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PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The human neuroblastoma cell line SK-N-BE was grown at 37 °C and 5% CO2 in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (100 U/mL; 100 μg/mL).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The cells were differentiated into neuronal-like cells with 10 μM retinoic acid (RA) (Sigma–Aldrich, Darmstadt, Germany, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. R2625) as previously described .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The cell line used in this study is a well-established model, routinely employed in our laboratories, and extensively characterised in previous studies also by our group .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The differentiation status of the cells was carefully monitored during RA exposure, with neuronal-like features becoming evident within a few days of treatment (see Supplementary Figure S1).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Immunodetection of specific antigens was performed via the following primary antibodies: Tau-1 (Millipore, Single Oak Dr, Temecula, CA, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. MAB3420; dilution 1:100) for unphosphorylated tau (Pro189/Gly207); AT8 (Thermo Scientific, Rockford, IL, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. MN1020; dilution 1:50) for pSer202/Thr205 tau; PHF-1 (Invitrogen, Rockford, IL, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. 44-758G, dilution 1:100) for pSer396/Ser404 tau; T181 (Invitrogen, Rockford, IL, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. MN1050; dilution 1:100) for pThr181 tau; S262 (Invitrogen, Rockford, IL, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. 44-750G; dilution 1:100) for pSer262 tau; Ki67 (Invitrogen, Rockford, IL, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. MA5-14520; dilution 1:100) as a cell proliferation marker; UBTF (Novus Biologicals, Briarwood Avenue, Centennial, CO, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
N. NBP1-82545; dilution 1:100) to highlight the nucleolus; anti-fibrillarin (Clone 38F3; Invitrogen, Rockford, IL, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. MA1-22000; dilution 1:800) for nucleolar activity; and anti-acetylated histone H4 (H4Ac, Santa Cruz Biotechnology, Dallas, TX, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. sc-377520; dilution 1:50) for nuclear chromatin status.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The secondary antibodies used included FITC-conjugated anti-mouse (Sigma–Aldrich, Saint Louis, MO, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. F6257; dilution 1:300), FITC-conjugated anti-rabbit (Sigma–Aldrich, Saint Louis, Missouri, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. F4890; dilution 1:100), and TRITC-conjugated anti-rabbit (Sigma–Aldrich, Saint Louis, Missouri, USA, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. T6778; dilution 1:400) antibodies.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
While some antibodies have been reported to be non-specific , this was excluded because the antibodies were validated by Federico et al. (2018) through Western blot analyses, including the TAU-5 antibody (total tau).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Indirect immunofluorescence (IIF) experiments were performed as previously described .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Briefly, SK-N-BE cells were cultured on glass chamber slides, fixed with 4% paraformaldehyde for 20 min at room temperature, and permeabilised with 0.5% Triton X-100 (Chemsolute, Hamburg, Germany, Cat.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No. 8059.0500).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Immunodetection was carried out by overnight incubation with the specific primary antibody, followed by incubation with the corresponding secondary antibody.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The cell nuclei were stained with DAPI (blue).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Images were acquired via a confocal laser scanning microscope (CLSM) (LSM700, Zeiss, Oberkochen, Baden-Württemberg, Germany) equipped with 40× and 63× objectives and ZEN 2010 software.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Human brain sections were obtained in an anonymised form from the Tissue Biobank of the Hospital Universitario Fundación Alcorcón (HUFA), C/Budapest 1, 28922 Alcorcón, Madrid, Spain.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
All experiments were performed in accordance with the Declaration of Helsinki and approved by the Clinical Research Ethics Committee of Hospital Universitario Fundación Alcorcón (no. 23/69, on 14 June 2023).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The brain tissue sections, which were prepared as previously described , were obtained from the Cornu Ammonis 1 (CA1) region of foetuses, young, and elderly individuals with no Alzheimer’s disease (AD) diagnosis (Supplementary Table S1).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
These subjects had not received previous radiation or chemotherapy.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Brain tissues from individuals with a post-mortem diagnosis of AD were classified into severity grades (AD-I to AD-IV) according to Braak’s classification .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Pyramidal neurons in the CA1 region of the hippocampus were identified based on morphological criteria assessed by DAPI staining, including: (i) localisation within the pyramidal layer, (ii) large euchromatin-rich nuclei, and (iii) a prominent, centrally positioned nucleolus (Supplementary Figure S2).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
These features reliably distinguish neurons from glial cells, which exhibit smaller nuclei with dense heterochromatin and lack a distinct nucleolus .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Paraffin-embedded sections for IIF experiments were processed as previously described .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Briefly, human tissue sections were deparaffinised in xylene, rehydrated in graded alcohols, and treated with citrate buffer and Sudan Black to reduce autofluorescence.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The samples were incubated overnight at 4 °C with a specific primary antibody, followed by incubation with a secondary antibody and staining with DAPI.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Each IIF experiment was repeated at least three times.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Images were acquired via CLSM (LSM700, Zeiss, Oberkochen, Baden-Württemberg, Germany) equipped with 40× and 63× objectives and ZEN 2010 software for image acquisition.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Cell counting for statistical analyses was performed as previously described .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Specifically, IIF-positive cells were counted in 0.5-μm scanned images acquired via CLSM.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The number and percentage of cells showing specific IIF signals were determined in six optical fields at 400× magnification per experiment.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The quantitative detection of the AT8 epitope was performed via fluorescence intensity profiles using ZEN-2010 software.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Statistical analyses were conducted via Prism v. 8.0 (GraphPad Software, San Diego, CA, USA).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Normality was tested with the Kolmogorov–Smirnov test, and significant differences between pairs of groups (epitope-positive cells vs. controls) were assessed via Student’s t-test.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The data are presented as the mean ± standard error of the mean (S.E.M.), with significance levels set at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The tau protein contains multiple phosphorylatable sites, giving rise to various epitopes.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
