Fig 2: USP13 deubiquitinates SARM1 and its axonal protective effect requires the deubiquitinase activity.(A, B) Representative Western blot images showing the ubiquitination levels of SARM1 in HEK293T cells co-expressed with WT USP13 or USP13AE (A) and the interaction between them and SARM1 (B). (C, D) Representative phase contrast images (C) and quantification (D) of injury-induced axon degeneration in primary mouse DRG neurons infected with the lentivirus expressing WT USP13 or USP13AE. Images were captured live at the indicated time points after axotomy. (E) Western blot assay confirming the lentiviral expression of WT USP13 or USP13AE in the in vitro cultured mouse DRG neurons. hpi, hours post injury. Mean ± SEM; n = 3. *p < 0.05, **p < 0.01; ns, no significance; one-way ANOVA. Scale bar: 50 µm.

Fig 3: SARM1 phosphorylation at Ser-548 is involved in rotenone-induced cell death and neurofilament degradation. (A-E) To establish the gene-engineered NSCs that overexpress SARM1s, 201B7 NSCs were transfected with a set of plasmids, including the 1B-SARM1-WT or S548A plasmid, the 1B-transposase plasmid, and the 1B-puromycin resistance gene plasmid, and were selected by puromycin. The 1B-empty-gene plasmid was used instead of the 1B-SARM1 plasmid to obtain the control NSCs. Control, SARM1-WT-overexpressing, and SARM1-S548A-overexpressing NSCs were seeded at a density of 1.5 × 10 5 cells/cm2 and differentiated into neurons for 6 days, and then treated with 0.1% DMSO or 10 μM rotenone for 24 hr. (A) Cell viability was measured by the CellTiter-Glo assay. (B) Western blot analysis of control, SARM1-WT-overexpressing, and SARM1-S548A-overexpressing neurons. The changes of cellular molecules 24 hr after the addition of DMSO or 10 μM rotenone were analysed. (C) Quantification of the band intensity of phosphorylated SARM1. The level of phosphorylated SARM1 was normalized by total SARM1 protein. The data are expressed in fold changes using the DMSO-treated SARM1-WT neurons as control. (D) Immunostaining of NF-L, MAP2, and nucleus at 24 hr after treatment with DMSO or 10 μM rotenone. Scale bar: 100 μm. (E) Quantification of NF-L immunoreactivity. The NF-L fluorescence intensities were normalized against nuclear numbers. The data are expressed in fold changes using the DMSO-treated control neurons as control. Error bars: mean ± SD of three independent experiments. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Graphical Abstract for SARM1 induction of photoreceptor cell death.

Fig 5: SARM1 regulates IL-1β secretion through NADase-dependent and -independent mechanisms(A and B) PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Supernatants were collected to measure IL-1β protein secretion by ELISA (Α) and LDH release (B). Data shown are mean ± SEM of 3 independent experiments, each performed in triplicate. Data were tested with a two-way ANOVA; Tukey’s multiple comparisons test, ∗∗∗∗p < 0.0001.(C) Left panel, PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Immunoblot of supernatants from PMA-differentiated THP-1 cells with or without stimulation to activate NLRP3 inflammasome. Cleaved caspase-1 (p20), cleaved GSDMD (p30) and cleaved IL-1β (p17) are shown. Representative of 3 independent experiments. Right panel, 3 independent experiments were performed and Caspase 1 p20 was quantified by using ImageJ. Data shown are mean ± SEM of 3 independent experiments, each dot represents an independent experiment. Data were tested with a one-way ANOVA: Šídák’s multiple comparisons test, ∗p < 0.05.(D) PMA differentiated THP-1 cells were primed with LPS for 4 h, followed by nigericin stimulation for 2 h. Immunoblots of cell lysates from PMA-differentiated THP-1 cells with or without stimulation to activate NLRP3 inflammasome. Representative of 3 independent experiments. β-actin was used as a loading control.(E) Quantification of pro-IL-1β from (D). Pro-IL-1β/β-actin ratios were normalised to PMA-treated EV control cells without stimulation. Data shown are mean ± SEM of 3 independent experiments, each dot represents an independent experiment. Data were tested with a one-way ANOVA: Šídák’s multiple comparisons test, ∗p < 0.05, ∗∗p < 0.01.(F) Left panel, PMA differentiated THP-1 cells were stimulated with LPS and/or CZ-48 for 6 h. Immunoblots of pro-IL-1β and SARM1 (to show no effect by NADase in other proteins) from cell lysates of PMA-differentiated THP-1 stimulated with LPS and/or CZ-48. Representative of 3 independent experiments. β-actin was used as a loading control. Right panel, quantification of pro-IL-1β and significance is assessed as described in (E). ∗∗p< 0.01, ∗∗∗p< 0.001, ∗∗∗∗p< 0.0001.