Abstract
Life stress can shorten lifespan and increase risk for aging-related diseases, but the biology underlying this phenomenon remains unclear. Here we assessed the effect of chronic stress on cellular senescence—a hallmark of aging. Exposure to restraint stress, a psychological non-social stress model, increased p21Cip1 exclusively in the brains of male, but not female mice, and in a p16Ink4a-independent manner. Conversely, exposure to chronic subordination stress (only males were tested) increased key senescent cell markers in peripheral blood mononuclear cells, adipose tissue and brain, in a p16Ink4a-dependent manner. p16Ink4a-positive cells in the brain of chronic subordination stress-exposed mice were primarily hippocampal and cortical neurons with evidence of DNA damage that could be reduced by p16Ink4a cell clearance. Clearance of p16Ink4a-positive cells was not sufficient to ameliorate the adverse effects of social stress on measured metrics of healthspan. Overall, our findings indicate that social stress induces an organ-specific and p16Ink4a-dependent accumulation of senescent cells, illuminating a fundamental way by which the social environment can contribute to aging.
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Data availability
All data reported in this paper are available via figshare at https://doi.org/10.6084/m9.figshare.26236583.v1 (ref. 90). The sequencing data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE278620. All data are available from the corresponding author upon request.
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Acknowledgements
We dedicate this paper to the memory of Judith (Judy) Campisi, a giant in the field who kindly shared the p16-3MR mice and provided feedback during the execution of the research described in this paper. We also thank P. Rothwell for training and crucial support with the RNAscope experiments and G. Caviola, M. Berg and N. Sabarinathan for their assistance with the in vivo studies. The spatial transcriptomics workflow was carried out with the resources of and substantial assistance from C. Forster at the University of Minnesota Histology Core, G. Barthel at the University of Minnesota Imaging Center (UIC) SCR.020997 and F. Rodriguez at the University of Minnesota Genomics Center. C. Forster is also acknowledged for help provided with the γH2AX staining. The staff of the Research Animal Resources at the University of Minnesota are also acknowledged for their instrumental role in animal care on a daily basis. The project was supported by the MN Partnership for Biotechnology and Molecular Genomics #18.04 (A.B., J.M.v.D. and D.J.B.) and #19.02 (L.J.N.), which purchased the Nanostring GeoMx Digital Spatial Profiler, NIH R61/R33 AG078520 (A.B.), R01 HL151740 (A.B.), T32 AG029796 (C.M.L.), T32 DK083250 (J.P.P.), U54 AG076041 (A.B. and L.J.N.), U54 AG079754 (A.B. and L.J.N.), R01 AG063543 (L.J.N.), R01 HL166843 (J.W.W.), R01 AI165553 (J.W.W.) and Glenn Foundation for Medical Research (D.J.B.).
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The study was conceived by C.E.L. and A.B. C.E.L., J.P.P., S.M., R.P.M., C.W.C., M.J.Y., P.R.S. and M.R. performed experiments under the supervision of A.B., J.W.W. and L.J.N. C.E.L. and M.R. analyzed the data and assembled the figures. D.J.B. and J.M.v.D. shared mouse models and supported the development of the project. C.E.L. and A.B. drafted and edited the manuscript with the input and review by all authors. All authors reviewed the manuscript.
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D.J.B. and J.M.v.D. have a potential financial interest related to this research. J.M.v.D. is a cofounder of Unity Biotechnology, D.J.B. and J.M.v.D. are co-inventors on patents held by Mayo Clinic and patent applications licensed to or filed by Unity Biotechnology, and D.J.B. and J.M.v.D. are Unity shareholders. Research in the Baker laboratory has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo Clinic Conflict of Interest policies. The other authors declare no conflicts of interest.
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Extended data
Extended Data Fig. 1 Flow cytometry gating scheme.
Flow cytometry gating scheme used to identify major immune cell subsets (CD45 + ) in peripheral blood; including neutrophils (Ly6G + CD11b + ), Ly6C+ classical monocytes (Ly6C + CD115 + CD11b + Ly6G-), Ly6C- nonclassical monocytes (Ly6C- CD115 + CD11b + Ly6G-), B cells (CD19 + Ly6G- CD115-), CD4 T cells (CD4+ TCRb+ Ly6G- CD115-), and CD8 T cells (CD4+ TCRb+ Ly6G- CD115-).
Extended Data Fig. 2 Senescence-associated β-galactosidase (SA β-gal).
