Role of Protease Activated Receptors in Spinal Cord Injury and Repair

描述(由申请人提供)：脊髓创伤会导致一系列复杂的病理生理事件，导致比最初所受的更大的伤害， 这会导致炎症、脱髓鞘、轴突损伤和不利于神经恢复的环境。继续查明和严格评估推动这一级联反应的因素，因为每一种因素都可能成为新疗法的基本设计目标，以减轻伤害并促进修复和再生。我们实验室在上一次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与脊髓损伤有关的复杂的级联酶之一，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通过裂解从而激活G蛋白偶联受体来发挥其细胞效应，这种受体被称为蛋白酶激活受体(PARs)。作为细胞表面受体，PARs赋予细胞对发生在中枢神经系统创伤、炎症和血脑屏障破坏部位的快速变化的蛋白分解微环境的反应或过度反应的能力。在拟议的研究中要检验的中心假设是，选择的部分蛋白水解性激活调节创伤损伤脊髓中独特的细胞反应，这些受体可以不同地被靶向，以防止继发性损伤和促进修复。如果这一假设是正确的，PAR可能成为新疗法开发的靶点。为了验证这一假说，提出了关注细胞、分子和系统结果的四个互补目标。在目标1中，我们将确定PARS的基因靶向对创伤性脊髓损伤小鼠神经行为恢复的影响。在目标2中，我们将通过遗传学和药理学的功能丧失和功能获得的方法来确定PARs在介导与脊髓损伤相关的PAR激动剂(KLKs、凝血酶和纤溶酶)在原代培养的脊髓神经元、星形胶质细胞和少突胶质细胞中的作用以及它们对神经毒性药物的敏感性。在AIM 3，我们将剖析每种蛋白酶在神经元和神经胶质细胞以及负责介导这些影响的PARs中引发的分子信号和基因表达谱。目的4，探讨PAR药物治疗对小鼠创伤性脊髓损伤后神经行为恢复的影响。这项拟议的研究将确定新的基于受体的调节脊髓损伤微环境的机制，这些机制可能高度服从治疗干预，并考虑到PARs在中枢神经系统的广泛表达，可能对理解广泛神经状况下的损伤和修复机制具有基础性意义。

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.