Calpastatin overexpression as a therapeutic approach to traumatic brain injury

描述(由申请人提供)：在美国，每年约有140万人患有创伤性脑损伤(TBI)。脑外伤后，多种细胞因子参与神经元死亡和功能障碍，包括半胱氨酸蛋白酶、钙蛋白酶等。由于细胞内游离钙的快速和持续上升，神经元内钙调蛋白的长期激活发生。尽管钙蛋白酶的内源性抑制物calastatin是共表达的，但这些钙依赖的蛋白水解酶的持续激活表明内源性calastatin水平可能不足。这一提议的总体假设是，过度表达calastatin将减少calain的蛋白分解活性和相关的神经元在创伤后的死亡，从而减轻运动和认知障碍。Calastatin的过度表达将通过两种方式诱导--转基因过表达人Calastatin(HCAST)(Aim 1)和通过慢病毒载体将Calastatin表达到易受脑创伤影响的脑区(Aim 2)。目的1将使用一种新的转基因小鼠系，在普遍存在的Prion启动子的控制下，使用人Calastatin。与野生型小鼠相比，该品系小鼠大脑皮质和海马区的钙调蛋白表达增加了9倍。HCAST转基因和野生型窝产仔将受到严重的可控皮质冲击(CCI)伤害或假处理。为了证实过度表达calastatin会降低calain的活性，我们将通过免疫印迹来评估皮质和海马匀浆中calain介导的细胞骨架和膜蛋白的分解。损伤后将评估运动和认知功能，之后将对小鼠进行安乐死，分析海马神经变性和皮质组织损伤，以评估hCAST过度表达的神经保护作用。人们的期望是，hCAST转基因小鼠将降低创伤后钙蛋白水解酶的活性，从而提供神经保护优势。目的2建立一种通过慢病毒载体传递hCAST过表达的替代方法。在将对照慢病毒或钙调蛋白慢病毒注射到大脑皮质或海马区后，小鼠将受到1.0 mM CCI脑损伤或假治疗，并按照目标1进行评估。在损伤前将钙调蛋白靶向脆弱的神经元区域应避免受影响的神经元并减少行为缺陷。 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，以便制定有效的治疗策略。我的研究目标不仅是寻求在脑损伤中建立钙调蛋白的功能性和神经保护作用，而且还将促进脑损伤治疗的翻译努力。

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.