Role of Protease Activated Receptors in Spinal Cord Injury and Repair

项目概要/摘要 脊髓创伤促进了一系列复杂的病理生理事件，导致更大的损伤 比最初持续的，这有助于炎症，脱髓鞘，轴突损伤和不利的 神经恢复的环境。继续查明和严格评价推动这一级联的因素 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， 再生我们实验室在上一个资助期的工作表明，激肽释放酶的丝氨酸蛋白酶 (KLK)家族是目前公认的在脊髓创伤中失调的酶的复杂级联， 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通过裂解从而激活G蛋白偶联受体来发挥其细胞效应， 蛋白酶激活受体（PAR）。作为细胞表面受体，PAR赋予细胞应答或调节细胞凋亡的能力。 过度反应，迅速变化的蛋白水解微环境发生在中枢神经系统创伤，炎症和 血脑屏障崩溃在拟议的研究中要测试的中心假设是，蛋白水解 选择性PAR的激活调节创伤性损伤脊髓中独特的细胞反应， 受体可被不同地靶向以防止继发性损伤并促进修复。如果这个假设是正确的， PAR可以作为开发新疗法的靶点。四个互补的目标，侧重于细胞， 提出了分子和系统结果来检验这一假设。在目标1中，我们将确定基因的影响。 PARs靶向作用于创伤性脊髓损伤小鼠模型的神经行为恢复。在目标2中，我们将使用 通过遗传学和药理学的功能丧失和获得方法来确定PAR在介导细胞凋亡中的作用， SCI相关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 少突胶质细胞及其对体外神经毒性剂的敏感性。在目标3中，我们将剖析分子信号传导， 每种蛋白酶在神经元和神经胶质细胞以及负责的PARs中引发的基因表达谱 介导这些效应。在目标4中，我们将确定PAR药物治疗对神经行为恢复的影响， 小鼠创伤性脊髓损伤拟议的研究将确定新的受体为基础的机制，调节脊髓损伤 这些微环境可能非常适合于治疗干预，并广泛表达 在中枢神经系统中的PAR，可能是理解损伤和修复机制的基础性意义， 一系列神经系统疾病

PROJECT SUMMARY/ABSTRACT 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.