Calpain in Axon Injury: Pathology & Therapeutic Issues

创伤性脑损伤是世界范围内导致死亡和残疾的主要原因。创伤性轴索损伤(TAI)与颅脑损伤密切相关，是导致其发病率和死亡率的重要因素。最近的观察表明，TAI不是由损伤瞬间轴突的即时破裂引起的。相反，它是一系列缓慢演变的病理事件的结果，导致轴突断开，从而为治疗干预提供了可能性。我们实验室的最新数据表明，钙标记的钙蛋白酶介导的轴膜通透性蛋白水解性修饰和由此导致的受损轴突段的钙超载是导致TAI相关的线粒体损伤的关键因素，它们也是导致轴浆运输停止、轴突肿胀和连接中断的酶修饰和轴突细胞骨架破坏的原因。这项应用的目的是阐明Calain介导的蛋白水解性变化在轴膜/轴突损伤发病机制中的作用，同时也评估针对Calain激活的治疗干预在TAI中的疗效，以破坏导致轴突断开的病理级联反应。使用一个具有良好特征的惯性冲击啮齿动物模型，我们将测试系统地给予钙蛋白酶抑制剂是否可以阻止轴膜通透性的变化，从而排除对辣根过氧化物酶和荧光示踪剂的摄取。利用不同的光镜和电子显微镜双重标记方法，我们还评估了Calain抑制剂是否阻止了与TAI相关的下游事件，如caspase死亡级联的激活和β淀粉样前体蛋白的积聚，β淀粉样前体蛋白是细胞骨架改变引起的轴突断开的标志。利用免疫组织化学和免疫印迹技术，辅以数字图像分析和统计数据比较，我们将比较一种细胞渗透性的选择性钙蛋白酶抑制剂在预防钙蛋白酶和半胱氨酸天冬氨酸氨基转移酶介导的结构蛋白光谱蛋白(组成腋下神经网络的一个组成部分)分解方面的相对有效性，同时还评估此类干预的运动/行为影响。这项工作不仅有助于更好地理解创伤性轴索损伤的病理生物学，而且可能有助于设计更合理有效的创伤性脑损伤和轴突损伤的治疗干预措施。

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Traumatic axonal injury (TAI) is associated with TBI and significantly contributes to its morbidity and mortality. Recent observations have demonstrated that TAI is not caused by the immediate rupture of the axon at the moment of injury. Rather it is the result of a slowly evolving sequence of pathological events leading to axonal disconnection thereby offering the potential for therapeutic intervention. Recent data from our lab suggests that calcium- indticed, calpain-mediated proteolytic modification of axolemmal permeability and the resultant calcium overload of damaged axonal segments are pivotally involved in the mitochondrial damage associated with TAI and they are also responsible for the enzymatic modification and disruption of the axonal cytoskeleton that leads to the halt of the axoplasmic transport, axonal swelling and disconnection. The goal of this application is to clarify the role of calpain-mediated proteolytic changes in the pathogenesis of the axolemmal/axonal damage while also evaluating the efficacy of therapeutic interventions targeting calpain activation, in TAI to disrupt the pathological cascade that leads to axonal disconnection. Using a well-characterized rodent model of inertial impact we will test whether the systemic administration of calpain-inhibitors prevents the axolemmal permeability changes precluding the uptake of horseradish peroxidase and fluorescent tracers. Utilizing different lightand electron microscopic double labeling approaches we also assess, whether calpain-inhibitors prevents downstream events associated with TAI such as the activation of the caspase death cascade and the accumulation of beta amyloid precursor protein, a marker of axonal disconnection caused by cytoskeletal alterations. Using immunohistochemistry and immunoblot-techniques assisted by digital image analysis and statistical data-comparison we will compare the relative efficacy of a cell-permeable selective calpain inhibitor in the prevention of calpain- and caspase-mediated breakdown of the structural protein spectrin, a constituent of the subaxolemmal network while also assessing the motor/behavioral effects of such interventions. Not only should the work proposed lead to better understanding of the pathobiology of traumatically induced axonal injury but also may prove helpful for designing more rational and effective therapeutic interventions for traumatic brain injury and axonal damage.