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Sestrin is a key regulator of stem cell function and lifespan in response to dietary amino acids

Nature Aging volume 1, pages 60–72 (2021)Cite this article

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Abstract

Dietary restriction (DR) promotes healthy aging in diverse species. Essential amino acids play a key role, but the molecular mechanisms are unknown. The evolutionarily conserved Sestrin protein, an inhibitor of activity of the target of rapamycin complex 1 (TORC1), has recently been discovered as a sensor of amino acids in vitro. Here, we show that Sestrin null mutant flies have a blunted response of lifespan to DR. A mutant Sestrin fly line, with blocked amino acid binding and TORC1 activation, showed delayed development, reduced fecundity, extended lifespan and protection against lifespan-shortening, high-protein diets. Sestrin mediated reduced intestinal stem cell activity and gut cell turnover from DR, and stem cell proliferation in response to dietary amino acids, by regulating the TOR pathway and autophagy. Sestrin expression in intestinal stem cells was sufficient to maintain gut homeostasis and extend lifespan. Sestrin is thus a molecular link between dietary amino acids, stem cell function and longevity.

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Fig. 1: Loss of Sestrin attenuates the response of lifespan to DR.
Fig. 2: Generation of an amino acid-sensing defective Sestrin fly mutant.
Fig. 3: The SesnR407A mutation impairs growth, increases lifespan and blunts high-protein-diet-induced lifespan shortening.
Fig. 4: Sestrin is important for maintenance of ISCs in response to DR.
Fig. 5: Sestrin overexpression in gut stem cells improves gut homeostasis.
Fig. 6: Sestrin regulates lifespan and stem cell maintenance via the TOR pathway.
Fig. 7: Sestrin increases autophagy in ISCs to regulate gut cell turnover.
Fig. 8: Sestrin overexpression in gut stem cells extends lifespan.

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Data availability

The structure of human Sestrin2 (Uniprot: P58004) used to model the structure of the fly Sestrin protein (Uniprot: Q9W1K5) is PDB 5DJ4 (https://doi.org/10.2210/pdb5DJ4/pdb). All data that support the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

We thank J. H. Lee, B. Edgar, G. Juhasz, the Bloomington Stock Center and the VDRC Stock Center for fly strains and reagents. We are also grateful to all members of the Partridge Lab for helpful insights, and to C. Demetriades for critical comments. Imaging was performed in the FACS and Imaging Core Facility, and amino acid concentrations were determined in the Metabolomics Core Facility at the Max Planck Institute for Biology of Ageing. The work was supported by a Swiss National Science Foundation (SNSF) postdoc fellowship (P2BEP3_162093) to J.L. and by funding from the Max Planck Society to L.P. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 268739.

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  1. Max Planck Institute for Biology of Ageing, Cologne, Germany

    Jiongming Lu, Ulrike Temp, Andrea Müller-Hartmann, Jacqueline Esser, Sebastian Grönke & Linda Partridge

  2. Institute of Healthy Ageing, Department of Genetics, Evolution and Environment, University College London, London, UK

    Linda Partridge

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  1. Jiongming Lu
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Contributions

J.L., S.G. and L.P. conceived and designed the study. J.L. conducted most experiments, U.T. and A.M.-H. provided assistance. J.E. contributed to the generation of transgenic flies. J.L. and S.G. analysed the data. J.L., S.G. and L.P. wrote the manuscript.

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Extended Data

Extended Data Fig. 1 Characterization of a Sestrin loss-of-function allele and its effects on fly development.

a, Genomic locus of the Sestrin gene. In Sesn3F6 mutant flies the first two non-coding exons of the Sestrin gene are deleted. b, qRT-PCR analysis showed strongly reduced Sestrin mRNA levels in Sesn3F6 mutant flies. Data are presented as mean ± SEM. N = 3 biologically independent samples. Unpaired two-tailed t-test. c, No Sestrin protein was detected in Sesn3F6 mutant flies by immunoblotting analysis. An additional control sample (Sesn8A11 null mutant) was loaded in between. d, Sesn3F6 mutant flies showed a decreased developmental time compared with wDah wild type control flies (N = 10 vials, 40 embryos each). Permutation test (R, statmod package) showed that Sesn3F6 mutants eclosed significantly earlier than wild type flies. P value was adjusted for multiple testing. e, Sesn3F6 mutant flies had increased body weight. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots. N = 20 pairs of flies, unpaired two-tailed t-test.

