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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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Microarray analysis of transgenic ataxin-3 mice. Microarray analysis of four experimental groups of mice was carried out and changes between pairs of experimental groups were identified. (A) Shows a representation of these comparisons as a Venn diagram. The number of genes with altered regulation are given for each pair of groups. The shaded region identifies the transcripts selected i.e. those altered in wild-type versus transgenic mice that were normalized following the treatment of mice with temsirolimus (see Table 1). Microarray changes were verified by quantitative PCR. (B) Shows mRNA relative expression levels of Tloc1, Usp15 and Grik2, expression levels are normalized to wild-type, placebo treated mice (black bars). Grey bars represent placebo treated, ataxin-3 transgenic mice and white bars temsirolimus treated ataxin-3 transgenic mice. *P < 0.05 and ***P < 0.001 by t-test. (C) Additional verification of transcript alterations in wild-type versus ataxin-3 transgenic mice. mRNA was extracted from brains of untreated mice and expression levels of measured relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase 2. Expression was normalized to levels in wild-type mice and the relative levels in ataxin-3 transgenic mice are shown. *P < 0.05 and **P < 0.01 by t-test, n = 3, other comparisons are not statistically significant. Protein levels of Usp15 were analysed in 3.5–4 month old wild-type (n = 4) and transgenic ataxin-3 (n = 6) mouse brain by western blotting (D). Protein levels were quantified by densitometry relative to the level of the loading control, actin (E). **P < 0.01 by t-test. (F) mRNA expression of Usp15 was measured in brains of 6 month old wild-type and huntingtin (Htt) transgenic mice. Expression levels are corrected for the housekeeping gene SDHA and are shown relative to wild- type mice. *P < 0.05 by t-test. (G) Protein levels of Usp15 were investigated by western blot with actin as a loading control. Protein levels were quantified by densitometry relative to the level of actin (H). ***P < 0.001 by t-test.
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Figure 4: Microarray analysis of transgenic ataxin-3 mice. Microarray analysis of four experimental groups of mice was carried out and changes between pairs of experimental groups were identified. (A) Shows a representation of these comparisons as a Venn diagram. The number of genes with altered regulation are given for each pair of groups. The shaded region identifies the transcripts selected i.e. those altered in wild-type versus transgenic mice that were normalized following the treatment of mice with temsirolimus (see Table 1). Microarray changes were verified by quantitative PCR. (B) Shows mRNA relative expression levels of Tloc1, Usp15 and Grik2, expression levels are normalized to wild-type, placebo treated mice (black bars). Grey bars represent placebo treated, ataxin-3 transgenic mice and white bars temsirolimus treated ataxin-3 transgenic mice. *P < 0.05 and ***P < 0.001 by t-test. (C) Additional verification of transcript alterations in wild-type versus ataxin-3 transgenic mice. mRNA was extracted from brains of untreated mice and expression levels of measured relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase 2. Expression was normalized to levels in wild-type mice and the relative levels in ataxin-3 transgenic mice are shown. *P < 0.05 and **P < 0.01 by t-test, n = 3, other comparisons are not statistically significant. Protein levels of Usp15 were analysed in 3.5–4 month old wild-type (n = 4) and transgenic ataxin-3 (n = 6) mouse brain by western blotting (D). Protein levels were quantified by densitometry relative to the level of the loading control, actin (E). **P < 0.01 by t-test. (F) mRNA expression of Usp15 was measured in brains of 6 month old wild-type and huntingtin (Htt) transgenic mice. Expression levels are corrected for the housekeeping gene SDHA and are shown relative to wild- type mice. *P < 0.05 by t-test. (G) Protein levels of Usp15 were investigated by western blot with actin as a loading control. Protein levels were quantified by densitometry relative to the level of actin (H). ***P < 0.001 by t-test.

