Figure 7. Mutations in the VMN binding pocket of hSARM1 ARM domain prevent vacor toxicity.
(A) Representative images of neurites from Sarm1-/- SCG dissociated neurons co-injected with plasmids encoding wild-type or W103A hSARM1 and DsRed (to label neurites) and treated with 100 µM vacor. (B) Quantification of healthy neurites and viable neurons in experiments in (A) is shown as a percentage relative to 0 hr (time of drug addition) (mean ± SEM; n = 3; repeated measures three-way ANOVA followed by Tukey’s multiple comparison test; ****, p < 0.0001). (C) Representative images of neurites from Sarm1-/- SCG dissociated neurons co-injected with plasmids encoding wild-type or R157A hSARM1 and DsRed (to label neurites) and treated with 100 µM vacor. (D) Quantification of healthy neurites and viable neurons in experiments in (C) is shown as a percentage relative to 0 hr (time of drug addition) (mean ± SEM; n = 3; repeated measures three-way ANOVA followed by Tukey’s multiple comparison test; ****, p < 0.0001). (E) Representative images of neurites from Sarm1-/- SCG dissociated neurons co-injected with plasmids encoding wild-type or K193R hSARM1 and DsRed (to label neurites) and treated with 100 µM vacor. (F) Quantification of healthy neurites and viable neurons in experiments in (E) is shown as a percentage relative to 0 hr (time of drug addition) (mean ± SEM; n = 3; repeated measures three-way ANOVA followed by Tukey’s multiple comparison test; ****, p < 0.0001). (G) Fold change of NADase activity of purified, recombinant K193R hSARM1 in the presence of NMN and VMN (wild-type hSARM1+ VMN is also shown for comparison) (mean ± SEM; n = 2–3). K193R hSARM1 average basal activity is 17.75 ± 2.47 milliU/mg (fold activation = 1). Source data for Figure 7—source data 1.
Figure 7—figure supplement 1. Expression of wild-type, W103A, R157A and K193R hSARM1 in SCG neurons.
