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Autophagy induction reduces mutant ataxin-3 levels and toxicity in a mouse model of spinocerebellar ataxia type 3.

Menzies FM, Huebener J, Renna M, Bonin M, Riess O, Rubinsztein DC - Brain (2009)

Bottom Line: The mutant protein forms intracellular aggregates in the brain.However, the cellular mechanisms causing toxicity are still poorly understood and there are currently no effective treatments.This identified ubiquitin specific peptidase-15, which showed expression changes at both the messenger ribonucleic acid and protein level.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge, UK.

ABSTRACT
Spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by the expansion of the polyglutamine repeat region within the ataxin-3 protein. The mutant protein forms intracellular aggregates in the brain. However, the cellular mechanisms causing toxicity are still poorly understood and there are currently no effective treatments. In this study we show that administration of a rapamycin ester (cell cycle inhibitor-779, temsirolimus) improves motor performance in a transgenic mouse model of spinocerebellar ataxia type 3. Temsirolimus inhibits mammalian target of rapamycin and hence upregulates protein degradation by autophagy. Temsirolimus reduces the number of aggregates seen in the brains of transgenic mice and decreases levels of cytosolic soluble mutant ataxin-3, while endogenous wild-type protein levels remain unaffected. Temsirolimus is designed for long-term use in patients and therefore represents a possible therapeutic strategy for the treatment of spinocerebellar ataxia type 3. Using this disease model and treatment paradigm, we employed a microarray approach to investigate transcriptional changes that might be important in the pathogenesis of spinocerebellar ataxia type 3. This identified ubiquitin specific peptidase-15, which showed expression changes at both the messenger ribonucleic acid and protein level. Ubiquitin specific peptidase-15 levels were also changed in mice expressing another mutant polyglutamine protein, huntingtin. In total we identified 16 transcripts that were decreased in transgenic ataxin-3 mice that were normalized following temsirolimus treatment. In this mouse model with relatively mild disease progression, the number of transcripts changed was low and the magnitude of these changes was small. However, the importance of these transcriptional alterations in the pathogenesis of spinocerebellar ataxia type 3 remains unclear.

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Aggregation and clearance of ataxin-3 with temsirolimus treatment. (A) Ataxin-3 staining in the motor cortex reveals the presence of aggregates in transgenic mice. Control (placebo) mice show more aggregates in the motor cortex than treated (temsirolimus, CCI-779) animals. Examples of aggregates are indicated by arrows in both treated and untreated brains. Scale bar represents 20 µm and is valid for all pictures. Quantification of the mean number of aggregates is shown in (B). Aggregates were counted in the motor cortex on three sections each for five mice in each treatment group (n = 5). *P < 0.05 placebo versus temsirolimus treated mice by t-test. Levels of endogenous, and transgenic mutant ataxin-3 in placebo (cont) and drug treated (CCI) mice were measured by western blotting (C). The expanded polyglutamine stretch of the mutant protein results in its slower migration in the gel (upper band). Tubulin is used as a control to ensure equal loading of cytoplasmic samples and Histone H3 for nuclear samples. The black line marks where lanes of the western have been omitted for the sake of clarity. Quantification by densitometry of ataxin-3 levels in nuclear protein extracts (D) and cytosolic extracts (E) from three mice corrected for the level of tubulin (n = 3). **P < 0.01 placebo versus temsirolimus treated mice by t-test.
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Figure 3: Aggregation and clearance of ataxin-3 with temsirolimus treatment. (A) Ataxin-3 staining in the motor cortex reveals the presence of aggregates in transgenic mice. Control (placebo) mice show more aggregates in the motor cortex than treated (temsirolimus, CCI-779) animals. Examples of aggregates are indicated by arrows in both treated and untreated brains. Scale bar represents 20 µm and is valid for all pictures. Quantification of the mean number of aggregates is shown in (B). Aggregates were counted in the motor cortex on three sections each for five mice in each treatment group (n = 5). *P < 0.05 placebo versus temsirolimus treated mice by t-test. Levels of endogenous, and transgenic mutant ataxin-3 in placebo (cont) and drug treated (CCI) mice were measured by western blotting (C). The expanded polyglutamine stretch of the mutant protein results in its slower migration in the gel (upper band). Tubulin is used as a control to ensure equal loading of cytoplasmic samples and Histone H3 for nuclear samples. The black line marks where lanes of the western have been omitted for the sake of clarity. Quantification by densitometry of ataxin-3 levels in nuclear protein extracts (D) and cytosolic extracts (E) from three mice corrected for the level of tubulin (n = 3). **P < 0.01 placebo versus temsirolimus treated mice by t-test.

Mentions: To assess the cellular effects associated with the improvement in behavioural testing seen in temsirolimus treated mice we looked at ataxin-3 positive aggregates in the brains of treated and control mice. Mice treated with placebo show a large number of aggregates throughout the brain. We scored aggregates in the motor cortex as this region demonstrated a high level of aggregates, while aggregate number could still be counted accurately, making it the most suitable region for this assay. Mice were treated for two months with temsirolimus or placebo beginning at 5 weeks of age (as for rotarod performance). Mice were then sacrificed and the number of aggregates in the motor cortex quantified. Aggregate number was significantly reduced in mice treated with temsirolimus compared to placebo treated mice (Fig. 3A and B).Figure 3


Autophagy induction reduces mutant ataxin-3 levels and toxicity in a mouse model of spinocerebellar ataxia type 3.

