Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria

  1. Minxian Qian
  2. Zuojun Liu
  3. Linyuan Peng
  4. Xiaolong Tang
  5. Fanbiao Meng
  6. Ying Ao
  7. Mingyan Zhou
  8. Ming Wang
  9. Xinyue Cao
  10. Baoming Qin
  11. Zimei Wang
  12. Zhongjun Zhou
  13. Guangming Wang
  14. Zhengliang Gao
  15. Jun Xu
  16. Baohua Liu  Is a corresponding author
  1. Shenzhen University Health Science Center, China
  2. Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, China
  3. University of Hong Kong, China
  4. Tongji University School of Medicine, China
  5. Shanghai Tenth People's Hospital, Tongji University School of Medicine, China
  6. Tongji University, China
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
ATM activation by chloroquine alleviates senescence.

(a) Immunoblots showing protein levels of ATM, NBS1, and RAP80 in human skin fibroblasts (HSFs). A gradually increased level of p16 indicates cellular senescence, while elevated γH2AX level …

https://doi.org/10.7554/eLife.34836.003
Figure 1—source data 1

Statistical analysis for SA-β-Gal positive staining.

https://doi.org/10.7554/eLife.34836.006
Figure 1—source data 2

Statistical analysis for EdU positive staining.

https://doi.org/10.7554/eLife.34836.007
Figure 1—figure supplement 1
Decline of ATM-centered DNA repair machinery during senescence.

(a) Real-time PCR analysis showing progressively elevated mRNA level of p21 in continuously cultured human endothelial cells (HUVEC). **p<0.01. (b) SA-β-Gal staining of HUVEC cells at indicated …

https://doi.org/10.7554/eLife.34836.004
Figure 1—figure supplement 2
ATM regulates replicative senescence.

(a) Representative images showing cells treated with Scramble (sh-NC) or sh-ATM. (b) Percent EdU-positive cells in sh-NC or sh-ATM treated HSFs. Views were randomly captured and at least 100 cells …

https://doi.org/10.7554/eLife.34836.005
Figure 2 with 2 supplements
ATM-SIRT6 axis regulates age-related metabolic reprogramming.

(a) Quantitative RT-PCR analysis of mRNA levels of indicated glycolytic genes in different passages of MEFs with or without treatment of CQ. Data represent means ± SEM. *p<0.05, **p<0.01, ***p<0.001.…

https://doi.org/10.7554/eLife.34836.008
Figure 2—source data 1

Differently expressed mRNA profiles of Atm-/- MEF cells.

https://doi.org/10.7554/eLife.34836.011
Figure 2—source data 2

Differentially expressed Metabolites in Atm KO MEFs.

https://doi.org/10.7554/eLife.34836.012
Figure 2—source data 3

Differently expressed mRNA profiles of Sirt6-/- MEF cells.

https://doi.org/10.7554/eLife.34836.013
Figure 2—figure supplement 1
Atm deficiency promotes glycolysis.

(a) Quantitative RT-PCR analysis of mRNA levels of indicated glycolytic genes in different passages of HSFs with or without treatment of CQ. Data represent means ± SEM. *p<0.05, **p<0.01, ***p<0.001.…

https://doi.org/10.7554/eLife.34836.009
Figure 2—figure supplement 2
SIRT6 reduction underlies age-related metabolic reprogramming triggered by ATM decline.

(a) Heatmap representation of RNA-Seq data showing relative changes of glycolytic genes in Sirt6-/- MEF cells. Red and green indicate up- and downregulation, respectively. (b) Quantitative RT-PCR …

https://doi.org/10.7554/eLife.34836.010
Figure 3 with 1 supplement
ATM interacts with and phosphorylates SIRT6.

(a) Immunoblots showing protein levels of SIRT6 in HEK293 cells expressing Flag-ATM or Flag-ATM S1981A. (b) Immunoblots showing endogenous ATM and p-S/TQ motif in anti-Flag immunoprecipitates in …

https://doi.org/10.7554/eLife.34836.014
Figure 3—figure supplement 1
ATM directly phosphorylates SIRT6.

(a) Immunoblots showing a significant increase in SIRT6 protein level, pS1981ATM, and pS824KAP-1 in the presence of DNA damage reagents-CPT and Dox in U2OS cells. (b) Immunoblots showing Sirt6 …

https://doi.org/10.7554/eLife.34836.015
Figure 4 with 1 supplement
ATM prevents ubiquitination and degradation of SIRT6.

(a) Immunoblots showing protein levels of Flag-SIRT6 in WT and ATM KO HEK293 cells treated with CHX (50 µg/ml) for indicated periods of time. (b) Quantification of protein levels in (a) by ImageJ. …

https://doi.org/10.7554/eLife.34836.016
Figure 4—figure supplement 1
ATM-mediated phosphorylation of SIRT6 prevents its ubiquitination and degradation.

(a) Immunoblots showing Sirt6 levels in wild-type and Atm null MEFs in the presence of CHX (50 μg/ml). (b–c) Immunoblots showing SIRT6 protein level in HEK293 cells in the presence of CHX (50 μg/ml) …

https://doi.org/10.7554/eLife.34836.017
Figure 5 with 2 supplements
CQ extends lifespan in an ATM- dependent manner.

