RABV 感染抑制Rac1活性和神经突触功能的机制

It is well established that neuronal dysfunction may be responsible for the development of rabies，the mechanism of which remains unclear. Our previous research revealed that Rac1 was remarkably inactivated after RABV infection, as well as the fluorescence intensity of F-actin impaired. Based on the research that Rac1 plays a pivotal role in synaptic function through modulating F-actin dynamics, the project will focus on RABV dyregulating the activity of Rac1. After RABV infection, we firstly demonstrate aberrant actin polymerization; secondly, we attempt to detect the alteration of synaptic vesicles clustering and dendritic spine morphogenesis, as well as action potential; thirdly, we try to detect the expression and phosphorylation of key molecules involved in Rac1-PAK signaling pathway; Finally, we elucidate the mechanism of which RABV infection influences synaptic function through downregulating Rac1 activity and obstructing actin cytoskeleton reorganization. It is helpful to further contribute to our understanding of the pathogenesis of rabies, and might be theoretical basis for rabies clinical therapy and drug development.

狂犬病病毒（RABV）感染引起严重的神经功能异常表征，但其机制尚不明确。我们前期研究发现，RABV感染导致Rac1活性下调，F-actin荧光强度降低。由于Rac1与细胞骨架动态变化密切相关，为此，本项目拟以Rac1活性为切入点，研究RABV感染对F-actin骨架形成、突触囊泡募集、树突棘形态及突触电位等的影响，并对Rac1-PAK信号通路的关键分子表达及其磷酸化水平进行检测，以阐释RABV抑制F-actin聚合及突触功能的分子机制。这一研究对于解析RABV致突触功能异常的机制具有重要意义，同时Rac1类调节剂也有望为狂犬病临床治疗和靶向药物开发提供方向。

狂犬病病毒（RABV）具有噬神经性、高致死率，感染引起严重的神经功能异常的机制尚不明确，肌动蛋白微丝细胞骨架（F-actin）的结构是维持神经突触信号正常传递的重要因素。本项目以Rac1为切入点，分别采用小鼠原代神经元细胞、小鼠神经母细胞瘤细胞（N2a），通过抑制剂处理、基因过表达、免疫共沉淀（IP）、Pull down、免疫荧光等一系列生物学手段，观察到RABV感染致使神经元树突棘数量减少、actin聚合强度显著降低，发现了Rac1、Cdc42活性影响RABV感染水平，检测到RABV感染下调Rac1活性、Rac1-PAK1信号通路下游分子PAK1、LIMK1、cofilin1磷酸化水平，且在病毒感染宿主细胞早期Rac1-PAK1信号通路先开启后关闭动态变化，鉴定了RABV结构蛋白P与Rac1之间蛋白-蛋白相互作用，RABV通过P蛋白反向调控Rac1-PAK1信号通路。从而创新性探究了RABV入胞脱壳后通过P蛋白结合Rac1、抑制Rac1-PAK信号通路、影响微丝细胞骨架生成，阐释了RABV感染动态调控树突棘骨架聚合解聚的分子机制，对于解析RABV致神经突触功能异常的机制具有重要意义，为基于Rac1的狂犬病的临床治疗和靶向药物的开发提供新的研究方向。