Gpr124 is essential for blood-brain barrier integrity in central nervous system disease

Abstract

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Figure 1

Endothelial Gpr124 deficiency induces rapid BBB breakdown and hemorrhagic transformation following brain ischemia and reperfusion. (a) Gross appearance (top) and TTC staining (bottom) of brains of global Gpr124-CKO and het mice after 1 h tMCAO and 1–5 d reperfusion; intracerebral hemorrhage and infarcted areas (white in TTC staining) can be visualized. (b) H&E histological analysis (left) and Perl’s Iron staining for red blood cells (right) to visualize microvascular hemorrhage. Scale bar, 100 μm. (c) Gross appearance (top) and TTC staining (bottom) of endothelial (EC) Gpr124-CKO and het mice after 1 h tMCAO and 1–5 d reperfusion. (d) The percentage of male mice of the indicated genotypes showing hemorrhage poststroke within 7 d reperfusion. (e) Quantification of hemorrhage area on the surface of the cortex and inside infarcted tissue of EC Gpr124-CKO mice and het mice after 1 h tMCAO and 1–7 d reperfusion. Het 1–2 d, n = 5 mice; CKO 1–2 d, n = 10; het 3–4 d, n = 4; CKO 3–4 d, n = 5; het 5–7 d, n = 8; CKO 5–7 d, n = 4. n.s., not significant, *P < 0.05, **P < 0.01, unpaired Student’s t-test. (f,g) Survival of male global Gpr124-CKO mice (f) or EC Gpr124-CKO mice (g), together with their respective het controls, after 1 h tMCAO and 7 d reperfusion. P < 0.05, log–rank test. (h) Quantification of infarct size determined by TTC staining of male global Gpr124-CKO mice or EC Gpr124-CKO mice, together with their respective het controls. Global Gpr124 het mice, n = 7; global Gpr124 CKO, n = 7; EC Gpr124 het, n = 5; EC Gpr124 CKO, n = 4. *P < 0.05, **P < 0.01, unpaired Student’s t-test. (i) Immunofluorescence staining of leaked endogenous plasma fibrinogen after 1 h tMCAO and 1 h reperfusion in EC Gpr124-CKO mice and het mice. Arrows point to plasma fibrinogen that has leaked out of vessels. Scale bar, 50 μm. (j) Quantification of plasma fibrinogen leakage in (i). Data are mean ± s.e.m., n = 3 mice per group. **P < 0.01, unpaired Student’s t-test.

