Axonal necroptosis triggers local axon degeneration. (A) Schematic diagram of microfluidic device. BB (100 nM) is applied to the axon compartment to dimerize MLKL.ND locally. (B) Representative bright-field and TMRM images of soma (left) and axons (right) 24 h after BB is applied to the axon compartment. Scale bar, 50 µm. (C) Quantification of axon degeneration (top) and TMRM intensity (bottom). Axons with a DI >0.3 are defined as degenerated (dashed line). Data are represented as mean ± SEM, n = 3. For axon degeneration; one-way ANOVA with post hoc Tukey test, F(3, 8) = 29.47, P = 0.0001; for TMRM, one-way ANOVA with post hoc Tukey test, F(3, 8) = 23.28, P = 0.0003; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (D) Representative fluorescent images of soma (left) and axons (right) from WT or SARM1 KO DRG neurons cultured in microfluidic devices. BB is only applied to the axon compartment. Scale bar, 30 µm. (E) Left: Schematic of cultured DRGs in which cell bodies have been removed before TNF-α treatment in order to obtain axon-only lysate. Middle: SARM1 KO severed axons are treated with DMSO or TNF-α (100 ng/ml) together with SMAC mimetic (LCL161, 10 µM) and Z.VAD (100 µM) with or without necrostatin-1s (Nec, 20 µM). Axonal extracts are immunoblotted for phosphorylated MLKL. TUJ1 is used as a loading control. Right: Quantification of pMLKL normalized to TUJ1 level. Data represent the mean ± SEM, n = 4; one-way ANOVA with post hoc Tukey test, F(2.014, 6.041) = 15.02, P = 0.0045; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. AxD, axon degeneration.