clock mechanisms and by controlling circadian rhythms in association with molecular timing genes.",Current Neuropharmacology,Melatonin,2023
Melatonin Receptors and Implications for Alzheimer’s Therapy,"The hypothalamic high-affinity G protein-coupled melatonin receptors MT1 and MT2 are involved in the regulation of circadian rhythms, suggesting clinical applications of melatonin receptor-targeting drugs. Moreover, findings from preclinical and clinical studies revealed some roles of melatonin in the control of the circadian clock-associated genes, thus suggesting that the hormone could have beneficial effects in AD patients via an improvement of their circadian rhythms given that AD pathogenesis involves atrophic alterations in the brain resulting in circadian disruptions. Indeed, melatonin treatment has beneficial effects in mild cognitive impairment and AD patients with sleep disorders in improving sleep quality and in regulating the sleep/wake rhythm.",Current Neuropharmacology,Melatonin,2023
Melatonin’s Multifaceted Roles in AD Pathogenesis,"Melatonin has been recognized to alleviate pathogenic mechanisms in AD by regulating several signalling pathways. Recently, it has also been demonstrated that melatonin, by modulating specific pathways, might also play a role in cell cycle functions related to AD. Moreover, a complete understanding of the interrelationship between AD and circadian rhythm disruption might concede for earlier identification of AD diagnostic. Therefore, the present review emphasizes these two particular mechanisms, which can probably portend the onset of neurodegenerative diseases like AD and determine further clinical interventions.",Current Neuropharmacology,Melatonin,2023
Melatonin and Normal Aging,"It is well established that melatonin secretion is maximum during brain development (from childhood to adolescence) and declines gradually over the lifespan with very low production at an advanced age. This drop in melatonin levels occurs due to dysfunction of the suprachiasmatic nucleus or of the neuronal pathways of transmission to the pineal gland. As a consequence, aging is tightly associated with a weakening of the circadian system. In addition, because melatonin is a highly pleiotropic regulatory molecule that not only acts through its antioxidant properties but also plays important physiological and pharmacological roles in the control of neuronal plasticity and neuroprotection, its drop in level is expected to reduce adult hippocampal neurogenesis. Moreover, the loss of amplitude of melatonin rhythm in advanced age is both an indicator as well as a cause of age-related disturbances in the circadian pacemaker leading to chronobiological disorders. The effect of melatonin on aging-associated pathologies, with an emphasis on data from aged organisms and senescence-accelerated animals, has been extensively and critically reviewed.",Current Neuropharmacology,Melatonin,2023
Exogenous Melatonin and Chronobiotic Effects,"In this context, the chronobiotic efficacy of exogenous melatonin has been demonstrated in both experimental models and clinical studies with validated recommendations for treatment purposes and slowing the process of aging and neurodegenerative diseases. Importantly, age-specific preventive clinical and therapeutic applications of melatonin in newborns, children and adults based on its physiological regulatory effects have been substantiated to better understand the short- and long-term effects of melatonin following its immediate or prolonged release. Moreover, the influence of endogenous and exogenous melatonin on the adolescent brain, with specific reference to the evolution of brain structure and functions, sleep regulation, and modulation of behaviors in health or disease, has been well described and a review on the ongoing clinical trials on the effects of melatonin on circadian rhythms in young adults with at-risk symptoms has been provided. However, the dosage paradigm and duration of exposure is the key to a better prognosis. Recently, the benefits and adverse events of melatonin use in the elderly have been extensively discussed.",Current Neuropharmacology,Melatonin,2023
Melatonin Levels and Therapeutic Efficacy in AD,"Disruptions in melatonin levels in the CSF, blood, saliva and urine, which occur with age and are more pronounced in AD, have been recently overviewed and summarized extensively. Moreover, studies assessing the effect of melatonin on transgenic models of AD and clinical evaluations of melatonin treatment of MCI and AD patients have been well documented. Furthermore, clinical analysis of melatonin therapy in AD patients and meta-analysis of randomized, double-blind, placebo-controlled trials of melatonin in AD pathology gave insight into the therapeutic potential of melatonin and how the dosage regimen should be applied. Additional clinical investigations provide evidence to support the beneficial effects of melatonin on sleep disorders and cognitive deficits in AD patients.",Current Neuropharmacology,Melatonin,2023
"Melatonin, Sleep Deprivation, and Oxidative Stress","On this point, it has been recently evidenced that sleep deprivation can be fatal due to an accumulation of reactive oxygen species (ROS) in the gut in drosophila and mice. Given the important roles played by melatonin in sleep regulation as well as its antioxidant power, these findings certainly reinforce the validity of the use of melatonin for the reduction of sleep disturbances and oxidative stress observed in AD. Besides, melatonin via its receptor activation can modulate the survival of newborn neurons in the adult hippocampus, making it the first known exogenously applicable substance with such specificity. The beneficial therapeutic effects of melatonin for neurogenesis impairment and the associated underlying molecular mechanisms in aging and neurological disorders, including AD, have been comprehensively reviewed. Moreover, because melatonin stimulates early and late stages of neurodevelopment in the adult brain and significantly enhances memory and cognitive functions, its beneficial effects in positively regulating neurogenesis in AD are more than likely.",Current Neuropharmacology,Melatonin,2023
Clinical Evidence for Melatonin in Cognitive Function,"The clinical efficacy of melatonin intervention for cognitive function in AD and its clinical effectiveness for cognitive function in healthy subjects has been systematically reviewed based on a meta-analysis of randomized controlled trials with specific emphasis on the duration of melatonin intervention. Recently, the developmental trends of melatonin therapies from preclinical studies for AD, the beneficial effects of melatonin on behavior in animal models of AD, and the clinical effects of melatonin on sleep, cognition, behavior, psychiatric symptoms, electroencephalography findings, and molecular biomarkers in patients with mild cognitive impairment and AD along with limitations of current melatonin therapies for AD from 1997 to 2021, have been examined.",Current Neuropharmacology,Melatonin,2023
Cell Cycle–Circadian Rhythm Interplay in AD,"Considering the underlying molecular mechanisms, circadian rhythms are conserved from drosophila to humans. The clock regulation of the cell cycle in the mammalian system is a multifactorial process, and any disruption in this system leads to changes in cell cycle characteristics. The functional clock components ubiquitously regulate optimal cell growth. Hence, any internal de-synchronization of the cerebral clocks results in pathophysiological alterations observed in many neurodegenerative disorders where the aetiology and pathogenesis of AD and disrupted circadian rhythm share common factors. First proposed to influence the timing of the cell cycle, the circadian clock later came to be known as a gating factor, the clock-dependent regulation of the cell cycle being an essential control mechanism.",Current Neuropharmacology,Melatonin,2023
Core Clock Genes and Shared Regulatory Proteins,"The reciprocal influence of the circadian clock and the cell cycle on each other suggests that these intertwined biological circuits are essential to ensure proper time-dependent mechanisms. A few examples of such common regulatory proteins are mammalian Timeless protein (TIM), the mammalian silent mating type information regulation 2 homolog 1 (SIRT1), Period (Per) and Cryptochrome (Cry) that are all involved in the modulation of both circadian and cell cycles with overlapping functions. The major ‘clock’ genes include the period genes, Per1 and Per2, the cryptochrome genes, Cry1 and Cry2, the clock (circadian locomotor output cycles kaput) gene, and the Bmal1 (aryl hydrocarbon receptor nuclear translocator-like) gene. Clock and Bmal1 heterodimers act on E-box components of the promoters of the Per and Cry genes to stimulate transcription.",Current Neuropharmacology,Melatonin,2023
Melatonin’s Effects on Circadian Rhythms and Gene Regulation,"Melatonin levels decline with advancing age, and aging alters the properties of the core transcriptional clock, as evidenced in flies. Recently, it has been observed that loss of rhythmic Klf4 expression, a crucial transcription factor, in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie the age-associated loss of protective immune responses. In this context, melatonin promotes the stabilization of core pluripotency factors, such as Nanog, Sox2, Klf4, and c-Myc, by preventing m6A-dependent mRNA decay. This draws particular attention to the age-associated decline of melatonin levels, which indicates the aging-associated disruption of circadian gene regulation and function.",Current Neuropharmacology,Melatonin,2023
Melatonin Regulation of Clock Genes Across Lifespan,"In addition, not only is this hormone a vital tool in the elderly, but inhibition of maternal melatonin also changes the expression of brain and muscle Arnt-like protein-1 (Bmal1), Per2 and melatonin type-1 receptor in the foetal SCN. These studies suggested that melatonin controls certain clock genes along with circadian rhythm. As such, the change in expression patterns of clock genes marks the rate of the aging process, and regulation of this phenomenon by melatonin signalling is yet another mechanism of how this indoleamine controls age-associated rhythmic processing.",Current Neuropharmacology,Melatonin,2023
Melatonin and Circadian Disruption in AD,"It has been well recognized in rodent models that learning and memory have defined circadian variation throughout the day. Recently, circadian learning and memory performance has been evaluated using the single cosinor-based method in AD mice. It has also been demonstrated in the 5XFAD model of AD that constitutive deletion of Rev-erbα (a circadian clock component and heme-responsive nuclear receptor protein) decreased amyloid plaque number and size and prevented plaque-associated amplification of disease-associated microglia markers, including Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), lymphocyte common antigen (CD45) and C-Type Lectin Domain Containing 7A (Clec7a). Melatonin’s interaction with the proteasome and its involvement in the feedback loops (CRY/PER and REV-ERBα) suggests another mechanism of how melatonin could possibly control Aβ clearance and neuroinflammation thus providing new insights into the role of circadian machinery in AD.",Current Neuropharmacology,Melatonin,2023
"Melatonin, REV-ERBs, and Learning Processes","Moreover, it has been previously shown that melatonin, at micromolar concentrations, potentiates the inhibitory effect of REV-ERB ligand SR9009 (which increases the constitutive repression of genes regulated by Rev-ErbA) in LX2 cells. Although more exploration is needed to fully understand the relationship between melatonin and REV-ERBs in the context of neurodegeneration, significant disruptions of these interactions lead to altered memory performance and learning abilities, thereby suggesting that, by regulating circadian rhythms, melatonin could modulate learning and memory. Circadian rhythm disruptions are more pronounced in neurodegenerative diseases like AD and related dementias, where they occur before the onset of typical clinical symptoms of neurodegeneration.",Current Neuropharmacology,Melatonin,2023
Early Circadian Alterations in AD Models,"Recently, the presence of circadian alterations and differences in long-term spatial memory, new object recognition memory and LTP, along with less robust locomotor activity rhythm, has been demonstrated in APP/PS1 mice. During the early development of AD, there is a disruption of the normal expression of genes regulating circadian function after exposure to light, particularly in the SCN but also in extra-hypothalamic brain areas supporting circadian regulation, suggesting a severe impairment of the functioning of the clock gene pathway as investigated in a triple transgenic model of AD (3×Tg-AD) and their wild type littermates. Importantly, the circadian redox regulation of neuronal excitability extends from the SCN to the hippocampus, which gives insights to understand the hippocampal circadian processes, such as learning and memory, along the course of aging and neurodegeneration.",Current Neuropharmacology,Melatonin,2023
Circadian Disturbances and Cognitive Impairment,"It has also been observed that epigenetic alterations in the SCN and hippocampus elicit cognitive decline and memory impairments in rodents, while a study in an APP/PS1 transgenic mouse model of AD has evidenced that circadian disturbances might occur early during the stage of AD pathogenesis. As such, an altered synchronization of circadian rhythmicity in the brain of AD patients has further reinforced the idea that circadian oscillators are key factors in the development of this devastating disorder. Recent clinical evaluation of the regional distribution of Aβ in the brain by positron emission tomography-computed tomography (PET-CT) standardized uptake value ratios (SUVRs) in normal healthy, MCI and AD dementia groups showed that in AD patients, the alterations in circadian rhythms are well associated with amyloid burden, which provides a clear-cut indication of how alterations in circadian rhythm result in neurodegeneration due to Aβ deposition. Importantly, this phenomenon runs parallel with the declining levels of melatonin observed in AD.",Current Neuropharmacology,Melatonin,2023
Melatonin Modulates Aβ Biology and Tau Pathology,"Moreover, the deregulated diurnal variation in clock gene expression in the hippocampus corresponds with a loss of normal day/night differences in membrane excitability, synaptic physiology and cognition in APP transgenic mice. As melatonin modulates circadian rhythms and regulates the Aβ biology in terms of production, conformational changes, oligomerization, fibrillation, and ultimately senile plaque formation, its therapeutic implementation seems legitimate. Interestingly, tau function is also essential for the regulation of the circadian network and associated behaviours. Indeed, alterations in tau homeostasis deregulate the structural plasticity of the ventral lateral circadian pacemaker neurons by disrupting the temporal cytoskeletal remodelling of its dorsal axonal projections and by inducing a slight increase in the cytoplasmic accumulation of core clock proteins. In this particular context, it has been shown that melatonin treatment in mouse and human ex vivo and in vivo tau-related models provides some beneficial effects and prevents cognitive decline.",Current Neuropharmacology,Melatonin,2023
Melatonin’s Regulatory Effects on Tau and Therapeutic Implications,"Additionally, melatonin regulates tau phosphorylation by affecting the function of kinases and phosphatases and reduces the aggregation propensity of tau by inhibiting its aggregation and dissolving the preformed aggregates. Because altered circadian clocks act as a risk factor for the development of neurodegenerative diseases via an altered production or clearance rates of toxic metabolites such as Aβ, they could be potential therapeutic targets for attenuating the onsets and progressions of these disorders. Supporting this possibility, a randomized clinical trial has revealed that interventions targeting the circadian system improve sleep, mood and behaviour in patients with AD and related dementias. Since not only dysfunction in clock gene but also altered melatonin secretion has been reported in AD and other neurodegenerative diseases and considering the interrelationship between the circadian clock and neurodegeneration, the prospects of using melatonin is of prime interest in the context of circadian disruption being an emerging link to the aetiology of AD.",Current Neuropharmacology,Melatonin,2023
Melatonin Regulates Circadian Rhythm and Neurogenesis (Figure Description),"Role of melatonin in regulating circadian synchronization, cell cycle machinery and neurogenesis. The zeitgeber-like light synchronizes the daily rhythms that are generated in the suprachiasmatic nucleus (SCN) located in the hypothalamus. Melatonin plays a major role in peripheral clock synchronization by controlling clock genes and circadian rhythm. On the one hand, the molecular clock in the melatonin-producing cells of the pineal gland plays a key role in regulating the expression of cell cycle regulator genes, including cyclins, CDKs, CKIs, as well as checkpoint protein encoding genes, thereby preventing cell cycle re-entry-mediated apoptosis and degeneration. On the other hand, melatonin regulates time-dependent diurnal processes of cell proliferation and differentiation by augmenting the levels of stem cell markers, thus maintaining a homeostatic balance in the adult neurogenic niches.",Current Neuropharmacology,Melatonin,2023
"Circadian Rhythm, Cell Cycle, and Melatonin Connection","Several mammalian cell cycle genes, such as c-Myc, cyclin D1 and Wee-1, are regulated in a circadian fashion and mutations in various clock genes have been shown to result in the modulation of several cell cycle-associated genes. As an example, Wee1 expression is controlled by the clock gene complex and contributes to cell-cycle progression. Wee1 is a critical coordinator of the transition between DNA replication and mitosis and yet another mitotic regulator that participates in the AD neurodegenerative process. Therefore, it could be speculated that an aberrant expression of circadian clock genes can lead to atypical expression of their downstream targets that are involved in cell proliferation and apoptosis. As melatonin is itself a circadian regulator, it could possibly affect the downstream effectors of the circadian pathway both in physiological and pathological conditions.",Current Neuropharmacology,Melatonin,2023
"Melatonin, Light, and Neurodevelopment","Importantly, the formation of the embryonic brain requires the production, migration, and differentiation of neurons to be timely and coordinated with both light and melatonin scheduling the differentiation of neurons and the formation of neural processes as seen in the habenular nuclei of zebrafish. Interestingly, neurogenesis in the adult hippocampus occurs in a time-of-day dependent fashion, which may dictate daily modifications of dentate gyrus physiology. The hippocampus is subjected to diurnal/circadian rhythms on both the morphological and molecular levels. A study in the animal model showed that melatonin receptor type 1 and 2 (MT1/2)-mediated signaling appears to be crucial for the generation and timing of zeitgeber time-dependent changes in cell proliferation and apoptosis and for differentiation of proliferating cells into neurons in the subgranular zone (SGZ).",Current Neuropharmacology,Melatonin,2023
Circadian Modulation of Neurogenesis and Therapeutic Timing,"Daily variations of neural progenitor divisions and neurogenesis in the adult mouse brain have been demonstrated. During night time, the progenitors actively enter M-phase, thereby giving rise to more neuronal progeny. The possibility that light-controlled rhythms are a primary regulator of neuronal proliferation exemplifies the complexity of the circadian control pathway of the neuronal cell cycle. Nevertheless, it has been shown in neurogenic niches of an adult diurnal vertebrate that the circadian modulation of cell cycle progression involves both systemic and niche-specific factors, implying that the cell cycle progression displays a robust circadian pattern.",Current Neuropharmacology,Melatonin,2023
Clock Gene Regulation of Neural Stem Cells,"Another study in the dentate gyrus of knock-out mouse models provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis. Therefore, understanding the circadian regulation of cell cycle machinery can help optimize the timing of therapeutic approaches in patients with neurodegenerative disorders. As the circadian molecular clock regulates adult hippocampal neurogenesis by controlling the timing of cell-cycle entry and exit, it is of utmost importance to investigate the underlying mechanisms of this regulation and the functional role of melatonin as this neurohormone acts as both circadian and cell cycle regulator. This is supported by the timing of gene expression for critical cell cycle regulators cyclins D, A2, and B2 and cyclin-dependent kinase inhibitor p20 in brain tissue.",Current Neuropharmacology,Melatonin,2023
Melatonin-Based Approaches for Neurodegeneration,"Also, cellular senescence is characterized by the altered expression of cell-cycle proteins, particularly the up-regulation of cyclin-dependent kinase inhibitors such as p16 and p21. Because the molecular clock in the melatonin-producing cells of the pineal gland plays a key role in modulating circadian behavior, since circadian clocks are considered potential therapeutic targets to attenuate onsets and progressions of neurodegenerative diseases like AD, and considering melatonin therapy as an advantageous modality in terms of therapeutics, using melatonin-based approaches to mitigate circadian rhythm disruptions in aging and neurodegenerative diseases would certainly delay the onset or abate the development of AD pathogenesis.",Current Neuropharmacology,Melatonin,2023
"Sleep, Hormonal Balance, and Ageing Overview","Sleep serves important biological functions, and influences health and longevity through endocrine and metabolic related systems. Sleep debt, circadian misalignment and sleep disruption from obstructive sleep apnea is widespread in modern society and accumulates with life because recovery sleep is not completely restorative. Accumulated disordered sleep throughout life impacts the ageing process and the development of age-related diseases. When epidemiological and interventional studies are considered collectively, sleep loss and lower sleep duration are associated with lower morning, afternoon and 24-h testosterone; as well as higher afternoon, but not morning or 24-h cortisol. These reciprocal changes imbalances anabolic-catabolic signaling because testosterone and cortisol are respectively the main anabolic and catabolic signals in man.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone-Cortisol,2022
Testosterone–Cortisol Balance and Metabolic Harm,"Fixing testosterone-cortisol balance by means of a novel dual-hormone clamp mitigates the induction of insulin resistance by sleep restriction and provided the first proof-of-concept that the metabolic harm from sleep loss can be ameliorated by approaches that do not require sleeping more. Obstructive sleep apnea is associated with lower testosterone, even after controlling for age and obesity whereas the conclusion that continuous positive airway pressure therapy has no effect on testosterone is premature because available studies are underpowered and better-quality studies suggest otherwise. High dose testosterone therapy induces OSA, but more physiological dosing may not; and this effect may be transient or may dissipate with longer term therapy.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone-Cortisol,2022
Sleep Physiology and Ageing,"Sleep restores neurobehavioral performance, improves immune function, and conserves whole body energy expenditure through metabolic processes that also restore brain energy stores and are important for neuronal plasticity and connectivity. These sleep-related benefits on metabolism, immunity and cognition are essential for healthy ageing. Conversely, the accumulation of sleep debt across the lifespan negatively impacts metabolic conservation, neurobehavioral performance, immunity and autoimmunity—impacts that may be responsible for the development of diseases that accrue with age.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone-Cortisol,2022
Sleep Debt Across the Lifespan,"Sleep debt accumulates throughout life from repetitive episodes of insufficient sleep, misaligned sleep (as occurs with jetlag or night shiftwork), and disrupted sleep (from obstructive sleep apnea, nocturia associated with ageing or sporadic environmental noise). This accumulation occurs because intermittent recovery or 'catch-up' sleep does not seem to completely reverse the adverse effects of sleep debt on multiple physiological processes including psychomotor performance, metabolism, blood pressure regulation and immune/adrenal response. Furthermore, the accumulation of sleep debt is widespread in modern society: approximately one in three sleep insufficiently, up to 20% are shiftworkers with misaligned sleep, and 10%–17% of men have at least moderate obstructive sleep apnea.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone-Cortisol,2022
Circadian and Endocrine Impacts of Sleep,"Sleep may influence health and longevity through endocrine and metabolic systems. This is because endocrine networks evolved to regulate whole-body metabolism in a diurnally appropriate manner, and to allow dynamic responses to external environmental insults and internal stress through the pulsatile nature of hormone secretion. The circadian pattern in hormones allows for anticipation of regular metabolic events, whereas the pulsatile nature allows for rapid responses to unpredictable events. In this fashion, endocrine networks coordinate growth, puberty and reproduction in an age- and environment-appropriate manner.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone-Cortisol,2022
"Testosterone, Cortisol, and Sleep Disturbance","This review primarily examines the effect of sleep disturbances on testosterone in the context of male gonadal ageing. To understand the full impact of restricted sleep on testosterone, it is necessary to determine the net effects on whole body metabolism and metabolic illnesses in conjunction with cortisol. This is because the hypothalamo-pituitary testicular and hypothalamo-pituitary adrenal axes that respectively control testosterone, the major anabolic hormone in men, and cortisol, a key catabolic signal, are intertwined in their regulation and sleep restriction imbalances both their signaling. Obstructive sleep apnea and its interaction with testosterone is of particular clinical relevance because sleep architecture is disrupted and sleep duration is reduced.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone-Cortisol,2022
Sleep Architecture and Biological Functions,"Sleep is highly organized and is divided into periods of non-rapid eye movement (NREM) sleep and periods of rapid eye movement (REM) sleep. NREM sleep is subdivided into 3 stages (1, 2 and 3) and transitions from stage 1 to 3 represent progressive slowing of the electroencephalogram that occurs as sleep deepens from wake. Stage 3 NREM sleep, also named slow wave sleep (SWS), mostly occurs during the first half of the biological night and is the stage of sleep that is most metabolically and hormonally active. On the other hand, REM sleep mainly occurs during the second half of the biological night and is required for memory consolidation and vivid dreaming. Sleep loss or disrupted sleep occurring at different biological times will therefore differentially affect sleep architecture. Since these different sleep stages likely serve different biological functions, the impact of sleep loss or disruption at different times of the biological night will also vary.",Reviews in Endocrine and Metabolic Disorders,Sleep Architecture,2022
Sleep Need and Age-Related Changes,"Expert opinion suggests that adults aged between 18–64 require 7–9 h of sleep per night, whereas adults over 65 need 7–8 h, including naps. This is because sleep need decreases with age, but only up to 60 years, after which sleep need remains stable. Meta-analyses of cross-sectional studies of healthy adults show that total sleep time decreases by 10–12 min for each decade of life from age 20 to 60 years, but does not change thereafter. Sleep architecture changes analogously over this age period: the proportion of SWS and REM sleep decreases while the proportion of stage 1 and 2 sleep increases before age 60, but is stable thereafter. An important limitation of the studies examining sleep architecture is the cross-sectional nature of the cohorts that were analyzed.",Reviews in Endocrine and Metabolic Disorders,Sleep Architecture,2022
Limitations of Sleep Architecture Research,"Available longitudinal studies are inconclusive due to small sample size and short follow up. For example, one such study of 11 community dwelling seniors (8 women and 3 men) aged 60 to 72 years showed no changes in sleep architecture 3 years later, other than a small increase in the number of sleep stage transitions. Large scale longitudinal studies are required to fully understand the changes in sleep architecture that occur with ageing, but are difficult to perform because in-laboratory polysomnography in large numbers of adults followed for decades is likely required to detect relevant changes.",Reviews in Endocrine and Metabolic Disorders,Sleep Architecture,2022
Testosterone Decline and Ageing,"Many longitudinal and cross-sectional epidemiological studies of multiple populations across the world have unequivocally shown that testosterone declines with age in men. More recently, the relevance of these declines in testosterone has been illustrated in the United Kingdom biobank cohort of 150,000 men followed for 11 years. Here, lower circulating testosterone concentrations were associated with higher future all-cause and cancer-related mortality. Many of the features of ageing such as increased fat, insulin resistance, falls and fractures, and decreased muscle mass, muscle strength, physical performance, bone mineral density, erectile function and libido are also reminiscent of androgen deficiency when considered as a whole. Therefore the decline in testosterone, irrespective of the underlying reasons for the decline, ultimately leads to testosterone levels that are so low that it triggers consideration of the diagnosis of hypogonadism because suggestive clinical features, which overlap with non-specific features of ageing, are likely to be present already.",Reviews in Endocrine and Metabolic Disorders,Testosterone and Ageing,2022
Definition and Causes of Late-Onset Hypogonadism,"A syndrome of late-onset hypogonadism has since been objectively defined as requiring three sexual symptoms (decreased sexual interest, fewer morning erections and erectile dysfunction) in conjunction with a total testosterone below 11 nmol/liter and free testosterone below 220 pmol/liter and is now widely accepted to properly identify individuals who may require testosterone replacement therapy. However, this decline of testosterone with age is now recognized to be largely, although not exclusively, due to factors associated with ageing such as concomitant obesity and illnesses, rather than from ageing itself. Notwithstanding these epidemiological conclusions, animal experiments and clinical investigations in healthy men have unveiled multiple alterations in the ageing hypothalamic-pituitary–testicular axis.",Reviews in Endocrine and Metabolic Disorders,Testosterone and Ageing,2022
Age-Related Changes in the GnRH–LH–Testosterone Axis,"These regulatory changes include: (1) smaller amplitude Luteinizing Hormone (LH) pulses which suggest reduced drive by Gonadotropin-Releasing Hormone (GnRH) and/or excessive sex-steroid inhibition; (2) more frequent LH pulses and less orderly patterns of LH release, pointing to reduced negative feedback; (3) preserved LH response to exogenous GnRH pulses, revealing intact gonadotrope responsiveness; (4) reduced pulsatile and total daily testosterone secretion; and, (5) impaired testosterone secretion in response to both elevated endogenous LH concentrations (stimulated by flutamide, tamoxifen, GnRH or anastrozole) and infused pulses of recombinant human LH. These regulatory alterations suggest that the decline in testosterone in older men reflects multisite failure of the GnRH-LH-testosterone axis.",Reviews in Endocrine and Metabolic Disorders,Testosterone and Ageing,2022
Ensemble Modeling of Hormonal Aging Mechanisms,"Dissecting the individual signaling failures that together result in this set of findings requires an integrative biomathematical model of reciprocal dynamic interactions among GnRH, LH and testosterone via estimable feedback and feedforward interactions. This ensemble model reconstructs ageing-related adaptations among all three interlinked signals simultaneously, rather than appraising any one signal in isolation. According to this ensemble model, the most parsimonious explanation of available data in humans is that ageing (1) diminishes hypothalamic GnRH outflow, (2) impairs testicular responsiveness to LH pulses and (3) decreases androgenic negative feedback.",Reviews in Endocrine and Metabolic Disorders,Testosterone and Ageing,2022
Experimental Verification of Ageing-Related Hormonal Changes,"Experimental verification of the findings from ensemble modeling was recently achieved in 40 community dwelling men aged 19 to 73 years with body mass indices from 20.0 to 34.3 kg/m2 in whom multiple mechanisms were explored in the same individual for the first time. Each individual underwent sequential testing in random order of hypothalamic GnRH outflow by administering a submaximal dose of ganirelix (a GnRH antagonist), hypothalamopituitary feedback by steroidogenic blockade with ketoconazole, and testicular responsivity with repeated pulsatile infusions of recombinant human LH. Advancing age was shown to be associated with diminished GnRH outflow, diminished testicular responsivity to infused LH pulses and partial compensation by reduced central gonadotropic response to testosterone feedback.",Reviews in Endocrine and Metabolic Disorders,Testosterone and Ageing,2022
Inflammation and Hormonal Ageing,"A possible unifying mechanism that could underpin these diverse associations is that specific proinflammatory cytokines could induce androgen depletion simultaneously through enhanced inhibition of GnRH and/or LH secretion as well as direct Leydig cell effects. In any case, longitudinal studies will be required to establish the magnitude, relative importance and timing of inferred regulatory deficits in healthy ageing men. This is because declines in testosterone per year of age is 2–threefold greater with longitudinal compared with cross-sectional analysis of the same cohort.",Reviews in Endocrine and Metabolic Disorders,Testosterone and Ageing,2022
Need for Longitudinal Studies,"However, longitudinal studies examining for any of these regulatory changes are not available and attempts to re-examine an adequate number of individuals after a sufficiently long period of time have not been successful. Thus, while age-related declines in testosterone and changes in hypothalamic, pituitary, and testicular signaling are highly suggestive, definitive timelines and causal mechanisms remain to be fully established.",Reviews in Endocrine and Metabolic Disorders,Testosterone and Ageing,2022
Sleep Duration and Testosterone: Cross-Sectional Evidence,"A consistent relationship between sleep duration and testosterone has not been apparent in large cross-sectional studies of healthy community-dwelling older and young and older men when divided according to studies that objectively measured sleep by actigraphy or polysomnography from those obtained by self-report. Longitudinal studies comparing changes in sleep duration with changes in testosterone would be valuable to resolve whether a relationship exists, but are not available in men of any age.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone Relationship,2022
Sleep Restriction and Testosterone: In-Laboratory Evidence,"Table 1 shows all the in-laboratory studies of sleep restriction on blood testosterone, divided into the 3 studies that assessed testosterone for a full 24 h from those that did not. Within each division, studies are ordered by more frequent blood sampling and then by larger sample size. Only one study intensively sampled blood frequently enough to allow determination of pulse characteristics by mathematical deconvolution. All studies when collectively examined show that sleep restriction decreases testosterone. An important limitation is that most of these studies assessed testosterone only in the morning. Another limitation is that only 3 studies have examined 24-h testosterone, and of these, one was confounded by strenuous exercise, which is known to increase testosterone. Accordingly, understanding time of day differences is also necessarily limited.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone Relationship,2022
Age-Specific Effects of Total Sleep Deprivation,"Only one study has examined the effect of manipulating sleep in a cohort that specifically included older men. In this study, 18 healthy older men (average age 63.9±4.0) as well as 17 healthy young male adults (average age 24±2.9 years) underwent total sleep deprivation (complete nighttime wakefulness) and 8 h of regular night sleep in random order. Blood was sampled every 10 min to allow unbiased, accurate and validated calculations of the timing and frequency of pulses; the mass per pulse; the basal, total and pulsatile secretion; and its biexponential elimination. Blood was also sampled over an entire 24 hour period to allow appraisal of time of day and diurnal effects.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone Relationship,2022
Effects of Sleep Restriction on Testosterone Pulsatility,"Whereas 24 h, morning, and afternoon testosterone concentrations were all decreased in both older and younger men by sleep restriction, a significant age group by sleep condition interaction revealed that sleep restriction decreased the testosterone pulse frequency and pulsatile secretion only in older men. Twenty four-hour LH concentrations and pulse characteristics did not change despite these changes in testosterone. However, morning but not afternoon, LH concentration, pulsatile secretion and mass per pulse were reduced by sleep restriction in both young and older men.",Reviews in Endocrine and Metabolic Disorders,Sleep-Testosterone Relationship,2022
Cortisol Rhythms and Circadian Regulation,"The 24-h rhythm of cortisol expressed in humans is driven by the central circadian pacemaker located in the suprachiasmatic nucleus, which entrains all other peripheral clocks throughout the body to coordinate bodily processes with the environment. This rhythm is important for its action because cortisol, not melatonin, is recognized to be the key metabolic central synchronizing signal. Melatonin is not discussed further in this review because cortisol, not melatonin, is the signal that has been shown to synchronize the timing of peripheral clocks in organs relevant for metabolism to the timing of the central circadian pacemaker. Experimental administration of glucocorticoid synchronizes the peripheral clocks located in liver, muscle, and adipose tissue which are the major storage sites for glycogen, protein, and fat, respectively, as well as other metabolically relevant organs such as pancreas and gut. Resetting of the peripheral clock occurs through well-defined molecular mechanisms that involve direct interaction of glucocorticoids, glucocorticoid response elements and the regulatory regions of core clock genes Bmal1, Cry1, Per1 and Per2.",Reviews in Endocrine and Metabolic Disorders,Cortisol and Ageing,2022
Need for High-Fidelity Cortisol Profiling,"Therefore, understanding the 24-h pattern of cortisol, and not just averaged levels, is required. Documenting these changes requires high fidelity sampling of circulating cortisol patterns across an entire 24-h day in large cohorts with expansive age ranges. Only two such studies, of 143 and 177 healthy men and women aged 20 to 80 years, respectively, are available. These and other studies are consistent in showing that age is associated with higher late-afternoon and early-evening cortisol, and an advance in the timing (i.e., phase) of the cortisol rhythm, but not with cortisol-binding protein. These findings are highly relevant because hypercortisolemia during the late-afternoon and early-evening is harmful, and is believed to cause the insulin resistance observed with advanced age, impair physical performance, and trigger neurocognitive deficits.",Reviews in Endocrine and Metabolic Disorders,Cortisol and Ageing,2022
Metabolic and Cognitive Consequences of Elevated Cortisol,"In fact, experimentally increasing late-afternoon and early-evening cortisol through hydrocortisone administration induces insulin resistance the following day. Premenopausal women, but not older women, had lower 24-h, and morning, cortisol concentrations compared with age-matched men. In contrast to the consistent findings previously outlined, 24-h cortisol concentrations were independent of age in one study, but increased with age in the other, for reasons that are not entirely apparent. Only one of these two studies examined the effect of age on pulsatile cortisol secretory characteristics. These analyses show that cortisol pulse frequency was higher, cortisol half-life was shorter, and basal secretion was lower in women compared to men, whereas total and pulsatile cortisol secretion did not differ by sex. None of these secretory parameters differed according to age in a multivariate analysis that adjusted for sex and obesity.",Reviews in Endocrine and Metabolic Disorders,Cortisol and Ageing,2022
Sleep Duration and Cortisol: Epidemiological Evidence,"Recently it has been established that actigraphically-measured sleep duration is not associated with higher daytime salivary cortisol levels (average of 3 samples collected from 9AM to 6PM) in a random subsample of 672 adults, representing a nationally representative probability sample of adults aged 62–90 years in the United States of America. This is consistent with another study of 325 community-dwelling men aged 65 years or more that reported no significant association between actigraphally-measured sleep duration and 24-h urinary free cortisol. These studies suggest that sleep duration does not influence 24-h or near 24-h cortisol. On the other hand, recurrent self-reported short sleep duration is associated with the development of higher late afternoon or bedtime salivary cortisol after 10 years of follow up in a large occupational cohort of 3314 adults initially aged 35–55 years.",Reviews in Endocrine and Metabolic Disorders,Sleep-Cortisol Relationship,2022
Time-of-Day Specific Cortisol Effects,"These epidemiological data show that the effect of short sleep may be on a specific time of day (i.e. late-afternoon and early-evening) where higher cortisol levels result in metabolic harm, without any impact on 24-h cortisol. These findings are consistent with studies where sleep was manipulated. Table 2 summarizes the twelve in-laboratory studies that have examined the effect of sleep restriction on blood cortisol determined over a full 24-h period, ordered by more frequent blood sampling and then by larger sample size. Twenty four-hour studies are essential because cortisol levels at specific times, particularly the late afternoon or early evening, are important for metabolic health.",Reviews in Endocrine and Metabolic Disorders,Sleep-Cortisol Relationship,2022
Sleep Restriction Increases Afternoon Cortisol,"When appraised collectively, these studies show that sleep restriction increases afternoon cortisol. Whereas 6 studies reported increased afternoon cortisol with sleep restriction, only 3 studies reported no change, and none reported a decrease. Furthermore, it has been directly shown that insufficient blood sampling of cortisol increases variability of averaged cortisol and reduces power so that only studies of sufficiently high frequency of sampling may be adequately powered. Also, sleep loss may need to be of sufficient magnitude to induce cortisol changes. In this regard, all studies that sampled blood more frequently than every 30 min or restricted sleep to 5.5 h/night or less reported that sleep restriction increased afternoon cortisol.",Reviews in Endocrine and Metabolic Disorders,Sleep-Cortisol Relationship,2022
Sleep Restriction and 24-h Cortisol Levels,"In contrast, sleep restriction does not seem to alter 24-h cortisol. Seven studies reported no change in 24-h cortisol with sleep restriction, 3 reported an increase and none reported a decrease. The effect of sleep restriction on morning cortisol is not interpretable because different studies show increases, decreases or no change. These discrepancies may be due to confounding by the cortisol awakening response which differs among studies according to the timing of scheduled wake and is of short duration so that confounding is compounded by infrequent sampling. With this in mind, all studies that sampled blood more frequently than every 30 min actually show no change in morning cortisol.",Reviews in Endocrine and Metabolic Disorders,Sleep-Cortisol Relationship,2022
Age-Specific Cortisol Responses to Sleep Loss,"Relevant for understanding the effects of ageing, only one study has included a cohort of older men. This study is also the only study of sufficient sampling fidelity to allow calculation of pulsatile secretory parameters by mathematical deconvolution. Total sleep loss did not alter any pulse characteristic when assessed over the full 24-h period or in the morning (0600 to 0900), but analysis of a specific 3-h time window in the afternoon (1500 to 1800) revealed an increase in cortisol concentrations and pulsatile secretion specifically in older men. One limitation of this study is that the afternoon time window was relatively early. Older men have a phase-advanced acrophase of almost 3 h, so the 1500 to 1800 time period represents a later circadian time compared to young men.",Reviews in Endocrine and Metabolic Disorders,Sleep-Cortisol Relationship,2022
Circadian Phase Considerations in Younger vs. Older Men,"This phase difference may explain why sleep restriction did not alter early afternoon cortisol in the young men examined in this study. Accordingly, if sampling had continued for an additional 3 h in the young men in this study (i.e. from 1800 to 2100), then increases in cortisol levels with sleep deprivation might have been detected, as it has been in other studies.",Reviews in Endocrine and Metabolic Disorders,Sleep-Cortisol Relationship,2022
Testosterone–Cortisol Imbalance and Metabolic Harm,"Cortisol and testosterone are respectively the major catabolic and anabolic signals in men. Changes in cortisol and testosterone signaling from sleep restriction, namely a reduction in testosterone and an increase in late-afternoon and early-evening cortisol, has long been postulated to be a mechanism by which sleep restriction induces insulin resistance. Although the hypothalamo-pituitary testicular and hypothalamo-pituitary adrenal alterations that cause these changes in testosterone-cortisol balance are yet to be fully elucidated, substantial evidence implicate testosterone and cortisol in the development of insulin resistance. Randomized controlled trials show that testosterone treatment improves insulin sensitivity in men at risk for type 2 diabetes mellitus (T2DM); prevents the development of T2DM in men at risk for T2DM; and improves glycemic control in men with T2DM. Administration of glucocorticoids to bolster cortisol levels in the late afternoon and early evening also induces insulin resistance through post-receptor mechanisms.",Reviews in Endocrine and Metabolic Disorders,Testosterone-Cortisol Balance,2022
Dual-Hormone Clamp Evidence,"Direct substantiation of the role of testosterone-cortisol balance in the induction of insulin resistance from sleep restriction has only recently become available in a study that utilized a novel dual hormonal clamp. Thirty-four healthy young mostly overweight men completed a randomized two-condition crossover study in a highly controlled in-laboratory environment. Under both conditions, participants underwent one night of 10 h baseline sleep followed by 4 consecutive nights where sleep was restricted to a 4 h opportunity. Under the dual hormonal clamp condition, mid-physiological cortisol and testosterone concentrations were maintained by co-administration of ketoconazole to block endogenous steroidogenesis in conjunction with simultaneous addback of physiologically appropriate doses of oral hydrocortisone and transdermal testosterone gel. In the placebo condition, cortisol and testosterone were not clamped. Fixing testosterone-cortisol balance mitigated the development of insulin resistance and hyperinsulinemia with 4 consecutive nights of sleep restriction by at least 50%, but did not prevent the development of hyperglycemia.",Reviews in Endocrine and Metabolic Disorders,Testosterone-Cortisol Balance,2022
Implications for Ageing and Chronic Sleep Debt,"These findings provide first proof of principle for the amelioration of metabolic harm from sleep restriction by a targeted approach that does not require sleeping more. These findings are highly relevant to ageing because (1) this pattern of sleep restriction (4 nights of 4 h/night sleep opportunity) is ecologically valid, and (2) accumulated sleep debt over a lifetime could also lead to prediabetes and type 2 diabetes mellitus through the induction of insulin resistance; obesity due to hyperinsulinemia; and retinopathy and nephropathy due to hyperglycemia. Furthermore, fixing testosterone-cortisol balance in older men to prevent the development of insulin resistance and hyperinsulinemia is plausible because sleep restriction is now known to decrease testosterone and increase late-afternoon and early-evening cortisol in older men.",Reviews in Endocrine and Metabolic Disorders,Testosterone-Cortisol Balance,2022
Circadian Drivers of Cortisol and Testosterone Rhythms,"Although the 24-h rhythm of cortisol is driven by the central circadian pacemaker, the origin of the 24-h pattern of testosterone in humans is not known. Prior studies have documented a marked 24-h rhythm in testosterone, but these rhythms could be attributable to external rhythmic influences because cycles of light/dark, sleep/wake, feeding/fasting, and activity/rest were present. Showing that the 24-h pattern of testosterone is driven specifically by the central circadian pacemaker, free of external influences, requires the use of the constant routine protocol as the gold standard method to remove or uniformly distribute these external influences so that only the endogenous rhythm is expressed. These findings will have implications for the assessment of hypogonadism, including late onset hypogonadism, in night shiftworkers.",Reviews in Endocrine and Metabolic Disorders,Circadian Misalignment,2022
Definition and Consequences of Circadian Misalignment,"Circadian misalignment occurs when the timing of behavioral rhythms differs from the timing of endogenous circadian rhythm. It occurs with jetlag and repetitively in night shiftworkers who typically revert back to a day-aligned schedule (to interact with family and friends, address domestic duties, attend daytime events, etc.). Circadian misalignment is harmful. It induces insulin resistance, and night shiftwork is associated with metabolic diseases such as obesity and T2DM. The effect of circadian misalignment on endocrine rhythms has not been adequately explored.",Reviews in Endocrine and Metabolic Disorders,Circadian Misalignment,2022
Circadian Misalignment Effects on Cortisol,Only one recent study utilized a constant routine to properly evaluate the effect of circadian misalignment on cortisol rhythms and concluded that 21 days of circadian misalignment through forced desynchrony reduced overall cortisol (24-h area under the curve) by 120 mcg/day. No equivalent studies are available to examine the effect of circadian misalignment on testosterone rhythms.,Reviews in Endocrine and Metabolic Disorders,Circadian Misalignment,2022
Need for Studies on Testosterone and Circadian Misalignment,"Such studies would be valuable because symptoms consistent with hypogonadism are common in night shiftworkers, particularly those with shiftwork sleep disorder, and understanding whether circadian misalignment per se can directly alter testosterone levels and induce hypogonadism would inform treatment strategies.",Reviews in Endocrine and Metabolic Disorders,Circadian Misalignment,2022
"OSA, Obesity, and Testosterone in Ageing Men","OSA is the most important clinical cause of disrupted sleep. It is a male-preponderant disease that is more prevalent with advanced age, particularly in the presence of obesity. Accordingly, low testosterone concentrations, OSA and obesity are known to frequently occur together, but the mechanisms that underpin these inter-relationships are not well established. Cohort studies that include older men show that higher degrees of hypoxemia, as a marker of more severe OSA, are associated with lower testosterone concentrations. This association may or may not be due to obesity. A recent meta-analysis of 18 studies involving 1823 men with or without OSA also found a significant inverse relationship between OSA and testosterone concentrations – a relationship which remained after controlling for age and obesity. Notably, a significantly lower testosterone was only observed in men with severe OSA. These findings collectively suggest that OSA, through hypoxemia or possibly through disrupted sleep architecture, most likely lowers testosterone, independently of obesity and age – although the reverse relationship is possible since these are cross-sectional analyses.",Reviews in Endocrine and Metabolic Disorders,OSA and Testosterone,2022
Limitations of CPAP Evidence on Testosterone,"The direction of this relationship would be better substantiated if reversing OSA with continuous positive airway pressure (CPAP) therapy increases testosterone. However, the two available meta-analyses have not concluded that CPAP therapy increases testosterone in men with OSA, but have instead concluded that CPAP has no effect on testosterone. This conclusion is problematic because these meta-analyses are likely underpowered, and meta-analyses consider all studies equally irrespective of quality. A notable limitation is that none of the available studies have assessed the effect of CPAP on 24-h testosterone. Only one study assessed testosterone frequently and reported that CPAP increased mean, incremental and area under the curve testosterone measured every 20 min during a 12-h period, although this study included only 5 men and had selection bias.",Reviews in Endocrine and Metabolic Disorders,OSA and Testosterone,2022
Evidence from Additional Clinical Studies,"The remaining 11 studies examined the effect of CPAP on morning testosterone, assessed on one or two occasions only, in relatively few men. Only one is a randomized sham-controlled trial, and this study reported that CPAP improved testosterone compared with sham – although this was due to a reduction in testosterone in the sham group rather than an increase in testosterone in the treated group. A complementary study not included in any meta-analyses examined 12 men in whom sleep disordered breathing was almost completely reversed by uvulopalatopharyngoplasty. Blood testosterone significantly increased after 3 months, while the number of apneas fell from an average of 40 events/hour to 5 events/hour.",Reviews in Endocrine and Metabolic Disorders,OSA and Testosterone,2022
Need for Better CPAP Studies,"Due to the limitations of the available literature, it seems premature to conclude that CPAP has no effect on circulating testosterone levels in men with OSA. The available meta-analyses included no more than 12 studies, of which only 2 were randomized controlled trials, and the higher quality studies and other complementary data suggest that CPAP increases testosterone. Nevertheless, it is plausible that CPAP has no effect on circulating testosterone levels. If true, this would point to irreversible neuroendocrine changes to the testicular axis arising from longstanding intermittent hypoxia – irreversible changes that are not found in the adrenal axis. By comparison, a meta-analysis of 22 studies was sufficiently powered to conclude that CPAP decreases cortisol. More studies, including randomized sham-controlled studies utilizing repetitive overnight or 24-h blood sampling and modern mathematical deconvolution techniques that better discriminate subtle regulatory changes, are needed to resolve this issue.",Reviews in Endocrine and Metabolic Disorders,OSA and Testosterone,2022
Testosterone Therapy and Risk of Inducing OSA,"Testosterone therapy is widely believed to induce or worsen sleep apnea, and recent European and North American societal guidelines recommend vigilance in the detection of new OSA and/or avoiding testosterone therapy in those with severe OSA. Surprisingly, other national guidelines from the United Kingdom make no mention of OSA at all. The issue of testosterone effects on OSA should not be ignored because two randomized controlled trials show that testosterone therapy can acutely (within 2–3 weeks) induce sleep-disordered breathing. Whether these adverse findings would have occurred with longer-term near-physiological testosterone replacement is uncertain because one of the studies utilized testosterone doses that resulted in sustained supraphysiological testosterone levels, and the other likely induced intermittent supraphysiological peaks and assessed for OSA during these peaks.",Reviews in Endocrine and Metabolic Disorders,Testosterone and OSA,2022
Longer-Term Testosterone Therapy Studies,"Two other randomized, placebo controlled, parallel group studies have partly addressed this uncertainty. The first study administered a testosterone patch or a matching dose-titrated placebo patch for 3 years to 108 healthy men over the age of 65 years. The initial dose was 6 mg/day, titrated every 3 months to maintain blood testosterone levels below 34.7 nmol/litre. No significant difference in sleep disordered breathing was detected between groups after 6, 12, 24 or 36 months of therapy. However, the method of detection was relatively insensitive and may have missed the development of mild or even moderate OSA. The second study remains the only study to purposefully administer testosterone to men with known moderate-severe OSA. Sixty-seven middle-aged obese men with OSA were treated with 3 doses of testosterone undecanoate 1000 mg every 6 weeks, or matching placebo, along with hypocaloric diet recommendations. Testosterone treatment significantly increased sleep-disordered breathing by about 10 events/hour at week 7, but not at week 18.",Reviews in Endocrine and Metabolic Disorders,Testosterone and OSA,2022
Mechanisms and Time Course of OSA Worsening,"Both studies allow for the possibility that the worsening in OSA could dissipate with longer term therapy. Another possibility is that OSA is only induced acutely, potentially due to transient effects on ventilatory drive. Despite these adverse effects on breathing during sleep, 18 weeks of testosterone therapy in men with OSA increased muscle mass, reduced liver fat, improved insulin sensitivity and heightened sexual desire compared with placebo therapy. Studies advancing knowledge regarding the relative risks and benefits of testosterone therapy in older men have recently become available, but further research is still needed.",Reviews in Endocrine and Metabolic Disorders,Testosterone and OSA,2022
Clinical Recommendations and Future Needs,"More studies examining the risks and benefits of testosterone therapy in men with OSA over the longer term are required. Until such data are available, expert opinion will continue to caution against the use of testosterone therapy in men with untreated severe OSA. Understanding whether testosterone's adverse effects on breathing are transient, dose-dependent, or mitigated over time remains essential for forming accurate treatment guidelines.",Reviews in Endocrine and Metabolic Disorders,Testosterone and OSA,2022
"Summary of Sleep, Aging, and Endocrine Health","Sleep is highly organized, serves important biological functions, and influences health and longevity through endocrine and metabolic regulated systems. Accumulated sleep debt is widespread in modern society and when accumulated throughout life likely impacts the ageing process, and the development of age-related diseases. Sleep loss and lower sleep duration are associated with lower morning, afternoon and 24-h testosterone, whereas they are associated with higher late afternoon and early evening, but not morning or 24-h cortisol. These reciprocal changes in testosterone and cortisol with sleep loss imbalances catabolic-anabolic signaling and is an important, but not exclusive, mechanism by which sleep loss induces insulin resistance.",Reviews in Endocrine and Metabolic Disorders,Sleep-Endocrine Interactions,2022
Metabolic Implications of Hormonal Imbalance,"By fixing testosterone-cortisol balance to prevent the induction of insulin resistance by sleep restriction, we provided the first proof-of-concept that the metabolic harm that occurs with sleep loss can potentially be mitigated by therapeutic approaches that do not require sleeping more. This approach is likely to be relevant also to older men since the changes in testosterone-cortisol balance that occur in young men have recently been shown to also occur in older men. Epidemiological studies when considered in unison show that OSA is associated with lower testosterone levels, independently of confounders such as age and obesity. Circumstantial evidence would favor the possibility that more severe OSA, due to greater hypoxemia, lowers testosterone although the opposite is plausible.",Reviews in Endocrine and Metabolic Disorders,Sleep-Endocrine Interactions,2022
"OSA, CPAP Therapy, and Hormonal Effects","It would be premature to conclude that CPAP therapy has no effect on testosterone in men with OSA since the available studies are underpowered, and the higher quality studies suggest otherwise. In contrast, the larger number of studies available, particularly higher quality studies, has allowed the conclusion to be made that CPAP decreases cortisol. High dose testosterone therapy induces OSA, but more physiological dosing may not; this effect may be transient or dissipate with longer term therapy. Important limitations are that only one interventional study has examined the effect of restricting sleep on testosterone and cortisol in a cohort of older men; few studies have been designed to examine changes in relevant testosterone and cortisol pulse characteristics with age and/or in response to sleep manipulation; and longitudinal epidemiological studies examining the age-related changes in sleep architecture, the impact of changes in sleep on testosterone and cortisol, are rudimentary.",Reviews in Endocrine and Metabolic Disorders,Sleep-Endocrine Interactions,2022
Future Directions and Need for Research,"Nevertheless, the available data are highly suggestive that restricted sleep, circadian misalignment and disrupted sleep from OSA are relevant to age and age-related diseases through alterations in testosterone and cortisol signaling. Therefore, society should prioritize and value sufficient and appropriately timed sleep. Future research to understand the molecular underpinnings of these findings (for example in circadian clocks) is needed to develop countermeasures to reduce the impact of insufficient sleep, disrupted sleep, or circadian misalignment on cardiometabolic health. This is because insufficient sleep and night shiftwork may sometimes be unavoidable. Such investigations are currently being planned.",Reviews in Endocrine and Metabolic Disorders,Sleep-Endocrine Interactions,2022
Magnesium in Aging and Health: Overview,"Magnesium ion (Mg) is the divalent intracellular cation most present in the human cell and the second cation after potassium. Mg has a crucial role in numerous biological processes, including oxidative phosphorylation, energy production, glycolysis, protein and nucleic acid synthesis. Mg plays a role in the mitochondrial synthesis of adenosine triphosphate (ATP) to form MgATP. Cell signaling needs MgATP for protein phosphorylation and activation of cyclic adenosine monophosphate (cAMP), which is involved in many biochemical processes. Mg participates in ion transport across membranes, muscle contraction, and neuronal excitability. Cellular Mg homeostasis is linked to the metabolism of K, Na and Ca through Na+/K+/ATPase, Ca++-activated K channels and other mechanisms. Mg is a cofactor for over 600 enzymatic reactions and is necessary for all phosphorylation processes and ATP-related reactions. Mild Mg deficits are generally asymptomatic, but chronic deficits increase free radical production, contributing to numerous age-related disorders.",Nutrients,Magnesium,2021
Magnesium Metabolism and Distribution,"The Mg content in the human body is around 24–29 g, with nearly two thirds stored in bone and one third in cells. Less than 1% is extracellular. Serum Mg levels range between 0.75 and 0.95 mmol/L and are tightly maintained by a balance between dietary intake, intestinal absorption, renal excretion, bone storage, and tissue requirements. Mg absorption increases during low intake, and bone stores release Mg to maintain serum levels during deficiency. Serum Mg below 0.75 mmol/L is considered low, while frank hypomagnesemia occurs below 0.7 mmol/L. However, total serum Mg is not a precise indicator of whole-body Mg status because serum levels may remain normal despite intracellular depletion. Optimal Mg intake is ~320 mg/day for women and 420 mg/day for men, though requirements may be higher during aging, pregnancy, exercise or diseases such as type 2 diabetes.",Nutrients,Magnesium,2021
Factors Affecting Magnesium Balance,"Many factors influence Mg balance, including diet, intestinal absorption, renal excretion, and bone storage. A healthy person needs to consume around 5–7 mg/kg/day to maintain Mg balance. Mg absorption varies from 25–60% depending on intake. The kidneys filter ~80% of circulating Mg and reabsorb ~60%, resulting in a net excretion of ~120 mg/day. Mg reabsorption increases during deficiency. Diuretics such as furosemide increase Mg wasting. No hormone specifically regulates Mg, but insulin, parathyroid hormone, calcitonin, and catecholamines affect Mg metabolism. Western diets high in refined foods and low in whole grains and vegetables often lead to inadequate Mg intake. Cooking and boiling cause Mg loss in foods. Phytic acid, pesticides such as glyphosate, and pathogenic gut microbiota may impair Mg absorption. Mg-rich drinking water can improve intake, with higher bioavailability than food-based Mg.",Nutrients,Magnesium,2021
Dietary Deficiencies and Environmental Factors,"Studies consistently show that Mg intake is often inadequate in Western populations. Two thirds of Americans consume less than the recommended daily allowance, with nearly 20% consuming less than half. European populations show similar deficits. Diets rich in refined or processed foods are particularly low in Mg because boiling and food processing significantly reduce Mg content. Soil depletion, pesticide use, and reduced mineral content in crops further contribute to low dietary Mg. Organic foods have been shown to contain significantly higher Mg levels. Drinking water is an important alternative source, especially Mg-rich mineral water, which has greater bioavailability. Desalinated seawater, increasingly used in many countries, removes Mg, and high consumption of low-Mg water has been associated with increased cardiac morbidity and mortality in regions relying heavily on desalinated water.",Nutrients,Magnesium,2021
Mg Deficits Associated with Aging,"Aging is often associated with a total body Mg deficit [19]. Serum Mg levels remain constant with age [28]. Alterations in serum Mg are usually associated with the existence of diseases and/or alterations in kidney function. In healthy older persons, an age-dependent decrease in cellular Mg concentration was previously shown [29] in the absence of alterations of total serum Mg. It has been confirmed that chronic latent Mg deficiency is quite common in older adults in western countries. Possible mechanisms of this demonstrated Mg insufficiency with aging are detailed in Table 3. This Mg shortage is frequently associated with a low Mg intake [30,31], while Mg requirements for the body processes do not change with age [32]. Table 3. Mechanisms of Mg insufficiency in the elderly. Primary Mg Deficiency: • Insufficient Mg dietary intake • Reduced Mg absorption (often in parallel with reduced vitamin D levels) • Increased urinary Mg excretion (often related to reduced kidney function and tubular reabsorption, which are common in old age) Secondary Mg Deficiency: • Linked to age-related diseases and comorbidities • Linked to drug action causing Mg loss in the urine (i.e., diuretics, proton pump inhibitors). Data from the National Health and Nutrition Examination (NHANES) III have confirmed that aging is an additional risk factor for inadequate Mg consumptions and progressive decrease with age [30].",Nutrients,Magnesium,2021
Age-Related Changes in Mg Absorption and Renal Handling,"Intestinal absorption of Mg tends to fall with age, and this decline may be one of the possible causes of Mg deficit with aging [33]. The alteration of the intestinal absorption of Mg in old age is often worsened by impairment of vitamin D homeostasis, common in old age. Renal reabsorption of Mg is an active process occurring in the loop of Henle and in the proximal convoluted tubule. Reduced kidney functionality, common in the elderly, is a possible additional cause of Mg loss. Secondary Mg deficiencies in older adults may be linked to the presence of several conditions and related polypharmacotherapy [34]. Diuretic therapy may cause excessive Mg urinary loss. Diuretic-induced hypomagnesemia is often accompanied by hypokalemia. Hypomagnesemia may be present in around 40% of patients with hypokalemia, and the correction of Mg deficit is needed to achieve the correction of the K deficits. It is thus advisable to evaluate Mg levels in patients with hypokalemia. Other medications commonly used in the elderly may contribute to Mg deficits (e.g., antacids, H2 blockers, proton pump inhibitors, antihistamines, antibiotics, antiepileptic drugs, and antivirals, among others).",Nutrients,Magnesium,2021
"Mg, Inflammation, and Oxidative Stress","Mg deprivation, low serum Mg levels, and reduced dietary Mg intake have all been associated in preclinical, epidemiological and clinical human studies with an increased production of free radicals of oxygen, with low-grade systemic inflammation, increased levels of inflammation markers and proinflammatory molecules (IL-6, TNF-alpha, IL-1-beta, VCAM-1, PAI-1, complement, alfa2-macroglobulin, fibrinogen) [16,35–42]. King et al., using the NHANES database, found that dietary Mg intake was inversely related to reactive protein C levels [16]. Similar results were found by Song et al. using data from the Women’s Health Study [42]. Mg depletion results in an increased production of oxygen-derived free radicals (ROS), increased oxygen peroxide production, and increased production of superoxide anion by inflammatory cells. Mg deficiency not only increases oxidative stress but also decreases the antioxidant defense competence [35,43]. Mg is required for the proper functioning of gamma-glutamyl transpeptidase, which plays a key role in the synthesis of antioxidant glutathione [44], confirming that Mg may have a mild antioxidant action [45].",Nutrients,Magnesium,2021
"Mg Deficiency, Oxidative Damage, and Inflammaging","In humans, a correlation between intracellular Mg and circulating reduced/oxidized glutathione ratio has been reported [46]. In another study, a negative correlation between Mg levels and oxidative stress markers (plasma superoxide anions and malondialdehyde) was detected in a population exposed to chronic stress [47]. Aging is accompanied by a low-grade inflammatory state that has been named “inflammaging” [48]. We have previously postulated that a chronic Mg insufficiency facilitating this inflammaging condition and an impairment of the redox status may facilitate the development of age-related illnesses [19,49]. In particular, we have suggested a link between the Mg inadequacy and the occurrence of an insulin resistance state, T2DM, and cardiometabolic syndrome [2]. Figure 2 describes the relationship of low Mg status, generated by multiple factors (low intake and absorption, Mg transport genetic defects, obesity, T2DM, cardiometabolic syndrome, polypharmacotherapy, alcohol abuse), which may trigger increased ROS production, oxidative damage, and activation of redox signaling (NF-KB, AP-1). The elevation in oxidative stress may lead to release of inflammatory mediators forming chronic low-grade inflammation.",Nutrients,Magnesium,2021
Mg and the Immune Responses,"Mg modulates both innate and acquired immune responses and acts as a mediator in the signaling pathways controlling immune cell development, homeostasis, and activation [35]. Mg is a crucial cofactor for T helper-beta cell adherence, immunoglobulin synthesis, antibody-dependent cytolysis, IgM lymphocyte binding, and macrophage response to lymphokines [37,50]. Mg influences acquired immunity by modulating the proliferation and development of lymphocytes [51]. TRPM7 is crucial for Mg homeostasis in immune cells. A fall in free cytosolic Mg and cell cycle arrest was found in TRPM7-deficient B cell lines, which was preserved by culturing the cells in a medium containing high Mg. An impaired development of T cells was observed in TRPM7 knockout mice [52]. Mg deficiency may accelerate thymus involution. In thymuses from Mg-deficient rats, higher levels of apoptosis were observed compared with controls [53]. A Mg-deficient diet caused alteration of polymorphonuclear cell number and functionality and activation of phagocytosis [38]. Mg is involved in the regulation of cell apoptosis. Fas-induced β-cell apoptosis requires Mg. A rise in cellular free Mg is necessary for expression of Fas molecule binding on the β-cell surface to initiate apoptosis [54]. Mg is also involved in vitamin D synthesis, transport and activation, an important immunomodulator in infectious diseases including SARS-CoV-2. Mg deficit may contribute to immune hyperresponsiveness, cytokine storm, endothelial dysfunction, thrombotic complications, and predisposing conditions such as old age, diabetes, and hypertension.",Nutrients,Magnesium,2021
Clinical Symptoms of Magnesium Deficits,"Clinical signs and symptoms are generally absent or non-specific in moderate Mg deficits and mild hypomagnesemic subjects are usually asymptomatic. Non-specific manifestations may include anxiety, insomnia, fatigue, hyperemotionality, depressive symptoms, headache, light-headedness, dizziness. Most of these symptoms are non-specific and common in older patients, and might be mistaken with normal age-related manifestations. Other symptoms may be associated, such as myalgias, acroparesthesias, and cramps. Other non-specific functional complaints may include chest pain, sine materia dyspnea, precordialgia, palpitations, extrasystoles and other arrhythmias, etc. [55]. Several signs and symptoms are connected with severe Mg deficits including weakness, tremor, muscle fasciculation, dysphagia, presence of Chvostek’s sign (facial twitching as a reaction to the tapping of the facial nerve), or Trousseau’s sign (spasm of muscles of the hand and forearm following the application of a pressure cuff, to transiently occlude the brachial artery), orthostatic hypotension and/or borderline hypertension [55]. Elin suggested naming the condition of subjects with this non-specific symptomatology associated with chronic, negative Mg balance as a syndrome of “Chronic Latent Mg Deficiency” (CLMD) [8]. Subjects affected by CLMD generally present lower normal total serum Mg levels (latent) and are generally clinically undiagnosed, not having hypomagnesemia, but may benefit from Mg supplementation.",Nutrients,Magnesium,2021
"Magnesium, Genomic Stability, and Cellular Aging","Mg is crucial to preserve genomic stability in cellular systems, because of its stabilizing effects on DNA and chromatin structures. Thus, Mg ion is needed in nucleotide excision repair, base excision repair, and mismatch repair, and is crucial for the removal of DNA damage caused by endogenous processes, environmental mutagens, and DNA replication [56]. Mg deficiency triggers the cell vulnerability to oxidation and may affect immune system performance; a lack of Mg would alter membrane integrity and functionality and may facilitate mitochondrial alterations (decreased number, morphology modifications, increased apoptosis, increased DNA mutations, decreased biogenesis, decreased autophagy) [19]. Mg has an important role in the modulation of protein synthesis and membrane repair [56,57]. DNA is incessantly altered by endogenous processes and environmental mutagens. An increase in cellular Mg has been demonstrated in the early stages of apoptosis, possibly linked to a mobilization of Mg from the mitochondria; Mg may act as a “second messenger” for downstream events in apoptosis [54]. Mg deprivation increases the susceptibility to oxidative damage facilitating alterations of the membrane integrity and function.",Nutrients,Magnesium,2021
Magnesium Deficiency and Accelerated Cellular Senescence,"Some alterations in cell physiology occurring in senescence in different cell types [58] are similar to those caused by Mg deficit. Mg-depletion-related alterations include reduced protection from oxidative stress damage, reduced cell cycle progression, reduced culture growth, and reduced cellular viability as well as triggering of the expression of proto-oncogene and of transcription factors [59]. Culturing primary fibroblasts in Mg-deficient media would lower the replicative capacity and accelerate the expression of biomarkers associated with senescence and in telomere attrition. A decreased replicative lifespan was observed vs. fibroblast cultured in normal Mg media conditions [60]. Because of the essential role of Mg in the stabilization of DNA, in defending the cell to the damage of ROS and in stimulating DNA replication and transcription, a Mg deficit may facilitate genomic instability, alter DNA repair, and reduce mitochondria functionality, thus facilitating an accelerated cellular senescence and aging [56,61]. Mg has a demonstrated protective role against these effects and contrasts the shortening of telomeres (that is associated with aging and a reduced life expectancy), which is observed in low Mg conditions. It has been suggested that because of this action to prevent telomere shortening, correcting Mg deficiencies may prolong life [62].",Nutrients,Magnesium,2021
Magnesium in Hypertension and Vascular Physiology,"In the last decades, Mg deficit has been connected with several cardiovascular conditions [2,63]. Kobayashi first noted in 1957 that the mineral composition of drinking water was associated with cardiovascular death rates; the stroke incidence was lower in regions with hard water (mainly linked to Mg and Ca content) [64]. Schroeder confirmed these data only a few years later, analyzing the relationship between water hardness and death rates. He found that cardiovascular death rates were significantly lower in states with hard water compared to states with soft water [65]. Mg is involved in blood pressure homeostasis. Although Mg has not a direct role in the biochemical mechanisms of contraction, classic studies from Altura et al. have demonstrated that Mg controls vascular tone and contractility by altering Ca levels, and that changes in Mg concentration modulates Ca-triggered vascular smooth muscle contraction [66–68]. Mg itself functions as nature’s weak physiologic Ca channel blocker [69], modulating Ca-channel activity in heart cells [70]. Mg deficit stimulates angiotensin II-mediated aldosterone synthesis, as well as thromboxane and vasoconstrictor prostaglandins production [71]. Mg has a favorable action on vascular endothelium modulating the release of nitric oxide, prostacyclin, and endothelin-1 [72]. In humans, oral Mg supplementation was shown to improve endothelial function in T2DM older adults [73].",Nutrients,Magnesium,2021
Magnesium Homeostasis in Hypertension and Cardiovascular Risk,"Mg ion, because of these actions on vascular smooth muscle tone, plays a modulatory action on blood pressure homeostasis, while Mg deficits may be relevant to the physiopathology of hypertensive disorders. Altura showed that the depletion of Mg would induce vascular hyper-reactivity and elevation of blood pressure [74]. Serum total Mg levels are usually normal in hypertensive subjects. However, numerous defects of Mg homeostasis have been documented in hypertension. Former epidemiologic studies already showed an inverse relationship between Mg dietary intake and blood pressure [75]. In older populations, the age-related increase in blood pressure was concomitant with a reciprocal suppression of intracellular free Mg [29,76], suggesting a possible role for Mg deficit in the age-related elevation of blood pressure. Intracellular free Mg concentrations were found to be significantly lower in hypertensive subjects vs. normotensive controls [77]. Mg urinary excretion was also found to be altered in an experimental model of hypertensive rats [78]. High salt diet was reported to elevate blood pressure in salt-sensitive subjects, reciprocally suppressing intracellular free Mg levels [79].",Nutrients,Magnesium,2021
Magnesium Therapy and Cardiovascular Outcomes,"Blackfan and Hamilton, as early as 1925, recommended Mg therapy to lower blood pressure in patients with malignant hypertension [80]. Intravenous (i.v.) Mg has been commonly used with consistent benefit in preeclampsia and in eclampsia [81], and in malignant hypertension [82]. However, the response to Mg oral supplement in essential hypertension is less clear [63,83–86]. In experimental hypertension, high or low Mg diets that, respectively, raised or reduced cellular free Mg, in parallel reduced and raised blood pressure. In humans, in some studies, Mg supplementation was found to have hypotensive effects, while in others it had no effect on blood pressure or may even worsen it [83,86]. Nevertheless, when quality studies are analyzed together in meta-analysis and systematic reviews the evidence is convincing, validating the key role of Mg in hypertension and of an inverse relationship of dietary Mg intake with the prevalence and incidence of hypertension [63]. However, a possible confounding factor of the evidence derived from observational studies is that diets containing elevated Mg intake are also usually low in Na and rich in K and of other elements with health benefits and thus high Mg intake may be, at least in part, a marker of a healthy diet [63].",Nutrients,Magnesium,2021
"Magnesium in Atherosclerosis, Arrhythmias, and Stroke","Mg deficit has been linked to the development of atherosclerosis. Low Mg status may trigger vascular calcification, alter lipid metabolism and facilitate lipid accumulation in vascular plaques [87]. Serum Mg was found to be positively [88] or negatively [89] associated with serum lipid levels. Mg supplementation has been suggested to improve lipid profiles, and prevent atherosclerotic plaque formation and to act as a weak inhibitor of hydroxyl-3-methylglutaryl-coenzyme A reductase, and of other enzymes of the lipid metabolism [90]. Mg is involved in the heart’s electrical conduction and hypomagnesemia, hypokalemia and other electrolyte disturbances may trigger cardiac arrhythmias. Mg and K depletion also increase the susceptibility to arrhythmogenic effects of cardiac glycosides. Mg effects on conduction comprise prolongation of atrial and atrioventricular nodal refractory periods, which may help in rate and rhythm control in atrial fibrillation (AF) [91]. Mg supplements may be used as a supportive non-pharmacological treatment for atrial and/or ventricular arrhythmias [92]. AF may be associated with hypomagnesemia [93] and reduced serum Mg concentration may facilitate the development of AF [94]. In postmenopausal women, dietary Mg deficiency was associated with heart rhythm abnormalities, including AF and flutter that may respond to Mg supplementation [95]. A meta-analysis has suggested that i.v. Mg administration may have a role in the acute management of AF [96]. Because of its rapid, effective and simple application, i.v. Mg administration has been indicated in the treatment of torsade de pointes [97,98]. Antiarrhythmic actions of elevated Mg dietary intake have been suggested to mediate the reduced risk of sudden death in women in the highest quartile of Mg intake [99]. Oral Mg supplements were suggested to help improving clinical symptoms and survival outcomes vs. placebo in patients with severe congestive heart failure [100]. A systematic review and meta-analysis of prospective studies that included over 300,000 individuals found that elevated Mg serum levels paralleled a reduced risk of cardiovascular disease; elevated dietary Mg intakes were shown to be inversely associated with ischemic heart disease [101]. Another meta-analysis of prospective trials including a total of 241,378 subjects reported an inverse association between Mg intake and the risk of stroke [102]. A recent umbrella review evaluating health outcomes connected with Mg intake and supplementation confirmed the link between higher Mg intake and decreased risk of stroke [86]. Mg sulfate has been found to be protective both in preclinical models of stroke and in humans. Mg has been suggested to have some potential efficacy, and a good safety profile if delivered i.v. early after stroke onset [103].",Nutrients,Magnesium,2021
Magnesium and Type 2 Diabetes,"A consistent body of evidence has linked Mg deficiency to alterations of insulin sensitivity and T2DM. Indeed, T2DM has been associated with several extra-and intra-cellular Mg abnormalities [2,104–106]. Lower cellular and/or ionized plasma Mg concentrations have been found in patients with T2DM despite still normal less sensitive total serum Mg levels [107,108]. Possible mechanisms favoring Mg depletion in T2DM include a low Mg dietary intake and an increased Mg urinary loss, while the absorption and retention of dietary Mg seem to be unchanged [109]. It has been reported an inverse relationship between Mg intake and the incidence of new cases of T2DM. Both hyperglycemia and hyperinsulinemia have been implicated in contributing to Mg depletion [110]. Both, hyperglycemia and hyperinsulinemia favor an excessive urinary Mg excretion, while insulin resistance may alter Mg transport [111]. Altered Mg metabolism may predispose to the development of T2DM and to an impairment of insulin-mediated glucose uptake [2]. Because of these pieces of evidence, Mg supplementation has been suggested as a possible non-pharmacologic, economic and safe treatment for the prevention and the metabolic control of T2DM. However, prospective trials concerning the effects of Mg supplementation in people with or at risk of T2DM are limited [112,113]. A modest beneficial effect of Mg supplements on glycemic profiles have been found in many, but not all, studies.",Nutrients,Magnesium,2021
Magnesium Supplementation and T2DM Outcomes,"A systematic review and meta-analysis including 18 double-blind randomized controlled trials (12 in subjects with diabetes and six in subjects at elevated risk of T2DM) reported that Mg supplementation may have some beneficial actions improving glucose parameters in people with T2DM and to improve insulin-sensitivity parameters in subjects at high risk of T2DM [114]. Using an umbrella review to map and grade health outcomes linked to Mg intake and supplementation, our group recently confirmed that an elevated Mg intake is associated with a decreased risk of T2DM [86].",Nutrients,Magnesium,2021
Magnesium and Cardiometabolic Syndrome,"There are also convincing proofs of a link of Mg deficit with metabolic syndrome [2,6,115]. In epidemiological studies, dietary Mg inadequacy has been connected with an increased risk of glucose intolerance, metabolic syndrome, and T2DM [115–117]. Intracellular Mg deficit, causing a defective activity of all the Mg-dependent kinases involved in the insulin signaling, and increasing oxidative stress would favor insulin resistance and resulting metabolic conditions, including glucose intolerance, metabolic syndrome and T2DM. Mg-deprivation in sheep caused an impairment of insulin-mediated glucose uptake [118], while Mg supplementation delayed the development of the disease in a rat model of diabetes [119]. Lower fasting insulin levels were found in healthy women without diabetes with higher Mg intakes [120]. Total dietary Mg intakes were inversely related to insulin responses to an oral glucose tolerance test [121].",Nutrients,Magnesium,2021
Magnesium in Asthma and Respiratory Insufficiency,"First, Haury in 1940 proposed a role for Mg in asthma showing a beneficial clinical response after i.v. Mg sulfate administration in two hospitalized patients having acute exacerbations of asthma [122]. In the following decades, other reports confirmed positive results of Mg i.v. treatment in acute airway constriction, suggesting a possible beneficial action of Mg in the mechanism of bronchial dilation [123,124], although other reports did not confirm the therapeutic effect [125,126]. Administration of i.v. Mg seems to increase, in an additive fashion, the bronchial dilating effect of terbutaline [127] and salbutamol [128] in improving functional pulmonary tests. Mg modulates the contractile state of bronchial smooth muscle cells; Mg depletion triggers bronchial contraction and spasm, while Mg restoration produces bronchial relaxation. Several possible mechanisms have been postulated for the positive Mg action to relax bronchial smooth muscle, such as the Ca channel blocking action of Mg [69], a decreased sensibility to the depolarizing action of acetylcholine [92], a stabilization of mast cells and T-lymphocytes [129], and a stimulation of nitric oxide [130] and prostacyclin [131]. In the general population, significant positive independent associations of dietary Mg intake with lung function and inverse associations with airway reactivity, inhaled methacholine, and respiratory symptoms (wheezing) have been reported, suggesting that a low Mg intake may be implicated in the etiology of asthma [132].",Nutrients,Magnesium,2021
Magnesium Status in Asthma,"However, total Mg serum levels are not clinically useful, since no differences were found in serum Mg in patients with asthma during acute exacerbation compared to a non-asthmatic population and serum total Mg is not predictive of the severity of the asthmatic attacks, or of the bronchial dilating response to Mg infusion [133]. Conversely, cellular Mg (more related to body Mg status) was found to be reduced in asthmatic subjects when compared to non-asthmatic controls [134]. In addition, our group demonstrated a direct correlation, in asthmatic patients, between cellular Mg levels and the methacholine bronchial reactivity confirming the presence of intracellular Mg alterations in asthma and have proposed an additive role for a non-pharmacological Mg supplementation in asthmatic patients [135]. Altogether, the available data suggest a role for cellular and body Mg deficit as a modulator of smooth muscle bronchial reactivity and contractility, facilitating bronchoconstriction in predisposed asthmatic subjects, and a possible preventive and/or therapeutic role for additive use of Mg administration in these persons [136].",Nutrients,Magnesium,2021
Magnesium and Psychiatric Disorders,"Several psychiatric disorders including anxiety, depression, irritability, insomnia, hypochondriasis, panic attacks, hyperexcitability, headache, dizziness, tremors, and psychotic behavior have been associated with Mg deficiency. Neuromuscular symptoms may be associated, including asthenia, muscular weakness, and myalgias (e.g., chronic fatigue syndrome and fibromyalgia) [137]. A number of enzymes and cellular reactions involved in stress responses are Mg-dependent [15]. Serum Mg levels have been proposed to be reduced in subjects with depression [138]. A recent study by Noah et al. showed that nearly half (forty-four percent) of patients screened for stress had a latent Mg insufficiency [139]. Mg deficit may produce electrophysiological evidence of hyperexcitability in the central nervous system (CNS). In Mg deficient rats, electroencephalogram (EEG) modifications were monitored during auditory stimuli. Several alterations with spike activity were found in the EEG, suggesting that auditory stimulation induced behavioral changes in Mg-deficient rats, which may be linked to Mg related-increased excitability of the CNS [140].",Nutrients,Magnesium,2021
Neuropsychiatric Effects of Magnesium Deficiency,"In humans, Mg insufficiency has been connected with neuro-muscular hyperexcitability [141]. Various possible mechanisms may link Mg deficiency to nervous hyperexcitability, such as the previously described Mg modulatory actions on cellular Ca, the increased peroxidation, the hyper-activation of some excitatory neurotransmitters (i.e., acetylcholine, catecholamines, NMDA and non-NMDA receptors of excitatory aminoacids), and a decreased activity of inhibitory neurotransmitters (i.e., gamma-aminobutyric acid (GABA), taurine, glutaurine, adenosine), as well as an increased production of neuropeptides, inflammatory cytokines, prostanoids, and a decreased activity of anti-oxidant defenses [137]. In relation to these links with the transduction and biological pathways implicated in depression, and because of the modulatory role of Mg on the ion channel of the NMDA-receptor complex [142], Mg supplements have been proposed to be helpful in the treatment of depression [143,144]. Some antidepressant drugs such as sertraline and amitriptyline have been suggested to increase intracellular Mg levels [145]. A systematic review showed that higher Mg intakes were associated with reduced depression symptoms [144]. Oral Mg supplements may provide advantage in the prevention of depressive symptoms and may be supportive as an adjunctive therapy. The effect of Mg supplementation on stress and anxiety is less documented. However, more interventional and prospective studies are needed in order to establish a clear role for Mg supplementation as a possible adjunct care in the treatment of depression, and other psychiatric disorders. Mg has also been suggested as an adjuvant treatment in the therapy of insomnia. Mg, in addition to being a natural NMDA antagonist and a GABA agonist, also has a relaxant action, and may increase melatonin levels, thus helping to improve sleep [146].",Nutrients,Magnesium,2021
Magnesium and Cognitive Decline,"A possible protective action of Mg in cognitive deterioration and AD was already suggested in 1990 [147]. Mg ion is important for a normal neuronal maturation and is present in the cerebrospinal fluid (CSF) in the CNS [148]. Mg passes the blood–brain barrier, and is actively transported by choroidal epithelial cells into the CSF [148]. Alterations of Mg metabolism are present in patients with dementia: total and ionized serum Mg levels, and various tissues Mg content have all consistently be found reduced in patients with AD [149–152]. Mg concentrations in the brain affect multiple biochemical processes involved in cognitive functions, including cell membrane stability and integrity, NMDA-receptor response to excitatory stimuli, and Ca-antagonist action [19]. It has been suggested that the neurotoxic effect of some metals, such as aluminum, may be related to an alteration of the incorporation of Mg into brain neurons, thus impairing Mg protective effects on brain tissue [147]. Mg has been reported to expedite toxin clearance, reduce neuroinflammation, inhibit the pathologic processing of amyloid protein precursor, inhibit abnormal tau protein phosphorylation, and reverse deregulation of NMDA receptors. However, the mechanisms of these effects are not completely clear [153].",Nutrients,Magnesium,2021
Magnesium Supplementation and Cognitive Health,"Mg-L-threonate administration has been reported to reduce neuroinflammation and decrease beta-amyloid deposition in experimental models of AD [154,155], and to enhance learning abilities, working and short- and long-term memory in rats [156]. Experimental animal studies are promising and may suggest that Mg supplementation if started at the early stages of cognitive deficits may decrease the slope of memory fall and cognitive decline [157]. In humans, only a few clinical trials have studied the role of Mg in cognitive health. Epidemiologically, it has been suggested that people consuming diets rich in Mg may have a reduced risk of cognitive decline. In 1400 healthy adult men followed for eight years, elevated dietary Mg intakes were associated with a reduced risk of developing mild cognitive impairment [158]. In another cohort study including more than 1000 community-dwelling Japanese participants aged over 60 years and followed for 17 years, it was found that those who were assuming more than 200 mg/day of Mg had thirty-seven percent less chances to develop any type of dementia and seventy four percent less chances to develop vascular dementia [159]. A short-term (12 weeks) randomized controlled trial suggested that Mg may help in improving cognitive abilities in elderly subjects with memory complaints [160]. Long-term prospective randomized clinical trials with Mg supplementation are needed to confirm if Mg-rich diets may help in preventing dementia and/or cognitive impairment.",Nutrients,Magnesium,2021
Magnesium and Osteoporosis,"Dietary Mg deficit has been hypothesized as a potential risk factor for osteoporotic disease and bone loss. Epidemiologic studies have shown that elevated dietary intakes of Mg were positively and significantly related to bone mineral density (BMD). On the opposite, inadequate dietary Mg intakes were linked to an increased rate of bone loss in postmenopausal osteoporotic women [161,162]. In the Health, Aging and Body Composition Study, it was observed that higher Mg intakes were associated with higher BMD in healthy white participants, aged 70 to 79 years at baseline [163]. Following a selective dietary Mg deprivation, Mg-depleted mice with frank hypomagnesemia developed osteoporosis, increased skeletal fragility associated with increased bone resorption, decreased bone formation, and impaired bone growth [164,165]. Elevated concentrations of inflammatory cytokines may play a role in explaining these bone alterations, although a clear pathophysiologic link remains undefined. Rude and Gruber showed that an increased osteoclastic bone resorption was associated with increased levels of inflammatory substance P and TNF-alfa in bone from Mg-deficient rats [166].",Nutrients,Magnesium,2021
"Magnesium Deficiency, Vitamin D, and Bone Fragility","In addition, Mg is necessary for vitamin D synthesis, transport, and activation; hence, Mg deficits would impair the production of the active form of vitamin D, 1,25-OH2 D3, and cause a resistance to PTH and vitamin D actions [167]. The effects of Mg deficiency added together with an altered PTH responsiveness and low 1,25-OH2 D3 synthesis would impair the bone formation and mineralization processes and would reduce the quality, and strength of the bone as well as the BMD. It has been hypothesized that Mg supplementation in doses sufficient to restore a normal bone turnover may reduce the bone loss and prevent the risk of osteoporosis [168,169]. In participants to the cohort “Osteoarthritis Initiative” followed for 8 years, it was found that women with the higher dietary Mg intake had a twenty-seven percent reduced risk for future fractures, confirming the positive role of maintaining an adequate Mg balance on the risk of osteoporosis and fragility fractures [170].",Nutrients,Magnesium,2021
Magnesium and Muscle Health,"Mg ion has a key role in all enzymes utilizing or synthesizing muscle ATP, and thus in the production of muscle energy, and indirectly in the contraction and relaxation processes. Mg deficit has been related to a poor muscle performance. Severe Mg deficits have been suggested to cause weakness, muscle pain and night cramps. It has been proposed that Mg deficit may contribute to the development of fibromyalgia [171]. Data on the effects of Mg supplements in fibromyalgia symptoms are scarce, although it was suggested that Mg supplements may be used to reduce tenderness, pain, and symptom severity in fibromyalgic subjects [172]. Dietary Mg deficiency in rats boosts the production of free radical in skeletal muscle and may cause several alterations in muscle cell metabolism together with structural impairments affecting the production of muscle energy needed for muscle contraction and relaxation [173]. In humans, Dominguez et al., showed a strong and independent relationship between serum Mg levels with muscle performance and several muscle parameters [174]. In young volunteers, Brilla et al. showed that Mg supplements (up to 8 mg/kg daily) were able to enhance muscle strength and endurance performance, and to reduce the oxygen consumption [175]. In older subjects, Veronese et al. showed that oral Mg supplementation (three hundred mg/day) was able to improve the physical performance, in particular in those subjects with a baseline low Mg dietary intake, proposing that Mg supplementation may help in preventing or delaying the decline in physical performance with age [176].",Nutrients,Magnesium,2021
Magnesium and Cancer,"In regard to cancer, Mg intake has been connected with the incidence of some cancers. However, the relation between Mg and cancer is complex, and nowadays there are more questions than answers [177]. In animal models, Mg may exert both anti- and pro-tumor effects such as inhibition of tumor growth at its primary site and facilitation of tumor implantation at its metastatic sites. In Mg-deficient mice, low Mg may both restrict and foster tumorigenesis, since inhibition of tumor growth at its primary site is observed in the face of increased metastatic colonization [177]. Oxidative stress and trace elements have been implicated in the development of breast cancer. However, how they impact the pathogenesis of the disease remain unclear [178]. Lower serum Mg levels in women with breast cancer may compromise the antioxidant defense systems involved in the carcinogenesis process. A study evaluated Mg metabolism, the superoxide dismutase activity, and its relation with oxidation stress in women with breast cancer. The authors reported that breast cancer patients display a complex alteration of Mg homeostasis, characterized by low dietary Mg intakes, reduced plasma, and erythrocytes Mg levels and an increase in Mg excretion in the urine [179]. Mg supplementation may have a protective effect on experimentally induced fibrosarcoma in rats [180], and may inhibit nickel-induced carcinogenesis in the rat kidney [181]. Mg has been suggested to have anti-tumor effects in colorectal cancer by inhibiting c-myc expression and ornithine decarboxylase activity in the mucosal epithelium of the intestine [182]. In human studies, high dietary Mg consumption has been suggested to be protective for the risk of developing colorectal cancer [183].",Nutrients,Magnesium,2021
Magnesium Intake and Cancer Risk,"In postmenopausal women, it has been proposed that a higher ratio of serum Ca to Mg may increase the risk for breast cancer [184]. Ca, Mg or Ca:Mg intake ratio may interact with polymorphisms in the SLC7A2 gene in associations with colorectal cancer [185]. Higher Mg intake was associated with a lower risk of liver cancer, based on an analysis of the National Institute of Health-American Association of Retired Persons (NIH-AARP) Diet and Health Study prospective cohort [186]. One of the reasons that the independent relationship of Mg intake and cancer protection is not easy to define is because dietary Mg content parallels fiber content and is mostly obtained from green leafy vegetables and whole cereals, rich sources of fiber, that are themselves cancer protective.",Nutrients,Magnesium,2021
Abstract and Background,"Background: Insomnia, affecting 30–40% of the global population, is a debilitating sleep disorder linked to significant health risks, including cardiovascular disease, metabolic syndrome, and neurodegeneration. Emerging evidence implicates dysregulation of circadian clock genes as a core molecular mechanism underlying its pathophysiology.
Methods and results: This review synthesizes current knowledge on how core clock genes regulate the sleep–wake cycle via transcription–translation feedback loops, incorporating recent insights into regulatory layers such as SUMOylation. We discuss how genetic polymorphisms and epigenetic modifications disrupt circadian rhythmicity, predisposing individuals to insomnia. The molecular pathways linking clock dysfunction to insomnia encompass dysregulation of neurotransmitter systems (melatonin, serotonin, GABA, dopamine), metabolic imbalance, neuroinflammation, mitochondrial oxidative stress, and altered synaptic plasticity. Chronic circadian misalignment, often driven by aberrant light exposure, exacerbates these disruptions.
Therapeutic implications: Targeting circadian pathways presents novel therapeutic avenues. Melatonin receptor agonists facilitate sleep initiation and phase alignment; synthetic REV–ERBα/β ligands enhance circadian amplitude; dopaminergic modulators address hyperarousal; and GABAergic drugs restore inhibitory balance. Notably, Traditional Chinese Medicine formulations exhibit multi-pathway regulatory effects on clock gene expression. However, treatment efficacy varies across insomnia subtypes, and challenges regarding pharmacokinetics and long-term safety remain.
Conclusion: Dysfunctional circadian clock genes are pivotal in insomnia pathogenesis via interconnected molecular pathways. Future research should focus on biomarker-driven, personalized chronotherapies targeting these genes and their downstream effects to improve clinical outcomes.",Annals of Medicine,Circadian Clock Genes & Insomnia,2025
Abbreviations and Article Information,"Abbreviations: ADHD: Attention-deficit/hyperactivity disorder; ATP: Adenosine triphosphate; BMAL1: Brain and muscle ARNT-like 1; CK1δ/ε: Casein kinase 1 delta/epsilon; CLOCK: Circadian locomotor output cycles kaput; CRY: Cryptochrome; CREB: cAMP response element-binding protein; DEC1/2: Differentially expressed in chondrocytes ½; DMH: Dorsomedial hypothalamus; DRN: Dorsal Raphe nucleus; DSPD: Delayed sleep phase disorder; EEG: Electroencephalogram; E-box: Enhancer box; FASPS: Familial advanced sleep phase syndrome; FBXL3: F-Box and leucine-rich repeat protein 3; GABA: γ-Aminobutyric acid; GABA-T: GABA transaminase; GWAS: Genome-wide association study; HDAC: Histone deacetylase; IL-6: Interleukin-6; ISI: Insomnia severity index; LC: Locus Coeruleus; lncRNA: Long noncoding RNA; MT1/MT2: Melatonin receptor 1/2; N24SWD: Non-24-hour sleep–wake disorder; NMDA: N-methyl-D-aspartate; NREM: Non-rapid eye movement; NR1D1/2: Nuclear receptor subfamily 1 group D member 1/2 (REV–ERBα/β); PER: Period circadian regulator; PTM: Post-translational modification; PVN: Paraventricular nucleus; REM: Rapid eye movement; RORα/γ: Retinoic acid receptor-related orphan receptor alpha/gamma; ROS: Reactive-oxygen species; SCN: Suprachiasmatic nucleus; SNP: Single nucleotide polymorphism; SSRI: Selective serotonin reuptake inhibitor; SUMOylation: Small ubiquitin-like modifier conjugation; SWS: Slow-wave sleep; TCM: Traditional Chinese medicine; TMN: Tuberomammillary nucleus; TTFL: Transcription–translation feedback loop; TNF-α: Tumor necrosis factor-alpha; VIP: Vasoactive intestinal peptide; VNTR: Variable number tandem repeat; 5-HT: 5-Hydroxytryptamine (serotonin).