Figure 2

Activation of endothelial Wnt–β-catenin signaling rescues the hemorrhagic-stroke phenotype of Gpr124-deficient mice. (a) Expression of Gpr124 and the Wnt target genes Axin2 and Apcdd1, as assessed by RT–qPCR, in primary brain ECs from adult global Gpr124-CKO and het mice; cells were infected with adenovirus expressing mouse IgG2α Fc (Ad Fc) or Wnt7b (Ad Wnt7b) for 2 d before determination of mRNA levels. Data are mean ± s.e.m. of triplicate experiments. **P < 0.01, unpaired Student’s t-test. (b) Expression of Gpr124, Wnt target genes and the BBB marker Cldn5, as assessed by RT–qPCR, in freshly isolated brain ECs of tamoxifen-treated global Gpr124-CKO mice versus het mice. Mean ± s.e.m., n = 4 mice per group. Individual mice are plotted. *P < 0.05, **P < 0.01, unpaired Student’s t-test. (c) Expression of Wnt-signaling target genes, as assessed by RT–qPCR, in FACS-sorted adult brain ECs from EC Gpr124-CKO versus het mice with or without constitutive β-catenin activation (β-cat). Mean ± s.e.m., n = 4 mice per group. Individual mice are plotted. This experiment was repeated with n = 4 mice per group. *P < 0.05, **P < 0.01 versus het, unpaired Student’s t-test. (d) Fold changes (log²) in expression of canonical Wnt-signaling target genes, as assessed by RNA-seq analysis, in FACS-sorted brain ECs from tamoxifen-treated mice, showing comparisons between Ctnnb1^lox(ex3)/+;Cdh5-CreER (β-cat) mice versus Ctnnb1^+/+;Cdh5-CreER (WT) mice (n = 3 mice per group), and global Gpr124-CKO mice versus het mice (n = 4 mice per group). (e) Relative expression levels of Wnt target genes, as assessed by RNA-seq analysis, of brain ECs from the stroke and nonstroke hemispheres of adult global Gpr124-CKO and het mice after 1 h tMCAO and 1 d reperfusion. For each gene, the level of gene expression is presented relative to the het nonstroke value and assigned to values between 0 and 1 to allow red–green color depiction. n = 4 mice per group. **P < 0.01, paired Student’s t-test. (f) Gross appearance (top) and TTC staining (bottom) of brains of EC Gpr124-CKO versus het mice with or without endothelial constitutive β-catenin activation to show intracerebral hemorrhage and infarcted areas (white in TTC staining) after 1 h tMCAO and 5 d reperfusion. (g) Quantification of infarct size determined by TTC staining. EC het, n = 5 mice; EC CKO, n = 3; EC het; β-cat, n = 4; EC CKO; β-cat, n = 7. **P < 0.01, unpaired Student’s t-test. (h) Survival of EC Gpr124-CKO versus het mice with or without endothelial constitutive β-catenin activation after 1 h tMCAO and 5 d reperfusion. *P < 0.05, log–rank test. (i) Representative images of BBB integrity in brains of the indicated mice after 1 h tMCAO and 1 d reperfusion, as assessed by the Sulfo-NHS-biotin tracer extravasation assay. Scale bar, 50 μm. (j) Quantification of extravasated exogenous tracer Sulfo-NHS-biotin or endogenous plasma fibrinogen. Biotin or fibrinogen signal density was measured with ImageJ and normalized to vessel area (CD31). Data are mean ± s.e.m., n = 4 mice per group. **P < 0.01, unpaired Student’s t-test.

Figure 3

Gpr124–Wnt signaling regulates endothelial tight junction, pericyte and extracellular matrix following stroke. (a) Expression of Cldn5 mRNA, as assessed by RT–qPCR, using FACS-sorted adult brain CD31⁺ ECs from EC Gpr124 CKO versus het mice, with or without constitutive β-catenin activation (β-cat). Data are mean ± s.e.m., EC het, n = 7 mice; EC CKO, n = 8; EC het; β-cat, n = 7; EC CKO; β-cat, n = 6. Individual mice are plotted. *P < 0.05, **P < 0.01, unpaired Student’s t-test. (b) Co-immunofluorescence staining for CD31 and desmin in infarcted brain regions after 1 h tMCAO and 1 d reperfusion of global Gpr124-CKO versus het mice. Arrows show desmin⁺ pericytes detached from CD31⁺ endothelial cells. Scale bar, 20 μm. (c,d) Co-immunofluorescence staining for Pdgfr-β and CD31 in infarcted brain regions (c) and quantification of pericyte coverage (d) after 1 h tMCAO and 5 d reperfusion in EC Gpr124 CKO versus het mice with or without constitutive β-catenin activation. The lengths of Pdgfr-β or desmin staining signal surface were normalized to that for CD31; 6–8 low-power fields per mouse, EC het, n = 4; EC CKO, n = 3; EC het; β-cat, n = 4; EC CKO; β-cat, n = 4. *P < 0.05, **P < 0.01, unpaired Student’s t-test. Scale bar in c, 100 μm. (e) Pdgfb expression, as assessed by in situ hybridization, in infarcted brain cortex of EC Gpr124-CKO mice and het controls after 1 h tMCAO and 1 d reperfusion. Representative images are shown from three mice. (f) Expression of Pdgfb, as assessed by RT–qPCR, in adult brain ECs from EC Gpr124 CKO versus het mice after 1 h tMCAO and 1 d reperfusion. Infarcted brain tissues from two mice were pooled to form one sample; EC het, n = 6, EC CKO, n = 7. Individual mice are plotted. *P < 0.05 versus EC het, unpaired Student’s t-test. (g) Expression of Pdgfb, as assessed by RT–qPCR, in adult brain ECs from mice with EC Gpr124-het mice with or without constitutive β-catenin activation (EC β-cat). Data are mean ± s.e.m.; n = 4 mice per group. *P < 0.05, unpaired Student’s t-test. (h,i) Co-immunofluorescence staining for laminin and CD31 and for collagen IV (col IV) and CD31 in infarcted brain regions (h) and quantification of ECM protein levels (i) after 1 h tMCAO and 5 d reperfusion. Laminin and col IV signal densities were normalized to CD31 signal area. 6–8 low-power fields per mouse were randomly selected. Data are mean ± s.e.m., n = 4 mice per group. **P < 0.01, unpaired Student’s t-test. Scale bar in h, 100 μm. (j) CD31 staining in infarcted brain cortex in EC Gpr124-CKO versus het mice with or without constitutive β-catenin activation after 1 h tMCAO and 5 d reperfusion. Scale bar, 100 μm. (k) Quantification of microvessel density, as assessed by the percentage of CD31 signal area to brain tissue area in (j). Five or six low-power fields per mouse were randomly selected. Data are mean ± s.e.m., n = 4 mice per group. Individual mice are plotted.

