Cytoskeletal processing in sublethal brain injury

描述(由申请人提供)： 新生儿的大脑对亚致死性缺氧损伤异常敏感。作为一种常见的 伴随着极早产，亚致死性缺氧期扰乱突触 新生儿大脑的重塑和成熟，从而可能解释长期的 这类儿童的认知缺陷。促进突触组织的一种机制 而功能就是神经元的幽灵骨架。最近的数据显示，特定的亚型 在轴突运输、受体运输和受体运输中发挥不同的作用 组织和受体的周转。总的来说，这些活动对神经元和 突触功能。由于血影蛋白骨架受钙信号通路的调节 参与钙调蛋白和钙蛋白酶的蛋白分解，以及丝氨酸和钙蛋白酶的磷酸化。 通过最近发现的酪氨酸磷酸化，我们假设这是不合适的 轻度缺氧性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。因此很重要的一点是要理解生理学 钙激活的蛋白水解酶介导的血影蛋白加工的病理后果 发育中的大脑。我们将使用体外分析来确定特定的Calain裂解位点 在BetaI、BetaIII和BetaIV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切割变构偶联(与BetaII血影蛋白切割一样)。这些 最近才在专门的神经元中发现了其他的β-血影蛋白亚型。 它们对蛋白质分解的敏感性尚不清楚，生物学上也是如此 这种乳沟的后果。使用切割特异性抗体，地形和 在经历轻度亚致死性缺氧的小鼠中，将使用wt小鼠和动物进行评估，通过使用cre-loxP重组酶系统选择性地删除血影蛋白基因的外显子28-30，来评估体内对这些血影蛋白和alphaII血影蛋白的瞬时处理。这些外显子编码高度敏感的钙蛋白和半胱氨酸天冬氨酸氨基转移酶3靶点，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，这个关键的血影蛋白中心区域是大脑中钙离子和磷酸化介导的信号通路的交汇点。通过产生这些行为中的每一个都被选择性地阻断的动物，将确定这些途径对神经元成熟和存活的贡献。项目核心资源将用于评估这些表型。

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.