Fig. 1.

Small-molecule inhibitors of DLK suppress axon degeneration by elevating NMNAT2/SCG10 protein levels. (A) DRG sensory neurons were pretreated with DMSO or GNE-3511 (500 nM); axons were then severed with a razor blade, and the fragmentation of distal axons was measured over time. An axon degeneration score above 0.3 indicates axons are fragmenting (dotted line). (B) Bright-field images of distal, severed axons 24 h after axotomy, pretreated with vehicle or GNE-3511. (Scale bar, 5 μm.) Pretreating DRG sensory neurons with 1 μM Tozarsertib (Toz.) suppresses injury-induced axon degeneration for ∼24 h after axotomy (C), with bright-field images of distal axons 12 h after axotomy (n = 3) (D). (Scale bar, 5 μm.) Western blots of endogenous NMNAT2 and SCG10 from axon-only extracts derived from DRG axons after treatment with GNE-3511 (E), Tozarsertib (F), or Sunitinib (G) are shown. (H) Western blots of phosphorylated MKK4 and JNK from axon-only extracts after treatment with GNE-3511. Western blots are representative of at least three independent experiments. (I) Cas9-expressing DRGs were transduced with sgRNAs targeting NMNAT2 or a nonspecific scrambled sgRNA control and pretreated with DMSO or GNE-3511 as in A. Loss of NMNAT2 suppresses axon protection afforded by GNE-3511 (n = 3). For A and C, statistical analysis was performed with a repeated-measures, two-way ANOVA, while in I, a standard two-way ANOVA was performed. Post hoc analysis was performed with Bonferroni correction for multiple comparisons, where *P < 0.05, **P < 0.005, and ****P < 0.0001. Error bars represent SEM.