Rapamycin modulates tissue aging and lifespan independently of the gut microbiota in Drosophila

Abstract

The FDA approved drug rapamycin can prolong lifespan in diverse species and delay the onset of age-related disease in mammals. However, a number of fundamental questions remain unanswered regarding the mechanisms by which rapamycin modulates age-related pathophysiology and lifespan. Alterations in the gut microbiota can impact host physiology, metabolism and lifespan. While recent studies have shown that rapamycin treatment alters the gut microbiota in aged animals, the causal relationships between rapamycin treatment, microbiota dynamics and aging are not known. Here, using Drosophila as a model organism, we show that rapamycin-mediated alterations in microbiota dynamics in aged flies are associated with improved markers of intestinal and muscle aging. Critically, however, we show that the beneficial effects of rapamycin treatment on tissue aging and lifespan are not dependent upon the microbiota. Indeed, germ-free flies show delayed onset of intestinal barrier dysfunction, improved proteostasis in aged muscles and a significant lifespan extension upon rapamycin treatment. In contrast, genetic inhibition of autophagy impairs the ability of rapamycin to mediate improved gut health and proteostasis during aging. Our results indicate that rapamycin-mediated modulation of the microbiota in aged animals is not causally required to slow tissue and organismal aging.

Figure 1

Rapamycin treatment improves gut health and modulates microbiota composition during aging. (A) Survival curves of w^Dahomey females with or without rapamycin treatment from day 4 onwards (post-eclosion). p < 0.0001, log rank test; n > 225 flies. Representative result of 4 separate lifespan trials. (B) Intestinal integrity during aging of w^Dahomey females with or without rapamycin treatment from day 4 onwards (post-eclosion). *p < 0.05, **p < 0.01, one-way ANOVA/Bonferroni’s multiple comparisons test; n = 300 flies on day 10 per treatment. (C) Bacterial levels assayed by qPCR of the 16S rRNA gene in surface sterilized, non-smurf w^Dahomey females with or without rapamycin treatment from day 4 onwards (post-eclosion). **p < 0.01, Mann-Whitney U-test; n = 6 replicates of five flies per timepoint. (D) Bacterial levels assayed by taxon specific primers in surface sterilized, non-smurf w^Dahomey females fed with or without rapamycin from day 4 onwards. **p < 0.01, Mann-Whitney U-test; n = 6 replicates of five flies per timepoint. Rapamycin was provided in the media at a concentration of 10 μg/ml for (A–D). All error bars represent SEM.

Figure 2

Rapamycin treatment requires autophagy to improve gut health during aging. (A) Survival curves of daGS > UAS-Atg1-RNAi female flies treated with (red) or without rapamycin (black), and with (dashed lines) or without RU486 (solid lines) from day 4 onwards. p < 0.0001, log rank test; n > 205 flies. Representative result of 2 separate lifespan trials shown. (B) Intestinal integrity of day 42 daGS > UAS-Atg1-RNAi female flies treated with (red) or without (black) rapamycin, and with (dashed lines) or without (solid lines) RU486 from day 4 onwards. *p < 0.05, one-way ANOVA/Bonferroni’s multiple comparisons test; n = 180 flies per treatment. Rapamycin was provided in the media at a concentration of 10 μg/ml and RU486 at a concentration of 25 μg/ml for (A,B). All error bars represent SEM.

Figure 3

Rapamycin treatment maintains proteostasis during aging in an autophagy-dependent manner. (A,B) Immunostaining of indirect flight muscles from 10 and 42 day old daGS > UAS-Atg1-RNAi female flies with or without both rapamycin and RU486 treatment from day 4 onwards (post-eclosion), showing polyubiquitinated aggregates (green channel, anti-FK2) and muscles (red channel stained with phalloidin/F-Actin). Scale bar is 10 µm. (C,D) Quantifications of polyubiquitin aggregates in muscle as shown in (A,B) respectively. **p < 0.01, one-way ANOVA w/ Newman-Keuls post-test; n > 12, one fly per replicate. Rapamycin was provided in the media at a concentration of 10 μg/ml and RU486 at a concentration of 25 μg/ml for (A–D). All error bars represent SEM.

Figure 4

Rapamycin treatment extends lifespan, improves barrier function and reduces TOR signaling in germ-free flies. (A) Survival curves of w^Dahomey female flies rendered germ free as embryos, and either maintained germ free (AX, dashed lines) or refed with a bacterial homogenate (HF, solid lines) and treated with (red) or without (black) rapamycin from day 4 onwards (post-eclosion). ***p < 0.0001, log rank test; n ≥ 200 flies. Representative result of 3 separate lifespan trials shown. (B) Intestinal integrity of day 10, 35 and 42 w^Dahomey flies with (HF, solid bars) or without homogenate feeding (AX, dashed bars) and with (red) or without rapamycin (black) treatment from day 4 onwards (post-eclosion). **p < 0.01 and ***p < 0.001, one-way ANOVA/Bonferroni’s multiple comparisons test; n = 300 flies at day 10 per treatment. (C) Western blot detection and densitometry of p-S6K (T398) and total S6K levels from day 10 homogenate fed (HF) w^Dahomey flies treated with or without rapamycin from day 4 onwards. n = 5 replicates, 10 flies per replicate; **p < 0.01; Mann-Whitney U-test. (D) Western blot detection and densitometry of p-S6K (T398) and total S6K levels from day 10 axenic (AX) w^Dahomey flies treated with or without rapamycin from day 4 onwards. n = 5 replicates, 10 flies per replicate; *p < 0.05; Mann-Whitney U-test. Rapamycin was provided in the media at a concentration of 10 μg/ml. All error bars represent SEM.

Figure 5

The efficacy of rapamycin treatment on proteostasis is enhanced in the absence of a microbiota. (A,B) Immunostaining of indirect flight muscles from 10 and 37 day old w^Dahomey female flies rendered germ free as embryos, and either maintained germ free (AX) or refed a bacterial homogenate (HF), and treated with or without rapamycin from day 4 onwards, showing polyubiquitinated aggregates (green channel, anti-FK2) and muscles (red channel stained with phalloidin/F-Actin). Scale bar is 10 µm. (C,D) Quantifications of polyubiquitin aggregates in muscle as shown in (A,B) respectively. *p < 0.05, ***p < 0.001, one-way ANOVA w/ Newman-Keuls post-test; n > 13, one fly per replicate. Rapamycin was provided in the media at a concentration of 10 μg/ml for (A–D). All error bars represent SEM.