Background: The 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 expression Objective: Here, we investigated if Netrin-1-binding to endothelial Unc5B also controlled BRB integrity, and if this process involved Norrin/beta-catenin signaling, which is the major known driver of BRB development and maintenance. Methods: We analyzed Tamoxifen-inducible loss- and gain- of-function alleles of Unc5B, Ntn1 and Ctnnb1 in conjunction with tracer injections and biochemical signaling studies. Results: Inducible endothelial Unc5B deletion, and inducible global Ntn1 deletion in postnatal mice reduced phosphorylation of the Norrin receptor LRP5, leading to reduced beta-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. Inducible Ctnnb1 gain of function rescued vascular leak in Unc5B mutants, and Ntn1 overexpression induced BRB tightening. Unc5B expression in pericytes contributed to BRB permeability, via regulation of endothelial Unc5B. Mechanistically, Netrin-1-Unc5B signaling promoted beta-catenin dependent BRB signaling by enhancing phosphorylation of the Norrin receptor LRP5 via the Discs large homologue 1 (Dlg1) intracellular scaffolding protein. Conclusions: The data identify Netrin1-Unc5B as novel regulators of BRB integrity, with implications for diseases associated with BRB disruption.
Glioblastoma (GBM) is the most common and fatal primary tumor of the central nervous system (CNS) and current treatments have limited success. Chemokine signaling regulates both malignant cells and stromal cells of the tumor microenvironment (TME), constituting a potential therapeutic target against brain cancers. Here, we investigated the C–C chemokine receptor type 7 (CCR7) and the chemokine (C–C-motif) ligand 21 (CCL21) for their expression and function in human GBM and then assessed their therapeutic potential in preclinical mouse GBM models. In GBM patients, CCR7 expression positively associated with a poor survival. CCL21–CCR7 signaling was shown to regulate tumor cell migration and proliferation while also controlling tumor associated microglia/macrophage recruitment and VEGF-A production, thereby controlling vascular dysmorphia. Inhibition of CCL21–CCR7 signaling led to an increased sensitivity to temozolomide-induced tumor cell death. Collectively, our data indicate that drug targeting of CCL21–CCR7 signaling in tumor and TME cells is a therapeutic option against GBM.
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