Figure 4

Endothelial Gpr124 deficiency increases tumor hemorrhage and reduces survival in experimental glioblastoma. (a,b) Co-immunofluorescence staining for Gpr124 and CD31 in mouse normal brain tissue (a) and GL261 glioblastoma (b). The images are representative of those obtained from five mice. Scale bar, 100 μm. (c) Survival of global Gpr124-CKO and het control mice with implanted GL261 glioblastoma cells. n = 15 mice per group. P < 0.02, log–rank test. (d) Gross appearance and H&E histology of brain tumors in global Gpr124-CKO and het control mice. Representative images from 15–20 tumors are shown. Scale bar, 100 μm. (e) Expression of Gpr124, as assessed by RT–qPCR, in FACS-sorted CD31⁺ ECs from normal brain and tumor tissue of EC Gpr124-CKO and het mice. n = 6 samples for EC het-normal, EC CKO-normal and EC CKO-tumor, and n = 5 samples for EC het-tumor; each sample consisted of FACS-sorted CD31⁺ ECs from pooled normal or tumor tissue from two mice of the same genotype. Data are mean ± s.e.m., *P < 0.05, **P < 0.01 versus EC het-normal, unpaired Student’s t-test. (f) Gross appearance and H&E analysis of brain tumors from EC Gpr124-CKO mice versus het controls. Representative images from 15–20 tumors are shown. Scale bar, 100 μm. (g) Intratumoral edema, quantified as the area of interstitial space within tumors absent of tumor cells but instead filled with blood cells or plasma, relative to total tumor area. Global Gpr124 het, n = 4; global Gpr124 CKO, n = 4; EC Gpr124 het, n = 6; EC Gpr124 CKO, n = 6. **P < 0.01, unpaired Student’s t-test. (h,i) Quantification of tumor volumes without (h) and with (i) compensation for edema. Global Gpr124 het, n = 4; global Gpr124 CKO, n = 4; EC Gpr124 het, n = 6; EC Gpr124 CKO, n = 6. Data are mean ± s.e.m. Individual mice are plotted. n.s., not significant, *P < 0.05, **P < 0.01, unpaired Student’s t-test.