© 2025 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. ARTICLE HISTORY: Received 6 June 2025, Accepted 13 October 2025. KEYWORDS: Circadian clock genes; insomnia; molecular mechanisms; therapeutic targets; pharmacology.",Annals of Medicine,Circadian Clock Genes & Insomnia,2025
Introduction: Prevalence and Health Consequences,"Insomnia is a common sleep disorder, which affects about 30–40% of the general population in the world. Its main manifestations are difficulty in falling asleep at night, difficulty in maintaining sleep or waking up early. Long-term insomnia not only impairs sleep quality, but also has a significant impact on daily functioning and overall health, increasing the risk of cardiovascular disease, metabolic disorders, and mental health problems such as depression and anxiety. In addition, insomnia may also induce digestive and respiratory diseases and metabolic syndrome, while increasing the incidence of neurodegenerative diseases such as mild cognitive impairment and dementia. These factors have made insomnia increasingly attracting attention from the medical community.",Annals of Medicine,Insomnia Epidemiology,2025
Primary vs Secondary Insomnia,"Insomnia can be divided into two types: primary and secondary. Primary insomnia refers to simple insomnia that still exists after excluding physical diseases, mental disorders and drug effects, there is no clear cause at present. Secondary insomnia is caused by other diseases, such as depression, anxiety, diabetes and cardiovascular disease. Despite the high prevalence of insomnia and its significant health consequences, its underlying molecular mechanisms are still not fully understood, emphasizing the need for further research to identify new treatment targets.",Annals of Medicine,Insomnia Classification,2025
Circadian Clock Genes and Sleep–Wake Regulation,"In recent years, advances in chronobiology have revealed the key role of biological clock genes in regulating the sleep–wake cycle and maintaining overall physiological homeostasis. The biological clock gene is the molecular basis of the endogenous circadian rhythm. External light is transmitted to the suprachiasmatic nucleus (SCN) through the retina–hypothalamic tract, forming a series of transcription and translation feedback loops. These circuits synchronously regulate the expression of the peripheral biological clock through the interaction and periodic expression of biological clock genes, thereby keeping the body synchronized with the outside world by alternating day and night. The core transcription and translation feedback loop is mainly composed of transcription factors Brain and Muscle ARNT-Like 1/Circadian Locomotor Output Cycles Kaput (BMAL1/CLOCK), Period Circadian Regulators (PERs) and Cryptochromes (CRYs). BMAL1 and CLOCK act as activators of the biological clock, forming heterodimers and activating the transcription of PERs and CRYs genes. PERs and CRYs are photoreceptor genes that act as inhibitors of the biological clock and form a positive and negative feedback loop with BMAL1 and CLOCK.",Annals of Medicine,Circadian Regulation,2025
Clock Gene Mutations and Sleep Disruption,"Studies have found that mutations in the BMAL1 gene can lead to increased conversion from non-rapid eye movement sleep (NREM) to rapid eye movement sleep (REM), shortened deep sleep time, reduced sleep, and increased night activity, which in turn leads to changes in diet patterns, glucose intolerance and liver steatosis. Mutations in the CLOCK gene can lead to reduced sleep time, continued nerve excitement, advanced phase, and circadian rhythm disorders. Mutations in the PERs and CRYs genes can also affect sleep. For example, mutations in the PERs gene impair the body’s ability to maintain the stability of the circadian cycle, shortening the circadian cycle. Mutations in the CRYs gene can reduce the number of sleep awakenings in mice, increase NREM sleep time and electroencephalogram (EEG) slow-wave activity. In addition, genetic variants in clock genes such as PER3 and CRY1 have been linked to changes in sleep patterns and increased susceptibility to sleep disorders. These findings highlight the potential importance of biological clock genes in the pathophysiology of insomnia and suggest that targeting these genes may provide new treatment opportunities.",Annals of Medicine,Clock Gene Mutations,2025
Review Objective and Scope,"The primary objective of this review is to delve into the molecular mechanisms linking circadian clock genes to insomnia. We place a strong emphasis on understanding how these genes regulate the neurotransmitter systems and cellular signaling pathways that are vital for sleep regulation. Furthermore, we explore the clinical significance of these findings, particularly the potential of circadian clock genes as targets for insomnia treatment. By integrating insights from molecular biology, neuropharmacology, and clinical research, this review aims to enhance our comprehension of insomnia’s pathophysiology and to identify promising avenues for future research and therapeutic development. It should be noted that the molecular mechanisms discussed herein, while particularly relevant to primary insomnia, also extend to secondary insomnia. This is because circadian misalignment, neurotransmitter dysregulation, and inflammatory-metabolic disturbances are commonly shared pathways in comorbid conditions such as depression, anxiety, cardiovascular disease, and metabolic syndrome, all of which frequently present with secondary insomnia.",Annals of Medicine,Circadian Mechanisms in Insomnia,2025
Core Circadian Clock Gene Functions,"The molecular circadian clock is orchestrated by a network of core genes, including CLOCK, BMAL1, PER1/2/3, and CRY1/2, which generate self-sustaining transcriptional–translational feedback loops with a 24-h periodicity. The CLOCK–BMAL1 heterodimer binds to Enhancer box (E-box) motifs in promoters of target genes, including PER and CRY, driving their transcription. Accumulated PER and CRY proteins form repressive complexes that translocate back to the nucleus, inhibiting CLOCK–BMAL1 activity and thereby closing the primary feedback loop. Secondary loops involve nuclear receptors REV–ERBα/β and Retinoic Acid Receptor-Related Orphan Receptor Alpha/Gamma (RORα/γ), which rhythmically regulate BMAL1 expression by competing for ROR response elements.",Annals of Medicine,Circadian Clock Genes,2025
Post-Translational Regulation of Clock Proteins,"Post-translational modifications (PTMs) critically regulate clock protein stability and subcellular localization. Phosphorylation of PER proteins by casein kinase 1δ/ε (CK1δ/ε) marks them for degradation via the ubiquitin–proteasome system, while F-Box and Leucine-Rich Repeat Protein 3 (FBXL3)-mediated ubiquitination targets CRY proteins for proteasomal turnover. Recent studies highlight the role of SUMOylation in modulating CLOCK–BMAL1 transcriptional activity, suggesting a novel layer of circadian regulation. SUMO modification of BMAL1 can enhance its transcriptional activation of E-box-driven target genes, whereas excessive SUMOylation promotes proteasomal degradation through crosstalk with ubiquitination pathways. SUMOylation of CLOCK influences its nuclear localization and stability, fine-tuning the amplitude and robustness of circadian oscillations. These dynamic PTMs ensure circadian precision and adaptability to environmental zeitgebers like light and feeding cycles.",Annals of Medicine,Circadian Regulation,2025
Circadian Disruption and Environmental Misalignment,"Circadian disruption is increasingly recognized as a key driver of insomnia, arising from complex interactions between environmental misalignment, genetic predisposition, and epigenetic modifications. The suprachiasmatic nucleus (SCN) regulates rhythmic output to peripheral clocks through hormonal and neural pathways, notably melatonin from the pineal gland and cortisol from the adrenal cortex. Aberrant light exposure, such as excessive nighttime blue light, suppresses melatonin secretion, delays sleep onset, and fragments sleep structure. Epidemiological studies reveal that shift workers exposed to chronic circadian misalignment show a two- to threefold higher prevalence of insomnia compared with day workers, underscoring the contribution of environmental disturbance to sleep disorders.",Annals of Medicine,Circadian Disruption,2025
Genetic Polymorphisms and Sleep Regulation,"Experimental models provide mechanistic insights into the link between circadian disruption and insomnia. Bmal1 knockout mice exhibit severe sleep fragmentation and reduced non-REM sleep, while Per2 mutations result in altered circadian phase and sleep instability. Human studies demonstrate that polymorphisms in circadian clock genes contribute to variability in sleep timing, duration, and vulnerability to insomnia. Among these, PER3 variable number tandem repeats (VNTRs) are associated with altered REM and slow-wave sleep duration; carriers of the long allele (PER35/5) tend to have prolonged deep sleep but shorter REM, whereas short allele (PER34/4) carriers show delayed sleep phase and higher insomnia severity under irregular schedules.",Annals of Medicine,Genetic Variants,2025
Additional Clock Gene Variants and Circadian Resilience,"Polymorphisms in other clock genes also influence sleep regulation. BMAL1 null alleles alter baseline sleep architecture in mice, and PER3 variants predict insomnia severity in alcohol-dependent patients. Epidemiological work by Viola and colleagues confirmed that PER3 polymorphisms affect sleep structure and waking performance. Meanwhile, the CLOCK 3111T/C variant has been linked to sleep initiation difficulties, early morning awakening, and maintenance problems in depressed cohorts, though other studies have failed to replicate this association. Large-scale cohort studies demonstrate that TIMELESS polymorphisms are associated with early morning awakening, with gender-specific effects. These findings collectively suggest that genetic variations in circadian clock genes modulate sleep architecture and insomnia risk, though their effects vary across populations. This variability reflects the concept of circadian resilience, namely the capacity of the circadian system to resist or adapt to environmental challenges.",Annals of Medicine,Clock Gene Polymorphisms,2025
Epigenetic Modifiers and Environmental Interactions,"Discrepancies between studies highlight the role of environmental and epigenetic factors as modifiers. Genetic predispositions may only manifest under specific conditions, such as shift work, alcohol dependence, or psychiatric comorbidities. Epigenetic modifications—including DNA methylation and microRNA regulation—have emerged as critical mechanisms linking environmental stressors to circadian gene expression. Methylation of circadian promoters can attenuate gene transcription and shift circadian phase, while histone acetylation and non-coding RNAs dynamically adjust circadian plasticity, further influencing susceptibility to insomnia.",Annals of Medicine,Epigenetics and Circadian Rhythm,2025
Epigenetic Aging and Insomnia,"Recent data from Rivero-Segura et al. emphasize the epigenetic consequences of insomnia in older adults. Their study found that insomnia is associated with acceleration of epigenetic clocks (GrimAGE, SkinBloodClock), global hypomethylation, and reduced methylation-inferred telomere length. These findings suggest that insomnia may not only disturb circadian gene expression but also contribute to biological aging via epigenetic modifications, oxidative stress, and impaired proteostasis. Such evidence strengthens the link between insomnia and systemic aging processes, particularly in older individuals, and underscores the importance of considering age when exploring interventional targets. In conclusion, circadian clock gene polymorphisms significantly impact sleep regulation, though their effects may vary across populations and interact with environmental factors. These genetic variations offer valuable insights for understanding individual differences in sleep patterns and developing personalized treatments for sleep disorders.",Annals of Medicine,Epigenetic Aging,2025
SCN and Transcription–Translation Feedback Loop,"The circadian clock command center of mammals is located in the SCN of the hypothalamus. At the molecular level, the SCN receives and integrates light signals to drive the transcription and translation of clock genes, forming a self-regulating transcription–translation feedback loop (TTFL). The core loop of TTFL consists of CLOCK, BMAL1, PER, and CRY genes. In the early morning, CLOCK and BMAL1 form a heterodimer CLOCK/BMAL1, bind to the E-box element in the promoter region of target genes, and activate the transcription of PER1-3 and CRY1-2. In the evening, after PER proteins are phosphorylated by casein kinase 1 (CK1), they form a CK1/PER/CRY complex with CRY proteins, which in turn inhibits the transcriptional activity of CLOCK/BMAL1, creating a negative feedback mechanism. In addition, other regulatory loops, such as NR1D1/REV–Erbα, RORα, and Differentially Expressed in Chondrocytes 1/2 (DEC1-2), also fine-tune the main loop to ensure molecular circadian rhythmicity and form a nearly 24-hour circadian cycle.",Annals of Medicine,Circadian TTFL,2025
SCN Outputs and Neural Circuit Regulation of Sleep,"The SCN regulates sleep rhythms through neural projections and neurotransmitter diffusion. During wakefulness, the SCN projects to arousal-promoting nuclei such as the locus coeruleus (LC), dorsal raphe nucleus (DRN), and tuberomammillary nucleus (TMN) via the ventral subparaventricular zone (vSPZ) and dorsomedial hypothalamus (DMH), while inhibiting the sleep-promoting ventral lateral preoptic nucleus (VLPO). During sleep, the VLPO inhibits the LC, DRN, and TMN, and SCN neurons project to the paraventricular nucleus (PVN) to regulate melatonin (MT) synthesis. Borbély’s ‘two-process model’ further explains circadian sleep regulation. It proposes that sleep is regulated by two interacting processes: the homeostatic sleep drive (process S) and the circadian pacemaker (process C). As wake time increases, process S promotes deep sleep in the early night. In the late night, process S declines and process C dominates, promoting arousal.",Annals of Medicine,SCN Neural Circuits,2025
Clock Gene Expression Variations in Insomnia,"Research shows that clock gene expression in insomniacs differs significantly. For instance, Attention-Deficit/Hyperactivity Disorder (ADHD) patients show rhythmic loss of PER2 and BMAL1 expression in oral mucosa. Some insomniacs carry CLOCK gene polymorphisms like rs1801260 T/C, linked to ADHD-related insomnia. Additionally, Per2Brdm1 mutant mice exhibit reduced glutamate transporter Eaat1 mRNA and protein levels. This causes decreased glial glutamate uptake and increased extracellular glutamate, possibly explaining alcohol-related insomnia mechanisms.",Annals of Medicine,Clock Gene Expression,2025
Melatonin and Circadian Feedback Regulation,"The interaction between circadian clocks and neurotransmitter systems is crucial for sleep regulation. Melatonin secretion, controlled strictly by the SCN, rises at night to promote sleep. The light–dark cycle synchronizes the circadian clock by regulating Per gene expression in the SCN, which in turn affects melatonin rhythm. Melatonin also provides feedback to regulate the circadian clock by binding to Melatonin Receptor 1/2 (MT1/2) in the SCN, inducing Per1 and Per2 expression, or by inhibiting glutamate activity to stabilize circadian rhythm.",Annals of Medicine,Melatonin Regulation,2025
Serotonin and Dopamine Interaction with Clock Genes,"Similarly, 5-Hydroxytryptamine (5-HT) system is influenced by the circadian clock, with its synthesis and secretion following a circadian rhythm likely regulated indirectly by the SCN. 5-HT modulates SCN activity through its receptors (e.g. 5-HT1B, 5-HT7, 5-HT2C) and reuptake transporters, thereby affecting circadian rhythm. SSRIs can adjust circadian rhythms by altering clock gene expression in the SCN. Abnormalities in circadian clock gene expression and neurotransmitter dysfunction can create a vicious cycle that exacerbates insomnia. For example, dopamine transport and degradation-related gene expression in clock gene-mutated mice shows circadian disorganization, potentially affecting reward-motivated behavior, and dopamine system abnormalities are linked to insomnia. Moreover, neurotransmitter level changes may further disrupt clock gene expression, destabilize rhythms, and worsen sleep disorders.",Annals of Medicine,Neurotransmitter Regulation,2025
Circadian Clock Genes and Cellular Pathways,"There are complex interactions between circadian clock genes and cellular pathways, and these interactions jointly contribute to the development of insomnia. These pathways encompass metabolic regulation, inflammatory signaling, mitochondrial dysfunction, and epigenetic modifications, all of which intersect with circadian rhythms to disrupt sleep homeostasis.",Annals of Medicine,Circadian Pathways,2025
Metabolic Dysregulation and Energy Homeostasis,"Circadian clock genes, particularly BMAL1 and CLOCK, govern metabolic processes by regulating genes involved in glucose metabolism, lipid synthesis, and mitochondrial function. Disruption of these genes impairs cellular energy balance, which is critical for maintaining sleep–wake cycles. Bmal1 knockout mice exhibit hypoglycemia and altered Adenosine Triphosphate (ATP) production in the hypothalamus, leading to fragmented sleep and reduced non-REM sleep duration. Similarly, Clock mutant models show dysregulated hepatic lipid metabolism, which correlates with increased nighttime activity and sleep fragmentation. These metabolic disturbances may impair the SCN signaling, thereby destabilizing circadian output to sleep-regulatory brain regions. Thus, metabolic dysregulation caused by circadian clock gene disruptions directly contributes to insomnia by compromising the energy-dependent processes necessary for stable sleep initiation and maintenance.",Annals of Medicine,Metabolic Dysregulation,2025
Inflammatory Signaling and Neuroimmune Crosstalk,"Chronic low-grade inflammation is increasingly recognized as a contributor to insomnia. Circadian clock genes modulate immune responses by regulating the expression of pro-inflammatory cytokines such as IL-6 and TNF-α. For example, PER2 deficiency in mice elevates systemic IL-6 levels, which disrupts SCN neuronal activity and promotes hyperarousal states characteristic of insomnia. In humans, polymorphisms in CRY1 are associated with elevated inflammatory markers and increased insomnia severity, suggesting a bidirectional relationship between circadian dysfunction and neuroinflammation. Furthermore, microglial activation in sleep-regulatory regions, driven by circadian misalignment, may exacerbate synaptic pruning abnormalities and sleep continuity issues. Therefore, neuroinflammatory processes stemming from circadian disruption play a direct role in insomnia pathogenesis by inducing hyperarousal and impairing sleep-regulatory neural circuits.",Annals of Medicine,Inflammation,2025
Mitochondrial Dysfunction and Oxidative Stress,"Mitochondrial rhythms, synchronized by circadian clock genes, are essential for maintaining redox balance. REV–ERBα, a key circadian nuclear receptor, regulates mitochondrial biogenesis and oxidative phosphorylation. Knockdown of REV–ERBα in mice increases reactive oxygen species (ROS) production in the SCN, impairing neuronal firing and melatonin secretion. Beyond their damaging effects at high levels, ROS also act as signaling molecules that modulate various cellular processes, including inflammatory responses and neuronal excitability, which are implicated in sleep regulation. Elevated ROS levels also inhibit BMAL1 transcription, creating a vicious cycle that exacerbates circadian disruption and sleep fragmentation. Clinical studies report elevated oxidative stress markers in insomniacs, correlating with reduced slow-wave sleep and impaired cognitive function. Consequently, mitochondrial dysfunction and oxidative stress directly contribute to insomnia by disrupting neuronal energy metabolism and promoting cellular damage in sleep-regulatory regions.",Annals of Medicine,Mitochondrial Dysfunction,2025
Epigenetic Modifications and Circadian Plasticity,"Epigenetic mechanisms, including DNA methylation and histone acetylation, dynamically regulate circadian gene expression. Hypermethylation of the CLOCK promoter reduces its transcriptional activity, leading to delayed sleep phase phenotypes. Specific histone marks such as H3K27ac (associated with active enhancers) and H3K4me3 (linked to active promoters) show circadian oscillations in the SCN and are altered in animal models of sleep disruption. Reduced H3K27ac levels at the Per2 promoter have been correlated with impaired sleep homeostasis. Histone deacetylase (HDAC) inhibitors, such as valproic acid, rescue Per2 expression in animal models of shift work disorder, restoring sleep architecture. Furthermore, the histone methyltransferase MLL1, which catalyzes H3K4me3, is regulated by CLOCK:BMAL1 and influences circadian period length. SIRT1, an NAD+-dependent deacetylase, also contributes to circadian regulation by modulating CLOCK activity and PER2 stability. Additionally, circadian lncRNAs (e.g. Per2AS) interact with chromatin modifiers to fine-tune rhythmic gene expression, offering novel targets for insomnia therapy.",Annals of Medicine,Epigenetic Regulation,2025
Synaptic Plasticity and Neuronal Excitability,"Circadian clocks regulate synaptic strength and neurotransmitter receptor trafficking. BMAL1 modulates glutamatergic signaling by controlling the expression of NMDA receptor subunits in the prefrontal cortex, a region critical for sleep initiation. Mice lacking Bmal1 exhibit hyperexcitability in cortical neurons, mirroring the hyperarousal observed in insomnia. CRY1 regulates GABAergic neurotransmission in the thalamus, and its mutation reduces sleep spindle density, impairing sleep maintenance. These findings underscore the role of clock genes in maintaining synaptic equilibrium necessary for stable sleep, and their dysfunction directly promotes insomnia by increasing neuronal excitability and disrupting inhibitory balance. In summary, insomnia arises from a confluence of cellular and molecular disruptions rooted in circadian clock dysfunction. Targeting these pathways—through metabolic interventions, anti-inflammatory agents, antioxidants, or epigenetic modulators—holds promise for developing precision therapies. Genetic, epigenetic, and environmental factors disrupt the core circadian feedback loop, leading to neurotransmitter dysregulation, metabolic imbalance, neuroinflammation, oxidative stress, and epigenetic alterations, which collectively contribute to insomnia.",Annals of Medicine,Synaptic Plasticity,2025
Melatonin Receptor Agonists,"Melatonin receptor agonists (MRAs) are a first-line circadian-aligned therapy for insomnia, leveraging melatonin’s role in synchronizing the SCN and peripheral clocks. By binding to MT1 and MT2 receptors, MRAs replicate endogenous melatonin’s dual effects: MT1 activation inhibits SCN neuronal firing to promote sleep initiation, while MT2 regulates phase shifts to stabilize circadian alignment. Ramelteon, a selective MT1/MT2 agonist, reduces sleep latency by 10–15min in chronic insomnia patients, particularly those with delayed sleep phase disorder (DSPD). A randomized controlled trial involving 829 adults demonstrated that ramelteon improved sleep efficiency by 8% compared to placebo, with effects persisting over 6months. Mechanistically, ramelteon enhances PER2 and CRY1 expression in the SCN, reinforcing disrupted feedback loops in circadian misalignment. However, its efficacy diminishes in shift workers with inconsistent light exposure, highlighting the need for adjunct light therapy. Agomelatine, a dual MT1/MT2 agonist and 5-HT2C antagonist, addresses both circadian misalignment and comorbid depression. A 12-week trial in 332 patients with depression-related insomnia reported a 30% reduction in nighttime awakenings and a 20% increase in slow-wave sleep (SWS) duration, likely via PER1 upregulation in the prefrontal cortex. For non-24-hour sleep–wake disorder (N24SWD) in blind individuals, tasimelteon demonstrates unique efficacy. In phase III trials, tasimelteon normalized circadian phase in 68% of participants by upregulating BMAL1 and PER1 expression, reducing sleep fragmentation by 40% over 6months.",Annals of Medicine,Melatonin Agonists,2025
Limitations and Clinical Considerations for MRAs,"Preclinical studies in rodents reveal tasimelteon’s neuroprotective effects, reducing hippocampal oxidative stress markers (e.g. malondialdehyde) by 50% through MT2-mediated activation of the Nrf2 pathway. Despite these advantages, MRAs face limitations. Approximately 15–20% of users report transient dizziness or daytime fatigue, likely due to off-target receptor interactions. Efficacy is also subtype-dependent; MRAs show minimal benefit in primary insomnia unrelated to circadian disruption. Long-term safety data (>1year) remain sparse, necessitating further studies on receptor desensitization and tolerance. In clinical practice, MRAs are most effective when combined with behavioral interventions (e.g. timed light exposure) and tailored to circadian phenotypes. Future research should prioritize biomarkers (e.g. PER3 polymorphisms) to predict individual responses and optimize dosing regimens.",Annals of Medicine,Melatonin Agonist Limitations,2025
REV–ERBα/β Synthetic Ligands,"REV–ERBα and REV–ERBβ, nuclear receptors encoded by NR1D1 and NR1D2, repress BMAL1 transcription by competitively binding to ROR response elements (ROREs), counteracting RORα/γ-mediated activation and stabilizing the secondary feedback loop essential for circadian oscillation. Synthetic ligands targeting these receptors have emerged as promising therapeutics for insomnia, particularly in cases driven by circadian misalignment and metabolic dysregulation. The REV–ERB agonist SR9009 enhances circadian amplitude by selectively binding to REV–ERBα/β, suppressing BMAL1 transcription, and phase-advancing peripheral clocks. In jet-lagged mice, SR9009 reduced sleep latency by 25% and increased non-REM sleep duration by 15%, accompanied by upregulated PER2 and CRY1 expression in the SCN. SR9011 improved sleep consolidation in a rodent shift work model, decreasing nighttime hyperactivity by 40% and restoring hepatic BMAL1 rhythmicity.",Annals of Medicine,REV–ERB Ligands,2025
"Efficacy, Challenges, and Future Development of REV–ERB Ligands","REV–ERB ligands also modulate metabolism. SR9009 normalized glucose tolerance and reduced oxidative stress markers (e.g. malondialdehyde) in the SCN of sleep-deprived mice. Combining REV–ERB ligands with behavioral interventions may enhance therapeutic outcomes. In a pilot trial, co-administration of SR9009 with timed blue light exposure in shift workers improved circadian alignment, reducing Insomnia Severity Index (ISI) scores by 35% over 4weeks. Emerging agonists like LY3045698, a brain-penetrant REV–ERBα ligand, selectively repress SCN BMAL1 without altering hepatic gene expression in primates, offering a strategy to minimize metabolic side effects. Current REV–ERB agonists face pharmacokinetic challenges, including poor oral bioavailability (15–20% in rodents) and short half-lives (~2h for SR9009), requiring frequent dosing. Strategies being explored include prodrug formulations, nanoparticle-based delivery systems, and novel brain-penetrant agonists with optimized pharmacokinetics. Additionally, REV–ERBα knockout mice exhibit impaired antiviral immunity, raising concerns about infection risk in long-term use.",Annals of Medicine,REV–ERB Ligand Challenges,2025
Dopamine Receptor Modulators,"Dopamine regulates arousal, reward processing, and circadian rhythms. Dysregulation of dopaminergic signaling, particularly via D1 and D2 receptors, is implicated in hyperarousal states and sleep fragmentation. Dopamine receptor modulators (DRMs) target these receptors to restore circadian alignment. Dopamine receptor partial agonists such as aripiprazole stabilize dopaminergic tone, balancing D2 receptor activation and inhibition. In rodent models of circadian disruption, aripiprazole reduced nighttime hyperactivity by 30% and increased non-REM sleep duration by 20% through upregulation of PER2 expression in the SCN. Cariprazine, a D3-preferring agonist, improved sleep continuity in mice with CLOCK mutations. Dopamine reuptake transporter antagonists such as modafinil indirectly enhance dopaminergic signaling by increasing extracellular dopamine; low-dose modafinil (50mg/day) advanced circadian phase by 1.5h in shift workers with insomnia.",Annals of Medicine,Dopamine Modulators,2025
DRM Limitations and Clinical Implications,"Selective D1 receptor antagonists such as ecopipam showed mixed results. Combined D2/D3 agonists like piribedil reduced nighttime awakenings by 40% in Parkinson’s disease-related insomnia. DRMs are associated with dose-dependent side effects including akathisia (15–20% with aripiprazole) and metabolic disturbances. Long-term use of dopamine reuptake inhibitors like modafinil may exacerbate anxiety or tachyphylaxis, limiting chronic application. Additionally, DRMs’ effects on reward pathways raise concerns about misuse potential in vulnerable populations.",Annals of Medicine,DRM Limitations,2025
GABAergic Drugs and Circadian Regulation,"GABA, the principal inhibitory neurotransmitter, suppresses neuronal excitability to promote sleep and interacts with circadian clock genes. SCN receives GABAergic inputs from interneurons and extra-SCN regions. GABA(A) receptor activation in the SCN regulates phase shifts and stabilizes circadian oscillations. Zolpidem is clinically effective for sleep-onset insomnia and may influence circadian timing by enhancing SCN neuronal synchronization and upregulating PER2 expression. Benzodiazepines modulate BMAL1 transcription in the SCN via GABA(A)-mediated inhibition of glutamatergic signaling, restoring rhythmicity in mice with Clock mutations. GABAergic drugs influence peripheral clocks as well; eszopiclone increases CRY1 expression in the liver and represses CLOCK-dependent metabolic genes, aligning peripheral clocks with SCN rhythm.",Annals of Medicine,GABAergic Drugs,2025
Bidirectional GABA–Circadian Interactions and Limitations,"GABAergic signaling and circadian genes form reciprocal regulatory loops. GABA(A) receptor activity in SCN neurons shows circadian rhythmicity driven by BMAL1/CLOCK-dependent transcription of GABA receptor subunits. Pharmacological enhancement of GABAergic tone can alter the phase and amplitude of PER1/2 expression, resetting circadian oscillations. CRY1 modulates GABA(A) receptor trafficking in thalamic neurons, linking clock gene function to inhibitory synaptic transmission. Long-term use of GABAergic drugs, however, may disrupt circadian plasticity. Chronic benzodiazepine exposure reduces PER3 expression in human fibroblasts, correlating with diminished circadian amplitude and non-REM fragmentation. Dependency risks and tolerance—linked to epigenetic changes at the CLOCK promoter—limit long-term utility.",Annals of Medicine,GABAergic Limitations,2025
Traditional Chinese Medicine (TCM) and Circadian Regulation,"TCM offers a holistic approach to insomnia treatment by targeting circadian rhythm regulation and systemic balance. Several TCM herbs and formulations demonstrate circadian-regulatory properties. Suanzaoren Decoction upregulates PER2 and CRY1 expression in the SCN of sleep-deprived rodents, enhancing circadian amplitude and consolidating non-REM sleep. Jiaotai Pills alleviate insomnia by inhibiting wakefulness-promoting substances (Glu, GAT3, GAT1, GABA-T) and increasing sleep-promoting ones (GABAA, GABAB, GAD). Anmeidan modulates the CREB/Per signaling pathway, upregulates Bmal1, Clock, Per1, and Cry1, and enhances circadian rhythmicity. Nobiletin, a polymethoxyflavonoid, acts as a RORs agonist and regulates BMAL1 and Per1. Modified Wen Dan Decoction improves neurotransmitters, inflammatory factors, sex hormones, and sleep quality in perimenopausal insomnia.",Annals of Medicine,Traditional Chinese Medicine,2025
TCM Summary and Integrated Therapeutic Perspective,"Acupuncture, a key TCM therapy, is effective for sleep disorders and may regulate circadian clock genes. Circadian clock gene-targeted therapies represent a promising avenue for insomnia management by addressing the molecular underpinnings of circadian disruption. From melatonin receptor agonists and REV–ERB modulators to dopamine receptor regulators, GABAergic drugs, and Traditional Chinese Medicine, these approaches illustrate the potential of circadian-aligned interventions. Each strategy has distinct advantages and limitations, highlighting the translational significance of circadian biology in guiding personalized treatment.",Annals of Medicine,Integrated Circadian Therapies,2025
Conclusion,"Insomnia, a pervasive sleep disorder with significant health implications, is increasingly recognized as a condition rooted in molecular dysregulation of circadian clock genes. This review synthesizes evidence highlighting the pivotal role of core circadian clock components—BMAL1, CLOCK, PER, CRY, and REV–ERBα/β—in orchestrating sleep–wake cycles and maintaining physiological homeostasis. Genetic polymorphisms, epigenetic modifications, and environmental disruptions in these genes contribute to circadian misalignment, hyperarousal states, and metabolic-inflammatory imbalances, all of which exacerbate insomnia. Mechanistically, clock genes regulate neurotransmitter systems (e.g. melatonin, serotonin, GABA, dopamine), mitochondrial function, and neuroimmune crosstalk, offering a molecular framework to explain sleep fragmentation and impaired sleep quality. Current therapeutic strategies targeting circadian pathways, such as melatonin receptor agonists (e.g. ramelteon, agomelatine), REV–ERB synthetic ligands (e.g. SR9009), and GABAergic drugs, demonstrate efficacy in specific subtypes of insomnia, particularly those linked to circadian disruption. However, limitations persist, including subtype-dependent responses, pharmacokinetic challenges, and long-term safety concerns. Traditional Chinese Medicine interventions, such as Suanzaoren Decoction and acupuncture, show promise in modulating clock gene expression and restoring circadian rhythms, though further mechanistic validation is required.",Annals of Medicine,Circadian Clock & Insomnia,2025
Future Research Directions,"To translate the promise of circadian medicine into clinical reality for insomnia treatment, future research must address several key priorities with a focus on biomarker-driven, personalized approaches. First, the discovery and validation of robust circadian biomarkers are essential for patient stratification. This includes identifying genetic variants beyond known polymorphisms (e.g. through large-scale pharmacogenomic studies) and utilizing physiological measures like dim light melatonin onset (DLMO) to predict individual response to timed light, melatonin, or novel clock-targeting drugs. Critically, biomarker-driven clinical trials that stratify insomnia patients based on circadian phenotypes are needed to optimize treatment matching and objectively measure therapeutic efficacy. Second, integration of multi-omics technologies (e.g. transcriptomics, metabolomics) is crucial for defining molecular subtypes of insomnia and uncovering novel therapeutic targets within the circadian–immune–metabolic axis. Third, overcoming pharmacokinetic limitations of next-generation compounds (e.g. REV–ERB agonists) requires innovative strategies such as prodrug development and nanoparticle-based delivery systems to enhance bioavailability and half-life. Fourth, there is a critical shortage of long-term safety and efficacy data for most circadian-targeted therapies, necessitating prospective longitudinal studies and real-world evidence to assess risks such as receptor desensitization and long-term physiological impacts. Finally, the mechanistic validation of TCM interventions requires a modern research framework involving computational target identification, preclinical models, multi-omics analyses, and biomarker-driven clinical trials incorporating molecular endpoints and objective sleep measures. Embracing this comprehensive and evidence-based roadmap will be crucial for developing precise and effective chronotherapeutic interventions for insomnia.",Annals of Medicine,Circadian Therapeutics,2025
Sleep Duration and Global Health,"Habitual sleep duration is consistently associated with many domains of overall health and functioning. Although the amount of sleep essential for optimal functioning and health may be difficult to ascertain at the individual level, more than 50 years of converging findings have demonstrated that sleeping too little or too much is associated with increased morbidity and mortality.1 Commensurate with the preponderance of evidence over the last 4 decades showing strong associations between sleep duration and health outcomes, several organizations have proposed age-based recommendations for healthy sleep, including the Sleep Research Society and American Academy of Sleep Medicine2,3 and the National Sleep Foundation.4 These recommendations have enabled meaningful comparisons across cross-sectional and longitudinal studies, which heretofore had been challenging, given that variable definitional criteria were used in published sleep reports. Moreover, the consensus that the amount of sleep needed over the lifespan is age dependent has informed policies to promote healthy sleep differently for different age groupings and to monitor progress made toward achieving the national mandate of healthy sleep at the individual and population levels.",JAMA Network Open,Sleep Duration,2021
"Sleep Duration, Mortality, and East Asian Populations","The significance of the results of the study by Svensson et al5 is 2-fold. First, investigators presented evidence of a strong curvilinear association of sleep duration with all-cause mortality using data from 4 East Asian countries (Japan, China, Singapore, and Korea) studied between 1984 and 2002. Findings from their cohort study, along with other previous epidemiologic studies enrolling Asian populations, make a compelling case that habitual sleep duration is an important harbinger of health and longevity among East Asian individuals. This suggests that adequate sleep is indeed a global health issue, rather than solely a concern of health policy makers in Western populations. Second, they reiterate the urgent need to generate guidelines governing recommended healthy sleep amounts for individuals across the globe, which should reflect differences in geographic areas as well as demographic and cultural factors. Just as sleep needs likely vary by age, recommendations may require calibration among various groups around the globe. It may be the case that the association between sleep and health in Western countries is different from that in other parts of the world, given differences in behaviors (eg, diet, physical activity, smoking), environmental exposures (eg, photoperiod, toxins), and sleep-related health risks (eg, diabetes, heart disease, infectious disease).",JAMA Network Open,Sleep Duration,2021
Age-Modified Effects of Sleep Duration on Mortality,"The observation that age modified the associations of sleep duration with mortality in the study by Svensson et al5 is especially noteworthy. It is consistent with recent analyses of data from the Chinese Longitudinal Healthy Longevity Survey, finding U-shaped associations of sleep duration with all-cause mortality, favoring the highest survival among individuals habitually sleeping 7 to 8 hours.6 These data align with the epidemiologic findings of age-associated effects on sleep duration among US residents, which have prompted the aforementioned recommendations.3,4 The results of this important study are not diminished by a lack of guidelines specifically for the populations studied, but notably, countries in Asia have not advanced many sleep education campaigns, especially toward groups with the highest risk. Delineating consensus around a sleep health scheme for Asian populations would enable a more culturally competent approach to the depiction of the potential consequences of sleep loss across different age groups. For example, campaigns could address sleep insufficiency in families, school-aged children and adolescents, working-age adults, and older adults. This differentiation might be necessary within the Asian population, given the observed differences in environmental factors that characterize these populations.",JAMA Network Open,Sleep Duration,2021
Cultural and Demographic Factors in Sleep Recommendations,"Furthermore, differing psychosocial and cultural factors might also influence sleep amount differentially among the 4 studied East Asian populations, and the relationships between sleep and health factors may differ among these groups.7 The finding that age modifies the associations of sleep duration with mortality risks particularly among men is equally important. This finding is not entirely novel. Similar data have been reported based on adjusted epidemiologic sleep models,1,3 but results of the study from Svensson et al5 suggest that while men might be at increased mortality risks if they sleep 5 or fewer hours or 10 or more hours, women sleeping 8 or 9 hours seem to have a greater mortality risk as well. In all, these findings show that recommendations of a healthy sleep amount must also consider sex, as it remained an independent factor for increased morbidity and mortality associated with sleep duration in fully adjusted multivariate models. Of note, available data indicate that individuals’ race and ethnicity as well as their place of birth should also be considered in such recommendations to enhance their applicability in various communities. Unfortunately, no definitive conclusions could be made regarding their effects on the analyses that Svensson et al5 reported, as the independent contributions of these factors in their models were not ascertained.",JAMA Network Open,Sleep Duration,2021
Global Need for Targeted Sleep Health Guidelines,"While great progress has been made regarding the implementation of policies informed by recommendations, more targeted strategies have been hampered by a lack of specifications for healthy sleep that consider potential differences in factors including an individual’s sex, race and ethnicity, and/or place of birth. The findings of this study serve as an essential call to action to sleep investigators and policy makers across the globe to think critically about the need for more targeted evidence-based recommendations. The field would benefit greatly from the development of new guidelines for healthy sleep amounts that consider all factors that adversely affect habitual sleep on a global scale. Furthermore, sleep duration is only 1 dimension of sleep and circadian health. Future work should expand the scope to include sleep timing, regularity, and quality as well as daytime consequences.",JAMA Network Open,Sleep Duration,2021
Study Overview and Objectives,"To clarify the association of sleep duration with all-cause and cardiovascular mortality, and further estimate the population attributable fraction (PAF) for the 10-year risk of cardiovascular disease (CVD) due to inappropriate sleep duration among US adults, we included data of the National Health and Nutrition Examination Survey (NHANES) from 2005 to 2014 by linkage to the National Death Index until December 31, 2015 in a prospective design. Cox proportional hazards models were used for multivariate longitudinal analyses. The Pooled Cohort Equations methods was adopted to calculate the predicted 10-year CVD risk. In the current study, sleep <5 h or longer than 9 h per day were significantly associated with elevated risks of all-cause mortality, and the multivariable-adjusted HRs across categories were 1.40 (95% CI, 1.14–1.71), 1.12 (95% CI, 0.91–1.38), 1 (reference), 1.35 (95% CI, 1.12–1.63), and 1.74 (95% CI, 1.42–2.12). Similarly, the HRs of cardiovascular mortality across categories were 1.66 (95% CI, 1.02–2.72), 1.15 (95% CI, 0.77–1.73), 1 (reference), 1.55 (95% CI, 1.05–2.29), and 1.81 (95% CI, 1.09–3.02). Under a causal–effect assumption, we estimated that 187000 CVD events were attributable to short sleep duration and 947000 CVD events were attributable to long sleep duration from 2018 to 2028.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Introduction and Epidemiological Context,"Sleep is a complex set of brain processes that supports physiological needs, and healthy sleep duration has been increasingly recognized as a public health issue. The American Academy of Sleep Medicine and the Sleep Research Society recommend adults obtain seven or more hours of sleep per night. However, the proportion of US adults with short sleep duration (<7 h/day) has increased over the past decades, while long sleep duration (≥9 h) shows an opposite trend. Experimental findings suggest short sleep duration is associated with adverse physiological and immunological consequences, but several researchers argue that long sleep may be a greater concern. A previous meta-analysis suggested that both short and long sleep durations were associated with increased risk of all-cause mortality and cardiovascular events, though other studies reported inconsistent results. Interpretation of associations has been restricted by diverse study designs, target populations, and differences in reference sleep duration (6–8 h, 7–8 h, or 7–9 h). Cardiovascular disease remains the leading cause of death in the United States. However, no study has yet translated prospective associations between sleep duration and cardiovascular health into measures of population-level impact such as PAF.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Study Design and NHANES Cohort Description,"NHANES is an ongoing national cross-sectional survey conducted by the National Center for Health Statistics to assess health and nutritional status in the US since 1999. The Research Ethics Review Board approved the protocol, and informed consent was obtained from all participants. Questions on sleep duration were added to NHANES beginning in 2005. We included adults aged ≥20 years from five NHANES cycles (2005–2014) linked to National Death Index records through December 31, 2015. We further estimated population attributable fraction for 10-year CVD risk using the NHANES 2017–2018 cycle. Sleep duration during 2005–2014 was based on the question: “How much sleep do you usually get at night on weekdays or workdays?” In 2017–2018, sleep duration was calculated from self-reported usual sleep and wake times. All-cause and CVD mortality were identified by National Death Index linkage and defined using ICD-10 codes I00–I09, I11, I13, I20–I51, and I60–I69. Follow-up time was from interview date to mortality date or end of follow-up. Covariates included demographic factors, lifestyle factors, BMI, diet quality, comorbidities, and family history of disease.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Study Overview and Objectives,"To clarify the association of sleep duration with all-cause and cardiovascular mortality, and further estimate the population attributable fraction (PAF) for the 10-year risk of cardiovascular disease (CVD) due to inappropriate sleep duration among US adults, we included data of the National Health and Nutrition Examination Survey (NHANES) from 2005 to 2014 by linkage to the National Death Index until December 31, 2015 in a prospective design. Cox proportional hazards models were used for multivariate longitudinal analyses. The Pooled Cohort Equations methods was adopted to calculate the predicted 10-year CVD risk. In the current study, sleep <5 h or longer than 9 h per day were significantly associated with elevated risks of all-cause mortality, and the multivariable-adjusted HRs across categories were 1.40 (95% CI, 1.14–1.71), 1.12 (95% CI, 0.91–1.38), 1 (reference), 1.35 (95% CI, 1.12–1.63), and 1.74 (95% CI, 1.42–2.12). Similarly, the HRs of cardiovascular mortality across categories were 1.66 (95% CI, 1.02–2.72), 1.15 (95% CI, 0.77–1.73), 1 (reference), 1.55 (95% CI, 1.05–2.29), and 1.81 (95% CI, 1.09–3.02). Under a causal–effect assumption, we estimated that 187000 CVD events were attributable to short sleep duration and 947000 CVD events were attributable to long sleep duration from 2018 to 2028.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Introduction and Epidemiological Context,"Sleep is a complex set of brain processes that supports physiological needs, and healthy sleep duration has been increasingly recognized as a public health issue. The American Academy of Sleep Medicine and the Sleep Research Society recommend adults obtain seven or more hours of sleep per night. However, the proportion of US adults with short sleep duration (<7 h/day) has increased over the past decades, while long sleep duration (≥9 h) shows an opposite trend. Experimental findings suggest short sleep duration is associated with adverse physiological and immunological consequences, but several researchers argue that long sleep may be a greater concern. A previous meta-analysis suggested that both short and long sleep durations were associated with increased risk of all-cause mortality and cardiovascular events, though other studies reported inconsistent results. Interpretation of associations has been restricted by diverse study designs, target populations, and differences in reference sleep duration (6–8 h, 7–8 h, or 7–9 h). Cardiovascular disease remains the leading cause of death in the United States. However, no study has yet translated prospective associations between sleep duration and cardiovascular health into measures of population-level impact such as PAF.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Study Design and NHANES Cohort Description,"NHANES is an ongoing national cross-sectional survey conducted by the National Center for Health Statistics to assess health and nutritional status in the US since 1999. The Research Ethics Review Board approved the protocol, and informed consent was obtained from all participants. Questions on sleep duration were added to NHANES beginning in 2005. We included adults aged ≥20 years from five NHANES cycles (2005–2014) linked to National Death Index records through December 31, 2015. We further estimated population attributable fraction for 10-year CVD risk using the NHANES 2017–2018 cycle. Sleep duration during 2005–2014 was based on the question: “How much sleep do you usually get at night on weekdays or workdays?” In 2017–2018, sleep duration was calculated from self-reported usual sleep and wake times. All-cause and CVD mortality were identified by National Death Index linkage and defined using ICD-10 codes I00–I09, I11, I13, I20–I51, and I60–I69. Follow-up time was from interview date to mortality date or end of follow-up. Covariates included demographic factors, lifestyle factors, BMI, diet quality, comorbidities, and family history of disease.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Sleep Duration Assessment and Mortality Ascertainment,"During the 5 cycles of NHANES from 2005 to 2014, sleep duration was determined using the question, “How much sleep do you usually get at night on weekdays or workdays?” The response was recorded as the number of hours of sleep obtained on a typical night and assessed as a continuous variable. In NHANES 2017–2018, participants reported usual sleep time and wake time from which hours of sleep were calculated. All-cause and CVD mortality were determined by linking NHANES to the National Death Index through December 31, 2015. Mortality data were linked using anonymized sequence numbers. Death from cardiovascular disease was defined according to ICD–10 codes I00–I09, I11, I13, I20–I51, and I60–I69. Follow-up time was defined as the period from the date of interview to the date of death or the end of follow-up, whichever occurred first. Covariates collected included demographic characteristics and lifestyle factors such as age, sex, race/ethnicity, smoking status, physical activity, alcohol consumption, body weight, and height. Additional variables included dietary quality via the Healthy Eating Index 2015, comorbidity conditions based on diagnosis or prescribed medication, and family history of chronic disease.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Statistical Analysis and Hazard Models,"Sleep duration was categorized into five groups: ≤5, 6, 7, 8, and ≥9 hours per day. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals with 7 hours as the reference category. Model 1 adjusted for age and sex. Model 2 additionally adjusted for race/ethnicity, BMI, educational level, physical activity, alcohol intake, smoking status, and diet quality. Model 3 further adjusted for family history of chronic disease and comorbidities. To examine potential nonlinear associations between sleep duration and all-cause mortality, sleep duration was treated as a continuous variable and fitted using restricted cubic splines with three knots. Likelihood ratio tests assessed non-linearity. Stratification analyses were performed across subgroups including age, sex, race/ethnicity, education, BMI, smoking, alcohol drinking, and physical activity. Wald F tests evaluated interaction effects with Bonferroni correction (P < 0.005). Sensitivity analyses excluded participants with heart disease or cancer, and those who died within the first year of follow-up.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Population Attributable Fraction and CVD Risk Modeling,"Under an assumption of causality, the study estimated the proportion of cardiovascular disease events attributable to short or long sleep duration in the United States. Sleep duration and risk factors for CVD were based on data from NHANES 2017–2018. Pregnant women and participants with self-reported cardiovascular disease were excluded. The American Heart Association’s Pooled Cohort Equations (PCE) were used to calculate 10-year CVD risk for adults aged 40 to 79 years. Risk variables included age, total cholesterol, HDL cholesterol, systolic blood pressure (treated or untreated), diabetes status, and smoking status. The natural logarithms of these variables were multiplied by race–sex–specific coefficients, and the predicted 10-year risk was calculated using validated formulae. Altered risk assuming 7 hours of sleep (Rq) was computed using hazard ratios for per-hour deviation from 7 hours based on meta-analysis data: HR 1.12 per hour above 7 and HR 1.06 per hour below 7. The number of CVD events attributable to sleep duration was estimated as Σ(Rp − Rq) multiplied by population size, and population attributable fraction as Σ(Rp − Rq)/Σ(Rp). All analyses incorporated NHANES sampling weights and design.",Frontiers in Public Health,Sleep Duration & Mortality,2022
Overview of Stress Management Interventions,"Effectiveness of stress management interventions to change cortisol levels: a systematic review and meta-analysis Olivia Rogerson, Sarah Wilding, Arianna Prudenzi, Daryl B. O’Connor. Stress has a damaging impact on our mental and physical health, and as a result, there is an on-going demand for effective stress management interventions. However, there are no reviews or meta-analyses synthesising the evidence base of randomised controlled trials testing the effectiveness of psychological interventions on changing cortisol levels (the stress hormone) in non-patient groups. Therefore, the primary aim of this systematic review and meta-analysis was to address this gap. Six databases (Medline, PsychInfo, Embase, CINAHL, Cochrane and Web of Science) were searched (1171 studies identified) with 58 studies (combined N = 3508) included in the meta-analysis. The interventions were coded into one of four categories; mind body therapies, mindfulness, relaxation or talking therapies. A random effects meta-analysis on cortisol as measured in blood, saliva or hair found that stress management interventions outperformed pooled control conditions with a medium positive effect size (g = 0.282). The studies that utilised cortisol awakening measures (g = 0.644) revealed larger effects of stress management interventions than those that measured diurnal cortisol (g = 0.255). Mindfulness and meditation (g = 0.345) and relaxation (g = 0.347) interventions were most effective at changing cortisol levels, while mind body therapies (g = 0.129) and talking therapies (g = 0.107) were shown to have smaller and non-significant effect sizes.",Elsevier,Cortisol,2023
Intervention Effects and Moderators,"Additionally, studies that utilised an active control group (g = 0.477) over passive control group (g = 0.129) were found to have stronger effects. Length of the intervention, study quality, risk of bias, age and gender did not influence the effectiveness of interventions and there was no evidence of publication bias. Overall, the current findings confirm that stress management interventions can positively influence cortisol levels. Future research should investigate the longer term implications for health and health outcomes. 1. Introduction Stress is a profound public health concern and an important mechanism through which the social and physical environment can impact later health outcomes (O’Connor et al., 2021). It is well established that experiencing stressful life events and reporting greater perceived stress over sustained periods of time are associated with poorer mental and physical health (Epel et al., 2018; O’Connor et al., 2021). Additionally, experiencing traumatic life events across one’s life have also been consistently found to be associated with poorer health outcomes (Howarth et al., 2020; Liu and Miller, 2014). A key mechanism regulating how the environment impacts the stress process is the stress hormone – cortisol. Cortisol is a product of the hypothalamic-pituitary adrenal (HPA) axis system which plays an essential role in regulating the body’s biological systems - from metabolic to immune systems (Lupien et al., 2009; Sapolsky et al., 2000). The dysregulation of the HPA axis is well documented to have links with negative health outcomes.",Elsevier,Cortisol,2023
HPA Axis Dysregulation and Allostatic Load,"The chronic over-activation of the HPA axis through experiencing acute stress or stressful life events can lead to allostatic load (McEwen, 1998). Most recently, allostatic overload was conceptualised referring to the detrimental impacts of stress on the body’s biological systems when stress mediators, such as cortisol, are released to respond to stress in one’s environment but their excessive and prolonged use, as well as dysregulation, leads to tissue damage (McEwen and Rasgon, 2018). Collectively, stress, and by part, cortisol, impacts psychological and physical body functioning; subsequently implicated in mental and physical health outcomes, suggesting cortisol regulation plays a key mediating role in the relationship between stress exposure and later negative health outcomes (Adam et al., 2017; Chrousos and Gold, 1992; O’Connor et al., 2021). 1.1. The stress response and health outcomes Low and high cortisol responses to stress may be associated with poor health outcomes; research has emerged to suggest that smaller increases, or a blunted cortisol response, to stress may be indicative of current ill-health or future health risks (Lovallo, 2016). Lower cortisol stress reactivity has been shown to be associated with the risk of obesity and with symptoms of depression and anxiety (de Rooij, 2013). In other research it was found that individuals who had previously made a suicide attempt exhibited low levels of cortisol in response to an acute stressor compared to control participants (O’Connor et al., 2017).",Elsevier,Cortisol,2023
Blunted and Heightened Cortisol Responses,"Moreover, the results of a meta-analysis found evidence of an association between early-life adversity and a blunted cortisol response to social stress (Bunea et al., 2017). Conversely, literature exists whereby heightened cortisol responses are associated with poorer health outcomes. Specifically, in trauma participants, it has been shown that there is an increase in cortisol to a stressor (Heim et al., 2000). Additionally, in another study, an elevated cortisol response to a stressor increased the odds of experiencing hypertension and progression to coronary artery calcification 3 years later (Hamer and Steptoe, 2012). Collectively, evidence points towards both heightened and blunted cortisol responses being associated with poorer health outcomes in the future. 1.2. Cortisol across the day The cortisol awakening response (CAR) is also implicated in later health status; linked to an array of health outcomes as confirmed in a meta-analysis whereby enhanced CAR is linked to job stress and general life stress. Conversely, reduced CAR has also been found to be associated with fatigue, exhaustion and burnout (Chida and Steptoe, 2009). The natural cortisol fluctuations throughout the day also play an important role in relation to later health. A flatter diurnal slope represented by low morning and high evening levels has also been suggested to be indicative of HPA dysregulation.",Elsevier,Cortisol,2023
Diurnal Cortisol Patterns and Health Outcomes,"Flatter diurnal cortisol slopes across the waking day may be one mechanism by which stress influences negative health outcomes (Adam and Kumari, 2009). A number of studies have found that there is an association between a flatter cortisol slope and negative health outcomes such as depression, cardiovascular disease, obesity and suicide attempt (Matthews et al., 2006; O’Connor et al., 2020; Ruttle et al., 2013). This is synthesised in a meta-analysis that found consistent evidence that flatter cortisol slopes were associated with numerous poor health outcomes, from cancer, to depression and even obesity (Adam et al., 2017). 1.3. Stress management interventions Therefore, taken together, it is clear that stress can be damaging for our mental and physical health, and as a result, there is an on-going demand for effective stress management interventions. An abundance of stress management interventions exist, however, which type of intervention is most effective? Is there evidence that they can influence cortisol? How do they perform in randomised controlled trials? For example, some of the most increasingly popular intervention approaches are mindfulness based (Khoury et al., 2013).",Elsevier,Cortisol,2023
"Mindfulness, Meditation, and Intervention Types","A previous systematic review reported varied success for mindfulness-based interventions on changing cortisol outcomes, finding mindfulness-based interventions had limited effectiveness but that they were more effective when standardised measures of cortisol were assessed such as the CAR and diurnal slope, instead of unstandardised measures such as averages of raw cortisol concentrations (Sanada et al., 2016). A recent meta-analysis found that meditation interventions were effective at lowering cortisol levels but only in highly stress samples that assessed cortisol in blood (Koncz et al., 2021). There is also evidence that psychological interventions can influence cortisol levels in patients with cancer, psychiatric conditions and other health issues (e.g., Antoni et al., 2023; Saban et al., 2022). However, there are no reviews or meta-analyses synthesising the evidence base of randomised controlled trials testing the effectiveness of psychological interventions on changing cortisol levels in non-patient groups. Therefore, the primary aim of the current systematic review and meta-analysis was to examine the effectiveness of psychological interventions to reduce cortisol levels in healthy adults that used randomised controlled trial designs.",Elsevier,Cortisol,2023
Aims and Methodological Considerations,"The secondary aim was to investigate the heterogeneity of any observed effects in terms of the type of cortisol measurement (in blood, hair or saliva), control group (active, inactive, waitlist or active/passive) and intervention together with exploring the moderating effects of sample size, study quality and risk of bias.",Elsevier,Cortisol,2023
Data Extraction Plan,"2.7. Data extraction plan The following data was extracted from each study: number of participants analysed with cortisol, the number of participants in the intervention and control group(s), the mean age of the entire sample and separate intervention/control groups (if available). The percentage of females in the study, the included control conditions (active, inactive, waitlist), pooled control conditions (active/passive), type of intervention, broad intervention category, length of intervention in absolute minutes (if available), an interpretation of length of intervention (as short (0 – 250 minutes), medium (251 − 800 minutes), long >801 minutes), type of cortisol sampling (blood/saliva/hair), categorisation of cortisol measurement (awakening/diurnal), number of days cortisol was measured on, number of times per day cortisol measured, timing of cortisol measurement (AM/PM/AM – PM), study quality (as described above) and whether the sample was stressed or non-stressed.",Elsevier,Cortisol,2023
Meta-Analytic Procedure,"2.8. Meta-analytic procedure All analyses were conducted using the Comprehensive Meta-Analysis 4.0 (CMA) software (Borenstein, 2022). The aim of the meta-analysis was to determine the effectiveness of stress management interventions on the change in cortisol levels from pre-intervention to post-intervention; meaning the dependent variable was the standardised mean difference change in cortisol from pre- to post-intervention between the intervention and comparator group. By utilising the standardised mean difference it permitted us to summarise evidence when studies used a variety of sampling strategies; from single measure, cortisol awakening response to diurnal cortisol. Following the procedure of Koncz et al. (2021) we devised a hierarchy of cortisol reporting, should different indices be available in a study; selecting the AUCg measure first, followed by the mean of multiple measures then choosing a single measurement. Additionally, if a study reported more than one control condition we included both contrasts. CMA software takes an average of multiple effects sizes in one study, as these are not independent of one another, before calculating a grand average. The current meta-analysis utilised the random effects model and Hedges g as a measure of effect size.",Elsevier,Cortisol,2023
Effect Size Interpretation and Bias Analyses,"When considering the direction of effect, a positive effect size indicates favouring the intervention condition, shown by a larger decrease, or a smaller increase, in change in cortisol levels from pre- to post-test. As the included studies varied in the samples, interventions, control conditions and cortisol sampling approaches, average effect sizes and corresponding 95% confidence intervals were calculated based on the random-effects model, which accounts for between-study variances (Borenstein et al., 2009). Funnel plots were inspected to determine the degree of publication bias. Egger’s regression coefficient was utilised to identify publication bias (Egger et al., 1997) and Duval and Tweedie’s trim and fill analysis to understand the number of missing studies to the left and the right of the mean (Duval and Tweedie, 2000). Sensitivity analyses were also performed by removing each study one at a time. Subgroup analyses investigated the effectiveness of intervention type, control group type, cortisol sampling type, intervention length, study quality, stress risk, risk of bias and cortisol measurement (awakening, diurnal). Meta-regressions identified moderating variables.",Elsevier,Cortisol,2023
Study Characteristics Overview,"3. Results 3.1. Study characteristics Of the 59 studies, 56 were RCTs and 3 were crossover trials (Benvenutti et al., 2017; Bittman et al., 2001; Lai and Li, 2011). 57 studies provided a baseline and post-intervention measure, 2 studies provided the pre-post intervention change in cortisol. In total, there were 3508 participants included in the meta-analysis, with sample sizes ranging from 12 to 154. There were 1648 participants allocated to the intervention condition and 1860 allocated to the control condition. There was a mean age of 35.84 years and 64.84% of participants were female. The average intervention length was 19 hours but ranged from 20 minutes to 4560 minutes. Fifteen studies included samples with individuals considered at stress risk such as caregivers or healthcare workers; the remaining 44 studies were considered non-stressed samples. For cortisol type, 13 studies used blood, 43 saliva and 3 hair. Cortisol was measured AM, PM, or both; categorised as awakening or diurnal.",Elsevier,Cortisol,2023
Control Groups and Risk of Bias,"We conceptualised control comparison groups as active, inactive or passive. We also followed previous meta-analyses (e.g., Koncz et al., 2021) to examine whether collapsing inactive and waitlist groups into a larger passive control group influenced subgroup differences. When considering the risk of bias, many included studies were categorised as having ‘some concerns’, with six studies labelled ‘high risk’. The greatest risk of bias stemmed from the category ‘missing outcome data’, due to participant dropout. Additional risk stemmed from lack of detail in cortisol sampling methods and failure to conduct sensitivity analyses to understand possible bias. There was also limited clarity in ‘selection of the reported result’, where despite standardised cortisol procedures, studies did not specify whether personnel were aware of group allocation.",Elsevier,Cortisol,2023
Cortisol Measurement Quality,"As the outcome of interest was cortisol measured in saliva, blood or hair, it was essential to recognise variability in cortisol measurement quality across studies. The current meta-analysis used the cortisol quality tool devised by Laufer et al. (2018), adapting it for hair cortisol. The measure uncovered patterns in cortisol sampling that may confound intervention effectiveness. Notably, many studies lacked reporting of state confounders that could influence cortisol measurement such as time of day, medication use, or menstrual phase in female samples. These were frequent indicators of poorer cortisol sampling quality. See Supplementary Table 1 for a summary of study characteristics.",Elsevier,Cortisol,2023
Categorising the Interventions,"3.2. Categorising the interventions There was a great variety of interventions included in the meta-analysis. For the purpose of analyses, and to improve understanding of differential effectiveness of different broad types of interventions, we summarised the underlying concepts of the interventions and this allowed us to categorise each intervention into one of four broad categories to allow meaningful comparison of key intervention components (see Fig. 2). We conceptualised four categories of intervention: 1) mindfulness and meditation, incorporating any mindfulness meditation, mindfulness based therapy, including mindfulness based stress reduction and mindfulness based cognitive therapy where the central core of the intervention is to gain a greater awareness of one’s physical, mental and emotional condition; 2) talking therapies included psychological interventions involving talking one-to-one, in a group, online, over the phone or with friends, family or co-workers, an example of talking therapy being cognitive behavioural therapy; 3) relaxation, included any intervention specifying muscle relaxation, biofeedback assisted relaxation and breathing exercises; 4) mind body training, incorporated yoga and biofeedback where there was an awareness of bodily movement to influence mental state.",Elsevier,Cortisol,2023
Grand Meta-analysis Results,"3.3. Grand meta-analysis This analysis is based on 58 studies that investigated the effect of stress management interventions on cortisol (as measured in blood, hair or saliva). The meta-analysis excluded one study, Danucalov et al. (2013), due to being identified as an outlier with inflated effect sizes. The grand meta-analysis found that stress management interventions led to a small-to-medium, and heterogeneous, positive effect on cortisol levels (g = 0.282, 95% CI = 0.166, 0.398, Z = 4.749, p < 0.001; I2 = 60.3%, Q(57) = 143.603, p < 0.001) reflecting a favourable outcome for the psychological intervention compared to the control condition. See Supplementary Fig. 1 for the high resolution plot of effect sizes.",Elsevier,Cortisol,2023
Publication Bias and Sensitivity Analysis,"3.4. Publication bias and sensitivity analysis Egger’s regression coefficient did not indicate presence of publication bias when all studies were considered together (see Fig. 3; intercept = 1.284, df = 56, p = .082). Duval and Tweedie’s trim and fill analyses indicated there were no missing studies either side of the mean. Sensitivity analyses were performed to determine the impact of removing each study from the analyses, one at a time. These analyses did not detect any studies that had a significant independent impact on the overall effect size at post-intervention (effect sizes (hedges g) ranged from 0.250 to 0.298).",Elsevier,Cortisol,2023
Subgroup Analysis: Cortisol Measurement Type,"3.5.1. Cortisol measurement type To compare the effectiveness of the interventions in studies utilising different cortisol outcomes, as measured in blood, hair or saliva, a subgroup analysis was conducted. As outlined earlier, there were only 3 studies utilising hair cortisol, therefore, this category was omitted from the analysis as there were too few studies to have adequate power to conduct the analysis. There was a main effect of the interventions, when compared to controls, in blood (g = 0.331, SE = 0.136, p = .015) and saliva (g = 0.284, SE = 0.074, p < .001). However, there was no evidence that the effect sizes varied as a function of cortisol outcome measure (Q = 0.093, p = .761).",Elsevier,Cortisol,2023
Subgroup Analysis: Types of Intervention,"3.5.2. Types of intervention We explored whether the type of intervention impacted the effectiveness of stress management interventions (see Supplementary Table 2). The interventions were grouped into one of four categories; mind body therapies, mindfulness, relaxation or talking therapies. The subgroup analysis revealed the largest, significant effect sizes for mindfulness (g = 0.345, SE = 0.085, p < .001) and relaxation (g = 0.347, SE = 0.125, p = .005). We observed much smaller, non-significant effect sizes for mind body therapies (g = 0.129, SE = 0.187, p = .492) and talking therapies (g = 0.107, SE = 0.162, p = .510). Overall, there was no evidence that the effect sizes varied as a function of the type of intervention received (Q = 2.643, p = .450).",Elsevier,Cortisol,2023
Subgroup Analysis: Comparison Groups,"3.5.3. Comparison group In this subgroup analysis we only included studies with one control group; for instance, a study that had two control groups would be excluded (e.g. Errazuriz et al., 2022). In studies where the intervention group was compared against an active control group, we observed a large, significant, effect size (g = 0.477, SE = 0.109, p < .001). In studies where the intervention was compared against a passive control group there was a much smaller, non-significant effect observed (g = 0.129, SE = 0.076, p = .093). Additionally, the effect sizes varied as a function of the type of comparison group and were significantly different across conditions (Q = 6.967, p = .009).",Elsevier,Cortisol,2023
Subgroup Analysis: Awakening vs Diurnal Cortisol,"3.5.4. Awakening or diurnal Next, analyses were conducted to explore whether the effectiveness of interventions on cortisol varied based on the type of cortisol measure – awakening or diurnal cortisol. The analyses found a large, significant effect when studies utilised awakening measures of cortisol (g = 0.644, SE = 0.153, p < .001), and smaller, but also significant, effects when using diurnal measures of cortisol (g = 0.225, SE = 0.063, p < .001). Moreover, the magnitude of effect was significantly different, indicating interventions were more effective at changing cortisol in the morning awakening measures (Q = 6.37, p = .012).",Elsevier,Cortisol,2023
Subgroup Analysis: Intervention Length,"3.5.5. Length of intervention One study was excluded as it did not provide detail on length (Johansson and Uneståhl, 2006). When considering length categories—short, medium, long—there was a significant effect for long interventions (>801 min; g = 0.348, SE = 0.093, p < .001) as well as short interventions (<250 min; g = 0.306, SE = 0.084, p < .001). No significant effect was found for medium length interventions (251–800 min; g = 0.150, SE = 0.147, p = .308). Overall, no significant difference between categories (Q = 1.299, p = .522).",Elsevier,Cortisol,2023
Subgroup Analysis: Study Quality and Bias,"3.5.6. Study quality Significant effects were observed in studies with moderate study quality (g = 0.346, SE = 0.080, p < .001). High study quality was not significant (g = 0.212, SE = 0.130, p = .103) and low quality showed the smallest, non-significant effect (g = 0.195, SE = 0.144, p = 0.178). No differences across study quality categories (Q = 1.272, p = 0.529). 3.5.7. Risk of bias Significant effects were observed in low risk (g = 0.295) and some risk (g = 0.303) studies. High risk studies showed smaller, non-significant effects (g = 0.207). No significant differences across bias categories (Q = 0.224, p = 0.894).",Elsevier,Cortisol,2023
Subgroup Analysis: Stress Risk and Meta-regressions,"3.5.8. Stress risk Interventions were effective in non-stressed samples (g = 0.351, SE = 0.075, p < .001). In stressed samples, effects were smaller and non-significant (g = 0.135, SE = 0.098, p = .169). No significant differences across stress categories (Q = 3.078, p = .079). 3.6. Meta-regressions 3.6.1. Time elapsed No significant relationships between time elapsed and cortisol change (B = −0.0002, p = .734). 3.6.2. Demographics No significant effect of sample size (B = −0.002, p = .273). No significant effect of age (B = 0.012, p = .1048) or gender (B = .0002, p = .955).",Elsevier,Cortisol,2023
Hormesis Overview and Definition,"Hormesis in health and chronic diseases Xin Li, Tingting Yang, Zheng Sun. Abstract What doesn’t kill you makes you stronger. Hormesis, the paradoxical beneficial effects of lowdose stressors, can be better defined as the biphasic dose-effect or time-effect relationship for any substance. Here we review hormesis-like phenomena in the context of chronic diseases for many substances, including lifestyle factors and endocrine factors. Intermittent or pulsatile exposure can generate opposite effects compared to continuous exposure. An initial exposure can elicit an adaptive stress response with long-lasting protection against subsequent exposures. Early-life stress can increase resilience in later life, and lack of stress can lead to vulnerability. Many stressors are naturally occurring and are required for healthy growth or homeostasis, which exemplifies how ‘illness is the doorway to health’. Keywords biphasic; dose-effect; time-effect; intermittent; resilience; stress response Introduction Hormesis was originally defined as a phenomenon in which exposure to a harmful substance gives beneficial effects to living organisms when the dose of the harmful substance is small. Radiation hormesis is among the first documented examples. Although high-dose radiation promotes mutagenesis and carcinogenesis, low-dose ionizing radiation such as X-ray has been shown to suppress tumor development.",Elsevier,Cortisol,2023
Early Examples and Stress-Response Hormesis,"Many chemical carcinogens such as DTT or α-benzene hexachloride, when given at a low dose, can protect against DNA damage and cytotoxic effects induced by subsequent exposures at a much higher dose. As illustrated in the aphorism ‘what doesn’t kill you makes you stronger’, this original idea of hormesis involves an adaptive response upon the initial exposure and can be referred to as ‘stress-response’ hormesis. There are three components in this scenario: the initial stress exposure that ‘tries to kill you’, the subsequent stress exposure that you are more resilient against, and a time interval in between. If the initial low-dose exposure protects against subsequent exposure to the higher dose of the same substrate, it is a ‘single-mode’ stress response. If the initial low-dose exposure protects against a different substance, it is a ‘cross-mode’ stress response. If the time interval between the two exposures involves a developmental process, it is a ‘developmental’ stress-response hormesis. The issue with the original definition is that ‘harmfulness’ is not an intrinsic feature of any substance; it is the dose and timing that determine toxicity.",Elsevier,Cortisol,2023
Redefining Hormesis and Biphasic Effects,"Many substances discussed in the context of hormesis are naturally occurring and the body has evolved mechanisms to respond to these stressors for healthy growth or homeostasis. Similarly, what is ‘beneficial’ is context-dependent. A beneficial effect of calorie restriction on glucose metabolism can come with sacrifice on muscle mass or bone mineral density. A thrifty phenotype can be beneficial when food is scarce but detrimental when food is abundant. Therefore, a more reasonable definition of hormesis is the biphasic dose-effect or time-effect relationship for any substance, defining a broad sense of hormesis. The biphasic time-effect can be mechanistically related to the stress response hormesis. Hormesis can be counterintuitive: if ‘too much of a good thing is a bad thing’, then a little bad thing can be good. A bell-shaped biphasic curve is expected for the dose-effect relationship. A dose-effect relationship consistent with classical hormesis appears only after defining the baseline. This transformation of an adverse effect into a favorable effect exemplifies the Yin and Yang concept that ‘illness is the doorway to health’.",Elsevier,Cortisol,2023
Stress-Response Hormesis Mechanisms,"Hormesis induced by environmental chemicals and radiation has been reviewed extensively in toxicology, with readouts including cell viability, proliferation, enzyme activity, or gene expression. This review focuses on lifestyle factors and endocrine factors in the context of chronic diseases, emphasizing in vivo studies in mammals. Stress-response hormesis Single-mode and cross-mode stress responses Many xenobiotic chemicals induce the expression of detoxification enzymes, which is protective against subsequent higher-dose exposures. Some xenobiotic chemicals directly serve as ligands for nuclear receptors such as CAR or PXR; others activate transcription factors such as Nrf2 by altering intracellular redox status. Activation of these transcription factors upregulates genes encoding detoxification enzymes across phase I (cytochrome P450), phase II (glutathione conjugation enzymes), and phase III (ABC transporters). Priming the body with a low-dose xenobiotic chemical can confer protection against subsequent exposures of the same chemical, constituting classic single-mode hormesis.",Elsevier,Cortisol,2023
Cross-Mode Hormesis and Adaptive Protection,"If the initial exposure confers protection against a different substance, it is referred to as ‘cross-mode’ hormesis. In xenobiotic-mediated detoxification, one induced set of detoxification enzymes can protect against many chemicals. This cross-mode hormesis is central to chemoprevention, where low-toxicity chemicals induce adaptive protective responses against carcinogens. Cross-mode hormesis appears in many contexts. For example, exposure of cells to mild heat stress can make them more protected from oxidative stress or toxins such as cyanide. Similarly, exposure to a low-dose mitochondrial uncoupling agent such as 2,4-dinitrophenol can make cells less vulnerable to ischemia. This cross-mode aspect of hormesis may contribute to broad benefits of exercise and dietary restriction. Below we examine a few other common stressors.",Elsevier,Cortisol,2023
Hormesis Overview and Definition,"Hormesis in health and chronic diseases Xin Li, Tingting Yang, Zheng Sun. Abstract What doesn’t kill you makes you stronger. Hormesis, the paradoxical beneficial effects of lowdose stressors, can be better defined as the biphasic dose-effect or time-effect relationship for any substance. Here we review hormesis-like phenomena in the context of chronic diseases for many substances, including lifestyle factors and endocrine factors. Intermittent or pulsatile exposure can generate opposite effects compared to continuous exposure. An initial exposure can elicit an adaptive stress response with long-lasting protection against subsequent exposures. Early-life stress can increase resilience in later life, and lack of stress can lead to vulnerability. Many stressors are naturally occurring and are required for healthy growth or homeostasis, which exemplifies how ‘illness is the doorway to health’. Keywords biphasic; dose-effect; time-effect; intermittent; resilience; stress response Introduction Hormesis was originally defined as a phenomenon in which exposure to a harmful substance gives beneficial effects to living organisms when the dose of the harmful substance is small. The radiation hormesis is among the first documented examples.",Trends in Endocrinology & Metabolism,Hormesis,2019
Early Examples and Stress-Response Hormesis,"Although high-dose radiation promotes mutagenesis and carcinogenesis, low-dose ionizing radiation such as X-ray has been shown to suppress tumor development. Many chemical carcinogens such as DTT or α-benzene hexachloride, when given at a low dose, can protect against DNA damage and cytotoxic effects induced by subsequent exposures at a much higher dose. As illustrated in the aphorism ‘what doesn’t kill you makes you stronger’, this original idea of hormesis involves an adaptive response upon the initial exposure and, therefore, can be referred to as ‘stress-response’ hormesis. There are three components in this scenario: the initial stress exposure that ‘tries to kill you’, the subsequent stress exposure that you are more resilient against, and a time interval in between. If the initial low-dose exposure protects against subsequent exposure to the higher dose of the same substrate, it is a ‘single-mode’ stress response. If the initial low-dose exposure protects against a different substance, it is a ‘cross-mode’ stress response. If the time interval between the two exposures involves a developmental process, it is a ‘developmental’ stress-response hormesis.",Trends in Endocrinology & Metabolism,Hormesis,2019
Redefining Hormesis and Biphasic Effects,"The issue with this original definition is that the ‘harmfulness’ or ‘stress’ is not an intrinsic feature for any substance. It is the dose, and the time of the exposure, that determines toxicity. Many substances discussed in the context of hormesis are naturally occurring substances that the body is exposed to during evolution. Therefore, our body has likely evolved mechanisms to respond to, adapt to, or even rely on, the stressors for healthy growth and homeostasis. Similarly, what is ‘beneficial’ is also context-dependent. A beneficial effect of calorie restriction on glucose metabolism can come with a sacrifice on muscle mass or bone mineral density. A thrifty phenotype can be beneficial when food is scarce but can be detrimental when food is in excess. Therefore, a more reasonable definition of hormesis seems to be the biphasic dose-effect or time-effect relationship for any substance, which defines a broad sense of hormesis. The biphasic time-effect can be mechanistically related to the stress response hormesis. Hormesis can be counterintuitive but not entirely surprising. If ‘too much of a good thing is a bad thing’, a little bad thing can be good.",Trends in Endocrinology & Metabolism,Hormesis,2019
"Hormesis, Dose Curves, and Conceptual Models","The golden mean doctrine in philosophy suggests a Goldilocks zone for everything. A bell-like biphasic curve, therefore, is expected for the dose-effect or time-effect relationship for any substance. A dose-effect relationship consistent with the original definition of hormesis appears only after we artificially define the baseline. The transformation of an adverse effect into a favorable effect exemplifies how ‘illness is the doorway to health’ as articulated in the philosophy of Yin and Yang. Hormesis induced by environmental chemicals and radiation has been nicely reviewed in the field of toxicology with the functional readout mainly on cell viability, cell proliferation, enzyme activity, or gene expression. In this review, we focus on lifestyle factors and endocrine factors in the context of chronic diseases with the emphasis on in vivo studies in mammals. The underlying molecular mechanisms are unclear for most hormesis phenomena.",Trends in Endocrinology & Metabolism,Hormesis,2019
Stress-Response Hormesis Mechanisms,"Stress-response hormesis Single-mode and cross-mode stress responses Many xenobiotic chemicals induce the expression of detoxification enzymes, which is protective against subsequent higher-dose exposures. Some xenobiotic chemicals can directly serve as ligands for nuclear receptors such as constitutive androstane receptor (CAR) or pregnane X receptor (PXR). Other xenobiotic chemicals can indirectly activate other transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf2) through altering the intracellular redox status or a variety of stress-response signaling pathways. Activation of these transcription factors upregulates the expression of genes that encode enzymes in xenobiotic metabolism. These enzymes are classified into three phases, including phase I enzymes such as cytochrome P450 family enzymes, phase II enzymes such as glutathione-based conjugation enzymes, and phase III enzymes such as ATP-binding cassette transporters, which are collectively responsible for detoxification and excretion of the xenobiotic chemicals from the body.",Trends in Endocrinology & Metabolism,Hormesis,2019
Cross-Mode Hormesis and Adaptive Protection,"Priming the body with a low-dose xenobiotic chemical can confer protection against subsequent exposures of the same chemical. This adaptive response in xenobiotic metabolism constitutes a classic example of single-mode stress-response hormesis. If the initial exposure confers protection against a different substance, it is referred to as ‘cross-mode’ hormesis. In the abovementioned example of xenobiotic-mediated upregulation of detoxification enzymes, a combination of detoxification enzymes induced by one chemical can protect against many other chemicals. Such cross-mode hormesis is the basis of chemoprevention, an approach to use low-toxicity chemicals to induce adaptive protective responses against many other carcinogenic chemicals. Cross-mode hormesis exists in many situations. Exposure of cells to mild heat stress can make them more protected from oxidative stress or toxins such as cyanide. Exposure to a low-dose mitochondrial uncoupling agent 2,4-dinitrophenol can make cells less vulnerable to being killed by ischemia. This cross-mode aspect of hormesis may contribute to the broad benefits of exercise and dietary restriction.",Trends in Endocrinology & Metabolism,Hormesis,2019
Reactive Oxygen Species and Oxidative Stress,"Reactive oxygen species (ROS) Oxidative stress refers to the high level of ROS that causes damage to DNA, protein, or other cellular components. The free radical theory of aging suggests that oxidative stress causes aging. As some DNA repair mediators are downregulated in aged organisms, DNA damage induced by oxidative stress can be left unrepaired, leading to genome instability. However, low-level ROS can function as signal molecules to initiate biological processes without the above detrimental effects and, therefore, can be beneficial. For example, the increased oxidative stress, caused by depletion of the mitochondrial superoxide dismutase, extended lifespan in C. elegans. Treatment with Paraquat, a superoxide generator, at a low dose can also increase the lifespan, while a high-dose Paraquat was deleterious. Randomized clinical trials have shown that antioxidants, such as Vitamin C and Vitamin E, do not prolong lifespan in human populations and can potentially increase the risk for cancer and diabetes.",Trends in Endocrinology & Metabolism,Hormesis,2019
Mitochondrial ROS and Mitohormesis,"In addition to the level, subcellular location also determines the role of ROS. While cytosolic ROS tends to shorten lifespan, mitochondrial ROS (mtROS) can extend the lifespan. For example, skin wounding triggers local production of mtROS. Lowering mtROS levels by mitochondrial superoxide-specific antioxidants blocked actin-based wound closure. Paraquat, a pro-oxidant that induces mitochondrial superoxide, promoted actin-based wound closure. Mutations of superoxide dismutase in worms elevated mitochondrial superoxide, promoted wound closure, and enhanced survival. Mechanistically, mtROS was increased by wound-triggered calcium influx, which locally inhibited Rho GTPase activity via a redox-sensitive motif. A similar role of mtROS was documented in mammalian skeletal muscle cells. Injury increased mitochondrial calcium uptake through the mitochondrial calcium uniporter, which transiently increased mtROS.",Trends in Endocrinology & Metabolism,Hormesis,2019
mtROS in Tissue Repair and Mitohormesis,"Mitochondrial respiratory chain inhibitor rotenone and antimycin A increased injury-triggered mtROS production and showed beneficial effects on plasma membrane repair in a dose-dependent manner. Mechanistically, mtROS locally activated GTPase RhoA, triggered F-actin accumulation at the site of injury, which facilitated membrane repair. Quenching mtROS using mitoTEMPO in myofibers during eccentric exercise prevented injury-triggered RhoA activation and actin polymerization, leading to increased damage to myofibers and a greater loss of force. Therefore, mtROS not only play a role in extending longevity but also is required for wound repair. This biphasic dose-effect of ROS and other substances in mitochondria is referred to as mitohormesis. ROS not only serves as an intracellular signal but can also mediate inter-cellular communications. ROS generated by neutrophils plays a vital role in promoting liver repair.",Trends in Endocrinology & Metabolism,Hormesis,2019
Neutrophil ROS and Inflammation Resolution,"Upon the tissue injury, neutrophils are recruited to the injured site, contributing to liver repair by causing the macrophage phenotypic conversion from a pro-inflammatory stage to a pro-regenerative stage. ROS from neutrophils acts as a mediator for the process because reducing neutrophil ROS by either depletion of neutrophils or genetically perturbation of neutrophil NADPH oxidase 2 (Nox2) blocked the macrophage phenotypic conversion. Conversely, transferring wild-type neutrophils, but not Nox2 knock-out neutrophils, can rescue the hepatic damage in neutrophil-depleted mice and promote the macrophage phenotypic conversion. Thus, ROS is a critical inter-cellular signaling molecule that mediates the resolution of inflammation. Hypoxia Obstructive sleep apnea (OSA) is a risk factor for cardiovascular and liver diseases. Intermittent hypoxia (IH) is a major component of OSA and contributes to the hepatic, metabolic, and vascular effects of OSA.",Trends in Endocrinology & Metabolism,Hormesis,2019
Intermittent Hypoxia and Metabolic Dysfunction,"Nocturnal IH is independently associated with metabolic dyslipidemia and steatosis. IH (1 min cycle, FiO2 5–6% for 30 s, FiO2 20.9% for 30 s; for 9 h) can also cause insulin resistance in lean mice. Chronic IH (1 min cycle, FiO2 6–7% for 30 s, FiO2 ~21% for 30 s) during the light phase (9 am–9 pm) for 4 weeks exacerbates insulin resistance and glucose intolerance in diet-induced obesity. Paradoxically, IH shows benefits in some conditions by facilitating the adaptation to reduced oxygen. Exposure to daily IH cycle for 4 days, each composed of 2-min at 6–8% O2 followed by 3-min re-oxygenation for 5 times, can protect the heart against ischemia-reperfusion injury. IH also increases exercise tolerance in elderly men and improves myocardial perfusion in patients with severe coronary heart disease. Mechanistically, IH improves nitric oxide (NO) bioavailability and storage.",Trends in Endocrinology & Metabolism,Hormesis,2019
Mechanistic Benefits of Intermittent Hypoxia,"Decreased O2 level can lead to less oxidization of NO to NO2− and NO3−, allowing more NO release from hemoglobin. In addition, hypoxia can induce the expression of nitric oxide synthase through hypoxia-inducible factor HIF-1. IH (9.5–10% O2, 5–10 min, 5–8 times/day, for 20 days) suppressed hypertension in spontaneously hypertensive rats. Endothelial function was sustained in the IH group but decreased in the control group. This was associated with enhanced capacity of aortic rings to store NO and increased NO availability in vascular walls. In a mouse model of metabolic syndromes, short-term IH (1 min cycle, FiO2 5% for 30 s, FiO2 21% for 30 s; 8 h/day) increased insulin and leptin levels, and prevented endothelial dysfunction caused by a high-fat diet. IH restored mitochondrial complex I activity and slightly increased complex II and IV activity, which may help boost mitochondrial oxidative-phosphorylation and reduce liver lipid accumulation.",Trends in Endocrinology & Metabolism,Hormesis,2019
Nitric Oxide and Biphasic Biological Effects,"Nitric oxide (NO) NO can lead to mutagenesis and cell death at high concentrations through inhibiting DNA synthesis, disrupting cell membrane integrity, arresting the cell cycle, causing DNA strand break, and apoptosis. Excess NO also impairs mitochondrial function and affects metabolic processes in neurons, contributing to neurodegenerative diseases. However, NO at low concentrations modulates glutamatergic neurotransmission. Depletion of neuronal NO synthase deletion impaired cognitive performance and synaptic plasticity. In the cardiovascular system, NO promotes new vessel formation, limits vessel constriction, suppresses inflammation, and promotes blood flow.",Trends in Endocrinology & Metabolism,Hormesis,2019
Amyloid-β Peptide Hormesis,"Amyloid-β peptide (Aβ) Excessive Aβ deposits causes synaptoxicity and memory dysfunction. Beyond this amyloid hypothesis, emerging evidence suggests that Aβ and its precursor protein APP have physiological functions at low concentrations in the healthy brain. APP is involved in cell proliferation, differentiation, neurite outgrowth, cell adhesion, and synaptogenesis. In contrast to the detrimental effects at nanomolar concentrations, picomolar concentrations of Aβ42 enhances hippocampal long-term potential (LTP) formation by increasing acetylcholine and activation of nicotinic acetylcholine receptors, which suggests a positive role of Aβ in synaptic plasticity. Infusion of mice with low dose Aβ in hippocampus improved the fear memory.",Trends in Endocrinology & Metabolism,Hormesis,2019
Physiological Roles of Aβ and Dose-Dependent Outcomes,"Conversely, APP knockout mice showed impaired LTP and memory. Aβ oligomers can also bind to the glycoproteins of the herpesvirus capsid, resulting in the entrapment of virus particles and protection of the brain from Herpesviridae infection. The biphasic dose-response effects of Aβ might contribute to the failure of Aβ-targeting drugs in clinical trials in treating Alzheimer’s disease.",Trends in Endocrinology & Metabolism,Hormesis,2019
Developmental Stress-Response Hormesis,"Developmental stress-response hormesis During the stress-response hormesis, the initial exposure can occur at an early developmental stage. A classic example is the hygiene hypothesis, in which a lack of early childhood exposure to infectious agents and parasites can suppress the natural development of the immune system and increase susceptibility to allergic diseases. The stress-inoculation hypothesis is a counterpart of the hygiene hypothesis in psychology. It suggests that mild or intermittent stress exposure early in life induces resilience to subsequent stress in adults. The stress-inoculation hypothesis is supported by many animal studies. In one study, male mice were subjected to a variety of stresses manipulations such as maternal separation, early weaning, reduced nesting, isolation, handling, restraining, daily swim stress in early life, and were then tested for depression- or anxiety-like behaviors in adulthood following exposure to chronic social defeat stress. Some of the manipulations mitigated the deleterious consequences of adult stress.",Trends in Endocrinology & Metabolism,Hormesis,2019
Early-Life Stress and Behavioral Outcomes,"Similarly, manipulations of female mice with early handling or limited nesting in early life can make them more resistant to similar aversive conditions in adult, as measured by the anxiety, depression, or sociability behaviors. Rats exposed to physical stress during early-adolescence showed increased anxiety behaviors in adulthood, while mid-adolescence stress paradoxically reduced the anxiety-like behaviors in adulthood. These results suggest that the timing of adolescent adversity is essential to long-term outcomes. In another study, early life stress of reduced bedding in mice led to resistance to social interaction deficits after chronic social defeat stress. It also mitigated acute restraint and tail-shock stress-induced impairments in hippocampal synaptic plasticity, an effect abolished by adrenalectomy. Short-term separation stress in early life altered histone modifications and expression of genes involved in dopamine receptor signaling in the brain, a distinct effect from long-term separation stress, suggesting the potential involvement of the dopamine signaling and epigenetic changes in the underlying mechanisms.",Trends in Endocrinology & Metabolism,Hormesis,2019
Neuropeptide Signaling and Human Evidence,"Signaling pathways of other neurotransmitters or neuropeptides were also implicated in the process, including AVP and oxytocin. The stress-inoculation theory also has support from human studies. Threat-related amygdala reactivity can be measured using task-based functional magnetic resonance imaging (fMRI) in human and is a biomarker of vulnerability to stress-related depression or anxiety. In a study with adolescents, increased amygdala reactivity to an interpersonal threat was positively associated with better familial affective responsiveness, especially in adolescents reporting lower recent stressful life events. The finding is in line with studies examining the parenting style and the mental wellbeing of adolescents. Parental overprotection refers to a restrictive or indulgent parenting style when it comes to protecting the child from potential harm or risk. Parental overprotection is positively associated with later psychopathology, including dysfunctional attitudes, depression or anxiety disorders, and suicide intent.",Trends in Endocrinology & Metabolism,Hormesis,2019
Stress-Inoculation Training and Brain Development,"Conversely, stress inoculation training (SIT) is a type of psychotherapy using intermittent exposure to mild stress to improve patients’ ability to deal with stress. SIT can reduce anxiety and depression for cancer patients and veterans with posttraumatic stress disorder or traumatic brain injury. The paradoxical beneficial effect of early-life stress makes sense from an evolutionary perspective, considering that the brain needs stimulation from the environment for proper mental development during the young age. What we assume as a stressor can be actually perceived as a positive stimulation by the brain. Indeed, the brain responds to a variety of stressors in a similar way as to the enriched environment (EE), positive stimuli known to enhance synaptogenesis and intellectual development. The resilience conferred by early-life stress likely represents an adaptation to the altered environment and is often a trade-off between different traits.",Trends in Endocrinology & Metabolism,Hormesis,2019
"Trade-offs: Fecundity, Longevity, and Environmental Match","Exposure of female zebra finches to time-restricted feeding and daily corticosterone injection from young ages reduced breeding performance during early adulthood but led to better breeding performance than the control when birds were bred in old adulthood. Birds exposed to short episodes of mild heat stress in early life had ameliorated oxidative damage in adulthood when given heat stress, as compared to the control group without the early-life priming. Interestingly, birds with the early-life heat stress, but did not experience it again later in life, had a shorter lifespan than any other group. Female blackbirds show decreased breeding success, but increased lifespan with increasing exposure to lead, but not cadmium. These studies suggest that some stress can cause a rebalance of the trade-off between fecundity and longevity. The trade-off can be either beneficial or harmful, depending on whether the late-life environment matches the early-life stress.",Trends in Endocrinology & Metabolism,Hormesis,2019
Developmental Origins and Mismatch Effects,"If the outcome is harmful, it falls into the ‘Developmental Origins of Health and Disease’ (DOHaD) paradigm. A classic example of the later is the thrifty phenotype hypothesis, in which undernutrition during the fetal or infant stage rebalances the metabolism towards a more thrifty phenotype that favors energy storage, which increases the risk of developing obesity, diabetes, and other metabolic syndromes in later life when the food becomes plenty. Therefore, the developmental stress-response hormesis and the DOHaD paradigm are two sides of the same coin.",Trends in Endocrinology & Metabolism,Hormesis,2019
Hormesis in Endocrine Factors: Parathyroid Hormone,"Biphasic dose-effect or time-effect of endocrine factors Parathyroid hormone (PTH) Many endocrine factors show dose-dependent or time-dependent opposite effects, which falls within the broad definition of hormesis. Here, time not only refers to the total duration of the exposure but also include the temporal pattern. Intermittent or bipolar treatment can produce the opposite effects as chronic continuous treatment. Parathyroid hormone (PTH) causes bone loss at a constantly high level as in chronic hyperparathyroidism. However, intermittent exposure to PTH or its paralogue, at a rate of once per day, increases bone mass. Such intermittent exposure strategy has been approved as a therapy for osteoporosis. The underlying mechanism is not completely understood. PTH promotes two opposite processes: bone formation by osteoblasts and bone reabsorption by osteoclasts, but with seemingly different kinetics. Even within one cell type, PTH can have opposite effects. For example, in osteoblast cells, PTH inhibits apoptosis but also inhibits differentiation.",Trends in Endocrinology & Metabolism,Hormesis,2019
PTH Hormesis and Insulin Secretion,"PTH also affects other cell types, including bone lining cells, osteoblast progenitor cells, osteoclast cells, lymphocytes, and macrophages. A variety of intracellular molecular signaling pathways, presumably downstream of the cell membrane receptors for PTH, are being characterized in different cell types upon PTH treatment. However, what seems lacking is a detailed characterization of the kinetics of the cellular phenotypic changes in response to PTH with different temporal patterns. PTH also shows biphasic dose-dependent effects on insulin secretion from pancreatic islets cells. Low-dose PTH stimulates glucose-induced insulin release, while high-dose PTH inhibits it. The PTH level for the maximal stimulatory effects is dependent on the extracellular calcium level. High-dose PTH reduces the intracellular ATP level and increases the resting intracellular calcium level, which contributes to impaired insulin release.",Trends in Endocrinology & Metabolism,Hormesis,2019
Glucocorticoid Hormesis,"Glucocorticoid Intermittent weekly glucocorticoids treatment can produce the opposite effect as chronic daily glucocorticoids treatment on muscle atrophy. In an acute focal muscle injury model in mice, pulsatile weekly treatment of glucocorticoids reduced injury area, macrophage infiltration, and injury associated fibrosis, which improved muscle performance recovery. However, chronic daily glucocorticoids worsened muscle performance. In mouse models of muscular dystrophy, daily glucocorticoids exacerbated muscle atrophy, while weekly glucocorticoids ameliorated it. The expression of several genes in muscle atrophy was upregulated in skeletal muscles upon daily glucocorticoids treatment but was downregulated upon weekly treatment. The effect of glucocorticoids on cognitive functions is also biphasic. Chronic high levels of glucocorticoids are associated with increased risk of cognitive decline and neurodegeneration. However, the acute rise of glucocorticoids improves memory consolidation in multiple models.",Trends in Endocrinology & Metabolism,Hormesis,2019
Glucocorticoids and Memory Processing,"This positive effect of glucocorticoids on memory consolidation is accompanied by an adverse effect on memory retrieval. Electrophysiological recording assessing long-term potentiation of rat CA1 population showed a positive correlation between low-level glucocorticoids with primed burst potentiation, but a negative correlation at high levels. Thus, dosage, duration, and the temporal pattern collectively determine the outcome of glucocorticoids on cognitive functions by acting on multiple stages of memory processing.",Trends in Endocrinology & Metabolism,Hormesis,2019
Thyroid Hormone Biphasic Effects,"Thyroid hormone Thyroid hormone is a classic example of biphasic dose-effect, as both hyperthyroidism and hypothyroidism are medical conditions. Thyroid hormone has cardioprotective potentials because it inhibits apoptosis, activates mitochondrial metabolism, decreases fibrosis, and increases neoangiogenesis. Therefore, thyroid hormone replacement therapy could be used for myocardial infarction to induce positive cardiac remodeling. However, hyperthyroidism increases the risk of coronary heart diseases, atrial fibrillation, and pulmonary hypertension. Conversely, hypothyroidism causes left ventricular diastolic dysfunction and increases carotid intima-media thickness. Thyroid hormone increases catabolism and energy expenditure, and therefore, is presumably negatively correlated with metabolic syndromes characterized by overnutrition.",Trends in Endocrinology & Metabolism,Hormesis,2019
Complex Dose–Response of Thyroid Hormone,"However, blood triiodothyronine (T3) and thyroid-stimulating hormone (TSH) levels in euthyroid human subjects are positively associated with metabolic syndromes such as higher body mass index and blood lipid or glucose levels. It is speculated that the elevated T3 can be a failed compensatory mechanism aiming to maintain metabolic health under overnutrition. It is also possible that the dose-effect curve of thyroid hormone on each component of the metabolic syndrome is biphasic, and certain unfavorable metabolic parameters can shift the dose-effect curve in a way that favors detection of only one side of the curve.",Trends in Endocrinology & Metabolism,Hormesis,2019
Adiponectin Paradox and Hormesis,"Adiponectin Adiponectin has an elusive relationship with mortality, known as ‘adiponectin paradox’. Adiponectin improves glucose tolerance, reduces inflammation, improves endothelial functions, and inhibits atherosclerosis. However, blood levels of adiponectin, both total adiponectin and the high molecular-weight isoform, are positively associated with mortality across many clinical conditions such as coronary heart disease. One obvious explanation is that the elevated adiponectin is a compensatory response to potential adiponectin resistance in the situation of deteriorated health although this hypothesis has not been rigorously tested. It is also possible that adiponectin has biphasic effects on some of the physiological processes. This possibility is supported by observations that adiponectin may exacerbate inflammation in chronic inflammatory conditions such as colitis, rheumatoid arthritis, or Crohn disease. It is unclear what determines such a switch from anti-inflammatory to pro-inflammatory effects for adiponectin.",Trends in Endocrinology & Metabolism,Hormesis,2019
Estrogen Biphasic Effects,"Estrogen Estrogen has biphasic effects on cell proliferation and tumor growth. It stimulates tumor cell proliferation at low doses but promotes cell apoptosis at high doses. In mouse models of prostate cancer and breast cancer, exposure to low-dose 17β-estradiol (E2) led to larger tumors than placebo, while exposure to high-dose E2 led to smaller tumors. Mechanistically, low-dose E2 decreases KLF5-dependent pro-apoptotic FOXO1 transcription through ERβ, which inhibits apoptosis and promotes tumor growth. High-dose E2 suppresses the expression of PDGFA and FOXO1, and thereby blocks angiogenesis and suppresses tumor growth. High-dose E2 also activates extrinsic and intrinsic apoptosis pathways.",Trends in Endocrinology & Metabolism,Hormesis,2019
Estrogen and Cardiovascular/Bone Hormesis,"In the cardiovascular system, low-dose E2 activates plasminogen activator in aortic endothelial cells, while high-dose E2 inhibits plasminogen activator, which may contribute to the increased risk of myocardial infarction or ischemic stroke in young women who receive higher doses of estrogen as oral contraception. Estrogen also has biphasic effects on bone remodeling. During bone development, both estrogen and androgen stimulate endochondral bone formation at the start of puberty but induce epiphyseal closure at the end of puberty. In treating Turner syndrome, intermittent low-dose estrogen induces maximal ulnar growth, while high-dose estrogen fails to stimulate ulnar growth.",Trends in Endocrinology & Metabolism,Hormesis,2019
Progesterone Biphasic Time-Effects,"Progesterone Progesterone has biphasic time-effects on estrogen-dependent regulation of memory. Intracranial infusion of progesterone in young ovariectomized mice increased dorsal hippocampal p42 ERK after 5 min but decreased its phosphorylation after 15 min, and intriguingly had no apparent effect after 30 min. Estradiol facilitates extinction recall, an effect potentiated by progesterone if progesterone administration occurred 6 h before extinction training. However, when given 24 h before the extinction training, progesterone abolished the estradiol effect on extinction recall. Similar time-dependent biphasic effects were found for spatial memory in estradiol-treated ovariectomy rats, progesterone augmented the beneficial effect of estradiol on spatial memory when administered 90 min before the test, but reversed estradiol’s effects when administered 24 h before the test. The biphasic effect of progesterone on the immune system is also time-dependent. Longer exposure time with a long-acting progestin formulation was associated with poor innate and adaptive immune responses to genital herpes HSV. In contrast, mice immunized shortly after progesterone treatment were protected from HSV challenge.",Trends in Endocrinology & Metabolism,Hormesis,2019
GH and IGF-1 Biphasic Effects,"Growth hormone (GH) and IGF-1 GH shares common ancestry with the insulin/insulin-like growth factor 1 (IGF-1). Epidemiological studies suggest that the relationship of IGF-1/GH levels with healthy aging is biphasic. Both low and high circulating IGF-I levels are associated with increased mortality in the general population or increased cancer mortality in older men. Interventional studies also revealed a perplexing relationship between GH/IGF-1 and aging. On one hand, GH administration in some elderly individuals can increase muscle mass, reduce adiposity, and improve bone mineral density, demonstrating anti-aging benefits. On the other hand, mice with deficiency in GH, GH receptor, GH releasing hormone (GHRH), GHRH receptor, IGF-1, IGF-1 receptor, insulin receptor, insulin receptor substrate, or downstream molecules such as mTOR or p70 ribosomal protein S6 kinase 1 (S6K1) all have increased lifespan or healthspan. The underlying mechanisms include improved antioxidant defenses, reduced inflammation, reduced insulin levels, reduced cell senescence, altered mitochondrial function and energy metabolism, and enhanced stress resistance.",Trends in Endocrinology & Metabolism,Hormesis,2019
GH/IGF-1 Paradox and Longevity,"Enhanced insulin sensitivity is particularly interesting, as mice with transgenic overexpression of GH receptor antagonist showed increased adiposity but improved glucose tolerance on a high-fat diet. Humans with genetic deficiencies in the GH/IGF-1 signaling pathway are characterized by proportional short stature, central obesity, delayed puberty, but are generally healthy and protected from aging-related diseases such as cancer, diabetes, and atherosclerosis. Thus, the general retardation of growth and reproduction seems to be a sacrifice for the longevity benefits. It may be possible to get the best from both sides through careful timing of the intervention, i.e. reducing the GH/IGF-1 signaling pathway only after mid-life. The trade-off between survival and fecundity, or between two other distinct physiological processes, can potentially explain many paradoxical hormesis phenomena.",Trends in Endocrinology & Metabolism,Hormesis,2019
