Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster.
Increase in life span by rapamycin was associated with increased resistance to both starvation and paraquat.Analysis of the underlying mechanisms revealed that rapamycin increased longevity specifically through the TORC1 branch of the TOR pathway, through alterations to both autophagy and translation.Rapamycin could increase life span of weak insulin/Igf signaling (IIS) pathway mutants and of flies with life span maximized by dietary restriction, indicating additional mechanisms.
Affiliation: Institute of Healthy Ageing, Department of Genetics, Evolution and Environment, University College London, UK.
- Drosophila Proteins/metabolism*
- Drosophila melanogaster/physiology*
- Protein Kinases/metabolism*
- Protein Biosynthesis/drug effects
- Proto-Oncogene Proteins c-akt/metabolism
- Ribosomal Protein S6 Kinases/metabolism
- TOR Serine-Threonine Kinases
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fig5: Rapamycin-Mediated Life Span Extension Is Blocked by the Ubiquitous Overexpression of Constitutively Active S6K, the Absence of 4E-BP, or Downregulation of Atg5(A) Ubiquitous overexpression of a constitutively active form of S6K (UAS-S6Kconst.act.) by the daughterless-GAL4 (daGAL4) driver abolishes rapamycin-mediated life span extension. Rapamycin increases median life span of wDah females (p < 0.0001, log-rank test), but not daGAL4 > UAS-S6Kconst.act· (p = 0.1083, log-rank test). On control 0 μM rapamycin food, overexpression of constitutively active S6K slightly decreases life span (p = 0.0463, log-rank test).(B) Flies overexpressing constitutively active S6K are more sensitive to starvation compared to wDah control flies (p < 0.0001, log-rank test), and their starvation resistance is improved by rapamycin (p < 0.0001, log-rank test).(C) Rapamycin does not extend the life span of 4E-BP mutant female flies (p = 0.4027, log-rank test) but does increase the life span of control yw flies (p = 0.0033, log-rank test).(D) 4E-BP flies are sensitive to starvation compared to control flies (p < 0.0001, log-rank test), but rapamycin improves their starvation resistance (p < 0.0001, log-rank test).(E) Downregulation of autophagy abolishes rapamycin-mediated life span extension (p = 0.5383, log-rank test). Autophagy was downregulated by ubiquitous overexpression of UAS-atg5-RNAi using daughterless-GAL4 (daGAL4 > UAS-atg5RNAi). Note that since the experiments were run in parallel, life span data for wild-type controls are the same as in Figure 4A.(F) Flies with ubiquitous overexpression of UAS-atg5-RNAi (daGAL4 > UAS-atg5RNAi) were more starvation sensitive compared to their controls (p < 0.0001, log-rank test), and their starvation resistance was improved by rapamycin treatment (p < 0.0001, log-rank test). Note that these experiments were run in parallel to those in Figures 4B and 4D, hence the life span data for controls are the same.
We next determined the processes that mediated life span extension by rapamycin. S6K mediates the downstream effects of TOR signaling on translation initiation, and downregulation of protein translation has been shown to increase life span in worms (Hansen et al., 2007; Kaeberlein and Kennedy, 2008; Syntichaki et al., 2007). We therefore examined the effects of rapamycin on flies ubiquitously overexpressing a constitutively active form of S6K (Barcelo and Stewart, 2002), and found that rapamycin had no significant effects on life span (Figure 5A), suggesting that rapamycin extended life span by downregulation of S6K activity. Downregulation of TOR signaling leads to 4E-BP activation, resulting in inhibition of cap-dependent translation (Sonenberg and Hinnebusch, 2009). We therefore administered rapamycin to 4E-BP mutant flies (Tettweiler et al., 2005) and found that it did not significantly extend their life span (Figure 5C), suggesting that 4E-BP also mediates extension of life span by rapamycin.