Source data

Extended Data Fig. 2 Sestrin mRNA and protein levels are not affected by the SesnR407A mutation.

a-c, qRT-PCR (a) and immunoblotting (b,c) analyses confirmed that there was no change in Sestrin mRNA or protein levels in SesnR407A mutant flies compared with Sesnwt control flies. The Myc tag was used to detect Sestrin proteins in immunoblotting. Data are presented as mean ± SEM. N = 3 biologically independent samples, unpaired two-tailed t-test.

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Extended Data Fig. 3 SesnR407A mutation reduces cell growth and decreases fecundity.

a,b, SesnR407A mutation did not regulate pAMPKα activity. Fat body tissues from 3rd instar larvae grown under standard conditions were subjected to immunoblotting analysis. pAMPKα was normalized to Tubulin. Data are presented as mean ± SEM. N = 3 biologically independent samples, unpaired two-tailed t-test. c,d, SesnR407A-dependent reduction of cell size was cell autonomous. Confocal microscopy images of SesnR407A mutant clones in larval fat bodies (c). DNA (DAPI, blue), Actin (Phalloidin, red), and GFP (green). GFP-negative cells, outlined with white dashed lines, indicate Sestrin homozygous clones. Size of homozygous cells was normalized to wild type twin clones (marked by bright GFP). Scale bar: 25 μm. (d) Quantification of cell size from confocal images. N = 13 (Sesnwt) and N = 14 (SesnR407A) independent fat body samples, unpaired two-tailed t-test. e, SesnR407A mutant flies showed reduced cumulative egg laying. N = 10 vials with 20 flies each, two-tailed, Mann–Whitney test. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots (d, e). Outliers are shown as open circles. f, Reduced fecundity was restricted to early-life fecundity. Data are presented as mean ± SEM. N = 10 vials, two-tailed, Mann–Whitney test.

Source data

Extended Data Fig. 4 Sestrin is required for better maintenance of gut homeostasis by DR.

a-d, Sestrin function was required for preventing gut dysplasia from a high-protein diet. Gut dysplasia of wild type and Sesn mutant flies on fully fed (2.0x) and DR (1.0x) food was measured. (a,c) Representative gut images from 45 days old flies. DNA (DAPI) was blue. Epithelial layers are indicated by dashed lines. Scale bar represents 20 μm. (b, d) Quantification of gut dysplasia in Sesn3F6 (b) and in SesnR407A (d) mutant females under DR. N = 13 guts (b), N = 11 guts (d). Interaction between diet and genotypes was significant: two-way ANOVA, P = 0.024 (b), P = 0.011 (d). e,f, Sestrin was also required for maintenance of gut epithelial barrier function under DR. Smurf phenotypes of wild type and Sesn mutant flies on fully fed (2.0x) and DR (1.0x) food were scored at the age of 50 days. Proportion of Smurf flies in Sesn3F6 (e) and in SesnR407A (f) mutant females. N = 15 vials (e, f). Interaction between diet and genotypes was significant: two-way ANOVA, P = 0.04 (e), P = 0.02 (f). Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots ((b, d-f)). Outliers are shown as open circles. Statistics in (b,d-f): two-way ANOVA followed by Bonferroni’s post-hoc test, P values were adjusted for multiple comparisons.

Source data

Extended Data Fig. 5 Sestrin over-expression induces autophagic flux in gut stem cells.

a,b, Sestrin mRNA expression levels in RNAi-mediated knockdown and over-expression conditions. The ubiquitous, constitutive da-Gal4 driver was used to drive expression of each construct. (a) RNAi-mediated Sestrin knockdown significantly reduced Sestrin mRNA level, whereas (b) Sestrin over-expression significantly increased Sestrin mRNA level. Data are presented as mean ± SEM. N = 3 biologically independent samples, unpaired two-tailed t-test. c, Autophagic flux in ISCs upon Sestrin over-expression. Sestrin and a GFP::mCherry::Atg8a reporter were co-expressed in ISCs using the esg-Gal4 driver. GFP in green, mCherry in red, and DAPI (DNA) in blue. A representative gut image from 6 guts was shown. A strong mCherry signal was detected when Sestrin was over-expressed. The lower panel shows the magnification of the inset in the upper panel. Scale bars: 20 μm.

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Lu, J., Temp, U., Müller-Hartmann, A. et al. Sestrin is a key regulator of stem cell function and lifespan in response to dietary amino acids. Nat Aging 1, 60–72 (2021). https://doi.org/10.1038/s43587-020-00001-7

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