(a) Survival analysis of C. elegans treated with the indicated dosage of CQ. **p<0.01. NS indicates no significant difference. (b) Survival analysis of wild-type and atm-1 null C. elegans cultured …

https://doi.org/10.7554/eLife.34836.018
Figure 5—source data 1

Lifespan analysis of chloroquine's effect on nematodes.

https://doi.org/10.7554/eLife.34836.021
Figure 5—source data 2

Lifespan analysis of chloroquine's effect on mice.

https://doi.org/10.7554/eLife.34836.022
Figure 5—figure supplement 1
ATM activation ameliorates aging-associated features.

(a) Survival analysis of sir-2.4 downregulated C. elegans exposed to medium with or without 1 µM CQ. ‘N.S.’ indicates no significant difference. (b) Quantitative RT-PCR analysis of mRNA levels of sir…

https://doi.org/10.7554/eLife.34836.019
Figure 5—figure supplement 2
Schematic model of ATM-SIRT6 axis in regulating aging and longevity.

Left, DNA damage activates DDR cascade, and its constant activation leads to permanent cell cycle exit and senescence. Right, defective ATM-SIRT6 axis underlies premature aging in mouse models …

https://doi.org/10.7554/eLife.34836.020
Author response image 1
Physiological (3%) oxygen condition is inappropriate for MEFs culture to assess the replicative lifespan-extending effect of CQ.

(a) MEFs at passage 1 were cultured in 20% O2 (black) or 3% O2 (grey) with treatment of 1 μM CQ (circles) or DMSO (squares), and cell number was determined at each passage. ***P < 0.01. (b) …

https://doi.org/10.7554/eLife.34836.025
Author response image 2
Lifespan analysis for worms overexpressing ATM-1.

(a) The strategy for constructing ATM-1 overexpressing plasmid with GFP-tag. (b) Representative images showing the detectable GFP expression in worms. (c) Survival analysis of worm line expressing …

https://doi.org/10.7554/eLife.34836.026

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (human)ATMNational Center for Biotechnology InformationGene ID: 472
Gene (mouse)AtmNational Center for Biotechnology InformationGene ID: 11920
Gene (Caenorhabditis elegans)atm-1National Center for Biotechnology InformationGene ID: 3565793
Gene (human)SIRT6National Center for Biotechnology InformationGene ID: 51548
Gene (mouse)Sirt6National Center for Biotechnology InformationGene ID: 50721
Gene (Caenorhabditis elegans)sir-2.4National Center for Biotechnology InformationGene ID: 182284
Gene (mouse)Zmpste24National Center for Biotechnology InformationGene ID: 230709
Gene (mouse)p53National Center for Biotechnology InformationGene ID: 230710
Cell line (human)HEK293ATCCCatalog number: ATCC CRL-1573; RRID:CVCL_0042
Cell line (human)HepG2ATCCCatalog number: ATCC HB-8065; RRID:CVCL_0027
Cell line (human)U2OSATCCCatalog number: ATCC HTB-96; RRID:CVCL_0042
Cell line (mouse)Atm-/-; p53-/- MEFfrom Dr. Yosef Shiloh (Tel Aviv University, Israel)
Cell line (mouse)Sirt6-/- MEFfrom Dr. Raul Mostoslavsky (Massachusetts General Hospital Cancer center, USA)
AntibodyATMAbcam (Cambridge, UK)Cat# ab78; RRID:AB_306089Applications: WB;Immunofluorescence
AntibodySIRT6Abcam (Cambridge, UK)Cat# ab62739; RRID:AB_956300Applications: WB; Immunofluoresce;Chromatin immunoprecipitation
AntibodyγH2AXAbcam (Cambridge, UK)Cat# ab81299; RRID:AB_1640564Applications: WB
Antibodyp21Santa Cruz BiotechnologyCat# sc-6246; RRID:AB_628073Applications: WB
AntibodyMDM2Santa Cruz BiotechnologyCat# sc-965; RRID:AB_627920Applications: WB
Antibodyp-ATM (Ser1981)EMD MilliporeCat# 05–740; RRID:AB_309954Applications: WB
AntibodyH3K9acEMD MilliporeCat# 07–352; RRID:AB_310544Applications: WB; Chromatin immunoprecipitation
Antibodyp-S/T QCell Signaling Technology (Beverly, MA)Cat #9607S; RRID:AB_10889739Applications: WB
Antibodycleaved caspase-3Cell Signaling Technology (Beverly, MA)Cat #9661; RRID:AB_2341188Applications: WB
AntibodyHASigma-AldrichCat# H3663; RRID:AB_262051Applications: WB
AntibodyFlagSigma-AldrichCat# F1804; RRID: AB_262044Applications: WB
AntibodyLC3BSigma-AldrichCat# L7543; RRID:AB_796155Applications: WB
Transfected construct (human)Flag-His-ATM wtAddgene (Cambridge, MA)Cat #31985
Transfected construct (human)Flag-SIRT6Addgene (Cambridge, MA)Cat #13817
Transfected construct (human)Flag-His-ATM S1981AAddgene (Cambridge, MA)Cat #32300
Commercial assay or kitSenescence beta- galactosidase staining KitCell Signaling Technology (Beverly, MA)Cat #9860
Commercial assay or kitLactate Colorimetric Assay KitBioVisionCat #K667-100
Commercial assay or kitClick-iT EdU Alexa Fluor 488 KitInvitrogenCat #C10425
Chemical compound, drugCycloheximideSigma-AldrichCat #66-81-9
Chemical compound, drugMG-132Sigma-AldrichCat #474787
Chemical compound, drugChloroquineSigma-AldrichCat #C6628

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