培養大鼠海馬迴神經細胞結構極化過程中軸突之tubulin修飾與MAPs分布變化的研究

A study of the changes in tubulin modifications and MAPs in the axon of cultured rat hippocampal neurons during structural polarization

游朝楷(Chao-Kai You)

指導教授 ： 周姽嫄

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2017年)

摘要

在神經細胞傳遞訊號的過程中，軸突和樹突有著不同的功能，訊號從樹突被接收然後從軸突被送出。軸突和樹突的差異源自於神經突在生長中受到不同的調控，軸突的MT (microtubule)上會有高於樹突MT的tubulin acetylation，而且軸突生長錐內的MT有較高的tubulin tyrosination；軸突內主要的MAP (microtubule associated-protein)是tau，而樹突內主要的MAP是MAP2。除了軸突與樹突間的差異，本實驗室過去利用特殊的培養裝置分離軸突的生長錐和軸桿(shaft)，發現兩個部位的mRNA種類和量也有所差異。為了進一步研究軸突內不同時間及不同位置的調控狀態，我以免疫螢光染色的方式分析MAP2、 dephosphorylated tau、 acetylated tubulin、 tyrosinated tubulin在軸突上分布的位置和量，結果顯示培養第八天的細胞四種染色的結果都是在軸突後半段(distal half)的螢光所佔的比例高於前半段(proximal half)，顯示四者在軸突上的表現都偏重於後半段。其中MAP2和acetylated tubulin在DIV4時於軸突上的表現較為平均，到DIV8則偏重表現於軸突後半段；dephosphorylated tau和tyrosinated tubulin則是從DIV4開始就已經偏重表現在軸突的後半段。另外我也比較了軸突和樹突上的螢光密度(fluorescence/pixel)，發現tyrosinated tubulin的密度在軸突和樹突上較為接近，而dephosphorylated tau和acetylated tubulin在軸突上的密度高於樹突，MAP2的密度則隨時間變化。我的研究成果用折線圖呈現了軸突的MT受到的調控在不同時間及不同位置的變化，並探討可能的原因及功能。

關鍵字

海馬迴 ； 神經 ； 微管 ； 微管修飾 ； 極化 ； 免疫螢光染色 ； 軸突

並列摘要

Axon and dendrite play different roles in neuronal signal transduction. A neuron receives signals in dendrites and sends signal out via axon. Differences between axon and dendrite are resulted from various regulation processes during development. Higher tubulin acetylation and tyrosination are observed in axonal microtubules. One of the major axonal microtubule-associated protein (MAP) is tau, while one major dendritic MAP is MAP2. To study the spatial and temporal variations of microtubules and MAPs in axon, immunofluorescence staining was used. By studying the fluorescence distribution of MAP2, dephosphorylated tau, acetylated tubulin and tyrosinated tubulin, I found that the contents of these antigens in the distal halves were higher than in the proximal halves of axons on 8th day in vitro (DIV8). On the other hand, dephosphorylated tau and tyrosinated tubulin were preferentially localized to distal halves of axons since DIV4. MAP2 and acetylated tubulin distributed rather evenly along axons on DIV4 and gradually distributed preferentially to distal axon until DIV8. Furthermore, fluorescence density (fluorescence/pixel) in axon and dendrite were also calculated. Tyrosinated tubulin immunoreactivity exhibited similar fluorescence density in axons and dendrites; dephosphorylated tau and acetylated tubulin immunoreactivities exhibited higher density in axons than in dendrites; the immunoreactivity density of MAP2 changed between DIV4 to DIV8. Conclusively, my study illustrated the changes of the distribution of microtubules and MAPs in axons during the in vitro development of cultured rat hippocampal neurons. The possible functions of these changes in the development of axons in neurons are also discussed.

並列關鍵字

hippocampus ； neuron ； microtubule modification ； microtubule-associated protein ； MAP ； polarization ； tau ； tyrosination ； acetylation ； immunofluorescence ； axon

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