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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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Reduced CAT levels, but increased levels of SOD1 and GSTA3 in ATXN7Q65-GFP expressing cells. A) Representative western blot analysis of GSTA3, SOD1 and CAT levels in FLQ65 cells induced to express ATXN7Q65-GFP for 0–12 days. B) Quantitative analysis of GSTA3 levels from three experiments as shown in A. C) Quantitative analysis of SOD1 levels from three experiments as shown in A. D) Quantitative analysis of CAT levels from three experiments as shown in A. E) NAC effect on the expression of GSTA3, SOD1 and CAT levels in ATXN7Q65-GFP expressing cells. FLQ65 cells not induced or induced to express ATXN7Q65-GFP for 9 days while growing in media with or without NAC (5 mM) were analyzed. F) Quantitative analysis of GSTA3 levels from three experiments with treatments as in E. G) Quantitative analysis of SOD1 levels from three experiments with treatments as in E. H) Quantitative analysis of CAT levels from three experiments with treatments as in E. All quantifications are shown as means ± SEM from three independent experiments with triplicates. * p <0.05, ** p <0.01 and *** p <0.001.
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Figure 5: Reduced CAT levels, but increased levels of SOD1 and GSTA3 in ATXN7Q65-GFP expressing cells. A) Representative western blot analysis of GSTA3, SOD1 and CAT levels in FLQ65 cells induced to express ATXN7Q65-GFP for 0–12 days. B) Quantitative analysis of GSTA3 levels from three experiments as shown in A. C) Quantitative analysis of SOD1 levels from three experiments as shown in A. D) Quantitative analysis of CAT levels from three experiments as shown in A. E) NAC effect on the expression of GSTA3, SOD1 and CAT levels in ATXN7Q65-GFP expressing cells. FLQ65 cells not induced or induced to express ATXN7Q65-GFP for 9 days while growing in media with or without NAC (5 mM) were analyzed. F) Quantitative analysis of GSTA3 levels from three experiments with treatments as in E. G) Quantitative analysis of SOD1 levels from three experiments with treatments as in E. H) Quantitative analysis of CAT levels from three experiments with treatments as in E. All quantifications are shown as means ± SEM from three independent experiments with triplicates. * p <0.05, ** p <0.01 and *** p <0.001.

Mentions: Mutant ATXN7 could induce oxidative stress by interfering with the anti-oxidant defense system or by causing an increase in free radical production. To investigate the status of the anti-oxidant defense system, we analyzed the expression levels of some key anti-oxidant enzymes; Glutathione transferase A3 (GSTA3), SOD1 and CAT in our stable PC12 model. After induction of ATXN7Q65-GFP, the expression levels of GSTA3 and SOD1 showed a progressively increasing trend with statistical differences in expression at day 9 and/or 12 after induction (Figure 5A-C). In contrast, the expression level of CAT showed a decreasing trend after induction of ATXN7Q65-GFP (Figure 5A and 5D). We also investigated whether anti-oxidant treatment could prevent the change in expression of these enzymes. Indeed, in induced FLQ65 cells grown in NAC supplemented media for 9 days, the increase in GSTA3 and SOD1 expression was reduced or completely gone (Figure 5E-G). However, the decreased expression of CAT was not restored (Figure 5H). Our results suggest that the cell is trying to cope with the oxidative environment by up-regulating at least some key anti-oxidant enzymes.


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)

Reduced CAT levels, but increased levels of SOD1 and GSTA3 in ATXN7Q65-GFP expressing cells. A) Representative western blot analysis of GSTA3, SOD1 and CAT levels in FLQ65 cells induced to express ATXN7Q65-GFP for 0–12 days. B) Quantitative analysis of GSTA3 levels from three experiments as shown in A. C) Quantitative analysis of SOD1 levels from three experiments as shown in A. D) Quantitative analysis of CAT levels from three experiments as shown in A. E) NAC effect on the expression of GSTA3, SOD1 and CAT levels in ATXN7Q65-GFP expressing cells. FLQ65 cells not induced or induced to express ATXN7Q65-GFP for 9 days while growing in media with or without NAC (5 mM) were analyzed. F) Quantitative analysis of GSTA3 levels from three experiments with treatments as in E. G) Quantitative analysis of SOD1 levels from three experiments with treatments as in E. H) Quantitative analysis of CAT levels from three experiments with treatments as in E. All quantifications are shown as means ± SEM from three independent experiments with triplicates. * p <0.05, ** p <0.01 and *** p <0.001.
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Related In: Results  -  Collection

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Figure 5: Reduced CAT levels, but increased levels of SOD1 and GSTA3 in ATXN7Q65-GFP expressing cells. A) Representative western blot analysis of GSTA3, SOD1 and CAT levels in FLQ65 cells induced to express ATXN7Q65-GFP for 0–12 days. B) Quantitative analysis of GSTA3 levels from three experiments as shown in A. C) Quantitative analysis of SOD1 levels from three experiments as shown in A. D) Quantitative analysis of CAT levels from three experiments as shown in A. E) NAC effect on the expression of GSTA3, SOD1 and CAT levels in ATXN7Q65-GFP expressing cells. FLQ65 cells not induced or induced to express ATXN7Q65-GFP for 9 days while growing in media with or without NAC (5 mM) were analyzed. F) Quantitative analysis of GSTA3 levels from three experiments with treatments as in E. G) Quantitative analysis of SOD1 levels from three experiments with treatments as in E. H) Quantitative analysis of CAT levels from three experiments with treatments as in E. All quantifications are shown as means ± SEM from three independent experiments with triplicates. * p <0.05, ** p <0.01 and *** p <0.001.
Mentions: Mutant ATXN7 could induce oxidative stress by interfering with the anti-oxidant defense system or by causing an increase in free radical production. To investigate the status of the anti-oxidant defense system, we analyzed the expression levels of some key anti-oxidant enzymes; Glutathione transferase A3 (GSTA3), SOD1 and CAT in our stable PC12 model. After induction of ATXN7Q65-GFP, the expression levels of GSTA3 and SOD1 showed a progressively increasing trend with statistical differences in expression at day 9 and/or 12 after induction (Figure 5A-C). In contrast, the expression level of CAT showed a decreasing trend after induction of ATXN7Q65-GFP (Figure 5A and 5D). We also investigated whether anti-oxidant treatment could prevent the change in expression of these enzymes. Indeed, in induced FLQ65 cells grown in NAC supplemented media for 9 days, the increase in GSTA3 and SOD1 expression was reduced or completely gone (Figure 5E-G). However, the decreased expression of CAT was not restored (Figure 5H). Our results suggest that the cell is trying to cope with the oxidative environment by up-regulating at least some key anti-oxidant enzymes.

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