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Overexpression of Atg5 in mice activates autophagy and extends lifespan.

Pyo JO, Yoo SM, Ahn HH, Nah J, Hong SH, Kam TI, Jung S, Jung YK - Nat Commun (2013)

Bottom Line: Autophagy has been implicated in the ageing process, but whether autophagy activation extends lifespan in mammals is unknown.We demonstrate that moderate overexpression of Atg5 in mice enhances autophagy, and that Atg5 transgenic mice showed anti-ageing phenotypes, including leanness, increased insulin sensitivity and improved motor function.Our observations suggest that the leanness and lifespan extension in Atg5 transgenic mice may be the result of increased autophagic activity.

View Article: PubMed Central - PubMed

Affiliation: Global Research Laboratory, School of Biological Sciences/Bio-MAX Institute, Seoul National University, Seoul 151-747, Korea.

ABSTRACT
Autophagy has been implicated in the ageing process, but whether autophagy activation extends lifespan in mammals is unknown. Here we show that ubiquitous overexpression of Atg5, a protein essential for autophagosome formation, extends median lifespan of mice by 17.2%. We demonstrate that moderate overexpression of Atg5 in mice enhances autophagy, and that Atg5 transgenic mice showed anti-ageing phenotypes, including leanness, increased insulin sensitivity and improved motor function. Furthermore, mouse embryonic fibroblasts cultured from Atg5 transgenic mice are more tolerant to oxidative damage and cell death induced by oxidative stress, and this tolerance was reversible by treatment with an autophagy inhibitor. Our observations suggest that the leanness and lifespan extension in Atg5 transgenic mice may be the result of increased autophagic activity.

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Enhanced autophagy in Atg5 Tg mice.(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a). (c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P<0.0001 versus control; Student’s t-test. (e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and Atg5/GFP-LC3 Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0). (f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.
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f2: Enhanced autophagy in Atg5 Tg mice.(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a). (c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P<0.0001 versus control; Student’s t-test. (e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and Atg5/GFP-LC3 Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0). (f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.

Mentions: We analysed the expression levels of endogenous and transgenic Atg5 in the tissues of WT and Atg5 Tg mice no. 25 using western blotting. Compared with WT mice, the expression of Atg5 was highly increased in the lung, heart, liver and muscle (≥2-fold) and marginally increased in the brain of Atg5 Tg mice (Fig. 2a). Accordingly, the levels of Atg12-Atg5 conjugate and LC3 I and the conversion of LC3 I to LC3 II, which are markers of autophagy14, were apparently higher in almost all of the tissues of Atg5 Tg mice compared with WT mice. Consistently, the levels of p62 protein, an indicator of ubiquitinated protein21, were drastically decreased in the tissues of Atg5 Tg mice (Fig. 2a). Although there were marginal differences in the expression levels of p62 and Atg5 during ageing of WT mice, autophagy activity was higher in the heart of all ages of Atg5 Tg mice than WT mice (Supplementary Fig. S2).


Overexpression of Atg5 in mice activates autophagy and extends lifespan.

Pyo JO, Yoo SM, Ahn HH, Nah J, Hong SH, Kam TI, Jung S, Jung YK - Nat Commun (2013)

Enhanced autophagy in Atg5 Tg mice.(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a). (c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P<0.0001 versus control; Student’s t-test. (e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and Atg5/GFP-LC3 Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0). (f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.
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Related In: Results  -  Collection

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f2: Enhanced autophagy in Atg5 Tg mice.(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a). (c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P<0.0001 versus control; Student’s t-test. (e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and Atg5/GFP-LC3 Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0). (f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.
Mentions: We analysed the expression levels of endogenous and transgenic Atg5 in the tissues of WT and Atg5 Tg mice no. 25 using western blotting. Compared with WT mice, the expression of Atg5 was highly increased in the lung, heart, liver and muscle (≥2-fold) and marginally increased in the brain of Atg5 Tg mice (Fig. 2a). Accordingly, the levels of Atg12-Atg5 conjugate and LC3 I and the conversion of LC3 I to LC3 II, which are markers of autophagy14, were apparently higher in almost all of the tissues of Atg5 Tg mice compared with WT mice. Consistently, the levels of p62 protein, an indicator of ubiquitinated protein21, were drastically decreased in the tissues of Atg5 Tg mice (Fig. 2a). Although there were marginal differences in the expression levels of p62 and Atg5 during ageing of WT mice, autophagy activity was higher in the heart of all ages of Atg5 Tg mice than WT mice (Supplementary Fig. S2).

Bottom Line: Autophagy has been implicated in the ageing process, but whether autophagy activation extends lifespan in mammals is unknown.We demonstrate that moderate overexpression of Atg5 in mice enhances autophagy, and that Atg5 transgenic mice showed anti-ageing phenotypes, including leanness, increased insulin sensitivity and improved motor function.Our observations suggest that the leanness and lifespan extension in Atg5 transgenic mice may be the result of increased autophagic activity.

View Article: PubMed Central - PubMed

Affiliation: Global Research Laboratory, School of Biological Sciences/Bio-MAX Institute, Seoul National University, Seoul 151-747, Korea.

ABSTRACT
Autophagy has been implicated in the ageing process, but whether autophagy activation extends lifespan in mammals is unknown. Here we show that ubiquitous overexpression of Atg5, a protein essential for autophagosome formation, extends median lifespan of mice by 17.2%. We demonstrate that moderate overexpression of Atg5 in mice enhances autophagy, and that Atg5 transgenic mice showed anti-ageing phenotypes, including leanness, increased insulin sensitivity and improved motor function. Furthermore, mouse embryonic fibroblasts cultured from Atg5 transgenic mice are more tolerant to oxidative damage and cell death induced by oxidative stress, and this tolerance was reversible by treatment with an autophagy inhibitor. Our observations suggest that the leanness and lifespan extension in Atg5 transgenic mice may be the result of increased autophagic activity.

Show MeSH
Related in: MedlinePlus