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Antioxidant Treatment and Induction of Autophagy Cooperate to Reduce Desmin Aggregation in a Cellular Model of Desminopathy.

Cabet E, Batonnet-Pichon S, Delort F, Gausserès B, Vicart P, Lilienbaum A - PLoS ONE (2015)

Bottom Line: We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model.Further, a combination of two or three of these treatments was more effective than any of them alone.These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.

View Article: PubMed Central - PubMed

Affiliation: Physiopathology of the striated muscle laboratory, Unit of Functional and Adaptive Biology, University Paris Diderot, Sorbonne Paris Cité, UMR CNRS 8251, Paris, France.

ABSTRACT
Desminopathies, a subgroup of myofibrillar myopathies (MFMs), the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the desmin gene (DES), encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant DES. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.

No MeSH data available.


Related in: MedlinePlus

Autophagy inducers and antioxidants cooperate to reduce aggregation in C2C12 myoblasts.(A) Expression of Rac1 DN protein and treatment with PP242 cooperate to reduce desmin aggregates. C2C12 cells were co-transfected with the GFP-Desmin WT- (left panel) and D399Y- (right panel) expressing vectors and Rac1 DN, PAK1 WT, PKC WT, or pcDNA3 (CNTL and PP242) for 4 h. Cells were washed and subsequently incubated with PP242 (5 μM) or DMSO for 16 h. Cells were then fixed and the numbers of cells with aggregates were counted under a microscope (n = 100). A box plot representing 3 independent experiments is shown. Statistical analysis showed significant differences from pcDNA3 (p < 0.05 with a non-parametric test) as indicated by an asterisk. A significant difference for Rac1 DN + PP242 treatment versus either PP242 or Rac1 DN alone is indicated by an asterisk (p < 0.01) over an horizontal bar. (B) PP242 and α-tocopherols cooperate to reduce desmin aggregation. C2C12 cells were transiently transfected with a GFP-Desmin WT (left panel) or D399Y (right panel) vectors for 4 h. They were washed and then treated with PP242 (5 μM), α-tocopherol (α-Toco, 150 μM), or both for 16 h. The box plot represents 3 independent experiments. An asterisk indicates a significant difference from control at p < 0.05, and an asterisk above an horizontal bar indicates a significant difference between the double and simple treatments (p < 0.05).
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pone.0137009.g008: Autophagy inducers and antioxidants cooperate to reduce aggregation in C2C12 myoblasts.(A) Expression of Rac1 DN protein and treatment with PP242 cooperate to reduce desmin aggregates. C2C12 cells were co-transfected with the GFP-Desmin WT- (left panel) and D399Y- (right panel) expressing vectors and Rac1 DN, PAK1 WT, PKC WT, or pcDNA3 (CNTL and PP242) for 4 h. Cells were washed and subsequently incubated with PP242 (5 μM) or DMSO for 16 h. Cells were then fixed and the numbers of cells with aggregates were counted under a microscope (n = 100). A box plot representing 3 independent experiments is shown. Statistical analysis showed significant differences from pcDNA3 (p < 0.05 with a non-parametric test) as indicated by an asterisk. A significant difference for Rac1 DN + PP242 treatment versus either PP242 or Rac1 DN alone is indicated by an asterisk (p < 0.01) over an horizontal bar. (B) PP242 and α-tocopherols cooperate to reduce desmin aggregation. C2C12 cells were transiently transfected with a GFP-Desmin WT (left panel) or D399Y (right panel) vectors for 4 h. They were washed and then treated with PP242 (5 μM), α-tocopherol (α-Toco, 150 μM), or both for 16 h. The box plot represents 3 independent experiments. An asterisk indicates a significant difference from control at p < 0.05, and an asterisk above an horizontal bar indicates a significant difference between the double and simple treatments (p < 0.05).

Mentions: Having shown that different treatments reduce desmin aggregation in muscle cells, we next asked whether applying two different treatments could further reduce the percentage of cells with aggregates. To test this, we used transient co-transfection of GFP-desmin constructs with Rac1 DN, PAK1 WT, or PKC WT expressing vectors for 16 h, combined with treatment with alpha-tocopherols or PP242. In a second type of test, using only pharmacological products, cells were transfected for 4 h with GFP-Desmin WT, Q389P or D399Y mutants and then treated with alpha-tocopherol and PP242 for 16 h. The most promising combinations are shown in Fig 8. Surprisingly, the most significant effect was induced by the combination of Rac1 DN expression and PP242 treatment, which cooperated to reduce the percentage of cells with aggregates by more than the effect of either individually (Fig 8A). PP242 reduced the percentage of cells with D399Y aggregates by 39%, Rac1 DN by 34%, and the combination by 60%. We also found that co-treatment with PP242 and alpha-tocopherol (α-Toco) produced a cooperative effect for both WT (left panel) and D399Y mutant (right panel)(Fig 8B). In particular, the individual treatments reduced the percentage of cells with D399Y aggregates by between 14 and 28%, while the combination of PP242 and α-Toco reduced the percentage of cells with aggregates by 45%. In that case, an antioxidant molecule combined with a pro-autophagic treatment cooperated to reduce the proportion of cells with aggregate. The effect, though significant, was less marked compared to that obtained with Rac1 DN and PP242 (60%). These results were confirmed using myc-tagged constructs in similar experiments (S9 Fig).


