Cytoskeletal processing in sublethal brain injury

亚致死性脑损伤中的细胞骨架加工

基本信息

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
项目类别：
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

项目摘要

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）：新生儿大脑对脱氧性低氧损伤异常敏感。常见伴随着极早出生的伴奏，一定的缺氧时期破坏了突触新生大脑的重塑和成熟，可能会说明长期这些孩子的认知缺陷。促成突触组织的一种机制功能是神经元光谱骨架。最近的数据表明特定的同工型 Spectrin在轴突运输，受体运输和受体中的效果不同组织和受体转换。这些活动统称对神经元和突触功能。由于光谱骨架受钙信号通路调节涉及钙调蛋白和钙蛋白酶蛋白水解，以及通过磷酸化的Ser和Ser和通过最近公认的酪氨酸磷酸化，我们假设这是不合适的轻度低氧损伤后神经元光谱骨架的修饰有助于早产脑功能障碍的病理学。因此，重要的是要理解生理钙激活蛋白酶介导的光谱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识别特定的钙蛋白酶切割位点在betai，betaiii和betaiv谱系中，并确定这些光谱对钙蛋白酶的敏感性是否在谱蛋白裂解上构成耦合（betaii spectrin cleavage）。这些直到最近才在专业神经元中鉴定出其他β-光谱同工型车厢及其对蛋白水解的敏感性尚不清楚，生物学也是如此这种乳沟的后果。使用切割特异性抗体，地形和使用WT小鼠和动物进行了遗传修饰，因此使用CRE-Lo-LoxP重新组合酶选择性地删除了外显子，将评估这些光谱蛋白对这些光谱和alphaii光谱的体内处理，并在患有轻度余心缺氧的小鼠中进行评估。这些外显子编码了超敏的钙蛋白酶和胱天蛋白酶3个靶位点，两个推定的酪氨酸磷酸化位点以及α谱的钙调蛋白结合结构域。因此，光谱蛋白的关键中央区域是CA ++和磷酸化介导的信号通路之间的收敛点。通过产生这些动作中的每一种动物，将确定这些途径对神经元成熟和生存力的贡献。项目核心资源将用于评估这些表型。