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PMC12514566
AGS and AGS-EBV cells were subjected to hypoxia for 24 h, and the expression levels of phosphorylated AKT (p-AKT) at Ser473 and Thr308 were assessed by Western blot. (
[ { "end": 3, "label": "CellLine", "start": 0, "text": "AGS" }, { "end": 15, "label": "CellLine", "start": 8, "text": "AGS-EBV" } ]
PMC12514566
AGS and AGS-EBV cells were cultured under normoxic or hypoxic conditions for 24 h, followed by RT-qPCR to measure the relative expression levels of HIF-1α.
[ { "end": 3, "label": "CellLine", "start": 0, "text": "AGS" }, { "end": 15, "label": "CellLine", "start": 8, "text": "AGS-EBV" } ]
PMC12514566
Data are presented as mean ± SD, with statistical significance indicated (∗p < 0.05, ∗∗∗∗p < 0.0001).
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PMC12514566
Beyond its effect on the proliferation of EBVaGC cells, we investigated the impact of 2-DG on the EBV life cycle within these cells.
[ { "end": 48, "label": "CellLine", "start": 42, "text": "EBVaGC" } ]
PMC12514566
Western blot analysis was performed to assess the expression of EBV lytic proteins, including BZLF1 and BMRF1 in AGS-EBV and SNU719 cell lines treated with 2-DG under hypoxic conditions.
[ { "end": 120, "label": "CellLine", "start": 113, "text": "AGS-EBV" }, { "end": 131, "label": "CellLine", "start": 125, "text": "SNU719" } ]
PMC12514566
Fig. 6 revealed that hypoxia induced the expression of these lytic proteins, promoting EBV lytic reactivation.
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PMC12514566
However, treatment with 2-DG significantly downregulated the expression of BZLF1, BMRF1 under hypoxic conditions.
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PMC12514566
These findings suggest that glycolysis is essential for hypoxia-induced EBV lytic reactivation, highlighting the critical role of metabolic pathways in regulating the EBV life cycle within EBVaGC cells.
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PMC12514566
Fig. 62-DG inhibits hypoxia-induced EBV lytic reactivation in EBVaGC cell lines.
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PMC12514566
Western blot analysis of EBV lytic proteins Zta (BZLF1) and BMRF1 in AGS-EBV and SNU719 cells under normoxic or hypoxic conditions, with or without 2-DG treatment.
[ { "end": 76, "label": "CellLine", "start": 69, "text": "AGS-EBV" }, { "end": 87, "label": "CellLine", "start": 81, "text": "SNU719" } ]
PMC12514566
Fig. 6 2-DG inhibits hypoxia-induced EBV lytic reactivation in EBVaGC cell lines.
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PMC12514566
Western blot analysis of EBV lytic proteins Zta (BZLF1) and BMRF1 in AGS-EBV and SNU719 cells under normoxic or hypoxic conditions, with or without 2-DG treatment.
[ { "end": 76, "label": "CellLine", "start": 69, "text": "AGS-EBV" }, { "end": 87, "label": "CellLine", "start": 81, "text": "SNU719" } ]
PMC12514566
Our results demonstrate that 2-DG effectively inhibits cell proliferation in both EBV-infected and uninfected gastric carcinoma cells, with EBVaGC cells showing greater sensitivity, particularly under hypoxic conditions.
[ { "end": 146, "label": "CellLine", "start": 140, "text": "EBVaGC" } ]
PMC12514566
This heightened sensitivity in EBVaGC cells was associated with increased glycolytic gene expression, primarily through upregulate HIF-1α mRNA-dependent mechanisms.
[ { "end": 37, "label": "CellLine", "start": 31, "text": "EBVaGC" } ]
PMC12514566
In addition to inhibiting cell proliferation, 2-DG treatment also inhibited EBV lytic reactivation in EBVaGC cells under hypoxia.
[ { "end": 108, "label": "CellLine", "start": 102, "text": "EBVaGC" } ]
PMC12514566
This study provides significant insights into the differential effects of the glycolysis inhibitor 2-DG on EBVaGC and EBVnGC and highlights the therapeutic potential of glycolysis inhibition in EBVaGC.
[ { "end": 113, "label": "CellLine", "start": 107, "text": "EBVaGC" }, { "end": 124, "label": "CellLine", "start": 118, "text": "EBVnGC" } ]
PMC12514566
Our study represents that the glycolysis inhibitor 2-DG exerts a significantly stronger anti-proliferative effect on EBV-positive AGS cells compared to their EBV-negative counterparts, a differential sensitivity that is further amplified under hypoxic conditions.
