Naturally occurring p16Ink4a-positive cells shorten healthy lifespan

Journal name:
Nature
Volume:
530,
Pages:
184–189
Date published:
DOI:
doi:10.1038/nature16932
Received
Accepted
Published online

Abstract

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16Ink4a (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16Ink4a-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16Ink4a-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16Ink4a-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

At a glance

Figures

  1. Clearance of senescent fat progenitor cells attenuates age-related lipodystrophy.
    Figure 1: Clearance of senescent fat progenitor cells attenuates age-related lipodystrophy.

    a, FACS profiles of single-cell suspensions from iWAT of the indicated 12- and 24-month-old (m) mice. b, GFP+ and GFP cell populations from iWAT of 12-month-old ATTAC mice (see a for sorting brackets) analysed by qRT–PCR (n = 6 mice). p21 is also known as Cdkn1a; Pai1 is also known as Serpine1. c, SA-β-Gal activity in GFP+ and GFP iWAT cells (n = 3 mice). d, GFP+ cells in the indicated iWAT (IAT) cell populations (‘rest’ represents the iWAT vascular stromal fraction minus leukocytes, endothelial cells and progenitors). ei, Fat-related analyses of C57BL/6 ATTAC mice before treatment (12 months) or after 6 months of treatment with vehicle (18 months −AP) or AP (18 months +AP). e, SA-β-Gal activity in iWAT and epididiymal WAT (eWAT). f, Electron micrograph showing perivascular X-Gal-positive cells from an 18-month-old vehicle-treated C57BL/6 ATTAC male. A, adipocyte; C, capillary. Arrows mark endothelial cells. g, Quantification of iWAT cells containing X-Gal crystals (n = 4 mice per treatment). h, Fat mass measurements. i, iWAT and eWAT depot weights. j, Mean adipocyte diameters in iWAT. k, Expression of adipogenesis markers in iWAT (n = 4 mice per group). Scale bars, 10 μm (c), 0.5 cm (e), 2 μm (f) and 200 nm (f, inset). Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (one-sample t-tests using a theoretical mean of 1 (b), and unpaired two-tailed t-tests (c, d, gk)).

  2. Senescent cell clearance extends lifespan.
    Figure 2: Senescent cell clearance extends lifespan.

    a, Study design for clearance of senescent cells in mixed and C57BL/6 mouse cohorts. Healthspan analysis was done at 18 months, an age at which relatively few mice in vehicle (Veh.)- or AP-treated groups have died, and bias owing to selection for long-lived animals is unlikely. b, c, Survival curves for vehicle-treated (−AP) and AP-treated (+AP) mixed (b) and C57BL/6 (C57; c) mice. Median survival (in days, d) and percentage increase in median survival are indicated. We note that median lifespans of our vehicle-treated cohorts are similar to those of wild-type mice administered AP (c). *P < 0.05; **P < 0.01; ***P < 0.001 (log-rank tests).

  3. Clearance of senescent cells prolongs healthspan.
    Figure 3: Clearance of senescent cells prolongs healthspan.

    a, Survival curves of ATTAC mice dying of cancer (mice that had an overt tumour at time of death; mice with lymphomas, sarcomas and carcinomas were included, mice without tumours were censored). Median survival and percentage increase are indicated. b, Representative images of aged mice with and without senescent cell clearance. c, Spontaneous movement and exploratory behaviour of ATTAC mice analysed by the open field test. Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (log-rank test (a) and unpaired two-tailed t-test (c)).

  4. Senescent cells cause glomerulosclerosis, kidney dysfunction and renal RAAS hyperactivity.
    Figure 4: Senescent cells cause glomerulosclerosis, kidney dysfunction and renal RAAS hyperactivity.

    a, Images of sclerotic (left) and normal (right) glomeruli from the indicated mice. H&E, haematoxylin and eosin; PAS, periodic acid–Schiff. b, Quantification of sclerotic glomeruli. c, Measurements of blood urea nitrogen levels. d, SA-β-Gal-stained kidney sections. e, Electron micrograph showing a X-Gal crystal-containing renal epithelial cell with brush border membrane (arrowheads). Insets show X-Gal crystal close-ups. f, Percentage of cells with X-Gal crystals in renal sections (n = 5 TEM grids for each treatment group). g, Renal expression of Agtr1a analysed by qRT–PCR (n = 4 mice per group). h, Western blot of kidney lysates probed for Agtr1a (n = 3 mice per treatment group). Ponceau S staining served as loading control. i, Immunostaining of kidney sections for Agtr1a. Yellow circles denotes glomeruli. Scale bars, 50 μm (a and i, bottom), 250 μm (d), 5 μm (e), 200 nm (e, insets) and 100 μm (i, top). Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests). For gel source data, see Supplementary Fig. 1. All mice in di were C57BL/6 ATTAC.

