從小鼠腦組織分離的突觸神經體之轉錄組學分析

Transcriptomics analysis of synaptoneurosomes isolated from mouse brain tissues

周偉倫(Wei-Lun Chou)

指導教授 ： 戴桓青

國立臺灣大學/理學院/化學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202000524

摘要

神經突觸在許多神經系統疾病的病因跟病理中扮演了重要的角色，包括阿茲海默症、帕金森氏症、精神分裂症等。其中，阿茲海默症是最常見的痴呆症類型，大約佔了百分之五十的案例，受到此疾病纏身的人，其神經元以及神經突觸會緩慢地受到損害，最後導致神經凋亡，進而影響到腦中的訊息傳遞。阿茲海默症的致病機制可能與三種蛋白質相關，分別為β-澱粉樣蛋白、Tau 蛋白和載脂蛋白，而這三種蛋白皆為神經突觸蛋白。
然而，除了突觸末梢蛋白質的異常表現之外，必定還有眾多的因素導致此疾病的產生，例如RNA分子，近年來，有越來越多的證據指出mRNA的定位過程和多種神經退化性疾病有關。但是大腦中的突觸非常小，直徑大約只有500奈米，也因此想要藉由傳統的螢光原位雜交技術鑑定定位於突觸周圍的mRNA是十分有難度的。因此，我們開發了一種方法，此方法是利用次世代定序科技去鑑定突觸末端的mRNA組成。我們透過簡單的沉澱法（粗製備法）製備含有較多突觸神經體的樣品，並通過蔗糖梯度離心或螢光激活突觸神經體分選的方式將粗製備法的樣品再進一步純化，以獲得高純度的突觸神經體。接著以高度敏感的轉錄組擴增試劑將它們的mRNA轉為cDNA，隨後我們會以qPCR初步檢驗cDNA的品質，確認之後會將樣品送給商業公司進行RNA定序，他們使用Illumina平台，以paired-end的方式測序，並且提供每個樣品至少約2000萬條reads，總共會有至少60億個base pair的數據量，用來確保足夠的定序深度。
根據定序結果做基因表現差異分析，我們發現在RNA level的實驗中，蔗糖梯度離心法的效果並不顯著，只有極少數的基因之表現量與粗製備法所獲得的樣品相比存在顯著差異，與前人以電子顯微鏡觀察到的現象不同。而螢光激活突觸神經體分選法以尺寸、雙重螢光標記篩選出突觸神經體，由基因表現差異分析得知，在近10000個基因中，約有7000個下調基因，3000個基因維持不變，於是我們利用生物資訊軟體分析這些未被螢光激活突觸神經體分選法排除的基因，發現這數千個基因與訊息的傳送以及神經退化性疾病的路徑相關，所以我們認為在突觸附近含有數千條mRNA。此外，我們也發現在突觸附近含有許多non-coding RNA，但其功能目前皆尚未明朗。

關鍵字

阿茲海默症 ； 突觸神經體 ； mRNA定位 ； 蔗糖梯度離心法 ； 螢光激活突觸神經體分選法 ； 次世代定序

並列摘要

The synapse plays a vital role in the pathogenesis and pathology of many neurological diseases, including schizophrenia, Parkinson’s disease, and Alzheimer’s disease (AD). Among them, AD is the most common type of dementia, accounting for over 50 percent of cases. AD is a neurodegenerative disease characterized by the loss of neurons and synapses. There are three major proteins that cause the loss of the neurons and synapses: Aβ, tau, and apolipoprotein E, and all of which are synaptic proteins.
However, the pathogenesis of AD is very complicated, and scientists firmly believe that many factors contribute to AD. Apart from protein abnormalities at synaptic terminals, the RNA molecules need to be taken into consideration in order to comprehensively study the pathogenesis of AD. Recently, there is increasing evidence indicates that the process of mRNA localization is related to multiple neurodegenerative diseases. However, because the size of the mammalian synapses is only about 500 nm in diameter, it is challenging to analyze mRNA localization in synapses by conventional fluorescence in situ hybridization (FISH) techniques. Therefore, we developed a method to identify the mRNA composition of synaptic terminals which utilizes next-generation sequencing (NGS) technology. From mouse cortical tissues, we prepared synaptically enriched biochemical fractions, synaptoneurosomes, by simple sedimentation (crude preparation) and did further purification by sucrose gradient centrifugation or fluorescence-activated synaptoneurosome sorting to obtain high-purity synaptoneurosomes. The crude synaptoneurosomes and purified synaptoneurosomes were then treated by highly sensitive transcriptome amplification kits to convert their mRNAs into cDNA and did qPCR to roughly check the cDNA quality. Afterwards, we submitted the samples for RNA sequencing. Using Illumina platform, they performed pair-end sequencing and provided each sample with at least 20 million reads (approximately 6Gb) to ensure sufficient sequencing depth.
According to differential gene expression analysis, we found that the enrichment effect of sucrose gradient centrifugation was not significant at the RNA level experiment, which was different from the previous studies based on electron microscopy. On the other hand, our method based on size and dual fluorescent labelling to sort the synapses did reduce the astrocyte and oligodendrocyte contamination. Among nearly 10,000 genes, about 7000 genes showed reduction after sorting and 3000 genes remained unchanged. We used bioinformatics software to analyze the 3000 genes and found that these genes were associated with signaling functions and neurodegenerative diseases. Thus, we believe that thousands of cortical synapses of mice mRNAs are localized around. In addition, we also found a lot of non-coding RNA around the cortical synapses, but their functions still remain unclear.

並列關鍵字

Alzheimer’s disease ； synaptoneurosome ； mRNA localization ； fluorescence-activated synaptoneurosome sorting ； sucrose gradient centrifugation ； NGS

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