Results highlight unexpected categories of essential genes and inhibitors.

Scientists have used a laser injury model in mechanosensory PLM neurons of the roundworm Caenorhabditis elegans to identify genes and pathways involved in the regulation of axonal growth. The investigators used null mutants to screen over 654 C. elegans genes, based on their orthology to human genes and potential neuronal function or known biochemical role, but which weren’t essential for overall health or growth rate.

The findings suggested that there were genes among all the structural and functional classes tested that affected PLM regrowth. Of particular interest was the finding that overexpression of the conserved Arf guanine nucleotide exchange factor (GEF), EFA-6, significantly inhibited axonal regrowth by destablilizing axonal microtubules. This activity of EFA-6 appeared independent to its Arf GEF activity.

The research, led by the University of California San Diego (UCSD)’s Andrew D. Chisholm, Ph.D., Lizhen Chen, Ph.D., and colleagues, is published in Neuron in a paper titled “Axon Regeneration Pathways Identified by Systematic Genetic Screening in C. elegans.

Collectively, and when analyzed as nine gene classes, those genes promoting regrowth (i.e., those that reduced growth in loss-of-function mutants) were more frequent in the cytoskeleton and motors, and “neurotransmission” classes, the team reports. Conversely, the few genes that were found to inhibit regrowth (i.e., increased regrowth in loss-of-function mutants) were concentrated in the cell adhesion/extracellular matrix class. A cluster of genes affecting both Ca2+ and Na+ ionic balance was notably critical for regrowth, and axonal regrowth was similarly strongly reduced in mutants affecting chemical neurotransmitters such as acetylcholine, GABA, and biogenic amines.

The DLK-1 MAPK cascade has previously been found to be essential for axon regrowth after injury, and the researchers’ screen for over 80 additional protein kinases and selected protein phosphatases identified the importance of several additional cytosolic kinases to axonal regrowth. Most of these had not previously been linked to axonogenesis.

Of 130 genes implicated in RNA metabolism, transcription, and translation, along with specific transcription factor genes, the Argonaute-like protein ALG-1 was critical for regrowth, “implying a regrowth-promoting role for microRNAs,” the authors note. Several proteins affecting chromatin remodeling were also crucial, including the SWI/SNF complex component XNP-1/ATR-X. In contrast, loss of function of the histone deacetylase HDA-3/HDAC3 improved regrowth. The authors suggest that as loss of HDA-3 function has previously been shown to be neuroprotective in a C. elegans model of polyglutamine toxicity, HDA-3 may act generally to repress neuroprotective genes.

Interestingly, axonal regrowth was strongly reduced in a cluster of mutants previously thought to be dedicated to synaptic vesicle (SV) recycling, whereas genes involved in SV exocytosis didn’t affect regrowth. The requirement for SV recycling genes seemed independent to their role in synaptic function, the authors note, as other genes critical for synaptic transmission didn’t affect regrowth. One possiblility for this finding is that SV endocytosis genes may be required for vesicles that function in injury signaling, they suggest.

Overall, some 10% of genes tested displayed significant effects on axonal regrowth, with a similar number of genes displaying smaller yet still significant effects. “A key outcome of our screen has been the identification of pathways with inhibitory influences on axon regrowth, indicating that PLM axon regrowth in the wild-type is restrained by intrinsic and extrinsic inhibitory influences,” the authors conclude.

“Overall, our analysis suggests the following model for PLM axon regrowth. Axonal injury triggers a calcium transient that activates cAMP and PKA signaling upstream of DLK-1. In parallel, SV endocytosis may be activated to form signaling vesicles. Such vesicles could transport DLK-1 itself, or other injury signals. DLK-1 kinase is activated and triggers local translation. Each of these pathways is critical either for competence of injured axons to regrow or for the initial stages of regeneration in which the proximal stump re-establishes a growth cone.” 

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