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. 2016 Sep 19;139(11):2891–2908. doi: 10.1093/brain/aww228

Figure 5.

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).