支架蛋白RIM1调控NMDA受体质膜插入的机制及功能研究

NMDA receptor is one of the most important excitatory neurotransmitter receptors in the brain. Its trafficking is precisely regulated and determines the amount and subtype of synaptic receptors, which in turn determines synaptic function. Previous research on the molecular mechanisms involved in NMDA receptor trafficking is mainly focused on the identification of the molecular motif within the receptor subunit, such as ER retention motif, or post-translational modification of the receptor subunit, such as phosphorylation and palmitoylation. As we know, NMDA receptor, similar with presynaptic neurotransmitter, is transported in form of vesicles. Howerver, it is still unclear as to the mechanisms mediating the trafficking of receptor-containing vesicles. In our present study,by using EM imaging, we found that the scaffolding protein RIM1, which plays key roles in the release of presynaptic neurotransmitter vesicles, was located both presynaptically and postsynaptically. In addition, biochemical results showed that RIM1 was specifically bound to NMDA receptor complex, but not to AMPA receptor complex. Moreover, RIM1 siRNA knockdown specifically decreased the surface distribution of NMDA receptor in cultured cortical neurons, indicating that RIM1 participated in the regulation of NMDA receptor trafficking. Combined with the previous work indicating that presynaptic RIM1 is critical for the vesicle release, we provide a hypothesis that postsynaptic RIM1 is involved in the regulation of NMDA receptor trafficking, especially NMDA receptor membrane insertion. Therefore, in this project, we will focus on this hypothesis and find out the molecular mechanisms underlying this process. Furthermore, we will examine whether this process is controlled by neuronal activity and the role of this process in some pathological conditions, such as chronic neuropathic pain. This research will be a great help for us to understand NMDA receptors trafficking in the view of receptor-containing vesicles.

NMDA受体是脑内重要的兴奋性递质受体，它的运输受到精确调控并决定突触上受体的表达量及亚型构成，进而决定突触功能。以往针对其运输调控机制的研究主要围绕受体亚单位分子内序列信号的识别及翻译后修饰位点的鉴定而展开。但是，NMDA受体与突触前神经递质类似，是以囊泡形式运输，目前尚不清楚含受体的运输囊泡是如何被调控。我们发现突触前调控递质囊泡释放的核心蛋白RIM1在突触后也有分布，并特异性地存在于NMDA受体复合物中，抑制RIM1的表达明显减少NMDA受体膜表面的分布，提示RIM1参与NMDA受体运输的调控。结合突触前RIM1在递质囊泡释放中的作用，我们提出假设，突触后RIM1参与调控NMDA受体运输，特别是质膜插入过程。本项目将验证该假设并研究其分子机制，以及此过程与神经元活动的关系，及其在某些神经疾病，如慢性神经痛中的作用。此研究将有助于我们从受体囊泡与质膜融合的角度来理解受体运输的调控机制。

N-甲基-D-门冬氨酸(N-Methyl-D-Aspartate, NMDA)受体是中枢神经系统中一类重要的离子型谷氨酸受体，对突触可塑性，学习记忆非常重要，其功能异常与许多神经疾病紧密相关。NMDA受体的激活可以引起AMPA受体的上膜和下膜，从而引起突触功能的可塑性，而NMDA受体本身在突触上的表达也受到调控。RIM1是一个经典的突触前定位的蛋白，其主要功能是介导突触前囊泡的释放，并参与调控突触前可塑性。在本研究中，我们通过免疫电镜和生化组分分离的方法确定RIM1不仅仅定位在突触前，在突触后也有分布。为了研究其在突触后的功能，我们将装载了shRNA的慢病毒定位注射到小鼠海马CA1脑区，结合电生理记录的手段建立突触后RIM1特异敲降模型。结果显示，突触后敲降RIM1不影响AMPA受体介导的兴奋性突触后电流，但是会下调NMDA受体介导的兴奋性突触后电流，并且导致NMDA受体依赖的LTP发生缺损。而且，海马CA1区的RIM1敲降的小鼠，还表现出海马依赖的空间记忆的缺损。提示，RIM1是一个调节NMDA受体膜运输的关键蛋白，并RIM1的缺失会影响NMDA受体介导的突触可塑性及小鼠的学习记忆功能。此外，我们利用生化手段确定了NMDA受体与RIM1的相互关系，我们发现，NMDA受体和RIM1在一个复合物中存在，但是NMDA受体亚单位与RIM1没有直接的相互作用，而是通过PSD95结合到一起的。接下来，我们用细胞成像的方法，证实了NMDA受体的运输受到RIM1的影响，RIM1参与NMDA受体的再循环过程，而不参与NMDA受体的内化过程。结合生化和SIM高分辨成像，我们分析了RIM1与RAB11的关系。我们发现RIM1是rab11的效应因子，从而确定，RIM1通过与RAB11的结合，参与调控NMDA受体的再循环过程。因此，本研究证实RIM1是在突触后也有定位的，并且突触后RIM1特异性参与NMDA受体的再循环过程，并影响NMDA受体介导的突触可塑性。