Regulation of NMDA Receptors in Excitotoxicity by Calpain and FYN

谷氨酸是中枢神经系统中主要的兴奋性递质，在突触传递中起着重要作用 和可塑性以及称为“兴奋性毒性”的病理生理过程。“兴奋性毒性通常需要 特异性谷氨酸受体，N-甲基-D-天冬氨酸（NMDA）受体的激活。治疗 但是，由于NMDA受体拮抗剂的不良反应， 方面的影响.更好地理解NMDA受体活性、定位和转换的调节， 提供了控制兴奋性毒性同时最小化NMDA受体阻断的有害作用的新方法。 NMDA受体的定位和功能受许多事件控制，包括NMDA受体C-末端的切割。 NR 2B亚基通过钙蛋白酶和NR 2B的磷酸化通过Src家族酪氨酸激酶（SFK）。我们 初步数据表明Fyn对NR 2B的磷酸化控制Caipain对NR 2B的切割， 下游信号传导事件的激活，包括p38 MAK激酶的激活。Fyn的激活 范式依赖于NMDA受体活化，允许Y1336磷酸化是正性和负性的一部分。 负反馈机制控制神经元的反应。在本提案中，我们将剖析 钙蛋白酶、Fyn和MAGUK蛋白在NMDA调控中相互作用的分子机制 受体特性，并研究其在NMDA受体生理学和兴奋性毒性模型中的重要性 人类疾病的机制。在目的1中，我们将检查NMDA受体激活的Fyn的能力， 磷酸化NMDA受体和其他底物上的不同位点。这将使我们能够了解 NMDA受体直接或间接激活SFK的机制以及这种活性如何被定向到 NMDA受体上的不同位点。目标2将定义结构决定因素和机制， 介导钙蛋白酶（及其亚型）与NMDA受体和SFK的相互作用。在目标3中，我们 评估检查钙蛋白酶切割的NMDA受体的电生理学特性， 通过钙蛋白酶改变下游MAP激酶的激活，以进一步连接钙蛋白酶介导的 NR 2B与生理和病理生理事件。最后的目的是调查是否 钙蛋白酶和SFK的相互作用改变HIV脑病体外模型中的病理生理学事件， 以及钙蛋白酶和SFK的激活是否有助于HIV诱导的神经元死亡。更好地了解 钙蛋白酶调节NMDA受体的机制以及MAGUK蛋白和SFK如何改变这种机制 这一过程应该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依据。

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) . Our preliminary data demonstrate that phosphorylation of NR2B by Fyn controls NR2B cleavage by caipain 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 HIV encephalopathy, and whether activation of calpain and SFK contribute to HIV induced neuronal death. 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.