Insulin Biphasic Metabolic Effects,"Insulin Insulin is widely used to lower blood glucose levels in diabetes patients. However, suppressing hyperglycemia can improve glucose control and protect against obesity, which implicates a potential biphasic effect of insulin in metabolic disorders. Insulin is encoded by two genes, Ins1 and Ins2, in mice. Female Ins1−/−; Ins2+/− mice showed reduced hyperinsulinemia compared to the control Ins1−/−; Ins2+/+ mice after high-fat diet feeding, which was associated with attenuated weight gain, lower glucose levels, improved insulin sensitivity, and extended lifespan. These results suggest that hyperglycemia contributes to insulin resistance in diet-induced obesity. Pharmacological reduction of insulin secretion also lowers body weight in obese people. However, on the leptin-deficient Lepob/ob background, loss of 2 or 3 insulin alleles reduced body fat, but resulted in exacerbated glucose intolerance as compared to the control Ins1+/+; Ins2+/−; Lepob/ob mice. These results suggest that reduced adiposity can be separated from improved glucose control. It also suggests that leptin is required for the effect on glucose control.",Trends in Endocrinology & Metabolism,Hormesis,2019
Irisin Biphasic Bone Effects,"Irisin Irisin is a myokine secreted as a cleaved product of fibronectin type III domain-containing protein 5 (FNDC5) from skeletal muscles in response to physical exercise. The effect of irisin on bone is biphasic. Recombinant irisin, given at a low dose weekly in mice, increased cortical bone mineral density and positively modified bone geometry, upregulated pro-osteoblastic genes in bone marrow, increased the activity of osteoblasts, and reduced osteoclast numbers. Consistently, irisin upregulated sclerostin in osteocyte-like cell line and in mice. However, FNDC5 null mice were resistant to ovariectomy (OVX)-induced trabecular bone loss and displayed a marked reduction in bone resorption. Deficiency of FNDC5 suppressed bone resorption by reducing osteoclast number and bone erosion, which ameliorated OVX-induced bone loss. Thus, although irisin could be a therapeutic target for osteoporosis, its other effects on bone remodeling should also be considered.",Trends in Endocrinology & Metabolism,Hormesis,2019
Costs and Benefits of Senotherapeutics,"Cellular senescence is a major contributor to age-related diseases in humans; however, it also has a beneficial role in physiological and pathological processes, including wound healing, host immunity, and tumour suppression. Reducing the burden of cell senescence in animal models of cardiometabolic disorders, inflammatory conditions, neurodegenerative diseases, and cancer using pharmaceutical approaches that selectively target senescent cells (ie, senolytics) or that suppress senescence-associated secretory phenotype (ie, senomorphics) holds great promise for the management of chronic age-associated conditions. Although studies have provided evidence that senolytics or senomorphics are effective at decreasing the number of senescent cells in humans, the short-term and long-term side-effects of these therapies are largely unknown. In this Review, we systematically discuss the senolytics and senomorphics that have been investigated in clinical trials or have been used off-label, presenting their various adverse effects. Despite the potential of senotherapeutics to transform anti-ageing medicine, a cautionary approach regarding unwanted dose-dependent side-effects should be adopted.",The Lancet Healthy Longevity,Senotherapeutics,2022
Introduction to Cellular Senescence,"Cellular senescence is a process that occurs in response to different triggers, including DNA damage, oncogene activation, and telomere dysfunction. This process has been linked to fundamental mechanisms, such as embryogenesis, regeneration, tissue repair, tumour suppression, and physiological ageing of organisms. In 1961, it was first shown that human fibroblasts divide a finite number of times before irreversibly arresting their growth. Although senescent cells exist in a state of permanent growth arrest, they remain metabolically active and undergo physiological transformations, including alterations of paracrine signalling. In this respect, the senescence-associated secretory phenotype (SASP), a hallmark of senescent cells that mediates their pathophysiological effects, is characterised by the increased secretion of some bioactive molecules, including cytokines, chemokines, proteases, and growth factors.",The Lancet Healthy Longevity,Senotherapeutics,2022
SASP and Age-Related Tissue Dysfunction,"Over the past decade, it has become clear that tissue ageing is caused by the accumulation of senescent cells, which alters the physiological responses in the surrounding microenvironment in an autocrine and paracrine fashion through SASP. Tools have since been developed, such as the colorimetric assay for senescence-associated β-galactosidase activity, proving that accumulation of senescent cells promotes organ and organismal ageing. In addition to marking ageing, cellular senescence can suppress tumorigenesis by limiting the malignant transformation of preneoplastic cells and by hampering the proliferation of tumour cells. Nevertheless, the incidence of cancer increases when people get older because of the increased burden of cell senescence in organs, partly due to an impaired immune system, which results in a reduced clearance of senescent cells. Furthermore, SASP contributes to persistent chronic inflammation (known as inflammaging).",The Lancet Healthy Longevity,Senotherapeutics,2022
Development of Senolytics and Senomorphics,"The body of evidence showing that elimination of senescent cells seemed to be largely beneficial led to huge research efforts to identify novel agents that eliminate senescent cells in humans. These senotherapeutic strategies can be broadly categorised into two categories: pharmacological agents termed senolytics, which eliminate senescent cells, and those termed senomorphics, which prevent the detrimental cell-extrinsic effects of senescent cells by selectively targeting and suppressing the development of SASP, which is associated with increased age and medical risk in humans. Senolytics decrease the number of naturally occurring senescent human cells in vitro, and improve physical function and increase the lifespan of aged mice. Over the past 5 years, senotherapeutic research has progressed exponentially with the demonstration that these drugs can be used as a potential approach to improve transplantation outcomes and transplant availability in both animal models and in humans, or to reduce mortality from a SARS-CoV-2-related murine-β-coronavirus in an aged mouse model.",The Lancet Healthy Longevity,Senotherapeutics,2022
Clinical Barriers and Limitations,"However, the repurposing of existing drugs and the use of new senotherapeutics are associated with various side-effects; incomplete functional characterisation of peripheral tissues at systemic administration; an absence of standardised guidelines for timing, dose, and route of administration; and a paucity of efficacy and safety data from clinical trials. Therefore, the full potential of senotherapeutics has been hampered in clinical applications. The scope of this Review is to summarise the state-of-art literature on the benefits and risks of senotherapeutics (particularly senolytics) and on their use in patients with ageing-related disease. We will not address the use of these agents in chemotherapy-induced senescence, given that this has already been reviewed by Prasanna and colleagues. We will break this evidence down to single physiological functions and organ systems, both in patients and in murine models.",The Lancet Healthy Longevity,Senotherapeutics,2022
Senotherapeutics and Cardiovascular Aging,"Cardiovascular disease has a prevalence of 70–75% in the older population (aged 60–69 years). It has been shown that senescent cells accumulate in the heart with age and contribute to related pathologies in animal models. Clearance of senescent cells in aged mice and in mice with atherosclerosis using genetic and pharmacological approaches improves vascular and myocardial function and attenuates age-dependent remodelling. In a 2019 study, mice aged 22 months were treated with the BCL2 and Bcl-xL inhibitor, ABT263 (Navitoclax, Selleckchem, Houston, TX, USA), or vehicle alone by oral gavage at 50 mg/kg per day for 7 days per cycle, for two cycles with a 1-week interval. Treatment with ABT263 showed a significant reduction in heart hypertrophy and fibrosis, together with a compensatory regeneration in cardiomyocytes. However, ABT263 had no significant effect on cardiac function, left ventricle mass, or ventricle wall rigidity. Subsequently, it was shown that ABT263 was able to rescue the functional decrease in ejection fraction occurring after myocardial infarction in aged mice, to improve left ventricular function, to increase myocardial vascularisation, to decrease scar size, and to attenuate the inflammatory response in a young mouse model of ischaemia–reperfusion.",The Lancet Healthy Longevity,Senotherapeutics,2022
Senolytics and Atherosclerosis Models,"The incidence of atherosclerotic coronary artery disease increases with age and is present in over 50% of people older than 60 years. The two most frequently used genetic mouse models of atherosclerosis are the Apoe-knockout model and the Ldlr-knockout model, which differ in their dietary conditions for developing atherosclerosis. A study in mice aged 10 weeks with Ldlr deficiency, fed a high-fat diet (a reliable model of atherosclerosis in mice), and treated once daily with 100 mg/kg ABT263 for 5 days followed by 14 days off treatment on a repeating cycle for 88 days, showed that elimination of senescent cells led to inhibited atherogenesis and reduced the number and average size of plaques. Similar results were obtained after administering the dasatinib (5 mg/kg) and quercetin (10 mg/kg) cocktail as senolytic treatment once monthly for 3 months by oral gavage in the Apoe-knockout mouse model of atherogenesis. Specifically, mice with Apoe deficiency on a high-fat diet developed atherosclerotic plaques containing an increased number of senescent cells. Treatment with dasatinib and quercetin decreased the burden of senescence and plaque calcification, even though no change in plaque size was observed.",The Lancet Healthy Longevity,Senotherapeutics,2022
Age-Related Muscle Decline and Senescence,"Skeletal muscle is among the most age-sensitive tissues in mammals. Considerable changes occur in resident stem cells, myofibers, and the extracellular matrix, leading to a decrease in tissue homoeostasis, function, and regenerative capacity. Regeneration of skeletal muscle is carried out by the satellite stem-cell population that remains in a quiescent state. In aged mice, resting satellite cells lose the ability to repress P16ink4a (Cdkn2a), switching from the reversible quiescent state to an irreversible pre-senescence state. When injured, these cells fail to activate and expand, entering full senescence. A study showed that administration of dasatinib and quercetin in a murine model of irradiation-induced senescence ameliorated muscle function. After 12 weeks of leg irradiation (10 Gy), mice aged 4 months showed impaired capacity in a treadmill exercise and an increased expression of senescent markers in the leg muscles. Five days after a single dose of dasatinib (5 mg/kg) and quercetin (50 mg/kg) by oral gavage, expression of senescent markers was reduced and exercise endurance was better than in vehicle-treated controls. These differences were maintained for 7 months following treatment.",The Lancet Healthy Longevity,Senotherapeutics,2022
Senotherapeutics in Muscular Dystrophy and Human Sarcopenia,"Senotherapy has also proven to be effective in a rat model of Duchenne muscular dystrophy, a progressive disease characterised by chronic muscle degeneration and inflammation. The administration of senolytic ABT263 (18.75 mg/kg per day for 7 days per cycle, for two cycles with a 2-week interval) in rats reduced the expression of senescence markers, prevented the loss of bodyweight and muscle strength, and increased muscle regeneration, even at the late stage of Duchenne muscular dystrophy. In humans, skeletal muscles are among the largest organs in the human body, generating movement and maintaining metabolic homoeostasis. Muscle regeneration and maintenance are facilitated by resident mesenchymal progenitors and muscle stem cells. Skeletal muscle mass and function decline with ageing, culminating in sarcopenia, and are linked to an increased burden of senescent cells with involvement of the immune system. The effects of senolytic therapy in the context of age-associated functional decline of skeletal muscle and sarcopenia in humans have not yet been investigated. However, a wealth of literature shows that exercise or physical activity might be the most cost-effective senolytic therapy, although there is large heterogeneity among published studies.",The Lancet Healthy Longevity,Senotherapeutics,2022
Immune Aging and Lung Senescence,"Lung ageing is associated with structural remodelling, decline of respiratory function, and increased susceptibility to acute and chronic lung diseases, including asthma, obstructive pulmonary disease, and idiopathic pulmonary fibrosis. Several external factors, such as cumulative exposures to environmental pollutants, allergens, smoke, and respiratory infections, accelerate lung impairment. Furthermore, advanced age is associated with many age-related changes in innate and adaptive immunity, including phagocytotic function altered by macrophages and neutrophils, reduced activity of natural killer cells, increased serum concentrations of proinflammatory cytokines, an increased number of airway neutrophils, and reduced T-cell activation. These changes support the development of chronic pulmonary diseases in older people, which is associated with a poor prognosis in cases of comorbidities, such as infection and inflammatory disease. The increased burden of senescent cells, together with the release of inflammatory cytokines associated with SASP, contribute to chronic inflammation in the lung. In particular, interleukin (IL)-6 and IL-8, the most common cytokines associated with SASP, are elevated in the bronchoalveolar lavage of patients aged 65 years and older.",The Lancet Healthy Longevity,Senotherapeutics,2022
"Cytokines, Macrophage Senescence, and Pulmonary Remodelling","IL-6 is a chemotactic and prosurvival factor for monocytes and neutrophils. In macrophages, p16 expression is associated with proliferation of and differentiation into the proinflammatory M1 macrophage, leading to increased IL-6 secretion. Senolytic drugs can eliminate p16-positive macrophages and senescent vascular cells, exerting a dual approach to reverse vascular remodelling. Van der Feen and colleagues showed that intraperitoneal administration of ABT263 (10 mg/kg daily for 7 days) reversed vascular remodelling and improved pulmonary haemodynamics in the end stage of shunt-induced pulmonary arterial hypertension in a rat model. A study linked the senescent fibroblast secretome to fibrogenic activity in a mouse model of idiopathic pulmonary fibrosis, a fatal disease characterised by interstitial remodelling and compromised lung function, induced by administration of bleomycin. The use of dasatinib (5 mg/kg) and quercetin (50 mg/kg) by oral gavage once a week for 3 weeks improved pulmonary function and physical health, although lung fibrosis was visibly unaltered. Interestingly, data show that other senotherapeutics, belonging to the cardiac glycoside family, resulted in effective elimination of senescence-induced lung fibrosis in female immunodeficient NMRI nude mice aged 8 weeks.",The Lancet Healthy Longevity,Senotherapeutics,2022
Human Trials in Idiopathic Pulmonary Fibrosis,"A two-centre, open-label study in 14 patients with idiopathic pulmonary fibrosis showed that intermittent dasatinib (100 mg/day) and quercetin (1250 mg/day) treatment for 3 days per week over 3 weeks had a tolerable safety profile and caused significant ameliorations in physical function, assessed with a 6-min walk distance and 4 m gait speed. By contrast, pulmonary function, circulating concentrations of biochemical markers of senescence and profibrotic factors, frailty index scores, and reported health were unchanged. Patients with idiopathic pulmonary fibrosis are extremely susceptible to developing COVID-19 after infection with SARS-CoV-2. Similarly, COVID-19 survivors are susceptible to developing pulmonary fibrosis-like symptoms. A shared genetic aetiology between idiopathic pulmonary fibrosis and severe COVID-19 has been proposed.",The Lancet Healthy Longevity,Senotherapeutics,2022
"Senolytics, COVID-19, and Antiviral Responses","A Science report showed that aged mice acutely infected with pathogens, including murine-β-coronavirus related to SARS-CoV-2, had increased senescence and 100% mortality. Targeting senescent cells with either fisetin (20 mg/kg per day) or dasatinib (5 mg/kg) plus quercetin (50 mg/kg) on days 3, 4, 11, and 12 after pathogen exposure significantly reduced mortality (by 50%), cellular senescence, and inflammation, and increased antiviral responses. This mouse study did not analyse lung function. A clinical trial funded by the National Institutes of Health is in progress to investigate whether fisetin is able to decrease pulmonary pathological progression and morbidity related to SARS-CoV-2 in hospitalised older patients with COVID-19 (NCT04537299). Supporting these data with larger randomised controlled trials in patients with pulmonary fibrosis would constitute a major breakthrough for public health.",The Lancet Healthy Longevity,Senotherapeutics,2022
"Bone Aging, Osteogenesis, and Haematopoiesis","Bone ageing is major risk factor for primary osteoporosis, an age-related disease characterised by altered bone metabolism that suppresses bone formation and promotes bone resorption, impairing bone homoeostasis. Bone homoeostasis relies on a dynamic balance between osteogenesis, carried out by osteoblasts, and osteoclastogenesis, carried out by osteoclasts. It is well established that haematopoiesis, the generation of new blood cells that takes place in the bone marrow, involves both haematopoietic and non-haematopoietic cells. Non-haematopoietic stromal cells, including osteoblasts and osteoprogenitor cells, promote the maintenance of haematopoietic stem cells. Ageing and genotoxic stress induce cellular senescence of these stem cells and osteoprogenitor cells, with subsequent decline in the functions of these cells in both mice and humans.",The Lancet Healthy Longevity,Senotherapeutics,2022
Senolytics in Haematopoietic Aging and Platelet Toxicity,"In the past few years, various studies have assessed the possible beneficial effects of senotherapeutic approaches on bone ageing and haematopoiesis ageing, yielding different findings. In 2016, Chang and colleagues showed that oral administration of ABT263 in sublethally irradiated mice and naturally aged mice resulted in a mitigation of premature ageing of the haematopoietic system induced by total body irradiation and a rejuvenation of aged haematopoietic stem cells. However, several other studies indicated that ABT263 and other Bcl-xL-inhibitory BH3 mimetics induced thrombocytopenia and a transient thrombocytopathy, which could impair the haemostatic function of platelets. Studies on the effects of senolytics on bone remodelling have also showed divergent results.",The Lancet Healthy Longevity,Senotherapeutics,2022
Senolytics in Bone Remodeling: Conflicting Mouse Data,"Some studies have shown that targeting senescent cells in aged mice (aged 20–24 months) with established bone loss by use of dasatinib (5 mg/kg) plus quercetin (50 mg/kg) by oral gavage once monthly for 4 months, or by ABT263 (41 μmol/kg) daily for 5 days, lowered bone resorption, with maintained trabecular bone formation and enhanced cortical bone formation. However, another study showed that, despite decreasing the burden of senescent cells, ABT263 treatment in aged female and male mice reduced both trabecular bone volume fraction (by 60.1% in females and by 45.6% in males) and the ability of osteoblasts derived from bone marrow stem cells to produce mineralised matrix (by 88% in females and by 83% in males). Therefore, it is not clear from these murine studies whether senotherapy could be a suitable approach to counteract age-related bone loss and impairments in haematopoietic renewing, and further studies are required to assess the safety and efficacy of these drugs.",The Lancet Healthy Longevity,Senotherapeutics,2022
Human Trials and Setbacks in Bone and Haematopoietic Disorders,"Nevertheless, several pilot studies in humans testing the potential beneficial effects of senolytics in recipients of haematopoietic stem-cell transplantation, patients with age-related osteoporosis, and patients with osteoarthritis are currently recruiting or ongoing. However, the leading compound, UBX0101 (Unity Biotechnology, San Francisco, CA, USA), failed a phase 2 study in patients with osteoarthritis in the knee, a major setback for this early stage research into senotherapy. It is not known whether UBX0101 was able to remove senescent cells; however, the drug did not improve clinical symptoms. Therefore, further studies are needed to evaluate the usefulness of senolytics in patients with haematopoietic disorders or bone disorders.",The Lancet Healthy Longevity,Senotherapeutics,2022
Discovery of New Senolytics and Engineering Approaches,"The safety profile and efficacy of senotherapeutics in patients are yet to be fully investigated in clinical trials, and it is likely that the best senotherapeutic against age-associated diseases and malignancies is yet to be discovered. Dasatinib, quercetin, and other senolytics were discovered using a mechanism-based approach. High throughput screening technology, which allows for automated testing of thousands of molecules present in chemical compound libraries in in-vitro senescence models, could assist with the discovery of new effective senolytics. To date, high throughput screening of commercial chemical compound libraries has led to the discovery of new families of senolytics: HSP90 inhibitors, the BET family protein degraders, and cardiac glycosides. Furthermore, the safety and potency of existing senolytics can be improved by molecular engineering and drug delivery approaches. For example, the use of ABT263 is limited due to dose-limiting platelet toxicity. He and colleagues devised a proteolysis-targeting chimera technology to reduce the platelet toxicity of ABT263 by converting it into PZ15227. Compared with ABT263, PZ15227 was shown to be less toxic to platelets, but was a more potent senolytic in vitro and in vivo. Similar strategies might be useful to improve the efficacy and the safety profile of other toxic or repurposed senolytic agents.",The Lancet Healthy Longevity,Senotherapeutics,2022
Limits of Senescent Cell Removal and Cell-Type Specificity,"Eradicating senescent cells in an adult organism is not always beneficial. For instance, senescence can be induced in macrophages as part of a polarisation in response to reversible immunomodulatory stimuli, and a senescence-like phenotype is present in various post-mitotic cells of mice entering middle age in the absence of disease or advanced ageing. Grosse and colleagues reported that genetic removal of liver sinusoidal endothelial cells with high expression of p16 hampered the healthspan of mice because the procedure induced fibrosis in the liver and systemic perivasculature. Interestingly, given the high expression of p16, dasatinib and quercetin treatment removed senescent macrophages, but was ineffective against senescent liver sinusoidal endothelial cells in mice. Future therapeutic approaches based on novel nanotechnology-based strategies for cargo delivery specific to cell type, biomarker, and phenotype are also being developed to increase the specificity and reduce the side-effects of senolytics.",The Lancet Healthy Longevity,Senotherapeutics,2022
Clinical Translation Challenges and Conclusions,"In conclusion, senolytic drugs have shown promising results in the elimination of senescent cells and in alleviating various diseases in animal models. However, in patients, there is a paucity in data on the efficacy and safety of senotherapeutics from clinical trials, including systemic effects and side-effects. In this regard, as highlighted in a workshop delivered by the National Institutes of Health on the consideration of senolytics for clinical trials, it is important to assess the specificity of senolytics in killing targeted senescent cells and their cytotoxic effects, to identify reliable markers for intervention responses, to elucidate interactions with comorbidities and other drugs, and to standardise administration protocols.",The Lancet Healthy Longevity,Senotherapeutics,2022
Study Overview and Abstract,"Exploring the effects of Dasatinib, Quercetin, and Fisetin on DNA methylation clocks: a longitudinal study on senolytic interventions. Edwin Lee, Natàlia Carreras-Gallo, Leilani Lopez, Logan Turner, Aaron Lin, Tavis L. Mendez, Hannah Went, Alan Tomusiak, Eric Verdin, Michael Corley, Lishomwa Ndhlovu, Ryan Smith, Varun B. Dwaraka. Keywords: senolytics, longitudinal studies, epigentic clocks, aging, immune system. Received: August 3, 2023 Accepted: January 19, 2024 Published: February 22, 2024. ABSTRACT Senolytics, small molecules targeting cellular senescence, have emerged as potential therapeutics to enhance health span. However, their impact on epigenetic age remains unstudied. This study aimed to assess the effects of Dasatinib and Quercetin (DQ) senolytic treatment on DNA methylation (DNAm), epigenetic age, and immune cell subsets. In a Phase I pilot study, 19 participants received DQ for 6 months, with DNAm measured at baseline, 3 months, and 6 months. Significant increases in epigenetic age acceleration were observed in first-generation epigenetic clocks and mitotic clocks at 3 and 6 months, along with a notable decrease in telomere length. However, no significant differences were observed in second and third-generation clocks.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
DQF Intervention and Abstract Findings,"Building upon these findings, a subsequent investigation evaluated the combination of DQ with Fisetin (DQF), a well-known antioxidant and antiaging senolytic molecule. After one year, 19 participants (including 10 from the initial study) received DQF for 6 months, with DNAm assessed at baseline and 6 months. Remarkably, the addition of Fisetin to the treatment resulted in non-significant increases in epigenetic age acceleration, suggesting a potential mitigating effect of Fisetin on the impact of DQ on epigenetic aging. Furthermore, our analyses unveiled notable differences in immune cell proportions between the DQ and DQF treatment groups, providing a biological basis for the divergent patterns observed in the evolution of epigenetic clocks. These findings warrant further research to validate and comprehensively understand the implications of these combined interventions.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Introduction to Senescence and Aging Mechanisms,"INTRODUCTION Senescence is defined as a stable growth arrest of cells that can limit the proliferation of damaged cells, which is important for tissue homeostasis. However, senescent cells also release harmful substances that can cause inflammation and damage to nearby healthy cells. Recent studies suggest that senescence may contribute to aging and age-related pathologies through the impossibility of tissue renewal by the stem cells caught in senescence or through the chronic inflammation of nearby cells that can lead to tissue dysfunction. In fact, studies in mice have demonstrated that injecting senescent cells can induce age-related conditions like osteoarthritis, frailty, and reduced lifespan. Aging is characterized by gradual functional decline. It is associated with increased risk of multiple chronic diseases, geriatric syndromes, impaired physical resilience, and mortality. For this reason, the pursuit of strategies to combat age-related diseases and promote healthy aging has increased in recent years.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Senolytics Background: DQ and Fisetin,"Given the potential role of senescence in aging, senolytic drugs have emerged as promising candidates for extending lifespan. Some initially identified senolytics were Dasatinib, Quercetin, and Fisetin. These molecules were drugs or natural products already used for other indications in humans, including anti-cancer therapies. Dasatinib is a tyrosine kinase inhibitor approved by the FDA to treat myeloid leukemia. Quercetin is a flavonoid compound that induces apoptosis in senescent endothelial cells. Combined treatment with Dasatinib and Quercetin (DQ) has been demonstrated to decrease senescent cell burden in humans in multiple tissues; improve pulmonary and physical function along with survival in mice while lessening their age-dependent intervertebral disc degeneration; and reduce senescence and inflammatory markers in non-human primates. In human studies, patients with idiopathic pulmonary fibrosis improved 6-minute walk distance, walking speed, chair rise ability and short physical performance battery after 9 doses of oral DQ over 3 weeks.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Fisetin Mechanisms and Epigenetic Biomarkers,"Fisetin is another flavonoid compound that has gained recognition for its anti-proliferative, anti-inflammatory, and anti-metastatic properties. Fisetin has the potential to reduce senescence markers in multiple tissues in murine and human subjects. Administration of Fisetin to old mice restored tissue homeostasis, reduced age-related pathology, and extended median and maximum lifespan. Notably, a comparative study has highlighted Fisetin as the safest and most potent natural senolytic among the tested compounds. To date, research has not determined the effect of senolytics in biological aging measured by molecular biomarkers, such as the length of the telomeres, the proportion of immune cells, and the alteration of DNA methylation. DNA methylation (DNAm) has emerged as a widely used biomarker for predicting health span and age-related diseases. In particular, multiple aging biomarkers, also known as clocks, have been developed since 2013.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Aging Clocks and Study Rationale,"Among them, the first-generation clocks, such as the Hannum clock and Horvath clocks, utilize CpG sites that are highly associated with chronological age to estimate an individual’s biological age. Second-generation clocks, including the DNAmPhenoAge and GrimAge, instead of being trained to predict chronological age, have been trained to predict biological phenotypes, such as clinical features or proteins highly associated with aging. This new methodology led to higher hazard ratios of age-related outcomes for second-generation clocks compared to first-generation. Finally, in 2023, a third-generation clock was developed, the DunedinPACE, which measures the rate of aging rather than providing an overall age estimation. Therefore, this study aims to comprehensively assess the impact of senolytic drugs on epigenetic aging through two longitudinal studies to address our research objective. The initial investigation focuses on a combination treatment of Dasatinib and Quercetin, while the subsequent phase incorporates Fisetin into the treatment regimen.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Effects on Telomere Length Predictors,"Impact of senolytic drugs on DNAm telomere length and mitotic clock epigenetic methylation prediction algorithms. Cells with critically short telomere lengths are also known to undergo senescence once they approach their Hayflick limit, therefore we investigated the potential changes due to telomere length using the DNAm predictor for telomere length (DNAmTL). We found significant alterations in the DQ group, but not in the DQF group. Specifically, we observed a significant decrease in PC DNAm telomere length after the whole treatment (p-value=0.01), which was even more significant after adjusting by age (p-value=4.4·10-4). Importantly, the difference in telomere length acceleration between baseline and 3 months was larger and more significant (p-value=7.5·10-5) than the difference between baseline and 6 months (p-value=4.4·10-4).",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Mitotic Clock Outcomes,"Mitotic clock metrics were also employed to evaluate relative changes in stem cell replication. At the 3-month mark of DQ treatment, we observed a decrease in both the total number of stem cell divisions and the intrinsic tissue stem cell divisions, although these changes were not statistically significant (p-value=0.22 and p-value=0.22, respectively). However, between 3-month and 6-month points, a significant increase was evident in both mitotic clocks (p-value=2.4·10-4 and p-value=7.8·10-4, respectively). In the case of DQF treatment, no significant differences were found between the baseline and the 6-month measurement.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Immune Cell Composition Changes Under DQ,"Impact of senolytic drugs on whole blood immune cell composition. We utilized EpiDISH (2023) to quantify 12 different immune cell subsets and assess changes within these subsets. During the 6-month period of DQ treatment, significant alterations were observed in CD4T Naive cells, B Naive cells, and monocytes (Table 4). The most notable and significant change was in CD4T Naive Cells, which exhibited a slight decrease at the 3-month mark (p-value=0.628) and experienced a more substantial decline between the 3 and the 6-month marks (p-value=0.029). B Naive cells displayed an insignificant increase for the global treatment (p-value=0.059), but we observed a significant increase between 3 and 6 months (p-value=0.001). Monocytes showed a global increase after 6 months of treatment (p-value=0.003) that was characterized by a significant decrease at 3 months (p-value=0.035) followed by a significant increase between 3 and 6 months (p-value=3.0·10-5).",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Immune Effects of DQF and Immune-Adjusted EAA,"Conversely, CD4T Memory, CD8T Naive, CD8T Memory, B Memory, basophil, regulatory T cells, eosinophil, Natural Killer, and Neutrophil did not exhibit significant changes. Regarding the impact of DQF on immune cells, B Naive cells (Bnv) demonstrated a significant decrease after 6 months (p-value=3.0·10-4), which contrasts with the observations from DQ treatment. No significant changes were observed in the proportions of other immune cell subsets (Table 5). Since most of the epigenetic clocks, especially the first-generation clocks, are dependent on immune subsets, we calculated the correlation between the EAA metrics and the immune cells proportions. As expected, we did not observe significant correlations between IntrinClock and immune cells. However, we observed high correlations between the other clocks and most of the immune cells. Thus, we decided to calculate immune EAA adjusting EAA values by all the immune cells that were significantly associated to the clocks (CD4T naive and memory cells, B naive and memory cells, CD8T naive and memory cells, natural killers, and neutrophils) and see whether the trends in first-generation clocks after DQ treatment were maintained. We found that the significance and direction of the associations were not modified after adjusting by immune cells, indicating that the increase of epigenetic age after DQ treatment was not due to the alteration of immune cell subsets.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Whole-Genome DNAm Modifications: EWAS Findings,"Impact of senolytic drugs on whole-genome DNA methylation. We also assessed global modification of DNAm in those individuals who were treated with DQ and those with DQF. To this end, we performed an Epigenome-Wide Association Study (EWAS) comparing the methylation levels for all the CpG sites in the genome at different timepoints in each trial (Supplementary Table 1). The first EWAS was performed between baseline and 3 months of DQ treatment. In this case, we identified 11 CpG sites differentially methylated, 4 of them hypermethylated and 7 hypomethylated after 3 months. These probes were mapped to 8 genes. Among them, TGIF1, SORBS2, and ZNF768 were implicated in senescence. Using a less restrictive threshold of p-value lower than 1·10-4, we performed an enrichment analysis. Among the 305 probes identified, we found three enriched processes highly related with senescence, such as glycolic process, vesicle recycling and endocytosis, and cytoskeletal organization.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
EWAS at 6 Months of DQ Treatment,"Second, we evaluated the differences in global methylation between baseline and 6 months after DQ treatment. In this case, we only saw 2 CpG sites differentially methylated with an adjusted p-value lower than 0.05. One of them was hypermethylated and the other hypomethylated after a period of 6 months. The GREAT analysis was performed with the 475 CpG sites with a nominal p-value lower than 1·10-4. Although multiple gene ontology terms were identified as enriched, none of them were directly associated with aging or senescence. Finally, when we compared the methylation levels between baseline and 6 months after DQF treatment, we identified 208 significant probes. Among them, approximately 50% were hypomethylated and 50% were hypermethylated. The GREAT analysis was performed using 556 probes with a p-value below 1·10-4 and revealed multiple enriched pathways associated with senescence, such as epithelial cell proliferation, platelet dense granule membrane, cell junction, and positive regulation of cardiac muscle cell apoptotic process.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Proteomic Surrogate Analysis: SASP-Related Signals,"Clinical and DNAm proteomic surrogate analysis. The major hypothesized mechanism for the negative impacts of senescence is through the increased senescence-associated secretory phenotypes (SASP) which lead to high inflammatory cytokine signaling from senescent cells in a paracrine fashion. As an alternative to robust clinical lab measurements of inflammatory mediators, we used methylation risk scores surrogates to predict and quantify predicted changes in circulating proteomic markers. The quantification of these markers and the comparison between the different timepoints are included in Supplementary Tables 2, 3. We paid special attention to inflammation and inflammatory proteomic EpiScore analysis and these are listed in Table 6. In this analysis, we see some increased inflammatory mediators at 3 months which decrease from the 3 to 6-month timepoints. These inflammatory-associated proteins include CRP, CXCL9, CXCL11, CCL17, and TGF-alpha. We see opposite trends with other inflammation-associated markers such as Complement C4 and Complement C5a. Many inflammatory mediators for the innate adaptive immune system were not registered as significant in this analysis.",Aging (Albany NY),Senolytics and Epigenetic Clocks,2024
Introduction to Cellular Senescence,"Cellular senescence is a critical biological process that contributes to aging and the onset of age-related diseases. Defined as an irreversible arrest in cell division in response to stress or damage, senescence serves as a protective mechanism to prevent the propagation of damaged cells. However, over time, the accumulation of senescent cells in tissues leads to a decline in cellular function and the development of a range of age-related conditions, including cardiovascular diseases, neurodegenerative disorders, and cancers.1,2 The presence of these cells is often accompanied by the senescence-associated secretory phenotype (SASP), a pro-inflammatory state that can exacerbate tissue dysfunction and promote chronic disease progression. As such, cellular senescence has become a focal point in aging research, offering new opportunities for therapeutic intervention.3 Recent advances in the field have introduced novel strategies aimed at targeting senescent cells to mitigate their harmful effects.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Senolytics and Senomorphics Overview,"Among these strategies, senolytics and senomorphics have emerged as two promising therapeutic approaches.4 Senolytics are compounds that selectively induce the death of senescent cells, thereby reducing their detrimental impact on tissues. In contrast, senomorphics work by modulating the SASP or altering the cellular environment to alleviate the negative effects of senescence without eliminating the senescent cells themselves.5 Both approaches offer a unique opportunity to delay the onset of age-related diseases and extend the healthspan, the period of life spent in good health. Despite promising preclinical findings, challenges such as specificity, safety, and clinical translation remain unresolved.6 This review aims to explore recent advancements in senolytics and senomorphics, their therapeutic potential, and the challenges in translating these strategies into clinical applications for enhancing longevity and healthspan.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Rationale and Scope of the Review,"This study is justified as it provides a comprehensive review of recent advancements in senescence-targeting therapies, highlighting their potential, limitations, and future directions for promoting healthy aging. Unlike previous reviews that often focus on either class of therapeutics, this study integrates recent advancements, compares their mechanisms, and discusses innovative combination strategies. Additionally, it explores emerging challenges in clinical translation, including precision targeting, safety concerns, and personalized interventions, offering new perspectives on optimizing these therapies for longevity and healthspan extension. We will discuss the underlying mechanisms of cellular senescence, the progress in developing senolytics and senomorphics, and the preclinical and clinical data supporting their potential as therapeutic agents.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Challenges and Future Perspectives,"Additionally, we will examine the challenges faced in the translation of these therapies from the laboratory to the clinic, as well as the future prospects for their application in aging-related interventions. By exploring these therapeutic avenues, this review seeks to highlight the potential of targeting cellular senescence as a strategy to promote healthy aging and reduce the burden of age-associated diseases. This review makes a distinct contribution to the field by integrating mechanistic insights into cellular senescence with a dual therapeutic focus on both senolytics and senomorphics. While previous reviews have often addressed these approaches separately, our synthesis brings together the most recent preclinical and clinical evidence, elucidates how oxidative stress is mechanistically linked to SASP activation, and critically evaluates pharmacokinetic and bioavailability challenges that influence therapeutic translation.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Research Gaps and Translational Barriers,"By consolidating evidence from 2014–2025 into comprehensive comparative tables and highlighting combination therapy strategies, we offer a novel framework for understanding how targeted senescence modulation can be optimized for healthy aging. Furthermore, the review identifies underexplored research gaps such as tissue-specific senescence heterogeneity, biomarker-driven patient selection, and advanced delivery systems, that, if addressed, could substantially advance both scientific knowledge and clinical application in geroscience.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Methodology,"This narrative review was conducted through a comprehensive literature search using databases such as PubMed, Scopus, and Web of Science. Peer-reviewed articles, review papers, and clinical studies published between 2014–2025 were prioritized. Keywords including cellular senescence, aging, senolytics, senomorphics, SASP, and age-related diseases were used to identify relevant studies. Articles were selected based on their relevance to the mechanisms of senescence, its role in aging and disease, and emerging therapeutic strategies. The findings of the study were presented and discussed concurrently. Thus, this review presents current knowledge, gaps in research, and discusses potential future directions for targeting cellular senescence in healthy aging. In total, our search initially retrieved 412 records. After removal of duplicates and screening titles and abstracts for relevance, 358 articles were selected for full-text review. Following eligibility assessment based on predefined inclusion criteria—relevance to cellular senescence mechanisms, therapeutic targeting (senolytics or senomorphics), and availability of clear mechanistic or clinical outcome data—261 peer-reviewed articles were finally included in this review. Only studies published in English between 2014–2025 were considered.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Inclusion and Exclusion Criteria,"Inclusion criteria prioritized mechanistic studies, preclinical and clinical trials, and high-quality reviews with explicit senescence-related endpoints, while those excluded were conference abstracts, editorials, and studies lacking primary data on senescence modulation. Cellular Senescence: Mechanisms and Implications Cellular senescence is a complex and highly regulated process that acts as a response to various cellular stresses, including DNA damage, oxidative stress, telomere shortening, and oncogenic signaling.7 This process, which can be triggered by a variety of internal and external factors, leads to a permanent cell cycle arrest. While senescence functions as a defense mechanism against the propagation of damaged cells and thus prevents the development of cancer and other diseases, it also contributes to the aging process when senescent cells accumulate over time in tissues.8","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Mechanisms of Senescence Overview,"Cellular senescence, a state of stable cell cycle arrest that prevents the proliferation of damaged or stressed cells, plays a crucial role in aging, tumor suppression, and tissue remodeling. Senescence is triggered by various intrinsic and extrinsic factors, including DNA damage, telomere attrition, oncogene activation, and oxidative stress, as illustrated in Figure 1. Once senescence is established, the cell enters a state of permanent arrest and exhibits specific markers, such as β-galactosidase activity, elevated levels of p53 and p16INK4a, and changes in the chromatin landscape that reinforce its non-proliferative state. Understanding the molecular mechanisms underlying senescence is critical for developing interventions in aging-related diseases and cancer. The primary molecular pathways involved in senescence include: i. DNA Damage Response (DDR) and Senescence Induction: When DNA damage occurs, the DDR pathway is activated to repair the damage. If the damage is too severe to repair, the p53-p21 pathway is triggered, inducing cell cycle arrest. This mechanism prevents the replication of damaged DNA and serves as an essential barrier against cancer.9 Additionally, the p16INK4a-Rb pathway is activated in response to cellular stress, causing another form of cell cycle arrest, particularly in older cells.10","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Telomere Shortening and Oncogene-Induced Senescence,"ii. Telomere Shortening: Telomeres are repetitive DNA sequences at the ends of chromosomes that protect them from damage and prevent the loss of genetic material during cell division.11 However, each cell division results in the shortening of telomeres. Once telomeres become critically short, the cell enters senescence as a protective measure to avoid chromosomal instability and potential malignant transformation.12 Telomeres shorten with each cell division due to the end-replication problem in DNA synthesis. Critically short telomeres activate the p53/p21 pathway, leading to an irreversible cell cycle arrest. The enzyme telomerase can counteract this process, but its activity is limited in most somatic cells.13 iii. Oncogene-Induced Senescence (OIS): Activation of certain oncogenes, such as RAS or MYC, can induce senescence in cells, thereby preventing the transformation of these cells into tumors.14 In other words, oncogenic activation (eg, RAS, RAF, and MYC) can induce senescence as a protective mechanism against uncontrolled proliferation. Hyperactivation of oncogenes leads to excessive replication stress and activation of the DDR pathway.15","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Oxidative Stress and SASP Activation,"iv. Oxidative Stress and Mitochondrial Dysfunction: Reactive oxygen species (ROS) generated from mitochondrial dysfunction or environmental stress contribute to cellular senescence.16 Oxidative stress leads to DNA damage, lipid peroxidation, and protein dysfunction.17 ROS can activate the p38 MAPK pathway, which enhances p53 and p16 signaling, reinforcing senescence.18 Importantly, oxidative stress is not an isolated mechanism but a central upstream driver of the SASP. Elevated ROS levels, whether from mitochondrial dysfunction, environmental insults, or chronic inflammation, activate redox-sensitive transcription factors such as NF-κB and p38 MAPK, which directly enhance SASP gene expression. This mechanistic link explains why oxidative stress can amplify SASP-mediated tissue damage, creating a self-reinforcing cycle of inflammation and cellular dysfunction. Senolytics reduce SASP not only by eliminating the senescent cells that produce high ROS but also by disrupting pro-survival signaling pathways that sustain SASP activity. For example, dasatinib and quercetin reduce SASP cytokines by promoting apoptosis of ROS-rich senescent cells, while fisetin decreases oxidative stress burden and downregulates NF-κB signaling, thereby dampening SASP output.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
SASP and Epigenetic Regulation,"v. Senescence-Associated Secretory Phenotype (SASP): Senescent cells develop a distinct secretory profile, known as the SASP. SASP includes pro-inflammatory cytokines (IL-6, IL-8, TNF-α), growth factors, and matrix metalloproteinases (MMPs), influencing the tissue microenvironment.19 While SASP plays roles in wound healing and tumor suppression, chronic SASP contributes to inflammation, fibrosis, and aging-related diseases.20 vi. Epigenetic Regulation of Senescence: Chromatin remodeling through histone modifications and DNA methylation influences senescence. H3K9me3 and H3K27me3 histone marks promote heterochromatin formation, reinforcing senescence-associated gene silencing.21 Senescent cells also exhibit DNA methylation changes, altering gene expression patterns.22","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Senescence in Aging and SASP,"The role of cellular senescence in aging is multifaceted. While senescence may initially serve as a protective mechanism, the long-term accumulation of senescent cells in tissues can contribute to various age-related pathological conditions. Over time, senescent cells secrete a variety of pro-inflammatory factors, collectively known as the senescence-associated secretory phenotype (SASP). The SASP includes cytokines, chemokines, growth factors, and extracellular matrix-degrading enzymes, which promote local inflammation and tissue degeneration.23 i. Tissue Dysfunction: Senescent cells accumulate in various tissues as organisms age, including the skin, skeletal muscle, adipose tissue, liver, and vasculature. The presence of these cells in tissues disrupts the normal tissue architecture and impairs regenerative capacities, leading to functional decline.2 For example, in skeletal muscle, the accumulation of senescent cells has been linked to sarcopenia, the age-related loss of muscle mass and function.24 ii. Chronic Diseases: The inflammatory environment created by senescent cells contributes to the pathogenesis of several chronic diseases. In the cardiovascular system, senescent cells in the blood vessels promote arterial stiffness and endothelial dysfunction, increasing the risk of hypertension and atherosclerosis.25","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Senescence in Chronic Disease,"In the brain, the buildup of senescent cells has been implicated in neurodegenerative diseases such as Alzheimer’s and Parkinson’s, where they exacerbate neuroinflammation and neuronal loss.26 Similarly, in adipose tissue, the accumulation of senescent cells contributes to insulin resistance and the development of metabolic disorders such as type 2 diabetes.27 Senescent chondrocytes promote cartilage degradation and joint inflammation. The SASP secreted by senescent chondrocytes increases matrix metalloproteinase (MMP) activity, leading to the breakdown of cartilage and the progression of osteoarthritis.28 iii. Cancer: While cellular senescence serves as a protective mechanism against tumorigenesis by halting the proliferation of damaged or oncogene-expressing cells, the chronic inflammatory environment created by senescent cells can promote cancer progression. The SASP can induce genomic instability in neighboring cells, creating a microenvironment conducive to tumor growth.29 Furthermore, the persistence of senescent cells in tissues can undermine the effectiveness of cancer therapies by altering immune surveillance and the tissue response to treatment.30","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Double-Edged Nature of Senescence,"The Role of Senescence in Aging: A Double-Edged Sword While senescence is essential for preventing the proliferation of damaged cells and the onset of cancer, its persistent presence in tissues throughout aging disrupts homeostasis. Initially protective, senescent cells transform into a detrimental factor that accelerates aging and facilitates the onset of specific chronic diseases.31 It functions as a double-edged sword, simultaneously inducing organismal deterioration and affecting health positively or negatively. Early life advantages from senescence partly by curtailing the proliferation of cancerous or damaged cells, promoting wound healing, and maintaining tissue homeostasis.32 The senescence-associated secretory phenotype (SASP) comprises bioactive substances generated by senescent cells that modify immune responses and tissue repair. Conversely, age-related senescence becomes detrimental when senescent cells accumulate due to diminished immune clearance. This results in chronic inflammation, tissue dysfunction, and age-associated diseases such as neurodegeneration, cardiovascular disorders, and cancer.3","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Accumulation and Impact of Senescent Cells,"The pro-inflammatory environment created by SASP exacerbates aging through systemic damage and diminished cellular function.33 The accumulation of senescent cells over time underscores the necessity of developing treatment strategies that selectively target these cells while preserving their beneficial roles in cancer suppression and tissue repair.34 The growing body of research on cellular senescence and its implications for aging has shifted the focus towards therapy techniques aimed at mitigating the adverse effects of senescent cells. Senolytic and senomorphic therapies have garnered significant interest as potential methods to promote healthy aging by either eliminating senescent cells or modifying their detrimental secretions.35 These medications represent a compelling domain in gerontology, aiming to restore tissue equilibrium, enhance organ functionality, and mitigate the impact of age-associated ailments.36 In greater detail, cellular senescence confers physiological benefits under specific conditions such as embryonic development, where transient senescence aids tissue patterning; wound healing, where short-term SASP signaling recruits immune cells for tissue repair; and fibrosis resolution, where senescent myofibroblasts limit excessive extracellular matrix deposition.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Pathological Persistence of Senescent Cells,"It also serves as a robust tumor-suppressive mechanism by halting proliferation of potentially malignant cells. However, under pathological conditions such as chronic infections, metabolic dysregulation, or age-related immune decline, senescent cells persist beyond their beneficial window. This persistence is often due to reduced immune clearance efficiency, leading to prolonged SASP activity that fuels chronic inflammation, extracellular matrix degradation, and tissue dysfunction. Mechanistically, this shift from beneficial to detrimental involves sustained activation of NF-κB and p38 MAPK pathways, elevated ROS production from dysfunctional mitochondria, and unresolved DNA damage response signaling. The chronic inflammatory milieu created by these mechanisms accelerates degenerative changes in organs such as the brain, vasculature, joints, and adipose tissue. Understanding these situation-dependent roles is critical for designing interventions that selectively suppress detrimental aspects of senescence while preserving its protective functions.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Introduction to Senolytics,"Senolytics represent a promising class of therapeutic agents that selectively induce the death of senescent cells, thereby reducing the harmful effects these cells have on surrounding tissues. Unlike traditional approaches aimed at mitigating the consequences of aging, senolytics directly target and eliminate senescent cells, potentially reversing the negative impact of their accumulation.37 This approach offers a novel means to delay the onset of age-related diseases and improve overall healthspan. A personalised strategy integrating senolytics with traditional therapies may provide optimal benefits for healthy aging and longevity while mitigating risks.38 Table 1 below compares senolytics and traditional methods targeting cellular senescence. Overview of Senolytics Senolytics are a class of therapeutic agents specifically designed to eliminate senescent cells, which are cells that have entered an irreversible condition of cell cycle arrest while remaining metabolically active.36 Numerous stressors, such as telomere attrition, oxidative stress, DNA damage, and oncogene activation, induce cellular senescence.1 Initially, senescence safeguards against cancer by inhibiting the proliferation of damaged cells; however, the continuous accumulation of senescent cells, through the secretion of pro-inflammatory molecules known as the senescence-associated secretory phenotype (SASP), ultimately leads to chronic inflammation, tissue dysfunction, and age-related diseases.34","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Senolytics vs Traditional Interventions,"Senolytic therapies are designed to exploit the vulnerabilities of senescent cells, particularly their altered molecular mechanisms that heighten their susceptibility to particular stressors.37 Distinctive features include modifications in the expression of pro-survival proteins and activation of specific survival pathways. Targeting these pathways enables senolytic therapies to specifically induce programmed cell death in senescent cells while sparing non-senescent, healthy cells, hence enhancing healthspan and mitigating age-related diseases.49 In contrast to conventional anti-aging therapies that just decelerate the aging process, senolytics actively eradicate dysfunctional cells, thereby rejuvenating tissues and improving physiological function.50 Mechanisms of Action of Senolytics Senolytics exert their effects by targeting the key survival pathways that allow senescent cells to evade apoptosis. These pathways include anti-apoptotic proteins, metabolic regulators, and inflammatory mediators, as illustrated in Figure 2 below. Different senolytic agents act through distinct molecular mechanisms, including: i. Inhibition of Anti-Apoptotic Pathways: The BCL-2 family regulates apoptosis, and senescent cells often exhibit increased expression of anti-apoptotic (pro-survival proteins) proteins such as BCL-2, BCL-XL, and BCL-W.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Anti-Apoptotic Pathways and FOXO4–p53,"These proteins prevent the activation of caspases, which are essential for apoptosis. Senolytic compounds can act by inhibiting these proteins, promoting cell death in senescent cells.51,52 For instance, navitoclax (ABT-263), venetoclax, and ABT-737 are BCL-2 inhibitors that disrupt pro-survival signaling, leading to senescent cell death. These agents have shown promise in preclinical studies for reducing senescent cell burden in aging, fibrosis, and neurodegeneration.53,54 ii. Disruption of the FOXO4-p53 Interaction: While p53 is a tumor suppressor protein that plays a crucial role in cellular stress responses, its prolonged activation in senescent cells contributes to cellular dysfunction.55 Senolytics can modulate the p53 pathway to promote the selective elimination of senescent cells.56 Additionally, the FOXO4 transcription factor can interact with p53 in senescent cells. This interaction blocks p53-mediated apoptosis, allowing senescent cells to survive.57 FOXO4-DRI, a synthetic peptide, disrupts the FOXO4-p53 interaction, reactivating apoptosis and clearing senescent cells. This approach has demonstrated effectiveness in reversing age-related decline in animal models.58","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
SASP Suppression and Heat Shock Protein Targeting,"iii. Suppression of Senescence-Associated Secretory Phenotype (SASP): Senescent cells secrete pro-inflammatory cytokines (IL-6, IL-8, TNF-α), growth factors, chemokines, and matrix metalloproteinases, which create a pro-inflammatory environment that accelerates aging and disease progression.59 For example, dasatinib and quercetin is a combination therapy that reduces SASP production and enhances the clearance of senescent cells.60 Similarly, fisetin and curcumin are flavonoids with senolytic properties that inhibit SASP secretion, reduce inflammation and improve tissue function.61,62 iv. Targeting Heat Shock Proteins (HSPs): Heat shock proteins (HSP90, HSP70) are molecular chaperones that stabilize key proteins involved in senescence.63 HSP90 inhibitors (eg, 17-AAG, Geldanamycin) cause degradation of senescence-associated proteins, leading to senescent cell death. This approach is being explored in fibrosis, neurodegenerative diseases, and cancer therapy.64,65","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Metabolic and Autophagy-Targeting Senolytics,"v. Modulation of Metabolic Pathways: Senescent cells exhibit altered metabolism, particularly increased reliance on glycolysis and mitochondrial dysfunction. When senescent cells exhibit mitochondrial dysfunction, it leads to an altered cellular redox state. This provides an opportunity for senolytic agents to exploit the altered metabolism of these cells, inducing oxidative stress and cell death.66 For instance, metformin reduces mitochondrial oxidative stress and modulates AMPK signaling, helping prevent the accumulation of senescent cells.67,68 Resveratrol on the other hand activates SIRT1 and AMPK, which promote mitochondrial health and energy balance while NAD+ boosters (such as nicotinamide riboside, nicotinamide mononucleotide) improve mitochondrial function, counteract cellular senescence, and extend lifespan in animal models.69 vi. Enhancement of Autophagy and Cellular Clearance: Autophagy is a process that removes damaged organelles and proteins, but senescent cells often exhibit impaired autophagy, leading to metabolic dysfunction. This can serve as a therapeutic target against aging and age-related disorders.70 For example, rapamycin, an mTOR inhibitor restores autophagy and delays senescence-related diseases.71,72 Similarly, spermidine induces autophagy and improves cellular renewal, thus promoting longevity.73","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Oxidative Stress and DNA Damage Response,"vii. Regulation of Oxidative Stress and DNA Damage Response: Oxidative stress and DNA damage are major drivers of senescence. Some senolytics mitigate these effects by enhancing antioxidant defenses. A common example is sulforaphane, found in cruciferous vegetables, which activates Nrf2, a master regulator of antioxidant pathways.74 Similarly, coenzyme Q10 supports mitochondrial health and reduces oxidative stress-related senescence.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Key Senolytic Compounds Overview,"Key Senolytic Compounds and Their Mechanisms of Action A variety of compounds have been identified as potential senolytics, each with distinct mechanisms of action. These compounds have shown promise in preclinical models and are currently undergoing evaluation in clinical trials to assess safety, effectiveness, and long-term benefits (Table 2). Challenges in Senolytic Therapy Despite the promising potential of senolytic therapy in combating aging and age-related diseases, several challenges must be addressed before widespread clinical application. These challenges include safety concerns, drug specificity, delivery methods, off-target effects, long-term consequences, and regulatory hurdles. Addressing these issues through better drug design, personalized treatment approaches, and rigorous clinical research will be crucial for making senolytics a viable therapeutic option in the future. i. Safety and Toxicity Concerns: Many senolytic drugs, such as navitoclax (ABT-263), target BCL-2 family proteins, which are also essential for the survival of non-senescent cells, including platelets and immune cells. This can lead to severe side effects, including thrombocytopenia (low platelet count) and immune suppression, increasing the risk of infections. Long-term use of senolytics could potentially cause tissue damage, impaired wound healing, or unintended apoptosis in healthy cells.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Senolytic Specificity and Targeting Challenges,"ii. Lack of Senescent Cell-Specific Targets: One of the major challenges in senolytic therapy is ensuring the selective elimination of senescent cells without affecting healthy cells. While certain senolytic compounds have shown specificity for senescent cells in preclinical models, achieving this selectivity in humans remains a significant hurdle. For example, some senescence-associated proteins (eg, BCL-2, p53, SASP factors) are also expressed in normal, proliferating cells, leading to potential off-target effects. Therefore, optimizing senolytic agents to minimize side effects while maintaining efficacy is crucial. Table 2 Key Senolytic Compounds, Their Mode of Action, and Evidence from Preclinical and Clinical Trials. Navitoclax (ABT-263): Inhibits BCL-2, BCLxL, and BCL-W. Preclinical evidence shows reduced senescent cell burden, improved lung function in IPF, suppression of osteoclastogenesis, and revitalization of aged hematopoietic stem cells. Clinical trials report thrombocytopenia concerns. Dasatinib + Quercetin (D+Q): Dasatinib inhibits tyrosine kinases; quercetin inhibits PI3K/AKT and SASP. Demonstrated senescent cell reduction, improved vasomotor function, and extended lifespan in mice, with early human trials showing reduced senescent burden.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
"FOXO4-DRI, Fisetin, Venetoclax, ABT-737","FOXO4-DRI: Disrupts FOXO4–p53 interaction, reactivating apoptosis in senescent cells. Preclinical studies show rejuvenation of aged tissues, improved testosterone secretion, and enhanced spermatogenesis in aged mice. Limited clinical trials exist. Fisetin: Modulates SASP, activates AMPK/SIRT1, and induces apoptosis. Extends lifespan in mice and reduces oxidative stress, inflammation, and apoptosis in diabetic cardiomyopathy. Multiple clinical trials are ongoing, and fisetin has reduced markers of senescence in human adipose tissue. Venetoclax: Selectively inhibits BCL-2, inducing senescent cell death. Effective against AML cells and promising against therapy-induced senescence. FDA-approved for CLL but not for aging. ABT-737: Mimics BH3-only proteins to inhibit BCL-2, promoting apoptosis. Eliminates senescent cells in fibrotic and aged tissue; no clinical trials due to off-target effects.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
"Additional Senolytics: 17-AAG, Curcumin, Resveratrol, Metformin","17-AAG (Tanespimycin): Inhibits HSP90, destabilizing proteins required for senescent cell survival. Suppresses senescent fibroblasts in fibrosis and cancer models; investigated mostly in cancer trials. Curcumin: Modulates NF-κB and p53, enhances autophagy, and prevents SASP accumulation. Reduces inflammation and oxidative stress in rats, with clinical studies supporting neuroprotective and cognitive benefits. Resveratrol: Activates SIRT1, reduces oxidative stress, and improves mitochondrial function. Extends lifespan in multiple species and improves metabolic and vascular function in rodents; senolytic-specific trials remain limited. Metformin: Activates AMPK, reduces mTOR signaling, and prevents senescence accumulation. Increases lifespan in rodents, reduces inflammation, and improves aging outcomes, with the TAME trial ongoing to evaluate its role in delaying aging in humans.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
"Rapamycin, Sulforaphane, Piperlongumine, Bardoxolone, UBX0101","Rapamycin: Inhibits mTOR, enhances autophagy, delays cellular aging, and extends lifespan in mice. Human trials show promising effects on immune function and aging biomarkers. Sulforaphane: Activates Nrf2, reduces SASP, and protects against inflammation and oxidative stress in osteoarthritis models. Piperlongumine: Induces ROS production, selectively killing senescent cells. Demonstrates anti-osteosarcoma effects and induces apoptosis through ROS/Akt pathways. Bardoxolone methyl: Modulates Nrf2 signaling, reduces oxidative damage, improves renal function and mitochondrial efficiency, and is under investigation in clinical trials for kidney disease. UBX0101: Blocks Mdm2–p53 interaction and promotes clearance of senescent chondrocytes, preventing osteoarthritis progression. Phase 1 trials showed some benefit; Phase 2 found no significant improvement, requiring further studies.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
"Drug Delivery, Long-Term Use, Variability, Dosing","iii. Drug Delivery Challenges: Senolytic drugs need to penetrate tissues efficiently, but bioavailability and tissue specificity remain hurdles. FOXO4-DRI requires peptide-based delivery, complicating clinical use. Blood–brain barrier penetration is difficult, limiting treatment of neurodegenerative diseases. iv. Long-Term Consequences: Eliminating senescent cells may disrupt tissue homeostasis. Because senescent cells accumulate over time, repeated treatment is necessary, raising concerns about chronic exposure and cumulative toxicity. v. Variability in Response: Genetics, lifestyle, and metabolic differences affect response to senolytics. Age-related changes in metabolism and drug clearance influence safety and efficacy. vi. Dosing and Scheduling: Determining optimal frequency is complex. Intermittent dosing appears safer, reducing thrombocytopenia risk from agents like navitoclax while preserving efficacy. Continuous dosing raises toxicity risks. Biomarker-guided scheduling represents a key translational task.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Regulatory Barriers and Bioavailability Challenges,"vii. Ethical and Regulatory Challenges: Aging is not recognized as a disease by the FDA or EMA, complicating approval. Ethical concerns include life-extension debates, equitable access, and high development costs. viii. Need for More Clinical Trials: Large-scale, long-term human trials are needed to establish safety, efficacy, and dosing. Ongoing trials such as TAME and D+Q provide important data, but broader human studies remain lacking. ix. Bioavailability Challenge: Many senolytics, especially polyphenols such as fisetin and quercetin, exhibit poor oral absorption, rapid metabolism, and low systemic retention. Preclinical mouse doses often range from 20–100 mg/kg PO (fisetin) and 5–10 mg/kg PO (dasatinib). Early human D+Q trials use 100 mg/day dasatinib plus 1000 mg/day quercetin for 3 days/month, with variable pharmacokinetics. These limitations highlight the need for nanoparticle encapsulation, liposomal formulations, and prodrug designs to improve absorption, protect compounds from first-pass metabolism, and enhance tissue targeting. Pharmacokinetic parameters such as Cmax, t½, and AUC are essential to guide optimal dosing and reduce toxicity.","Drug Design, Development and Therapy",Senolytics & Senomorphics,2025
Introduction to Senomorphics,"Senomorphics: Modulating the Senescence-Associated Secretory Phenotype (SASP) Unlike senolytic therapies that aim to directly eliminate senescent cells, senomorphics work by modulating the senescence-associated secretory phenotype (SASP), which consists of pro-inflammatory cytokines, chemokines, growth factors, and extracellular matrix-degrading enzymes secreted by senescent cells. By targeting the SASP, senomorphic therapies aim to alleviate the detrimental effects of senescence without necessarily removing the senescent cells.128 This approach provides a novel strategy to mitigate the harmful inflammation, tissue degradation, and chronic diseases associated with cellular senescence, while preserving the beneficial aspects of senescence, such as tumor suppression and tissue repair.129","Drug Design, Development and Therapy",Senomorphics,2025
Overview of Senomorphics,"Overview of Senomorphics Senomorphic agents target the SASP or the molecular pathways that drive it. These compounds aim to reduce inflammation, restore tissue homeostasis, and promote tissue regeneration by modulating the environment surrounding senescent cells.130 Unlike senolytics, which focus on cell death, senomorphics maintain the senescent cell’s presence but modify its behavior, particularly the pro-inflammatory and tissue-damaging components of the SASP.129 By reducing the detrimental effects of senescent cells, such as the SASP, senomorphics hold promise for treating a range of diseases, including cancer, neurodegenerative disorders, cardiovascular diseases, and metabolic syndromes.26 At the molecular level, senomorphics exert their effects primarily by modulating the signaling pathways that govern SASP production without inducing apoptosis in senescent cells. This often involves inhibition of pro-inflammatory transcription factors such as NF-κB and C/EBPβ, suppression of p38 MAPK and mTOR signaling, and enhancement of autophagic flux to reduce the accumulation of damaged organelles and protein aggregates.","Drug Design, Development and Therapy",Senomorphics,2025
Mechanistic Examples of Senomorphics,"For instance, rapamycin attenuates SASP by inhibiting mTORC1-dependent translation of IL-1α, a key upstream SASP regulator, while metformin activates AMPK to indirectly suppress NF-κB activity and lower pro-inflammatory cytokine secretion. Flavonoids like apigenin and luteolin downregulate MAPK and JAK/STAT pathways, leading to broad SASP suppression. By altering the secretory profile of senescent cells, senomorphics can mitigate tissue inflammation and promote a microenvironment more conducive to regeneration, while preserving the beneficial growth-arrest functions of senescence. This mechanistic distinction from senolytics is crucial for situations where complete removal of senescent cells may be undesirable, such as in wound healing or tissue repair. Figure 3 Mechanisms of Action of Senomorphics (Created in BioRender. Basajja, M. (2025) https://BioRender.com/z3l9g0q).","Drug Design, Development and Therapy",Senomorphics,2025
Senomorphic Mechanisms of Action,"Mechanisms of Actions of Senomorphics Senomorphics target various molecular pathways involved in cellular senescence, effectively modulating the detrimental aspects of the senescent phenotype while preserving beneficial aspects. Simply put, senomorphics work by modulating key pathways that regulate the SASP. Some of the key mechanisms include the following and illustrated in Figure 3. i. NF-κB Pathway: The nuclear factor-kappa B (NF-κB) pathway plays a central role in regulating the expression of pro-inflammatory cytokines and other SASP factors. In senescent cells, NF-κB is often constitutively activated, driving chronic inflammation.131 Senomorphic agents can inhibit this pathway, reducing the secretion of inflammatory cytokines and alleviating the deleterious effects of chronic inflammation.132 ii. JAK/STAT Pathway: The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is involved in the inflammatory signaling of senescent cells. Inhibition of the JAK/STAT pathway can reduce the secretion of pro-inflammatory cytokines and mitigate the impact of senescence-induced inflammation.133 iii. mTOR Pathway: The mechanistic target of rapamycin (mTOR) pathway is implicated in aging and senescence. Activation of mTOR in senescent cells contributes to cellular dysfunction and the pro-inflammatory SASP.134 Inhibition of mTOR has been shown to reduce the expression of SASP factors and improve tissue function in animal models of aging.135","Drug Design, Development and Therapy",Senomorphics,2025
"SASP Modulation, Epigenetics, Mitochondria, DDR","iv. Modulation of the SASP: Senescent cells secrete a variety of pro-inflammatory cytokines, chemokines, growth factors, and proteases collectively known as the SASP. This secretory profile contributes to chronic inflammation, which is a hallmark of aging and a driver of multiple diseases. Senomorphics reduce SASP activity, thereby lowering inflammation, preventing tissue damage, and mitigating age-related diseases.3 v. Epigenetic and Transcriptomic Regulation: Cellular senescence is associated with widespread epigenetic changes, including alterations in DNA methylation, histone modifications, and chromatin remodeling. Senomorphics target these epigenetic regulators to reverse or slow down senescence.136 Some compounds reactivate silenced genes or suppress harmful gene expression, thus modifying the aging process at the molecular level.137 vi. Maintenance of Mitochondrial and Metabolic Homeostasis: Mitochondrial dysfunction is a major driver of cellular senescence, leading to increased reactive oxygen species (ROS) production, metabolic decline, and energy deficits.138 Senomorphics enhance mitochondrial function by stimulating mitophagy, improving oxidative phosphorylation, and reducing oxidative stress, thereby promoting healthier cellular metabolism.66 vii. Modulation of DNA Damage Response (DDR) and p53/p21 Pathways: Cellular senescence is often triggered by DNA damage, which activates the DNA damage response (DDR) and key regulatory pathways such as p53/p21 and p16INK4a/Rb.55 Senomorphics modulate these pathways to restore cell cycle control and improve tissue regeneration while maintaining the tumor-suppressive benefits of senescence.139","Drug Design, Development and Therapy",Senomorphics,2025
Differences Between Senolytics and Senomorphics,"Differences Between Senolytics and Senomorphics The major difference between senolytics and senomorphics lies in their mechanism of action on senescent cells. Senolytics selectively eliminate senescent cells by inducing apoptosis, thereby reducing their harmful effects on aging and disease. Senomorphics modulate the behavior of senescent cells, suppressing their harmful secretions (SASP) without killing them, aiming to restore function and reduce inflammation.36,37 In short, senolytics kill senescent cells, while senomorphics reprogram them to be less harmful. A combination of both strategies may provide a more balanced approach to mitigating aging-related pathologies while preserving essential cellular functions.140 Table 3 highlights some major differences between senolytics and senomorphics. Key Senomorphic Compounds and Their Mechanisms of Action Several compounds have been identified as potential senomorphics, each targeting different components of the SASP or senescence-associated signaling pathways. These agents aim to modify the senescent cell’s secretory phenotype, suppress inflammation, and promote tissue homeostasis. While animal models have shown encouraging results, translation to humans remains in early stages.","Drug Design, Development and Therapy",Senomorphics,2025
Challenges and Opportunities in Senomorphic Therapy,"Challenges and Opportunities in Senomorphic Therapy Senomorphic therapy offers a promising approach to mitigating the harmful effects of cellular senescence without eliminating the beneficial functions of senescent cells and thus holds great promise in combating age-related diseases. However, several challenges remain in its development and application. i. Limited Clinical Evidence: While preclinical studies in animal models suggest that senomorphic compounds can effectively modulate senescence, clinical trials in humans are still limited. Large-scale RCTs are needed to confirm long-term safety and efficacy, particularly in diseases like Alzheimer’s disease, osteoarthritis, and cardiovascular disorders.123 Most evidence comes from short-term or observational studies, making long-term effects unclear. ii. Translating Preclinical Findings to Humans: Species differences, variations in aging processes, and complexity of human biology complicate successful translation.192,193 More clinical trials are required to validate senomorphic efficacy in humans.","Drug Design, Development and Therapy",Senomorphics,2025
"Biomarkers, Bioavailability, and Off-Target Effects","iii. Variability in Senescence Markers: Senescence features differ by tissue, age, and stressors. Identifying consistent biomarkers for SASP or senescence burden remains difficult. Variability complicates the development of standardized outcome measures for senomorphic therapy.194 iv. Bioavailability and Pharmacokinetics: Many senomorphics, such as curcumin and resveratrol, have poor oral bioavailability due to low absorption and rapid metabolism.195 Researchers are exploring liposomal formulations, nanoencapsulation, and prodrug strategies to improve delivery and tissue targeting.196 v. Potential Off-Target Effects: Senescence has protective roles such as tumor suppression and wound healing. Chronic SASP suppression may reduce immune surveillance or impair tissue repair.194 Inhibition of mTOR may weaken immunity, and NAD+ restoration may interfere with metabolic pathways, raising safety concerns that require ongoing monitoring.198","Drug Design, Development and Therapy",Senomorphics,2025
Senescent Cell Heterogeneity and Regulatory Challenges,"vi. Heterogeneity of Senescent Cells: Senescent cells differ widely across tissues. For example, mechanisms driving senescence in muscle differ from those in endothelial cells. This heterogeneity complicates development of broad-spectrum senomorphic therapies.31 Compounds effective in one tissue may not work in another, necessitating tissue-specific approaches.199 vii. Regulatory Hurdles: Senomorphic therapies, especially natural compounds such as resveratrol or curcumin, may face challenges in regulatory approval. Many are categorized as nutraceuticals or supplements, not pharmaceuticals, and thus bypass rigorous testing.200 However, to be used therapeutically, they will likely require clinical evaluation and approval from regulatory bodies such as FDA or EMA.126 Regulatory frameworks may need updates to accommodate senescence-targeting therapies.","Drug Design, Development and Therapy",Senomorphics,2025
Drug Synergy and Combination Strategies,"viii. Drug Synergy and Combinatorial Approaches: Combining senomorphic compounds with other therapeutic strategies—such as senolytics, caloric restriction mimetics, or antioxidants—may enhance therapeutic outcomes. However, interactions between these treatments are not fully understood. More systematic research is needed to evaluate how senomorphics and senolytics can be used together to optimize treatment for aging and age-related diseases. Long-term safety of combination therapies remains unclear.36 Table 4 Key Senomorphic Compounds, Their Mode of Action, and Evidence from Preclinical and Clinical Trials lists major senomorphic compounds including metformin, rapamycin, resveratrol, curcumin, EGCG, quercetin, fisetin, apigenin, pterostilbene, nicotinamide riboside, spermidine, α-ketoglutarate, berberine, sulforaphane, and ginsenosides—along with their SASP-modulating mechanisms and emerging human evidence.","Drug Design, Development and Therapy",Senomorphics,2025
Opportunities in Senomorphic Therapy,"Opportunities in Senomorphic Therapy i. Targeting Age-Related Diseases: Senomorphic therapy offers tremendous potential in treating a wide range of chronic age-related diseases. By modulating the harmful effects of senescence, these therapies could slow down or even reverse conditions such as neurodegenerative diseases (eg, Alzheimer’s, Parkinson’s), cardiovascular disease, arthritis, and metabolic disorders (eg, type 2 diabetes).193 Since many of these diseases are associated with the accumulation of senescent cells and their inflammatory secretions, senomorphic compounds have the potential to treat the root cause of these diseases, rather than just alleviating symptoms.201 ii. Personalized Medicine Approaches: As our understanding of individual aging profiles and senescence mechanisms deepens, there is an opportunity to design personalized treatments based on a person’s genetic makeup, lifestyle factors, and specific senescence-related conditions. Biomarker-based diagnostics will allow for more precise identification of individuals who would benefit most from senomorphic therapy.202 Personalized medicine has proven to help in disease diagnosis, minimize side effects and maximize therapeutic approaches.203","Drug Design, Development and Therapy",Senomorphics,2025
Combination Approaches and Drug Delivery,"iii. Combination with Senolytics: Senomorphic therapy could be used in combination with senolytic compounds (which selectively kill senescent cells). While senolytics eliminate the senescent cells that accumulate over time, senomorphics can help modulate the negative effects of the remaining senescent cells. The combination of these therapies could provide a synergistic effect, improving tissue function, reducing inflammation, and slowing down aging processes. This approach could enhance the overall effectiveness of treatments for age-related diseases.65 iv. Advancements in Drug Delivery Systems: Nanotechnology and advanced drug delivery systems offer the potential to improve the bioavailability, stability, and targeted delivery of senomorphic compounds. By encapsulating these compounds in nanoparticles or using liposomal carriers, researchers can enhance the efficiency of drug delivery to specific tissues or organs. These delivery technologies may allow for more precise treatment of senescence in key tissues (such as the brain, heart, or joints), thereby improving therapeutic outcomes.204","Drug Design, Development and Therapy",Senomorphics,2025
Preventive and Nutraceutical Potential,"v. Expansion into Preventive Medicine: Instead of focusing solely on treating diseases, senomorphic therapies could be used for preventive purposes. Early intervention with senomorphic compounds could delay the onset of age-related diseases by modulating senescence in at-risk individuals, potentially leading to an extension of healthspan (the period of life spent in good health).35,130 This preventive approach could be especially beneficial for populations with genetic predispositions to certain conditions or those already showing early signs of aging-related decline. vi. Potential for Nutraceutical Development: Many senomorphic compounds are naturally occurring and are already consumed as part of a healthy diet (eg, resveratrol, curcumin, quercetin). These compounds could be developed as dietary supplements or functional foods that support healthy aging and reduce the risk of age-related diseases. Their natural origin may make them appealing to consumers seeking less invasive, more holistic health solutions.130,205 As research progresses, these compounds could be integrated into preventive health regimens.","Drug Design, Development and Therapy",Senomorphics,2025
AI and Systems Biology in Senomorphic Discovery,"vii. Integration with AI and Systems Biology: The application of artificial intelligence (AI) and systems biology approaches in drug discovery can help accelerate the identification of new senomorphic compounds. AI-driven analyses can uncover novel compounds and predict their senescence-modulating properties based on biological data.206 Moreover, systems biology approaches that analyze the complex networks of cellular aging processes could help uncover new targets for senomorphic therapies and optimize their clinical applications.207","Drug Design, Development and Therapy",Senomorphics,2025
Preclinical Evidence Supporting Combination Therapies,"Preclinical Evidence Supporting Combination Therapies In preclinical animal models, the combination of senolytic and senomorphic agents has shown promising results. For example, the combination of dasatinib and quercetin (senolytic agents) with fisetin (a senomorphic agent) has been tested in mice and humans and demonstrated enhanced effects compared to either treatment alone. In this combination, dasatinib and quercetin effectively reduced the number of senescent cells in adipose tissue and the vasculature, while fisetin modulated the inflammatory SASP, improving overall tissue function and reducing the risk of age-related diseases such as cardiovascular disease and osteoporosis.18 In another study, the combination of fisetin (a senolytic flavonoid) with sorafenib (a senomorphic agent) showed better synergistic effects in vitro and in vivo than either agent used alone against human cervical cancer.218 These findings highlight the potential of combination therapies to improve multiple aspects of aging simultaneously, rather than focusing on a single target. Table 5 highlights some preclinical animal models where the combination of senolytic and senomorphic agents have shown promising results.","Drug Design, Development and Therapy",Combination Therapies,2025
Preclinical Combination Therapy Models,"Table 5 Some Preclinical Evidence Supporting Combination Therapies of Senolytic and Senomorphic Agents Senolytic Agent Senomorphic Agent Disease Model Key Findings Reference Dasatinib + Quercetin Metformin Aging, Osteoarthritis Reduced senescent cell burden, improved cartilage integrity in mice [219] Navitoclax (ABT-263) Rapamycin Pulmonary Fibrosis Decreased lung fibrosis, improved respiratory function [220] Fisetin Resveratrol Cardiovascular Aging Improved vascular function, reduced oxidative stress [87] Curcumin Quercetin Neurodegenerative Disorders Enhanced cognitive function, reduced neuroinflammation [153] Piperlongumine N-Acetylcysteine Liver Fibrosis Reduced hepatic senescence, improved liver function [221,222] UBX0101 Senomorphics (IL-1β inhibitors) Osteoarthritis Reduced inflammation and improved joint function [117,223] FOXO4-DRI Metformin Age-Related Sarcopenia Increased muscle regeneration, improved mitochondrial function [224,225] ABT-737 Tocotrienols Alzheimer’s Disease Reduced amyloid plaques, improved cognitive function [226,227] Dasatinib + Quercetin NAD+ Precursors (NR, NMN) Systemic Aging Increased lifespan, improved metabolic function [60,228] HSP90 Inhibitors Resveratrol Cancer-Associated Senescence Reduced tumor progression, enhanced chemotherapy response [95,229] Fisetin Sulforaphane Metabolic Syndrome Improved insulin sensitivity, reduced inflammation [230] BCL-XL Inhibitor (A1331852) Rapamycin Age-Related Cognitive Decline Enhanced synaptic plasticity, reduced neuroinflammation [231,232] Quercetin Omega-3 Fatty Acids Cardiovascular Disease Reduced arterial stiffness, lowered oxidative stress [233,234] Navitoclax Melatonin Hematopoietic Aging Improved stem cell function, reduced DNA damage [235,236] Piperlongumine Pterostilbene Kidney Disease Decreased renal fibrosis, improved kidney function [237,238]","Drug Design, Development and Therapy",Combination Therapies,2025
Clinical Validation of Senescence Biomarkers,"Clinical Validation of Senescence Biomarkers The translation of senescence-targeted therapies into clinical practice is hindered by the lack of fully validated, standardized biomarkers for senescence burden. Commonly used markers such as p16^INK4a, SA-β-galactosidase, and SASP cytokines (eg, IL-6, IL-8) show promise in experimental models but have variable sensitivity and specificity across tissues and disease contexts.1 Their expression can be transient, context-dependent, and influenced by non-senescent cellular states, complicating interpretation in human studies. Furthermore, the invasive nature of current tissue-based assays limits their clinical utility. Advancements in liquid biopsy technologies, molecular imaging, and multi-omics profiling may enable non-invasive, longitudinal tracking of senescence burden, improving patient selection and therapeutic monitoring. However, large-scale, prospective studies are urgently needed to establish reproducibility, predictive validity, and regulatory acceptance of these biomarkers.","Drug Design, Development and Therapy",Clinical Translation,2025
Clinical Trials and Translational Research,"Clinical Trials and Translational Research Although preclinical data are promising, the translation of combination therapies into clinical practice is still in its early stages. A few early-phase clinical trials are exploring the use of senolytics and senomorphics in combination for various aging-related conditions. For instance, a clinical trial is currently investigating the effects of dasatinib and quercetin combined with rapamycin in patients with idiopathic pulmonary fibrosis (IPF), a condition marked by senescence-driven fibrosis and inflammation.239 Preliminary results suggest that this combination can reduce fibrosis and improve lung function, demonstrating the potential of this combined approach to target both cellular senescence and its deleterious effects in humans. Similarly, clinical trials have evaluated the effects of combining NMN with other senolytic compounds in elderly populations and proved improved muscle strength, metabolism, and overall health.240,241 These studies aimed to assess whether the synergistic effects of combining senolytics and senomorphics translate into meaningful clinical outcomes, such as improved mobility, cognitive function, and quality of life.","Drug Design, Development and Therapy",Clinical Translation,2025
Clinical Evidence Tables for Combination Therapies,"Table 6 Clinical Evidence Supporting Combination Therapies of Senolytic and Senomorphic Agents Combination Therapy Mechanism of Action Target Disease/Condition Clinical Evidence References Dasatinib + Quercetin Senolytic: Induces apoptosis in senescent cells Idiopathic Pulmonary Fibrosis (IPF) Improved physical function and reduced senescent cell burden in patients [242] Navitoclax + Quercetin Senolytic: BCL-2/BCL-xL inhibition; Quercetin as an anti-inflammatory agent Osteoarthritis Reduced senescent chondrocytes, improved cartilage integrity [143] Fisetin + Quercetin + Resveratrol Senolytic: Removes senescent cells; Senomorphic: mTOR inhibition reduces inflammation Age-Related Cognitive Decline Improved cognitive function and reduced neuroinflammation [141] Dasatinib + Curcumin Senolytic: Dasatinib-induced apoptosis; Senomorphic: Curcumin inhibits NF-κB Metabolic Syndrome Enhanced insulin sensitivity and reduced inflammatory markers [243] ABT-737 + Metformin Senolytic: BCL-2 inhibitor; Senomorphic: Metformin reduces mitochondrial ROS Type 2 Diabetes and Aging Improved metabolic profiles and reduced inflammation [244] Quercetin + Resveratrol Senolytic: Inhibits survival pathways in senescent cells; Senomorphic: SIRT1 activation Cardiovascular Aging Reduced arterial stiffness and improved endothelial function [245]","Drug Design, Development and Therapy",Clinical Translation,2025
Extended Clinical Evidence and Challenges,"Table 6 (Continued). Fisetin + NAC (N-Acetylcysteine) Senolytic: Induces apoptosis; Senomorphic: Antioxidant protection Neurodegenerative Diseases (Alzheimer’s, Parkinson’s) Decreased neuroinflammation and improved synaptic plasticity [240] Dasatinib + Fisetin Senolytic: Eliminates senescent cells; Senomorphic: Antioxidant and anti-inflammatory effects Osteoarthritis and Frailty Improved mobility and reduced pain in preclinical models [246] Hydrogen Sulfide (H2S) Donors + Metformin Senomorphic: Enhances mitochondrial function and reduces oxidative stress Aging-Associated Sarcopenia Preserved muscle function and improved mitochondrial health [247,248] Navitoclax + Palbociclib Senolytic: Targets BCL-2; Senomorphic: Reduces oxidative stress Breast Cancer-Associated Senescence Increased cancer cell apoptosis and reduced senescent tumor cells [249] Ginsenosides + Quercetin Senolytic: Apoptotic induction in senescent cells; Senomorphic: NF-κB inhibition Metabolic and Cardiovascular Aging Lowered oxidative stress and improved lipid metabolism [250] Bromodomain Inhibitors (JQ1) + Fisetin Senolytic: Epigenetic modulation; Senomorphic: Antioxidant properties Fibrosis and Chronic Lung Diseases Reduced lung fibrosis and improved respiratory function [251,252] Curcumin + Senolytic Peptides Senolytic: Selectively targets senescent cells; Senomorphic: Anti-inflammatory effects Age-Related Macular Degeneration Reduced retinal degeneration and improved visual function.","Drug Design, Development and Therapy",Clinical Translation,2025
Challenges in Clinical Translation,"Clinical Translation and Challenges in the Development of Senolytic and Senomorphic Therapies The clinical translation of senolytic and senomorphic therapies is a promising but challenging endeavor. Addressing safety concerns, developing reliable biomarkers, optimizing dosing regimens, and overcoming regulatory hurdles are key to the success of these therapies in clinical settings. As ongoing clinical trials continue to yield valuable insights, the path toward effective senescence-targeting interventions for healthy aging becomes clearer. The successful development of these therapies could revolutionize aging medicine, offering novel treatments for a range of age-related diseases and enhancing the quality of life for aging populations. Challenges in Clinical Translation While preclinical studies have demonstrated promising results for both senolytic and senomorphic therapies, translating these findings into effective and safe clinical treatments presents several challenges. The path from bench to bedside is often complex and fraught with regulatory, safety, and efficacy concerns. i. Safety and Toxicity: The safety of senolytic and senomorphic therapies is one of the foremost concerns in clinical translation. Although many senolytic agents, such as dasatinib and quercetin, have shown efficacy in preclinical models, their long-term effects in humans are not yet fully understood.","Drug Design, Development and Therapy",Clinical Translation,2025
"Biomarker Challenges, Dosing, and Strategies","The risk of off-target effects, immune system disruption, or toxicity when these agents are used in combination or over extended periods is still a significant challenge.254 For example, the selective removal of senescent cells by senolytics may inadvertently affect healthy cells that share certain senescent-like characteristics or may lead to inflammatory responses in tissues as a result of the clearance process.194 Additionally, long-term inhibition of pathways such as mTOR could have unintended consequences that need to be carefully studied.255 ii. Standardization of Senescence Biomarkers: A major challenge in both the development and application of senescence-targeting therapies is the identification of reliable biomarkers. Current biomarkers such as p16^INK4a, β-galactosidase activity, and SASP factors remain limited in clinical applicability. Heterogeneity across tissues and individuals complicates universal biomarker development.257 iii. Dosing and Timing: Finding optimal dosing schedules for senolytics and senomorphics is complex. Senolytics may require intermittent dosing, whereas senomorphics may be suited for continuous administration.36 Combination timing adds further complexity.258 Strategies for Overcoming Developmental Challenges Strategies include personalized medicine, advanced delivery systems such as nanoparticles and liposomes, and collaborative frameworks between academia, industry, and regulatory agencies to streamline development.260,204,126,119","Drug Design, Development and Therapy",Clinical Translation,2025
Methods,"Methods: A panel of flavonoid polyphenols was screened for senolytic activity using senescent murine and human fibroblasts, driven by oxidative and genotoxic stress, respectively. The top senotherapeutic flavonoid was tested in mice modeling a progeroid syndrome carrying a p16INK4a-luciferase reporter and aged wild-type mice to determine the effects of fisetin on senescence markers, age-related histopathology, disease markers, health span and lifespan. Human adipose tissue explants were used to determine if results translated.",EBioMedicine,Fisetin,2018
Findings,"Findings: Of the 10 flavonoids tested, fisetin was the most potent senolytic. Acute or intermittent treatment of progeroid and old mice with fisetin reduced senescence markers in multiple tissues, consistent with a hit-and-run senolytic mechanism. Fisetin reduced senescence in a subset of cells in murine and human adipose tissue, demonstrating cell-type specificity. Administration of fisetin to wild-type mice late in life restored tissue homeostasis, reduced age-related pathology, and extended median and maximum lifespan.",EBioMedicine,Fisetin,2018
Interpretation and Funding,"Interpretation: The natural product fisetin has senotherapeutic activity in mice and in human tissues. Late life intervention was sufficient to yield a potent health benefit. These characteristics suggest the feasibility to translation to human clinical studies.
Fund: NIH grants P01 AG043376 (PDR, LJN), U19 AG056278 (PDR, LJN, WLL), R24 AG047115 (WLL), R37 AG013925 (JLK), R21 AG047984 (JLK), P30 DK050456 (Adipocyte Subcore, JLK), a Glenn Foundation/American Federation for Aging Research (AFAR) BIG Award (JLK), Glenn/AFAR (LJN, CEB), the Ted Nash Long Life and Noaber Foundations (JLK), the Connor Group (JLK), Robert J. and Theresa W. Ryan (JLK), and a Minnesota Partnership Grant (AMAY-UMN#99)-P004610401–1 (JLK, EAA).
© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.",EBioMedicine,Fisetin,2018
Introduction – Senescence and Aging,"1. Introduction
Pharmacologically targeting fundamental mechanisms of aging is anticipated to reduce the severity or delay the onset of multiple age-associated co-morbidities simultaneously [5–7]. One key mechanism demonstrated to drive aging is cellular senescence, whereby accumulation of DNA damage and/or other cellular stressors [1–4] cause proliferating [8,9] or terminally differentiated non-dividing cells [10–13] to enter a state characterized by profound chromatin and secretome changes, increased expression of the cell cycle inhibitor p16Ink4a, replicative arrest, and resistance to apoptosis [1,14]. Senescent cells can develop a senescence-associated secretory phenotype (SASP), consisting of pro-inflammatory cytokines, chemokines, and extracellular matrix-degrading proteins [15–18], which has deleterious paracrine and systemic effects [19–21]. Indeed, even a relatively low abundance of senescent cells is sufficient to cause tissue dysfunction [22]. Senescent cells are rare in young individuals, but increase with age in multiple tissues, including adipose tissue, skeletal muscle, kidney, and skin of all vertebrates tested [22,23].",EBioMedicine,Fisetin,2018
INK-ATTAC Models and Senolytic Rationale,"The role of senescent cells in age-related decline was identified by studies demonstrating the therapeutic benefits of clearing of senescent cells from progeroid or naturally-aging INK-ATTAC mice using a suicide gene expressed only in p16Ink4a expressing cells (J.L.K., T.T., J.M. van Deursen, and D.J. Baker [all Mayo Clinic] designed the INK-ATTAC strategy [19,20,24–26]). Conversely, injection of senescent cells is sufficient to drive age-related conditions such as osteoarthritis, frailty, and decreased survival [26,27]. Thus, the development of therapies that selectively kill senescent cells was anticipated to delay the onset of aging phenotypes, attenuate severity of age-related diseases, improve resiliency, and enhance survival. Importantly, it was also predicted that senolytic therapies could be administered intermittently, serving to reduce the senescent cell burden by treating quarterly or even annually, which minimizes the risk of side effects [28,29].",EBioMedicine,Fisetin,2018
Previous Senolytic Discoveries – SCAPs and Drug Effects,"We previously identified drugs that selectively kill senescent cells using a hypothesis-driven discovery paradigm [30]. Senescent cells are resistant to apoptosis due to upregulation of Senescent-Cell Anti-Apoptotic Pathways (SCAPs) [28,29]. Targeting SCAPs in cell culture using a combination of dasatinib and quercetin, an inhibitor of BCL-2 pro-survival pathway members, Navitoclax, or the more specific BCLxL inhibitor, A1331852, results in apoptosis of some but not all senescent cell types [30–33]. Treatment of mice with dasatinib plus quercetin (D + Q) improves cardiac ejection fraction and increases vascular reactivity in old mice after a single, 3 day treatment course [30,34]. In addition, D + Q treatment decreases vascular calcification and increases vascular reactivity in hypercholesterolemic, high fat diet fed ApoE−/− mice after three monthly 3 day treatment courses [34]. Intermittent oral D + Q treatment improves pulmonary function and reduces pulmonary fibrosis in a bleomycin-induced murine model of idiopathic pulmonary fibrosis [35], reduces high fat diet-induced liver steatosis [36], alleviates gait impairment caused by leg irradiation [30] and reduces osteoporosis in aged mice [10]. Finally, D + Q also decreases frailty, osteoporosis, loss of intervertebral disc glycosaminoglycans, and spondylosis in the Ercc1−/Δ mouse model of a human progeroid syndrome after intermittent treatment [30].",EBioMedicine,Fisetin,2018
"Navitoclax, A1331852, and Limitations of Existing Senolytics","Similarly, Navitoclax, which decreases abundance of some but not all human and mouse senescent cell types in vitro [33], reduces hematologic dysfunction caused by whole body radiation [31] and reduces senescent cell-like, intimal foam cell/macrophages in vascular plaques in high fat fed LdlR−/− mice [37]. Treatment with A1331852 reduces senescent cholangiocytes and liver fibrosis in Mdr2−/− mice [38]. Taken together, these studies demonstrate that senolytic compounds can have significant effects on chronic degenerative diseases and age-related pathology. However, not all senescent cells are the same. Senescent cells may express different SASP factors, senescence markers, and more importantly use different mechanisms to resist apoptosis [30,39]. Furthermore, certain cancer therapeutics target SCAPs, e.g. Navitoclax, and could be repurposed as senolytics, but cause considerable toxicity including neutropenia and platelet deficiency [40,41]. Thus, new and improved senotherapeutic drugs and combinatorial approaches are needed to eliminate senescent cells safely from multiple organs or even within a single tissue [28–30,42].",EBioMedicine,Fisetin,2018
Fisetin Screening and Senotherapeutic Activity,"Here, we screened a panel of flavonoids for senotherapeutic activity to determine if we could improve upon quercetin. In primary murine embryonic fibroblasts induced to senescence through oxidative stress and in human fibroblasts induced to senescence with the genotoxin etoposide, fisetin was most effective at reducing senescent markers. Fisetin also reduced senescence markers in progeroid Ercc1−/Δ mice and aged WT mice, as well as human explants of adipose tissue. Fisetin treatment extended the health and lifespan in WT mice even when treatment was initiated in aged animals. This flavonoid is a natural compound present in many fruits and vegetables such as apples, persimmon, grapes, onions, cucumbers and strawberries [43,44], suggesting that it is imminently translatable. Importantly, no adverse effects of fisetin have been reported, even when given at high doses [45]. Thus, our results suggest that supplementation or even intermittent treatment with this safe, natural product could improve healthy aging, even in elderly individuals.",EBioMedicine,Fisetin,2018
Research in Context – Evidence Before This Study,"Research in Context
Evidence before this study
Pharmacological targeting of fundamental mechanisms of aging has the ability to reduce the severity or delay the onset of multiple age-associated co-morbidities simultaneously. One key mechanism demonstrated to drive aging is cellular senescence, whereby accumulation of DNA damage and/or other cellular stressors cause proliferating or terminally differentiated non-dividing cells to enter a state characterized by profound chromatin and secretome changes, increased expression of the cell cycle inhibitor p16Ink4a in many but not all senescent cells, replicative arrest, and resistance to apoptosis. Senescent cells can develop a senescence-associated secretory phenotype (SASP), which has deleterious paracrine and systemic effects. Senescent cells are rare in young individuals, but increase with age in multiple tissues. Drugs able to selectively kill senescent cells, termed senolytics, have been identified including the combination of dasatinib and quercetin (D ± Q), which improves many aspects of aging in mouse models of accelerated and natural aging. However, safer and improved drugs targeting senescence likely are needed to eliminate senescent cells safely from multiple organs or even within a single tissue.",EBioMedicine,Fisetin,2018
Added Value of the Study,"Added value of the study
This study identifies the flavonoid polyphenol fisetin as having greater senotherapeutic activity in cultured cells than quercetin. In addition, fisetin had potent senotherapeutic activity in vivo. Treatment of progeroid and aged wild-type mice acutely or intermittently with fisetin reduced senescence markers in multiple tissues and a subset of cell types in adipose tissue. Importantly, chronic administration of fisetin to wild-type mice late in life improved tissue homeostasis, suppressed age-related pathology, and extended median and maximum lifespan. This result, similar to a recent report on the combination of D ± Q, is the first to document extension of both health span and lifespan by a senolytic with few side effects, even though administration was started late in life.",EBioMedicine,Fisetin,2018
Implications of the Available Evidence,"Implications of all the available evidence
Taken together, these data establish the natural product fisetin as a potent senotherapeutic, able to reduce the burden of senescent T, NK, progenitor, and endothelial cells from fat tissue, and demonstrate that reducing the senescent cell burden in mice even late in life is sufficient to have a significant health impact. Given the known safety profile of fisetin in humans, clinical trials are beginning in order to test if fisetin can be used effectively to reduce senescent cell burden and alleviate dysfunction in elderly subjects. M.J. Yousefzadeh et al. / EBioMedicine 36 (2018) 18–28 treatment with this safe, natural product could improve healthy aging, even in elderly individuals.",EBioMedicine,Fisetin,2018
Chemicals,"2. Materials and methods
2.1. Chemicals
Chemicals were from Sigma-Aldrich (St. Louis) unless otherwise noted. The flavonoids were purchased from Selleckchem (Houston, TX): resveratrol (Cat #S1396), fisetin (Cat #S2298), luteolin (Cat #S2320), rutin (Cat #S2350), epigallocatechin gallate (EGCG, Cat #S2250), curcumin (Cat #S1848), pirfenidone (Cat #S2907), and myricetin (Cat #S2326). Apigenin, catechin, and quercetin were purchased from Sigma-Aldrich (Cat #1760595, #1096790 and 1,592,409, respectively).",EBioMedicine,Fisetin,2018
Animal Models and Diets,"2.2. Animals
All animal studies were conducted in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and were approved by the Scripps Florida or Mayo Clinic Institutional Animal Care and Use Committees. Ercc1−/Δ mice were bred as previously described [46]. p16-luciferase reporter mice were obtained from Ohio State University [47] and bred to create an albino C57BL/6 p16Luc/+;Ercc1+/− and FVB/n p16+/Luc;Ercc1+/Δ strain. These mice were further crossed to create f1 p16+/Luc;Ercc1−/Δ mice with white fur for imaging. All animals were genotyped from an ear punch by TransnetYX (Cordova, TN). For diet studies, mice were fed Teklad 2020 chow (Envigo, Madison, WI) prepared with or without 500 ppm (500 mg/kg) of fisetin (Indofine Chemical Co., Hillsborough, NJ) by Envigo. Co. (Tampa, FL). For oral administration of fisetin, mice were dosed with 100 mg/kg of fisetin in 60% Phosal 50 PG:30% PEG400:10% ethanol or vehicle only by gavage. Studies in aged wild-type mice were conducted in both f1 C57BL/6;FVB/n and inbred C57BL/6 genetic backgrounds.",EBioMedicine,Fisetin,2018
MEF Isolation and Culture,"2.3. MEF isolation
The Ercc1−/− MEFs were isolated from pregnant females at embryonic day 13 (E13) and cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F10 with 10% fetal bovine serum, 1× nonessential amino acids, penicillin, and streptomycin and incubated at 3% O2 initially, followed by a shift to 20% for 5 passages to induce senescence [48]. Cells were genotyped by TransnetYX (Cordova, TN) and routinely tested for mycoplasma contamination using the MycoAlert PLUS mycoplasma detection kit (Lonza, Walkersville, MD).",EBioMedicine,Fisetin,2018
Senotherapeutic Screening Assays,"2.4. Assays to identify senotherapeutics
Ercc1−/− MEFs were passaged 5 times at 20% O2 to induce senescence then seeded at 5000 cells per well in 96 well plates at least 6 h prior to treatment. Following the addition of drugs, the MEFs were incubated for 24–48 h at 20% O2. Subsequently SA-β-gal activity was measured in three independent experiments, as previously described [49]. Briefly, cells were washed with PBS then 10 μM C12FDG added in fresh culture medium and incubated for 2 h. Ten min prior to analysis, 2 μg/mL Hoechst dye was added. An IN Cell Analyzer 6000 was used to quantitate total number of viable cells (Hoechst+) and the number of senescent cells (C12FDG+). All samples were analyzed in duplicate with 3–5 fields per well and reported as the mean ± S.D. Senotherapeutic activity was confirmed in human fibroblasts (IMR90). The cells were obtained from American Type Culture Collection (ATCC) and cultured in EMEM medium with 10% FBS and antibiotics. To induce senescence, the cells were treated for 24 h with 20 μM etoposide. Two days after etoposide removal, ~70% of the cells were SA-β-gal+. Cells were treated for 48 h with different concentrations of fisetin (1–15 μM) and the percentage of SA-β-gal+ cells was determined using C12FDG, as described above.",EBioMedicine,Fisetin,2018
Luciferase Imaging,"2.5. IVIS in vivo imaging detection of luciferase activity
Isoflurane-anesthetized mice (n = 2–10 mice per group) were injected intraperitoneally with 10 μL per gram body weight D-luciferin substrate (Caliper Life Sciences, Hopkinton, MA; 15 mg/mL diluted in sterile PBS) and were imaged using an IVIS Lumina (PerkinElmer, Billerica, MA), as previously described [47,50].",EBioMedicine,Fisetin,2018
Lipid Peroxidation and Glutathione,"2.6. Measurement of lipid peroxidation
Levels of 4-hydroxynonenal-protein adducts of liver lysates (n = 4–6 mice per group) prepared in RIPA buffer were measured in the livers of mice using the OxiSelect HNE Adduct Competitive ELISA kit (Cell Biolabs, San Diego, CA), as described [50].
2.7. Measurement of glutathione
Murine livers (n = 4–7 mice per group) fixed in 5% sulfosalicylic acid were prepared and analyzed for the concentration of reduced (GSH) and oxidized (GSSG) glutathione using the Glutathione Assay Kit (Cayman Chemical, Ann Arbor, MI), as described [50]. Sample absorbance was measured at 405 nm using a plate reader and the ratio of GSH:GSSG was reported for each sample.",EBioMedicine,Fisetin,2018
Clinical Chemistries and Serum MCP-1,"2.8. Clinical chemistries
Whole blood (n = 3–6 mice per group) was collected immediately following animal euthanasia via cardiac puncture into heparinized tubes for analysis of clinical chemistries utilizing VetScan Comprehensive Diagnostic Profile rotors on a VestScan VS2 (Abaxis, Union City, CA).
