Unbiased screen identifies aripiprazole as a modulator of abundance of the polyglutamine disease protein, ataxin-3

Abstract

No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits. Aripiprazole increased longevity in a Drosophila model of Machado-Joseph disease and effectively reduced aggregated ATXN3 species in flies and in brains of transgenic mice treated for 10 days. The aripiprazole-mediated decrease in ATXN3 abundance may reflect a complex response culminating in the modulation of specific components of cellular protein homeostasis. Aripiprazole represents a potentially promising therapeutic drug for Machado-Joseph disease and possibly other neurological proteinopathies.

Keywords: Machado-Joseph disease; drug screen; neurodegeneration; spinocerebellar ataxia type 3; therapeutics.

There are as yet no disease-modifying treatments for Machado-Joseph disease (spinocerebellar ataxia type 3). Costa et al. report that the atypical antipsychotic aripiprazole reduces levels of the mutant protein ATXN3 in animal models of this disease. Aripiprazole may have therapeutic potential for Machado-Joseph disease and possibly other neurodegenerative proteinopathies.

Figure 1

Cell-based screen identifies small molecules that reduce levels of mutant ATXN3. (A) Schematic of ATXN3 fusion proteins expressed in stably transfected HEK 293 cell lines. (B) Immunoblots using anti-ATXN3 (1H9), anti-Luciferase (Luc) and anti-FLAG antibodies show the expression of ATXN3 fusion proteins in 293.ATXN3Q26Luc and 293.ATXN3Q81Luc cell lines. (C) Schematic view of the primary high throughput screen of 2880 small molecules using 293.ATXN3Q81Luc cells. Percentage of luminescence inhibition by plate (nine plates) is shown relative to the positive control (red squares) corresponding to parental HEK 293 cells treated with vehicle (DMSO). 293.ATXN3Q81Luc cells treated with small molecules at 8 μM (samples) are represented as green squares, and cells treated with DMSO (negative control) as blue squares. Z-factor is also represented for each plate in purple (average plate Z = 0.81). (D) Distribution of compounds in primary screen sorted by percentage of luminescence inhibition (n = 2880). (E) Stepwise strategy followed to select nine small molecules that reduce levels of mutant ATXN3 for follow-up studies. FF = firefly.

Figure 2

Five small molecules decrease levels of expanded ATXN3 in confirmation screens using 293.ATXN3Q81.Luc cells. (A) Representative anti-ATXN3 immunoblots show the efficacy of sodium salinomycin (Na Salinomycin), AM251, aripiprazole, clotrimazole and mifepristone to reduce the amount of mutant ATXN3 fusion protein after 48 h treatment of 293.ATXN3Q81Luc cells. Compounds were dissolved in DMSO except aripiprazole, which was dissolved in 1:1 DMSO/Tween-80. Quantification of bands corresponding to ATXN3Q81Luc and endogenous ATXN3 (endATXN3) is shown in B and C, respectively. Bars represent the mean percentage of each protein relative to vehicle-treated cells and normalized to α-tubulin (±SEM) in three independent experiments. Comparisons between cells treated with a specific compound concentration and cells treated with vehicle were performed using Student’s t-test with statistical significance, as indicated: *P < 0.05 and **P < 0.01.

Figure 3

Sodium Salinomycin, AM251, and aripiprazole reduce human mutant ATXN3 levels in organotypic brain slice cultures from YACMJD84.2 transgenic mice (Q84). (A) Anti-ATXN3 immunoblots show that sodium salinomycin (Na Salinomycin), AM251 and aripiprazole decrease human mutant ATXN3 levels in brainstem and cerebellar fractions of brain slices from hemizygous YACMJD84.2 mice (Q84). Sagittal brain slices (300 μM) from the same mouse were cultured with specific concentrations of the compound or its vehicle at 37°C/5% CO₂. After 48 h of treatment, brainstem and cerebellar regions from each slice were processed separately. Quantification of bands corresponding to human mutant ATXN3 and mouse ATXN3 shown in B and C, respectively. Bars represent the mean percentage of protein relative to levels in vehicle-treated slices and normalized to α-tubulin (±SEM) for three independent experiments using different mice. Comparison between slices treated with a specific compound/concentration and slices treated with vehicle was performed using Student’s t-test with statistical significance, as indicated: *P < 0.05 and **P < 0.01.

Figure 4

Aripiprazole modestly enhances the longevity of flies expressing mutant ATXN3 and reduces HMW ATXN3 species. (A) We generated new transgenic flies that express full-length human ATXN3 with a pathogenic expansion of 77 repeats (MJD). Kaplan-Meier survival curves show that, when ATXN3 expression is driven throughout the fly by the sqh-Gal4 driver, MJD flies (n = 318) have a markedly shortened lifespan compared to flies containing empty vector control (CTRL) (n = 284). Genotypes: w*; sqh-Gal4/+; Empty-Vector-Ctrl/+, w*; sqh-Gal4/+; UAS-ATXN3Q77(MJD)/+. (B) Upon eclosion, MJD flies were placed in instant formula food containing either the vehicle (1:1 DMSO:Tween-80) or aripiprazole (50 μM). Aripiprazole-treated flies (Arip) (n = 242) show a modest but significant increase in mean survival compared to vehicle-treated flies. Genotype: w*; sqh-Gal4/+; UAS-ATXN3Q77(MJD)/+. (C) ATXN3 immunoblotting of lysates from whole flies reveals decreased HMW ATXN3 species in flies treated with aripiprazole (A) compared to flies treated with vehicle (V) (n = 10 per group, except n = 3 for Day 7). Histogram shows the relative amount of HMW ATXN3 species to total ATXN3 quantified by band intensity shown in the immunoblot. Hash symbol represents a non-specific ATXN3 band. Kaplan-Meier survival curves were compared using the Log-Rank Mantel-Cox test. **P < 0.01 and ***P < 0.001.