Menzies FM, Huebener J, Renna M, Bonin M, Riess O, Rubinsztein DC - Brain (2009)

Aggregation and clearance of ataxin-3 with temsirolimus treatment. (A) Ataxin-3 staining in the motor cortex reveals the presence of aggregates in transgenic mice. Control (placebo) mice show more aggregates in the motor cortex than treated (temsirolimus, CCI-779) animals. Examples of aggregates are indicated by arrows in both treated and untreated brains. Scale bar represents 20 µm and is valid for all pictures. Quantification of the mean number of aggregates is shown in (B). Aggregates were counted in the motor cortex on three sections each for five mice in each treatment group (n = 5). *P < 0.05 placebo versus temsirolimus treated mice by t-test. Levels of endogenous, and transgenic mutant ataxin-3 in placebo (cont) and drug treated (CCI) mice were measured by western blotting (C). The expanded polyglutamine stretch of the mutant protein results in its slower migration in the gel (upper band). Tubulin is used as a control to ensure equal loading of cytoplasmic samples and Histone H3 for nuclear samples. The black line marks where lanes of the western have been omitted for the sake of clarity. Quantification by densitometry of ataxin-3 levels in nuclear protein extracts (D) and cytosolic extracts (E) from three mice corrected for the level of tubulin (n = 3). **P < 0.01 placebo versus temsirolimus treated mice by t-test.
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Figure 3: Aggregation and clearance of ataxin-3 with temsirolimus treatment. (A) Ataxin-3 staining in the motor cortex reveals the presence of aggregates in transgenic mice. Control (placebo) mice show more aggregates in the motor cortex than treated (temsirolimus, CCI-779) animals. Examples of aggregates are indicated by arrows in both treated and untreated brains. Scale bar represents 20 µm and is valid for all pictures. Quantification of the mean number of aggregates is shown in (B). Aggregates were counted in the motor cortex on three sections each for five mice in each treatment group (n = 5). *P < 0.05 placebo versus temsirolimus treated mice by t-test. Levels of endogenous, and transgenic mutant ataxin-3 in placebo (cont) and drug treated (CCI) mice were measured by western blotting (C). The expanded polyglutamine stretch of the mutant protein results in its slower migration in the gel (upper band). Tubulin is used as a control to ensure equal loading of cytoplasmic samples and Histone H3 for nuclear samples. The black line marks where lanes of the western have been omitted for the sake of clarity. Quantification by densitometry of ataxin-3 levels in nuclear protein extracts (D) and cytosolic extracts (E) from three mice corrected for the level of tubulin (n = 3). **P < 0.01 placebo versus temsirolimus treated mice by t-test.
Mentions: To assess the cellular effects associated with the improvement in behavioural testing seen in temsirolimus treated mice we looked at ataxin-3 positive aggregates in the brains of treated and control mice. Mice treated with placebo show a large number of aggregates throughout the brain. We scored aggregates in the motor cortex as this region demonstrated a high level of aggregates, while aggregate number could still be counted accurately, making it the most suitable region for this assay. Mice were treated for two months with temsirolimus or placebo beginning at 5 weeks of age (as for rotarod performance). Mice were then sacrificed and the number of aggregates in the motor cortex quantified. Aggregate number was significantly reduced in mice treated with temsirolimus compared to placebo treated mice (Fig. 3A and B).Figure 3

Bottom Line: The mutant protein forms intracellular aggregates in the brain.However, the cellular mechanisms causing toxicity are still poorly understood and there are currently no effective treatments.This identified ubiquitin specific peptidase-15, which showed expression changes at both the messenger ribonucleic acid and protein level.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge, UK.

ABSTRACT
Spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by the expansion of the polyglutamine repeat region within the ataxin-3 protein. The mutant protein forms intracellular aggregates in the brain. However, the cellular mechanisms causing toxicity are still poorly understood and there are currently no effective treatments. In this study we show that administration of a rapamycin ester (cell cycle inhibitor-779, temsirolimus) improves motor performance in a transgenic mouse model of spinocerebellar ataxia type 3. Temsirolimus inhibits mammalian target of rapamycin and hence upregulates protein degradation by autophagy. Temsirolimus reduces the number of aggregates seen in the brains of transgenic mice and decreases levels of cytosolic soluble mutant ataxin-3, while endogenous wild-type protein levels remain unaffected. Temsirolimus is designed for long-term use in patients and therefore represents a possible therapeutic strategy for the treatment of spinocerebellar ataxia type 3. Using this disease model and treatment paradigm, we employed a microarray approach to investigate transcriptional changes that might be important in the pathogenesis of spinocerebellar ataxia type 3. This identified ubiquitin specific peptidase-15, which showed expression changes at both the messenger ribonucleic acid and protein level. Ubiquitin specific peptidase-15 levels were also changed in mice expressing another mutant polyglutamine protein, huntingtin. In total we identified 16 transcripts that were decreased in transgenic ataxin-3 mice that were normalized following temsirolimus treatment. In this mouse model with relatively mild disease progression, the number of transcripts changed was low and the magnitude of these changes was small. However, the importance of these transcriptional alterations in the pathogenesis of spinocerebellar ataxia type 3 remains unclear.

Show MeSH
Related in: MedlinePlus