使用螢光顯微鏡和流式細胞儀分析神經突觸Apo E載脂蛋白分布

Analysis of synaptic Apolipoprotein E localization by immunofluorescence microscopy and flow cytometry

鄒怡鳳(Yi-Fong Zou)

指導教授 ： 戴桓青

國立臺灣大學/理學院/化學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601390

摘要

阿茲海默症是一種神經退化疾病，佔老人失智症中五成以上的成因。其主要特徵為β-類澱粉蛋白錯誤折疊和tau蛋白異常堆積、神經元凋亡和神經突觸喪失。比起神經細胞死亡，阿茲海默症患者腦中神經末端的失去和認知功能退化更有相關性。載脂蛋白Ｅ的Ɛ4對偶基因是罹患阿茲海默症的危險因子，載脂蛋白Ｅ會增加腦中β-類澱粉蛋白堆積並且被認為是引發神經毒性的開端，導致神經細胞退化與死亡。載脂蛋白Ｅ在突觸的分佈或許跟阿茲海默症的病理有關，因此我們希望能直接分析完整的突觸體中載脂蛋白Ｅ的分佈。我們發展出一套高通量分析的方法，藉由流式細胞術及螢光顯微鏡，我們可以直接分析神經突觸前端及後端。
我們的策略是藉由異性電荷相吸來捕捉神經突觸。神經突觸細胞膜表面富含帶負電的磷脂,再加上帶負電的多醣鏈和醣脂使得神經突觸在生理條件下帶負電。我們在突觸表面修飾二(N-琥珀醯亞胺)乙烯乙二醇二琥珀酸酯 (EGS)。EGS是一個具有12個原子長的分子，同時兩端具有和一級胺有高度反應性的N-琥珀醯亞胺，在這邊扮演兩個重要的角色。第一，EGS會交聯蛋白質間的氨基以達到突觸固定。第二，EGS和突觸表面的氨基反應後會消除一個正電荷，且EGS的其中一端N-琥珀醯亞胺水解後會帶負電。綜合EGS的修飾，加上細胞膜表面原本就帶負電的磷脂和多聚醣，使得突觸表面的淨負電荷增加。
經由EGS反應過後的神經突觸更容易因靜電力而被表面有3-氨基丙基三乙氧基矽烷 (APTES) 處理的玻璃捕捉。吸附在表面的突觸體可以在溫和的條件下進行免疫染色，染完色後再使用螢光顯微鏡或超解析螢光顯微鏡觀察。這個捕捉的策略使得我們能以最少樣品量做突觸分析。同時，突觸表面的負電荷也避免突觸間的聚集及減少在流式細胞術的實驗步驟中離心破壞的現象。
綜合流式細胞術及螢光顯微鏡下的分析，結果一致證明載脂蛋白Ｅ是一個神經突觸蛋白，同時位於完整的神經突觸和單獨的神經前突觸和神經後突觸，並且平均分佈於前後突觸。

關鍵字

阿茲海默症 ； 突觸體 ； 二(N-琥珀醯亞胺)乙烯乙二醇二琥珀酸酯 ； 3-氨基丙基三乙氧基矽烷 ； 螢光顯微鏡 ； 流式細胞術

並列摘要

Alzheimer’s disease (AD), a protein misfolding disorder of beta amyloid (Aβ) and tau, accounts for > 50% of cases of senile dementia. AD is a neurodegenerative disease characterized by the loss of neurons and synapses. Loss of synapses in the neocortex and limbic system correlates strongly with cognitive impairment, more than other known pathological markers. The ApoE ε4 allele is known to be a very strong genetic risk factor for developing AD. ApoE4 increases the level of Aβ oligomers in the brain and is thought to initiate neurotoxic events that lead to synaptic dysfunction. Direct visualization of intact synaptic terminals is one of our approaches to investigate the distribution of ApoE. We developed a novel high-throughput method for analyzes pre- and post-synaptic terminals by flow cytometry, as well as direct capturing of synaptosomes followed by fluorescence imaging.
Our new strategy to capture synapses is based on electrostatic attraction. Synaptosomes surfaces are negatively charged, caused by the presence of negatively charged phospholipids, protein side chains, and glycans already present on the plasma membrane. We also modified synaptic surfaces with ethylene glycol bis(succinimidyl succinate) (EGS), a crosslinker that contains two amine-reactive NHS esters and a 12-atom spacer, which serves two important functions. EGS may crosslink two amine groups on different proteins to achieve synaptosome fixation. Alternatively, EGS may increase the net negative charge by neutralizing a single amine group on the synapse surface, and later imparting a negative charge after the free NHS ester is hydrolyzed. After EGS modification, synaptosomes thus carry high negative charge density.
EGS-treated synaptosomes are easily captured via electrostatic attraction over amine-derivatized glass surfaces treated with (3-aminopropyl)triethoxysilane (APTES). Surfaced-attached synaptosomes can be immunostained under gentle conditions just like fixed cells and imaged by fluorescence microscopy or super-resolution optical microscopy. This capture strategy enables detailed analysis of brain synapses with minimal tissue samples. The negative surface charge also prevents synaptosome aggregation and centrifugation damages in flow cytometry protocols. This allows us to immunostain synaptosomes with primary and secondary antibodies just like ordinary cells before flow cytometry and sorting.
Using a combination of flow cytometry and single-synapse image analysis, we determined ApoE as being localized to both pre- and post-synaptic terminals. It is a fairly abundant protein at synapses, present in over half of the synaptic terminals by either characterization method. Therefore it is very likely that the synapse is one of the critical sites of interaction between ApoE and Aβ, but the synaptic function of ApoE is still poorly understood.

並列關鍵字

Alzheimer’s disease ； synaptosome ； EGS ； APTES ； fluorescence microscopy ； flow cytometry

參考文獻

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