We analysed the AT8 and Tau-1 epitopes, representing the unphosphorylated (Tau-1) and the phosphorylated (AT8) condition of the same protein region, Pro189/Gly207 and pSer202/Thr205, respectively, and the PHF1, T181, and S262 epitopes, which correspond to hyperphosphorylated tau, tau phosphorylated at threonine 181, and tau phosphorylated at serine 262, respectively.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
IIF was performed on neuroblastoma cells (replicative and neuronal-like differentiated conditions), and in CA1 brain tissue sections from subjects of different ages and at different AD stages.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
In untreated replicating SK-N-BE cells, the Tau-1, PHF1, T181, and S262 epitopes were detected in the nucleolus, whereas AT8 was absent (Figure 1, upper images).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
However, in cells differentiated with retinoic acid (Supplementary Figure S1), all analysed epitopes were observed in the nucleolus (Figure 1, bottom images).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
For Tau-1 and AT8, the present results are consistent with previous findings .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The nucleolar localisation of tau epitopes was further confirmed by co-localisation of AT8 and Tau-1 with the nucleolar marker upstream binding transcription factor (UBTF) (Figure 2).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Tau-1 and AT8 were present in nearly all nucleoli, with fewer than 5% of nuclei lacking detectable signal.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Thus, among the five nuclear tau epitopes analysed, AT8 was the only one showing a clear presence/absence pattern between differentiated and replicative SK-N-BE cells.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The nuclear localisation of the Tau-1, AT8, PHF1, T181, and S262 epitopes was analysed in CA1 neurons in brain sections from foetal, young, and aged individuals, as well as from AD patients at stages I and IV of disease progression (Figure 3).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
We observed the presence of all epitopes in the nucleus of CA1 neurons with an age-dependent decrease in the percentage of cells positive for the analysed epitopes.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
More precisely, Tau-1, PHF1, T181, and S262 epitopes showed a gradual decrease in the percentage of positive cells from foetus to senile ages and AD stages.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No statistically significant differences were detected when compared with the young brain.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
For the AT8 epitope, we observed the highest percentage of AT8-positive neurons in young subjects.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Compared to this condition, the percentage significantly decreased in aged samples (p < 0.01) and even more markedly in AD samples (p < 0.0001).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No statistically significant differences were detected for Tau-1, PHF1, T181, or S262 in any group compared to the young brain (Figure 3).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
A summary table (Supplementary Table S2) provides a comparative overview of AT8, Tau-1, PHF1, T181, and S262 nuclear positivity in SK-N-BE cells (proliferative and differentiated) and in human hippocampal tissue across different ages and AD stages.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Considering the unique behaviour of AT8 compared with the other analysed epitopes, both in neuroblastoma cells (Figure 1 and Figure 2) and in CA1 brain sections (Figure 3), we further investigated its presence in CA1 neurons in more detail, including stages II and III of AD (Figure 4).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Quantitative analysis confirmed that the percentage of AT8-positive neurons in AD brains was markedly reduced (p < 0.0001) to ~26% in AD samples, compared to ~68% in young and ~50% in senile individuals.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
No significant differences were observed among the three senile samples or among the four AD stages.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
All elderly samples showed a significant reduction (p < 0.01) compared to young samples, and all AD stages confirmed the strongest reduction (p < 0.0001) compared with both young and senile individuals (Figure 4).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Brain sections from individuals of different ages and at various stages of AD progression were analysed for the presence of cells with an active cell cycle using Ki67 as a marker.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Specifically, Ki67 and the AT8 epitope of nuclear tau were co-detected by IIF (Figure 5).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The results revealed a highly significant increase in the number of Ki67-positive cells in senile and AD brain sections compared to young subjects (p < 0.0001).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
In young brain sections, Ki67-positive neurons were rare (less than 3%), whereas their frequency increased in senile (23%) and AD (38%) brain sections.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Beyond the variation in the number of Ki67-positive cells, differences were also observed in Ki67 signal intensity within individual cells (Figure 5).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
In young neurons, Ki67 was detected at very low levels (<10 fluorescence intensity units, measured with ZEN2010 software), while AT8 intensity was consistently higher (>50).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
In senile neurons, Ki67 intensity increased (>20), along with an even higher AT8 signal (>80).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
In AD neurons, Ki67 reached its highest levels (>50), becoming comparable to AT8 intensity.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Importantly, all Ki67-positive neurons also exhibited nuclear AT8 immunoreactivity, and no cells displayed Ki67 signal in the absence of AT8, suggesting a strict association between aberrant cell cycle re-entry and AT8-positive tau in the nucleus.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The transcriptional status of neurons in the CA1 region was assessed through the detection of fibrillarin and acetylated histone H4 (H4Ac) in the nucleus.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The results revealed a gradual and statistically significant age-related decrease in fibrillarin levels in the nucleolus of neurons.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
However, no significant differences were observed between senile and AD subjects (Figure 6).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
With respect to H4Ac detection, the number of H4Ac-positive cells did not differ significantly across groups.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
However, notable variations were observed in the nuclear distribution of H4Ac: in foetal and young neurons, the signal was confined to relaxed chromatin regions, whereas in senile and AD neurons, it appeared more diffusely distributed throughout the nucleus (Figure 7).
