大腦皮質神經元發育期間生長相關蛋白 GAP-43 與 Gephyrin 之相互作用探討

1. 大腦皮質神經元發育期間生長相關蛋白GAP-43與Gephyrin之相互作用探討大腦皮質神經元發育期間生長相關蛋白GAP-43與Gephyrin之相互作用探討 生長相關蛋白(Growth association protein 43 ;GAP-43) 是一種位於突觸前末梢的蛋白，在神經發育以及神經修復過程中參與軸突的延伸、以及引導生長錐生長方向。先前的研究發現，PKC可促進GAP-43的磷酸化，進而對神經生長以及細胞可塑性有促進的作用。我們使用免疫沉澱和質譜儀(MALTI-TOF)的實驗方法，發現GAP-43會與一個位於突觸後膜上，具有聚集抑制性受器功能的蛋白質—gephyrin結合。Gephyrin藉著與glycine receptor  subunit 以及GABAA receptor2 subunit結合，來聚集glycine 及GABAA receptor。我們的實驗顯示在畸胎瘤分化的神經細胞或是發育中的大腦皮質神經元細胞， 其細胞膜表面皆可發現有gephnyrin的表現及聚集現象。然而，我們更進一步探討PKC抑制劑Ro-318220對於GAP-43以及gephyrin表現量的影響，結果發現，Ro-318220會增加發育初期的神經細胞(4 DIV)GAP-43的表現量，但在發育晚期神經細胞 (8 DIV)則有抑制的效果。Ro-318220對於gephyrin的表現則無顯著的影響。再者，在共同免疫沉澱(co-immunoprecipitation)的實驗結果發現，投予發育中的神經細胞Ro-318220 24小時會增加GAP-43和gephyrin結合的程度，且在共軛螢光免疫雙染的實驗中亦發現，PKC抑制劑會使GAP-43集中在細胞體，並與gephyrin有共位(colocolization)的現象。反之，Ro-318220會造成gephyrin與GABAAR2 subunit的結合程度降低，而且，我們進一步發現給予Ro-318220會造成GABA在發育神經元所引發的胞內鈣離子濃度升高反應消失。以上結果顯示，GAP-43受PKC調控的活性，不但會影響神經生長，也可能會藉著與gephyrin的交互作用，影響GABAA receptor的功能。

2. Protein-Protein Interaction of GAP-43 and Gephyrin in Developing Cortical Neurons • Growth association protein (GAP-43) is a presynaptic protein that involved in axon elongation, growth cone guidance and regeneration during development. Previous studies indicate that protein kinase C (PKC) could facilitate GAP-43 phosphorylation to promote neurite outgrowth and plasticity. In this study, we applied immunoprecipitation followed by MALTI-TOF (Matrix-assisted laser desorption/ ionzation time-of-flight) mass spectrometry to examine if other proteins could interact with GAP-43 to mediate neuronal development. Our results show that one of the GAP-43-interacting proteins is an inhibitory receptor clustering protein namely gephyrin. Gephyrin cluster the inhibitory receptors such as glycine receptor and GABAA receptor (-amino-butyric acid type A receptor) by binding with glycine receptor subunit and GABAA receptor 2 subunit. We found that gephyrin was expressed on the surface of both teratocarcinoma-derived neurons and primary cultured developing cortical neurons. We further examined the effect of PKC inhibitor Ro-31-8220 on the expression of GAP-43 and gephyrin expression. It was found that Ro-31-8220 increased the expression GAP-43 at early stage (4 days in vitro) but decreased at late stage (8 days in vitro ), but had no effect on gephyrin expression. Furthermore, co-immunoprecipitation studies show that Ro-31-8220 and MAP kinase inhibitor PD-98059, but not PKA inhibitor Rp-8-Br-cAMPs promoted association of GAP-43 and gephyrin. Furthermore, immunofluorescent double labeling with confocal microscopy shows that Ro-31-8220 treatment result in loss of axonal distribution of GAP-43 and the colocolization of GAP-43 and gephyrin become more apparent in soma. On the other hand, inhibition of PKC resulted in dissociation of gephyrin from GABAA R 2. Indeed PKC inhibitor abolished GABA-induced elevation of intracellular calcium concentration. These results implicate that GAP-43 with the activity governed by PKC, is not only crucial for neurite outgrowth, but might also contribute to GABAergic synaptogenenesis control via PKC-dependent dissociation from the GABAA receptor clustering protein gephyrin.