Fig. 5: RET inhibition differentially impacts SOD1G93A MNs compared to WT.
A Representative western blot showing WT and SOD1G93A primary MN lysates (mass culture) following treatment with DMSO, 1 µM C3 and 100 nM LOXO-292 for 1 h. Full WT blot is shown in Fig. 2G. Samples were run twice on two gels to analyse phosphorylated and total proteins of the same molecular weight. Bands were normalised to corresponding GAPDH loading control (phospho- “p”, total “t”). B Quantification of RET activation from western blots of WT and SOD1G93A primary MN lysates (1 µM C3, 100 nM LOXO-292; see also Fig. 2). There was no significant difference in RET activation comparing DMSO to either RET inhibitor (two-way ANOVA p = 0.1629); however, SOD1G93A cultures have reduced RET activation compared to WT (two-way ANOVA; *p = 0.0181). C Quantification of AKT activation. C3 significantly inhibits AKT activation compared to DMSO (two-way ANOVA with Tukey’s multiple comparison test; ****p < 0.0001), but LOXO-292 has no significant effect. D Quantification of ERK1/2 activation. C3 significantly inhibits ERK1/2 activation compared to DMSO (two-way ANOVA with Tukey’s multiple comparison test; **p = 0.0018). In contrast, LOXO-292 has no significant effect. RET, AKT and ERK1/2 activation was determined by first normalising all bands to GAPDH to control for loading, then normalising phosphoprotein levels to total protein. Bars represent mean ± SEM. E Experimental set up for SOD1G93A MNs transport analysis following treatment with the C3 inhibitor. In all, 50 ng/ml BDNF was added to both MFC chambers, whereas HCT was added to the axonal compartment only. F Representative kymographs from axonal transport videos in SOD1G93A primary MN cultures. Scale bars, 20 µm. G–I Average endosome speeds, p = 0.1646 (G), percentage of pausing, p = 0.2002 (H) and maximum endosome speed, p = 0.2175 (I) in SOD1G93A primary MN cultures following DMSO or 1 µM C3 treatment. Statistics are determined by Student’s t tests. N = 4 biological replicates. Bars represent mean ± SEM. DMSO results were collected from 295 endosomes in 26 axons for a total of 29,667 single endosomal movements. C3 results were collected from 354 endosomes in 23 axons for a total of 30,664 single endosomal movements.
