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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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The phenotypes of Atg7-deficient mice. (A) PCR analysis of genomic DNA extracted from wild-type, Atg7+/−, and Atg7−/− mice tail. The amplified fragments derived from wild and mutant alleles are indicated. (B) Expression of Atg7 transcript. Atg7 transcript was detected by RT-PCR analysis. The region amplified was between exons 12 and 13. G3PDH cDNA was amplified as an internal control. (C) ATG conjugation systems in Atg7−/− mice liver. The liver homogenate was centrifuged at 800 g for 10 min and the postnuclear supernatant (PNS) was immunoblotted with antibodies against Atg7, Atg5, LC3, and actin as a loading control. The bottom panel of Atg5 blotting is the long exposure of the top panel to detect free Atg5. Data shown are representative of three separate experiments. (D and E) Deficiency of autophagosome formation in Atg7−/− heart. Atg7+/− (D) and Atg7−/− (E) mice expressing GFP-LC3 were obtained by caesarean delivery and analyzed by fluorescence microscopy. Representative results obtained from each neonatal heart at 3 h after caesarean delivery. (F) Morphology of Atg7+/− and Atg7−/− mice. (G) Kaplan-Meier curves of survival of newborn mice. Control and Atg7−/− mice were delivered by caesarean section, and their survival was followed up to 26 h.
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fig2: The phenotypes of Atg7-deficient mice. (A) PCR analysis of genomic DNA extracted from wild-type, Atg7+/−, and Atg7−/− mice tail. The amplified fragments derived from wild and mutant alleles are indicated. (B) Expression of Atg7 transcript. Atg7 transcript was detected by RT-PCR analysis. The region amplified was between exons 12 and 13. G3PDH cDNA was amplified as an internal control. (C) ATG conjugation systems in Atg7−/− mice liver. The liver homogenate was centrifuged at 800 g for 10 min and the postnuclear supernatant (PNS) was immunoblotted with antibodies against Atg7, Atg5, LC3, and actin as a loading control. The bottom panel of Atg5 blotting is the long exposure of the top panel to detect free Atg5. Data shown are representative of three separate experiments. (D and E) Deficiency of autophagosome formation in Atg7−/− heart. Atg7+/− (D) and Atg7−/− (E) mice expressing GFP-LC3 were obtained by caesarean delivery and analyzed by fluorescence microscopy. Representative results obtained from each neonatal heart at 3 h after caesarean delivery. (F) Morphology of Atg7+/− and Atg7−/− mice. (G) Kaplan-Meier curves of survival of newborn mice. Control and Atg7−/− mice were delivered by caesarean section, and their survival was followed up to 26 h.

Mentions: To examine the Atg7-deficient phenotype, we bred Atg7F/F mice with a line of transgenic mice that express the Cre recombinase under the control of the Zp3 promoter in the oocyte (Lewandoski et al., 1997). The heterozygous mice (referred to as Atg7+/−) were obtained from female Atg7F/+:Zp3 mice. Atg7+/− mice were born healthy and fertile without any noticeable pathological phenotypes for 1 yr. The Atg7−/− mice, obtained by breeding Atg7+/− mice, were born at Mendelian frequency (+/+: +/−: −/− = 21: 38: 19). The results of PCR genotyping are shown in Fig. 2 A. Neither Atg7 mRNA nor protein was detected in the homozygous mice (Fig. 2, B and C). We also tested the loss of Atg7 activity by examining the ATG conjugation systems in the neonate liver. A 56-kD protein, equivalent to Atg5–Atg12 conjugate, was detected by Atg5 antibody in the control Atg7+/− but not Atg7−/− liver (Fig. 2 C). In contrast, free Atg5 of 30 kD, which was faintly observed in the Atg7+/− liver, increased in Atg7−/− liver (Fig. 2 C). Mammalian Atg8p homologue LC3 has two forms (i.e., LC3-I and LC3-II; Kabeya et al., 2000). It is generally accepted that LC3-I is the free mature form whereas LC3-II is the lipidated form, in analogy to yeast Atg8p (Ichimura et al., 2000; Kabeya et al., 2000). Both forms were detected in Atg7+/− liver whereas only the LC3-I form was detected and increased in Atg7−/− liver (Fig. 2 C). When crossed with GFP-LC3 transgenic mice (Mizushima et al., 2004), the punctate structures representing autophagosomes were detected in Atg7+/− but not in Atg7−/− heart (Fig. 2, D and E). These results indicate that Atg7 is essential for ATG conjugation systems and autophagosome formation in mice.


