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(申请人提供)：我们建议确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，快速的缺血耐受是通过泛素-蛋白酶体系统选择性地降解蛋白质而介导的。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：许多蛋白参与了突触后密度的调节或功能。缺血预适应后，突触后关键结构元素的降解导致肌动蛋白细丝的重排和树突棘的回缩。这些预适应缺血后神经元形态的改变导致NMDA细胞信号的改变，并降低了NMDA诱导的耐受细胞的神经毒性。因此，进一步了解快速缺血耐受的机制可以确定突触的形态和细胞信号，并揭示神经保护治疗的新靶点。我们对这一提议的中心假设是：-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，从而保护其免受有害缺血的伤害。这些事件发生迅速--在一小时内--并与急性中风治疗有关。这个项目验证了我们的假设，并调查了形态重组是否是对预处理剂的普遍反应。该项目利用了体内和体外的缺血模型。该提案有4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说。尤其是缺血预适应对波-1、细胞周期调节蛋白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，还将研究药理和病毒转移介导的Arp2/3复合体的多肽抑制剂和上游调节蛋白在快速缺血耐受中的作用。具体目的二：验证预适应缺血引起NMDA受体功能改变的假说。我们将研究预适应对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，我们还将研究再适应对NMDA受体与酪氨酸激酶src和pYK相互作用和激活的影响。具体目的三：验证树突棘突丢失、肌动蛋白重组和NMDA保护是药物预适应和缺血预适应后常见表型的假设。我们将测试两种快速耐受的药物诱导剂(腺苷和二氮嗪)是否具有类似于快速缺血耐受的保护性表型。具体地说，我们将确定腺苷和二氮嗪对肌动蛋白细丝重组、树突棘密度和对NMDA兴奋毒性的耐受性的影响。具体目的四：在活体缺血模型中验证预适应缺血诱导突触结构重组的假说。为了确定我们观察到的治疗潜力，我们将在局灶性缺血模型中确定预适应是否会导致树突形态、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了一种保护状态，并可能揭示适合药物治疗的靶点。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、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。公共卫生相关性：我们的新观察表明，短暂缺血(中风)后，神经元的形态发生了变化，这是保护机制的一部分。我们的目标是研究这些机制，以帮助开发中风的新疗法，或可以预测缺血的情况，如心脏搭桥手术。