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Expanded ataxin-7 cause toxicity by inducing ROS production from NADPH oxidase complexes in a stable inducible Spinocerebellar ataxia type 7 (SCA7) model.

Ajayi A, Yu X, Lindberg S, Langel U, Ström AL - BMC Neurosci (2012)

Bottom Line: In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity.Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7.Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurochemistry, Stockholm University, Svante Arrhenius väg 21A, Stockholm, Sweden.

ABSTRACT

Background: Spinocerebellar ataxia type 7 (SCA7) is one of nine inherited neurodegenerative disorders caused by polyglutamine (polyQ) expansions. Common mechanisms of disease pathogenesis suggested for polyQ disorders include aggregation of the polyQ protein and induction of oxidative stress. However, the exact mechanism(s) of toxicity is still unclear.

Results: In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity. The increase in ROS could be completely prevented by inhibition of NADPH oxidase (NOX) complexes suggesting that ATXN7 directly or indirectly causes oxidative stress by increasing superoxide anion production from these complexes. Moreover, we could observe that induction of mutant ATXN7 leads to a decrease in the levels of catalase, a key enzyme in detoxifying hydrogen peroxide produced from dismutation of superoxide anions. This could also contribute to the generation of oxidative stress. Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7. In contrast, ATXN7 aggregation was aggravated by treatments promoting oxidative stress.

Conclusion: Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity.

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Related in: MedlinePlus

Inhibition of NOX complexes reduces ROS production, increase the GSH level and ameliorates toxicity. A) Measurement of mitochondrial superoxide levels using MitoSox in cells induced to express ATXN7Q65-GFP for 0, 6, 9 or 12 days. Antimycin A treatment was used as a positive control. B) Effect of NOX complex inhibition on ATXN7Q65-GFP cells. ROS levels (top panel), GSH levels (middle panel) and cell viability (lower panel) was analyzed in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without the NOX complex inhibitor apocynin (50 μM). C) Effect of NOX inhibition by apocynin on ATXN7 aggregation and ATXN7Q65-GFP expression in FLQ65 cells grown and treated as in A. Top panel; representative western blot and quantification of expression. Lower panel; representative dot blot and quantification of aggregation. D) Effect of NOX complex inhibition by gp91ds-TAT on ATXN7Q65-GFP cells. ROS levels (top panel) and cell viability (lower panel) was analyzed in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without 10 μM of the inhibitor peptide. All quantifications are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05, ** p <0.01 and *** p <0.001.
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Figure 6: Inhibition of NOX complexes reduces ROS production, increase the GSH level and ameliorates toxicity. A) Measurement of mitochondrial superoxide levels using MitoSox in cells induced to express ATXN7Q65-GFP for 0, 6, 9 or 12 days. Antimycin A treatment was used as a positive control. B) Effect of NOX complex inhibition on ATXN7Q65-GFP cells. ROS levels (top panel), GSH levels (middle panel) and cell viability (lower panel) was analyzed in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without the NOX complex inhibitor apocynin (50 μM). C) Effect of NOX inhibition by apocynin on ATXN7 aggregation and ATXN7Q65-GFP expression in FLQ65 cells grown and treated as in A. Top panel; representative western blot and quantification of expression. Lower panel; representative dot blot and quantification of aggregation. D) Effect of NOX complex inhibition by gp91ds-TAT on ATXN7Q65-GFP cells. ROS levels (top panel) and cell viability (lower panel) was analyzed in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without 10 μM of the inhibitor peptide. All quantifications are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05, ** p <0.01 and *** p <0.001.

Mentions: Mitochondria are a major source of ROS and damage to mitochondria resulting in increased mitochondrial superoxide anion production has been implicated in neurodegeneration. To investigate whether this mechanism plays a role in the elevation of the ROS levels in our ATXN7Q65-GFP expressing cells, we measure the level of mitochondrial superoxide anions at various time points after induction of ATXN7Q65-GFP expression. No increase in mitochondrial superoxide production could be observed at any time point after ATXN7Q65-GFP induction (Figure 6A). Antimycin A treatment was used as a positive control and elevated the levels of mitochondrial ROS as expected (Figure 6A).


