Astrocytic neogenin/netrin-1 pathway promotes blood vessel homeostasis and function in mouse cortex

Abstract

Astrocytes have multiple functions in the brain, including affecting blood vessel (BV) homeostasis and function. However, the underlying mechanisms remain elusive. Here, we provide evidence that astrocytic neogenin (NEO1), a member of deleted in colorectal cancer (DCC) family netrin receptors, is involved in blood vessel homeostasis and function. Mice with Neo1 depletion in astrocytes exhibited clustered astrocyte distribution and increased BVs in their cortices. These BVs were leaky, with reduced blood flow, disrupted vascular basement membranes (vBMs), decreased pericytes, impaired endothelial cell (EC) barrier, and elevated tip EC proliferation. Increased proliferation was also detected in cultured ECs exposed to the conditioned medium (CM) of NEO1-depleted astrocytes. Further screening for angiogenetic factors in the CM identified netrin-1 (NTN1), whose expression was decreased in NEO1-depleted cortical astrocytes. Adding NTN1 into the CM of NEO1-depleted astrocytes attenuated EC proliferation. Expressing NTN1 in NEO1 mutant cortical astrocytes ameliorated phenotypes in blood-brain barrier (BBB), EC, and astrocyte distribution. NTN1 depletion in astrocytes resulted in BV/BBB deficits in the cortex similar to those in Neo1 mutant mice. In aggregate, these results uncovered an unrecognized pathway, astrocytic NEO1 to NTN1, not only regulating astrocyte distribution, but also promoting cortical BV homeostasis and function.

Keywords: Angiogenesis; Neurodevelopment; Neuroscience.

Figure 1. Reduced DiI⁺ vessels, but increased PECAM-1⁺ vessels, in astrocytic, but not pyramidal neuronal, Neo1-KO cortex.

(A) Schematic of the protocol for DiI perfusion. (B) Representative images of BVs in the cortex of control (Neo^fl/fl) and Neo^GFAP-Cre mice (at P60). Brain sections were subjected to immunostaining analysis using antibodies against PECAM-1 (green). Images were captured from 6 slides per animal. Average number of DiI⁺ vessel length per cubic millimeters was calculated and is presented in C, and PECAM-1⁺ vessel length was quantified and is presented in D. BV tracing and quantification of BV branches are shown in E and F, respectively. (G) Representative images of BVs in cortices of littermate control Neo^fl/fl and Neo^GFAP-CreER mice. Mice were injected with TAM at P30 and perfused with DiI at P60 before sacrifice. (H and I) Quantitative analyses of data in G. (J) Representative images of BVs in cortices of littermate control and Neo^Nex-cre mice at P60. (K and L) Quantitative analyses of data in J. Scale bars: 50 μm. Data are represented as mean ± SEM (n = 3 to 6 mice/group). *P < 0.05; **P < 0.01, Mann-Whitney U statistical test.

Figure 2. Reduced blood flow, but increased BBB leakage, in Neo^GFAP-CreER cortex.

(A) Representative images and (B) quantification of laser Doppler measurement of blood flow signals in control (Neo^fl/fl) and Neo^GFAP-CreER cortices (TAM at P30, imaged at P60). (C) Schematic of the protocol for EB and dextran injections. (D and E) Representative images of 10 kDa dextran (green) and EB (red) coimmunostained with anti-PECAM-1 in P60 control (neo^fl/fl) and Neo^GFAP-CreER cortex. (F) Quantitative analysis of data in D. (G) Quantitative analysis of EB extravasation. (H and I) Representative images of immunostaining using indicated antibodies (H) and the quantification (I) of 3 kDa dextran (green) in control (neo^fl/fl and GFAP-CreER) and Neo^GFAP-CreER cortex. (J) Pearson’s analysis of the correlation between EB leakage and vessel density in Neo^GFAP-CreER cortex. Scale bars: 20 μm. Data are represented as mean ± SEM (n = 3 mice/group). *P < 0.05; **P < 0.01, Student’s t test (B, F, and G); 1-way ANOVA plus post hoc analysis (I).

