Fig 2: 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 3: Schematic mechanism of JM17 on Nrf2 activation in MJD78 cells. ROS increased cell death and mitochondrial dysfunction by oxidative stress and ameliorated antioxidant enzyme expression and GSH levels in MJD78 cells expressing mutant ataxin-3 protein. JM17 administration reduced total and mitochondrial ROS and promoted antioxidant enzyme expression by upregulating Nrf2 activation. On the other hand, JM17 diminished protein aggregation by activating autophagy and enhanced mitochondrial respiratory function. NRF2, nuclear factor erythroid-2; ARE, antioxidant response element; ROS, reactive oxygen species; HO-1, heme oxygenase 1; NQO-1, NAD(P)H: quinone oxidoreductase 1; GSH, glutathione; SOD, superoxidase dismutase; CAT, catalase.

Fig 4: Study of the mRNA and protein levels of the most relevant antioxidant enzymes. (A) RT-PCR analysis of the genes that codify for CAT (Cat), GPX isoform 1 (Gpx1), GR (Gsr), CuZnSOD (Sod1), and MnSOD (Sod2) in db/db mice topically treated with vehicle (black bars) or sitagliptin (gray bars) and in db/+ mice (white bars). Results are presented as fold change vs. control mice; n = 4. (B,C) Densitometric analysis and Western blot bands of CAT, GPX isoform 1 (GPX1), GR, CuZnSOD and MnSOD protein levels in retinas of vehicle-treated db/db mice (black bars), sitagliptin-treated db/db mice (grey bars), and nondiabetic mice (white bars); n = 3. (D,E) Representative images of the retinal immunofluorescence of CuZnSOD (red) (D) and MnSOD (red) (E) in diabetic mice treated with vehicle or sitagliptin eye drops and in nondiabetic mice. Hoechst staining (blue) was used for nuclei labeling. Scale bars, 20 µm; n = 4. GCL (ganglion cell layer), IPL (inner plexiform layer), INL (inner nuclear layer), OPL (outer plexiform layer), ONL (outer nuclear layer). * p < 0.05; ** p < 0.01.

Fig 5: Effect of the C57D/C146D mutation on the biochemical properties of purified SOD1 proteins.a Structural comparison of cysteine-sulfinic acid and cysteine-sulfonic acid to aspartic acid. Cysteine-sulfinic acid and -sulfonic acid are the overoxidized forms of cysteine in the presence of ROS. Note that aspartic acid mimics Cys-sulfinic acid and Cys-sulfonic acid in terms of the electrostatic charge and the atomic arrangement. Detailed descriptions are in Supplementary Fig. 1. b SEC-MALS profiles of the purified wild-type (blue) and C57D/C146D mutant (red) SOD1 proteins. The UV absorbance at 280 nm (A280 at the right Y-axis) of the SEC is represented by solid lines. The molecular mass (the left Y-axis) based on MALS is represented by a dotted line. The average molar mass of wild-type or C57D/C146D mutant SOD1 is indicated under the dotted lines. c Tm measurement by the thermal shift assay of the wild-type SOD1 protein (WT, blue line), C57A/C146A mutant SOD1 protein (green line), and C57D/C146D mutant SOD1 protein (red line). Individual raw data points displayed for each temperature represented the average of three endpoint readings with ±SD. According to the analyzing results (Supplementary Fig. 2c), the Tm values of the wild-type, C57A/C146A, and C57D/C146D were 56 °C, 47 °C, and 38 °C, respectively. d Top, far-UV CD spectra of the wild-type SOD1 (WT, blue line) and C57D/C146D mutant SOD1 (red line) at the wavelength range of 190-260 nm. Bottom, the estimated secondary structure of wild-type and mutant protein calculated based on CD spectra. The proportion of each secondary structure constituting the SOD1 protein is expressed as a percentage. e Proteolytic digestion of the wild-type (WT) and C57D/C146D mutant SOD1 proteins by trypsin. The proteins were digested at 37 °C for 1 h in the presence or absence of 5 mM DTT and then subjected to SDS-PAGE. The redox states of SOD1 proteins are indicated by arrows to the right of the SDS polyacrylamide gel image. All the cysteines in SOD1 are reduced (red), or an intramolecular bond is formed between Cys57 and Cys146 (Ox). f Relative superoxide dismutase activities of the wild-type (blue), C57D/C146D (red), and C57A/C146A (green) mutant SOD1 protein. Inhibition of cytochrome c reduction by superoxide in the presence of SOD1 was represented in bar graphs with mean ± SD from six individual experiments. Statistical comparisons were performed using an unpaired two-tailed Student’s t-test. Differences were considered statistically significant at P-values < 0.05. **** denote p < 0.0001. Raw data are presented in Supplementary Fig. 2e.