Fig 2: Rilmenidine treatment increases the burden of pathological and aggregated SOD1 in motor neurons of SOD1G93A mice. (A) Immunoblot analysis of soluble SOD1 protein levels in spinal cords of vehicle- and rilmenidine-treated SOD1G93A mice at end stage. Quantification of (B) human SOD1 (HsSOD1) and (C) misfolded SOD1 (misSOD1) protein levels normalized to vehicle-treated mice. (D) Immunoblot analysis of insoluble SOD1 protein levels in spinal cord pellet fractions of vehicle- and rilmenidine-treated SOD1G93A mice at end stage. (E) Quantification of HsSOD1 protein level in pellet fraction normalized to vehicle group. Data represent mean ± SD, n = 5 mice per group, **p<0.01 compared to vehicle-treated mice using an unpaired t-test. (F, H and I) Misfolded SOD1 immunohistochemical analysis of lumbar spinal cords of vehicle- and rilmenidine-treated SOD1G93A mice at 90 days of age. (F-H) In vehicle-treated mice, misfolded SOD1 shows diffuse staining in the cytoplasm of RBFOX3/NeuN-positive motor neurons (arrows). In rilmenidine-treated mice, SOD1 immunoreactivity is increased with accumulation of SOD1 into cytoplasmic aggregates in motor neurons. SOD1 accumulates into (H) Lewy body-like or (I) skein-like inclusions (arrows) in motor neurons. (G) Quantification of misfolded SOD1 aggregates in motor neurons. Data represent mean ± SD, n = 5 mice per group, **p<0.01 compared to vehicle-treated mice using an unpaired t-test.

Fig 3: Autophagy is activated in NSC-34 cells expressing ALS-linked misfolded proteins. (A) Immunoblot analysis of macroautophagy and CMA markers in NSC-34 cells stably transfected with wild-type (WT) or mutant G85R or G93A SOD1. Quantification of (B) LC3-II, (C) SQSTM1, (D) LAMP2A and (E) HSPA8 protein levels from immunoblots normalized to WT SOD1-expressing cells. (F) Immunoblot analysis of macroautophagy and CMA markers in NSC-34 cells stably transfected with WT or mutant Q331K or M337V TARDBP. Quantification of (G) LC3-II, (H) SQSTM1, (I) LAMP2A and (J) HSPA8 protein levels from immunoblots normalized to WT TARDBP-expressing cells. Data represent mean ± SD, n = 5-6 independent experiments, *p<0.05 and **p<0.01 compared to cells expressing the WT form of relevant protein using one-way ANOVA with Tukey's posthoc test.

Fig 4: Rilmenidine-induced SOD1 inclusions accumulate outside the autophagy system in motor neurons of SOD1G93A mice. (A) Misfolded SOD1 and LC3 immunohistochemical analysis in lumbar spinal cords of vehicle- and rilmenidine-treated SOD1G93A mice at 90 days of age. Vehicle-treated mice show colocalization of SOD1 and autophagosomes (arrowheads). Large SOD1 aggregates in rilmenidine-treated mice (arrows) do not colocalize with autophagosomes (arrowheads). (B) Misfolded SOD1 and LAMP2 immunohistochemical analysis in lumbar spinal cords of vehicle- and rilmenidine-treated SOD1G93A mice at 90 days of age. Vehicle-treated mice show colocalization of SOD1 and lysosomes (arrowheads). Large SOD1 aggregates in rilmenidine-treated mice (arrows) do not colocalize with lysosomes (arrowheads).

Fig 5: Rilmenidine has no effect on the CMA pathway in NSC-34 cells. (A) Immunoblot analysis of CMA markers and substrate SNCA in NSC-34 cells transiently transfected with wild-type (WT) SOD1 and treated with rilmenidine (Ril) for 18 h post-transfection. Quantification of (B) LAMP2A, (C) HSPA8 and (D) SNCA protein levels from immunoblots normalized to untreated cells. Data represent mean ± SD, n = 3 independent experiments.