Rapid ischemic tolerance: Synaptic re-organization and reduced excitotoxicity

快速缺血耐受：突触重组和兴奋性毒性降低

• 批准号: 8142097
• 负责人: ROBERT MELLER
• 金额: $ 30.33万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8142097
• 关键词: Actins; Address; Adenosine; Biological; Brain; Calcium Signaling; Cardiopulmonary Bypass; Cells; Complex; Cyclic AMP-Responsive DNA-Binding Protein; Cytoskeleton; DNA Sequence Rearrangement; Data; Dendrites; Dendritic Spines; Diazoxide; Elements; Event; Family; Filament; Fragile X Syndrome; Grant; Health; Homologous Protein; Hour; Injury; Ischemia; Ischemic Preconditioning; Mass Spectrum Analysis; Mediating; Microfilaments; Modeling; Molecular; Morphology; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Neurons; Neuroprotective Agents; Nitric Oxide; Operative Surgical Procedures; Pattern; Peptides; Phenotype; Phosphorylation; Proteins; Proteomics; Publishing; Receptor Activation; Receptor Signaling; Regulation; Role; Signal Transduction; Stroke; Synapses; System; Testing; Therapeutic; Time; Translating; Ubiquitin; Ubiquitination; Viral; acute stroke; attenuation; density; excitotoxicity; genetic regulatory protein; in vitro Model; in vivo; in vivo Model; inhibitor/antagonist; multicatalytic endopeptidase complex; neuroprotection; neurotoxicity; novel; novel therapeutics; preconditioning; protein complex; protein degradation; receptor function; response; scaffold; src-Family Kinases; stroke therapy

DESCRIPTION (provided by applicant): We propose to identify molecular and cellular mechanisms that regulate the endogenous neuroprotective phenomenon of rapid ischemic tolerance. Our prior studies have shown that rapid ischemic tolerance is mediated by selective protein degradation via the ubiquitin-proteasome system. Using proteomics we have identified a pattern of ubiquitinated proteins in rapid tolerance: many of the proteins are involved with the regulation or function of the post synaptic density. Following preconditioning ischemia protein degradation of key post-synaptic structural elements results in rearrangement of actin filaments and the retraction of dendritic spines. These changes in neuronal morphology following preconditioning ischemia result in altered NMDA cell signaling and decreased NMDA-induced neurotoxicity in tolerant cells. Therefore further understanding the mechanisms of rapid ischemic tolerance may identify morphological and cell signaling at the synapse, and reveal novel targets for neuroprotective therapies. Our central hypothesis for this proposal is thus: - Rapid ischemic tolerance following preconditioning ischemia results in a) actin cytoskeletal re-arrangement and synaptic reorganization leading to b) the disruption of NMDA receptor anchoring to the cytoskeleton, c) thus altering NMDA receptor function with resultant protection from harmful ischemia. These events occur rapidly- within one hour- and have relevance for acute stroke therapy. This project with test our hypothesis and well as investigating whether morphological re-organization is a generalized response to preconditioning agents. The project utilizes both in vivo and in vitro models of ischemia. The proposal has 4 aims. SPECIFIC AIM ONE: Test the hypothesis that proteins regulating actin cytoskeleton reorganization mediate neuroprotection following preconditioning ischemia. Specifically the effect of preconditioning ischemia on WAVE-1, CYFIP, and actin related protein 2/3 (Arp2/3) complex protein levels and interactions. In addition both pharmacological and viral transfer mediated peptide inhibitors of the Arp2/3 complex and upstream regulatory proteins will be investigated for their role in rapid ischemic tolerance. SPECIFIC AIM TWO: Test the hypothesis that preconditioning ischemia induces a change in NMDA receptor function. We will investigate the effect of preconditioning on NMDA receptor-mediated electrophysiological responses, calcium signaling, nitric oxide synthesis and cyclic AMP response element binding protein (CREB) phosphorylation. In addition we will investigate the effect of reconditioning on NMDA receptor interactions with, and activation of, the tyrosine kinases src and pyk. SPECIFIC AIM THREE: Test the hypothesis that dendritic spine loss, actin re-organization and NMDA protection is a common phenotype following pharmacological as well as ischemic preconditioning. We will test whether two pharmacological inducers of rapid tolerance (adenosine and diazoxide) have a protective phenotype similar to rapid ischemic tolerance. Specifically we will determine the effect of adenosine and diazoxide on actin filaments reorganization, dendritic spine density and tolerance to NMDA excitotoxicity. SPECIFIC AIM FOUR: Test the hypothesis that preconditioning ischemia induces synaptic structural re-organization in an in vivo model ischemia. To determine the therapeutic potential of our observations we will determine, in a focal model of ischemia, whether preconditioning induces changes to dendrite morphology, actin organization, NMDA receptor scaffolding and NMDA mediated excitotoxicity. The rapid and selective degradation of specific brain proteins induces a protective state and may reveal suitable targets for pharmacological therapeutics. Indeed, the long-term aim of these studies is to discover endogenous protective mechanisms that can be translated into effective rapid-acting, but long lasting, neuroprotective agents for stroke or where ischemia may be predicted for example, heart bypass surgery. PUBLIC HEALTH RELEVANCE: Our novel observations suggest that the morphology of neurons change as part of a protective mechanism following brief ischemia (stroke). It is our aim to investigate these mechanisms to help develop new therapeutics for stroke, or for circumstances where ischemia can be predicted, such as heart bypass surgery.

