胺活性交聯試劑在神經突觸體研究的應用

神經突觸在許多神經系統疾病，包括阿爾茲海默症、帕金森氏症、精神分裂症等的病因跟病理中扮演了重大角色。近年來，利用免疫細胞化學來研究孤立突觸體越來越受重視，因為它提供了透過消除來自樹突跟軸突的訊號來觀察最細緻的突觸變化。要利用免疫螢光法來研究神經突觸體，突觸蛋白的化學交聯是必須的。傳統上是使用福馬林(甲醛)來當作固定劑，但是它有著使突觸體聚集的缺點。我們利用對胺具有反應性的試劑來取代甲醛，在免疫螢光顯微鏡跟流式細胞術中都得到了更好的結果。此外，我們發展了可以研究突觸的RNA組成的方法。在一次實驗中，我們意外的發現，沒有經過任何處理的突觸體可以吸附在帶正電的玻璃表面上。我們認為這是靜電力的作用。在突觸表面上的唾液酸，天門冬胺酸和麩胺酸等胺基酸，以及帶負電的磷脂都會讓突觸表面帶負電。我們使用二(N-琥珀酰亞胺)乙烯乙二醇二琥珀酸酯(EGS)來取代甲醛，它擁有兩個N-羥基琥珀酰亞胺(NHS)脂可以跟離胺酸反映達到蛋白質交聯的效果。又當我們用甘氨酸來中止反應時，EGS可以增加突觸表面的負電荷，因此增加了靜電力作用，達到減少突觸體聚集並增強與玻璃表面之間的吸引力。基於上述發現，我們設計了一種簡單有效的方法來固定並捕捉突觸體以進行免疫螢光研究。利用這個方法我們能夠透過直接隨機光學重建顯微鏡(dSTORM)來得到三色超解析度成像。在研究突觸體的轉錄組學時，我們使用二硫代琥珀酰亞胺基丙酸脂(DSP)作為固定試劑。DSP一樣能增強突觸體的負表面電荷，預防在流式細胞術和分選流程中的突觸體聚集和離心損傷的現象。而DSP的分子中間具有可切斷的雙硫鍵，使固定具有可逆性。因此我們能夠從分選出來的突觸體中分離出mRNA並研究。

關鍵字

神經突觸體；二(N-琥珀酰亞胺)乙烯乙二醇二琥珀酸酯；二硫代琥珀酰亞胺基丙酸脂；超解析度螢光顯微鏡；流式細胞儀

並列摘要

The synapse plays important roles in the pathogenesis and pathology of many neurological diseases, including Alzheimer’s disease, Parkinson’s disease, schizophrenia, etc. In recent years, there is growing interest in immunocytochemistry studies of isolated synaptosomes, because it provides the ability to observe the subtle biochemical changes by eliminating signals from the shaft of dendrites and axons. To use immunofluorescence to study synaptosomes, chemical crosslinking of synaptic proteins is necessary. Commonly, formaldehyde has been used as the fixative to for crosslinking, but it has the apparent drawback of causing synaptosome aggregation. Here, we used amine-reactive crosslinking agents instead of formaldehyde, thereby improving the results in immunofluorescence microscopy and flow cytometry. Furthermore, we are able to develop a new strategy for studying synaptic transcriptomics. We accidentally discovered that crude synaptosomes adhered to positively-charged glass surfaces without any additional manipulation, due to electrostatic interaction. Acidic glycans (e.g. sialic acids), acidic protein side chains (Asp and Glu), and negatively charged phospholipids give synaptic surface a negative Zeta potential which is -15.0 ± 0.4 mV. We used ethylene glycol bis(succinimidyl succinate) (EGS) as an alternative to formaldehyde, which has two N-hydroxysuccinimidyl (NHS) esters that react with lysines and promote protein crosslinking. EGS can increase the negative charge of synaptic surfaces when combined with glycine quenching, thereby reducing synaptosomal aggregation and enhancing attraction between the synaptosomes and the glass surface. Based on these principles, we devised a very simple and effective method for fixing and capturing synaptosomes for immunofluorescence studies. We can carry out three-channel super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to simultaneously image tau, synaptophysin (pre-synapse) and PSD-95 (post-synapse). In studying the transcriptome of synaptosomes, we chose dithiobis(succinimidyl propionate) (DSP) as the fixative due to its reversible nature. DSP also enhances negative surface charges of synaptosomes to prevent synaptosome aggregation in the flow cytometry and sorting protocol. DSP has a cleavable disulfide bond in its spacer arm, which makes fixation reversible. Thus, DSP fixation way allow mRNAs to be isolated from sorted synaptosomes after reducing disulfide bonds.

並列關鍵字

Synaptosome ； EGS ； DSP ； super-resolution microscopy ； flow cytometry ； tau

參考文獻

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胺活性交聯試劑在神經突觸體研究的應用

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