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SIRT1 is downregulated by autophagy in senescence and ageing

Abstract

SIRT1 (Sir2) is an NAD+-dependent deacetylase that plays critical roles in a broad range of biological events, including metabolism, the immune response and ageing1,2,3,4,5. Although there is strong interest in stimulating SIRT1 catalytic activity, the homeostasis of SIRT1 at the protein level is poorly understood. Here we report that macroautophagy (hereafter referred to as autophagy), a catabolic membrane trafficking pathway that degrades cellular components through autophagosomes and lysosomes, mediates the downregulation of mammalian SIRT1 protein during senescence and in vivo ageing. In senescence, nuclear SIRT1 is recognized as an autophagy substrate and is subjected to cytoplasmic autophagosome–lysosome degradation, via the autophagy protein LC3. Importantly, the autophagy–lysosome pathway contributes to the loss of SIRT1 during ageing of several tissues related to the immune and haematopoietic system in mice, including the spleen, thymus, and haematopoietic stem and progenitor cells, as well as in CD8+CD28 T cells from aged human donors. Our study reveals a mechanism in the regulation of the protein homeostasis of SIRT1 and suggests a potential strategy to stabilize SIRT1 to promote productive ageing.

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Fig. 1: SIRT1 protein is reduced during cellular senescence.
Fig. 2: SIRT1 is subjected to autophagosome–lysosome degradation during cellular senescence.
Fig. 3: SIRT1 associates with the autophagy protein LC3.
Fig. 4: SIRT1 interacts with LC3 through an LIR motif.
Fig. 5: SIRT1 undergoes lysosomal degradation during ageing in mice and humans.

Data availability

RNA sequencing data were referenced to the Gene Expression Omnibus under accession numbers GSE52848 and GSE53356. The mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD020081. The authors declare that the data that support the findings of this study are available within the manuscript. No restriction on data availability applies. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Zhao for help with confocal microscopy and L. Zhang for help with cell sorting by flow cytometry. We thank P. Klein, E. J. Wherry, F. Mourkioti and M. G. Vizioli for their help with the mouse experiments; R. Marmorstein for help with the SIRT1–LC3 interaction mapping experiments; and N. Raman and the Gladstone Flow Cytometry Core for assistance with FACS. C.X. acknowledges the Glenn/AFAR Scholarship for Research in the Biology of Aging. Z.D. is supported by NIH grant no. K99AG053406. S.L.B. and P.D.A. are supported by NIH grant no. P01AG031862. S.L.B. acknowledges the Glenn Foundation Award for Research in Biological Mechanisms of Aging.

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C.X., Z.D. and S.L.B. conceived the project. C.X. performed most of the experiments. Z.D. and L.W. performed cell-culture experiments. L.W., P.F. and M.O. performed human T-cell experiments. V.C., J.J. and W.T. performed HSPC isolation. M.N., J.D.W. and R.A. contributed autophagy reagents. T.J. and G.E. performed peptide arrays. C.L. and B.A.G. performed mass spectrometry analysis. C.B. and P.D.A. contributed to the mouse experiments. C.X., L.W., P.D.A., Z.D. and S.L.B. contributed to the experimental design. C.X., L.W., Z.D. and S.L.B. wrote the manuscript. All authors discussed the results and reviewed the manuscript.

Corresponding authors

Correspondence to Zhixun Dou or Shelley L. Berger.

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Extended data

Extended Data Fig. 1 Characterization of SIRT1 role and mRNA levels in cellular senescence.

