Indispensable roles of Vilse in cell viability, synaptic structure and function

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.