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）是中枢神经系统中的主要谷氨酸受体，并由GlUR1，GlUR2，Glur3和Glur4亚基组装。在海马锥体神经元上表达的大多数AMPAR都包含glur2的编辑形式，因此CA2不渗透？入口。最近的研究表明，AMPARS在促进缺血后损伤后延迟神经元死亡方面起着关键作用。这些谷氨酸能受体在缺血后再灌注期间发生了亚基组成的变化，从含Glur2的Ca2？不渗透的AMPAR变为glur2占用的Ca2？渗透受体。有大量证据表明，瞬时全球缺血引起的海马锥体神经元的延迟死亡涉及这些CA2？渗透性AMPAR的激活。目前，尚不清楚导致缺血/再灌注诱导的GLUR2累积AMPAR表达的机制。然而，最近的研究表明，氧化应激信号传导途径是AMPAR的亚基组成的变化。研究表明，NADPH氧化酶可能是在缺血后再灌注过程中启动氧化应激信号级联反应的来源。该提案的目的是检验以下假设：再灌注过程中NADPH氧化酶的活性增加会触发GLUR2 AMPAR亚基的隔离和随后降解，从而导致GLUR2占用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：NADPH氧化酶活性的抑制可防止磷酸化的增加和随后通过缺血再灌注对GLUR2 AMPAR亚基的内在化。在特定的目标2中，将进行实验以检验以下假设：NADPH氧化酶活性会加速GLUR2 AMPAR亚基的早期内吞运输。最后，设计用于特定目标3的实验，以检查NADPH氧化酶活性的抑制是否阻止了与短暂性缺血相关的AMPAR的功能变化，这导致了造成海马神经元延迟死亡的延迟死亡。该建议旨在以强调蒙大拿大学卫生专业和生物医学科学学院的研究生和本科培训的方式执行这些目标。因此，该项目将纳入研究生，Pharm。 D.学生以及本科生。总而言之，该项目试图表征氧化应激信号级联反应，这是由NADPH氧化酶活性增加触发的，导致瞬时缺血后Ca2？渗透性AMPAR的表面表达增加。如提案描述中所示，将重点放在学生培训上，包括动手替补研究经验，研讨会和小组会议演讲，以及最终在同行评审期刊中传播结果。公共卫生相关性：缺血后再灌注损伤的标志是突触AMPAR的亚基组成的变化。 AMPAR亚基组成的这种变化导致Ca2表面表达的增加？可渗透的Ampars。这些CA2？渗透性AMPAR与介导瞬时缺血诱导的神经元死亡有关。我们提出了一种新型的机械概念，其中缺血后再灌注期间增加的NADPH氧化酶活性是突触AMPARS亚基组成的变化的触发因素。从CA2？不渗透到Ca2？可渗透的AMPAR。我们假设超氧化物发电机NADPH氧化酶的激活在亚基组成的突触变化中具有关键作用，而AMPAR在缺血后神经元中的功能。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的Ca2？渗透性AMPAR表面表达增加的关键。