a, Western blot showing SIRT1 expression in IMR90 cells stably expressing LPC vector or LPC-HA-SIRT1 construct; n = 3 independent experiments. b, β-gal-stained cells at day 9 post etoposide treatment were imaged by microscopy; n = 2 independent experiments. OE: overexpression. c, Percentages of β-gal staining-positive cells were quantified at indicated days after etoposide treatment. Data are mean ± s.d.; more than 500 cells and 5 fields were counted; two-way ANOVA with Sidak’s multiple comparisons test (p values). d, Western blot showing SIRT1 expression in primary BJ fibroblasts treated with 100 μM etoposide for 48 h in time course as indicated; n = 2 independent experiments. e, Western blot showing SIRT1 expression in IMR90 cells cultured at 100% confluency for indicated days; n = 2 independent experiments. f-i, RT-qPCR time course analysis of proliferating and senescent IMR90 cells induced by overexpressing ER:HRasV12 treated with 4OHT (f), by etoposide for 48 h (g) and BJ cells treated with etoposide for 48 h (h), and in IMR90 replicative senescent cells (i). Data were normalized to GAPDH; the bar indicates the average of three technical replicates. PD, population doubling. j, SIRT1 deacetylation activity assay of endogenous SIRT1 protein immunoprecipitated from extracts of proliferating or senescent IMR90 cells expressing inducible hairpins of shNTC and shAtg7. For senescent cells, cells were induced by Dox for 5 days, and then were subjected to etoposide treatment for 48h; Cells at Day 8 after etoposide treatment were harvested for analysis. For proliferating cells, cells were induced by Dox for 5 days and then were harvested for analysis. Proteins loaded were analysed by western blotting. This experiment has been repeated for two times. Statistical information and unprocessed blots are provided as source data.

Source data

Extended Data Fig. 2 Characterization of the SIRT1–LC3 interaction.

a, IP of extracts from proliferating and senescent IMR90 cells. Quantification: SIRT1 IP bands were normalized to LC3 IP and SIRT1 input bands. Mean ± s.d.; n = 6 independent experiments; paired two-tailed Students’ t-test. b, IP of extracts from proliferating and contact-inhibited IMR90 cells (at 100% confluency for 8 days). This experiment has been repeated once. Excessive beads and antibodies were used in the IP to capture nearly 100% of LC3 protein in the lysates. Flow: flow-through. c, IP of extracts from proliferating and contact-inhibited cells; n = 3 independent experiments. d, IP of nuclear extracts from proliferating and senescent cells. Excessive beads and antibodies were used to capture nearly 100% of LC3 protein in the lysates. This experiment has been repeated once. Flow: flow-through. e, IP of nuclear extracts from proliferating and senescent cells; n = 2 independent experiments. In a,d,e, senescent cells were harvested at day 8 after etoposide treatment. f, Western blotting of nuclear (Nuclear) and cytoplasmic (Cyto) extracts from proliferating and senescent cells; n = 2 independent experiments. g, Endogenous LC3 IP of IMR90 cell extracts with or without protein phosphatase Lambda treatment. Quantification: SIRT1 IP bands were normalized to LC3 IP and SIRT1 input bands. Mean ± s.d.; paired one-tailed Students’ t-test; n = 5 independent experiments. h-i, Mass spectrometry analysis of SIRT1 immunoprecipitated from proliferating and senescent IMR90 cells. h, Boxplot showing the peptide intensity distribution of SIRT1. N = 54 peptides; p value = 0.25; unpaired two-tailed Student’s t-test. The median of the data was indicated as the line in the box, and edges stand for the 25th/75th percentile. i, Phosphorylated peptides identified by mass spec and their phosphorylation levels in proliferating and senescence states. AA: amino acid. Source data are provided.

Source data

Extended Data Fig. 3 Characterization of the SIRT1 deacetylation role in starvation and senescence.

a-c, IMR90 cells that undergo CRISPR/Cas9-mediated gene inactivation of non-targeting control (PRM1, as PRM1 is involved in spermatogenesis and is not expressed in IMR90 cells) or SIRT1 were analysed under starvation and senescence conditions. This experiment has been repeated for two times. a, Cells were analysed by western blotting. b, Cells were subjected to 250 μM Torin 1 and 5 μM Lys05 treatment for 24 h, and analysed by western blotting. Relative LC3-II intensities to GAPDH were quantified. c, Cells at day 6 after etoposide-treated senescence were subjected to 2 μM or 5 μM Lys05 treatments for 24 h, and analysed by western blotting. Relative LC3-II intensities to GAPDH were quantified. d-e, IMR90 cells under proliferating, starvation (Torin 1 250 μM for 24 h) and senescence (induced by etoposide treatment, harvested at day 7) conditions were stained with LC3 antibody and analysed. d, Cells were imaged by confocal microscopy. Scale bar: 10 μm. e, Percentages of cells with nuclear LC3 signals were quantified. Starv: starvation. Mean ± s.d.; more than 500 cells were counted; each data point (n) represents cells in 10 random fields, n = 5 for all conditions; one-way ANOVA coupled with Turkey’s multiple comparisons test. f, IMR90 were treated as indicated ways for 24 h and analysed by western blotting; n = 2 independent experiments. CT: control. A.A.: amino acids. 2-DG: treatment of 10 mM 2-DG. Torin 1: treatment of 250 μM Torin 1. Statistical information and unprocessed blots are provided as source data.