2.9. Serum MCP-1
Serum concentrations of MCP-1 (n = 5 mice per group) were measured using a mouse-specific MCP-1 ELISA (Raybiotech, Norcross, GA), as described [51].",EBioMedicine,Fisetin,2018
Histopathology Scoring,"2.10. Histopathology
Mouse tissues were collected at necropsy (n = 3–8 mice per group) and placed in 10% neutral buffered formalin for 48 h, transferred to 70% alcohol, and subsequently processed into paraffin blocks for sectioning and hematoxylin and eosin staining. Tissue sections were scored for the presence and severity of a well-defined panel of age-related lesions by a veterinary pathologist to create a composite tissue lesion score for each animal that reflects healthspan, as previously described [52].",EBioMedicine,Fisetin,2018
RNA Isolation and qPCR,"2.11. RNA isolation and qPCR
Tissues were harvested from mice (n = 4–10 mice per group) and snap frozen in liquid nitrogen. Total RNA was harvested from tissues and the expression of several markers of senescence was measured, as previously described in [51]. Total RNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher, Waltham, MA) and 1 μg of total RNA was used to generate cDNA using the Transcriptor First Strand cDNA synthesis kit (Roche, Basel, Switzerland). Gene expression changes were carried out in 20 μL reactions using the Universal SYBR Green master mix with ROX (Roche) and a StepOne thermocycler (Thermo Fisher). Primers for the genes of interest are as follows: Cdkn1a (p21Cip1) Fwd 5’-GTCAGGCTGGTCTGCCTCCG-3′, Cdkn1a (p21Cip1) Rev. 5’-CGGTCCCGTGGACAGTGAGCAG-3′; Cdkn2a (p16Ink4a) Fwd 5′- CCCAACGCCCCGAACT-3′, Cdkn2a (p16Ink4a) Rev. 5′- GCAGAA GAGCTGCTACGTGAA-3′; Gapdh Fwd 5’-AAGGTCATCCCAGAGCTGAA-3′, Gapdh Rev. 5’-CTGCTTCACCACCTTCTTGA-3′; Il6 Fwd 5’-CTGGGAAATCGTGGAAT-3′, Il6 Rev. 5’-CCAGTTTGGTAGCATCCATC-3′; Mcp1 Fwd 5’-GCATCCACGTGTTGGCTCA-3′, Mcp1 Rev. 5’-CTCCAGCCTACTCATTGGGATCA-3′. Data were analyzed by ΔΔCt method and gene expression was normalized to Gapdh.",EBioMedicine,Fisetin,2018
Isolation of CD3+ T Cells,"2.12. Isolation of peripheral blood CD3+ T lymphocytes
Blood was obtained from mice (n = 4–10 mice per group) postmortem by cardiac puncture, immediately placed into 1/10th volume of 0.5 M EDTA, and gently mixed to prevent coagulation. Samples were centrifuged at 300 g for 10 min in a table top centrifuge. The supernatant was discarded and the cell pellet was resuspended in 1 mL ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4) and incubated at room temperature for 10 min to lyse red blood cells. The cells were spun down and ACK lysis repeated. The cells were spun down, washed with PBS, and resuspended in PBS containing 0.5% FBS and 2 mM EDTA. Fifty μL of CD3-Biotin conjugate (Miltenyi Biotech, San Diego, CA) were added to the cell suspension and incubated for 30 min on ice. The cells were centrifuged at 100 g for 10 min and washed twice in resuspension buffer. The cell pellet was resuspended in 500 μL of resuspension buffer and 100 μL of anti-biotin microbeads were added followed by a 15 min incubation on ice. The cells were washed twice and then resuspended in 500 μL of resuspension buffer and applied to a MACS column attached to a magnet. The cells were washed with 3 column volumes of resuspension buffer before elution. The cells were centrifuged and RNA isolation conducted using an RNeasy kit (Qiagen, Germantown, MD) according to the manufacturer's specifications. qPCR analysis of senescence markers was performed as indicated above.",EBioMedicine,Fisetin,2018
SA-β-gal Staining,"2.13. Senescence-associated β-galactosidase (SA-β-gal) staining of tissue
Fresh fat tissues (n = 6–7 mice per group) were fixed and stained to detect senescence-associated β-galactosidase activity, as described [53].",EBioMedicine,Fisetin,2018
Mass Cytometry of Adipose Tissue,"2.14. Mass cytometry/CyTOF in adipose tissue
This high dimensional single-cell proteomics technique combines time-of-flight mass spectrometry with metal-labelling technology to detect up to 40 protein targets per cell [54,55]. A panel of antibodies based on cell surface markers and transcription factors (see Supplemental Table 1) was designed for CyTOF analysis of adipose tissues. Each antibody was tagged with a rare metal isotope and its function verified by mass cytometry according to the factory manual (Multi Metal labeling Kits, Fluidigm, CA). A CyTOF-2 mass cytometer (Fluidigm, South San Francisco, CA) was used for data acquisition. Acquired data were normalized based on normalization beads (Ce140, Eu151, Eu153, Ho165, and Lu175). One gram of subcutaneous fat tissue (n = 6–9 mice per group) was dissociated into a single-cell suspension using an adipose tissue dissociation kit (Adult Adipose Tissue Dissociation Kit, Miltenyi Biotec Inc.CA). Collected cells were incubated with metal-conjugated antibodies for cell surface markers and intracellular proteins. Fixation and permeabilization were conducted according to the manufacturer's instructions (Transcription Factor Staining Buffer Set, eBioscience, San Diego, CA). CyTOF data were analyzed by Cytobank (Santa Clara, CA).",EBioMedicine,Fisetin,2018
Human Adipose Tissue Explants,"2.15. Human adipose tissue explants
The protocol was approved by the Mayo Clinic Foundation Institutional Review Board for Human Research. Informed consent was obtained from all subjects (n = 3). Human greater omental adipose tissue was resected during surgery from 2 lean (BMI 25.5 and 26.2) and 1 obese (BMI 45.6) female subjects, ages ranging from 55 to 66 years. No subject was known to have a malignancy. The adipose tissue was cut into small pieces and washed with PBS 3 times. The adipose tissue was then cultured in medium containing 1 mM sodium pyruvate, 2 mM glutamine, MEM vitamins, MEM non-essential amino acids, and antibiotics with 20 μM of fisetin or DMSO. After 48 h, the adipose explants were washed 3 times with PBS and was then maintained in the same media without drugs for 24 h to collect conditioned medium (CM) for multiplex protein analysis. The adipose explants then were fixed and stained to detect senescence-associated β-galactosidase activity [85].",EBioMedicine,Fisetin,2018
Multiplex Protein Analysis,"2.16. Multiplex protein analyses
Pro-inflammatory cytokine and chemokine protein levels in CM from the adipose tissue explants (n = 3) were measured using Luminex xMAP technology. The multiplexing analysis was performed using the Luminex™ 100 system (Luminex, Austin, TX) by Eve Technologies Corp. (Calgary, Alberta, Canada). Human multiplex kits were from Millipore (Billerica, MA). The secreted protein levels in CM were normalized to the tissue weights and plotted as a percent relative to the vehicle control.",EBioMedicine,Fisetin,2018
Fisetin Identified as Potent Senotherapeutic In Vitro,"3. Results
We previously demonstrated that the flavonoid quercetin, an antioxidant, which also targets PI3 kinase delta as well as certain BCL-2 family members, reduces senescence in primary human umbilical vein endothelial cells (HUVECs) and murine embryonic fibroblasts (MEFs), especially when used in combination with the tyrosine kinase inhibitor dasatinib [30]. To determine if other flavonoids might have more potent senotherapeutic activity than quercetin, a panel of flavonoids was screened for effects on senescence induced by oxidative stress [49]. Primary MEFs from Ercc1−/− mice were used. These cells undergo premature senescence if grown at atmospheric oxygen [56]. Ercc1−/− MEF cultures were established at 3% O2 then shifted to 20% O2 for three passages to induce senescence. To quantify senescent cells, SA-ß-gal activity was measured using the fluorescent substrate C12FDG [57] using an IN Cell Analyzer 6000 confocal imager. At a dose of 5 μM, fisetin was most effective in reducing the fraction of SA-ß-gal positive MEFs (Fig. 1A). Luteolin and curcumin also showed weak activity at a dose where quercetin was ineffective. In addition, fisetin reduced senescence in MEFs and IMR90 cells in a dose-dependent manner (Fig. 1B and C). These results are consistent with our previous finding that fisetin selectively reduces the viability of senescent HUVECs without affecting proliferating cells [32]. In HUVECs, fisetin induces apoptosis as measured by caspase3/7 activity, whereas in MEFs, fisetin suppressed markers of senescence without evidence of cell killing [32].",EBioMedicine,Fisetin,2018
Fisetin Reduces Senescence In Vivo in Progeroid Mice,"To test the senotherapeutic activity of fisetin in vivo, initially progeroid Ercc1−/Δ mice carrying a p16Ink4a-luciferase reporter transgene were used [47,50]. These mice show accelerated accumulation of senescent cells compared to WT mice, but the overall level of senescence never exceeds that of naturally aged mice [50]. Ercc1−/Δ; p16Ink4a-luciferase mice were fed a standard Teklad 2020 chow diet with or without supplementation with 500 ppm (500 mg/kg) of fisetin, ad libitum (approximately 60 mg/kg fisetin per day). The mice were exposed to a fisetin diet intermittently from 6 to 8 then 12–14 wks of age. Whole body luciferase activity was measured before starting the fisetin diet then weekly thereafter. Animals in the two treatment groups had an equivalent luciferase signal prior to administration of the experimental diet. Dietary fisetin suppressed the luciferase signal of Ercc1−/Δ; p16Ink4a-luciferase mice significantly (Fig. 2A-B). The luciferase signal was lower at every time point after initiation of the fisetin diet (Fig. 2B-C). Notably, the level of p16Ink4a expression remained significantly lower in the fisetin-treated mice throughout the 4 week period when the animals were not exposed to fisetin (8–12 wks of age, Fig. 2B). This is consistent with a mechanism of action where senescent cells are cleared (senolytic) or senescence is reversed (senomorphic) but not a mechanism in which fisetin must be chronically present to suppress senescence.",EBioMedicine,Fisetin,2018
Reduction of Senescence and Oxidative Stress Markers,"To validate the imaging data, Ercc1−/Δ mice were treated with the 500 ppm fisetin diet for 10 wks beginning at 10 wks of age, then tissues collected for measurement of multiple markers of senescence including p16Ink4a and p21Cip1 and SASP factors. Expression of p21Cip1 was included since not all senescent cells are p16Ink4a positive: some are p21Cip1 positive, but p16Ink4a negative. As shown in Fig. 3A-D, p16Ink4a and p21Cip1 mRNA, as well as SASP markers, were significantly elevated in the fat, spleen, liver, and kidney of Ercc1−/Δ mice compared to age-matched WT mice. Fisetin reduced expression of senescence and SASP markers significantly in all tissues. Similarly, there was a reduction in the expression of p16Ink4a, p21Cip1 and the SASP factors in peripheral blood CD3+ T cells (Fig. 3E), a cell type that demonstrates a robust increase in p16INK4a expression as humans age [58]. In addition, fisetin reduced oxidative stress in the liver as determined by measuring the lipid peroxidation product 4-hydroxynonenal (HNE) adducts and an increase in the ratio of reduced to oxidized glutathione (Fig. 3F-G), consistent with data indicating fisetin has antioxidant activity as well as increasing intracellular glutathione [45].",EBioMedicine,Fisetin,2018
Fisetin Clears Senescent Cells in Aged Mice,"To confirm further the data obtained in progeroid mice, we employed naturally aged C57BL/6 mice and different methods of detecting senescence in tissue. 22–24-month-old mice were treated with 100 mg/kg fisetin for 5 consecutive days by oral gavage, or vehicle only. Mice were sacrificed 3 days after the last dose and the number of SA-ß-gal+ cells present in inguinal fat was determined by staining tissue sections to measure SA-β-gal activity. Fat tissue was chosen for the analysis since there is a clear upregulation of senescence markers including SASP in our mouse models, the tissue has a significant increase in the fraction of senescent cells including senescent immune cells, such as T and endothelial cells and macrophages, and injection of senescent pre-adipocytes is sufficient to induce frailty in young mice [26,50,59,60]. Short-term treatment with fisetin significantly reduced the fraction of senescent cells in white adipose tissue (WAT). To determine which cells become senescent in WAT and which cell types are cleared by fisetin, CyTOF analysis was performed on subcutaneous adipose tissue from aged INK-ATTAC mice expressing a Flag-tagged FKBPCasp8 protein from the p16Ink4a promoter (Fig. 4B). The Flag tag enabled identification of senescent (p16Ink4a-expressing) cells using an anti-Flag antibody. CyTOF analysis revealed a significantly elevated fraction of senescent cells in fat from old mice compared to young and identified these cells as mesenchymal stem/progenitor cells, T lymphocytes, natural killer cells, and endothelial cells (Fig. 4C). The short-course treatment with fisetin resulted in a significant reduction in the fraction of senescent cells in each of these populations (Fig. 4C).",EBioMedicine,Fisetin,2018
Lineage-Specific Reduction of Senescent Cells,"Fisetin reduced the fraction of p16Ink4a-expressing, c-Kit+ stem/progenitor cells, CD4+ and CD8+ T cells, NK-1.1+ NK cells, and CD146+CD31+ endothelial cells (Fig. 4C). These data are also shown in Spanning-tree Progression Analysis of Density-normalized Events (SPADE) analysis (Supplemental Fig. 1). To confirm senescence in these cell populations, CENP-B protein was measured by CyTOF (Fig. 4D). CENP-B binds centromeric satellite DNA [61], which becomes distended in senescent cells. The fraction of CENP-B+ cells in WAT was significantly increased in old mice compared to young and suppressed by treating the mice with a short-course of fisetin, in the same manner as p16Ink4a-expressing/FLAG+ cells. In contrast, p21Cip1 (another cell-cycle regulator that is often up-regulated in senescent cells) expression was not significantly elevated in these cell populations (Supplemental Fig. 2). While FLAG+ dendritic cells and macrophages were increased in WAT from old mice consistent with a previous report [62], fisetin treatment had no substantial effect on the fraction of macrophages or dendritic cells with high p16Ink4a, CENP-B, or p21Cip1 (Supplemental Fig. 3). Taken together, these data demonstrate that a short-course of fisetin reduces the number of p16Ink4a-expressing cells in subcutaneous WAT including mesenchymal stem/progenitor, immune, and endothelial cells. This is the first time a senotherapeutic has been demonstrated to differentially affect senescent cells of different lineages in vivo.",EBioMedicine,Fisetin,2018
Fisetin Reduces Senescence in Human Adipose Tissue,"To determine if fisetin also reduces senescence in human adipose tissue, greater omental adipose explants resected during surgery were treated with fisetin ex vivo. The tissue explants were treated for 48 h with 20 μM fisetin, washed, and cultured for an additional 24 h before measuring SASP factors by multiplex protein analysis [26]. Fisetin treatment caused a significant reduction in the percent of SA-ß-gal positive cells (Fig. 4E) as well as in expression of the SASP factors IL-6, IL-8, and MCP-1 in human WAT (Fig. 4F). These data support the translational potential of fisetin to reduce senescent cell burden and associated inflammation.",EBioMedicine,Fisetin,2018
Fisetin Extends Lifespan and Improves Pathology in Aged Mice,"To determine if fisetin-mediated clearance of senescent cells impacts the health or lifespan of mice, WT f1 C57BL/6:FVB mice were fed a diet containing 500 ppm fisetin beginning at 85 wks of age, roughly equivalent to age 75 years in humans. This resulted in an extension of median as well as maximal lifespan (Fig. 5A-B). Amylase and alanine aminotransferase (ALT) were significantly lower in serum of aged WT mice fed the diet supplemented with fisetin, consistent with improved pancreatic and liver homeostasis (Fig. 5C). Brain, kidney, liver, lung, and forepaw tissue sections were stained with hematoxylin and eosin and evaluated by a veterinary pathologist. Using the Geropathology Grading Platform to score age-related lesions [63], several tissues had reduced age-related pathology in the fisetin diet group compared to the control diet (Fig. 5D). An example of this is illustrated in a representative image from renal sections in Fig. 5E. Similar to the progeroid mice, fisetin reduced the expression of senescence and SASP markers in multiple tissues of aged WT mice exposed to oral fisetin (Fig. 5F-I). Furthermore, there was a reduction in senescence and SASP factor expression in peripheral CD3+ T cells (Fig. 5J). There was also a reduction in levels of circulating MCP-1 (Fig. 5K), a SASP factor [51]. Finally, fisetin reduced oxidative stress in the liver of old WT mice (Fig. 5L-M).",EBioMedicine,Fisetin,2018
Introduction to Aging and Cellular Senescence,"Aging is defined as a physiological dysfunction in an organism and is closely related to many chronic human diseases, including metabolic syndrome, cancer, and neurodegenerative diseases (Campisi, 2013; Munoz-Espin and Serrano, 2014; Hoang et al., 2021). Senescent cells generally accumulate in various tissues during aging. Cellular senescence is a heterogeneous process characterized by genetic, epigenetic, and environmental factors (van Deursen, 2014; Lagoumtzi and Chondrogianni, 2021). Senescence occurs in response to various stimuli, such as telomere shortening, DNA damage by radiation, oncogene activation, mitochondrial dysfunction, and endoplasmic reticulum stress. Senescence is classified as replicative senescence and stress-induced premature senescence. Replicative senescence, known as the Hayflick limit, was first found in human primary fibroblasts to stop dividing after many divisions. This type of cellular senescence is involved in the shortening of telomeric DNA. Stress-induced premature senescence is caused by various intrinsic and extrinsic factors such as oxidative stress, ionizing radiation, genotoxic stress, oncogenic transformation, and various pathogens (Coppe et al., 2010; Lopez-Otin et al., 2013; Kennedy et al., 2014; Munoz-Espin and Serrano, 2014; He and Sharpless, 2017; Borghesan et al., 2020).",Biomolecules & Therapeutics,Senescence,2022
Dual Roles of Cellular Senescence,"Cellular senescence has both beneficial and harmful effects on neighboring cells. The senescence process is fundamental for avoiding uncontrolled cellular replication. The accumulation of senescent cells during the aging process is critical for the tumor suppressor mechanism, which is a state of cell cycle arrest that suppresses abnormal proliferation of damaged cells and oncogenic development (Coppe et al., 2010; Munoz-Espin and Serrano, 2014). However, many recent studies have also demonstrated that cellular senescence can cause various deleterious processes, such as chronic inflammation, immune surveillance, and organismal aging (Lopez-Otin et al., 2013; Munoz-Espin and Serrano, 2014; He and Sharpless, 2017). Despite growth inhibition, senescent cells are metabolically active and produce a senescence-associated secretory phenotype (SASP) that contains pro-inflammatory cytokines, bioactive lipids, and other factors that mediate the autocrine/paracrine effects of senescent cells (Childs et al., 2017; Birch and Gil, 2020). In addition, these factors can induce chronic inflammation and age-associated disorders (Childs et al., 2017; He and Sharpless, 2017).",Biomolecules & Therapeutics,Senescence,2022
Characteristics of Senescent Cells,"These factors stimulate senescence-associated signaling pathways and result in cell cycle arrest, increase SA-β-galactosidase activity, and affect neighboring young cells in a paracrine manner (Bang et al., 2019). Senescent cells respond less to external signals, such as growth factors, abnormal structures, and mitochondrial dysfunction. Senescent cells are enlarged, vacuolated, and flattened. In addition, the lack of lamin A/B and accumulation of lipofuscin have been observed in cellular senescence (Kirkland and Tchkonia, 2017; Hernandez-Segura et al., 2018; Gorgoulis et al., 2019). Resistance to apoptosis is another hallmark of cellular senescence (Baar et al., 2017; Childs et al., 2017; Demaria et al., 2017). Apoptosis plays a key role in the clearance of both damaged and cancerous cells. Apoptosis is regulated by pro-apoptotic and anti-apoptotic proteins, such as the B-cell lymphoma 2 (Bcl-2) family, caspase, and death receptors. Enhanced reactive oxygen species (ROS) levels, another characteristic of senescent cells, accelerate chronic inflammation and autoimmunity, which are highly related to age-related diseases, such as metabolic syndrome, cognitive decline, and frailty (Munoz-Espin and Serrano, 2014; Hernandez-Segura et al., 2018).",Biomolecules & Therapeutics,Senescence,2022
Development of Senotherapeutics,"Much research effort has recently been made to therapeutically target the harmful effects of cellular senescence (Baker et al., 2011, 2016; Childs et al., 2017; Krimpenfort and Berns, 2017). The targeting of senescent cells by several pharmacological interventions, known as senotherapeutics, has been reported to ameliorate many senescence-associated diseases and delay the development of age-related disorders. Senotherapeutics can be classified into three development strategies. First, senolytics selectively eliminate senescent cells. Second, senomorphics induce senescent cells to obtain the functions and morphology of young cells or delay the progression of young cells to senescent cells. Third, senescence-targeting immunotherapeutics mediate the clearance of senescent cells. Some senolytics and senomorphics have been reported to prevent or treat age-related diseases in animal models (Baar et al., 2017; Childs et al., 2017; Kirkland and Tchkonia, 2017; Borghesan et al., 2020). Therefore, this review provides insights into the development of various senotherapeutics for improving age-related diseases and eventually increasing the health span.",Biomolecules & Therapeutics,Senotherapeutics,2022
Senescence in Aging and Chronic Inflammation,"Senescent cells accumulate because of either continuous stress stimuli or a reduction in immune function during the aging process. This phenomenon causes chronic inflammation through uncontrolled secretion of SASP (He and Sharpless, 2017; Hernandez-Segura et al., 2018; Gorgoulis et al., 2019). There are increased levels of interleukin-1A (IL-1A) and interleukin-6 (IL-6), one of several SASP factors, during aging. Suppression of the inflammatory key protein, nuclear factor-kappa B (NF-κB), can prevent DNA damage-induced senescence in mice (Salminen et al., 2012). SASP leads to pathological angiogenesis in a retinopathy mouse model and contributes to atherosclerosis progression (Oubaha et al., 2016; Ferrucci and Fabbri, 2018). Conversely, selective ablation of senescent cells ameliorates some age-associated symptoms and extends the health span of mice (Baker et al., 2011; Krimpenfort and Berns, 2017; van Deursen, 2019).",Biomolecules & Therapeutics,Senescence and Aging,2022
INK-ATTAC Model and Senescent Cell Clearance,"Baker et al. (2011) first revealed a direct relationship between cellular senescence and age-related symptoms. They designed a novel transgene using a senescence biomarker, p16Ink4a and generated an INK-ATTAC (apoptosis through targeted activation of caspase) transgenic mouse model in the BubR1 progeroid mouse background for inducible elimination of p16Ink4a-positive senescent cells upon the administration of AP20187. AP20187, an inducer of dimerization of the FK506 binding protein-caspase 8 fusion protein, was used to trigger senolysis. This approach delayed the progression of p16Ink4a-mediated age-related symptoms in adipose tissue and muscle, implying that cellular senescence is implicated in the generation of age-related disorders and that the elimination of senescent cells can prevent tissue dysfunction and extend the health span. Baker et al. (2016) further demonstrated a direct association between cellular senescence and age-related disorders. They revealed that the treatment of AP20187 eliminated p16Ink4a-positive senescent cells in INK-ATTAC transgenic mice with two different genetic backgrounds (C57BL/6 and mixed) and led to enhanced median health span in both mice.",Biomolecules & Therapeutics,Senotherapeutics,2022
Organ-Level Benefits of Senescent Cell Removal,"These data implied that AP20187 diminished age-related abnormal function and structural destruction of various organs, such as the adipose tissue, kidney, and heart. Several studies have demonstrated that the genetic clearance of p16Ink4a-positive senescent cells in INK-ATTAC transgenic mice ameliorated the symptoms of lipodystrophy (Xu et al., 2015a), hepatic steatosis (Ogrodnik et al., 2017), cardiac dysfunction (Lewis-McDougall et al., 2019), and cerebral disorders (Ogrodnik et al., 2021). In previous studies, p16Ink4a-positive senescent macrophages in the p16Ink4a-trimodality reporter (p16-3MR) transgenic mice were cleared by treatment with ganciclovir, which contributed to diminishing the formation of atherosclerotic plaques in low-density lipoprotein receptor-deficient (LDLR−/−) mice (Childs et al., 2016) and osteoarthritis model mice (Jeon et al., 2017). Conversely, transplantation of senescent ear fibroblasts into the knee caused osteoarthritis in a mouse model (Xu et al., 2017).",Biomolecules & Therapeutics,Senolytics,2022
Senescent Cell Transplantation and Health Span Effects,"Transplanting a small number of senescent cells into young mice was also sufficient to induce physical dysfunction, eventually leading to reduced survival (Xu et al., 2018). These results suggest that senotherapeutics can either prevent tissue dysfunction or contribute to extending the health span of aged models.",Biomolecules & Therapeutics,Senescence and Health Span,2022
Overview of Senolytics Strategy,"Chronic/periodic administration of senolytics eliminates senescent cells present in aged tissues, and the immune response contributes to discarding apoptotic bodies for subsequent regeneration. Senolytics target signaling pathways that are activated in senescent cells and specifically kill chronically senescent cells (Baar et al., 2017; Kirkland and Tchkonia, 2017). These senolytic compounds are extensive and are continuously found (Table 1, Fig. 1). Various senolytics, such as Bcl-2 family inhibitors, histone deacetylase (HDAC) inhibitors, forkhead box protein O4 (FOXO4), p53 binding inhibitor, and heat shock protein 90 (HSP90) inhibitors, have been identified (Zhu et al., 2015, 2016; Baar et al., 2017; Jeon et al., 2017; Samaraweera et al., 2017; Xu et al., 2018). Senolytics were first identified as combinations of quercetin and dasatinib (Zhu et al., 2015). Zhu et al. (2015) examined the gene expression profiles of young and senescent cells. They found that senescent cells increased the expression levels of anti-apoptotic biomarkers such as BCL-2 family members and the phosphoinositide 3-kinase (PI3K)/Akt pathway. In addition, they screened 46 candidate drugs having the ability to preferentially kill senescent cells in vitro (Zhu et al., 2015).",Biomolecules & Therapeutics,Senolytics,2022
Dasatinib and Quercetin as First-Generation Senolytics,"They found that the combination of dasatinib and quercetin efficiently accelerated apoptosis of senescent cells in naturally aged, irradiated, and even Ercc1−/Δ-progeroid mice and led to an extended lifespan and decreased age-associated symptoms. Dasatinib, a protein tyrosine kinase inhibitor used clinically for cancer therapy, effectively eliminated senescent preadipocytes. Quercetin, a natural plant flavonoid that targets the BCL-2 and PI3K/AKT pathways, ablated senescent endothelial cells and mouse bone marrow-derived mesenchymal stem cells (Zhu et al., 2015). In addition, transplantation of senescent cells into healthy mice causes physical abnormalities, which could be antagonized by an oral combination of dasatinib and quercetin (Xu et al., 2018). These senolytic effects of dasatinib and quercetin have also been demonstrated in various in vivo models, where intervention treatment ameliorated the symptoms of several age-related diseases, such as physical dysfunction, hepatic steatosis, insulin resistance, neurodegeneration, and skeletal muscle dysfunction (Xu et al., 2018; Wissler Gerdes et al., 2020).",Biomolecules & Therapeutics,Dasatinib and Quercetin,2022
Clinical Trials Involving Dasatinib and Quercetin,"The combination of dasatinib and quercetin is currently in progress in human clinical trials for idiopathic pulmonary fibrosis (NCT02874989), Alzheimer’s disease (NCT04685590), frailty (NCT04733534), chronic kidney disease (NCT02848131), hematopoietic stem cell transplant survivors (NCT02652052), skeletal health (NCT04313634), and adult cancer survivors (NCT04733534). Upregulation of Bcl-2 and Bcl-xL contributes to the resistance of senescent cells to apoptosis (Chang et al., 2016; Zhu et al., 2016), implying that inhibitors of these anti-apoptotic proteins can be effective candidates for senolytics. ABT-263 (known as Navitoclax), ABT-737, A1331852, and A1155463, which inhibit BCL-2 family members, were identified as candidate senolytics in vitro and in vivo animal models (Chang et al., 2016; Yosef et al., 2016; Zhu et al., 2016, 2017). ABT-263 eliminated senescent IMR-90 human lung fibroblasts and human umbilical vein epithelial cells (HUVECs) (Zhu et al., 2016).",Biomolecules & Therapeutics,Senolytics Clinical Trials,2022
BCL-2 Family Inhibitors as Senolytics,"ABT-263 also rejuvenated aged hematopoietic stem cells and muscle stem cells by clearing senescent cells (Chang et al., 2016). By binding the inhibitory domain of anti-apoptotic Bcl2 and Bcl-xL, ABT-263 specifically eliminated both senescent muscle stem cells (MuSCs) and senescent bone marrow hematopoietic stem cells (HSCs) and eventually contributed to the amelioration or rejuvenation of MuSCs and HSCs in aged mice (Miura et al., 2022). In addition, ABT-263 cleared senescent foam cell macrophages in atherosclerotic lesions, preventing atherosclerosis progression in LDLR−/− mice (Garrido et al., 2022). However, this compound caused trabecular bone loss and damages the function of osteoprogenitors in aged mice (Sharma et al., 2020). ABT-737 specifically eliminated etoposide-induced senescence in IMR-90 fibroblasts. ABT-737 treatment also efficiently eliminated senescent lung epithelial cells in irradiated mice and senescent epidermal cells in p14ARF transgenic mice. Furthermore, it stimulated hair follicle stem cell proliferation (Yosef et al., 2016). Both A1331852 and A1155463, selective Bcl-xL inhibitors, preferentially stimulated the clearance of irradiation-induced senescent HUVECs and IMR-90 fibroblasts (Zhu et al., 2017).",Biomolecules & Therapeutics,BCL-2 Senolytics,2022
HDAC and FOXO4 Inhibitors as Senolytics,"Panobinostat, an HDAC inhibitor approved by the FDA, was found to possess senolytic activity in the chemotherapy-induced senescence of squamous cell carcinoma cell lines and non-small cell lung cancer (Samaraweera et al., 2017). The senolytic effect of panobinostat was related to the upregulation of histone H3 acetylation and downregulation of BCL-XL expression. The potential application of senolytics targeting these senescent cancer cells can be a novel strategy for improving cancer metastasis and stemness. FOXO4 is an important protein that is involved with the viability of senescent cells (Baar et al., 2017; Krimpenfort and Berns, 2017; Zhang et al., 2020). A previous study demonstrated that FOXO4-induced nuclear localization of p53 repressed its association with the mitochondrial apoptotic pathway (Baar et al., 2017). They designed a FOXO4 inhibitor peptide (FOXO4-DRI peptide) to mimic the binding surface of both FOXO4 and p53 for disrupting their interaction. This disruption induced the translocation of p53 to the cytosol and caused caspase-3/7-dependent apoptosis in senescent IMR-90 fibroblasts and HUVECs.",Biomolecules & Therapeutics,FOXO4 and HDAC Senolytics,2022
FOXO4-DRI and MDM2/p53 Inhibition,"When administered in vivo, FOXO4-DRI improved hair density and renal dysfunction in both naturally aged mice and fast-aging XpdTTD/TTD-progeroid (Baar et al., 2017). In addition, another study demonstrated that FOXO4-DRI ameliorated age-related progression of hypogonadism in aged mice by inducing apoptotic cell death of senescent Leydig cells, known as interstitial cells, in the testes (Zhang et al., 2020). These findings suggest that it is possible to regulate cellular senescence by targeting the mutual interaction between FOXO4-p53. Murine double minute 2 (MDM2) E3 ligase, a major negative regulator of p53, promotes proteasome-dependent degradation of p53 (Fu et al., 2009). UBX0101, an inhibitor of the MDM2/p53 protein interaction, was known to be a senolytic candidate (Jeon et al., 2017). Intra-articular injection of UBX0101 ablated senescent cells in the articular cartilage and synovium. This administration led to attenuation of the onset of post-traumatic osteoarthritis in an osteoarthritis mouse model (Jeon et al., 2017).",Biomolecules & Therapeutics,FOXO4 and MDM2 Senolytics,2022
HSP90 Inhibitors and Senolytic Activity,"UBX0101 also eliminated senescent cells by inducing apoptosis and contributed to the ability of chondrocytes to form cartilage in osteoarthritis tissues (Jeon et al., 2017). UBX0101 was the first senolytic drug in phase 1 clinical trial to treat patients suffering from osteoarthritis. However, intra-articular injection of UBX0101 for treating osteoarthritis failed to meet its clinical requirements in phase 2 clinical trials. HSP90 is an ATP-dependent chaperone that is involved in the folding, stabilization, and degradation of various proteins. Most of these proteins are important for cellular processes, including cell survival and responses to cellular stress (Taipale et al., 2010). Ganetespib, geldanamycin, 17-DMAG (alvespimycin), and 17-AAG (tanespimycin), which are inhibitors of HSP90, were selected as new senolytic candidates by screening a library of autophagy regulators in senescent Ercc1−/− MEFs.",Biomolecules & Therapeutics,HSP90 Senolytics,2022
HSP90 Inhibitors in Models of Senescence,"These HSP90 inhibitors showed senolytic activity in a senescent cell-type-specific manner. Ganetespib exhibited senolytic activity in senescent HUVECs, whereas 17-DMAG exhibited senolytic activity in senescent human fibroblasts (IMR-90 and WI-38). In addition, 17-DMAG accelerated apoptosis by blocking the HSP90-AKT interaction to destabilize the active state of AKT in senescent MEFs. Treatment of 17-DMAG in the Ercc1-/Δ mouse model significantly decreased tissue senescence and attenuated the progression of several age-related pathologies (Fuhrmann-Stroissnigg et al., 2017). Previous studies also demonstrated that other natural and synthetic HSP90 inhibitors have senolytic activities (Fuhrmann-Stroissnigg et al., 2018; Dutta Gupta and Pan, 2020).",Biomolecules & Therapeutics,HSP90 Pathways,2022
Fisetin as a Natural Senolytic,"Fisetin is a natural flavonoid present in many plants, including fruits, vegetables, and flowers. It also has various pharmacological effects, including anti-inflammatory, antioxidant, antidiabetic, anticarcinogenic, and neuroprotective properties (Khan et al., 2013; Sundarraj et al., 2018). The biological activities of fisetin are involved with various molecular targets and signaling pathways, including the PI3K/Akt, NF-κB, and NRF2 pathways (Chiang et al., 2015; Pal et al., 2015). Fisetin preferentially eliminated ionizing radiation (IR)-induced senescent HUVECs but not preadipocytes or senescent IMR90 (Zhu et al., 2017). Furthermore, it preferentially eliminated genotoxin-induced senescence in human fibroblasts and oxidative stress-induced senescence in MEFs. Fisetin significantly decreased the characteristics of senescence in human adipose tissue (Zhu et al., 2017; Yousefzadeh et al., 2018).",Biomolecules & Therapeutics,Fisetin,2022
Fisetin in Animal Models and Clinical Trials,"Administration of fisetin significantly downregulated the accumulation of senescent cells and senescence markers in several organs of accelerated aged Ercc1-/Δ mice. Fisetin intervention in progeroid and old mice significantly decreased cellular senescence in several tissues, recovered tissue homeostasis, ameliorated frailty, and extended the lifespan (Yousefzadeh et al., 2018). Fisetin is currently being tested in clinical trials for chronic kidney disease (NCT03325322), skeletal health (NCT04313634), osteoarthritis (NCT04210986), frailty (NCT03675724), and adult cancer survivors (NCT04733534). Fisetin exhibits a chemical structure similar to that of quercetin, which is only different in the 5-hydroxy group, implying that more effective analogs can be developed by the structural optimization process.",Biomolecules & Therapeutics,Fisetin Clinical Research,2022
Piperlongumine as a Senolytic,"Piperlongumine, a natural amide alkaloid isolated from long peppers, was known to have senolytic activity (Wang et al., 2016; Zhang et al., 2018). Piperlongumine specifically caused apoptosis in ionizing radiation-induced or senescent human WI-38 fibroblasts by increasing ROS production and inhibiting the PI3K/Akt/mTOR pathway (Wang et al., 2016). In addition, piperlongumine could bind to oxidation resistance 1 (OXR1), a protein that regulates the expression levels of antioxidant enzymes and leads to their degradation (Zhang et al., 2018).",Biomolecules & Therapeutics,Piperlongumine,2022
Overview of Senomorphics,"Senomorphics is an agent that transforms the characteristics of senescent cells into those of young cells by intervening with senescence-associated signaling pathways and SASP without causing apoptosis of senescent cells (Kirkland and Tchkonia, 2017; Lagoumtzi and Chondrogianni, 2021). Senomorphics suppress the function of the SASP by targeting senescence-related signaling pathways, such as MAPKs, NF-κB, mTOR, and IL-1α (Yun et al., 2018; Birch and Gil, 2020; Lagoumtzi and Chondrogianni, 2021). Senomorphics are also related to well-known anti-aging compounds (Table 2, Fig. 1). Ataxia-telangiectasia mutated (ATM) kinase, a serine/threonine protein kinase stimulated by DNA double-strand breaks, modulates cellular senescence (Fausti et al., 2013). The ATM kinase inhibitor KU-60019 exhibited a possible senomorphic activity by recovering the lysosome/autophagy system and metabolic reprogramming (Kang et al., 2017).",Biomolecules & Therapeutics,Senomorphics,2022
"NF-κB, JAK/STAT, and Ruxolitinib as Senomorphics","NF-κB is a transcription factor that mediates senoinflammation or inflammaging, and its activation is related to the aging process. Thus, suppression of NF-κB is considered a possible target of senomorphics (Mato-Basalo et al., 2021). The 8K-NBD peptide, an inhibitor of NF-κB, decreased cellular senescence and attenuated age-related pathologies in Ercc1−/Δ-progeroid mice (Tilstra et al., 2012). Janus family tyrosine kinase (JAK)-signal transducers and activators of transcription (STAT) modulate the expression levels of some pro-inflammatory cytokines (Novakova et al., 2010). The phosphorylation of JAK tyrosine kinase leads to the phosphorylation of STAT3, which is related to the expression levels of the Bcl-2 family and increases levels of IL-6 and IL-8 receptors (Yu et al., 2009; Banerjee and Resat, 2016). STAT3 persistently activates NF-κB during chronic inflammation (Liang et al., 2013). Thus, JAK inhibitors were applied to improve the anti-tumor response by reprogramming the SASP (Toso et al., 2014). Ruxolitinib is a selective JAK1/2 inhibitor approved by the FDA for improving myelofibrosis (Harrison et al., 2016). Ruxolitinib prevented fat loss, decreased lipotoxicity, and increased insulin sensitivity in middle-aged mice (22-month-old) (Xu et al., 2015a).",Biomolecules & Therapeutics,JAK/STAT Senomorphics,2022
Ruxolitinib and Metformin as Senomorphic Agents,"Another study revealed that ruxolitinib repressed the expression level of SASP in IR- and replicative-induced senescent preadipocytes and IR-induced senescent HUVECs. Ruxolitinib treatment also significantly reduced both systemic and adipose tissue inflammation and improved physical function in aged mice, implying its senomorphic characteristics (Xu et al., 2015b). Metformin is a well-known drug derived from French lilac for treating type 2 diabetes (Sanchez-Rangel and Inzucchi, 2017). Senescent IMR-90 fibroblasts treated with metformin exhibited the inhibition of SASP by decreasing the expression levels of IL-1β, IL-6, and C-X-C motif chemokine 5 (CXCL5) (Moiseeva et al., 2013). Metformin exhibited senomorphic action by suppressing the phosphorylation of IκB, the cytoplasmic inhibitor of NF-κB, and IκB kinase, which inhibits NF-κB (Moiseeva et al., 2013; Barzilai et al., 2016). Currently, metformin is being tested in clinical trials as a next-generation drug for improving the aging process and will be approved by the FDA (Kulkarni et al., 2020). Recently, metformin was shown to ameliorate senescent lens epithelial cells by activating AMPK in aged mice (Chen et al., 2022).",Biomolecules & Therapeutics,Metformin Senomorphics,2022
MDM2 Inhibitors and Anti-SASP Antibodies,"Administration of nutlin-3a or MI-63, MDM2 inhibitors, causes growth arrest, significantly reducing the expression levels of IL-6, IL-1α, and SASP factors in genotoxic-induced senescent cells, eventually repressing the ability of senescent fibroblasts to stimulate the excessive proliferation of breast cancer cells (Wiley et al., 2018). Another repression strategy may be accomplished using specific neutralizing antibodies against each SASP factor, including IL-1α, IL-6, and IL-8. IL-1α plays an important role in the modulation of SASP. Thus, targeting either the IL-1α receptor or IL-1α diminished the expression of SASP during senescence (Orjalo et al., 2009). Several studies demonstrated that the neutralizing anti-human IL-1α monoclonal MABp1 antibody was efficient against type 2 diabetes, inflammation, and colorectal cancer in clinical trials (Dinarello et al., 2012; Timper et al., 2015; O’Sullivan Coyne and Burotto, 2017).",Biomolecules & Therapeutics,SASP Neutralization,2022
Neutralizing IL-6 and IL-8 to Suppress SASP,"IL-6 is a representative pro-inflammatory cytokine in SASP and is associated with tumor proliferation and immunosuppression. A neutralizing Mab-IL-6.8 monoclonal antibody against IL-6, named olokizumab, significantly blocked STAT signaling (Kuilman et al., 2008) and ameliorated pathological symptoms of arthritis, which is associated with senescent cells, in a primate animal model (Shaw et al., 2014). Silencing of the SASP or a decrease in senescent cells must be detected by the immune system for subsequent elimination and regeneration. IL-8 is a representative CXC motif chemokine in SASP and is associated with some types of cancer (Coppe et al., 2010; Birch and Gil, 2020). The administration of ABX-IL-8, a humanized monoclonal antibody against IL-8, diminished the growth rate of several cancer xenograft models (Huang et al., 2002; Waugh and Wilson, 2008). The composites of SASP are complex, and future studies are required to classify SASP in a senescence-dependent manner.",Biomolecules & Therapeutics,IL-6 and IL-8 Neutralization,2022
Introduction to Senescence-Targeting Immunotherapeutics,"Another strategy for targeting senescent cells is to strengthen the function of the immune system, a process termed senescence-targeting immunotherapeutics (Table 3). During aging, a decline in immunological function is strongly related to the accumulation of senescent cells. Thus, the physiological role of the immune system in the elimination of senescent cells is crucial (van Deursen, 2014; Ovadya et al., 2018). A previous study demonstrated that perforin knockout mice, which were impaired with the cytotoxic function of natural killer (NK) and T cells, accelerated the accumulation of senescent cells and chronic inflammation (Ovadya et al., 2018). The reduction in the CD28 receptor is a characteristic of human CD8+ T cell senescence.",Biomolecules & Therapeutics,Immunotherapeutics,2022
Immune Cells in Senescent Cell Clearance,"Senescent T cells have been observed in aged individuals and patients with cancer and arthrosis (Vicente et al., 2016), implying that senescent immune cells play a central role in aging-related diseases. Immune cell systems, such as NK cells, macrophages, and CD8+ T cells recognize and eliminate senescent cells (Birch and Gil, 2020; Borghesan et al., 2020; Salminen, 2021). It is possible to diminish the number of senescent immune cells using specific antibodies that recognize senescence surface markers. Therefore, it is important to identify senescent cell surface markers (Kim et al., 2017; Burton and Stolzing, 2018). The expression of CD44, a senescence-induced cell adhesion gene, was increased during the aging process in rat aorta endothelium. Antibodies against CD44 could significantly reduce the recruitment of monocytes to senescent lesion sites in senescent endothelial cells (Mun and Boo, 2010).",Biomolecules & Therapeutics,Immune Clearance of Senescent Cells,2022
"NK Cells, DPP4, and CAR-T Approaches","NK cells recognized the CD58/ICAM1 receptor that exists in senescent cells (Vicente et al., 2016). Another study found that CD26/dipeptidyl peptidase 4 (DPP4) was expressed on the membrane surface of replicative senescent fibroblasts (Kim et al., 2017). Kim et al. (2017) utilized DPP4 as a membrane target to facilitate antibody-dependent cell-mediated cytotoxicity, a mechanism of cell-mediated immune defense in NK cells, to eliminate senescent cells. They revealed that DPP4 helped NK cells recognize and kill senescent cells, implying a possible immunotherapeutic approach for clearing senescent cells. For macrophages, these modified membrane receptors, including glycans and lipids, in senescent cells were recognized by receptors such as CD36 and IgM (Burton and Stolzing, 2018). In addition, it is possible to increase the binding affinity of associated receptors. The use of chimeric antigen receptor T cell therapy to target specific senescent-associated molecules can be a meaningful strategy.",Biomolecules & Therapeutics,NK and CAR-T Immunosenotherapeutics,2022
Rejuvenation of Senescent Immune Cells,"This strategy has been currently applied in anticancer therapy (Amor et al., 2020; Marofi et al., 2021). Another strategy is to rejuvenate senescent immune cells by reversing their abnormal functions to acquire immune functions. The functions of NK and T cells significantly decrease during aging. Stimulation of the nutrient-sensing component AMPK seemed to play a key role in the aging process (Akbar, 2017). Thus, inhibition of AMPK activity enhanced the functions of senescent immune cells (Di Mitri et al., 2011). Similarly, suppression of p38 signaling, which is characteristic of senescent CD8+ T cells, recovered proliferation and mitochondrial biogenesis (Henson et al., 2014). In addition, the decline in the CD28 receptor was a representative characteristic of CD8+ T cell senescence (Vicente et al., 2016). Thus, the increase in CD28 by ectopic expression or other receptors associated with T cell activation could improve the senescence of CD8+ T cells. These results suggest that various immune cells play pivotal roles in delaying the onset of diseases caused by the accumulation of senescent cells.",Biomolecules & Therapeutics,Immune Rejuvenation,2022
Publication Information,"Application of the Yamanaka Transcription Factors Oct4, Sox2, Klf4, and c-Myc from the Laboratory to the Clinic
by Marisol Aguirre 1,2ORCID, Manuela Escobar 3, Sebastián Forero Amézquita 3ORCID, David Cubillos 3ORCID, Camilo Rincón 3, Paula Vanegas 3, María Paula Tarazona 3, Sofía Atuesta Escobar 3, Juan Camilo Blanco 3 and Luis Gustavo Celis 3,*ORCID
1 Department of Genetics, Fundación Valle del Lili, Cali 760026, Colombia
2 Faculty of Medicine, Universidad Icesi, Cali 760031, Colombia
3 Faculty of Medicine, Universidad de La Sabana, Km 7, Autopista Norte de Bogotá, Chía 250001, Colombia
* Author to whom correspondence should be addressed.
Genes 2023, 14(9), 1697; https://doi.org/10.3390/genes14091697
Submission received: 28 February 2023 / Revised: 6 August 2023 / Accepted: 14 August 2023 / Published: 26 August 2023
(This article belongs to the Topic Stem Cell Differentiation and Applications)",Genes,Yamanaka Factors,2023
Abstract Overview,"The transcription factors Oct4, Sox2, Klf4, and c-Myc enable the reprogramming of somatic cells into induced pluripotent cells. Reprogramming generates newly differentiated cells for potential therapies in cancer, neurodegenerative diseases, and rejuvenation processes. In cancer therapies, these transcription factors lead to a reduction in the size and aggressiveness of certain tumors, such as sarcomas, and in neurodegenerative diseases, they enable the production of dopaminergic cells in Parkinson’s disease, the replacement of affected neuronal cells in olivopontocerebellar atrophy, and the regeneration of the optic nerve.",Genes,Yamanaka Factors,2023
Therapeutic Potential and Limitations,"However, there are limitations, such as an increased risk of cancer development when using Klf4 and c-Myc and the occurrence of abnormal dyskinesias in the medium term, possibly generated by the uncontrolled growth of differentiated dopaminergic cells and the impairment of the survival of the new cells. Therefore, the Yamanaka transcription factors have shown therapeutic potential through cell reprogramming for some carcinomas, neurodegenerative diseases, and rejuvenation. However, the limitations found in the studies require further investigation before the use of these transcription factors in humans.
Keywords: cell- and tissue-based therapy; Oct4; Sox2; Klf4; c-Myc; cancer; neurodegenerative diseases; induced pluripotent stem cells; rejuvenation",Genes,Yamanaka Factors,2023
OSKM Factors in Cancer Biology,"A literature review found several applications of OSKM transcription factors in cancer-related experiments [15]. Technologies based on cell regeneration and reprogramming allow cancer stem cells to be reprogrammed into pluripotent stem cells using the described transcription factors [15]. However, this may be controversial, since c-Myc and Klf4 factors increase the risk of tumorigenesis [15]. Oct4 overexpression was present in multiple cancers, such as ovarian, cervical, colorectal, liver, breast, and bladder cancers. Sox2 was present in at least 25 types of cancers, including breast, gastric, and pancreatic cancers; adenocarcinoma of the ampulla of Vater; malignant gliomas; and other brain tumors. Klf4 overexpression was found in squamous cells and other cancer-forming processes [16], and c-Myc was the main oncogenic factor related to tumorigenesis and glycolysis as well as being linked to Wnt/β-catenin signaling in lung cancer [17].",Genes,Yamanaka Factors in Cancer,2023
Mouse Model Studies with OSKM Factors,"Likewise, a study combining gene and cell therapy was performed in a mouse model with anemia to recognize the therapeutic potential of pluripotent stem cells by using mouse embryonic fibroblasts, but this is not possible in human treatments. However, human dermal fibroblasts have been reported, with potential clinical applications in various fields, for example, genetic diseases. The study aimed to compare the results obtained from using bone marrow stromal cells and embryonic fibroblasts. Mice aged 12 to 24 weeks were isolated, and mouse bone marrow mononuclear cells were collected using centrifugation and fused. Then, hybrid cell induction was performed, followed by the use of a transfer method involving retroviruses containing the Yamanaka transcription factors. The result was that embryonic fibroblasts did not work well for generating pluripotent cells, but after using a higher transduction efficiency, it was possible. Bone marrow mononuclear cells did not require such interventions. Subsequently, these were transplanted subcutaneously to test the pluripotency of the bone marrow cells. Four weeks after injection, encapsulated cystic tumor growth was observed in all mice, and microscopic examinations showed that these tumors contained various tissues, including neural tissue, epidermal tissue, muscle fiber cells, cartilage, and pancreas-like cells, and the differentiation of three germ layers was confirmed in vitro [16].",Genes,Yamanaka Factors in Cancer,2023
CHD4 Interactions with OSKM and Cancer Pathways,"Yamanaka transcription factors show participation in some processes of tumorigenesis and pluripotent stem cells when interacting with other molecules that can offer new research options. For example, the helicase DNA-binding protein (CHD4) suppresses the expression of Sox2 and regulates cancer stem cells, targeting different molecules such as SANIL1, CCND1, CCND3, P21, P7, and c-Myc. CHD4 is part of the nucleosome remodeling and deacetylase complex that affects the pluripotency and differentiation of embryonic stem cells. CHD4 expression is associated with developing glioblastomas and colorectal, hepatocellular, endometrial, and breast cancers. A high CHD4 expression is associated with a worse survival prognosis, a larger tumor size, resistance to drugs used in chemotherapy, and a lower migration rate without affecting tumor cell proliferation. A low CHD4 expression causes increasing levels of Sox2 and paclitaxel resistance [18].",Genes,Yamanaka Factors in Cancer,2023
Reprogramming Sarcomas Using OSKM and Related Factors,"On the other hand, this technology could help to reduce the malignant potential of certain tumors such as sarcomas. In in vivo mice experiments, Nanog and Lin28 were used in addition to the four Yamanaka factors in five sarcomatous cell lines, and these experiments showed a decrease in the tumor growth rate compared with the controls. A smaller size and a decrease in the number of tumor cells was observed in mice with reprogrammed cells compared with the control group. In addition, the experiments demonstrated that the reprogrammed sarcomas could differentiate into mature connective tissue and red blood cells, which is evidence that the use of transcription factors decreases tumor aggressiveness and changes the morphology [19].",Genes,Yamanaka Factors in Cancer,2023
Cancer Stem Cell Reprogramming and Lung Cancer Diagnostics,"In an experiment in which cancer stem cells were split from samples taken from the peripheral blood mononuclear cells of a patient with neuroendocrine carcinoma of the lung that was given episomal vectors to generate pluripotent stem cells, at a follow-up two weeks later, colonies with the typical morphology of embryonic stem cells, a good in vitro differentiation potential, and a high expression of pluripotent markers were detected, which made it possible to determine that a treatment could be carried out in the same place where the disease is found. However, it is also relevant to evaluate the safety and effectiveness of these therapies according to each tumor [20]. Sox2, Oct4, and Nanog also demonstrated a high diagnostic potential and a potential future use in targeted therapy for adenocarcinoma and squamous cell lung cancer. For example, a clinical study revealed that the mentioned transcription factors were present in 76% of the 30 lung tissue samples collected before the treatment. Oct4 overexpression was associated with more advanced stages of lung cancer and tumors with little differentiation. Another study with 147 subjects showed a correlation of Sox2 with squamous cell lung cancer, with Sox2 being present in 79% of the samples, demonstrating its crucial role in the diagnostic approach to this pathology with a high incidence and mortality in the world today. Sox2 in non-small cell lung cancer has demonstrated the ability to preserve the pluripotent capacity of these cells, generating resistance to anticancer therapy with paclitaxel. Thus, inhibiting Sox2 and its effector CIC-3 increases the sensitivity of the drug to the affected cells, providing a new therapeutic option for this type of lung cancer [21]. Applications of the Yamanaka transcription factors in cancer play a role in treatment, diagnosis, prognosis, and drug resistance, but more research is needed on these factors to understand their safety, effectiveness, and interaction with other molecules.",Genes,Yamanaka Factors in Cancer,2023
OSKM-Induced Neural Differentiation and Parkinson’s Models,"Yamanaka transcription factors have impacted the development of cell replacement therapies in neurodegenerative diseases due to their ability to reprogram somatic cells (Figure 1). A study with rats achieved differentiation into neural stem cells, neurons, and dopaminergic neurons with iPSCs from the OSKM transcription factors, generating a slight reduction in the classic motor impairments of Parkinson’s disease [22]. In another study, for olivopontocerebellar atrophy, iPSCs were developed from fibroblasts to obtain neural cells identical to those of the patient with a 99% accuracy, which may be key for the treatments of this neurodegenerative disease [23]. While there are positive impacts, unfortunately, there were also adverse effects in both studies mentioned above. A few cell colonies were reported, with the growth of teratomas in mice with a severe combined immunodeficiency [22,23]. Figure 1. OSKM–iPSC positive outcomes in neurodegenerative diseases.",Genes,Yamanaka Factors Neurodegeneration,2023
Dopaminergic Neuron Replacement and Dyskinesia Concerns,"These transcription factors show a potential application for the development of a possible treatment for Parkinson’s disease. In in vivo studies, it is possible to obtain pluripotent cells differentiated into dopaminergic cells that could treat this neurodegenerative disease [24]. Additionally, iPSCs transplanted into a mouse brain can migrate to various brain regions and differentiate into dopaminergic, GABAergic, and glutamatergic neuronal cells and glial cells that are functional, providing hope for a treatment for multiple diseases [24]. Moreover, transplanting stem cells converted into dopaminergic neurons in the striatum of mice with motor abnormalities generated a decrease in motor symptoms [24]. However, in the medium term, dyskinesia was evident in the transplanted mice, which raised doubts about the safety of this therapy for improving motor symptoms in Parkinson’s disease [24]. Additionally, the long-term cell survival, functionality, and efficacy are unknown.",Genes,Yamanaka Factors Neurodegeneration,2023
Schizophrenia iPSCs and iNSC Transplantation Studies,"IPSCs obtained from the peripheral blood mononuclear cells (PBMCs) of schizophrenia patients and healthy control patients showed that these cells presented higher and more severe inflammatory responses than normal iPSCs, indicating that inflammation and the immune system could play a role in the etiology of schizophrenia disorders [25]. An analysis of these data helped to understand the pathogenesis of some neurodegenerative diseases. In a study, induced neural stem cells (iNSCs) generated from mesenchymal stromal cells (MSCs) encoding OSKM from a cynomolgus monkey were transplanted into an immunodeficient model of Parkinson’s disease induced by 6-hydroxydopamine (6-OHDA), a neurotoxin used to generate the destruction of dopaminergic neurons in the nigrostriatal region and its consequent neurodegeneration [26]. They demonstrated a good plasticity that allowed them to differentiate into dopaminergic neurons and glial cells such as astrocytes, oligodendrocytes, and pan-neurons, resulting in effective results in the improvement of motor functions in comparison with a control group six months post-transplantation, with a high statistical significance [26]. This was probably due to the increase in dopamine production and the reduction in the loss of dopaminergic neurons, indicating a neuroprotective factor against the progressive degeneration caused by the disease.",Genes,Yamanaka Factors Neurodegeneration,2023
"ALS iPSCs, Motor Neuron Differentiation, and Drug Discovery","Neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) generate the loss of lower and upper neurons, which produces progressive weakness, spasticity, muscle atrophy, and ultimately, death [27], and they have gained more importance and relevance since iPSCs obtained from ALS patients were found to differentiate into motor neurons [28]. Studies carried out with iPSCs, especially using the transcription factors Oct4 and Klf4, have resulted in an increase in the preservation of spinal cord neurons at the injection site and an increase in the expression of neuronal growth factor (NGF). It has also been evidenced that iPSCs are neuroprotective, with neuromodulator effects, and that they can reduce disease progression [29]. Additionally, thanks to their implementation in this disease, the modeling of ALS with iPSC-derived neurons has elucidated their potential role in drug discovery [30]. In 2021, a study described the role of cell lines in the pathogenesis and progression of ALS and how to use these cells derived from human iPSCs to identify new therapeutic targets [31]. Another study described how to produce motor neurons from human iPSCs, their application in drug discovery for this disease, and their potential use in cell therapy by performing cell transplantation [32].",Genes,Yamanaka Factors Neurodegeneration,2023
iPSCs for Dementia and Alzheimer’s Disease,"For frontotemporal dementia and Alzheimer’s disease modeling and drug discovery, studies show favorable results [33]. iPSC therapy improves neuronal plasticity and memory by increasing proteins responsible for cognition. For example, in a mouse model, iPSC therapy could achieve an increased release of brain-derived neurotrophic factors that improve cognitive function. iPSCs differentiate into different brain cells, promoting brain neuroplasticity, decreasing the production of inflammatory cytokines, and stimulating growth factor release. However, tumor growth could appear with this therapy, so more studies are still needed [34]. Likewise, recent studies have investigated and understood the pathophysiology of neurodegenerative diseases to provide timely treatment. According to these investigations, the excessive activation of microglia in the brain and increased levels of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-10 were present.",Genes,Yamanaka Factors Neurodegeneration,2023
"BIG1, Klf4, and Neuroinflammation Mechanisms","A novel protein called BIG1 (brefeldin A-inhibited guanine nucleotide-exchange protein 1), responsible for the cell migration of iNSCs and neuronal soma growth and formation, was also discovered. RT-qPCR and Western blot techniques found that silencing BIG1 reduced the expression of TNF-α, IL-1β, and IL-6, decreasing neuroinflammation, and the inhibition of this protein generated a decrease in the cell migration of iNSCs. Based on the above, the transcription factor Klf4 binds to the BIG1 promoter, positively regulating it, thus mediating neuroinflammation and allowing cell migration through the PI3K/Akt/NF-kB signaling pathway that regulates proliferation, apoptosis, and differentiation, controlling a wide range of target proteins and, thus, improving neuroinflammation and neuroplasticity [35]. With the passage of time and further research, these OSKM transcription factors could be applied to improve the modeling of many more diseases and to gain a deeper understanding of disease pathogenesis, progression, and therapeutic targets.",Genes,Yamanaka Factors Neurodegeneration,2023
Developmental vs Age Reprogramming Concepts,"In the years following the discovery of the Yamanaka transcription factors, efforts were made to implement a method of rejuvenating cells, tissues, and even organisms using them. Senescent cells can be reprogrammed in two ways: first, through developmental reprogramming using OSKM or somatic cell nuclear transfer (SCNT), and second, through age reprogramming by bypassing the de-/re-differentiation cycle, reducing or silencing age-related markers, and retaining their original identity. In the proposed model for age reprogramming, the old cell transiently de-differentiates after OSMK introduction. Then, a phase of epigenetic instability occurs in which aging-related markers are reduced or silenced. Later, OSKM expression stops, and ESs/iPSCs re-differentiate into an original young cell without embryonic features (Figure 2). The time that the cell spends in the phase of epigenetic instability can determine the degree of rejuvenation. An optimal time results in a younger reprogrammed cell without losing its somatic identity, but a very long time generates a reprogrammed cell that is less young. The difference in the results of chronological reprogramming may be due to the accumulation of senescence-associated gene products at the end of the critical reprogramming time [36]. However, more research is needed. Figure 2. Characteristics of senescent cells and developmental and age reprogramming pathways.",Genes,Yamanaka Factors Rejuvenation,2023
Early Partial Reprogramming and Epigenetic Reversal,"In 2010, the possibility of inducing cell regression without generating stem cells was proposed to avoid the risk of cancer, giving rise to partial cell reprogramming and epigenetic rejuvenation [37,38], which allowed its subsequent use in human fibroblasts to restore their heterochromatin to non-senescent levels [39] and in mouse fibroblasts to decrease age-related changes such as DNA damage, nuclear damage, stress, and senescence factors, among others [40,41,42]. In 2016, a study used OSKM to generate partial reprogramming in vivo in progeroid mice, extending their lifespan without the appearance of teratomas. When applied in adult mice, it improved the regenerative capacity of muscles and the pancreas after injury, as well as glucose tolerance [43]. However, this study did not have a method to quantify the degree of rejuvenation [44]. In another study, it was not possible to demonstrate the duration of rejuvenation over time at the cellular or organism level [45], which gave rise to subsequent studies aimed at improving the phenotype of mice with Hutchinson–Gilford progeria syndrome and promoting cell regeneration in middle-aged mice by administering OSKM and producing partial cellular reprogramming. However, these effects remained transient [46].",Genes,Yamanaka Factors Rejuvenation,2023
Rejuvenation of Neurons and Optic Nerve Regeneration,"Thanks to previous discoveries, OSKM was used with Lin28 and Nanog in other cell lines, such as mature neurons or neurons with aging lesions, to rejuvenate them through their total or partial reprogramming [47]. At the level of retinal ganglion cells (RGCs), the overexpression of at least three of the transcription factors, including Lin28, was performed using the AAV2 viral vector, resulting in the significant regeneration of the optic nerve and axons, as well as a decrease in injury-induced neuronal cell death. However, using these factors individually or using only two did not regenerate the cell [46]. Another significant point is that the experimental approach and the modified transcripts in mature neurons may have potential problems. For example, some of the transcription factors used are oncoproteins, such as Sox2, Lin28, and c-Myc, which may favor tumor creation when overexpressed in glial cells with a cell division capacity [47].",Genes,Yamanaka Factors Rejuvenation,2023
Muscle Stem Cell Niche and Regenerative Modulation,"Additionally, the short-term expression of Yamanaka factors promotes tissue regeneration in vivo. For example, in myofibers, it favors muscle regeneration in young mice, inducing the activation of muscle stem cells or satellite cells (SCs), thus accelerating their regeneration. This occurs through the modification of the stem cell niche, as the regenerative capacity of SCs is influenced by intrinsic modulators and the extrinsic microenvironment. In this case, the expression of the factors in myofibers regulates the expression of genes for the SCs microenvironment, including the up-regulation of p21, which, in turn, down-regulates Wnt4. Myofibers secrete Wnt4 to maintain SCs quiescence, favor muscle regeneration, and slow SCs aging. In contrast, the expression of Yamanaka factors directly in SCs does not enhance muscle regeneration [48].",Genes,Yamanaka Factors Rejuvenation,2023
Lifespan Extension Studies and Remaining Challenges,"Finally, in this manuscript, two ongoing investigations are dealing with the administration in mice of Oct4, Sox2, and Klf4 factors in combination through a lentivirus and doxycycline, with the latter being used to activate the factors administered in the lentivirus, which has resulted in a reversal of biomarkers of aging, such as a decreased frailty index and an increased epigenetic age calculated from methylation patterns, thereby prolonging the lifespan of the mice to 109% and maintaining it over time [49,50]. However, there are limitations, such as the difficulty with the combined administration of these transcription factors due to their size, which exceeds the capacity of lentiviruses; the formation of teratomas with the use of c-Myc; and the possibility of replicating these results in humans (Figure 3) [49]. Finally, the Yamanaka transcription factors have a high potential for use in the aging field. However, the mechanism and risks must be well known before these factors can be safely implemented as a treatment in humans [51].",Genes,Yamanaka Factors Rejuvenation,2023
Pluripotency and Differentiation Overview,"Embryonic stem cells exhibit pluripotency: they can differentiate into all types of somatic cells. Pluripotent genes such as Oct4 and Nanog are activated in the pluripotent state, and their expression decreases during cell differentiation. Inversely, expression of differentiation genes such as Gata6 and Gata4 is promoted during differentiation. The gene regulatory network controlling the expression of these genes has been described, and slower-scale epigenetic modifications have been uncovered. Although the differentiation of pluripotent stem cells is normally irreversible, reprogramming of cells can be experimentally manipulated to regain pluripotency via overexpression of certain genes. Despite these experimental advances, the dynamics and mechanisms of differentiation and reprogramming are not yet fully understood.",PLoS Computational Biology,Pluripotency,2015
Gene Regulatory Network Model,"Based on recent experimental findings, we constructed a simple gene regulatory network including pluripotent and differentiation genes, and we demonstrated the existence of pluripotent and differentiated states from the resultant dynamical-systems model. Two differentiation mechanisms, interaction-induced switching from an expression oscillatory state and noise-assisted transition between bistable stationary states, were tested in the model. The former was found to be relevant to the differentiation process. We also introduced variables representing epigenetic modifications, which controlled the threshold for gene expression. By assuming positive feedback between expression levels and the epigenetic variables, we observed differentiation in expression dynamics.",PLoS Computational Biology,Pluripotency,2015
Reprogramming and Yamanaka Factors,"Additionally, with numerical reprogramming experiments for differentiated cells, we showed that pluripotency was recovered in cells by imposing overexpression of two pluripotent genes and external factors to control expression of differentiation genes. Interestingly, these factors were consistent with the four Yamanaka factors, Oct4, Sox2, Klf4, and Myc, which were necessary for the establishment of induced pluripotent stem cells. These results, based on a gene regulatory network and expression dynamics, contribute to our wider understanding of pluripotency, differentiation, and reprogramming of cells, and they provide a fresh viewpoint on robustness and control during development.",PLoS Computational Biology,Pluripotency,2015
Publication Information,"PLOS Computational Biology | DOI:10.1371/journal.pcbi.1004476 August 26, 2015 1 / 25. Citation: Miyamoto T, Furusawa C, Kaneko K (2015) Pluripotency, Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation. PLoS Comput Biol 11(8): e1004476. doi:10.1371/journal.pcbi.1004476. Editor: John C Marioni, EMBL, UNITED KINGDOM. Received: March 10, 2015. Accepted: July 22, 2015. Published: August 26, 2015. Copyright: © 2015 Miyamoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",PLoS Computational Biology,Pluripotency,2015
Author Summary on Pluripotency and Reprogramming,"Characterization of pluripotent states, in which cells can both self-renew and differentiate, and the irreversible loss of pluripotency are important research areas in developmental biology. In particular, an understanding of these processes is essential to the reprogramming of cells for biomedical applications, i.e., the experimental recovery of pluripotency in differentiated cells. Based on recent advances in dynamical-systems theory for gene expression, we propose a gene-regulatory-network model consisting of several pluripotent and differentiation genes. Our results show that cellular-state transition to differentiated cell types occurs as the number of cells increases, beginning with the pluripotent state and oscillatory expression of pluripotent genes.",PLoS Computational Biology,Pluripotency,2015
Epigenetic Barriers and Reprogramming,"Cell-cell signaling mediates the differentiation process with robustness to noise, while epigenetic modifications affecting gene expression dynamics fix the cellular state. These modifications ensure the cellular state to be protected against external perturbation, but they also work as an epigenetic barrier to recovery of pluripotency. We show that overexpression of several genes leads to the reprogramming of cells, consistent with the methods for establishing induced pluripotent stem cells. Our model, which involves the inter-relationship between gene expression dynamics and epigenetic modifications, improves our basic understanding of cell differentiation and reprogramming.",PLoS Computational Biology,Pluripotency,2015
Stemness and Differentiation,"In multicellular organisms, cells that exhibit stemness during development can both self-renew and differentiate into other cell types. In contrast, differentiated cells lose the ability to further differentiate into other cell types and terminally differentiated cells can only self-renew. Currently, how stemness and the irreversible loss of differentiation potential are characterized by gene expression patterns and dynamics are key questions in developmental biology. Cells with stemness include embryonic stem cells (ESCs), which are derived from the inner cell mass of a mammalian blastocyst and are pluripotent, i.e., they can differentiate into all the types of somatic cells [1, 2]. To maintain pluripotency, pluripotent genes such as Pou5f1 (also known as Oct4) [3, 4] and Nanog [5, 6] are activated in ESCs. Expression of these genes gradually decreases during cell differentiation, whereas expression of differentiation marker genes increases. Understanding these changes in gene expression patterns over the course of cell differentiation is important for characterizing the loss of pluripotency.",PLoS Computational Biology,Pluripotency,2015
Recovery of Pluripotency and Yamanaka Factors,"During normal development, the loss of pluripotency is irreversible. However, the recovery of pluripotency in differentiated cells was first achieved by experimental manipulation in plants, and then in Xenopus laevis via cloning by Gurdon [7]. More recently, the overexpression of four genes that are highly expressed in ECSs, Oct4, Sox2, Klf4, and Myc (now termed Yamanaka factors), has been used to reprogram differentiated cells. Overexpression of these genes leads to cellular-state transition and changes in gene expression patterns, and the transition generates cells known as induced pluripotent stem cells (iPSCs) [8]. Previous studies have also uncovered the gene regulatory network (GRN) related to the differentiation and reprogramming of cells [9, 10]. To understand the differentiation process theoretically, Waddington proposed a landscape scenario in which each stable cell-type is represented as a valley and the differentiation process is represented as a ball rolling from the top of a hill down into the valley [11]. In this scenario, the reprogramming process works inversely to push the ball to the top of the hill [12–14].",PLoS Computational Biology,Pluripotency,2015
Dynamical Systems and Attractor Models,"As a theoretical representation of Waddington’s landscape, the dynamical-systems approach has been developed over several decades, pioneered by Kauffman [15] and Goodwin [16]. In this approach, the cellular state is represented by a set of protein expression levels with temporal changes that are given by GRNs. According to gene expression dynamics, the cellular state is attracted to one of the stable states, which is termed an attractor. Each attractor is assumed to correspond to each cell type. Indeed, this attractor view has become important for understanding the diversification of cellular states and their robustness. Both theoretical and experimental approaches have been developed to assign each cell-type to one of the multi-stable states [17–19]. In these approaches, a pluripotent state is regarded as a stationary attractor with relatively weak stability, and the loss of pluripotency is the transition by noise to attractors with stronger stability.",PLoS Computational Biology,Pluripotency,2015
Oscillatory Dynamics and Cell-Cell Interaction,"An alternative approach investigated how the interplay between intra-cellular dynamics and interaction leads to differentiation and the loss of pluripotency [20–23]. Specifically, the pluripotent state is represented by oscillatory states following the expression dynamics of more genes, whereas the loss of pluripotency is represented by the decrease in the degree of expressed genes necessary for oscillatory dynamics. Here, differentiation is triggered by cell-cell interactions, which lead to robustness in developmental paths and the final distribution of cell types [20, 24, 25]. By using several GRNs, cells with oscillatory intracellular gene expression dynamics are found to differentiate into other cell types by cell-cell interactions [21, 26–28]. Indeed, the recovery of pluripotency by gene overexpression is a process predicted to facilitate recovery of lost degrees of freedom and oscillation [20]. However, of the question of whether this theory applies to realistic GRNs has yet to be explored. Despite these earlier studies, pluripotency has not yet been confirmed in a realistic GRN observed in experiments, and the mechanism of reprogramming remains elusive.",PLoS Computational Biology,Pluripotency,2015
Epigenetic Modifications and Memory,"Epigenetic modifications such as DNA methylation and histone modification are now also recognized as important in cell differentiation. Epigenetic change solidifies differentiated-cellular states by altering chromatin structure to generate transcriptionally active and inactive regions [29, 30]. With epigenetic change, the activity of gene expression is preserved in a process known as epigenetic memory [31]. Indeed, epigenetic modification is suggested as a barrier to reprogramming [32]. However, the theoretical inter-relationship between expression dynamics and epigenetic modification has yet to be fully explored. The aim of the present study was three-fold. First, by using GRNs obtained from a previous experimental study, we examined the validity of two differentiation scenarios: 1) oscillation + cell-cell interaction and 2) multistability + noise.",PLoS Computational Biology,Pluripotency,2015
Study Aims and Modeling Approach,"Second, to demonstrate that differentiation by gene expression dynamics is solidified by epigenetic modification, we introduced a mathematical model for epigenetic feedback regulation. Third, we investigated how overexpression of some genes leads to reprogramming, i.e., regaining pluripotency from differentiated states (scenario 1) by initializing epigenetic changes. Below, we have first introduced a simple model extracted from an experimentally observed GRN. This model consists of several genes, including pluripotent and differentiation genes, with mutual activation and inhibition. We then examined the oscillatory dynamics and multistable states scenarios to show that differentiation with the loss of pluripotency progresses from a stem cell state with oscillatory expression through cell-cell interactions. Additionally, the two scenarios were compared with regard to their robustness to noise and the regulation of the ratio of differentiated cells.",PLoS Computational Biology,Pluripotency,2015
Reprogramming via Overexpression,"We also investigated the epigenetic process by introducing variables that give the threshold for the expression of genes to demonstrate that the cellular state derived from gene expression dynamics is fixed by epigenetic feedback regulation. Differentiation by gene expression is fixed according to these threshold variables; thus, the pluripotent and differentiated states are fixed. Finally, we investigated reprogramming by temporally imposing overexpression of genes and examining whether the differentiated state is reversed to the pluripotent state. Via overexpression of several genes, epigenetic fixation was relaxed and the expression levels and dynamics of the pluripotent state were recovered. This reprogramming was shown to require the overexpression of several genes, including pluripotent genes, over a sufficient period beyond the time scale of epigenetic fixation. Indeed, by using a model with five relevant genes, we found that four genes corresponding to the Yamanaka factors must be overexpressed for reprogramming to occur. It was also demonstrated that insufficient overexpression of genes, i.e., overexpression of pluripotent genes only, results in partially reprogrammed cells (which, experimentally, are known as pre-iPSCs).",PLoS Computational Biology,Pluripotency,2015
Epigenetic Barrier and Pluripotency Overview,"Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier
Akira Watanabe, Yasuhiro Yamada and Shinya Yamanaka
Center for iPS Cell Research and Application, Kyoto University
The differentiation and reprogramming of cells are accompanied by drastic changes in the epigenetic profiles of cells. Waddington’s classical model clearly describes how differentiating cells acquire their cell identity as the developmental potential of an individual cell population declines towards the terminally differentiated state. The recent discovery of induced pluripotent stem cells as well as of somatic cell nuclear transfer provided evidence that the process of differentiation can be reversed. The identity of somatic cells is strictly protected by an epigenetic barrier, and these cells acquire pluripotency by breaking the epigenetic barrier by reprogramming factors such as Oct3/4, Sox2, Klf4, Myc and LIN28.",Philosophical Transactions of the Royal Society B,Epigenetic Regulation / iPS Cells,2013
Spatio-Temporal Epigenetic Regulation,"This review covers the current understanding of the spatio-temporal regulation of epigenetics in pluripotent and differentiated cells, and discusses how cells determine their identity and overcome the epigenetic barrier during the reprogramming process.
1. Introduction
Induced pluripotent stem (iPS) cells are generated by the enforced expression of embryonic transcription factors, most commonly Oct3/4, Sox2, c-Myc and Klf4. In addition to pluripotency, they have infinite capacity for self-renewal [1,2]. iPS cells have been generated from multiple cell types, including keratinocytes [3], mesenchymal cells in fat [4], the oral mucosa [5], dental pulp cells [6], peripheral blood [7] and cord blood [8], as well as skin fibroblasts [2]. The characteristics of fully reprogrammed cells are functionally and molecularly very similar to those of embryonic stem (ES) cells in terms of their morphology, gene expression profile and capacity to differentiate into any of the three germ layers: endoderm, mesoderm and ectoderm.",Philosophical Transactions of the Royal Society B,Epigenetic Regulation / iPS Cells,2013
Pluripotency and Waddington’s Landscape,"iPS cells could be a useful source for cell transplantation therapy, drug screening and disease modelling [9]. iPS cells are also highlighted as a cell model for epigenetic research. Pluripotent stem cells differentiate into any of the 200–300 specialized cell types with distinct properties. Waddington clearly described how differentiating cells acquire their cell identity, by illustrating differentiating cells as marbles rolling down valleys, with the developmental potential of individual cell populations declining towards the terminally differentiated state at the lowest elevation [10]. Recent genome-wide analyses using high-performance sequencers have uncovered key differences in the epigenetic landscape of pluripotent stem cells compared with that of lineage-committed cells. Waddington’s classical model is now widely accepted; it appears that ‘Waddington’s marbles’ are present in different valleys and that the different elevation levels have distinct epigenetic profiles, which are likely to play a role in the irreversibility of the properties of lineage-committed cells and the maintenance of their identity [11].",Philosophical Transactions of the Royal Society B,Epigenetic Regulation / iPS Cells,2013
Reprogramming and Epigenetic Reversal,"Cellular reprogramming induces differentiated cells to revert back to undifferentiated cells including pluripotent stem cells. On the basis of Waddington’s model, somatic cells in differentiated states maintain their own cell fate and do not normally change from one differentiation pathway to another, although cell fate can be altered by nuclear reprogramming [11–15]. This reversal process can be achieved by breaking the barrier of the differentiated state, and it provides one of the strategies for investigating the molecular basis of cell identity governed by epigenetic regulation. Because of its observed lower efficiency, the reprogramming process has been depicted as climbing a mountain, because it is much harder to achieve than differentiation, which is a spontaneous process, as sliding down a hill [11]. Therefore, a molecular understanding of the reprogramming process may address the question of how differentiated cells maintain their identity.",Philosophical Transactions of the Royal Society B,Epigenetic Regulation / iPS Cells,2013
Epigenetic Events During Induced Pluripotency,"Induced pluripotency is a process associated with gradual epigenetic changes [16], and thus can be exploited to obtain a molecular understanding of the determination of cell fate, which is mediated by epigenetic changes such as the silencing of retroviral transgenes upon the establishment of pluripotency [17,18], the reactivation of endogenous pluripotency genes [1], the establishment of bivalent chromatin domains in the promoters of developmentally regulated genes [12,19], global DNA hypomethylation, DNA hypermethylation of imprinted gene loci [17], reactivation of the inactive X chromosome in female iPS cells and reorganization of chromatin fibres [20,21]. This review summarizes studies performed to understand the epigenetic signatures associated with pluripotent and differentiated states, and addresses how their unique signatures contribute to the maintenance of pluripotency and how they are established during the reprogramming process.",Philosophical Transactions of the Royal Society B,Epigenetic Regulation / iPS Cells,2013
Histone Modification Mapping and Key Complexes,"2. Distinct histone modification profile in pluripotent cells
Recent technical advances have allowed us to map chromatin modifications throughout the genome by combining chromatin immunoprecipitation with DNA microarray analysis (ChIP-chip) or high-performance sequencing (ChIP-seq). Pluripotent stem cells have a unique expression pattern for histone modifiers and distinct distributions of modified histones. The Polycomb group (PcG) complexes with the activity of H3K27 methylation to repress the expression of developmentally regulated genes in pluripotent stem cells [22,23], whereas the Trithorax group (TrxG) complexes with the activity of H3K4 methylation to activate the expression of genes associated with self-renewal [24]. An active mark, H3K4me3, is frequently observed in promoter regions of pluripotent stem cells, and is linked to transcriptional activation in general [25–27]. The methylation of H3K4 is mediated by TrxG members such as Set/mixed lineage leukaemia (MLL) methyltransferases. Wdr5, a key component of TrxG, interacts with H3K4me2, and mediates the transition of H3K4me2 to H3K4me3 [28]. The expression of Wdr5 is the highest in undifferentiated ES and iPS cells, and the level decreases during the differentiation process.",Philosophical Transactions of the Royal Society B,Histone Modifications / Pluripotency,2013
"Wdr5, LSD1 and H3K4/H3K27 Regulation","The expression of Wdr5 along with the reprogramming factors enhances the efficiency of iPS cell generation [24]. Wdr5 physically interacts with Oct3/4, and co-occupies the DNA-binding sites of Oct3/4. Silencing of Wdr5 expression results in decreased expression of Oct3/4 target genes and the loss of self-renewal capacity of ES cells. H3K4 demethylase LSD1 stabilizes global DNA methylation [29] and also maintains an appropriate balance between H3K4 and H3K27 methylation in the regulatory regions of several developmental genes in pluripotent stem cells [30]. The recently reported interaction between LSD1 and Dnmt1 indicated that LSD1 mediates the linkage between DNA methylation and H3K4 demethylation [31]. The methylation of H3K27 is mediated by Polycomb repressive complex 2 (PRC2), which is composed of PcG proteins such as enhancer of zeste 2 (Ezh2), embryonic ectoderm development (Eed) and suppressor of zeste 12 homolog (Suz12) [32,33]. ES cells lacking a single component of the PRC2 complex, such as Ezh2, Eed or Suz12, show partial disruption of self-renewal accompanied by complete depletion of H3K27me3 [23,34].",Philosophical Transactions of the Royal Society B,Histone Modifications / Pluripotency,2013
"PRC2, PRC1 and Noncoding RNA Interactions","These findings indicate that each component of the PRC2 complex collaboratively executes H3K27 trimethylation and regulates pluripotency and differentiation [35–38]. The histone methyltransferase activity of Ezh2 is responsible for maintaining H3K27 trimethylation in pluripotent stem cells [36,38,39]. Suz12 interacts with Ezh2, and inhibits protein degradation of Ezh2 [37]. A genome-wide analysis showed that Suz12 is co-localized with H3K27 trimethylation at key development regulators, as well as with highly conserved non-coding elements in ES cells [22]. A subset of Suz12-bound and H3K27me3-enriched genes are co-occupied by Oct3/4, Sox2 and Nanog. They are preferentially activated during ES cell differentiation, indicating that PRC2 poises differentiation-related genes for rapid gene activation during differentiation [22,40]. The PRC1 complexes composed of RING1A, RING1B, BMI1 and other proteins exhibit diverse functions in a PRC2-independent manner, such as ubiquitination of lysine 119 of H2A [41,42], and are also involved in transcriptional repression [43,44]. Previous studies in Drosophila melanogaster and Caenorhabditis elegans demonstrated that PcG proteins bind cis-acting DNA sequences and repress transcription, facilitating heterochromatin formation by binding to RNA.",Philosophical Transactions of the Royal Society B,Histone Modifications / Pluripotency,2013
Heterochromatin and H3K9 Regulation in Pluripotency,"For example, the incorporation of non-coding RNAs into PRC2 complexes has been observed. The PRC2 complexes interact with Xist RNA in mouse ES cells [51], whereas interaction between HOTAIR and SUZ12 has been observed in human fibroblasts. Such a gene repression mechanism may also be employed by mammalian pluripotent stem cells. Transcriptionally inactive heterochromatin is usually accompanied by H3K9 di- and tri-methylation (H3K9me2/3). Oct3/4 upregulates demethylases for H3K9me2/3, such as Jmjd1a and Jmjd2c, by interacting with their promoters. Demethylation of H3K9me2/3 by these demethylases contributes to the self-renewal of ES cells [52,53]. In fact, depletion of Jmjd1a and Jmjd2c leads to decreased expression of pluripotency genes and differentiation of ES cells. In contrast, H3K9 methyltransferases play important roles in early embryogenesis. G9a is an H3K9 methyltransferase essential for embryonic development [54], and prevents reprogramming by recruiting Dnmt3a and Dnmt3b to the promoters of Oct3/4 and HP1b [55]. Treatment with a G9a inhibitor increases iPS cell generation [56].",Philosophical Transactions of the Royal Society B,Histone Modifications / Pluripotency,2013
"ERV Silencing, Histone Acetylation, and Reprogramming","Although the molecular significance of silencing is unknown, ES cells are considered a good model for studying the relationship between DNA methylation and histone modifications because of their high de novo DNA methyltransferase activity [57]. Endogenous retroviruses (ERVs) are transcriptionally silenced in ES cells. The silencing of ERVs is initiated by the H3K9 methyltransferase ESET/SETDB1, with KRAB-associated protein 1 (KAP1/TRIM28) in a DNA methylation-independent manner [58,59]. This suggests that not only the global level of H3K9me2/3, but also context-dependent regulation of H3K9 methylation is involved in the maintenance of pluripotency and differentiation. The acetylation of histones is a significant modification in pluripotent stem cells. Acetylation is generally correlated with transcriptional activation, and is regulated by the balanced actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs) [62]. RNA interference screening identified HAT complexes including Tip60/p400 as ES cell development regulators [63,64]. HDAC inhibitors such as valproic acid and trichostatin A improve nuclear reprogramming efficiency by both nuclear transfer [65,66] and pluripotency factor transduction [67].",Philosophical Transactions of the Royal Society B,Histone Modifications / Pluripotency,2013
"Bivalent Domains, Poised Chromatin, and Reprogramming Barriers","One of the most distinctive features of histone modifications in pluripotent stem cells is ‘bivalent domains’, where both the active mark H3K4me3 and the repressive mark H3K27me3 are observed [25,68,69]. These marks occur at promoters of lineage-specific genes in pluripotent stem cells but rarely in differentiated cells [19,25,27,68,70,71]. Target genes in bivalent domains are ‘poised’ for expression—kept silent by H3K27me3 and dependent on H3K4 trimethylation. Genes in bivalent domains show low expression in pluripotent cells but switch to conventional activation or repression patterns by erasing opposite marks during differentiation [69,72]. ES cells lacking PRC2 components show derepression of lineage-specific genes [22,23,68]. Incomplete bivalent domain formation occurs in partially reprogrammed cells [19]. The Ink4/Arf locus is silenced early in reprogramming with formation of bivalent domains, and shRNA silencing of Ink4/Arf increases iPS cell generation, suggesting Ink4/Arf acts as a reprogramming barrier [73].",Philosophical Transactions of the Royal Society B,Histone Modifications / Pluripotency,2013
Histone Variants and Chromatin Architecture in Pluripotency,"In the last decade, the bivalent domain has been widely accepted as a key feature of pluripotent stem cells. However, recent findings revised this role. Mouse ES cells cultured in LIF with two small-molecule kinase inhibitors (2i) exhibit ground-state pluripotency and show decreased H3K27me3 on bivalent domains compared with ES cells in conventional media. H3K4me3 distribution remains similar, indicating that the bivalent domain is transiently formed during differentiation [74]. Replacement of canonical histones with specific variants modulates nucleosome dynamics and chromatin structure. H2AZ, a conserved H2A variant, is enriched in bivalent domains of developmentally important genes [78,79]. H2AZ depletion expels PcG proteins from bivalent domains, leading to derepression, though H2AZ is not essential for stemness [78]. MacroH2A, incorporated mainly into heterochromatin [80], was identified as a regulator of reprogramming [81]. Differential linker histone H1 composition has also been observed [82], and roles of additional histone variants in differentiation or reprogramming require further investigation.",Philosophical Transactions of the Royal Society B,Histone Modifications / Pluripotency,2013
DNA Methylation as a Barrier to Reprogramming,"3. DNA methylation and demethylation: modulating the barrier for reprogramming
DNA methylation maintains long-lasting cell memories, and is therefore considered to be a pivotal epigenetic barrier to cellular reprogramming [83]. During reprogramming, the activation of endogenous pluripotency genes including Oct3/4 and Nanog is accompanied by erasing the methylation of cytosines at their promoter regions. Insufficient DNA demethylation at the promoter regions, which is occasionally observed in partially reprogrammed iPS cells, fails to produce the robust reactivation of pluripotency genes [1,84–86]. In addition, the differential patterns of DNA methylation that are associated with genomic imprinting, retrotransposon silencing and X chromosome inactivation are observed between differentiated and pluripotent stem cells and among a series of pluripotent stem-cell lines [27,86–89], indicating that DNA methylation may be a suitable epigenetic marker for characterizing pluripotent stem-cell lines. Although it is unclear how such differential levels of DNA methylation arise, functional linkage between DNA methylation and reprogramming has been demonstrated. The inhibition of DNA methylation by chemical compounds or RNA interference targeting DNA methyltransferase can increase the efficiency of iPS cell generation [19].",Philosophical Transactions of the Royal Society B,DNA Methylation / Reprogramming,2013
CpG Density and Methylation Patterns in Pluripotent Cells,"Recent analyses using a high-performance sequencer have enabled mapping of DNA methylation with high resolution and have revealed an intriguing distribution of methylated cytosine in pluripotent stem cells. Since DNA methylation is frequently observed at CpG islands, which contain a high frequency of CpG sites, it is considered that the frequency of CpG sequences was positively correlated with the susceptibility to DNA methylation. However, the most recent studies of genome-wide DNA methylation status in pluripotent stem cells have produced observations that differ from the widely accepted model. The methylation levels of CpGs in pluripotent stem cells were negatively correlated with the local CpG density. In ES and iPS cells, regions with high CpG density exhibited low DNA methylation, whereas those with low CpG density exhibited high DNA methylation [27,87,90,91]. Regions with low CpG density are frequently observed in the promoters of tissue-specific genes [91], implying that the mechanism responsible for DNA methylation in the regulation of tissue-specific genes is different from that for DNA methylation in the regulation of other genes.",Philosophical Transactions of the Royal Society B,DNA Methylation / Reprogramming,2013
Non-CpG Methylation and Dnmt Function,"Intriguingly, DNA hypermethylation at the promoters of these tissue-specific genes with low CpG density is accompanied by bivalent chromatins in ES and iPS cells [91,92]. The relevance of this uniquely low CpG methylation level in pluripotent stem cells with bivalent domains is yet to be investigated at the molecular level; such information would provide important clues regarding the mechanisms of epigenetic regulation during differentiation. A single-base-resolution methylome analysis by whole-genome bisulphite sequencing (WGBS) also highlighted the significance of non-CG methylation in pluripotent stem cells [86,89]. Surprisingly, approximately one-quarter of all methylated cytosines in ES and iPS cells occurred in a non-CpG context, whereas most of the methylated cytosines in somatic cells were observed in CpG sequences. These pluripotent stem cell-specific non-CpG methylation sites tend to be located in the exonic regions of actively transcribed genes [86]. Studies using mice harbouring mutant DNA methyltransferases showed the importance of strict regulation of DNA methylation during normal development. Dnmt1 and Dnmt3a/Dnmt3b are essential enzymes for maintenance and establishment of DNA methylation [87,94,95].",Philosophical Transactions of the Royal Society B,DNA Methylation / Reprogramming,2013
Dnmt Mutants and Reprogramming Independence,"The loss of Dnmt1 causes the loss of two-thirds of total DNA methylation, leading to embryonic lethality [96]. Embryos with mutant Dnmt3b appear normal during early stages but exhibit developmental defects later [97]. The conditional deletion of Dnmt3b in mouse embryonic fibroblasts leads to a partial loss of DNA methylation [98]. However, although the Dnmt family plays essential roles in both development and germ cell reprogramming, de novo methylation by Dnmt3a and Dnmt3b is dispensable for the induction of iPS cells [99]. The mechanism by which methylated cytosine is converted into unmodified cytosine during reprogramming is elusive. Two global mechanisms have been proposed: replication-independent ‘active’ DNA demethylation and replication-dependent ‘passive’ DNA demethylation. Active demethylation is demonstrated by BER (base excision repair) machinery in plants and animals. Candidates for mammalian DNA demethylases include TDG [100,101], MBD4 [102], AID/APOBEC [103], GADD45A [104] and MBD2B [105–107], requiring coordinated action through the BER pathway [112]. However, their role in mammals remains controversial.",Philosophical Transactions of the Royal Society B,DNA Methylation / Reprogramming,2013
Tet Proteins and 5hmC in Pluripotency,"The recent findings of Tet family proteins as candidate DNA demethylases have advanced our understanding of DNA demethylation in pluripotent stem cells and other tissues [113–119]. Tet proteins catalyze the conversion of methylcytosine to 5-hydroxymethylcytosine (5hmC) in an Fe(II)- and α-ketoglutarate-dependent manner. Tet1 and Tet2 are highly expressed in mouse ES cells and are downregulated upon differentiation [116,119]. RNA interference silencing of Tet1 decreases expression of pluripotency genes such as Nanog, Esrrb, Klf4, Prdm14, Lefty1 and Lefty2, and increases trans-differentiation potential toward extra-embryonic lineages [93,116,119–121]. Genome-wide analyses revealed that Tet1 preferentially binds gene bodies and GC-rich promoter regions of both active and repressed genes [122]. Tet1-binding sites overlap with PcG targets [123], and proteomic analyses identified Sin3a as a Tet1-binding partner [93]. Knockdown of Tet1 reduces expression of PcG-target genes and pluripotency genes [120], indicating functions beyond PcG association.",Philosophical Transactions of the Royal Society B,DNA Methylation / Reprogramming,2013
"Tet1, 5hmC Readers, and Demethylation Pathways","Chromatin remodellers were also implicated in Tet1-mediated regulation. The Mbd3/NURD complex was identified as a ‘reader’ of 5hmC and directly recognizes 5hmC to control Tet1-target gene expression [124]. Mbd3/NURD functioning as a 5hmC reader may affect regulation of Tet1-target genes. Knockdown of Tet1 produces phenotypes similar to Mbd3 knockdown, including increased expression of trophectoderm markers, implying a functional link. In addition to 5hmC, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) were detected as intermediates of Tet1-mediated oxidation [125], and 5caC undergoes base excision by TDG in ES cells [126], suggesting that Tet1 oxidation followed by TDG excision is a possible active demethylation pathway. Tet1’s relevance to active demethylation and pluripotency has been demonstrated in vitro; however, Tet1 mutant mice are viable and fertile, and their ES cells show no defects in maintaining pluripotency [127]. Further studies are necessary to elucidate Tet1’s role in pluripotent stem cells.",Philosophical Transactions of the Royal Society B,DNA Methylation / Reprogramming,2013
ATP-Dependent Chromatin Remodeling in Pluripotency,"4. Implication of other epigenetic regulations: chromatin remodelling, high-order structure and non-coding RNA
Adenosine triphosphate (ATP)-dependent chromatin remodelling factors, which are capable of mobilizing or displacing nucleosomes at both the global and the locus-specific level [128–130], regulate gene expression programmes in early development and cell fate decisions [129,131,132]. SWI/SNF (switch/sucrose non-fermentable) complexes trigger the ejection of nucleosomes. The SWI/SNF factor is composed of two complexes Brg/Brahma-associated factors (BAF) and polybromo BAF (PBAF), and contributes to the self-renewal, proliferation or differentiation of ES cells. BRG1, a catalytic subunit of BAF, regulates the self-renewal and pluripotency of ES cells [133]. Knockdown of BRG1 by RNA interference results in morphological changes and a decreased proliferation of ES cells. BRG1 binds to the promoter regions of pluripotency genes such as Oct3/4, Sox2 and Nanog. Decreased expression of BRG1 downregulates the expression of pluripotency genes including Oct3/4, Sox2 and Sall4, accompanied by upregulation of differentiation-related genes such as Gata4 and Gata6. The involvement of esBAF, an ES cell-specific BAF complex, in self-renewal and pluripotency has also been reported [134–137]. esBAF enhances the binding of Oct3/4 to the target promoters, and facilitates the reprogramming of fibroblasts.",Philosophical Transactions of the Royal Society B,Chromatin Remodeling / Epigenetic Regulation,2013
"BAF, CHD, NuRD Complexes and Pluripotency","Although the molecular mechanism of esBAF-mediated facilitation of reprogramming has been explained, Brg1, the subunit of esBAF, also facilitates PcG function and represses the expression of classical PcG targets such as Hox genes, the expression of which is essential for the maintenance of pluripotency [139]. The CHD (chromodomain helicase DNA-binding) family complexes, as well as SWI/SNF complexes, trigger the ejection of nucleosomes, and are involved in the cell identity and function of ES cells [140–146]. CHD complexes are composed of the CHD enzymes, methyl-CpG-binding domain 3 (MBD3) and HDACs. The NuRD complex includes CHD3, CHD4 and MBD3, and is responsible for the deacetylation and trimethylation of H3K27. In addition, the NuRD complex is essential for maintaining both pluripotency and developmental transitions in early embryogenesis [140,147,148]. The roles of each component have been reported: CHD1 targets Oct3/4-binding sites and is required for efficient reprogramming of fibroblasts; silencing CHD1 blocks normal differentiation and causes accumulation of heterochromatin [144]. HDAC1 deletion results in aberrant ES cell differentiation toward mesoderm and ectoderm and leads to embryonic lethality [149–153]. ES cells lacking MBD3 express trophectoderm markers and show sustained high Oct3/4 expression [140,146].",Philosophical Transactions of the Royal Society B,Chromatin Remodeling / Epigenetic Regulation,2013
INO80 Complexes and High-Order Chromatin Architecture,"The restriction of interaction of MBD3 with the SWI/SNF component BRG1 to pluripotent stem cells [137] implies that crosstalk among chromatin remodelling complexes regulates pluripotency. The TIP60/p400 complexes belonging to the INO80 family regulate gene transcription by depositioning histone variants H2A.Z into chromatin [130]. Knockdown of Tip60/p400 expression in ES cells resulted in aberrant morphology and a loss of pluripotency. The expression profile of Tip60/p400-silenced cells was similar to that of Nanog-silenced cells [63], suggesting that Nanog and Tip60/p400 cooperatively maintain pluripotency in ES cells. Bprt, a member of the ISWI family proteins, is also involved in early embryonic growth and represses differentiation markers in ES cells [154]. The organization of high-order chromatin structures has emerged as key machinery of genome regulation [155–157]. ES cells possess loosely compacted euchromatin and develop highly condensed heterochromatin during differentiation [158,159]. Another study using fully reprogrammed iPS cells with high Nanog expression and partially reprogrammed iPS cells morphologically similar to ES cells but lacking Nanog expression revealed that fully reprogrammed cells lose the ability to form heterochromatin [21].",Philosophical Transactions of the Royal Society B,Chromatin Remodeling / Epigenetic Regulation,2013
"Nuclear Lamina, miRNAs and LIN28","Not only loci-specific heterochromatin formation but also nuclear lamina, nucleolus and nuclear speckles may affect chromatin architecture. The role of the lamina in pluripotency remains controversial: one report indicates morphological differences between pluripotent and differentiated cells [20], while another suggests B-type laminas are not required for ES cells [160]. RNA occasionally acts as a chromatin regulator. MicroRNAs (miRNAs) regulate post-transcriptional gene expression [161]. Their involvement in pluripotency was suggested by the finding that miRNA expression is regulated by the core transcriptional circuit in ES cells [162]. Mice lacking Dicer or Dgcr8 show essential roles of miRNAs in ES cell proliferation and differentiation [163–165]. However, Dgcr8-deficient ES cells maintain self-renewal despite cell cycle defects and aberrant differentiation [163]. This complex regulation is partially explained by antagonism between miRNA-294, a cell-cycle regulator, and the let-7 family, abundant in somatic cells [166]. miRNA-294 downregulates let-7 via stabilization of LIN28, a let-7 inhibitor highly expressed in pluripotent cells [167,168]. LIN28 facilitates somatic cell reprogramming with Oct3/4, Sox2 and Nanog [169].",Philosophical Transactions of the Royal Society B,Chromatin Remodeling / Epigenetic Regulation,2013
"miRNA Feedback Loops, Imprinting and XCI","let-7 downregulates MYC in cancer cells via let-7 binding sites on MYC 3′UTR, whereas MYC overexpression decreases let-7 expression [170,171]. These observations reveal a direct double-negative feedback loop and imply similar capacities of MYC and LIN28 to promote induction of pluripotency. However, further study is needed to characterize the autoregulatory loop between MYC and LIN28. Although previous studies showed similar mRNA and miRNA profiles among ES and iPS clones, others reported differential expression of transcripts from imprinted regions or pluripotency-related genes. Some iPS clones show aberrant silencing at the Dlk-Dio3 gene cluster on mouse chromosome 12F1, associated with poor contribution to chimeric mice [172]. Another report found contradictory evidence, showing that reprogramming factor stoichiometry, not Dlk1-Dio3 imprinting, determines iPS cell quality [160]. Further studies are needed to identify molecular features predictive of pluripotent cell quality. X chromosome inactivation (XCI) is a regulatory mechanism by which one X chromosome is silenced in female cells [173,174]. Mouse ES cells or fully reprogrammed iPS cells carry two active X chromosomes (XaXa), and differentiation initiates XCI to equalize X-linked gene expression between sexes.",Philosophical Transactions of the Royal Society B,Chromatin Remodeling / Epigenetic Regulation,2013
X Chromosome Reactivation and Pluripotency States,"Reprogramming of female mouse fibroblasts faithfully reactivated the silenced X chromosome [175]. However, some human ES and iPS cells retain an inactive X chromosome [176]. Human pluripotent stem cells share characteristics with mouse epiblast stem cells (EpiSCs), suggesting that human pluripotent cells are in a ‘primed’ pluripotent state, whereas mouse ES/iPS cells are in a ‘naive’ state [177]. Since diverse mechanisms for XCI initiation exist among mammals, these differences may explain inconsistencies in X chromosome reactivation between mouse and human pluripotent cells [178]. Recent reports describe human iPS cells with ground-state pluripotency and X chromosome reactivation under specific culture conditions [179,180]. Further studies are required to understand the molecular mechanisms underlying X chromosome inactivation and reactivation during reprogramming [181].",Philosophical Transactions of the Royal Society B,Chromatin Remodeling / Epigenetic Regulation,2013
Epigenetic Models and Reprogramming Insights,"5. Conclusions and perspectives
Pluripotent stem cells have been used as a cell model for understanding the molecular mechanism of cellular differentiation and a source of cells for regenerative medicine. Studies of stem-cell identity and the fate of pluripotent stem cells upon differentiation have advanced remarkably over the last few decades. Many studies, including a recent genome-wide analysis of epigenetic modifications, support the classical ‘landscape model’ of Waddington, which describes irreversible cell differentiation. Our growing understanding of epigenetic regulation in pluripotent stem cells and their dynamic changes during differentiation can be used to update this model, which represents not only cell fate but also the coupling of developmental potential with the epigenetic status of the cells during differentiation. The recent discovery of iPS cells has enabled us to dissect epigenetic regulation during reprogramming and differentiation [1,182]. Reprogramming, the reverse of differentiation, is achieved by breaking the barrier of the differentiated state. Dissection of epigenetic regulation during the reprogramming process may provide a description of how cells sustain their fate and may provide candidates for molecules that act as guardians of differentiation.",Philosophical Transactions of the Royal Society B,Pluripotency / Epigenetic Regulation,2013
Guardian Molecules and Quality Control in Pluripotent Cells,"The identification of guardian molecules that are responsible for the differentiated state of cells will be of use in the efficient generation of iPS cells. Reprogramming factors, including Oct3/4 and Sox2, are thought to be inducers of pluripotency and may also act as ‘destroyers’ of the differentiated state. It will be of interest to know how reprogramming factors contribute to the destruction of the epigenetic barrier in the early stage of the reprogramming process, and whether such a mechanism directly regulates the master regulators that maintain the differentiated state, or break the differentiated state through genome-wide alteration of epigenetic status. Pluripotent stem cells are now suggested as an artificial source of tissues, and consequently it is necessary to be able to guarantee their safety in the human body after transplantation. However, both ES cells and iPS cells are produced after long-term culture, and thus harbour clone-to-clone variations in their epigenetic profiles as well as DNA sequences and copy numbers. Differences in iPS cells among clones have been reported [85], and it is therefore important to validate the quality of pluripotent stem cells including ES and iPS cells by genomic and epigenomic analyses.",Philosophical Transactions of the Royal Society B,Pluripotency / Epigenetic Regulation,2013
Epigenetic Memory and Evaluating iPS Cell Quality,"Methylome analysis may be a good candidate for evaluating the quality of pluripotent stem cells, since DNA methylation is stable and acts as a source of long-term memory. Some residual DNA methylation signatures observed in iPS cells show characteristics of their somatic tissues of origin, implying the presence of epigenetic memory [85,183]. Considering the recent reports of variation among clones in terms of the characteristics of pluripotent stem cells, it is crucial to establish methods for the reliable evaluation of the quality of iPS cells, which should eventually be useful for generating clinical-grade iPS cells for use in regenerative medicine. Analysis using deep sequencers has also revealed non-negligible differences among individuals in the genome and epigenome. One of the advantages of using iPS cells as pluripotent stem cells is the ability to analyse and compare established iPS cells with the original somatic cells. In other words, with these cells, it is possible to distinguish whether the observed alterations in the genome/epigenome represent aberrations acquired during long-term cell culture or the individual variation that is normally observed among the individuals.",Philosophical Transactions of the Royal Society B,Pluripotency / Epigenetic Regulation,2013
Future Directions in Epigenetic Profiling and Regenerative Medicine,"There is increasing evidence of the importance of epigenetic regulation in maintaining pluripotency and the reprogramming process. Current high-performance sequencers make it possible to screen for genomic alterations at the whole-genome level, and can be used to guarantee that the cells are of clinical grade, on the basis of their genomic sequence. It is also important to examine the epigenetic profile of pluripotent stem cells, because the epigenetic landscape represents both the past and the current developmental state, and may be a useful indicator to predict their future potential. Further advances in the understanding of epigenetic regulation hold promise for the molecular understanding of cell fate and the realization of regenerative medicine using pluripotent stem cells.",Philosophical Transactions of the Royal Society B,Pluripotency / Epigenetic Regulation,2013
Abstract and Overview of Epigenetic Rejuvenation,"Rejuvenation of cells by reprogramming toward the pluripotent state raises increasing attention. In fact, generation of induced pluripotent stem cells (iPSCs) completely reverses age-associated molecular features, including elongation of telomeres, resetting of epigenetic clocks and age-associated transcriptomic changes, and even evasion of replicative senescence. However, reprogramming into iPSCs also entails complete de-differentiation with loss of cellular identity, as well as the risk of teratoma formation in anti-ageing treatment paradigms. Recent studies indicate that partial reprogramming by limited exposure to reprogramming factors can reset epigenetic ageing clocks while maintaining cellular identity. So far, there is no commonly accepted definition of partial reprogramming, which is alternatively called interrupted reprogramming, and it remains to be elucidated how the process can be controlled and if it resembles a stable intermediate state. In this review, we discuss if the rejuvenation program can be uncoupled from the pluripotency program or if ageing and cell fate determination are inextricably linked. Alternative rejuvenation approaches with reprogramming into a pluripotent state, partial reprogramming, transdifferentiation, and the possibility of selective resetting of cellular clocks are also discussed.",BioEssays,Partial Reprogramming,2023
Introduction to Cellular Ageing Reversal,"Cellular ageing is reversed by reprogramming in induced pluripotent stem cells. Ageing is a complex process that entails continuously increasing dysfunction and degeneration, susceptibility to disease, and, ultimately, death. While this process is inevitable and associated with continuous molecular changes, it is remarkable that most effects of ageing can be reversed at the cellular level. The groundbreaking work by Takahashi and Yamanaka in 2006 demonstrated that somatic cells could be reprogrammed into induced pluripotent stem cells (iPSCs) by the overexpression of four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc, now referred to as the “Yamanaka factors” or “OSKM” factors), altering the epigenetic landscape of these cells.[1] While in the pluripotent state, iPSCs can be passaged extensively, without any signs of cellular ageing. Seminal work by Marion et al. showed that even reprogramming of cells from elderly animals resulted in iPSCs with elongated telomeres, efficient telomere capping, and a heterochromatin profile similar to embryonic stem cells (ESCs).[2] Fibroblasts from very old donors and centenarians could also be reprogrammed into iPSCs by OSKM overexpression – and even more effectively in combination with Nanog and LIN28.[3]",BioEssays,Partial Reprogramming,2023
Evidence for Reversal of Ageing Features in iPSCs,"For example, B-lymphoblastoid cell lines derived from a supercentenarian (114-year-old donor) were reprogrammed, and the iPSCs exhibited complete erasure of the age-associated transcriptomic signature and resetting of telomeres.[4] Last but not least, iPSCs derived from elderly mice could give rise to healthy newborn mice that pursue normal ageing.[5] Aging may also involve accumulation of somatic mutations, which are certainly not repaired by the reprogramming procedure. Yet, the acquisition of mutations may rather be a side effect than the underlying mechanism of aging. Either way, there is compelling evidence that molecular features of ageing can be reversed by reprogramming into the pluripotent state. On the other hand, if the iPSCs are re-differentiated toward mature cell types, they keep this rejuvenated phenotype. For example, when T cell-derived iPSCs were re-differentiated into CD8+ T cells, they maintained high proliferation and elongated telomeres[6]; when iPSCs that were derived from skin fibroblasts of aged humans were re-differentiated into neurons[7] or fibroblasts,[8] their age-associated transcriptomic changes, nucleo-cytoplasmic compartmentalization, and oxygen consumption levels remained rejuvenated; and iPSC-derived mesenchymal stromal cells (iMSCs) also stayed epigenetically rejuvenated.[9]",BioEssays,Partial Reprogramming,2023
Limitations of Using iPSC-Derived Cells in Ageing Research,"This aspect should be considered, because iPSC-derived cells are often used as model systems for age-associated diseases, for example, iPSC-derived neurons for research in Alzheimer’s disease.[10] However, these in vitro differentiated cells somewhat resemble a fetal phenotype with regard to telomere length or epigenetic age. So far, it is impossible to accelerate these age-associated cellular changes in the iPSC-derived cells to facilitate better mimicking of the physiological disease state of the elderly.",BioEssays,Partial Reprogramming,2023
Resetting DNA Methylation Clocks,"DNA methylation (DNAm) is one of the central epigenetic modifications, which emerged across different kingdoms of life and revealed a range of essential biological functions.[11 ] DNAm was initially described in the context of cellular differentiation and development,[12 ] but epigenetic patterns changes also continuously with aging of the organism.[13 ] In the mammalian system, DNAm occurs particularly in the context of CG dinucleotides (CpG sites). So far, it is largely unclear how the tight regulation of DNAm patterns at specific genome sites is governed. While DNAm is generally considered to be associated with gene repression, it may also activate gene expression – depending on where it occurs within a gene and how it affects the binding of transcription factors and enhancers.[14 ] Furthermore, there is evidence that DNAm patterns reflect modifications in the histone code.[15 ] Even if the direct functional relevance of DNAm remains unclear, it can undoubtedly provide beneficial biomarkers for cellular composition, various diseases, and particularly for the ageing process.[16 ] More than a decade ago, the first epigenetic age-predictors based on DNAm, called epigenetic clocks, were described.[17,18 ] Since then, numerous epigenetic clocks have been developed to predict chronological age. They were particularly trained for blood,[19–21 ] while others have even proven to be applicable across many tissues.[22 ] Notably, there is evidence that the deviation of predicted and chronological age is associated with all-cause mortality and prevalence of age-associated features, indicating that epigenetic clocks provide a measure for biological age rather than chronological age.[13,21,23 ] Interestingly, upon reprogramming into iPSCs, virtually all age-associated DNAm changes are reversed.[20,22,24 ] The predicted epigenetic age is thus close to zero years, even if reprogramming was performed on cells from elderly donors. This resetting occurs parallel to DNAm changes in pluripotency-associated CpGs,[25,26 ] indicating that resetting cellular identity to a pluripotent state and rejuvenation might be directly linked.",BioEssays,Epigenetic Clocks,2023
Resetting Culture-Associated DNA Methylation Changes,"In addition to the ageing of the organism, long-term in vitro cell culture is also reflected by specific DNAm changes. Cells can only be propagated for a limited number of passages until they reach a state of replicative senescence, and DNAm changes are found in specific senescence-associated CpGs.[27 ] In fact, they are continuously acquired during cell culture and can therefore be utilized to estimate the in vitro culture time. Epigenetic clocks and culture-associated DNAm changes tick independently, albeit with some overlap in these signatures. Notably, the culture-associated epigenetic clocks can also be reversed by reprogramming into the pluripotent state.[24,25,28 ] However, in contrast to the epigenetic ageing clocks, the culture-associated DNAm changes are gradually reacquired as soon as the cells exit from the pluripotent state.[9] In the future, it will be interesting to understand better if specific culture conditions, co-culture, or matrices can modulate the gradual re-acquisition of culture-associated DNAm patterns.",BioEssays,Epigenetic Clocks,2023
Resetting Other Epigenetic Marks,"In addition to DNAm, other epigenetic modifications have also been associated with ageing. DNA is packaged by core histone proteins that are subject to numerous post-translational modifications.[29 ] These histone modifications regulate gene expression by governing the accessibility of the transcription machinery. Many studies demonstrated that the histone code is also altered in an age-dependent manner.[30 ] These changes include a reduction in bulk histone levels, a global decrease in repressive histone modifications such as H3K9me3, H4K20me3 (reduced in ageing but increased in senescent cells),[31 ] and H3K27me3. Furthermore, there is age-associated remodeling of activating histone modifications such as H3K4me3 and H3K36me2/me3. Interestingly, while H3K27me3 marks reduce with age in Caenorhabditis elegans and human progeria model systems, they seem to increase in mouse quiescent satellite cells.[32–34 ] Similarly, H3K4me3 has also been shown to promote or inhibit longevity in a context-dependent manner.[35 ] Cells from patients with premature ageing disorders, such as Hutchinson–Gilford Progeria and Werner Syndrome, showed reduced expression of the H3K9me3 methyltransferase SUV39H1 as well as the heterochromatin protein HP1.[36,37 ] Recently a novel epigenetic ageing clock was developed using chromatin accessibility profiles (ATAC-Seq) generated from peripheral blood mononuclear cells (PBMCs) isolated from young and old donors, which correlated with age-associated gene expression changes more significantly than the DNAm-based ageing clocks.[38 ] It would be interesting to correlate age-associated changes in histone modifications, chromatin accessibility, and DNAm, given the strong association between these regulatory pathways,[15,39 ] potentially leading to the development of more comprehensive epigenetic-ageing signatures.",BioEssays,Epigenetic Clocks,2023
Possibility of Uncoupling Reprogramming and Rejuvenation,"While, in principle, reprogramming has been shown to reverse age-related cellular phenotypes and epigenetic changes this is still not applicable for rejuvenation in the clinical setting. The process of reprogramming into pluripotent state also reverses the features of cellular differentiation – which notoriously affects functional integrity of the rejuvenated tissue. Furthermore, reprogramming toward iPSCs bears the risk of teratoma formation.[40,41 ] It has been suggested that transient expression of the OSKM factors can result in a de-differentiated progenitor-like state, that still maintain some of the cellular characteristics without reaching pluripotent state.[42,43 ] This approach of partial reprogramming gains a lot of attention for possible rejuvenation approaches. Despite the many encouraging reports, it remains to be further validated if intermediate or partial reprogramming that stably reverses epigenetic ageing over a long period while retaining cell identity can really be reproducibly achieved. If such an intermediate state is stable, it might successfully uncouple cell fate from age-associated changes and serve as a novel paradigm for understanding and applying rejuvenation through reprogramming in a clinical setting. To this end, epigenetic ageing and epigenetic modifications during cellular differentiation must also be uncoupled.",BioEssays,Partial Reprogramming,2023
Four Scenarios for Reprogramming–Rejuvenation Relationship,"Here, the following four scenarios are conceivable (Figure 1):
1. Ageing and cellular differentiation during development are directly linked and cannot be uncoupled. In this scenario, complete reprogramming and the resulting de-differentiation are essential for resetting the epigenetic clock, and further differentiation of the iPSCs is necessary to re-establish function. The observations of partial reprogramming might then instead resemble a transient state toward pluripotency or reflect heterogeneity in reprogramming. In fact, it has been suggested that the reversal of epigenetic clocks occurs simultaneously with changes in pluripotency-associated CpGs.[25 ]
2. Partial uncoupling of age-associated changes and cell fate determination: In this scenario, age-related epigenetic changes, cellular senescence, and cell fate would be co-regulated but not directly interlinked. This might be achieved by interrupted reprogramming, for example, by not changing to culture conditions that maintain a pluripotent state or limiting the duration of exposure to reprogramming factors. The possibility of partial reprogramming is supported by studies indicating that loss of cell-type specific gene expression, for example, for fibroblast lineage, follows different kinetics than the reversal of epigenetic clocks, suggesting a safe time window for rejuvenation without complete erasure of somatic identity.[26,44 ]
3. Age-associated changes can be entirely uncoupled from cellular specification and differentiation. Here, it would be possible to maintain all cellular characteristics, including epigenetic features for the specific tissue and anatomic location, while resetting epigenetic ageing. It might even be feasible to titrate the rejuvenation to a particular age without resetting all the way back to the embryonic state. However, cell-type-specific epigenetic patterns and ageing-associated patterns overlap. While it is still unclear how these exact genome-wide DNAm changes are regulated, it appears unlikely that the two processes can be entirely uncoupled.
