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Time-Point Dependent Activation of Autophagy and the UPS in SOD1G93A Mice Skeletal Muscle.

Oliván S, Calvo AC, Gasco S, Muñoz MJ, Zaragoza P, Osta R - PLoS ONE (2015)

Bottom Line: In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue.Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages.The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Genética y Bioquímica (LAGENBIO), Facultad de Veterinaria, Instituto Agroalimentario de Aragón (IA2), Instituto de Investigación Sanitaria Aragón, Universidad de Zaragoza, Zaragoza, Spain.

ABSTRACT
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by a selective loss of motor neurons together with a progressive muscle weakness. Albeit the pathophysiological mechanisms of the disease remain unknown, growing evidence suggests that skeletal muscle can be a target of ALS toxicity. In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue. In this study we investigated the activation of autophagy and the UPS as well as apoptosis in the skeletal muscle from SOD1G93A mice along disease progression. Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages. The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy. Furthermore, no apoptosis activation was observed along disease progression. The combined data provided clear evidence for the first time that there is a time-point dependent activation of autophagy and UPS in the skeletal muscle from SOD1G93A mice.

No MeSH data available.


Related in: MedlinePlus

Autophagy protein expression.(A) Western blots for Beclin1, LC3-II, LC3-I and p62 protein levels in SOD1G93A mice (grey bars) and age-matched wild type mice (WT, black bars) along disease progression. The cropped blots portrayed are representative of independent experiments. All gels were run under exact same experimental conditions for best comparison. Data showed mean ± SEM, n = 12 animals per time-point and genotype. *p <0.05 and ***p <0.001 versus age-matched WT. (B) Results of LC3-II were normalised to the corresponding LC3-I signal to generate ratio LC3-II/LC3-I in SOD1G93A mice (light grey bars) and age-matched wild type mice (WT, black bars). Data showed mean ± SEM. *p <0.1, **p <0.01 and ***p <0.001. (C) Relative expression levels of LC3-II from SOD1G93A mice at P120, before (light grey bars) and after chloroquine treatment (dark grey bars). Data showed mean ± SEM. n = 6 animals. *p <0.05.
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pone.0134830.g002: Autophagy protein expression.(A) Western blots for Beclin1, LC3-II, LC3-I and p62 protein levels in SOD1G93A mice (grey bars) and age-matched wild type mice (WT, black bars) along disease progression. The cropped blots portrayed are representative of independent experiments. All gels were run under exact same experimental conditions for best comparison. Data showed mean ± SEM, n = 12 animals per time-point and genotype. *p <0.05 and ***p <0.001 versus age-matched WT. (B) Results of LC3-II were normalised to the corresponding LC3-I signal to generate ratio LC3-II/LC3-I in SOD1G93A mice (light grey bars) and age-matched wild type mice (WT, black bars). Data showed mean ± SEM. *p <0.1, **p <0.01 and ***p <0.001. (C) Relative expression levels of LC3-II from SOD1G93A mice at P120, before (light grey bars) and after chloroquine treatment (dark grey bars). Data showed mean ± SEM. n = 6 animals. *p <0.05.

Mentions: Moreover, we analyzed the protein expression of three widely used autophagy markers, Beclin1, LC3 and p62 along disease progression. The expression of Beclin1 was significant increased in all stages except at P90 when no differences were detected between WT and SOD1G93A. In case of LC3-II and p62, the expression levels of both proteins remained almost unchanged until P90. A statistically significant downregulation of p62 levels were observed at P40, while a significant impairment was found in LC3-I levels at the first stages of the disease. However, at P120, a significant upregulation was found in Beclin1, LC3-I, LC3-II and p62 (Fig 2A). Furthermore, the ratio LC3-II/LC3-I showed that the autophagy flux was significant increased at P40 and P120 while decreased at P60 (Fig 2B).


Time-Point Dependent Activation of Autophagy and the UPS in SOD1G93A Mice Skeletal Muscle.

Oliván S, Calvo AC, Gasco S, Muñoz MJ, Zaragoza P, Osta R - PLoS ONE (2015)

Autophagy protein expression.(A) Western blots for Beclin1, LC3-II, LC3-I and p62 protein levels in SOD1G93A mice (grey bars) and age-matched wild type mice (WT, black bars) along disease progression. The cropped blots portrayed are representative of independent experiments. All gels were run under exact same experimental conditions for best comparison. Data showed mean ± SEM, n = 12 animals per time-point and genotype. *p <0.05 and ***p <0.001 versus age-matched WT. (B) Results of LC3-II were normalised to the corresponding LC3-I signal to generate ratio LC3-II/LC3-I in SOD1G93A mice (light grey bars) and age-matched wild type mice (WT, black bars). Data showed mean ± SEM. *p <0.1, **p <0.01 and ***p <0.001. (C) Relative expression levels of LC3-II from SOD1G93A mice at P120, before (light grey bars) and after chloroquine treatment (dark grey bars). Data showed mean ± SEM. n = 6 animals. *p <0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526523&req=5

pone.0134830.g002: Autophagy protein expression.(A) Western blots for Beclin1, LC3-II, LC3-I and p62 protein levels in SOD1G93A mice (grey bars) and age-matched wild type mice (WT, black bars) along disease progression. The cropped blots portrayed are representative of independent experiments. All gels were run under exact same experimental conditions for best comparison. Data showed mean ± SEM, n = 12 animals per time-point and genotype. *p <0.05 and ***p <0.001 versus age-matched WT. (B) Results of LC3-II were normalised to the corresponding LC3-I signal to generate ratio LC3-II/LC3-I in SOD1G93A mice (light grey bars) and age-matched wild type mice (WT, black bars). Data showed mean ± SEM. *p <0.1, **p <0.01 and ***p <0.001. (C) Relative expression levels of LC3-II from SOD1G93A mice at P120, before (light grey bars) and after chloroquine treatment (dark grey bars). Data showed mean ± SEM. n = 6 animals. *p <0.05.
Mentions: Moreover, we analyzed the protein expression of three widely used autophagy markers, Beclin1, LC3 and p62 along disease progression. The expression of Beclin1 was significant increased in all stages except at P90 when no differences were detected between WT and SOD1G93A. In case of LC3-II and p62, the expression levels of both proteins remained almost unchanged until P90. A statistically significant downregulation of p62 levels were observed at P40, while a significant impairment was found in LC3-I levels at the first stages of the disease. However, at P120, a significant upregulation was found in Beclin1, LC3-I, LC3-II and p62 (Fig 2A). Furthermore, the ratio LC3-II/LC3-I showed that the autophagy flux was significant increased at P40 and P120 while decreased at P60 (Fig 2B).

Bottom Line: In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue.Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages.The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Genética y Bioquímica (LAGENBIO), Facultad de Veterinaria, Instituto Agroalimentario de Aragón (IA2), Instituto de Investigación Sanitaria Aragón, Universidad de Zaragoza, Zaragoza, Spain.

ABSTRACT
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by a selective loss of motor neurons together with a progressive muscle weakness. Albeit the pathophysiological mechanisms of the disease remain unknown, growing evidence suggests that skeletal muscle can be a target of ALS toxicity. In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue. In this study we investigated the activation of autophagy and the UPS as well as apoptosis in the skeletal muscle from SOD1G93A mice along disease progression. Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages. The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy. Furthermore, no apoptosis activation was observed along disease progression. The combined data provided clear evidence for the first time that there is a time-point dependent activation of autophagy and UPS in the skeletal muscle from SOD1G93A mice.

No MeSH data available.


Related in: MedlinePlus