Figure 3. Swollen astrocytes, disrupted vBMs, reduced pericytes, and increased EC caveolae in Neo^GFAP-CreER cortex by EM analysis.

(A) Representative EM images of BVs in control (Neo^fl/fl) and Neo^GFAP-CreER cortex (TAM at P30, EM at P60). Astrocytes are highlighted in pink, pericytes (PC) in light green, and EC in light yellow. Scale bars: 1 μm. (B) Quantification of astrocyte area. (C) Quantification of pericyte coverage of BVs. (D) Quantification of disrupted vBMs of BVs. (E) Quantification of EC caveolae per vessel. Data are represented as mean ± SEM (n = 7 to 13 vessels from 2 mice per group). *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 4. Disrupted vBMs and reduced pericytes in Neo^GFAP-CreER cortex by coimmunostaining analysis.

(A–F) Coimmunostaining analyses using indicated antibodies. Representative images of laminin-γ1 (A), laminin-α5 (C), and collagen IV (E) are shown. Quantification of laminin-γ1 (B), laminin-α5 (D), and collagen IV (F) in control (Neo^fl/fl) and Neo^GFAP-CreER cortex are presented. In A, arrowheads indicate absent laminin-γ1 coverage and arrows indicate laminin-γ1 aggregates. In E, arrowheads show detached collagen IV from BVs. (G–J) Representative images of desmin (G) and PDGFR-β (I) marked pericytes, and quantification of desmin (H) and PDGFR-β (J) in control (Neo^fl/fl) and Neo^GFAP-CreER cortex are shown. (K and L) Representative images (K) and quantification (L) of active caspase-3⁺ pericytes in BVs of control and Neo^GFAP-CreER cortex. Scale bars: 20 μm. Data are represented as mean ± SEM (n = 5 mice/group). *P < 0.05, Mann-Whitney U test.

Figure 5. Impaired EC barrier, increased vein capillaries, and thinner arterioles in Neo^GFAP-CreER cortex.

(A, B, E, and F) Representative images of coimmunostaining analyses using indicated antibodies in control (Neo^fl/fl) and Neo^GFAP-CreER cortex. (C, D, G, and H) Quantification analyses of data in A, B, E, and F. PLVAP is an EC fenestrae marker, caveolin-1 is a marker for caveolae, Slc16a1 is a venous-capillary marker, and labels arteriole. Scale bars: 20 μm. Data are represented as mean ± SEM (n = 5 mice/group). *P < 0.05; **P < 0.01, Mann-Whitney U test. (I) Schematic summary of vessel deficits in control and Neo^GFAP-CreER cortex.

Figure 6. Altered astrocyte distribution in Neo^GFAP-CreER cortex.

(A) Schematic of the protocol performed in control (GFAP-CreER;Ai9) and Neo^GFAP-CreER;Ai9 mice. (B) Representative images of td-Tomato⁺ astrocytes and PECAM-1⁺ BVs. (C) Quantification analysis of td-Tomato⁺ astrocyte association with PECAM-1⁺ (white) BVs in control and Neo^GFAP-CreER cortex. (D) Representative images showing td-Tomato⁺ astrocyte distribution. 3D reconstruction images are included in right panels. (E) Profile of rectangle area of td-Tomato⁺ fluorescence in D analyzed by ZEN 2.3 lite. Cell-cell nuclear distance was quantified and is shown in F and G. (F) Histogram of average distance. Scale bars: 10 μm. Data are represented as mean ± SEM (n = 3 mice/group). *P < 0.05, Mann-Whitney U test (C); F test for variance analysis (G).

Figure 7. Elevated tip EC proliferation in Neo^GFAP-CreER cortex.

