Among the many signals that control BBB functionality, neural progenitor-derived Wnt7a/b ligands play arguably important roles. During embryogenesis, they control CNS vascularization. In adults, they contribute to maintaining BBB function. In this project, we explored if these endogenous BBB-inducing signals can be repurposed as BBB repair agents in disease settings in which the BBB dysfunctions. To that end, we implemented a two-step strategy. In step one, we investigated the molecular mechanisms by which Wnt7a/b ligands trigger Wnt signaling in CNS endothelial cells. Prior evidence indicated that two membrane proteins, Reck and Gpr124 played critical roles. Our observations suggest that the two proteins share tasks in the process. The GPI-anchored Reck binds to Wnt7a/b but is unable to relay a signal within the endothelial cell. Signal transduction requires Gpr124. This member of the adhesion class of G protein-coupled receptors acts as a linker molecule between Reck and the signaling initiating Frizzled receptors. Our observations reveal three distinct linking mechanisms, of varying importance among vertebrate species. In step two, based on the molecular understanding of the Gpr124/Reck mechanism of action, we explored whether the endogenous Wnt7a/b ligands can be engineered into BBB-specific repair agents. By introducing subtle mutations in the sequence of these ligands, we could shift their signaling properties, making them strictly specific to the Gpr124/Reck receptor complex of the BBB. Thereby, the engineered ligands became safer and could be used to reduce glioblastoma progression and stroke infarct volumes, when delivered in the mouse brain.
