Fig. 2: SARM1 BEIs induce SARM1 activation, cell death and neuronal damage at low doses in vitro.

A Chemical structures of different SARM1 BEIs used in this study. B SARM1 NADase activity measured in a biochemical assay via mass spectrometry detection of NAD and linear ADPR levels. Substrate concentrations of 20, 60, and 200 µM NAD were assessed. The ratio of linear ADPR to NAD peak area was normalized against negative control (2% DMSO) and positive control (100 µM NB-3) and then plotted against compound concentration. Boxes indicate increased SARM1 activity. C SARM1 activation-induced cell death in SH-SY5Y cells. Cell death following vacor exposure was assessed by measurement of ATP levels. Luminescent signals were normalized against untreated negative controls (0 µM vacor) and positive controls (40 µM vacor). Green arrows indicate increased cell death in the presence of both sub-µM BEI concentrations and a subactivating vacor concentration of 5 µM. Significance is indicated by ***P < 0.001, **P < 0.01 and *P < 0.05, determined by two-way ANOVA followed by Šidák’s multiple comparisons test to assess differences between treatment with compound +5 µM Vacor and 5 µM Vacor alone. NfL release from iPSC-derived motor neurons following exposure to 5 µM (D) or 25 µM (E) vacor and treatment with increasing concentrations of SARM1 BEIs. F Phase contrast (top) and Neurotrack (bottom) images of iPSC-derived motor neurons treated with vacor and low (1 nM) or high (100 nM) concentration of Ex. 27. Neurotrack images show the neurite mask in magenta and cell-body cluster mask in yellow. Images taken at 72 h post-vacor exposure. G Relative neurite area quantified using the Neurotrack module at 72 h post-vacor exposure. For (D, E and G), data are shown as mean ± standard deviation (SD) and results are representative of two to three independent experiments. Significance is indicated by ****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05, determined by one-way ANOVA and Dunnett’s post-hoc test.