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Senolytic vaccination improves normal and pathological age-related phenotypes and increases lifespan in progeroid mice

Abstract

Elimination of senescent cells (senolysis) was recently reported to improve normal and pathological changes associated with aging in mice1,2. However, most senolytic agents inhibit antiapoptotic pathways3, raising the possibility of off-target effects in normal tissues. Identification of alternative senolytic approaches is therefore warranted. Here we identify glycoprotein nonmetastatic melanoma protein B (GPNMB) as a molecular target for senolytic therapy. Analysis of transcriptome data from senescent vascular endothelial cells revealed that GPNMB was a molecule with a transmembrane domain that was enriched in senescent cells (seno-antigen). GPNMB expression was upregulated in vascular endothelial cells and/or leukocytes of patients and mice with atherosclerosis. Genetic ablation of Gpnmb-positive cells attenuated senescence in adipose tissue and improved systemic metabolic abnormalities in mice fed a high-fat diet, and reduced atherosclerotic burden in apolipoprotein E knockout mice on a high-fat diet. We then immunized mice against Gpnmb and found a reduction in Gpnmb-positive cells. Senolytic vaccination also improved normal and pathological phenotypes associated with aging, and extended the male lifespan of progeroid mice. Our results suggest that vaccination targeting seno-antigens could be a potential strategy for new senolytic therapies.

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Fig. 1: GPNMB is a potential candidate of seno-antigen.
Fig. 2: Elimination of Gpnmb-positive cells attenuates metabolic abnormalities and atherosclerosis in mice fed a HFD.
Fig. 3: Gpnmb vaccination improves HFD-induced metabolic abnormalities.
Fig. 4: Gpnmb vaccination decreases tissue senescence and alleviates normal and pathological age-related phenotypes.

Data availability

All data are available from the authors upon reasonable request. Additional materials, including the source data, are available online. RNAseq data and gene expression data obtained in these studies were deposited in the Gene Expression Omnibus database (GSE155680 for HUVECs and GSE155596 for ApoE KO mice).

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Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research (A) (20H00533) from MEXT, AMED under grant number JP20ek0210114, and AMED-CREST under grant number JP20gm1110012, and Moonshot Research and Development Program (21zf0127003s0201), MEXT Supported Program for the Strategic Research Foundation at Private Universities Japan, Private University Research Branding Project, and Leading Initiative for Excellent Young Researchers, and grants from the Takeda Medical Research Foundation, the Vehicle Racing Commemorative Foundation, Ono Medical Research Foundation and the Suzuken Memorial Foundation (to T.M.). Support was also provided by a Grant-in-Aid for Scientific Research (C) from MEXT (18K08063) and a grant from the Suzuken Memorial Foundation, the SENSHIN Medical Research Foundation and the MSD Life Science Foundation (to M.S.). All funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

T.M. contributed to establishment of the research concept and research design, wrote the manuscript and supervised all experiments. M. Suda wrote the manuscript, contributed to designing the experiments and performed most of the experiments. I.S. contributed to designing the experiments, as well as the in vitro and in vivo studies. G.K. contributed to the in vitro and in vivo studies. Yohko Yoshida contributed to analyses of the genetic mouse models. Y.H. and R.I. assisted with the in vivo studies. A.K. and J.W. contributed to analysis of Gpnmb KO mice. M. Seki, Y.S. and A.I. contributed to epigenetic and transcriptome analyses. H.N. and R. Morishita contributed to experiments using the peptide vaccine. R. Mikawa and M. Sugimoto contributed to analysis of Gpnmb-DTR-luciferase mice and p19ARF-DTR-luciferase mice. A.N. and M.T. contributed to histological examination. S.O. contributed to bioinformatic analysis. K.O. contributed to analysis of human samples. Yutaka Yoshida, N.M. and M.N.M. contributed to analysis of lysosomes.

Corresponding author

Correspondence to Tohru Minamino.

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The authors declare no competing interests.