(A) Schematic of TAM and EdU injection protocol in control (GFAP-CreER;Ai9) and Neo^GFAP-CreER;Ai9 mice. (B) Representative images of cortical brain sections coimmunostained with EdU (green), PECAM-1 (magenta), and td-Tomato (red). (C and D) Quantification analyses of data in B. (E and H) Representative images of cortical brain sections coimmunostained with indicated antibodies. (F–J) Quantitative analyses of data in E and H, respectively. Scale bars: 20 μm. Data in C, D, F, G, I, and J are represented as mean ± SEM (n = 5 mice/group). *P < 0.05; **P < 0.01, Mann-Whitney U test

Figure 8. Increased EC proliferation and migration in HUVEC cultures exposed to Neo1-KO astrocytes or their CM.

(A–C) Increased EC proliferation in HUVECs cocultured with Neo1-KO astrocytes. (A) Schematic of coculture of HUVECs with cortical astrocytes of control and Neo-KO. BrdU was incubated for 4 hours. (B) Representative images of BrdU⁺ HUVECs. (C) Quantitative analysis of data in B. Data are represented as mean ± SEM (n = 4 experiments of astrocytes from Neo^GFAP-CreER with 4-OH-TAM). (D–F) Increased EC proliferation in HUVECs exposed to CM of Neo1-KO astrocytes. (D) Schematic of the culture. BrdU was incubated for 6 hours. (E) Representative images of BrdU⁺ HUVECs. (F) Quantification analysis of data in E. Data are represented as mean ± SEM (n = 4 experiments with astrocytes from Neo^GFAP-CreER [+TAM] and its control group). (G–K) Increased EC migration in HUVECs exposed to CM of Neo1-KO astrocytes. (G) Schematic of HUVEC migration assay. The HUVECs were cultured with CM of astrocytes in a 6-well plate (left panel), and were scratched for the wound-healing migration assay (right panel). (H) Representative images of HUVEC migration 24 hours after wound scratching. (I) Representative images of HUVECs that were cultured with CM of astrocytes and immunostained with indicated antibodies. (J) Quantification analyses of data in H. Data are represented as mean ± SEM (n = 4 experiments from Neo^GFAP-CreER and its control group astrocytes). (K) Quantification of data in I. Data are represented as mean ± SEM (n = 3 experiments from Neo^GFAP-CreER and its control group astrocytes). Scale bars: 20 μm. *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 9. Reduced NTN1 in Neo1-KO cortical astrocytes.

(A–D) Antibody array analysis. The nitrocellulose membranes containing 26 antibodies (see Supplemental Figure 10A) were first incubated with biotin-conjugated proteins from CM of control and Neo1-KO astrocytes, respectively, washed, and then incubated with HRP-streptavidin, according to the manufacturer’s instructions. (A) Representative images of the antibody array blots. NTN1 is highlighted in red. (B) Volcano plot analysis showing reduced NTN1 in the CM of Neo1-KO astrocytes. (C) Heatmap analysis (n = 4). (D) Quantification analysis. Data are represented as mean ± SEM (n = 4). (E and F) Quantitative PCR (qPCR) analysis of indicated gene expression in cortical (E) and hippocampal astrocytes (F) (cultured in the presence of 10% FBS). Values were normalized to GAPDH levels and controls. Data are represented as mean ± SEM (n = 3 experiments /group). (G and H) FISH analysis of NTN1 mRNAs in td-Tomato⁺ astrocytes in control (GFAP-CreER;Ai9) and Neo^GFAP-CreER;Ai9 cortex and hippocampus. (G) Representative images. Scale bar: 5 μm. (H) Quantification analysis of data in G. Data are represented as mean ± SEM (n = 6 slides/group). (I–M) BMP2 induction of phospho-Smad_1/5/8 and NTN1 expression in cortical and hippocampal astrocytes (cultured in the presence of 1% FBS) from control and Neo1-KO mice. (I) Illustration of protocol of BMP2 treatment.(J) Western blot analyses using indicated antibodies. (K) Quantification of data in J. (L and M) qPCR analyses of BMP2-induced NTN1 mRNAs in cortical (L) and hippocampal (M) astrocytes. Data are represented as mean ± SEM (n = 3 experiments). *P < 0.05; **P < 0.01, 2-way ANOVA.

