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Proteasome dysfunction induces muscle growth defects and protein aggregation.

Kitajima Y, Tashiro Y, Suzuki N, Warita H, Kato M, Tateyama M, Ando R, Izumi R, Yamazaki M, Abe M, Sakimura K, Ito H, Urushitani M, Nagatomi R, Takahashi R, Aoki M - J. Cell. Sci. (2014)

Bottom Line: The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes of protein and organelle clearance.The autophagy pathway was upregulated, but the process of autophagosome formation was impaired.Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Department of Medicine and Science in Sports and Exercise, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

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Autophagy is reduced in the gastrocnemius muscles of Rpt3−/− mice. (A) Immunoblotting revealed increased levels of p62, LC3, beclin-1 and Atg5 proteins in Rpt3−/− mice at the age of 6 weeks. Quantitative data are also presented (n = 3). Note that p62 was increased and the LC3II∶LC3I ratio was decreased in Rpt3−/− mice. (B) Upregulation of p62 mRNA in Rpt3−/− mice at the age of 6 weeks. RNA was extracted from the gastrocnemius muscles, and quantitative PCR analysis was performed in triplicate using specific primers (supplementary material Table S1). Data were normalized to the GAPDH content and expressed as the fold increase over the level of expression in Rpt3+/+ mice (n = 5). (C) Immunohistochemical examination revealed p62-positive myofibers in Rpt3−/− mice at the age of 6 weeks. Scale bar: 50 µm. (D) Immunohistochemical examination revealed LC3-positive myofibers in Rpt3−/− mice at the age of 6 weeks. TOTO3, nuclei; OVL, overlay. Scale bar: 50 µm. (E) Autophagy flux was investigated using C2C12 cells. Representative immunoblotting showing autophagy flux assay with reduced LC3II levels following Rpt3 knockdown under chloroquine inhibition (n = 4/treatment). con, control. (F) The protein level of LC3II was significantly different between vehicle- and chloroquine-treated samples (*P<0.05). The protein level of LC3II was significantly different between samples treated with both Rpt3 siRNA and chloroquine and those treated with control siRNA and chloroquine (†P<0.05). The protein level of LC3II was not significantly different between samples treated with control siRNA without chloroquine those treated with Rpt3 siRNA without chloroquine (P = 0.058). Quantitative data in A,B,F show the mean+s.e.m.; *P<0.05 (Student's t-test).
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f06: Autophagy is reduced in the gastrocnemius muscles of Rpt3−/− mice. (A) Immunoblotting revealed increased levels of p62, LC3, beclin-1 and Atg5 proteins in Rpt3−/− mice at the age of 6 weeks. Quantitative data are also presented (n = 3). Note that p62 was increased and the LC3II∶LC3I ratio was decreased in Rpt3−/− mice. (B) Upregulation of p62 mRNA in Rpt3−/− mice at the age of 6 weeks. RNA was extracted from the gastrocnemius muscles, and quantitative PCR analysis was performed in triplicate using specific primers (supplementary material Table S1). Data were normalized to the GAPDH content and expressed as the fold increase over the level of expression in Rpt3+/+ mice (n = 5). (C) Immunohistochemical examination revealed p62-positive myofibers in Rpt3−/− mice at the age of 6 weeks. Scale bar: 50 µm. (D) Immunohistochemical examination revealed LC3-positive myofibers in Rpt3−/− mice at the age of 6 weeks. TOTO3, nuclei; OVL, overlay. Scale bar: 50 µm. (E) Autophagy flux was investigated using C2C12 cells. Representative immunoblotting showing autophagy flux assay with reduced LC3II levels following Rpt3 knockdown under chloroquine inhibition (n = 4/treatment). con, control. (F) The protein level of LC3II was significantly different between vehicle- and chloroquine-treated samples (*P<0.05). The protein level of LC3II was significantly different between samples treated with both Rpt3 siRNA and chloroquine and those treated with control siRNA and chloroquine (†P<0.05). The protein level of LC3II was not significantly different between samples treated with control siRNA without chloroquine those treated with Rpt3 siRNA without chloroquine (P = 0.058). Quantitative data in A,B,F show the mean+s.e.m.; *P<0.05 (Student's t-test).

Mentions: The autophagy pathway is another important mechanism that is responsible for protein degradation and processing within cells. The protein p62 (also known as SQSTM1) has a role in the aggregation of intracellular ubiquitin-related protein (Komatsu et al., 2007). LC3 (also known as MAP1LC3A and MAP1LC3B) is a post-translational modifier that is essential for autophagosome formation. The protein and transcriptional levels of p62 were markedly increased in Rpt3−/− mouse gastrocnemius muscle compared with the same muscle from Rpt3+/+ mice (Fig. 6A,B). There was also an increase in LC3II and a marked decrease in the LC3II∶LC3I ratio in Rpt3−/− mice (Fig. 6A). Immunohistochemical analyses detected a marked increase in the amount of p62 and LC3 in the myofibers of Rpt3−/− mutant animals (Fig. 6C,D).