[ { "end": 133, "label": "CellLine", "start": 130, "text": "AGS" } ]
PMC12514566
Currently, there are many studies exploring the impact of EBV infection on glycolysis in tumor cells under normoxic conditions.16, 17, 18 For example, LMP1 enhances glucose metabolism by activating the metabolic enzyme PDHE1α in NPC cells.
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PMC12514566
However, there is still a lack of research on the effects of EBV infection on glycolysis in tumor cells under hypoxic conditions.
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PMC12514566
We thought that this phenomenon is not merely a simple additive effect of EBV infection and hypoxia as two independent glycolysis-promoting factors.
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PMC12514566
Rather, it profoundly reveals that EBV infection fundamentally reshapes the host cell's metabolic architecture.
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PMC12514566
We propose that EBV infection acts as a potent biological “stressor”, compelling gastric cancer cells to rewire their metabolic network, driving a heightened reliance on glycolytic pathways for carbon skeletons and ultimately pushing the cells into a state of “glycolytic addiction”.
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PMC12514566
Hypoxia, a key feature of the tumor microenvironment, amplifies the metabolic vulnerability of EBV-positive cells, leading to an extreme dependence on glycolysis.
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PMC12514566
Perturbation of this metabolic pathway, such as hexokinase inhibition by 2-DG, can trigger cell proliferation arrest and death.
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PMC12514566
This suggests that 2-DG may have enhanced cytotoxic effects in aggressive, radio/chemotherapy-resistant hypoxic regions of EBVaGC.
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PMC12514566
Our investigation into the molecular mechanisms of glycolysis upregulation by EBV and hypoxia found that in AGS-EBV cells under hypoxic conditions, the upregulation of glycolysis-related genes is HIF-1α-dependent.
[ { "end": 115, "label": "CellLine", "start": 108, "text": "AGS-EBV" } ]
PMC12514566
Knocking down HIF-1α reversed this effect, while knocking down HIF-2α had no impact.
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PMC12514566
This aligns with the known functions of HIF-α subtypes, as HIF-1α is the primary regulator of metabolic reprogramming in response to acute hypoxia, directly activating glycolytic enzyme and glucose transporter gene transcription, thus rapidly switching cellular metabolism to glycolysis.
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PMC12514566
Conversely, although structurally similar to HIF-1α, HIF-2α regulates a vastly different set of target genes, primarily involved in long-term adaptive responses such as angiogenesis, cell fate determination, and iron metabolism, without directly participating in glycolysis pathway regulation.
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PMC12514566
In fact, previous studies have explicitly stated that HIF-2α does not regulate the glycolytic pathway.
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PMC12514566
Therefore, our results suggest that EBV infection amplifies the host cell's HIF-1α-mediated hypoxic response, driving a more pronounced glycolytic phenotype, rather than creating a novel signaling pathway.
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PMC12514566
Our findings indicate that EBV infection enhances HIF-1α activity in gastric cancer cells under hypoxic conditions, a process that doesn't depend on increased protein stability.
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PMC12514566
To investigate HIF-1α protein stability, we used CoCl2, a well-established hypoxia-mimicking agent.
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PMC12514566
CoCl2 works by substituting the ferrous iron (Fe) in the active site of prolyl hydroxylase (PHD) enzymes, thereby inhibiting their function.
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PMC12514566
Since PHDs need oxygen to tag HIF-1α for proteasomal degradation, their inhibition by CoCl2 leads to HIF-1α stabilization and accumulation, effectively mimicking the primary consequence of cellular oxygen deprivation.
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PMC12514566
We recognize that chemical mimetics don't perfectly replicate the complex environment of physiological hypoxia, which includes broad changes in cellular metabolism and redox status in addition to HIF-1α stabilization.
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PMC12514566
Despite these differences, the use of CoCl2 remains a valid and targeted approach for specifically investigating the post-translational regulation of HIF-1α stability, which was the precise goal of this experiment.
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PMC12514566
Under normoxic conditions, where EBV latent proteins like LMP2A activate the PI3K/AKT/mTOR pathway, promoting HIF-1α mRNA translation and a hypoxia-independent increase in HIF-1α protein.27, 28, 29 Our results found that under hypoxic conditions, EBV does not activate PI3K/AKT signaling.
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PMC12514566
In true hypoxic environments, HIF-1α regulation shifts: hypoxia directly inhibits PHD enzymes, stabilizing and accumulating HIF-1α.
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PMC12514566
We hypothesize that gastric cancer cells prioritize this potent hypoxic signal over the indirect PI3K/AKT pathway for HIF-1α upregulation, potentially suppressing PI3K/AKT due to energetic stress.
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PMC12514566
In this context, EBV acts as a "transcriptional amplifier," working with hypoxia-induced protein stability to drive HIF-1α gene expression.
