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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

Expression of ATXN7Q65-GFP leads to increased ROS levels prior to toxicity. A) Western blot analysis of ATXN7 in FLQ10 and FLQ65 stable PC12 cell lines induced to express GFP-tagged ATXN7 with 10 (ATXN7Q10-GFP, top panel) or 65 (ATXN7Q65-GFP, lower panel) glutamines for the indicated number of days. Actin was used as loading control. B) Analysis of ATXN7 aggregation in FLQ65 cells induced to express ATXN7Q65-GFP for 1–12 days using the filter trap assay. C) Viability measured by the WST-1 assay and normalized against the protein content in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. D) Toxicity measured by the LDH membrane leakage assay in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. E) Total cellular ROS levels measured using DCHF-DA in cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for 0–12 days. F) Analysis of GSH levels in cells induced (−Dox) to express ATXN7Q65-GFP for 9 days and non-induced control cells (+Dox). For quantifications all data 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 1: Expression of ATXN7Q65-GFP leads to increased ROS levels prior to toxicity. A) Western blot analysis of ATXN7 in FLQ10 and FLQ65 stable PC12 cell lines induced to express GFP-tagged ATXN7 with 10 (ATXN7Q10-GFP, top panel) or 65 (ATXN7Q65-GFP, lower panel) glutamines for the indicated number of days. Actin was used as loading control. B) Analysis of ATXN7 aggregation in FLQ65 cells induced to express ATXN7Q65-GFP for 1–12 days using the filter trap assay. C) Viability measured by the WST-1 assay and normalized against the protein content in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. D) Toxicity measured by the LDH membrane leakage assay in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. E) Total cellular ROS levels measured using DCHF-DA in cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for 0–12 days. F) Analysis of GSH levels in cells induced (−Dox) to express ATXN7Q65-GFP for 9 days and non-induced control cells (+Dox). For quantifications all data are shown as means ± SEM from three independent experiments with triplicates. * p <0.05, ** p <0.01 and *** p <0.001.

Mentions: To study the impact of mutant ATXN7 on cellular functions we used two recently generated stable inducible PC12 cell lines expressing N-terminal FLAG- and C-terminal GFP-tagged ATXN7 with 10 (FLQ10 line) or 65 (FLQ65 line) glutamines [28]. In these cell lines the expression of the corresponding transgenic proteins named ATXN7Q10-GFP and ATXN7Q65-GFP is controlled by the Tet-off expression system and induced by removal of doxycycline from the cell culture media. The induction timing, expression levels and sub-cellular localization of the transgenic ATXN7-GFP proteins have previously been extensively characterized and showed not to differ in these two cell lines [28]. Immunoblotting with an ATXN7 antibody revealed weak expression after three days of induction, but clear expression of both constructs from day 6 onwards (Figure 1A and [28]. No ATXN7 aggregation was detected in ATXN7Q10-GFP expressing cells [28]. However, in cells expressing ATXN7Q65-GFP filter trap analysis revealed aggregation from day 3 onwards and from day 9 the level of aggregated ATXN7 material was stable (Figure 1B and [28]).


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)

Expression of ATXN7Q65-GFP leads to increased ROS levels prior to toxicity. A) Western blot analysis of ATXN7 in FLQ10 and FLQ65 stable PC12 cell lines induced to express GFP-tagged ATXN7 with 10 (ATXN7Q10-GFP, top panel) or 65 (ATXN7Q65-GFP, lower panel) glutamines for the indicated number of days. Actin was used as loading control. B) Analysis of ATXN7 aggregation in FLQ65 cells induced to express ATXN7Q65-GFP for 1–12 days using the filter trap assay. C) Viability measured by the WST-1 assay and normalized against the protein content in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. D) Toxicity measured by the LDH membrane leakage assay in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. E) Total cellular ROS levels measured using DCHF-DA in cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for 0–12 days. F) Analysis of GSH levels in cells induced (−Dox) to express ATXN7Q65-GFP for 9 days and non-induced control cells (+Dox). For quantifications all data 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 1: Expression of ATXN7Q65-GFP leads to increased ROS levels prior to toxicity. A) Western blot analysis of ATXN7 in FLQ10 and FLQ65 stable PC12 cell lines induced to express GFP-tagged ATXN7 with 10 (ATXN7Q10-GFP, top panel) or 65 (ATXN7Q65-GFP, lower panel) glutamines for the indicated number of days. Actin was used as loading control. B) Analysis of ATXN7 aggregation in FLQ65 cells induced to express ATXN7Q65-GFP for 1–12 days using the filter trap assay. C) Viability measured by the WST-1 assay and normalized against the protein content in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. D) Toxicity measured by the LDH membrane leakage assay in FLQ10 and FLQ65 cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for the indicated number of days. E) Total cellular ROS levels measured using DCHF-DA in cells induced to express ATXN7Q10-GFP or ATXN7Q65-GFP for 0–12 days. F) Analysis of GSH levels in cells induced (−Dox) to express ATXN7Q65-GFP for 9 days and non-induced control cells (+Dox). For quantifications all data are shown as means ± SEM from three independent experiments with triplicates. * p <0.05, ** p <0.01 and *** p <0.001.
Mentions: To study the impact of mutant ATXN7 on cellular functions we used two recently generated stable inducible PC12 cell lines expressing N-terminal FLAG- and C-terminal GFP-tagged ATXN7 with 10 (FLQ10 line) or 65 (FLQ65 line) glutamines [28]. In these cell lines the expression of the corresponding transgenic proteins named ATXN7Q10-GFP and ATXN7Q65-GFP is controlled by the Tet-off expression system and induced by removal of doxycycline from the cell culture media. The induction timing, expression levels and sub-cellular localization of the transgenic ATXN7-GFP proteins have previously been extensively characterized and showed not to differ in these two cell lines [28]. Immunoblotting with an ATXN7 antibody revealed weak expression after three days of induction, but clear expression of both constructs from day 6 onwards (Figure 1A and [28]. No ATXN7 aggregation was detected in ATXN7Q10-GFP expressing cells [28]. However, in cells expressing ATXN7Q65-GFP filter trap analysis revealed aggregation from day 3 onwards and from day 9 the level of aggregated ATXN7 material was stable (Figure 1B and [28]).

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