Activated protein C mechanisms of brain white matter protection and new therapies for brain white matter ischemic injury

摘要 皮质下白质(WM)区域的小脑血管卒中占所有卒中的25%。它会导致血管认知障碍和痴呆(VCID)，是导致痴呆症的第二大原因。尽管有如此重要的临床意义，但对缺血性WM损伤(WMI)和VCID的病理生理学仍知之甚少。此外，目前还没有一种被批准的治疗方法来预防和/或治疗西医中风和VCID。在这里，我们建议Zlokovic实验室和Griffin实验室合作研究激活蛋白C(APC)在西医中的通路，并使用西医中小脑血管收缩的模型来评估基于APC的治疗缺血性WMI的治疗潜力。我们以前使用大动脉梗死、脑损伤和神经退行性变模型的研究发现，APC及其细胞保护选择性突变体具有血管保护、稳定血脑屏障、神经保护和抗炎活性。2019年，这些发现已经成功地完成了3K3A-APC治疗缺血性中风的第二阶段试验，这是一种第二代细胞保护选择性APC类似物，抗凝活性丧失90%。然而，在缺血性WMI期间，激活WM中的APC通路是否有益仍是未知的。我们的目标包括：1)为APC在西药中保护活性的假想机制提供概念证据；2)表征与蛋白水解酶激活受体1(PAR1)相关的新的P1-47和PAR3-42 APC模拟多肽；以及3)测试用于治疗和预防缺血性WMI和WM卒中的改良的第三代APC R-46选择性生物制品。我们的初步数据支持我们的假设：1)APC通过切割Arg46位的PAR1，保护WM纤维束、少突胶质细胞和血脑屏障免受缺血性WMI(AIM1)的影响；ii)来自PAR1和PAR3序列的类似APC的多肽(如P1-47和P3-42)(即由APC裂解产生的拴系的PAR激动剂)显示协同偏向的激动剂，并将在体外和体内诱导WM卒中后脑内皮细胞和少突胶质细胞(AIM2)依赖的-arrestin 2依赖的细胞保护信号；和iii)E56K-APC和D180E-APC新设计的APC突变体增强了在Arg46位切割PAR1的能力，并将为西医卒中治疗提供改进的APC生物制剂(AIM 3)。为了解释我们的假设，我们将使用：i)卒中的WM模型；ii)携带R41Q-PAR1和R46Q-PAR1点突变的新的小鼠系，以及-arrestin 2-/-和G12-/-小鼠；iii)新的模拟APC的PAR1和PAR3相关多肽，分别具有PAR1和PAR3的拴系氨基酸序列；iv)新的APCR46-裂解位点选择性生物制剂；v)体内WM损伤体积、血脑屏障完整性、血流、结构和连接变化的多参数磁共振成像，以及用于电路水平分析的基于轨迹的连接；vi)行为测试；vii)组织学、神经病理学；少突胶质细胞培养和体外血脑屏障模型。如果成功，该项目产生的新知识将作为治疗西医中风和VCID的新疗法应用于临床。

ABSTRACT Stroke in small brain vessels in subcortical white matter (WM) regions account for 25% of all strokes. It leads to vascular cognitive impairment and dementia (VCID), and is the second leading cause of dementia overall. Despite such clinical importance, the pathophysiology of ischemic WM injury (WMI) and VCID is still poorly understood. Moreover, there is no yet an approved therapy for prevention and/or treatment of WM strokes and VCID. Here, we propose collaborative studies between the Zlokovic and Griffin labs on activated protein C (APC) pathways in the WM, and to evaluate therapeutic potential of APC-based therapies for ischemic WMI using a model of vasoconstriction of small brain vessels in the WM. Our previous studies using models of large artery infracts, brain trauma and neurodegeneration led to discovery of vasculoprotective, blood-brain barrier (BBB)-stabilizing, neuroprotective, and anti-inflammatory activities of APC and its cytoprotective-selective mutants. In 2019, these findings have been translated into successfully completed phase 2 trial for ischemic stroke of 3K3A-APC, a 2nd generation cytoprotective-selective APC analog with >90% loss of anticoagulant activity. However, whether activation of APC pathways in the WM is beneficial or not during ischemic WMI, remains unknown. Our goals include: 1) providing proof of concept for hypothesized mechanisms for protective activities of APC in the WM; and 2) characterizing novel protease activated receptor 1 (PAR1)-related P1-47 and PAR3-related P3-42 APC-mimetic peptides, and 3) testing improved 3rd generation APC R-46-selective biologics for treating and preventing ischemic WMI and WM stroke. Our pilot data support our hypotheses that: i) APC will be beneficial for ischemic WMI via PAR1 cleavage at Arg46 to protect WM fiber tracts, oligodendrocytes and BBB from ischemic WMI (AIM 1); ii) APC-mimetic peptides derived from PAR1 and PAR3 sequences (e.g,, P1-47 and P3-42, (i.e., the tethered PAR agonists created by APC cleavages) exhibit synergistic biased agonism, and will elicit -arrestin 2-dependent cytoprotective signaling in brain endothelium and oligodendrocytes in vitro and in vivo after WM stroke (AIM 2); and iii) E56K-APC and D180E-APC newly engineered APC mutants have enhanced ability to cleave PAR1 at Arg46 and will provide improved APC biologics for WM stroke therapy (AIM 3). To address our hypotheses, we will use i) WM model of stroke; ii) new mouse lines carrying R41Q-PAR1 and R46Q-PAR1 point mutations, and -arrestin 2-/- and G12-/- mice; iii) new APC-mimetic PAR1- and PAR3-related peptides with the respective PAR1 and PAR3 tethered-ligand amino acid sequences; iv) new APC R46-cleavage site selective biologics; v) in vivo mutiparametric longitudinal MRI of WM lesion volume, BBB integrity, blood flow, structural and connectivity changes, and tract-tracing based connectomics for circuit level analysis; vi) behavior tests; vii) immunohistology, neuropathology; and viii) oligodendrocyte cultures and in vitro BBB model. If successful, new knowledge generated from this project could translate to the clinic as new therapies for WM stroke and VCID.