  5. Senescent cells promote age-related cardiomyocyte hypertrophy and loss of cardiac stress tolerance.
    Figure 5: Senescent cells promote age-related cardiomyocyte hypertrophy and loss of cardiac stress tolerance.

    a, SA-β-Gal-stained hearts. Insets show aortic roots (ar) from a transverse plane (arrow marks the aortic root wall) or close-ups of the ventricular (v) and arterial (a) boxed areas. b, Electron micrographs of X-Gal-positive cells in the pericardium (red asterisk marks cilia). Insets show close-ups of X-Gal crystals. c, Quantification of cells with X-Gal crystals in the visceral pericardium (n = 4 mice per treatment). d, Measurements of left ventricle wall thickness (n = 4 mice per group). e, Representative cardiomyocyte cross-sectional images (n = 4 mice per group). f, Quantification of e. g, Analysis of Sur2a expression in hearts by qRT–PCR (n = 4 mice per group). h, Cardiac stress resistance determined by measuring the time to death after injection of a lethal dose of isoproterenol. i, Change in left ventricular (LV) mass in response to sublethal doses of isoproterenol (10 mg kg−1) after ten doses administered over 5 days. Scale bars, 1 mm (a), 2 μm (b) and 200 nm (b, inset). Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests). All mice, except for h, were C57BL/6 ATTAC.

  6. ATTAC transgene expression tracks with expression of senescence markers in iWAT and induces apoptosis of senescent cells after AP administration.
    Extended Data Fig. 1: ATTAC transgene expression tracks with expression of senescence markers in iWAT and induces apoptosis of senescent cells after AP administration.

    a, Comparative analysis of SA-β-Gal activity in intact iWAT. Scale bar, 0.5 cm. b, Analysis of endogenous Ink4a and ATTAC transcript SA-β-Gal activity in iWAT by qRT–PCR. H/H denotes BubR1H/H mice (n = 4 mice per group). c, FACS-based quantification of iWAT progenitor cell numbers in 18-month-old ATTAC mice treated with vehicle or AP. ASC, adipocyte stem cells; PAC, preadipocytes. d, Expression of the ATTAC transgene and senescence markers in iWAT as determined by qRT–PCR (n = 4 mice per group). Asterisks above individual bars denote significant changes to 2-month-old mice; asterisks above brackets denote significant differences between 18-month-old vehicle and AP-treated mice. e, Perirenal, mesenteric, subscapular and brown adipose tissue depot weights. SSAT, subscapular adipose tissue. f, SA-β-Gal activity in iWAT from 2-month-old ATTAC mice treated with vehicle or AP beginning at weaning age. g, p16Ink4a levels in iWAT from the mice described in f. Actin was used a loading control. h, Expression of ATTAC and senescence marker mRNA in the mice described in f (n = 3 mice per group). ik, Early passage non-senescent ATTAC MEFs express p16Ink4a but are not susceptible to FKBP–Casp8-mediated elimination when cultured in the presence of AP. i, Levels of p16Ink4a in passage 3 ATTAC MEFs, with and without AP treatment. j, Growth curves of passage 3 ATTAC MEFs (n = 4 independently generated MEF lines per group), with or without AP treatment. k, Expression of ATTAC and senescence marker mRNA in passage 3 ATTAC MEFs (n = 3 independently generated MEF lines per group), with or without AP treatment. Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests). For gel source data, see Supplementary Fig. 1.

