Intermittent clearance of p21-highly-expressing cells extends lifespan and confers sustained benefits to health and physical function

Abstract

A key challenge in aging research is extending lifespan in tandem with slowing down functional decline so that life with good health (healthspan) can be extended. Here, we show that monthly clearance, starting from 20 months, of a small number of cells that highly express p21^Cip1 (p21^high) improves cardiac and metabolic function and extends both median and maximum lifespans in mice. Importantly, by assessing the health and physical function of these mice monthly until death, we show that clearance of p21^high cells improves physical function at all remaining stages of life, suggesting healthspan extension. Mechanistically, p21^high cells encompass several cell types with a relatively conserved proinflammatory signature. Clearance of p21^high cells reduces inflammation and alleviates age-related transcriptomic signatures of various tissues. These findings demonstrate the feasibility of healthspan extension in mice and indicate p21^high cells as a therapeutic target for healthy aging.

Keywords: aging; cellular senescence; frailty; inflammation; morbidity compression.

Figure 1.. Intermittent clearance of p21^high cells extends both median and maximum lifespan in mice

(A) Experimental design. p21-Cre transgene contains a p21 promoter driving a Cre fused to a tamoxifen-inducible estrogen receptor (ER) element as well as an enhanced GFP. Both P and P/D mice received 2 doses of tamoxifen monthly starting at the age of 20 months. For each month, physical function assessments were performed 25-30 days after the last dose of tamoxifen. (B) Kaplan-Meier survival curves of P (n = 90; 42 males, 48 females) and P/D (n = 88; 41 males, 47 females) mice starting at 20 months of age. Male only and female only survival curves are shown as well. Median lifespan (days) are indicated. (C) Body weight of P (n = 90; 42 males, 48 females) and P/D (n = 88; 41 males, 47 females) mice from 20 months to natural death. (D) Body weight, fat mass, lean mass (relative to baseline) of P (n = 20) and P/D (n = 27) mice. (E) Tumor burden, tumor severity, disease burden, lethal disease number of P (n = 81; 40 males, 41 females) and P/D (n = 84; 39 males, 45 females) mice at death. (F) Causes of death of P (n = 81; 40 males, 41 females) and P/D (n = 84; 39 males, 45 females) mice. n represents the number of biological replicates with one technical replicate. The results are shown as mean ± SEM. Significance was determined by log-rank (Mantel-Cox) test and Boschloo’s test (B); two-tailed unpaired Student’s t test (C and D); Mann–Whitney U test (E); or chi-square test (F). n.s., not significant. See also Figure S1 and S2.

Figure 2.. Intermittent clearance of p21^high cells improves physical function throughout the post-treatment lifespan and extend healthspan

(A) Grip strength over post-treatment lifespan and area under curve (AUC) for each mouse (n = 41 for P; n = 41 for P/D). Each line indicates the grip strength change over the whole post-treatment lifespan for each mouse. (B) Maximal walking speed over post-treatment lifespan and AUC for each mouse (n = 42 for P; n = 39 for P/D). Each line indicates the maximal walking speed change over the whole post-treatment lifespan for each mouse. (C) Grip strength over the percentage of remaining lifespan after treatment and normalized AUC for each mouse (n = 41 for P; n = 41 for P/D). (D) Maximal walking speed over the percentage of remaining lifespan after treatment and normalized AUC for each mouse (n = 42 for P; n = 39 for P/D). (E) Frailty index scores over post-treatment lifespan starting at 24 months of age (n = 42 for P; n = 52 for P/D). For A-E, each dot represents the measurement for each mouse at the specified age or specific percentage of remaining post-treatment life (0 indicates death). (F) Frailty index slope of P (n = 42) and P/D (n = 52) mice. (G) Representative images of P and P/D mice at 31 months of age. n represents the number of biological replicates with one technical replicate. The results are shown as mean ± SEM. Significance was determined by two-tailed unpaired Student’s t test (A to F); or linear regression (E). *P < 0.05. See also Figure S3, S9, and S10.

