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: 7583695
• 负责人: ROBERT MELLER
• 金额: $ 34.5万
• 依托单位: LEGACY EMANUEL HOSPITAL AND HEALTH CENTER
• 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/7583695
• 关键词: Actins; Address; Adenosine; Biological; Brain; Calcium Signaling; Cardiopulmonary Bypass; Cells; Chromosome Pairing; Complex; Cyclic AMP-Responsive DNA-Binding Protein; Cytoskeleton; DNA Sequence Rearrangement; Data; Dendrites; Dendritic Spines; Diazoxide; Disruption; 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; Public Health; 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 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）：我们建议确定调节内源性快速缺血耐受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，我们已经确定了快速耐受中泛素化蛋白的模式：许多蛋白质参与突触后密度的调节或功能。预处理缺血后，关键突触后结构元件的蛋白质降解导致肌动蛋白丝的重排和树突棘的缩回。预处理缺血后神经元形态的变化导致NMDA细胞信号传导改变，耐受性细胞中NMDA诱导的神经毒性降低。因此，进一步了解快速缺血耐受的机制可能有助于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，我们的中心假设是：预适应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)锚定在细胞骨架上的NMDA受体的破坏，c)从而改变NMDA受体的功能，从而保护NMDA受体免受有害缺血。这些事件发生迅速-在一小时内-与急性中风治疗有关。该项目旨在验证我们的假设，并研究形态重组是否是对预处理因子的广义反应。该项目利用体内和体外缺血模型。该提案有四个目标。具体目的一：验证调节肌动蛋白细胞骨架重组的蛋白质介导预适应缺血后神经保护的假设。特别是预处理缺血对WAVE-1、CYFIP和肌动蛋白相关蛋白2/3 （Arp2/3）复合物蛋白水平和相互作用的影响。此外，还将研究Arp2/3复合物的药理学和病毒转移介导的肽抑制剂以及上游调节蛋白在快速缺血耐受中的作用。目的二：验证预适应缺血诱导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的相互作用和激活的影响。具体目的三：验证树突棘丢失、肌动蛋白重组和NMDA保护是药物和缺血预处理后的常见表型的假设。我们将测试两种快速耐受的药物诱导剂（腺苷和二氮氧化合物）是否具有类似于快速缺血耐受的保护性表型。具体来说，我们将确定腺苷和二氮氧化物对肌动蛋白丝重组、树突棘密度和对NMDA兴奋毒性耐受性的影响。目的四：在体内缺血模型中验证预适应缺血诱导突触结构重组的假设。为了确定我们观察的治疗潜力，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介导的兴奋毒性的变化。特定脑蛋白的快速和选择性降解诱导保护状态，并可能揭示合适的药物治疗靶点。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，用于中风或可能预测缺血的地方，例如心脏搭桥手术。公共卫生相关性：我们的新观察表明，神经元形态的改变是短暂缺血（中风）后保护机制的一部分。我们的目标是研究这些机制，以帮助开发新的治疗中风的方法，或在缺血可以预测的情况下，如心脏搭桥手术。