Calpain in Axon Injury: Pathology & Therapeutic Issues

轴突损伤中的钙蛋白酶：病理学

• 批准号: 6401874
• 负责人: John T Povlishock
• 金额: $ 4.03万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6401874
• 关键词: amyloid proteins; axon; biomarker; brain injury; calcium flux; calpain; chemoprevention; digital imaging; disease /disorder model; electron microscopy; enzyme activity; fluorescent dye /probe; horseradish peroxidase; immunocytochemistry; laboratory rat; light microscopy; membrane permeability; neuronal transport; pathologic process; protease inhibitor; proteolysis; spectrin; trauma; western blottings

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.

创伤性脑损伤（TBI）是世界范围内死亡和残疾的主要原因。创伤性轴索损伤（TAI）与TBI相关，并显著增加其发病率和死亡率。最近的观察表明，TAI不是由损伤时轴突的立即破裂引起的。相反，它是一系列病理事件缓慢演变的结果，导致轴突断开，从而提供了治疗干预的可能性。来自我们实验室的最新数据表明，轴膜渗透性的钙诱导的、钙蛋白酶介导的蛋白水解修饰和受损轴突节段的所得钙过载在与TAI相关的线粒体损伤中起关键作用，并且它们还负责轴突细胞骨架的酶促修饰和破坏，其导致轴浆运输的停止、轴突肿胀和断开。本申请的目的是阐明钙蛋白酶介导的蛋白水解变化在轴膜/轴突损伤发病机制中的作用，同时还评估针对钙蛋白酶激活的治疗干预在TAI中破坏导致轴突断开的病理级联的功效。使用一个良好的特点啮齿动物模型的惯性影响，我们将测试是否钙蛋白酶抑制剂的全身给药防止轴膜通透性的变化，排除辣根过氧化物酶和荧光示踪剂的摄取。利用不同的光和电子显微镜双标记的方法，我们还评估，钙蛋白酶抑制剂是否防止下游事件与TAI如激活的caspase死亡级联和β淀粉样前体蛋白，轴突断开的标记物的积累引起的细胞骨架改变。使用免疫组织化学和免疫印迹技术辅助数字图像分析和统计数据比较，我们将比较相对有效性的细胞渗透性选择性钙蛋白酶抑制剂在预防钙蛋白酶和半胱天冬酶介导的结构蛋白血影蛋白，一个组成部分的轴膜下网络，同时也评估运动/行为的影响，这样的干预措施。所提出的工作不仅可以更好地了解创伤性轴突损伤的病理生物学，而且可能有助于为创伤性脑损伤和轴突损伤设计更合理、更有效的治疗干预措施。