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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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RORα and SOD1 reduce ATXN7Q65-Myc protein aggregation. A) HEK 293 cells were co-transfected to express ATXN7Q65-Myc and RORα. After 48 hours RORα and ATXN7Q65-Myc expression (left panel) and ATXN7 aggregation (right panel) were analyzed and quantified. Actin was used as loading control for western blots. B) HEK 293 T cells were co-transfected with ATXN7Q65-Myc and 0–1 μg of a plasmid encoding wild-type (WT), A4V mutant or H48Q mutant SOD1. Empty vector (Myc) was used to allow the same amount of plasmids to be transfected in each well. Forty-eight hours after transfection ATXN7Q65-Myc aggregation was analyzed by filter trap in cells co-transfected with WT SOD1 (left), A4V SOD1 (middle) and H48Q SOD1 (right). All quantifications are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05 and ** p <0.01.
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Figure 3: RORα and SOD1 reduce ATXN7Q65-Myc protein aggregation. A) HEK 293 cells were co-transfected to express ATXN7Q65-Myc and RORα. After 48 hours RORα and ATXN7Q65-Myc expression (left panel) and ATXN7 aggregation (right panel) were analyzed and quantified. Actin was used as loading control for western blots. B) HEK 293 T cells were co-transfected with ATXN7Q65-Myc and 0–1 μg of a plasmid encoding wild-type (WT), A4V mutant or H48Q mutant SOD1. Empty vector (Myc) was used to allow the same amount of plasmids to be transfected in each well. Forty-eight hours after transfection ATXN7Q65-Myc aggregation was analyzed by filter trap in cells co-transfected with WT SOD1 (left), A4V SOD1 (middle) and H48Q SOD1 (right). All quantifications are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05 and ** p <0.01.

Mentions: To investigate the relationship between oxidative stress and ATXN7 aggregation, we analyzed whether anti-oxidant treatment of our stable FLQ65 cells induced to express ATXN7Q65-GFP also had an effect on the level of ATXN7 aggregation. Results showed that both α-tocopherol and NAC treatment during the induction of ATXN7Q65-GFP expression in the stable PC12 cell model lowered the level of aggregated ATXN7 material with circa 40–80% (Figure 2C and 2F). To further confirm the connection between oxidative stress and ATXN7 aggregation and make sure that the effect seen in Figure 2 was not influenced by the GFP-tag on ATXN7 or specific to PC12 cells, we did further experiments in HEK 293 T cells transfected to express myc-tagged ATXN7 with 10 (ATXN7Q10-myc) or 65 (ATXN7Q65-myc) glutamines. We first investigated whether support of the anti-oxidant system could also ameliorate the aggregation of ATXN7Q65-myc, by co-transfecting the HEK 293 T cells with ATXN7Q65-myc and RORα or SOD1. Over-expression of RORα, a transcription factor known to activate anti-oxidant genes [29], ameliorated the aggregation of ATXN7Q65-Myc (Figure 3A). So did over-expression of wild-type SOD1 with full dismutase activity, whereas co-expression of mutant forms of SOD1 with reduced (A4V) [30,31] or no enzymatic activity (H48Q) [31] showed reduced or no ability, respectively, to reduce ATXN7Q65-Myc aggregation (Figure 3B). Neither RORα nor SOD1 co-expression affected the expression of soluble ATXN7Q65-Myc (Figure 3A and data not shown). We next investigated whether promotion of an oxidative environment could aggravate the aggregation of ATXN7Q65-myc by treating transfected HEK 293 T cells with increasing concentrations of H2O2 (Figure 4A-B) or BSO (Figure 4C-D) an inhibitor of GSH biosynthesis [32]. No trace of aggregated material could be detected in ATXN7Q10-Myc expressing cells under control or treated conditions (data not shown). However, aggregated ATXN7 material was detected in ATXN7Q65-Myc cells and both H2O2 and BSO treatment led to an increase in aggregated material without affecting the expression of soluble ATXN7Q65-Myc (Figure 4). Taken together, these data suggest that there is a clear connection between oxidative stress and aggregation of mutant ATXN7.


