Calpain Mediated Cleavage of NR2 in Excitotoxicity

描述（由申请人提供）：谷氨酸受体称为n -甲基- d -天冬氨酸（NMDA）受体是多种形式的兴奋性毒性的重要介质，是神经系统疾病的病理生理机制。NMDA受体由NR1和NR2亚基组合而成，每个亚基具有不同的性质。不同的亚型可能在兴奋性毒性中发挥不同的作用，这可能是基于它们胞内结构域的不同特征。这些结构域将受体与细胞内信号传导过程联系起来，并且是钙蛋白酶水解修饰的潜在位点。这种修饰可能在控制NMDA受体的亚型特异性方面起着至关重要的作用。之前我们已经证明NR2A亚基是calpain的选择性底物，而NR1亚基不被calpain切割。这种分裂可能产生具有新特性的受体，这些受体可能在兴奋毒性和缺氧期间以亚基选择性方式进行调节。在本研究中，我们将评估钙蛋白酶裂解对不同NMDA受体亚型的生理特性和定位的影响，以及这些事件在兴奋性毒性中的作用。我们将首先确定calpain在体外、异种系统和神经元中每个NR2亚基的切割位点。在这些实验之后，我们将定义calpain切割对NMDA受体生理特性的影响，并确定calpain切割是否会改变NMDA受体在异源表达系统和神经元中的定位。在最后的Aim中，我们将研究calpain在兴奋性毒性和缺氧模型中是否会切割NMDA受体，以及calpain切割NMDA受体是否会改变细胞损伤的严重程度。此外，我们将确定钙蛋白酶裂解是否在一种特定的癫痫模型中起作用，即点燃模型。总的来说，本研究将定义NMDA受体的钙蛋白酶蛋白水解如何调节受体特性，使我们能够确定这一过程在神经元功能和疾病过程中的作用。

DESCRIPTION (provided by applicant): The glutamate receptor called the N-methyl-D-aspartate (NMDA) receptor is a crucial mediator of many forms of excitotoxicity, a pathophysiological mechanism of neurologic disease. The NMDA receptor is constructed from combinations of NR1 and NR2 subunits, with each subunit conveying distinct properties. Different subtypes may play distinct roles in excitotoxicity, possibly based on the variable features of their intracellular domains. These domains link the receptor to intracellular signaling processes, and are potential sites for proteolytic modification by the enzyme calpain. Such modifications could play a crucial role in control of the subtype specific properties of NMDA receptors. Previously we have shown that the NR2A subunit is a selective substrate for calpain while the NR1 subunit is not cleaved by calpain. This cleavage may create receptors with novel properties, which could be regulated in a subunit selective manner during excitotoxicity and hypoxia. In this proposal, we will assess the effect of calpain cleavage on the physiological properties and localization of different NMDA receptor subtypes and the role of these events in excitotoxicity. We will initially determine the sites of cleavage by calpain in each NR2 subunit in vitro, in heterologous systems and in neurons. Following these experiments, we will define the effect of calpain cleavage on physiological properties of the NMDA receptor, and determine whether cleavage by calpain alters NMDA receptor localization in heterologous expression systems and in neurons. In the final Aim, we will investigate whether calpain cleavage of NMDA receptors occurs during models of excitotoxicity and hypoxia, and whether calpain cleavage of NMDA receptors alters the severity of cellular damage. In addition, we will ascertain whether calpain cleavage plays a role in a specific model of epilepsy, the kindling model. Overall, the present I proposal will define how calpain proteolysis of the NMDA receptor modulates receptor properties, allowing us to determine the role of this process in neuronal function and disease processes.