A-D) Representative gross images of subcutaneous white adipose tissue (scWAT), Liver, Kidney, Hippocampus, and Cortex of control (ctrl) and Chronic Subordination Stress (CSS) mice following senescence-associated β-galactosidase staining. A-C) CSS mice have significantly more SA β-gal positive cells in the scWAT than control mice (p = 0.001). Unpaired, two-sided t test. Ctrl N = 3; CSS N = 4. A-D) No significant difference between control and CSS mice in SA β-gal staining in the liver, kidney, liver, hippocampus or cortex. Unpaired, two-sided t test. Ctrl N = 3; CSS N = 4 * indicates p < 0.05. Scale bars represent 50 μm unless otherwise specified. Histogram bars represent group mean. Error bars represent standard error of the mean (SEM). Scale bars represent 1 mm.
Extended Data Fig. 3 RNAscope representative images in animals exposed to Chronic Subordination Stress (CSS).
A) 10x DAPI stitched images of entire sagittal sections showing boxed regions of interest at various medial-lateral positions in which they appear. B) 60x fields of view captured from regions of high p16/p21 expression (CA3 – hippocampus and somatosensory cortex) and regions with negligible p16/p21 expression (BST – bed nucleus of stria terminalis | PVN – paraventricular nucleus | LC – locus coeruleus | MEA – medial amygdala nucleus) showing DAPI, Rbfox3 (neurons), p21, and p16. Scale bars represent 50μM. Experiment was repeated twice with similar results; second pass was to have a cohort large enough for valid statistical analysis.
Extended Data Fig. 4 Effect of Chronic Subordination Stress (CSS) or Chronic Restraint Stress (CRS) on gene expression.
A-D) Senescence/senescence associated secretory phenotype (SASP)-related gene expression in the CSS exposed mice. E-F) Senescence/SASP-related gene expression in the lung and liver in animals exposed to CRS. Control + vehicle (Ctrl+veh) N = 6; ctrl + ganciclovir (GCV) N = 5; CSS+veh N = 14; CSS + GCV N = 11; CRS+veh N = 9; CRS + GCV N = 5; G) CSS + GCV group had significantly higher expression of IL1B than ctrl (p = 0.046). G-H) Senescence/SASP-related gene expression in the hippocampus and cortex of CRS exposed female mice. Ctrl+veh N = 5; ctrl+GCV N = 4; CRS+veh N = 6; CRS + GCV N = 5. 2-way ANOVA, Tukey post hoc. * indicates p < 0.05. Histogram bars represent group mean. Shaded bars represent GCV treatment groups. Error bars represent standard error of the mean (SEM).
Extended Data Fig. 5 Histological analysis of p16CreERT2;Ai14 mice.
A) Tamoxifen (Tam) is required for tdTomato (TdTom) expression in p16CreERT2;Ai14 mice. Control (Ctrl) Tam- N = 5, ctrl Tam+ N = 5 (p = 0.017). Two-sided, unpaired t-test. Histogram bars represent group mean. Error bars represent standard error. B) Representative images of tdTom+ cells co-stained with NeuN (neurons), and GFAP+ (astrocytes), Iba1+ (microglia), and NG2+ (oligodendrocytes precursors, OPCs) cells which were all negative for TdTom; multi-staining repeated twice with consistent results. Scale bars represent 30 µm. **p < 0.01.
Extended Data Fig. 6 Gene set enrichment analysis (GSEA).
GSEA for the Hernandez-Segura et al.70 gene set (genes represented in Hernandez-Segura’s list = 52) in mouse whole brain (A-C) or hippocampus (B-D). GSEA mRNA expression levels using the SenMayo9 gene set (genes represented in SenMayo’s list = 118) in mouse whole brain (E-F) or hippocampus (G-H). X axis represents: (A-B and E-F) chronic subordination stress (CSS)/control level; (C-D and G-H) CSS high p16 expressing/CSS low p16 expressing. Y axis represents the enrichment score of these mRNAs.
Extended Data Fig. 7 Gene expression analysis.
A-F) Select genes in the NLRP3 inflammasome pathway and G-L) select genes in the RAF:RAS pathway in the hippocampus or cortex of male mice. Control (Ctrl)=4; Chronic Subordination Stress (CSS) = 5. Unpaired, two-sided t test (A: p = 0.0169; B: p < 0.0001). Bars represent group mean and error bars represent standard error of the mean (SEM). * p < 0.05, ****p < 0.0001.
Extended Data Fig. 8 Behavioral and physiological characterization of mice exposed to Chronic Subordination Stress (CSS) or Chronic Restraint Stress (CRS).
