Rapid ischemic tolerance: Synaptic re-organization and reduced excitotoxicity

点击翻译按钮获取中文摘要

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.