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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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Temsirolimus inhibits the mTOR pathway in vivo, as rapamycin does in cultured primary neurons. (A) Sections from mouse brains treated with either temsirolimus (CCI-779) or placebo were immunostained for phosphorylated S6 protein (Phospho-S6, top panels) or total S6 protein (bottom panels), nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Total S6 immunoreactivity was seen in the perinuclear region of all cells. In placebo treated animals some cells stained positive for phosphorylated S6, however, this staining was not seen in sections from animals treated with temsirolimus. Scale bar represents 5 µm and is valid for all panels. In cultured primary neurons, LC3-II levels were assessed by western blot (B). Two different exposures are shown to allow comparison of weaker bands in untreated lanes (−Baf A1) and stronger bands in Bafilomycin A1 (+Baf A1) lanes without saturation. Densitometric quantification of LC3-II levels relative to actin in triplicate experiments is shown in (C). **P < 0.01 and *P < 0.05 by t-test. Effect of rapamycin treatment of phosphorylation of downstream mTOR targets was investigated by western blotting, (D) phosphorylated p70 S6 kinase levels and phosphorylated S6 ribosomal protein and (E) phosphorylated eukaryotic initiation factor 4E-binding-protein (EBP)-1.
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Figure 2: Temsirolimus inhibits the mTOR pathway in vivo, as rapamycin does in cultured primary neurons. (A) Sections from mouse brains treated with either temsirolimus (CCI-779) or placebo were immunostained for phosphorylated S6 protein (Phospho-S6, top panels) or total S6 protein (bottom panels), nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Total S6 immunoreactivity was seen in the perinuclear region of all cells. In placebo treated animals some cells stained positive for phosphorylated S6, however, this staining was not seen in sections from animals treated with temsirolimus. Scale bar represents 5 µm and is valid for all panels. In cultured primary neurons, LC3-II levels were assessed by western blot (B). Two different exposures are shown to allow comparison of weaker bands in untreated lanes (−Baf A1) and stronger bands in Bafilomycin A1 (+Baf A1) lanes without saturation. Densitometric quantification of LC3-II levels relative to actin in triplicate experiments is shown in (C). **P < 0.01 and *P < 0.05 by t-test. Effect of rapamycin treatment of phosphorylation of downstream mTOR targets was investigated by western blotting, (D) phosphorylated p70 S6 kinase levels and phosphorylated S6 ribosomal protein and (E) phosphorylated eukaryotic initiation factor 4E-binding-protein (EBP)-1.

Mentions: Rapamycins, including temsirolimus, up-regulate autophagy by inhibiting the kinase mTOR. We therefore confirmed that the mTOR pathway was inhibited in the brains of treated mice. As we previously observed in a mouse model of Huntington’s disease (Ravikumar et al., 2004), mice treated with temsirolimus showed a decrease in immunoreactivity for phosphorylated S6 ribosomal protein, a downstream target of the mTOR pathway, whilst immunoreactivity for total S6 ribosomal protein did not change (Fig. 2A).Figure 2


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)

Temsirolimus inhibits the mTOR pathway in vivo, as rapamycin does in cultured primary neurons. (A) Sections from mouse brains treated with either temsirolimus (CCI-779) or placebo were immunostained for phosphorylated S6 protein (Phospho-S6, top panels) or total S6 protein (bottom panels), nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Total S6 immunoreactivity was seen in the perinuclear region of all cells. In placebo treated animals some cells stained positive for phosphorylated S6, however, this staining was not seen in sections from animals treated with temsirolimus. Scale bar represents 5 µm and is valid for all panels. In cultured primary neurons, LC3-II levels were assessed by western blot (B). Two different exposures are shown to allow comparison of weaker bands in untreated lanes (−Baf A1) and stronger bands in Bafilomycin A1 (+Baf A1) lanes without saturation. Densitometric quantification of LC3-II levels relative to actin in triplicate experiments is shown in (C). **P < 0.01 and *P < 0.05 by t-test. Effect of rapamycin treatment of phosphorylation of downstream mTOR targets was investigated by western blotting, (D) phosphorylated p70 S6 kinase levels and phosphorylated S6 ribosomal protein and (E) phosphorylated eukaryotic initiation factor 4E-binding-protein (EBP)-1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2801325&req=5

Figure 2: Temsirolimus inhibits the mTOR pathway in vivo, as rapamycin does in cultured primary neurons. (A) Sections from mouse brains treated with either temsirolimus (CCI-779) or placebo were immunostained for phosphorylated S6 protein (Phospho-S6, top panels) or total S6 protein (bottom panels), nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Total S6 immunoreactivity was seen in the perinuclear region of all cells. In placebo treated animals some cells stained positive for phosphorylated S6, however, this staining was not seen in sections from animals treated with temsirolimus. Scale bar represents 5 µm and is valid for all panels. In cultured primary neurons, LC3-II levels were assessed by western blot (B). Two different exposures are shown to allow comparison of weaker bands in untreated lanes (−Baf A1) and stronger bands in Bafilomycin A1 (+Baf A1) lanes without saturation. Densitometric quantification of LC3-II levels relative to actin in triplicate experiments is shown in (C). **P < 0.01 and *P < 0.05 by t-test. Effect of rapamycin treatment of phosphorylation of downstream mTOR targets was investigated by western blotting, (D) phosphorylated p70 S6 kinase levels and phosphorylated S6 ribosomal protein and (E) phosphorylated eukaryotic initiation factor 4E-binding-protein (EBP)-1.
Mentions: Rapamycins, including temsirolimus, up-regulate autophagy by inhibiting the kinase mTOR. We therefore confirmed that the mTOR pathway was inhibited in the brains of treated mice. As we previously observed in a mouse model of Huntington’s disease (Ravikumar et al., 2004), mice treated with temsirolimus showed a decrease in immunoreactivity for phosphorylated S6 ribosomal protein, a downstream target of the mTOR pathway, whilst immunoreactivity for total S6 ribosomal protein did not change (Fig. 2A).Figure 2

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