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Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T - J. Cell Biol. (2005)

Bottom Line: Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation.Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies.Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.

ABSTRACT
Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

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Impaired autophagosome formation in Atg7-deficient liver. (A–H) Electron micrographs of liver from Atg7F/+:Mx1 (A–D) and Atg7F/F:Mx1 (E–H) mice fed ad libitum (A and E) or fasted for 1 d (B and F). (C and D) Early stages of autophagic vacuoles observed in B are highlighted. (E and F) Autophagosome was not induced in mutant hepatocytes upon fasting. Insets show higher magnification views of glycogen granules. (G and H) Occasionally observed autophagosome-like structures in mutant hepatocytes. Bars: (A, B, E, and F) 5 μm; (C, D, G, and H) 0.5 μm. (I) Number of autophagosomes per hepatocyte (n = 20) in each genotype was counted and their averages are shown. (J–M) Immunofluorescent analysis of LC3 in primary cultured hepatocytes. Hepatocytes isolated from Atg7F/+:Mx1 (J and K) and Atg7F/F:Mx1 mice (L and M) were cultured in Williams' E (J and L) or Hanks' solution (K and M). Inset highlights the cup-like structure of LC3 observed in K. (N) Immunoblot analysis of Atg8 homologues in the liver. Atg7F/+:Mx1 (lanes 1 and 2) and Atg7F/F:Mx1 mice (lanes 3 and 4) were fed ad libitum (lanes 1 and 3) or fasted for 1 d (lanes 2 and 4), and then PNS fractions of liver were analyzed by immunoblotting with anti-LC3, GABARAP, GATE-16, and actin antibodies. Asterisk denotes a nonspecific band. Data shown are representative of three separate experiments.
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fig3: Impaired autophagosome formation in Atg7-deficient liver. (A–H) Electron micrographs of liver from Atg7F/+:Mx1 (A–D) and Atg7F/F:Mx1 (E–H) mice fed ad libitum (A and E) or fasted for 1 d (B and F). (C and D) Early stages of autophagic vacuoles observed in B are highlighted. (E and F) Autophagosome was not induced in mutant hepatocytes upon fasting. Insets show higher magnification views of glycogen granules. (G and H) Occasionally observed autophagosome-like structures in mutant hepatocytes. Bars: (A, B, E, and F) 5 μm; (C, D, G, and H) 0.5 μm. (I) Number of autophagosomes per hepatocyte (n = 20) in each genotype was counted and their averages are shown. (J–M) Immunofluorescent analysis of LC3 in primary cultured hepatocytes. Hepatocytes isolated from Atg7F/+:Mx1 (J and K) and Atg7F/F:Mx1 mice (L and M) were cultured in Williams' E (J and L) or Hanks' solution (K and M). Inset highlights the cup-like structure of LC3 observed in K. (N) Immunoblot analysis of Atg8 homologues in the liver. Atg7F/+:Mx1 (lanes 1 and 2) and Atg7F/F:Mx1 mice (lanes 3 and 4) were fed ad libitum (lanes 1 and 3) or fasted for 1 d (lanes 2 and 4), and then PNS fractions of liver were analyzed by immunoblotting with anti-LC3, GABARAP, GATE-16, and actin antibodies. Asterisk denotes a nonspecific band. Data shown are representative of three separate experiments.

Mentions: To delete Atg7 gene in the adult mice, we bred the Atg7F/F mice with Mx1-Cre transgenic mice that express the Cre recombinase in response to interferon γ or its chemical inducer, polyinosinic acid–polycytidylic acid (pIpC). The Mx1-Cre transgenic mice can excise Flox allele completely in the liver and spleen and partially in the kidney and heart (Kuhn et al., 1995). Intraperitoneal injections of pIpC resulted in effective recombination of the Atg7Flox allele in the liver and spleen (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200412022/DC1; and not depicted). No Atg7 transcript, protein, and activity were detected, similar to Atg7−/− mice (Fig. S2). Next, we tested the autophagosome formation under fasting condition. 1-d fasting resulted in induction of typical autophagosomes in control Atg7F/+:Mx1 mice (Fig. 3, A–D and I). In contrast, no such induction of autophagosome formation was noted in the liver of fasted Atg7F/F:Mx1 mice (Fig. 3, E, F, and I). Although some autophagosome-like structures were occasionally observed both in fed and fasted mutant mice livers (Fig. 3, G and H), they tended to be smaller than those observed in fasted control liver and hardly contained large cytoplasmic organelles (compare with Fig. 3, C and D). The number of autophagosomes per hepatocyte was counted and the mean values are shown in Fig. 3 I. The mutant hepatocytes lacked typical glycogen area, in contrast to the fed hepatocytes (Fig. 3, A and E); however, well-developed glycogen granules (α granules) were observed between numerous smooth endoplasmic reticula (Fig. 3 E, inset). Immunofluorescent analysis also revealed the presence of many cup-shaped and ringlike structures representing autophagosomes in the control hepatocytes (Fig. 3, J and K). Although several LC3-positive dots were observed in the mutant hepatocytes, they were not induced in response to starvation and did not form cup-shaped and ringlike structures (Fig. 3, L and M).


Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T - J. Cell Biol. (2005)

Impaired autophagosome formation in Atg7-deficient liver. (A–H) Electron micrographs of liver from Atg7F/+:Mx1 (A–D) and Atg7F/F:Mx1 (E–H) mice fed ad libitum (A and E) or fasted for 1 d (B and F). (C and D) Early stages of autophagic vacuoles observed in B are highlighted. (E and F) Autophagosome was not induced in mutant hepatocytes upon fasting. Insets show higher magnification views of glycogen granules. (G and H) Occasionally observed autophagosome-like structures in mutant hepatocytes. Bars: (A, B, E, and F) 5 μm; (C, D, G, and H) 0.5 μm. (I) Number of autophagosomes per hepatocyte (n = 20) in each genotype was counted and their averages are shown. (J–M) Immunofluorescent analysis of LC3 in primary cultured hepatocytes. Hepatocytes isolated from Atg7F/+:Mx1 (J and K) and Atg7F/F:Mx1 mice (L and M) were cultured in Williams' E (J and L) or Hanks' solution (K and M). Inset highlights the cup-like structure of LC3 observed in K. (N) Immunoblot analysis of Atg8 homologues in the liver. Atg7F/+:Mx1 (lanes 1 and 2) and Atg7F/F:Mx1 mice (lanes 3 and 4) were fed ad libitum (lanes 1 and 3) or fasted for 1 d (lanes 2 and 4), and then PNS fractions of liver were analyzed by immunoblotting with anti-LC3, GABARAP, GATE-16, and actin antibodies. Asterisk denotes a nonspecific band. Data shown are representative of three separate experiments.
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Related In: Results  -  Collection

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fig3: Impaired autophagosome formation in Atg7-deficient liver. (A–H) Electron micrographs of liver from Atg7F/+:Mx1 (A–D) and Atg7F/F:Mx1 (E–H) mice fed ad libitum (A and E) or fasted for 1 d (B and F). (C and D) Early stages of autophagic vacuoles observed in B are highlighted. (E and F) Autophagosome was not induced in mutant hepatocytes upon fasting. Insets show higher magnification views of glycogen granules. (G and H) Occasionally observed autophagosome-like structures in mutant hepatocytes. Bars: (A, B, E, and F) 5 μm; (C, D, G, and H) 0.5 μm. (I) Number of autophagosomes per hepatocyte (n = 20) in each genotype was counted and their averages are shown. (J–M) Immunofluorescent analysis of LC3 in primary cultured hepatocytes. Hepatocytes isolated from Atg7F/+:Mx1 (J and K) and Atg7F/F:Mx1 mice (L and M) were cultured in Williams' E (J and L) or Hanks' solution (K and M). Inset highlights the cup-like structure of LC3 observed in K. (N) Immunoblot analysis of Atg8 homologues in the liver. Atg7F/+:Mx1 (lanes 1 and 2) and Atg7F/F:Mx1 mice (lanes 3 and 4) were fed ad libitum (lanes 1 and 3) or fasted for 1 d (lanes 2 and 4), and then PNS fractions of liver were analyzed by immunoblotting with anti-LC3, GABARAP, GATE-16, and actin antibodies. Asterisk denotes a nonspecific band. Data shown are representative of three separate experiments.
Mentions: To delete Atg7 gene in the adult mice, we bred the Atg7F/F mice with Mx1-Cre transgenic mice that express the Cre recombinase in response to interferon γ or its chemical inducer, polyinosinic acid–polycytidylic acid (pIpC). The Mx1-Cre transgenic mice can excise Flox allele completely in the liver and spleen and partially in the kidney and heart (Kuhn et al., 1995). Intraperitoneal injections of pIpC resulted in effective recombination of the Atg7Flox allele in the liver and spleen (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200412022/DC1; and not depicted). No Atg7 transcript, protein, and activity were detected, similar to Atg7−/− mice (Fig. S2). Next, we tested the autophagosome formation under fasting condition. 1-d fasting resulted in induction of typical autophagosomes in control Atg7F/+:Mx1 mice (Fig. 3, A–D and I). In contrast, no such induction of autophagosome formation was noted in the liver of fasted Atg7F/F:Mx1 mice (Fig. 3, E, F, and I). Although some autophagosome-like structures were occasionally observed both in fed and fasted mutant mice livers (Fig. 3, G and H), they tended to be smaller than those observed in fasted control liver and hardly contained large cytoplasmic organelles (compare with Fig. 3, C and D). The number of autophagosomes per hepatocyte was counted and the mean values are shown in Fig. 3 I. The mutant hepatocytes lacked typical glycogen area, in contrast to the fed hepatocytes (Fig. 3, A and E); however, well-developed glycogen granules (α granules) were observed between numerous smooth endoplasmic reticula (Fig. 3 E, inset). Immunofluorescent analysis also revealed the presence of many cup-shaped and ringlike structures representing autophagosomes in the control hepatocytes (Fig. 3, J and K). Although several LC3-positive dots were observed in the mutant hepatocytes, they were not induced in response to starvation and did not form cup-shaped and ringlike structures (Fig. 3, L and M).

Bottom Line: Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation.Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies.Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.

ABSTRACT
Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

Show MeSH
Related in: MedlinePlus