Figure 6
From: Ebselen analogues delay disease onset and its course in fALS by on-target SOD-1 engagement
MR6-26-2 ameliorated a neuromuscular junction pathology of SOD1G93A mice and improved proteostasis in vitro and in vivo. (A,B) The MR6-26-2 treatment reduced poly-ubiquitin- (poly-Ubi) and p62/SQSTM1-positive inclusions in the 120-days-old SOD1G93A lumbar spinal cord. Representative immunofluorescent images are shown (A). The number of the inclusions per anterior horn (AH) quantified from three mice are plotted as mean ± SEM (n = 3) (B). Scale bar: 75 µm. (C–E) Immunoblot analyses of poly-Ubi and p62 expressions in insoluble fractions of spinal cords from the control or MR6-26 treated SOD1G93A mice at 120 days old. Representative immunoblotting images are shown (C), and the quantification results of insoluble poly-Ubi (bold line in C) and insoluble p62 (black rectangle) are expressed as mean ± SEM in (D) and (E), respectively. An asterisk indicates non-specific bands. (F,G) MR6-26-2 reduced denervation at neuromuscular junctions (NMJs) of 120-days-old SOD1G93A mice. Representative immunofluorescent images of tibialis anterior muscles are shown (F). Bungarotoxin (BTX) and synaptophysin indicate postsynaptic acetylcholine receptors on muscles and motor nerve terminal ends, respectively. Innerved NMJ ratio was quantified as a co-localization ratio of BTX and synaptophysin and plotted as mean ± SD (n = 3) (G). (H–L) MR6-26-2 improved proteostasis in cultured Neuro2a cells. Representative immunoblotting images are shown (H), and the quantification results of insoluble SOD1 ratio against soluble SOD1 (I), insoluble poly-Ubi (indicated by a double line in H) (J), and insoluble (K) or soluble (L) p62 are expressed as mean ± SEM (n = 3, each), respectively. Three independent experiments were performed, and the amount of each protein was quantified as relative to the DMSO-treated controls with SOD1G93A expression. Scale bar: 10 µm.