Figure 3.. Intermittent clearance of p21^high cells results in better cardiac function, metabolic function and liver function

(A and B) Representative M-mode echocardiographic images for the left ventricle (LV) of the male (A) and female (B) mice as well as results for the ejection fraction (EF, %), fractional shortening (FS, %), corrected LV mass (mg), end-diastolic LV volume (d-LV Vol, μL) and end-systolic LV volume (s-LV Vol, μL). For male, n = 9 for Y (3-4 months old); n = 10 for P (26-27 months old); n = 12 for P/D (26-27 months old). For female, n = 8 for Y (3-5 months old); n = 10 for P (26-27 months old); n = 12 for P/D (26-27 months old). (C and D) GTT curve and area under curve (AUC) (C), ITT curve and AUC (D) in young mice (Y, 3-5 months old; n = 15), P (23-25 months old; n = 19), and P/D (23-25 months old; n = 19) mice. (E) Representative images of insulin immunostaining in pancreas from P and P/D mice. Red: insulin, Blue: DAPI. (F) Quantification of islet area and islet size in pancreas from P (23-25 months old; n = 6) and P/D mice (23-25 months old; n = 6). (G) Serum levels of ALT and AST. n = 12 for Y (3-4 months old); n = 19 for P (23-25 months old); n = 12 for P/D (23-25 months old). n represents the number of biological replicates with one technical replicate. The results are shown as mean ± SEM. Significance was determined by two-tailed unpaired Student’s t test (A and B, AUC in C and D, F and G); two-way ANOVA (GTT and ITT curves in C and D). *P < 0.05 for P vs P/D, ^#P < 0.05 for P vs Y. See also Figure S3.

Figure 4.. p21^high cells have a relatively conserved pro-inflammatory signature

(A) Schematic of the workflow for scRNAseq of visceral fat from 5 old female P mice (25-30 months old). (B) Uniform manifold approximation and projection (UMAP) plots of sorted p21^high cells and non-p21^high cells. A total of 10 cell populations were identified. (C) Percentages of p21^high cells and non-p21^high cells in 10 cell populations. (D) UMAP plots of cell cycle phases in p21^high cells and non-p21^high cells. (E) Percentages of G1, G2M, S phase cells in p21^high cells and non-p21^high cells. (F) Violin plots of p21 (Cdkn1a) expression and p16 (Cdkn2a) expression in p21^high cells and non-p21^high cells. (G) Enriched gene sets in p21^high cells versus non-p21^high cells in preadipocytes, macrophages, endothelial cells, T cells, B cells. −log₁₀(P-value) (−log(p)) and normalized enrichment score (NES) were shown. Hallmarks with P-value <0.05 and False discovery rate (FDR) <0.25 were considered as significant, and thus were shown. Higher NES indicates higher upregulation of indicated hallmarks in p21^high cells. n represents the number of biological replicates with one technical replicate. The results are shown as mean ± SEM. Significance was determined by two-tailed paired Student’s t test (E). *P < 0.05. See also Figure S4–S6.

Figure 5.. Intermittent clearance of p21^high cells reduces inflammation and alleviates age-related transcriptomic signatures in multiple tissues

Tissues were collected from young mice (Y, 3-4 months old), P (23-25 months old), and P/D (23-25 months old) mice, male and female mixed. (A) Gene set enrichment analysis (GSEA) of liver in P versus Y mice, and P versus P/D mice (n = 4 for Y; n = 4 for P; n = 4 for P/D). (B) GSEA of visceral fat in P versus Y mice, and P versus P/D mice (n = 4 for Y; n = 6 for P; n = 6 for P/D). (C) GSEA of muscle in P versus Y mice, and P versus P/D mice (n = 4 for Y; n = 6 for P; n = 6 for P/D). (D) GSEA of heart in P versus Y mice, and P versus P/D mice (n = 4 for Y; n = 6 for P; n = 6 for P/D). (E) GSEA of visceral fat, liver, muscle, heart in P versus P/D mice. −log₁₀(P-value) (−log(p)) and normalized enrichment score (NES) were shown. Higher NES indicates higher upregulation of indicated hallmarks in P mice. (F) Circulating pro-inflammatory cytokines level in Y (n = 12), P (n = 21), and P/D (n = 18) mice. (3-4 months old for Y; 23-25 months old for P and P/D). (G) Overlapped gene sets of each tissue in P versus Y mice, and P versus P/D mice. n represents the number of biological replicates with one technical replicate. The results are shown as mean ± SEM. Significance was determined by two-tailed unpaired Student’s t test (F). *P < 0.05. See also Figure S7 and S8.