Activated protein C system in stroke models

描述(由申请人提供)：对于这个竞争性更新项目(R01HL63290-16)，我们建议继续Zlokovic和Griffin实验室之间的合作研究，旨在促进中枢神经系统(CNS)中蛋白C/激活蛋白C(APC)途径的基础知识，总体目标是从基础知识到翻译到临床。由R01项目资助的先前结果导致在中风、脑创伤和慢性神经元变性的啮齿动物模型中发现了APC及其细胞保护选择性突变体的直接血管保护、稳定血脑屏障(BBB)、神经保护和抗炎活性。本项目确定了中枢神经系统中的APC受体及其下游通路，这些通路在神经元、脑内皮细胞和血脑屏障中介导细胞保护性APC信号，并表明蛋白酶激活的受体-1(PAR1)在保护中起主要作用。我们的进展在2014年转化为第二代细胞保护性APC 3K3A-APC治疗缺血性中风的临床II期试验。为了扩大这个项目，我们的目标包括：1)为APC神经保护活性的假想机制提供概念证明，2)表征新的第三代APC模拟药物，以及3)发现具有治疗其他神经疾病潜力的缺血性中风的改进生物制剂。我们令人兴奋的新发现和初步数据表明，APC激活PAR1涉及到该受体的N-末端结构域在Arg46处的新的裂解，这揭示了在残基Asn47处终止的新的拴系配体，它导致了APC的偏向的、？？arrestin依赖的细胞保护信号。相比之下，经典的凝血酶激活PAR1涉及Arg41的裂解，导致G蛋白信号转导和细胞毒性。APC可以同时在Arg41和Arg46上切割PAR1，而APC的神经保护作用是基于Arg46上的PAR1切割的这一有吸引力的假设缺乏体内概念的证据。我们假设：i)Arg46位的PAR1的APC裂解保护CNS、BBB和神经元(AIM 1)；ii)含有PAR1残基47-66(TR47)序列的APC模拟肽(即Arg46裂解后产生的拴系激动剂)在体外和体内缺血性卒中后在脑内皮和神经元中诱导依靠？arrestin 2的细胞保护信号(AIM 2)；以及iii)APC突变体在Arg46裂解PAR1的能力增强可能提供第三代神经保护生物制剂。初步数据包括：i)培育了携带R41Q-PAR1和R46Q-PAR1的新小鼠品系，以防止Arg41或Arg46处的裂解；ii)证明了研究TR47肽神经保护作用的可行性；iii)成功设计出新型APC变异体，其突变可增强Arg46上的PAR1裂解。将使用血脑屏障和神经元损伤的体外模型、小鼠和大鼠的中风模型、磁共振成像、神经病理学分析、共聚焦显微镜和行为测试来评估神经保护。我们期望从这个项目中产生的新知识可能会对翻译到缺血性中风的临床产生重大的、直接的影响，并进而对其他神经疾病的治疗产生影响。

DESCRIPTION (provided by applicant): For this competing renewal project (R01HL63290-16), we propose to continue collaborative studies between the Zlokovic and Griffin labs aimed at advancing basic knowledge on protein C/activated protein C (APC) pathways in the central nervous system (CNS) with an overall goal of advancing from basic knowledge to translation to the clinic. Previous results funded by this R01 project led to discovery of direct vasculoprotective, blood-brain barrier (BBB)-stabilizing, neuroprotective and anti-inflammatory activities of APC and its cytoprotective-selective mutants using rodent models of stroke, brain trauma and chronic neuronal degeneration. This project identified APC receptors and downstream pathways in the CNS that mediate cytoprotective APC signaling in neurons, brain endothelium and at the BBB and showed that protease activated receptor-1 (PAR1) has a major role in protection. Our progress was translated in 2014 into clinical Phase II trial for ischemic stroke of 3K3A-APC, a 2nd generation cytoprotective APC. For extending this project, our goals include: 1) providing proof of concept for hypothesized mechanisms for neuroprotective activities of APC, 2) characterizing novel 3rd generation APC-mimetic agents, and 3) discovering improved biologics for ischemic stroke with potential for treating other neurologic diseases. Our exciting new findings and preliminary data show that activation of PAR1 by APC involves a novel cleavage of this receptor's N-terminal domain at Arg46 which reveals a novel tethered ligand ending at residue Asn47 that causes APC's biased, ß-arrestin-dependent cytoprotective signaling. In contrast, classical activation of PAR1 by thrombin involves cleavage at Arg41 resulting in G-protein signaling and cellular toxicity. APC can cleave PAR1 at both Arg41 and Arg46, and in vivo proof of concept is lacking for the attractive hypothesis that APC's neuroprotection is based on PAR1 cleavage at Arg46. We hypothesize that: i) APC cleavage of PAR1 at Arg46 protects CNS, BBB and neurons (AIM 1); ii) an APC-mimetic peptide with the sequence of PAR1 residues 47-66 (TR47) (i.e., the tethered agonist generated after Arg46 cleavage) elicits ß-arrestin 2-dependent cytoprotective signaling in brain endothelium and neurons in vitro and in vivo after ischemic stroke (AIM 2); and iii) APC mutants with enhanced ability to cleave PAR1 at Arg46 may provide 3rd generation neuroprotective biologics. PRELIMINARY DATA include: i) generation of new mouse lines carrying R41Q-PAR1 and R46Q-PAR1 to prevent cleavages at either Arg41 or Arg46; ii) proof for feasibility of studying the neuroprotective actions of the TR47 peptide; and iii) success at engineering novel APC variants with mutations that enhance PAR1 cleavage at Arg46. Neuroprotection will be assessed using in vitro models of BBB and neuronal injury, stroke models in mice and rats, magnetic resonance imaging, neuropathological analysis, confocal microscopy and behavioral testing. We expect that new knowledge generated from this project will likely have significant, direct impact for translation to the clinic for ischemic stroke and, by extension, implications for therapies for othr neurological disorders.