A) Diagram of the experimental protocol. B) CSS increases body weight gain irrespective of GCV treatment. Ctrl+veh vs CSS+veh p = 0.0091. ctrl+veh vs CSS + GCV p = 0.0412. ctrl+GCV vs CSS+veh p = 0.0007. ctrl+GCV vs CSS + GCV p = 0.0030. 2-way ANOVA, Tukey post hoc. Ctrl+veh N = 15, Ctrl+GCV N = 7, CSS+veh N = 28, CSS + GCV N = 14. C) CSS increases body weight gain irrespective of GCV treatment. Ctrl+veh vs CSS+veh p = 0.0010. Ctrl+veh vs CSS + GCV p = 0.0178. ctrl+GCV vs CSS+veh p = 0.0036. ctrl+GCV vs CSS + GCV p = 0.0229. 2-way ANOVA, Tukey post hoc. Ctrl+veh N = 15, Ctrl+GCV N = 7, CSS+veh N = 28, CSS + GCV N = 14.D) CSS affects expression of select Hypothalamus Pituitary Adrenocortical (HPA)-axis related genes in the hypothalamus, cortex or hippocampus irrespective of GCV treatment. 1-way ANOVA. CRH= Corticotropin-releasing hormone, GR=glucocorticoids receptor. Ctrl+veh=3-4, CSS+veh=4-5, CSS + GCV = 6. E-F) CRS decreases body weight in male mice irrespective of GCV treatment. Ctrl+veh vs CSS+veh p < 0.0001. ctrl+veh vs CSS + GCV p < 0.0001 ctrl+GCV vs CSS+veh p < 0.0001. ctrl+GCV vs CSS + GCV p < 0.0001. 2-way ANOVA. Tukey post hoc. Ctrl+veh=15, Ctrl+GCV = 7, CRS+veh=15, CRS + GCV = 9. G-H) CRS exerts no effect in female mice. Ganciclovir (GCV) decreases body weight gain in control but not CRS treated mice. Ctrl+veh vs ctrl+GCV p = 0.0246. 2-way ANOVA. Tukey post hoc. Ctrl+veh=5, Ctrl+GCV = 5, CRS+veh=6, CRS + GCV = 5. * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. Shaded bars indicate GCV treatment groups. Histograms represent group mean and error bars represents standard error of the mean (SEM).
Extended Data Fig. 9 Behavioral and physiological characterization of mice exposed to lifelong Chronic Subordination Stress (CSS) up to 16 months of age.
A) Diagram of the experimental protocol. B) while treatment group didn’t affect body weight (age: F(1.66, 94.73) = 391.1, p < 0.0001; group: F(3,63) = 0.24, p = 0.86; age x group: F(12, 228) = 1.12, p = 0.35), CSS causes hyperphagia. C) (age: F(2.74, 150.7) = 11.1, p < 0.0001; group: F(3,63) = 10.72, p < 0.0001; age x group: F(12, 220) = 2.51, p = 0.0042) and increases clinical frailty index (CFI). D) (age: F(2, 110) = 149.6, p < 0.0001; group: F(3,63) = 2.39, p = 0.076; age x group: F(6, 110) = 2.05, p = 0.06) – including loss of fur color). E) (age: F(1.71, 94.03) = 46.76, p < 0.0001; group: F(3,63) = 6.14, p = 0.001; age x group: F(6, 110) = 4.17, p = 0.0008) irrespective of Ganciclovir (GCV) treatment. Ctrl+veh=14, Ctrl+GCV = 15, CSS+veh=14-19 (range due to animal death during the experiment), CSS + GCV = 9-19 (range due to animal death during the experiment). * indicates p < 0.05, ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. 2-way ANOVA, Tukey post hoc. Data are represented as group means and error bars represent standard error of the mean (SEM).
Extended Data Fig. 10 RNAscope representative images validating mRFP expression, and colocalization with p16 and p21.
Representative RNAscope Fluorescent In Situ Hybridization images showing probes for mRFP, p21, and p16 expression in Hippocampus-CA3 (A) and somatosensory cortex (B) in vehicle (Veh) treated control animals (Ctrl), veh treated chronic Subordination Stress (CSS) animals, and Ganciclovir (GCV) treated CSS animals. The results here were consistent across two repeated experiments, with the second pass having a more substantial n per group. The mRFP signal that is observed is not due to autofluorescence (any autofluorescent signal gets quenched due to the protease reagents in FISH protocol), but rather RNAscope mRFP probes detecting mRFP transcripts and that signal then being amplified. The inserts in the p16 panels show the merge of the 3 signals on one representative nucleus. Scale bars represent 30 µm.
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Lyons, C.E., Pallais, J.P., McGonigle, S. et al. Chronic social stress induces p16-mediated senescent cell accumulation in mice. Nat Aging 5, 48–64 (2025). https://doi.org/10.1038/s43587-024-00743-8
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DOI: https://doi.org/10.1038/s43587-024-00743-8
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