Biochem. Basis of Axonal & Myelin Injury In Head Trauma

描述（由申请人提供）：相当多的创伤性脑损伤（TBI）研究集中在机械和化学损伤（例如钙稳态紊乱）对轴突的病理意义上。然而，轴索损伤的确切生化机制尚不清楚。脑外伤中白质损失和脱髓鞘的研究还不够充分，髓鞘损伤的生化机制也知之甚少。在过去，我们的实验室和其他人已经记录了脑损伤后各种蛋白酶（如钙蛋白酶、半胱天冬酶和基质金属蛋白酶）的过度表达和激活。在这项拟议的研究中，我们假设轴突和髓鞘中的一组结构蛋白在脑外伤后对蛋白水解的易感性不同。不可避免地，这种蛋白质水解会导致降解，严重损害轴突和髓鞘的结构和功能完整性，导致长期轴突损伤和脱髓鞘。提出的研究代表了轴突和运动蛋白结构蛋白的蛋白质降解机制的第一个系统检查。我们的初步数据已经确定了几种易受蛋白质水解的轴突富集蛋白（神经丝- H和- l，微管相关蛋白tau，轴突相关淀粉样前体蛋白[APR]和细胞骨架蛋白α -和β -谱蛋白）。重要的是，我们还确定了至少两种髓鞘蛋白在TBI后被蛋白水解（髓鞘碱性蛋白[MBP]和髓鞘少突胶质细胞特异性蛋白[MOSP]）。独特的体内裂解位点在蛋白质水解倾向的蛋白质底物将鉴定与国家的最先进的蛋白质组学技术。这些知识将使我们能够产生新的“片段特异性”抗体，用于免疫组织化学检查这些蛋白水解产物的精确亚细胞分布。同样的抗体工具将用于配置酶联免疫测定（elisa），以定量脑组织和脑脊液（CSF）中的这些轴突和髓磷脂蛋白水解产物。后一种方法，如果成功，将提供一种新的、微创的量化tbi相关轴突和髓鞘损伤的方法。这些在了解脑外伤中轴突和髓磷脂病理背后的蛋白质机制方面的进展将极大地促进保持轴突和髓磷脂结构和功能完整性的疗法的发展。

DESCRIPTION (provided by applicant): A considerable amount of traumatic brain injury (TBI) research has focused on the pathologic significance of mechanical and chemical insults (e.g. disturbed calcium homeostasis) to the axons. Yet the precise biochemical mechanisms of axonal injury remain unclear. White matter loss and demyelination have been insufficiently studied in TBI, and we have little knowledge of the biochemical mechanisms of myelin damage. In the past, our laboratories and others have documented over-expression and activation of various proteases (e.g. calpains, caspases, and matrix metalloproteases) following brain injury. In this proposed research, we hypothesize that a subset of structural proteins in axons and myelin are differentially vulnerable to proteolysis after TBI. Inevitably, such proteolysis would result in degradation that significantly compromises the structural and functional integrity of axons and myelin sheaths leading to long-lasting axonal damage and demyelination. The proposed research represents the first systematic examination of the mechanisms of proteolvtic degradation of structural proteins in both axons and mvelin. Our preliminary data have already identified several axonally-enriched proteins that are vulnerable to proteolysis (neurofilament- H, and -L, microtubule-associated protein tau, axolemma-associated amyloid precursor protein [APR] and cytoskeletal proteins alphall- and (betaII-spectrin). Importantly, we have also identified at least two myelin proteins that are proteolysed after TBI (myelin basic protein [MBP] and myelin oligodendrocyte specific protein [MOSP]). Unique in vivo cleavage sites in proteolysis-prone protein substrates will be identified with state-of-the- art proteomic techniques. This knowledge will then allow us to generate novel "fragment-specific" antibodies, which will be used to immunohistochemically examine the precise subcellular distribution of these proteolytic products. The same antibody tools will be used to configure enzyme-linked immunoassays (ELISAs) to quantify these axonal and myelin proteolytic products in both brain tissue and cerebrospinal fluid (CSF). The latter method, if successful, will provide a novel and minimally-invasive way of quantifying TBI-associated axonal and myelin damage. These advances in understanding the proteolvtic mechanisms underlying axonal and myelin pathology in TBI will greatly accelerate development of therapies preserving the structural and functional integrity of axons and myelin.