Vilse在細胞存活與神經發育中功能之研究

Abstract

Rho GTPases 在細胞運動、胞內運輸、神經細胞分化與移動等生理功能均扮演重要角色， 當與GTP結合時，RhoGTPase具有酵素活性，可以催化下游反應；反之，GTP被水解成GDP時，RhoGTPase不具有酵素活性，無法啟動下游反應。這種GTP/GDP結合態的循環，分別受到 GTPase activating proteins (GAPs) 和 guanine nucleotide exchange factors (GEFs)的調控。 Vilse/CrGAP/Arhgap39 具有保守程度極高的 RhoGAP區域，還含有可與其他蛋白質作用的 WW和MyTH4區域，先前在果蠅的研究發現 Vilse利用WW區域與 Robo 受體作用，調控 Rac/cdc42相關的細胞骨架重整，進而影響軸突導向的 midline repulsion。此外，小鼠與大鼠NG108融合細胞中，Vilse同樣利用WW區域與connector enhancer of KSR-2 (CNK2) 作用，調控 Rac 活性，進而影響海馬迴神經元的生成。 為了研究Vilse在神經發生所扮演的角色，我們構築去除Vilse不同區域的表現載體以及Y448/S604點突變的表現載體，分別送進神經母細胞瘤 Neuro2a細胞中，在低血清和 retinoic acid誘導分化狀況下，發現除了去除MyTH4區域和Y448F不被磷酸化的Vilse不會影響N2a細胞的分化以外，其餘的Vilse表現載體皆可抑制N2a細胞的分化；為了瞭解Vilse是否參與胚胎神經發育過程，我們建立Vilse基因剔除鼠，結果發現缺乏Vilse表現的小鼠都死於胚胎時期，顯示全身性剔除Vilse表現會造成胚胎死亡 N2a細胞中Vilse表現量下降亦會造成細胞凋亡，證明Vilse對細胞存活有重要影響。我們進一步利用Camk2a-Cre轉殖小鼠產生Vils基因在前腦和海馬迴組織特異性剔除小鼠，其中，利用HA-iCre剔除體外培養海馬迴神經細胞Vilse-LoxP基因後發現神經細胞的樹突有發育不良之情形 而Vilse基因在海馬迴組織特異性剔除小鼠進行Y-maze實驗後發現其空間記憶能力下降，Morris water maze也發現Vilse cKO小鼠空間記憶能力與學習都較差，顯示Vilse剔除確實會造成空間記憶能力缺失。我們測量Vilse cKO小鼠海馬迴電生理反應並記錄海馬迴在受刺激後產生的long-term potentiation (LTP)現象，結果顯示Vilse剔除造成海馬迴LTP現象明顯下降，證明Vilse剔除影響海馬迴神經傳導與神經可塑性。利用Golgi stain觀察Vilse cKO小鼠海馬迴神經細胞的樹突外觀，結果顯示Vilse剔除造成神經細胞樹突較短、複雜度較低和大量未成熟的樹突小棘 (dendritic spine)。綜合以上結果可以證明Vilse在神經與胚胎發育上扮演相當重要的角色。

Vilse/CrGAP/Arhgap39 is a newly identified GAP that contains a conserved RhoGAP domain accompanied with two protein-protein interaction domain, namely WW domain and MyTH4 domain. Previous studies show that Vilse interacts with Robo receptor through WW domain and acts downstream of Robo receptor to regulate Rac/cdc42-dependent cytoskeletal changes. Additionally, Vilse associates with connector enhancer of KSR-2 (CNK2) through WW domain to modulate Rac cycling during spine morphogenesis in hippocampal neurons. We constructed MyTH4, WW and RhoGAP-deleted mutants of Vilse and introduced these mutants to neuroblastoma N2a cells. The presence of Vilse impaired neurite growth in N2a cells. MyTH4-deleted mutant accumulated in the nucleus, but not the plasma membrane, and the neurite outgrowth of N2a cells was not hindered. In addition, Vilse Y448 phosphorylation is also important to localization of Vilse. Y448 mutate Vilse accumulated in cytosol and failed to impair neurite growth in N2a cells. To decipher Vilse’s function in vivo, homozygous knockout of Vilse mice has been generated and macroscopic examinations reveal an essential role of Vilse for embryonic survival. Vilse knockdown in N2a cells also result in apoptosis. Ablating Vilse-LoxP alleles using HA-Cre in hippocampal neurons in vitro supported the notion that Vilse is indispensable for neurite development. Further studies on the consequences of condition knockout Vilse (cKO) using Camk2a-Cre were performed. Interestingly, Morris water maze analysis showed that Vilse cKO mice displayed a longer latency to find the platform than wild type mice after repeated training. Y-maze test showed robust result support Vilse cKO interfered spatial memory. Furthermore, electrophysiological studies showed that the EPSPs in the hippocampus of Vilse cKO mice decreased quickly to a normal level compared with control mice, which remained in a high amplitude at 60 min post stimulation. Finally, Golgi stain results revealed smaller cell bodies, shorter dendrite lengths, less complexity and more immature spin in Vilse cKO mice. Collectively, Vilse is essential for embryonic development and required for the acquisition of spatial memory.