  7. ATTAC lacks promoter elements required for expression in replication-competent cells or aged lymphocytes expressing high levels of endogenous p16Ink4a.
    Extended Data Fig. 2: ATTAC lacks promoter elements required for expression in replication-competent cells or aged lymphocytes expressing high levels of endogenous p16Ink4a.

    ac, SV40 large-T-antigen-immortalized ATTAC MEFs robustly express endogenous p16Ink4a (owing to SV40 large-T-antigen-mediated inactivation of Rb) but fail to engage the ATTAC transgene and are not subject to FKBP–Casp8-mediated elimination. a, p16Ink4a protein levels in passage 4 (P4) primary MEFs and MEFs immortalized with SV40 large T antigen. Actin was used as a loading control. b, p16Ink4a protein levels in immortalized MEFs treated with vehicle or two concentrations of AP. Actin was used as a loading control. c, Expression of ATTAC and senescence marker transcripts in passage 4 primary MEFs, vehicle-treated immortalized MEFs, and AP-treated immortalized MEFs (n = 3 independently generated MEF lines per group). d, Schematic representation of the endogenous Ink4a locus and the various Ink4a promoter regions driving ATTAC, 3MR and firefly luciferase (FLUC). ATTAC and p16-3MR mice have 2.6 kb and ~50 kb Ink4a promoter fragments driving transgene activity, respectively. p16-FLUC has firefly luciferase knocked into the endogenous Ink4a locus, which keeps the entire promoter region intact but ablates p16Ink4a protein expression. e, p16Ink4a protein levels in early passage primary and SV40 large-T-antigen-immortalized p16-3MR MEFs. f, Expression of senescence marker mRNA in early and late passage primary MEFs and SV40 large-T-antigen-immortalized p16-3MR MEFs (n = 1 independently generated MEF line per group performed in triplicate). g, Expression of senescence marker mRNA in early and late passage primary MEFs and SV40 large-T-antigen-immortalized p16-FLUC MEFs (n = 1 independently generated MEF line per group performed in triplicate). h, Expression of ATTAC and senescence markers in CD3+ T cells from 12- and 18-month-old ATTAC mice (n = 5 mice per group). Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests). We note that all values in f and g have P < 0.05 compared to passage 4 MEFs, with the exception of the one marked NS for not significant. For gel source data, see Supplementary Fig. 1.

  8. ATTAC-mediated clearance of senescent cells is partial and tissue selective and attenuates expression of inflammation markers.
    Extended Data Fig. 3: ATTAC-mediated clearance of senescent cells is partial and tissue selective and attenuates expression of inflammation markers.

    a, Expression of the ATTAC transgene and a senescence marker panel, as determined by RT–PCR, in gastrocnemius, eye, kidney, heart (atria), spleen, lung, liver and colon (n = 4 females per group). b, Expression of Il6, Il1a and Tnfa as determined by qRT–PCR in mouse iWAT, kidney and skeletal muscle at different ages (n = 4 females per group). Il6 values are as indicated in Extended Data Fig. 1d (iWAT) and in a (kidney and gastrocnemius). Expression levels of inflammation markers in unmanipulated 18-month-old C57BL/6 females suggests that repeated vehicle injections were not a source of tissue inflammation. Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests). Asterisks above individual bars in a denote significant changes to 2-month-old mice; asterisks above brackets denote significant differences between 18-month-old vehicle and AP-treated mice.

  9. Comparison of lifespans under different diets and housing facilities.
    Extended Data Fig. 4: Comparison of lifespans under different diets and housing facilities.

    a, Survival curves of unmanipulated wild-type C57BL/6-129Sv mice fed a 5% versus 9% fat diet. Median lifespan (in days) are indicated. b, Survival curves of unmanipulated wild-type C57BL/6-129Sv mice fed a 9% fat diet plotted against those of vehicle-treated (−AP) and AP-treated (+AP) C57BL/6-129Sv-FVB ATTAC mice from Fig. 2b. These data suggest that the lifespans of vehicle-injected C57BL/6-129Sv-FVB control mice were quite normal for the diet that they were on, and unlikely to be negatively affected by repeated intraperitoneal injections. *P < 0.01; **P < 0.001; ***P < 0.001 (log-rank tests). c, d, Median survival data of unmanipulated C57BL/6 male (c) and female (d) mice from various laboratories for comparsion to the results obtained from our facility.

  10. Senescent cell clearance delays tumour and cataract formation.
    Extended Data Fig. 5: Senescent cell clearance delays tumour and cataract formation.

    a, b, Survival curves of mixed (a) and C57BL/6 (b) ATTAC mice dying of cancer (mice that had an overt tumour at time of death; only mice with lymphomas, sarcomas and carcinomas were included). Median survival (in days) and percentage increase are indicated. c, d, Incidence of macroscopically detectable neoplasms (lymphomas, sarcomas and carcinomas) at time of death in mixed (c) and C57BL/6 (d) ATTAC mice from survival cohorts. e, Survival curves of C57BL/6-129Sv-FVB mice dying without cancer (mice that had an overt tumour at time of death, including lymphoma, sarcoma and/or carcinoma, were excluded). Median survival (in days) and percentage increase are indicated. f, g, Cataract incidence for mixed (f) and C57BL/6 (g) ATTAC mouse cohorts. *P < 0.05; **P < 0.01; ***P < 0.001 (log-rank tests).

