Regulation of Blood-Brain Barrier Function by the RECK/GPR124/Wnt7 Pathway

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This application investigates regulation of central nervous system (CNS) angiogenesis and BBB integrity by the membrane protein Reck, a glycophosphatidylinositol (GPI)- anchored protein with large extracellular domain (ECD). In a fascinating convergence of data from many groups including ourselves, substantial overlap exists between the knockout mouse phenotypes for Reck, the G protein-coupled receptor family member Gpr124 and the Wnt7a/7b ligands. These phenotypes are all unified by marked deficits in developmental CNS angiogenesis with glomeruloid vascular malformations and forebrain-specific embryonic lethal hemorrhage. Further, Reck and Gpr124 synergistically promote canonical Wnt signaling with pronounced specificity for Wnt7a/7b and not other Wnt family members. Here, we perform mechanistic exploration of the contribution of Reck to Gpr124- and Wnt7a/7b-mediated signaling and blood-brain barrier (BBB) function. Here we investigate regulation of BBB integrity by the RECK/GPR124/Wnt pathway in vitro and in vivo. Aim 1 biochemically characterizes RECK/GPR124- regulated Wnt7a/7b signaling by defining domains of Reck and Gpr124 that underlie their physical interaction and functional promotion of Wnt7a/7b signaling. Aim 1 also investigates potential complex formation with Wnt7a/7b and the established Wnt receptors Frizzled (Fzd) and LRP. Aim 2 creates an in vitro culture model of the RECK/GPR124/Wnt pathway using primary brain endothelial cells (ECs) isolated from genetically modified mice and analyzes effects of pathway modulation on BBB marker expression and function. Aim 3 investigates the role of the RECK/GPR124/Wnt pathway in BBB integrity post-stroke using endothelial-specific and inducible knockout of Reck genetically modified mice and the tMCAO stroke model, and superimposed effects of Gpr124 knockout or Wnt pathway manipulation. Overall, these studies utilize complementary biochemical, cell biological, and genetic strategies to explore RECK/GPR124/Wnt7 regulation of Wnt/β-catenin signaling and BBB function.