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

本申请研究中枢神经系统（CNS）血管生成的调节 和BBB完整性的膜蛋白Reck，糖磷脂酰肌醇（GPI）- 锚定蛋白与大细胞外结构域（ECD）。在一个迷人的融合， 包括我们在内的许多团体的数据， Reck基因敲除小鼠表型，Reck是G蛋白偶联受体家族成员 Gpr 124和Wnt 7a/7 b配体。这些表型都是统一的显着赤字 在肾小球样血管畸形的发育性CNS血管生成中， 前脑特异性胚胎致死性出血此外，Reck和Gpr 124 协同促进对Wnt 7a/7 b具有显著特异性典型Wnt信号传导 而不是其他Wnt家族成员。在这里，我们进行机械探索的 Reck对Gpr 124和Wnt 7a/7 b介导的信号传导和血脑的贡献 屏障（BBB）功能。 在此，我们研究了RECK/GPR 124/Wnt对BBB完整性的调节作用。 在体外和体内的途径。目的1生物化学表征RECK/GPR 124- 通过定义Reck和Gpr 124的结构域调节Wnt 7a/7 b信号传导， 它们的物理相互作用和Wnt 7a/7 b信号传导的功能促进。目标1 研究Wnt 7a/7 b和已建立的Wnt的潜在复合物形成 受体Frizzled（Fzd）和LRP。Aim 2建立了一个体外培养的模型， RECK/GPR 124/Wnt通路，使用分离自 基因修饰小鼠，并分析通路调节对BBB标志物的影响 表达和功能。目的3研究RECK/GPR 124/Wnt的作用 使用内皮特异性和可诱导敲除的 Reck转基因小鼠和tMCAO中风模型，以及叠加效应 Gpr 124敲除或Wnt通路操纵。总的来说，这些研究利用 互补的生物化学，细胞生物学和遗传学策略， RECK/GPR 124/Wnt 7调节Wnt/β-catenin信号传导和BBB功能。

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.