Cytoskeletal processing in sublethal brain injury

描述（由申请人提供）： 新生儿脑对亚致死性缺氧损伤异常敏感。作为共同 伴随着极度早产，亚致死缺氧期破坏了突触， 新生儿大脑的重塑和成熟，从而可能解释长期的 这些孩子的认知缺陷。突触组织的一种机制 功能是神经元血影蛋白骨架。最近的数据显示， 血影蛋白在轴突运输、受体运输和受体转运中起着不同的作用。 组织和受体周转。总的来说，这些活动对神经元和 突触功能由于血影蛋白骨架是由钙信号通路调节的， 包括钙调蛋白和钙蛋白酶蛋白水解，以及通过丝氨酸和丝氨酸蛋白酶上的磷酸化， 通过最近认识到的酪氨酸磷酸化，我们假设， 轻度缺氧损伤后神经元血影蛋白骨架的修饰有助于 早产儿脑功能障碍的病理学重要的是要了解生理 钙激活蛋白酶介导的血影蛋白加工的病理后果 大脑发育。我们将使用体外分析来确定特定的钙蛋白酶切割位点 在β I、β III和β IV血影蛋白中，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偶联（如β II血影蛋白裂解）。这些 另外的β-血影蛋白同种型最近才在专门的神经元细胞中被鉴定， 隔室，它们对蛋白质水解的敏感性是未知的，生物学上也是如此。 这种分裂的后果。使用切割特异性抗体， 在经历轻度亚致死性缺氧的小鼠中，使用野生型小鼠和经遗传修饰的动物来评估钙蛋白酶对这些血影蛋白和α II血影蛋白的暂时体内加工，所述经遗传修饰的动物使得血影蛋白基因的外显子28-30已经使用Cre-loxP重组酶系统选择性缺失。这些外显子编码过敏性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，和α II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脑中Ca ++和磷酸化介导的信号传导途径之间的汇聚点。通过产生这些作用中的每一个都被选择性阻断的动物，将确定这些通路对神经元成熟和活力的贡献。项目核心资源将用于评价这些表型。

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.