研究Tau的結構與在神經退化性疾病中的細胞轉移現象

Abstract

阿茲海默症是一種慢性神經退化性疾病，並佔失智症病例的60-70%，乙型類澱粉蛋白 (Amyloid β) 聚集而成的老年斑塊以及tau蛋白不正常堆積形成的神經纖維糾結 (Neurofibrillary tangles, NFTs) 是阿茲海默症的兩大病理特徵。Tau蛋白具有穩定微管的功能，但不正常折疊的tau會脫離微管並堆積沉澱形成神經纖維糾結，先前的研究證明突變型ΔK280相較於野生型 (wild-type) 的tau更易形成纖維狀沉積，近期的研究也指出tau蛋白和prion蛋白相似具有在細胞間轉移的性質，不正常tau蛋白的轉移會造成疾病的惡化。本研究透過對tau單體與纖維狀沉積物進行分析，了解野生型與突變型ΔK280在結構上的差異，此外為了進一步了解野生型與突變型ΔK280在細胞轉移上的差異，本研究也建立一個平台可以分析tau蛋白轉移的現象，該平台利用螢光共振能量轉移 (Fluorescence resonance energy transfer, FRET) 偵測tau的交互作用，或是以流式細胞儀分析tau在細胞間的轉移率，而為了進一步研究O-GlcNAcylation對tau的影響，我們也完成初步O-GlcNAc tau純化系統的建立。我們的實驗結果指出在tau單體的結構上突變型ΔK280較野生型鬆散，而在細胞實驗中也發現突變型ΔK280較野生型更易在細胞間轉移，此外加入tau的纖維種子 (fibril seeds) 也會增加tau蛋白的轉移作用。了解tau在細胞間的轉移特性以及結構上的差異可以幫助我們對tau的病理機制有進一步的認識，並且對於減緩阿茲海默症的惡化提供可能的治療方向。

Alzheimer’s disease (AD) is a chronic neurodegenerative disease. It is known that β-amyloid plaques and tau neurofibrillary tangles (NFTs) are two main hallmarks of AD. Tau protein is a soluble microtubule-associated protein that stabilizes microtubules under normal physiological conditions. In brain of AD patients, tau becomes abnormally hyperphosphorylated and loses its ability to bind microtubules. The detached tau accumulates and aggregates into insoluble NFTs. Previous studies indicated ΔK280 mutant tau has higher tendency to form aggregates. Another research found tau protein transmits from cell to cell as prion-like behavior. Abnormal tau spreads throughout the brain may lead to disease progression. In this study, we investigated the structural differences between wild-type (WT) and ΔK280 tau in monomer or aggregated tau. To further study cellular transmission properties of WT and ΔK280 tau, we established a cell-to-cell tau transmission platform. Forster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) was used to confirm interaction of tau and flow cytometry for tau transmission rate. In order to investigate the effect of O-GlcNAcylation on tau in the future, we also established an O-GlcNAc tau purification protocol. In this study, we found ΔK280 tau monomer is structurally looser than WT tau. Furthermore, ΔK280 mutant tau has higher transmission propensity, and tau fibril seeds may increase tau transmission. Characterization of tau transmission and structure can help us to understand more about tau pathology and could provide a possible therapeutic strategy to slow down AD progression.