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Axon Regeneration Requires a Conserved MAP Kinase Pathway

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Axon Regeneration Requires a Conserved MAP Kinase Pathway

Marc Hammarlund et al. Science.

Abstract

Regeneration of injured neurons can restore function, but most neurons regenerate poorly or not at all. The failure to regenerate in some cases is due to a lack of activation of cell-intrinsic regeneration pathways. These pathways might be targeted for the development of therapies that can restore neuron function after injury or disease. Here, we show that the DLK-1 mitogen-activated protein (MAP) kinase pathway is essential for regeneration in Caenorhabditis elegans motor neurons. Loss of this pathway eliminates regeneration, whereas activating it improves regeneration. Further, these proteins also regulate the later step of growth cone migration. We conclude that after axon injury, activation of this MAP kinase cascade is required to switch the mature neuron from an aplastic state to a state capable of growth.

Figures

Fig. 1
Fig. 1
dlk-1 is required for axon regeneration in β-spectrin mutants. (A) Cartoon showing development of axon morphology in control β-spectrin mutant (left) and in β-spectrin mutant lacking hypothetical regeneration gene (right). (B and C) GABA neurons in representative L4-stage β-spectrin mutants (unc-70) under control conditions or after dlk-1 RNAi. Scale bars: 20 µm. (D) High-magnification view of boxed region in (C). Arrows indicate inert axon stumps. Scale bar: 10 µm. (E and F) GABA neurons in representative L4-stage wild-type and dlk-1 mutant animals. Scale bars: 20 µm.
Fig. 2
Fig. 2
dlk-1 is required in severed axons for growth cone initiation. (A) Regenerating axons 18–20 hours after laser surgery in a wild-type animal. Both severed axons have generated a growth cone (arrows). Scale bar: 10 µm. (B) Axons in dlk-1 mutants fail to generate growth cones 18–24 hours after surgery. Scale bar: 10 µm. (C) DLK-1 acts cell intrinsically to mediate regeneration. (D) RPM-1 controls DLK-1 activity in axon regeneration. (E) Regeneration requires dlk-1 at all ages and over expression of DLK-1 rescues age- associated decline. (F) DLK-1 acts at the time of injury to mediate regeneration. Time of heat shock relative to surgery is indicated in hours. No heat shock is indicated by ‘no’. ‘L2’ indicates surgery at L2-stage. Bars in panels C–F show percentage of axons that initiated regeneration and 95% confidence interval (CI).
Fig. 3
Fig. 3
dlk-1 controls growth cone initiation and morphology during axon regeneration. (A) Transient filopodium in a wild-type animal. Images were taken at 165 (left), 170 (center), and 180 (right) minutes after surgery. Scale bar: 5 µm. (B) Transient filopodium in a dlk-1 mutant animal. Images were taken at 475 (left), 480 (center), and 490 (right) minutes after surgery. Scale bar: 5 µm. (C) Representative axons in a wild-type animal 120 minutes after axotomy. Proximal and distal ends have retracted away from site of surgery, but proximal ends (arrows) show no evidence of regeneration. Scale bar: 10 µm. (D) A representative axon in an animal over expressing DLK-1 120 minutes after axotomy. The proximal end (arrow) has already regenerated past the retracted distal end. Scale bar: 10 µm. (E) Representative growth cones in a wild-type animal. Although these axons successfully initiated regeneration, the growth cones (arrows) have a dystrophic morphology. Scale bar: 10 µm. (F) Representative growth cones in an animal over expressing DLK-1 under the unc-47 promoter. These growth cones (arrows) have a compact morphology similar to growth cones observed during development. Scale bar: 10 µm. (G) Distribution of all times of filopodia initiation in wild type and dlk-1 . Each dot represents a filopodium. (H) Time of first filopodium initiation in wild type and dlk-1. Bars show mean and standard error. (I) Rate of filopodia initiation in wild type and dlk-1. Bars show mean and standard error. (J) Time to initiate regeneration after surgery in wild type and dlk-1 over expressing (OE) animals. Initiation is defined as the appearance of the filopodia that becomes a growth cone. Each dot represents a single axon. (K) Percentage of regenerating axons that reached the dorsal cord after 18–24 hours. Error bars indicate 95% CI.
Fig. 4
Fig. 4
MAP kinase signaling is required for axon regeneration. (A) Regeneration is eliminated by mutations in the DLK-1/MKK-4/PMK-3 MAP kinase module. (B) Other MAP kinase elements contribute to regeneration, but are not essential. (C) Activated DLK-1 has targets in addition to MKK-4 and PMK-3. (D) Model for function of MAP kinase signaling during axon regeneration.

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