Mechanistic and Therapeutic Studies of GPR124/RECK/WNT7-Regulated Blood-Brain Barrier Function

项目总结 脑血管系统是一个高度专业化的血管床，在那里细胞和分子成分 血脑屏障(BBB)严格控制进入中枢神经系统(CNS)。血脑屏障中断 发生在中风、脑瘤和多发性硬化症等疾病中，因此改善了机制 理解和指导治疗是迫切需要的。通过融合的遗传和生物化学研究， 我们和其他人已经定义了GPR124/RECK/WNT7通路，它对BBB功能是必不可少的 胚胎发生以及中风和胶质母细胞瘤等病理状态。在之前的授权期内，我们 证明GPI锚定的膜蛋白RECK结合并稳定新分泌的WNT7用于 介绍给规范的WNT受体FrizzledFZD。值得注意的是，GPCR样的7遍跨膜 GPR124蛋白与RECK协同作用有效放大WNT7A/WNT7B的信号，但不能放大其他17个信号 WNTS。而GPR124在血脑屏障中的功能已经通过体内基因敲除(KO)和在 体外转染法的研究，其分子机制仍然是个谜，临床翻译一直难以捉摸。 在这里，我们对GPR124/RECK/WNT7进行机械和翻译调查 路径，建立在大量初步数据的基础上。目标1研究了GPR124是至关重要的假设 将配体WNT7和受体RECK偶联到下游的FZD/LRP信号复合体所需。 我们利用多西环素诱导的WNT7表达来启动WNT7信号，克服了历史上的困难 WNT7蛋白的溶解和产生，覆盖在具有和不具有GPR124的同基因脑内皮细胞上 表达，通过免疫共沉淀确定RECK、FZD和LRP的信号复合体动力学 单分子分辨率实时成像研究。目的2探索WNT7-和GPR124- 依赖信号可诱导人脑血管生成和神经血管单位(NVU)的形成， 利用我们新的成人大脑器官系统，它可以发展广泛的血管网络和 准确地概括了NVU细胞在神经元环境中的相互作用。这些来自成人的大脑 在前一个授权期开发的有机化合物，与传统的无血管IPSC来源的有机化合物形成了强烈的对比 脑器官。最后，目标3研究了GPR124/RECK/WNT7途径的翻译潜力 通过假设具有和不具有新的生物工程FZD4的激动型GPR124胞外区 卒中后应用激动剂可改善短暂性大脑中动脉闭塞的预后 笔划模型。总而言之，这次续签申请利用了前一批准期内的实质性进展来进行 GPR124/RECK/WNT7机制和临床前的综合多学科研究 翻译，朝着以血脑屏障为靶点的中风疗法的发展。

PROJECT SUMMARY The cerebrovasculature is a highly specialized vascular bed where cellular and molecular components of the blood-brain barrier (BBB) stringently regulate entry into the central nervous system (CNS). BBB disruption occurs in diseases such as stroke, brain tumors and multiple sclerosis and thus improved mechanistic understanding and directed therapies are urgently needed. Through convergent genetic and biochemical studies, we and others have defined a GPR124/RECK/WNT7 pathway that is essential for BBB function during embryogenesis and during pathologic states such as stroke and glioblastoma. In the prior granting period, we demonstrated that the GPI-anchored membrane protein RECK binds and stabilizes newly secreted WNT7 for presentation to Frizzled (FZD), the canonical WNT receptor. Remarkably, the GPCR-like 7-pass transmembrane protein GPR124 synergizes with RECK to potently amplify signaling by WNT7A/WNT7B but not by the other 17 WNTs. While GPR124 function in the BBB has been unequivocally established by in vivo knockout (KO) and in vitro transfection studies, its molecular mechanism remains an enigma and clinical translation has been elusive. Here, we pursue both mechanistic and translational investigations into the GPR124/RECK/WNT7 pathway, building upon substantial preliminary data. Aim 1 investigates the hypothesis that GPR124 is crucially required for coupling of the ligand WNT7 and receptor RECK to the downstream FZD/LRP signaling complex. We utilize doxycycline-inducible WNT7 expression to initiate WNT7 signaling, surmounting historical difficulties with WNT7 protein solubility and production, overlaid upon isogenic brain endothelium with and without GPR124 expression, to defining signaling complex dynamics with RECK, FZD and LRP by co-immunoprecipitation and single molecule resolution live imaging studies. Aim 2 explores the hypothesis that WNT7- and GPR124- dependent signaling can induce human cerebral angiogenesis and neurovascular unit (NVU) formation, leveraging our novel adult human brain organoid system that develops extensive vascular networks and accurately recapitulates NVU cellular interactions within a neuronal context. These adult-derived brain organoids, developed in the previous granting period, contrast strongly with conventional avascular iPSC-derived brain organoids. Lastly, Aim 3 investigates the translational potential of the GPR124/RECK/WNT7 pathway through the hypothesis that agonistic GPR124 ectodomains with and without novel bioengineered FZD4 agonists, administered post-stroke, can improve outcomes in the transient middle cerebral artery occlusion stroke model. In all, this renewal application leverages substantial progress in the prior granting period to pursue a comprehensive and multidisciplinary approach to GPR124/RECK/WNT7 mechanism and preclinical translation, towards the development of BBB-targeted stroke therapeutics.