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Nature Aging thanks Jeffrey Miller and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expression of Gpnmb in various tissues.

a, qPCR analysis of relative Gpnmb expression in the aorta or lung endothelial cells (EC) of mice fed NC or an HFD (aorta, n = 8 for NC and n = 10 for HFD; EC, n = 6 for NC and n = 4 for HFD). b, qPCR analysis of relative Gpnmb expression in various tissues of young mice (12 weeks old) and aged mice (109–110 weeks old) (heart, n = 10 for young mice and n = 11 for aged mice; aorta, n = 4 for young mice and n = 7 for aged mice; lung, n = 10 for young mice and n = 11 for aged mice; liver, n = 5 for young mice and n = 10 for aged mice; kidney, n = 9 for young mice and n = 11 for aged mice; gWAT, n = 7 for young mice and n = 10 for aged mice; brown adipose tissue (BAT) n = 10 for both mice; skeletal muscle (SM), n = 10 for young mice and n = 9 for aged mice; spleen, n = 10 for young mice and n = 11 for aged mice; bone marrow (BM), n = 10 for young mice and n = 11 for aged mice). Gpnmb expression in the hearts of young mice was set as 1. c, Western blot analysis of Gpnmb in visceral adipose tissue of mice fed NC or the HFD. The similar results were obtained from three independent experiments. d, Clinical characteristics of patients with or without atherosclerotic diseases (AD). The data were analyzed by the 2-tailed Student’s t-test (a and b). *P < 0.05, **P < 0.01, NS (not significant). The data are shown as box and whisker plots that display the range of the data (whiskers), the 25th and 75th percentiles (box), and the median (solid line) (a and b).

Source data

Extended Data Fig. 2 Gpnmb vaccination improves HFD-induced metabolic abnormalities.

a, Serum titer of anti-Gpnmb antibody (n = 5, 5, 4 for Cont, A, and B). b, Western blot analysis of Gpnmb expression in senescent cells of the stromal vascular fraction (SVF) from wild-type mice (WT) and Gpnmb knockout (KO) mice. The similar results were obtained from three independent experiments. c, ADCC activity of purified IgG antibody from mice immunized with Gpnmb vaccine (Gpnmb vac) or control vaccine (Cont vac) (n = 3 each). d, Serum titer of anti-Gpnmb antibody in mice administered Gpnmb vaccine plus a neutralizing antibody (n = 8, 8, 6, and 6 for Cont vac, Gpnmb vac+Cont, Gpnmb vac+NK, and Gpnmb vac+CD8, respectively). e, FACS analysis for SPiDER-beta-gal and Gpnmb expression in the SVF of HFD-fed mice. f, The number of SPiDER-beta-gal-positive cells among Gpnmbhigh cells in the SVF (n = 5). g, Cellular populations expressing Gpnmb in the SVF from visceral adipose tissue (n = 5). h, The number of Gpnmblow SPiDER-beta-gallow cells in the SVF (Cont vac, n = 5; Gpnmb vac, n = 9). i, FACS analyses of the SVF from HFD-fed mice (Cont vac, n = 5; Gpnmb vac, n = 9). The data were analyzed by the 2-tailed Student’s t-test (h and i) or repeated measures analysis followed by Tukey’s multiple comparison test (a, c, and d). *P < 0.05; **P < 0.01; NS, not significant. In Extended Data Fig. 2a, **P < 0.01 Cont vac vs. Gpnmb vac (peptide A). In Extended Data Fig. 2d, ##P = 0.026 Cont vac vs. Gpnmb + NK, $P = 0.005 Cont vac vs. Gpnmb vac + CD8, **P = 0.004 Cont vac vs. Gpnmb vac + Cont. The data are shown as box and whisker plots that display the range of the data (whiskers), the 25th and 75th percentiles (box), and the median (solid line) (fi), the mean ± SEM with plots of all individual data (a and d), or the mean ± SD with plots of all individual data (c).

