Targeting Protease Activated Receptor 1 for Repair of the Injured Spinal Cord

靶向蛋白酶激活受体 1 修复受损脊髓

• 批准号: 10471261
• 负责人: ISOBEL A SCARISBRICK
• 金额: $ 48.58万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10471261
• 关键词: Acute; Address; Appearance; Astrocytes; Axon; Biological Assay; Bone Marrow; Brain; Cell Surface Receptors; Cell physiology; Central Nervous System Diseases; Chimera organism; Chronic; Clinical; Coculture Techniques; Coupling; Disease; Enzymes; Event; Exhibits; Experimental Models; F2R gene; FDA approved; Fostering; G-Protein-Coupled Receptors; Growth Factor; Hormones; Immune; In Vitro; Individual; Inflammation; Inflammatory Response; Injury; Kininogenase; Knock-out; Knowledge; Laboratories; Link; Locomotor Recovery; LoxP-flanked allele; MAP Kinase Gene; Messenger RNA; Microglia; Modeling; Molecular; Mus; Myelin; Myelin Sheath; Myelinated nerve fiber; Neuroglia; Neurons; Oral; Outcome; PAR-1 Receptor; Pathogenesis; Pathology; Pathway interactions; Peptide Hydrolases; Peripheral; Pharmacology; Physiological; Play; Positioning Attribute; Property; Proteinase-Activated Receptors; Publishing; Recovery; Recovery of Function; Research Design; Ribosomes; Role; STAT3 gene; Sensorimotor functions; Serine Protease; Signal Transduction; Spinal cord injury; Techniques; Testing; Therapeutic; Thrombin; Thrombin Receptor; Time; Translations; Treatment Efficacy; United States; astrogliosis; base; brain health; cell injury; central nervous system injury; clinical translation; combinatorial; design; disability; experimental study; functional improvement; functional outcomes; genetic approach; improved; improved outcome; injury and repair; insight; knockout gene; loss of function; monocyte; nerve injury; neural repair; neurobehavioral; neuroinflammation; neuroprotection; receptor; relating to nervous system; repair strategy; repaired; response; response to injury; restoration; small molecule inhibitor; targeted treatment; therapeutic target; tool

PROJECT SUMMARY/ABSTRACT Secreted serine proteases are abundant in the intact CNS and become deregulated by injury and disease, yet we lack knowledge regarding their physiological functions and contributions to pathology. Several years ago, the discovery of a set of enzyme-activated G protein-coupled receptors, the Protease Activated Receptors (PARs), led to a new conceptual framework for understanding the physiological impact of proteases. PARs permit activating enzymes to signal in a hormone-like fashion to modulate key cellular functions, but when overactivated can contribute to pathology. The PI’s team recently discovered that mice with global PAR1 gene knockout exhibit significant improvements in locomotor recovery after spinal cord injury (SCI). Functional improvements were accompanied by reductions in inflammation and astrogliosis and improvements in the appearance of myelin and axons, all integral substrates to support restoration of function. We also documented that CNS injury relevant proteases, such as thrombin and kallikrein 6 elicit Ca2+, MAPK and STAT3 signaling linked to neuroinflammation and pro-injury responses across neurons and neuroglia in a PAR1-dependent manner. Together, these studies highlight the likely multifactorial roles played by PAR1 in key cellular and molecular events positioned to govern outcomes after SCI. These findings also highlight the potential to target PAR1 for neural protection and repair. Despite these encouraging findings the cellular mechanisms by which blocking PAR1 improves recovery after SCI have not been defined and this knowledge gap hampers progress towards translation of existing FDA approved and orally bioavailable PAR1 small molecule inhibitors. Additionally, whether blocking PAR1 therapeutically at acute or chronic time points after SCI are both capable of improving neural recovery is unknown. Based on recently published findings, taken with new preliminary results, we propose 3 integrated Aims to test the Central Hypothesis that PAR1 is an essential regulator of reactivity across the microglial- astrocyte compartments and can be selectively blocked to improve glial-neuronal trophic coupling, neuroprotection and repair after SCI. In Aim 1, we will determine the impact of pharmacologic PAR1 inhibition initiated at acute or chronic time points after injury on signs of neuroprotection, neural repair and recovery of sensorimotor function and use ribosomal mRNA capture techniques to document cellular and molecular mechanisms engaged across the astroglial, microglial/monocyte and neuron compartments. In Aim 2, we will determine whether conditional deletion of PAR1 selectively in astrocytes, microglia or peripheral monocytes is sufficient to enhance recovery. In Aim 3, we will use glial-neuron co-cultures as bioassays to establish PAR1- regulated glial-neural trophic coupling mechanisms relevant to neuroprotection and repair. The studies proposed address key mechanistic questions regarding the functional roles of PAR1 in neural injury and will provide new information needed to optimize therapeutic targeting strategies for recovery of function.

项目摘要/摘要 分泌的丝氨酸蛋白酶在完整的中枢神经系统中含量丰富，并因损伤和疾病而失去调节。 我们缺乏关于它们的生理功能和对病理学的贡献的知识。几年前， 发现一组酶激活的G蛋白偶联受体，即蛋白酶激活的受体(PARs)， 为理解蛋白水解酶的生理影响提供了一个新的概念框架。PARS许可 激活酶，以荷尔蒙般的方式发出信号，调节关键的细胞功能，但当过度激活时 可能对病理学有贡献。PI的团队最近发现，具有全局PAR1基因敲除的小鼠表现出 显著改善脊髓损伤(SCI)后的运动恢复。功能改进是 伴随着炎症和星形胶质细胞增生症的减轻以及髓鞘和 轴突，所有支持功能恢复的完整底物。我们还记录了中枢神经系统损伤与 凝血酶和激肽释放酶6等蛋白酶可诱导与神经炎症相关的钙、丝裂原活化蛋白激酶和信号转导通路 并以PAR1依赖的方式跨神经元和神经胶质细胞进行促损伤反应。总而言之，这些研究 强调PAR1在关键细胞和分子事件中可能发挥的多因素作用 脊髓损伤后的预后。这些发现也强调了以PAR1为靶点进行神经保护和修复的潜力。 尽管有这些令人鼓舞的发现，但阻断PAR1的细胞机制促进了患者的康复 SCI尚未定义，这一知识差距阻碍了翻译现有FDA的进展 批准的和口服生物可用的PAR1小分子抑制剂。此外，是否阻止PAR1 治疗在急性或慢性脊髓损伤后的时间点都有能力促进神经恢复 未知。基于最近发表的研究结果，结合新的初步结果，我们提出了3个综合的 目的是检验中央假说，即PAR1是跨小胶质细胞反应性的重要调节因子。 星形胶质细胞隔间，可以选择性地阻断，以改善神经胶质细胞-神经元营养偶联， 脊髓损伤后的神经保护与修复。在目标1中，我们将确定药物抑制PAR1的影响 在损伤后的急性或慢性时间点启动，以显示神经保护、神经修复和恢复的迹象 感觉运动功能，并使用核糖体mRNA捕获技术记录细胞和分子 涉及星形胶质细胞、小胶质细胞/单核细胞和神经元的机制。在目标2中，我们将 确定在星形胶质细胞、小胶质细胞或外周单核细胞中选择性地删除PAR1的条件 足以提高采收率。在目标3中，我们将使用神经胶质细胞-神经元共培养作为生物测试来建立PAR1- 与神经保护和修复相关的调节神经胶质-神经营养偶联机制。这项研究建议 解决有关PAR1在神经损伤中的功能作用的关键机制问题，并将提供新的 优化治疗靶向策略以恢复功能所需的信息。