Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice

doi:10.1038/s41586-018-0162-7

. 2018 Jun;558(7708):136-140.

doi: 10.1038/s41586-018-0162-7. Epub 2018 May 30.

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice

Álvaro F Fernández^{1

2}, Salwa Sebti^{1

2}, Yongjie Wei^{1

2

3}, Zhongju Zou^{1

2

3}, Mingjun Shi⁴, Kathryn L McMillan⁴, Congcong He⁵, Tabitha Ting^{1

2}, Yang Liu^{1

2

3}, Wei-Chung Chiang^{1

2}, Denise K Marciano², Gabriele G Schiattarella², Govind Bhagat⁶, Orson W Moe^{2

4

7}, Ming Chang Hu^{8

9}, Beth Levine^{10

11

12

13}

Affiliations

¹ Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, TX, USA.
² Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.
³ Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁴ Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁵ Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
⁶ Department of Pathology and Cell Biology, Columbia University Medical Center and New York Presbyterian Hospital, New York, NY, USA.
⁷ Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁸ Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA. ming-chang.hu@utsouthwestern.edu.
⁹ Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, TX, USA. ming-chang.hu@utsouthwestern.edu.
¹⁰ Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, TX, USA. beth.levine@utsouthwestern.edu.
¹¹ Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA. beth.levine@utsouthwestern.edu.
¹² Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA. beth.levine@utsouthwestern.edu.
¹³ Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA. beth.levine@utsouthwestern.edu.

PMID: 29849149
PMCID: PMC5992097
DOI: 10.1038/s41586-018-0162-7

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice

Álvaro F Fernández et al. Nature. 2018 Jun.

. 2018 Jun;558(7708):136-140.

doi: 10.1038/s41586-018-0162-7. Epub 2018 May 30.

Authors

Affiliations

¹ Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, TX, USA.
² Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.
³ Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁴ Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁵ Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
⁶ Department of Pathology and Cell Biology, Columbia University Medical Center and New York Presbyterian Hospital, New York, NY, USA.
⁷ Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁸ Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA. ming-chang.hu@utsouthwestern.edu.
⁹ Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, TX, USA. ming-chang.hu@utsouthwestern.edu.
¹⁰ Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, TX, USA. beth.levine@utsouthwestern.edu.
¹¹ Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA. beth.levine@utsouthwestern.edu.
¹² Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA. beth.levine@utsouthwestern.edu.
¹³ Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA. beth.levine@utsouthwestern.edu.

PMID: 29849149
PMCID: PMC5992097
DOI: 10.1038/s41586-018-0162-7

Erratum in

Author Correction: Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.
Fernández ÁF, Sebti S, Wei Y, Zou Z, Shi M, McMillan KL, He C, Ting T, Liu Y, Chiang WC, Marciano DK, Schiattarella GG, Bhagat G, Moe OW, Hu MC, Levine B. Fernández ÁF, et al. Nature. 2018 Sep;561(7723):E30. doi: 10.1038/s41586-018-0270-4. Nature. 2018. PMID: 29921925

Abstract

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established^1,2. 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 (Becn1^F121A/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 Becn1 ^F121A/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 klotho ³ 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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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

**Extended Data Figure 1. Increased basal autophagy in tissues of beclin 1 F121A knock-in mice**
a, Representative images and quantitation of GFP-LC3 puncta (autophagosomes) in glomeruli from *Becn1^WT/WT*:GFP-LC3 (WT) and *Becn1^F121A/F121A*:GFP-LC3 (KI) mice +/− chloroquine (CQ) (50 mg kg⁻¹, 6 h). Scale bars, 10 μm. Bars represent mean ± s.e.m for three mice per genotype. b, Enlarged versions of the images shown in Fig. 1c of main text. P-values, one-sided unpaired t-test. Arrows denote representative GFP-LC3 puncta.

**Extended Data Figure 2. Sustained increase in basal autophagy during adulthood in beclin 1 F121A knock-in mice**
a, Co-immunoprecipitation of beclin 1 and Bcl-2 in representative samples of hearts and kidneys from 8 month-old *Becn1^WT/WT* (WT) and *Becn1^F121A/F121A* (KI) animals. b, Quantitation of beclin 1 co-immunoprecipitated with Bcl-2 in indicate tissues of 8 month-old WT and KI mice (n = 3 mice per genotype). c, Quantitation of GFP-LC3 puncta in hearts and tissues from 6 month-old in *Becn1^WT/WT*:GFP-LC3 (WT) and *Becn1^F121A/F121A*:GFP-LC3 (KI) mice +/− CQ (50 mg kg⁻¹, 6 h). d, Western blot analysis of autophagy markers in hearts and kidneys from 8 month-old WT and KI mice. Each lane represents a different mouse. e, Quantitation of p62 and total LC3 levels (normalized to β-Actin), as well as LC3-II/I ratios from samples in **(d)**. Bars represent mean ± s.e.m for three mice per genotype. P-values, one-sided unpaired t-test. WCL, whole cell lysate. For uncropped gels, see Supplementary Fig. 1.