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)

The phenotypes of Atg7-deficient mice. (A) PCR analysis of genomic DNA extracted from wild-type, Atg7+/−, and Atg7−/− mice tail. The amplified fragments derived from wild and mutant alleles are indicated. (B) Expression of Atg7 transcript. Atg7 transcript was detected by RT-PCR analysis. The region amplified was between exons 12 and 13. G3PDH cDNA was amplified as an internal control. (C) ATG conjugation systems in Atg7−/− mice liver. The liver homogenate was centrifuged at 800 g for 10 min and the postnuclear supernatant (PNS) was immunoblotted with antibodies against Atg7, Atg5, LC3, and actin as a loading control. The bottom panel of Atg5 blotting is the long exposure of the top panel to detect free Atg5. Data shown are representative of three separate experiments. (D and E) Deficiency of autophagosome formation in Atg7−/− heart. Atg7+/− (D) and Atg7−/− (E) mice expressing GFP-LC3 were obtained by caesarean delivery and analyzed by fluorescence microscopy. Representative results obtained from each neonatal heart at 3 h after caesarean delivery. (F) Morphology of Atg7+/− and Atg7−/− mice. (G) Kaplan-Meier curves of survival of newborn mice. Control and Atg7−/− mice were delivered by caesarean section, and their survival was followed up to 26 h.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171928&req=5

fig2: The phenotypes of Atg7-deficient mice. (A) PCR analysis of genomic DNA extracted from wild-type, Atg7+/−, and Atg7−/− mice tail. The amplified fragments derived from wild and mutant alleles are indicated. (B) Expression of Atg7 transcript. Atg7 transcript was detected by RT-PCR analysis. The region amplified was between exons 12 and 13. G3PDH cDNA was amplified as an internal control. (C) ATG conjugation systems in Atg7−/− mice liver. The liver homogenate was centrifuged at 800 g for 10 min and the postnuclear supernatant (PNS) was immunoblotted with antibodies against Atg7, Atg5, LC3, and actin as a loading control. The bottom panel of Atg5 blotting is the long exposure of the top panel to detect free Atg5. Data shown are representative of three separate experiments. (D and E) Deficiency of autophagosome formation in Atg7−/− heart. Atg7+/− (D) and Atg7−/− (E) mice expressing GFP-LC3 were obtained by caesarean delivery and analyzed by fluorescence microscopy. Representative results obtained from each neonatal heart at 3 h after caesarean delivery. (F) Morphology of Atg7+/− and Atg7−/− mice. (G) Kaplan-Meier curves of survival of newborn mice. Control and Atg7−/− mice were delivered by caesarean section, and their survival was followed up to 26 h.
Mentions: To examine the Atg7-deficient phenotype, we bred Atg7F/F mice with a line of transgenic mice that express the Cre recombinase under the control of the Zp3 promoter in the oocyte (Lewandoski et al., 1997). The heterozygous mice (referred to as Atg7+/−) were obtained from female Atg7F/+:Zp3 mice. Atg7+/− mice were born healthy and fertile without any noticeable pathological phenotypes for 1 yr. The Atg7−/− mice, obtained by breeding Atg7+/− mice, were born at Mendelian frequency (+/+: +/−: −/− = 21: 38: 19). The results of PCR genotyping are shown in Fig. 2 A. Neither Atg7 mRNA nor protein was detected in the homozygous mice (Fig. 2, B and C). We also tested the loss of Atg7 activity by examining the ATG conjugation systems in the neonate liver. A 56-kD protein, equivalent to Atg5–Atg12 conjugate, was detected by Atg5 antibody in the control Atg7+/− but not Atg7−/− liver (Fig. 2 C). In contrast, free Atg5 of 30 kD, which was faintly observed in the Atg7+/− liver, increased in Atg7−/− liver (Fig. 2 C). Mammalian Atg8p homologue LC3 has two forms (i.e., LC3-I and LC3-II; Kabeya et al., 2000). It is generally accepted that LC3-I is the free mature form whereas LC3-II is the lipidated form, in analogy to yeast Atg8p (Ichimura et al., 2000; Kabeya et al., 2000). Both forms were detected in Atg7+/− liver whereas only the LC3-I form was detected and increased in Atg7−/− liver (Fig. 2 C). When crossed with GFP-LC3 transgenic mice (Mizushima et al., 2004), the punctate structures representing autophagosomes were detected in Atg7+/− but not in Atg7−/− heart (Fig. 2, D and E). These results indicate that Atg7 is essential for ATG conjugation systems and autophagosome formation in mice.

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