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: 7915525
• 负责人: ROBERT MELLER
• 金额: $ 30.64万
• 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/7915525
• 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; Figs - dietary; Filament; Fragile X Syndrome; Grant; 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; actin-related protein 3; 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; public health relevance; 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）：我们提出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。我们先前的研究表明，快速缺血耐受是通过泛素-蛋白酶体系统介导的选择性蛋白质降解介导的。利用蛋白质组学，我们已经确定了一种模式的泛素化蛋白质的快速耐受：许多蛋白质参与的调节或功能的突触后密度。缺血预处理后，突触后关键结构元件的蛋白质降解导致肌动蛋白丝重排和树突棘回缩。预处理缺血后神经元形态学的这些变化导致耐受细胞中NMDA细胞信号转导的改变和NMDA诱导的神经毒性的降低。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学和细胞信号传导，并揭示神经保护治疗的新靶点。因此，我们对这一建议的中心假设是：-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）NMDA受体锚定到细胞骨架的破坏，c）因此改变NMDA受体功能，从而保护免受有害的缺血。这些事件发生迅速-在1小时内-并与急性卒中治疗相关。本研究旨在验证我们的假设，并探讨形态学重组是否是预处理剂的一种普遍反应。该项目利用体内和体外缺血模型。该提案有四个目标。具体目标一：验证调节肌动蛋白细胞骨架重组的蛋白质介导缺血预处理后的神经保护作用的假设。特别是预处理缺血对WAVE-1、CYFIP和肌动蛋白相关蛋白2/3（Arp 2/3）复合物蛋白水平和相互作用的影响。此外，将研究Arp 2/3复合物和上游调节蛋白的药理学和病毒转移介导的肽抑制剂在快速缺血耐受中的作用。目的二：验证缺血预处理诱导NMDA受体功能改变的假设。我们将研究预处理对NMDA受体介导的电生理反应、钙信号、一氧化氮合成和环磷酸腺苷反应元件结合蛋白（CREB）磷酸化的影响。此外，我们将调查NMDA受体的相互作用和激活，酪氨酸激酶src和pyk的再调节的效果。具体目标三：验证以下假设：树突棘丢失、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性表型。具体来说，我们将确定腺苷和二氮嗪对肌动蛋白丝重组，树突棘密度和耐受NMDA兴奋性毒性的影响。具体目的4：在体内缺血模型中检验缺血预处理诱导突触结构重组的假设。为了确定我们的观察结果的治疗潜力，我们将确定，在局部缺血模型中，预处理是否诱导树突形态，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诱导保护性状态，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速效但持久的神经保护剂，用于中风或可预测缺血的地方，例如心脏搭桥手术。公共卫生关系：我们新的观察结果表明，神经元的形态变化作为短暂缺血（中风）后保护机制的一部分。我们的目标是研究这些机制，以帮助开发新的治疗中风的方法，或用于可以预测缺血的情况，如心脏搭桥手术。