Rapamycin treatment during development extends life span and health span of male mice and Daphnia magna

Abstract

Development is tightly connected to aging, but whether pharmacologically targeting development can extend life remains unknown. Here, we subjected genetically diverse UMHET3 mice to rapamycin for the first 45 days of life. The mice grew slower and remained smaller than controls for their entire lives. Their reproductive age was delayed without affecting offspring numbers. The treatment was sufficient to extend the median life span by 10%, with the strongest effect in males, and helped to preserve health as measured by frailty index scores, gait speed, and glucose and insulin tolerance tests. Mechanistically, the liver transcriptome and epigenome of treated mice were younger at the completion of treatment. Analogous to mice, rapamycin exposure during development robustly extended the life span of Daphnia magna and reduced its body size. Overall, the results demonstrate that short-term rapamycin treatment during development is a novel longevity intervention that acts by slowing down development and aging, suggesting that aging may be targeted already early in life.

Fig. 1.. Rapamycin inhibits growth and delays reproduction of genetically heterogeneous mice.

(A) Experimental design for evaluating the effect of EL rapamycin treatment on mouse longevity and health. RNA-seq, RNA sequencing. GTT, glucose tolerance test; ITT, insulin tolerance test. (B) Representative images of 2-month-old UMHET3 mice subjected to eudragit-encapsulated rapamycin or control (eudragit-containing) diets during the first 45 days of their lives. (C and D) Body weight change of male (C) and female (D) mice subjected to rapamycin (n = 30 per sex) or control (n = 34 to 39 per sex) diets from 0 to 45 days of age. The shaded area shows the period of rapamycin treatment. The data are means ± SE. (E) Experimental design for evaluating the effects of EL rapamycin treatment on pups’ nutrition. (F) Percentage of 7-day-old pups’ body weight gain calculated as weight of pup after it consumed milk divided by the mean weight of the group after a 6-hour starving period and exactly before pups were returned to dams and multiplied by 100. Mean weight was used for normalization within each group because of the baseline difference in weight between rapamycin and control pups. (G) Milk protein concentration in dams subjected to rapamycin or control diets starting from the day they give birth. (H) Number of pups born to breeders subjected to rapamycin or control diets during development. (I) Time between pregnancies for rapamycin-treated and control groups of breeders. *P < 0.05; ****P < 0.0001; ns, not significant.

Fig. 2.. Rapamycin treatment during development extends the life span of UMHET3 mice.

(A) Survival curve of treated and untreated UMHET3 mice, sexes combined. (B and C) Survival curves of (B) male and (C) female UMHET3 mice that were subjected to encapsulated rapamycin or encapsulating material as a control from birth until 45 days of age. P values were calculated with the log-rank test.

Fig. 3.. Rapamycin treatment during development extends the health span of mice.

(A) Frailty index score of 20- to 29-month-old UMHET3 mice subjected to diets containing encapsulated rapamycin or encapsulating material (eudragit) from birth until 45 days of age. P value was calculated with repeated-measures analysis of variance (ANOVA) test. (B) Frailty index score of 20- to 29-month-old male (M) and female (F) UMHET3 mice subjected to rapamycin or control diets from birth until 45 days of age. P value was calculated with repeated-measures ANOVA test. (C) Predicted survival (in months) based on frailty index calculated using the AFRAID (analysis of frailty and death) clock. (D) Scheme of gait speed measurement in mice. (E) Median time mice spent in seconds to reach the finish line. P value was calculated with repeated-measures ANOVA test. (F and G) Glucose tolerance test results for (F) females and (G) males (G) (n = 4 to 6 per group per sex). Insulin tolerance test result for (H) females and (I) males (n = 4 to 6 per group per sex). P values were calculated with repeated-measures ANOVA test. (J and K) Area under the curve (AUC) for glucose tolerance and insulin tolerance tests. P values were calculated with a one-tailed Student’s t test.

Fig. 4.. Rapamycin treatment during development attenuates aging of the transcriptome and DNA methylation (DNAm) of young animals, with males preserving these younger transcriptomes across the life span.

(A) Number of differentially expressed genes (DEGs) [false discovery rate (FDR) < 0.1] in the liver and kidney of young, middle-aged, and old animals treated with EL rapamycin and compared to controls. (B) Association between gene expression changes induced by EL rapamycin and measured in young (red), middle-aged (green), and old (blue) animals, and signatures of aging and life span extension in males (top plot) and females (bottom plot). The intervention signatures include gene signatures of individual interventions [caloric restriction (CR), growth hormone (GH) deficiency, and rapamycin), common gene expression signatures across different interventions (common), and signatures associated with the effect of interventions on maximum or median life span (max and median life span). Aging signatures include age-related gene expression changes across different tissues of humans, mice, and rats (human, mouse, and rat) as well as liver- and brain-specific changes (liver, brain). (C) DNAm age of livers estimated by the Liver epigenetic aging clock in young, middle-aged, and old treated and untreated animals. P values are calculated with a two-tailed Student’s t test. (D) GSEA for pathways from the KEGG database using ranked DEGs in livers and kidneys from young treated animals. Only significant enrichments (FDR < 0.1) for at least two conditions are shown. Dots are colored according to normalized enrichment score (NES) and sized according to −log₁₀ of P value. (E) GSEA for hallmark pathways from the MSigDB using ranked differentially expressed and (F) differentially methylated genes in treated animals. Only significant enrichments (FDR < 0.1) for at least two conditions are shown. Dots are colored according to NES and sized according to −log₁₀ of P value.

Fig. 5.. Rapamycin treatment during development extends life span and reduces the body size of Daphnia magna.

(A) Representative images of D. magna at 12 to 14 days following rapamycin exposure. Also shown is a control animal at the same chronological age. (B) Daphnia body size measurement at days 14 to 16, days 59 to 63, and days 81 to 83. P values are calculated using two-sided Student’s t test. The data are means ± SD. Kruskal-Wallis test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). n.s., not significant. (C) Survival curves for control and three different concentrations of rapamycin (control, n = 379; 0.01 nM, n = 107; 1 nM, n = 97; and 0.1 nM, n = 63).