**Extended Data Figure 3. Increased autophagy, but not endocytosis, in beclin 1 F121A murine embryonic fibroblasts (MEFs)**
a, Co-immunoprecipitation of beclin 1 and Bcl-2 in MEFs derived from *Becn1^WT/WT* (WT) and *Becn1^F121A/F121A* (KI) animals. b, Representative images (top) and quantitation (bottom) of GFP-LC3 puncta in WT and KI cells +/− bafilomycin A1 (BafA1) (10 nM, 3 h). Scale bars, 10 μm c, Western blot analysis of autophagy markers in WT and KI MEFs +/− Baf A1 (100 nM, 2 h). d, Representative images and quantitative electron microscopic analysis of autophagic structures in WT and KI MEFs +/− Baf A1 (100 nM, 3 h). Insets show representative autophagosome (arrowhead) and autolysosome (arrow). Scale bars, 1 μm. e, Representative images and quantitation of transferrin uptake kinetics in WT and KI cells. Scale bars, 20 μm. Results shown are representative of 2 and 4 independent experiments respectively for (a) and (c). Bars represent mean ± s.e.m for 3 replicates in (b) and for 50 cells per genotype and condition in (d). Data points on line graph in (e) represent 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 for indicated comparisons, unpaired t-test one-sided for (b) and two-sided for (d) and (e). WCL, whole cell lysate. For uncropped gels, see Supplementary Fig. 1.

**Extended Data Figure 4. Apoptosis and autophagy analyses in kidneys and hearts of aged mice**
Representative images and quantitation of active caspase 3-positive cells in kidneys (a) and hearts (b) from *Becn1^WT/WT* (WT) and *Becn1^F121A/F121A* (KI) animals. Two month-old WT and KI mouse kidneys and hearts (n=6 per genotype) were analysed. For kidney analyses, aged (20 month-old) WT (n=20) and KI (n=26) mice were used. For heart analyses, aged (20 month-old) WT (n=19) and KI (n=26) mice were used. Scatter plot bars represent median ± interquartile ranges. P-values, two-sided Mann-Whitney test. c–d, Enlarged versions of the endogenous LC3 puncta (autophagosome) images shown in Fig. 3d (c) and 3h (d) of main text. Arrows denote represent LC3 puncta.

**Extended Data Figure 5. *In vitro* Klotho treatment disrupts beclin 1/Bcl-2 interaction**
Co-immunoprecipitation of beclin 1 and Bcl-2 in HeLa cells treated with PBS or indicated concentrations of recombinant full-length mouse Klotho for 24 h. Result shown is representative of 2 independent experiments. For uncropped gels, see Supplementary Fig. 1.

**Figure 1. Effects of beclin 1 F121A mutation on beclin 1/Bcl-2 interaction and basal autophagy**
a, Co-immunoprecipitation of beclin 1 and Bcl-2 in indicated tissues from two month-old *Becn1^WT/WT* (WT) and *Becn1^F121A/F121A* (KI) animals. b, Quantitation of beclin 1 co-immunoprecipitated with Bcl-2 in **(a). c,** Representative images of GFP-LC3 puncta (autophagosomes) in indicated tissues from *Becn1^WT/WT*:GFP-LC3 (WT) and *Becn1^F121A/F121A*:GFP-LC3 (KI) mice +/− chloroquine (CQ) (50 mg kg⁻¹, 6 h). Scale bars, 10 μm. (See enlarged images, Extended Data 1). White arrows denote representative GFP-LC3 puncta. d, Quantitation of GFP-LC3 puncta +/− CQ in indicated tissues. e, Western blot analysis of autophagy markers in hearts and kidneys of 2 month-old WT and KI mice. f, Quantitation of p62 and total LC3 levels (normalized to β-Actin) and LC3-II/I ratio in **(e)**. Bars represent mean ± s.e.m for three mice per genotype. For (a) and (e), each lane represents a different mouse. One-sided unpaired t-test. PCT, renal proximal convoluted tubules. WCL, whole cell lysate. For uncropped gels, see Supplementary Fig. 1.

**Figure 2. Beclin 1 F121A KI mutation extends lifespan in mice**
Kaplan-Meier survival curves for *Becn1*^WT/WT (WT) and *Becn1^F121A/F121A* (KI) mice, showing the lifespan of all mice in cohort **(a)**, females alone **(b)** or males alone **(c)**. N = number of mice per group. P-values are calculated by log-rank (Mantel-Cox) test.

