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

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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.