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)

RORα and SOD1 reduce ATXN7Q65-Myc protein aggregation. A) HEK 293 cells were co-transfected to express ATXN7Q65-Myc and RORα. After 48 hours RORα and ATXN7Q65-Myc expression (left panel) and ATXN7 aggregation (right panel) were analyzed and quantified. Actin was used as loading control for western blots. B) HEK 293 T cells were co-transfected with ATXN7Q65-Myc and 0–1 μg of a plasmid encoding wild-type (WT), A4V mutant or H48Q mutant SOD1. Empty vector (Myc) was used to allow the same amount of plasmids to be transfected in each well. Forty-eight hours after transfection ATXN7Q65-Myc aggregation was analyzed by filter trap in cells co-transfected with WT SOD1 (left), A4V SOD1 (middle) and H48Q SOD1 (right). All quantifications are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05 and ** p <0.01.
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Related In: Results  -  Collection

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Figure 3: RORα and SOD1 reduce ATXN7Q65-Myc protein aggregation. A) HEK 293 cells were co-transfected to express ATXN7Q65-Myc and RORα. After 48 hours RORα and ATXN7Q65-Myc expression (left panel) and ATXN7 aggregation (right panel) were analyzed and quantified. Actin was used as loading control for western blots. B) HEK 293 T cells were co-transfected with ATXN7Q65-Myc and 0–1 μg of a plasmid encoding wild-type (WT), A4V mutant or H48Q mutant SOD1. Empty vector (Myc) was used to allow the same amount of plasmids to be transfected in each well. Forty-eight hours after transfection ATXN7Q65-Myc aggregation was analyzed by filter trap in cells co-transfected with WT SOD1 (left), A4V SOD1 (middle) and H48Q SOD1 (right). All quantifications are shown as means ± SEM from three independent experiments with triplicates. NS: not significant, * p <0.05 and ** p <0.01.
Mentions: To investigate the relationship between oxidative stress and ATXN7 aggregation, we analyzed whether anti-oxidant treatment of our stable FLQ65 cells induced to express ATXN7Q65-GFP also had an effect on the level of ATXN7 aggregation. Results showed that both α-tocopherol and NAC treatment during the induction of ATXN7Q65-GFP expression in the stable PC12 cell model lowered the level of aggregated ATXN7 material with circa 40–80% (Figure 2C and 2F). To further confirm the connection between oxidative stress and ATXN7 aggregation and make sure that the effect seen in Figure 2 was not influenced by the GFP-tag on ATXN7 or specific to PC12 cells, we did further experiments in HEK 293 T cells transfected to express myc-tagged ATXN7 with 10 (ATXN7Q10-myc) or 65 (ATXN7Q65-myc) glutamines. We first investigated whether support of the anti-oxidant system could also ameliorate the aggregation of ATXN7Q65-myc, by co-transfecting the HEK 293 T cells with ATXN7Q65-myc and RORα or SOD1. Over-expression of RORα, a transcription factor known to activate anti-oxidant genes [29], ameliorated the aggregation of ATXN7Q65-Myc (Figure 3A). So did over-expression of wild-type SOD1 with full dismutase activity, whereas co-expression of mutant forms of SOD1 with reduced (A4V) [30,31] or no enzymatic activity (H48Q) [31] showed reduced or no ability, respectively, to reduce ATXN7Q65-Myc aggregation (Figure 3B). Neither RORα nor SOD1 co-expression affected the expression of soluble ATXN7Q65-Myc (Figure 3A and data not shown). We next investigated whether promotion of an oxidative environment could aggravate the aggregation of ATXN7Q65-myc by treating transfected HEK 293 T cells with increasing concentrations of H2O2 (Figure 4A-B) or BSO (Figure 4C-D) an inhibitor of GSH biosynthesis [32]. No trace of aggregated material could be detected in ATXN7Q10-Myc expressing cells under control or treated conditions (data not shown). However, aggregated ATXN7 material was detected in ATXN7Q65-Myc cells and both H2O2 and BSO treatment led to an increase in aggregated material without affecting the expression of soluble ATXN7Q65-Myc (Figure 4). Taken together, these data suggest that there is a clear connection between oxidative stress and aggregation of mutant ATXN7.

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