  11. Senescent cell clearance does not affect coordination, memory or exercise ability of 18-month-old ATTAC mice.
    Extended Data Fig. 6: Senescent cell clearance does not affect coordination, memory or exercise ability of 18-month-old ATTAC mice.

    a, Time spent balanced during a fixed speed rotarod test for 18-month-old ATTAC mice (n = 6 male and 8 female mice per group). b, Novel object investigation test. The percentage of investigations of a novel object divided by the total investigations is graphed. Key and animal numbers are as indicated in a. ce, Time-to-exhaustion (c), distance (d) and work (e) during a treadmill exercise test. Animal numbers are as indicated in c. f, Gastrocnemius muscle weight of ATTAC mice (n = 6 12-month-old males and females; n = 4 18-month-old −AP males and females; n = 4 18-month-old +AP males and females). gi, Myofibre diameter measurements on isolated gastrocnemius (g), abdominal (h) and paraspinal muscle (i). Animal numbers are as indicated in f. j, Analysis of forelimb grip strength of ATTAC mice. Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests).

  12. Senescent cell clearance has no effect on haematological parameters and age-related changes in leukocyte populations.
    Extended Data Fig. 7: Senescent cell clearance has no effect on haematological parameters and age-related changes in leukocyte populations.

    al, Haematology results of 3- and 10-month-old untreated ATTAC C57BL/6 mice and 18-month-old vehicle- and AP-treated ATTAC C57BL/6 mice. White blood cell count (a), platelet count (b), red blood cell count (c), haemaglobin concentration (d), haematocrit (e), mean corpuscular volume (f), mean corpuscular haemoglobin (g), neutrophils (h), lymphocytes (i), basophils (j), monocytes (k) and eosinophils (l). mq, Assessment for leukocyte subpopulations in 3- and 10-month-old untreated ATTAC C57BL/6 mice and 18-month-old vehicle- and AP-treated ATTAC C57BL/6 mice. CD4+ T cells (percentage of peripheral blood mononuclear cells, PBMC) (m), CD8+ T cells (percentage of PBMC) (n), CD44hi CD4+ T cells (percentage of CD4+) (o), CD44hi CD8+ T cells (percentage of CD8+) (p), and NK1.1+ cells (percentage of PBMC) (q). Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests).

  13. Senescent cell removal does not affect somatotrophic axis signalling in vivo.
    Extended Data Fig. 8: Senescent cell removal does not affect somatotrophic axis signalling in vivo.

    a, Glucose levels following intraperitoneal glucose administration after an overnight fast in 18-month-old vehicle- and AP-treated ATTAC C57BL/6 females. b, Normalized glucose levels after intraperitoneal insulin administration following a 4-h fast in 18-month-old vehicle- and AP-treated ATTAC C57BL/6 females. c, Serum Igf1 levels in ATTAC C57BL/6 mice (n = 4 mice of each group). d, Representative western blots for phospho-S6K, total S6K, phospho-AKTS473 and total AKT in iWAT, kidney and skeletal muscle tissue lysates from 18-month-old vehicle- and AP-treated ATTAC C57BL/6 females. e, Quantification of phospho-S6K to total S6K ratio in blots from d, n = 4 mice of each group. f, Quantification of phospho-AKTS473 to total AKT ratio in blots from d, n = 4 mice of each group. Error bars indicate s.e.m. No statistically significant differences were observed in ac, e and f using unpaired two-tailed t-tests. For gel source data, see Supplementary Fig. 1.