Source data

Extended Data Fig. 3 Gpnmb vaccination improves phenotypes of pathological aging.

a, Aortic samples obtained from ApoE KO mice treated with Gpnmb or control vaccine were subjected to RNA sequencing analysis (n = 3, respectively). b, Body weight (BW) of ApoE KO mice (Cont vac, n = 19; Gpnmb vac, n = 20). c, Cell populations expressing Gpnmb in the aorta from ApoE KO mice (n = 5). Outlier values were detected by boxplot analysis and was excluded from statistical assessment as indicated in Source data. d, FACS analyses of the aorta of ApoE KO HFD-fed mice treated with Gpnmb vaccine (Gpnmb vac) or control vaccine (Cont vac) (n = 5 for Cont vac and n = 4 for Gpnmb vac). e, FACS analysis with SPiDER-beta-gal of the aorta from ApoE KO mice treated with Gpnmb vaccine (Gpnmb vac) or control vaccine (Cont vac) (n = 5 for Cont vac and n = 4 for Gpnmb vac). f, The number of Gpnmblow SPiDER-beta-gallow cells in the aorta of ApoE KO HFD-fed mice (Cont vac, n = 5; Gpnmb vac, n = 4). g, Lifespan of Zmpste24 KO mice treated with Gpnmb vaccine (Gpnmb vac) or control vaccine (Cont vac) at 10 weeks of age (Cont vac, n = 19 for male mice and n = 18 for female mice; Gpnmb vac, n = 19 for male mice and n = 16 for female mice). The data were analyzed by the 2-tailed Student’s t-test (b, d, e, f, and g). *P < 0.05; **P < 0.01; NS, not significant. The data are shown as box and whisker plots that display the range of the data (whiskers), the 25th and 75th percentiles (box), and the median (solid line) (b–g).

Source data

Extended Data Fig. 4 Treatment with senolytics and dietary metabolic abnormalities.

a, Experimental protocol. b, Body weight (BW) and food intake of mice treated with vehicle (Cont), dasatinib and quercetin (D + Q), Navitoclax (Nav), or Gpnmb vaccine (Gpnmb vac) (body weight n = 8, 8, 7, 8, food intake 4, 4, 4, 5). c, Energy expenditure (n = 7, 8, 8, 4). d, Luciferase activity. e, Relative luciferase activity (n = 3, 5, 5, and 3). f, Serum titer of anti-Gpnmb antibody. (n = 5, 5 for 0, 8, 12, 16 weeks, n = 3, 3 for 20 weeks). g, Assay of SA-beta-gal (n = 11, 10, 12 and 6 for 16 weeks, and n = 8, 8, 7 and 10 for 24 weeks). h, Relative expression of Cdkn2a (n = 4, 5, 5, and 3 for 16 weeks and n = 7, 8, 7, and 7 for 24 weeks). i, Glucose tolerance test (GTT, left) and insulin tolerance test (ITT, right) (n = 6, 8, 7, and 7). Changes from basal glucose levels are shown in the ITT graph. j, White blood cell and platelet counts, and bleeding time (n = 3, 4, 4, and 4 for white cell counts, n = 3, 4, 4, and 4 for platelet counts, and n = 5, 3, 3, and 3 for bleeding time). The data were analyzed by repeated measures analysis followed by one-way ANOVA followed by Tukey’s multiple comparison test (b, c, g, and j), by the 2-tailed Student’s t-test (h), or by univariate analysis of variance or repeated measures analysis followed by Tukey’s multiple comparison test (f and i). *P < 0.05, **P < 0.01, NS (not significant). In Extended Data Fig. 4i, *P = 0.024, **P < 0.001 Cont vs. Gpnmb vac, $P = 0.018 Cont vs. D + Q, ##P = 0.007 Cont vs. Nav. The data are shown as box and whisker plots that display the range of the data (whiskers), the 25th and 75th percentiles (box), and the median (solid line) (b, c, g, h, and j) or the mean ± SEM with plots of all individual data (h and j).

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Suda, M., Shimizu, I., Katsuumi, G. et al. Senolytic vaccination improves normal and pathological age-related phenotypes and increases lifespan in progeroid mice. Nat Aging 1, 1117–1126 (2021). https://doi.org/10.1038/s43587-021-00151-2

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