Synaptic Modification of AMPARs by Oxidative Stress

描述(申请人提供)：AMPA受体(AMPAR)是中枢神经系统中一种主要的谷氨酸受体，由GLUR1、GLUR2、GLUR3和GluR4亚基组装而成。在海马锥体神经元上表达的大多数AMPAR都含有GLUR2的编辑形式，因此对钙离子不能通透。进入。最近的研究表明，AMPAR在促进脑缺血后迟发性神经元死亡中起关键作用。在缺血再灌流期间，这些谷氨酸能受体的亚基组成发生了变化，从含有GLUR2的不通透性AMPAR转变为缺乏GLUR2的通透性受体。有大量证据表明，短暂性全脑缺血诱导的海马锥体神经元迟发性死亡与这些钙通透性AMPAR的激活有关。目前，GLUR2缺失的AMPAR在缺血/再灌注诱导表达的机制尚不清楚。然而，最近的研究表明，氧化应激信号通路是导致AMPAR亚单位组成变化的原因。研究表明，NADPH氧化酶可能是脑缺血再灌流过程中启动氧化应激信号级联的来源。这项建议的目的是验证这样的假设，即再灌注期间NADPH氧化酶的活性增加触发了GLUR2 AMPAR亚单位的隔离和随后的降解，从而导致GLUR2缺失的AMPAR表面表达增加。这项拟议的研究将使用急性成年大鼠的海马脑片。为特定目的设计的实验1将检验这样一种假设，即抑制NADPH氧化酶活性可防止缺血-再灌流增加GLUR2 AMPAR亚单位的磷酸化和随后的内化。在具体目标2中，将进行实验来验证NADPH氧化酶激活加速GLUR2 AMPAR亚基早期内吞转运的假设。最后，针对特定目的3设计实验，以检验抑制NADPH氧化酶活性是否阻止与短暂性缺血相关的AMPAR的功能变化，这是导致海马神经元延迟性死亡的原因。这项提案旨在以强调蒙大拿大学保健专业和生物医学学院研究生和本科生培训的方式实现这些目标。因此，这个项目将纳入研究生药业。D.学生，以及本科生。综上所述，该项目试图描述由NADPH氧化酶活性增加所触发的氧化应激信号级联，该活性导致短暂性脑缺血后细胞表面钙离子通透性AMPAR表达增加。如提案说明所示，将把重点放在学生培训上，包括实际操作的板凳研究经验、研讨会和小组会议介绍，以及最终在同行评议的期刊上传播结果。 公共卫生相关性：缺血再灌注后损伤的一个标志是突触AMPAR亚单位组成的变化。这种AMPAR亚基组成的变化导致细胞表面钙离子的表达增加。透气性AMPAR。这些钙离子通透性的AMPAR参与了短暂性脑缺血诱导的迟发性神经元死亡。我们提出了一个新的机制概念，即缺血再灌注期间NADPH氧化酶活性的增加是突触AMPAR亚单位组成变化的触发因素，即从钙不通透转变为钙通透。我们推测，超氧化物歧化酶NADPH氧化酶的激活在脑缺血后神经元中AMPARs亚单位组成和功能的突触改变中起关键作用。这些研究将验证NADPH氧化酶的激活是缺血-再灌注引起的钙通透性AMPAR表面表达增加的关键的假说。

DESCRIPTION (provided by applicant): AMPA receptors (AMPAR) are a major glutamate receptor in the CNS and are assembled from GLUR1, GLUR2, GLUR3, and GLUR4 subunits. Most AMPARs expressed on hippocampal pyramidal neurons contain the edited form of GLUR2, and are thus impermeable to Ca2? entry. Recent studies have shown that AMPARs play a key role in promoting delayed neuronal death following post-ischemic injury. These glutamatergic receptors undergo a change in subunit composition during post-ischemic reperfusion, changing from a GLUR2-containing Ca2?impermeable AMPAR to a GLUR2-lacking Ca2?permeable receptor. There is substantial evidence that transient global ischemia induced delayed death of hippocampal pyramidal neurons involves activation of these Ca2?permeable AMPARs. At present, the mechanisms responsible for the ischemia/reperfusion-induced expression of GLUR2-lacking AMPARs are not known. However, recent studies indicate that an oxidative stress signaling pathway is responsible for the change in subunit composition of AMPARs. Studies indicate that NADPH oxidase may be the source that initiates the oxidative stress-signaling cascade during post- ischemic reperfusion. The goal of this proposal is to test the hypothesis that increased activity of NADPH oxidase during reperfusion triggers the sequestration and subsequent degradation of the GLUR2 AMPAR subunit leading to an increased surface expression of GLUR2-lacking AMPARs. The proposed study will use acute adult rat hippocampal brain slices. Experiments designed for Specific Aim 1 will test the hypothesis that suppression of NADPH oxidase activity prevents the increase in phosphorylation and subsequent internalization of the GLUR2 AMPAR subunit by ischemia- reperfusion. In Specific Aim 2, experiments will be performed to test the hypothesis that NADPH oxidase activation accelerates the early endocytic trafficking of GLUR2 AMPAR subunit. Lastly, experiments are designed for Specific Aim 3 to examine whether suppression of NADPH oxidase activity prevents the functional change in AMPARs associated with transient ischemia that is responsible for the delayed death in hippocampal neurons. This proposal seeks to carry out these aims in a manner that emphasizes both graduate and undergraduate training at the College of Health Professions and Biomedical Sciences at The University of Montana. Therefore, this project will incorporate graduate students, Pharm. D. students, as well as undergraduate students. In summary, this project seeks to characterize the oxidative stress-signaling cascade, triggered by increased NADPH oxidase activity that leads to an increased surface expression of Ca2?permeable AMPARs following transient ischemia. As indicated within the proposal description, an emphasis will be placed on student training, including hands-on bench research experiences, seminar and group meeting presentations, as well as eventual dissemination of results in peer-reviewed journals. PUBLIC HEALTH RELEVANCE: A hallmark of post-ischemic reperfusion injury is a change in subunit composition of synaptic AMPARs. This change in AMPAR subunit composition leads to an increase in surface expression of Ca2? permeable AMPARs. These Ca2?permeable AMPARs have been implicated in mediating transient ischemia induced-delayed neuronal death. We propose a novel mechanistic concept in which increased NADPH oxidase activity during post-ischemic reperfusion is the trigger that underlies the change in subunit composition of synaptic AMPARs; changing from Ca2?impermeable to Ca2?permeable AMPARs. We postulate that activation of the superoxide generator NADPH oxidase has a critical role in the synaptic alteration of subunit composition and function of AMPARs in post-ischemic neurons. The proposed studies will test the hypothesis that activation of NADPH oxidase is key to the ischemia- reperfusion induced increase in surface expression of Ca2?permeable AMPARs.