Calpain in Axon Injury: Pathology & Therapeutic Issues

轴突损伤中的钙蛋白酶：病理学

• 批准号: 6530092
• 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 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6530092
• 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）是全世界死亡和残疾的主要原因。创伤性轴突损伤 (TAI) 与 TBI 相关，并显着增加其发病率和死亡率。最近的观察表明，TAI 并不是由受伤时轴突立即断裂引起的。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，从而提供了治疗干预的潜力。我们实验室的最新数据表明，钙诱导的钙蛋白酶介导的轴突通透性蛋白水解修饰以及由此产生的受损轴突节段的钙超载在与 TAI 相关的线粒体损伤中起着关键作用，它们还负责轴突细胞骨架的酶促修饰和破坏，从而导致轴浆运输停止。 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突/轴突损伤发病机制中的作用，同时评估 TAI 中针对钙蛋白酶激活的治疗干预措施的功效，以破坏导致轴突断开的病理级联。使用特征良好的惯性影响啮齿动物模型，我们将测试全身施用钙蛋白酶抑制剂是否可以防止轴膜通透性变化，从而阻止辣根过氧化物酶和荧光示踪剂的吸收。我们还利用不同的光和电子显微镜双标记方法评估钙蛋白酶抑制剂是否可以防止与 TAI 相关的下游事件，例如半胱天冬酶死亡级联的激活和 β 淀粉样前体蛋白的积累，β淀粉样蛋白前体蛋白是细胞骨架改变引起的轴突断开的标志。利用数字图像分析和统计数据比较辅助的免疫组织化学和免疫印迹技术，我们将比较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（轴膜下网络的组成部分）分解方面的相对功效，同时评估此类干预措施的运动/行为效应。所提出的工作不仅应该有助于更好地理解创伤性轴突损伤的病理学，而且可能有助于设计针对创伤性脑损伤和轴突损伤的更合理和有效的治疗干预措施。