Rap1GAP对GDNF/RET转运及其神经营养功能的影响

Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson's disease. GDNF binds to its coreceptor GFRa1 and signals through the transmembrane receptor tyrosine kinase RET. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. We previously identified a new interaction between RET and the adapter protein Rap1GAP, they were co-expressed in TH positive mesencephalic neurons.Through gain or loss of Rap1GAP gene expression, functional analysis indicated that Rap1GAP suppressed GDNF-induced neurite outgrowth in PC12-GFRα1-RET cells (that stably overexpress GDNF receptor GFRα1 and RET). More over, mechanism study performed from two way, firstly proved Rap1GAP could inactive downstream Rap1/ERK by it's specific Rap1 GTPase-activating activity, the other way, which could prevent RET internalizing into cell body. We speculated that the absence of Rap1GAP, and thus potentially altered GDNF activity, might lead to abnormal functionality of the DA system. To assess the significance of Rap1GAP on GDNF downstream, some tests were designed as follows: firstly, to investigate the impact of Rap1GAP expression on internalization and trafficking of GNDF and RET; secondly, Rap1GAP(N290A) mutant that are lose of GAP catalytic activity were recited to separate the two function manner, to evaluate the importance of Rap1GAP in GDNF signaling from inhibiting RET internalization; thirdly, we using Rap1GAP knockout mice to figure out the effects of Rap1GAP loss on DA system in vivo. By all these systemic analysis, we expect to provide a potential target for the treatment of pathological conditions related to the DA system such as drug abuse and Parkinson's disease.

胶质细胞源性神经营养因子GDNF目前发现的最强的促进多巴胺神经元存活的神经营养因子。GDNF通过共受体GFRα1，和跨膜受体RET进行信号转导，下游通路已得到大量证实，而调控其内化和转运的机制却知之甚少。项目组前期确定了一个新的RET结合蛋白Rap1GAP,它抑制GDNF诱导的神经细胞突起生长。进一步研究发现，Rap1GAP至少通过两种途径起作用，一是抑制下游Rap1/ERK途径活化；另一方面抑制RET受体复合物内化。我们设想Rap1GAP表达改变，会潜在影响GDNF的转运，进而影响多巴胺系统功能，为此设计以下实验：1明确Rap1GAP对GDNF、RET转运的影响；2利用丧失Rap1催化酶活性的Rap1GAP突变体，衡量其抑制RET内化的意义；3，利用基因敲除小鼠探索Rap1GAP在多巴胺系统的生理意义。希望通过此工作，为GDNF应用于帕金森等多巴胺系统疾病的治疗提供理论基础和新的靶点。

GDNF支持中枢神经系统中脑多巴胺能神经元与运动神经元等多种神经元的存活和发育，目前发现的GDNF受体有三个:GFRα1、RET和NCAM，而RET是其最主要的功能受体。酪氨酸激酶类受体RET被GDNF激活后，发生二聚化和自磷酸化，继而募集下游信号分子，通过一系列的酶促级联反应，最终激活Ras/MAPK、PI3-K以及PLCγ等信号转导通路，调控神经元的生长、分化、存活等生物学效应。RET和其它RTK类受体一样，被配体激活，经过经典的clathrin包被的途径发生内化和细胞内转运，部分在溶酶体降解，部分重新运输至细胞膜再利用。我们在前期研究中，RET与Rap1GAP结合, Rap1GAP过表达抑制GDNF诱导的神经元突起生长，而Rap1GAP基因敲减可以显著促进 GDNF诱导的神经元突起生长。.为了明晰Rap1GAP是否通过其它机制调控GDNF信号通路，利用表达内源性RET，Rap1GAP, GFRα1蛋白的SH-SY5Y细胞系，观察GDNF刺激后RET和Rap1GAP分布变化。免疫荧光细胞化学实验显示，GDNF不刺激情况下，Rap1GAP弥散分布在SH-SY5Y细胞胞浆里，RET大部分分布在细胞膜上。GDNF刺激SH-SY5Y细胞10分钟后，RET有大量内化现象，Rap1GAP和RET可以在同一早期内吞体里检测到；GDNF刺激细胞不同时间，用strepavidin-biotin沉淀细胞膜蛋白，western检测膜蛋白里RET的剩余量。结果显示，Rap1GAP过表达情况下，GDNF刺激细胞后，RET在细胞膜内表达维持时间最长，GDNF刺激15分钟后膜上仍能检测到RET：而Rap1GAP沉默组，GDNF刺激5分钟后，细胞膜里几乎检测不到RET。以上结果说明，Rap1GAP的表达量明显影响GDNF引起的RET内化过程。