Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

Abstract

ABSTRACTBackgroundThe blood brain barrier (BBB) preserves neuronal function in the central nervous system (CNS) by tightly controlling metabolite exchanges with the blood. In the eye, the retina is likewise protected by the blood-retina barrier (BRB) to maintain phototransduction. We showed that the secreted guidance cue Netrin-1 regulated BBB integrity, by binding to endothelial Unc5B and regulating canonical β-catenin dependent expression of BBB gene expressionObjectiveHere, we investigated if Netrin-1-binding to endothelial Unc5B also controlled BRB integrity, and if this process involved Norrin/β-catenin signaling, which is the major known driver of BRB development and maintenance.MethodsWe analyzed Tamoxifen-inducible loss- and gain-of-function alleles ofUnc5B, Ntn1andCtnnb1in conjunction with tracer injections and biochemical signaling studies.ResultsInducible endothelialUnc5Bdeletion, and inducible globalNtn1deletion in postnatal mice reduced phosphorylation of the Norrin receptor LRP5, leading to reduced β-catenin and LEF1 expression, conversion of retina endothelial cells from a barrier-competent Claudin-5+/PLVAP-state to a Claudin-5-/PLVAP+ leaky phenotype, and extravasation of injected low molecular weight tracers. InducibleCtnnb1gain of function rescued vascular leak inUnc5Bmutants, andNtn1overexpression induced BRB tightening. Unc5B expression in pericytes contributed to BRB permeability, via regulation of endothelial Unc5B. Mechanistically, Netrin-1-Unc5B signaling promoted β-catenin dependent BRB signaling by enhancing phosphorylation of the Norrin receptor LRP5 via the Discs large homologue 1 (Dlg1) intracellular scaffolding protein.ConclusionsThe data identify Netrin1-Unc5B as novel regulators of BRB integrity, with implications for diseases associated with BRB disruption.