Mentions: From the 91 transcripts altered in wild-type versus transgenic animals we selected those that were also changed in the opposite direction (i.e. returned to normal levels) in transgenic animals treated with temsirolimus (Fig. 4A). This analysis identified only 16 genes (Table 1) whose expression was decreased in transgenic ataxin-3 mice and increased in transgenic mice treated with temsirolimus. We did not identify any genes whose expression increased in transgenic ataxin-3 mice and decreased in temsirolimus treated mice.Figure 4


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)

Microarray analysis of transgenic ataxin-3 mice. Microarray analysis of four experimental groups of mice was carried out and changes between pairs of experimental groups were identified. (A) Shows a representation of these comparisons as a Venn diagram. The number of genes with altered regulation are given for each pair of groups. The shaded region identifies the transcripts selected i.e. those altered in wild-type versus transgenic mice that were normalized following the treatment of mice with temsirolimus (see Table 1). Microarray changes were verified by quantitative PCR. (B) Shows mRNA relative expression levels of Tloc1, Usp15 and Grik2, expression levels are normalized to wild-type, placebo treated mice (black bars). Grey bars represent placebo treated, ataxin-3 transgenic mice and white bars temsirolimus treated ataxin-3 transgenic mice. *P < 0.05 and ***P < 0.001 by t-test. (C) Additional verification of transcript alterations in wild-type versus ataxin-3 transgenic mice. mRNA was extracted from brains of untreated mice and expression levels of measured relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase 2. Expression was normalized to levels in wild-type mice and the relative levels in ataxin-3 transgenic mice are shown. *P < 0.05 and **P < 0.01 by t-test, n = 3, other comparisons are not statistically significant. Protein levels of Usp15 were analysed in 3.5–4 month old wild-type (n = 4) and transgenic ataxin-3 (n = 6) mouse brain by western blotting (D). Protein levels were quantified by densitometry relative to the level of the loading control, actin (E). **P < 0.01 by t-test. (F) mRNA expression of Usp15 was measured in brains of 6 month old wild-type and huntingtin (Htt) transgenic mice. Expression levels are corrected for the housekeeping gene SDHA and are shown relative to wild- type mice. *P < 0.05 by t-test. (G) Protein levels of Usp15 were investigated by western blot with actin as a loading control. Protein levels were quantified by densitometry relative to the level of actin (H). ***P < 0.001 by t-test.
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Figure 4: Microarray analysis of transgenic ataxin-3 mice. Microarray analysis of four experimental groups of mice was carried out and changes between pairs of experimental groups were identified. (A) Shows a representation of these comparisons as a Venn diagram. The number of genes with altered regulation are given for each pair of groups. The shaded region identifies the transcripts selected i.e. those altered in wild-type versus transgenic mice that were normalized following the treatment of mice with temsirolimus (see Table 1). Microarray changes were verified by quantitative PCR. (B) Shows mRNA relative expression levels of Tloc1, Usp15 and Grik2, expression levels are normalized to wild-type, placebo treated mice (black bars). Grey bars represent placebo treated, ataxin-3 transgenic mice and white bars temsirolimus treated ataxin-3 transgenic mice. *P < 0.05 and ***P < 0.001 by t-test. (C) Additional verification of transcript alterations in wild-type versus ataxin-3 transgenic mice. mRNA was extracted from brains of untreated mice and expression levels of measured relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase 2. Expression was normalized to levels in wild-type mice and the relative levels in ataxin-3 transgenic mice are shown. *P < 0.05 and **P < 0.01 by t-test, n = 3, other comparisons are not statistically significant. Protein levels of Usp15 were analysed in 3.5–4 month old wild-type (n = 4) and transgenic ataxin-3 (n = 6) mouse brain by western blotting (D). Protein levels were quantified by densitometry relative to the level of the loading control, actin (E). **P < 0.01 by t-test. (F) mRNA expression of Usp15 was measured in brains of 6 month old wild-type and huntingtin (Htt) transgenic mice. Expression levels are corrected for the housekeeping gene SDHA and are shown relative to wild- type mice. *P < 0.05 by t-test. (G) Protein levels of Usp15 were investigated by western blot with actin as a loading control. Protein levels were quantified by densitometry relative to the level of actin (H). ***P < 0.001 by t-test.
Mentions: From the 91 transcripts altered in wild-type versus transgenic animals we selected those that were also changed in the opposite direction (i.e. returned to normal levels) in transgenic animals treated with temsirolimus (Fig. 4A). This analysis identified only 16 genes (Table 1) whose expression was decreased in transgenic ataxin-3 mice and increased in transgenic mice treated with temsirolimus. We did not identify any genes whose expression increased in transgenic ataxin-3 mice and decreased in temsirolimus treated mice.Figure 4

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