  14. Senescent cell clearance does not alter cardiac morphology and function in ‘resting’ mice and AP treatment has no effect on healthspan of mice lacking the ATTAC transgene.
    Extended Data Fig. 9: Senescent cell clearance does not alter cardiac morphology and function in ‘resting’ mice and AP treatment has no effect on healthspan of mice lacking the ATTAC transgene.

    a, Electron micrographs of X-Gal crystal containing cells in the aortic root. VSMC, vascular smooth muscle cell. Scale bars, 1 μm (main panel) and 200 nm (inset). bg, Echocardiography measurements of heart rate (b), left ventricular mass (c), posterior wall thickness (d), left ventricular inner diameter (e), ejection fraction (f), and the fractional shortening of the heart (g) in 12-month-old untreated mice and 18-month-old ATTAC mice treated with vehicle or AP. h, Fat mass (n = 9 mice per group). 18-month-old ATTAC vehicle-treated mouse values are the same as indicated in Fig. 1. i, iWAT and eWAT depot weight (n = 4 mice per group). 18-month-old ATTAC vehicle-treated mouse values are the same as indicated in Fig. 1. j, k, Kidney sclerosis (j) and blood urea levels (k) (n = 4 mice per group). 18-month-old ATTAC vehicle-treated mouse values are the same as indicated in Fig. 4. l, Time to death after isoproterenol administration (n = 4 mice per group). 18-month-old ATTAC vehicle-treated mouse values are the same as indicated in Fig. 5. Error bars indicate s.e.m. No statistically significant differences were observed using unpaired two-tailed t-tests.

  15. Effect of senescent cell clearance on wound healing and tissue fibrosis.
    Extended Data Fig. 10: Effect of senescent cell clearance on wound healing and tissue fibrosis.

    a, Closure of 3-mm punch biopsy wounds in 18-month-old ATTAC females after treatment with vehicle or AP for 6 months and if drug treatment was stopped 2 days before skin puncture or continued during wound closure (n = 6 wounds for −AP;−AP and +AP; −AP and n = 10 wounds for −AP;+AP and +AP;+AP). AP administration during the wound healing process significantly attenuates the rate of wound closure independently of whether senescent cell removal had occurred before wounding. b, Closure of 3-mm punch biopsy wounds in 4-month-old ATTAC females after treatment with vehicle or AP following wounding (n = 10 wounds per group). Similar to 18-month-old mice, AP administration during the wound healing process dramatically attenuated the rate of wound closure. c, Quantification of total GFP+ cells isolated from 3-mm punch biopsy wounds of 4-month-old mice two days into the wound healing process treated with vehicle (black) or AP (red, n = 3 mice per group). d, PTAH-stained tissues sections from 18-month-old ATTAC mice for detection of fibrosis. Scale bars, 100 μm. Error bars indicate s.e.m. Mice receiving AP during the healing process in a and b are significantly different from those treated with vehicle from day 1.5 to day 9.5. *P < 0.05 (unpaired two-tailed t-tests).

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Author information

Affiliations

  1. Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    • Darren J. Baker,
    • Matej Durik,
    • Melinde E. Wijers,
    • Jian Zhong,
    • Rachel A. Saltness,
    • Karthik B. Jeganathan &
    • Jan M. van Deursen
  2. Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    • Bennett G. Childs,
    • Cynthia J. Sieben &
    • Jan M. van Deursen
  3. Division of Cardiovascular Surgery, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    • Grace Casaclang Verzosa &
    • Jordan D. Miller
  4. Department of Immunology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    • Abdulmohammad Pezeshki &
    • Khashayarsha Khazaie

Contributions

D.J.B. performed all lifespan and most healthspan analyses on ATTAC mice. B.G.C. designed and conducted experiments to identify and quantify X-gal-positive cells by TEM, and analysed mice for cardiomyocyte hypertrophy and local RAAS activity in kidney. M.E.W., J.Z. and R.A.S. assisted with various aspects of healthspan analyses: the extent of their contributions is reflected in the authorship order. C.J.S. conducted the lifespan analysis of C57BL/6-129Sv/E hybrid mice on diets containing 5% or 9% fat. K.B.J. investigated somatotrophic axis signaling. G.C.C.V., J.D.M. and M.D. analysed resting heart functions by echocardiography. M.D. designed and conducted cardiac stress tests. A.P. and K.K. analysed leukocyte subpopulations. J.M.v.D., D.J.B. and B.G.C. wrote the paper with input from all co-authors. J.M.v.D. directed and supervised all aspects of the study in collaboration with D.J.B.

Competing financial interests

J.M.v.D. and D.J.B. are inventors on patents licensed to Unity Biotechnology by Mayo Clinic and J.M.v.D. is a co-founder of Unity Biotechnology.

