Title

使用螢光顯微鏡和流式細胞儀分析神經突觸中tau蛋白分布

Translated Titles

Analysis of synaptic tau protein localization by immunofluorescence microscopy and flow cytometry

DOI

10.6342/NTU.2014.00449

Authors

王柏堯

Key Words

神經突觸 ; tau蛋白 ; 螢光顯微鏡 ; 流式細胞術 ; 阿茲海默症 ; synapse ; Alzheimer disease ; tau ; flow cytometry ; immunofluorescence microscopy

PublicationName

臺灣大學化學研究所學位論文

Volume or Term/Year and Month of Publication

2014年

Academic Degree Category

碩士

Advisor

戴桓青

Content Language

英文

Chinese Abstract

阿茲海默症病患的神經纖維糾結的聚集在病人的腦中顯示會沿著腦的神經網路傳遞和蔓延,這暗示著tau 蛋白所造成的疾病可能是藉由穿透神經突觸而傳遞。Tau 蛋白傳遞理論最重要的觀察是錯誤摺疊的tau 蛋白會在神經突觸中發現,不論是神經前突觸或視神經後突觸中。一般來說tau 蛋白是存在於軸突中的蛋白,然而廣為科學家接受的概念是在阿茲海默症病患腦中的tau 蛋白會會因為錯置或是傳遞而出現在神經樹突中。在我們的實驗中成功的從阿茲海默症病患腦組織分離出完整的神經突觸和單獨的神經前突觸和神經後突觸。使用螢光顯微鏡觀察錯誤摺疊的tau 蛋白在完整神經突觸中的份部比例為15.4%、16.4%、2.9% (神經前突觸、神經後突觸、神經突觸的雙邊)。觀察多重磷酸化tau蛋白在完整神經突觸中的份部比例為23.1%、26.9%、3.8%。觀察所有形式的tau 蛋白在完整神經突觸中的份部比例為34.6、47.1、3.8。對一般人和阿茲海默症病患的神經突觸中所有形式tau蛋白染色結果顯示兩者的分布相似,而且在一般人的腦中可以觀察到微量的多重磷酸化tau 蛋白和錯誤摺疊的tau 蛋白。在神經突觸中我們也觀察到抗十二烷基硫酸钠的寡聚物。經由以上的觀察我們認為tau 蛋白並不是錯置,而是神經前突觸和神經後突觸吸收的多重磷酸化、錯誤摺疊或是寡聚型態的tau 蛋白。因此我們也提出了兩種可行的傳遞途徑。此後我們也著力於發展以流式細胞儀分析神經突觸中tau蛋白分布的技術,現階段我們分析得神經前突觸和神經後突觸占所有分析物比例為35.2%和33.5%,這樣的結果和我們以螢光顯微鏡的觀察結果是一致的。

English Abstract

The accumulation of neurofibrillary tangles in Alzheimer disease (AD) propagates with characteristic spatiotemporal patterns following brain network connections, which may imply trans-synaptic transmission of tauopathy. A prerequisite of this transmission theory would require misfolded tau to accumulate at synapses—i.e. that it be present in both pre- and post-synaptic locations. However, tau is thought to normally be primarily an axonal protein, and it is widely hypothesized that tau is mislocalized or mistrafficked to the neuronal somatodendritic compartment in AD. We isolated intact, bipartite synapses from cortical tissues of AD subjects and detected misfolded tau by immunofluorescence microscope with a distribution ratio of 15.4%:16.4%:2.9% (presynaptic-only/postsynaptic-only/both), while hyperphosphorylated tau exhibited a ratio of 23.1%:26.9%:3.8%; total tau (any form) exhibited a ratio of 34.6%:47.1%:3.8%. Non-demented controls showed total tau distribution similar to that of AD subjects, but with little phosphorylation or misfolding. In AD-affected synapses, we observed tau misfolded into SDS-resistant oligomers. Thus tau appears not to be mislocalized, but instead adopts misfolded, oligomeric, and phosphorylated forms within both pre- and post-synaptic sites. Based on these observations, we propose two models for the transmission of misfolded tau at synapses. We are also develop a high throughput method to analyze tau protein localization in synaptic terminals. We analyzed synaptosomes of mouse brains and immunostaining for pre- and post-synaptic terminals with synaptophysin and PSD-95 antibody. We found 35.2% and 33.5% of total particles to be pre- and post-synaptic terminals, respectively consistent with immunofluorescence counting under the microscope.

Topic Category 基礎與應用科學 > 化學
理學院 > 化學研究所
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