Figure 5

Subchronic treatment of Q84 mice with aripiprazole reduces soluble HMW species of ATXN3 in the brainstem/midbrain. Twelve-week-old Q84 mice were injected daily with aripiprazole 15 mg/kg (10–18) or vehicle (1–9) for 10 days (males 1–4 and 10–13; females 5–9 and 14–18). (A) Anti-ATXN3 immunoblotting of solubilized protein extracts from brainstem reveals decreased HMW ATXN3 species in aripiprazole-treated mice. (B) Quantification of ATXN3 species (in A) shows that aripiprazole reduced HMW ATXN3 species to 44% of levels found in vehicle-treated mice. Bars represent the average percentage of protein species relative to vehicle-treated mice, corrected for α-tubulin (±SEM). Comparison between groups was made using Student’s t-test and statistical significance is indicated as *P < 0.05. (C) Aripiprazole and vehicle-treated mice show similar levels of human ATXN3 and mouse Atxn3 transcripts in brainstem. Values were normalized for Gapdh expression and referenced to the average of vehicle-treated mice of the correspondent gender. Bars represent the average of transcript fold change per mouse group (n = 9) ± SEM. (D) Filter trap assay using anti-MJD antibody shows insoluble ATXN3 in the brainstem/midbrain of aripiprazole and vehicle-treated mice. (E) Quantification of bands in D, normalized for total protein levels revealed by Ponceau staining (Supplementary Fig. 6), shows no differences of insoluble ATXN3 between the two groups of mice. Bars represent the average of insoluble ATXN3 relative to vehicle-treated mice (±SEM). (F) Confocal single plan images of pontine neurons from aripiprazole or vehicle-treated mice labelled for ATXN3 (green), NeuN (red) and nuclei with DAPI (blue). No major differences of ATXN3 staining between the two groups of mice are noted except for a slight reduction of ATXN3 in the cytoplasm in mice treated with aripiprazole. Scale bar = 20 μm. (G) Both groups of mice display similar number of ATXN3-positive puncta in ventral pontine nuclei. Bars represent the average of puncta (±SEM). (H) Quantification of nuclear ATXN3 fluorescence in pontine neurons reveals no differences between mice treated with aripiprazole (n = 10) or vehicle (n = 7). Bars correspond to the average corrected total cell fluorescence (CTCF) of ATXN3 (±SEM).

Figure 6

Aripiprazole does not interfere with ATXN3 fibril formation in vitro. (A) Thioflavin T fluorescence assay shows that ATXN3Q55 fibril formation is not affected by aripiprazole. Curves of recombinant ATXN3Q55 (10 μM) incubated with 40 times molar excess of aripiprazole (400 μM) in (red) or vehicle (black) were normalized by fluorescence values for the blank control (buffer). Each point on thioflavin T fluorescence assays corresponds to the average of three replicates in two independent experiments. (B) Blue native PAGE gel analysis of samples at the end of Thioflavin T assay shows similar ATXN3 species in samples of ATXN3Q55 incubated with aripiprazole or vehicle. Two major species are present equally in both samples: HMW species and ATXN3Q55 dimers. (C) Transmission electron microscopy images of the same samples used in B show spheroidal and short fibrillar ATXN3 complexes formed in the presence of aripiprazole or vehicle. Scale bar = 100 nm.

Figure 7

Q84 mice show dysregulation of key proteostasis components. Western blot analysis of total soluble protein lysates from brainstems of 12-week-old Q84 mice (n = 6; mouse number 25–30) and wild-type littermate controls (wt) (n = 6; mouse number 19–24) (A) show decreased Hsp40 (B), increased Hsp90β (C), increased HSF1 (D), and increased RAD23A (E) in Q84 mice. (A) Immunoblot showing ATXN3 expression in the brainstems of tested mice. (B–E) Left panels show immunoblots the indicated proteins in soluble fractions of brainstem extracts from Q84 or wild-type littermate mice. Right panels display quantification of band intensity, with values normalized to GAPDH. Bars represent the average percentage of protein relative to vehicle-treated mice (±SEM). Comparison between groups was made using Student’s t-test and statistical significance is indicated as **P < 0.01, and ***P < 0.001.

Figure 8

Aripiprazole affects components of the protein quality control machinery in brains of treated Q84 mice. In mice treated with aripiprazole evaluation of chaperone machinery components shows decreased levels of Hsp70 (A), Hsp90α and Hsp90β (B), increased levels of HSF1 (C), and a trend for decreased amount of RAD23A and RAD23B (D). (A–D) Left panels show immunoblots detecting the indicated proteins in soluble fractions of brainstem extracts from aripiprazole and vehicle-treated mice (same animals as in Fig. 5). Right panels display the quantification of band intensity, with values normalized for α-tubulin or GAPDH. Bars represent the average percentage of protein relative to vehicle-treated mice (±SEM). Comparison between groups was made using Student’s t-test and statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.