Corresponding authors

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Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: ATTAC transgene expression tracks with expression of senescence markers in iWAT and induces apoptosis of senescent cells after AP administration. (452 KB)

    a, Comparative analysis of SA-β-Gal activity in intact iWAT. Scale bar, 0.5 cm. b, Analysis of endogenous Ink4a and ATTAC transcript SA-β-Gal activity in iWAT by qRT–PCR. H/H denotes BubR1H/H mice (n = 4 mice per group). c, FACS-based quantification of iWAT progenitor cell numbers in 18-month-old ATTAC mice treated with vehicle or AP. ASC, adipocyte stem cells; PAC, preadipocytes. d, Expression of the ATTAC transgene and senescence markers in iWAT as determined by qRT–PCR (n = 4 mice per group). Asterisks above individual bars denote significant changes to 2-month-old mice; asterisks above brackets denote significant differences between 18-month-old vehicle and AP-treated mice. e, Perirenal, mesenteric, subscapular and brown adipose tissue depot weights. SSAT, subscapular adipose tissue. f, SA-β-Gal activity in iWAT from 2-month-old ATTAC mice treated with vehicle or AP beginning at weaning age. g, p16Ink4a levels in iWAT from the mice described in f. Actin was used a loading control. h, Expression of ATTAC and senescence marker mRNA in the mice described in f (n = 3 mice per group). ik, Early passage non-senescent ATTAC MEFs express p16Ink4a but are not susceptible to FKBP–Casp8-mediated elimination when cultured in the presence of AP. i, Levels of p16Ink4a in passage 3 ATTAC MEFs, with and without AP treatment. j, Growth curves of passage 3 ATTAC MEFs (n = 4 independently generated MEF lines per group), with or without AP treatment. k, Expression of ATTAC and senescence marker mRNA in passage 3 ATTAC MEFs (n = 3 independently generated MEF lines per group), with or without AP treatment. Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests). For gel source data, see Supplementary Fig. 1.

  2. Extended Data Figure 2: ATTAC lacks promoter elements required for expression in replication-competent cells or aged lymphocytes expressing high levels of endogenous p16Ink4a. (330 KB)

    ac, SV40 large-T-antigen-immortalized ATTAC MEFs robustly express endogenous p16Ink4a (owing to SV40 large-T-antigen-mediated inactivation of Rb) but fail to engage the ATTAC transgene and are not subject to FKBP–Casp8-mediated elimination. a, p16Ink4a protein levels in passage 4 (P4) primary MEFs and MEFs immortalized with SV40 large T antigen. Actin was used as a loading control. b, p16Ink4a protein levels in immortalized MEFs treated with vehicle or two concentrations of AP. Actin was used as a loading control. c, Expression of ATTAC and senescence marker transcripts in passage 4 primary MEFs, vehicle-treated immortalized MEFs, and AP-treated immortalized MEFs (n = 3 independently generated MEF lines per group). d, Schematic representation of the endogenous Ink4a locus and the various Ink4a promoter regions driving ATTAC, 3MR and firefly luciferase (FLUC). ATTAC and p16-3MR mice have 2.6 kb and ~50 kb Ink4a promoter fragments driving transgene activity, respectively. p16-FLUC has firefly luciferase knocked into the endogenous Ink4a locus, which keeps the entire promoter region intact but ablates p16Ink4a protein expression. e, p16Ink4a protein levels in early passage primary and SV40 large-T-antigen-immortalized p16-3MR MEFs. f, Expression of senescence marker mRNA in early and late passage primary MEFs and SV40 large-T-antigen-immortalized p16-3MR MEFs (n = 1 independently generated MEF line per group performed in triplicate). g, Expression of senescence marker mRNA in early and late passage primary MEFs and SV40 large-T-antigen-immortalized p16-FLUC MEFs (n = 1 independently generated MEF line per group performed in triplicate). h, Expression of ATTAC and senescence markers in CD3+ T cells from 12- and 18-month-old ATTAC mice (n = 5 mice per group). Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests). We note that all values in f and g have P < 0.05 compared to passage 4 MEFs, with the exception of the one marked NS for not significant. For gel source data, see Supplementary Fig. 1.

