Synaptic Modification of AMPARs by Oxidative Stress

氧化应激对 AMPAR 的突触修饰

基本信息

批准号：
8101475
负责人：
DARRELL A JACKSON
金额：
$ 42.51万
依托单位：
UNIVERSITY OF MONTANA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8101475
关键词：
AMPA Receptors Ablation Acute Adult Biological Assay Biotinylation Brain Cessation of life Co-Immunoprecipitations Complex Confocal Microscopy Fluorescence GRIA3 gene Genetic Glucose Glutamate Receptor Glutamates Goals Group Meetings Guanine Nucleotide Dissociation Inhibitors Health Occupations Hippocampus (Brain)Immunoblotting Injury Ischemia Ischemic Stroke Journals Laboratories MAP Kinase Gene MAPK14 gene Measures Mediating Modification Montana NADPH Oxidase Neurons Oxidative Stress Oxygen Peer Review Phase Phosphorylation Phosphotransferases Play Proteins Rattus Reactive Oxygen Species Receptor Activation Reperfusion Injury Reperfusion Therapy Research Role Science Serine Signal Pathway Signal Transduction Slice Source Students Superoxides Surface Synapses System Testing Therapeutic Training Tyrosine Universities caspase-3 caspase-9 clinically significant college cytochrome c deprivation design experience glutamate receptor interacting protein graduate student hippocampal pyramidal neuron inhibitor/antagonist neuron loss neutrophil cytosol factor 67K novel prevent receptor research study small hairpin RNA trafficking

项目摘要

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.

描述（由申请人提供）：AMPA 受体（AMPAR）是 CNS 中的主要谷氨酸受体，由 GLUR1、GLUR2、GLUR3 和 GLUR4 亚基组装而成。大多数在海马锥体神经元上表达的 AMPAR 包含 GLUR2 的编辑形式，因此对 Ca2+ 是不可渗透的。入口。最近的研究表明，AMPAR 在促进缺血性损伤后迟发性神经元死亡中发挥着关键作用。这些谷氨酸能受体在缺血后再灌注期间经历亚基组成的变化，从含有GLUR2的Ca2+不可渗透的AMPAR变为缺乏GLUR2的Ca2+可渗透的受体。有大量证据表明，短暂性全身缺血诱导的海马锥体神经元延迟死亡涉及这些 Ca2+ 渗透性 AMPAR 的激活。目前，缺血/再灌注诱导缺乏 GLUR2 的 AMPAR 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 AMPAR 亚基组成变化的原因。研究表明，NADPH 氧化酶可能是缺血后再灌注期间启动氧化应激信号级联的来源。该提案的目的是检验以下假设：再灌注期间 NADPH 氧化酶活性的增加会触发 GLUR2 AMPAR 亚基的隔离和随后的降解，从而导致缺乏 GLUR2 的 AMPAR 的表面表达增加。拟议的研究将使用急性成年大鼠海马脑切片。为特定目标 1 设计的实验将检验以下假设：抑制 NADPH 氧化酶活性可防止缺血再灌注引起的 GLUR2 AMPAR 亚基的磷酸化增加和随后的内化。在具体目标 2 中，将进行实验来检验 NADPH 氧化酶激活加速 GLUR2 AMPAR 亚基早期内吞运输的假设。最后，针对特定目标 3 设计了实验，以检查 NADPH 氧化酶活性的抑制是否会阻止与短暂性缺血相关的 AMPAR 功能变化，而短暂性缺血是导致海马神经元延迟死亡的原因。该提案旨在以强调蒙大拿大学健康专业和生物医学科学学院研究生和本科生培训的方式实现这些目标。因此，该项目将纳入研究生，药学。 D.学生，以及本科生。总之，该项目旨在表征氧化应激信号级联反应，该级联反应是由 NADPH 氧化酶活性增加触发的，导致短暂缺血后 Ca2+ 渗透性 AMPAR 的表面表达增加。正如提案描述中所指出的，重点将放在学生培训上，包括实践台架研究经验、研讨会和小组会议演示，以及最终在同行评审期刊上传播结果。公共卫生相关性：缺血再灌注损伤的一个标志是突触 AMPAR 亚基组成的变化。 AMPAR 亚基组成的这种变化导致 Ca2? 表面表达增加。渗透性 AMPAR。这些 Ca2+ 渗透性 AMPAR 与介导短暂性缺血诱导的延迟性神经元死亡有关。我们提出了一个新的机制概念，其中缺血后再灌注期间 NADPH 氧化酶活性的增加是突触 AMPAR 亚基组成变化的触发因素；从 Ca2+ 不可渗透的 AMPAR 变为 Ca2+ 可渗透的 AMPAR。我们假设超氧化物生成剂 NADPH 氧化酶的激活在缺血后神经元 AMPAR 亚基组成和功能的突触改变中具有关键作用。拟议的研究将检验以下假设：NADPH 氧化酶的激活是缺血再灌注诱导 Ca2+ 渗透性 AMPAR 表面表达增加的关键。