Fig. 5: Knockdown of SARM1 protects from Vacor toxicity and AIND.

A Schematic indicating how Vacor is metabolized intracellularly by NAMPT to the toxic metabolite Vacor mononucleotide (VMN), which acts as a strong SARM1 activator. NAMPT can be specifically inhibited by FK866. B LUHMES cells were seeded on DoD2 and were grown until DoD6. Cells were exposed to Vacor on DoD6 for 18 h. Cells were stained with calcein-AM and H-33342 and imaged by epifluorescence imaging. The neurite area and cell viability were quantified by an image analysis algorithm. Each data point is the means ± SEM of three biological replicates with three technical replicates each. Data was normalized to untreated controls. C Cells were treated and analyzed as described in (B), but were treated with 500 nM FK866 30 min before exposure to Vacor. D LUHMES cells were left untransfected or were transfected as cell suspensions with siRNA (negative control (Silencer Select non-targeted siRNA) or targeting SARM1 (Silencer Select; ID: s23031)) on DoD2. Then, cells were plated and grown until DoD6. These cells were exposed to Vacor for 18 h, or were left untreated. Cells were stained with calcein-AM before epifluorescence imaging (cf. Fig S4A). Representative images are shown. Scale bar = 50 µm. E Neurite area of LUHMES cells transfected with either negative siRNA, or siRNA targeting SARM1, was quantified after 18 h exposure to different concentrations of Vacor (note the logarithmic scaling). Significance between conditions was evaluated by ANOVA with Bonferroni’s post hoc test. ##p < 0.01, ###p < 0.001. Significance between treatments and control was evaluated by ANOVA with Dunnet’s post hoc test. **p < 0.01, ***p < 0.001. F LUHMES cells were transfected with siRNA on DoD2 (in suspension) and cultured for four days in low-adherence round-bottom plates to generate spheroids. These were transfected a second time and then plated. Axotomy was performed after 3 days of neurite outgrowth (on DoD9). Neurites were stained 18 h later with calcein-AM and TMRE and images were obtained by epifluorescence microscopy (cf. Fig. S5A). Scale bar = 200 µm. G, H Quantification of (E) neurite fragmentation and integrity and (F) number of TMRE+ mitochondria (as in Fig. 4C). Data were normalized to untransfected controls. **p < 0.01, ***p < 0.001 by ANOVA with Dunnet’s post hoc test.