Figure 5

Endothelial activation of Wnt–β-catenin signaling reduces tumor hemorrhage and edema in endothelial-specific Gpr124-deleted mice with glioblastoma. (a) Expression of Wnt-signaling target genes Axin2 and Apcdd1, as assessed by RT–qPCR, in FACS-sorted CD31⁺ ECs from normal brain and tumor tissue of EC Gpr124-CKO and het mice. n = 6 samples for EC het-normal, EC CKO-normal, EC CKO-tumor and n = 5 samples for EC het-tumor; each sample was FACS-sorted CD31⁺ ECs from pooled normal or tumor tissue from two mice of the same genotype. Data are mean ± s.e.m., *P < 0.05, **P < 0.01, unpaired Student’s t-test. (b) Gross appearance, H&E histology and co-immunofluorescence staining of mouse IgG (mIgG) and CD31 of brain tumors in EC Gpr124-CKO versus het mice with or without constitutive β-catenin activation. EC het, n = 8; EC CKO, n = 6; EC het; β-cat, n = 6; EC CKO; β-cat, n = 6. Representative images are shown. Scale bar, 100 μm. (c) Quantification of mouse IgG signal density relative to that of CD31 signal in b. Five or six low-power fields per mouse were randomly selected. a.u, arbitrary unit. n = 4 mice per group. Data are mean ± s.e.m., **P < 0.01, unpaired Student’s t-test. (d) Intratumoral edema, quantified as the area of interstitial space within tumors absent of tumor cells but instead filled with blood cells or plasma, relative to total tumor area. EC het, n = 5; EC CKO, n = 5; EC het; β-cat, n = 5; EC CKO; β-cat, n = 6. *P < 0.05, **P < 0.01, unpaired Student’s t-test. (e,f) Quantification of tumor volumes in EC Gpr124 CKO versus het mice with or without constitutive β-catenin activation without (e) or with compensation (f) for edema. EC het, n = 5; EC CKO, n = 5; EC het; β-cat, n = 5; EC CKO; β-cat, n = 6. (g) Tumor-vessel density, quantified as CD31-positive signal area relative to tumor tissue area, with correction for edema. 6–8 low-power fields per mouse were randomly selected. EC het, n = 6; EC CKO, n = 5; EC het; β-cat, n = 5; EC CKO; β-cat, n = 4. *P < 0.05, unpaired Student’s t-test. Data are mean ± s.e.m.

Figure 6

Gpr124–Wnt signaling increases BBB integrity in glioblastoma by regulating tight-junction protein, pericyte coverage, Glut1 and the ECM. (a) Co-immunofluorescence staining for Cldn5 and CD31 in tumors of GL261-implanted EC Gpr124 CKO versus het mice with or without constitutive β-catenin activation. Scale bar, 100 μm. (b) Quantification of Cldn5 signal density in (a) normalized to CD31 signal area. 6–8 low-power fields per tumor were randomly selected. n = 4 mice per group. Data are mean ± s.e.m. **P < 0.01, unpaired Student’s t-test. (c,d) Co-immunofluorescence staining for Pdgfr-β and CD31 of tumors (c) and quantification of pericyte coverage (Pdgfr-β⁺ and desmin⁺, d) in endothelial Gpr124 CKO versus het mice with or without constitutive β-catenin activation. Pdgfr-β and desmin signal areas were normalized to CD31 signal area. 6–8 low-power fields per mouse were randomly selected. n = 4 mice per group. *P < 0.05, **P < 0.01, unpaired Student’s t-test. (e) Expression of Pdgfb, as assessed by RT–qPCR, in FACS-sorted brain tumor ECs from EC Gpr124-CKO and het mice. n = 5 samples for EC het, n = 6 samples for EC CKO; tumor tissues from two mice were pooled as one sample. Data are mean ± s.e.m. *P < 0.05, unpaired Student’s t-test. (f,g) Co-immunofluorescence staining for laminin and CD31 and for collagen IV (col IV) and CD31 in tumor regions (f) and quantification of ECM protein abundance (g). Laminin and col-IV signal densities were normalized to CD31 signal area. 6–8 low-power fields per mouse were randomly selected. n = 4 mice per group. *P < 0.05, unpaired Student’s t-test. (h) Co-immunofluorescence staining for Glut1 and CD31 in tumors of EC Gpr124-CKO versus het mice with or without constitutive β-catenin activation. Note the strong expression of Glut1 in tumor cells in both Gpr124 het and CKO tumors. Scale bar, 100 μm. (i) Quantification of the number of Glut1⁺ tumor vessels. 100–200 vessels were counted per tumor. n = 4 mice per group. Data are mean ± s.e.m. **P < 0.01, unpaired Student’s t-test. (j) Schematic diagram illustrating the mechanisms underlying pathologic BBB breakdown and hemorrhage caused by Gpr124 deficiency. In ischemic stroke or glioblastoma, endothelial Gpr124 deficiency reduces Wnt7-dependent canonical β-catenin signaling, resulting in β-catenin activation-reversible effects: loss of tight-junction protein (Cldn5) and Pdgfb expression, pericyte loss, ECM disruption (laminin, collagen IV) and downregulation of endothelial Glut1 expression (in the glioblastoma setting only). Gpr124 deletion impairs angiogenesis only in the GL261 GBM, but not in acute stroke setting.