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

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.

Figure 1

Temsirolimus administration improves rotarod performance in ataxin-3 transgenic mice. (A) Mice expressing mutant ataxin-3 (black line) demonstrate a decreased latency to fall on the accelerating rotarod test compared with non-transgenic littermate controls (grey line). Data shown represent mean values and standard error. P < 0.001 by ANOVA. (B) Mice administered temsirolimus (CCI-779; black line) show an increase in performance on the rotarod over placebo treated control animals (grey line) throughout the period of the study. P < 0.001 by ANOVA. For placebo treated mice n = 17 at each time point. For temsirolimus treated mice n = 17 up to and including 14 weeks, n = 15 at 16 weeks and n = 14 thereafter.

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.

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.

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.