Calpastatin overexpression as a therapeutic approach to traumatic brain injury

钙蛋白酶抑制素过度表达作为创伤性脑损伤的治疗方法

基本信息

批准号：
8386581
负责人：
Kathleen Marie Schoch
金额：
$ 0.92万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-05 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8386581
关键词：
Address Affect Area Attenuated Behavior Behavioral Brain Injuries Brain region C-terminal Calcium Calpain Cells Cessation of life Cleaved cell Cognitive Cognitive deficits Convection Cysteine Protease Cytoskeletal Proteins Diffusion Elements Exhibits Functional disorder Genes Goals Hippocampus (Brain)Human Image Immunoblot Analysis Immunoblotting Immunohistochemistry Individual Infusion procedures Injection of therapeutic agent Injury Lentivirus Vector Location Measurement Mediating Mediator of activation protein Membrane Membrane Proteins Methods Mitochondrial Proteins Motor Mus Nerve Degeneration Neurologic Neurons Pathogenesis Peptide Hydrolases Performance Placebos Prions Proteolysis Regulatory Element Research Role Severities Subfamily lentivirinae Testing Therapeutic Tissues Transgenic Mice Transgenic Organisms Trauma Traumatic Brain Injury Variant Viral behavior measurement brain tissue calpain inhibitor calpastatin cognitive function cohort controlled cortical impact effective therapy expectation improved inhibitor/antagonist injured morris water maze motor deficit novel overexpression promoter public health relevance receptor response treatment strategy

项目摘要

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.

描述（由申请人提供）：美国每年约有 140 万人遭受创伤性脑损伤 (TBI)。 TBI 后，多种细胞介质会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持续增加，神经元内钙蛋白酶的长时间激活发生。尽管钙蛋白酶的内源性抑制剂钙蛋白酶抑制素是共表达的，但这些钙依赖性蛋白酶的持续激活表明内源性钙蛋白酶抑制素水平可能不足。该提议的总体假设是，钙蛋白酶抑制素的过度表达将减少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。 Calpastatin 过度表达将以两种方式诱导：人 Calpastatin (hCAST) 转基因过度表达（目标 1）和通过慢病毒载体递送到易受 TBI 影响的大脑区域表达 Calpastatin（目标 2）。目标 1 将使用一种新型转基因小鼠品系，其含有受普遍存在的朊病毒启动子控制的人钙蛋白酶抑制剂。与野生型小鼠相比，该小鼠系的皮质和海马中钙蛋白酶抑制素的表达量高出 9 倍。 hCAST 转基因和野生型同窝动物将受到严重的受控皮质撞击 (CCI) 损伤或假治疗。为了确认钙蛋白酶抑制素过度表达会降低钙蛋白酶活性，将通过免疫印迹评估皮质和海马匀浆中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，之后将小鼠安乐死以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 hCAST 转基因小鼠将减少创伤后钙蛋白酶的蛋白水解活性，从而提供神经保护优势。目标 2 建立了一种通过慢病毒载体递送实现 hCAST 过表达的替代方法。将对照慢病毒或卡蛋白酶抑制剂慢病毒注射到皮层或海马后，小鼠将接受 1.0mm CCI 脑损伤或假治疗，并按照目标 1 进行评估。在损伤前将钙蛋白酶抑制剂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。