描述（由申请人提供）：我们建议鉴定调节快速缺血耐受的内源性神经保护现象的分子和细胞机制。我们之前的研究表明，快速缺血耐受是通过泛素-蛋白酶体系统选择性蛋白质降解介导的。使用蛋白质组学，我们已经确定了快速耐受的泛素化蛋白质的模式：许多蛋白质与突触后密度的调节或功能有关。预处理后，关键突触后结构元件的缺血蛋白降解导致肌动蛋白丝重新排列和树突棘回缩。预处理缺血后神经元形态的这些变化导致 NMDA 细胞信号传导改变，并降低耐受细胞中 NMDA 诱导的神经毒性。因此，进一步了解快速缺血耐受的机制可能会识别突触的形态和细胞信号传导，并揭示神经保护治疗的新靶点。因此，我们对该提议的中心假设是： - 预处理缺血后的快速缺血耐受导致 a) 肌动蛋白细胞骨架重排和突触重组，导致 b)​​ 锚定到细胞骨架的 NMDA 受体破坏，c) 从而改变 NMDA 受体功能由此产生的针对有害缺血的保护。这些事件在一小时内迅速发生，并且与急性中风治疗相关。该项目测试了我们的假设，并调查形态重组是否是对预处理剂的普遍反应。该项目利用体内和体外缺血模型。该提案有 4 个目标。具体目标一：检验调节肌动蛋白细胞骨架重组的蛋白质在预处理缺血后介导神经保护作用的假设。具体来说，预处理缺血对 WAVE-1、CYFIP 和肌动蛋白相关蛋白 2/3 (Arp2/3) 复合蛋白水平和相互作用的影响。此外，将研究药物和病毒转移介导的 Arp2/3 复合物肽抑制剂和上游调节蛋白在快速缺血耐受中的作用。具体目标二：检验预处理缺血会引起 NMDA 受体功能变化的假设。我们将研究预处理对 NMDA 受体介导的电生理反应、钙信号传导、一氧化氮合成和环 AMP 反应元件结合蛋白 (CREB) 磷酸化的影响。此外，我们将研究修复对 NMDA 受体与酪氨酸激酶 src 和 pyk 相互作用以及激活的影响。具体目标三：检验以下假设：树突棘损失、肌动蛋白重组和 NMDA 保护是药理学和缺血预处理后的常见表型。我们将测试两种快速耐受的药理学诱导剂（腺苷和二氮嗪）是否具有与快速缺血耐受相似的保护表型。具体来说，我们将确定腺苷和二氮嗪对肌动蛋白丝重组、树突棘密度和对 NMDA 兴奋性毒性的耐受性的影响。具体目标四：检验预处理缺血会在体内缺血模型中诱导突触结构重组的假设。为了确定我们观察到的治疗潜力，我们将在局部缺血模型中确定预处理是否会诱导树突形态、肌动蛋白组织、NMDA 受体支架和 NMDA 介导的兴奋性毒性的变化。特定脑蛋白的快速和选择性降解会诱导保护状态，并可能揭示药物治疗的合适靶点。事实上，这些研究的长期目标是发现内源性保护机制，这些机制可以转化为有效的快速作用但持久的神经保护剂，用于治疗中风或可预测缺血的情况，例如心脏搭桥手术。公共健康相关性：我们的新观察结果表明，神经元形态的变化是短暂缺血（中风）后保护机制的一部分。我们的目标是研究这些机制，以帮助开发针对中风或可预测缺血的情况（例如心脏搭桥手术）的新疗法。