**Figure 3. Beclin 1 F121A KI mutation improves healthspan in mice**
**a–d**, Representative images and quantitation of pathological score (a); TUNEL-positive nuclei (b); interstitial fibrosis (c) and endogenous LC3 puncta (autophagosomes) (see enlarged images, Extended Data Fig. 4c) (d) in the cortical region of the kidney. e–h, Representative images and quantitation of TUNEL-positive nuclei (e); cardiomyocyte cross-sectional fiber size (f); cardiac interstitial fibrosis (g); and endogenous LC3 puncta (see enlarged images, Extended Data Fig. 4d) (h) in the heart. Two month-old WT and KI mouse kidneys and hearts (n=6 per genotype) were analyzed. For kidney analyses, 20 month-old WT (n=20 and n=16) and KI (n=26 and n=19) mice were used for histopathological and autophagy analyses, respectively. For heart analyses, 20 month-old WT (n=19 and n=15) and KI (n=22 and n=19) mice were used for histopathological and autophagy analyses, respectively. Scatter plot bars represent median ± interquartile ranges. P-values for indicated comparisons, Mann-Whitney test. Autophagy analyses were one-sided and all other analyses were two-sided. **i–j,** Percentage of WT and KI mice (aged 20 months) with spontaneous tumors, including lymphoproliferative disease (LPD), lymphomas, and non-lymphoid malignancies (i) and representative images of most frequently observed neoplastic lesions from WT mice (j). See text for statistical analyses of data. Scale bars, 50 μm (a–c, e–g, j) and 10 μm (d, h). For (d) and (h), red arrows denote representative endogenous LC3 puncta.

**Figure 4. Expression of beclin 1 F121A prevents lethality of Klotho-deficient mice**
a, Co-immunoprecipitation of beclin 1 and Bcl-2 in kidneys from mice of indicated genotype (n = 3 animals per genotype). b, Quantitation of beclin 1 co-immunoprecipitated with Bcl-2 in **(a). c**, Western blot analysis of autophagy markers in kidney from mice of indicated genotype. d, Quantitation of western blot analysis of p62 levels and LC3-II/I ratios (combined results are for six mice per genotype; each independent experiment had 1 mouse per genotype). Bars represent mean ± s.e.m. e–g, Kaplan-Meier survival curves for mice of indicated genotype, showing the lifespan of all mice in cohort **(e)**, females alone **(f)** or males alone **(g)**. N = number of mice per group. h, Representative images of 10 week-old mice of indicated genotype for body size comparison. Scale bars, 1 cm. i, Serial body weights in female and male mice of indicated genotype. Data points and error bars represent mean ± s.e.m for 12 mice per genotype. P-values in (b) and (d), one-sided unpaired t-test. P-values in (e–g), log-rank (Mantel-Cox) test. WCL, whole cell lysate. WT, wild-type mice; beclin 1 KI, *Becn1^F121A/F121A* mice; Klotho HM, *klotho^HM/HM* mice and beclin 1 KI/Klotho HM, *Becn1^F121A/F121A; klotho^HM/HM* mice. For uncropped gels, see Supplementary Fig. 1.

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Comment in

Self-Digestion for Lifespan Extension: Enhanced Autophagy Delays Aging.
Arensman MD, Eng CH. Arensman MD, et al. Mol Cell. 2018 Aug 16;71(4):485-486. doi: 10.1016/j.molcel.2018.08.002. Mol Cell. 2018. PMID: 30118677

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References

1. Madeo F, Zimmermann A, Maiuri MC, Kroemer G. Essential role for autophagy in life span extension. J Clin Invest. 2015;125:85–93. - PMC - PubMed
1. Rubinsztein DC, Marino G, Kroemer G. Autophagy and aging. Cell. 2011;146:682–695. - PubMed
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[1] Madeo F, Zimmermann A, Maiuri MC, Kroemer G. Essential role for autophagy in life span extension. J Clin Invest. 2015;125:85–93. - PMC - PubMed

[2] Madeo F, Zimmermann A, Maiuri MC, Kroemer G. Essential role for autophagy in life span extension. J Clin Invest. 2015;125:85–93. - PMC - PubMed

[3] Rubinsztein DC, Marino G, Kroemer G. Autophagy and aging. Cell. 2011;146:682–695. - PubMed

[4] Rubinsztein DC, Marino G, Kroemer G. Autophagy and aging. Cell. 2011;146:682–695. - PubMed

[5] Kuro-o M, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997;390:45–51. - PubMed

[6] Kuro-o M, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997;390:45–51. - PubMed

[7] Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42. - PMC - PubMed

[8] Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42. - PMC - PubMed

[9] Melendez A, et al. Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science. 2003;301:1387–1391. - PubMed

[10] Melendez A, et al. Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science. 2003;301:1387–1391. - PubMed

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Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice

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Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice

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