Senolytics improve physical function and increase lifespan in old age

Abstract

Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.

Conflict of interest statement

Competing financial interests: J.L.K, T.T., M.X., T.P., N.G., and A.K.P. have a financial interest related to this research. Patents on senolytic drugs (PCT/US2016/041646) are held by Mayo Clinic. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies. None of the other authors has a relevant financial conflict of interest.

Figures

Figure 1

Transplanting small numbers of senescent cells induces physical dysfunction in younger mice. (a) Experimental design for transplantation and physical function measurements. (b,c) Representative images of LUC activity of various organs from LUC-negative male mice (n = 3) 5 d post-transplantation with SEN (induced by radiation) and CON preadipocytes from LUC-positive transgenic mice. Scale bars, 10 mm. (d-j) Maximal walking speed (relative to baseline) (d), hanging endurance (e), grip strength (f), daily activity (g), treadmill endurance (h), food intake (i), and change in body weight (BW) (j) of 6-month-old male C57BL/6 mice 1 mo after being injected with PBS, 1×106 non-senescent control (1M CON), 0.2 x106 SEN (0.2M SEN), 0.5×106 SEN (0.5M SEN), or 1×106 SEN (1M SEN) preadipocytes (n = 6 for all groups). Results are means ± s.e.m. (k-m). SA-βgal+ cell numbers (n = 6) (k), p16Ink4a mRNA levels (n = 7) (l), and cells from recipient mice that were TAF+ (>2 TAFs/nucleus) and LUC− (n = 4 mice) (m) in 6-month-old male wildtype (LUC−) C57BL/6 mice 2 mo after being transplanted with 1×106 SEN or CON transgenic constitutively-expressing LUC (LUC+) preadipocytes from transgenic mouse donors. Results are shown as box and whiskers plots, where a box extends from the 25th to 75th percentile with the median shown as a line in the middle, and whiskers indicate smallest and largest values. *P < 0.05; ANOVA with Tukey’s post-hoc comparison (d-j) and two-tailed, unpaired Student’s t-test (k-m).

Figure 2

Aging exacerbates effects of senescent cell transplantation. (a) Experimental design for transplantation and physical function measurements. (b-h) Maximal walking speed (relative to baseline) (b), hanging endurance (c), grip strength (d), body weight change from baseline (e), treadmill endurance (f), daily activity (g), and food intake (h) of 17-month-old male C57BL/6 mice 1 mo after being injected with 0.5 × 106 SEN or CON preadipocytes (n = 8 for both groups). (i) Percent changes in RotaRod (in 6-month-old mice, n = 21 for both SEN and CON; in 17-month-old mice, n = 22 for SEN, n = 20 for CON) and hanging test (in 6-month-old mice, n = 6 for both SEN and CON; in 17-month-old mice, n = 8 for both SEN and CON) in mice transplanted with 0.5 × 106 SEN cells relative to the average of mice transplanted with 0.5 × 106 CON cells at both ages. Results are shown as box and whiskers plots, where a box extends from the 25th to 75th percentile with the median shown as a line in the middle, and whiskers indicate smallest and largest values. (j) One year survival curves of 17-month-old non-transplanted mice (n = 33, N/A) and mice transplanted with 0.5 × 106 SEN (n = 23) or CON (n = 24) preadipocytes. (k) Tumor burden, disease burden, and inflammation at death are shown as means ± s.e.m. after transplanting SEN or CON cells (n = 10 for SEN, n = 7 for CON). (l) Causes of death (n = 10 for SEN, n = 7 for CON). *P <0.05; Two-tailed unpaired Student’s t-test (b-i), Cox proportional hazard regression model (j) and chi-square and Fisher’s exact tests (l).

Figure 3

Senescent cells reduce resilience to metabolic stress in mice. (a) Experimental design for transplantation and physical function measurements. (b-h) Maximal walking speed (relative to baseline) (b), hanging endurance (c), grip strength (d), daily activity (e), food intake (f), body weight change from baseline (g), and treadmill endurance (h) of 8-month-old male C57BL/6 mice 1 mo after being on HFD and injected with 0.4 × 106 SEN or CON preadipocytes (n = 6 for both groups). (i) Percent changes in RotaRod (on NCD, n = 21 for both SEN and CON; on HFD, n = 12 for both SEN and CON) and hanging test (on NCD, n = 6 for both SEN and CON; on HFD, n = 6 for both SEN and CON) in mice transplanted with 0.4-0.5 × 106 SEN cells relative to the average of mice transplanted with 0.4-0.5 × 106 CON cells. (j) Experimental design for transplantation and physical function measurements. (k-q) Maximal walking speed (relative to baseline) (k), hanging endurance (l), grip strength (m), body weight change from baseline (n), treadmill endurance (o), daily activity (p), and food intake (q) of 8-month-old male C57BL/6 mice 1 mo after being on HFD and injected with 1 × 106 SEN or CON autologous ear fibroblasts (n = 10 for both groups). All results are shown as box and whiskers plots, where a box extends from the 25th to 75th percentile with the median shown as a line in the middle, and whiskers indicate smallest and largest values. *P <0.05; Two-tailed unpaired Student’s t-tests (a-q).