4. Rejuvenation during transdifferentiation into other cell types: It has been suggested that direct conversion, for example, of blood cells into neurons, gives rise to specified cells with a rejuvenated transcriptomic and epigenetic state.[45 ] It is yet unclear if epigenetic modulation in direct reprogramming may briefly touch a pluripotency-associated state. Furthermore, there is evidence that the rejuvenated intermediate state is not stable but further declines during culture expansion (Flitch et al., unpublished findings). It will be necessary to better understand cellular heterogeneity in direct cell fate conversion and if the new cellular state is stable.",BioEssays,Partial Reprogramming,2023
Kinetics of Rejuvenation and Influence of Reprogramming Factors,"In the following sections, we want to highlight some relevant studies that support the general feasibility of partial reprogramming. For a historic time line of major discoveries, we would like to refer to other review articles.[46–48 ]
Evidence for partial reprogramming from mouse in vivo studies
In a landmark study, the group of Carlos Izpisua Belmonte[49 ] induced partial in vivo cellular reprogramming in a Hutchison-Gilford Progeria Syndrome mouse model. They expressed the OSKM factors cyclically (2 days on and 5 days off) using a doxycycline-inducible reprogramming cassette. This method resulted in the reversal of multiple hallmarks of ageing, such as reduced accumulation of DNA damage and cellular senescence, demonstrating the potential of partial reprogramming in rejuvenation. At the organism level, cyclical expression of OSKM led to an extended lifespan in progeroid mice, which was closer to a normal murine life span. Furthermore, it improved age-associated histological changes in multiple organs such as skin, spleen, kidney, and heart, expansion in muscle satellite cells, and an improvement in muscle regeneration after injury in naturally aged and progeroid mice. While the findings of this study led to crucial insights into partial reprogramming and rejuvenation, the authors did not quantify epigenetic age.",BioEssays,Partial Reprogramming,2023
OSKM-Induced Rejuvenation Across Mouse Organs,"Using the same OSKM expression system, a more recent study indicated that a single cycle of transient OSKM expression in naturally aged mice could partially reverse epigenetic and transcriptional changes in the pancreas, liver, spleen, and blood cells that are maintained after the termination of the OSKM expression.[50 ] Consistent with this data, analysis of serum metabolites revealed a reversal of the metabolomics profiles toward a younger profile. Specifically, 4-hydroxyproline and thymine, that declined with age, showed an increase upon OSKM treatment. This study provides evidence that low levels of OSKM are sufficient to induce transcriptomic and epigenetic rejuvenation, thus minimizing the risk of teratoma formation associated with long-term OSKM treatment. The authors also argue that the recovery period after the OSKM expression was critical as they detected many rejuvenation changes a week after stopping of OSKM expression, indicating that rejuvenation events continue even during recovery period post-OSKM termination. In analogy, another recent study demonstrated that single short reprogramming induction is sufficient to prevent musculoskeletal deterioration in mice when applied in early life.[51 ] These authors also suggested the reversal of age-associated epigenetic changes but did not test established epigenetic clocks.",BioEssays,Partial Reprogramming,2023
Organ-Specific Effects of Transient OSKM Expression,"At the organ level, Chen et al. expressed OSKM in cardiomyocytes of mice for 6 or 12 days and saw that the functional, morphological, and gene expression profile reversed to neonatal stages.[52 ] Transient OSKM expression also extended the regenerative window of juvenile murine hearts and enabled adult heart repair through the proliferation of preexisting cardiomyocytes, indicating a strong potential for partial reprogramming in achieving functional cardiac recovery in a clinical setting. Similarly, Lu et al. used Tet-Off AAV2s carrying OSK as a single polycistron injected into the vitreous body to induce OSK in murine retinal ganglion cells (RGCs) of the central nervous system.[53 ] They observed significant regeneration, survival, and sprouting of RGC axon fibers upon induction of OSK, and this was reversed upon the suppression of OSK. Furthermore, OSK induction also reversed global transcription and DNAm changes caused by injury or natural ageing, specifically at light detection and synaptic transmission genes. Similar effects were seen in differentiated human neurons where OSK expression reversed the axonal loss and DNAm age after injury. OSK expression was also shown to reverse vision impairment in glaucomatous eyes and during natural ageing. However, this restoration of visual acuity was not seen beyond a certain age, indicating a temporal cut-off.",BioEssays,Partial Reprogramming,2023
Limitations and Translational Challenges in Mouse Studies,"While the above studies provide important insights suggesting that limited exposure to OSKM can successfully rejuvenate cells and organs without alteration in cell identity, it must be noted that the premature ageing murine genetic model does not fully recapitulate the complexity of natural ageing. Additionally, these studies do not account for cellular heterogeneity or analyze the long-term rejuvenation effects to determine stability. Notably, this murine model’s doxycycline-inducible OSKM expression system can hardly be translated for clinical application. It is challenging to examine efficacy and safety in human patients based on animal studies. This necessitates alternative approaches and further work on understanding the role of reprogramming in rejuvenating cells derived from human donors.",BioEssays,Partial Reprogramming,2023
Partial Reprogramming in Human Cells: Early Evidence,"Recently, Sarkar et al.[54 ] indicated that transient expression of reprogramming factors could reverse ageing hallmarks without erasing cell identity in naturally aged human cells. Their experiments in fibroblasts and endothelial cells showed that OSKLMN expression promoted activation of a more youthful gene expression profile, heterochromatin state, autophagy levels, and mitochondrial membrane potential. Epigenetic clock analysis indicated that transient reprogramming had a moderate effect on methylation age, which was more pronounced in endothelial cells (average age difference = −4.94 years) than in fibroblasts (average age difference = −1.84 years). Expression levels of cell identity genes and telomere length remained unaltered, indicating that the cells did not de-differentiate. This study was also extended to aged chondrocytes from osteoarthritic human donors as well as murine skeletal muscle stem cells and showed a partial reversal of gene expression and cellular physiology to a more youthful state in chondrocytes and improved regenerative potential in MuSc. By identifying day four of transfection as the point of no return during cellular reprogramming when age-associated changes are reversed before epigenetic reprogramming of cellular identity occurs, this study provides insights into the temporal sequence of partial reprogramming. However, further validation may be required since the epigenetic rejuvenation was only very moderate, and there is notorious offset between such experiments. Either way, a rejuvenation of only 4 years might not be sufficient for many clinical applications. It is also unclear if the rejuvenation remains stable or if a subset of de-differentiated cells might ultimately outgrow the other cells.",BioEssays,Partial Reprogramming,2023
Extended Human Cell Studies and Optimization of Reprogramming Duration,"Gill et al. used a novel strategy where OSKM factors were expressed using a doxycycline-inducible polycistronic cassette encoding OSKM and GFP in fibroblasts isolated from middle ages donors by lentiviral transduction.[44 ] This expression was terminated after the maturation phase of reprogramming at different lengths of time (10, 13, 15, or 17 days). They observed robust transcriptional rejuvenation (approximately 20 years), partial functional rejuvenation, and substantial reduction of DNAm age (approximately 30 years) whilst retaining overall cell identity. Interestingly, the authors reported that 13 days of reprogramming was optimal for rejuvenation and lower or higher reprogramming periods led to reduced rejuvenation. It should be noted that this rejuvenation was based on DNAm clocks, which may be affected by the heterogeneity within the samples and the presence of outliers among the age-associated CpGs. Also, the rejuvenation was calculated based on the Horvath clock,[22 ] which is a multi-tissue clock trained for 353 CpGs. It is difficult to determine whether the change in DNAm occurs over all the CpGs or rather in a subset of these CpGs. Analyzing the changes at individual CpGs would better elucidate the mechanism of epigenetic rejuvenation. Interestingly, transient reprogramming did not lead to telomere elongation,[44 ] indicating that the senescence pathway is not reversed. It would be interesting to compare this strategy with the cyclical expression of reprogramming factors, as described by Ocampo et al.,[49 ] to narrow down the temporal window of partial reprogramming to permit efficient rejuvenation without loss of cell function.",BioEssays,Partial Reprogramming,2023
Introduction and Context of Aging Research,"Life span extension by resveratrol, rapamycin, and metformin: The promise of dietary restriction mimetics for an healthy aging. Laurent Mouchiroud, Laurent Molin, Nicolas Dallière, and Florence Solari. Life expectancy at the turn of the 20th century was 46 years on average worldwide and it is around 65 years today. The correlative increase in age-associated diseases incidence has a profound public health impact and is an important matter of concern for our societies. Aging is a complex, heterogeneous, and multifactorial phenomenon, which is the consequence of multiple interactions between genes and environment. In this review, we survey animals models that have been of great help for both investigating mechanism of aging and identifying molecules, which slow down the onset of age-related diseases. Resveratrol (RSV) is one of those. We will report evidences supporting RSV as a molecule that acts by mimicking the beneficial effects of dietary restriction, and may share common downstream targets with rapamycin and metformin. Although those molecules do not reveal all the secrets of the fountain of youth, they may help us maintaining the quality of life in the old age.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Genetic Pathways Conserved Across Evolution,"Aging-related research was primarily focused on understanding the molecular processes that cause aging. This has been first addressed by looking for genetic manipulations that extend life span of simple model organism. Initial evidence for gene impact on life span came from the work done in late 80s with the nematode Caenorhabditis elegans, in which mutations that reduce the activity of the PI3 kinase ortholog AGE-1 or of the insulin/IGF-1 receptor (IIR) can double average life span. Later on, the role of the IIR pathway in life span regulation was shown to be conserved in higher organisms such as mice. Another important, nongenetic, intervention that increases life span throughout the animal kingdom is the reduction of caloric intake, also called dietary restriction (DR), to 30–50% below ad libitum levels without malnutrition. DR has been shown to increase life span in yeast, nematode, flies, rodents, and also more recently in primates. Furthermore, DR delays the onset of age-related diseases, improves stress resistance, and decelerates functional decline, and its beneficial effects have been observed to some extent in human.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Resveratrol Extends Lifespan by Mimicking Dietary Restriction,"A number of observations suggested that DR beneficial effects result from highly regulated processes, rather than passive metabolic changes, which depend on the activation of specific effectors. The Guarente laboratory reported in 2000 that the life span increase induced by calorie restriction in the budding yeast Saccharomyces cerevisae was no longer effective in SIR2 mutants. SIR2 belongs to the sirtuin family of NAD+-dependent deacetylases homologous to SIRT1 in mammals. Extra copies of SIR2 or its homologs extend life span in yeast, worms, and flies. Furthermore, SIRT1 is required for the induction of physiological changes associated to DR in mice and its overexpression mimics DR beneficial effects. Those observations supported the idea that if a drug can alter the activity of sirtuins, then DR health benefits may be recapitulated pharmacologically. Resveratrol (RSV) was initially identified in 2003 from an in vitro screen aimed at isolating molecules that activate SIRT1. RSV treatment extends the life span between 18 and 56% of S. cerevisae, C. elegans, and Drosophila melanogaster and its effect depends on SIR2, although modulation of life span by RSV may require specific experimental conditions.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Resveratrol-Induced Physiological Changes in Mice,"Studies in rodents have confirmed beneficial effects of RSV treatment on several physiological parameters in obese mice. It reversed multiple deleterious effects induced by high calorie diet including impaired glucose homeostasis, insulin resistance, dyslipidemia, endurance exercise, and cardiovascular dysfunction. Those effects account for increased survival of mice to the point where it was not significantly different from nonobese standard diet mice. In 2008, two independent studies reported beneficial effects of RSV on health and life span in standard diet conditions compared to dietary restriction. They investigated gene expression changes during aging in several tissues and physiological signs of aging, showing that both DR and RSV have overlapping effects on gene expression in liver, heart, skeletal muscle, adipose tissue, and neocortex. RSV slowed age-related decline of several organs and functions and its effects were not dependent on weight loss. Additionally, RSV protects against neurodegenerative diseases, cancer, and cardiovascular disease, although under standard diet RSV did not increase overall survival or maximum life span. Nevertheless, RSValong with every-other-day feeding increased life span significantly compared with standard diet alone.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Resveratrol Molecular Targets Beyond Sirtuins,"4. Resveratrol molecular targets and effectors that mediate its effect on health and life span: From sirtuins to AMPK and PGC-1alpha. Until recently, sirtuins appeared as the best candidate for being both a direct target and an important effector of RSV beneficial health effects. Regarding obesity, other sirtuins activators have also shown similar protection in mice, suggesting that this effect of reveratrol is likely mediated by sirtuins. However, the extent to which the sirtuin-activating actions of RSV are direct has been questioned during the last years. Indeed, biochemical approaches indicated that RSV may not activate sirtuins toward biological relevant substrates and that target activation requires the presence of a covalently bound fluorophore. Thus, although a number of evidences indicate that sirtuins mediate at least some of RSV effects, they may not be RSV direct target(s). Other potential RSV substrates have been identified in vitro and/or in vivo which includes AMP-activated protein kinase (AMPK), which has also been shown to activate SIRT1. AMPK plays a central role in cellular energy homeostasis and is activated by conditions that deplete ATP and increase AMP:ATP ratio, such as hypoxia, hypoglycaemia, and ischaemia. Furthermore, AMPK is also a direct substrate for the oncosuppressive serine threonine kinase LKB1.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
"AMPK, PGC-1α, and Lifespan Extension Mechanisms","AMPK activation leads to the phosphorylation of a large number of downstream targets leading to an overall inactivation of energy-using/anabolic pathways while energy-generating/catabolic pathways are switched on, thus helping to restore the energy balance within the cell. AMPK has very recently emerged as another potentially important aging-related target of RSV. RSV activates AMPK in vivo and overexpression of AMPK has been shown to increase life span in nematode. The essential role of AMPK for RSV in vivo response has been recently demonstrated by Um et al. who used mice deficient in the catalytic subunit of AMPK (alpha1 or alpha2). These mice did not benefit from RSV treatment under high calorie regimen. Furthermore, in embryonic fibroblast isolated from those mice, RSV failed to increase peroxisome proliferators-activated receptor gamma coactivator-1 alpha (PGC-1alpha) target genes. AMPK phosphorylates and activates PGC-1alpha, resulting in the upregulation of mitochondrial biogenesis, which is a conserved feature in caloric restriction. Activation of PGC-1alpha also improves mitochondria function by inducing genes expression for mitochondrial and fatty acid oxidation and by increasing mitochondrial membrane potential. Interestingly, SIRT1 may also control PGC1alpha activation via its deacetylation.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
"Interplay Between AMPK, SIRT1, and Additional RSV Targets","Future studies should examine whether the same is true for other mutual substrate of AMPK and SIRT1, such as Forkhead box class O. The functional interplay between AMPK and SIRT1 is complex and depends on the cellular context. Indeed, data obtained in condition of chronic treatment by RSV of hepatocytes suggest that SIRT1 can also lay upstream LKB1/AMPK to control lipid metabolism. This finding is supported by the capacity of SIRT1 to activate LKB1 via its direct deacetylation. Furthermore, AMPK activation by RSV in neurons is LKB1 dependent but SIRT1 independent. Additionally, AMPK activation may also be secondary to the direct inhibitory RSV interaction with the mitochondrial F1 ATPase, which would result in ATP depletion. Overall, although the mechanisms responsible for AMPK activation by RSV are not completely clear, overall data suggest an important function of AMPK as an effector of RSV-induced beneficial effects. RSV has been shown to have a number of potential targets, which can be either activated or inhibited. Their respective role in the regulation of both health and life span would deserve to be tested. Among other, PI3K, which is inhibited by RSV, would be a good candidate given its powerful life span lengthening effect in worms and its important role in human tumors.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Metformin as a Dietary Restriction Mimetic,"5. Other potential dietary restriction mimetics: Metformin and rapamycin. Two other molecules, rapamycin and metformin, have been shown to improve age-associated diseases and may act through similar mechanisms of action as RSV.
5.1. Metformin. Metformin is a drug in the biguanides class, widely used as first-line therapy for type 2 diabetes. Physiological consequences associated to metformin treatment are similar to those observed in animals under DR. Metformin treatment lowers blood glucose predominantly by decreasing its production by the liver. It also increases peripheral tissue insulin-dependent glucose uptake and promotes fatty acid usage leading to lowering of animals fat mass. Comparative transcriptomic analysis from the liver of mice treated by metformin or under DR revealed consistent homologies between profiles, which support the similarity of physiological features shared by both treatments. Furthermore, epidemiological analyses indicate that it may reduce cancer incidence in diabetic type 2 patients. Those observations suggest that metformin may have a broader positive effect on health, although in the case of obese patients one cannot exclude that the decrease in cancer occurrence results primarily from its effect on weight loss, and thus may be restricted to overweight patients.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Metformin Lifespan Effects and AMPK/SKN-1 Pathways,"However, the beneficial effect of metformin on healthy life span have been reported in different models, including cancer prone mice which showed an extended life span and a delay in the appearance of tumors. The role of metformin in the life span regulation of healthy animals has been recently reported in two studies. Smith et al. have shown that when rats were fed in parallel either a DR regimen or metformin treatment, although DR extended early life span (25th quantile), metformin treatment did not significantly affect life span. Conversely, the Driscoll laboratory has shown that metformin increases C. elegans average life span by 40%. When metformin treatment was applied to eat-2 mutants, a genetic model of DR in C. elegans, in which eat-2 mutation lowers food intake by slowing down pharyngeal pumping, then the life span extension was no longer observed. They next took genetic approaches toward identifying metformin molecular effectors. The life span promoting action of metformin was suppressed in mutants for AMPK, called aak-2, and its upstream activating kinase LKB1, called par-4 in C. elegans. Further analysis revealed that the extension of life span by metformin treatment also relies on the activity of the NRF2/SKN-1 transcription factor which is well known for its role in the response to oxidative stress in both mammals and C. elegans.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Metformin Molecular Mechanisms and AMPK/TOR Crosstalk,"Although our understanding of metformin’s effects at the tissue and organ level has progressed, its direct molecular target remains unknown. It was initially proposed that metformin inhibits the mitochondrial electron transporting enzyme complex I of the respiratory chain in isolated hepatocytes and it has also been shown to activate the enzyme AMPK in the same cells. As AMPK can be activated by an increased AMP/ATP ratio and given that inhibition of the respiratory chain eventually leads to a decrease in ATP production, both mechanisms may be tightly linked in response to metformin. Interestingly, AMPK is also activated by its upstream kinases LKB1, and thus life span data in C. elegans support a model in which AMPK/AAK-2 and LBK1/PAR-4 would mediate metformin effect on general health. Overall, experimental evidences associate a key role to AMPK in the response to metformin but whether this kinase is activated by metformin directly or indirectly, via the alteration of cell’s energy status and AMP/ATP ratio and/or through its upstream kinase remains to be elucidated. Given that AMPK has been shown to activate the sirtuin enzyme SIRT1, thus metformin may also behave as a SIRT1 activator via its effect on AMPK.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Metformin Interaction with TOR and Genetic Dissection,"However, a recent report challenged this view by demonstrating that metformin could also inhibit the serine threonine kinase target of rapamycin (TOR) in a rag GTPase-dependent and AMPK-independent manner. Thus, metformin targets may be wider than expected but those targets may not function at the same time and in the same tissue. Metformin treatment of mice that do not express AMPK and/or TOR and/or SIRT1 in certain tissues important for overall metabolic homeostasis may help to better understand the functional relationship between its different direct/indirect targets and its physiological effects.
5.2. Rapamycin. Conversely to RSV and metformin, rapamycin substrate has been clearly identified and its impact on life span of healthy animals has been demonstrated. Rapamycin was initially identified as a new antibiotic with strong antifungal activity, secreted by bacteria isolated from Easter Island (Rapa Nui). A genetic screen aimed at determining rapamycin molecular target for its antiproliferative activity in yeast led to the identification of the serine threonine kinase TOR. Rapamycin derivates are now used in clinical practice as an immunosuppressors to prevent graft rejection but also for cancer treatment.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
"Rapamycin, TOR Inhibition, and Longevity Pathways","The role of rapamycin and/or TOR inactivation in life span extension has been clearly demonstrated in both simple model organisms and more recently in mammals. TOR inhibition is sufficient to increase life span in yeast, nematode, and flies. Furthermore, rapamycin was shown to increase life span in mice and strikingly, this effect was reported for mice treated at an advanced age corresponding roughly to 60 years in people. Epistasis studies in yeast and C. elegans indicate that TOR is required for life span extension by DR. TOR is a nutrient-sensing protein which plays a key role in starvation response. Decreased nutrient availability will inhibit TOR (in part through the AMPK) and this will result in translational inhibition and autophagy activation via, among other effectors, phoshorylation of S6K. Interestingly, Selman et al. recently reported that deletion of ribosomal S6 protein kinase 1 (S6K1), led to increased life span and resistance to age-related pathologies such as bone, immune, and motor dysfunction and loss of insulin sensitivity. S6K homologs also modulate aging in yeast, nematodes, and flies, thus making of the TOR/S6K axis a conserved longevity modifying pathway.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
TORC1 vs TORC2 and Rapamycin Effects Relative to Dietary Restriction,"Furthermore, comparison of gene expression profiles in muscle of S6K knockout mice revealed overlapping upregulated targets both with DR mice and with mice treated with the AMPK activator aminoimidazole carboxamide ribonucleotide (AICAR). Thus, the extension of life span in those mice may also rely on AMPK activation. Overall, studies aimed at directly testing the impact of rapamycin on life span are limited while a number of experimental evidences have been accumulated on the role of TOR in life span regulation. TOR can be associated with two different complexes, TORC1 and TORC2, which show both overlapping and independent biological activities. Given that rapamycin appears to be specific for TORC1, whether treating animals with rapamycin will completely recapitulate the benefits of TOR inhibition at the level of the whole organism remains to be assessed in details. Furthermore, experiments performed in fly show that rapamycin can further extend the life span of DR animals, thus indicating that additional mechanisms to those induced by DR may mediate TOR role in life span.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Toward a Multistep Pro-Longevity Drug Pathway,"6. Toward the definition of a multistep drugable prolongevity/health beneficial molecular pathway. Potential molecular targets for RSV and metformin, and the well-known rapamycin target TOR define an interconnected network in which PGC-1alpha may be an important readout of the complex interplay between AMPK, TOR, and SIRT1. Furthermore, there is increasing evidence that the antiaging action of DR, at least in part, stems from the attenuation of the age-associated increase in oxidative damages. Transcription factors and coactivators involved in the regulation of mitochondrial biogenesis and energy metabolism with PGC-1alpha are thus strong candidates for playing a central role in life span extension by DR.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
Translating Dietary Restriction Benefits to Humans,"Whether the beneficial effects on life span and the delay of age-related pathologies can be transposed to human remains to be demonstrated. Furthermore, paradoxically, low body weight in middle aged and elderly humans is associated with increased mortality. Thus, rather than DR regimen, pharmaceutical interventions that recapitulate DR beneficial effect without body weight lost would be of great value. Regarding this matter, RSV holds good promise.",BioFactors (Wiley),Resveratrol / Rapamycin / Metformin / Dietary Restriction Mimetics,2010
"Rapamycin, Longevity and Aging Overview","Longevity, aging and rapamycin. Dan Ehninger, Frauke Neff, Kan Xie. Received: 12 February 2014 / Revised: 30 June 2014 / Accepted: 30 June 2014 / Published online: 12 July 2014. The Author(s) 2014. This article is published with open access at Springerlink.com. Abstract. The federal drug administration (FDA)-approved compound rapamycin was the first pharmacological agent shown to extend maximal lifespan in both genders in a mammalian species. A major question then is whether the drug slows mammalian aging or if it has isolated effects on longevity by suppressing cancers, the main cause of death in many mouse strains. Here, we review what is currently known about the effects that pharmacological or genetic mammalian target of rapamycin (mTOR) inhibition have on mammalian aging and longevity. Currently available evidence seems to best fit a model, wherein rapamycin extends lifespan by suppressing cancers. In addition the drug has symptomatic effects on some aging traits, such as age-related cognitive impairments.",Springerlink,Rapamycin,2014
"Aging, Disease Risk, and Rationale for Targeting mTOR","Keywords: mTOR, Mammalian target of rapamycin, Longevity, Lifespan, Aging, Rapamycin, Mice, Mammals, Disease, Treatment, Drug, Prevention, Mechanism, Neurodegeneration, Cardiovascular disease, Cancer, Anti-aging. Introduction. Aging is a major risk factor for a range of diseases in numerous organ systems, including cardiovascular disease, neurodegenerative diseases, cancers, diabetes mellitus type II, osteoporosis etc. Interventions that target the molecular processes underlying aging could therefore provide novel entry points for the development of innovative preventatives and/or therapeutics for a range of age-related diseases. Much research over the past ~2 decades has focused on the identification of genetic mutations that extend lifespan in invertebrate model organisms. Pathways involved in the control of cell growth and metabolism have emerged as important players of lifespan regulation. Mammalian target of rapamycin (mTOR) is a kinase at a key signalling node that integrates information regarding extracellular growth factor stimulation, nutrient availability and energy supplies.",Springerlink,Rapamycin,2014
mTOR Pathway and Early Evidence from Model Organisms,"A number of studies in yeast, worms and flies have initially implicated mTOR in lifespan control. The recent discovery that the mTOR inhibitor rapamycin extends mammalian lifespan has created much excitement because it represented the first demonstration of pharmacological extension of maximal lifespan in a mammalian species. Rapamycin’s mammalian longevity effects have since then been confirmed by a number of additional studies. Rapamycin is FDA-approved and considerable experience exists with the clinical application of this drug: Rapamycin and derivates of this compound are used clinically to prevent organ rejection after kidney transplantation and also to prevent occlusion of cardiac stents. mTOR inhibitors are also clinically tested for the treatment of cancers and neurogenetic disorders, such as tuberous sclerosis. Translational studies that assess rapamycin’s effects on human aging and age-related disease are therefore thought to be within reach and have actually been initiated at some sites.",Springerlink,Rapamycin,2014
Evidence for mTOR Inhibition Effects on Aging and Longevity,"Although mTOR inhibition has been clearly shown to extend murine lifespan and also to have beneficial effects on a set of murine aging traits, there is currently little evidence available to support the notion that mTOR inhibitors slow the rate of mammalian aging. In this article, we review what is currently known about the effects that pharmacological or genetic mTOR inhibition have on mammalian aging and longevity.",Springerlink,Rapamycin,2014
mTOR Complexes and Upstream Signaling,"Rapamycin and mTOR signaling. mTOR occurs in two distinct protein complexes, namely mTORC1 and mTORC2 (see Fig. 1; for abbreviations, see Fig. 1 legend). The mTORC1 protein complex includes raptor (regulatory-associated protein of mTOR) and mLST8 in addition to mTOR. Upstream regulatory signaling inputs that converge onto mTORC1-related cell signaling include the PI3K/AKT and Ras/MAPK pathways, as well as AMPK signaling. mTORC1 is therefore well-positioned to coordinate cellular growth-related processes and integrate them with the availability of nutrients, energy and the appropriate stimulation of growth factor receptors.",Springerlink,Rapamycin,2014
mTORC1 Regulation of Protein Synthesis and Longevity Links,"mTORC1 plays an important role in the regulation of a range of cellular processes, including de novo protein synthesis: mTORC1 stimulates the translation of mRNAs with a highly structured 5' untranslated region (5' UTR) by phosphorylating 4E-BPs, thereby derepressing eIF4E and, consecutively, promoting translational initiation. Additionally, mTORC1 controls protein synthesis via the p70S6 kinase/ribosomal protein S6 pathway, which stimulates the translation of mRNAs with a 5' terminal oligopyrimidine tract (5' TOP), many of which encode for components of the translational machinery. Experiments in C. elegans showed that a number of different genetic manipulations affecting the protein synthesis machinery (such as genetic deletion or siRNA-mediated knock-down of ribosomal subunits and translation factors, respectively) are associated with extended lifespan, indicating that altered translational rates could contribute to longevity effects of mTOR inhibition. In mice, lifespan extension was observed in female mice with a homozygous mutation in ribosomal S6 protein kinase 1 (S6K1). Whether mammalian aging rates are slowed by translational modulation remains unknown.",Springerlink,Rapamycin,2014
"mTORC1, Autophagy, and Aging","Another important cellular process regulated by mTORC1 signaling is autophagy. Autophagy, a process by which the cell recycles macromolecules and organelles, allows for the removal of damaged cellular constituents and enables the cell to mobilize substrate under nutrient-poor conditions. mTORC1 regulates autophagy by phosphorylating and inhibiting the autophagy-initiating kinase Ulk1. In C. elegans, autophagy has been reported to be required for the lifespan extension caused by genetic mTOR inhibition. Available evidence in mice indicates that aspects of liver aging (age-related histopathological and functional liver changes) are improved when an inducible genetic system is employed to increase autophagy levels in aged mice. Whether murine lifespan extension, caused by rapamycin, is dependent on autophagy effects has not been addressed to date.",Springerlink,Rapamycin,2014
mTORC2 Function and Rapamycin-Mediated Inhibition,"In the context of the mTORC2 protein complex, mTOR is associated with rictor (rapamycin-insensitive companion of mTOR), GbL and mSIN1 (mammalian stress-activated protein kinase interacting protein). mTORC2 is involved in regulating the activity of AKT. In contrast to mTORC1, which is inhibited by rapamycin, mTORC2 is rapamycin-insensitive. Prolonged rapamycin treatment, however, inhibits mTORC2 indirectly. The mechanisms by which rapamycin inhibits mTOR are not fully understood; it is, however, established that rapamycin associates with FKBP12 to bind to mTOR’s FRB (FKBP12-rapamycin-binding) domain. Binding of the rapamycin-FKBP12 complex to mTOR may destabilize the mTORC1 complex and, in addition, interfere with the activation of mTOR by phosphatidic acid.",Springerlink,Rapamycin,2014
Next-Generation mTOR Inhibitors,"Novel mTOR inhibitors are available that either inhibit mTOR by interfering with mTORC1 complex formation (FKBP12-dependent or FKBP12-independent) or by directly inhibiting mTOR’s catalytic domain. These distinct modes of inhibition allow for selective targeting of different mTOR-related processes and may ultimately help dissect the specific contributions of mTORC1 and mTORC2 to aging, longevity, and disease phenotypes.",Springerlink,Rapamycin,2014
Rapamycin Extends Lifespan in UM-HET3 Mice,"Rapamycin extends lifespan in mice. In their important 2009 paper, the NIA’s intervention testing program (ITP) identified rapamycin as the first pharmacological agent to extend maximal lifespan in a mammalian species, with effects in both males and females. Rapamycin was encapsulated (to increase bioavailablity of the drug) and delivered via the mouse chow at a concentration of 14 parts per million (ppm). In this study, treatment was started at two different ages: 270 days and 600 days, respectively. Both treatments were found to effectively extend maximal lifespan in a genetically heterogeneous stock of mice (UM-HET3 mice) and the effect size did not differ in any obvious way between the earlier-onset and the later-onset treatment. More recently, the longevity effect of two additional doses of rapamycin has been examined: in this follow-up study, the ITP assessed rapamycin orally administered to initially 9 months old UM-HET3 mice at the concentrations of 4.7, 14 or 42 ppm. All three doses extended maximal and median lifespan in females and the two higher doses (14, 42 ppm) caused significant lifespan extension in males.",Springerlink,Rapamycin,2014
Sex Differences and Pharmacokinetics in Rapamycin Response,"Lifespan-extending effects were larger in females than in males, most likely because of higher rapamycin blood concentrations found in females at a given rapamycin chow concentration, although these gender differences in rapamycin blood concentrations were not observed in all studies. It remains to be determined whether these possible gender differences in rapamycin blood levels are related to sex differences in food intake, resorption of the drug, tissue distribution, metabolism of the drug or a combination of these factors. Rapamycin has also been shown to extend lifespan in mice of other genetic backgrounds, such as C57BL/6 and 129/Sv. Additionally, there is also genetic evidence implicating loss of mTOR function in murine lifespan extension: animals homozygous for a hypomorphic mTOR mutation (decreasing mTOR expression to 25% of wildtype levels) show a lifespan extension that is also seen across both males and females.",Springerlink,Rapamycin,2014
Summary of Rapamycin Longevity Studies (Table 1),"Table 1 Mammalian longevity studies using rapamycin or genetic mTOR inhibition. Oral rapamycin (encapsulated, 14 ppm) initiated at 270 days or 600 days of age in male and female UM-HET3 mice extended median and maximal lifespan in both genders. Both treated animals and controls die due to cancers in >80% of cases, but treated animals do so later in life. Oral rapamycin (encapsulated; 4.7, 14 or 42 ppm) initiated at 9 months of age in male and female UM-HET3 mice extended median and maximal lifespan in females; the two higher doses extended lifespan in males. Rapamycin injected i.p. (4 mg/kg once every other day for 6 weeks, starting at 22–24 months) increased survival in male C57BL/6 mice. Oral rapamycin (14 ppm) for approx. 1 year starting at 4, 13 or 25 months extended lifespan in male C57BL/6J Rj mice. Rapamycin injected s.c. (1.5 mg/kg, 3× weekly with intermittent 2-week breaks) starting at 2 months extended lifespan specifically in tumor-bearing female 129/Sv mice but had no significant effect in tumor-free animals.",Springerlink,Rapamycin,2014
Genetic mTOR Inhibition and Longevity Outcomes,Hypomorphic mTOR allele (mTOR^D/D); mice on a mixed 129S1 and C57BL/6Ncr background were compared to wildtype littermate controls. Extension of median survival was observed in both male and female mTOR^D/D mice; probable extension of maximal lifespan was also noted (but with low sample size). Necropsies showed reduced incidence of malignant tumors but higher rates of infections in hypomorphic mTOR animals. These findings support the view that both pharmacological and genetic mTOR inhibition extend lifespan in mammals and suggest that cancer suppression is a major contributor to the longevity benefits of rapamycin.,Springerlink,Rapamycin,2014
Rapamycin Longevity Studies and Cancer as Primary Cause of Death,"Rapamycin longevity studies: why do treated animals live longer? As mentioned above, the rapamycin longevity studies in mice published to date examined several genetic backgrounds, namely inbred C57BL/6 backgrounds, 129/Sv and the genetically heterogeneous UM-HET3 stock of animals (the stock used by the NIA’s Intervention Testing Program). In all these backgrounds and across sexes, neoplastic lesions represent a major cause of death. For example, approx. 70% of C57BL/6 animals naturally die due to neoplastic disease with lymphomas and hematopoietic neoplasms representing the leading causes of death. Similarly, in UM-HET3 mice, neoplastic lesions are the natural cause of death in >80% of cases. Lymphomas and hematopoietic tumors also represent the most common neoplastic lesions that naturally limit life in UM-HET3 mice. Any intervention extending lifespan in these strains is, therefore, expected to do so primarily by counteracting these common life-limiting neoplastic pathologies.",Springerlink,Rapamycin,2014
Rapamycin as an Anti-Cancer Longevity Agent,"Lifespan extension via inhibition of carcinogenesis is indeed a plausible scenario for rapamycin-mediated longevity effects because rapamycin has well-known antineoplastic properties, including inhibitory effects on de novo cancer formation, as well as suppression of established tumors via inhibition of cancer growth, promotion of apoptosis of neoplastic cells and/or a modification of the host response to the tumor (for example, inhibiting angiogenesis). In line with this, rapamycin was found to suppress cancers and extend life in a range of genetic early-onset cancer models, such as p53 mutant mice, Apc mutant animals, Rb mutant mice and HER-2/neu transgenic mice, strongly implicating direct anti-cancer action in the longevity effects seen in these studies. Detailed cause-of-death analyses in rapamycin-treated UM-HET3 mice and controls indicated that both groups die primarily (i.e., in >80% of cases) due to cancers, but rapamycin-treated animals do so later in life than controls, indicating that rapamycin postpones lethal neoplastic disease in treated animals.",Springerlink,Rapamycin,2014
Neoplastic vs. Non-Neoplastic Death in Rapamycin Studies,"In the context of this study, it was not possible to determine if rapamycin also extends lifespan in those animals that die due to non-neoplastic disease because non-neoplastic disease accounted only for a small fraction (approx. 10%) of deaths in UM-HET3 mice. An analysis of rapamycin’s lifespan effects in aging 129/Sv female mice showed a clear lifespan extension and a reduced tumor burden in treated animals. Further analyses in 129/Sv mice indicated that rapamycin-mediated lifespan extension is only seen in animals that eventually die due to neoplastic disease; in contrast, rapamycin did not significantly extend lifespan in those animals that die due to non-neoplastic disease, supporting the notion that rapamycin extended lifespan by specifically inhibiting neoplasia-related lethality (without effects on lethality related to non-neoplastic disease).",Springerlink,Rapamycin,2014
Genetic mTOR Inhibition and Tumor Reduction,"Similarly, a study analyzing lifespan and end-of-life pathology in hypomorphic mTOR mutant mice reported a clear reduction of malignant tumors in the mutants, while infections were more common in these animals. Reduced numbers of precancerous lesions and cancers were also found in rapamycin-treated aging C57BL/6J mice. Together, the data available indicate that rapamycin primarily extends mammalian lifespan by inhibiting lethal neoplastic disease. It will be important to assess rapamycin’s effects on additional mouse strains and/or other mammalian species that show a broader spectrum of additional non-neoplastic pathology as contributory factors to death.",Springerlink,Rapamycin,2014
From Lifespan Studies to Healthspan Concepts,"Aging research: from lifespan to healthspan measures. Studies over the past ~20 years have identified a large number of genetic manipulations that extend life in invertebrate model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. Some of the pathways identified were also shown to be involved in the regulation of mammalian lifespan (using mice), although mechanisms of lifespan extension in these different species are likely very different (at least the proximal mechanisms; see discussion above). Lifespan extension does not necessarily indicate effects on aging. With regards to lifespan-extending interventions in mice two general scenarios are possible: (a) interventions may have isolated effects on lifespan by inhibiting specific life-limiting pathology, such as cancers, without broadly modulating aging traits; (b) lifespan extension occurs by inhibition of life-limiting pathologies, such as cancers, and this effect represents one aspect of a more general effect that the respective intervention has on aging.",Springerlink,Rapamycin,2014
Evaluating Interventions Through Aging Phenotypes,"During the course of aging most mammalian tissues and organ systems undergo characteristic molecular, structural and functional alterations. To assess whether a pharmacological or genetic intervention slows the rate of mammalian aging it is necessary to examine its effects on a broad range of mammalian aging phenotypes in different cell types, tissues and organ systems. Comprehensive analyses of aging traits may then identify aging traits that are ameliorated by specific genetic or pharmacological interventions.",Springerlink,Rapamycin,2014
Mechanisms of Modulating Aging Traits,"Modulation of aging traits: slowing aging vs. symptomatic effects on aging traits. A given intervention that ameliorates aging traits could in principle do so via one of two ways: (a) causally, by slowing the rate of aging, that is by slowing the process(es) that underlie the age-dependent development of the respective aging trait(s); (b) symptomatically, i.e., by modifying the respective traits in ways that are mechanistically distinct from the processes that underlie the aging traits’ development. Experimentally, one can reveal the symptomatic nature of the modulation of an aging trait by administering the intervention well before the respective aging trait has developed. C57BL/6J mice, for example, develop aging-associated deficits in spatial reference memory tasks, such as the Morris water maze, at some point during their second year of life or thereafter. If a one-month treatment with a drug enhances spatial memory in 4 months old C57BL/6J mice, then it is clear that this drug improves memory by tapping into mechanisms that are distinct from those that underlie aging-associated cognitive decline.",Springerlink,Rapamycin,2014
Distinguishing Aging Rate Reduction from Symptomatic Effects,"If it is then found that a long-term treatment with this same drug enhances memory in 2 year old mice, the most parsimonious explanation of this set of findings is that the drug affects age-dependent memory impairments symptomatically (i.e., without affecting the rate of cognitive aging). This principle is illustrated in Fig. 2: interventions that slow the rate of aging are expected to specifically interfere with the age-related change of specific traits (e.g., age-related decline in performance on certain cognitive tasks), but do not have the same effects on young organisms that did not yet express the respective aging trait. Other interventions, in contrast, may have symptomatic effects on an aging trait without slowing the rate of aging itself: These interventions would be expected to influence the respective trait in young, as well as in old individuals.",Springerlink,Rapamycin,2014
Rapamycin Effects Across Aging Traits,"Rapamycin effects on aging traits in mice. Several studies have assessed rapamycin’s effects on aging traits and they cover numerous aging phenotypes in different cell types, tissues and organ systems. Most of these studies have employed the ITP’s original rapamycin treatment regimen (i.e., encapsulated rapamycin administered via the mouse chow at a concentration of 14 ppm) that has been repeatedly shown to extend maximal and median lifespan in both males and females. Rapamycin shows extensive tissue distribution and is therefore available for mTOR inhibition in numerous organs. The ITP’s rapamycin treatment regimen was shown to inhibit phosphorylation of ribosomal protein S6 at the mTOR-sensitive sites Ser240/244 in a number of tissues of both male and female mice, including heart, liver, kidney and fat.",Springerlink,Rapamycin,2014
Autophagy Activation and Comprehensive Aging Analysis,"Similarly, this treatment protocol was also found to increase the LC3II/LC3I ratio in the tissues mentioned above, which is indicative of enhanced autophagy in treated animals. The by far most comprehensive assessment of rapamycin’s effects on murine aging was performed by Neff et al. in a study that covered >150 aging traits across >25 tissues. The Neff et al. study is the only published report that took aging-independent drug effects into consideration when assessing rapamycin’s effects on aging traits. In the sections below, we review the current state of knowledge regarding rapamycin effects and effects of genetic mTOR inhibition on murine aging traits.",Springerlink,Rapamycin,2014
Neurological Aging Traits and Rapamycin Overview,"Aging is associated with a number of neurobehavioral and neurological changes, such as cognitive decline, alterations in motor coordination, balance and reduced muscle strength. Rapamycin effects on neurological aging traits were assessed in several studies and the observations are summarized below. Aging in mice is typically associated with reduced levels of exploration and locomotor activity. One robust finding, seen across a number of studies, is rapamycin’s stimulatory effect on locomotor activity in treated animals. Miller et al. and Wilkinson et al. measured spontaneous motor activity of animals in their cages for an extended time period (50 h). They examined animals at two time points, first at 7 months of age and, in those that survived, again at 18 months of age. Rapamycin or vehicle control treatment was initiated at 9 months of age. Their findings indicated that the age-dependent reduction of motor activity is ameliorated in animals aged on rapamycin. Stimulatory rapamycin effects on motor activity were also noted in another study, in which voluntary wheel running was measured in aged animals subjected to oral rapamycin or vehicle control chow for 3 months starting at the age of 24 months.",Springerlink,Rapamycin,2014
"Rapamycin Effects on Exploration, Motor Activity, and Cognitive Tasks","Neff et al. examined exploratory activity in an open field assay in three cohorts of animals that were aged on rapamycin or vehicle control for approx. 1 year, starting at either 4, 13 or 25 months. Exploratory activity levels were substantially reduced in aged mice relative to young controls and rapamycin treatment significantly elevated exploratory activity in aged animals. The same regimen, however, also increased exploratory activity in young mice treated for 12 weeks before testing, indicating that these drug effects were probably not related to a modulation of aging, but aging-independent effects on locomotor activity. Collectively, the studies mentioned above demonstrate stimulatory effects of rapamycin on locomotor activity observable across genetic backgrounds and affecting both sexes. Relatively shorter-term rapamycin treatment is sufficient to increase motor activity and this is also the case for young animals, indicating age-independent effects. Another robust neurobehavioral finding is enhanced performance on learning and memory tasks. Reduced escape latencies and improved probe trial performance in the Morris water maze in rapamycin-treated animals was observed across different genetic backgrounds. Rapamycin improved learning and memory not only in animals aged on the drug but also in young adult mice treated well before the onset of cognitive decline, indicating symptomatic cognitive enhancement rather than slowed cognitive aging.",Springerlink,Rapamycin,2014
"Motor Coordination, Balance, Muscle Strength, and Nociception","Additional neurological studies in rapamycin-treated aged animals included assessments of motor coordination and balance, gait, muscle strength, nociception and histopathological assessments of aging traits in muscle and brain. The ITP rapamycin regimen applied for 1 year did not measurably improve performance on an accelerating rotarod in animals treated beginning at 4 or 13 months. Another study employed a longitudinal assessment of rotarod performance at 25 and 31 months in mice subjected to treatment starting at 19 months. Although there was no decline in rotarod performance between 25 and 31 months in controls, rapamycin treatment improved performance at 31 months. Rotarod performance was also examined in hypomorphic mTOR mutant mice. Aged mutants performed better than aged wildtype controls, while no genotype effect was observed in young animals. Limitations include small sample sizes and unaccounted-for body weight differences. The ITP rapamycin regimen did not measurably affect age-related impairments in muscle strength or sarcopenia. Wu et al. reported increased muscle strength in aged hypomorphic mTOR mice while young animals were unaffected, although sample size limitations remain.",Springerlink,Rapamycin,2014
Summary of Neurological Effects and Symptomatic Nature of Rapamycin,"In sum, oral rapamycin has stimulatory effects on locomotor behavior and improves learning and memory. These are robust findings seen across mouse strains and genders. Because rapamycin has similar effects in young animals and aging cohorts, it is the most parsimonious explanation of the data that these rapamycin effects are not related to a modulation of aging but instead represent symptomatic enhancements. The oral rapamycin ITP protocol had limited effects on motor coordination, balance, muscle strength, sarcopenia and age-related nociceptive dysfunction. Preliminary evidence suggests that genetic mTOR inhibition in hypomorphic mTOR mutant mice may result in preserved motor coordination and muscle strength in aged animals. A common aging-associated ophthalmological pathology is cataract formation. Two studies assessed rapamycin effects on age-related lens density alterations. Wilkinson et al. reported exacerbation of age-related lens density alterations in UM-HET3 mice, while Neff et al. found no clear effects in C57BL/6J mice. Rapamycin did not prevent age-related decline in visual acuity. Together, the data indicate that lifespan-extending rapamycin treatment does not beneficially influence age-related ophthalmological changes and may even have adverse effects on specific aging traits of the eyes.",Springerlink,Rapamycin,2014
Cardiac Structure and Function Under Rapamycin,"Cardiological findings. Aging is associated with structural and functional changes affecting the heart. Echocardiography was employed in two studies to analyze the effects of the ITP rapamycin protocol on age-related structural and functional cardiac alterations. In both studies, rapamycin was found to decrease a subset of heart dimensional measures, such as diastolic left ventricular internal diameter (LVIDd), and also decreased overall heart mass. Analyses in young animals demonstrated similar effects after a 3 months rapamycin treatment starting at 12 weeks of age, indicating age-independent drug effects on heart weight and heart dimensional measures. Echocardiography also demonstrated the expected aging-associated decrease in functional cardiac measures, such as cardiac output, ejection fraction, fractional shortening, as well as blood flow measurements and pressure gradients across the aortic, pulmonary and mitral valves. Neff et al. assessed 26 months old male C57BL/6J Rj mice treated for 13 months and found no significant rapamycin effects on these measures, suggesting limited effects on cardiac function.",Springerlink,Rapamycin,2014
Cardiac Function: Study Designs and Rapamycin Outcomes,"Flynn et al. used a within-subjects design in female C57BL/6J mice, measuring cardiac function at 24 months of age and then again after 3 months of rapamycin or vehicle treatment. These authors report a small beneficial rapamycin effect on the ejection fraction and on fractional shortening. Possible explanations for differences between studies include differences in study design (between-subjects versus within-subjects), gender (male versus female) and treatment duration (with transient effects of treatment on these cardiac measures).",Springerlink,Rapamycin,2014
Rapamycin and Age-Related Skeletal Changes,"Effects on the skeletal system and tendons. During aging, typical changes affecting bones and the skeletal system occur, including decreases in trabecular bone volume and progressive kyphotic changes affecting the spine. The effect of a short-term rapamycin treatment (the ITP protocol for 3 months, starting at 24 months of age) on spine kyphosis was examined using whole body micro CT. Measures were taken before and after treatment. While a progressive change of spine kyphotic index was detected over this 3 months period, rapamycin had no measurable effects on the degenerative changes. Micro CT analyses were also employed to measure tibial bone density in young and aging mTORD/D mice, as well as corresponding wildtype controls. These analyses showed the expected loss of trabecular bone volume in the tibiae of aged mice. The mTORD/D genotype had no beneficial effects on age-related loss of bone volume, but appeared to lead to a further exacerbation of this aging phenotype. No difference in trabecular bone volume was detected between young mutants and controls.",Springerlink,Rapamycin,2014
Rapamycin Improves Tendon Biomechanics but Has Limited Skeletal Effects,"Together, the data available suggest that pharmacological and genetic mTOR inhibition may have limited effects on specific age-related skeletal and bone changes. Wilkinson et al. examined age-related changes in the mechanical properties of tibialis anterior tendons. Aged mice showed a significant increase in maximum tangent modulus (indicating resistance to stretching) and a decrease in hysteresis (indicating reduced recovery to the original length in the unstretched state). Rapamycin treatment significantly improved these age-related biomechanical tendon properties. Rapamycin effects on tendons in young animals were not included in the analyses. Future studies have to determine whether rapamycin has symptomatic effects on tendon properties in aged mice or if it slows the age-related development of these alterations.",Springerlink,Rapamycin,2014
Hematological Changes and Rapamycin Effects,"Clinical chemistry, hematology and immunology. Murine aging is associated with an altered cellular composition of the peripheral blood. Blood cell counts were measured in three aging mouse cohorts that were treated with rapamycin or vehicle control for approx. 1 year (starting at either 4, 13 or 25 months) before analyses commenced. Aged animals showed elevated white blood cell counts and elevated platelet counts, both of which were not modified by rapamycin treatment. Additionally, old animals showed reduced red blood cells counts associated with decreased mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and increased red blood cell distribution width (RDW). These findings are reminiscent of hematology findings associated with iron deficiency anemia, anemia of chronic diseases and anemia caused by chronic bleeding. Rapamycin treatment significantly elevated red blood cell counts in aged animals and tended to have similar effects in young animals, indicating that rapamycin effects were likely linked to aging-independent effects on erythrocyte production and/or turnover.",Springerlink,Rapamycin,2014
Clinical Chemistry Profiles in Rapamycin-Treated Mice,"Detailed clinical chemistry assessments showed clear aging-associated changes in plasma concentrations of sodium, calcium, and chloride (increased), glucose (decreased), and triglycerides (decreased), which is in agreement with previously reported murine aging traits. Additionally, old mice showed increased alkaline phosphatase and a-amylase levels in their plasma. Rapamycin treatment had by and large no detectable effects on these clinical chemistry aging traits (except for an elevation of blood glucose).",Springerlink,Rapamycin,2014
Immune Cell Population Changes During Aging and Rapamycin Response,"Ten-color polychromatic flow cytometry was used to examine quantitative aging and/or rapamycin effects on various immune cell populations in the three aging mouse cohorts mentioned above. Aged animals showed the expected strong reduction in the frequency of CD4+ T cells, an increase in the frequency of CD44hi T cells (indicative of an activated/memory T cell phenotype) and a strong reduction in the frequency of NK cells, among additional alterations. Rapamycin treatment had no measurable effects on the frequency of CD4+ T cells and NK cells, but counteracted the age-related change in CD44hi T cells. Rapamycin did not influence the frequency of CD44hi T cells in the young group of animals examined. Nevertheless, it remains unclear if rapamycin specifically has this effect on CD44hi T cells in aged animals because genetic mTOR inhibition has been shown previously to lead to reduced CD44hi T cell counts in young animals.",Springerlink,Rapamycin,2014
Immunoglobulin Alterations and Rapamycin Treatment,Immunoglobulin plasma concentrations are often robustly elevated in aged mice. Rapamycin tended to decrease plasma immunoglobulin concentrations in several cases. These analyses were only carried out in the aging cohorts and it remains to be determined if a more acute rapamycin treatment may have similar consequences in young mice.,Springerlink,Rapamycin,2014
Metabolic Aging Traits and Rapamycin,"Metabolism. Indirect calorimetry was used to assess metabolic changes in aged animals and their possible modulation by rapamycin. These studies showed that certain parameters seen in aged mice, such as reduced oxygen consumption and lower body temperature, were not restored by rapamycin treatment. Rapamycin had an effect on the respiratory exchange ratio, at least in one of the examined cohorts, that deserves further attention in future studies.",Springerlink,Rapamycin,2014
Histopathology: Neurogenesis and Protein Damage,"Pathology findings. One of the well-documented age-related alterations is the strong decline in adult hippocampal neurogenesis. Neff et al. determined if a 1 year rapamycin treatment (the ITP protocol), initiated at 4 months, prevents the age-related decline in hippocampal neurogenesis. They observed the expected clear reduction in doublecortin immunoreactivity in the dentate gyrus, which was not significantly modulated by rapamycin, indicating no measurable effects on age-related alterations in adult hippocampal neurogenesis. There is an accumulation of polyubiquitinated and nitrotyrosinylated proteins in the aging mouse brain. Wu et al. employed immunohistochemical analyses of brain sections to measure effects of a hypomorphic mTOR mutation. Their data showed reduced immunoreactivity for polyubiquitinated and nitrotyrosinylated proteins in aged mTOR mutants compared to wildtype controls, suggesting suppression of these aging traits in the mutants.",Springerlink,Rapamycin,2014
"Sarcopenia, Myocardial Aging and Rapamycin","The aging-associated loss in muscle strength is paralleled by an atrophy of skeletal muscle fibers (sarcopenia). Neff et al. quantitatively analyzed cross-sectional muscle fiber surface area of the quadriceps femoris muscle of aged animals treated with rapamycin or vehicle control. Rapamycin did not ameliorate the expected reduction in cross-sectional muscle fiber area. Aging of the cardiovascular system is associated with ventricular dilation, myocardial hypertrophy, fibrosis and thickening of the heart valves. Detailed histopathological analyses showed no preventative effects of rapamycin treatment on these aging traits. Wilkinson et al. assessed morphological changes in cardiomyocytes: their findings suggest rapamycin may have reduced the frequency of abnormalities in nuclear size and chromatin conformation in aged animals, although significance was borderline and age-independent effects were not excluded.",Springerlink,Rapamycin,2014
"Liver, Kidney, and Endocrine Aging Under Rapamycin","Age-related liver phenotypes (periportal fibrosis, polyploidy, steatosis) were examined in Neff et al. and Wilkinson et al. Neff observed no preventative effects of rapamycin on periportal fibrosis and polyploidy, but rapamycin decreased the frequency of hepatic microgranulomas. Wilkinson reported that multifocal macrovesicular lipidosis in aged male UM-HET3 mice was reduced by rapamycin in a dose-dependent manner. Rapamycin’s effect could reflect prevention of liver aging or more immediate effects on hepatic lipid metabolism. Renal aging includes glomerulosclerosis and tubular alterations. Rapamycin caused no benefit on glomerulosclerosis but significantly increased vacuolization of renal tubulus epithelial cells, indicating nephrotoxic side effects. In the thyroid gland, aging is associated with increased follicle size; rapamycin reduced follicle size in both aged and young animals, indicating an aging-independent drug effect. Rapamycin did not ameliorate adrenal lipofuscin deposition.",Springerlink,Rapamycin,2014
Reproductive Aging and Rapamycin Effects,"Rapamycin effects on the male reproductive tract were examined histopathologically. Both Wilkinson et al. and Neff et al. found significant testicular degeneration in rapamycin-treated animals across different doses and genetic backgrounds, indicating a robust side effect. Aging-associated changes in the uterus include endometrial hyperplasia. Wilkinson reported a significant decrease in endometrial hyperplasia in aged female UM-HET3 mice treated with the highest rapamycin dose (42 ppm). It is unclear if this reflects slowed uterine aging or an aging-independent effect, given evidence that Tsc2 inactivation induces endometrial hyperplasia which can be suppressed by mTOR inhibition.",Springerlink,Rapamycin,2014
Rapamycin-Induced Gene Expression Changes in Liver,"Gene expression. Using the ITP rapamycin administration protocol, two complementary studies investigating rapamycin-induced gene expression changes in the liver of aged mice were published recently. The first performed transcriptome analysis of liver tissues derived from 25 months old C57BL/6 mice fed 14 ppm encapsulated rapamycin starting at either 4 months (for 21 months) or 19 months (for 6 months). Despite considerably extended lifespan in both genders, gene expression changes induced by long-term rapamycin treatment were much greater in females (2,504 genes up-regulated and 2,257 down-regulated) than in males (159 genes up-regulated and 129 down-regulated). Chronically rapamycin-fed males showed strong heterogeneity: half resembled controls, half showed female-like expression profiles. Shorter-term treatment (6 months) altered 100 genes in males (32 up-regulated, 68 down-regulated) and 1,427 genes in females (675 up-regulated, 752 down-regulated). Ingenuity pathway analysis identified 13 pathways modulated by chronic treatment, including mitochondrial function and protein degradation also affected by 6-month treatment.",Springerlink,Rapamycin,2014
Comparing Rapamycin and Dietary Restriction Gene Expression Effects,"The second study compared liver transcriptome profiles of rapamycin-fed male mice with those under a 40% dietary restriction (DR) regimen initiated at 2 months until 8 months of age. Most gene expression modifications were unique to either DR or rapamycin, with only a small overlap (490 up-regulated genes = 26%; 74 down-regulated genes = 9%). Ubiquitination was the only top pathway shared by both treatments. Interestingly, DR combined with rapamycin caused additional gene expression changes (1,049 up-regulated and 767 down-regulated) not found with DR or rapamycin alone. In summary, these studies provide initial insights into rapamycin-associated gene regulation in the liver. However, whether these changes are directly linked to aging remains unknown because young mice were not included in one study, and the other was limited to young males. Broader transcriptome analyses across tissues are needed to clarify how rapamycin-regulated gene expression relates to aging.",Springerlink,Rapamycin,2014
Rapamycin: Summary of Aging Trait Modulation,"Conclusions and future directions. Rapamycin effects on numerous aging traits have been analyzed to date, primarily using the original ITP regimen that extends lifespan in mice. Although many aging traits were not modified by treatment, rapamycin improved a subset of examined traits. These included immune system changes (plasma immunoglobulins, T cell subsets, cytokine concentrations, vaccination response), age-related changes in body mass and organ metrics (body weight, fat mass, lean mass, thyroid follicle size, cardiac dimensions, heart weight), tumors and pre-cancerous lesions, and neurobehavioral changes (motor activity, learning and memory). Where available, the data show similar effects in young adult mice under shorter-term treatment, suggesting many rapamycin effects are aging-independent symptomatic improvements. More research is needed to determine to what extent rapamycin’s benefits in aged animals reflect symptomatic effects versus true slowing of aging. Additionally, higher doses should be evaluated to test whether more complete mTOR inhibition produces more robust anti-aging outcomes.",Springerlink,Rapamycin,2014
Rapamycin Resistance Mechanisms,"tance to rapamycin, and expression of the human homolog FKBP12 (FK-506 binding protein 12) restoring drug sensitivity. Mutations in TOR1 and TOR2, originally designated DDR1 and DDR2 (dominant rapamycin resistance), were found to confer resistance to the antiproliferative effects of rapamycin in yeast. Wild-type Tor1 and Tor2 are bound and inhibited by the Fpr1/rapamycin complex [1]. This mechanism was subsequently found to be conserved in mammals with the rapamycin-FKBP12 complex binding to and inhibiting mTOR [2].",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR / Rapamycin,2015
mTOR and Longevity in Lower Eukaryotes,"A link between mTOR signaling and aging was first established in yeast when studies in Saccharomyces cerevisiae demonstrated that deletion of Sch9, the yeast homolog of the mTORC1 substrate S6K (see 'mTOR Downstream Signaling'), results in a significant increase in chronological life span, defined as the duration of time that a yeast population retains viability when in a nondividing state [3]. Studies in the nematode Caenorhabditis elegans subsequently revealed that mTORC1 can negatively regulate longevity in multicellular organisms; knockdown of daf-15 (the nematode homolog of Raptor, a component of mTORC1) or let-363 (the nematode homolog of mTOR) by RNAi can extend life span in this model [4]. Reports from Drosophila melanogaster and yeast replicative aging studies further supported the role of mTOR in regulating longevity in lower eukaryotes.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR / Rapamycin,2015
mTOR Inhibition and Mammalian Lifespan Extension,"Direct evidence of a role for mTOR in mammalian life span has been provided by studies showing life span extension in mice resulting from deletion of S6K, by double heterozygosity for mTOR and mlst8 (a component of mTORC1), and by treatment with rapamycin [5]. Intriguingly, life span extension in each of these studies was strongly sex-specific, with males receiving no longevity benefit from S6K deletion or double heterozygosity of mTORC1 components. Female mice also experienced a more robust response to treatment with rapamycin with an 18% increase in median life span, compared to a 10% increase in male animals [5]. Notably, intervention with rapamycin resulted in an increase in life span even when rapamycin treatment began late in life, suggesting that mTOR inhibition may prove an attractive target for intervening in human aging.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR / Rapamycin,2015
mTORC1 and mTORC2 Overview,"mTORC1 and mTORC2 both play essential roles in eukaryotic biology, as complete loss of either Raptor, an mTORC1 specific component, or Rictor, an mTORC2 component, results in embryonic lethality. While both are essential for development, the two mTOR complexes differ in their components, relative regulatory roles, and upstream regulation of their activity. The upstream regulators and downstream effectors of mTORC1 are generally better characterized than those related to mTORC2. Although mTOR signaling is affected by numerous intra- and extracellular growth cues and conditions, and it affects numerous downstream pathways and processes, only the best characterized of these pathways, in terms of aging and disease, are described below.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
Insulin and IGF Regulation of mTORC1,"mTOR is activated by a variety of growth factors and mitogens. Among the canonical regulators of mTORC1 signaling in mammals are insulin and the insulin-like growth factors (IGFs). Insulin and IGFs are recognized at the cell surface by tyrosine kinase receptors and provide the primary extracellular regulation of mTOR. Signaling through insulin/IGF is partially through PI3-mediated activation of PI3-dependent kinase (PDK) and subsequent activating phosphorylation of AKT on T308. IGF-1 signaling represents a longevity-regulating pathway in its own right, acting through both mTOR and FoxO. IGF receptor loss has been shown to increase life span in mice and worms; serum IGF levels correlate with life span among mouse strains, and FOXO gene variants are strongly associated with extreme longevity in humans. AKT stimulates mTORC1 through phosphorylation of the mTORC1 inhibitor TSC2.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
TSC–Rheb Axis and mTORC1 Activation,"In its active form, the TSC1/2 complex is a GTPase-activating factor for the small guanine nucleotide-binding protein Rheb. Stimulation of Rheb by active TSC1/2 results in conversion of loaded GTP to GDP, inactivating the protein. Active GTP-bound Rheb is a necessary component of mTORC1; thus, inhibition of TSC1/2 results in downstream activation of mTORC1 through an increase in active Rheb. While mTORC2 is not activated by insulin and growth factors through canonical signaling, an intriguing mechanism has been described which couples regulation of mTORC2 activity by extracellular signals to intracellular ribosomal capacity. mTORC2 is activated by PI3K through increased physical association with ribosomes, where it phosphorylates S473 on Akt, priming it for activation by PDK1. Ribosome-associated mTORC2 also phosphorylates SGK and PKC, linking translation capacity to growth signaling.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
mTORC2–AKT–FoxO Crosstalk,"mTORC2 lies both upstream and downstream of AKT, a relationship that may explain otherwise perplexing observations in mTOR biology. One example is the uncoupling of mTORC1 and AKT signaling by FoxO induction. FoxO upregulates Rictor, the mTORC2-specific binding partner of mTOR, and activates AKT while decreasing assembled mTORC1. This decrease results from sequestration of mTOR into mTORC2, while increased mTORC2 activity directly activates AKT. The downstream effects of mTORC2-specific signaling are not well characterized in aging, but its interconnected relationship with mTORC1 and AKT highlights mTORC2 as a key determinant of cellular outcomes following mTOR perturbation.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
AMPK as an Energy-Sensing Regulator of mTOR,"mTOR activity is modulated not only by extracellular signals but also by intracellular energy and nutrient-sensing pathways. A well-characterized regulator is the AMP-activated kinase AMPK, an ancient sensor of cellular energy status. AMPK is activated when AMP:ATP ratios increase. Upon activation, AMPK drives catabolic processes and inhibits anabolic pathways to restore energy balance. AMPK activates TSC2 through phosphorylation at T-1227 and S-1345 and directly inhibits mTORC1 through phosphorylation of Raptor at serine 722 and 792. Pharmaceutical AMPK activators such as phenformin and metformin influence this axis, though metformin appears to act partly through AMPK-independent mechanisms.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
Amino Acid Sensing via the Lysosomal Ragulator,"mTORC1 is directly activated by amino acids through an interaction at the surface of the lysosome, enabling intracellular amino acid abundance to regulate growth and metabolism. This sensing is facilitated by the ragulator complex, a vacuolar ATPase-binding complex with guanine nucleotide exchange factor activity for Rheb. Under high amino acid conditions, mTORC1 and Rheb are recruited to the ragulator at the lysosomal surface, where Rheb activates mTORC1. These regulatory interactions define mTORC1 as a central sensor integrating growth conditions and nutrient cues.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
Caloric Restriction and mTORC1,"Caloric restriction (CR) is a robust life span-extending intervention found in species including yeast, flies, rodents, and primates. Defined as nutrient intake reduction without malnutrition, CR varies widely in implementation. In nematodes it can be achieved by limiting bacterial food; in yeast through glucose deprivation or carbon source substitution; in mice through controlled food intake. Despite variability, CR consistently extends life span, suggesting highly conserved downstream regulators. mTORC1 has been implicated as a key component of the CR response. CR reduces mTORC1 activity in invertebrates and mammals. CR does not additively increase life span in mTOR or S6K mutants, supporting mTORC1 involvement, though interpretation of lifespan complementation studies is complex due to longevity's multifactorial nature.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
mTORC1-Regulated Processes in Caloric Restriction,"A substantial body of literature links mTORC1-regulated processes—including autophagy and mRNA translation—to the beneficial effects of caloric restriction. Given mTOR’s role in nutrient signaling, decreased mTORC1 signaling during CR, and known CR effects on downstream processes, there is consensus that altered mTOR signaling significantly contributes to CR-mediated life span extension, although additional pathways also play roles in the overall intervention response.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
mTORC1 Downstream Biological Roles,"As a major energy and growth signaling sensor, mTORC1 acts as a central coordinator of proliferative and maintenance programs. mTORC1 activity drives growth through activation of mRNA translation, regulation of metabolic pathways including glycolysis and fatty acid metabolism, and repression of cellular catabolic pathways, primarily the autophagy/lysosomal degradation pathway. Inhibition of mTORC1 results in reduced mRNA translation, increased catabolic processes, and a shift in metabolic substrate preference. Many of the effects of mTORC1 activity or inhibition are mediated by activation or suppression of downstream transcriptional regulators and the complex crosstalk between these factors. The effects of mTORC1 modulation on metabolism are highly context dependent on organism, tissue type, intervention duration and severity, and environmental interactions. Dissecting pathways and targets of importance in aging and disease is a significant challenge where future systems biology approaches may be especially valuable. While mTOR signaling is complex, several conserved and well-defined pathways have been identified downstream of mTORC1 and mTORC2.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
mRNA Translation via p70S6K and 4E-BP1,"mTORC1 has been studied extensively, and mTORC1-regulated processes are generally better described. Hormonal signaling and abundant nutrient availability promote mTORC1 activation, which upregulates processes required for growth. One critical process driven by active mTORC1 is mRNA translation. mTORC1 kinase activity promotes translation through at least two substrates. It phosphorylates p70S6K, the 70-kDa ribosomal S6 kinase, an activator of ribosome biogenesis. It also phosphorylates eIF4E-BP1, causing its release from eIF4E and allowing formation of the cap-dependent translation initiation complex. Activation of mRNA translation likely accounts for many mTOR-driven disease phenotypes, while decreased translation mediates positive effects of mTOR inhibition. Antiproliferative effects of mTOR inhibitors in cancer and immune diseases may largely derive from reduced protein synthesis. Decreased mRNA translation is a major pro-longevity intervention in multiple organisms; caloric restriction is thought to act largely through reduced translation. Deletion or knockdown of ribosomal components or initiation factors increases life span in yeast, flies, and nematodes. Deletion of S6K extends life span and decreases body size in mice.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
Selective mRNA Translation and Longevity,"While mRNA translation is globally decreased in caloric restriction and some forms of mTOR inhibition, studies suggest that beneficial effects may result from differential translation of specific mRNAs rather than reduced global translation alone. This model is best developed in budding yeast, where life span extension from ribosomal protein subunit deletion is linked to increased translation of the transcription factor Gcn4. Gcn4 regulates genes involved in low nutrient response and stress resistance. Gcn4 is necessary for life span extension from ribosomal mutants and appears necessary for full life span extension from mTOR or S6K deletion. Similar findings have been described in nematodes and flies, but not yet in mammals. A report using the mTORC1 catalytic inhibitor Torin 1 in p53−/− mouse embryonic fibroblasts suggested that differential translation following mTOR inhibition could largely be explained by the presence of a 5′ terminal oligopyrimidine motif, though additional regulatory 5′ or 3′ elements could not be excluded. Interpretation is complicated by claims that effects of mTOR inhibition were mediated solely through 4E-BPs; deletion of individual 4E-BPs does not alter translation rates or body size in mice, whereas S6K deletion reduces body mass, indicating its role in growth.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
"Proteostasis, Translation Rates, and Longevity","Global reductions in mRNA translation may directly contribute to beneficial effects of mTOR inhibition on age-related diseases involving proteotoxic stress. Lowering translation rates may enhance translation fidelity, and decreased protein synthesis is thought to improve proteostasis by reducing the workload on protein repair and degradation systems. A reduced steady-state demand for proteostasis machinery may increase cellular capacity to respond to stresses such as oxidative damage, protein aggregation, or heat and cold shock. Loss of proteostasis is a critical component of many age-related diseases, and maintaining protein homeostasis is essential for organism survival. Decreased translation may promote longevity partly through improved proteostasis and increased protein degradation, though separating these effects from increased autophagy, antioxidant defense, or other consequences of mTOR inhibition is difficult.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR Signaling,2015
Autophagy Regulation by mTORC1,"In addition to promoting protein synthesis and cell growth, active mTOR inhibits the intracellular catabolic process of autophagy. As a major intracellular recycling pathway in eukaryotes, the autophagy-lysosomal pathway plays an essential role in degrading damaged organelles and macromolecules. Nutrient deprivation decreases mTOR activity, relieving the inhibition of autophagy by active mTOR and resulting in an increase in the catabolism of proteins and organelles. This increased catabolic activity provides amino acids and allows for cell survival when nutrients are limiting. The observed accumulation of damaged and aggregated proteins, oxidized lipids, and damaged organelles with age suggest that basal levels of autophagy decline or are insufficient to prevent the accumulation of damaged macromolecules associated with aging. Lipofuscin, the complex granular pigment that accumulates in aged tissue, is a highly conserved phenotype of cellular aging observed in all multicellular eukaryotes. Longevity-promoting interventions slow the rate of lipofuscin accumulation, and lipofuscin is often used as a biomarker of relative age.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),Autophagy and mTOR,2015
"Autophagy, Longevity, and Age-Related Disease","Given the correlation between longevity and damaged macromolecule accumulation, a major prediction is that accumulated damaged macromolecules drive aging and that modulating their accumulation could attenuate aging. Although this hypothesis is difficult to test due to challenges in selectively inducing the autophagy-lysosomal system, evidence from yeast and C. elegans supports the model that induction of autophagy is a necessary downstream effector of mTORC1 inhibition in mediating life span extension. Autophagy induction is also necessary for caloric restriction-mediated longevity, potentially via mTOR. While its necessity is accepted, it is not clear whether autophagy induction alone is sufficient to increase life span. Dysfunctional autophagy is implicated in numerous pathologies, and activation of autophagy attenuates age-related diseases. Autophagy induction has been targeted clinically for cardiovascular disease, age-related macular degeneration, diabetes, and neurodegenerative disorders including Parkinson’s disease and Alzheimer’s disease. Increasing autophagic degradation prevents neurodegeneration in models of AD, PD, and Huntington’s disease, which is associated with proteotoxic stress.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),Autophagy and mTOR,2015
mTORC1 and Mitochondrial Metabolism,"Mitochondria are key organelles in metabolism, disease, and aging. They are the major producers of energy, a primary site of metabolic reactions, a major source of toxic products including reactive oxygen species, and important regulators of cellular signaling. Mitochondria have been linked to diverse pathological states, diseases, and aging. mTORC1 appears to influence mitochondrial function through multiple mechanisms and downstream regulatory factors. Hypoxia-inducible factor 1 (Hif-1), a transcription factor promoting glycolytic processes, can be activated through mTOR signaling in mammals. Hif-1 is linked to longevity in model organisms via unclear mechanisms, and in mammals is associated with vascular tumor growth, wet macular degeneration, and rheumatoid arthritis via effects on VEGF. Intracellularly, Hif-1 promotes glycolysis, downregulates mitochondrial oxygen consumption, and partially mediates the Warburg effect in neoplasia. Reduced mTOR signaling influences tumor vascularization and metabolism partly through decreased Hif-1 activation.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),Mitochondria and mTOR,2015
mTOR Inhibition and Mitochondrial Respiration,"mTOR inhibition has been associated with increased mitochondrial respiration in yeast and worms, and caloric restriction has been associated with increased mitochondrial content and respiration in many organisms. This effect is directly associated with longevity in yeast, with adaptive signaling from increased mitochondrial superoxide production implicated. Mice lacking mTORC1 components in white adipose tissue show increased mitochondrial content and respiration, suggesting conservation in mammals. Mitochondrial metabolism and mitochondrial mass have been reported to increase under mTORC1 inhibition through downstream activation of PGC-1α and the transcription factor Ying-Yang 1. The role of mitochondrial metabolism as a downstream mediator of mTOR remains unclear, but available data suggest it is a critical component of aging and disease, warranting further attention.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),Mitochondria and mTOR,2015
Mitophagy and the Mitochondrial-Lysosomal Axis,"mTOR affects mitochondrial function through autophagic degradation of mitochondria, or mitophagy. The mitochondrial-lysosomal axis theory of aging proposes that maintaining a functional mitochondrial pool depends on continuous removal of damaged components through fission and mitophagy. Reduced turnover of mitochondria, as may occur in aging, would lead to accumulation of dysfunctional mitochondria, increased basal ROS production, damage accumulation, loss of tissue homeostasis, and potential senescence or cell death. mTORC1 inhibition increases basal autophagy and would be predicted to preserve or restore mitochondrial function with age, improving overall mitochondrial performance. While this model has not been directly tested, it offers an attractive mechanistic link between mitochondrial function and mTOR signaling in aging and disease.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),Mitophagy and mTOR,2015
Stem Cell Maintenance and mTORC1,"Stem cell loss and dysfunction are likely a significant factor in mammalian aging and age-related diseases. This is particularly likely in proliferative tissues such as dermis, the immune and gastrointestinal system, as well as in wound repair or response to ischemic injury in which proliferative capacity is required. While the exact role for stem cells in aging is currently unknown, evidence suggests that mTORC1 is central in the maintenance of stem cells with age. As discussed below, mTOR inhibition has been shown to protect immune function with age in murine models of infection, and this has been attributed to enhanced hematopoietic stem cell capacity. mTOR inhibition with rapamycin has also been recently reported to improve intestinal stem cell function, although in this case the improvement was linked to alterations in mTOR signaling in the adjacent Paneth cells, which are responsible for maintaining the stem cell niche, rather than a direct effect on intestinal stem cells. CR has been shown to enhance the function of skeletal muscle stem cells, presumably related, at least in part, to the concomitant decrease in mTOR activity. The role of stem cells in aging and of mTOR in regulating their function remains an exciting and largely uncharted avenue of research.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR and Stem Cells,2015
Longevity and Health Span,"Extension of health span, defined as the duration of life for which an organism is free from major age-related disease or loss of function, is considered by many to be the critical goal of aging research. Longevity studies in model organisms can intrinsically include health span components. C. elegans viability determination in standard plate or liquid-based life span studies is based on the ability of the animals to respond to mechanical stimulus. Yeast studies, both replicative and chronological, depend on the cell capacity to successfully produce progeny. In both cases, the individual organism may remain viable beyond the point that they are considered deceased by the assay standards. Thus, the nematode and yeast models are tied to neurological, muscular health, and reproductive capacity, respectively. In the murine model animal welfare regulations generally prevent expiration of mice by natural causes, requiring euthanasia if animals decline past a set cutoff in body mass, appear immobile, hunched, or in pain, or if they show signs of severe and untreatable diseases. These restrictions may complicate accurate determination of life span but they compel longevity-promoting interventions to also protect health span.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),"mTOR, Longevity, Health Span",2015
Health Span–Longevity Relationship and CR Studies,"Current data suggest longevity-enhancing interventions extend the health of populations and decrease or delay incidence of age-related disease rather than increase survival of unhealthy individuals. Regulation of health span and life span appear closely linked. Few examples exist where health span improves without longevity benefits. A notable exception is the recent (2013) NIA study of CR in rhesus monkeys, which observed decreased appearance of age-related diseases without a change in survival. This contrasts with a prior study reporting increases in both life span and health span. Differences in diet, housing conditions, and severity of CR may distinguish the studies, and lack of positive controls limits conclusions. Longevity interventions may more sensitively affect health span than life span. Efforts have grown to define health parameters influenced by aging interventions. Each model organism has distinct health span metrics: in yeast, replicative capacity, mitochondrial function, cell morphology; in nematodes, proteostasis, neurological decline, proteotoxic disease models, muscle function; in D. melanogaster, neurological, muscle, sensory, stem cell, and cardiac function; in mammals, diverse physiological and health parameters relevant to human aging.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),"mTOR, Health Span, Caloric Restriction",2015
mTOR in Age-Related Disease,"Concurrent with demonstrations of a role for mTOR in regulating longevity, it became increasingly apparent that mTOR signaling plays a central role in regulating health span and a variety of age-related and non-age-related pathologies. mTOR inhibition attenuates specific age-related changes in lower organisms. In addition, mTOR inhibition slows or delays many age-related and age-associated changes conserved from lower eukaryotes to mammals. These include lipofuscin accumulation, DNA damage accumulation, age-related mitochondrial dysfunction, and loss of proteostasis. Cardiac, neuronal, and stem cell functions are also improved with age in multicellular invertebrates and mammals by inhibition of mTOR. Table 1 summarizes mTOR inhibition across diseases: neurodegeneration (PD, AD, HD), age-related cognitive decline, cancer, heart disease, diabetes and obesity, immune function, kidney disease, age-related macular degeneration, and Hutchinson-Gilford progeria. Altered mTOR signaling has been investigated as a therapeutic strategy in numerous age-related pathologies. While efficacy in humans remains to be determined, extensive literature suggests mTOR is a clear potential target for diseases of aging.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR and Age-Related Disease,2015
"mTOR, Rapamycin and Immune Function","Rapamycins are used clinically as immunosuppressive or immunomodulatory drugs. There are, however, also reports that rapamycins can enhance immune system efficacy in certain settings, including tuberculosis, anti-tumor vaccine responses in mice, and vaccinia vaccination in nonhuman primates. In the context of age-related immune function, treating 22- to 24-month-old mice with rapamycin for only 6 weeks doubled the percentage and number of B (but not T) cells in the bone marrow, and restored the capacity of the aged animals’ immune system to mount an effective response to influenza vaccination, which was protective against subsequent infection. The apparent contradiction between these observations and the use of rapamycins as immunosuppressive drugs may be explained by observations that mTOR can exert divergent immunoregulatory functions during immune cell activation and differentiation, depending on the cell subset type. Rapamycins may limit immune activation in acute settings but appear to prevent age-related immune decline.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR and Immune Function,2015
mTOR and Inflammation,"Inflammation is strongly associated with aging and drives a multitude of age-associated disorders. Cardiovascular disease, obesity and metabolic disorders, cancer, and neurodegenerative diseases all include inflammatory components, and attenuation of inflammation has been implicated as a clinical target in each of these disease settings. Hyperactive mTOR has been linked to inflammation, and inhibition of mTOR by rapamycins has been demonstrated to be anti-inflammatory in renal disease, lung infection, and in vascular inflammation in atherosclerosis and following angioplasty. Furthermore, caloric restriction strongly attenuates age-related inflammatory signaling, and this effect appears to be at least partly mediated through mTOR. Reduced mTOR thus seems a strong candidate for treating or preventing age-related inflammatory processes, and reduced inflammation seems to play a mechanistic role in the pro-longevity effects of mTOR inhibitors.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR and Inflammation,2015
mTOR and Renal Disease,"Rapamycins are used clinically to reduce nephrotoxicity in chemotherapy, prevent allograft rejection, and as a treatment for renal cell carcinoma. Activation of mTOR signaling has been associated with several common forms of kidney disease, suggesting that inhibition of mTOR might have broad therapeutic benefits for renal health. Consistent with this, rapamycins have been shown to reduce kidney fibrosis, attenuate diabetic nephropathy, and improve outcome in animal models of polycystic kidney disease. These results indicate that mTOR inhibitors may hold wide therapeutic potential in renal pathology and could be effective in addressing diverse kidney disorders.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR and Renal Disease,2015
mTOR and Age-Related Macular Degeneration,"Age-related macular degeneration is the leading cause of blindness in Western countries. Capillary overgrowth in the choroid layer of the eye, a contributing factor, has been attributed to excessive production of VEGF. Rapamycin has been shown to reduce VEGF expression in retinal pigment epithelium and inhibit angiogenesis in vitro. In a rat model of age-related macular degeneration, rapamycin decreased the incidence and severity of retinopathy, and in human patients rapamycin appeared to decrease the need for anti-VEGF intravitreal injections by approximately half. Thus, age-related macular degeneration appears to be a promising clinical target for mTOR-inhibiting interventions, with evidence supporting benefits in both animal models and human patients.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),mTOR and Macular Degeneration,2015
Rapamycin in Progeria and Laminopathies,"Hutchinson-Gilford progeria syndrome (HGPS) is typically caused by a de novo mutation in the lamin A/C gene (LMNA) that activates a cryptic splice site, producing an abnormal lamin A protein termed progerin. Accumulation of progerin leads to aberrant nuclear morphology and is believed to be the causal factor in the pathogenesis of disease. The precise mechanism linking progerin accumulation to the phenotypes associated with this disease is unclear, but likely involves disruption of nuclear DNA binding proteins, including transcription factors and DNA repair components. Treatment of cells from HGPS patients with rapamycin corrects the nuclear morphology defect, delays the onset of cellular senescence, and enhances the clearance of progerin through autophagic degradation. No effective treatment for HGPS currently exists, and these data provide hope that rapamycins might slow disease progression. Success in HGPS would suggest possible efficacy in atypical Werners’ syndrome and lamin-related muscular dystrophies.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),Rapamycin and Progeria,2015
High-Throughput Methods in mTOR and Aging Research,"Genome-wide approaches, such as yeast single-gene mutant screens and RNAi screens in nematodes, have been critical to uncovering genes involved in the regulation of life span. The advent of proteomics, metabolomics, whole-genome sequencing, and RNAseq has fundamentally altered the way that aging studies are designed and executed. As the accessibility of these high-throughput methods has improved, these techniques are being increasingly utilized. The study of mTOR in aging has historically been largely led by reductionist experiments, with researchers focusing primarily on the use of single gene or small molecule perturbations to study mTOR and aging in model organisms. While these approaches have been, and continue to be, very fruitful in defining the regulation of aging and age-related processes by mTOR, they are limited in their ability to model the complexities of aging.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),Systems Biology and mTOR,2015
Integrating Multi-Omics for mTOR Systems Biology,"As large datasets produced using these methods become more widely available, it becomes increasingly important that the systems biology paradigm is applied to produce a comprehensive picture of the information. Modern aging research will greatly benefit from collaborations with bioinformatics experts that extend beyond analysis of high-throughput data and into systems-based integration of diverse datasets into models that combine genetic, proteomic, transcriptomic, and metabolomics data into informative and approachable descriptions of aging processes. These models should provide novel targets and strategies for intervention in the aging process as new nodes are described in genetic, proteomics, transcriptional, and metabolic paradigms. In addition, the systems approach to aging research may provide clear answers to difficult scientific queries, such as the nature of the similarities and differences between caloric restriction and CR mimetics (such as rapamycin). Thus, while classic methods for studying aging are far from exhausted, it is clear that systems approaches have a role to play in our understanding of aging, the influence of genotype on aging, and the mechanisms of interventions, such as mTOR inhibition, on the aging process.",Interdisciplinary Topics in Gerontology and Geriatrics (OAPEN),Systems Biology and mTOR,2015
Rapamycin and Rapalogs as Anti-Aging Therapeutics,"Rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), has the strongest experimental support to date as a potential anti-aging therapeutic in mammals. Unlike many other compounds that have been claimed to influence longevity, rapamycin has been repeatedly tested in long-lived, genetically heterogeneous mice, in which it extends both mean and maximum life spans. However, the mechanism that accounts for these effects is far from clear, and a growing list of side effects make it doubtful that rapamycin would ultimately be beneficial in humans. This Review discusses the prospects for developing newer, safer anti-aging therapies based on analogs of rapamycin (termed rapalogs) or other approaches targeting mTOR signaling. Rapamycin was discovered in the soil of Easter Island as a compound produced by Streptomyces hygroscopicus that inhibited proliferation of Candida albicans but did not affect bacterial growth. In mammals, rapamycin was found to inhibit the immune response and became a standard therapy to prevent graft rejection and treat autoimmune disorders.",Journal of Clinical Investigation,Rapalogs and mTOR Inhibitors,2013
Discovery of mTOR and Rapamycin Mechanism,"Rapamycin broadly inhibits the growth and proliferation of mammalian cells, spurring interest in its use as a cancer therapy. Mechanistically, rapamycin binds FKBP12, an immunophilin with prolyl isomerase activity. Two additional proteins required for its effects in yeast were identified in 1991 and termed target of rapamycin 1 (TOR1) and TOR2. During 1994 and 1995, three groups isolated a 289-kDa kinase bound and inhibited by the rapamycin-FKBP12 complex in mammalian cells. This kinase, mechanistic target of rapamycin (mTOR), is approximately 40% homologous to Saccharomyces cerevisiae TOR proteins and highly conserved among eukaryotes. mTOR forms two complexes with distinct functions and different sensitivities to rapamycin. mTORC1 (mTOR, raptor, mLST8/GβL, PRAS40, DEPTOR) regulates translation and cell growth through phosphorylation of S6K and 4E-BP and is potently inhibited by rapamycin. In contrast, mTORC2 (mTOR, rictor, mLST8/GβL, mSIN1, protor, DEPTOR) regulates AKT S473, serum/glucocorticoid–regulated kinase, and PKC-α and is acutely resistant to rapamycin.",Journal of Clinical Investigation,Rapalogs and mTOR Inhibitors,2013
mTOR Complexes and Signaling Inputs,"mTORCs receive inputs through a wide variety of signaling mechanisms with roles in many aspects of physiology. Briefly, mTORC1 responds to amino acids, glucose, WNT ligands, oxygen, cAMP, and insulin/IGF-1. Regulation of mTORC2 activity is less clear but may involve interaction with ribosomes. Insulin/IGF-1 signaling to mTORC1 is mediated in part by mTORC2 via AKT phosphorylation. In turn, mTORC1 activation feeds back to attenuate insulin/IGF-1 signaling via S6K1 and GRB10. A role for TOR signaling in aging was first revealed in 2003, when RNAi against let-363/CeTor significantly extended life span in C. elegans independently of daf-16, a FOXO homolog. This was followed by genetic evidence in Drosophila and budding yeast showing that inhibition of TOR signaling extends life span. Genetic inhibition of mTOR signaling in mammals is difficult, as mTOR, raptor, rictor, and mLST8 are essential for development.",Journal of Clinical Investigation,Rapalogs and mTOR Inhibitors,2013
Genetic Evidence Linking mTOR to Longevity,"Recent studies demonstrated that female Mtor+/– Mlst8+/– mice have reduced mTORC1 activity and increased longevity, similar to the phenotype of mice lacking S6K1, a principal mTORC1 substrate. Thus, the link between mTOR signaling and longevity appears to be conserved from yeast to mammals. Rapamycin extends life span in yeast, worms, and flies. In 2009, rapamycin was shown to extend both mean and maximum life spans in male and female genetically heterogeneous mice, with treatment initiated only at 20 months of age—roughly equivalent to 60-year-old humans. In a follow-up study beginning at 9 months, rapamycin extended median life span in males and females by 10% and 18% and maximum life span by 16% and 13%, respectively. Rapamycin was microencapsulated in an enteric coating enabling food delivery, and blood levels achieved were roughly three-fold higher than the therapeutic range for human immunosuppression.",Journal of Clinical Investigation,Rapalogs and mTOR Inhibitors,2013
Rapamycin Longevity Mechanisms: Anticancer and Multisystem Effects,"Anticancer effects. Cancer is the most common cause of death for laboratory mice, and rapamycin is an anticancer drug. Therefore, it remains possible that life span extension by rapamycin is secondary to tumor suppression and unrelated to the underlying aging process. There are several reasons why we do not favor this model. First, the initial experiments linking rapamycin and mTOR inhibition to longevity were performed in organisms that are mainly postmitotic (worms and flies) or single celled (yeast) and therefore do not experience cancer. Second, rapamycin increases maximum longevity, providing support for the idea that it slows multiple age-related pathologies. Targeting a single disease should not substantially increase the life spans of the longest-lived individuals unless the underlying aging process has been postponed. Third, rapamycin has been shown to delay multiple age-related changes in mice, including loss of stem cell function, cognitive decline, retinopathy, subcellular alterations in myocardium, liver degeneration, endometrial hyperplasia, tendon stiffening, and decline in physical activity. Rapamycin is also therapeutic in rodent models of cardiac hypertrophy and neurodegenerative diseases. Thus, cancer prevention is only one aspect of its anti-aging action.",Journal of Clinical Investigation,Rapamycin Longevity Mechanisms,2013
"Translation, mTORC1, and Longevity","Translation. mTORC1, via S6K and 4E-BP, plays a central role in the regulation of translation, and it is worth considering whether reduced protein synthesis per se might mediate the effects of rapamycin on longevity. Decreasing translation might improve fidelity and relieve stress on protein quality control systems. Experiments in S. cerevisiae, C. elegans, and D. melanogaster show that deletion or knockdown of ribosomal subunits, S6K, or translation initiation factors increases life span, and S6K1 deletion extends life span in female mice, whereas 4E-BP deletion blocks CR-induced life span extension in flies. However, recent findings challenge the idea that translation per se is the main driver. Female mice lacking S6K1 have extended life spans without detectable changes in overall translation in skeletal muscle. Worms lacking a key initiation factor can live even longer when TOR is deleted, suggesting distinct mechanisms. Life span extension from initiation factor deletion depends on daf-16, whereas extension by TOR, S6K, or ribosomal subunit depletion does not. Reducing TOR by RNAi does not extend life in eat-2 CR-model worms despite further lowering protein synthesis. These findings suggest a complex relationship between translation and longevity.",Journal of Clinical Investigation,Rapamycin Longevity Mechanisms,2013
Selective mRNA Translation and Longevity,"Translation of specific mRNAs may influence life span. While complete loss of mTOR function strongly reduces general translation, rapamycin has a more subtle effect because some functions of 4E-BP are rapamycin resistant. Both rapamycin and full mTOR inhibition preferentially suppress translation of mRNAs with 5′ terminal oligopyrimidine motifs. CR in flies decreases overall protein synthesis but specifically enhances translation of mRNAs with short, unstructured 5′ UTRs, including nuclear-encoded mitochondrial genes. TOR substrate 4E-BP is required for this effect and for CR-induced life span extension. In yeast lacking ribosomal subunits or TOR, full life span extension requires increased translation of the GCN4 transcript. Under low TOR signaling, upstream ORFs are bypassed more often, allowing translation of GCN4. These examples highlight regulatory subtleties in translational control that impact longevity.",Journal of Clinical Investigation,Rapamycin Longevity Mechanisms,2013
"Autophagy, Rapamycin, and Aging","Autophagy. mTOR inhibition induces autophagy, which recycles proteins and organelles. Autophagy is essential for survival during nutrient scarcity and removes damaged components. mTOR phosphorylates and inhibits ULK1 under nutrient sufficiency. Loss of autophagy genes decreases life span in yeast, C. elegans, and Drosophila, and promoting autophagy in the fly nervous system extends life span. Autophagy is required for rapamycin-induced life span extension in yeast and for CR- or mTOR-inhibition-dependent longevity in worms. In mammals, autophagy is strongly linked to aging. Induction of autophagy rejuvenates liver structure and function in aged mice. Autophagy mediates CR benefits in heart, liver, and kidney, and is elevated in long-lived Snell dwarf mice. Cardiomyocytes from aged mice show reduced autophagy and calcium defects, both corrected by rapamycin ex vivo. However, excessive autophagy may contribute to progeroid phenotypes. Rapamycin improves nuclear defects and premature senescence in Hutchinson–Gilford progeria cells by stimulating progerin clearance via autophagy. Autophagy regulation appears central to healthy aging.",Journal of Clinical Investigation,Rapamycin Longevity Mechanisms,2013
Rapamycin and Stem Cell Function,"Stem cell maintenance. Rapamycin affects stem cell function in multiple ways. Hyperactive signaling upstream of mTORC1 due to Pten deletion, Tsc1 deletion, or constitutive AKT activation reduces hematopoietic stem cell (HSC) number and function. Rapamycin restores normal self-renewal in HSCs with high oxidative stress and reduced capacity. mTORC1 activity is elevated in HSCs from aged mice, which show functional deficits similar to Tsc1 deletion, and rapamycin restores HSC capacity and enhances influenza immune responses in aged mice. Rapamycin also increases intestinal stem cell self-renewal via inhibition of mTORC1 in adjacent Paneth cells, similar to CR effects. Rapamycin enhances reprogramming of somatic cells into induced pluripotent stem cells. Conversely, rapamycin impairs pluripotency, reduces proliferation, and promotes differentiation in human embryonic stem cells, and in mouse ESCs it reduces size, slows proliferation, and enhances differentiation. Rapamycin also depletes leukemia-initiating cells and inhibits self-renewal of hemangioma-derived stem cells. Overall, rapamycin generally favors retention of “stemness” in adult stem cells.",Journal of Clinical Investigation,Rapamycin Longevity Mechanisms,2013
"Inflammation, mTORC2, and Stress Resistance","Antiinflammatory mechanisms. Rapamycin’s effects on immunity are complex: chronic low-grade inflammation is a hallmark of aging, and nearly all chronic diseases have inflammatory components. Rapamycin has both positive and negative effects on innate and adaptive immunity but can enhance immune responses in aged mice. mTORC2-dependent mechanisms. Although rapamycin acutely inhibits mTORC1, chronic exposure can also inhibit mTORC2 in cell lines and in vivo in liver, muscle, and adipose tissue. It remains unclear whether mTORC2 inhibition contributes to longevity. Female S6K1–/– and Mtor+/–Mlst8+/– mice appear long-lived due to impaired mTORC1 signaling, but C. elegans data suggest mTORC2 inhibition can also extend life span. Worm longevity from mTORC1 disruption requires skn-1 (NRF1/2) and daf-16 (FOXO), whereas longevity from rapamycin or mTORC2 disruption requires only SKN-1. Worms and flies with impaired TOR function are stress resistant, and NRF1/2 and FOXO target genes are induced in livers of rapamycin-treated mice.",Journal of Clinical Investigation,Rapamycin Longevity Mechanisms,2013
mTOR-Independent Mechanisms and Endocannabinoid Links,"mTOR-independent mechanisms. Some in vivo effects of rapamycin may be independent of mTOR. FKBP12 proteins influence sodium and calcium currents in multiple excitable cell types, in part through binding to ryanodine receptors. Moreover, rapamycin also binds to FKBP52, and analogs that favor interaction with FKBP52 over FKBP12 exhibit neuroprotective properties. Endocannabinoid signaling. Although rapamycin itself has not yet been tested, an intriguing connection between TOR and endocannabinoid signaling was recently described. Small molecules analogous to a mammalian endocannabinoid were identified in C. elegans, and depletion of these molecules was associated with life span extension by CR. One specific molecule, eicosapentaenoyl ethanolamide (EPEA), was also found to be lower in worms lacking S6K, and treatment with EPEA suppressed life span extension in both models while conferring increased susceptibility to heat stress. These findings highlight complex nutrient–signaling interactions with potential relevance for rapamycin and longevity.",Journal of Clinical Investigation,Rapamycin and mTOR-independent mechanisms,2013
"Rapamycin Side Effects: Immunity, Dermatology, Fertility, Metabolism","Rapamycin side effects. Rapamycin is FDA approved as an immunosuppressant after transplant surgery and for treatment of renal cell carcinoma, and is used in coronary stents and clinical trials for lymphangioleiomyomatosis and autoimmune disorders. Although clinically useful, it is unlikely to be approved preventatively in healthy individuals due to substantial side effects. A major concern is immune suppression. While rapamycin extends mouse life span in pathogen-free facilities and can boost immunity against certain pathogens, human data remain complicated by concomitant immunosuppressants. A controlled renal transplant study found that 34% of patients experienced viral infection and 16% fungal infection. Dermatological adverse events are common: edema in 60% of patients, aphthous ulcers in 55%, frequent mucositis and rash, hair and nail disorders including alopecia in ~90%. Rapamycin is associated with loss of testicular function and reduced male fertility in both humans and mice. Metabolic effects include hyperlipidemia, decreased insulin sensitivity, glucose intolerance, and increased diabetes risk. Chronic treatment is also associated with gastrointestinal events such as diarrhea, along with anemia, renal toxicity, impaired wound healing, and joint pain.",Journal of Clinical Investigation,Rapamycin side effects,2013
Prospects for Safer mTOR Inhibitors: Rapalogs and Kinase Inhibitors,"Prospects for safer mTOR inhibitors. Based on its ability to inhibit cell proliferation, rapamycin has attracted interest for cancer therapy. Rapalogs with improved pharmacokinetics include temsirolimus, everolimus, ridaforolimus, 32-deoxo-rapamycin, and zotarolimus. Despite promising preclinical results, rapalogs have generally disappointed in human cancer trials and are approved only for renal cell carcinoma and certain pancreatic cancers or tuberous sclerosis. One explanation is that rapalogs primarily inhibit mTORC1, causing compensatory increases in PI3K/AKT signaling via loss of negative feedback through S6K and GRB10. AKT activity may later be attenuated by mTORC2 disruption, but insufficiently to prevent tumor-promoting signals. New compound classes include ATP-competitive mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2, and dual PI3K/mTOR inhibitors. These reveal rapamycin-resistant mTORC1 functions but likely have similar or greater side effects due to broad pathway inhibition. An interesting exception is caffeine, a weak TOR inhibitor; TOR inhibition mediates life span extension in yeast exposed to caffeine, raising the possibility of mild mTOR effects in humans at dietary doses.",Journal of Clinical Investigation,Rapalogs and mTOR inhibitors,2013
"Indirect Inhibitors of mTOR: Aspirin, AMPK Activators, Autophagy Modulators","Indirect inhibitors. Studies showing that S6K1–/– mice and Mtor+/–Mlst8+/– mice exhibit increased longevity suggest that specific inhibition of mTORC1 or S6K1 may provide many benefits of rapamycin. S6K1 inhibitors are being developed but remain years from clinical use. Multiple FDA-approved compounds reduce mTORC1 activity. Aspirin decreases S6K phosphorylation and may act by inhibiting TSC1 phosphorylation by IKKβ or by activating AMPK. AMPK inhibits mTORC1 via activating phosphorylation of TSC2 and inhibitory phosphorylation of raptor. Other AMPK activators (e.g., AICAR) likewise reduce mTORC1 signaling. Aspirin extends average but not maximum life span in male mice and decreases cancer-related and all-cause mortality in humans. A screen for autophagy regulators identified perhexiline, niclosamide, rottlerin, and amiodarone as mTORC1 inhibitors without affecting mTORC2. Rottlerin regulates mTORC1 in a TSC-dependent manner; the others act independently of TSC. Natural compounds such as phenethyl isothiocyanate also inhibit mTORC1. Many drugs that act on insulin/IGF-1 signaling additionally influence mTOR pathways.",Journal of Clinical Investigation,Indirect mTOR inhibitors,2013
Metformin as a Safer mTOR Modulator and Longevity Candidate,"Metformin and safer mTOR inhibition. Metformin, a widely used AMPK activator and first-line therapy for type 2 diabetes, lowers glucose, inhibits lipolysis, decreases circulating free fatty acids, and has few side effects. Metformin inhibits phosphorylation of mTORC1 substrates S6K1 and 4E-BP1 and decreases translation. While originally thought to act solely through AMPK, metformin also regulates mTORC1 directly by inhibiting Rag GTPases and indirectly by upregulating REDD1 to enhance TSC2 activity. Metformin extends life span and health span in C. elegans via AMPK and SKN-1/NRF2, independent of insulin signaling. It extends life span in HER2/neu tumor-prone mice and female SHR mice. Ongoing ITP studies are testing metformin in genetically heterogeneous mice. Long-term human studies show metformin treatment decreases all-cause mortality, including diabetes-related mortality, cancer, and myocardial infarction. However, effects on maximum life span in humans or long-lived rodents have not been demonstrated.",Journal of Clinical Investigation,Metformin and mTOR modulation,2013
Conclusion: Future Directions for Rapamycin and mTOR-Targeting Therapies,"Conclusion. Rapamycin shows significant promise in animal models as a pharmaceutical agent for the treatment of age-related disease. However, the significant side effects limit its long-term utility in humans. Similar problems are likely to emerge for rapalogs and mTOR kinase inhibitors. Moving forward, mTORC1-specific inhibitors that avoid disruption of mTORC2 signaling or that only reduce, rather than abolish, the activity of the mTORC1 pathway, may offer a safer method for the treatment of age-related diseases. The exploration of different dosing regimens for rapamycin and further testing of metformin have significant promise in this regard, but further research will be required to determine whether any of the available strategies for targeting mTOR will ultimately prove beneficial to human longevity and protect against age-related diseases.",Journal of Clinical Investigation,mTOR inhibition and longevity,2013
Abstract Overview of NAD and CD38,"Nicotinamide adenine dinucleotide (NAD) acts as a cofactor in several oxidation-reduction (redox) reactions and is a substrate for a number of nonredox enzymes. NAD is fundamental to a variety of cellular processes including energy metabolism, cell signaling, and epigenetics. NAD homeostasis appears to be of paramount importance to health span and longevity, and its dysregulation is associated with multiple diseases. NAD metabolism is dynamic and maintained by synthesis and degradation. The enzyme CD38, one of the main NAD-consuming enzymes, is a key component of NAD homeostasis. The majority of CD38 is localized in the plasma membrane with its catalytic domain facing the extracellular environment, likely for the purpose of controlling systemic levels of NAD. Several cell types express CD38, but its expression predominates on endothelial cells and immune cells capable of infiltrating organs and tissues. Here we review potential roles of CD38 in health and disease and postulate ways in which CD38 dysregulation causes changes in NAD homeostasis and contributes to the pathophysiology of multiple conditions. Indeed, in animal models the development of infectious diseases, autoimmune disorders, fibrosis, metabolic diseases, and age-associated diseases including cancer, heart disease, and neurodegeneration are associated with altered CD38 enzymatic activity. Many of these conditions are modified in CD38-deficient mice or by blocking CD38 NADase activity. In diseases in which CD38 appears to play a role, CD38-dependent NAD decline is often a common denominator of pathophysiology. Thus, understanding dysregulation of NAD homeostasis by CD38 may open new avenues for the treatment of human diseases.",American Journal of Physiology – Cell Physiology,CD38 / NAD metabolism,2022
