可裂解之交聯試劑在突觸神經體分選及RNA偵測之應用

The application of a cleavable crosslinking reagent in synaptoneurosome sorting and RNA detection

林書煒(Shu-Wei Lin)

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/NTU202000436

摘要

突觸的功能障礙被認為在許多神經性疾病的病理中扮演重大的角色，其中阿茲海默症（Alzheimer’s disease）是最常見的失智症，其主要特徵為β-類澱粉蛋白（amyloid beta，Aβ）的沉積斑塊和被過度磷酸化後的tau蛋白異常堆積形成的神經纖維糾結（neurofibrillary tangles，NFTs），導致神經突觸的損失，造成認知功能退化。近年來越來越多證據顯示，在正常的腦中，mRNA在突觸中的定域化（localization），使特定蛋白質能在突觸局部快速被轉譯，這樣的機制對突觸可塑性（synaptic plasticity）和記憶鞏固（memory consolidation）至關重要。然而，目前還沒有一套有力的方法來研究突觸中RNA的組成。
突觸神經體（Synaptoneurosome）是被使用來研究突觸功能的一種重要模型，它是由突觸終端被分離並重新閉合的顆粒，其仍保有大部分神經末梢的特徵功能。為了研究突觸的RNA組成，我們發展了一套能成功得到高純度突觸神經體的純化法，此方法的關鍵在於使用具有雙硫鍵結構的交聯試劑3,3’-二硫代二丙酸二（N-琥珀醯亞胺）酯（Dithiobis(succinimidyl propionate)，DSP）進行可逆式的固定作用，實驗結果發現其可以避免傳統以甲醛固定造成突觸神經體聚集的現象，以DSP的固定為基礎，我們優化了對突觸神經體進行免疫染色的流程，並成功應用於流式細胞術（flow cytometry）的偵測及分選，最後根據流式細胞術測得之側向散射訊號作為顆粒大小的指標，加上對前後突觸的標誌蛋白進行染色而得的螢光訊號作為分選的三重條件，收集獲得高純度的突觸神經體以進行後續RNA的分析。
由於DSP分子中間為雙硫鍵結構，使用還原劑解開即可解開其固定效果，我們優化了DSP固定以及三（2-羧乙基）膦（tris(2-carboxyethyl)phosphine，TCEP）切割的反應條件，在分選得到高純度的突觸神經體後，再利用TCEP將DSP之雙硫鍵切斷，便能將突觸神經體裂解並從中萃取出含有多腺苷酸（polyadenylic acid）的RNA，再以定量聚合酶連鎖反應（quantitative polymerase chain reaction，qPCR）進行分析。我們在每次實驗中能夠回收數百萬個突觸神經體，並藉由qPCR確認了其中突觸RNA如Actb（beta-actin）和Arc（activity-regulated cytoskeleton-associated protein）的表現。

關鍵字

阿茲海默症 ； 突觸神經體 ； 3,3’-二硫代二丙酸二（N-琥珀醯亞胺）酯 ； 流式細胞術 ； 定量聚合酶連鎖反應

並列摘要

Synaptic dysfunction is thought to play important roles in the pathology of many neurological diseases. Alzheimer’s disease is the most common type of dementia. The pathological hallmarks of Alzheimer’s disease are the presence of the deposition of senile plaques which consist of beta-amyloid peptide and the neurofibrillary tangles composed of abnormal aggregates of hyperphosphorylated tau, which lead to the loss of synapses and the cognitive disorders. In normal brains, there has been increasing evidence showing that mRNAs localized at the synapses allow the rapid production of specific proteins through local translation, which critically contributes to synaptic plasticity and memory consolidation. However, there is no effective method to study the RNA composition of synapses.
Synaptoneurosomes have become one of the important model systems for studying synaptic functions, which are the detached-and-resealed synaptic terminals remaining most of the functional characteristics of nerve terminals. To study the RNA composition of the synapse, we devised a novel purification method for obtaining high-purity synaptoneurosomes. The key to this method is the use of the cross-linking reagent dithiobis(succinimidyl propionate) (DSP), which contains a disulfide linker to carry out the reversible fixation. Results showed that DSP helps circumvent the aggregation of synaptoneurosomes frequently induced by formaldehyde. Based on the DSP fixation, we optimized the protocols for the immunostaining of synaptoneurosomes and their application in flow cytometry and sorting. According to the side scatter signals measured with the flow cytometer as an indicator of particle size and the fluorescent signals obtained by immunostaining the presynaptic and postsynaptic marker proteins, we can collect highly purified synaptoneurosomes for subsequent RNA analysis.
Owing to the disulfide structure in DSP, the fixation can be reversed by reducing agents. We optimized the reaction conditions of DSP fixation and Tris(2-carboxyethyl)phosphine (TCEP) cleavage. After sorting, we can unfix the purified synaptoneurosomes and extract poly (A)-containing RNAs for quantitative polymerase chain reaction (qPCR) analysis. In a typical experiment, we can recover several million synaptoneurosomes, and the presence of synaptic RNAs Actb and Arc are confirmed by qPCR.

並列關鍵字

Alzheimer’s disease ； synaptoneurosome ； dithiobis(succinimidyl propionate) ； flow cytometry ； quantitative polymerase chain reaction

參考文獻

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