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PMC12514566
Our study revealed that 2-DG not only inhibited the proliferation of AGS-EBV cells but also significantly suppressed EBV lytic reactivation under hypoxic conditions.
[ { "end": 76, "label": "CellLine", "start": 69, "text": "AGS-EBV" } ]
PMC12514566
Notably, while hypoxia, through HIF-1α upregulation, can initiate the EBV lytic program, 2-DG remained effective in suppressing lysis even under hypoxic conditions.
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This suggests that successful lytic initiation requires not only “transcriptional permission” but also “metabolic permission”.
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On one hand, the EBV lytic cycle is highly dependent on metabolic energy, including ATP generation, nucleotide synthesis, and protein translation.
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PMC12514566
By inhibiting hexokinase, 2-DG cuts off the essential energy and raw material supply required for viral lysis.
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This establishes a "metabolic checkpoint," effectively blocking viral replication at the energy level.
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PMC12514566
On the other hand, 2-DG also interferes with N-linked glycosylation, leading to misfolded viral envelope glycoproteins that accumulate in the endoplasmic reticulum.
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This triggers endoplasmic reticulum stress and the unfolded protein response (UPR).
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Once activated, the UPR, via mechanisms like eIF2α phosphorylation, globally inhibits protein translation, further preventing viral protein synthesis and the formation of new viral particles.
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PMC12514566
While lytic induction is explored as a potential "suicide" therapy for EBV-positive cancers, its uncontrolled, spontaneous activation within the tumor microenvironment is often detrimental.
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PMC12514566
Spontaneous lytic reactivation can create a pro-tumorigenic microenvironment by driving chronic inflammation, promoting angiogenesis through the release of viral factors like BZLF1, and facilitating immune evasion.37, 38, 39, 40 Furthermore, the expression of lytic proteins has been associated with resistance to conventional treatments such as chemotherapy.
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PMC12514566
Therefore, this approach ingeniously transforms the risk of hypoxia-induced lytic reactivation into a therapeutic advantage.
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PMC12514566
By allowing 2-DG to exert a dual mechanism—simultaneously targeting host cell metabolism and viral pathogenesis—it strengthens the rationale for using glycolysis inhibitors in the treatment of EBVaGC.
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PMC12514566
In conclusion, this study demonstrates that the glycolysis inhibitor 2-DG has a stronger inhibitory effect on the proliferation of EBVaGC cells compared to EBVnGC.
[ { "end": 137, "label": "CellLine", "start": 131, "text": "EBVaGC" }, { "end": 162, "label": "CellLine", "start": 156, "text": "EBVnGC" } ]
PMC12514566
Additionally, 2-DG also inhibits EBV lytic reactivation.
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PMC12514566
These findings underscore the potential of targeting glycolysis as a therapeutic strategy for EBVaGC and reveal the role of EBV-driven glycolytic metabolism.
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PMC12514566
Yu Du: Writing – review & editing, Writing – original draft, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.
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PMC12514566
Wangsheng Zuo: Methodology, Investigation, Data curation, Conceptualization.
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PMC12514566
Yiting Shao: Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.
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PMC12514566
Yanying Ji: Methodology, Investigation.
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PMC12514566
Bojin Su: Methodology, Investigation, Funding acquisition.
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PMC12514566
Sihong Liang: Methodology, Investigation.
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PMC12514566
Deyu Wang: Methodology, Investigation.
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PMC12514566
Bin Li: Methodology, Investigation.
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PMC12514566
Yujie Feng: Methodology, Investigation.
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PMC12514566
Liping Gong: Writing – review & editing, Writing – original draft, Funding acquisition, Data curation, Conceptualization.
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PMC12514566
Jianning Chen: Writing – review & editing, Writing – original draft, Data curation, Conceptualization.
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PMC12514566
Chunkui Shao: Writing – review & editing, Funding acquisition, Data curation.
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PMC12514566
During the preparation of this work the author(s) used Google Gemini in order to improve the readability and language of the manuscript.
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PMC12514566
After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article.
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PMC12514566
This work was supported by the 10.13039/501100001809National Natural Science Foundation of China (82403764, 82202835) and the 10.13039/501100002858China Postdoctoral Science Foundation (2022M713568, 2023M744005), 10.13039/501100021171Guangdong Basic and Applied Basic Research Foundation (2024A1515012426, 2022A1515012555), Guangzhou Science and Technology Project on Fundamental and Applied Basic Research (2023A04J1098).
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PMC12514566
I have nothing to declare.
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PMC10158546
Prions are misfolded proteins that accumulate within the brain in association with a rare group of fatal and infectious neurological disorders in humans and animals.
