Role of calpastatin in modulating function following TBI

创伤性脑损伤（TBI）导致钙蛋白酶的激活延长，这有助于细胞骨架的破坏。 损伤、神经元死亡和行为功能障碍。然而，令人惊讶的是，很少有体内细胞基质的 钙蛋白酶已在创伤性脑损伤中被鉴定，因此，对钙蛋白酶的作用知之甚少。 钙蛋白酶介导创伤后发病的途径。钙蛋白酶抑制剂，内源性抑制剂 是唯一已知的专门抑制钙蛋白酶的蛋白质。因此，钙蛋白酶抑制蛋白代表了 理想的分子工具，用于分离受损大脑中钙蛋白酶的作用。努力翻译 令人兴奋的临床前数据表明，脑损伤的啮齿动物的功能改善， 外源性钙蛋白酶抑制剂的溶解性、特异性和生物利用度受到挑战， 小分子抑制剂。增强内源性钙蛋白酶抑制蛋白活性可能代表了一种新的有效的 治疗方法那么，项目1的总体目标是评估钙蛋白酶抑制蛋白在以下方面的作用 调节创伤后钙蛋白酶介导的蛋白水解，并评估神经保护和行为 在TBI的情况下增加钙蛋白酶抑制蛋白活性的功效。使用基因改变的老鼠， 过表达人钙蛋白酶抑制蛋白或钙蛋白酶抑制蛋白缺乏，项目1将：1）评估钙蛋白酶抑制蛋白的作用 在调节局灶性或弥漫性脑损伤后的行为结果中，2）量化改变的 局灶性或弥漫性脑损伤后钙蛋白酶抑制蛋白表达对神经元存活和轴突损伤的影响，3）确定 钙蛋白酶抑制素在限制创伤诱导的神经元细胞骨架蛋白质水解中的体内作用，以及4） 评估创伤后钙蛋白酶激活在修饰与钙有关的膜蛋白中的作用， 内流和调节与细胞死亡相关的事件。我们的中心假设是钙蛋白酶抑制蛋白 过表达将阻止钙蛋白酶介导的对细胞存活至关重要的神经元底物的切割， 从而减轻创伤后神经元死亡和功能障碍。这些实验将提供 钙蛋白酶抑制蛋白在创伤后病理学中的功能作用的第一个证据， 钙蛋白酶/钙蛋白酶抑制素系统在局灶性和弥漫性TBI中的作用。此外，该项目将提供 新的治疗方法的基础，基于钙蛋白酶抑制蛋白系统的操作，旨在 减轻由于TBI以及其他CMS损伤和疾病状态引起的脑损伤和功能障碍。

Traumatic brain injury (TBI) results in the prolonged activation of calpains, which contributes to cytoskeletal damage, neuronal death and behavioral dysfunction. However, surprisingly few in vivo cellular substrates of calpains have been identified in the traumatically injured brain and, consequently, little is understood about the pathways through which calpains mediate posttraumatic morbidity. Calpastatin, the endogenous inhibitor of calpains, is the only known protein that exclusively inhibits calpains. As such, calpastatin represents an ideal molecular tool with which to isolate the actions of calpains within the injured brain. Efforts to translate exciting preclinical data demonstrating functional improvement in brain-injured rodents treated with exogenous calpain inhibitors have been slowed by challenges with solubility, specificity and bioavailability of small molecule inhibitors. Enhancing endogenous calpastatin activity may represent a novel and potent therapeutic approach. The overall goals of Project 1, then, are to evaluate the role of calpastatin in regulating posttraumatic calpain-mediated proteolysis and to assess the neuroprotective and behavioral efficacy of increasing calpastatin activity in the setting of TBI. Using genetically altered mice that either overexpress human calpastatin or are calpastatin deficient, Project 1 will: 1) evaluate the role of calpastatin in modulating behavioral outcome following focal or diffuse brain injury, 2) quantify the effects of altered calpastatin expression on neuronal survival and axonal injury after focal or diffuse brain injury, 3) determine the in vivo role of calpastatin in limiting trauma-induced proteolysis of neuronal cytoskeletal proteins, and 4) evaluate the role of posttraumatic calpain activation in modifying membrane proteins involved in calcium influx and in modulating mitochondria-related cell death events. Our central hypothesis is that calpastatin overexpression will prevent calpain-mediated cleavage of neuronal substrates critical for cell survival, thereby attenuating posttraumatic neuronal death and dysfunction. The proposed experiments will provide the first evidence for a functional role for calpastatin in posttraumatic pathology and elucidate differential roles for the calpain/calpastatin system in focal and diffuse TBI. In addition, this Project will provide the groundwork for novel therapeutic approaches, based on manipulation of the calpastatin system, aimed at attenuating brain damage and dysfunction due to TBI as well as other CMS injury and disease states.