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Extensive knowledge exists concerning the cellular and tissue changes that occur in the hippocampal region and are associated with Alzheimer’s disease (AD), such as the accumulation of neurofibrillary tangles with hyperphosphorylated tau protein and beta-amyloid deposits .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
However, the initial triggers of neurodegeneration in AD remain elusive.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The tau protein and its post-translational modifications, particularly hyperphosphorylation, play significant roles in neuronal dysfunction and death.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
The amount and type of neurofibrillary tangles observable in the hippocampus are key indicators of disease progression .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Tau, encoded by the microtubule-associated protein tau (MAPT) gene, is known primarily for stabilising microtubules, but increasing evidence suggests that it also has nuclear functions in chromatin organisation and protection .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Among the nuclear epitopes of tau, Tau-1 and AT8 are particularly relevant for its interaction with DNA.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
These epitopes, which mark the same protein region but in different phosphorylation states, are linked to structural changes that could influence the chromatin-binding properties of tau.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
AT8 phosphorylation at Ser202/Thr205 is particularly important for nuclear tau function, suggesting roles in maintaining nuclear integrity and transcriptional regulation .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
While AT8 shows marked and progressive nuclear changes with ageing and AD, other phosphorylated tau epitopes such as T181, S262, and PHF1 also localise to the nucleus and may regulate nuclear processes.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
This suggests that nuclear tau regulation and involvement in cell cycle re-entry are controlled by a coordinated phosphorylation programme across multiple sites.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Therefore, AT8 could serve as a key marker within a complex phosphorylation landscape modulating nuclear functions and neuronal vulnerability.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Present and previous data indicate that the AT8 epitope is absent in proliferative neuroblastoma cells but appears upon differentiation or actinomycin-D (Act-D) treatment, supporting its association with reduced transcriptional activity .
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Other tau epitopes (Tau-1, PHF1, T181, and S262) do not exhibit this pattern, reinforcing the specificity of AT8 in the function of nuclear tau.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Therefore, although differentiation and transcriptional blockade are mechanistically distinct, both conditions reduce nucleolar activity and may promote the accumulation or redistribution of tau within the nucleus.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
These observations support the hypothesis that nuclear tau responds dynamically to nucleolar stress or changes in transcriptional homeostasis.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Although in the present study we compared proliferative and differentiated conditions as distinct experimental endpoints, a more detailed time-course analysis of SK-N-BE differentiation could provide valuable insights into the dynamics of tau nuclear localization.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
Future investigations should therefore include longitudinal differentiation studies to better capture the temporal progression of nuclear AT8 accumulation and redistribution.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
In human pyramidal neurons of the hippocampal CA1 region across different ages and AD stages, we observed a significant decline in nucleolar AT8-positive neurons from youth to senility, with a further marked decrease in AD cases.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
This progressive loss parallels changes in fibrillarin and histone acetylation, indicating alterations in transcriptional regulation and chromatin organisation during ageing and neurodegeneration.
PMC12650037
Loss of AT8 Nuclear Tau as a Marker of Neuronal Ageing and Alzheimer’s Disease Progression
AT8 phosphorylation at Ser202/Thr205 may influence chromatin structure by modulating tau’s interaction with nuclear proteins involved in transcriptional regulation, DNA repair, or nucleolar integrity, suggesting that specific nuclear partners of AT8-phosphorylated tau—such as fibrillarin, histone-modifying enzymes, and transcription factors—could mediate the observed effects on chromatin organisation and nucleolar function.