A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease

Abstract

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntington's disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD.

Figure 1.

Genetic and pharmacologic modulation of Sir2 affects mHTT phenotypes in Drosophila. (A) Reducing Sir2 dose by half (Sir2 +/−) protects photoreceptor neurons from degeneration in flies challenged with mHttex1p Q93, but complete loss of Sir2 (Sir2 −/−) is deleterious (*P < 0.05, ***P < 0.005). (B) Animals heterozygous for Sir2 (+/−, black) show better climbing behavior than Htt-challenged animals with two doses of Sir2 (+/+, gray solid), while Sir2 −/− animals (dashed) have severely reduced climbing ability. (C) Selisistat rescues retinal neuron degeneration of mHttex1p Q93-challenged flies in a dose-dependent manner (*P < 0.05, ***P < 0.005). (D) Treatment with 10 µm selisistat (black squares) tends to improve survival of mHtt-challenged animals with the greatest effect seen in the 10–20 day survival rates (**P < 0.01) compared with no drug controls (gray diamonds). Control males not expressing mHtt (gray circles) survive much longer than mHtt-challenged females and are not affected by selisistat (not shown).

Figure 2.

Target validation: dSir2 is the in vivo target of selisistat in mHtt-challenged flies. (A) In mHTT flies challenged with Httex1p Q93 and bearing the normal two doses of Sir2 (+/+), selisistat rescues photoreceptors, while in the absence of Sir2 (−/−), the beneficial effects of selisistat on photoreceptor neurodegeneration are eliminated. Retinal neuron survival that is typically improved by treatment with selisistat is abolished if the presumed target gene is removed genetically (***P < 0.005). (B) Elimination of the Sir2 target also eliminates the effect of selisistat treatment on motor phenotype amelioration. Treatment of mHtt-expressing animals harboring two doses of Sir2 (+/+) with 10 µm selisistat (gray solid line) improved motor function compared with untreated animals (gray dashed line). Genetic reduction of Sir2 (−/−) in mHtt-challenged animals (black dashed line) decreased climbing ability and was not rescued by treatment with 10 µm selisistat (black solid line). (**P < 0.01 at 5 s).

Figure 3.

Selisistat inhibits the deacetylation activity of both human SirT1 and Drosophila Sir2. (A and D) WB analysis of total cell lysates from HEK293 cells transiently transfected with GCN5, p65 subunit and SirT1 (A) or Sir2 (D) (ratio 1: 0.5: 1). p65 acetylation was measured using a specific antibody against acetylated K310 and normalized to the total p65 content. The acetylation level in cells transfected with p65 and GCN5 only served as the control to evaluate the deacetylation activity of SirT1 or Sir2. The acetylation level in cells transfected with p65, GCN5 and SirT1 or Sir2 and treated with DMSO served as the control to evaluate the effect of selisistat treatment. The WBs shown are representative of three independent experiments. (B and E) Band intensity ratios normalized within each independent experiment (n = 3) show the extent of deacetylation by transfected SirT1 (B) or Sir2 (E). p65 deacetylation by SirT1 and Sir2, tested by applying the one-sample t-test versus the theoretical mean of 100, found the samples significantly different from respective controls (**P < 0.01). (C and F) Band intensity ratios normalized within each experiment (n = 3) show the extent to which p65 acetylation is restored by the drug treatment in cells transfected with SirT1 (C) or Sir2 (F). One-way ANOVA analysis followed by the Tukey–Kramer test for multiple comparisons found all concentrations of selisistat tested significantly different from DMSO control in SirT1-transfected cells (***P < 0.005) (C) and selisistat tested at 10 µm was significantly different from DMSO controls in Sir2-transfected cells (**P < 0.01) (F).

Figure 4.

Pharmacological inhibition of SirT1 rescues mHTT-mediated toxicity in mammalian cells. (A) In PC12 cells inducibly expressing a human HTT exon 1 fragment bearing 72 glutamine repeats, selisistat suppresses toxicity induced by expression of the mHtt transgene. Data are represented as mean and SEM of normalized data (induced control only—cited in the figure as ctrl = 100%; uninduced ctrl = 0% not shown) (controls are DMSO-treated only). (*P < 0.05, **P < 0.01 post-ANOVA Tukey–Kramer test for multiple comparisons). (B) In primary rat striatal cultures infected with lentiviral vectors expressing mutant (82Q) or wild type (18Q), selisistat treatment abolished 82Q-HTT-induced neuronal loss in a concentration-dependent manner. Data are displayed as mean and SEM (n = 4). (*P < 0.05, **P < 0.01 by two-sample Student's t-test for indicated comparisons).

Figure 5.

Effect of chronic administration of selisistat (5 and 20 mg/kg, PO, QD) on survival, body weight and behavioral parameters in mice. (A) R6/2 mice show premature death. Improved survival was associated with selisistat administration (one-sided log-rank Mantel–Cox statistic: *P < 0.05). Pairwise comparison of individual dose groups with vehicle-treated mice indicated no effect in the 5 mg/kg group (P > 0.5) but significant increase in survival in the 20 mg/kg group (P < 0.05) increasing median survival of 3 weeks. (B) The effect of chronic administration of selisistat (5 and 20 mg/kg, PO, QD) on body weights of the R6/2 mice from 4 to 25 weeks of age is depicted. R6/2 mice display lowered body weight compared with wild-type mice (data not shown). There was no statistically demonstrable effect of treatment on body weight although statistical analysis of body weight could only be carried out to 19 weeks due to the need for multiple subjects in all groups to satisfy conditions for ANOVA. (C and D) The effect of chronic administration of selisistat (5 and 20 mg/kg, PO, QD) in R6/2 mice on two different parameters of Open Field performance, distance traveled in center (C) and average velocity (D). (C) A significant effect of treatment on distance traveled in the center of the open field was observed with compound-treated mice. Individual comparison of drug-treated groups with the vehicle group across all ages revealed significant increases in center locomotion in the 5 mg/kg group (**P < 0.01). (D) A strong trend for treatment to increase velocity in the open field was detected in the 5 mg/kg group (P = 0.08), and individual group comparisons with vehicle revealed significant effects for the 20 mg/kg group (*P < 0.05).

Figure 6.

Pharmacological inhibition of SirT1 prevents the enlargement of ventricular volume and lowers Htt inclusion load in R6/2 mice. Brain slices from 12-week-old mice were analyzed for ventricular volume and inclusions using image analysis software. (A) Representative photomicrographs of brains from R6/2 mice treated with vehicle or 5 or 20 mg/kg selisistat. Note the reduction of ventricular enlargement in treated animals. (C) Ventricular volume is reduced in R6/2 animals treated with selisistat compared with untreated R6/2 animals. Data are displayed as mean and SEM (*P < 0.05 post-ANOVA Tukey–Kramer test for multiple comparisons). (B and D) Pharmacological inhibition of SirT1 lowers aggregation load in R6/2 mice striata. Brain slices from 12-week-old mice were stained to detect EM48-positive striatal inclusions. (B) Representative photomicrographs of striatal neurons from R6/2 mice treated with vehicle or 5 or 20 mg/kg selisistat. (D) Analysis of aggregate load in R6/2 mice striata in the different groups of animals expressed as percent of cells containing aggregates. Data are displayed as mean and SEM (*P < 0.05 post-ANOVA Tukey–Kramer test for multiple comparisons).