Source data

Extended Data Fig. 4 Characterization of SIRT1 mutants and peptides.

a, Information of potential SIRT1–LC3 interaction regions identified in the peptide array as in Fig. 4a, and the corresponding synthetic peptides and mutants. Key amino acid residues are labelled in red. Potential region: LC3-binding regions on SIRT1 identified in the peptide array as in Fig. 4a. Peptide region: synthetic peptides tested in the peptide competition IP as in Fig. 4b. Peptide competition: results of the peptide competition IP as in Fig. 4b. Substitution generated: SIRT1 mutants tested in the IP as in Fig. 4c. b, SIRT1 deacetylation activity assay of SIRT1 WT or WV mutant immunoprecipitated from extracts of HEK293T expressing corresponding HA-tagged constructs. Proteins loaded were analysed by western blotting. This experiment has been repeated for two times. c, IP of HEK293T cells expressing HA-SIRT1 and Flag-LC3 constructs; n = 2 independent experiments. Cells were pre-treated with 20 μM resveratrol for 6 h. d, IMR90 cells at day 6 after etoposide-initiated senescence were subjected to treatment with 20 μM resveratrol for 48 h; n = 2 independent experiments. Cells were then harvested for western blotting. e, HEK293T cells expressing HA-SIRT1 and Flag-LC3 were treated with 20 μM resveratrol for 6 h, and were then harvested for SIRT1 activity assay. This experiment has been repeated for two times. f, SIRT1 deacetylation activity assay of SIRT1 WT or I347A mutant immunoprecipitated from extracts of HEK293T expressing corresponding HA-tagged constructs. Proteins loaded were analysed by western blotting; n = 2 independent experiments. g, IP of HEK293T cell lysates expressing Flag-tagged LC3 and HA-tagged SIRT1 WT or I347A, WV+I347A, or WV mutants. This experiment has been repeated for two times. Statistical information and unprocessed blots are provided as source data.

Extended Data Fig. 5 Analysis of SIRT1 in mouse tissues and HSPCs.

a,b, Thymus from young (2-4 months) and aged (19-26 months) C57BL/6 mice were lysed and analysed by western blotting (a) and RT-qPCR (b); n = 3 biologically independent animals in each group. RT-qPCR data were normalized to 18S; mean ± s.e.m.; unpaired two-tailed Students’ t-test. c. Indicated organs and tissues of young (3 months) and aged (19 months) mice were dissected and analysed by western blotting; n = 2 independent experiments. SE: short exposure; LE: long exposure. d,e, Young (3 months) mice were fed or fasted for 24 h. Spleens (d) and Testes (e) were harvested for western blotting; n = 2 biologically independent animals in each group. f,g, Young (2-4 months) mice were subjected to daily i.p. injection of 10 mg/kg Lys05 in PBS or PBS control in 100 μL volume for two weeks. Spleens (f) and testes (g) were analysed by western blotting. Western blot quantification: SIRT1 bands were normalized to GAPDH bands. For spleens, data are mean ± s.e.m.; control group n = 3 animals, Lys05 group n = 5 animals; two-tailed Mann-Whitney test. For testes, data are mean ± s.e.m.; n = 4 animals; two-tailed Mann-Whitney test. h, Representative flow cytometry plots of cell sorting of lineage-depleted bone marrow cells from young and aged mice to isolate Lin-Sca-1+c-Kit+ cells (HSPC populations). Boxes indicate cell populations isolated. i, HSPC populations were isolated from young (2-4 months) mice, cultured with or without 2 μM Lys05 for 24 hours and analysed by western blotting. This experiment has been repeated once. Statistical information and unprocessed blots are provided as source data.

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Supplementary information

Supplementary Information

Supplementary Fig. 1. Gating strategies used for cell sorting.

Reporting Summary

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Xu, C., Wang, L., Fozouni, P. et al. SIRT1 is downregulated by autophagy in senescence and ageing. Nat Cell Biol 22, 1170–1179 (2020). https://doi.org/10.1038/s41556-020-00579-5

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