斑馬魚四穿膜醣蛋白M6Aa和M6Ab蛋白在PC-12細胞和斑馬魚胚胎之功能研究

Functional studies of tetraspanin glycoprotein M6Aa and M6Ab in PC-12 cells and zebrafish embryo

黃凱筠(Kai-Yun Huang)

指導教授 ： 李明亭

國立臺灣大學/生命科學院/生化科學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.03414

摘要

M6A是一個脂質蛋白PLP/DM20家族的一員，它主要表現在神經細胞。M6A最早被發現是以小鼠腦部為抗原製成的單株M6抗體，當時發現M6抗體在小鼠大腦培養的細胞中可以抑制神經纖維的生長而辨識之抗原稱M6A。在斑馬魚這個物種上可以找到兩個M6A，分別是M6Aa跟M6Ab。在活體跟細胞試驗中，我們分別用了PC-12細胞跟斑馬魚來當成我們的研究M6A在絲狀偽足形成(filopodium formation)跟神經纖維生長的材料。我們的研究發現在以神經生長因子(NGF)分化的PC-12細胞中，M6Aa或M6Ab可以促進絲狀偽足的形成數目，而這樣的結果跟哺乳類的M6A的研究是一致的。此外，我們發現M6Ab蛋白上的磷酸化絲氨酸263位置是主要造成M6Ab可以促進在絲狀偽足形成的因素。當絲氨酸263位置被置換成無法進行磷酸化的丙胺酸，則不會促進絲狀偽足的數目的增加。而當絲氨酸263位置被置換成模擬磷酸化的天門冬胺酸時，在斑馬魚胚胎中發現會促進神經纖維的生長，然而在正常的M6Ab卻不行，而且還會造成抑制。在利用特定核酸序列位置的原位雜交技術跟反轉錄聚合酶鏈式反應中，發現在組織表現上M6Aa跟M6Ab都大量表現在成魚的眼睛跟腦部。在胚胎時期的表現中，M6Aa跟M6Ab當出生後二十四到九十六小時時表現在眼睛、端腦、後腦。在四十八小時後則集中在腦部。利用反義核酸合成(morpholino)發現當減少M6Aa跟M6Ab表現的時候，發育上的斑馬魚會有腦部跟尾巴的畸形以及身軀的縮短。利用反義核酸合成減少M6Aa跟M6Ab表現時，鈣離子的細胞流入會減少，一些鈣調控的基因表現會增加並且CaMKII的活性也會增加。總結上述，我們的研究發現Ｍ6Ab絲氨酸263這個位置是影響M6Ab再造成絲狀偽足形成數目增加的主要變因。當反義核酸合成減少M6Aa跟M6Ab表現時會引發細胞自然凋亡增加，鈣離子細胞流入會減少並增加一些鈣調控的基因表現及CaMKII的活性，在此暗示M6A對於鈣離子的一些調控扮演很重要的角色。

關鍵字

醣蛋白 ； 偽足運動 ； 神經纖維生長

並列摘要

M6A is a member of the proteolipid protein (PLP/DM20) family and expressed specifically in neurons. M6A was first identified in the adult mouse brain as an antigen reacting with the monoclonal M6 antibody that was shown to cause disruption of normal neurite extension in cultured neurons from mouse cerebellum. There are duplicated forms of zebrafish M6A proteins, M6Aa and M6Ab. In in vivo experiments, we used both PC-12 cells and zebrafish to investigate the role of zebrafish M6Aa and M6Ab in filopodium formation and neurite outgrowth. We provide evidence demonstrating that zebrafish M6Aa and M6Ab were able to promote extensive filopodium formation in NGF-treated PC-12 cells, similar to the function of mammalian M6A. Furthermore, we showed that phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Abolishing serine 263 phosphorylation site on M6Ab would greatly affect the extensive filopodium formation in PC-12 cells. On the other hand, only S263D could induce neurite outgrowth, while the wild-type M6Ab also induced neurite outgrowth only in the presence of a constitutively active CaMKII β1 in zebrafish embryos. The expression profile of M6Aa and M6Ab were characterized by whole-mount in situ hybridization and RT-PCR. Expression patterns are similar between M6Aa and M6Ab. RT-PCR results show that M6Aa and M6Ab are both abundant in brain and eye in zebrafish adult tissues. We observed that M6Aa and M6Ab are highly expressed in eye, telencephalon and hiddbrain from 24 to 96 hours post fertilization (hpf), through the whole-mount in situ hybridization. Morpholino-mediated knockdown of either M6Aa or M6Ab expression caused similar defects in brain, anteroposterior (AP) axes and tail development in zebrafish embryo. Knock-down of M6Aa or M6Ab causes decrease in Ca2+ influx and increase in the CaMKII kinase activity which results in increased expression of Ca2+ regulation-related genes. Taken together, our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical in regulating filopodium formation. Morpholino knockdown of M6Aa or M6Ab triggers apoptosis, decreases Ca2+ influx and increase in CaMKII kinase activity suggesting that M6A protein could act as a Ca2+ regulator and is prerequisite for zebrafish neuronal development.

並列關鍵字

M6A ； glycoprotein ； filopodia ； PLP ； neurite outgrowth ； PC-12

參考文獻

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