以質譜分析阿茲海默症小鼠模型中tau蛋白過磷酸化

阿茲海默症是最為常見的神經退化性老年失智症，而由Aβ胜肽堆積形成的斑塊以及tau蛋白堆積形成的神經纖維糾結為該病症最主要的病理特徵。其中，發生於新皮層的Aβ胜肽病變通常會早於tau蛋白的病變，然而兩者之間的關聯性是最為關鍵但尚未能解決的議題。在先前的研究中我們觀察到：患有阿茲海默症的大腦中在神經突觸的位置有過磷酸化並且錯誤折疊的tau蛋白沉積物。於此研究中，主要探討的議題為Aβ胜肽堆積是否會引發神經突觸中的tau蛋白過磷酸化產生病變，因此我們使用有大量斑塊而沒有神經纖維糾結的APP/PS1老鼠模型來進行研究。為了要進行磷酸化蛋白質體的分析，我們從老鼠的大腦中分離出突觸體，並利用固定化金屬離子親和層析(IMAC)將磷酸化胜肽進行濃縮。接著，我們使用無標記的液相層析串聯式質譜儀定量技術(label-free LC-MS/MS)對tau蛋白不同的磷酸化位置進行定量。在控制組和APP/PS1的老鼠中，鑑定出19個tau蛋白的磷酸化位置，其中有16個在人類tau蛋白序列中存在有相對應之磷酸化位置。比較兩組老鼠整體的磷酸化結果可發現APP/PS1老鼠整體磷酸化現象比起控制組有些微上升，而其中有8個位置磷酸化增加的量在統計上有顯著的差異。這些在磷酸化上有顯著增加的位置主要位於微管結合結構域(microtubule binding domain)的附近，可能因此增加tau蛋白與微管表面之間的電子斥力。為了進一步地去確認這8個磷酸化的結果，我們利用免疫墨點法語質譜之數據相比較，發現：在細胞溶質中有5個位置 (T181, S199/S202, S396/S404) 有過磷酸化，而在突觸體中有2個位置 (S199/S202) 有過磷酸化。同時我們也檢驗了一般認為和tau蛋白的調控最有關聯的其中一個激酶(GSK3β)以及磷酸酶(calcineurin Aα)之修飾或含量，皆未發現顯著的變化。我們的數據表明，Aβ所引起突觸的tau蛋白病變可能是起始於tau蛋白中S199/S202位置的磷酸化，而該位置可能是早期生物標誌物檢測或治療性干預的重要潛在目標。

關鍵字

阿茲海默症； tau蛋白；質譜分析；過磷酸化

並列摘要

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.

並列關鍵字

Alzheimer disease ； tau protein ； hyperphosphorylation ； mass spectrometry

參考文獻

30. Hanger, D.P., and Noble, W. (2011). Functional implications of glycogen synthase kinase-3-mediated tau phosphorylation. Int. J. Alzheimers Dis. 2011, 352805.

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以質譜分析阿茲海默症小鼠模型中tau蛋白過磷酸化

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