Regulation and Function of Kallikreins in Spinal Cord Injury and Repair

描述（由申请人提供）：本提案的目的是研究缓激肽在创伤性脊髓损伤中的活性和潜在作用机制，重点研究它们在介导轴突损伤、星形胶质增生和神经再生环境改变中的作用。对这个新发现的基因家族的所有15个成员的初步研究表明，选择性钾化酶在中枢-免疫轴上有差异表达，并受到损伤的差异调节。我们对激肽激酶6 （K6）的活性特别感兴趣，因为我们已经表明：1)在动物模型和人类创伤性脊髓损伤病例中，损伤后急性和慢性时间点，K6在常驻脊髓细胞和浸润性巨噬细胞中上调；2) K6是一种胰蛋白酶样酶，能快速水解髓磷脂和细胞外基质蛋白；3) K6水解生长促进底物（如层粘连蛋白）抑制神经突生长，而水解抑制底物（如聚集蛋白）促进神经突延伸；4) k6功能阻断抗体可减轻炎性脊髓损伤小鼠模型的临床和病理病变。此外，初步数据表明，钾化钾可能不仅在底物水平上发挥作用，而且还可能切割蛋白酶激活受体（PARs），从而触发细胞内信号级联反应，包括钙通量和Erk-磷酸化，这可能直接导致神经突生长和星形胶质细胞形成的改变。基于这些结果，我们假设钾化钾素介导的蛋白水解在脊髓对损伤的反应中起着重要作用，包括继发性组织破坏事件和神经再生能力的改变。为了验证这一假设，我们打算实现以下具体目标：1)确定人类和小鼠创伤性脊髓损伤中所有15种钾化钾蛋白表达的动态和细胞特异性；2)利用细胞培养系统比较2个钾化钾激酶（K1或K6）水平升高对轴突完整性和神经突生长、星形胶质形成的影响，以及潜在的作用机制；3)在野生型或PAR缺陷小鼠脊髓中直接注射后，确定K1和K6在脊髓发病中的作用范围，以及它们的作用是否由PAR介导；4)在小鼠夹压模型中，确定单独或联合改变脊髓损伤时K1、K6或PAR的活性是否会改变继发组织破坏并促进运动恢复。我们的长期目标是了解钾激肽参与脊髓创伤继发事件的作用和可能的机制，包括轴突再生失败。这项研究将产生新的和重要的信息，为开发新的治疗方案提供动力，以促进脊髓损伤的康复。公共卫生相关性：该项目的目标是描述人类创伤性脊髓损伤中由蛋白酶家族（称为钾化酶）介导的新型病理生理机制，并在小鼠中建立这些机制的模型，并确定改变其活性是否促进神经行为恢复。这些研究有可能为急性和慢性损伤的脊髓损伤患者确定新的治疗干预措施。

DESCRIPTION (provided by applicant): The objective of this proposal is to investigate the activity and potential mechanism of action of kallikreins in traumatic spinal cord injury with a focus on their roles in mediating axon injury, astrogliosis, and alteration of the environment available for nerve regeneration. Preliminary studies of all 15 members of this newly identified gene family show select kallikreins are differentially expressed across the CNS-immune axis and therein are differentially regulated by injury. We are particularly interested in the activity of kallikrein 6 (K6) since we have shown that: 1) K6 is up regulated in resident spinal cord cells and infiltrating macrophages at acute and chronic time points post-injury in both animal models and cases of human traumatic spinal cord injury; 2) K6 is a trypsin-like enzyme that rapidly hydrolyzes myelin and extracellular matrix proteins; 3) K6 hydrolyzes growth facilitatory substrates such as laminin to inhibit neurite outgrowth, while hydrolysis of inhibitory substrates, such as aggrecan, promote neurite extension; 4) K6-function blocking antibodies attenuate clinical and pathological disease in murine models of inflammatory spinal cord injury. Further, preliminary data demonstrate that kallikreins may exert their effects not only at the level of the substrate, but in addition may cleave select protease activate receptors (PARs) to trigger intracellular signaling cascades including calcium flux and Erk- phosphorylation, which may directly contribute to altered neurite outgrowth and astrogliosis. Based on these results, we hypothesize that kallikrein-mediated proteolysis plays fundamental roles in the response of the spinal cord to injury, including secondary tissue destructive events and modification of the capacity for nerve regeneration. To test this hypothesis, we intend to fulfill the following specific aims: 1) determine the dynamics and cellular specificity of expression of all 15 kallikreins in human and murine traumatic spinal cord injury; 2) using cell culture systems compare the consequences of elevations in the level of 2 kallikreins, K1 or K6, on axon integrity and neurite outgrowth, astrogliosis, and the potential mechanism of action; 3) determine the range of action of K1 and K6 in spinal cord pathogenesis and whether their effects are mediated by PAR following direct injection into the spinal cord of wild-type or PAR-deficient mice; and 4) determine whether altering the activity of K1, K6, or PAR at the time of SCI, alone or in combination, alters secondary tissue destruction and promotes locomotor recovery in a murine clip compression model. Our long-term goal is to understand the role and possible mechanisms by which kallikreins participate in events secondary to spinal cord trauma, including failure of axon regeneration. This study should generate new and important information providing the impetus for development of novel therapeutic regimes to promote recovery in cases of SCI. PUBLIC HEALTH RELEVANCE: The goal of this project is to delineate novel pathophysiologic mechanisms mediated by a family of proteases referred to as kallikreins, in human traumatic spinal cord injury, to model these in mice, and to determine whether altering their activity promotes neurobehavioral recovery. These studies have the potential to identify new therapeutic interventions for SCI patients with both acute and more chronic injuries.