Fig 1: 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.
Fig 2: Low dose administration of a SARM1 BEI worsens neurodegeneration during the acute inflammatory phase of EAE.A Design of the EAE pharmacology study testing RO-7529 at 2, 10, and 50 mg/kg starting from time of immunization. Schematic was generated using Biorender. B Mean disability clinical scores of EAE mice. Green arrows indicate worsening disability with RO-7529 at 2 mg/kg during the acute inflammatory phase. C Serum NfL levels in EAE mice on days 8, 12, 16, 21, and 28 post-immunization. Serum NfL levels at day 16 (D) and day 21 (E) from the same study. F Inflammation in lumbar spinal cord at day 28 quantified as the number of inflammatory foci containing at least 20 cells per section per mouse. G Demyelination in lumbar spinal cord at day 28 quantified using a demyelination scoring system based on anti-MBP staining. H Illustrative lumbar spinal cord sections stained with: top - hematoxylin (nuclei: blue) and eosin (cytoplasm: pink), bottom - anti-MBP (brown) and hematoxylin (nuclei: blue). Magnified white matter regions from (H, E)-stained sections demonstrate inflammatory infiltrates. The anti-MBP stained section from a RO-7529 (2 mg/kg)-treated mouse depicts a demyelination score of 3. For (B–G), n = 12 animals per group; data are shown as mean ± standard error of the mean (SEM). In (D–G), dots represent individual animals. Significance is indicated by ****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05, determined by two-way ANOVA and Dunnett’s post-hoc test.
Supplier Page from Abcam for Recombinant Human SARM protein (DDDDK tag N-Terminus)