Fig 2: ROS derived from NOX complexes activate Ca2+ flux and axonal degeneration in vitro. A, DRG explants cultured in NGF were either maintained in NGF or withdrawn from trophic factor for 15 h before staining with Fluo-4 and imaged by confocal microscopy. B, Antioxidant NAC (20 mM) or NOX complex inhibition using VAS2870 (10 μM) significantly impaired axonal Ca2+ influx induced by trophic factor withdrawal (n = 3 pooled experiments standardized to the NGF condition, one-factor ANOVA and Dunnett’s post hoc comparison; mean and SEM are indicated). C–E, Antioxidant or NOX complex inhibition significantly rescued DRG axonal cytoskeleton (visualized with βIII-tubulin immunostaining) when added after 12 h of NGF deprivation for the final 12 h. D, Axoquant2.0 axon density output curves with mean and SEM. E, Axon density was analyzed within 1000-μm bins using two-factor ANOVA and Tukey’s post hoc analysis. Median, min/max, and 25/75% are indicated; *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig 3: TRPV1 mediates Ca2+ flux and cytoskeletal fragmentation during trophic factor deprivation. A, TRPV1 inhibition by capsazepine rescued axons from Ca2+ influx. Axons were loaded with Fluo-4 after the indicated treatments and imaged by confocal microscopy. A, Fifteen hours of NGF deprivation induced robust activation of Ca2+ sensor Fluo-4 in axons, but co-application of 10 μM capsazepine (TRPV1 inhibitor, CPZ) ablated the response as compared to deprived controls (quantified in B; n = 7 pooled experiments, one-factor ANOVA and Dunnett’s post hoc comparison to the deprived condition). C, D, NGF deprivation for 24 h resulted in a significant loss of tubulin-stained axons, but addition of 10 μM CPZ after 12 h of trophic withdrawal for the final 12 h resulted in a rescue of axon density to a level not significantly different from healthy controls (n = 8 embryos in NGF and capsazepine conditions, n = 7 in DMSO; one-factor ANOVA and Dunnett’s post hoc comparison were performed on axon density in a bin between 1000 and 1999 μm from soma. E, TRPV1 knock-out rescued axons from cytoskeletal degeneration; neurons cultured from mixed-genotype litters were deprived of NGF for 24 h, after which TRPV1-null axons were significantly more dense than axons in cultures derived from wild-type animals. Axon density between 1000 and 1999 μm from soma was compared by two-factor ANOVA and Tukey’s post hoc comparison. Indicated are median, min/max, and 25/75% in each panel; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: PKC mediates Ca2+ flux and axonal degeneration in vitro. A, Representative micrographs of DRG axons loaded with Fluo-4 and treated with PKC activator PMA (100 nM). B, Direct PKC stimulation by PMA is sufficient to activate axonal Ca2+ influx (n = 14 embryos pooled from PMA and buffer controls and compared using an unpaired, two-tailed t test.) C, D, Ca2+ influx activated by 15 h of NGF deprivation is significantly rescued when PKC is inhibited by Gö6976 and Gö6983 (10 μM; n = 6 embryos each condition standardized to NGF values and tested by one-factor ANOVA and Dunnett’s post hoc comparison). E, F, PKC inhibition after 12 h rescues axons from cytoskeletal degeneration induced by 24 h of NGF deprivation. E, Axoquant2.0 axon density output curves are presented with mean and SEM (n = 9 for all conditions except n = 7 for Gö6983). F, Axon density was analyzed within 1000-μm bins using two-factor ANOVA and Tukey’s post hoc analysis. Median, min/max, and 25/75% are indicated; ***p < 0.001, ****p < 0.0001.

Fig 5: Crmp4 deletion delays Wallerian degeneration in vivo and in vitro. A, Semithin cross-sections of the degenerating sciatic nerve at 36 HPI, 3 DPI, or 7 DPI following sciatic nerve transection, or of the intact contralateral nerve, in Crmp4+/+ or Crmp4−/− mice. Scale bar, 50 μm. B, Quantification of the mean number of intact myelin sheaths per square micrometer ± SEM in the Crmp4−/− sciatic nerves compared with the Crmp4+/+ controls displayed in A (n = 3–7; two-way ANOVA with Bonferroni’s multiple-comparison test). C, D, Representative pictures of the axons extending from DRG explants at a distance of 500 μm from the center of the explant stained with Tuj1 (C) and quantification (D) of the extent of degeneration in E12.5 Crmp4+/+ or Crmp4−/− DRG explants at 5 h after axotomy. Data in the graph are represented as the mean index of degeneration ± SEM (n = 9–10; two-tailed Student t test). Scale bar, 100 μm. E, Representative images of intact E12.5 DRG explants or at 24 h following NGF withdrawal (anti-NGF). F, Curves of the area covered by axons of Crmp4+/+ and Crmp4−/− DRG explants in the presence (NGF) or absence (anti-NGF) of NGF. The curves represent the mean ± SEM. C, Mean ± SEM of the area covered by the axons at a distance of 700 μm from the center of the explants (as indicated by a dotted line in G) in the presence or absence of NGF. Data are normalized to Crmp4+/+ grown in the presence of NGF (n = 3; one-way ANOVA, Tukey’s multiple-comparison test).