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

Abstract

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/β-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 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. 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 β-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.

Keywords: Blood-Retina Barrier; Dlg1; Netrin-1; Norrin/β-catenin signaling; Unc5B.

Figure 1:. Unc5B expression in endothelial cells and mural cells of the developing retina.

(a) UMAP plot of retinal cells. (b) Unc5B dot plot expression levels and frequency among retina cell clusters. Color scale: red: high expression; green: low expression. (c) UMAP plot of EC sub-clusters. (d) Unc5B dot plot expression levels and frequency among ECs subclusters. Color scale: red: high expression; green: low expression. (e-h) Immunofluorescence staining and confocal imaging on whole-mount P5 or P12 retinas with the indicated antibodies.

Figure 2:. Endothelial Unc5B regulates retinal angiogenesis and BRB formation.

(a) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (b) IB4 immunofluorescence staining and confocal imaging on whole-mount P5 retinas. (c) Quantification of IB4+ vascular outgrowth and density in P5 retinas. (d) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (e) Immunofluorescence staining of IB4+ vessel and confocal imaging on whole-mount P12 retinas. Superficial, intermediate and deep plexus are color-coded. (f) Quantification of superficial, intermediate and deep plexus vascular density in P12 retinas. (g) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (h) Confocal imaging on whole-mount P5 retinas, 2h after i.p. cadaverine injection. (i) quantification of cadaverine leakage. (j) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies, 2h after i.p. cadaverine injection. (k) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (l,m) Western blot (l) and quantification (m) of P5 retina protein extracts. (n) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (o) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies, 10min after i.v. sulfo-NHS-biotin injection. All data are shown as mean+/−SEM. Two-sided Mann-Whitney U test was performed for statistical analysis.

Figure 3:. Endothelial Unc5B regulates BRB permeability via Norrin/β-catenin signaling.

(a) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (b,c) Western blot (b) and quantification (c) of P5 retina protein extracts. (d) Unc5B and Ctnnb1 gene deletion strategy using tamoxifen injection in P5 retinas. (e) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (f) Unc5B and Ctnnb1 gene deletion strategy using tamoxifen injection in P12 retinas. (g) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies. (h) Unc5B gene deletion and Ctnnb1^flex/3 gene overexpression strategy using tamoxifen injection in P5 retinas. (i) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (j) Unc5B gene deletion and Ctnnb1^flex/3 gene overexpression strategy using tamoxifen injection in P12 retinas. (k) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies, 10min after i.v. sulfo-NHS-biotin injection. All data are shown as mean+/−SEM. Two-sided Mann-Whitney U test was performed for statistical analysis.

Figure 4:. Netrin-1 regulates BRB development and integrity.

(a) Ntn1 gene deletion strategy using tamoxifen injection in P5 neonates. (b) IB4 immunofluorescence staining and confocal imaging on whole-mount P5 retinas. (c) Quantification of IB4+ vascular outgrowth and density in P5 retinas. (d) Ntn1 gene deletion strategy using tamoxifen injection in P12 neonates. (e) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies. (f) Netrin1 binding to Unc5B blockade strategy using anti-Unc5B-3 antibody. (g) Confocal imaging on P12 retinas i.p. injected with anti-Unc5B-3 (2h, 10mg/kg) followed by i.v. sulfo-NHS-biotin injection for 10min. (h) Ntn1 gene deletion strategy using tamoxifen injection in P5 neonates. (i,j) Western blot (i) and quantification (j) of P5 retina protein extracts. (k) Ntn1 gene overexpression strategy using tamoxifen injection in P5 neonates. (l) qPCR analysis of P5 retina mRNA extracts. (m) IB4 immunofluorescence staining and confocal imaging on whole-mount P5 retinas. (n) Quantification of IB4+ vascular outgrowth and density in P5 retinas. (o) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (p) Ntn1 gene overexpression strategy using tamoxifen injection in P5 neonates. (q) Confocal imaging on P5 retinas i.v. injected with sulfo-NHS-biotin for 10mim. All data are shown as mean+/−SEM. Two-sided Mann-Whitney U test was performed for statistical analysis.

Figure 5:. Mural cell Unc5B contributes to retinal angiogenesis.

(a) Global or Pericyte Unc5B gene deletion strategy using tamoxifen injection. (b,c) Survival curve after neonatal global (b) or pericyte (c) Unc5B gene deletion. (d) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (e,f) Quantification on IB4+ vascular outgrowth and density in P5 retinas. (g) Immunofluorescence staining of IB4+ vessel and confocal imaging on whole-mount P12 retinas. Superficial, intermediate and deep plexus were color-coded. (h,i) Quantification of superficial, intermediate and deep plexus vascular density in P12 retinas. (j,k) Immunofluorescence staining and confocal imaging on the vascular front of whole-mount P5 retinas with the indicated antibodies. All data are shown as mean+/−SEM. Mantel-cox test was performed for survival curve statistical analysis. Mann-Whitney U test was performed for statistical analysis between two groups. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups.

Figure 6:. Mural cell Unc5B contributes to BRB regulation.

(a) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (b) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies, 10min after i.v. sulfo-NHS-biotin injection. (c) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (d) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies. (e) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (f,g) Western blot (f) and quantification (g) of P5 retina protein extracts. All data are shown as mean+/−SEM. Mann-Whitney U test was performed for statistical analysis between two groups.

Figure 7:. Netrin-1 and Norrin cooperate to induce β-catenin signaling

(a-c) Western blot (a) of mouse brain EC protein extracts treated for the indicated times (min) with Netrin-1, Norrin, or a combination of both. (b,c) Quantification of LRP5 phosphorylation levels over time (d) Mouse brain ECs were transfected with the β-catenin transcriptional luciferase reporter TOPflash or FOP flash control and stimulated with Netrin-1, Norrin, or a combination. Cells were harvested in 1x cell culture lysis reagent and luciferase activity was quantified using a plate-based luminometer. (e) CTRL IgG and Unc5B immunoprecipitation from cultured mouse brain ECs. (f) CTRL IgG and Unc5B immunoprecipitation on CTRL or Dlg1 SiRNA-treated mouse brain ECs. (g,h) Western blot (g) and quantification (h) of CTRL, Unc5B or Dlg1 SiRNA-transfected mouse brain ECs treated with Netrin-1 (250ng/ml). All data are shown as mean+/−SEM. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups.