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Reversal of prolonged obesity-associated cerebrovascular dysfunction by inhibiting microglial Tak1

Nature Neuroscience volume 23, pages 832–841 (2020)Cite this article

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Abstract

Prolonged obesity is associated with cerebrovascular dysfunction; however, the underlying mechanisms remain largely unclear. In the present study, using a prolonged obesity mouse model that suffers from basilar artery (BA) abnormalities, we find that microglial transforming growth factor β-activated kinase 1 (Tak1) is over-activated in the brainstem. Both pharmacological inhibition primarily in the brainstem and genetic microglia-selective deletion of Tak1 ameliorated BA vascular dysfunction. Conversely, microglia-specific activation of Tak1 in the brainstem was sufficient to cause an impairment in BA function in chow-fed mice. Mechanistically, Tak1 activation leads to increased interleukin-18 (IL-18) production, whereas blockade of IL-18 receptor in the brain helped protect against cerebrovascular dysfunction despite prolonged obesity. Microglia-selective deletion of Tak1 also protects against ischemic stroke in prolonged obesity. Taken together, these findings provide evidence that microglial Tak1 in the brain, and particularly the brainstem, contributes to the pathogenesis of obesity-associated cerebrovascular dysfunction.

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Fig. 1: Vascular dysfunction of the BA and activation of Tak1 in the brainstem microglia of DIO mice.
Fig. 2: Pharmacological blockade of Tak1 improves vascular dysfunction of the BA in prolonged DIO mice.
Fig. 3: Microglia-selective deletion of Tak1 ameliorates vascular dysfunction of the BA in prolonged DIO mice.
Fig. 4: Tak1 regulates the production of IL-18 in brainstem microglia.
Fig. 5: Blockade of IL-18Rα ameliorates vascular dysfunction in prolonged DIO mice.
Fig. 6: Microglia-selective deletion of Tak1 improves the outcome of ischemic stroke in prolonged DIO mice.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank M. Li (Washington University in St. Louis) for sharing the LV-GFAP-GFP plasmid. The present study was supported by the National Natural Science Foundation of China (grant no. 91539125, 81573146), the Junior Thousand Talents Program of China and the Huazhong University of Science and Technology startup fund (all to G.Z.). J.L. was supported by the National Natural Science Foundation of China (grant no. 81570774) and the National Key Research and Development Program (grant no. 2018YFC1003504).

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Authors and Affiliations

  1. Key Laboratory of Environmental Health, Ministry of Education, Department of Toxicology, School of Public Health, Tongji Medical College, Wuhan, China

    Qing Shen, Zhuo Chen, Faming Zhao, Susu Pan, Tingting Zhang, Xueer Cheng, Shanshan Zhang & Guo Zhang

  2. Institute for Brain Research, Wuhan, China

    Qing Shen, Zhuo Chen, Faming Zhao, Susu Pan, Tingting Zhang, Xueer Cheng, Shanshan Zhang & Guo Zhang

  3. Collaborative Innovation Center for Brain Science, Huazhong University of Science and Technology, Wuhan, China

    Qing Shen, Zhuo Chen, Faming Zhao, Susu Pan, Tingting Zhang, Xueer Cheng, Shanshan Zhang & Guo Zhang

  4. State Key Laboratory of Reproductive Medicine, Nanjing, China

    Lei Zhang, Junxia Qi & Juxue Li

  5. Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing, China

    Lei Zhang, Junxia Qi & Juxue Li

  6. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, USA

    Dongsheng Cai

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Contributions

Q.S. designed the study, performed the experiments, analyzed the data and drafted the manuscript. Z.C., F.Z., S.P., T.Z., X.C., L.Z., S.Z. and J.Q. performed the experiments and analyzed the data. J.L. designed the experiments and analyzed the data. D.C. co-designed the study and co-wrote the paper. G.Z. conceived the study, designed the experiments, analyzed the data and co-wrote the paper. All authors commented on the manuscript.

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Correspondence to Dongsheng Cai or Guo Zhang.

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Extended data

Extended Data Fig. 1 Characterization of the basilar artery in DIO mice.

a-c, Body weight (a), fat mass (b) and lean mass (c) of C57 BL/6 mice fed a normal chow or a high-fat diet (HFD) for 32 weeks. *P = 0.000001, t(14) = 8.52 (a) or P = 0.00001, t(14) = 6.75 (b). n = 8 mice per group. d, Plasma total cholesterol (TC) levels. *P = 0.002, t(14) = 3.91. n = 8 mice per group. e, The internal perimeter of basilar artery. n = 5 mice per group. f, Representative images of Verhoeff’s Van Gieson elastic (EVG) staining of basilar artery. Elastic fiber stains blue-black. Scale bars, 20 μm. n = 7 (Chow) or 6 (HFD) mice. g, The relative level of elastin in basilar artery. au, arbitrary unit. n = 7 (Chow) or 6 (HFD) mice. h, Male C57 BL/6 mice (6 weeks old) were fed a chow or HFD for 12 weeks. Immunofluorescent staining for α-smooth muscle actin (α-SMA, green), a protein marker for smooth muscle cells, was performed. Representative images show the immunostaining of basilar artery. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. n = 3 mice per group. i, Relative content of α-SMA in the wall of basilar artery. au, arbitrary unit. n = 3 mice per group. j, Immunofluorescent staining for Vimentin (green), a protein marker for fibroblasts, was performed. Representative images show the immunostaining of basilar artery. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. n = 3 mice per group. k, Relative content of Vimentin in the wall of basilar artery. au, arbitrary unit. n = 3 mice per group. l, The dose-response curves to sodium nitroprusside (SNP). n = 7 (Chow) or 8 (HFD) mice. Data are presented as mean ± SEM. Two-tailed Student’s t-test was used for statistical analysis (a, b, d).

Extended Data Fig. 2 Expression and activity of Tak1 in the neurons of brainstem.

a, Double immunofluorescence staining for Tak1 (red) and Hu C/D (a neuronal marker, green). The region of brainstem close to basilar artery is shown. Arrows indicate double positive cells. Scale bars, 20 µm. n = 4 mice per group. b, Male C57 BL/6 mice (6 weeks old) were fed a chow or a HFD for 12 weeks, and then double immunofluorescence staining for pTak1 (red) and Hu C/D (green) was performed. The region of brainstem adjacent to the basilar artery is shown. Scale bars, 20 µm. n = 4 mice per group. c, Density of neuron in the brainstem. n = 4 mice per group. d, Percentage of neurons that were pTak1+ in the brainstem. n = 4 mice per group. Data are presented as mean ± SEM.

Extended Data Fig. 3 Vascular dysfunction of basilar artery in chow-fed ob/ob mice.

a, Body weight of male wt/wt and ob/ob mice (12 weeks old) fed a chow diet. ***P = 0.00000001, t(8) = 23.62. n = 5 mice per group. b, Representative images of H&E, Masson’s trichrome and EVG stainings of basilar artery. Collagen stains blue in Masson’s trichrome staining. Elastic fiber stains blue-black in EVG staining. Scale bars, 20 μm. For H&E or EVG staining, n = 4 (wt/wt) or 5 (ob/ob) mice. For Masson’s trichrome staining, n = 4 mice per group. c, The wall thickness of basilar artery. *P = 0.00001, t(7) = 11.13. n = 4 (wt/wt) or 5 (ob/ob) mice. d, Percentage of collagen (blue) area over wall area in the basilar artery. *P = 0.001, t(6) = 6.01. n = 4 mice per group. e, The relative elastin level in basilar artery. au, arbitrary unit. n = 4 (wt/wt) or 5 (ob/ob) mice. f, Immunofluorescent staining for p-eNOS (green) in the basilar artery. Scale bars, 50 μm. n = 4 mice per group. g, Relative p-eNOS level in the basilar artery. au, arbitrary unit. *P = 0.0003, t(6) = 7.66. n = 4 mice per group. h, The basilar artery of ob/ob mice is less sensitive to Ach in wire myography. F(1, 40) = 26.8 for column factor. F(4, 40) = 8.92 for row factor. *P = 0.020, logM (Ach) = -6; P = 0.023, logM (Ach) = -5 or P = 0.012, logM (Ach) = -4. n = 5 mice per group. i, Double immunofluorescence staining for pTak1 (red) and Iba1 (green) in the brainstem of wt/wt and ob/ob mice. Scale bars, 20 µm. n = 4 mice per group. (j) Percentage of pTak1+ microglia in the brainstem. *P = 0.0002, t(6) = 8.35. n = 4 mice per group. Data are presented as mean ± SEM. Two-tailed Student’s t-test (a, c, d, g, j) and two-way ANOVA with Bonferroni’s post hoc test (h) were used for statistical analysis.

Extended Data Fig. 4 Expression of Tak1 in the BA and perivascular macrophages in Cx3cr1CreER, Tak1Loxp/Loxp mice.

a, Male C57 BL/6 mice (12 weeks old) were perfused and then the brains were sectioned. Double immunofluorescence staining for Tak1 (red) and CD31, α-SMA or Vimentin (green) of basilar artery was performed. Cell nuclei were counterstained with DAPI (blue). Scale bars, 10 µm. n = 3 mice per group. b, Adult male Cx3cr1CreER, Tak1Loxp/Loxp and Cx3Cr1CreER, Tak1Loxp/Loxp (KO) mice were anesthetized and perfused with 4% PFA. Double immunofluorescence staining for Tak1 (red) and CD31 (green) of basilar artery was performed. Cell nuclei were counterstained with DAPI (blue). Scale bars, 10 µm. n = 3 mice per group. c, Double immunofluorescence staining for Tak1 (red) and CD11b (green, a macrophage marker). Arrows indicate double positive cells. Cell nuclei were counterstained with DAPI (blue). Scale bars, 10 µm. n = 3 mice per group.

Extended Data Fig. 5 Effect of microglial Tak1 on energy balance, and the internal perimeter or elastin content in basilar artery.

a, Male Cx3cr1CreER, Tak1Loxp/Loxp and Cx3cr1CreER, Tak1Loxp/Loxp (KO) mice were fed a chow or high-fat diet (HFD) starting at 6 weeks of age. Body weight was then monitored. F(5, 196) = 31.6 for column factor. F(6, 196) = 27.7 for row factor. *P < 0.05 (HFD versus Chow). **P < 0.01 (HFD versus Chow). n = 5 (Chow), 6 (Cx3cr1CreER, HFD), 7 (Tak1Loxp/Loxp, HFD) or 6 (KO, HFD) mice per group. b,c, Fat mass (b) and lean mass (c) immediately before HFD treatment. n = 5 mice per group. d,e, Fat mass (d) and lean mass (e) after 24 weeks of chow or HFD treatment. In d, F(5, 38) = 6.52. *P < 0.05. n = 6 (Chow), 8 (Cx3cr1CreER, HFD), 9 (Tak1Loxp/Loxp, HFD) or 9 (KO, HFD) mice per group. (f) Cumulative food intake. n = 6 (Cx3cr1CreER, Chow), 7 (Tak1Loxp/Loxp, Chow), 6 (KO, Chow), 8 (Cx3cr1CreER, HFD), 10 (Tak1Loxp/Loxp, HFD) or 10 (KO, HFD) mice. g, Internal perimeter of basilar artery. n = 4 (Cx3cr1CreER, Chow), 3 (Tak1Loxp/Loxp, Chow), 4 (KO, Chow), 3 (Cx3cr1CreER, HFD), 3 (Tak1Loxp/Loxp, HFD) or 4 (KO, HFD) mice. h, EVG staining of basilar artery. Blue-black color represents elastin fibers. Scale bars, 20 μm. n = 5 (Cx3cr1CreER, Chow), 6 (Tak1Loxp/Loxp, Chow), 4 (KO, Chow), 6 (Cx3cr1CreER, HFD), 5 (Tak1Loxp/Loxp, HFD) or 5 (KO, HFD) mice. i, Relative elastin level in basilar artery. au, arbitrary unit. n = 5 (Cx3cr1CreER, Chow), 6 (Tak1Loxp/Loxp, Chow), 4 (KO, Chow), 6 (Cx3cr1CreER, HFD), 5 (Tak1Loxp/Loxp, HFD) or 5 (KO, HFD) mice. Data are presented as mean ± SEM. One-way (d) or two-way (a) ANOVA with Bonferroni’s post hoc test.

Extended Data Fig. 6 Effect of Tak1 activation in the microglia of brainstem on energy balance.

a, Schematics of the CD11b-Ctrl-Lenti and CD11b-Tak1CA-Lenti plasmids. HA, Hemagglutinin epitope tag. Tak1CA, constitutively active Tak1. 2A, 2A peptide. EGFP, Enhanced green fluorescent protein. LTR, Long terminal repeat. b, Representative images showing the expression of EGFP in the brainstem region close to basilar artery after the injection of CD11b-Ctrl-Lenti or CD11b-Tak1CA-Lenti (CD11b-Tak1CA-L) viruses. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. n = 3 mice per group. c, Adult male mice were injected CD11b-Ctrl-Lenti viruses into the bottom region of brainstem. A week later, mice were perfused with saline and then the brainstem was dissected out. After tissue digestion, cells were isolated by using the Percoll gradients, blocked by FcX, and then labelled with PE-conjugated anti-mouse Cx3cr1 antibody. A representative flow cytometry plot is shown. n = 6 mice per group. d,e, Male mice (12 weeks old) were injected CD11b-Ctrl-Lenti or CD11b-Tak1CA-Lenti viruses into the brainstem region close to basilar artery. Body weight (d) and cumulative food intake (e) were then assessed. n = 8 (d) or 10 (e) mice per group. Data are presented as mean ± SEM.

Extended Data Fig. 7 Effects of Tak1 activation in the microglia of brainstem on BA morphology and function.

a, H&E and Masson’s trichrome stainings of basilar artery. Ctrl-Lenti, CD11b-Ctrl-Lenti. Tak1CA-Lenti, CD11b-Tak1CA-Lenti. Scale bars, 20 µm. For H&E staining, n = 5 mice per group. For Masson’s trichrome staining, n = 4 (Ctrl-Lenti) or 3 (Tak1CA-Lenti) mice. b, Thickness of basilar arterial wall. Ctrl-L, CD11b-Ctrl-Lenti. Tak1CA-L, CD11b-Tak1CA-Lenti. *P = 0.024, t(8) = 2.77. n = 5 mice per group. c, Percentage of collagen area over vessel wall area. *P = 0.01, t(5) = 4.0. n = 4 (Ctrl-L) or 3 (Tak1CA-L) mice. d, Immunofluorescent staining for p-eNOS (green) in the basilar artery. Scale bars, 50 µm. n = 4 mice per group. e, Relative p-eNOS fluorescence level in the basilar artery. au, arbitrary unit. *P = 0.002, t(6) = 5.14. n = 4 mice per group. f, C57 BL/6 mice (12 weeks old) were injected the indicated lentiviruses into brainstem. Four weeks later, basilar arteries were collected and lysed. The lysates were immunoblotted with p-eNOS and eNOS antibodies. Gapdh was used as a loading control. To prepare each sample, tissues from 8 mice were combined. g, Impairment of Ach-evoked relaxation of basilar artery of CD11b-Tak1CA-Lenti virus-injected mice. Ctrl-Lenti, CD11b-Ctrl-Lenti. F(1, 100) = 51.03 for column factor. F(4, 100) = 88.33 for row factor. *P = 0.002, logM (Ach) = -6; P = 0.001, logM (Ach) = -5; or P = 0.0008, logM (Ach) = -4. n = 11 mice per group. Uncropped western blots can be found in Source Data Extended Data Fig. 7. Data are presented as mean ± SEM. Two-tailed Student’s t-test (b, c, e) and two-way ANOVA with Bonferroni’s post hoc test (g) were used for statistical analysis.

Source data

Extended Data Fig. 8 IL-18 suppresses eNOS activity in RAEC cells.

a, Quantitative RT-PCR analysis of the mRNA levels of cytokines and chemokines in the brainstem of chow- or HFD-fed mice. au, arbitrary unit. n = 6 (IL-1β, HFD or IL-23α, Chow) or 7 (all other groups) mice per group. b, Immunofluorescent staining for iNOS (red) and Iba1 (green). Tissue sections were counterstained with DAPI (blue) to visualize cell nuclei. Brainstem region close to basilar artery is shown. Scale bars, 20 µm. n = 3 mice per group. c, Percentage of iNOS+ microglia in chow- or HFD-fed mice. n = 3 mice per group. d, Rat aortic endothelial cells (RAECs) were incubated with vehicle (Ctrl) or IL-18 (20 ng ml-1) for 4 hrs. Total proteins were prepared and subjected to western blot analysis. β-Actin was used as a loading control. n = 3 cell cultures per group. e, RAECs were transfected with scramble RNA (Ctrl) or siRNA targeting IL-18Rα. The cells were further cultured for 4 days and then harvested for protein preparation. The proteins were subjected to western blot analysis. β-Actin was used as a loading control. n = 3 cell cultures per group. f, Quantification of the immunoblots shown in d and e. For p-eNOS/eNOS, *P = 0.005, t(4) = 5.55, Ctrl versus IL-18; or P = 0.03, t(4) = 3.3, Ctrl versus IL-18Rα siRNA. For IL-18Rα/β-Actin, *P = 0.0002, t(4) = 13.95. n = 3 cell cultures per group. Uncropped western blots can be found in Source Data Extended Data Fig. 8. Data are presented as mean ± SEM. Two-tailed Student’s t-test was used for statistical analysis (f).

Source data

Extended Data Fig. 9 Validation of pTak1 Thr 187 antibody.

a, HEK293T cells were transfected with vector, Tak1 wild type (WT) or Tak1 T187A plasmids for 48 h. Both the Tak1 WT and Tak1 T187A cDNAs were Flag-epitope tagged at the COOH-terminus. Cells were then serum-starved overnight, stimulated with vehicle or TGFβ1 (10 ng ml-1, 15 mins) and fixed with 4% PFA. Double immunofluorescence staining for pTak1 Thr 187 (red) and Flag (green) was performed. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. Data shown are a representative of 3 independent experiments. b, HEK293T cells were transfected with the indicated plasmids. After stimulated with TGFβ1, the cells were lysed. Lysates were immunoblotted with anti-pTak1 Thr 187 (pTak1) and anti-Flag antibodies, respectively. β-Actin was used as a loading control. A representative of 3 independent experiments is shown. c, Quantification of the immunoblots for pTak1. au, arbitrary unit. F(5, 12) = 15.5. *P = 0.0002 (Vector, Vehicle versus Tak1 WT, TGFβ1); P = 0.0003 (Vector, TGFβ1 versus Tak1 WT, TGFβ1); P = 0.0007 (Tak1 WT, Vehicle versus Tak1 WT, TGFβ1); P = 0.0003 (Tak1 WT, TGFβ1 versus Tak1 T187A, Vehicle); or P = 0.0002 (Tak1 WT, TGFβ1 versus Tak1 T187A, TGFβ1). n = 3 independent experiments. Uncropped western blots can be found in Source Data Extended Data Fig. 9. Data are shown as mean ± SEM. One-way ANOVA with Bonferroni’s post hoc test.

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Extended Data Fig. 10 Validation of p-eNOS antibody.

a, RAECs were transfected with scramble RNA or eNOS siRNA for 48 h. Cells were then serum-starved, stimulated with vehicle or VEGF (100 ng ml-1, 30 mins) and lysed. Lysates were immunoblotted with anti-p-eNOS and anti-eNOS antibodies, respectively. β-Actin was used as a loading control. n = 3 cell cultures per group. b,c, Quantification of the western blots. au, arbitrary unit. In b, F(3, 8) = 263.5. *P = 0.0000001 (scramble RNA, Vehicle versus scramble RNA, VEGF); P = 00000008 (scramble RNA, VEGF versus eNOS siRNA, Vehicle); or P = 0.00000007 (scramble RNA, VEGF versus eNOS siRNA, VEGF). In c, F(3, 8) = 87.6. *P = 0.00003 (scramble RNA, Vehicle versus eNOS siRNA, Vehicle); P = 0.00004 (scramble RNA, Vehicle versus eNOS siRNA, VEGF); P = 0.00001 (scramble RNA, VEGF versus eNOS siRNA, Vehicle); or P = 0.00001 (scramble RNA, VEGF versus eNOS siRNA, VEGF). n = 3 cell cultures per group. d, Immunofluorescent staining for p-eNOS (green) in RAEC cells. Scale bars, 20 µm. A representative of 3 independent experiments is shown. e, A schematic representation displaying the impact of microglia Tak1 in obesity-related cerebrovascular dysfunction. Obesity results in microgliosis and Tak1 activation in the microglia of brainstem, which then increases the production and secretion of IL-18. IL-18 acts on endothelial cells to decrease the activity of eNOS and reduces the production of nitric oxide (NO), thereby resulting in endothelial dysfunction. Action of IL-18 on other cell types leads to wall thickening and collagen deposition in basilar artery. Vascular dysfunction worsens the outcome of ischemic stroke in DIO mice. Yellow, microglia; grey, neuron; blue, astrocytes; green, endothelial cells. Uncropped western blots can be found in Source Data Extended Data Fig. 10. Data are presented as mean ± SEM. One-way ANOVA with Bonferroni’s post hoc test is used for statistical analysis.

Source data

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Supplementary Fig. 1 and Supplementary Tables 1 and 2.

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Source Data Fig. 1

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Source Data Fig. 4

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Source Data Extended Data Fig. 7

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Source Data Extended Data Fig. 8

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Source Data Extended Data Fig. 9

Uncropped western blots.

Source Data Extended Data Fig. 10

Uncropped western blots.

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Shen, Q., Chen, Z., Zhao, F. et al. Reversal of prolonged obesity-associated cerebrovascular dysfunction by inhibiting microglial Tak1. Nat Neurosci 23, 832–841 (2020). https://doi.org/10.1038/s41593-020-0642-6

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