Figure 4

D+Q reduces senescent cell abundance and decreases pro-inflammatory cytokine secretion in human adipose tissue. (a) Experimental design. (b) Percent TAF+ cells (n = 5). Blue arrows indicate TAFs. Scale bars, 5μm. (c) Percent p16INK4A-high cells (red arrows), percent p16INK4A+ cells (expressing any detectable level of p16INK4A, green arrows), percent p16INK4A− cells (black arrows), and cell number per field (n = 6). Scale bar, 100μm. (d) Percent SA-βgal+ cells (red arrows) (n = 6). Scale bar, 100μm. (e) Percent cleaved caspase-3+ cells (red arrows) (n = 5). Scale bar, 100μm. (f) Secreted cytokine and adipokine levels in conditioned media (CM) (n = 8). Results are means ± s.e.m. (g) The relative mRNA abundance of key SASP components and markers for adipose tissue function (n = 7). All results are shown as box and whiskers plots, where a box extends from the 25th to 75th percentile with the median shown as a line in the middle, and whiskers indicate smallest and largest values. *P <0.05; Two-tailed Student’s t-tests (a-g).

Figure 5

Eliminating senescent cells both prevents and alleviates physical dysfunction. (a) Experimental design for transplantation and physical function measurements. (b) Representative images of LUC activity in mice 2 days after the last treatment. Scale bars, 15mm. (c) Luminescence of transplanted cells as percent relative to the average of mice treated with V (n =16 for SEN-DQ vs. SEN-V; n =13 for CON-DQ vs. CON-V). (d-f) Maximal walking speed (relative to baseline) (d), hanging endurance (e), and grip strength (f) of 5-month-old male C57BL/6 mice 1 mo after the last drug treatment (n = 7 for SEN-V, CON-V, and SEN-DQ; n = 6 for CON-DQ). (g) Experimental design for transplantation and physical function measurements. (h-j) Maximal walking speed (relative to baseline) (h), hanging endurance (i), and grip strength (j) of 5-month-old male C57BL/6 mice 2 weeks after the last drug treatment (n = 10 for SEN-DQ and SEN-V; n = 14 for CON-V). All results are shown as box and whiskers plots, where a box extends from the 25th to 75th percentile with the median shown as a line in the middle, and whiskers indicate smallest and largest values. *P <0.05; Two-tailed Student’s t-tests (a-j).

Figure 6

Senolytics extend both health- and life-span in aged mice. (a) Experimental design for physical function measurements in 20-month-old male mice treated with D+Q once every 2 weeks (bi-weekly) for 4 months. (b-h) Maximal walking speed (relative to baseline) (b), hanging endurance (c), grip strength (d), body weight change from baseline (e), treadmill endurance (f), daily activity (g), and food intake (h) of 20-month-old male C57BL/6 mice 4 mo after drug initiation (n = 20 for D+Q; n = 13 for V). (i) The relative mRNA abundance for target genes of visceral adipose tissue from 6-month-old non-treated (6m, n = 7), 24-month-old V-treated (24m-V, n = 8), and 24-month-old D+Q-treated (24m-DQ, n = 8) mice. (j) Experimental design for lifespan analyses. (k,l) Post-treatment survival curves (k) and whole-life survival curves (l) of C57BL/6 mice treated bi-weekly with D+Q (n = 71; 40 males, 31 females) or V (n = 76; 41 males, 35 females) starting at 24-27 months of age. Median survival is indicated for all curves. (m) Maximal walking speed and hanging endurance averaged over the last 2 months of life and lifespan for the longest living mice (top 40%) in both groups for both sexes. For male mice, n = 12 for D+Q and n = 12 for V. For female mice, n = 13 for D+Q and n = 13 for V. (n) Disease burden and tumor burden at death. For both sexes, n = 59 for D+Q, n = 62 for V. For males, n = 30 for D+Q, n = 29 for V. For females, n = 29 for D+Q, n = 33 for V. (b-i, m) Results are shown as box and whiskers plots, where a box extends from the 25th to 75th percentile with the median shown as a line in the middle, and whiskers indicate smallest and largest values. (n) Results are shown as mean ± s.e.m. *P <0.05; n.s., not significant; Two-tailed Student’s t-tests (b-i, m-n) and Cox proportional hazard regression model (k-l).

Methods-Only References