Fig 2: IGF-1 reduced the ataxin-3 protein level in the cerebellum of the SCA3 84Q mice. (a) Immunochemical staining of ataxin-3 in the cerebellum. The black arrows indicate PCs (right panel). Histograms show the means ± SEM (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 5. (b) Western blot confirming ataxin-3 expression in the mouse cerebellum (left panel). Quantification of the ataxin-3 level relative to the total protein level (mean ± SEM) (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 5. (c) Slices of the cerebellum of two mice in each group were selected and double-labeled using an aggresome detection kit (red) and an Alexa 488-conjugated secondary IgG against the anti-ataxin-3 antibody (green), and fluorescence intensities of 30–40 PCs in each mouse were examined using the ImageJ software. The white arrows indicate PCs. SCA3 15Q, n = 2; SCA3 84Q, n = 2; SCA3 84Q + IGF-1, n = 2. Note: * p < 0.05 indicates a significant difference.

Fig 3: Nrf2 activation-dependent regulation in raising antioxidation and decreasing mutant ataxin-3 expression. MJD78 cells were transiently transfected with si-NTC or si-Nrf2 and with or without an ARE-luciferase reporter construct for 16 h. They were then treated with or without DMSO vehicle, 1 µM JM17 and 0.3 µM RTA-408 for 24 h, respectively. (A) Protein expression of Nrf2, NQO1, HO-1, SOD1, SOD2 and mutant and normal ataxin3. Levels of (B) ARE luciferase activity, (C) total GSH, (D) reduced GSH and (E) catalase are analyzed after treatments. Data are presented as the mean ±SD of at least three independent experiments. Values from the treated cells were normalized to those of the MJD78 cells treated with si-NTC transfection vehicle only. * p < 0.05, compare to non-treated MJD78 with si-NTC group; # p < 0.05, compare to non-treated MJD78 with si-Nrf2 group.

Fig 4: Expression of the autophagic influx in the SCA3 mice. Representative Western blots of the autophagy-related markers (right panel). Quantitative results of the autophagy-related proteins were normalized to those of total protein (mean ± SEM) (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. Note: * p < 0.05 indicates a significant difference.

Fig 5: IGF-1 prevented impairment of the motor function in the SCA3 mice. (a) Latency to fall (time in seconds for which the mice persisted on the rotarod) for the SCA3 15Q mice and the saline- and IGF-1-treated SCA3 84Q mice during the 9 months of treatment. (b) Within the same group, the latency to fall at pretreatment was normalized to 100%. (c) EthoVision XT 7.0 software was used to analyze trajectories of the mice in the behavioral test. (d) The distance of movement, time of movement, frequency of zone change, and average velocity were included in transformed indices. (e) Captured images of the single stance for each paw. (f) Catwalk parameters included the step cycle, stride length, stand, and average speed. The data are presented as the means ± SEM. Note: # p < 0.05 denotes statistical significance in the saline-treated SCA3 84Q mice compared with the SCA3 15Q mice; * p < 0.05 indicates a significant difference.