Figure 10. NTN1 inhibition of EC proliferation and migration induced by CM of Neo1-KO astrocytes.

(A–C) NTN1 (1 μg/mL) inhibition of MBMEC proliferation. (A) Schematic of NTN1 administration in MBMEC cultures in the presence of CM of astrocytes. BrdU (3 μg/mL) was incubated for 6 hours. (B) Representative images of BrdU⁺ MBMECs. (C) Quantification analysis of data in B. (D–F) NTN1 inhibition of MBMEC migration. (D) Schematic of NTN1 administration in a Transwell assay to access MBMEC migration. (E) Representative images of MBMECs (stained with crystal violet). (F) Quantification analysis of data in E. Data are represented as mean ± SEM (n = 5–6 coverslips /group). *P < 0.05, 2-way ANOVA. Scale bars: 20 μm.

Figure 11. NTN1 amelioration of phenotypes of BV increase and BBB leakage in Neo1-KO cortex.

(A) Schematic of protocol of AAV-GFP and AAV-GFAP-NTN1 viruses and TAM injections. AAV-GFAP-NTN1 encoding NTN1 fusion protein (NTN1-myc-his-P2A-mCherry) under control of GFAP promoter was injected into left side of Neo^GFAP-CreER cortex (ipsilateral side) at P30. (B) Representative images of immunostaining using indicated antibodies. P60 Neo^GFAP-CreER cortices were injected with AAV-GFP or AAV-GFAP-NTN1, respectively. Scale bars: 100 μm. (C and D) Quantitative analyses of BV length (C) and branches (D). (E) Representative images showing 10 kDa dextran leakage in ipsilateral and contralateral cortices injected with AAV-GFP or AAV-GFAP-NTN1, respectively. (F) Quantitative analyses of data in E. (G) Representative images showing PLVAP staining in ipsilateral and contralateral cortices injected with AAV-GFP or AAV-GFAP-NTN1, respectively. (H) Quantitative analyses of data G. Data are represented as mean ± SEM (n = 4 mice/group). *P < 0.05; **P < 0.01, 2-way ANOVA. Scale bars: 20 μm.

Figure 12. Increased BV density, leaky BBB, and fewer pericytes and vBMs in astrocytic Ntn1-KO cortex.

(A) Schematic of experiments. NTN1^GFAP-CreER and littermate control (NTN1^fl/fl) mice (at P30) were injected with TAM, and mice at P60 were anesthetized, tail injected with 3 kDa dextran, and sacrificed 30 minutes after dextran injection. (B) Representative images of BVs (magenta) and dextrans (green) in cortices of control and NTN1^GFAP-CreER mice. (C and D) Quantitative analyses of PECAM-1⁺ BV density (C) and dextron⁺ intensity (D). (E and G) Representative images (E) and quantification analysis (G) of BV-EC proliferation in the cortices of control and NTN1^GFAP-CreER mice. Arrowheads indicate Ki67⁺ cells. (F and H) Representative images (F) and quantification analysis (H) of caveolin1⁺ capillary-venous in cortices of control and NTN1^GFAP-CreER mice. (I and K) Representative images (I) and quantification (K) of PDGFRβ⁺ pericytes in cortexes of control and NTN1^GFAP-CreER mice. (J and L) Representative images (J) and quantification (L) of laminin-α5⁺ BVs in cortices of control and NTN1^GFAP-CreER mice. (M) Summary of phenotypes and illustration of a working model for astrocytic Neo1 to regulate BV hemostasis through NTN1. Scale bars: 20 μm. Data are represented as mean ± SEM (n = 5 mice). *P < 0.05; **P < 0.01 Mann-Whitney U test.