Regulation of NMDA Receptors in Excitotoxicity by Calpain and FYN

描述（由申请人提供）：谷氨酸，中枢神经系统中主要的兴奋性递质，对突触传递和可塑性以及称为“兴奋毒性”的病理生理过程至关重要。兴奋性毒性通常需要激活特定的谷氨酸受体，即n -甲基- d -天冬氨酸（NMDA）受体。NMDA受体拮抗剂在人类中的治疗应用由于不良反应一直不成功。更好地了解NMDA受体活性、定位和转换的调节可能为控制兴奋性毒性提供新的方法，同时最大限度地减少NMDA受体阻断的有害影响。NMDA受体的定位和功能受许多事件控制，包括calpain对NR2B亚基c端的切割和Src家族酪氨酸激酶（SFK）对NR2B的磷酸化(1)。我们的初步数据表明，Fyn对NR2B的磷酸化控制了calpain对NR2B的裂解，并激活了下游信号事件，包括p38 MAK激酶的激活。在这种情况下，Fyn的激活依赖于NMDA受体的激活，这使得Y1336的磷酸化成为控制神经元反应的正反馈和负反馈机制的一部分。在本研究中，我们将分析calpain、Fyn和MAGUK蛋白相互作用控制NMDA受体特性的分子机制，并探讨它们在NMDA受体生理学和人类疾病兴奋毒性机制模型中的重要性。在目的1中，我们将研究NMDA受体激活的Fyn磷酸化NMDA受体和其他底物上不同位点的能力。这将使我们了解NMDA受体直接或间接激活SFK的机制，以及这种活性如何定向到NMDA受体上的不同位点。目的2将定义钙蛋白酶（及其亚型）与NMDA受体和SFK相互作用的结构决定因素和机制。在Aim 3中，我们将评估calpain切割NMDA受体的电生理特性，以及calpain切割是否会改变下游MAP激酶的激活，从而进一步将calpain介导的NR2B切割与生理和病理生理事件联系起来。最终目的是在体外兴奋性毒性模型中研究钙蛋白酶和SFK的相互作用是否会改变病理生理事件。更好地了解钙蛋白酶调节NMDA受体的机制，以及MAGUK蛋白和SFK如何改变这一过程，将为在神经系统疾病中使用调节这些事件的药物提供合理的基础。公共卫生相关性：该提案解决了n -甲基- d -天冬氨酸受体降解的机制。通过了解这一过程，该建议可能有助于预防神经系统疾病（如中风）损害的治疗。

DESCRIPTION (provided by applicant): Glutamate, the major excitatory transmitter in the central nervous system is crucial for synaptic transmission and plasticity as well as the pathophysiological process termed "excitotoxicity." Excitotoxicity usually requires activation of a specific glutamate receptor, the N-methyl-D-aspartate (NMDA) receptor. Therapeutic applications of NMDA receptor antagonists in humans though have been unsuccessful due to adverse effects. Better understanding of the modulation of NMDA receptor activity, localization, and turnover may provide novel ways to control excitotoxicity while minimizing deleterious effects of NMDA receptor blockade. NMDA receptor localization and function is controlled by many events including cleavage of the C-terminal of the NR2B subunit by calpain and phosphorylation of NR2B by Src family tyrosine kinases (SFK) (1). Our preliminary data demonstrate that phosphorylation of NR2B by Fyn controls NR2B cleavage by calpain and activation of downstream signaling events including activation of p38 MAK kinase. Activation of Fyn in this paradigm depends on NMDA receptor activation, allowing Y1336 phosphorylation to be part of positive and negative feedback mechanisms controlling neuronal responses. In this proposal, we will dissect the molecular mechanism of the interactions of calpain, Fyn and MAGUK proteins in the control of NMDA receptor properties, and investigate their importance in NMDA receptor physiology and models of excitotoxic mechanisms of human diseases. In aim 1, we will examine the ability of NMDA receptor-activated Fyn to phosphorylate distinct sites on the NMDA receptor and other substrates. This will allow us to understand the mechanism by which NMDA receptors directly or indirectly activate SFK and how such activity is directed to distinct sites on the NMDA receptor. Aim 2 will define the structural determinants and mechanisms that mediate the interactions of calpain (and its subtypes) with NMDA receptors and SFK. In Aim 3, we will assess examine electrophysiological properties of calpain-cleaved NMDA receptors and whether cleavage by calpain alters activation of downstream MAP kinases in order to link further calpain mediated cleavage of NR2B with physiological and pathophysiological events. The final aim will investigate whether the interactions of calpain and SFK alter pathophysiological events in an in vitro model of excitotoxicity. Better understanding of the mechanisms by which calpain modulates NMDA receptors and how MAGUK proteins and SFK alter this process should allow a rational basis for use of agents modulating these events in neurological disorders. PUBLIC HEALTH RELEVANCE: The proposal addresses the mechanisms of degradation of the N-methyl-D-aspartate receptor. Through understanding of this process, the proposal may facilitate therapies for preventing damage in neurologic disorders such as stroke.