Caspase mediated cell death after brain trauma

描述（由申请人提供）：创伤性脑损伤（TBI）是美国45岁以下个体死亡的主要原因，幸存者面临慢性脑损伤，导致衰弱的行为功能障碍。脑损伤和行为功能障碍可能部分归因于TBI后的神经元死亡。在该提议中待测试的假设是创伤后神经元细胞死亡是半胱氨酸蛋白酶的促凋亡半胱天冬酶家族激活的结果。本提案的目的是阐明（1）实验性TBI和头部损伤患者死后组织中的神经元死亡与“执行者”半胱天冬酶-3活化之间的关联，（2）半胱天冬酶-3活化是否直接由于“起始者”半胱天冬酶-8活化而发生，和/或间接由于需要Bax易位的线粒体途径而发生，细胞色素c释放和半胱天冬酶-9活化，（3）半胱天冬酶-8活化是否由于死亡受体的肿瘤坏死因子家族的活化而发生，（4）Bax在介导创伤诱导的半胱天冬酶-9活化和随后的半胱天冬酶-3活化和细胞死亡中的作用，和（5）半胱天冬酶-3的创伤后抑制，-8和-9将降低损伤诱导的细胞死亡的程度。使用特异性和选择性抗体的免疫印迹和免疫组织化学分析将用于caspase-3，-8和-9活化的时间和区域模式，Bax和细胞色素c的重新分布，以及细胞凋亡神经元损伤，如细胞DNA片段化和形态学分析所示。TNF缺陷的小鼠将用于通过使用免疫印迹、免疫组织化学和组织学分析来确定TNF在介导半胱天冬酶-8和-3活化以及最终凋亡细胞死亡中的作用。Bax在创伤诱导的caspase-9和-3活化、细胞色素c再分布、凋亡性神经元死亡和行为功能障碍中的作用将使用免疫印迹和免疫组织化学技术进行检查，并通过测试脑损伤、Bax缺陷小鼠的认知和运动功能，然后通过细胞死亡的组织学分析进行检查。用对半胱天冬酶-3（DEVD）、半胱天冬酶-8（IETD）和半胱天冬酶-9（LEHD）具有选择性的肽抑制剂进行创伤后治疗对局部细胞死亡和行为功能障碍的程度的影响将提供半胱天冬酶活化与TBI诱导的病理学之间的机制联系。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is the leading cause of death among individuals under the age of 45 in the United States and survivors are faced with chronic brain damage leading to debilitating behavioral dysfunction. Brain damage and behavioral dysfunction may be, in part, due to neuronal death following TBI. The hypothesis to be tested in this proposal is that post-traumatic neuronal cell death is a result of activation of the pro-apoptotic caspase family of cysteine proteases. The objectives of this proposal are to elucidate (1) the association between neuronal death in experimental TBI and in postmortem tissue from head-injured patients and the activation of the "executor" caspase-3, (2) whether caspase-3 activation occurs directly as a result of activation of the "initiator" caspase-8, and/or indirectly as a result of mitochondrial pathway which requires Bax translocation, cytochrome c release and caspase-9 activation, (3) whether caspase-8 activation occurs as a result of activation of the tumor necrosis factor family of death receptors, (4) the role of Bax in mediating trauma-induced caspase-9 activation and subsequent caspase-3 activation and cell death, and (5) whether post-traumatic inhibition of caspases-3, -8 and -9 will reduce the extent of injury-induced cell death. Immunoblot and immunohistochemical analyses using specific and selective antibodies will be utilized to temporal and regional patterns of activation of caspases-3, -8 and -9, resdistribution of Bax and cytochrome c, and, apoptotic neuronal damage, as indicated by the presence of cellular DNA fragmentation and morphologic analyses. Mice deficient in TNF will be used to determine the role of TNF in mediating activation of caspases-8 and -3, and eventual apoptotic cell death by using immunoblot, immunohistochemical and histological analyses. The role of Bax in trauma-induced activation of caspase-9 and -3, cytochrome c redistribution, apoptotic neuronal death and behavioral dysfunction will be examined using immunoblot and immunohistochemical techniques, and by testing cognitive and motor function in brain-injured, Bax-deficient mice, followed by histological analysis of cell death. The effect of post-traumatic treatment with peptide inhibitors selective for caspases-3 (DEVD), -8 (IETD) and -9 (LEHD), on the extent of regional cell death and behavioral dysfunction will provide the mechanistic link between caspase activation and TBI-induced pathology.