TNF-α induces neuronal necroptosis and SARM1-dependent axon degeneration in sensory neurons. (A) Schematic of embryonic day 13.5 DRG culture system. (B) Left: DRG neurons are treated with the indicated compounds and axon-only extracts are immunoblotted for pMLKL. TNF-α (100 ng/ml) is applied together with SMAC mimetic (LCL161, 10 µM) and pan caspase inhibitor Z.VAD (100 µM). Necrostatin-1s (Nec), 20 µM. Right: Quantification of pMLKL protein levels normalized to TUJ1 levels. Data are represented as mean ± SEM, n = 3; one-way ANOVA with post hoc Tukey test, F(3,8) = 13.04, P = 0.0019; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (C) Representative bright-field and TMRM images of cultured DRG axons from WT or SARM1 KO neurons treated with TNF-α (together with 10 µM LCL161, 100 µM Z.VAD) and necrostatin as indicated. Scale bar = 50 µm. (D) DRG neurons from WT or SARM1 KO mice were treated with TNF-α (together with 10 µM LCL161 and 100 µM Z.VAD) and necrostatin where indicated. Axon degeneration was quantified from bright-field images at the indicated time points after TNF-α application. Axons with a DI >0.3 are defined as degenerated (dashed line). Data represent the mean ± SEM, n = 5; two-way ANOVA with post hoc Bonferroni’s multiple comparison test; time, F(2.217, 35.48) = 256, P < 0.0001; interaction, F(16,48) = 13.41, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (E) Quantification of TMRM intensity in axons after 72 h of TNF-α treatment (as described in D). TMRM (50 nM) was applied 30 min before image acquisition. Data represent the mean ± SEM, n = 3; one-way ANOVA with post hoc Tukey test, F(3,8) = 71.72, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.