  3. Extended Data Figure 3: ATTAC-mediated clearance of senescent cells is partial and tissue selective and attenuates expression of inflammation markers. (426 KB)

    a, Expression of the ATTAC transgene and a senescence marker panel, as determined by RT–PCR, in gastrocnemius, eye, kidney, heart (atria), spleen, lung, liver and colon (n = 4 females per group). b, Expression of Il6, Il1a and Tnfa as determined by qRT–PCR in mouse iWAT, kidney and skeletal muscle at different ages (n = 4 females per group). Il6 values are as indicated in Extended Data Fig. 1d (iWAT) and in a (kidney and gastrocnemius). Expression levels of inflammation markers in unmanipulated 18-month-old C57BL/6 females suggests that repeated vehicle injections were not a source of tissue inflammation. Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests). Asterisks above individual bars in a denote significant changes to 2-month-old mice; asterisks above brackets denote significant differences between 18-month-old vehicle and AP-treated mice.

  4. Extended Data Figure 4: Comparison of lifespans under different diets and housing facilities. (591 KB)

    a, Survival curves of unmanipulated wild-type C57BL/6-129Sv mice fed a 5% versus 9% fat diet. Median lifespan (in days) are indicated. b, Survival curves of unmanipulated wild-type C57BL/6-129Sv mice fed a 9% fat diet plotted against those of vehicle-treated (−AP) and AP-treated (+AP) C57BL/6-129Sv-FVB ATTAC mice from Fig. 2b. These data suggest that the lifespans of vehicle-injected C57BL/6-129Sv-FVB control mice were quite normal for the diet that they were on, and unlikely to be negatively affected by repeated intraperitoneal injections. *P < 0.01; **P < 0.001; ***P < 0.001 (log-rank tests). c, d, Median survival data of unmanipulated C57BL/6 male (c) and female (d) mice from various laboratories for comparsion to the results obtained from our facility.

  5. Extended Data Figure 5: Senescent cell clearance delays tumour and cataract formation. (530 KB)

    a, b, Survival curves of mixed (a) and C57BL/6 (b) ATTAC mice dying of cancer (mice that had an overt tumour at time of death; only mice with lymphomas, sarcomas and carcinomas were included). Median survival (in days) and percentage increase are indicated. c, d, Incidence of macroscopically detectable neoplasms (lymphomas, sarcomas and carcinomas) at time of death in mixed (c) and C57BL/6 (d) ATTAC mice from survival cohorts. e, Survival curves of C57BL/6-129Sv-FVB mice dying without cancer (mice that had an overt tumour at time of death, including lymphoma, sarcoma and/or carcinoma, were excluded). Median survival (in days) and percentage increase are indicated. f, g, Cataract incidence for mixed (f) and C57BL/6 (g) ATTAC mouse cohorts. *P < 0.05; **P < 0.01; ***P < 0.001 (log-rank tests).

  6. Extended Data Figure 6: Senescent cell clearance does not affect coordination, memory or exercise ability of 18-month-old ATTAC mice. (358 KB)

    a, Time spent balanced during a fixed speed rotarod test for 18-month-old ATTAC mice (n = 6 male and 8 female mice per group). b, Novel object investigation test. The percentage of investigations of a novel object divided by the total investigations is graphed. Key and animal numbers are as indicated in a. ce, Time-to-exhaustion (c), distance (d) and work (e) during a treadmill exercise test. Animal numbers are as indicated in c. f, Gastrocnemius muscle weight of ATTAC mice (n = 6 12-month-old males and females; n = 4 18-month-old −AP males and females; n = 4 18-month-old +AP males and females). gi, Myofibre diameter measurements on isolated gastrocnemius (g), abdominal (h) and paraspinal muscle (i). Animal numbers are as indicated in f. j, Analysis of forelimb grip strength of ATTAC mice. Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests).

  7. Extended Data Figure 7: Senescent cell clearance has no effect on haematological parameters and age-related changes in leukocyte populations. (318 KB)

    al, Haematology results of 3- and 10-month-old untreated ATTAC C57BL/6 mice and 18-month-old vehicle- and AP-treated ATTAC C57BL/6 mice. White blood cell count (a), platelet count (b), red blood cell count (c), haemaglobin concentration (d), haematocrit (e), mean corpuscular volume (f), mean corpuscular haemoglobin (g), neutrophils (h), lymphocytes (i), basophils (j), monocytes (k) and eosinophils (l). mq, Assessment for leukocyte subpopulations in 3- and 10-month-old untreated ATTAC C57BL/6 mice and 18-month-old vehicle- and AP-treated ATTAC C57BL/6 mice. CD4+ T cells (percentage of peripheral blood mononuclear cells, PBMC) (m), CD8+ T cells (percentage of PBMC) (n), CD44hi CD4+ T cells (percentage of CD4+) (o), CD44hi CD8+ T cells (percentage of CD8+) (p), and NK1.1+ cells (percentage of PBMC) (q). Error bars indicate s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t-tests).