Introduction: NAD Discovery and Classical Functions,"A series of studies by Warburg and others between the 1920s and the 1950s led to the discovery that nicotinamide adenine dinucleotide (NAD) is an essential cofactor in oxidation-reduction (redox) reactions (1). These discoveries shed light on reactions that drive the transfer of reducing equivalents from energy-rich substrates such as glucose, fatty acids, and amino acids to the mitochondrial electron transport system (ETS), resulting in the synthesis of ATP (2). The NAD+-to-NADH ratio (NAD+/NADH) is considered a readout of the cell’s redox state (3). The phosphorylated form of NADH (NADPH) is also a critical substrate for enzymes charged with scavenging reactive oxygen species (ROS) and preventing oxidative damage to cells (4, 5). In addition to these “classical” functions, NAD plays a crucial role in cell signaling as a substrate for protein-modifying enzymes (e.g., ADP-ribosylation and deacetylation) and the formation of putative second messengers such as cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) (6). This review critically discusses the mounting literature demonstrating that NAD dysregulation is consequential to cellular homeostasis. In fact, dysregulation of NAD metabolism is currently reported in preclinical animal models of age-related disease and increasingly identified in human diseases (7). NAD homeostasis relies on several enzymes including those associated with NAD degrading and synthetic pathways. This review pays particular attention to the role of the NAD-catabolizing enzyme CD38, which plays critical roles in the pathogenesis of diseases related to infection, inflammation, fibrosis, metabolism, and aging (Fig. 1) (7–9).",American Journal of Physiology – Cell Physiology,CD38 / NAD metabolism,2022
NAD Synthesis and Degradation Overview,"Cellular NAD levels are dynamically controlled by the balance between its synthesis and degradation (6). As a redox carrier, NAD is interconverted between its oxidized form (NAD+) and its reduced form (NADH) by dehydrogenases or oxidoreductases that catalyze hydride transfer. In redox reactions, the ratio between the oxidized and reduced forms (NAD+/NADH) is changed but no NAD is consumed and the total NAD+/NADH pool is maintained. However, NAD-degrading enzymes such as CD38, ARTCs (ADP-ribosyltransferase C2 and C3 toxin-like), sirtuins (SIRTs), and poly(ADP-ribose) polymerases (PARPs) break the glycosidic bond between the nicotinamide ring and the ribose of the dinucleotide. This generates free nicotinamide and an ADP-ribosyl moiety and contributes to a decrease in the levels of NAD (6). The activity of these enzymes is reflected by high turnover of NAD in some tissues (10). NAD half-life varies across tissues from 15 min to 15 h in mice, averaging 2–4 h in most tissues (10). The dynamic turnover of NAD in tissues highlights the previously underappreciated metabolic fluxes of NAD in vivo. Why NAD turnover is especially rapid is not entirely understood (11). The total NAD pool is recycled in hours and requires a significant expenditure of energy to maintain cellular levels (Table 1). Conservative estimates predict that the mouse recycles its NAD pool nearly three times a day to maintain its steady-state level (Table 1).",American Journal of Physiology – Cell Physiology,NAD Metabolism,2022
"De Novo, Salvage, and Preiss–Handler Pathways","NAD can be synthesized by multiple pathways (Fig. 2). De novo NAD synthesis requires the essential amino acid tryptophan and proceeds through the kynurenine pathway (KP) mainly in the liver (10, 12). A portion of de novo-synthesized NAD is likely metabolized in the liver to nicotinamide (NAM) and then distributed organism-wide. It is speculated that NAM released from the liver or NAM formed as a product of NAD breakdown in cells is used to generate NAD in other tissues through a second pathway called the NAD salvage pathway (10). The first step of the salvage pathway is catalyzed by the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT), which synthesizes nicotinamide mononucleotide (NMN) from NAM and a phospho-ribosyl group from 5-phospho-a-D-ribosyl 1-diphosphate (PRPP), a metabolite that originates from the pentose-phosphate pathway (13). NMN is a substrate for NMN-adenylyltransferases (NMNATs 1–3), which catalyze the conversion of NMN and ATP to NAD and pyrophosphate. Interestingly, the synthesis of NAD from NAM is energetically costly (Table 1), particularly given the requirement of 2–4 molecules of ATP per molecule of NMN (14, 15). Based on 24-h NAD turnover, one would expect a mouse to use 1.25 mmol of ATP each day to recycle its NAD pool via the salvage pathway (Table 1). This amount would correspond to 0.7 g of ATP per day, 1–3% of the weight of a young mouse, to maintain steady-state levels of NAD.",American Journal of Physiology – Cell Physiology,NAD Metabolism,2022
Preiss–Handler Pathway and Microbiota Influence,"NAD is alternatively generated from nicotinic acid (NA), a dietary form of vitamin B3, through a third pathway called the Preiss–Handler pathway. Here the enzyme nicotinate phosphoribosyltransferase 1 (NAPRT1) catalyzes the reaction between NA and PRPP using energy derived from ATP and produces nicotinic acid mononucleotide (NaMN), ADP, and inorganic pyrophosphate (PPi) (16, 17). The next steps involve the incorporation of AMP (from ATP) by NMNATs and an amidation in the pyridine ring of NA by the NAD-synthetase, which uses L-glutamine or ammonia as a nitrogen source (18) to generate NAD. Interestingly, changes in the intestinal microbiota can affect NAD production via the Preiss–Handler pathway (19). In the gut, metabolism of NA by the intestinal microbial nicotinamidase PncA generates NAM (19). As expected, germfree mice are more vulnerable to the inhibition of the salvage pathway, indicating that gut microbiota indeed contribute to NAD production through the Preiss–Handler pathway. Furthermore, gut microbiota potentiates the NAD-boosting effect of NAM supplementation by providing deaminated intermediates for NAD synthesis. Gut microbiota also participates in conversion of NR to NAM, NA, and nicotinic acid riboside (NAR) in mice (19). Taken together, gut microbiota contributes to the energy balance of the organism by maintaining NAD levels within a healthy range mainly by diversifying the array of NAD precursors for NAD synthesis available to the host.",American Journal of Physiology – Cell Physiology,NAD Metabolism,2022
"NR, NRH, and Additional NAD Precursors","NMN, another NAD precursor, can be generated intracellularly from NR via a phosphorylation reaction catalyzed by nicotinamide riboside kinase 1 (NRK1) (20–22). A reduced form of NR (NRH) can also serve as a potent NAD precursor (23, 24). NRH is phosphorylated to NMNH by adenosine kinase and results in enhanced NAD boosting (23–25). The interplay between these multiple pathways for NAD synthesis has not been completely elucidated.",American Journal of Physiology – Cell Physiology,NAD Metabolism,2022
Overview of NAD-Degrading Enzymes,"The common feature of enzymes that degrade NAD is that each breaks the glycosidic bond between the NAM ring and the ribose of the dinucleotide. This section highlights ADP-ribosyltransferases (PARPs), NAD-dependent protein deacylases (SIRTs), and the sterile a and toll/IL-1 receptor motif containing 1 enzyme (SARM1).",American Journal of Physiology – Cell Physiology,NAD Consumers,2022
ADP-Ribose Polymerases (PARPs),"ADP-ribosyltransferases such as PARPs [poly(ADP-ribose) polymerases] and tankyrases catalyze the transfer of ADP-ribose from NAD to a number of amino acids in substrate proteins (i.e., arginine, aspartate, glutamate, cysteine, serine, lysine, or tyrosine residues) and release NAM (26, 27). This posttranslational modification can then be recognized by other proteins that possess specific interaction domains such as PIN, PEPPAR, PBZ, BRCT, MDPAR, WWE, OB-fold, and/or FHA. A well-known example of poly ADP-ribosylation (PAR) is the PARylation of proteins by ribosyltransferases that sense single- or double-strand DNA breaks (26). The resulting PAR chains recruit DNA repair machinery. ADP-ribosylation additionally controls many other important cellular processes such as transcription (28, 29), cell cycle progression (30), proteasome regulation (31), and metabolism (32). Among the ADP-ribosyltransferases, PARP1 is the most abundant and significantly impacts NAD levels when overactivated (33–36). Another example of ADP-ribosylation is the recently described ADP-ribosylation of DNA on thymidine, which is performed by DNA ADP-ribosyltransferase (DarT) and is induced by bacterial toxins (e.g., DarT-DarG-toxin antitoxin system) requiring NAD consumption (37).",American Journal of Physiology – Cell Physiology,PARPs,2022
NAD-Dependent Protein Deacetylases (Sirtuins),"Largely known as NAD-dependent protein deacetylases, sirtuins (SIRTs) transfer the acetyl group of an acetylated substrate protein to the ADPR moiety of NAD, forming 2'-O-acetyl-ADPR, NAM, and deacetylated proteins (38). Some sirtuins such as SIRT4–7 display lower deacetylase activity (39–42). Apart from protein deacetylation activity, sirtuins also perform other reactions such as defatty-acylation by SIRT1–3 and -6 (43–46), deacylation by SIRT4 (47), and desuccinylation, demalonylation, and deglutarylation by SIRT5 (48–50) in an NAD-dependent manner. NAD levels regulate these activities as well as biological processes related to these enzymes (51, 52). Having low binding affinity to NAD+ (27), sirtuins are more likely to be influenced by changes in NAD homeostasis as a consequence of the activity of other NAD-dependent enzymes.",American Journal of Physiology – Cell Physiology,Sirtuins,2022
SARM1 as an NADase,"Sterile a and Toll/interleukin-1 receptor motif-containing 1 (SARM1) has been identified in neurons as a new member of the NADase enzyme family (53, 54). It was shown to degrade NAD and generate ADPR, cADPR, and NAM as products (53). SARM1 may also generate the second messenger NAADP via the base-exchange reaction (55). In neurons, SARM1-dependent NAD depletion plays a key role in the axonal degeneration pathway in vitro and in vincristine-induced traumatic injury models in vivo (53). SARM1 is an important NADase of the nervous system, yet it is still not known whether the enzyme contributes to NAD depletion in other tissues.",American Journal of Physiology – Cell Physiology,SARM1,2022
CD38 Expression and Regulation,"CD38 was first observed on thymocytes and T lymphocytes (56) and is widely reported on immune, endothelial, and smooth muscle cells (9, 57–64). CD38 is upregulated in a cell-dependent manner by several stimuli in the presence of 1) proinflammatory or secreted senescence factors (9, 64–66) or in response to a bacterial infection (63, 67); 2) retinoic acid (68); or 3) gonadal steroids (60, 69). CD38 is stimulated in a cell-specific manner by lipopolysaccharide (LPS), tumor necrosis factor a (TNF-a), interleukin-6 (IL-6), and interferon-c (IFN-c) (9, 66, 70, 71). Among the NAD-degrading enzymes, CD38 is the main regulator of NAD levels in mouse tissues (51, 52, 72–74).",American Journal of Physiology – Cell Physiology,CD38,2022
CD38 Enzymatic Activities,"CD38 exhibits three main functions using NAD as a substrate. ADP-ribosyl cyclase activity produces cADPR and NAM, which represents <1% of the enzymatic activity of CD38. cADPR is a putative second messenger that induces calcium release from the endoplasmic reticulum by binding to ryanodine receptors (75). CD38 also catalyzes a rare base-exchange reaction that substitutes the NAM group with an NA moiety, which produces NAADP and free NAM (76, 77). NAADP is a putative second messenger that mediates Ca2+ signaling acting on two-pore channel receptor (TPC) in acid endolysosomes (78) and is generated by CD38-dependent base-exchange reaction in vitro (76, 77, 79) with uncertain physiological relevance in vivo (7, 80, 81). Because the base-exchange reaction in vitro occurs in excess of NA and low pH (82), it may have some importance in specific cellular compartments such as the endolysosomal system (83). NAADP is also shown to mediate calcium release from the endoplasmic reticulum through its interaction with the NAADP binding protein HN1L/JPT2, which forms complex with ryanodine receptor 1 (84–86). Upon T-cell receptor stimulation, the dual NADPH oxidases DUOX1 and DUOX2 produce NAADP from NAADPH, eliciting calcium signaling leading to T-cell activation (87). CD38 does not participate in NAADP formation in this context but appears to coordinate NAADP degradation in T cells (88).",American Journal of Physiology – Cell Physiology,CD38,2022
CD38 NAD Glycohydrolase Activity,"Most significantly, CD38 NAD glycohydrolase activity produces ADPR and NAM and represents >90% of enzyme activity (89). ADPR acts on an intracellular domain of transient receptor melastatin 2 (TRPM2) channels, thus eliciting Ca2+ influx across the plasma membrane (90). CD38 can also degrade NMN, producing NAM and presumably phosphoribose (52, 91), regulating NMN levels in vivo (64). Although CD38 can be found in the cytoplasm and in the membranes of intracellular organelles, the vast majority of CD38 activity is in the plasma membrane, facing outside the cell (7, 92). That CD38 regulates NAD homeostasis inside the cell while its catalytic site faces the extracellular space has been known as the CD38 “topological paradox.” One explanation for this paradox is that the roles of extracellular and intracellular CD38 differ. Whereas intracellular CD38 targets NAD for degradation, extracellular-facing CD38 additionally degrades the NAD precursor NMN, limiting the production of NAD intracellularly (51, 52, 64, 91).",American Journal of Physiology – Cell Physiology,CD38,2022
CD38 in Purinergic Signaling and NAM Accumulation,"In addition to its function in NAD catabolism, CD38 is also required in purinergic signaling pathways, where it interacts with extracellular nucleotidases and modulates the production of the immunosuppressive molecule adenosine (93). Neither a-NAD derivatives, NR and NRH, nor reduced forms of NAD can be efficiently hydrolyzed by CD38 (24, 52, 94). CD38 also produces NAM as a product of intracellular NAD breakdown and as a product of NMN extracellular breakdown. Considering the importance of CD38 as the main NAD-consuming enzyme in the body (74), it may also be responsible for local NAM accumulation (36, 95) and subsequent inhibition of NAD-utilizing enzymes, but this possibility remains to be explored.",American Journal of Physiology – Cell Physiology,CD38,2022
Interaction Between CD38 and Sirtuins,"CD38 interferes with the activity of other NAD-dependent enzymes such as sirtuins. For example, CD38 inhibits sirtuins both by reducing NAD levels and by generating NAM, a well-characterized sirtuin inhibitor (36, 51, 96). Inhibition of CD38 by 78c, a specific and potent CD38 inhibitor, promotes protein deacetylation (51). Genetic deletion or inhibition of CD38 also protects mice from conditions that impair sirtuin activity such as a high-fat diet (97), dysregulation of glucose and lipid homeostasis in obesity (72), age-associated mitochondrial dysfunction (52), and D-galactose-induced myocardial cell senescence (98). Thus, the interplay between CD38 and sirtuins is an important component of the pathophysiology of diseases associated with NAD decline (Fig. 3).",American Journal of Physiology – Cell Physiology,CD38 and Sirtuins,2022
CD38 in Autoimmunity: Overview,"CD38 plays a critical role in inflammation, migration, and immunometabolism, but equally important is the resolution of the inflammatory response, if left unchecked, leads to loss of self-tolerance (115), tissue infiltration of lymphocytes, and circulation of autoantibodies (116). Mounting evidence suggests that CD38 acts as a double-edged sword in the formation of autoimmunity. Depending upon context, CD38 can either promote or protect against an autoimmune response (117–121). Here, the role of CD38 in four autoimmune disorders is discussed briefly and outstanding questions highlighted.",American Journal of Physiology – Cell Physiology,CD38 Autoimmunity,2022
CD38 in Systemic Lupus Erythematosus (SLE),"Systemic lupus erythematosus (SLE) involves formation of autoantibodies against nuclear and cytoplasmic antigens (122, 123), resulting in tissue damage in multiple organs including skin and kidneys (124). Initial observations of dysregulated NAD metabolism in SLE show decreased ADP-ribosylation in patient-derived peripheral blood lymphocytes (125). Individuals with SLE have CD8+ T cells with high CD38 expression (CD8+ CD38high) (117, 121, 126, 127) and accompanying NAD decline, Sirt1 inactivation, acetylation of the methyl-transferase EZH2, and subsequent suppression of cytotoxicity-related transcription factors (128). The result is a lower T-cell cytotoxic response observed in SLE, which predisposes patients to infection. Pharmacological inhibition of EZH2 restores cytotoxic capacity of CD8+ CD38high T cells in SLE. What triggers increased CD38 expression in CD8+ T cells in this condition remains unknown.",American Journal of Physiology – Cell Physiology,CD38 Autoimmunity,2022
CD38 in Multiple Sclerosis,"Multiple sclerosis (MS) is a chronic autoimmune degenerative disease of the central nervous system (CNS) characterized by inflammation, demyelination, and destruction of the blood-brain barrier (129). The pathogenesis of MS involves activation of microglia and macrophages, which when reversed significantly decreases disease severity (129). In a cuprizone (CPZ)-induced demyelination mouse model, CD38 is upregulated in both astrocytes and microglia (130). CD38 inhibition in this model attenuates glial activation and demyelination by restoring NAD levels. Furthermore, in a myelin-oligodendrocyte-glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mouse model, CD38 is involved in T-cell activation (131), which is attenuated in CD38-knockout mice. Further studies are required to evaluate whether CD38 expression on inflammatory cells also disrupts NAD homeostasis in EAE.",American Journal of Physiology – Cell Physiology,CD38 Autoimmunity,2022
"Diet, Demyelination, and CD38","Interestingly, a Western diet potentiates demyelination in the CNS of mice, which is improved by genetic ablation or pharmacological CD38 inhibition (132). Furthermore, CD38 levels increase in mouse spinal cord after chronic high-fat diet exposure, after focal toxin-mediated demyelinating injury, and in reactive astrocytes in active MS lesions (132). CD38-catalytically inactive mice are protected from high-fat-induced NAD depletion, oligodendrocyte loss, oxidative damage, and astrogliosis. Likewise, the CD38 inhibitor 78c increases NAD and attenuates neuroinflammatory changes in astrocytes treated with saturated fats in vitro (132). Taken together, a high-fat diet impairs oligodendrocyte survival and differentiation by a CD38-mediated mechanism and underscores the potential therapeutic value of CD38 inhibitors in myelin regeneration (132).",American Journal of Physiology – Cell Physiology,CD38 Autoimmunity,2022
CD38 in Inflammatory Bowel Disease (IBD),"Inflammatory bowel disease (IBD) is an idiopathic chronic and progressive inflammatory condition of the gastrointestinal tract mucosa that includes Crohn’s disease and ulcerative colitis (133). The chronic mucosal inflammation and tissue damage characteristic of the disease predispose IBD patients to the development of colorectal cancer, and the risks increase with duration, extent, and severity of inflammation (134). CD38 expression is increased in macrophages from intestinal tissues of individuals with IBD (135). Additionally, IBD shows systemic chronic inflammation with increased activation of CD38+ T lymphocytes in the blood compared with healthy individuals (136, 137). Dysregulation of NAD metabolism partially explains the pathogenesis of IBD, which includes upregulation of the NAD consumers CD38, PARP9, PARP14, and SIRT1; the NAD synthesis enzyme NAMPT; and the NAD excretion enzyme nicotinamide N-methyltransferase (NNMT) (135).",American Journal of Physiology – Cell Physiology,CD38 Autoimmunity,2022
CD38 Knockout Phenotypes in Colitis Models,"The direct link between CD38 and intestinal inflammation is demonstrated in CD38-knockout mice in a dextran sulfate sodium (DSS)-induced colitis model (119). Wild-type mice exposed to DSS display dense CD38+ cells in the mucosa, including resident T cells, granulocytes, and inflammatory monocytes, and exhibit weight loss, shortening of the colon, and alterations in the morphology of the epithelium, crypts, and submucosa. Conversely, CD38-knockout mice displayed only mild disease during DSS treatment (119). CD38 depletion is likely to be protective in DSS-treated mice because of impaired migration of immune cells to mucosal tissue. It is proposed that ADPR produced by CD38 breakdown of NAD acts on TRPM2, a plasma membrane Ca2+-permeable cation channel (119). Curiously, DSS-induced ulcerative colitis is also suppressed in TRPM2-knockout mice (138). It remains to be established whether CD38-induced cell migration, CD38-dependent secretion of proinflammatory cytokines, and/or CD38 NADase activity contribute to the role of CD38 in intestinal inflammation.",American Journal of Physiology – Cell Physiology,CD38 Autoimmunity,2022
CD38 in Rheumatoid Arthritis (RA),"Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease more frequently observed in females and the elderly and associated with progressive disability and premature death. RA mainly targets the lining of synovial joints, includes extra-articular involvement, and results in inflammatory arthritis (139, 140). Antibody-producing plasma cells and their B cell precursors contribute considerably to the pathogenesis of RA by synthesizing autoantibodies that either bind to tissue antigens or form immune complexes within tissues (9). CD38 is highly expressed in plasma cells compared with other immune cell populations in synovial biopsies and peripheral blood mononuclear cells (PBMCs) (121). In fact, pathogenic autoantibodies such as anti-cyclic citrullinated peptide (anti-CCP) antibodies and rheumatoid factor (RF), synthesized by infiltrating plasma cells, are considered biomarkers of rheumatoid synovitis and serve to gauge the severity of RA and disease activity (141). Importantly, B cells, T cells, and macrophages, all of which express CD38, infiltrate the joint tissue (synovium), producing various proinflammatory cytokines that facilitate inflammation and ultimately destroy surrounding tissue (142).",American Journal of Physiology – Cell Physiology,CD38 Autoimmunity,2022
Immune Cell CD38 in RA Pathogenesis,"CD38 expression is higher in synovial tissues of individuals with RA compared with other inflammatory conditions like ankylosing spondylitis (AS) and osteoarthritis (OA) (143). In the peripheral blood of RA patients, the proportions of CD38+ cells and CD38+ CD56+ cells [which represents CD38+ natural killer (NK) cells] are significantly higher, and the level of CD38+ cells correlates with the level of autoantibodies (143). CD38+ NK cells release proinflammatory cytokines IFN-c and TNF-a, which contribute to RA progression (144). Conversely, suppression of CD38 in cultured patient-derived synovial fibroblasts results in decreased IL-1a and IL-1b secretion (143). Several drugs targeting CD38 show promise in the treatment and management of RA. Daratumumab, a CD38-targeting monoclonal antibody approved for the treatment of multiple myeloma (MM), reduces autoantibody levels (145) and depletes autoreactive plasma cells (145). Additionally, the anti-CD38 monoclonal antibody TAK-079 prevents arthritis by decreasing NK cells, B cells, and T cells in primates (146). Likewise, cyanidin-3-O-glucoside, a competitive inhibitor of CD38 cyclase activity, ameliorates RA synovial fibroblast proliferation, IL-6 and IFN-c levels, and the percentage of CD38+ NK cells in rats (144). Future studies will seek to understand the implications of dysregulated NAD homeostasis in autoimmune conditions like RA and the ways in which targeting CD38 may reverse NAD-related metabolic imbalance (147).",American Journal of Physiology – Cell Physiology,CD38 Autoimmunity,2022
CD38 and Fibrosis: Overview,"Differentiation of quiescent progenitor cells into activated myofibroblasts and their persistence is a common feature to all forms of fibrosis. However, what triggers the process remains obscure. Mounting evidence suggests that dysregulation of the NAD metabolome may underlie processes leading to fibrosis and, importantly, that CD38 may be a promising therapeutic target.",American Journal of Physiology – Cell Physiology,CD38 Fibrosis,2022
CD38 in Systemic Sclerosis (SSc),"Systemic sclerosis (SSc) is a chronic systemic orphan disease associated with high mortality (148). A hallmark of SSc is synchronous fibrosis in multiple tissues including skin, lung, heart, and muscle that leads to permanent and irreversible organ dysfunction with no effective treatment (8). One hypothesis is that dysregulation of NAD metabolism resulting from the interplay of immune cells and fibroblasts plays a key role in SSc pathogenesis. Indeed, skin biopsies of SSc patients that show signatures of fibrosis have increased expression of key NAD-depleting enzymes including CD38 and NNMT (8). Conversely, genetic ablation or pharmacological inhibition of CD38 in mice increases NAD levels in skin and lung and substantially attenuates bleomycin-induced fibrosis (8).",American Journal of Physiology – Cell Physiology,CD38 Fibrosis,2022
CD38 in Pulmonary Fibrosis and Airway Disease,"Inflammatory processes are pathognomonic for common disorders of the lung [e.g., asthma, chronic obstructive pulmonary disease (COPD)]. Therefore, the link between CD38 and inflammatory diseases of the lung may shed light on early events contributing to fibrosis. Asthma pathophysiology involves a series of events including increased airway smooth muscle contractility, mucus hypersecretion, impaired lung elasticity, and disruption of epithelial integrity, which lead to airway narrowing and impact lung function (149). The influx of inflammatory cells contributes to airway hyperresponsiveness (AHR), which is the main characteristic of asthma and defined as an exacerbated response to nonspecific stimuli present in the environment. Studies in CD38-knockout mice using different models of AHR such as inhaled methacholine, TNF-a, IL-13, or ovalbumin (OVA) challenge reveal a dual role for CD38 in airway smooth muscle (ASM) reactivity via cADPR-dependent Ca2+ release from sarcoplasmic reticulum and modulation of inflammation (150–153). There is a paucity of data elucidating the role of CD38 as an NAD consumer in this context.",American Journal of Physiology – Cell Physiology,CD38 Fibrosis,2022
CD38 in Pulmonary Fibrosis and EMT,"Pulmonary fibrosis occurs in the presence of unresolved inflammation and dysregulated tissue repair and results from an array of injurious stimuli including infection, toxicant exposure, adverse effects of drugs, and autoimmune response. Fibrosis is irreversible and is associated with a poor prognosis (154). Interestingly, CD38+ cells are found in the peripheral blood and in the bronchoalveolar space of individuals with pulmonary fibrosis (155, 156), although the contribution of CD38 remains unexplored. One possibility is that CD38 is involved in the epithelial-to-mesenchymal transition (EMT) (157). EMT is a phenotypic transition of epithelial cells necessary for normal tissue repair (158) but can be induced by stress and lead to loss of apical/basal polarization, loss of cell-cell adhesion, and acquisition of a fibroblast-like phenotype (158). Interestingly, it is reported that postinfluenza viral lung fibrosis observed in old mice is mediated by a subset of CD8+ cells (159). Additionally, CD8+ cells are shown to be enriched with CD38 in some studies (128, 160, 161). Thus, it is possible that CD38+ CD8+ cells may contribute to lung fibrosis observed after viral infections such as influenza or coronavirus.",American Journal of Physiology – Cell Physiology,CD38 Fibrosis,2022
CD38 in Renal Fibrosis: Hemodynamics and Podocyte Function,"CD38 plays an important role in kidney physiology, namely renal vasoconstriction responsible for regulating both renal blood flow and glomerular filtration (162, 163). Renal vasoconstriction is controlled hormonally by angiotensin II (ANG II), endothelin-1 (ET-1), and norepinephrine (NE) (164), which in turn are modulated by CD38 expressed on preglomerular resistance arterioles (162). CD38-knockout mice display attenuated hemodynamic response to administration of ANG II, ET-1, and NE, which impact renal microcirculation functioning by reducing basal renal blood flow and urine excretion (162). Furthermore, thromboxane prostanoid (TP)-induced vasoconstriction is also mediated by CD38 (163). Additionally, CD38 is important for differentiation and function of podocytes, which are epithelial cells of the glomerulus that act as a glomerular filtration barrier (165, 166). Although the mechanisms are unknown, CD38 deficiency leads to podocyte EMT, resulting in enhanced glomerular injury and sclerosis (166).",American Journal of Physiology – Cell Physiology,CD38 Renal Fibrosis,2022
"CD38, NAD Levels, and Renal Disease","CD38 deficiency significantly elevates levels of renal NAD (74, 167). The kidney is among the organs with the highest mitochondrial abundance (168). Therefore, regulation of NAD is critical for maintaining renal homeostasis. Reduced NAD+-to-NADH ratios (NAD+/NADH) are reported in numerous renal diseases including diabetic nephropathy (169) and acute kidney injury (AKI) (170–172) and in high-glucose-induced mesangial cell hypertrophy (173). By inhibiting CD38 or boosting NAD to increase NAD+/NADH and reduce mitochondrial stress, an improvement of renal conditions is observed. For example, inhibiting CD38 with apigenin restores NAD+/NADH and SIRT3 function in renal cells and improves renal injury in a model of diabetic nephropathy (174). In an LPS-induced AKI mouse model, inhibition of CD38 with quercetin ameliorates LPS-induced AKI and improves kidney function and inflammation by inhibiting LPS-induced M1 polarization and activation of NF-κB signaling in kidney macrophages (172). Supplementation with NR (175) and NMN promotes NAD boosting and protects against age-associated susceptibility to AKI (170). Similarly, NAM supplementation is associated with reduced AKI in cardiac surgery patients (171). These studies demonstrate that modulation of CD38 and NAD levels in kidney disease may provide therapeutic approaches for the prevention of inflammatory conditions of the kidney that predispose the kidney to fibrosis and altered function.",American Journal of Physiology – Cell Physiology,CD38 Renal Fibrosis,2022
CD38 in Liver Fibrosis and Cirrhosis,"Fibrosis is a critical step in the progression of most chronic liver diseases and a precursor of cirrhosis (176). Liver cirrhosis arises from a variety of conditions including alcoholism, chronic hepatitis virus infection, nonalcoholic fatty liver disease (NAFLD), exposure to drugs and toxins, and inherited diseases among others (177, 178). Despite many etiologies of cirrhosis, some common pathological findings include degeneration and necrosis of hepatocytes, appearance of regenerative nodules, and fibrotic tissue (176). The specific role of CD38 in the development of liver cirrhosis is still unclear. An association between CD38 and cirrhosis is shown in a thioacetamide-induced rat model of liver cirrhosis. Cirrhotic rat livers demonstrate increased CD38 expression and cyclase activity resulting in higher cADPR levels in liver microsomes (179). These findings raise the possibility that increased CD38 activity and cADPR could be involved in the pathogenesis of cirrhosis. Immunohistochemical studies in liver biopsies from patients with chronic liver disease show an increased number of CD38+ hepatic stellate cells (HSCs), which are cells known to contribute to hepatic fibrosis (180). Furthermore, there is a positive correlation between the number of CD38+ HSCs and the METAVIR score, which is based on the intensity of necroinflammatory activity, interface hepatitis, and lobulitis. These findings highlight CD38+ HSCs as a potential biomarker of fibrosis in chronic liver diseases (180).",American Journal of Physiology – Cell Physiology,CD38 Liver Fibrosis,2022
"CD38, Senescence, and Liver Disease Progression","Moreover, markers of inflammation and senescence are present in the pathophysiology of chronic liver diseases that progress to cirrhosis (181–186). CD38 plays a role in both inflammation and senescence. Age-related NAD decline and increased inflammation are partially mediated by senescence-induced accumulation of CD38+ inflammatory cells in tissues (64, 65). This may offer new insight into a role for CD38 in cirrhosis.",American Journal of Physiology – Cell Physiology,CD38 Liver Fibrosis,2022
CD38 in Obesity and Metabolic Syndrome,"Obesity is a disease of epidemic proportions and represents a major public health problem worldwide (187). Besides being a risk factor for increased morbidity and mortality, obesity is a feature of metabolic syndrome, which is a cluster of conditions that increase risk of cardiovascular disease (CVD), type 2 diabetes, and stroke (188). CD38 plays a key role as a regulator of the obesity phenotype (97, 189), and inhibition of CD38 has the potential to ameliorate obesity and metabolic syndrome. In mice, CD38 deficiency results in a higher metabolic rate and resistance to high-fat diet-induced obesity (97). In humans, several lines of evidence positively associate CD38 with metabolic phenotypes including CD38 methylation and adiposity and linkage between CD38 and cholecystokinin A receptor genes and lipid levels (190–192). Previous studies also demonstrate a relationship between obesity and NAD decline in multiple metabolic tissues including liver, pancreas, and adipose (193, 194). Most notably, inhibition of CD38 ameliorates high-fat diet-induced hepatic steatosis and protects against obesity and metabolic syndrome by increasing availability of NAD (72, 194, 195). Taken together, elevation of NAD levels by genetic ablation of CD38 affords protection from the diet-induced insulin resistance, accumulation of fat, and metabolic inflexibility observed in wild-type mice (195), which is likely to translate to metabolic syndrome observed in humans.",American Journal of Physiology – Cell Physiology,CD38 Metabolic Diseases,2022
"CD38, Sirtuins, and Metabolic Regulation","In addition to its role as the primary NADase in mammalian tissues, CD38 regulates SIRT enzymes by regulating NAD availability (189). CD38-knockout mice show increased NAD levels compared with wild type and are protected against high-fat diet (HFD)-induced obesity, metabolic syndrome, glucose intolerance, and liver steatosis through a SIRT1-dependent mechanism. CD38-knockout mice also have higher energy expenditures compared with wild-type mice, with increased basal and activity-induced metabolic rates (97). The mechanism underlying these changes is mediated at least in part by NAD-dependent activation of SIRT1-peroxisome proliferator-activated receptor-c coactivator a (PGC1a) axis and downstream effects on energy metabolism (97). Furthermore, SIRT1 regulates mitochondrial biogenesis and response to stress through the deacetylation of PGC-1a (196). Consistent with these findings, inhibition of CD38 by the small-molecule inhibitor 78c in mice ameliorates age-related glucose intolerance and insulin resistance, suggesting CD38 as a possible pharmacological target for aging-related metabolic dysfunction (51). A possible mechanism for amelioration of obesity through inhibition of CD38 is impaired adipogenesis and lipogenesis through the activation of a SIRT1-peroxisome proliferator-activated receptor c (PPARc) or a SIRT1-sterol regulatory element binding protein 1 (SREBP1) signaling pathway, respectively (197). Expression of adipogenic genes including PPARc, fatty acid-binding protein 4 (FABP4), and CCAAT/enhancer-binding protein a (C/EBPa) is attenuated in CD38-knockout mice. In addition, in vitro expression of CD38 is increased during adipocyte differentiation in mouse embryonic fibroblasts (MEFs). CD38-deficient MEFs show increased expression of SIRT1 and downregulation of SREBP1-mediated FASN (fatty acid synthase) expression, suggesting that CD38 may also influence lipogenesis. Altogether, these results suggest an important link between CD38 deficiency and the development of obesity through activation of Sirt1 signaling (197).",American Journal of Physiology – Cell Physiology,CD38 Metabolic Diseases,2022
"CD38, SIRT3, and Energy Metabolism","NAD-dependent SIRT3, which is localized in mitochondria, also plays a CD38-dependent role in obesity and other features of metabolic syndrome (197). To illustrate, CD38-knockout mice demonstrate improved glucose tolerance profiles compared with wild-type mice, but this observation is reversed in CD38/SIRT3 double-knockout mice (52). Furthermore, CD38-knockout mice exhibit increased oxygen consumption coupled to ATP synthesis compared with wild-type mice, a phenomenon that is also reversed in double-knockout mice. Taken together, these observations suggest that ablation of SIRT3 in CD38-knockout mice abrogates the protective effect of the CD38 inhibition in HFD-induced obesity, suggesting a role for NAD-dependent enzymes in obesity (52). Alterations in white adipose tissue (WAT), in particular, may play a part in the pathogenesis of obesity and metabolic diseases (198). Low-grade chronic inflammation is characteristic of obesity and accompanied by macrophage infiltration in WAT (199, 200). Moreover, the observed macrophage burden and proinflammatory secretome of adipose tissue are correlated with obesity-associated metabolic derangements (201). Macrophage-induced inflammation and insulin resistance in obesity are attenuated by quercetin, a CD38 inhibitor (202), which highlights the importance of CD38+ immune cells in the pathogenesis of metabolic diseases.",American Journal of Physiology – Cell Physiology,CD38 Metabolic Diseases,2022
"CD38, Thermogenesis, and Brown Fat","Curiously, CD38 is implicated in browning of white fat and the development of brown fat in mice. CD38 downregulation occurs during cold-induced thermogenesis and results in increased NAD+ and NADP(H) levels in brown fat (203). Thus, the role of CD38 in obesity and energy expenditure is linked to thermogenesis in brown fat through SIRT1-dependent mechanisms including the inactivation of the NAD/SIRT1/caveolin-1 axis (204, 205). Taken together, CD38 inhibition resulting in increased NAD availability to SIRT1 and 3 may have therapeutic potential in the treatment of obesity-related metabolic syndrome.",American Journal of Physiology – Cell Physiology,CD38 Metabolic Diseases,2022
CD38 in NAFLD and NASH,"A common disease closely associated with metabolic syndrome is nonalcoholic fatty liver disease (NAFLD). NAFLD may lead to nonalcoholic steatohepatitis (NASH), the most common form of chronic liver disease characterized by inflammation and hepatocellular damage. Progression to fibrosis, the first stage of liver scarring, occurs in 32–37% of individuals with NASH and may increase risk of hepatocellular carcinoma (119, 206, 207). Comorbidities that accompany NAFLD including cardiovascular diseases, type 2 diabetes, and dyslipidemia often contribute to the morbidity and mortality of this disease (208). SIRT1 and 3 appear to be important targets of NASH pathophysiology. Overexpression of SIRTs not only protects the liver from steatosis and progression to NASH but also can reverse effects of this disease (209–214). In a methionine and choline diet-deficient mouse model of NASH (209), sirtuins are downregulated, resulting in increased expression of lipogenic genes such as fatty acid synthase (215). Increased gene expression is observed as well in mice fed a HFD, which results in decreased NAD levels, reduced SIRT3 activity in liver, hyperacetylation of liver proteins, and reduced activity of mitochondrial complexes III and IV (213). A role for CD38 in the pathophysiology of NASH has also been proposed. In a HFD model, CD38-knockout mice show significantly lower liver fat infiltration in comparison to wild-type control (97). Additionally, CD38 inhibition in a mouse model of HFD-induced obesity treated with the flavonoid apigenin demonstrates decreased lipid accumulation in liver through increased lipid oxidation, NAD boosting, and SIRT1 activation (72).",American Journal of Physiology – Cell Physiology,CD38 Metabolic Diseases,2022
Therapeutic Potential of CD38 and NAD Boosting,"Taken together, these studies show a role for sirtuins and CD38 in metabolic syndrome and NAFLD and suggests that therapeutic interventions targeting SIRT1 activators or CD38 inhibitors or supplementation with NAD synthesis precursors may be beneficial for management of metabolic diseases (216–218). For example, NMN administration improves glucose metabolism in obese mice and muscle insulin sensitivity in prediabetic women (216, 217), whereas NR supplementation in mice activates both SIRT1 and SIRT3 and ameliorates metabolic dysfunction by improving mitochondrial function (219). In patients with mitochondrial myopathy, administration of niacin improves NAD metabolism and muscle function and decreases liver fat infiltration (220). Furthermore, NAD precursors, alone or in combination with other metabolic activators, have the potential to reverse obesity, enhance exercise performance, and ameliorate NAFLD (221–224).",American Journal of Physiology – Cell Physiology,CD38 Metabolic Diseases,2022
Inflammaging and Age-Related NAD Decline,"Tissue NAD levels decline with age and in progeroid syndromes (52, 225–228) despite NAD synthesis being relatively maintained (229). Boosting NAD levels in vivo is protective against some age-associated disorders in animal models (36, 230–233). One of the causes of NAD decline during aging is increase of NAD breakdown in the presence of increased CD38 expression and activity on immune cells, thus linking inflammaging with tissue NAD decline (51, 52, 64, 65). Other sources of NAD decline include increased DNA damage requiring PARP1 activation and decreased NAMPT levels leading to diminished NAD synthesis through the salvage pathway (216, 225, 226, 234–237). Although age-related NAD decline seems to be multifactorial, CD38 appears to have a significant contribution in this process (51, 52, 64–66). Among the NAD-degrading enzymes, CD38 expression is upregulated with chronological aging in mice, whereas PARP1 and SIRT1 are downregulated (52). In addition, no changes are observed in the expression of NAD-synthesizing enzymes in multiple tissues in mice including liver, white adipose tissue (WAT), spleen, and skeletal muscle (52). Interestingly, CD38-knockout mice demonstrate a delayed age-related NAD decline with age compared with wild-type animals (52). This observation indicates that CD38 plays a major role in NAD dysregulation during aging.",American Journal of Physiology – Cell Physiology,CD38 Aging,2022
"CD38, SIRT3, and Mitochondrial Dysfunction in Aging","Moreover, CD38-dependent age-related NAD decline occurs as a consequence of SIRT3-mediated mitochondrial dysfunction. SIRT3, a key modulator of mitochondrial metabolism, undergoes an age-related decrease in activity, causing increased acetylation of mitochondrial proteins and impaired mitochondrial function independent of mitochondrial biogenesis (52, 238). Aged CD38-knockout mice display more robust mitochondrial function including increased oxygen consumption rates, higher mitochondrial membrane potential, and a higher NAD+ /NADH compared with wild-type aged mice (52). Administration of the highly potent and specific CD38 inhibitor 78c restores NAD levels and ameliorates metabolic dysfunction in aged mice, thus improving glucose tolerance, muscle function, exercise capacity, and cardiac function (51). 78c-induced NAD boosting also increases sirtuin and PARP activity; prevents the accumulation of telomere-associated foci (TAFs), a marker of DNA damage associated with aging; and promotes the activation of longevity pathways in tissues of aged mice (51). Taken together, CD38 is a critical regulator of NAD levels during the aging process in rodents.",American Journal of Physiology – Cell Physiology,CD38 Aging,2022
"NAMPT, NNMT, and Additional Mechanisms of Age-Related NAD Decline","Whereas CD38 plays a major role in aging-related systemic NAD decline, other mechanisms such as the downregulation of the NAD salvage pathway likely contribute to the NAD decline observed in specific tissues during aging. NAMPT is found inside the cells (iNAMPT) as well as in the extracellular environment (eNAMPT). Plasma eNampt released from adipocytes increases NAD levels in remote tissues such as the hypothalamus by increasing the capacity of the NAD salvage pathway. Increased NAD levels in the hypothalamus regulate SIRT1 activity and neural activation in response to fasting (239). The circulating levels of eNAMPT-containing extracellular vesicles (EVs) decrease with age both in mice and in humans (235). In addition, overexpression of NAMPT in adipose tissue increases NAD biosynthesis in the hypothalamus, hippocampus, pancreas, and retina. Exposure to circulating eNAMPT-containing vesicles results in increased physical activity and sleep quality, delayed aging, and extended life span (235). Conversely, NAMPT decline is correlated with decreased Sirt1 expression/activity in retinal pigment epithelium cells from old mice and in skeletal muscle of aged rats (236, 240).",American Journal of Physiology – Cell Physiology,CD38 Aging,2022
"NNMT, Methylation Sink, and NAD Decline","In instances where there is an increase in NAD breakdown by CD38, there will be an excess of NAM. NNMT catalyzes the N-methylation of NAM using S-adenosylmethionine (SAM) as a methyl donor, resulting in the generation of methyl-NAM and S-adenosyl-homocysteine (SAH) (Ref. 95, Fig. 1). Thus, increased NNMT expression has been associated with decreased cellular methylation capacity, reduced histone methylation, and alterations to the epigenetic landscape (95, 241). Also, by limiting the availability of NAM to the NAD salvage pathway, NNMT overexpression is associated with reduced NAD levels in specific contexts such as in high-fat diet-induced fatty liver disease (242) and obesity (243), thereby impacting NAD levels both in liver and in WAT. Accordingly, NNMT inhibitors are shown to increase NAD levels in adipocytes (244). NNMT expression increases in skeletal muscle of old mice, whereas NNMT inhibition enhances muscle regeneration in aging by rescuing muscle stem cell (muSC) function (245). Downregulation of Sirt1 activity as a result of NAD decline is a possible mechanism by which NNMT induces muSC activation in aged muscle tissue. Since NNMT is mostly associated with specific tissues such as liver and fat, it is unlikely that NNMT upregulation contributes to systemic NAD decline in aging. As a NAM generator, CD38 may upregulate NNMT activity in tissues such as fat and liver, thus contributing to a methylation sink, NAD decline, and metabolic dysfunction in specific organs.",American Journal of Physiology – Cell Physiology,CD38 Aging,2022
"Senescent Cells, SASP, and Immune CD38 Accumulation","Whereas modulation of both NAMPT and NNMT expression seems to be context specific, the age-related increase in CD38 expression/activity appears to have the greatest impact on NAD levels. CD38 also appears to play a wider role in aging by disrupting telomere integrity. In patient-derived cells from individuals with congenital telomere shortening, CD38 expression is higher, resulting in lower NAD levels and reduced PARP and SIRT1 activity (246). However, modification of systemic NAD metabolism by CD38 also occurs, at least partially, by way of circulating CD38-expressing immune cells that infiltrate tissues under inflammatory conditions. Indeed, inflammation is among the major risk factors that predispose organisms to age-associated diseases. During aging, the accumulation of senescent cells (SCs) creates an environment rich in proinflammatory signals, leading to “inflammaging” (247). SCs are metabolically active cells that lose their replicative capacity by entering an irreversible quiescent state and are considered both a cause and a consequence of inflammaging (248). SCs secrete several factors collectively known as the senescence-associated secretory phenotype (SASP), which in turn serves as a source of chemotactic factors for immune cell recruitment. SCs and SASP factors upregulate CD38 in bone marrow-derived macrophages and endothelial cells in culture as well as in vivo (64–66). Increased accumulation of CD38+ immune cell clusters is observed in WAT and liver during aging (64, 65). Furthermore, CD38-knockout mice receiving wild-type donor bone marrow cells accumulate CD38+ cells in tissues, indicating that these cells infiltrate from the circulation (64).",American Journal of Physiology – Cell Physiology,CD38 Aging,2022
"SASP, Immune Cell Recruitment, and NAD Loss","In tissues where high levels of immune cells typically reside, such as spleen and intestine, NAD levels decline in CD38-knockout mice transplanted with bone marrow from WT mice. LPS treatment in this model leads to a decline in NAD levels as well (64). Furthermore, clearance of p16ink4a-positive cells from aged INK-ATTAC mice decreases CD38 expression and promotes recovery of NAD levels in WAT and liver (64). Blocking SASP production also leads to restored tissue NAD levels (64, 65). Moreover, the senescence-inducing agent doxorubicin reduces NAD levels in WT mice but has no effect in mice expressing a catalytically inactive form of CD38, thereby linking NAD decline, senescence, and CD38 activity. Taken together, these studies support a role for senescent cells and their SASP factors in the accumulation of CD38+ cells and age-related NAD decline (11, 64).",American Journal of Physiology – Cell Physiology,CD38 Aging,2022
CD38 and Cardiovascular Disease,"Cardiovascular disease is a leading cause of death among the aged and by 2030 will result in 40% of all deaths in individuals aged 65 yr and older. An abundance of mitochondria in cardiac muscle ensures high levels of NAD, which is required to sustain the metabolic demands of the heart (249). Thus, the NAD-consuming CD38, which is present on nonparenchymal cells of the heart, is emerging as a target of pathogenesis as well as a druggable target in heart disease.",American Journal of Physiology – Cell Physiology,CD38 Aging,2022
"NAD Decline, Aging, and Neurodegeneration","The relationship between pellagra pathophysiology and its effect on the nervous system (NS), when first described, revealed a link between NAD metabolism and proper NS functioning (268, 269). Functional pellagra is likely to cause premature aging and is a risk factor for neurodegeneration. Healthy aged brains demonstrate a decline of NAD+/NADH compared with the brains of younger subjects, and this decline has a potential impact on mitochondrial function (228). Furthermore, the hippocampus of 10- to 12-mo old mice shows a decline in NAD levels and the NAD rate-limiting enzyme NAMPT compared with 1-mo-old mice (270). Considering that CD38 expression and activity increase in multiple tissues and organs with age (52) and are detected in the CNS (130, 271–273), CD38 is proposed to have a role in age-associated neurodegenerative diseases like Alzheimer’s disease (AD) and multiple sclerosis (MS).",American Journal of Physiology – Cell Physiology,CD38 Neurodegeneration,2022
"CD38, Microglia Activation, and Alzheimer’s Disease","AD is the most prevalent form of dementia (274) and is characterized by accumulation of β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) (275). CD38 promotes activation of microglia, the resident immune cells of the CNS. Furthermore, CD38 knockdown or 8-Br-cADPR treatment results in reduced secretion of proinflammatory cytokines and nitric oxide (NO) production in LPS-stimulated murine microglial BV2 cells (276, 277). In one study, knockout of CD38 in AD-prone mice (APP.PS.CD38-KO) results in decreased microglia/macrophage accumulation (278), reduced activity of α-, β-, and γ-secretases, less amyloidogenic Aβ peptide formation, and improved spatial learning (278). However, the role of CD38 in AD and other neurodegenerative diseases has not been well established. In fact, no functional or physiological data are presented in the studies described above (278). Although a direct association with AD pathology remains to be determined, CD38 appears to play an important role in the nervous system.",American Journal of Physiology – Cell Physiology,CD38 Neurodegeneration,2022
"CD38, Mitochondrial Transfer, and Neuroprotection","For instance, CD38 improves neuronal survival after cerebral ischemia by facilitating mitochondrial transfer from astrocytes to neurons, neurons to astrocytes, and astrocytes to astrocytes (272, 279). Mitochondria transfer is proposed to release extracellular mitochondrial particles mediated by CD38/cADPR signaling. The introduction of Alexander disease-associated point mutations into the GFAP (glial fibrillary acidic protein) gene of astrocytes results in impaired mitochondrial transfer from astrocytes and decreased CD38 expression (279). It is not yet known how mutations in the GFAP gene affect CD38 expression. Moreover, changes in NAD levels are not shown to affect mitochondrial transfer among neuronal cells. Thus, CD38 participation in mitochondrial transfer deserves further investigation, particularly because of its potential neuroprotective function. Interestingly, transcriptome-wide association data identify CD38 as a possible gene associated with susceptibility to Parkinson’s disease (280). However, akin to AD, the role of CD38 in this condition is not well explored.",American Journal of Physiology – Cell Physiology,CD38 Neurodegeneration,2022
"NAD Metabolism, Cancer, and CD38","Cancer is the quintessential disease of aging, occurring on a backdrop of inflammation, failed DNA repair, senescence, and impaired immune surveillance (281). Because of high metabolic demand and reliance on NAD-dependent signaling, cancer cell growth is tied to NAD metabolism (282, 283). In fact, lowering levels of NAD/NADP by NAMPT inhibition is a promising anticancer strategy in both NAPRT-depleted and CD38-overexpressing tumors (283–285). In addition, NAMPT inhibition sensitizes cancer cells to oxidative stress and chemotherapeutics (283, 285). Interestingly, CD38 is shown to participate in both tumor progression and tumor suppression, depending on tumor type. CD38 appears to play an important role in multiple myeloma (MM), the second most common hematological malignancy (286, 287). MM cells strongly express CD38, which is used as a marker for myeloma cell immunophenotyping (288, 289). A possible role for CD38 in MM appears to be related to the transfer of mitochondria from bone marrow stromal cells to MM cells via tunneling nanotubes, a process that supports oxidative phosphorylation in MM cells and contributes to cancer cell survival (290). It is still not clear, however, how CD38 facilitates this process.",American Journal of Physiology – Cell Physiology,CD38 Cancer,2022
CD38 in Multiple Myeloma and CLL,"In astrocytes, CD38/cADPR/Ca2+ signaling is proposed to induce release of extracellular mitochondrial particles, promoting mitochondria transfer to adjacent neurons (272). Whether the CD38/cADPR/Ca2+ signaling axis is also part of tunneling nanotube formation in MM remains to be established. Therapeutically, anti-CD38 monoclonal antibodies such as isatuximab and daratumumab are being investigated and used as therapies in patients with MM and other hematological malignancies (291, 292). The process by which anti-CD38 antibodies exert their anticancer effects is not completely understood and comprises several different mechanisms including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) (291, 293–296). Another hematological malignancy in which CD38 plays an important role is chronic lymphocytic leukemia (CLL) (297, 298). CD38 positivity in B-CLL cells is a prognostic factor, indicating decreased patient survival (299). Overexpression of CD38 increases aggressiveness of CLL, and CD38 inhibition by the flavonoid kuromanin results in downstream disruption of calcium signaling necessary for CLL chemotaxis, adhesion, and homing (300).",American Journal of Physiology – Cell Physiology,CD38 Cancer,2022
"CD38, CLL Microenvironment, and Purinergic Immunosuppression","Furthermore, migration and homing of CLL lymphocytes requires interaction between CD38 and its endothelial cell coreceptor CD31, which favors localization of neoplastic cells to growth-permissive sites (301). In addition to the CD38/CD31 axis, ADPR and NAADP signaling appear to mediate CD38-dependent effects of CLL migration via activation of the Ras family GTPase Rap1 through regulation of the Ca2+-sensitive Rap1 guanine-nucleotide exchange factor RasGRP2 (302). Not surprisingly, the anti-CD38 MAb daratumumab has antitumor effects in a partially humanized CLL xenograft model (303). Furthermore, anti-CD38 targeting agents modulate the tumor microenvironment by inducing apoptosis of B regulatory cell-like CLL cells and Treg cells, which have immunosuppressive roles. CD38 inhibition also increases activated cytotoxic CD8+ T cells, which target CLL cells (304). Thus, CD38 likely regulates the CLL microenvironment by modulating migration and proliferation of CLL cells, in addition to having immunomodulatory effects on B and T cells.",American Journal of Physiology – Cell Physiology,CD38 Cancer,2022
"CD38, Adenosinergic Signaling, and Tumor Immune Evasion","An alternative CD38-mediated mechanism of tumorigenesis relies on the interplay of adenosine (ADO)-generating ectonucleotidases expressed on tumor cells, stromal cells, and/or tumor-infiltrating immune cells (305). ADO is a signaling molecule that suppresses multiple immune subsets such as cytotoxic T cells via activation of purinergic receptors (306, 307). ADPR generated by CD38 can be used as a substrate by CD203a to generate AMP, a substrate for the primary adenosine-producing ectoenzyme CD73, which then produces ADO. ADO-producing ectoenzymes are expressed on multiple cell types within the tumor microenvironment or specifically on tumor cells (305). For instance, CD38 expressed on melanoma cells plays a prominent role in adenosinergic signaling leading to suppression of T-cell proliferation (308). Kuromanin-mediated inhibition of CD38 activity on melanocytes partially restores T-cell proliferation in coculture experiments. The immunosuppressive quality of the adenosinergic pathway highlights CD38 as a promising target for cancer immunotherapy (93, 305).",American Journal of Physiology – Cell Physiology,CD38 Cancer,2022
"CD38, PD-L1 Resistance, and Myeloid-Derived Suppressor Cells","Cancer cells elude or modify the immune system by several mechanisms. One mechanism involves increased expression of cell surface immune checkpoint proteins such as the programmed cell death ligand 1 (PD-L1), which binds to the programmed cell death 1 (PD1) receptor present on the surface of T cells (309). PD1/PD-L1 binding triggers T-cell suppression and prevents an antitumor cytotoxic response. By blocking PD1/PD-L1 binding, the immune system targets cancer cells that express high levels of PD-L1. Increased CD38 expression on tumors and the subsequent inhibition of tumor-infiltrating lymphocytes via purinergic signaling is the main mechanism for reversing PD1/PD-L1 blockade in preclinical models of lung cancer (93, 310). Accordingly, coinhibition of CD38 and PD1/PD-L1 improves antitumor immune response (93, 310). Another CD38-mediated protumor immune response is the expansion of myeloid-derived suppressor cells (MDSCs), which are an immature cell population with immunosuppressive functions. Cancer-related inflammation alters myelopoiesis and stimulates generation of MDSCs, which in turn are recruited to tumor tissues in response to chemokines and act as a barrier to antitumor immunity (311). In a murine esophageal cancer model, administration of a CD38 antibody inhibits the expansion and survival of MDSCs (312). Moreover, CD38+ MDSCs are found in peripheral blood of advanced-stage cancer patients (312).",American Journal of Physiology – Cell Physiology,CD38 Cancer,2022
Cytokine-Induced CD38 in MDSCs and Tumor Progression,"Cytokines such as IFNγ, TNF-α, IGFBP3, CXCL16, and IL-6 induce CD38 expression in MDSCs, leading to expansion and maintenance of undifferentiated cells with greater immunosuppressive capacity and higher tumor-promoting activity (312). This mechanism is proposed to be mediated by CD38 induction of inducible nitric oxide synthase (iNOS), presumably through binding of CD38 to surface receptors. Another possibility is that the CD38 glycohydrolase activity modulates NAD-dependent signaling and alters gene expression to favor MDSC expansion. Taken together, CD38 is an important immune checkpoint for carcinogenesis with promising therapeutic potential. CD38 may facilitate interaction between a tumor and its microenvironment including cancer-associated fibroblasts and tumor-associated blood vessels by a mechanism yet to be elucidated. Tumor outgrowth induced by subcutaneous injection of melanoma cells is reduced in CD38-knockout mice compared with wild-type mice and is partially explained by fewer cancer-associated fibroblasts and reduced density of tumor-associated blood vessels in CD38-knockout mice (313).",American Journal of Physiology – Cell Physiology,CD38 Cancer,2022
Context-Dependent Roles of CD38 in Tumor Biology,"In an opposing scenario, CD38 expression is negatively correlated with disease progression in prostate cancer, in which low CD38 mRNA is prognostic for tumor recurrence and metastasis (314, 315). CD38 is epigenetically regulated in prostate cancer. Suppression of CD38 by methylation may increase the availability of extracellular NAD in prostate cancer and lead to disease progression (314). Higher CD38 expression results in lower NAD and leads to cell cycle arrest. The presence of CD38 also reduces glycolytic and mitochondrial metabolism, inhibits fatty acid metabolism, activates AMPK, and promotes a nonproliferative phenotype (316). Similarly, the presence of CD38+ cells in hepatocarcinomas improves clinical outcome in cancer patients. Infiltration of CD38+ leukocytes in human hepatocarcinomas is correlated with better patient survival (317). Also, the presence of CD38+ M1 macrophages is associated with a positive prognosis after surgery (318). Although there is no direct evidence, CD38 may contribute to an antitumor immune response.",American Journal of Physiology – Cell Physiology,CD38 Cancer,2022
CD38-Dependent NAD Decline and Tumor Suppression,"Furthermore, CD38 expression sensitizes pancreatic cancer cells to NAMPT-mediated metabolic collapse (284). In pancreatic cancer cells, CD38 overexpression reduces NAD levels and inhibits cell growth both in vitro and in a xenograft mouse model. In addition, CD38 knockdown in the pancreatic cancer cell line PaTu8988t results in increased NAD levels and resistance to NAMPT inhibition (284). Therefore, CD38-dependent NAD decline has possible antitumor properties. Akin to what is observed in an infection, CD38 plays a complex role in cancer biology that is highly cell and context dependent. These opposing effects of CD38 on cancer cells, inflammatory cells, and cancer stromal cells, which can be pro- or antitumor, can be explained by the multifunctionality of the ectoenzyme, which seems to be tissue specific and/or depend on interactions between the tumor and its microenvironment. Collectively, these findings highlight the importance of understanding the role of CD38 in different types of cancer, in immune cells, and in the tumor microenvironment.",American Journal of Physiology – Cell Physiology,CD38 Cancer,2022
Article Overview and Abstract,"Therapeutic potential of boosting NADþ in aging and age-related diseases
Yahyah Aman a, Yumin Qiu b, Jun Tao b, Evandro F. Fang a, b, c, *
a Department of Clinical Molecular Biology, University of Oslo, Akershus University Hospital, 1478, Lørenskog, Norway
b Department of Hypertension and Vascular Disease, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510080, China c Institute of Geriatric Immunology, School of Medicine, Jinan University, 510632, China
article info
Article history:
Received 20 July 2018
Received in revised form 10 August 2018
Accepted 10 August 2018
Available online 18 August 2018
Keywords:
NADþ Aging
Age-related diseases
Autophagy
Alzheimer's disease
Neurodegeneration
Therapeutic potential
abstract
Nicotinamide adenine dinucleotide (NADþ) is an essential cofactor in all living cells that is involved in fundamental biological processes. NADþ depletion has been associated with hallmarks of aging and may underlie a wide-range of age-related diseases, such as metabolic disorders, cancer and neurodegenerative diseases. Emerging evidence implicates that elevation of NADþ levels may slow or even reverse the aspects of aging and also delay the progression of age-related diseases. Here we discuss the roles of NADþ-synthesizing and -consuming enzymes in relationships to aging and major age-related diseases. Specifically, we highlight the contribution of NADþ depletion to aging and evaluate how boosting NADþ levels may emerge as a promising therapeutic strategy to counter aging-associated pathologies and/or accelerated aging. © 2020 KeAi Communications Co., Ltd. Publishing Services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).",Translational Medicine of Aging,NAD+,2018
Introduction to NAD+ Biology,"1. Introduction
Nicotinamide adenine dinucleotide (NADþ) is an important cofactor in all living cells that is involved in fundamental biological processes, namely metabolism, cell signalling, gene expression, DNA repair, among others [1e4]. Originally, Harden and Young described NADþ in 1906 as a molecular fraction (“cozymase”) that accelerated fermentation in yeast extracts. Over subsequent years, NADþ was identified as a nucleoside sugar phosphate, which plays a role in redox reactions [5]. However, evidence stemming from recent studies have unveiled numerous roles of NADþ metabolism on aging and longevity. In particular, an age-dependent decline in NADþ levels have consistently been reported, possibly due to an imbalance in the synthesis and consumption of NADþ. Decreased levels of NADþ are associated with the hallmarks of aging as well as several age-related diseases, including metabolic disorders, cancer and neurodegenerative diseases. Replenishment of NADþ levels via administration of its precursors have been demonstrated to display beneficial effects against aging and age-related diseases. Importantly, boosting NADþ levels have been shown to extend lifespan of various laboratory animal models including worms, flies, and rodents [3,5e8].",Translational Medicine of Aging,NAD+,2018
NAD+ Cellular Roles and Consumption,"As a cofactor, NADþ is found in abundance in the mitochondria, cytoplasm, and nucleus. It is essential for many cellular metabolism pathways that include: glycolysis, fatty acid b-oxidation, and the tricarboxylic acid cycle. Whilst the reduced form of NADþ (NADH) is a primary hydride donor in the production of ATP via anaerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) [6,9]. On the other hand, NADþ is consumed by the NADþ-dependent sirtuins and the DNA damage sensors poly (ADP-ribose) polymerases (PARPs) in the processes of protein deacetylation and poly-ADP-ribosylation (PARylation), respectively. In addition, NADþ glycohydrolases (i.e. CD38 and CD157) also consume NADþ via conversion of NADþ into ADP-ribose (ADPR) or cyclic-ADPR [1e3]. Thus, the importance of NADþ has expanded from a key element in intermediate metabolism to a critical regulator of multiple cell signalling pathways; and is now a major player contributing to aging and age-related diseases [6].",Translational Medicine of Aging,NAD+,2018
NAD+ Synthesis Pathways,"In mammals, NADþ is synthetized from a variety of dietary sources, including NADþ itself (it is metabolized in the gut, then synthesized again in cells) as well as from one or more of its major precursors that include: tryptophan (Trp), nicotinic acid (NA), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and nicotinamide (NAM). Based upon the bioavailability of its precursors, there are three pathways for the synthesis of NADþ in cells: (i) from Trp by the de novo biosynthesis pathway or kynurenine pathway; (ii) from NA in the PreisseHandler pathway; and (iii) from NAM, NR, and NMN in the salvage pathway [5,6]. Accumulating evidence demonstrates an age-dependent decline in NADþ levels and associate its depletion to several hallmarks of aging and age-related diseases (Fig. 1) [6]. Here, we summarize the roles of NADþ-synthesizing and -consuming enzymes in aging and age-related diseases. Specifically, we highlight the contribution of NADþ depletion to mammalian aging and evaluate how boosting endogenous NADþ levels might emerge as a promising therapeutic strategy to counter aging-associated pathologies and/or accelerated aging.",Translational Medicine of Aging,NAD+,2018
Age-Related Decline of NAD+,"2. Recent progress on the roles of NADþ in aging
Mounting evidence has indicated that NADþ levels decline with age in multiple types of tissues, which include the liver, skeletal muscle, adipose tissue, heart, brain, kidney, pancreas, lungs, spleen, skin, as well as extracellular fluids [3,10]. In addition, an age-dependent decline in NADþ levels in Caenorhabditis elegans (C. elegans), mice, and human post-mortem tissues are reported. Thus, highlighting a universal age-dependent decrease of NADþ across species. However, it remains elusive whether this is due to increased NADþ consumption and/or decreased synthesis [6]. In this section, we provide an overview of methods that could potentially boost endogenous levels of NADþ.",Translational Medicine of Aging,NAD+,2018
NMN: Synthesis and NAD+ Boosting,"2.1. NMN and aging
NMN is a physically stable natural compound that serves as an efficient NADþ precursor. In mammals, NMN is synthesized from nicotinamide, a form of water-soluble vitamin B3 and 5′-phosphoribosyl-1-pyrophosphate (PRPP), by the rate-limiting enzyme nicotinamide phosphoribosyl transferase (NAMPT). In addition, it can also be synthesized from NR via NR kinases (NRKs)-mediated phosphorylation reactions. NMN is subsequently converted into NADþ by NMN adenylyl transferases (NMNATs) [5]. Over the years, it has become increasingly evident that systemic administration of NMN in rodents enhances the biosynthesis of NADþ in various peripheral tissues including liver, pancreas, adipose tissue, heart, skeletal muscle, kidney, eyes, and blood vessels [5,6,11–19]. Furthermore, NMN has also been shown to elevate levels of NADþ in hypothalamus and hippocampus following an intraperitoneal injection, thereby indicating its ability to penetrate the blood-brain barrier (BBB) [20,21]. More importantly, long-term (1-year) oral administration of NMN (up to 300 mg/kg) has recently been shown to be well tolerated without any obvious deleterious or toxic effects in normal wild type C57BL/6 mice [22].",Translational Medicine of Aging,NMN,2018
NMN Benefits in Aging Models,"NMN has been shown to have remarkable beneficial effects that counter normal aging. In models of aging, long-term administration of NMN protects against age-associated functional decline as demonstrated by increases in energy metabolism, insulin sensitivity, lipid metabolism, mitochondrial oxidative metabolism, eye function, bone density and immune function [10,22]. On the other hand, it suppressed age-related changes in gene expression and adipose tissue inflammation [22]. Moreover, it has been shown to maintain neural stem/progenitor cell population and restore skeletal muscle mitochondrial function as well as arterial function in aged mice [15,19,20]. In addition, loss of enzymes involved in NADþ synthesis, namely NAMPT during the process of aging, led to decrease in NADþ content and reduced SIRT1 activity; consequently, promoted cellular senescence in retinal pigment epithelium, which performs numerous functions critical to retinal health and visual function [23].",Translational Medicine of Aging,NMN,2018
NMN in Disease and Premature Aging,"NMN administration may counteract age-predisposed metabolic diseases and neurodegeneration. In age-related pathophysiological conditions, NMN ameliorated impairments in glucose tolerance and promoted insulin secretion/sensitivity in age- or diet-induced diabetic mice, Nampt+/− mice, as well in aged wild-type and β cell-specific Sirt1-overexpressing (BESTO) mice [5,6,11,24–26]. Likewise, promotion and overexpression of the mitochondrial Nmnat3 in mice, also involved in NADþ biosynthesis, resulted in improved glucose tolerance during the process of aging as well as in models of high-fat induced obesity [27]. The beneficial effect was suggested to be a result of improved mitochondrial function and by an independent mechanism of NADþ–SIRT1–PGC1α axis, which despite previously being reported to contribute to improved mitochondrial function, was not activated in these transgenic mice despite elevated levels of NADþ [27]. Furthermore, NMN protects the heart and brain from ischaemia-induced damage [28,29]. In rodent models of Alzheimer's disease (AD), administration of NMN decreased AD-associated β-amyloid (Aβ) pathology and improved cognitive function. In addition, it restored mitochondrial function and ameliorated inflammation, synaptic loss as well as protected against neuronal cell death [30–32].",Translational Medicine of Aging,NMN,2018
NMN in Premature Aging Models,"The beneficial effect of NMN was also evident in premature aging conditions as demonstrated by extended lifespan and improved healthspan in the C. elegans model of xeroderma pigmentosum group A (XPA, a nucleotide excision DNA repair (NER) disorder with severe neurodegeneration) [33], and Ataxia telangiectasia (A-T, due to mutation of ATM which encodes a master regulator of DNA damage response) [34,35]. Moreover, in mice with hypomorphic BubR1 (a mitotic check-point kinase) exhibited characteristics of premature aging as evidenced by shorter lifespan, which was restored by NMN supplementation [17]. Altogether, these findings highlight NMN induced restoration of NADþ levels serve as beneficial therapeutic strategy in countering aging as well as age-related pathological conditions in animal models.",Translational Medicine of Aging,NMN,2018
NR: Mechanisms and Benefits,"2.2. NR and aging
NR is a natural NADþ precursor. It can directly be converted into NMN via the activity of NRKs, thereby bypassing the requirement of the NAMPT in the salvage pathway, and therefore offers to provide an additional pathway for elevation of NADþ levels. Similar to NMN, NR also exhibited beneficial effects in protection against aging and age-related diseases. It has been shown to promote longevity as well improve healthspan in multiple laboratory animal models [36–41]. In age-related disease, in particular obesity, diabetes and cardiovascular conditions NR was able to decrease weight gain, improve glucose tolerance and increase survival rates, respectively in rodents [42–44]. In addition, in models of diabetes and high-fat diet, NR was able to improve metabolic function and reduce lipid accumulation as well as increasing lifespan [40,44–46]. Furthermore, supplementation of NR reversed the progressive wasting syndrome and restored endurance in Nampt skeletal muscle knockout mice, mdx model of Duchenne's muscular dystrophy [47,48].",Translational Medicine of Aging,NR,2018
NR in Neurodegeneration,"NR ameliorates neurodegeneration in animal models. In animal models of age-related neurodegenerative diseases such as AD and Parkinson's disease (PD), NR has been shown to improve memory, learning, motor function and mitochondrial function as well as protected against neuronal cell death [49–52]. In particular, chronic NR administration in the amyloidogenic models of AD delayed the development and progression of Aβ pathology in AD mice, SH-SY5Y cells, and AD C. elegans [50,52]. In addition, it was able to promote longevity and inhibit/delay cognitive decline in AD C. elegans and mice, with the enhanced process of mitochondrial proteostasis and modulation of β-secretase 1 (BACE-1) activity via peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC)-1alpha highlighted as possible underlying mechanisms [50,52].",Translational Medicine of Aging,NR,2018
NR in DNA Damage and PD Models,"Further evidence reinforcing the beneficial effects of NR as a therapeutic strategy in AD was provided by a recent study using triple transgenic model of AD (3×Tg), which exhibited not only reduced phosphorylated tau pathology and inhibited cognitive decline; but also, normalised AD-associated neuroinflammation and synaptic dysfunction. Moreover, DNA damage was reduced, in addition, following chronic administration of NR in a DNA repair-deficient 3×Tg/Polβ+/− mouse model [6,51]. The underlying mechanism proposed was the reduction in DNA damage results in reduced activity of NADþ-consuming enzyme PARPs that is involved in DNA repair; thereby increase in the levels of NADþ, which in turn contributes to neurogenesis and inhibits AD-associated pathology, neuroinflammation and mitochondrial dysfunction [51]. Likewise, NR treated induced pluripotent stem cells (iPSCs) derived from PD patients harbouring mutations in the lysosomal enzyme β-Glucocerebrosidase (GBA) gene (GBA-PD), the most common genetic risk for PD, resulted in elevated levels of NADþ and NAM which coincided with improved mitochondrial morphology and function [49,53].",Translational Medicine of Aging,NR,2018
"NR in PD, Mitophagy & Premature Aging","Mitophagy was suggested to be a possible mechanism promoted by NR, which may underlie improved mitochondrial quality control [49]. In addition, flies model of GBA-PD expressing human N370S GBA raised on food containing NR displayed improved motor function and significantly decline in loss of dopamine-containing neuronal population [49]. Additionally, NR has shown significant neuroprotection in a series of DNA repair-deficient premature aging diseases, including XPA, A-T, and Cockayne syndrome (CS, due to impairment of NER) [33,34,54].",Translational Medicine of Aging,NR,2018
NAM: Effects on Aging and Metabolism,"2.3. NAM and aging
NAM is also a precursor for NADþ and a key molecule involved in energy metabolism. Low doses of NAM have been shown to increase lifespan in yeast and C. elegans, however, higher doses have been associated with reduced lifespan via inhibition of Sir2 activity [39,55–58]. In models of aging and high fat diet-induced obesity, NAM improved healthspan although it failed to extend lifespan as illustrated by comparable mean and maximum lifespan [59]. In the model of obesity, it has able to restore glucagon storage to similar levels as age-matched standard-diet mice as well as ameliorate diet-induced hepatosteatosis, oxidative stress and inflammation [59]. It was suggested that the beneficial impact of NAM may be attributes to improved mitochondrial function and countering age and high fat diet induced DNA damage [59].",Translational Medicine of Aging,NAM,2018
NAM in Glaucoma and Nmnat1 Therapy,"Further evidence reinforcing the beneficial impact of NAM stemmed from a mouse model of glaucoma, which inhibited the development glaucoma in the eyes [60]. Moreover, these findings were replicated by Nmnat1 gene therapy whereby an intravitreal administration of adeno-associated virus AAV2.2 carrying a plasmid to overexpress murine Nmnat1 under a CMV promoter was performed in D2 eyes [60]. The improvement of mitochondrial health and metabolism was suggested to be the underlying mechanism for countering glaucoma mediated by NAM supplementation and Nmnat1 gene therapy [60].",Translational Medicine of Aging,NAM,2018
PARP Inhibition and NAD+ Conservation,"3.1. PARP inhibition
The NADþ consuming enzymes, PARPs, cleave NADþ into NAM and ADP-ribose (ADPR), as a result generating a chain of ADPR. PARP1 is the most abundant PARPs, which is ubiquitously expressed and is a major consumer of NADþ in response to DNA damage whereby it contributes to facilitation of the DNA repair process [61]. Both PARPs and sirtuins share NADþ as a common substrate thus compete for its consumption. It has been reported that PARP1 activity increased with the inevitable process of aging possibly due accumulation of DNA damage [33]. Consequently, the NADþ pool is depleted which results in reduced activity of sirtuins [62]. Evidence stemming from genetic deletion of PARP1 in mice as well as pharmacological inhibition of PARPs revealed increase in NADþ content and enhanced activities of sirtuins, in particular SIRT1 and SIRT6 [33,34,39]. Elevated SIRT1 activity was associated with increased mitochondrial content and oxidative metabolism as well as protection against metabolic dysfunction, DNA damage, and neurodegeneration [4,10,62].",Translational Medicine of Aging,PARP inhibition,2018
PARP1 in Neurodegeneration and Premature Aging,"In addition, increased PARP1 activity has been reported in animal models of age-related neurodegenerative diseases, namely AD and PD [63–65]. Deletion of PARP1 in AD mice protected against cognitive decline as well as attenuated neuroinflammation and Aβ-induced neurotoxicity [66]. In PD rodents, PARP1 pharmacological inhibitors or deletion resulted in resistance to the toxic effects and loss of dopamine-containing neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA), respectively [67–69]. Thus, implicating the contribution of PARP1 hyperactivity in processes leading to neurodegeneration. Moreover, hyperactivity of PARP1 is reported in models of premature aging that resulted decrease in SIRT1 activity, a feature that was reversed by replenishment of NADþ using its precursors [33,34]. Therefore, a potential therapeutic counter for depleted NADþ pool in aging and age-related diseases could be inhibition of its consumer, PARPs, which will enable activity of sirtuins that plays a pivotal role in regulating cellular processes.",Translational Medicine of Aging,PARP inhibition,2018
CD38 Inhibition and NAD+ Restoration,"3.2. CD38 inhibition
CD38 is one of the primary NADases in mammals. It can modulate the levels of NADþ via hydrolysis of NADþ itself as well degradation of its precursors, NMN and NR. Primarily, CD38 hydrolyses NADþ to produce ADPR and NAM [70–72]. It is a key player in several physiological processes, including nuclear Ca2þ homoeostasis, immunity, inflammation, glucose and lipid homoeostasis, transferring of mitochondria from astrocytes to neurons, as well as social behaviour [6,73–77]. An age-dependent increase in levels of CD38 protein has been reported in multiple tissues and organs, which as a result contributes to NADþ decline [78]. Therefore, CD38-dependent modulation of NADþ can alter the activity of NADþ-consuming enzymes and affect cellular signalling and metabolism [71,72]. Inhibition of CD38 can also promote NADþ levels and improve glucose and lipid metabolism, which protects against age- and diet-induced diabetes and obesity [72,75]. In APP/PS1 model of AD, CD38 depletion resulted in elevation of NADþ levels that were associated with decrease in Aβ pathology and associated neuroinflammation accompanied by improvement spatial learning behaviour [79]. However, due to the reported important neuroprotective activities of CD38, further stringent and comprehensive evaluation of the procedures of CD38 inhibition as a safe anti-aging strategy [76,77].",Translational Medicine of Aging,CD38 inhibition,2018
NNMT Knockdown and NAD+ Preservation,"3.3. NNMT knockdown
Nicotinamide N-methyltransferase (NNMT) catalyses the methylation of NAM to N1-methyl-2-pyridone-5-carboxamide (2py) and N1-methyl-4-pyridone-3-carboxamide (4py) using the universal methyl donor S-adenosyl methionine (Met) (SAM). Both products of NAM methylation are eventually excreted in the urine; thus, NNMT removes NAM from the NADþ biosynthesis pathway and thereby contributes to decrease in levels of NADþ [80]. It is predominantly expressed in the liver and adipose tissue but is also found in other tissues including kidney, lung, muscle, heart, brain, and tumour cells. NNMT has been shown to be involved in various disease conditions such as metabolic disorders, neurodegenerative diseases and cancer [80]. In conditions such as obesity and diabetes, NNMT levels have been reported to be upregulated significantly, which in turn are associated with the disease phenotype [81,82]. In fact, genetic knockdown as well as pharmacological inhibition of NNMT was shown to be beneficial in protection against obesity in rodent models of obesity [83,84].",Translational Medicine of Aging,NNMT knockdown,2018
Genetic Enhancement of NAD+ Biosynthesis,"3.4. Genetic promotion of NADþ biosynthesis
In addition to abovementioned techniques that have shown to enhance NADþ biosynthesis, genetic tools in the form of Lactobacillus brevis (LbNOX), a water-forming NADH oxidase, has been demonstrated to also induce an increase in compartment-specific levels of NADþ/NADH ratio in HeLa cells [85]. It catalyses a four-electron reduction of oxygen to water (2 NADH + 2Hþ + O2 → 2 NADþ + 2 H2O) [85,86]. The NADþ/NADH ratio plays a role in cellular metabolism by affecting the activity of NADþ-dependent enzymes such as sirtuins. LbNOX was shown to ameliorate proliferative and metabolic defects induced by a dysfunctional electron transport chain (ETC) by recycling the pool of NADþ [85]. This, therefore offers to be a novel approach whereby NADþ levels could potentially be boosted via genetic manipulation in order to understand the fundamental molecular mechanisms in models of aging and age-related diseases.",Translational Medicine of Aging,Genetic NAD+ enhancement,2018
Challenges in Detecting Subcellular NAD+,"4. Methods to detect subcellular NADþ
In view of the importance of NADþ in life, aging, and diseases, it is necessary to accurately detect subcellular NADþ levels to further unveil its intracellular functions as well as to develop sub-cellular organelle-targeted therapeutic approaches. Traditionally, several assays, such as high-performance liquid chromatography (HPLC)-based methods (e.g., HPLC/MALDI/MS) and fluorometric-based commercial kits have been utilised to detect whole NADþ [26,48] at both cellular levels and/or sub-cellular levels (by isolating subcellular fractions ahead). There have some challenges of using these methods to accurately detect sub-cellular NADþ levels because of the highly instability of NADþ as well as impossibility of NADþ detection in live cells/tissues. The recent development of genetically encoded fluorescent biosensors such as SoNar and a biosensor with a bipartite NADþ-binding domain have enabled imaging of relative levels of free NADþ in the subcellular compartments [87,88].",Translational Medicine of Aging,NAD+ detection,2018
Biosensor-Based NAD+ Quantification,"Quantification of NADþ using fluorescent biosensor targeted to different compartments of mammalian cells showed that mitochondria contain more than twice as much free NADþ as other compartments [87]. Specifically, the concentration of NADþ was reported to be approximately 110 mM in the cytoplasm and the nucleus relative to 230 mM in the mitochondrion [87]. These levels are consistent with other reports demonstrating that, in highly metabolically active, post mitotic cells, such as neurons, mitochondria have higher NADþ levels compared with other subcellular compartments [1]. It was further demonstrated genetic and pharmacologic inhibition of NAMPT result in a reduction in NADþ concentration in all compartments, but depletion of mitochondrial NADþ occurred at a slower rate. Furthermore, the nuclear and cytoplasmic pools were shown to be readily exchangeable, whilst the mitochondrial pool may maintain mitochondrial NADþ levels via NADþ biogenesis by mitochondrial isoform NMNAT3 and import from the cytoplasm [87].",Translational Medicine of Aging,NAD+ detection,2018
NAD+ Flux Quantification Approaches,"Another recent development has been offered in the form of NADþ flux quantification that is isotope-tagged and used for analysis of NADþ metabolism. It demonstrated that approximately 50% decrease in NADþ consumption following treatment with SIRT1/2 and PARP1/2 inhibitors; thereby implicating both are major consumers of NADþ [89]. The use of such approaches in models of aging and age-related diseases in combination with NADþ promoting methods would allow the identification of subcellular localisation as well as consumption, which in turn would allow to identify the underlying molecular mechanisms and pathways attributed to NADþ benefits.",Translational Medicine of Aging,NAD+ detection,2018
Clinical Translation of NAD+ Precursors,"5. Clinical translation
Encouraged by significant and replicable benefits of NADþ precursors, NR and NMN, in aging and disease animal models, a series of clinical trials of NR and NMN in normal aged population and individuals with diseases have been performed [10]. NADþ precursors, in particular NR has been demonstrated to elevate blood concentration of NADþ in healthy individuals in a dose-dependent manner and without any toxic effects [90]. In particular, single oral self-administration of NR (1000 mg) over a period of seven days in a 52 years old male increased blood concentration of NADþ by 2.7-folds and 45.5-fold increase in nicotinic acid adenine dinucleotide (NAAD), an NADþ biosynthesis intermediate [90]. In addition, a randomized double-blind pharmacokinetic study of single oral administration NR (doses: 100 mg, 300 mg, and 1000 mg) with seven-days gap conducted in 12 healthy patients (aged 30–55 years old) revealed a dose-dependent increase in NADþ and NAAD levels, with no reported adverse effects [90]. The encouraging results in animal models of aging and age-related diseases of chronic administration of NADþ precursors have led to studies in humans [91,92].",Translational Medicine of Aging,Clinical translation,2018
NR Human Trials and Cardiovascular Benefits,"An eight-week randomized, double-blinded, placebo-controlled study in 120 healthy adults (60–80 years old) demonstrated NR (250 mg and 500 mg) induced dose-dependent increase of blood NADþ level that becomes apparent after 4-weeks and is sustained till the end of the study [91]. Importantly, no serious adverse effects were reported, thereby implicating the chronic administration of NR is a safe and effective way to increase NADþ levels [91]. These findings are reinforced by a 2 × 6-week randomized, double-blind, placebo-controlled crossover clinical trial conducted in 55–79 years old individuals that showed NR (oral 500 mg, twice a day) to be well tolerated and able to effectively elevates NADþ levels in healthy adults [92]. Moreover, it was able to reduce systolic blood pressure and aortic stiffness, which are considered measures of cardiovascular disease [92]. Thus, not only the NADþ precursors are safely administrated but they may also recapitulate the beneficial effects that were evident in animal models, which is an exciting prospect for future clinical trials.",Translational Medicine of Aging,Clinical translation,2018
Considerations for NAD+ Precursors in Disease,"However, in conditions such as pancreatic cancer, cell growth has been shown to be dependent of the NADþ salvage pathway [93]. Hence, inhibition of NADþ synthesis (via Nampt inhibition) and/or promotion of its consumption (via CD38 NADase) prevented cancer cell growth [93,94]. Therefore, implicating that there should be a thorough evaluation for the use of approaches that promote NADþ biosynthesis as it may be contributor rather than a counter-mechanism in certain conditions. Clinical trials of NADþ precursors on age-related diseases, such as diabetes, premature aging diseases, and neurodegenerative diseases are in progress [10].",Translational Medicine of Aging,Clinical translation,2018
Outstanding Questions in NAD+ Biology,"6. Outstanding questions and future perspectives
Age is the primary cause of the majorly of human diseases and interventional strategies/therapeutics targeting on human aging is arguably the most efficient approach to achieve healthy aging and the improvement of the quality of life worldwide. Although, maintaining a healthy diet, fasting, and exercise may improve the quality of life, it may not be feasible option for all individuals. Therefore, the beneficial effects of NADþ discussed in the present review, highlight possible ways for improving the quality of life via hindering numerous pathological hallmarks of aging and thereby improving the quality of life and delay age-related diseases (summarized in Fig. 2). Preclinical evidence of NADþ replenishment that could potentially delay and/or prevent metabolic conditions, hearing loss, muscle atrophy, and cognitive decline are really encouraging for future perspectives. Moreover, NADþ precursors, in particular NR has been shown to be safely administrated and also able to demonstrate improvement of cardiovascular functions in human. Thus, implicating a possible translational aspect of preclinical benefits of NADþ supplementation, which is an exciting prospect and opens avenues for future studies to test the impact of elevated NADþ biosynthesis in aging and age-associated diseases in human.",Translational Medicine of Aging,Future perspectives,2018
Future Research Directions for NAD+,"Despite extensive research on NADþ biosynthesis and its implications in health and disease, there are major questions that are yet to be explored. Firstly, what levels of NADþ are to be associated with healthy aging and age-related diseases? In particular, it is of great relevance to elucidate organ and sub-cellular localisation as well levels of NADþ in health and disease. Such observations would allow to map health- and disease-specific alterations of NADþ, which could be utilised to develop therapeutic interventions that promote NADþ in a region-specific manner as a counter-mechanism. Decline in NADþ has been implicated during the process of aging and age-associated diseases, thereby may be affect various processes that are likely to key contributors and drivers of associated dysfunction. Thus, preclinical studies driven towards unveiling the pathways and mechanisms underlying the beneficial effects of NADþ replenishment in healthy aging and models of disease are required in order to understand how NADþ contributes to delay and/or prevent hallmarks associated with aging.",Translational Medicine of Aging,Future perspectives,2018
Mechanistic Insights and Clinical Needs,"In particular, it is important to elucidate whether individual or multiple hallmark(s) mechanisms associated with aging are countered by NADþ replenishment. This would allow understanding of the mode of NADþ action and the interconnection and contribution of the various hallmarks of aging. NADþ precursors have thus far shown to be safely and effectively administrated in healthy old humans, elevating NADþ levels in blood, but its safety and tolerance is yet to be determined in individuals with age-related diseases. Therefore, future clinical trials are required to assess the safety of NADþ precursors in patients with age-associated diseases such as diabetes and AD. Though, as abovementioned, careful evaluation of the role of NADþ, whether friend or foe in disease, must be taken into account for each disease-condition. Altogether, NADþ replenishment may serve as a potential therapeutic strategy for aging and multiple conditions to improve the quality of life of the increasing aged population.",Translational Medicine of Aging,Future perspectives,2018
Overview of NAD+ and Its Biological Roles,"Pharmacology and Potential Implications of Nicotinamide Adenine Dinucleotide Precursors
Jing She, Rui Sheng*, Zheng-Hong Qin*
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Jiangsu Key Laboratory of Neuropsychiatric Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
[Received March 16, 2021; Revised May 22, 2021; Accepted May 23, 2021]
ABSTRACT: Coenzyme I (nicotinamide adenine dinucleotide, NAD+/NADH) and coenzyme II (nicotinamide adenine dinucleotide phosphate, NADP+/NADPH) are involved in various biological processes in mammalian cells. NAD+ is synthesised through the de novo and salvage pathways, whereas coenzyme II cannot be synthesised de novo. NAD+ is a precursor of coenzyme II. Although NAD+ is synthesised in sufficient amounts under normal conditions, shortage in its supply due to over consumption and its decreased synthesis has been observed with increasing age and under certain disease conditions. Several studies have proved that in a wide range of tissues, such as liver, skin, muscle, pancreas, and fat, the level of NAD+ decreases with age. However, in the brain tissue, the level of NADH gradually increases and that of NAD+ decreases in aged people. The ratio of NAD+/NADH indicates the cellular redox state. A decrease in this ratio affects the cellular anaerobic glycolysis and oxidative phosphorylation functions, which reduces the ability of cells to produce ATP. Therefore, increasing the exogenous NAD+ supply under certain disease conditions or in elderly people may be beneficial. Precursors of NAD+ have been extensively explored and have been reported to effectively increase NAD+ levels and possess a broad range of functions. In this review article, we discuss the pharmacokinetics and pharmacodynamics of NAD+ precursors.",Aging and Disease,NAD+ precursors,2021
Introduction to Coenzyme I and Coenzyme II,"1. Introduction
1.1 A introduction to coenzyme I and coenzyme II
Numerous research results have confirmed that nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADPH) participate in mitochondrial energy and redox metabolism, reductive biosynthesis and cell signalling transduction [1-3], calcium homeostasis [4], gene expression [5], aging [6, 7], cell death [8], and other biological processes. NAD+ and NADPH exert preventive and protective effects in various diseases such as ischaemic stroke, cardiovascular disease, neurodegenerative disease, and liver damage [9-13]. NAD+ plays a central role in the biosynthesis of NADH, NADP+, and NADPH, all of which require NAD+ as a precursor. NAD+ and NADH are transformed into each other under the action of NAD+-dependent dehydrogenase and NADH-dependent oxidase, and NAD+ can generate NADP+ under the action of NAD+ kinase. Under the action of glucose-6-phosphate dehydrogenase, NADPH-dependent isocitrate dehydrogenase, NADPH-dependent malate dehydrogenase, and transhydrogenase, NADP+ is converted to NADPH [14]. NAD+ is synthesised through the kynurenine pathway, Preiss-Handler pathway, and salvage pathway from tryptophan, nicotinic acid, and nicotinamide, respectively.",Aging and Disease,NAD+ biochemistry,2021
"NAD+ Functions, Homeostasis, and Age-Related Decline","NAD+ is an essential cofactor for redox reactions and energy metabolism. NAD+ is also an important cofactor for NAD+-consuming enzymes including sirtuins, poly(ADP-ribose) polymerase (PARP) and CD38. NAD+ thus directly or indirectly regulates many key cellular functions, including energy metabolism, redox, DNA repair, cellular senescence and immune regulation, which are essential for maintaining metabolic homeostasis and health [15, 16]. With aging, the body's NAD+ content decreases [17,18]. Alteration in NAD+ homeostasis is found in a variety of age-related diseases, including neurodegenerative diseases, cardiovascular diseases, diabetes, and cancer [19,20]. The age-related decline in NAD+ is considered to be a driving force for these aging-related diseases. The level of NAD+ is strictly regulated by CD38 (one major NADase). However, the expression and activity of CD38 increase with aging, while inhibition or knockout of CD38 can partially prevent the decline of NAD+ [21,22]. During aging, senescent cells gradually accumulate in the white adipose tissue and liver. Then the inflammatory cytokines are secreted by senescent cells, the senescence-related secretory phenotype (SASP), can induce immune cells to proliferate and to express CD38, thereby consume more NAD+ in tissues [23,24]. These results reveal a causal relationship between cellular senescence and NAD+ decline during aging.",Aging and Disease,NAD+ and aging,2021
NAD+ Replenishment and Therapeutic Potential,"Correspondingly, to increase intracellular NAD+ can prevent age-related metabolic decline [25], improve the function of mitochondria and stem cells [26], maintain skeletal muscle function and exercise capacity [27]. Therefore, elevation of NAD+ may slow down or even reverse the progression of many aging-related diseases such as neurodegenerative diseases [28], metabolic dysfunction [29-31], immune disorders [32], mitochondrial dysfunction [33], and vascular aging [34], and extend the life span of animals [26,35-37]. However, several studies have found that the oral administration of NAD+ cannot effectively increase the level of NAD+ in plasma or in tissues. On one hand, the intestinal effect of NAD+ lowers its bioavailability; on the other hand, the excessively large polarity of NAD+ inhibits its passive transport through the plasma membrane. Therefore, the direct absorption of NAD+ by cells is believed to be unfeasible; however, this viewpoint may be challenged because a NAD+ transporter has been recently identified [38, 39].",Aging and Disease,NAD+ therapy,2021
Limitations of Direct NAD+ and NADH Supplementation,"Furthermore, the direct administration of high doses of NAD+ can cause insomnia, fatigue, anxiety, and other adverse reactions [40]. NAD+ levels in plasma or tissues do not increase significantly after oral administration of NADH mainly because orally administered NADH cannot be oxidised to NAD+, which inhibits its effective absorption in the intestine, although a NADH transporter has also been identified [41]; another possible reason is that NADH, before being absorbed by the gastrointestinal system in the human body, is transformed into a product that cannot produce NAM [42, 43]. Currently, intravenous infusion of NAD+ is the only clinically recognised method to increase the level of NAD+ in humans [44].",Aging and Disease,NAD+ supplementation,2021
NAD+ Precursors as an Alternative Strategy,"In recent years, more and more researchers have turned their attention to NAD+ precursors, namely nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), and nicotinamide ribose (NR). These precursors may have potential health and/or longevity benefits by increasing the level of NAD+ in the body and may be a promising strategy for alleviating aging-related diseases (Fig. 2).",Aging and Disease,NAD+ precursors,2021
Cell Entry Limitations of NAD+ and Transport Mechanisms,"2. Pharmacokinetics of NAD+ precursors
2.1 Cell entry modes of NAD+ and its precursors
Current knowledge suggests that all precursors and NAD+ must enter cells to produce biochemical and physiological actions. Increasing evidence indicates that NAD+ cannot enter cells directly through the plasma membrane and that it must be converted into smaller, less charged molecules to enter cells [58]. NAD+ can be degraded into NAM by membrane-bound CD38 and CD157 outside the cells, and the produced NAM can further produce NMN under the action of extracellular nicotinamide phosphoribosyl transferase (ENAMPT); NAD+ can also generate NMN directly under the action of membrane-bound CD73 outside the cells [59-61]. Some studies have identified a special NAD+ transporter, the connexin 43 (Cx43) channel, through which NAD+ can enter cells. Cx43 is highly expressed in cardiomyocytes [62, 63]. NADH has been reported to enter cells through the P2X7 receptor [41]; however, the same has not been confirmed by other researchers. Further research is required to determine the cell-specific efficiency of NAD+/NADH transporters and other NAD+/NADH transporters, if any, in addition to the Cx43 and P2X7 channels.",Aging and Disease,NAD+ transport,2021
"Cell Entry of Trp, NA, NAM, and NR","In the de novo pathway, Trp enters cells through carrier proteins (SLC7A5 and SLC36A4), which transport large, neutral amino acids [64]. NA and NAM, two forms of vitamin B3, can directly pass through the plasma membrane. Of these forms, the entry of NA into cells is mediated by a membrane carrier system, which includes a pH-dependent anion antiporter and a proton cotransporter (SLC5A8 or SLC22A13) [65, 66]. NAM can enter cells through two pathways; it may either be directly transported into the cell in its intact form or be converted into a metabolite of the salvage pathway and taken up by cells. The presence of the enzyme NAMPT, which converts NAM to NMN both inside and outside the cells, indicates that both pathways are feasible [67, 68]. Relevant studies on rodents have reported that NAM can be directly absorbed by the intestine [69]. The third form of vitamin B3, NR, does not require conversion to enter cells, which accounts for the high bioavailability of NR. NR enters cells through equilibrative nucleoside transporters (ENTs) and is phosphorylated into NMN by nicotinamide ribose kinase (NRK1/2) in cells [70, 71].",Aging and Disease,NAD+ precursor uptake,2021
NMN Uptake Pathways and Transport Controversies,"The routes through which NMN enters cells are described in literature as being highly complicated. First, NMN is transformed into NAM under the action of membrane-bound CD38, and then, it directly passes through the plasma membrane [21]; second, under the action of membrane-bound CD73, NMN is transformed into NR, which enters cells through ENTs [72]; third, NMN can directly enter cells through an NMN-specific transporter, a recently discovered transporter, which is highly expressed in the small intestine and encoded by the Slc12a8 gene [73]. Therefore, the uptake of NMN may be cell- or tissue-specific. However, a recent study in yeast showed that dephosphorylation of NMN into NR is necessary for the production of NAD+, whereas another study reported that the conversion of NMN to NAD+ can be inhibited by silencing the gene CD73, indicating that NMN must be converted into NR [4, 69, 70]. Therefore, investigating the uptake mode and kinetics of cell- or tissue-specific NAD+ precursors is essential. Figure 4 illustrates the routes through which NAD+ and its precursors enter cells.",Aging and Disease,NMN transport,2021
Overview of Pharmacokinetics and NAD+ Synthesis,"2.2. Pharmacokinetics of NAD+ precursors
To maintain the level of NAD+ in vivo, most of NAD+ is synthesised through the salvage synthesis pathway rather than the de novo pathway. Trp can produce kynurenic acid and serotonin in addition to producing QA for further synthesis of NAD+ [74]. Trp is considered to be the main precursor of NAD+ production in the liver [75]. NA and NAM are the only precursors that are increased in the liver 15 min after oral administration of NA or NAM, suggesting that the liver can use both de novo and salvage pathways to synthesis NAD+ [76]. We hereafter discuss the pharmacokinetics of NA and NAM. At high doses, the half-life of NA is 1 h, whereas that of NAM is 4 h. Studies have shown that the administration of high doses of NA will increase the level of NAM, but whether the administration of high doses of NAM influences the content of NA is still unclear [77]. However, the administration of NAM has been reported to cause skin flushing, which is a common adverse reaction that occurs after the administration of NA, indicating that the administration of high doses of NAM may also increase the NA content [78].",Aging and Disease,NAD+ pharmacokinetics,2021
Pharmacokinetics of NA and NAM,"In a study, the ability of NA and NAM to increase NAD+ was compared by orally administering NAD+ precursors to mice and NA was reported to produce the lowest level of NAD+ [76]. Oral administration of NA has been shown to result in a two-fold increase in NAD+ levels in the liver along with an increase in the NAAD level [76,79]. In another study, 30 mg/kg NA and 4000 mg/kg NA were administered to rats, and 4000 mg/kg NA was found to increase the level of NAD+ in the bone marrow of rats [79]. In a recent clinical study, after 10 or 4 months of administration of NA (750-1,000 mg/day), the blood and muscle NAD+ levels of human subjects were significantly increased. NA can also alleviate systemic NAD+ deficiency and improve muscle performance in adult-onset mitochondrial myopathy [80]. In another study, six healthy male subjects who took the upper level of NAM that can be tolerated per day (200 mg) in a single oral administration caused the maximum NAM blood concentration to increase by 30 times at 0.5 h, and then continued to decrease until 6 h, and the NAD+ blood concentration also increased significantly with the maximum concentration at 12 h [81]. The pharmacokinetics of oral administration of 3–6 g NAM in humans has been studied, and high doses have been shown to produce adverse reactions such as nausea and vomiting [82]. Although the capacity of NAM to increase NAD+ levels offer an advantage over NA, the less accumulation of ADP-ribose (ADPR) mediated by NAM also indicates a disadvantage. ADPR is a marker for NAD+-consuming enzymes activities [76], and NAM inhibits the activities of NAD+-consuming enzymes, such as PARP and sirtuin.",Aging and Disease,NA & NAM pharmacokinetics,2021
Pharmacokinetics of NMN,"Although limited pharmacokinetic data are available on NMN and NR compared with those on NA and NAM, a few studies have demonstrated that NMN and NR can effectively increase the NAD+ content in various tissues. Limited evidence shows that the administration of NMN can enhance NAD+ levels in various peripheral tissues such as pancreas [29], liver [83], adipose tissue [84], heart [85], skeletal muscle [33], and kidney [86]. Reports also indicate that after the administration of NMN, NAD+ levels in the testes [87] and eyes [25] are significantly increased. Furthermore, NMN has been reported to rapidly increase the level of NAD+ in the hippocampus, hypothalamus, and other brain regions within 15 min of intraperitoneal administration [88,89], which further suggests that NMN can pass through the blood–brain barrier, thereby contributing to the biosynthesis of NAD+ in the brain. Studies have reported that NMN can be detected in the mouse plasma, liver, adipose tissue, and pancreas within 15 min of the administration of 500 mg/kg NMN to wild-type mice through intraperitoneal injection; NMN is then used for NAD+ biosynthesis, which increases the level of NAD+ in the liver by 2–3 times [29].",Aging and Disease,NMN pharmacokinetics,2021
Time-Course Distribution and Metabolism of NMN,"A study also reported that the administration of 300 mg/kg NMN to mice through gavage increases the plasma NMN level significantly within 2.5 min and further increases the level after 10 min; however, the plasma NMN level returned to the original level within 15 min. Simultaneously, an increase in NAD+ levels in the liver, skeletal muscle, and cerebral cortex was observed. Results of the study indicated that NMN reaches the blood circulation from the intestine within 2–3 min and reaches the tissue from the blood circulation within 15 min [25]. Another study reported that the retention time of NMN in the body after intraperitoneal injection may be longer than that of NAM [90]. Some studies have shown that the plasma NAM content and hippocampal NR level are significantly increased after NMN injection, suggesting that at least a part of NMN is transformed into NAM and NR [40,91]. In a recent clinical study, after a single oral administration of 100–500 mg of NMN in 10 healthy men, the plasma concentrations of NMN and NAD+ metabolites (N-methyl-2-pyridone-5-carboxamide and N-methyl-4-pyridone-5-carboxamide) increased significantly [92]. NMN is generally believed to show good chemical stability. More than 90% of NMN can remain stable for 7–10 days in drinking water at room temperature [25]. NMN is also relatively stable in human HEK293 cell culture in FBS-free medium, and only 5% of NMN is dephosphorylated to NR by cells in 24 h [93]. However, during aging, senescence-induced inflammation promotes the accumulation of CD38 in immune cells. Then CD38 degrades the extracellular NMN through its ecto-enzyme activity, resulting in the decrease of intracellular NAD+ [24].",Aging and Disease,NMN pharmacokinetics,2021
Pharmacokinetics of NR and Biomarkers of NAD+ Synthesis,"Trammell et al. administered NR orally to study its effect on human peripheral blood mononuclear cells (PBMCs) and mouse liver NAD+ metabolism [76]; results of the study showed that the concentrations of all NAD+ metabolites, except NAM, were elevated in PBMCs. Moreover, the concentration of NAAD, a metabolite that is supposed to increase after the administration of NA instead of NR [94], was also assessed, which was found to be significantly increased, although a slight delay in the increase in the concentration of NAAD was observed relative to other metabolites, NAAD may be a biomarker of NAD+ biosynthesis and indicate the conversion of NR to NAD+ over time. In the liver of mice, the oral administration of 185 mg/kg NR increased the levels of NAM and NAD+ by approximately four times. Similarly, the level of NAD+ and NAAD in blood cells of a healthy 52-year-old man who took NR (1000 mg/kg) for 7 days was found to increase by 2.7 times. The study also found that the ability of NR to increase ADPR is 2–3 times that of NAM and that ADPR is a marker of the activity of NAD+-depletion enzymes such as sirtuin [76].",Aging and Disease,NR pharmacokinetics,2021
"Summary, Gaps, and Need for Further Research","The pharmacokinetics of NMN and NR have not been fully studied. NMN and NR exhibit better pharmacological properties compared with NA and NAM; however, a deeper dosage and mechanistic research are required to compare pharmacokinetics between NMN and NR. Both NMN and NR undergo primary metabolism before being absorbed in the body and are rapidly converted into intermediates [27]. Exploration of the pharmacokinetics of NMN and NR in the body can help determine their optimal concentration for different applications and provide insights into their pharmacological mechanisms of action.",Aging and Disease,NAD+ pharmacokinetics,2021
Natural Food Sources and General Roles of NAD+ Precursors,"3. Pharmacological actions of NAD+ precursors
NAD+ precursors are widely present in natural foods such as meat, eggs, dairy products, and whole wheat [25, 95]. NA is produced in plants and algae; NAM is the main form of vitamin B3 that can be absorbed from foods, and it is also a byproduct of deacetylation and ADP-ribosylation mediated by NAD+-metabolising enzymes such as SIRT, PARP, and CD38. NMN and NR are found in vegetables (such as broccoli and cucumber), fruits (such as avocados), and meats (such as beef) [96]. NR, the third discovered NAD+ precursor, is naturally present in milk and is considered a nutritious food source [97]. Several studies have shown that relying more on nutritious plant foods rather than meat may be the most effective strategy for obtaining health benefits and extending the lifespan [98].",Aging and Disease,NAD+ precursors,2021
Pharmacological Actions of NA and NAM,"Both NA and NAM are the forms of vitamin B3 that were introduced more than 50 years ago for the prevention and treatment of pellagra. Usually, 15 mg/day of NA, the acidic form of niacin, is commonly used in clinics to treat hyperlipidemia [44]. Intake of 1–3 g of NA per day has been reported to effectively regulate the ratio of low-density lipoprotein to high-density lipoprotein (LDL: HDL) [99, 100]. Similarly, increased NA levels have been shown to improve the genome integrity, and NA deficiency has been shown to cause chromosomal instability [101, 102]. High-dose NAM, the amide form of niacin, is used in radiotherapy and chemotherapy to promote microvascular blood flow in the brain [103, 104]. Studies have also reported that in several types of animal models of diabetes, NAM can prevent and reduce the progression of diabetes [105, 106]. NAM has been shown to inhibit cell apoptosis caused by glutamate-induced excitotoxicity [107, 108]. Moreover, it has been reported to maintain the genomic stability and reduce the incidence of skin cancer [109]. NAM can also improve remyelination after stroke [110]. In addition, NAM is widely used to treat skin diseases including autoimmune vesicular diseases.",Aging and Disease,NA & NAM,2021
Pharmacological Actions of NMN,"Researchers have paid considerable attention to the precursors NMN and NR in recent years because these are highly efficient in increasing NAD+ levels. These two precursors are involved in the DNA repair and ATP production and also play roles in cell signal transmission [98]. NMN was reported to improve insulin sensitivity and exert a positive effect on insulin levels [111]. Some studies have reported that NMN may be an effective intervention for patients with hypoglycaemia [112]. NMN participates in mitochondrial energy metabolism by improving mitochondrial respiration. NMN also has a hepatoprotective effect. NMN supplementation can prevent liver fibrosis by promoting the degradation of prostaglandin E2 and inhibiting the activation of hepatic stellate cells [113]. The application of NMN alone can restore the cardiac systolic function of elderly mice, while the combined application of NMN and SS-31 (a drug that targets mitochondria) in mice improved both systolic and diastolic function, and reduced myocardial hypertrophy [114]. Studies have shown that NMN can improve cognitive impairment in the Alzheimer disease (AD) mice model [115]. NMN can also improve the depressive behavior in animal models [116] and improve the survival rate in the Parkinson disease (PD) model in vitro [117]. NMN has also been reported to exert a protective effect on secondary brain damage caused by cerebral haemorrhage [118], haemorrhagic transformation in the MCAO model [119] and haemorrhagic transformation caused by ischaemic stroke with tPA [120].",Aging and Disease,NMN,2021
"NMN in Aging, Fertility, Metabolism, and Vascular Health","Additionally, NMN protects against cardiac ischaemia and ischaemic stroke [121, 122]. Therefore, NMN may be a potential drug for the treatment of age-related neurodegenerative diseases. Low doses of NMN were shown to improve the quality of female oocytes [123], thereby improving female fertility. Because NMN can effectively improve the quality of aging oocytes, NMN may be a potential drug for the treatment of fertility problems in older women [123–126]. Studies have shown that NMN can reverse age-related weight gain and cognitive impairment [25, 116]. Moreover, administration of NMN in aging mice has been shown to improve vascular oxidative stress and energy metabolism, restore the activity of sirtuin, and reverse age-related arterial dysfunction [29, 127]. NMN also improved the impaired neurovascular coupling response in the aged cortex and the resulting vascular cognitive impairment by the induction of genes involved in mitochondrial regeneration, anti-inflammation and anti-apoptosis [128]. All these initial studies suggest that NMN exhibits certain therapeutic prospects in aging-related diseases.",Aging and Disease,NMN,2021
Pharmacological Actions of NR,"NR is the main precursor of NAD+ in the central nervous system and the preferred precursor in mitochondria. It maintains the function of mitochondria by regulating the activity of sirtuin [44]. NR is also the preferred precursor for supplementing NAD+ levels in animal models of heart failure [129] and was shown to reduce cholesterol in obese mice [130]. It has also been shown to exert a certain ameliorating effect on alcohol-induced liver disease and depressive behaviour [28, 131, 132] and improve diabetic lesions and hepatic steatosis in mice with high-fat diet-induced obesity [133, 134]. NR can also ameliorate angiotensin II–induced cerebral small vessel disease in mice [135] and prevent noise-induced hearing loss [132, 136]. Similar to NMN, NR can also improve female fertility [137, 138]. NR is the only precursor that can prevent axon degeneration [139] as well as the oxidative stress and organ damage caused by sepsis [140]. Moreover, NR has been shown to exert a certain degree of therapeutic effect in the pathological progress of neurodegenerative diseases such as AD [28, 141], PD [142], aging [138, 143], cerebral apoplexy [129], and hypertension and cardiovascular diseases [76, 129, 144]. Numerous studies have shown that NR can increase the lifespan of all species tested so far, including mice [35, 37, 145].",Aging and Disease,NR,2021
Unresolved Questions and Distinct Actions of Precursors,"Although NR, NMN, NA, and NAM can effectively improve NAD+ levels, many issues still remain to be explored. As an NAD+ precursor enters the body, it is converted into NAD+, which is further converted into NADH, NADP+, and NADPH. Therefore, whether the beneficial effects are produced by the precursor itself or the transformed NAD+ or other coenzymes is unclear. Canto et al. reported that the ability of NA to lower cholesterol levels can be attributed to its ability to increase NAD+ levels [130]. NAD+ depletion increases the skin’s sensitivity to ultraviolet light, increases the DNA damage response, and eventually increases the instability of the genome and incidence of skin cancer. Conversely, NAM increases the genomic stability and decreases skin cancer incidence, which can also be attributed to its ability to increase NAD+ levels. Moreover, the administration of NA and NAM did not show the same physiological results compared with those of NMN and NR [76, 95, 146]. Liver damage caused by diet can be reversed by endogenous metabolism of NR rather than NAM [147]. Oral administration of NR can significantly improve the survival rate of immune-deficient mice and regeneration of haematopoietic stem cells, which cannot be achieved by administrating NA or NAM [148]. The effects that are observed after the administration of precursors may not necessarily be produced by NAD+, and different functions of the precursor itself or its transformation into other coenzymes may also play a role.",Aging and Disease,NAD+ precursor mechanisms,2021
Decline of NAD+ Synthesis With Aging and Dual Nature of the de novo Pathway,"4. Safety and side effects of NAD+ precursors
Studies have found that with aging, the body's intake of L-Trp [149, 150] and the de novo synthesis of NAD+ decrease. Among all organs of the body, only the liver contains all the synthetases involved in the de novo pathway, including the rate-limiting enzyme QPRT. Studies have shown that most Trp is consumed in the liver when the body is not aging [151]. However, the absence of QPRT has no significant effect on NAD+ levels in tissues, including the liver [152]. These results indicate that mammals may synthesise NAD+ mainly through the salvage synthesis pathway. The de novo pathway synthesises two basic neurotransmitters, namely glutamate and acetylcholine, in addition to generating NAD+. Some intermediates are also produced that regulate the activity of N-methyl-D-aspartic acid (NMDA) [153], for example, the antagonist of NMDA receptor kynurenic acid exerts a protective effect, and the agonist of NMDA receptor QA induces excitatory toxicity through glutamate receptors; hence, the de novo pathway is a double-edged sword that regulates the neuronal function [12].",Aging and Disease,NAD+ safety,2021
Safety Concerns and Side Effects of NA and NAM,"NA has been clinically used to treat dyslipidemia because it can lower blood lipid levels. However, the dosage of NA should be carefully used. If the daily dosage exceeds 50 mg, it will not only cause headaches and dizziness but also induce the production of prostaglandins, cause irritation to skin immune cells, and dilate skin capillaries, leading to skin flushing and itching [154]. Studies have found that this side effect is due to the activation of the G protein-coupled receptor, GPR109A (HM74A). NA can also cause spontaneous skin flushing reactions even at therapeutic doses because it acts as an agonist of this G protein-coupled receptor [155, 156], and this side effect greatly limits its clinical application [157, 158]. In addition, some of the animal studies have used a much higher NA dosage than those used in clinical patients to assess the effects of NA. For example, NA improves the neuronal function after hypoxic injury at a concentration of 250 μM–1000 μM in the culture medium, which exceeds the usual therapeutic concentration achievable in humans [159, 160]. Therefore, an optimal dosage of NA required for the elevation of NAD+ in the human body should be investigated.

