| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 102 === The pathological hallmarks of Alzheimer’s disease (AD), the most common cause of neurodegenerative senile dementia, are aggregates of Aβ peptide (plaques) and aggregates of tau protein (tangles). The link between Aβ pathology and tau pathology is a critical but unresolved issue, with the former generally preceding the latter in the neocortex. We previously observed that neuronal synapses in AD-affected brains showed deposits of hyperphosphorylated and misfolded tau protein. In this study, we used the APP/PS1 mouse model (high plaque burden, no tangles) to study if Aβ aggregation may induce tau hyperphosphorylation inside synapses to trigger tauopathy. We isolated synaptosomes from mouse brains for phosphoproteomics analysis, using immobilized metal ion affinity chromatography to enrich all phosphopeptides. Then we used label-free liquid chromatography-tandem mass spectrometry method to quantify phosphorylation sites on tau protein. In both APP/PS1 and control mice, 19 tau phosphosites were consistently identified in mouse tau protein, 16 of which are conserved in human tau. The overall pattern suggests a subtle increase in phosphorylation in APP/PS1 mice compared to wild type littermates, with 8 sites showing statistically significant increased level. These upregulated sites are located in flanking regions of the microtubule binding domain, possibly increasing electrostatic repulsion between tau and the microtubule surface. Further confirmation using immunoblotting verified hyperphosphorylation at 5 of these sites in the cytosol (T181, S199/S202, S396/S404), and 2 sites in synaptosomes (S199/S202). We also checked a kinases (GSK3β) and a phosphatase (calcineurin Aα) commonly associated with tau regulation but found no significant changes in their levels or modifications. Our data suggest that synaptic tau pathology induced by Aβ may be initiated by phosphorylation in the central region of the protein (S199/S202), which may be a potential target for early biomarker detection or therapeutic intervention.
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