Expanded ataxin-7 cause toxicity by inducing ROS production from NADPH oxidase complexes in a stable inducible Spinocerebellar ataxia type 7 (SCA7) model.

Ajayi A, Yu X, Lindberg S, Langel U, Ström AL - BMC Neurosci (2012)

Inhibition of NOX complexes reduces ROS production, increase the GSH level and ameliorates toxicity. A) Measurement of mitochondrial superoxide levels using MitoSox in cells induced to express ATXN7Q65-GFP for 0, 6, 9 or 12 days. Antimycin A treatment was used as a positive control. B) Effect of NOX complex inhibition on ATXN7Q65-GFP cells. ROS levels (top panel), GSH levels (middle panel) and cell viability (lower panel) was analyzed in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without the NOX complex inhibitor apocynin (50 μM). C) Effect of NOX inhibition by apocynin on ATXN7 aggregation and ATXN7Q65-GFP expression in FLQ65 cells grown and treated as in A. Top panel; representative western blot and quantification of expression. Lower panel; representative dot blot and quantification of aggregation. D) Effect of NOX complex inhibition by gp91ds-TAT on ATXN7Q65-GFP cells. ROS levels (top panel) and cell viability (lower panel) was analyzed in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without 10 μM of the inhibitor peptide. All quantifications are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05, ** p <0.01 and *** p <0.001.
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Related In: Results  -  Collection

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Figure 6: Inhibition of NOX complexes reduces ROS production, increase the GSH level and ameliorates toxicity. A) Measurement of mitochondrial superoxide levels using MitoSox in cells induced to express ATXN7Q65-GFP for 0, 6, 9 or 12 days. Antimycin A treatment was used as a positive control. B) Effect of NOX complex inhibition on ATXN7Q65-GFP cells. ROS levels (top panel), GSH levels (middle panel) and cell viability (lower panel) was analyzed in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without the NOX complex inhibitor apocynin (50 μM). C) Effect of NOX inhibition by apocynin on ATXN7 aggregation and ATXN7Q65-GFP expression in FLQ65 cells grown and treated as in A. Top panel; representative western blot and quantification of expression. Lower panel; representative dot blot and quantification of aggregation. D) Effect of NOX complex inhibition by gp91ds-TAT on ATXN7Q65-GFP cells. ROS levels (top panel) and cell viability (lower panel) was analyzed in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without 10 μM of the inhibitor peptide. All quantifications are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05, ** p <0.01 and *** p <0.001.
Mentions: Mitochondria are a major source of ROS and damage to mitochondria resulting in increased mitochondrial superoxide anion production has been implicated in neurodegeneration. To investigate whether this mechanism plays a role in the elevation of the ROS levels in our ATXN7Q65-GFP expressing cells, we measure the level of mitochondrial superoxide anions at various time points after induction of ATXN7Q65-GFP expression. No increase in mitochondrial superoxide production could be observed at any time point after ATXN7Q65-GFP induction (Figure 6A). Antimycin A treatment was used as a positive control and elevated the levels of mitochondrial ROS as expected (Figure 6A).

Bottom Line: In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity.Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7.Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurochemistry, Stockholm University, Svante Arrhenius väg 21A, Stockholm, Sweden.

ABSTRACT

Background: Spinocerebellar ataxia type 7 (SCA7) is one of nine inherited neurodegenerative disorders caused by polyglutamine (polyQ) expansions. Common mechanisms of disease pathogenesis suggested for polyQ disorders include aggregation of the polyQ protein and induction of oxidative stress. However, the exact mechanism(s) of toxicity is still unclear.

Results: In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity. The increase in ROS could be completely prevented by inhibition of NADPH oxidase (NOX) complexes suggesting that ATXN7 directly or indirectly causes oxidative stress by increasing superoxide anion production from these complexes. Moreover, we could observe that induction of mutant ATXN7 leads to a decrease in the levels of catalase, a key enzyme in detoxifying hydrogen peroxide produced from dismutation of superoxide anions. This could also contribute to the generation of oxidative stress. Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7. In contrast, ATXN7 aggregation was aggravated by treatments promoting oxidative stress.

Conclusion: Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity.

Show MeSH
Related in: MedlinePlus