In recent years, the results of various clinical studies on NA and NAM have shown that NAM is safer and more easily absorbed by the gastrointestinal tract than NA; although NAM reaches a serum peak 1 h after its oral administration, high doses of NAM can cause adverse reactions such as nausea and vomiting [161]. As mentioned earlier, NAM is a byproduct of NAD+ catabolism and a natural feedback inhibitor of NAD+-dependent enzymes such as sirtuin. Several studies have shown that the activities of PARP, sirtuin, and CD38 are inhibited at high doses of NAM [162]. The inhibition of NAD+-dependent enzymes produces side effects in the body. For example, a study demonstrated that the administration of NAM increases the accumulation of liver fat in a rat model of choline deficiency [44]. In addition, NAM consumes methyl groups and leads to a decrease in epigenetic methylation [163, 164]. Therefore, NAM is not considered to be an ideal precursor for supplementing NAD+ due to its feedback inhibition of NAD+-dependent enzymes and side effects of methyl depletion [165].",Aging and Disease,NA & NAM safety,2021
Safety Profile and Risks of NMN,"According to reports, low-dose NMN may be effective and safe. Single oral administration of 500 mg of NMN in healthy individuals is also safe and does not cause adverse reactions [92]. Following the oral administration of 300 mg/kg of NMN to normal wild-type mice (C57BL/6) for up to one year, the mice did not display any harmful or toxic effects, suggesting the superior safety and tolerability of NMN [25]. However, high-dose NMN may have adverse effects. Although low-dose NMN can improve the quality of female oocytes, high-dose NMN can reduce sperm quality [166]. In particular, the brain is highly sensitive to NMN, and high doses of NMN may exert adverse effects on neurons after ischaemia [121]. Studies have shown that high doses of NMN promote axonal degeneration in case of nerve damage [167–170]. NMN may also exacerbate in vitro axonal degeneration caused by a chemotherapy drug, vincristine [171]. Although numerous studies have proved the potential of NMN in the treatment of metabolic and aging-related diseases, its toxicological and clinical effects have not been sufficiently studied, and further studies are required to investigate the optimum dose range of NMN and long-term safety to humans.",Aging and Disease,NMN safety,2021
Safety Profile and Efficacy Considerations for NR,"Clinical studies on the short-term and long-term administration of NR have demonstrated the superior bioavailability and safety of NR. It is considered safe even when administered at a dose of 2000 mg a day for 12 weeks, and no adverse symptoms, such as nausea and vomiting, or undesirable skin flushing have been reported [143, 172]. Supplementation of NR neither inhibits NAD+-dependent enzymes nor causes side effects such as liver damage [76]. A study indicated that with an increase in the NR level in tissues following NR administration, the activity of the enzyme sirtuin is significantly increased compared with NAM administration [76]. Compared with other precursors, NR is gradually becoming a preferred candidate precursor because of its high bioavailability, safety, and ability to increase NAD+ levels. It offers many potential health benefits in diseases such as cardiovascular diseases [173, 174], neurodegenerative diseases [130, 175, 176], and metabolic diseases [177]. In summary, NR is a more effective precursor for synthesising NAD+ and increasing the activity of NAD+-dependent enzymes than NA and NAM. However, further research is required to explore whether NR can cause skin flushing or other adverse symptoms. Although NR produces no serious adverse reactions, it has not been shown to improve insulin sensitivity, endogenous glucose production, glucose disposal, and oxidation [172, 178]. Therefore, further studies are required to determine the benefits of NR.",Aging and Disease,NR safety,2021
Potential Cancer Risks and Complex Role of NAD+ in Tumors,"NAD+ participates in thousands of biochemical reactions in the body and thus maintains and regulates various physiological processes such as DNA repair, calcium homeostasis, and energy metabolism. Whether the supplementation of NAD+ precursors to increase the NAD+ content produces, in addition to the aforementioned effects, other side effects, especially diseases involving cell proliferation, such as tumours and atherosclerotic plaques, remains unclear. Energy metabolism not only plays an important role in the growth of normal cells but also promotes the growth of tumour cells. Moreover, aerobic glycolysis and other energy metabolism pathways in tumour cells are abnormally upregulated, thus generating a large amount of energy and metabolic intermediates to satisfy the rapid proliferation of tumour cells [179]. In addition, NAD+ not only acts a key coenzyme in aerobic glycolysis but also plays a central role in other energy metabolism pathways, including the TCA cycle [16]. Tumour cells have higher NAD+ levels than normal cells; therefore, NAD+ poses a risk of driving the growth of tumours.

For chemotherapy drugs under development, some researchers have turned their attention to drugs that can consume NAD+ [180]. The consumption of NAD+ in tumour cells inhibits the ability of NAD+ to repair DNA and participate in energy metabolism, thereby inhibiting the rapid proliferation of tumour cells. The consumption of NAD+ also promotes the production of reactive oxygen species, which in turn causes the disruption of tumour cells due to autophagy and apoptosis [181]. Inhibition of the rate-limiting enzyme NAMPT in the salvage synthesis pathway in tumour cells and animal tumour models has been shown to reduce the growth of tumour cells and enhance survival of animals [182–184]. However, some researchers believe that the decline in NAD+ levels may be related to aging-related diseases including tumours. Lack of NA in rats along with carcinogen exposure has been shown to increase the incidence of tumours [185, 186]. Moreover, the incidence of skin tumours in mice was shown to reduce with the topical application of NAM or supplementation of NA in the diet [187]. Recently, NR was reported to reduce the proliferation and activation of liver progenitor cells involved in liver tumour heterogeneity [188]. NR treatment can also reduce the size of the established liver tumour [189]. Increasing NAD+ levels have been shown to play an important role in the prevention of liver cancer and pancreatic cancer in mice [188, 190, 191]. The expression of CD38 increases with cell aging, thereby degrading NMN, which is one of the main reasons for the decline in NAD+ levels in senescent cells. According to a study, the proliferation of gliomas can be inhibited by inhibiting CD38, thereby prolonging the survival time of glioma mice [192]. Daratumumab, a CD38 monoclonal antibody, is a drug used for the treatment of multiple myeloma [193].",Aging and Disease,NAD+ and cancer,2021
Polyamine Synthesis and Fasting-Induced Metabolic Remodeling,"Polyamine synthesis is required for efficient metabolic remodelling and TORC1 inhibition during fasting. Next, we investigated the cellular consequences of impaired polyamine anabolism on acute fasting responses. We generated yeast lacking the rate-limiting enzyme ornithine decarboxylase (ODC1; yeast Spe1), which are characterized by polyamine depletion (Supplementary Fig. 1a). This strain (∆spe1) showed no elevation of polyamines upon starvation, and SPD supplementation (100 µM) fully replenished the intracellular SPD pool (Fig. 2a). We subjected ∆spe1 cells with and without SPD to proteomic analyses after 6 h nitrogen starvation (Supplementary Fig. 1b). Principal-component analysis (PCA) of the proteome revealed a clear distinction between the genotypes and that SPD could revert ∆spe1-associated global differences, whereas it did not affect the wild-type (WT) proteome (Fig. 2b). Mapping the identified proteins to Kyoto Encyclopedia of Genes and Genomes (KEGG) terms, we found several pathways that have been implicated in the starvation response dysregulated in ∆spe1 (Supplementary Fig. 1c). This included, for example, the metabolism of several amino acids (including ARG), lipids and fatty acids, as well as energy-relevant pathways (tricarboxylic acid (TCA) cycle and oxidative phosphorylation). Notably, we also found a disturbed starvation response of the proteostasis-associated pathways autophagy and TORC1/2, the yeast homologues of mechanistic target of rapamycin complex 1/2 (mTORC1/2), in ∆spe1 versus WT cells (Fig. 2c and Supplementary Fig. 1c).",Nature Cell Biology,Spermidine,2024
Metabolomic Changes Caused by Loss of Polyamine Synthesis,"The prominent dysregulation in metabolic pathways was supported by unbiased metabolomic profiling by nuclear magnetic resonance (NMR) spectroscopy, revealing substantial differences in the intracellular metabolomes after nitrogen deprivation (Supplementary Fig. 2). The metabolic disturbances affecting starved ∆spe1 cells (Fig. 2d) confirmed findings from the proteome analysis, including increased citric acid and reduced levels of nicotinamide adenine dinucleotide (NAD+) and adenosine/guanosine-X-phosphate (AXP/GXP; where X stands for mono-, di- or triphosphate), suggesting a disrupted energy metabolism secondary to the loss of intracellular polyamine synthesis (Supplementary Fig. 2). Our analysis also indicated dysregulated amino acid homoeostasis, which is normally maintained by autophagy in starving yeast. Notably, exogenously supplemented SPD reversed the metabolic dysregulations in ∆spe1 cells, both in control and nitrogen-starvation medium, whereas it hardly affected the general WT metabolomes (Fig. 2e and Extended Data Fig. 2a,b). This included normalized amino acid metabolism (Fig. 2f and Extended Data Fig. 2c,d), which has been critically linked to TOR and autophagy regulation (for example, for arginine and serine) and metabolites central to energy metabolism (glucose, NAD+ and citric acid, among others) (Fig. 2f and Extended Data Fig. 2e). Overall, Spe1 was required for the metabolic switch from glycolysis to oxidative phosphorylation, a key event in the cellular adaptation to nitrogen starvation, which partly depends on functional autophagy.",Nature Cell Biology,Spermidine,2024
Polyamine Regulation of TORC1 Signaling,"For instance, glucose levels were less increased under −N, and the ratios of TCA cycle metabolites were heavily dysregulated (for example, citric acid to succinic acid). Given the implication of TORC1 in the fasting response of ∆spe1 yeast cells, we next asked whether ∆spe1 cells would functionally alter the nitrogen deprivation-induced inhibition of TORC1. Sch9 (the yeast equivalent of mammalian p70S6K) dephosphorylation was significantly delayed in ∆spe1 cells (Fig. 3a,b). Focusing on TORC-associated proteins in our proteome data, we found dysregulated TORC subunits, including Tco89, Avo1/2 and Tsc11, under both conditions in the ∆spe1 strain (Extended Data Fig. 3a). Of note, low levels of polyamines (100 µM) completely reverted the delayed TORC1 inhibition in ∆spe1 cells (Fig. 3c,d). On the other hand, high levels of additional SPD (5 mM) did not affect TORC1 activity in the WT strain (Fig. 3e,f). Similar to −N, acute pharmacological inhibition of TORC1 with rapamycin led to a rapid increase of ORN (likely due to the known activation of arginase expression) as well as elevated SPD and SPM levels (Extended Data Fig. 3b). Thus, the efficient shutdown of yeast TOR signalling upon −N, a key event for autophagy induction, requires intact polyamine metabolism, whereas TOR inhibition promotes the anabolism of polyamines.",Nature Cell Biology,Spermidine,2024
In Vivo Translation of TOR-Polyamine Interactions,"To translate these findings in vivo, we measured polyamines and precursors in hearts from young male mice overexpressing the human insulin-like growth factor 1 receptor (IGF1Rtg) or carrying a dominant negative phosphoinositide 3-kinase mutant (dnPI3K) specifically in cardiomyocytes. IGF1Rtg causes increased IGF1R signalling, leading to elevated mTOR activity and autophagy inhibition, as well as age-associated heart failure, which can be overridden by SPD. Conversely, the cardiomyocyte-specific dnPI3K mutation inhibits mTOR and enhances autophagic flux. We observed a trend towards lower cardiac SPD levels in IGF1Rtg mice (P = 0.222) and significantly elevated levels of SPD in dnPI3K mice (Extended Data Fig. 3c). Collectively, blocking polyamine synthesis caused a defective cellular response to −N in yeast, thus compromising, inter alia, energy metabolism and amino acid homoeostasis, both of which interface with TOR signalling, an integral hub for sensing and relaying nutrient information to cellular responses and functional autophagy. SPD supplementation reversed the metabolic inflexibility of ∆spe1 cells.",Nature Cell Biology,Spermidine,2024
Autophagy Dysregulation Caused by Loss of Polyamine Synthesis,"Spermidine is vital for autophagy induction during fasting. mTOR is a major repressor of autophagic flux and proteomics revealed a profound dysregulation of autophagy-relevant proteins upon SPE1 loss (Extended Data Fig. 3d–f). SPD has been previously shown to induce ATG7 expression in yeast, and accordingly, SPE1 knockout caused reduced Atg7 protein expression (Fig. 3g). Furthermore, several autophagy-relevant proteins were differently modulated in the ∆spe1 strain upon −N (Extended Data Fig. 3d–f). This included the transcription factors Gcn4 (for amino acid biosynthesis), Msn4 (stress response), several autophagy-related proteins (Atg2/8/11/16/38), as well as vacuolar proteinases (Prb1, Ysp3 and Pep4) and proteins involved in intracellular vesicle trafficking (Vps33, Arc19, Vti1 and Ykt6), among others.",Nature Cell Biology,Spermidine,2024
Functional Autophagy Impairment in Polyamine-Deficient Yeast,"As a functional consequence, ∆spe1 cells exhibited reduced autophagy induction in response to −N. We observed a diminished autophagy-dependent proteolytic liberation of green fluorescent protein (GFP) from GFP fused to autophagy-related protein 8 (GFP-Atg8, Atg8 being the yeast orthologue of the mammalian LC3 family) (Extended Data Fig. 4a–d), which was rescued by SPD supplementation (Fig. 3h,i). This was confirmed with additional autophagy assays, including the reduced redistribution of GFP-Atg8 towards autophagic vacuoles, compared with WT cells (Extended Data Fig. 4e–g) and the Pho8∆N60 assay (Fig. 3j and Extended Data Fig. 4h). However, supplementing SPD could not further elevate autophagic flux under −N in WT cells (Fig. 3k,l). Similarly, rapamycin-induced autophagy was significantly curtailed in ∆spe1 cells (Extended Data Fig. 4i–l), which again could be partly rescued by SPD (Extended Data Fig. 4m,n).",Nature Cell Biology,Spermidine,2024
Polyamine Synthesis Required for Autophagy in Human Cells,"The ODC1 inhibitor difluoromethylornithine (DFMO) depleted polyamines in human U2OS and H4 in nutrient-rich (Extended Data Fig. 5a) and starved (Extended Data Fig. 5b) conditions to <5% of control levels, hence excluding the possibility that ODC1-independent mechanisms (such as uptake of exogenous SPD) would account for the starvation-induced elevation of SPD. DFMO reduced the translocation of GFP–LC3 to autophagosomes and autolysosomes in response to starvation in the presence of the lysosomal inhibitor chloroquine (CQ), indicating that polyamine synthesis is required for autophagic flux (Fig. 3m,n and Extended Data Fig. 5c–f). These phenotypes could be rescued by co-treatment with 10 µM SPD (Extended Data Fig. 5g–m). Three siRNAs targeting ODC1 phenocopied the effects of DFMO, hence depleting intracellular polyamines from U2OS cells (Extended Data Fig. 5n) and dampening starvation-induced autophagic flux similar to ATG5 siRNA (Extended Data Fig. 5o–q). As found in yeast, SPD supplementation failed to further enhance autophagy in starved U2OS GFP–LC3 cells (Extended Data Fig. 5r,s). Moreover, mTORC1 inhibition by rapamycin or torin-1 induced DFMO-inhibitable autophagy in U2OS and H4 cells (Supplementary Fig. 3a–f), which was rescued by SPD (Supplementary Fig. 3g–l).",Nature Cell Biology,Spermidine,2024
ODC1 Requirement for Autophagy in C. elegans,"C. elegans subjected to acute fasting exhibited elevated messenger RNA levels of odc-1, amx-3 and hpo-15 (PAOX and SMOX orthologues), whereas spermidine synthase (spds-1) and the AMD1 orthologue smd-1 decreased. Among the tested polyamine-relevant genes, argn-1 (ARG1/2 orthologue) and d2023.4 (SAT1/2) remained unaffected (Extended Data Fig. 6a). Knockdown of odc-1 by RNA interference (Extended Data Fig. 6b) reduced fasting-induced autophagic flux assessed by a tandem-tagged (mCherry/GFP) LGG-1 fluorescent reporter, the worm equivalent of yeast Atg8 and human LC3 (Fig. 3o,p and Extended Data Fig. 6c,d). This was confirmed in a strain expressing an alternative autophagy-sensitive biosensor, GFP-fused SQST-1 (the worm orthologue of human sequestosome 1 (SQSTM1)/p62, an autophagy substrate), which was less degraded in fasted odc-1 knockdown worms than in control nematodes (Extended Data Fig. 6e,f). Notably, SPD feeding reverted the autophagic deficit of odc-1 worms (Extended Data Fig. 6g,h). Similar to yeast, odc-1 knockdown also caused a trend (P = 0.140) towards reduced number of rapamycin-induced autolysosomes (Extended Data Fig. 6i,j).",Nature Cell Biology,Spermidine,2024
Conclusion: ODC1 Enables Fasting-Induced Autophagy Across Species,"Altogether, these findings indicate that ODC1 is required for optimal fasting-induced autophagy across species.",Nature Cell Biology,Spermidine,2024
Polyamine Synthesis Required for Lifespan Extension in Yeast,"Spermidine is required for fasting-mediated lifespan extension. Nitrogen deprivation reduces the fraction of dead cells in chronological aging experiments performed on yeast, a model for post-mitotic aging, in an autophagy-dependent manner. This longevity-extending effect was abolished in ∆spe1 cells, indicating that polyamine synthesis is required for longevity upon −N (Fig. 4a,b). The knockout of spermidine synthase (∆spe3) and that of SAM decarboxylase (∆spe2), which are both required for SPD generation, phenocopied ∆spe1 with respect to the loss of the longevity during −N (Fig. 4c). In contrast, the knockout of spermine synthase (∆spe4) failed to affect survival under nitrogen-deprived conditions (Fig. 4c). Of note, survival deficits triggered by the loss of Spe1 could be fully rescued by the addition of PUT, SPD or SPM (P > 0.05 against each other under nitrogen starvation) (Supplementary Fig. 4b). However, increasing concentrations of SPD only improved the nitrogen starvation-prolonged chronological lifespan of WT cells on early time points (Supplementary Fig. 4c).",Nature Cell Biology,Spermidine,2024
Spermidine and Multiple Longevity Pathways in Yeast,"We next investigated the involvement of SPE1 in other lifespan-increasing interventions in yeast. Inhibition of TOR with rapamycin extends yeast lifespan in an autophagy-dependent fashion, and this effect was diminished in the ∆spe1 strain (Supplementary Fig. 4d) but rescued by polyamine supplementation (Supplementary Fig. 4e). The extension of chronological lifespan by glucose restriction was partially compromised by the spe1 knockout (Supplementary Fig. 4f). Replicative aging, which reflects the diminished replicative capacity of aging mother cells, is especially responsive to glucose restriction. Knockout of SPE1 diminished the survival, median (WT 23 versus ∆spe1 19 days) and maximal replicative lifespan (68 versus 44 days) in low-glucose (0.05%) cultures but did not affect replicative lifespan when glucose concentrations were kept at standard levels (2%) (Supplementary Fig. 4g). In summary, SPE1 and SPD are essential for longevity induction by nitrogen starvation, rapamycin and glucose depletion.",Nature Cell Biology,Spermidine,2024
Spermidine Enables Fasting-Induced Longevity in Flies,"Testing these findings’ relevance, we subjected fruit flies to an IF regime that improves healthspan and lifespan. We followed the survival of female and male w1118 flies under IF12:12, during which increased daytime food intake compensated for the nightly calorie loss after 10 cycles and at later time points. DFMO lowered whole-body SPD levels and reduced the effects of IF12:12 on improved survival in both sexes (Fig. 4d, Extended Data Fig. 7c, Supplementary Table 2). Generally, IF12:12 seemed more effective in female flies, which we further tested for age-sensitive locomotor capacity with a modified negative geotaxis assay. DFMO prevented locomotion improvement by IF12:12 (Fig. 4e,f). DFMO did not affect food consumption during the first cycles of IF12:12 or body weight during acute fasting and generally seemed non-toxic for flies. Female flies lacking one functional copy of Odc1 (Odc1MI10996 mutant) were also unresponsive to IF12:12, and nightly SPD feeding could re-instate IF12:12-mediated longevity in such flies (Extended Data Fig. 7f and Supplementary Table 2).",Nature Cell Biology,Spermidine,2024
Spermidine Drives Fasting-Mediated Longevity in C. elegans,"In C. elegans, genetic inhibition of odc-1 reduced the lifespan extension and heat stress resistance conferred by IF48:48, but did not affect the body size of fasted worms (Extended Data Fig. 7g). Knockdown of spds-1 (spermidine synthase) or smd-1 (adenosylmethionine decarboxylase 1), which are critical for SPD synthesis, as well as argn-1 reduced the lifespan extension elicited by IF48:48 in worms (Fig. 4i and Supplementary Table 2). SPD significantly extended the lifespan of intermittently fasted odc-1 knockdown worms towards that of WT controls. However, SPD did not modulate the IF48:48 effect on the lifespan of WT worms and was less potent than IF alone when fed ad libitum. Similarly, rapamycin-triggered longevity was blunted upon odc-1 knockdown in worms.",Nature Cell Biology,Spermidine,2024
Spermidine Required for IF Healthspan Benefits in Mice,"Altogether, these findings unravel a pathway in which endogenous polyamine biosynthesis mediates the lifespan-extending effects of nitrogen starvation in yeast and IF in flies and worms. Cardioprotective and antiarthritic effects of intermittent fasting are blunted by DFMO feeding in mice. We next determined whether endogenous SPD biosynthesis in mice was essential for IF-induced healthspan improvements, focusing on cardioprotection and suppression of inflammation, knowing that both are elicited by SPD supplementation. In aged male mice, DFMO abolished favourable cardiac effects of an IF16:8 protocol (daily 16 h fasting and 8 h ad libitum food access during the light phase). This concerned improvements in cardinal signs of cardiac aging, including left ventricular (LV) diastolic dysfunction (E/e′), LV hypertrophy and LV remodelling index, whereas other cardiac parameters remained unaffected by DFMO and IF16:8. DFMO did not alter the IF16:8-induced reduction of body weight and food intake.",Nature Cell Biology,Spermidine,2024
Polyamine Synthesis Required for Systemic Health Benefits of IF,"In a second cohort of aged male mice, we tested the effects of DFMO on general and muscular healthspan improvements elicited by an IF + CR (30%) combination (IFCR; one meal a day, provided shortly before the dark phase). DFMO prevented IFCR-mediated improvements in a visual frailty index, grip strength and wire hanging ability, whereas it did not affect body weight, body composition or body surface temperature. Additionally, in young mice, both IF24:24 and oral SPD supplementation ameliorated the progression of autoantibody-induced arthritis, and the antiarthritic effects of IF24:24 were blunted by parallel DFMO administration, with comparable results in female and male mice. Collectively, genetic or pharmacologic ODC1 inhibition attenuated or abolished the healthspan-promoting, cardioprotective and inflammation-regulating effects of IF regimens in multiple distantly related species. Therefore, SPD is probably a dominant pro-autophagic and anti-aging effector metabolite accounting for the beneficial effects of various forms of IF.",Nature Cell Biology,Spermidine,2024
EIF5A and the Polyamine–Hypusination Axis in Autophagy,"Increased eIF5A hypusination is required for fasting-induced longevity. U2OS GFP–LC3 cells were screened for the effects of small interfering RNAs (siRNAs) targeting genes previously linked to SPD effects that hence might modulate starvation-induced autophagy. Besides the knockdown of ODC1 and the autophagy-essential genes ATG5 and ULK1, we found that depletion of EIF5A (eukaryotic translation initiation factor 5A) significantly reduced the number of GFP–LC3 dots after starvation (Extended Data Fig. 8o,p). SPD is required to hypusinate eIF5A via a conserved reaction involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The resulting covalently modified and active hypusinated eIF5A (eIF5AH) is involved in the pro-autophagic and antiaging effects of SPD administration. We thus investigated whether the polyamine–eIF5AH axis played a role in autophagy and lifespan regulation during −N and IF.",Nature Cell Biology,Spermidine,2024
Fasting Elevates eIF5A Hypusination Across Species,"Nitrogen deprivation of yeast enhanced eIF5AH (Hyp2 in yeast) in a Spe1-dependent fashion (Fig. 6b,c and Extended Data Fig. 9a,b). The hypusination defect observed in Spe1-deficient cells was reversed by SPD, which had no additional impact on hypusine levels in starved WT cells. Similarly, immunostaining of brains revealed increased eIF5AH and total eIF5A in female and male flies after 12 h fasting, as confirmed by immunoblotting of whole-head lysates from female flies. Notably, female heterozygous Odc1MI10996 mutants, which failed to show lifespan extension upon IF12:12, also lost their eIF5AH fasting response. We also found elevated eIF5AH in lysates from fasted worms and livers from fasted male mice. Of note, dnPI3K hearts, which had increased SPD levels, showed highly upregulated eIF5AH levels, while the IGF1Rtg mutation caused no alterations in eIF5AH or SPD levels. Increased eIF5AH, but not total eIF5A, levels were detected in starved human U2OS cells and in PBMCs from healthy human volunteers over several days of fasting, which persisted after food re-introduction.",Nature Cell Biology,Spermidine,2024
Regulation of Hypusination Machinery During Fasting,"We next explored the mechanisms of increased eIF5AH levels. In yeast, protein levels of Dys1 (DHS) tendentially, but not significantly, increased after 6 h and decreased after 24 h nitrogen deprivation, whereas Lia1 (yeast DOHH) was elevated significantly at both time points, suggesting a superior role for DOHH in driving or maintaining the observed effects on eIF5AH. Compared with WT cells, ∆spe1 cells exhibited increased Lia1 protein levels in control but not −N conditions, and this phenotype could be corrected by SPD. In double knockout ∆spe1∆lia1 cells, SPD failed to enhance eIF5AH levels in control and −N medium, indicating that Lia1 is indeed responsible for the hypusination response. Similarly, increased mRNA transcript levels of dohh-1 (C. elegans DOHH) and dhps-1 (DHS), but not iff-1 (EIF5A), were found in worms after 24 h fasting. Only DOHH levels stayed elevated after prolonged 48 h fasting. In U2OS cells, 6 h starvation increased the abundance of mRNAs transcribed from all three genes.",Nature Cell Biology,Spermidine,2024
"Hypusination Suppression Disrupts Metabolism, TOR Signaling and Autophagy","Next, we compared the proteomic landscape during nitrogen deprivation in yeast cells treated with GC7, a specific pharmacological inhibitor of DHS/DYS1, and/or lacking SPE1. As previously observed, proteins involved in autophagy, TOR signalling, translation and amino acid metabolism were dysregulated when polyamine synthesis or hypusination were suppressed, implying a multipronged effect of intracellular polyamine metabolism on these processes. Targeting hypusination directly with GC7 phenocopied many of the proteomic perturbations observed in the ∆spe1 strain, supporting a role of eIF5AH as a major downstream mediator of polyamine effects on energy and amino acid metabolism, as well as on TOR signalling and autophagy. Additionally, ribosomal processes, which are known to require polyamines, were strongly decreased when SPE1 was lacking and/or eIF5AH was inhibited by GC7. Accordingly, deleting the starvation-responsive LIA1 gene significantly decreased the long-term survival benefits conferred by nitrogen deprivation.",Nature Cell Biology,Spermidine,2024
EIF5A Hypusination Required for Longevity Across Species,"Similarly, a temperature-sensitive mutation of eIF5A (hyp2-1) entirely abolished the beneficial effect of nitrogen deprivation on chronological lifespan at the restrictive temperature (28 °C), when eIF5AH was blocked, but only partially at the permissive temperature (20 °C). Moreover, GC7 reduced eIF5AH and chronological lifespan extension by nitrogen depletion. A heterozygous point mutation in eIF5A mutating lysine 51, the target of hypusination in flies (eIF5AK51R), has previously been shown to render SPD supplementation ineffective on the climbing ability of aging flies. Likewise, IF12:12-conferred lifespan extension was lost in female and male eIF5AK51R/+ flies, while not affecting food consumption during the first cycles of IF12:12. Similarly to SPD supplementation, IF12:12 did not improve the climbing ability of aged female eIF5AK51R/+ flies. In C. elegans, the knockdown of dhps-1 attenuated the induction of autophagy and strongly reduced the beneficial effect of IF48:48 on lifespan without affecting body size. GC7 reduced autophagic flux in starved U2OS cells. In summary, fasting induced SPD-dependent increases in eIF5AH in multiple species and this effect on eIF5AH was required for the pro-longevity effects of nitrogen starvation or IF in yeast, worms and flies.",Nature Cell Biology,Spermidine,2024
"Fasting, Nutrient Sensors, and the Polyamine–eIF5A Axis","Discussion. Acute fasting stimulates autophagy by inhibiting nutrient sensors (such as TORC1 and EP300) and activating signal transducers for nutrient scarcity (such as AMPK and SIRT1). Adult-onset IF with CR represents a translatable tool to improve age-associated diseases and systemic health of humans. Still, mechanistic details into the molecular and metabolic relay of nutritional information to lifespan regulation are missing but mandatory for successful clinical implementation. Here, we focused on closing a major gap in our understanding of the metabolic control of fasting-induced autophagy and longevity that apparently involves an increase in SPD-dependent hypusination of eIF5A. Although fasting reduces the levels of amino acids, including ARG (together with its product ORN) and MET (and its product SAM), it also stimulates metabolic flux through the polyamine synthesis pathway, favouring an increase in SPD levels across different species, which requires ODC1. SPD is needed to hypusinate and activate eIF5A, a translation factor known to stimulate autophagy. Indeed, we detected fasting-induced eIF5A hypusination across all analysed species, supporting the concept that SPD is universally implicated in the fasting response.",Nature Cell Biology,Spermidine,2024
SPD and Hypusination Regulate Autophagy and Longevity,"Accordingly, genetic or pharmacological inhibition of SPD elevation or eIF5A hypusination curbed autophagy induction by fasting, and the longevity-promoting, cardioprotective and antiarthritic effects of IF across the phylogenetic spectrum. Among the three ODC1-dependent polyamines, SPD seems to be crucial for mediating many fasting responses, mainly because it represents the sole co-factor for eIF5A hypusination. Nevertheless, supplementation with PUT or SPM also reversed ODC1 deficiencies, likely because they are interconvertible with SPD. However, PUT and SPM may partly function by other, yet-to-be-identified mechanisms. We also found that autophagy induction by pharmacological mTOR inhibition partly depends on SPD synthesis in yeast and human cells, arguing in favour of a general role of SPD in autophagy stimulation.",Nature Cell Biology,Spermidine,2024
"Polyamines, TOR Signaling, and Trophic Pathways","Notably, in yeast, nitrogen deprivation-induced inhibition of TORC1 was partially delayed in ∆spe1 cells, indicating a reciprocal relationship between polyamines and TOR signalling. Reflecting a complex crosstalk between polyamines and mTOR, SPD treatment was previously shown to activate mTORC1 in the white adipose tissue (WAT) of young mice, but to inhibit mTORC1 in the WAT of aged mice fed a high-fat diet, and not to affect liver mTOR signalling, suggesting organ- or cell type-specific circuitries that remain to be explored. Of note, in long-lived dilp2-3,5 mutant flies (which lack three of the seven insulin-like peptides), autophagy is induced as a result of enhanced levels of glycine N-methyltransferase (GNMT), which also results in enhanced synthesis of the pro-autophagic metabolite SPD. GNMT overexpression is sufficient to increase lifespan in flies, and lifespan extension via dietary restriction partially depends on GNMT in flies. Similarly, ODC-1 is highly upregulated in the well-studied long-lived C. elegans daf-2 mutant, which encodes for the IGF1 receptor, further suggesting that the polyamine and insulin signalling pathways are intertwined.",Nature Cell Biology,Spermidine,2024
"Polyamines, Circadian Rhythms, and Future Research","Moreover, the liver-specific knockout of insulin receptor substrate 1 (IRS1) causes an increase in GNMT levels in mice, pointing to a phylogenetically conserved pathway linking reduced trophic signalling to SPD elevation. However, it remains to be determined whether this pathway is active in response to fasting. Recently, the longevity effects of IF were linked to the circadian regulation of autophagy in flies. As polyamines are subject to, and regulate, circadian rhythms, this adds yet another possible intersection of lifespan regulation by SPD and hypusination in the context of fasting regimes. The exact kinetics of the polyamine-eIF5AH response to IF or CR remains unclear on a cell- and tissue-specific level. In future long-term studies, it will be important to weigh the impact of fasting length/periodicity versus the level of CR on polyamine metabolism. In rodents and humans, isocaloric IF studies have produced mixed results on health outcomes and lifespan and it remains to be studied how polyamines mechanistically integrate into this complexity.",Nature Cell Biology,Spermidine,2024
Polyamine Metabolism in Fasting and Its Clinical Relevance,"Given the observation that serum SPD levels were increased in mice after eight months of CR, it will be important to determine how pharmacological or genetic inhibition of polyamine metabolism affects the extension of healthspan and lifespan conferred by CR rather than by IF. While our work focused on the effects downstream of polyamine metabolism, additional studies should dissect the molecular relays connecting nutrient status to the transcriptional, translational and post-translational regulation of polyamine metabolism and spermidine-dependent eIF5A hypusination. Currently, the biochemical mechanism through which IF and CR stimulate polyamine synthesis and subsequent eIF5A hypusination remain elusive, limiting the novelty of our study. Future work must elucidate the crosstalk between polyamines and nutrient-responsive factors, such as insulin/IGF1 and mTOR that inhibit autophagy and accelerate aging or AMPK that induces autophagy and favourably influences healthspan and lifespan. It will also be important to determine whether polyamines induced by IF always confer health benefits.",Nature Cell Biology,Spermidine,2024
"Risks, Benefits, and Summary of the SPD–eIF5A Axis","While oral spermidine supplementation can elicit anticancer effects, elevated polyamine levels are detected in many cancer types and may stimulate cellular proliferation. Thus, the impact of IF on patients with cancer remains to be carefully evaluated. Our study reveals that fasting-induced longevity and improved healthspan partially rely on SPD-dependent eIF5A hypusination and ensuing autophagy induction in multiple species.",Nature Cell Biology,Spermidine,2024
Overview of Calorie Restriction Mimetics and Inflammation Control,"Control of Inflammation by Calorie Restriction Mimetics: On the Crossroad of Autophagy and Mitochondria. Abstract: Mitochondrial metabolism and autophagy are two of the most metabolically active cellular processes, playing a crucial role in regulating organism longevity. In fact, both mitochondrial dysfunction or autophagy decline compromise cellular homeostasis and induce inflammation. Calorie restriction (CR) is the oldest strategy known to promote healthspan, and a plethora of CR mimetics have been used to emulate its beneficial effects. Herein, we discuss how CR and CR mimetics, by modulating mitochondrial metabolism or autophagic flux, prevent inflammatory processes, protect the intestinal barrier function, and dampen both inflammaging and neuroinflammation. We outline the effects of some compounds classically known as modulators of autophagy and mitochondrial function, such as NAD+ precursors, metformin, spermidine, rapamycin, and resveratrol, on the control of the inflammatory cascade and how these anti-inflammatory properties could be involved in their ability to increase resilience to age-associated diseases.",Cells,Calorie Restriction Mimetics,2020
"Inflammation, Aging, and Immune System Dynamics","1. Introduction. Inflammation is a protective response that is triggered under certain threatening conditions promoting the elimination of damage, tissue repair, and the recovery of homeostasis. An acute inflammatory response is typically activated upon infection or tissue injury and involves the recruitment of immune cells to the site of damage. The coordination of immune cells mobilized from the bloodstream, in conjunction with tissue resident immune cells, ensures the elimination of the damage followed by the resolution of the inflammatory process. Both the innate immune system, that quickly responds to the initial damage, and the adaptive immune system, that confers immunological memory and promotes faster responses to repeated infections, are essential to coordinate a successful inflammatory response. Under certain circumstances such as aging, there is a failure in the resolution mechanisms leading to the chronic activation of immune cells and persistent inflammation. This state of low-grade but chronic inflammation is known as inflammaging, and is characterized by increased levels of pro-inflammatory cytokines in the circulation. Notably, inflammaging is considered a risk factor for many age-related diseases.",Cells,Calorie Restriction Mimetics,2020
"Mitochondria, Autophagy, and the Inflammatory Response","Even in certain tissues like the brain, that possesses a privileged protection against inflammation, certain signs of inflammation appear gradually with age, and this neuroinflammation can anticipate the appearance of some neurodegenerative diseases. In addition, the integrity of the intestinal barrier is compromised due to inflammatory stress during aging and contributes to the development of several diseases. Finding drugs that protect against inflammaging, the disruption of the intestinal barrier, and neuroinflammation should be a priority for geroscience in the next years. Mitochondrial metabolism and autophagy are two of the most metabolically active cellular processes, playing a crucial role in regulating organism longevity. It is well known that an intense crosstalk exists between mitochondria and autophagosomes, and the activity or stress status of either one of these organelles may affect the other. A mitochondrial or autophagy decline compromises cellular homeostasis and induces inflammation.",Cells,Calorie Restriction Mimetics,2020
Immunometabolism and Mitophagy in Aging,"Mitochondria control a plethora of processes in the cell, not only by controlling ATP production, but also by serving as biosynthetic and signaling centers. In the last decade, it has become evident that mitochondria are essential organelles that direct the fate of immune cells, giving rise to the field of Immunometabolism. Moreover, the outcome of the inflammatory response can be controlled by modulating the metabolism of immune cells. Macroautophagy is a cellular process responsible for the degradation of protein aggregates and damaged organelles. During autophagy, autophagosomes engulf and transport cargo, which is subsequently degraded following fusion with lysosomes. Mitophagy—the selective autophagy of mitochondria—plays an important role in cellular homeostasis. Defects in PINK1-Parkin-mediated mitophagy, impaired organelle recognition, and defective LC3-II-mediated engulfment all contribute to mitochondrial dysfunction.",Cells,Calorie Restriction Mimetics,2020
Mitochondrial Theory of Aging and Autophagy Decline,"Remarkably, both mitochondria and autophagy function dramatically impact the aging process. Harman’s mitochondrial theory of aging proposed that accumulation of mtDNA mutations impairs mitochondrial respiration and leads to the accumulation of mitochondrial reactive oxygen species (mtROS). This accelerates the appearance of new mtDNA mutations, resulting in a vicious cycle that would cause cellular aging. Indeed, mutations affecting the stability, transcription, or translation of the mtDNA induce progeroid-like phenotypes by destabilizing the mitochondrial pool. Autophagy induction has been proposed as a way to fight aging symptoms. Mice with tissue-specific deletion of autophagy genes or whole-body downregulation of autophagy present degenerative signs resembling those associated with aging and age-related diseases, such as neurodegeneration, Alzheimer’s disease, increased lipid storage, muscle atrophy, hyperglycemia, and cardiac or renal dysfunction. Autophagy is downregulated during aging in worms, Drosophila, and mammals, and its induction promotes longevity and healthspan.",Cells,Calorie Restriction Mimetics,2020
"Crosstalk Between Mitochondria, Autophagy, and Inflammation","Recent findings exemplify the clear interconnection that exists between both mitochondria and autophagy. An impairment of this crosstalk favors activation of several inflammatory pathways. For instance, inhibition of autophagy promotes the accumulation of dysfunctional mitochondria facilitating the release of mtDNA to the cytoplasm. Cytosolic mtDNA increases Caspase-1 activation and IL-1β production, exemplifying the importance of mitochondria–autophagy interconnection in controlling IL-1β production. Autophagy modulates activation of the inflammatory transcription factor NF-κB. Mitochondrial dysfunction and rewiring of metabolism toward glycolysis favors acquisition of a pro-inflammatory phenotype in different immune cells. Mitochondria modulate intracellular pools of Ca2+ and ROS, which are classical mediators of inflammation. Mitochondria–ER contact sites regulate leukocyte migration, lymphocyte activation, and autophagy induction through Ca2+ homeostasis. ROS overproduction supports the oxidation–inflammation theory of aging, describing a vicious cycle of increased ROS and inflammatory mediators.",Cells,Calorie Restriction Mimetics,2020
"Calorie Restriction, Autophagy Induction, and Anti-Inflammatory Effects","Due to the clear importance of autophagy and mitochondria in conferring resilience to age-related diseases, the potential anti-aging effects of interventions which activate these pathways have been explored in detail. Calorie restriction induces autophagosome formation, mitophagy and increases NAD+ levels, improving autophagy function and mitochondrial fitness. CR protocols have shown potent anti-inflammatory properties. These findings encouraged efforts to develop CR mimetics that replicate CR’s benefits without the burden of food restriction. However, the role of inflammation downregulation as a mechanism mediating the effects of these compounds has been underrated. Here, we outline the effects of compounds classically known as modulators of autophagy and mitochondrial function on the control of the inflammatory cascade. These anti-inflammatory effects could mitigate inflammaging, intestinal barrier disruption, and neuroinflammation, improving resilience to aging and age-related diseases.",Cells,Calorie Restriction Mimetics,2020
Calorie Restriction as an Anti-Inflammatory Longevity Strategy,"2. Anti-Inflammatory Effects of Calorie Restriction. CR is probably the oldest strategy known to promote healthspan. First evidences dated from AD 1000, when the Persian polymath Avicenna already taught the elderly to eat less than when they were young. More recent research on aging has focused on unraveling the molecular mechanism by which CR acts. Several reports have characterized that the effects of CR on lifespan extension are mediated by the activation of Sirtuin-1 (Sirt1) and autophagy. In addition, CR reduces adiposity and alters Insulin-like Growth factor 1 (IGF-1) signaling. Thus, insulin receptor knockout in the adipose tissue extends longevity in mice. Indeed, insulin downstream signaling inhibits the Forkhead Box proteins (FOXO). Therefore, PHA-4, the C. elegans orthologue of the human FoxA transcription factors, is required for CR-induced longevity. In addition to autophagy promotion, CR promotes mitochondrial biogenesis in humans and corrects the expression of several genes affected by aging, whose function is related to mitochondrial biogenesis and function. CR also exerts anti-aging effects by reducing oxidative stress through a Sirt3-dependent activation of superoxide dismutase 2.",Cells,Calorie Restriction,2020
CR Suppression of Systemic Inflammation and Inflammasome Signaling,"One of the mechanisms that has emerged as a potential candidate of CR action is the downregulation of inflammatory pathways. Indeed, CR is able to decrease inflammation in several experimental models. For example, CR normalizes TNF-α and IL-6 serum levels in old mice up to young mice levels, and promotes a youthful transcriptional profile that includes downregulation of inflammatory pathways in rats and middle age humans. Likewise, β-hydroxybutyrate, a ketone metabolite that accumulates during CR, mediates an anti-inflammatory effect by blocking the NLRP3 inflammasome and the subsequent IL-1β/IL-18 production in human monocytes and mouse models of different inflammatory diseases. Recently, a very elegant study has shown that fasting increases AMP-activated protein kinase levels (AMPK), modulates the metabolic activity of monocytes, including a decrease in OXPHOS, and reduces the steady state levels of CCL2, thus precluding monocytes from leaving the bone marrow (BM). By doing so, fasting improves the phenotype of mice undergoing experimental autoimmune encephalomyelitis (EAE) without altering the immune response to bacterial infections.",Cells,Calorie Restriction,2020
Immunomodulatory Effects of Calorie Restriction on Adaptive Immunity,"CR has also shown important immunomodulatory effects on adaptive immune cells. Upon CR, high levels of glucocorticoids (GC) in the circulation favor the reorganization of the BM and the recruitment of memory T cells into it, in an attempt to avoid high levels of circulating GC. Once in the BM, the memory T cells activate the expression of pro-survival factors such as Bcl-2 or mTOR, which license them to remain longer in the BM stroma. Upon a second infection, in CR conditions, the increased reservoir of memory T cells that survive in the BM is once again mobilized into the bloodstream, improving fighting against infection. Considering the decrease in the efficiency of the immune response during aging, CR could improve resistance to infection in old organisms by this mechanism. Additionally, a recent report has shed light on the mechanisms by which autophagy and caloric restriction confer potent anti-tumor activity to T cells. High levels of K+ in the extracellular space of tumors activate a functional CR molecular program in surrounding effector T cells, promoting autophagy activation and metabolic reprogramming of CD8+ T cells, encouraging mitochondrial respiration and activation of the TCA cycle.",Cells,Calorie Restriction,2020
Neuroinflammatory Protection and Barrier Integrity Under CR,"CR has also demonstrated important anti-inflammatory properties in the context of neurological diseases. CR attenuates microglia activation in lipopolysaccharide-injected mice and in cortical injury rat models. Furthermore, CR ameliorates behavioral deficits in an Alzheimer’s disease (AD) mouse model. CR also improved behavioral and molecular signs in a MPTP-induced mouse model of Parkinson’s disease by increasing the levels of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor. The anti-neuroinflammatory properties of CR have also been extrapolated to EAE models where it decreases IFN-γ and IL-6 levels and ameliorates demyelination. The effect of CR on preserving the integrity of the intestinal barrier has been tested in rats and humans. In rats, long-life CR does not reverse the loss of permeability observed in old animals. In contrast, a mild four-week CR improves systemic inflammation and the permeability of the intestinal barrier in obese humans.",Cells,Calorie Restriction,2020
NAD+ Precursors as Calorie Restriction Mimetics,"3. Calorie Restriction Mimetics. 3.1. NAD+ Precursors. NAD+ synthesis is required for the effects of CR on lifespan extension. The molecular mechanisms underlying NAD+ precursor effects have been largely attributed to the activation of Sirt1, an important molecule that modulates the crosstalk between autophagy and mitochondrial function. Thus, NAD+ levels decline during aging, disrupting mitochondrial respiration through the impaired transcription of nuclear encoded mitochondrial subunits. Moreover, NAD+ activates Sir2p, the yeast homolog of the mammalian Sirt1, which de-acetylates Ku70, a DNA repair factor that sequesters Bax away from mitochondria, thereby inhibiting apoptosis and inducing lifespan extension. In humans, SNP variants of the FOXO3 gene, a well-known regulator of autophagy, are associated with longevity in centenarians. Interestingly, Sirt1 modulates the levels of the Forkhead Box O3A (FOXO3A). Accordingly, NAD+ precursor supplementation induces mitophagy and improves healthspan and lifespan in Drosophila, C. elegans and in mouse models of accelerated aging. Thus, NAD+ has emerged as a potent anti-aging factor, and the use of NAD+ precursors has been proposed as a potent strategy to target age-associated diseases in old organisms and invertebrate models of premature aging syndromes.",Cells,NAD+ Precursors,2020
Anti-Inflammatory Properties of NAD+ Precursors,"Importantly, NAD+ precursor compounds are potent anti-inflammatory drugs. Nicotinamide mononucleotide (NMN) supplementation, a product of the nicotinamide phosphoribosyltransferase (NAMPT), which acts as the rate limiting enzyme in NAD+ biosynthesis, decreases the levels of inflammatory cytokines in mice fed on a high fat diet (HFD) and in a mouse model of aged-induced type 2 diabetes. This effect seems to be mediated by a Sirt1 dependent de-acetylation of NF-κB and correlates with improved glucose and lipid homeostasis. NMN treatment also decreases IL-1β production in mouse models of diabetes and in the muscle of old mice. Additionally, NAM is also effective in various inflammatory skin conditions. Intriguingly, Van Gool et al. reported that, in LPS-stimulated macrophages, inhibition of NAMPT blunts the production of TNF-α, but not IL-6 or IL-1β, in a Sirt6-dependent manner, exemplifying the complexity of the relationship between NAD+ metabolism and the regulation of inflammatory cascades.",Cells,NAD+ Precursors,2020
NAD+ Precursors in Neuroinflammation and Nervous System Protection,"The anti-inflammatory effect of NAD+ boosting compounds is also extended to diseases affecting the nervous system. Nicotinamide riboside (NR) supplementation reduces microgliosis, astrogliosis and cytokine secretion in a mouse model of AD. Interestingly, NAD+ supplementation activates mitophagy and inhibits the NLRP3 inflammasome, which is hyperactivated in disease. In addition, during AD progression, microglia phagocytose Aβ thus protecting from its toxic effects. Similarly, NR treatment induces mitophagy, DNA repair and improves lifespan and healthspan in mouse models of ataxia telangiectasia. NAD+ decline during aging, due to reduced de novo synthesis, impairs phagocytosis and the resolution of inflammation in macrophages, suggesting this could be another potential mechanism by which NAD+ precursors reduce neuroinflammation in AD models. Likewise, supplementation with NMN decreased microglia activation, neutrophil infiltration and TNF-α and IL-6 levels in mouse models of intracerebral hemorrhage.",Cells,NAD+ Precursors,2020
NAD+ Supplementation and Immune Regulation,"NAD+ supplementation also efficiently worked in mouse models of EAE, in which it blocks disease progression by regulating T cell differentiation through the activation of the tryptophan hydroxylase-1 (Tph1). The activation of Tph1 skews T cell differentiation towards CD4+IFNγ+IL-10+ subsets, favoring immune homeostasis and preventing demyelination. Similarly, NAM reduces the infiltration of immune cells and demyelination in EAE models. Recently, the anti-inflammatory properties of NAD+-boosting compounds have been extended to the human scenario. The short-term administration (21 days) of NR to aged individuals increases NAD+ levels in the muscle and reduces the expression of inflammaging-associated cytokines IL-6, IL-5 and IL-2. This study opens a door for longer NR interventions in aged humans.",Cells,NAD+ Precursors,2020
Resveratrol as a Calorie Restriction Mimetic,"3.2. Resveratrol. Resveratrol is a natural phenol produced by several plants in response to injury or infections. Natural sources of resveratrol include grapes, blueberries or peanuts. Pioneer studies showed that resveratrol is able to extend longevity in Saccharomyces cerevisiae and Drosophila. In mice, resveratrol administration improves healthspan without extending longevity. However, in mice under a high-caloric diet, resveratrol extends both healthspan and lifespan and improves insulin sensitivity. Moreover, in humans, short term administration (30 days) to obese individuals improves several metabolic parameters, probably by promoting mitochondrial function in the muscle. Regarding the molecular mechanism, resveratrol is considered a CR mimetic that works by activating Sirt1. Park et al. showed that resveratrol increases the intracellular levels of cAMP, resulting in an increase in the intracellular levels of Ca2+. This activates the CamKK-AMPK pathway, via phospholipase C, and the ryanodine receptor Ca2+-release channel which increases NAD+ levels and Sirt1 activation.",Cells,Resveratrol,2020
"Resveratrol, Autophagy, Mitochondria, and Oxidative Stress","Notably, Sirt1 dependent activation of autophagy mediates longevity extension in C. elegans. Furthermore, resveratrol can also impact on mitochondrial function by inducing mitophagy and by modulating mitochondrial dynamics both in vitro and in vivo. In addition to Sirt1 activation, resveratrol works as a scavenger of different ROS, thus protecting from oxidative stress. Cumulative studies have demonstrated the anti-inflammatory properties of resveratrol. In old mice, resveratrol treatment reverses the increase in the secretion of the pro-inflammatory cytokines TNF-α, IL-6, IL-1β as well as the levels of ICAM-1 and the inducible nitric oxide synthase (iNOS). In addition, it decreases inflammation in the adipose tissue of rhesus monkeys under high fat and sugar diets. Similarly, resveratrol treatment downregulates the production of some circulating inflammatory markers such as IL-1β, TNF-α, IL-6 and IL-8, conceivably, by decreasing the number of circulating leukocytes in obese humans.",Cells,Resveratrol,2020
Innate and Adaptive Immune Modulation by Resveratrol,"Coinciding with the importance of Sirt1 in the regulation of macrophage activation, strong evidence supports the anti-inflammatory effects of resveratrol, in part, due to its action on innate immune cells. Resveratrol attenuates the activation of the NF-κB pathway in vitro, suggesting that the decrease in circulating cytokines and inflammatory markers is not an indirect, beneficial effect derived from its effect on non-immune cells. Actually, resveratrol inhibits NF-κB and Nitric Oxide production in macrophages in vitro. Additionally, resveratrol could regulate the activation of important inflammatory pathways, such as the NF-kB pathway, by scavenging ROS. The effect of resveratrol on adaptive immune cells has been tested in different models of T cell-dependent inflammation, like EAE, in which it effectively decreases inflammation. It was shown that at least some of the effects of resveratrol on EAE could be mediated by an increase in the number of IL-17+ IL-10+ T cells. However, other authors have stated that this effect could rather be due to an induction of T cell apoptosis via activation of the aryl hydrocarbon receptor and a concomitant decrease in the number of T cells, or due to decreased T cell proliferation.",Cells,Resveratrol,2020
Resveratrol in Endothelial and Neuroinflammatory Pathways,"The anti-inflammatory properties of resveratrol go beyond its action on immune cells. For instance, resveratrol modulates the adhesion of leukocytes to endothelial cells, a crucial step in the inflammatory cascade. Thus, resveratrol attenuates inflammation in human umbilical vein endothelial cells (HUVEC) cells by a mechanism that depends on the Sirt1 mediated activation of autophagy. Resveratrol also decreases the levels of important adhesion molecules, such as ICAM-1, in TNF-α stimulated endothelial progenitor cells and HUVEC cells. The beneficial effects of resveratrol extend to neuroinflammation. Sirt1 activation protects microglia cells from activation upon Aβ exposure. Thus, in those conditions, resveratrol promotes microglia quiescence by inhibiting NF-κB activation and the subsequent production of pro-inflammatory cytokines, protecting neurons from dying in co-culture experiments. In vivo, resveratrol ameliorates disease signs and astrocytosis in the p25 transgenic model of AD and in cellular models of amyotrophic lateral sclerosis.",Cells,Resveratrol,2020
Metformin as a Calorie Restriction Mimetic,"3.3. Metformin. Metformin is a drug derived from galegine, a natural product from the plant Galega officinalis used as a standard treatment for diabetes. While the exact mechanism by which metformin improves diabetes is not fully understood, cumulative evidences show that this compound can target a wide range of molecular pathways related to aging and therefore, it has been postulated as a potential treatment for aging-associated diseases. One of the preferential targets that is activated by metformin is AMPK. As mentioned, AMPK regulates autophagy and mitochondrial dynamics, and is also activated by CR, thus mediating at least some of the beneficial effects that CR has on healthspan and lifespan extension. Accordingly, metformin extends lifespan and healthspan in C. elegans and mice. While AMPK activation is the most characterized effect of metformin, several reports have shown that it also inhibits the mitochondrial complex I, which is essential during the OXPHOS. This leads to a decrease in mtROS production that can be protective in certain situations.",Cells,Metformin,2020
"Metformin, Autophagy, Mitochondria, and Inflammation Control","Because AMPK and mitochondrial respiration are two of the multiple potential pathways that metformin can target, it has been postulated that autophagy and mitochondrial function might be mediators of its beneficial effects on lifespan and healthspan extension. Interestingly, metformin is a potent anti-inflammatory drug and, thus, it has been postulated that some of the effects of metformin on healthspan and lifespan extension could be mediated by the control of the immune system response. Some evidence from in vitro experiments have shown that metformin is able to diminish the expression of adhesion molecules such as E-selectin, ICAM-1, VCAM-1 in TNF-activated endothelial cells. The underlying mechanism would be mediated by an inhibition of the NF-κB pathway. The vascular NF-κB-dependent anti-inflammatory effect of metformin has been further confirmed in IL-1β stimulated endothelial cells and vascular smooth muscle cells. By targeting the NF-κB pathway, metformin also inhibits the pro-inflammatory senescence associated secretory phenotype (SASP) of senescent cells.",Cells,Metformin,2020
In Vivo Anti-Inflammatory Effects of Metformin,"Importantly, several evidences suggest that metformin can also act as an anti-inflammatory drug in vivo. Old mice treated with metformin show decreased activation of the pro-inflammatory pathways NF-κB and JNK and increased levels of the anti-inflammatory cytokine IL-10. In addition, mice presenting non-alcoholic fatty liver disease (NAFLD), a disease that is clearly associated with aging, present an improved profile of inflammatory markers following metformin administration. An additional potential mechanism could be mediated by a Sirt1-dependent activation of the autophagy pathway. Another possible anti-inflammatory effect of metformin arises from the modulation of lymphocyte differentiation. During aging, an increased number of antigen-naïve but semi-differentiated memory T cells, known as virtual memory T cells, accumulate and impair proper response to antigens. Strikingly, metformin promotes CD8+ memory T cell differentiation by activating a TRAF-6-dependent induction of fatty acid metabolism.",Cells,Metformin,2020
Metformin in Neuroinflammation and Neurodegenerative Disease,"Interestingly, metformin has also been proven to be effective in combating neurological diseases associated with inflammation. Metformin restricts the entry of mononuclear cells into the central nervous system of a mouse model of EAE, downregulating the expression of several inflammatory markers and cytokines like TNF-α, IFN-γ, IL-6 or IL-17. Similar efforts have been made using metformin for the treatment of AD, which is the most common age associated neurodegenerative disease worldwide. However, while it increases the production of the AD-associated neurotoxic peptide Aβ by upregulation of BACE-1 activity, it results protective in Aβ-driven models of AD, in which it efficiently decreases microglia and astrocyte activation and NF-κB signaling. Moreover, it reduces the phosphorylation of Tau protein by activating the phosphatase PP2A and induces autophagy-mediated clearance of toxic aggregates. Therefore, the use of metformin in AD patients, even though it could have potential neuroinflammatory properties, should be taken with caution.",Cells,Metformin,2020
Metformin in Parkinson’s Disease and Intestinal Inflammation,"PD is a neurodegenerative disease affecting dopaminergic neurons of the substantia nigra. Mutations in the mitophagy associated genes PINK1 and PARKIN are responsible for early onset PD. Recent evidence has shown that Parkin and Pink1 deficient mice develop activation of the STING pathway and inflammation which is directly responsible for dopaminergic neuron death. Metformin supplementation ameliorates signs of stroke, cytokine levels, and neutrophil infiltration in the brains of mice subjected to medial cerebral artery occlusion by activating the AMPK pathway and inhibiting NF-κB signaling. In an inflammatory intranigral LPS model of PD, metformin ameliorates microglial activation and cytokine secretion, but exacerbates dopaminergic neuron death—possibly due to its weak inhibition of mitochondrial complex I. Metformin can also improve remyelination in the spinal cord of old rats by enhancing mitochondrial function in oligodendrocytes via AMPK activation. Some studies have also addressed the potential of metformin to preclude intestinal barrier disruption. Metformin treatment in IL-10 knockout mice increased AMPK signaling, decreased M1 macrophages and reduced TNF-α, IL-1β and IFN-γ levels. Similar effects were found in a DSS-induced intestinal inflammation model, though age-related intestinal permeability effects remain unaddressed.",Cells,Metformin,2020
Spermidine as a Longevity-Promoting Polyamine,"3.4. Spermidine. Spermidine is a natural polyamine, usually administered as a dietary compound, that extends longevity in yeast, worms, and flies by inducing autophagy. Interestingly, spermidine is also able to improve mitochondrial function, possibly by increasing the degradation of damaged mitochondria through mitophagy. These intriguing properties have led to several studies demonstrating the protective effect of spermidine treatment on different age-associated diseases. Spermidine administration demonstrated to be cardioprotective in old mice, reducing cardiac hypertrophy and improving diastolic function and, most importantly, spermidine intake negatively correlates with cardiovascular risk in humans. Of note, spermidine reduces the levels of the inflammaging-associated cytokines IFN-γ, IL-1β, IL-6, and TNF-α in mice.",Cells,Spermidine,2020
Spermidine Effects on Leukocyte Adhesion and Epigenetic Regulation,"An important step in the inflammatory cascade involves the adhesion of immune cell molecules to the endothelium and the directional migration of these cells towards the injured tissue. One of the key molecules mediating this process is the adhesion molecule LFA-1. Notably, the transcription of Itgal, the gene encoding for LFA-1, is increased in blood cells from old human donors. Interestingly, spermidine induced hypermethylation of the Itgal gene and suppressed LFA-1 expression in lymphocytes, suggesting that another anti-inflammatory effect of spermidine could involve epigenetic inhibition of leukocyte migration.",Cells,Spermidine,2020
Spermidine and Immune Cell Modulation,"The direct effects of spermidine have also been well characterized in immune cells. In aged humans, T cell function declines with age alongside with a decrease in autophagy. In old mice, impaired CD8+ T cell response against influenza vaccination is restored by spermidine in an autophagy dependent manner. The induction of autophagy is however independent of mTORC1 activation, suggesting that spermidine and rapamycin act through different pathways in T cells. Spermidine blocks the production of the pro-inflammatory cytokines IL-12 and IFN-γ and enhances the production of the anti-inflammatory cytokine IL-10 in LPS-stimulated mouse macrophages. The anti-inflammatory effects of spermidine on macrophages were further validated in LPS-stimulated human peripheral blood mononuclear cells, in which it inhibits the production of TNF-α, IL-1, MIP-1a and MIP-1b and in mouse models of carrageenan-induced footpad inflammation.",Cells,Spermidine,2020
Spermidine in Intestinal and Neural Inflammation,"Another age-associated feature that is very sensitive to increased inflammation is the maintenance of the intestinal barrier, which controls the entry of pathogens and toxic compounds into the circulation as well as the absorption of ingested nutrients. Intriguingly, spermidine protects epithelial cells from the inflammatory effects of IFN-γ in vitro, and is able to restore transepithelial electrical resistance through the activation of the T cell protein-tyrosine phosphatase (TCPTP). Spermidine also has direct in vitro anti-inflammatory properties in microglia, at least when stimulated by LPS through a mechanism that involves the downregulation of NF-κB activation. Furthermore, spermidine rescues age-associated loss of memory in flies by promoting autophagy and it was found to be neuroprotective in mice undergoing EAE, a model that highly depends on immune cell activation. Surprisingly, the effect on EAE was T cell-independent, but due to skewed differentiation of macrophages towards the M2 phenotype, a blockage in the production of IL-1, IL-12, and CD80 and induced the expression of arginase-1.",Cells,Spermidine,2020
Rapamycin as a Longevity-Promoting mTOR Inhibitor,"3.5. Rapamycin. Rapamycin, also known as sirolimus, is a macrolide compound isolated from Streptomyces hygroscopicus that inhibits the mammalian target of rapamycin (mTOR) complex. Overall, rapamycin has probably been the most studied anti-aging drug. This molecule extends lifespan in both male and female mice of genetically heterogeneous background and in Drosophila. Accordingly, genetic deletion of the S6K1, an mTOR downstream effector, increases mouse lifespan and protects from age and diet induced obesity and cardiac dysfunction. While most of the anti-aging effects of rapamycin have been attributed to an induction of autophagy, which is inhibited by mTOR signaling, these effects could also be mediated by an induction of mitonuclear protein imbalance and activation of the mitochondrial Unfolded Protein Response (UPRmt) in C. elegans. However, the effect of the UPRmt on lifespan extension in C. elegans remains controversial.",Cells,Rapamycin,2020
"mTOR, Metabolic Regulation, and Immunomodulation by Rapamycin","Chin et al. found a further connection between mTOR, mitochondrial function and lifespan extension. These authors found that mTOR inactivation partially mediates the lifespan extension effects of α-ketoglutarate, an intermediate of the TCA cycle, in C. elegans. Importantly, rapamycin possesses potent immunomodulatory properties and is actually used as an immunosuppressant drug in patients receiving organ transplantation. The phenotype of several immune cells is determined by a balance between OXPHOS and glycolysis. Resting macrophages mainly rely on OXPHOS for energy production, whereas pro-inflammatory activated macrophages show high glycolytic rates. mTOR signaling controls this metabolic switch by inducing the expression of HIF-1α. Accordingly, rapamycin inhibits TNF-α production in β-glucan-stimulated monocytes. Rapamycin has also been shown to inhibit natural killer cell activation through IL-15 signaling. Furthermore, chronic rapamycin treatment alters gene expression in immune cells, reduces PD1+ T cells, and increases the lifespan of non-infected immunocompromised mice.",Cells,Rapamycin,2020
"Rapamycin and Adaptive Immunity, SASP, and Inflammaging","Rapamycin treatment increases the number of memory precursors during the T cell expansion phase and accelerates memory cell differentiation of CD8+ T cells during the effector to memory transition, thus increasing the number and quality of memory CD8+ T cells. This points to rapamycin as an efficient treatment to improve responses after vaccination and against viral infections, which are known to be impaired during aging. The anti-inflammatory properties of rapamycin go beyond immune cells, as it also blocks SASP-associated cytokine production in senescent cells. In the nfkb1−/− mouse model of inflammaging, rapamycin prevents age-related frailty without altering inflammation by decreasing the number of senescent cells.",Cells,Rapamycin,2020
Rapamycin in Neuroinflammation and Neurodegenerative Disease,"Rapamycin has been shown to exert beneficial effects against neurological diseases. Initial seminal work attributed these benefits to autophagy enhancement and clearance of toxic proteins. However, targeting inflammation associated with toxic aggregate accumulation may also be critical. Rapamycin ameliorates microglia activation and disease signs in models of Leigh syndrome, cerebral palsy, traumatic brain injury, and tauopathy. In EAE, mTOR activation in T cells promotes HIF-1α signaling and glycolysis, directing differentiation toward pathogenic Th17 cells; thus, rapamycin improves EAE symptoms by inhibiting this pathway. Rapamycin also reduces infiltration of gamma delta T cells and granulocytes in ischemic brain injury models, decreases M1 microglia and increases regulatory T cells, attenuating brain damage. Dzamko et al. found that rapamycin reduces TLR2-dependent α-synuclein accumulation, highlighting autophagy–inflammation crosstalk.",Cells,Rapamycin,2020
Rapamycin and Intestinal Barrier Integrity,"Rapamycin treatment has shown properties in preserving age-associated loss of intestinal barrier integrity. In aged Drosophila, rapamycin-dependent inhibition of TOR activates the FoxA transcription factor homolog Fork Head (FKH) in the gut and promotes longevity extension. These effects are not mediated by altered microbiota composition but due to enhanced autophagy.",Cells,Rapamycin,2020
Introduction to Klotho and Aging,"Klotho was first discovered as an anti-ageing protein linked to a number of age-related disease processes, including cardiovascular, renal, musculoskeletal, and neurodegenerative conditions. Emerging research has also demonstrated a potential therapeutic role for Klotho in cancer biology, which is perhaps unsurprising given that cancer and ageing share similar molecular hallmarks. In addition to functioning as a tumour suppressor in numerous solid tumours and haematological malignancies, Klotho represents a candidate therapeutic target for patients with these diseases, the majority of whom have limited treatment options. Here, we examine contemporary evidence evaluating the anti-neoplastic effects of Klotho and describe the modulation of downstream oncogenic signalling pathways, including Wnt/β-catenin, FGF, IGF1, PIK3K/AKT, TGFβ, and the Unfolded Protein Response. We also discuss possible approaches to developing therapeutic Klotho and consider technological advances that may facilitate the delivery of Klotho through gene therapy.",Cancers,Klotho,2020
Discovery of Alpha-Klotho and Its Role in Aging,"The alpha-KLOTHO gene, named after the Goddess who spins the thread of life in Greek mythology, was first discovered as an anti-ageing gene in 1997. Kuro-o et al. demonstrated that mice with a deletion of approximately eight kilobases in the kl locus of the alpha-KLOTHO gene died prematurely at 8–9 weeks of age, and displayed phenotypes similar to advanced ageing, including arteriosclerosis, skin atrophy, osteoporosis, infertility, and emphysema. In contrast, transgenic mice overexpressing alpha-Klotho had an extended life span of up to 30 percent compared to wild type mice. These discoveries sparked immense interest into the role of alpha-Klotho in ageing, and many studies have since linked alpha-Klotho deficiency or mutations to cardiovascular, renal, musculoskeletal, and neurodegenerative conditions. Emerging research has also highlighted the potential role of alpha-Klotho in the pathogenesis of human malignancies.",Cancers,Klotho,2020
"Aging, Cancer, and Genomic Instability","Cancer and ageing share comparable principles; the time-dependent accumulation of DNA damage is a contributing factor in ageing and also drives cancer progression. DNA damage, alongside genomic instability, is an established hallmark of most cancers. This results in an increased frequency of mutagenesis during cell division due to the malfunction of the genomic maintenance machinery and surveillance systems that usually drive damaged cells into either senescence or apoptosis. Genetic defects driving genomic instability include loss of function mutations in caretaker genes responsible for detecting DNA damage and repairing damaged DNA, as well as gain of function mutations activating downstream signalling pathways. These pathways influence capabilities acquired by cancer cells including evasion of apoptosis, tumour invasion and metastasis, angiogenesis, and unlimited replicative potential. Alpha-Klotho potentially influences these phenotypes through inhibition of signalling pathways such as IGF-1R, FGF, TGFβ, and WNT.",Cancers,Klotho,2020
"Structure, Isoforms, and Expression of Alpha-Klotho","Alpha-Klotho was originally identified in the distal convoluted tubules of the kidney in mice. Since then, expression has been observed in arterial, epithelial, endocrine, reproductive, and neuronal tissues in humans by targeted proteomic analysis and antibody-based methods. The human alpha-KLOTHO gene consists of five exons and four introns and spans 50 kb on chromosome 13q12. It encodes a type I single-pass 135 kDa membrane protein composed of an N-terminal signal sequence, two extracellular glycosidase domains (KL1 and KL2), a transmembrane helix, and a short intracellular domain. Soluble Klotho (sKL) is generated via α- or β-cleavage of the membrane-bound form by ADAM10/17 or γ-secretase, producing circulating 130 kDa or 65 kDa isoforms. Secreted Klotho is generated by alternative mRNA splicing and resembles the KL1 domain.",Cancers,Klotho,2020
Alpha-Klotho vs Beta-Klotho,"While alpha-Klotho and beta-Klotho share sequence similarity, their genes are distinct; alpha-KLOTHO is located on chromosome 13 and beta-KLOTHO on chromosome 4. Beta-Klotho is membrane-bound without a secreted form, expressed in liver and white adipose tissue. Its functions differ significantly from alpha-Klotho and involve bile acid synthesis, glucose uptake, and fatty acid metabolism. Beta-Klotho forms complexes with FGFR1c and FGFR4, facilitating activation of downstream signalling. FGF21 binds the beta-Klotho–FGFR1c complex to activate ERK1/2 phosphorylation and reduce bile acid synthesis. FGF15/19 act through beta-Klotho–FGFR4 to suppress Cyp7a1, reducing bile acid synthesis. For this review, the focus remains on alpha-Klotho’s role in human malignancies and therapeutic potential in cancer treatment.",Cancers,Klotho,2020
Klotho as a Tumor Suppressor Across Malignancies,"2. The Tumour Suppressor Role of Klotho
The pleiotropic functions of Klotho have been increasingly recognised, including its importance in relation to tumorigenesis, cancer progression and prognosis. In addition to being described as a tumour suppressor gene in numerous solid tumours and haematological malignancies, Klotho represents a possible therapeutic target for patients with these diseases, the majority of whom have limited treatment options. Klotho may play a role in inhibiting tumour initiation and progression. Several studies have evaluated protein expression in malignant tissue compared with matched normal tissue in different tumour groups. Klotho expression has been shown to be significantly down-regulated in malignant tissue compared to adjacent non-malignant tissue using immunohistochemical techniques, including colorectal, pancreatic, gastric, oesophageal, breast, hepatocellular, ovarian, and renal cancers.",Cancers,Klotho,2020
KLOTHO Gene Polymorphisms and Cancer Risk,"Furthermore, more than ten single nucleotide polymorphisms (SNPs) have been identified in the human KLOTHO gene, and a number of studies have been performed to evaluate the associations between allelic variations in the KLOTHO gene and the aetiology of ageing-related diseases, including the effect of Klotho’s functional variants on cancer predisposition. A meta-analysis of 29 case-control studies has been undertaken to derive a pooled estimate of effect and to quantify the variability observed between individual studies. Relevant to cancer, the F allele of the F352V polymorphism was observed to protect against breast and ovarian cancer susceptibility. In a subgroup analysis stratified according to BRCA1/2 mutation carrier status, the F352V polymorphism was associated with the overall cancer risk in BRCA1 mutation carriers but not in BRCA2 mutation carriers.",Cancers,Klotho,2020
Circulating Klotho Levels and Clinical Prognosis,"In contrast to the sizeable body of literature describing the loss of Klotho protein expression in human tumour tissues, there is a relative paucity in published data relating to circulating Klotho in solid tumours. In a study of 160 patients who underwent nephrectomy for renal cell carcinoma, pre-surgery serum alpha-Klotho levels were lower in patients with more advanced disease; statistically significant differences were observed with tumour size (p = 0.003) and clinical stage (p = 0.0004). Further, a multivariate analysis showed that a low serum level of alpha-Klotho was an independent adverse prognostic factor for cancer-specific and progression-free survival in this study. Tang et al. measured serum Klotho levels in 40 patients with oesophageal squamous cell carcinoma and matched controls. Despite the limitations in the size of this study, analysis demonstrated significantly higher levels of serum Klotho in patients with OSCC compared with the control group (p < 0.001). However, a study assessing plasma alpha-Klotho levels in newly diagnosed lung cancer patients compared with control subjects did not reveal any difference between the two groups.",Cancers,Klotho,2020
Clinical Potential of Klotho as a Therapeutic Target,"Considering these data together, it is clear that further efforts are required to ascertain whether circulating Klotho has a role as a serum marker that could aid in the early diagnosis of different tumour types. Although circulating Klotho levels may not be altered compared with cancer tissues in all tumour contexts, functional data confirming tumour regression in several in vivo models that are not Klotho deficient supports further evaluation of Klotho as a candidate therapeutic target.",Cancers,Klotho,2020
Cellular Susceptibility and Rejuvenation Pathways,"One study indicated that out of a total of 20 tissues and 122,280 cells, approximately 49 cell types are susceptible to the accelerated aging effects of parabiosis, while 51 cell types are susceptible to induced rejuvenation11. Circulating factors present in the blood of elderly subjects have been found to accelerate the typical changes of aging, contributing to various age-related processes. In contrast, the blood of young individuals possesses a remarkable rejuvenating potential, capable of reversing age-related profiles. Specifically, adipose mesenchymal stromal cells, hematopoietic stem cells and hepatocytes stand out as cell types that show increased sensitivity to the effects of parabiosis-induced rejuvenation and parabiosis-accelerated aging. At the pathway level, studies have indicated that young blood not only reverses established aging patterns, but also triggers the activation of new sets of genes. For example, in parabiosis-induced rejuvenation, there is evidence of enhanced mitochondrial function, as evidenced by the complete rescue of genes encoding subunits of the electron transport chain11,13. Similarly, it has been observed that many cell-cell communication networks, which are disrupted during the aging process, undergo alterations in response to heterochronic parabiosis13.",npj Aging,Heterochronic Parabiosis,2024
Extracellular Vesicles and Mitochondrial Improvements,"Circulating small extracellular vesicles (EVs) from young mice increase peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) expression in aged mice through their miRNA cargoes, including miR-144-3p, miR-149-5p and miR-455-3p. These miRNAs target amyloid-β precursor protein (APP), poly[ADP-ribose] polymerase 2 (PARP2), and hypoxia-inducible factor 1α inhibitor (HIF1AN), respectively, which result in improved mitochondrial function and alleviates mitochondrial deficits in aged tissues10. Heterochronic parabiosis decreases senescent cell levels and prevents the inhibition of senescent cell elimination in old mice exposed to a young systemic milieu. Conversely, it increases senescent cell levels in young mice exposed to a young systemic milieu18. In preclinical models of aging, the accumulation of senescent cells is associated with a number of chronic diseases, geriatric syndromes, multimorbidity and accelerated aging. In contrast, the reduction of these cells by genetic and/or pharmacological methods has been shown in animal studies to prevent, delay and alleviate various aging-related diseases and their effects19.",npj Aging,Heterochronic Parabiosis,2024
"Senescence, SASP Reduction and Immune Rejuvenation","Expression of markers of the senescence-associated secretory phenotype (SASP) factors, including cyclin-dependent kinase inhibitor 1 (CDKN1A), cyclin-dependent kinase inhibitor 2A (CDKN2A), C-C motif chemokine 2 (CCL2) and proinflammatory cytokines such as interleukin-1β (IL1B), interleukin-6 (IL-6) and tumor necrosis factor α (TNFα), was significantly reduced in multiple tissues of old mice exposed to a young environment. In contrast, senescence markers were simultaneously increased in heterochronic young parabionts20. Recent research has shown that the removal of senescent immune cells significantly improves the immune function of old animals. By eliminating these cells, the animals prevent the release of cytokines that promote chronic inflammation and their immune cells respond more effectively to infectious agents and tumor cells, similar to the responses observed in young individuals. This improvement not only results in a higher quality of life, but also extends their lifespan21–23. The aging process often involves intricate alterations in iron metabolism, such as reduced serum iron levels and increased tissue iron deposition in various tissues of aged mice.",npj Aging,Heterochronic Parabiosis,2024
"Iron Regulation, Accelerated Aging and Study Variability","Surprisingly, exposure of old animals to the systemic milieu of young mice has been observed to reverse these age-related changes. Correlation analysis further underscores the importance of iron metabolism in the aging process, revealing a negative correlation between tissue iron levels and telomerase expression in vital organs such as liver, kidney and heart of parabiotic mice24. Accelerated aging has been reported to induce more cell-specific changes, reflecting divergent pathways in different cell types. In contrast, induced rejuvenation involves a more coordinated process, suggesting a conserved response in various cell types11. Currently, it is uncertain whether the beneficial effects observed in older individuals during heterochronic parabiosis are of the same level as the detrimental effects observed in younger individuals exposed to this same procedure. In this context, there is controversy about whether the negative effects of accelerated aging outweigh the positive effects of induced rejuvenation. Some studies support this assertion25–29, while others have reported contradictory findings30. In addition, some studies indicate that the result may vary depending on the cell type11.",npj Aging,Heterochronic Parabiosis,2024
"Parabiosis Duration, Age Gap Effects and Stressors","It is also crucial to consider the possible influence of other factors and experimental differences that have contributed to the conclusions reached in these studies. Furthermore, it has been mentioned that short-term parabiosis results in smaller and less enduring effects compared to long-term parabiosis. Following the separation of the individuals, long-term parabiosis showed more pronounced and sustained effects over time31. Similarly, it has been mentioned that the magnitude of induced rejuvenation or accelerated aging is positively correlated with the age difference between mice. Apparently, the greater the age difference, the greater the observed benefits or detriments32. Although this phenomenon does not occur in all cases. For example, although statistically significant changes in certain SASP factors were observed in the liver of 67-week-old mice paired with their 4-week-old and 8-week-old counterparts, it is not definitively demonstrated that older mice paired with 4-week-old animals experienced more pronounced overall parabiosis-induced rejuvenation33. Furthermore, the effects of parabiosis-accelerated aging may be exacerbated by various stressors, such as traumatic surgery and infectious diseases34.",npj Aging,Heterochronic Parabiosis,2024
Bone Aging and Parabiosis Overview,"Aging is a widely recognized risk factor associated with reduced bone density and deterioration of bone quality, resulting in increased susceptibility to fractures. Bones, as dynamic living tissues, undergo a perpetual cycle of formation and decomposition. This dynamic process is orchestrated by two types of cells, osteoblasts, which promote bone formation, and osteoclasts, which facilitate bone resorption. Maintaining a delicate balance between these activities is crucial to preserving optimal bone health54. Heterochronic parabiosis exerts various effects on bone, affecting factors such as bone density, microarchitecture and remodeling processes (Fig. 2). One study reported that exposure to blood from aged mice markedly promoted osteoclastic activity, reduced bone mineral density and skeletal stem cell abundance in young heterochronic mice27. In contrast, increased chondrocyte proliferation and matrix synthesis were observed in cartilage from old mice exposed to a systemic milieu of a young mouse.",npj Aging,Heterochronic Parabiosis – Bone,2024
GDF11 and Chondrocyte Proliferation,"This phenomenon was related to the action of growth differentiation factor 11 (GDF11), which attenuates age-related knee degeneration and promotes chondrocyte proliferation in the cartilage of old mice. GDF11 achieves these effects by up-regulating the SMAD2/3 pathway and promoting type II collagen (COL2A1) secretion55. Heterochronic parabiosis has been shown to reverse the impaired fracture repair phenotype observed in aged animals, effectively restoring their diminished capacity for osteoblastic differentiation. This rejuvenating effect is facilitated by hematopoietic cells, which modulate the β-catenin signaling pathway during the initial stages of the repair process56. β-catenin triggers osteoprotegerin (OPG) transcription in osteoblasts. OPG is a cytokine receptor within the tumor necrosis factor (TNF) receptor superfamily. Its main function is to safeguard bone by binding to receptor activator of NF ligand (RANKL), thus preventing excessive bone resorption. This mechanism promotes osteoblast differentiation and helps maintain bone density57,58.",npj Aging,Heterochronic Parabiosis – Bone,2024
"Fracture Repair, β-Catenin, and Neutrophils","However, with aging, β-catenin levels are elevated in the early phase of repair, and its reduction improves the quality of fracture healing56. In particular, young macrophage cells secrete lipoprotein receptor-related protein 1 (LRP1), a crucial molecule for reducing β-catenin signaling, thereby enhancing fracture repair in old bone marrow stromal cells59. Another study revealed that when aged mice were exposed to the juvenile circulation by heterochronic parabiosis, there was a marked increase in the neutrophil population, especially CD11b + Ly6C + Ly6G++ neutrophils. This increase was accompanied by a corresponding increase in the levels of plasma extracellular vesicles associated with these neutrophils. It is believed that binding of the lymphocyte antigen 6 complex of the G6D locus (Ly6G+) to its ligand on neutrophils may exert an inhibitory effect on local immune responses. Consequently, these changes have been found to be related to the enhanced fracture healing observed in aged mice participating in heterochronic parabiosis couples60.",npj Aging,Heterochronic Parabiosis – Bone,2024
DMD Model and Cytokine Changes,"In a murine model of Duchenne muscular dystrophy (DMD) subjected to heterochronic parabiosis, remarkable effects were observed. Individuals with DMD often experience reduced bone density and weakened bones. In particular, older heterochronic mice exposed to blood from young mice experienced a significant reduction in trabecular bone loss and improved healing of bone lesions in the proximal tibia. However, these positive results were accompanied by detrimental changes in bone health observed in young mice exposed to blood from old mice, although to a lesser extent. The benefits observed in the older mice were potentially associated with an increase in the number of bone marrow hematopoietic stem cells and a reduction in cytokines such as fibroblast growth factor 21 (FGF21), leukemia inhibitory factor (LIF), IL-6, myostatin and RANKL30. All of these cytokines promote bone resorption through osteoclast activity and contribute to the creation of an inflammatory environment through various mechanisms61–",npj Aging,Heterochronic Parabiosis – Bone,2024
Muscle Regulatory Factors in Young Circulation,"Heterochronic parabiosis exerts various effects on muscle cells (Fig. 3). A study found that numerous factors present in the juvenile circulation contribute to improved health of aging tissues. Specifically, some of these factors are thought to enhance myogenic proliferation by activating insulin-like growth factor 1 (IGF-1) and NOTCH signaling pathways, such as LIF. Others, such as cerberus (CER1), CRIPTO and dickkopf-related protein 1 (DKK1), counteract the TGF-β or WNT pathways, which usually impede the repair of aged muscle. Another subset of young proteins identified improving old muscle comprises tissue remodeling factors such as metalloproteinases and their inhibitors. Notable examples are tissue inhibitor of metalloproteinases 2 (TIMP2), cadherin-5 (CDH5) and vascular cell adhesion protein 1 (VCAM1), which facilitate cell-cell interactions. In addition, growth/differentiation factor 5 (GDF5) plays a role in regulating reinnervation, while factors involved in blood coagulation and vascular remodeling, such as serum amyloid protein A-1 (SAA1), fractalkine (CX3CL1), collagen α-1(XVIII) chain (COL18A1) and thromboplastin, are also found in this subset. Finally, leukocyte-specific proteins such as interleukin-27 (IL27), interleukin-10 (IL10), interleukin-22 (IL22), interleukin-22 receptor subunit α2 (IL22RA2) and lymphotoxin α (LTA) contribute to adult myogenesis66.",npj Aging,Heterochronic Parabiosis – Muscle,2024
Satellite Cell Rejuvenation and GDF11 Effects,"Satellite cells isolated from old mice subjected to heterochronic parabiosis with young mice showed enhanced colony-forming myogenic capacity and restored genomic integrity. GDF11 was also associated with these benefits. In addition, administration of this protein by injections to aged mice produced several positive results: restoration of myofiber size in regenerated muscle, increased mean size of regenerated myofibers, improved mitochondrial morphology, elevated levels of PGC1α, a key regulator of mitochondrial biogenesis, promotion of autophagy and mitophagy, improved regenerative potential of satellite cells, and increased neuromuscular junction size67. Although these findings are noteworthy, it is important to approach them with caution, as there are studies suggesting contrary effects of GDF11 on muscle. Some research indicates that GDF11 may hinder, rather than enhance, muscle regeneration by activating SMAD2/3 signaling and promoting inhibition of the differentiation process68.",npj Aging,Heterochronic Parabiosis – Muscle,2024
NOTCH Pathway Activation and Testosterone Influence,"In another study, it was observed that exposure of satellite cells from old mice to serum from young mice increased the activity of the NOTCH pathway and thus enhanced their proliferation69. Many components of the canonical NOTCH signaling pathway are known to play important roles in skeletal muscle development or function. Although the exact mechanisms by which this signaling pathway regulates muscle stem cells (MuSCs) are not fully understood, a crucial component appears to be ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1). This protein, secreted by macrophages, targets NOTCH1, thereby stimulating MuSC activation and promoting muscle regeneration70. High testosterone levels in the young mouse have also been identified as a stimulator of this signaling pathway in the old mouse after heterochronic parabiosis, playing a crucial role in the improvement of muscle mass and ultrastructure71.",npj Aging,Heterochronic Parabiosis – Muscle,2024
Mitochondrial Decline Under Accelerated Aging,"In contrast to previous findings, exposure of a young mouse to blood from an older mouse leads to a decrease in muscle functional performance, indicating detrimental effects of circulating factors72. Mitochondria play a fundamental role in the physiology of skeletal muscle, as they are responsible for regulating the metabolic state of the tissue and providing the energy necessary for its correct functioning73. In this sense, muscle samples obtained from young heterochronic mice showed significant reductions in both total mitochondrial area and mean mitochondrial size. In addition, a trend toward decreased expression of mitochondrial complex IV was observed. Furthermore, there was a marked decrease in oxygen consumption and activity of mitochondrial complexes I, II and coenzyme Q10 (CoQ10), as well as the entire electron transport system (ETS) in general72.",npj Aging,Heterochronic Parabiosis – Muscle,2024
Inflammatory SASP Factors and Muscle Decline,"The systemic milieu of an aged mouse triggers an increase in SASP factors in a young mouse, including IL-1a, IL-6, CCL5 and TNF-α. In this manner, the inflammatory process induced by these circulating factors can trigger tissue dysfunction. These factors primarily affect skeletal muscle, but also other organs such as the kidneys and liver. These circulating factors cause a decrease in muscle strength, physical endurance and lipid accumulation in muscle tissue. They also induce an increase in senescent satellite cells and senescent muscle interstitial cells in young mice. However, these effects can be mitigated by pre-treating the older mouse with senolytic drugs such as dasatinib plus quercetin or navitoclax before subjecting it to heterochronic parabiosis26.",npj Aging,Heterochronic Parabiosis – Muscle,2024
miR-199-3p and Myogenic Differentiation,"miR-199-3p is significantly decreased in the blood of aged mice compared to young mice. Restoration of its plasma levels by heterochronic parabiosis enhances myogenic differentiation and muscle regeneration by targeting LIN28 homolog B (LIN28B) and suppressor of zeste 12 protein homolog (SUZ12). This, in turn, enhances the expression of myogenic differentiation 1 (MYOD1) and myogenic factor 5 (MYF5)74.",npj Aging,Heterochronic Parabiosis – Muscle,2024
Transcriptional Plasticity in Hematopoietic Cells,"A study focused on hematopoietic and immune cells found that they exhibit considerable transcriptional plasticity in the context of heterochronic parabiosis (Fig. 4). This flexibility is particularly evident in hematopoietic stem and progenitor cells, which encompass both short- and long-term self-renewing hematopoietic stem cells and multipotent progenitor cells. Up-regulated genes associated with parabiosis-accelerated aging were consistently associated with neutrophil activation and antimicrobial response, whereas down-regulated genes were associated with reduced cytokine production, hematopoiesis, chromatin organization, circadian rhythm regulation, and cell cycle regulation. In contrast, parabiosis-induced rejuvenation produced opposite effects on these gene expressions75. The observed changes were attributed to several factors. These included transcription factors such as activating transcription factor 3 (ATF3) and activating transcription factor 4 (ATF4), as well as chromatin organization and remodeling factors such as lysine-specific demethylase 6B (KDM6B), Jumonji domain-containing protein 1C (JMJD1C) and yin and yang 1 (YY1)75.",npj Aging,Heterochronic Parabiosis – Hematopoietic & Immune,2024
Epigenetic Regulators and Aging Pathways,"ATF3 and ATF4 play a vital role in inducing specific senescence-associated mRNAs, either by directly acting on their promoters or by modulating chromatin accessibility76,77. Meanwhile, KDM6B plays a key role in the aging process by modulating several signaling pathways. It regulates the expression of factors involved in key cellular processes such as CDKN2A, TGF-β/SMADs and SASP factors. These factors act as transcriptional regulators, inhibiting or activating gene expression to promote cell cycle arrest and other aging-related changes78,79. YY1 recruits sirtuin 6 (SIRT6) to the transcription factor A, mitochondrial (TFAM) gene via YY1-associated factor 2 (YAF2), which ultimately contributes to the decline in mitochondrial function associated with aging1,80. In addition, parabiosis-accelerated aging was observed to affect cell-cell communication pathways involving cytokine-cytokine receptor interactions, in particular CCL3, CCL4 and VEGFA. However, exposure to young blood restored these pathways.",npj Aging,Heterochronic Parabiosis – Hematopoietic & Immune,2024
Rejuvenation of Immune Communication,"Especially striking was the rejuvenation-induced reestablishment of communication between hematopoietic stem cells, dendritic cells, immature NK cells and pro-B cells, which had been disrupted during aging81. These dysfunctions in intercellular communication play an important role in the aging process. They contribute to the chronic inflammation, as well as to deficiencies in the immune system’s ability to detect and eliminate pathogens and precancerous cells4. Heterochronic parabiosis did not enhance the cellularity of peripheral lymph nodes of old mice exposed to the systemic milieu of young mice. This suggests that the environment itself limits cellularity in these organs. Interestingly, the T cells in the peripheral lymph nodes of these old animals showed a higher proportion of self T cells with a peripheral maintenance advantage than T cells of the young mouse82.",npj Aging,Heterochronic Parabiosis – Hematopoietic & Immune,2024
Erythrocyte Metabolism and Partial Reversal,"Heterochronic parabiosis experiments demonstrated that the young systemic milieu only partially corrected the abnormalities of erythrocyte metabolism in old mice. Specifically, it normalized methionine levels and elevated erythrocyte choline levels83. Methionine enhances antioxidant capacity in erythrocytes, while choline enhances erythrocyte deformability, reduces erythrocyte aggregation, and increases membrane lipid fluidity84,85. In addition, parabiosis reduced levels of early glycolytic metabolites in erythrocytes and partially restored levels of late glycolytic products (pyruvate and lactate) to those observed in young mice. However, these interventions showed minimal or no effect on erythrocyte glutathione homeostasis, the pentose phosphate pathway, and the oxidation of purines and tryptophan, which are all associated with maintaining redox balance83.",npj Aging,Heterochronic Parabiosis – Hematopoietic & Immune,2024
Hepatic Progenitor Cell Proliferation and CEBPA–SMARCA2,"Heterochronic parabiosis exerts various effects on hepatocytes (Fig. 5). Heterochronic parabiosis elevated the proliferation of aged hepatic progenitor cells by reducing the levels of CCAAT/enhancer-binding protein α (CEBPA)-SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2) complex to levels comparable to those seen in young animals, thereby facilitating E2F-driven gene expression69. Aging leads to elevated levels of SMARCA2, which interacts with CEBPA, initiating the formation of the CEBPA-retinoblastoma-associated protein (RB1)-E2F transcription factor 4 (E2F4) complex. This complex binds to E2F-regulated promoters, suppressing the expression of their target genes. Consequently, it inhibits MYC promoter activation, causing a decrease in the proliferative response86.",npj Aging,Heterochronic Parabiosis – Liver,2024
MANF and Anti-Inflammatory Rejuvenation,"Notably, mesencephalic astrocyte-derived neurotrophic factor (MANF), a stress response protein secreted by cells with immunomodulatory properties, is also important for rejuvenating the liver by heterochronic parabiosis. After parabiosis-induced rejuvenation, MANF is crucial for decreasing markers of inflammation, reducing the accumulation of fibrotic areas and preventing hepatocyte apoptosis in the liver. However, it does not have the ability to reverse hepatosteatosis. These effects are closely linked to an enrichment of circadian rhythm and metabolic pathways87. In mice undergoing heterochronic parabiosis-induced rejuvenation, levels of miR-29c-3p, a non-coding RNA (ncRNA) strongly associated with aging in various organs, plasma, and extracellular vesicles (EVs), were significantly decreased. This reduction brought its levels close to those observed in the liver of young mice.",npj Aging,Heterochronic Parabiosis – Liver,2024
miR-29c-3p Targets and Aging Pathways,"Specifically, miR-29c-3p targets genes related to the extracellular matrix and secretory pathways, such as elastin (ELN), collagen α1(I) chain (COL1A1), collagen α2(I) chain (COL1A2), collagen α1(III) chain, disintegrin and metalloproteinase domain-containing protein 12 (ADAM12) and ADAM metalloproteinase with thrombospondin motifs 17 (ADAMTS17), all of which play roles in the aging process8. Exposure of young animals to the systemic environment of old mice resulted in a rapid decline in hepatic functional performance. Moreover, when muscle injury was introduced into these animals, these detrimental effects were further exacerbated28. In addition, these mice also exhibit hepatic fibrosis, highlighting the impact on several systems26.",npj Aging,Heterochronic Parabiosis – Liver,2024
Cardiac Hypertrophy Regression,"After 4 weeks of exposure to the circulation of young mice, a notable regression of cardiac hypertrophy was observed in old mice. This regression was characterized by a reduction in the size of cardiomyocytes and molecular remodeling. Specifically, there was a decrease in the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), along with an increase in levels of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2)88. Cardiac natriuretic peptides, such as ANP and BNP, are vital for maintaining cardiovascular balance and heart health. They not only influence blood pressure regulation, but also the management of glucose and lipid metabolism89. Meanwhile, SERCA2a facilitates the movement of calcium ions (Ca+2) from the cytoplasm to the sarcoplasmic reticulum, a process crucial for myocardial relaxation. This mechanism directly influences the heart’s ability to contract efficiently and maintain a regular rhythm, essential for overall cardiac function. With aging, the regulation of SERCA2a activity can become dysregulated, potentially contributing to age-related changes in cardiac performance and the development of cardiovascular conditions90.",npj Aging,Heterochronic Parabiosis – Heart,2024
Cardiosphere Vesicles and GDF11 Studies,"Furthermore, young cardiosphere-derived extracellular vesicles induce structural and functional improvements in the hearts, lungs, skeletal muscles, and kidneys of old rats. Indeed, these vesicles positively modulate glucose metabolism and activate anti-senescence pathways, contributing to overall rejuvenation91. One study reported that GDF11, as well as its effects on other tissues, plays a role in mediating the positive changes elicited by heterochronic parabiosis. This protein is abundant in the blood of young mice and decrease with age. Research showed that when its levels are increased in old mice subjected to heterochronic parabiosis, it contributes to reversing cardiac hypertrophy88. However, a subsequent study failed to replicate the impact on cardiac hypertrophy observed previously and found minimal effects of GDF11 injections92. Significantly, there were notable discrepancies between the two studies, including variations in the origin and sex of the mice used, as well as differences in the source of the recombinant active domain of GDF11 administered in the injections.",npj Aging,Heterochronic Parabiosis – Heart,2024
GDF11 Overexpression and Angiogenesis,"A third study shed new light on the matter, revealing that overexpression of GDF11 led to a reduction of senescent cells in the hearts of aged mice. Moreover, it stimulated cardiac stem cell proliferation and facilitated the recruitment of endothelial progenitor cells, ultimately resulting in increased angiogenesis in aged ischemic hearts93. Benefits were noted in mitigating age-related cardiac fibrosis and enhancing cardiac pump function under pressure overload conditions. Nevertheless, it was also noted that administering high doses of GDF11 could lead to severe cachexia and mortality94.",npj Aging,Heterochronic Parabiosis – Heart,2024
Vascular Endothelium Improvements,"Parabiosis has been shown to improve vascular endothelial dysfunction in aged female mice. In the group of aged mice subjected to heterochronic parabiosis, there was a marked decrease in the maximal relaxation response to acetylcholine during the third week. However, this effect was no longer evident by the ninth week after the parabiosis protocol95. This observation suggests that the improvement in vascular function may not be sustained over time, possibly indicating that the circulating factors responsible for the improvement lose their efficacy over time. Blood from young mice markedly enhanced the ability of blood vessels from old mice subjected to heterochronic parabiosis to relax in response to endothelial signals, and also reduced the production of ROS. These effects appeared to be mediated by a number of protective effects on mitochondria. These effects included improvements in oxidative phosphorylation and the tricarboxylic acid cycle, as well as anti-inflammatory and antioxidant effects96.",npj Aging,Heterochronic Parabiosis – Vascular Endothelium,2024
Vascular Pathology in Young Parabionts,"Contrary to earlier findings, genes affected by old mouse blood in young parabiont mice could potentially play a role in inducing pathological changes in the vascular structure of the aortic arch. This could occur through the suppression of pathways controlled by serum response factor (SRF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor A (VEGF-A)97. SRF-associated genes encode proteins essential for processes such as proliferation, repair and regeneration in vascular endothelial cells, which are critical for maintaining the health and function of blood vessels98. Meanwhile, IGF-1 plays a protective role against various cardiovascular conditions, including endothelial dysfunction and atherosclerotic plaque formation99. Lastly, VEGF is an important growth factor known for its crucial proangiogenic properties. It exerts mitogenic and antiapoptotic effects on endothelial cells, stimulating cell proliferation and preventing cell death. In addition, VEGF increases vascular permeability and facilitates cell migration, processes essential for angiogenesis and tissue repair100.",npj Aging,Heterochronic Parabiosis – Vascular Endothelium,2024
Kidney Effects and Rejuvenation,"Parabiosis-induced acceleration of aging triggers various adverse effects in the kidneys, including necrosis of tubular epithelial cells, interstitial inflammation, and abnormalities in the glomeruli26. In another study, researchers observed a decrease in renal tubular degeneration and the number of tubular cell detachments, along with reductions in markers of senescence and inflammation in aged mice. Furthermore, there was an increase in autophagy factors and antioxidant markers in the kidneys. These findings underscore the significant mitigation of renal aging achieved by exposure to a young blood environment101.",npj Aging,Heterochronic Parabiosis – Kidney,2024
Eye Inflammation and TPM1 Role,"A study on lacrimal glands revealed that young mice exposed to the systemic milieu of older mice exhibited increased lymphocytic infiltration, especially of marginal zone B cells and plasma cells. In addition, there was an increase in inflammatory cytokines such as interferon-γ (IFNγ), IL1b and C-X-C motif chemokine 9 (CXCL9), along with elevated focal scores indicating the severity of inflammation. Interestingly, male mice showed more pronounced effects than females during heterochronic parabiosis. In contrast, aged mice exposed to the systemic milieu of their young counterparts showed minimal improvements in this tissue25. Studies of accelerated aging by parabiosis have shown the role of tropomyosin α1-chain protein (TPM1) in the regulation of age-related inflammatory responses, glial cell activation, and abnormal dendrite sprouting in the aged retina. This regulation involves activation of protein kinase A (PKA) signaling and subsequent modulation of the fatty acid-binding protein 5 (FABP5)/NF-κB signaling pathway. Further heterochronic parabiosis experiments with old mice lacking TPM1 protein, which did not induce similar effects in young mice, supported this role102.",npj Aging,Heterochronic Parabiosis – Eyes,2024
Visceral Adipose Tissue Rejuvenation,"In a heterochronic parabiosis model of induced rejuvenation, a significant reduction of proinflammatory cytokine levels and an alteration of the adipokine profile was observed in the VAT of old mice exposed to blood from young mice. This alteration of the adipose tissue microenvironment was found to be protective against cellular senescence. Cells of the stromovascular fraction derived from aged adipose tissue showed reduced production of proinflammatory cytokines such as CCL2 and IL-6. In addition, reduced expression of senescence-related markers such as CDKN2A and CDKN1A was observed. Furthermore, levels of senescence-related adipokines, such as C-reactive protein (CRP), endothelial cell-specific molecule 1 (ESM1), intercellular adhesion molecule 1 (ICAM1), IL-11, leptin, pentraxin 2, and serpin E1/plasminogen activator inhibitor 1 (PAI-1), were reduced in old mice exposed to blood from young mice. These results demonstrate that the young milieu has the ability to protect aged adipose tissue from senescence and inflammation103.",npj Aging,Heterochronic Parabiosis – Adipose Tissue,2024
Adipose miRNAs and SIRT7,"One study identified 233 uniquely deregulated miRNAs in different organs associated with induced rejuvenation and 43 with accelerated aging, mainly in gonadal and mesenteric adipose tissues8. Another study found that upregulation of miR-25-93-106b in old mice due to parabiosis resulted in decreased fat mass by reducing SIRT7 expression104. SIRT7 is a key regulator of adipogenesis, promoting differentiation and maturation of early adipocyte precursors3.",npj Aging,Heterochronic Parabiosis – Adipose Tissue,2024
"Other Tissues: Thymus, Intervertebral Disc, Pancreas","The thymus serves as a primary lymphoid organ crucial for the maturation of T lymphocytes, a key component of the immune system. However, as individuals age, the thymus undergoes a process known as thymic involution, which is characterized by a decline in its functionality and size. This age-related decline in thymic function is a significant aspect of immune senescence, contributing to a decline in immune responsiveness with age105. Despite attempts with heterochronic parabiosis, thymic involution proved irreversible82,106. When young mice were paired with old mice, significant elevations were observed in the levels of the intervertebral disc markers matrix metalloproteinase-13 (MMP13) and ADAM metalloproteinase with thrombospondin motifs 4 (ADAMTS4), as well as in aggrecan core protein (ACAN) fragmentation and histological tissue degeneration. However, there were negligible changes in cellular senescence markers such as CDKN2A and CDKN1A. The result was accelerated disc matrix imbalance and tissue degeneration, with minimal impact on disc cellular senescence. In contrast, when old mice were paired with young mice, the effects were opposite. This pairing significantly suppressed disc cellular senescence but produced only a slight decrease in disc matrix imbalance and degeneration107. As people age, the accumulation of senescent cells increases, contributing to pancreatic β-cell dysfunction and insulin resistance in peripheral tissues. These effects play an important role in the development of type II diabetes mellitus108. Old mice parabiosed to young mice show increased pancreatic β-cell replication. In contrast, young β cells transplanted into old mice decrease their replication rate109.",npj Aging,Heterochronic Parabiosis – Other Tissues,2024
Article Information and Study Aim,"International Journal of Nanomedicine 2018:13 5335–5345. Effect of young exosomes injected in aged mice. Bo-Ram Lee, Jung-Hee Kim, Eun-Sook Choi, Jung-Hoon Cho & Eunjoo Kim. Introduction: Exosomes, nano-sized extracellular vesicles, are known to circulate through the blood stream to transfer molecular signals from tissue to tissue. Methods: To determine whether exosomes affect aging in animals, we primarily identified the changes in exosomal miRNA contents during the aging process. In exosomes from 12-month-old mice, mmu-miR-126-5p and mmu-miR-466c-5p levels were decreased and mmu-miR-184-3p and mmu-miR-200b-5p levels were increased significantly compared with those of 3-month-old mice. Their levels in exosomes were partially correlated with those in tissues: levels of only mmu-miR-126-5p and mmu-miR-466c-5p in lungs and/or liver were decreased, but those of mmu-miR-184-3p and mmu-miR-200b-5p in tissues did not coincide with those of exosomes.",International Journal of Nanomedicine,Young Exosomes and Aging,2018
Reverse Aging Effects in Tissues,"Results and discussion: In the aged tissues injected with young exosomes isolated from serum, mmu-miR-126b-5p levels were reversed in the lungs and liver. Expression changes in aging-associated molecules in young exosome-injected mice were obvious: p16Ink4A, MTOR, and IGF1R were significantly downregulated in the lungs and/or liver of old mice. In addition, telomerase-related genes such as Men1, Mre11a, Tep1, Terf2, Tert, and Tnks were significantly upregulated in the liver of old mice after injection of young exosomes. Conclusion: These results indicate that exosomes from young mice could reverse the expression pattern of aging-associated molecules in aged mice. Eventually, exosomes may be used as a novel approach for the treatment and diagnosis of aging animals. Keywords: exosome, injection, reverse aging, telomerase, biomarker, molecular therapy.",International Journal of Nanomedicine,Young Exosomes and Aging,2018
Background on miRNAs and Aging,"Understanding the regulatory mechanisms and the involved molecules underlying aging has aroused interest to prevent or delay aging or aging-associated diseases. It has been reported that the upregulated or downregulated miRNAs induce cellular senescence. Downregulated miR-24 in diploid fibroblasts, upregulated miR-203 in melanoma cells, and downregulated miRNA group miR-26b, miR-181a, miR-201, and miR-424 in mammary epithelial cells are the examples of miRNAs inducing senescence of cells. In cell-to-cell signaling in systemic aging, miRNAs are reported to be released in circulation and transferred to remote tissues. The released miRNAs can affect their levels in circulation in aged individuals, and in a recent study, they served as regulatory molecules to control aging speed. Therefore, they are strongly considered as aging-associated biomarkers, possibly determined by minimally invasive or noninvasive methods.",International Journal of Nanomedicine,Young Exosomes and Aging,2018