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PMC10158546
A current challenge to research is a lack of in vitro model systems that are compatible with a wide range of prion strains, reproduce prion toxicity, and are amenable to genetic manipulations.
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PMC10158546
In an attempt to address this need, here we produced stable cell lines that overexpress different versions of PrP through lentiviral transduction of immortalized human neural progenitor cells (ReN VM).
[ { "end": 199, "label": "CellLine", "start": 193, "text": "ReN VM" } ]
PMC10158546
Differentiated cultures made from the neural progenitor cell lines overexpressed PrP within 3D spheroid-like structures of TUBB3 neurons and we observed evidence that PrP modulates formation of these structures, consistent with PrP’s role in neurogenesis.
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PMC10158546
However, through repeated measurements of amyloid seeding activity in 6-week time course experiments, we failed to observe any evidence of prion replication within the differentiated ReN cultures following challenge with four prion isolates (human sCJD subtypes MM1 and VV2, and rodent adapted scrapie strains RML and 263K).
[ { "end": 186, "label": "CellLine", "start": 183, "text": "ReN" } ]
PMC10158546
We attributed amyloid seeding activity detected within the cultures to residual inoculum and concluded that PrP overexpression was insufficient to confer permissiveness of ReN cultures to prion infection.
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PMC10158546
While our ReN cell prion infection model was unsuccessful, additional efforts to develop cellular models of human prion disease are highly warranted.
[ { "end": 13, "label": "CellLine", "start": 10, "text": "ReN" } ]
PMC10158546
Prion diseases are rare infectious neurological disorders that afflict humans and animals, and are characterized by long pre-clinical incubation periods prior to rapidly progressive neurocognitive decline culminating in death .
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PMC10158546
The disease causing agent is thought to comprise of misfolded prion proteins (PrP) that are deposited within the brain as plaques in association with neuropathological changes .
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PMC10158546
Accumulation and spread of prions are mediated by prion replication whereby PrP recruits and converts the host-encoded cellular prion protein (PrP) into the disease-associated conformation .
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PMC10158546
Prion diseases are diverse with widely varying clinical signs and symptoms encoded by prion strains – defined as infectious isolates with distinct pathological features and are thought to adopt unique 3D conformations of misfolded PrP .
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PMC10158546
In the laboratory, prion infection is usually achieved through intracerebral inoculation of mice in vivo followed by monitoring of clinical signs over several months to years, faithfully recapitulating the disease seen in humans.
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PMC10158546
However, in vivo models are long, laborious, costly, and wildtype mice are often infected with synthetic prion isolates that have been adapted to the host through repeated serial passaging .
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PMC10158546
Synthetic mouse-adapted prion strains are needed because of transmission barriers that are mainly attributed to differences between the amino acid sequence of host-encoded PrP and PrP present in the inoculum .
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PMC10158546
As such, specialized transgenic mice that express the PrP version of the natural host are often required to overcome transmission barriers and study naturally occurring prion isolates .
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PMC10158546
Alternatively, bank voles are considered a ‘universal prion acceptor’ because they are susceptible to a wide range of prion isolates including those from humans .
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PMC10158546
Nonetheless, in vivo mouse and other animal models enable correlation of neuropathological changes with onset of clinical signs and are considered the ‘gold standard’ prion bioassay.
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PMC10158546
In contrast, development of in vitro models for prion infection has proven much more challenging.
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PMC10158546
A lack of accessible and useful in vitro models of prion infection has hampered progress towards identifying links between prion replication and toxicity, screening putative therapeutics, and characterizing prion strain diversity.
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PMC10158546
This is particularly true for human prion isolates that have been notoriously difficult to culture.
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PMC10158546
Indeed, despite numerous efforts over the years, an immortalized neuron-like cellular model has never successfully replicated naturally occurring human prion isolates .
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PMC10158546
Certain cell culture systems that propagate prions are often used to examine PrP trafficking, but are mostly limited to isolates of mouse-adapted scrapie and usually do not reproduce prion toxicity .
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PMC10158546
In contrast to immortalized cell cultures, primary neuronal cultures can be used in the study of acute prion toxicity but do not have the longevity required for prion replication .
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PMC10158546
Cultured organotypic brain sections have proven to be one of the best in vitro models of prion infection because following several weeks of PrP replication, they reproduce the pathological hallmarks of the disease [14–16].
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PMC10158546
However, this system requires a steady supply of live animals, specialized equipment and a high level of technical expertise, reducing accessibility to researchers.
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PMC10158546
Thus, there is much room for improvement with respect to in vitro models of prion infection.
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PMC10158546
Cultures derived from differentiated neural progenitor cells have successfully been used to replicate prions in vitro and in some cases have reproduced prion toxicity [17–24].
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