Axon regeneration mechanisms: insights from C. elegans

Abstract

Understanding the mechanisms of axon regeneration is of great importance to the development of therapeutic treatments for spinal cord injury or stroke. Axon regeneration has long been studied in diverse vertebrate and invertebrate models, but until recently had not been analyzed in the genetically tractable model organism Caenorhabditis elegans. The small size, simple neuroanatomy, and transparency of C. elegans allows single fluorescently labeled axons to be severed in live animals using laser microsurgery. Many neurons in C. elegans are capable of regenerative regrowth, and can in some cases re-establish functional connections. Large-scale genetic screens have begun to elucidate the genetic basis of axon regrowth.

Figure I. Distal degeneration and fusion in mechanosensory axons

Mechanosensory (PLM) axons typically undergo any of three types of regrowth. Usually, the proximal axon regrows without contacting the distal fragment; the distal fragment undergoes degeneration. Less often, the regrowing axon fuses with the distal fragment, preventing distal degeneration. Fusion can be ‘end to end’ or ‘end to side’; the frequency of reconnection by fusion is somewhat sensitive to the transgenic background and the method of axotomy [14].

Figure 1. Types of neurons used to study regeneration

(A) Diagram (right) and images (left) of GABAergic motor neuron axotomy and regrowth at 24 h postaxotomy. df, distal fragment; gc, growth cone. Regrown process shown in green. Transgenic marker Punc-25-GFP(juIs76). (B) Diagram (right) of the PLM mechanosensory neuron showing position of laser axotomy relative to the synaptic branch and cell body. Confocal images (left) of PLM regrowth at 6 h, 14 h and 24 h postaxotomy (different animals). Transgenic marker, Pmec-7-GFP(muIs32). Scales, 10 μm.

Figure 2. Injury-triggered signaling pathways

Axon injury triggers elevation of axonal Ca²⁺ by several mechanisms. Ca²⁺ elevation leads to activation of adenylyl cyclase and elevated cAMP levels, leading to PKA activation. PKC may be also activated by the injury-induced Ca²⁺ transient. Injury signals, possibly Ca²⁺ or MT depolymerization, result in activation of DLK-1 and the entire DLK-1/MKK-4/PMK-3 cascade. The MLK-1/MEK-1/KGB-1 pathway is activated in parallel by unknown signals. The DLK-1 pathway stabilizes CEBP-1 mRNA in axons, and is likely to have other targets.

Figure 3. The MT cytoskeleton in axon regrowth

Highly simplified overview of possible changes in the MT cytoskeleton during regrowth, based on work in many organisms. Mature axons have stable polarized MT arrays with MT plus ends located distally from the soma. Axotomy disrupts MTs directly, and may trigger additional local severing of MTs into tubulin subunits, either via calpains or specific MT-severing enzymes. MT depolymerization may be sensed by the DLK-1 pathway. New axonal MTs are then nucleated, possibly from the newly formed plus ends or from as yet unknown noncentrosomal MT organizing centers (MTOCs). How polarity is re-established in the regrowing axon is not yet clear. Newly formed MTs may be highly dynamic (i.e. undergoing repeated catastrophe and regrowth) but for successful growth cone extension, MT growth must become more stable. In C. elegans neurons the plus end binding protein EBP-1 is required for regrowth, and the putative MT catastrophe factor EFA-6 inhibits regrowth [40].