Calpastatin overexpression as a therapeutic approach to traumatic brain injury

描述（由申请人提供）：在美国，创伤性脑损伤（TBI）每年折磨着大约140万人。创伤性脑损伤后，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的增加，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pastatin共同表达，但这些钙依赖性蛋白酶的持续激活表明内源性钙pastatin水平可能不足。该建议的总体假设是，钙pastatin的过表达会降低创伤后钙蛋白酶的蛋白水解活性和相关的神经元死亡，从而减轻运动和认知缺陷。Calpastatin的过表达将通过两种方式诱导：转基因人Calpastatin （hCAST）的过表达（Aim 1）和通过慢病毒载体将Calpastatin传递到脑外伤易感区域（Aim 2）。在普遍存在的朊病毒启动子的控制下，Aim 1将使用一种具有人类calpastatin的新型转基因小鼠系。与野生型小鼠相比，该小鼠系在皮质和海马体中calpastatin的表达增加了9倍。hCAST转基因和野生型的幼崽将遭受严重的控制性皮质冲击（CCI）损伤或假治疗。为了证实钙pastatin过表达会降低钙蛋白酶活性，我们将通过免疫印迹对皮质和海马匀浆进行钙蛋白酶介导的细胞骨架和膜蛋白分解评估。损伤后评估小鼠的运动和认知功能，然后对小鼠实施安乐死，分析海马神经变性和皮质组织损伤，以评估hCAST过表达的神经保护作用。预期是hCAST转基因小鼠将减少创伤后钙蛋白酶的蛋白水解活性，提供神经保护的优势。目的2建立了一种通过慢病毒载体传递hCAST过表达的替代方法。将对照慢病毒或calpastatin慢病毒注入小鼠皮质或海马后，给予1.0mm的CCI脑损伤或假治疗，并按照Aim 1进行评估。在损伤前将calpastatin靶向易感神经元区域，可以避免受损神经元，减少行为缺陷。

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.