miRNAs in Body Fluids and Biomarker Potential,"Several studies comparing miRNA expression profiles from the blood of young and old animals have revealed differences in the expression levels of several miRNAs with aging: miR-17-5p, miR-16-5p, miR-21a-5p, and miR-34a in mice and let-7 and miR-34a in humans. One of the ways by which miRNAs are released in circulation is via vesicles blebbed out from cellular membranes. A representative type of these vesicles is exosomes, which are tens to hundreds of nanometers in diameter. The exosomes released from parent cells enter systemic circulation, which thus explains the signaling process among remote tissues. Cells under stress would release more exosomes in vitro to dispose unnecessary molecules or communicate their signals to the surrounding cells. Aging is a type of cellular stress; thus, exosomes are secreted at higher levels from senescent cells than from normal cells.",International Journal of Nanomedicine,Young Exosomes and Aging,2018
Exosomal miRNAs in Aging and Disease,"It has been reported on cell-to-cell signaling via exosomes to explain systemic aging as a whole-body phenomenon. miRNAs are also contained in exosomes and released to various body fluids such as the saliva, synovial fluid, and blood; therefore, these miRNAs displaying changes in expression levels can be considered aging or aging-associated disease biomarkers. Upregulated miR-24-3p in exosomes was proposed as an aging biomarker in human saliva. In patients with osteoarthritis, expression levels of miR-16-2-3p, miR-6821-5p, miR-26a-5p, and miR-146a-5p in women and miR-201-5p and miR-6878-3p in men were altered in synovial exosomes in a sex-specific manner. In Alzheimer’s disease, induced miR-342-3p and miR-139b expression was considered a probable biomarker in plasma exosomes. However, limited information is available on changes in the miRNA contents of exosomes in naturally aged individuals and their effects in the aging process.",International Journal of Nanomedicine,Young Exosomes and Aging,2018
Study Design and Goals,"Therefore, the identification of miRNA molecules deregulated in exosomes in the aging process would be required to understand the mechanisms underlying aging and may have potential applications in evaluating or reversing the aging status of an individual. In this study, we primarily identified differentially expressed miRNAs (DERs) in exosomes from aged mice and compared them with those from young mice. In addition, we determined whether the exosomal level of DERs coincided with their levels in tissues, particularly in the lungs and liver. If the miRNAs in exosomes have regulatory capability in systemic aging, their increased levels in young exosomes were expected to exert a reversing effect on tissues of old mice. Therefore, after intravenously injecting exosomes from young mice to aged mice, changes in aging-associated molecule levels were analyzed in aged mice.",International Journal of Nanomedicine,Young Exosomes and Aging,2018
Biomarker Analysis and Telomerase,"To support the direct/indirect relationship of reverse-aging with injected young exosomes, we determined the expression level of previously reported molecular biomarkers for aging, such as p16Ink4A and TOR. IGF1R is involved in metabolic changes in the aging process, and upregulation of IGF1R was reported; thus, we also analyzed its expression level following the injection of young exosomes to old mice. As another biomarker of aging, the expression of telomerase-associated genes could be considered based on previous studies. The absence of telomerase activity in most human somatic cells resulted in telomere shortening during aging. In addition, the telomerase expression in human bone marrow stromal cells prevented senescence-associated impairments, and transfection of a telomerase gene to old mice delayed aging and increased longevity. In this study, we analyzed the expression of telomerase gene expression as a feasible indicator of reverse-aging. The aim of this study is to identify aging-associated molecules in exosomes and whether they have an ability to induce reverse-aging in animals.",International Journal of Nanomedicine,Young Exosomes and Aging,2018
Animals and Ethical Approval,"Animals: Male C57BL/6 mice aged 3 weeks to 23 months were purchased from Central Lab Animal Inc. (Seoul, Korea). The animals were housed after purchase at the DGIST Animal Laboratory in accordance with the Institutional Animal Care Guidelines. The Animal Care and Use Committee of DGIST approved all experimental protocols to be applied to the mice. After 1 month of acclimatization, animals were either euthanized or used for exosome injection. In all cases, tissues (blood, liver, and lung) were sampled immediately.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Exosome Isolation Procedures,"Isolation of exosomes: Whole blood was sampled by retro-orbital bleeding, according to the previous protocols. Blood samples were collected in commercially available tubes with EDTA (BD Microtainer, BD, Franklin Lakes, NJ, USA). After collection of the whole blood samples, they were allowed to clot by leaving them at room temperature for 30 minutes. The clots were removed by centrifugation for 10 minutes at 1,000×g using a refrigerated centrifuge. The samples were maintained at -20°C until the exosome isolation was performed. Exosomes were isolated by ExoQuick™ Exosome Precipitation Solution (System Biosciences, Inc., Palo Alto, CA, USA) in accordance with the manufacturer’s instructions. Briefly, 200 µL of serum was collected and centrifuged at 3,000×g for 15 minutes to eliminate cells and cell debris. The supernatant was transferred to a microtube, and an appropriate volume of precipitation solution was added. The mixture was incubated at 4°C for 30 minutes, and then centrifuged at 1,500×g for 30 minutes. The exosome pellet was resuspended in 100–500 µL of PBS.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Exosome Characterization,"Characterization of exosomes: The isolated exosomes were observed through transmission electron microscopy (TEM, Tecnai™ G2 Spirit, FEI Company, Hillsboro, OR, USA) to identify their morphology and the extent of dispersion. Prior to TEM observation, the samples were stained with methanolic uranyl acetate and lead citrate. The size distribution of exosomes was analyzed through dynamic light scattering (DLS; ZetaSizer Nano ZS, Malvern Instruments, Malvern, UK). The enrichment of exosomes was confirmed by the Western blotting analysis of exosome markers, such as CD63, CD9, and Flotillin-1.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Screening of Aging-Associated miRNAs,"Screening of aging-associated miRNAs in exosomes: For high-throughput screening of DERs in exosomes of aged mice, next-generation sequencing (NGS) was performed with small RNAs isolated from the exosomes of 3-, 8-, and 12-month-old mice (n=3). Total RNA was isolated from exosomes using the Hybride-R RNA Kit (GeneAll®, Seoul, Korea), in accordance with the manufacturer’s instructions. RNA integrity was confirmed with a bioanalyzer, using an Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA) with an RNA integrity number value >8 as a cutoff value. The isolated total RNA (50 ng) was processed for preparing a small RNA sequencing library using the TruSeq Small RNA Sample Preparation Kit (Illumina, San Diego, CA, USA).",International Journal of Nanomedicine,Young Exosomes Methods,2018
NGS Library Preparation and Sequencing,"The total RNA sample ligated RNA 3′ and 5′ adapter with T4 RNA ligase (Promega, Madison, WI, USA) in the ATP-free buffer. The ligated RNAs were reverse transcribed to single-stranded cDNAs, using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) with reverse transcription primers recommended by Illumina. The cDNAs were amplified in 11 cycles of PCR, using Illumina’s Primer Set. PCR products were run on a 12% tris-borate-EDTA (TBE) polyacrylamide gel, and a slice of gel containing fragments of 145–160 nt was excised. This fraction was eluted and the recovered cDNAs were precipitated, and quantification of library was performed through RT-PCR analysis, using a CFX96 real-time system (Bio-Rad Laboratories Inc., Hercules, CA, USA). The libraries were pooled in equimolar amounts and loaded on the flow cell of a HiSeq 2000 sequencing system (Illumina). Sequencing was performed to generate 1×50 bp length read.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Quantification of miRNA Expression via ddPCR,"Quantification of miRNA expression in exosomes: The amount of miRNAs in exosomes is usually not high; thus, miRNA levels in exosomes were validated with QX200 droplet digital PCR (ddPCR, Bio-Rad Laboratories Inc.). Total RNA was isolated from tissues using the Hybride-R RNA Kit (GeneAll), according to the manufacturer’s instruction. cDNA for miRNA analysis was synthesized from 5 ng of total RNA using the Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark). PCR was performed in a 20 µL volume containing 10 µL 1× ddPCR EvaGreen Supermix, 2 µL cDNA, 350 nM primer sets, and deionized water to fill the remaining volume. miRNA LNA™ PCR primer sets (Qiagen, Hilden, Germany) were used for the detection of target miRNAs. 5S rRNA was used as an internal standard to determine the relative expression of target miRNAs.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Droplet Generation and Thermal Cycling,"The prepared ddPCR assay mixture was loaded into a QX200 disposable droplet generator cartridge according to the manufacturer’s instruction. Briefly, 70 µL of Droplet Generation Oil (Bio-Rad Laboratories Inc.) for probes was loaded into each well. The cartridge was then placed inside the QX200 droplet generator. When droplet generation was complete, the droplets were transferred to a 96-well PCR plate by using a multichannel pipet (INTEGRA Biosciences, Zizers, Switzerland). The plate was heat-sealed with foil and placed in a conventional thermal cycler. Thermal cycling conditions for EvaGreen assays were as follows: 95°C for 10 minutes, 40 cycles of 95°C for 30 seconds, and 58°C for 1 minute (ramping rate to 2°C/s), and three final steps at 4°C for 5 minutes, 90°C for 5 minutes, and a 4°C indefinite hold to enhance stabilization.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Quantification of miRNA Expression in Tissues,"Quantification of miRNA expression in tissues: The amount of miRNAs in tissues was determined by quantitative real-time PCR. Total RNA was isolated from tissues using the Hybride-R RNA Kit (GeneAll), according to the manufacturer’s instruction. cDNA for miRNA analysis was synthesized from 5 ng of total RNA using the Universal cDNA Synthesis Kit II (Exiqon). For the determination of miRNA expression by quantitative real-time PCR, the miRCURY LNA Universal RT micro RNA PCR LNA PCR primer sets for mmu-miR-126-5p, mmu-miR-466c-5p, mmu-miR-184-3p, and mmu-miR-200b-5p (Exiqon) and the SYBR Green PCR Kit (Exiqon) in an ABI7900HT Real-Time PCR System (Thermo Fisher Scientific) was used. The expression of miRNAs was determined by comparison of 5S rRNA expression in each sample.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Injection of Young Exosomes into Old Mice,"Injection of young exosomes into old mice: Before investigating the effect of exosome injection from young to old mice, exosomes isolated from whole serum of a 3-month-old mouse was stained with exosome-specific dye (ExoGlow™-RNA, System Biosciences, Inc.) according to the manufacturer’s instruction, and then the exosome distribution in whole body was observed following the injection of the stained exosomes intravenously using an in vivo imaging system (IVIS® Spectrum, PerkinElmer Inc., Waltham, MA, USA; excitation/emission wavelength = 485 nm/538 nm). After 72 hours from exosome injection, lungs and liver tissues were dissected and observed by confocal microscopy (FV1200, Olympus Corporation, Tokyo, Japan). To investigate the effect of young exosomes to old mice, exosomes from 4-week-old C57BL/6N male mice were injected intravenously in 18-month-old male mice (n=3). Exosomes for injecting into an aged mouse were prepared from 400 µL serum each (almost all serum from a 4-week-old mouse was used to inject into an aged mouse). As a control experiment (n=3), PBS was injected with the same volume of the exosome sample injected (averaging 100 µL per injection). After 24 hours from exosome injection, animals were euthanized, and the lungs and liver were sampled.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Western Blot Analysis of Aging-Related Molecules,"Western blotting analysis for aging-related molecules: Proteins were extracted from the lungs and liver of mice using extraction buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride). After incubation on ice for 15 minutes, tissue extracts were centrifuged at 10,000×g for 30 minutes, and the supernatant was collected for analysis. Protein concentrations were determined using a BCA Protein Assay Kit (Bio-Rad Laboratories Inc.). Proteins (30 µg) were separated via SDS-PAGE on a 10% resolving gel and electroblotted onto nitrocellulose membranes using a transfer apparatus (Trans-Blot SD semidry transfer cell, Bio-Rad Laboratories Inc.) in accordance with the manufacturer’s instructions. Anti-MTOR, -p16Ink4A, and -IGF1R antibodies were obtained from Abcam, Inc. Secondary antibodies conjugated with horseradish peroxidase were used, and the substrate for enhanced chemiluminescence (ECL) was purchased from Thermo Fisher Scientific. The intensity of protein bands was quantified by ImageJ 1.X developed by the National Institutes of Health. The expression level of proteins was determined in comparison with that of ACTB and GAPDH.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Telomerase-Related Gene Expression Analysis,"Analysis of telomerase-related gene expression: Total RNA from tissues was isolated using TaKaRa MiniBEST Universal RNA Extraction Kit (Takara Bio Inc., Shiga, Japan) in accordance with the manufacturer’s instructions. Single-strand cDNA was synthesized from 1 µg total RNA, using PrimeScript™ first strand cDNA Synthesis Kit (Takara Bio Inc.). For the determination of mRNA expression, RT-PCR was performed using gene-specific primer pairs specified in previous report10 and the Roche SYBR-Green® master mix in an ABI7900HT Real-Time PCR System (Thermo Fisher Scientific). The relative expression level of genes was determined in comparison with that of β-actin.",International Journal of Nanomedicine,Young Exosomes Methods,2018
Statistical Analysis,"Statistical analysis: For all experiments, data from three independent experiments were analyzed using Student’s t-test and are reported as mean±SD. SigmaPlot version 12.3 was used (Systat Software, Inc., Chicago, IL, USA) to determine the P-values. A P-value <0.05 was considered statistically significant.",International Journal of Nanomedicine,Young Exosomes Results,2018
Characterization of Serum Exosomes,"Characterization of exosomes from mouse serum: The isolated exosomes were visualized through TEM, after negative staining. Figure 1A shows that vesicles <100 nm in diameter were isolated by the methods used in this study. The size distribution was determined through DLS, which indicates that the average exosome size (z-average) in the main peak was 70±6 nm (average±SD, Figure 1B). To confirm the enrichment of exosomes by the isolation procedure, the representative proteins expressed on exosomes were analyzed by Western blotting (Figure 1C). The enrichment of CD63, CD9, and Flotillin-1 was clearly enhanced in the isolated exosomes in comparison with those in the same amount of serum proteins.",International Journal of Nanomedicine,Young Exosomes Results,2018
Aging-Associated miRNAs in Exosomes,"Aging-associated miRNAs in exosomes: miRNA contents of exosomes isolated from 200 µL of serum of 3-, 8-, and 12-month-old mice (n=3) were analyzed using NGS analysis. The total number of miRNAs determined in exosomal fraction was 1,033. Among these miRNAs, the selection criteria for DERs were as follows: 1) continuously upregulated or downregulated in 8- and 12-month-old mice compared with that in 3- and 8-month-old mice, respectively, in >0.5 of log2 (Ratio); 2) statistically significant changes in expression, ie, P<0.05. Table 1 shows the list of DERs that satisfy both aforementioned criteria. In total, five miRNAs were selected as DERs: mmu-miR-126b-5p and mmu-miR-466c-5p were downregulated and mmu-miR-184-3p, mmu-miR-200b-5p, and mmu-miR-708-5p were upregulated depending on the age of the mice. Expression changes in each miRNA in exosomes were confirmed by ddPCR analysis. Figure 2 shows the expression level of four DERs in 3-, 8-, and 12-month-old mice.",International Journal of Nanomedicine,Young Exosomes Results,2018
Tissue Levels of Aging-Related miRNAs,"Tissue levels of aging-related miRNAs from exosomes: Until now, it is not clearly defined whether exosomal levels of miRNAs are closely linked to those of tissues. Figure 3 shows the expression of DERs in the lungs and liver. mmu-miR-126b-5p was downregulated in exosomes on aging and also showed a decreasing trend in the lungs. The expression of mmu-miR-126b-5p in 12- and 24-month-old mice was significantly decreased compared with that in 3-month-old mice (P<0.05). In liver, the decreased expression of mmu-miR-126b-5p was significantly observed in 12- and 24-month-old mice only when compared with that in 8-month-old mice. The other downregulated miRNA in exosomes, mmu-miR-466c-5p, was not significantly downregulated in both lungs and liver. However, downregulation of mmu-miR-466c-5p in lungs was obvious in 12- and 24-month-old mice compared with that in 8-month-old mice.",International Journal of Nanomedicine,Young Exosomes Results,2018
miRNA Regulation After Exosome Injection,"miRNA levels changed by injection of exosomes: The isolated exosomes stained with RNA-specific dye ExoGlow were clearly observed by fluorescent emission at a wavelength of 538 nm, after intravenously injecting them into another mouse. The amount of exosomes stained with ExoGlow was time-dependently reduced but remained until 72 hours postinjection. The ex vivo image shows that exosomes were transferred to liver and lungs much higher than spleen, kidney, and heart. Following the injection of exosomes from 3-month-old mice, the DER levels were determined through ddPCR analysis in 18-month-old recipient mice. mmu-miR-126-5p was significantly upregulated in the lungs and liver of young exosome-injected old mice. mmu-miR-466c-5p did not significantly change after injection. mmu-miR-200b-5p in the liver significantly changed, while mmu-miR-184-3p and mmu-miR-200b-5p in lungs were not significantly altered.",International Journal of Nanomedicine,Young Exosomes Results,2018
Reversal of Aging-Related Molecular Markers,"Reverse expression of aging-related molecules in the lungs and liver: The injection of young exosomes in old mice was performed to examine the possibility of the ability of exosomes to alter the expression of aging-associated signaling molecules in tissues. As aging-related signaling molecules, the expression of p16Ink4A, MTOR, and IGF1R was analyzed by Western blotting and compared with that of ACTB and GAPDH. IGF1R and p16Ink4A were significantly downregulated in both the lungs and liver of aged mice after injection of exosomes from young mice, while MTOR was significantly downregulated only in the liver. In lungs, MTOR showed a downward trend (P<0.10). As other aging biomarkers, telomerase-related proteins were also analyzed in the tissues of injected mice. Among the six genes involved in the telomerase complex (Men1, Mre11a, Tep1, Terf2, Tert, and Tnks), two genes (Men1 and Mre11a) were reversed significantly in the lungs and liver of aged mice by injection of young exosomes. Tnks showed a trend toward significance. Overall, the reversed expression of aging-related molecules was observed in aged mice after injection of young exosomes.",International Journal of Nanomedicine,Young Exosomes Results,2018
Heat and Cold Stress Overview,"Environmental stress such as extremely warm or cold temperature is often considered a challenge to human health and body homeostasis. However, the human body can adapt relatively well to heat and cold environments, and recent studies have also elucidated that particularly heat stress might be even highly beneficial for human health. Consequently, the aim of the present brief review is first to discuss general cardiovascular and other responses to acute heat stress, followed by a review of beneficial effects of Finnish sauna bathing on general and cardiovascular health and mortality as well as dementia and Alzheimer's disease risk. Plausible mechanisms included are improved endothelial and microvascular function, reduced blood pressure and arterial stiffness, and possibly increased angiogenesis in humans, which are likely to mediate the health benefits of sauna bathing. In addition to heat exposure with physiological adaptations, cold stress-induced physiological responses and brown fat activation on health are also discussed. This is important to take into consideration, as sauna bathing is frequently associated with cooling periods in cold(er) environments, but their combination remains poorly investigated. We finally propose, therefore, that possible additive effects of heat- and cold-stress-induced adaptations and effects on health would be worthy of further investigation.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Bathing,2017
Introduction to Heat and Cold Adaptation,"Environmental stress such as warm or cold temperature is often considered a challenge to human health and homeostasis, but the human body also physiologically adapts to heat and cold environments at least to some extent. Furthermore, recent studies have also elucidated that particularly heat stress may even be beneficial for human health, independently of other lifestyle factors. Consequently, after a brief review of general acute cardiovascular responses and long-term adaptations in response to heat stress, we will discuss recent results of a long-term prospective study that indicates that frequency and duration of Finnish sauna bathing plays a role in preventing cardiovascular and all-cause mortality, sudden cardiac death (SCD), dementia, and Alzheimer’s disease risk (72, 73). While other types of heat stress studies such as warm water immersion studies have revealed some plausible mechanisms that might mediate health effects of repetitive heat stress, it is also emphasized that unique features of the Finnish sauna environment with its typical sauna tradition may not be directly comparable with other heat therapy means.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Bathing,2017
Finnish Sauna Characteristics,"A typical sauna includes relatively high temperatures (80–100°C) with dry air, which also circulates well in a Finnish sauna, making it easier to tolerate and even enjoy high sauna temperature. Another important feature is that the protective effects of the Finnish sauna may reflect life-long habits, which usually begin very early in the childhood, as children of only 0.5–1 yr old are gradually introduced to the sauna, meaning that benefits in disease end points may not been obtained by short-term or temporal heat stress activities. Sauna bathing is a tradition embedded in the culture in Finland, and it is accessible basically to everyone. As a result of this, a typical Finn takes sauna baths at least once a week, the average being about twice a week (72). A typical duration of sauna bathing can vary between 5 and 20 min (or even 30 min) and is often intermittent, including short periods in colder environment(s). These are the typical Finnish sauna features that are important to bear in mind with regard to the aims of the present review with its results and discussion.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Bathing,2017
"Cooling, Cold Exposure and Brown Fat","Another tradition that is often combined with sauna bathing is intermittent cooling periods between sauna bathing sessions, sometimes including a hot sauna with a short stay in ice-cold water. In this review, we will therefore also briefly discuss possible health effects of a cold environment with respect to brown fat activation and its potential to combat body adiposity and related cardiovascular and metabolic disorders.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Bathing,2017
Cardiovascular Adjustments to Acute Heat Stress,"As comprehensively discussed previously (23), heat challenges the human body, which is however normally capable of responding to heat to maintain normal internal temperature that is critical for normal internal organs to function. This is obtained by directing the blood, and thus heat, to the skin, where heat is released by sweating allowed by nervous innervation and control of skin blood flow. Skin blood flow and sweating can increase even up to 7–8 l/min and 2–4 l/h, respectively, in response to acute heat stress, particularly when exposure is combined with physical activity (28, 99). This is accompanied by reduced blood pressure and increased heart rate, whereas stroke volume is largely maintained even if blood is moved away from the thorax region (24) to the periphery, which decreases venous return, particularly if a subject is at rest and venous return is not facilitated by muscle pump either. Stroke volume is maintained or is even slightly increased as systolic function and ejection fraction increase during heat stress in healthy subjects (11, 24). During prolonged heat stress, ventilation is also increased, which contributes to decreased brain blood flow in the heat as more carbon dioxide is exhaled (3), a condition that may lead to unconsciousness if this condition becomes too severe.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Heat Stress Physiology,2017
Heat-Induced Muscle and Tissue Blood Flow,"For many years, it was believed that as blood is directed to the periphery, it goes almost solely to skin, and muscle blood flow is not altered. However, it has now been shown that muscle blood flow also increases to some extent in response to local heat stress (44, 90). Increase in blood flow is not actually limited only to skeletal muscle, as bone marrow blood flow is also increased (44). Acute beneficial effects of hot water immersion-induced therapy in peripheral artery disease patients (82, 112) and aging (98) are likely to be mediated by increased heat-induced muscle perfusion in addition to the vasodilation in the skin. Interestingly, leg heating is also able to eliminate the detrimental effects of prolonged sitting (97), the effect that is also most likely mediated by increased skin and muscle blood flow. Increased muscle blood flow by hot water heat stress was also suggested to be the mechanism for the improved glucose balance in type 2 diabetic patients as a result of heat stress intervention (54).","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Heat Stress Physiology,2017
Thermoregulatory and Cardiovascular Adaptations to Heat,"Human thermoregulatory adaptations to heat have recently been comprehensively reviewed (109). It is evident that not only the cardiovascular system but also body metabolism is activated due to heat stress adaptations (see online Supplementary Table 1). The beneficial changes of heat therapy could include some similar target organs and functional adaptations, although not so well characterized, as it is documented to occur in response to long-term exercise training (45). Indeed, comparable with exercise-induced adaptations, repetitive heat stress could improve endothelial function and arterial stiffness and might lower resting blood pressure in sedentary humans (13). Because of strongly increased skin blood flow under heat stress conditions, cutaneous microvascular and conduit artery function and blood flow are also improved by passive, repeated heat therapy (12, 16, 17, 41, 81), although improved cutaneous vascular function by heat stress intervention has not been repeated in all studies (35). Perhaps the strongest adaptation together with enhanced sweating capacity and its sensitivity is, however, lowered core temperature (88) as a response to repetitive heat sessions, which further increases body heat tolerance, as maximal tolerable temperature cannot be increased due to fundamental effects of heat on protein folding. Blood volume, particularly plasma volume, may also increase, which is related to improved heat tolerance.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Heat Stress Adaptations,2017
"Heat Stress, Muscle Perfusion, and Myocardial Adaptations","Although sauna bathing cannot induce a similar increase in muscular perfusion to exercise training (47, 49, 51), increased skeletal muscle blood flow during heat stress (44) is likely closely coupled with increased skeletal muscle gene expression of angiogenic factors due to heat therapy (66). If this response occurs also in cardiac muscle, as has been suggested from some experimental studies focusing on healthy (40), hypertensive (57), and infarcted (106) myocardium, it would be interesting to investigate whether repeated heat stress exposure could lead to favorable exercise training-linked adaptations in the human heart muscle (48, 50) and improve myocardial blood flow reserve and even build up new cardiac collateralization. Heat stress-induced myocardial metabolic adaptations are also largely unexplored to the best of our knowledge, but it can be speculated that repetitive heat stress sessions might lead to comparable adaptations that have been observed in exercise training (32, 48, 50, 53). Heat stress improves skeletal muscle contractility (95), but it remains to be shown whether contractility is improved also in heart.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Heat Stress Adaptations,2017
"Heat Stress, Bone Marrow Perfusion, and Stem Cell Responses","It was found that, in addition to increased muscle blood flow, bone marrow blood flow may also increase in response to heat stress (44). Bone marrow blood flow also increases in response to acute exercise (46), which may be coupled to exercise-induced stem cell release (83). Consequently, exercise-like increases in bone marrow perfusion during heat stress may also stimulate the release of stem cells such as endothelial progenitor cells, which could have a wide range of tissue repair and health effects; to the best of our knowledge, however, this remains to be directly shown experimentally. Although the source of the cells has not been investigated previously, repeated heat stress treatment by sauna bathing has been shown to increase CD34-positive cells in the circulation (86), supporting the idea of heat therapy also as a possible stem cell stimulation therapy. Furthermore, reduced circulating endothelial and platelet microparticles may be contributing to the health benefits of heat exposure (2).","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Heat Stress Adaptations,2017
Finnish Sauna Characteristics and Therapy Context,"Sauna bathing, a form of passive heat therapy, is an activity that is a tradition, especially in Finland, and is commonly used mainly for relaxation and pleasure purposes and is becoming increasingly popular in many other countries and cultures (91, 113). Unique features of Finnish sauna include hot temperature and dry air conditions with good ventilation. Temperature and humidity can be temporarily increased by throwing water on the hot rocks of the sauna heater, which is the heating source of the 80–100°C temperature in a sauna. A typical Finnish sauna differs from far-infrared sauna bathing, which has been tested in Japanese studies using a far infrared-ray dry sauna (111). In these studies with dry sauna (58, 64, 65, 111), patients underwent regular sauna therapy at 60°C for 15 min and were then kept supine on a bed outside the bath room for 30 min with sufficient warmth provided by blankets.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna,2017
Sauna Bathing and Cardiovascular and Mortality Risk,"Previous studies have suggested important positive effects of sauna bathing on cardiovascular health. Indeed, recent observations demonstrate that sauna bathing of only 5–20 min duration a few times per week would be associated with a decreased risk of SCD, cardiovascular and all-cause mortality (72). In a prospective population-based study, it has been reported that sauna bathing is associated with a significantly lowered risk of fatal cardiovascular disease (CVD) events and all-cause mortality (72). The higher frequency of the sauna bathing (4–7 times/wk) was related to a considerably decreased risk of SCD, fatal coronary heart disease (CHD), and CVD events and all-cause mortality independently of conventional risk factors as shown in Fig. 1. Cardiovascular risk reduction was strongest in participants with the highest duration and frequency of sauna bathing, but shorter and less frequent sauna bathing sessions also provided some cardiovascular benefits. Furthermore, a follow-up study of this cohort has also shown that sauna bathing lowers the risk of neurodegerative diseases, dementia, and Alzheimer’s disease (73). The risk reductions also in regard to these brain disorders were substantial; there is a risk reduction of 66% of dementia and a 65% risk reduction of Alzheimer’s disease when sauna bathing four to seven times a week is compared with subjects having only one sauna session per week (73).","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna,2017
Cardiac and Metabolic Responses to Sauna,"Heart rate can increase close to 100 beats per minute during moderate-temperature sauna bathing sessions and even to higher levels (~150 beats/min) during much hotter sauna bathing (43, 67). Heart rate response corresponds to low- and moderate-intensity physical activity. Although there are no active contractions of skeletal muscles during the sauna bathing, sauna bathing may also increase the general metabolic rate (75). This is in contrast to the training response experienced during physical activity, although increased heart rate also increases myocardial oxygen demand similarly to physical exercise, which is likely to account for cardiac-specific health effects, while increased blood flow stimulates arterial health by shear stress and other mechanisms. It has been documented that cardiac output is increased mainly due to the increase in heart rate during sauna bathing. A typical Finnish sauna can positively modulate the autonomic nervous system (heart rate variability), which may contribute to the strong reduction in SCD. In the typical Finnish sauna bath session, skin blood flow increases, which leads to the higher cardiac output, whereas blood flow to internal organs decreases with an increased body temperature (63). Rectal temperature increases by ~1°C at 80°C sauna heat (75), and sweat is secreted at a rate of 0.6 to 1.0 kg/h during a hot sauna bath, with an average total secretion of 0.5 kg during a typical sauna bathing session of 15–30 min duration (116), which has effects in body fluid balance during and after sauna.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna,2017
Overview of Mechanisms Behind Sauna Health Benefits,"It is important to understand the mechanism by which health benefits of sauna bathing are mediated. In addition to being a relaxing lifestyle habit, it remains a potential strategy that could be useful in improving cardiovascular function and subsequently also prevent or delay the development of neurogenerative diseases such as dementia. The possible health-enhancing effects of sauna bathing are likely mainly those adaptations that have already been presented and discussed in the earlier section(s), including improved endothelial and microvascular function, reduced arterial stiffness and blood pressure, and increased angiogenesis in sedentary humans (see online Supplementary Table 1). However, some Finnish sauna-specific adaptations may also occur, which may not be obtained by other passive heat therapies such as warm water (leg) immersion, which has been applied in some studies. This is because of the relatively high (80–100°C) sauna temperatures and dry air (15–20% humidity) during Finnish sauna bathing sessions, but particularly its practicality in normal daily life as a heat stress therapy, by which mortality and neurodegenerative disease risk reductions have been described.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna Mechanisms,2017
"Sauna Effects on Arterial Stiffness, Blood Pressure, and Cardiovascular Function","Heat exposure of the sauna may improve cardiovascular function, which has been previously documented in subjects with CHD risk factors (58, 64), indicating a protective role of heat therapy on arterial stiffness (13, 74). Arterial stiffness can be modified by various underlying vessel-related factors such as elastic fiber degeneration, increased collagen content, and structural changes with vascular smooth muscle cell hypertrophy and hyperplasia (101). In addition to these long-term structural adaptations, improved arterial compliance is acutely improved through changes in hydration status as a result of sweating and thus the loss in plasma volume (14, 36). Consistent with existing evidence based on the effect of heat therapy such as sauna, there are also some findings showing that blood pressure may be decreased because of increased ambient temperature (68, 77, 117), including sauna bathing (75, 118). It has also been shown that even a short dry sauna session favorably modifies heart rate variability in patients with untreated hypertension (37). In patients with slightly elevated blood pressure, a single sauna session produced positive effects on systemic blood pressure as assessed by 24-h blood pressure levels (86), although a reduction in 24-h blood pressure is not a consistent finding (38). It has also been found that sauna exposure is associated with lower levels of cardiac propeptide concentrations with improved left ventricular function (86, 96). Repeated sauna treatment has been shown to improve endothelial function in patients with CHD risk factors and heart failure, suggesting a preventive role of thermal sauna therapy for vascular endothelium (86) as well as for myocardial perfusion in patients suffering from ischemia (105). However, additional well-controlled studies are needed to show whether regular long-term sauna bathing could produce longer-term changes in cardiovascular function.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna Mechanisms,2017
Neurohumoral and Cardiometabolic Mechanisms of Sauna Therapy,"Only a limited number of studies have reported that repeated sauna treatment or thermal therapy is associated with improvement in neurohumoral factors, which are widely used markers of prognosis in patients with CVDs (86, 110). Ohori et al. (86) demonstrated that 3 wk of repeated far-infrared sauna treatment (Waon therapy) in patients with chronic heart failure was associated with lower concentrations of brain natriuretic peptide and plasma norepinephrine. In patients with heart failure who were treated with dry sauna for 2 wk, Kihara et al. demonstrated a significant decrease in concentrations of circulating brain natriuretic peptides, which are associated with better prognosis among heart failure patients (64). The beneficial effects of sauna bathing on cardiometabolic health outcomes have been linked to its impact on circulatory and cardiovascular function. Sauna therapy may improve microvascular and endothelial dysfunction (64), which has been suggested to be involved in the pathophysiology of outcomes such as hypertension, CVDs, and diabetes (62, 89, 107). Sauna bathing has been shown to produce systemic blood pressure-lowering effects, increase cardiac output via an increase in heart rate, and decrease peripheral vascular resistance. Sauna therapy may exert its effects via changes in levels of blood-based cardiovascular biomarkers such as markers of glucose metabolism and insulin resistance, natriuretic peptides, cardiac troponin T, and inflammatory markers such as interleukins and C-reactive protein, although data on this topic are still very limited and further studies are warranted to elucidate these potential mechanisms.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna Mechanisms,2017
"Lipid Profile, Immunity, Oxidative Stress, and Antioxidant Adaptations","However, it has been documented in both men and women that Finnish sauna has positive effects on cholesterol and lipid levels, including total cholesterol and LDL and HDL (42, 93) and transiently also triglycerides (42). There is also some evidence that Finnish sauna can boost the immune system (92), which may partly explain why hot-cold bathing reduces susceptibility to colds and prevents infections in healthy subjects (31). Furthermore, although one sauna bath session may acutely increase oxidative stress, sauna bathing may also reduce exercise-induced oxidative stress and lead to improved antioxidant capacity after repeated sauna heat exposures (94, 108). This is plausible, as antioxidative enzymes can be boosted by heat shock proteins, which will be increased as a response to heat stress and their changes correlate with heat acclimatization (79).","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna Mechanisms,2017
"Brain Perfusion, Neurodegeneration, and Tissue-Specific Sauna Effects","With regard to tissue-specific effects of sauna bathing, is it evident that improvements in many of the “general” cardiovascular risk factors, which are underlying factors for both cardiac and neurodegerative disorders, such as endothelial function, arterial stiffness, blood pressure, and lipid profile, are largely responsible also for these organ-specific influences. However, sauna-induced improvements in cardiovascular function may also directly translate to improvements in brain function due to important interactions between these organs (60, 61). Furthermore, it is also likely that shear stress-induced mechanisms are one of those factors that lead to improvements in brain perfusion and brain arterial health (15). Regarding changes in brain perfusion and prevention of neurodegerative diseases by sauna bathing, it is somewhat paradoxical that heat stress is consistently shown to decrease brain blood flow acutely (3). However, to the best of our knowledge these studies are almost solely based on transcradial Doppler measurements, which can derive only blood flow velocity in one artery. Since even minimal changes in arterial diameter, which could be modified during heat stress, can lead to fairly large changes in total brain blood flow, it remains to be determined whether total blood flow is altered during heat stress and as an adaptation to long-term sauna bathing. In this regard, it is important to note that repeated warm water immersion can induce similar cerebrovascular adaptations to exercise training with moderate exercise intensity (1). In addition to total blood flow, it would also be important to determine regional distribution of brain perfusion in these experiments (34). These investigations have been performed at the level or large arteries (4, 85) but, to the best of our knowledge, not at the brain tissue perfusion level. It is also possible that there are direct influences of angiogenesis and neurogenesis in the brain (55, 102) that also contribute to explain the preventive effects of neurodegerative diseases due to Finnish sauna bathing.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna Mechanisms,2017
Respiratory and Musculoskeletal Effects of Sauna,"Finally, sauna bathing may improve symptoms of lung disease (22, 71) and has also been used in treating musculoskeletal pain (59, 84). It is suggested that sauna bathing increases the vital capacity, minute ventilation, and forced expiratory volume of the lungs (71), as heat stress increases minute ventilation leading to these favorable effects in regard to lung function. These effects of sauna on lung function may explain recent findings indicating that Finnish sauna bathing is associated with a decreased risk of respiratory diseases (70).","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Finnish Sauna Mechanisms,2017
General Safety and Cardiovascular Risks of Sauna Bathing,"Most people in general can tolerate a typical warm dry Finnish sauna, which is a pleasurable activity with an additional health benefit (100). It is, however, also well known that survival and heat stroke severity are related to the severity and duration of hyperthermia (56). There are therefore some concerns that sauna bathing might have some risks, particularly in patients with particular disease conditions; the very hot sauna exposure could be potentially harmful (67). Particularly. persons with unstable coronary artery disease or ischemic heart failure should be cautious with sauna bathing, as it might cause myocardial ischemia in patients with reversible exercise-induced ischemia, although it is still relatively less than that induced by exercise (39). Furthermore, persons who are prone to orthostatic hypotension or severe valvular disease should also be cautious of hot sauna bathing because of a possible decrease in blood pressure, which typically occurs just after a hot sauna bath during to the cooling down period (17, 44). However, dry common sauna bathing seems to be safe. Even patients who have recovered from myocardial infarction and patients with stable angina pectoris or heart failure can enjoy sauna bathing without any significant adverse cardiovascular effects (63), as also shown with heart failure patients who can gain substantial health benefits even if heat therapy has been started fairly late in life (65). However, it is likely that those who are not able to exercise even at a low-intensity level should be advised to apply sauna therapy cautiously.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Risks,2017
Far-Infrared Sauna Therapy and Heart Failure Outcomes,"Far infrared sauna intervention studies have been conducted in Japan to use sauna bathing as one component of the treatment of congestive heart failure. Cardiac events due to heart failure or cardiac death occurred in 68.7% of the control group but only 31.3% of the Waon therapy group after 60 mo of follow-up (65). In that study, hospitalization due to heart failure occurred in 20 patients in the sauna group and 44 patients in the control group, suggesting the beneficial effects of far infrared saunas among patient with heart failure.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Risks,2017
Limitations of Epidemiological Evidence on Sauna Habits,"In epidemiological studies, there is a lack of repeat and regular evaluations of sauna bathing habits that enable us to confirm the long-term health effects of sauna. Epidemiological evidence relies only on a questionnaire-based baseline assessment of sauna bathing habits during everyday life. These studies have used an exposure of sauna habits during a typical weekly sauna sessions (69, 70, 72, 73, 118). Second, it is possible that sauna bathing habits have changed during long-term follow-up due to possible changes in lifestyle habits or other diseases of participants occurring over a long period of time, which could have introduced some biases in the epidemiological studies. However, this is unlikely, given that sauna bathing is a tradition embedded in the culture in Finland, and even subjects with cardiovascular and metabolic diseases such as diabetes take sauna baths regularly. Given that sauna bathing is a commonly used relaxation habit in the Finnish population, it is anticipated that the correlation between measured sauna habits taken several years apart is high, and therefore analysis using baseline assessments is unlikely to considerably underestimate the associations.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Risks,2017
"Demographics, Socioeconomic Status, and Independent Benefits of Sauna","Even though socioeconomic status (SES) has been shown to influence future risk of CVD and may be associated with access to sauna bathing, previous studies have shown that adjustment for SES does not materially change the association of sauna bathing with outcomes (69, 70, 72, 73, 118) after an additional adjustment for a wide range of CVD risk factors and possible confounders, including a comprehensive panel of lifestyle and clinical factors. Thus, the associations persisted significant for SCD, fatal CHD and CVD events, suggesting the additional and independent health benefits of sauna bathing when SES was also taken into consideration. Indeed, strengths of the published follow-up studies on sauna habits and health-related outcomes (42, 43) include the rigorous measurement of other risk factors, the large community-based study sample (2,315 participants), and the long-term of follow-up with data on major health outcomes. Based on the available evidence, sauna bathing can be considered a recommended habit as part of a healthy lifestyle for the prevention strategies of CVDs.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Risks,2017
Cold Exposure Practices and Health Considerations,"In addition to heat stress induced by sauna bathing, intermittent heat and cold exposures are a quite common practice, for instance in Finland and other Nordic countries, when sauna bathing. They are normally practiced by going outside in a cold environment between sauna sessions. Other common ways are to sit or roll in snow or simply take a cold shower for several minutes. In an extreme form, they can also be practiced by dipping in ice-cold water three to five times from five seconds to a few minutes between sauna bathing sessions of 5–15 min, but it is currently not well known whether this kind of extreme activity promotes positive health effects even better than a single hot sauna bathing session alone. In addition to the effects of heat exposure on the human body and the common utilization of cold exposure between sauna bathing sessions, it is thus also important to consider the possible effects of cold temperatures in health. As with acute exercise, heat stress, and hypoxia, cold stress also challenges physiological systems and may be detrimental to human health acutely (18). When entering a cool environment, the human body will adjust to maintain heat balance. Heat balance can be restored by reducing heat loss and/or increasing heat production. Heat loss is minimized by cutaneous vasoconstriction, induced by reflex and local mechanisms (19), which decrease heat transfer from the core to the skin and other distal parts of the body. On the other hand, heat production can be increased by nonshivering and shivering thermogenesis.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Cold Stress,2017
"Cold Thermogenesis, Energy Expenditure, and Adaptation","Shivering especially is a very efficient way to produce heat. Although not commonly practiced as a part of body weight management, its efficiency in energy production and consumption is highlighted by the fact that peak shivering intensity can increase heat production close to five times over basal metabolic rate (33). The physiological and health effects of cold exposures such as winter swimming in ice-cold water have been investigated in many studies (30, 76, 103, 104). They indicate that they are generally not harmful for health when, for instance, antioxidant capacity or hormonal function is considered (30, 76, 103), but they can lead to reduction in skinfold thickness (104), meaning that cold exposures can be beneficial in reducing body adiposity. They are also well tolerated as thermal sensation and comfort is increased after first exposure to cold, meaning that subjects become habituated to cold (104). Numerous hormonal and nervous system adaptations contribute to this adaptation to cold (21, 27, 78). From the cardiovascular health point of view, one of these mechanisms can be called cold-induced vasodilation, which means that, after initial exposure to cold and resulting vasoconstriction in limbs, a period of vasodilation is observed that enables the return of warm blood to the fingers and other distal body parts (20, 21, 26). It is known that this periodic cold-induced vasodilation reflects a vasodilation in both muscle and cutaneous vasculature (29).","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Cold Stress,2017
Vascular Shear Stress and Temperature Variation Effects,"It is likely to stimulate shear stress-mediated improvements in vascular function leading to better health of the peripheral circulation. It is also likely that repeated extreme temperature variations such as sauna bathing and ice-cold swimming are particularly strong mediators of this effect. When an individual returns to the sauna after a swim in ice-cold water, this effect is evidenced by a feeling of swollen hands and legs together with strong pulsation sensations as blood flow is strongly stimulated by these extreme variations in temperature. Furthermore, with respect to cold stress it is important to consider that two types of adipose tissue exist in humans, white and brown adipose tissue. White adipose tissue is distributed mainly subcutaneously throughout the body, and in most of the subjects, it has the capacity to expand substantially when energy intake has been excessive and consumption minimal. In contrast to white adipose tissue, brown adipose tissue is localized only in special small depots, mostly in the neck area, and is activated by cold exposure (115). In contrast to white fat, which stores fat, brown fat mainly burns energy, which is released as heat. Brown fat is activated by cold, and colder environment relates to higher cold fat activation and lower body weight (25, 114).","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Cold Stress,2017
"Brown Fat Activation, Metabolic Effects, and Limitations","Although skeletal muscles are also very important contributors (8), brown adipose tissue plays a role in energy expenditure in response to acute cold exposure (87). Brown adipose tissue oxidative capacity and activity increase in response to repeated cold exposure (5, 9), leading to changes in lipid metabolism (6, 10). These effects on metabolism may contribute to human health, although glucose metabolism also plays a role, as that has been shown to be impaired in brown adipose tissue of type 2 diabetic patients (7). Therefore, as activation of brown fat may prevent body adiposity and related metabolic and cardiovascular disorders, repeated cold exposure may also be beneficial for health. However, in this respect it is noteworthy that as actual brown fat depots are only a few grams, browning of white fat, and that to a substantial extent, would be needed to show physiologically relevant effects on whole body metabolism, as an investigation in humans has shown that purely brown fat thermogenesis can only account for energy consumption of less than 20 kcal/day (80). This amount of energy consumption can be obtained by doing moderate-intensity physical activity, such as brisk walking or moderate-intensity running for only 2 min (80). This emphasizes the importance and potential of physical activity in the prevention and treatment of excessive body adiposity and related cardiometabolic disorders, although activation of brown fat by cold could certainly be applied as an adjunct therapy.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Cold Stress,2017
Combined Heat–Cold Exposure and Future Research Needs,"Moreover, the combined and possibly additive effects of alternating heat and cold exposures, such as sauna plus cold water immersion, on human health are currently not well known but should be investigated in the future.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Cold Stress,2017
"Conclusions on Heat, Cold, and Sauna Exposure","In conclusion, although acute warm and hot temperatures are stressful for humans, the human body also adapts physiologically to repetitive heat sessions, leading to improved heat tolerance and ultimately also decreased mortality and reduced risks for brain disorders such as dementia and Alzheimer’s disease (Fig. 2). Therefore, similarly to hypoxia or altitude (52), heat or cold stress could also be applied as an independent or adjunct therapy to exercise and physical activity to maintain or improve human health, particularly among those people who do not want to or cannot exercise. It seems that the best benefits in cardiovascular and all-cause mortality will, however, be obtained by increasing physical fitness combined with sauna bathing (69), highlighting the fact that several lifestyle habits affect human health.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Health Conclusions,2017
Perspectives and Significance of Sauna Bathing,"Although environmental stressors such as extremely warm or cold temperature are often considered a challenge to human health and body homeostasis, the human body can adapt relatively well to heat and cold environments. Furthermore, recent studies have elucidated that heat stress in particular might even be highly beneficial for human health. Indeed, heat stress particularly in the form of Finnish sauna bathing has been shown to improve general and cardiovascular health and mortality as well as preventing against dementia and Alzheimer's disease risks. Therefore, sauna bathing or other heat stress activities are recommended habits for the improvement of population health.","American Journal of Physiology-Regulatory, Integrative and Comparative Physiology",Sauna Health Conclusions,2017
All-Cause Mortality Study Overview,"All-Cause Mortality Events
Tanjaniina Laukkanen, MSc1; Hassan Khan, MD, PhD2; Francesco Zaccardi, MD3 et al
JAMA Intern Med
Published Online: April 2015
2015;175;(4):542-548. doi:10.1001/jamainternmed.2014.8187

Abstract
Importance  Sauna bathing is a health habit associated with better hemodynamic function; however, the association of sauna bathing with cardiovascular and all-cause mortality is not known.

Objective  To investigate the association of frequency and duration of sauna bathing with the risk of sudden cardiac death (SCD), fatal coronary heart disease (CHD), fatal cardiovascular disease (CVD), and all-cause mortality.

Design, Setting, and Participants  We performed a prospective cohort study (Finnish Kuopio Ischemic Heart Disease Risk Factor Study) of a population-based sample of 2315 middle-aged (age range, 42-60 years) men from Eastern Finland. Baseline examinations were conducted from March 1, 1984, through December 31, 1989.

Exposures  Frequency and duration of sauna bathing assessed at baseline.",JAMA Internal Medicine,Sauna Bathing Mortality,2015
Mortality Outcomes and Sauna Frequency,"Results  During a median follow-up of 20.7 years (interquartile range, 18.1-22.6 years), 190 SCDs, 281 fatal CHDs, 407 fatal CVDs, and 929 all-cause mortality events occurred. A total of 601, 1513, and 201 participants reported having a sauna bathing session 1 time per week, 2 to 3 times per week, and 4 to 7 times per week, respectively. The numbers (percentages) of SCDs were 61 (10.1%), 119 (7.8%), and 10 (5.0%) in the 3 groups of the frequency of sauna bathing. The respective numbers were 89 (14.9%), 175 (11.5%), and 17 (8.5%) for fatal CHDs; 134 (22.3%), 249 (16.4%), and 24 (12.0%) for fatal CVDs; and 295 (49.1%), 572 (37.8%), and 62 (30.8%) for all-cause mortality events.

After adjustment for CVD risk factors, compared with men with 1 sauna bathing session per week, the hazard ratio of SCD was 0.78 (95% CI, 0.57-1.07) for 2 to 3 sauna bathing sessions per week and 0.37 (95% CI, 0.18-0.75) for 4 to 7 sauna bathing sessions per week (P for trend = .005). Similar associations were found with CHD, CVD, and all-cause mortality (P for trend ≤ .005).",JAMA Internal Medicine,Sauna Bathing Mortality,2015
Effects of Sauna Duration and Study Conclusions,"Compared with men having a sauna bathing session of less than 11 minutes, the adjusted hazard ratio for SCD was 0.93 (95% CI, 0.67-1.28) for sauna bathing sessions of 11 to 19 minutes and 0.48 (95% CI, 0.31-0.75) for sessions lasting more than 19 minutes (P for trend = .002). Significant inverse associations were also observed for fatal CHDs and fatal CVDs (P for trend ≤ .03) but not for all-cause mortality events.

Conclusions and Relevance  Increased frequency of sauna bathing is associated with a reduced risk of SCD, CHD, CVD, and all-cause mortality. Further studies are warranted to establish the potential mechanism that links sauna bathing and cardiovascular health.

Introduction
Most sudden cardiac deaths (SCDs) occur in the general population, and most SCDs occur outside the hospital with few or no early warning signs. Therefore, ascertainment of lifestyle characteristics that could be protective against SCD is important.",JAMA Internal Medicine,Sauna Bathing Mortality,2015
Rationale and Study Aim,"Although some studies have found sauna bathing to be associated with a better cardiovascular and circulatory function, the association between regular sauna bathing and the risk of SCD and fatal cardiovascular diseases (CVDs) is not known. Long-term sauna bathing has been associated with lower blood pressure and enhanced left ventricular function and thus potentially with reduced CVD risk. Sauna bathing leads to skin sweating–induced fluid loss and increase in heart rate, which are physiologic responses to warm temperature.

Previous studies have found some positive effects of thermal exposure on CVD risk factors; however, the long-term effects of sauna bathing on risk of cardiovascular events, including the association between the frequency and duration of sauna bathing and the risk of SCD, are not well defined. The aim of this prospective study was to investigate the association between exposure to sauna bathing and the risk of SCD, fatal coronary heart disease (CHD), fatal CVD, and all-cause mortality events in the general male population.",JAMA Internal Medicine,Sauna Bathing Mortality,2015
Study Population and Design,"Methods
Study Population
The study was approved by the Research Ethics Committee of the University of Eastern Finland, Kuopio. Each participant gave written informed consent. This study (Finnish Kuopio Ischemic Heart Disease Risk Factor Study) was designed to investigate risk predictors for atherosclerotic cardiovascular outcomes in a population-based sample of men from Eastern Finland. Participants were a randomly selected sample of 3433 men aged 42 to 60 years who resided in Kuopio, Finland, or its surrounding rural communities. Of those invited, 2682 (78.1%) participated in the study, and those with complete information on sauna bathing were included (N = 2327). Twelve men who did not use a sauna were excluded, leaving 2315 participants for the analyses. Baseline examinations were conducted from March 1, 1984, through December 31, 1989.",JAMA Internal Medicine,Sauna Bathing Mortality,2015
Sauna Exposure and Risk Factor Assessment,"Assessment of Sauna Bathing
A traditional Finnish sauna has dry air (humidity 10%-20%) with a relatively high temperature. The recommended temperature for a sauna is usually 80°C to 100°C at the level of the bather’s face. Humidity is temporarily increased by throwing water on the hot rocks of the sauna heater. Sauna bathing was assessed by a self-administrated questionnaire based on weekly sauna bathing sessions, duration, and temperature. The assessment represents typical sauna use during the week. The temperature in the sauna room was measured using a thermometer in the sauna and self-reported.

Assessment of Risk Factors
Risk factors were assessed at baseline. The lifelong effect of smoking (cigarette pack-years) was estimated. Resting blood pressure was measured between 8 and 10 am with a random-zero sphygmomanometer. Cholesterol fractions and triglycerides were measured enzymatically. Serum high-density lipoprotein cholesterol was separated using ultracentrifugation. C-reactive protein was measured with an immunometric assay.",JAMA Internal Medicine,Sauna Bathing Mortality,2015
Outcome Classification Procedures,"Classification of Outcomes
All deaths that occurred by the end of 2011 were checked against hospital documents and death certificates. There were no losses to follow-up. Sources of information were interviews, hospital documents, autopsy reports, and medicolegal reports. A death was determined to be an SCD when it occurred within 1 hour of symptom onset or within 24 hours when no noncardiac cause was found. Deaths due to aortic aneurysm rupture, cardiac rupture, pulmonary embolism, cancer, or noncardiac comorbidities were excluded as SCDs. CHD and CVD deaths were coded using ICD-9 and ICD-10 codes. Documents related to the death were cross-checked by 2 physicians. The Independent Events Committee, masked to clinical data, performed classification of deaths.",JAMA Internal Medicine,Sauna Bathing Mortality,2015
Statistical Methods and Hazard Modeling,"Statistical Analysis
Differences in baseline characteristics were examined using analysis of variance and the χ2 test. Risk factors for outcomes were analyzed using multivariable Cox models. Men were divided into groups on the basis of sauna frequency (1, 2-3, 4-7 times/week) and duration (<11, 11-19, >19 minutes). Cox models were adjusted for age, BMI, systolic blood pressure, LDL cholesterol, smoking, alcohol consumption, previous myocardial infarction, type 2 diabetes, cardiorespiratory fitness, resting heart rate, physical activity, and socioeconomic status. Cumulative survival from SCD was calculated using Kaplan-Meier curves. Sensitivity analyses excluded participants who died within 5 years and restricted follow-up to 10 years. Hazard ratios were estimated as antilogarithms of coefficients. Proportional hazards assumptions were checked using Schoenfeld residuals.",JAMA Internal Medicine,Sauna Bathing Mortality,2015
Characteristics of Finnish Sauna Bathing,"SAUNA BATHING
Sauna bathing is a form of passive heat therapy that is characterized by exposure to high environmental temperature for a brief period. The typical Finnish sauna is characterized by dry air and relatively high temperature. Temperature and humidity can be temporarily increased by throwing water on the hot rocks of the sauna heater, which is the heating source with temperature settings from 80°C to 100°C in sauna. The sauna is usually made of log or wood with wooden benches well above the floor for bathers to sit on. The recommended temperature for a sauna bath is from 80°C to 100°C at the level of the bather’s head, but it is lower at the floor level, which ensures efficient ventilation and makes sure the conditions are comfortable for sauna bathers. The relative humidity of sauna usually varies from 10% to 20%. Typical sauna sessions consist of short stays in the sauna room, which is interspersed with cooling-off periods (swim, shower, or a cooling-off period at room temperature). The duration of stay in the sauna room depends on the comfort and temperature of the sauna bather, but it usually ranges from 5 to 20 minutes, although longer sauna bathing sessions may be used depending on the individual. Sauna bathing habits may have changed over time, but still a typical Finnish person has a sauna bath at least once per week, with the average habitual frequency being 2 to 3 times/wk.",Mayo Clinic Proceedings,Sauna Bathing,2018
Physiological Responses During Sauna,"During a sauna session, the heart rate may increase from baseline up to 120 to 150 beats/min. There is no active function of skeletal muscles during sauna bathing, which is in contrast to the training response experienced during physical activity. A part of blood volume is diverted from the internal organs to body peripheral parts with decreasing venous return, which is not facilitated by active skeletal muscle work. However, it has been proposed that muscle blood flow may increase, at least to some extent, in response to heat stress.
SAUNA BATHING AND VASCULAR OUTCOMES
Blood Pressure or Hypertension
Evidence from a number of experimental and epidemiological studies implicates sauna bathing to have a positive effect on blood pressure (BP) modulation. However, it appears that most of these reports were conducted in patients with preexisting vascular disease and/or evaluated only the short-term effects of sauna exposure on BP. Two recent experimental studies by Lee et al and Laukkanen et al in 100 men and women (56% men; age, 32–75 years) with at least 1 cardiovascular risk factor reported reductions in both systolic BP and diastolic BP after 30-minute sauna bathing sessions. In addition to reductions in BP, sauna bathing led to positive alterations in measures of arterial stiffness such as pulse wave velocity.",Mayo Clinic Proceedings,Sauna Bathing,2018
Effects of Sauna on Arterial Stiffness and Hypertension Risk,"The mean carotid-femoral pulse wave velocity was 9.8±2.4 m/s before sauna and decreased to 8.6±1.6 m/s immediately after sauna (P<.0001). The mean systolic BP decreased after sauna exposure from 137±16 to 130±14 mm Hg (P<.0001) and diastolic BP from 82±10 to 75±9 mm Hg (P<.0001). Systolic BP after 30-minute recovery remained lower than presauna levels. Gayda et al studied the effects of sauna alone as an intervention vs the combination of exercise and sauna on ambulatory BP monitoring and central hemodynamic variables in 16 patients with slightly elevated BP. There were 8 prehypertensive patients (systolic BP, 120–139 mm Hg) and 8 stage I hypertensive patients (systolic BP, 140–159 mm Hg). A single sauna session produced positive effects on systemic BP assessed using 24-hour BP recordings. On the basis of this relatively small sauna intervention study, Gayda et al proposed that both exercise and sauna were important nonpharmacological strategies to reduce systolic and mean BP in patients with untreated hypertension.
There is limited evidence on the long-term effects of habitual sauna bathing on BP or the risk of hypertension. In the only long-term prospective cohort study by Zaccardi et al in 1621 men (age, 42–60 years), white men who took frequent sauna baths (4–7 sessions/wk) had an approximately 47% reduced risk of developing hypertension when followed for 24.7 years.",Mayo Clinic Proceedings,Sauna Bathing,2018
Sauna Bathing and Neurocognitive Disease,"Neurocognitive Disease
The etiology of neurocognitive disease is multifactorial, with impaired cardiovascular function, inflammation, and oxidative stress postulated as being major contributors in its pathogenesis in addition to high systemic BP with elevated levels of common cardiovascular risk factors. Emerging recent evidence suggests that sauna exposure may have protective effects on neurocognitive disease. In a population-based prospective cohort study by Laukkanen et al conducted in 2315 apparently healthy Finnish men aged 42 to 60 years at baseline, men who had 4 to 7 sauna sessions/wk compared with those who had 1 sauna session/wk had a 66% and 65% reduced risk of dementia and Alzheimer disease, respectively. Whether sauna exposure exerts its neurocognitive protective effects via mediation in the pathways contributing to these diseases or it is just an enjoyable activity that prevents or delays the development of these memory diseases is not clearly understood.",Mayo Clinic Proceedings,Sauna Bathing,2018
Sauna Bathing and Pulmonary Disease,"SAUNA BATHING AND NONVASCULAR DISEASES
Pulmonary Disease
In addition to the potential beneficial effects of sauna bathing on several vascular outcomes, sauna bathing has been suggested to have beneficial effects on some nonvascular conditions. Evidence suggests that sauna bathing improves lung function by improving vital capacity and volume, ventilation, and forced expiratory volume. Cox et al studied the influence of sauna on pulmonary function in 12 male participants with obstructive pulmonary disease and concluded that sauna caused transient improvement in lung function in these patients, whereas Laitinen et al in a review evaluated previous research on the topic, finding sauna exposure to improve breathing in patients with asthma or chronic bronchitis. Ernst et al in a trial of 25 volunteers who were exposed to sauna and 25 controls, sauna bathing was observed to halve the incidence of common colds in the sauna group during the last 3 months of the study period. In the first prospective evaluation of the long-term effect of sauna bathing on the risk of pulmonary disease, Kunutsor et al found moderate (2–3 sessions/wk) to high (4–7 sessions/wk) frequency sauna bathing to be associated with a reduced risk of respiratory diseases (defined as chronic obstructive pulmonary disease, asthma, or pneumonia). In a separate analysis limited to pneumonia cases, having regular sauna baths was also associated with a reduced risk of pneumonia.",Mayo Clinic Proceedings,Sauna Bathing,2018
Other Health Benefits of Sauna Bathing,"SAUNA BATHING AND OTHER HEALTH BENEFITS
Sauna bathing has been linked to an improvement in pain and symptoms associated with musculoskeletal disorders such as osteoarthritis, rheumatoid arthritis, and fibromyalgia. Having sauna baths also improves headache disorders. In an RCT by Kanji et al, 37 people with chronic tension-type headache were randomized to regular sauna bathing or advice and education for a period of 8 weeks, and sauna therapy was found to substantially improve headache intensity. Although there is some evidence from a Japanese study that thermal therapy improved the symptoms of patients with mild depression, to our knowledge, no study has as yet reported the effects of Finnish sauna bathing on depression. However, we have recently shown that men who had 4 to 7 sauna sessions/wk had a 78% reduced risk of developing psychosis in the future as compared with men who had only 1 sauna session/wk. Although there is no robust evidence to suggest that sauna bathing can be used to treat or prevent skin disease, a study has suggested that sauna bathing may be of benefit to patients with psoriasis, as it facilitates the removal of hyperkeratotic scales. Indeed, a previous study has suggested a protective effect of regular Finnish sauna on skin physiology, as evidenced by stability of the epidermal barrier function, increase in hydration of the stratum corneum, and faster recovery of both elevated water loss and skin pH. Regular sauna baths have also been reported to be associated with better health-related quality of life, including improved physical function, vitality, social functioning, and general health.",Mayo Clinic Proceedings,Sauna Bathing,2018
Mechanistic Pathways in Sauna Health Benefits,"PATHWAYS IMPLICATED IN HEALTH BENEFITS OF SAUNA BATHING
Traditionally, sauna baths have been used for the purposes of pleasure and relaxation, which evidently reduce the stresses of everyday life. In addition, several mechanistic pathways have been proposed to underlie the effects of sauna bathing on vascular and nonvascular disease conditions. Evidence suggests that the responses produced by an ordinary sauna bath correspond to those produced by moderate- or high-intensity physical activity such as walking. Pathways implicated in the effects of sauna bathing on vascular disease and mortality risk include reduction in systemic BP; improvement in endothelial function; reduction in oxidative stress and inflammation; beneficial modulation of the autonomic nervous system; positive alteration in levels of circulating vascular risk factors such as natriuretic peptides and lipids; hormonal changes; improved arterial stiffness, arterial compliance, and intima media thickness; and improvement in the cardiorespiratory system as well as cardiovascular function. These pathways are involved in the pathophysiology of chronic disease outcomes such as type 2 diabetes and CVD as well as mortality.",Mayo Clinic Proceedings,Sauna Bathing,2018
Study Overview and Background,"Metabolism and Aging: Effects of Cold Exposure on Metabolic Rate, Body Composition, and Longevity in Mice
Physiological and Biochemical Zoology 82(4):314–324. 2009.
Lobke M. Vaanholt, Serge Daan, Kristin A. Schubert, G. Henk Visser
ABSTRACT
The proposition that increased energy expenditure shortens life has a long history. The rate-of-living theory (Pearl 1928) states that life span and average mass-specific metabolic rate are inversely proportional. Originally based on interspecific allometric comparisons between species of mammals, the theory was later rejected on the basis of comparisons between taxa (e.g., birds have higher metabolic rates than mammals of the same size and yet live longer). It has rarely been experimentally tested within species. Here, we investigated the effects of increased energy expenditure, induced by cold exposure, on longevity in mice. Longevity was measured in groups of 60 male mice maintained at either 22C (WW) or 10C (CC) throughout adult life. Forty additional mice were maintained at both of these temperatures to determine metabolic rate (by stable isotope turnover, gas exchange, and food intake) as well as the mass of body and organs of subsets of animals at four different ages. Because energy expenditure might affect longevity by either accumulating damage or by instantaneously affecting mortality rate, we included a third group of mice exposed to 10C early in life and to 22C afterward (CW). Exposure to cold increased mean daily energy expenditure by ca. 48% (from 47.8 kJ d–1 in WW to 70.6 kJ d–1 in CC mice, with CW intermediate at 59.9 kJ d–1).",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
"Cold Exposure, Energy Expenditure, and Lifespan Results","However, we observed no significant differences in median life span among the groups (WW, 832 d; CC, 834 d; CW, 751 d). CC mice had reduced body mass (lifetime mean 30.7 g) compared with WW mice (33.8 g), and hence their lifetime energy potential (LEP) per gram whole-body mass had an even larger excess than per individual. Greenberg (1999) has pointed out that the size of the energetically costly organs, rather than that of the whole body, may be relevant for the rate-of-living idea. We therefore expressed LEP also in terms of energy expenditure per gram dry lean mass or per gram “metabolic” organ mass (i.e., heart, liver, kidneys, and brain). No matter how it was expressed, LEP in CC mice significantly exceeded that of WW mice. This result demonstrates that increased energy expenditure does not shorten life span and adds evidence to the intraspecific refutation of the rate-of-living theory. We suggest that increased energy expenditure has both positive and negative effects on different factors determining life span and that the relationship between energy turnover and longevity is fundamentally nonmonotonic.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Introduction: Rate-of-Living and Free-Radical Theories,"Introduction
Since the beginning of the twentieth century, there has been much debate about the influence of energy expenditure on life span. The rate-of-living theory proposed in 1928 by Pearl states that there is a negative relationship between energy expenditure and life span. The mechanism linking metabolic rate and aging may be the inevitable production of free radicals during oxygen consumption as proposed by the so-called free-radical theory of aging postulated half a century ago by Harman (Harman 1956; Beckman and Ames 1998). Free radicals (or reactive oxygen species [ROS]) can cause damage to macromolecules, which could eventually result in cell death. Whereas the free-radical theory has gained much support in recent years, the rate-of-living theory has been discounted by many researchers based on interspecific comparisons and the lack of effects on lifetime energy expenditure in calorically restricted animals. Interspecific comparisons within the subgroups of birds and mammals do show that larger animals, with lower rates of energy expenditure (per gram body mass), generally live longer. This association does not hold across taxa. Birds spend up to three times more energy per day and yet live longer than mammals with similar body mass.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Challenges in Testing Rate-of-Living Theory,"Marsupials should live longer than eutherian mammals based on their metabolic rates, but they do not. Such counterexamples do not preclude the possibility that for the individual animal, increased energy expenditure shortens life. This relationship cannot be studied by interspecific comparisons and therefore requires experimental studies. Experiments addressing the effect of energy expenditure on mortality have manipulated workload (honeybees; Wolf and Schmid-Hempel 1989) and family size (kestrels; Daan et al. 1996) in the field. In laboratory conditions, caloric restriction (CR) has long been established as a manipulation that increases mean and maximum life span in rodents. Sacher (1977) proposed that CR extends life span by decreasing metabolic rate. However, most studies investigating these effects have not shown significant reductions in lifetime energy expenditure in CR animals. Interpretation is confounded by metabolic rate typically being expressed per gram body or lean mass, whereas the relative sizes of metabolically active organs differ between CR and ad libitum–fed animals. Speakman has pointed out multiple challenges in testing the rate-of-living theory, including inaccurate lifespan metrics, scaling errors, single-time metabolic rate measurement, and heterogeneous organ energetics.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Materials and Methods: Animal Groups and Housing,"Material and Methods
Animals and Housing
Male C57BL/6JOlaHsd mice were obtained from Harlan Netherlands (Horst) at 4 wk of age. Animals were housed in groups of three at 22C during the second month of life to keep developmental conditions equal between the groups. At 2 mo of age, when animals had reached adulthood and sexual maturity, they were caged individually and housed at different ambient temperatures. At this time, all mice were randomly allocated to three experimental groups of 100 mice each. Group WW was housed at 22C (warm) throughout adult life. Group CC was housed at 10C (cold) from the age of 2 mo onward. The third group (CW) was housed at 10C from age 2 mo until 15 mo and at 22C from age 15 mo onward. Animals were housed in Macrolon type II cages with Hemparade as bedding and EnviroDry as nesting material. Mice had ad lib. food (RMH-B chow) and water and were on a 12L:12D cycle throughout their lives. Body mass was measured monthly, and cages were cleaned every 2 weeks.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Experimental Design and DLW Metabolic Rate Measurement,"Each group of 100 mice was split into two subgroups of 60 and 40. The 60-animal subset (lifespan group) was undisturbed except for cage cleaning and monthly weight; spontaneous death time was recorded. The remaining 40 mice (test subgroup) were sampled at 3, 11, 19, and 27 months for food intake and metabolic rate measurements. Food intake (g/day) was measured over 3 days, correcting for evaporation. Daily energy expenditure (DEE) was determined by doubly labeled water (DLW) technique. Mice were injected with ~0.1g of DLW (2H/18O), equilibrated for 1h, blood sampled from the tail, and final samples taken after 48h. Background isotope levels were also measured. 18O/16O was analyzed via CO2 equilibration, and 2H/1H from H2 gas produced over uranium. rCO2 was calculated with Speakman’s formula using body water pool size and isotope turnover. Energy expenditure was derived assuming a respiratory quotient of 0.7 and an energetic equivalent of 22 kJ/L CO2.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Respirometry and Tissue Analysis Procedures,"Three days after DLW, the same mice were placed in an eight-channel open-flow respirometry system to measure CO2 production and O2 consumption at 22C and then 10C. Measurements occurred every 10 minutes over 24h. RMR was defined as the lowest running mean of energy output during inactivity. MR was calculated as: MR = 16.18 × VO2 – 5.02 × VCO2. After respirometry, mice were euthanized with CO2 and decapitated. Blood was collected, centrifuged, and plasma stored for later hormone assays. Organs (heart, liver, kidney, brain, muscles, adipose tissues, etc.) were dissected and weighed. Dry mass was determined by oven-drying and fat was extracted with soxhlet and petroleum ether. Lean mass of total organs was inferred from subsamples. Standardized water and fat content for tissues was assumed based on 6 reference animals.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Survival Outcomes and Mortality Modeling,"Results
Survival
As shown in Figure 1A, survival curves had similar shapes for the three experimental groups. Both mean and median age at death were virtually identical for mice exposed to 10C (CC) and 22C (WW) throughout their life (see Table 1). In mice that were exposed to cold only early in life (CW), median age at death was 82 d less than in groups CC and WW. This may be due to increased mortality in the CW group between 600 and 800 d of age. Maximal life span was similar in all groups. Overall and pairwise comparisons of the survival curves using Kaplan-Meijer survival analysis showed no significant differences in survival between the groups (P > 0.05).
Instantaneous mortality rates (mx) were calculated for each group over intervals of 100 d using the formula mx = ln(1 - Ne/Nb), where Ne and Nb are the numbers of animals at the end and the beginning of the interval, respectively. We tested Gompertz and logistic models of increasing mortality with age using the WinModest program. The Gompertz model includes parameters A (initial mortality rate) and b (rate of exponential increase). The logistic model contains an additional deceleration parameter (s). Both models were also tested with a constant Makeham term (c). Maximum log likelihoods were used to determine the best-fitting model. The Gompertz model was never rejected (P > 0.1), indicating no deceleration of age-specific mortality and suggesting mortality increased exponentially from early adulthood onward.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Mortality Parameter Comparisons and Body Mass Trajectories,"Once models were fitted, likelihood ratio tests were used to compare Gompertz parameters among treatment groups. For each pair of treatment groups, we compared models assuming common A, common b, or common A and b to models estimating both parameters independently. There were no differences in mortality rates between WW and CC mice (χ² = 1.1, P = 0.58). Comparing WW with CC mice showed the highest likelihood for the model with both parameters estimated independently, but this model did not fit significantly better than the model with common A and b (χ² = 5.7, P = 0.057).
Body Mass
In all groups, the development of body mass was characterized by a strong increase early in life followed by a slower increase later in life and then by a decrease at the end of life. Initially, CC mice had a mean increase in body mass similar to WW mice. After approximately 300 d, CC mice did not increase in body mass further, whereas WW mice kept increasing until about 500 d of age. When the temperature was switched from 10 to 22C, CW mice immediately increased their body mass and reached a plateau between CC and WW levels. For statistical analyses, average body mass was calculated over three intervals (0–250 d, 251–500 d, and 501–750 d). One-way ANOVA showed WW mice had significantly higher mean body mass than CW and CC at all ages. In the oldest group (501–750 d), CW had significantly higher mass than CC (P < 0.05).",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Body Composition Changes and Organ Mass Differences,"Body Composition
At four ages, subsets of mice from each treatment group were killed for body composition analysis (Tables 2, 3). Significant variation among groups was assessed with general linear models including group, age, and the group × age interaction, with body mass as a covariate. Body composition differences therefore reflect proportional changes. As shown in Figure 2, CC mice had decreased body mass compared with WW mice (GLM effect of group: F₂,₇₅ = 5.8, P < 0.001). Reduced mass was mainly due to lower fat mass (GLM effect of group: F₂,₇₄ = 2.0, P = 0.06; WW vs CC: P < 0.01). In contrast, dry lean mass was slightly increased in CC mice (F₂,₇₄ = 6.3, P = 0.003; WW vs CC: P < 0.01). All variables showed significant increases with age (P < 0.001).
Organ Masses
Several changes in organ mass occurred in cold-exposed mice. Differences were most pronounced at 19 mo of age (Table 3). GLM analyses showed CC mice had significantly increased heart and kidney weights, while skin mass was significantly decreased. Liver, brain, stomach, intestines, and lung masses showed no significant differences. CW mice resembled CC mice at 3 and 11 mo, and WW mice at 19 and 27 mo.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
"Corticosterone, Food Intake, RMR, and DEE","Corticosterone Levels
Basal plasma corticosterone was measured at 3, 11, 19, and 27 mo (Table 4). Levels did not differ among groups and were not affected by age (GLM, P > 0.05).
Food Intake and Energy Expenditure
Cold exposure significantly affected food intake, resting metabolic rate (RMR), and daily energy expenditure (DEE). Mean food intake was significantly increased in CC compared with WW mice at 11, 19, and 26 mo (P < 0.05). RMR of cold-exposed mice was measured at 10C (RMR_EXP) and 22C (RMR_22C); WW RMR was measured only at 22C. There were no significant differences in RMR between CC and WW at 22C. RMR_EXP was increased by ~60% in CC mice. Age significantly affected RMR in both groups, with a slightly faster decline in cold-exposed animals (significant interaction; Table 5). Mean DEE measured in the home cage was markedly increased in CC compared with WW and decreased with age. CW mice resembled CC at 3 and 11 mo and WW at 19 and 27 mo.
Lifetime Energy Potential
Lifetime energy potential (LEP) was estimated using DEE and survival (median or 90%). DEE measurements at 3, 11, 19, and 27 mo were used to calculate average lifetime expenditure. LEP was considerably higher in CC mice than in WW and CW, and this remained true when normalized to body mass, dry lean mass, or organ mass.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Discussion – Rate-of-Living Theory and Energy Expenditure,"Discussion
Despite a 48% increase in overall DEE and a 64% increase in mass-specific energy expenditure throughout adult life, mice in the cold lived just as long on average as mice in warm temperatures. These results strengthen the existing doubts about the rate-of-living theory, which states that increased mass-specific metabolic rate reduces life span (Rubner 1908; Pearl 1928). It has been pointed out by Greenberg (1999) that a more precise formulation of the theory should take changes in body mass and composition into account. In Table 6, we have therefore calculated the LEP for all groups based on measurements of DEE and the median (50%) or maximum (90%) life span in four different ways: per animal (LEP), per gram body mass (LEPBM), per gram dry lean mass (LEPDL), and per gram mass of metabolically highly active organs (heart, liver, kidneys, brain; LEPOM). Rubner (1908) originally suggested that LEPBM was size invariant in interspecific comparison. In our intraspecific experiment manipulating energy metabolism, LEPBM clearly increased in the cold due to an increase in energy turnover without reducing longevity. Expressing metabolic rate relative to dry lean mass or metabolic organ mass did not change this conclusion.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Discussion – Contradictory Rat Studies and Stress Considerations,"Studies investigating the effects of cold exposure on life span in rats have yielded conflicting results. Kibler and Johnson (1961; Johnson et al. 1963; Kibler et al. 1963) showed that rats kept at 9C continuously had shorter life spans than rats at 28C, and these studies have been cited as support for the rate-of-living theory. Holloszy and Smith (1986) argued that continuous cold exposure in Johnson et al.’s study was a chronic stressor, potentially causing deleterious effects on health and longevity mediated by chronic elevation of stress hormones. In our study, we applied a protocol similar to that of Kibler et al. (1963) and did not find differences in corticosterone levels (Table 4) or in life spans between cold-exposed or control mice. Apparently, cold exposure was not a chronic stressor, or at least it did not result in increased corticosterone. As in Holloszy and Smith’s study of rats, we found a strong increase in energy expenditure with no effect on life span. Artificial selection for high activity (increased voluntary wheel running) also increased lifetime energy expenditure (~20%) without affecting life span. These findings join growing evidence that in mammals, lifetime energy expenditure per se does not determine life span.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
"Discussion – ROS, Mitochondrial Uncoupling, and Antioxidant Defense","In apparent contrast to our conclusions, metabolic rate is known to influence aging via production of reactive oxygen species (ROS). ROS generated during oxidative phosphorylation can damage lipids, DNA, and proteins. If ROS were increased in our cold-exposed mice due to higher energy turnover, how did they protect themselves? Animals have several defense systems, including antioxidant enzymes and increased protein turnover. The rate of radical production depends on mitochondrial uncoupling. Uncoupling oxidative phosphorylation through uncoupling proteins (UCPs) reduces ROS production while dissipating energy as heat. This would be beneficial in cold exposure where thermogenesis is required. Several studies show increased uncoupling protein expression, particularly in BAT, in cold-exposed rodents. Cold exposure may evoke multiple physiological responses that counteract ROS-related aging, such as reduced tumor incidence linked to lower fat content. Thus, metabolic rate is not a simple predictor of life span.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Discussion – Comparative Biology of ROS and Maintenance Systems,"Across taxa, evolutionary adaptations reveal differences in ROS production and antioxidant defense. Birds expend ~1.5× more energy than similar-sized mammals yet live longer. Their mitochondria produce less ROS per mL O2 and are better protected against oxidative damage. Among mammals, long-lived species generally show lower ROS production and higher antioxidant protection than short-lived ones. Life-span–extending manipulations such as caloric restriction enhance maintenance systems, including antioxidant enzymes, protein turnover, and DNA repair. Aging can therefore be described as failure of maintenance and repair systems. Many mechanisms decline with age and require metabolic resources to maintain. Mechanistic theories of aging highlight different aspects of the same multifactorial process. No manipulation affects a single aging pathway in isolation; changes in hormones, fat content, antioxidants, and protein turnover complicate attribution of longevity differences to metabolic rate alone. Our study refutes the strict quantitative form of the rate-of-living theory but does not deny the foundational relevance of energetics in aging.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
Discussion – Non-Monotonic Model and Final Interpretation,"Taking multiple consequences of energy turnover into account implies that the relationship between energy expenditure and life span cannot be unidirectional. Figure 4 illustrates a model where increasing energy expenditure elevates ROS and decreases survival, while decreasing energy expenditure may also reduce survival via excess body fat. These opposing processes produce an intermediate optimum for longevity. The energy expenditure that maximizes survival is not necessarily the one that maximizes fitness, which also includes reproductive output. Thus, evidence for or against the rate-of-living theory depends on where species fall on this curve. In summary, although interspecific comparisons often show inverse relationships between metabolic rate and life span, our within-species manipulation demonstrates that increasing energy expenditure in laboratory mice (via cold exposure) does not reduce life span. The expectation of constant lifetime energy potential was not upheld. These results refute the strict rate-of-living formulation but emphasize the importance of understanding how multiple systems interact in aging.",Physiological and Biochemical Zoology,Cold Exposure and Longevity,2009
"Temperature, Metabolism, and Longevity Overview","Being cool: how body temperature influences ageing and longevity
Temperature is a basic and essential property of any physical system, including living systems. Even modest variations in temperature can have profound effects on organisms, and it has long been thought that as metabolism increases at higher temperatures so should rates of ageing. Here, we review the literature on how temperature affects longevity, ageing and life history traits. From poikilotherms to homeotherms, there is a clear trend for lower temperature being associated with longer lifespans both in wild populations and in laboratory conditions. Many life-extending manipulations in rodents, such as caloric restriction, also decrease core body temperature. Nonetheless, an inverse relationship between temperature and lifespan can be obscured or reversed, especially when the range of body temperatures is small as in homeotherms. An example is observed in humans: women appear to have a slightly higher body temperature and yet live longer than men. The mechanisms involved in the relationship between temperature and longevity also appear to be less direct than once thought, with neuroendocrine processes possibly mediating complex physiological responses to temperature changes.",Biogerontology,Body Temperature and Longevity,2015
"Thermodynamics, Entropy, and Ageing","Introduction
The second law of thermodynamics states that within a closed thermodynamic system the entropy will increase over time until it reaches thermodynamic equilibrium. This increase in molecular entropy over time within living organisms has been proposed, under the assumption that the second law of thermodynamics applies to open systems, to increase susceptibility to age-related disorders and thus essentially equate, at a high level of abstraction, to the ageing process (Hayflick 2007). Other authors have argued that ageing is due to an increase in molecular disorder due to an increase in thermodynamic entropy (Demetrius 2013). One of the factors known to affect thermodynamics is temperature, and therefore it has been long speculated that it may influence the ageing process with organisms ageing faster at higher temperatures due to more molecular damage being generated (Conti 2008; Liu and Walford 1972; Rikke and Johnson 2004). Temperature is an essential property of biological systems and various studies in many species have associated temperature with ageing and longevity. Early studies focused on animals that rely on external sources of heat (ectotherms), and whose internal temperature varies considerably (poikilothermy).",Biogerontology,Body Temperature and Longevity,2015
Historical Studies in Poikilotherms,"A century ago, Loeb and Northrop showed that lifespan correlates negatively with temperature in fruit flies Drosophila melanogaster (Loeb and Northrop 1916). Another early study to observe the effects of temperature on longevity was in the cladoceran crustacean Daphnia magna (MacArthur and Baillie 1929). Later studies in other poikilotherms (in particular fishes) demonstrated that even mild changes in temperature over long periods of time can influence lifespan (Walford and Liu 1965). Walford and Liu pioneered the use of the South American fish Cynolebias as a model organism in laboratory temperature and longevity studies. These insights were then expanded to species that can generate their own body heat (endotherms) such as mice, in which marijuana derivatives were used to induce hypothermia. However, this approach did not successfully reduce core body temperature long-term and could not fully establish the effect of temperature on mouse longevity. Walford instead examined the body temperatures of yoga masters in India, finding that a low-calorie intake could reduce core body temperature by 1–2°C. This observation motivated the use of caloric restriction to induce low Tb in homeotherms to investigate effects on lifespan.",Biogerontology,Body Temperature and Longevity,2015
Temperature Effects Across Normal Ranges,"Life-extension and temperature
Before exploring thermal effects on ageing and longevity, a distinction needs to be made between impacts at thermal extremes compared with body temperature (Tb) within the normal physiological range. In poikilotherms, very low temperatures cause severe enzyme inhibition and sharp declines in biological function, whereas extreme high temperatures lead to insufficient respiratory and circulatory capacity, protein denaturation, and membrane dysfunction. Homeotherms suffer hypothermia or hyperthermia when Tb deviates too far from regulated limits. However, it is the effects of temperature on ageing and longevity across the normal temperature range, rather than under thermal stress, that is the focus of this review. Figure 1 illustrates predicted influences of body temperature on longevity in both poikilotherms and homeotherms: away from extremes, longevity tends to decline exponentially with increased body temperature as predicted by metabolic rate theory.",Biogerontology,Body Temperature and Longevity,2015
Temperature and Longevity in Invertebrates,"Invertebrates
Early studies focused on temperature effects on invertebrate longevity because their body temperature is determined by environmental conditions and is easy to manipulate in laboratory settings. One benchmark study showed that Drosophila melanogaster live about twice as long at 21°C compared to 27°C (Miquel et al. 1976). In C. elegans, a 5°C drop in temperature produced a 75% increase in lifespan between both 15–20°C and 20–25°C (Van Voorhies and Ward 1999). Numerous other insect studies have reported negative correlations between temperature and lifespan, including wasps (Trichogramma platneri), beetles, and other species. Although variation in husbandry may influence results in poorly studied species, no invertebrate has been shown to live longer at higher temperatures except where very low temperatures cause pathology. These laboratory observations align with findings in the wild, despite confounding ecological factors such as predation.",Biogerontology,Body Temperature and Longevity,2015
"Caloric Restriction, Mortality Patterns, and Body Temperature","Caloric restriction (CR), defined as limiting calorie intake without malnutrition, has been shown to extend lifespan in multiple model systems. When comparing mortality curve trajectories under CR versus lowered body temperature (Tb), studies in Drosophila have revealed differences between the two interventions. Under CR, mortality is initially delayed, increasing overall lifespan, but the long-term slope of the mortality trajectory remains similar to controls. In contrast, lowered Tb not only extends lifespan but also reduces the slope of the mortality trajectory, suggesting distinct biological pathways between CR and temperature-mediated longevity in flies. Whether these distinctions hold for homeotherms is still unknown. In rodents, CR is generally associated with a 1–1.5°C drop in core Tb. Data from the LSXSS recombinant inbred series show that different strains exhibit varied Tb reductions under CR, ranging from 1–2°C to 3–5°C depending on genotype. Some rodent studies failing to detect Tb reduction likely suffered from measurement artefacts, such as stress induced by rectal probes. More reliable methods, including infrared non-contact thermometry calibrated with implanted microchip transponders, provide more accurate assessments of Tb changes under CR.",Biogerontology,Caloric Restriction and Temperature,2015
"CR-Induced Hypothermia, Metabolic Rate, and Strain Variability","CR in mammals can reduce metabolic rate, potentially lowering heat production and causing a fall in Tb—an adaptive mechanism during periods of food scarcity. However, the extent to which CR reduces metabolic rate remains debated, with some rodent studies suggesting CR can extend lifespan without decreasing metabolic output. Environmental temperature modifies these effects: lymphoma-prone B6 mice kept at thermoneutrality (30°C) did not exhibit CR-mediated reductions in Tb, diminishing CR's life-extending and anti-lymphoma benefits. In contrast, autoimmune-prone MRL mice maintained CR-related disease delay even at 30°C. These findings suggest that CR-induced hypothermia may contribute to specific anti-cancer effects but may not universally explain CR's longevity benefits. Strain-specific effects further complicate the picture: across 31 mouse strains, a greater CR-induced Tb reduction surprisingly predicted shorter lifespan extension. Strains with smaller decreases in Tb—and smaller reductions in body fat—showed better survival under CR. These results contrast sharply with findings in Hcrt-UCP2 mice, where modest reductions in Tb alone were sufficient to extend lifespan, illustrating that different mechanisms may operate across genetic backgrounds.",Biogerontology,Caloric Restriction and Temperature,2015
Caloric Restriction Effects in Primates and Humans,"Emerging primate studies suggest CR confers substantial health benefits but modest, if any, lifespan extension compared to rodents. In rhesus monkeys subjected to CR for six years, a 30% reduction in calorie intake lowered Tb by approximately 0.5°C compared to age-matched controls. Short-term CR (one month) produced a larger drop of ~1°C and reduced 24-hour energy expenditure by 24%. These effects appear linked to energy conservation, potentially mediated by reductions in circulating triiodothyronine. However, some long-term primate studies measured Tb only at specific times of day, limiting interpretability. Additional studies confirmed CR-associated Tb decreases of ~0.7°C alongside reductions in insulin and increases in DHEAS. Human CR studies provide similar evidence: in a cohort undergoing 6-year CR, mean 24-hour Tb was 36.64°C, compared to 36.86°C in endurance runners and 36.83°C in sedentary individuals. Importantly, the CR and endurance groups had similar low body fat, suggesting Tb reductions were driven by caloric intake rather than adiposity. Further human studies showed that six months of 25% CR reduced fasting insulin, thyroid hormone levels, and both total and sleeping energy expenditure, alongside a 0.2–0.3°C decrease in Tb. Reduced DNA damage was observed across CR and very-low-calorie diet groups, suggesting possible mechanisms for longevity benefits, though long-term maintenance remains unknown.",Biogerontology,Caloric Restriction and Temperature,2015
Thermodynamic and Metabolic Explanations of Temperature-Linked Longevity,"Mechanisms of temperature life-extending effects
Thermodynamic explanations to ageing have been long proposed, in which ageing is the result of an increase in molecular disorder and a decrease in metabolic stability. The intuitive interpretation for how low body temperature extends longevity is that it affects metabolic rates and decreases the rate of biochemical reactions, thereby retarding ageing processes. Because metabolic rate increases exponentially with temperature, it is reasonable to postulate that temperature acts exponentially on 1/lifespan, especially over a small temperature range, and this has been demonstrated in some poikilotherm studies. A study in the housefly Musca domestica supported this idea by showing that animals at 15°C lived longer but were much less active than at 23°C, and that lower physical activity itself correlated with increased lifespan. Mild heat stress can also increase lifespan in D. melanogaster and C. elegans by stimulating genome stability pathways in a hormetic response. A recent study showed that repeated heat shocks in young male flies induced long-term upregulation of heat shock response genes, suggesting the involvement of HSP70 pathways in longevity. Although the mechanisms responsible for the long lifespan of mice with lower core Tb are unknown, an increase in energy efficiency related to reduced metabolic requirements has been observed, potentially influencing molecular damage, oxidative stress, and DNA damage.",Biogerontology,Temperature and Longevity Mechanisms,2015
Challenges to the Rate-of-Living Theory and ROS-Based Explanations,"The fact that caloric restriction does not always extend lifespan, and that in diverse mouse strains its longevity effect is associated with smaller decreases in temperature, suggests that factors beyond Tb influence ageing. Women have higher temperatures than men yet live longer, while female C57BL/6 mice have higher temperatures than males and live shorter lives, further complicating temperature–longevity relationships. Although it has long been argued that drops in Tb reduce ROS production and thereby extend lifespan, the free radical theory of ageing has faced significant criticism due to numerous studies showing inconsistent or negative results. Temperature-associated reductions in oxidative damage and enhanced antioxidant defenses have been observed in ectothermic fish kept at lower temperatures, but these findings may not generalize to mammals. Recent evidence suggests that specific molecular mechanisms, rather than passive thermodynamic effects, mediate temperature-induced lifespan extension. In C. elegans, the cold-sensitive TRPA-1 channel promotes longevity at lower temperatures via calcium influx, PKC activation, SGK-1 signaling, and the DAF-16/FOXO transcription factor. Thermosensory neurons also modulate lifespan at warmer temperatures by regulating daf-9 expression. These findings demonstrate that temperature effects on longevity are not passive and challenge classical rate-of-living predictions.",Biogerontology,Temperature and Longevity Mechanisms,2015
"Endocrine, Immune, and Thermogenic Mechanisms in Temperature-Mediated Longevity","It has been proposed that caloric restriction reduces Tb and subsequently alters hormonal axes with downstream targets affecting lifespan, with endocrine roles being plausible contributors. In ectotherms, temperature can influence hormones controlling growth and development. Studies in fish suggest that the GH/IGF system mediates temperature effects on growth. In humans, exercise-induced increases in Tb correlate with GH secretion, though the causal direction remains unclear. Lower Tb has been suggested to promote longevity via enhanced metabolic pathways that suppress autoimmunity in old age. Hypothermic rodents show increased resistance to irradiation and endotoxins, and Drosophila raised at lower developmental temperatures show enhanced stress resistance. In homeotherms, brown adipose tissue plays a central role in thermogenesis via uncoupling proteins such as UCP1. Mice deficient in UCP1 show increased susceptibility to obesity during ageing. Polymorphisms reducing UCP1 expression in humans correspond to increased body mass and fat levels. UCPs also reduce ROS production when upregulated, linking thermogenesis to oxidative stress defense. Cold exposure upregulates UCP3 in rodents and increases BAT mass and cell proliferation in young but not old rats, indicating age-dependent thermogenic responsiveness. BAT mass and activity decline with age, more sharply in men. Cold-exposed mice tend to have lower body fat, which may reduce tumor incidence. Although low temperature may reduce molecular damage, this view is overly simplistic, especially in mammals where adipose, neuroendocrine, and other systems integrate temperature and ageing processes.",Biogerontology,Temperature and Longevity Mechanisms,2015
"Temperature, Torpor, and Hibernation Effects on Longevity","Species correlations between temperature and longevity
In homeotherms undergoing torpor or hibernation, body temperature (Tb) falls and endothermy is temporarily replaced with ectothermy. Hibernation can increase survival up to five-fold when comparing similar-sized hibernating and non-hibernating mammals. A phylogenetic study using a Chiroptera supertree revealed strong correlations between lifespan and the length of hibernation, body mass, and occasional cave use. Another comparative analysis showed that smaller mammals that hibernate tend to have longer maximum lifespans (≈50% longer for 50-g species), lower reproductive rates, and longer generation times than similar-sized non-hibernators. Hibernation provides predator avoidance: reduced metabolism lowers scent emissions, motion, and body heat, reducing detection risk. Because hibernation often occurs even when food is available, predator avoidance may be a stronger evolutionary driver than energy conservation. Some evidence suggests insulin may trigger hypothermia during hibernation, though this remains unproven. Since hibernation also occurs in times of limited food supply, its physiological role may overlap with caloric restriction (CR). In bats, hibernation reduces Tb by up to 85% (from ~40°C to ~6°C) for weeks at a time, dropping metabolic rate to ~5% of normal, potentially reducing oxidative damage and extending lifespan. However, simplistic interpretations—such as “lower Tb reduces ROS and thus extends life”—have been increasingly challenged.",Biogerontology,Temperature and Species Longevity,2015
"Comparative Biology: Temperature, Metabolic Rate, and Life History Across Species","Comparisons across species have largely disproved the classical rate-of-living theory in homeotherms. A low Tb is not required for evolution of longevity nor always associated with it. Birds exhibit higher Tb than similar-sized mammals yet live longer; marsupials have lower Tb than placentals but typically live shorter. A large comparative study examining body temperature, basal metabolic rate (BMR), body size, and lifespan across mammals found: (1) a strong positive correlation between Tb and BMR, (2) no significant correlation between BMR and lifespan, (3) a negative correlation between Tb and time to maturity, and (4) a weak, borderline-significant negative correlation between Tb and lifespan. Because mammals maintain a narrow thermal range and ecological constraints shape life histories, temperature may influence timing of life history events independently of metabolic rate. These findings point to more complex roles for Tb in lifespan evolution than predicted by classical thermodynamic or metabolic-rate models.",Biogerontology,Temperature and Species Longevity,2015
Naked Mole-Rat Longevity and the Role of Low Body Temperature,"The naked mole-rat (Heterocephalus glaber) is the longest-lived rodent (~30+ years) and highly cancer-resistant. It is unique among mammals for being poikilothermic, with a low metabolic rate and unusually low Tb. This has prompted speculation that low Tb may contribute to its exceptional lifespan. Wild-derived Mus musculus have a Tb of ~36.9°C; naked mole-rats are typically reported near 32.1°C, with some studies showing ~30.6°C. Breeding females have higher Tb (~+1.5°C), but queens do not appear to live shorter lives. Estimating continuous pregnancy cycles yields an upper Tb estimate of ~34.2°C, establishing a realistic lifetime average between ~32.1–34.2°C. Using lifespan–temperature relationships from other species, linear regression of ln(lifespan) versus temperature predicts that mice with naked mole-rat Tb would live ~4.5–7.3 years. Excluding questionable fish data, adjusted estimates indicate mice might live 30–65% longer (~5.3–6.6 years) if their Tb matched naked mole-rats. Conversely, naked mole-rats hypothetically raised to ~37°C might only live 20–25 years. Although simplistic, these calculations suggest low Tb contributes modestly to the species’ longevity. Mole-rats as a group have low Tb, likely reflecting stable, protected burrow environments conducive to evolving longer life. Naked mole-rats have both the longest lifespan and lowest Tb among studied mole-rats.",Biogerontology,Temperature and Species Longevity,2015
Broad Mammalian Comparisons and Additional Long-Lived Examples,"Other long-lived mammals provide additional context. The Eastern gray squirrel (Sciurus carolinensis), a small rodent capable of living ~23.6 years, actually has a higher Tb (~38.7°C) than average mammals, indicating that high Tb is not inherently detrimental for longevity. The bowhead whale (Balaena mysticetus) is the longest-lived mammal (~211 years). Interestingly, its Tb (~33.8°C) is lower than other large non-hibernating eutherians, and its metabolic resting rate is below that of other cetaceans. This raises the possibility—though still speculative—that relatively low Tb contributes to extreme longevity in some taxa. While body temperature is clearly not the primary factor determining maximal lifespan, these observations suggest it may play a supporting role in the evolution of longevity, warranting deeper comparative and mechanistic studies.",Biogerontology,Temperature and Species Longevity,2015
Concluding Remarks on Temperature and Longevity,"Concluding remarks
It is clear that temperature influences longevity across a wide range of species—from invertebrates to mammals—both within species and likely across evolutionary lineages. Early interpretations of this relationship focused on thermodynamics: lower temperatures were thought to extend lifespan simply by slowing metabolic rate, decreasing biochemical reaction rates, and reducing damage accumulation such as oxidative stress or DNA lesions. However, as evidence accumulated across taxa, this simple metabolic-rate–based view appears insufficient to explain the diversity of observed effects. Studies in ectotherms and endotherms increasingly show that temperature affects complex physiological systems rather than acting solely through passive thermodynamic principles. Neuroendocrine pathways, temperature-sensitive ion channels, hormonal regulation, and adaptive thermogenic responses all appear to shape how organisms respond to changes in environmental or core body temperature. These mechanisms may influence stress resistance, developmental timing, metabolic efficiency, immune function, and repair processes, ultimately modulating ageing trajectories. Thus, the relationship between temperature and longevity is emerging as a multifactorial network of interactions rather than a single mechanistic axis. While lower body temperature can extend lifespan in some controlled contexts, its effects depend on species, life history strategies, ecological pressures, and specific molecular pathways activated in response to thermal change. This underscores the broader principle that ageing is an integrative, systemic process influenced by numerous interacting biological systems rather than any single factor.",Biogerontology,Temperature and Longevity - Concluding Remarks,2015