Abstract
Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1–BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.
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Change history
19 June 2018
In this Letter, the graphs in Fig. 2a and c were inadvertently the same owing to a copy and paste error from the original graphs in Prism. The Source Data files containing the raw data were correct. Fig. 2c has been corrected online.
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Acknowledgements
We thank S. Sciarretta for discussions; N. Mizushima for reagents; L. Nguyen for technical assistance and H. Smith for assistance with manuscript preparation. This work was supported by NIH grants RO1-CA109618 (B.L.), U19-AI199725 (B.L.), RO1-DK091392 and RO1-DK092461 (M.C.H. and O.W.M.), P30-DK07938 (O.W.M.), K99R00-DK094980 (C.H.), Cancer Prevention Research Institute of Texas grant RP120718 (B.L.) and a Fondation Leducq grant 15CBD04 (B.L., A.F.F. and S.S.).
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Nature thanks D. Harrison and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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A.F.F., S.S., Y.W., C.H., T.T., Y.L., M.C.H. and B.L. designed the study. A.F.F., S.S., Y.W., Z.Z., M.S., K.L.M. and W.-C.C. performed biochemical analyses. A.F.F., S.S. and Z.Z. performed autophagy microscopic analyses. A.F.F. and S.S. performed renal and cardiac histopathological analyses. G.B. characterized malignancies. A.F.F., S.S., D.K.M., G.G.S, G.B., O.W.M., M.C.H. and B.L. discussed and analysed data. A.F.F., S.S., M.C.H. and B.L. wrote the manuscript. A.F.F. and S.S. contributed equally and the order of these authors was determined arbitrarily.
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B.L. is a Scientific Founder of Casma Therapeutics, Inc.
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Extended data figures and tables
Extended Data Fig. 1 Increased basal autophagy in tissues of beclin 1(F121A) knock-in mice.
a, Representative images and quantification of GFP–LC3 puncta (autophagosomes) in glomeruli from Becn1+/+;GFP-LC3 (wild-type, WT) and Becn1F121A/F121A;GFP-LC3 (knock-in, KI) mice with or without 50 mg kg−1 chloroquine for 6 h. Scale bars, 10 μm. Data are mean ± s.e.m. for three mice per genotype. b, Enlarged versions of the images shown in Fig. 1c. P values determined by one-sided unpaired t-test. Arrows denote representative GFP–LC3 puncta.
Extended Data Fig. 2 Sustained increase in basal autophagy during adulthood in beclin 1(F121A) knock-in mice.
a, Co-immunoprecipitation of beclin 1 and BCL2 in representative samples of hearts and kidneys from eight-month-old Becn1+/+ (wild-type) and Becn1F121A/F121A (knock-in) animals. b, Quantification of beclin 1 co-immunoprecipitated with BCL2 in indicated tissues of eight-month-old wild-type and knock-in mice (n = 3 mice per genotype). c, Quantification of GFP–LC3 puncta in hearts and tissues from six-month-old Becn1+/+;GFP-LC3 (wild-type) and Becn1F121A/F121A;GFP-LC3 (knock-in) mice with or without chloroquine (50 mg kg−1, 6 h). d, Western blot analysis of autophagy markers in the hearts and kidneys from eight-month-old wild-type and knock-in mice. Each lane represents a different mouse. e, Quantification of p62 and total LC3 levels (normalized to β-actin), as well as LC3-II/LC3-I ratios from samples in d. Data are mean ± s.e.m. for three mice per genotype. P values were determined by one-sided unpaired t-test. For uncropped gels, see Supplementary Fig. 1.
Extended Data Fig. 3 Increased autophagy, but not endocytosis, in beclin 1(F121A) MEFs.
a, Co-immunoprecipitation of beclin 1 and BCL2 in MEFs derived from Becn1+/+ (wild-type) and Becn1F121A/F121A (knock-in) animals. b, Representative images (top) and quantification (bottom) of GFP–LC3 puncta in wild-type and knock-in cells with or without 10 nM bafilomycin A1 (BafA1) for 3 h. Scale bars, 10 µm. c, Western blot analysis of autophagy markers in wild-type and knock-in MEFs with or without BafA1 (100 nM, 2 h). d, Representative images and quantitative electron microscopic analysis of autophagic structures in wild-type and knock-in MEFs with or without BafA1 (100 nM, 3 h). Insets show representative autophagosome (arrowhead) and autolysosome (arrow). Scale bars, 1 µm. e, Representative images and quantification of transferrin uptake kinetics in wild-type and knock-in cells. Scale bars, 20 µm. Results shown are representative of two and four independent experiments respectively for a and c. Data are mean ± s.e.m. for three replicates in b and for 50 cells per genotype and condition in d. Data points on line graph in e denote mean ± s.e.m. for cells at 7 min (n = 63, WT; n = 54, KI), 15 min (n = 58, WT; n = 52 KI), and 30 min (n = 76, WT; n = 83, KI). P values determined by unpaired one-sided (b) and two-sided (d, e) t-test. For uncropped gels, see Supplementary Fig. 1.
Extended Data Fig. 4 Apoptosis and autophagy analyses in kidneys and hearts of aged mice.
a, b, Representative images and quantification of active caspase 3-positive cells in kidneys (a) and hearts (b) from Becn1+/+ (wild-type) and Becn1F121A/F121A (knock-in) animals. Two month-old wild-type and knock-in mouse kidneys and hearts (n = 6 per genotype) were analysed. For kidney analyses, aged (20-month-old) wild-type (n = 20) and knock-in (n = 26) mice were used. For heart analyses, aged (20-month-old) wild-type (n = 19) and knock-in (n = 26) mice were used. Scatter plot bars represent median ± interquartile ranges. P values were determined by two-sided Mann–Whitney test. c, d, Enlarged versions of the endogenous LC3 puncta (autophagosome) images shown in Fig. 3d (c) and Fig. 3h (d). Arrows denote representative LC3 puncta.
Extended Data Fig. 5 In vitro klotho treatment disrupts the beclin 1–BCL2 interaction.
Co-immunoprecipitation of beclin 1 and BCL2 in HeLa cells treated with PBS or the indicated concentrations of recombinant full-length mouse klotho protein for 24 h. Result shown is representative of two independent experiments. For uncropped gels, see Supplementary Fig. 1.
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This file contains Supplementary Figure 1 which shows the uncropped gels for all the main and Extended Data Figures
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Fernández, Á.F., Sebti, S., Wei, Y. et al. Disruption of the beclin 1–BCL2 autophagy regulatory complex promotes longevity in mice. Nature 558, 136–140 (2018). https://doi.org/10.1038/s41586-018-0162-7
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DOI: https://doi.org/10.1038/s41586-018-0162-7
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