Proteasome dysfunction induces muscle growth defects and protein aggregation.

Kitajima Y, Tashiro Y, Suzuki N, Warita H, Kato M, Tateyama M, Ando R, Izumi R, Yamazaki M, Abe M, Sakimura K, Ito H, Urushitani M, Nagatomi R, Takahashi R, Aoki M - J. Cell. Sci. (2014)

Autophagy is reduced in the gastrocnemius muscles of Rpt3−/− mice. (A) Immunoblotting revealed increased levels of p62, LC3, beclin-1 and Atg5 proteins in Rpt3−/− mice at the age of 6 weeks. Quantitative data are also presented (n = 3). Note that p62 was increased and the LC3II∶LC3I ratio was decreased in Rpt3−/− mice. (B) Upregulation of p62 mRNA in Rpt3−/− mice at the age of 6 weeks. RNA was extracted from the gastrocnemius muscles, and quantitative PCR analysis was performed in triplicate using specific primers (supplementary material Table S1). Data were normalized to the GAPDH content and expressed as the fold increase over the level of expression in Rpt3+/+ mice (n = 5). (C) Immunohistochemical examination revealed p62-positive myofibers in Rpt3−/− mice at the age of 6 weeks. Scale bar: 50 µm. (D) Immunohistochemical examination revealed LC3-positive myofibers in Rpt3−/− mice at the age of 6 weeks. TOTO3, nuclei; OVL, overlay. Scale bar: 50 µm. (E) Autophagy flux was investigated using C2C12 cells. Representative immunoblotting showing autophagy flux assay with reduced LC3II levels following Rpt3 knockdown under chloroquine inhibition (n = 4/treatment). con, control. (F) The protein level of LC3II was significantly different between vehicle- and chloroquine-treated samples (*P<0.05). The protein level of LC3II was significantly different between samples treated with both Rpt3 siRNA and chloroquine and those treated with control siRNA and chloroquine (†P<0.05). The protein level of LC3II was not significantly different between samples treated with control siRNA without chloroquine those treated with Rpt3 siRNA without chloroquine (P = 0.058). Quantitative data in A,B,F show the mean+s.e.m.; *P<0.05 (Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f06: Autophagy is reduced in the gastrocnemius muscles of Rpt3−/− mice. (A) Immunoblotting revealed increased levels of p62, LC3, beclin-1 and Atg5 proteins in Rpt3−/− mice at the age of 6 weeks. Quantitative data are also presented (n = 3). Note that p62 was increased and the LC3II∶LC3I ratio was decreased in Rpt3−/− mice. (B) Upregulation of p62 mRNA in Rpt3−/− mice at the age of 6 weeks. RNA was extracted from the gastrocnemius muscles, and quantitative PCR analysis was performed in triplicate using specific primers (supplementary material Table S1). Data were normalized to the GAPDH content and expressed as the fold increase over the level of expression in Rpt3+/+ mice (n = 5). (C) Immunohistochemical examination revealed p62-positive myofibers in Rpt3−/− mice at the age of 6 weeks. Scale bar: 50 µm. (D) Immunohistochemical examination revealed LC3-positive myofibers in Rpt3−/− mice at the age of 6 weeks. TOTO3, nuclei; OVL, overlay. Scale bar: 50 µm. (E) Autophagy flux was investigated using C2C12 cells. Representative immunoblotting showing autophagy flux assay with reduced LC3II levels following Rpt3 knockdown under chloroquine inhibition (n = 4/treatment). con, control. (F) The protein level of LC3II was significantly different between vehicle- and chloroquine-treated samples (*P<0.05). The protein level of LC3II was significantly different between samples treated with both Rpt3 siRNA and chloroquine and those treated with control siRNA and chloroquine (†P<0.05). The protein level of LC3II was not significantly different between samples treated with control siRNA without chloroquine those treated with Rpt3 siRNA without chloroquine (P = 0.058). Quantitative data in A,B,F show the mean+s.e.m.; *P<0.05 (Student's t-test).
Mentions: The autophagy pathway is another important mechanism that is responsible for protein degradation and processing within cells. The protein p62 (also known as SQSTM1) has a role in the aggregation of intracellular ubiquitin-related protein (Komatsu et al., 2007). LC3 (also known as MAP1LC3A and MAP1LC3B) is a post-translational modifier that is essential for autophagosome formation. The protein and transcriptional levels of p62 were markedly increased in Rpt3−/− mouse gastrocnemius muscle compared with the same muscle from Rpt3+/+ mice (Fig. 6A,B). There was also an increase in LC3II and a marked decrease in the LC3II∶LC3I ratio in Rpt3−/− mice (Fig. 6A). Immunohistochemical analyses detected a marked increase in the amount of p62 and LC3 in the myofibers of Rpt3−/− mutant animals (Fig. 6C,D).

Bottom Line: The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes of protein and organelle clearance.The autophagy pathway was upregulated, but the process of autophagosome formation was impaired.Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan Department of Medicine and Science in Sports and Exercise, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

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Related in: MedlinePlus