Nck1在NMDA受体介导的缺血性神经损伤中的作用及其机制研究

Stroke is a major course of death in the world as a neurological disease. Excessive activation of NMDA receptor is a primary factor of neuronal death caused by excitotoxicity after ischemia. However, inhibition of the total NMDA receptor has not been successful in treating ischemia of brain. Otherwise, it is reported that intervention of certain type of NMDA receptor and its corresponding signal pathway is succeed in rodent and primate animal model of stroke. Therefore, for developing specific and effective treatment of ischemia, more efforts are needed to better and deeply understand the roles of different subtypes of NMDA receptor complex and their mediating signal pathway in ischemia. Based on our preliminary results, we proposed that Nck1, a member of NMDA receptor complex, played a scaffolding role in recruiting Src kinase for facilitating its phohorylating NMDA receptor, especially for a novel tyrosine phosphorylation site of 1246 of GluN2A, a subunit of NMDA receptor. Combining molecular cloning, cell imaging, biochemistry and animal model, the project herein will provide comprehensive as well as in-depth illustration of the role of Nck1 as a member of NMDA receptor complex in ischemia, hoping the exploration carried out in the project will enable us to promote the advance of dealing with ischemia by targeting NMDA receptor.

缺血性脑卒中是致死率极高的神经系统疾病。缺血后由NMDA受体介导的兴奋性毒是神经元死亡的主要原因。然而，在脑缺血损伤过程中全面阻断NMDA受体功能来保护神经元的治疗方案迄今未获得成功。近年来，以特定NMDA受体亚型为治疗靶点的干预方案已取得了有希望的进展。因此，深入理解不同亚型的NMDA受体复合物的组成及相应介导的信号通路有助于开发特异且有效的脑缺血损伤治疗手段。我们前期的发现提示Nck1蛋白可能在NMDA受体复合物中作为支架蛋白起到征募Src激酶从而调控NMDA受体磷酸化，尤其是GluN2A酪氨酸1246位点磷酸化的功能。我们希望综合运用一系列实验技术手段，如分子生物学、细胞成像、生化、整体动物模型等全面并深入地解析Nck1作为NMDA受体复合物中的成员在脑缺血过程中的作用，同时希望这种探索有助于推进以特定亚型的NMDA受体为靶点的脑缺血治疗进展。

全球范围内有相当多的人群受害于缺血性卒中引起的残疾或死亡，不仅如此，缺血性中风导致的损伤还给社会带来极大的经济负担。在神经元内，缺血再灌注损伤会过度激活NMDA受体，而NMDA受体的超激活被认为是诱导神经元死亡的重要信号通路之一。然而，过去二十年来以NMDA受体为靶点的治疗缺血再灌注损伤的药物开发全都宣告失败。因此，进一步全面细致地了解脑缺血中NMDA受体的调控机制，将极大地推进相关药物的研发。在本研究中，我们发现并鉴定了海马组织里NMDA受体复合物中新的蛋白质成员——Nck1。然后，我们通过免疫共沉淀进一步明确Nck1和NMDA受体相互作用的位点是受体中GluN2A亚单位上的Y1246位点。Y1246位点的磷酸化水平在神经元糖氧剥夺和小鼠中动脉栓塞模型后都可以被Src激酶调控而升高。如用干扰Nck1和GluN2A相互作用的多肽进行处理，则可以对受到糖氧剥夺或中动脉栓塞的神经元起到保护作用。此外，如果在海马神经元中通过慢病毒介导过表达Nck1的SH2结构域，在干扰Nck1和GluN2A相互作用的同时还会起到保护受糖氧剥夺处理的神经元的效果。更进一步的shRNA介导的Nck1敲减实验表明，降低Nck1在海马神经元中的表达，也可以使受糖氧剥夺处理的神经元减少死亡，达到保护效果。我们的实验数据表明以Y1246位点或Nck1为靶点进行干预，可能对缓解缺血性损伤带来有益的作用。最后，我们的研究为治疗卒中提供了新的潜在靶点和方法。