Fig. 1: Axotomy triggers a wave of DRP1-dependent mitochondrial fission, axon degeneration and cell death.

a Schematic representation of laser axotomy. Created in BioRender. Le Pichon, C. E. (2022) BioRender.com/z21b395. b Representative images of WT and SARM1 KO axons expressing cytoplasmic mApple (red) pre- and 4 h post-axotomy (PA). Scalebar 100 µm. c Axon degeneration index (ADI) in WT and SARM1 axons 4 h post-axotomy. n = 38. Two-way ANOVA (p = 0.0086 **, not significant, ns). d Representative images of mitoGFP (green) in axons pre- and 5 min post-axotomy (PA). Scalebar 20 µm. e Representative time-lapse images of mitochondria undergoing fission after axon injury. Scalebar 2 µm (valid for all 5 examples). f Schematic. g Quantification of mitochondrial length pre- (green) and 5 min post-axotomy (gray). n = 29. Two-way ANOVA (10 min post-axotomy, 0–100 µm vs 200–300 µm p = 0.0021; vs 400–500 µm p < 0.0001. 20 min post-axotomy, 0–100 vs 200–300 µm p = 0.0019). h Axonal mitochondrial particles in control and DRP1 knockdown (KD) neurons. n = 26 WT, n = 25 DRP1 KD. Two-way ANOVA (p ≤ 0.05 *). i Axonal mitochondrial particles 3 min post-axotomy in DMSO and P110 treated neurons. n = 27 DMSO n = 26 P110. Two-way ANOVA (p = 0.0467 *). j Mitochondrial particles 0–100, 200–300 and 400–500 µm from axotomy. n = 23. One-way ANOVA (*p ≤ 0.05, 0–100 µm vs 200–300 µm, # p ≤ 0.05, 0–100 µm vs 400–500 µm). k Representative images of axotomy-induced mitochondrial fission wave. mitoGFP (green). Scalebar 25 µm. l Axon degeneration index (ADI) 0–100, 200–300 and 400–500 µm from axotomy. n = 14. One-way ANOVA (*p ≤ 0.05, 0–100 vs 200–300 µm, ^## p ≤ 0.01, ^### p ≤ 0.005, 0–100 vs 400–500 µm). m Representative images of retrograde axon degeneration 0, 10-, 30- and 120-min post-axotomy. Cyto-mApple (red). Scalebar 25 µm. n Representative images of axotomy-induced neuron death, pre- and 12-h post axotomy. Arrowhead indicates axotomized neuron; the other neuron is uninjured. Scalebar 40 µm. o Quantification of axotomy-induced neuron death. Percentage of dead neurons 12 h post-axotomy. n = 30. Unpaired, two-tailed, t test (p = 0.004 **). Graphical representations show mean ± SEM. Dashed box in (a, f) indicates field of view. Asterisks * in (a, b, d) mark site of axotomy. All experiments include axotomies from N = 3 independent differentiations. Bonferroni correction applied to all ANOVAs. Schematics in a and f made using Biorender.