Limits...
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

Apoptosis activity.(A) Protein expression profile of apoptotic markers, Bax, Bcl-2 and procaspase-3, in SOD1G93A mice (grey bars) and age-matched wild type mice (WT, black bars) along disease progression. Data showed mean ± SEM, n = 12 animals per time-point and genotype. *p <0.05 and ***p <0.001 versus age-matched WT. (B) Detection of apoptotic nuclei by TUNEL assay at each stage of the disease. Data showed mean ± SEM, n = 8 animals per time-point and genotype.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526523&req=5

pone.0134830.g005: Apoptosis activity.(A) Protein expression profile of apoptotic markers, Bax, Bcl-2 and procaspase-3, in SOD1G93A mice (grey bars) and age-matched wild type mice (WT, black bars) along disease progression. Data showed mean ± SEM, n = 12 animals per time-point and genotype. *p <0.05 and ***p <0.001 versus age-matched WT. (B) Detection of apoptotic nuclei by TUNEL assay at each stage of the disease. Data showed mean ± SEM, n = 8 animals per time-point and genotype.

Mentions: To further study in depth the role of the time-dependent activation of autophagy and proteasome in the disease, we determined the role of apoptosis in the progression of the disease. Western blot analysis for the total and the cleaved form of caspase-3, caspase-9, Bax, Bcl-2 and the poly ADP ribose polymerase (PARP) profile expression pattern was performed. The caspase-3 and caspase-9 has been described as the final effector of the apoptotic cell death mechanism meanwhile the proteolytic cleavage (activation) of PARP-1 has been considered as a hallmark biochemical feature of apoptosis,. Interestingly, the proteins caspase-3 and caspase-9 in its active form were no detected in WT or SOD1G93A mice as well as PARP-1 (S1 Fig). However, the highest procaspase-3 and procaspase-9 expression levels were found at P90 (p>0.001) and P120 (p>0.001) respectively. Moreover, the activity of Bcl-2, an anti-apoptotic protein, decreased significantly at P40 and increased significantly at the terminal stage, P120 (p<0.05) (Fig 5A). These data suggested that apoptosis was not exerting a decisive role in the skeletal muscle tissue from SOD1G93A mice. In addition, to verify the western blot results, the apoptosis was visualized by TUNEL assay. As shown in Fig 5B, the percentage of TUNEL positive cells was very low at any stage of the disease (less than 1%) in all the tested samples from WT or SOD1G93A mice. Moreover, not significant differences were detected between WT and SOD1G93A mice, supporting the fact that the apoptotic cell death was not happening in the skeletal muscle from SOD1G93A mice along disease progression.


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)

Apoptosis activity.(A) Protein expression profile of apoptotic markers, Bax, Bcl-2 and procaspase-3, in SOD1G93A mice (grey bars) and age-matched wild type mice (WT, black bars) along disease progression. Data showed mean ± SEM, n = 12 animals per time-point and genotype. *p <0.05 and ***p <0.001 versus age-matched WT. (B) Detection of apoptotic nuclei by TUNEL assay at each stage of the disease. Data showed mean ± SEM, n = 8 animals per time-point and genotype.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134830.g005: Apoptosis activity.(A) Protein expression profile of apoptotic markers, Bax, Bcl-2 and procaspase-3, in SOD1G93A mice (grey bars) and age-matched wild type mice (WT, black bars) along disease progression. Data showed mean ± SEM, n = 12 animals per time-point and genotype. *p <0.05 and ***p <0.001 versus age-matched WT. (B) Detection of apoptotic nuclei by TUNEL assay at each stage of the disease. Data showed mean ± SEM, n = 8 animals per time-point and genotype.
Mentions: To further study in depth the role of the time-dependent activation of autophagy and proteasome in the disease, we determined the role of apoptosis in the progression of the disease. Western blot analysis for the total and the cleaved form of caspase-3, caspase-9, Bax, Bcl-2 and the poly ADP ribose polymerase (PARP) profile expression pattern was performed. The caspase-3 and caspase-9 has been described as the final effector of the apoptotic cell death mechanism meanwhile the proteolytic cleavage (activation) of PARP-1 has been considered as a hallmark biochemical feature of apoptosis,. Interestingly, the proteins caspase-3 and caspase-9 in its active form were no detected in WT or SOD1G93A mice as well as PARP-1 (S1 Fig). However, the highest procaspase-3 and procaspase-9 expression levels were found at P90 (p>0.001) and P120 (p>0.001) respectively. Moreover, the activity of Bcl-2, an anti-apoptotic protein, decreased significantly at P40 and increased significantly at the terminal stage, P120 (p<0.05) (Fig 5A). These data suggested that apoptosis was not exerting a decisive role in the skeletal muscle tissue from SOD1G93A mice. In addition, to verify the western blot results, the apoptosis was visualized by TUNEL assay. As shown in Fig 5B, the percentage of TUNEL positive cells was very low at any stage of the disease (less than 1%) in all the tested samples from WT or SOD1G93A mice. Moreover, not significant differences were detected between WT and SOD1G93A mice, supporting the fact that the apoptotic cell death was not happening in the skeletal muscle from SOD1G93A mice along disease progression.

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