  8. Extended Data Figure 8: Senescent cell removal does not affect somatotrophic axis signalling in vivo. (335 KB)

    a, Glucose levels following intraperitoneal glucose administration after an overnight fast in 18-month-old vehicle- and AP-treated ATTAC C57BL/6 females. b, Normalized glucose levels after intraperitoneal insulin administration following a 4-h fast in 18-month-old vehicle- and AP-treated ATTAC C57BL/6 females. c, Serum Igf1 levels in ATTAC C57BL/6 mice (n = 4 mice of each group). d, Representative western blots for phospho-S6K, total S6K, phospho-AKTS473 and total AKT in iWAT, kidney and skeletal muscle tissue lysates from 18-month-old vehicle- and AP-treated ATTAC C57BL/6 females. e, Quantification of phospho-S6K to total S6K ratio in blots from d, n = 4 mice of each group. f, Quantification of phospho-AKTS473 to total AKT ratio in blots from d, n = 4 mice of each group. Error bars indicate s.e.m. No statistically significant differences were observed in ac, e and f using unpaired two-tailed t-tests. For gel source data, see Supplementary Fig. 1.

  9. Extended Data Figure 9: Senescent cell clearance does not alter cardiac morphology and function in ‘resting’ mice and AP treatment has no effect on healthspan of mice lacking the ATTAC transgene. (296 KB)

    a, Electron micrographs of X-Gal crystal containing cells in the aortic root. VSMC, vascular smooth muscle cell. Scale bars, 1 μm (main panel) and 200 nm (inset). bg, Echocardiography measurements of heart rate (b), left ventricular mass (c), posterior wall thickness (d), left ventricular inner diameter (e), ejection fraction (f), and the fractional shortening of the heart (g) in 12-month-old untreated mice and 18-month-old ATTAC mice treated with vehicle or AP. h, Fat mass (n = 9 mice per group). 18-month-old ATTAC vehicle-treated mouse values are the same as indicated in Fig. 1. i, iWAT and eWAT depot weight (n = 4 mice per group). 18-month-old ATTAC vehicle-treated mouse values are the same as indicated in Fig. 1. j, k, Kidney sclerosis (j) and blood urea levels (k) (n = 4 mice per group). 18-month-old ATTAC vehicle-treated mouse values are the same as indicated in Fig. 4. l, Time to death after isoproterenol administration (n = 4 mice per group). 18-month-old ATTAC vehicle-treated mouse values are the same as indicated in Fig. 5. Error bars indicate s.e.m. No statistically significant differences were observed using unpaired two-tailed t-tests.

  10. Extended Data Figure 10: Effect of senescent cell clearance on wound healing and tissue fibrosis. (1,320 KB)

    a, Closure of 3-mm punch biopsy wounds in 18-month-old ATTAC females after treatment with vehicle or AP for 6 months and if drug treatment was stopped 2 days before skin puncture or continued during wound closure (n = 6 wounds for −AP;−AP and +AP; −AP and n = 10 wounds for −AP;+AP and +AP;+AP). AP administration during the wound healing process significantly attenuates the rate of wound closure independently of whether senescent cell removal had occurred before wounding. b, Closure of 3-mm punch biopsy wounds in 4-month-old ATTAC females after treatment with vehicle or AP following wounding (n = 10 wounds per group). Similar to 18-month-old mice, AP administration during the wound healing process dramatically attenuated the rate of wound closure. c, Quantification of total GFP+ cells isolated from 3-mm punch biopsy wounds of 4-month-old mice two days into the wound healing process treated with vehicle (black) or AP (red, n = 3 mice per group). d, PTAH-stained tissues sections from 18-month-old ATTAC mice for detection of fibrosis. Scale bars, 100 μm. Error bars indicate s.e.m. Mice receiving AP during the healing process in a and b are significantly different from those treated with vehicle from day 1.5 to day 9.5. *P < 0.05 (unpaired two-tailed t-tests).

Supplementary information

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Additional data