Calpastatin overexpression as a therapeutic approach to traumatic brain injury

描述（由申请人提供）：创伤性脑损伤（TBI）每年在美国折磨大约140万人。TBI后，多种细胞介质导致神经元死亡和功能障碍，包括半胱氨酸蛋白酶、钙蛋白酶。由于细胞内游离钙的快速和持续上升，钙蛋白酶的长期激活发生在神经元内。尽管钙蛋白酶的内源性抑制剂钙蛋白酶抑制剂共表达，但这些钙依赖性蛋白酶的持续活化表明内源性钙蛋白酶抑制剂水平可能不足。该建议的总体假设是钙蛋白酶抑制蛋白的过表达将降低钙蛋白酶的蛋白水解活性和创伤后相关的神经元死亡，从而减轻运动和认知缺陷。钙蛋白酶抑制蛋白过表达将以两种方式诱导-人钙蛋白酶抑制蛋白（hCAST）的转基因过表达（Aim 1）和钙蛋白酶抑制蛋白通过慢病毒载体递送到易受TBI影响的脑区域中的表达（Aim 2）。 目的1将使用一种新的转基因小鼠品系与人钙蛋白酶抑制蛋白的普遍存在的朊病毒启动子控制下。与野生型小鼠相比，该小鼠系在皮质和海马中表现出9倍更高的钙蛋白酶抑制蛋白表达。hCAST转基因和野生型同窝仔将经受严重的受控皮质撞击（CCI）损伤或假处理。为了证实钙蛋白酶抑制蛋白过表达降低钙蛋白酶活性，将通过免疫印迹评价皮质和海马匀浆的钙蛋白酶介导的细胞骨架和膜蛋白分解。损伤后将评估运动和认知功能，之后将对小鼠实施安乐死以分析海马神经变性和皮质组织损伤，从而评估hCAST过表达的神经保护作用。预期hCAST转基因小鼠将具有降低的创伤后钙蛋白酶蛋白水解活性，提供神经保护优势。目的2建立一种通过慢病毒载体递送hCAST过表达的替代方法。在将对照慢病毒或钙蛋白酶抑制蛋白慢病毒注射到皮质或海马中后，将对小鼠进行1.0mm CCI脑损伤或假处理，并如目的1中所述进行评估。在损伤前将钙蛋白酶抑制蛋白靶向于脆弱的神经元区域，可以使受影响的神经元免于损伤，并减少行为缺陷。 公共卫生相关性：鉴于创伤性脑损伤的范围，必须了解神经元损伤和死亡的机制，以制定有效的治疗策略。我的研究目标不仅是寻求建立一个功能，神经保护作用的钙蛋白酶抑制蛋白在脑创伤，但也推进翻译努力治疗脑损伤。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) afflicts approximately 1.4 million individuals in the U.S. each year. Following TBI, a variety of cellular mediators contribute to neuronal death and dysfunction including the cysteine proteases, calpains. Prolonged activation of calpains occurs within neurons due to a rapid and sustained rise of intracellular free calcium. Although an endogenous inhibitor of calpains, calpastatin, is co- expressed, the sustained activation of these calcium-dependent proteases suggests endogenous calpastatin levels may be insufficient. The overall hypothesis of this proposal is that overexpression of calpastatin will reduce the proteolytic activity of calpains and associated neuronal death after trauma, thereby attenuating motor and cognitive deficits. Calpastatin overexpression will be induced in two ways-transgenic overexpression of human calpastatin (hCAST) (Aim 1) and calpastatin expression via lentiviral vector delivery into brain regions vulnerable to TBI (Aim 2). Aim 1 will use a novel transgenic mouse line with human calpastatin under control of the ubiquitous prion promoter. This mouse line exhibits a 9-fold greater expression of calpastatin in the cortex and hippocampus compared to wildtype mice. hCAST transgenic and wildtype littermates will be subjected to severe controlled cortical impact (CCI) injury or sham treatment. To confirm that calpastatin overexpression decreases calpain activity, cortical and hippocampal homogenates will be evaluated for calpain-mediated cytoskeletal and membrane protein breakdown via immunoblot. Both motor and cognitive functions will be assessed after injury, after which mice will be euthanized for analysis of hippocampal neurodegeneration and cortical tissue damage to assess the neuroprotective actions of hCAST overexpression. The expectation is that hCAST transgenic mice will have reduced posttraumatic calpain proteolytic activity, offering a neuroprotective advantage. Aim 2 establishes an alternative approach to hCAST overexpression through lentiviral vector delivery. After injection of control lentivirus or calpastatin lentivirus into the cortex or hippocampus, mice will be subjected to 1.0mm CCI brain injury or sham treatment and assessed as in Aim 1. Targeting of calpastatin to vulnerable neuronal regions prior to injury should spare affected neurons and reduce behavioral deficits. PUBLIC HEALTH RELEVANCE: Given the scope of traumatic brain injury, it is imperative to understand the mechanisms of neuronal damage and death in order to develop effective treatment strategies. My research aims will not only seek to establish a functional, neuroprotective role for calpastatin in brain trauma but also advance translational efforts for the treatment of brain injury.