Cytoskeletal processing in sublethal brain injury

亚致死性脑损伤中的细胞骨架加工

• 批准号: 6740731
• 负责人: JON S MORROW
• 金额: $ 29.38万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6740731
• 关键词: brain injury; calpain; cerebral ischemia /hypoxia; chemical cleavage; cysteine endopeptidases; cytoskeleton; genetically modified animals; immunocytochemistry; laboratory mouse; neuropathology; phosphorylation; protein structure function; proteolysis; spectrin; staurosporine; synaptogenesis; tissue /cell culture; transfection; tyrosine

DESCRIPTION (provided by applicant): The neonatal brain is unusually sensitive to sublethal hypoxic injury. As a common accompaniment of extremely premature birth, periods of sublethal hypoxia disrupt synaptic remodeling and maturation in the newborn brain, and may thereby account for the long-term cognitive deficiencies of such children. One mechanism contributing to synaptic organization and function is the neuronal spectrin skeleton. Recent data has revealed that specific isoforms of spectrin subserve distinct roles in axonal transport, receptor trafficking, and receptor organization and receptor turnover. Collectively these activities are crucial to neuronal and synaptic function. Since the spectrin skeleton is regulated by calcium signaling pathways involving calmodulin and calpain proteolysis, as well as by phosphorylation on both ser and thru and by a recently recognized tyrosine phosphorylation, we hypothesize that inappropriate modification of the neuronal spectrin skeleton following mild hypoxic injury contributes to the pathology of premature brain dysfunction. It is important to thus understand the physiologic and pathologic consequences of calcium activated protease-mediated spectrin processing in the developing brain. We will use in vitro analysis to identify the specific calpain cleavage sites in betaI, betaIII, and betaIV spectrin, and determine whether the susceptibility of these spectrins to calpain is allosterically coupled to spectrin cleavage (as is betaII spectrin cleavage). These additional beta-spectrin isoforms have only recently been identified in specialized neuronal compartments, and their susceptibility to proteolysis is unknown, as is the biologic consequences of such cleavage. Using cleavage-specific antibodies, the topographic and temporal in vivo processing of these spectrins and of alphaII spectrin by calpain will be assessed in mice experiencing mild sublethal hypoxia, using both wt mice and animals genetically modified such that exons 28-30 of the spectrin gene have been selectively deleted using the cre-loxp recombinase system. These exons encode the hypersensitive calpain and caspase 3 target sites, two putative sites of tyrosine phosphorylation, and the calmodulin binding domain of alphaII spectrin. This critical central region of spectrin thus serves as a point of convergence between Ca ++ and phosphorylation-mediated signaling pathways in the brain. By generating animals in which each of these actions has been selectively blocked, the contributions of these pathways to neuronal maturation and viability will be determined. Project Core resources will be used to evaluate these phenotypes.

描述（由申请人提供）： 新生儿大脑对亚致死性缺氧损伤异常敏感。作为一个常见的 伴随着极度早产，亚致死性缺氧时期会破坏突触 新生儿大脑的重塑和成熟，从而可能解释长期的 此类儿童的认知缺陷。有助于突触组织的一种机制 功能是神经元血影蛋白骨架。最近的数据表明，特定亚型 血影蛋白在轴突运输、受体运输和受体中发挥不同的作用 组织和受体更新。总的来说，这些活动对于神经元和 突触功能。由于血影蛋白骨架受钙信号通路调节 涉及钙调蛋白和钙蛋白酶的蛋白水解，以及丝氨酸和磷酸化 通过最近认识到的酪氨酸磷酸化，我们假设不适当的 轻度缺氧损伤后神经元血影蛋白骨架的修饰有助于 早产性脑功能障碍的病理学。因此了解生理学很重要 钙激活蛋白酶介导的血影蛋白加工的病理后果 正在发育的大脑。我们将使用体外分析来识别特定的钙蛋白酶切割位点 血影蛋白 betaI、betaIII 和 betaIV 中的血影蛋白，并确定这些血影蛋白对钙蛋白酶的敏感性是否与血影蛋白裂解变构耦合（如 betaII 血影蛋白裂解）。这些 最近才在专门的神经元中发现了其他 β-血影蛋白亚型 隔室，以及它们对蛋白水解的敏感性是未知的，生物制剂也是如此 这种分裂的后果。使用切割特异性抗体，拓扑和 将在经历轻度亚致死缺氧的小鼠中评估这些血影蛋白和αII血影蛋白的时间体内加工，使用野生型小鼠和基因修饰的动物，使得血影蛋白基因的外显子28-30已使用cre-loxp重组酶系统选择性删除。这些外显子编码超敏感钙蛋白酶和半胱天冬酶 3 靶位点、两个假定的酪氨酸磷酸化位点以及 αII 血影蛋白的钙调蛋白结合域。因此，血影蛋白的这一关键中心区域充当大脑中 Ca ++ 和磷酸化介导的信号通路之间的汇聚点。通过培育其中每种作用都被选择性阻断的动物，将确定这些途径对神经元成熟和活力的贡献。项目核心资源将用于评估这些表型。