Role of Protease Activated Receptors in Spinal Cord Injury and Repair

描述（由申请人提供）：脊髓创伤促进了一系列复杂的病理生理事件，导致比最初持续的损伤更大的损伤， 其导致炎症、脱髓鞘、轴突损伤和不利于神经恢复的环境。驱动这一级联的因素继续被识别和严格评估，因为每一个都可以作为新疗法的基本原理设计的目标，以减轻损伤并促进修复和再生。我们实验室在上一个资助期间的工作表明，激肽释放酶（KLK）家族的丝氨酸蛋白酶是目前公认的脊髓创伤失调的酶的复杂级联反应之一，此外，几种KLK是神经毒性，星形胶质细胞增生和脱髓鞘的新介质。重要的是，我们发现KLK通过切割从而激活称为蛋白酶激活受体（PAR）的G蛋白偶联受体来发挥其细胞效应。作为细胞表面受体，PAR赋予细胞对发生在CNS创伤、炎症和血脑屏障破坏部位的快速变化的蛋白水解微环境作出反应或过度反应的能力。在拟议研究中待检验的中心假设是，选择PAR的蛋白水解激活调节创伤性损伤脊髓中的独特细胞反应，并且这些受体可以差异靶向，以防止继发性损伤并促进修复。如果这一假设是正确的，PAR可以作为新疗法开发的靶点。四个互补的目标，重点是细胞，分子和系统的结果，提出了测试这一假设。在目的1中，我们将确定PAR基因靶向对创伤性脊髓损伤小鼠模型神经行为恢复的影响。在目标2中，我们将使用遗传和药理学的功能丧失和获得的方法来建立PAR在介导SCI相关的PAR激动剂（KLK，凝血酶和纤溶酶）在原代脊髓神经元，星形胶质细胞和少突胶质细胞的细胞效应中的作用，以及它们对体外神经毒性药物的敏感性。在Aim中 3，我们将剖析每种蛋白酶在神经元和神经胶质细胞中引起的分子信号和基因表达谱，以及负责介导这些效应的PAR。在目标4中，我们将确定PAR药物治疗对小鼠创伤性SCI神经行为恢复的影响。拟议的研究将确定新的受体为基础的机制，调节SCI微环境，是潜在的高度服从治疗干预，并考虑到广泛表达的PAR在中枢神经系统，可能是根本的意义，了解损伤和修复机制，在广泛的神经系统疾病。

DESCRIPTION (provided by applicant): Trauma to the spinal cord promotes a complex cascade of pathophysiological events that result in greater injury than was initially sustained and which contribute to inflammation, demyelination, axon injury and an unfavorable environment for neural recovery. The factors which drive this cascade continue to be identified and critically evaluated since each may serve as a target for the rationale design of new therapies to mitigate injury and to promote repair and regeneration. Work in our laboratory during the previous funding period indicates that serine proteases of the kallikrein (KLK) family are among the complex cascade of enzymes now recognized to be deregulated with spinal cord trauma and furthermore that several KLKs are novel mediators of neurotoxicity, astrogliosis and demyelination. Importantly, we discovered that KLKs exert their cellular effects by cleaving thereby activating G-protein coupled receptors termed Protease Activated Receptors (PARs). As cell surface receptors, PARs endow the cell with the ability to respond, or to over respond, to the rapidly changing proteolytic microenvironment that occurs at sites of CNS trauma, inflammation and blood brain barrier breakdown. The CENTRAL HYPOTHESIS to be tested in the proposed studies is that proteolytic activation of select PARs regulates unique cellular responses in the traumatically injured spinal cord and that these receptors can be differentially targeted to prevent secondary injury and to promote repair. If this hypothesis is correct, PARs may serve as targets for the development of new therapies. Four complementary Aims that focus on cellular, molecular and systems outcomes are proposed to test this hypothesis. In Aim 1, we will determine the effects of genetic targeting of PARs on neurobehavioral recovery in a murine model of traumatic spinal cord injury. In Aim 2, we will use genetic and pharmacologic loss and gain of function approaches to establish the role of PARs in mediating the cellular effects of SCI-related PAR agonists (KLKs, thrombin and plasmin) in primary spinal cord neurons, astrocytes and oligodendroglia and their sensitivity to neurotoxic agents in vitro. In Aim 3, we will dissect the molecular signaling and gene expression profiles that are elicited by each protease across neurons and neuroglia and the PARs responsible for mediating these effects. In Aim 4, we will determine the effects of PAR-pharmacotherapy on neurobehavioral recovery in murine traumatic SCI. The proposed studies will identify new receptor based mechanisms regulating the SCI microenvironment that are potentially highly amenable to therapeutic intervention and given the widespread expression of PARs in the CNS, are likely to be of fundamental significance to understanding injury and repair mechanisms in a wide range of neurological conditions.