Antioxidant Treatment and Induction of Autophagy Cooperate to Reduce Desmin Aggregation in a Cellular Model of Desminopathy.

Cabet E, Batonnet-Pichon S, Delort F, Gausserès B, Vicart P, Lilienbaum A - PLoS ONE (2015)

Autophagy inducers and antioxidants cooperate to reduce aggregation in C2C12 myoblasts.(A) Expression of Rac1 DN protein and treatment with PP242 cooperate to reduce desmin aggregates. C2C12 cells were co-transfected with the GFP-Desmin WT- (left panel) and D399Y- (right panel) expressing vectors and Rac1 DN, PAK1 WT, PKC WT, or pcDNA3 (CNTL and PP242) for 4 h. Cells were washed and subsequently incubated with PP242 (5 μM) or DMSO for 16 h. Cells were then fixed and the numbers of cells with aggregates were counted under a microscope (n = 100). A box plot representing 3 independent experiments is shown. Statistical analysis showed significant differences from pcDNA3 (p < 0.05 with a non-parametric test) as indicated by an asterisk. A significant difference for Rac1 DN + PP242 treatment versus either PP242 or Rac1 DN alone is indicated by an asterisk (p < 0.01) over an horizontal bar. (B) PP242 and α-tocopherols cooperate to reduce desmin aggregation. C2C12 cells were transiently transfected with a GFP-Desmin WT (left panel) or D399Y (right panel) vectors for 4 h. They were washed and then treated with PP242 (5 μM), α-tocopherol (α-Toco, 150 μM), or both for 16 h. The box plot represents 3 independent experiments. An asterisk indicates a significant difference from control at p < 0.05, and an asterisk above an horizontal bar indicates a significant difference between the double and simple treatments (p < 0.05).
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pone.0137009.g008: Autophagy inducers and antioxidants cooperate to reduce aggregation in C2C12 myoblasts.(A) Expression of Rac1 DN protein and treatment with PP242 cooperate to reduce desmin aggregates. C2C12 cells were co-transfected with the GFP-Desmin WT- (left panel) and D399Y- (right panel) expressing vectors and Rac1 DN, PAK1 WT, PKC WT, or pcDNA3 (CNTL and PP242) for 4 h. Cells were washed and subsequently incubated with PP242 (5 μM) or DMSO for 16 h. Cells were then fixed and the numbers of cells with aggregates were counted under a microscope (n = 100). A box plot representing 3 independent experiments is shown. Statistical analysis showed significant differences from pcDNA3 (p < 0.05 with a non-parametric test) as indicated by an asterisk. A significant difference for Rac1 DN + PP242 treatment versus either PP242 or Rac1 DN alone is indicated by an asterisk (p < 0.01) over an horizontal bar. (B) PP242 and α-tocopherols cooperate to reduce desmin aggregation. C2C12 cells were transiently transfected with a GFP-Desmin WT (left panel) or D399Y (right panel) vectors for 4 h. They were washed and then treated with PP242 (5 μM), α-tocopherol (α-Toco, 150 μM), or both for 16 h. The box plot represents 3 independent experiments. An asterisk indicates a significant difference from control at p < 0.05, and an asterisk above an horizontal bar indicates a significant difference between the double and simple treatments (p < 0.05).
Mentions: Having shown that different treatments reduce desmin aggregation in muscle cells, we next asked whether applying two different treatments could further reduce the percentage of cells with aggregates. To test this, we used transient co-transfection of GFP-desmin constructs with Rac1 DN, PAK1 WT, or PKC WT expressing vectors for 16 h, combined with treatment with alpha-tocopherols or PP242. In a second type of test, using only pharmacological products, cells were transfected for 4 h with GFP-Desmin WT, Q389P or D399Y mutants and then treated with alpha-tocopherol and PP242 for 16 h. The most promising combinations are shown in Fig 8. Surprisingly, the most significant effect was induced by the combination of Rac1 DN expression and PP242 treatment, which cooperated to reduce the percentage of cells with aggregates by more than the effect of either individually (Fig 8A). PP242 reduced the percentage of cells with D399Y aggregates by 39%, Rac1 DN by 34%, and the combination by 60%. We also found that co-treatment with PP242 and alpha-tocopherol (α-Toco) produced a cooperative effect for both WT (left panel) and D399Y mutant (right panel)(Fig 8B). In particular, the individual treatments reduced the percentage of cells with D399Y aggregates by between 14 and 28%, while the combination of PP242 and α-Toco reduced the percentage of cells with aggregates by 45%. In that case, an antioxidant molecule combined with a pro-autophagic treatment cooperated to reduce the proportion of cells with aggregate. The effect, though significant, was less marked compared to that obtained with Rac1 DN and PP242 (60%). These results were confirmed using myc-tagged constructs in similar experiments (S9 Fig).

Bottom Line: We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model.Further, a combination of two or three of these treatments was more effective than any of them alone.These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.

View Article: PubMed Central - PubMed

Affiliation: Physiopathology of the striated muscle laboratory, Unit of Functional and Adaptive Biology, University Paris Diderot, Sorbonne Paris Cité, UMR CNRS 8251, Paris, France.

ABSTRACT
Desminopathies, a subgroup of myofibrillar myopathies (MFMs), the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the desmin gene (DES), encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant DES. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.

No MeSH data available.


Related in: MedlinePlus