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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

Antioxidant treatment rescues cells from ATXN7Q65-GFP induced toxicity and decrease ATXN7 aggregation. A) Viability measured by WST-1 in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without 1 μM Vitamin E (α-tocopherol). B) Analysis and quantification of soluble ATXN7Q65-GFP by western blot in FLQ65 cells grown and treated as in A. C) Analysis and quantification of ATXN7 aggregation by filter trap assay in FLQ65 cells grown and treated as in A. D) Viability in cells grown as in A but treated with or without 5 mM NAC. E) Analysis and quantification of soluble ATXN7Q65-GFP by western blot in FLQ65 cells grown and treated as in D. F) Analysis and quantification of ATXN7 aggregation by filter trap assay in FLQ65 cells grown and treated as in D. For all western blots actin was used as loading control. For quantifications data 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 2: Antioxidant treatment rescues cells from ATXN7Q65-GFP induced toxicity and decrease ATXN7 aggregation. A) Viability measured by WST-1 in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without 1 μM Vitamin E (α-tocopherol). B) Analysis and quantification of soluble ATXN7Q65-GFP by western blot in FLQ65 cells grown and treated as in A. C) Analysis and quantification of ATXN7 aggregation by filter trap assay in FLQ65 cells grown and treated as in A. D) Viability in cells grown as in A but treated with or without 5 mM NAC. E) Analysis and quantification of soluble ATXN7Q65-GFP by western blot in FLQ65 cells grown and treated as in D. F) Analysis and quantification of ATXN7 aggregation by filter trap assay in FLQ65 cells grown and treated as in D. For all western blots actin was used as loading control. For quantifications data 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: To confirm that the oxidative stress conditions induced by ATXN7Q65-GFP played a role in the decreased cell viability, FLQ65 cells induced to express ATXN7Q65-GFP for nine days while growing in media supplemented or not supplemented with the anti-oxidants α-tocopherol (Vitamin E) or NAC were analyzed. Treatment with either anti-oxidant rescued the viability of ATXN7Q65-GFP expressing cells and there was no longer any difference in viability between non-induced and induced FLQ65 cells expressing ATXN7Q65-GFP (Figure 2A and 2D). The positive effects of the anti-oxidant treatments were not due to changes in ATXN7 expression, as neither treatment did alter the expression level of soluble ATXN7Q65-GFP (Figure 2B and 2E). This result suggests that oxidative stress is contributing to the toxicity in SCA7 and this effect can be counteracted by application of an anti-oxidant.


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)

Antioxidant treatment rescues cells from ATXN7Q65-GFP induced toxicity and decrease ATXN7 aggregation. A) Viability measured by WST-1 in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without 1 μM Vitamin E (α-tocopherol). B) Analysis and quantification of soluble ATXN7Q65-GFP by western blot in FLQ65 cells grown and treated as in A. C) Analysis and quantification of ATXN7 aggregation by filter trap assay in FLQ65 cells grown and treated as in A. D) Viability in cells grown as in A but treated with or without 5 mM NAC. E) Analysis and quantification of soluble ATXN7Q65-GFP by western blot in FLQ65 cells grown and treated as in D. F) Analysis and quantification of ATXN7 aggregation by filter trap assay in FLQ65 cells grown and treated as in D. For all western blots actin was used as loading control. For quantifications data are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05, ** p <0.01 and *** p <0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412756&req=5

Figure 2: Antioxidant treatment rescues cells from ATXN7Q65-GFP induced toxicity and decrease ATXN7 aggregation. A) Viability measured by WST-1 in FLQ65 cells not induced (+Dox) or induced (−Dox) to express ATXN7Q65-GFP for 9 days while growing in media with or without 1 μM Vitamin E (α-tocopherol). B) Analysis and quantification of soluble ATXN7Q65-GFP by western blot in FLQ65 cells grown and treated as in A. C) Analysis and quantification of ATXN7 aggregation by filter trap assay in FLQ65 cells grown and treated as in A. D) Viability in cells grown as in A but treated with or without 5 mM NAC. E) Analysis and quantification of soluble ATXN7Q65-GFP by western blot in FLQ65 cells grown and treated as in D. F) Analysis and quantification of ATXN7 aggregation by filter trap assay in FLQ65 cells grown and treated as in D. For all western blots actin was used as loading control. For quantifications data 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: To confirm that the oxidative stress conditions induced by ATXN7Q65-GFP played a role in the decreased cell viability, FLQ65 cells induced to express ATXN7Q65-GFP for nine days while growing in media supplemented or not supplemented with the anti-oxidants α-tocopherol (Vitamin E) or NAC were analyzed. Treatment with either anti-oxidant rescued the viability of ATXN7Q65-GFP expressing cells and there was no longer any difference in viability between non-induced and induced FLQ65 cells expressing ATXN7Q65-GFP (Figure 2A and 2D). The positive effects of the anti-oxidant treatments were not due to changes in ATXN7 expression, as neither treatment did alter the expression level of soluble ATXN7Q65-GFP (Figure 2B and 2E). This result suggests that oxidative stress is contributing to the toxicity in SCA7 and this effect can be counteracted by application of an anti-oxidant.

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