Fig. 1: Axotomy- induced neurite degeneration is affected by NAD⁺ metabolism.

A LUHMES neurons (human CNS) were cultured as attached spheroids with a corona of neurites (2.5D setup). Axotomy was performed by physical removal of the cell bodies. Neurite cultures were analyzed at different time points after the axotomy in presence or absence of metabolic modifiers. B ATP was measured in isolated neurites at different time points after axotomy. Cells were either cultured in standard medium (glucose containing), or in medium containing galactose instead of glucose. Control experiments showed that neurites represented about 35% of the whole cell culture mass (protein levels) and that ATP levels immediately before and after the cut were similar. C The change of total NAD⁺/NADH (NAD) was measured in isolated neurites after axotomy. Data are means from biological triplicates with ≥5 technical replicates. Significance was evaluated by ANOVA relative to 0 h. **p < 0.01, ***p < 0.001; ^#p < 0.01 between time points. D NMNAT2 protein levels were analyzed by western blots. Average protein levels are indicated in %±SD of control (0 h), N = 3. E, F Plated spheroids were treated with different NAD⁺ concentrations immediately before axotomy. Neurites were fixed at 18 h after axotomy. E Images were recorded by scanning electron microscopy. Scale bar = 10 µm. F Neurites were stained with calcein-AM at 18 h after axotomy and imaged by epifluorescence microscopy. Scale bar = 200 µm. G, H Neurite integrity and fragmentation were quantified 18 h after cut in the presence of (G) different NAD⁺ concentrations. H Cells were treated with nicotinic acid (NA) alone or in combination with 500 nM FK866 (dotted line). Data are means from biological triplicates with 5 technical replicates each. Significance was evaluated by ANOVA followed Dunnet’s post hoc test (relative to solvent control). *p < 0.05, **p < 0.01, ***p < 0.001. I Schematic model of the synergistic effect of nicotinic acid (NA) and FK866 on SARM1 activity. Gray arrows indicate metabolic conversion; green/red arrows indicate activation/inhibition. Box sizes indicate levels of the metabolites. NMNAT2 is assumed to be depleted after axotomy so that NMN does not react to NAD⁺. In absence of FK866, nicotinic acid mononucleotide (NMN) activates SARM1, which leads to NAD⁺ depletion. NA cannot counteract this depletion. When NMN synthesis is blocked by FK866, then nicotinamide (Nam) accumulates and inhibits SARM1. In this situation, NAD⁺ levels, supported by NA supplementation, remain high. See Fig. S1 for more details.