Marc Hammarlund et al. (2008) C.elegans Neuronal Development Meeting "A Genetic Screen for Axon Regeneration"

Erik Jorgensen, Marc Hammarlund, Paola Nix, Michael Bastiani, Linda Hauth

[C.elegans Neuronal Development Meeting, 2008]

Damaged axons can sometimes regenerate and recover function, but the requirements for successful regeneration are poorly understood. This lack of understanding limits development of treatments for nerve damage (such as traumatic spinal cord injury and neurodegenerative disease). We are performing a genetic screen in C. elegans for genes involved in axon regeneration. We are using a mutant background (unc-70) in which axons spontaneously break and regenerate (1). Since most GABA commissures in the unc-70 background are produced by regeneration, knockdown of a regeneration gene should reduce GABA commissure number. We used OrthoMCL to select approximately 5,500 genes for screening that are orthologous between C. elegans and human (2). For each of these genes, we are assaying the effect of RNAi knockdown on regeneration in the unc-70 background. The GABA motor neurons are visualized by GFP. We also include the eri-1 lin-15 genotype to sensitize neurons to RNAi (3, 4). In each experiment, GABA commissures in approximately 10 animals are scored under a compound microscope. To date, this screen has identified over 60 genes that are likely required for regeneration. To confirm the regeneration phenotype we are using laser axotomy to trigger regeneration rather than the unc-70 genetic background. So far, these experiments confirm that most genes identified in the screen are required for regeneration. Genes identified in our screen are associated with a variety of cellular processes, including signal transduction, transcription, the cytoskeleton, and the extracellular matrix. Understanding how each of these genes contributes to the cell biology of successful regeneration will add to our understanding of how neurons regenerate, and suggest potential therapeutic targets. One essential component of regeneration that our screen has identified is the MAPKKK dlk-1. Please see the abstract by Nix et al. for details on these results.

Paola Nix et al. (2008) C.elegans Neuronal Development Meeting "Nerve Regeneration Requires MAPK Signaling"

Paola Nix, Marc Hammarlund, Linda Hauth, Erik Jorgensen, Michael Bastiani

[C.elegans Neuronal Development Meeting, 2008]

An understanding of how nerves regenerate is an important key to understanding certain neurological disorders. Currently, only a handful of molecules are known to be essential for neuronal regeneration, and very few of these actually enhance regeneration. We identified the gene dlk-1 from an RNAi-based screen for mutants defective in neuronal regeneration (see poster by Hammarlund et al.). We confirmed this mutant phenotype by cutting individual axons with a laser. In the wild type, about 70% of axons initiate and extend growth cones within 12 hours post-axotomy. In contrast, axons in a dlk-1 null background never regenerate after surgery. dlk-1 encodes a MAPKKK of the dual leucine zipper subfamily. Regulation of DLK-1 activity was previously shown to be required for presynaptic development as part of a MAP kinase signaling pathway that includes the MAPKK MKK-4 and the p38alpha-like MAPK PMK-3 (1). We assayed mkk-4 and pmk-3 neurons for their ability to regenerate following laser axotomy. Similar to dlk-1, 100% of mkk-4 and pmk-3 axons fail to regenerate. Importantly, MAPK activation is not essential for axon outgrowth during development as dlk-1, mkk-4, and pmk-3 mutant adults display relatively normal neuronal morphology. It is only when axons are challenged to regenerate that activity of this MAPK pathway is essential. DLK-1 is normally targeted for degradation by the presynaptically localized E3 ubiquitin ligase RPM-1 (1). Our data suggest a model in which DLK-1 is continually trafficked to synaptic regions and then degraded. Once the neuron has been severed, accumulation of DLK-1 in the proximal fragment initiates a signal to induce the machinery required for neuronal growth. Consistent with this model, overexpression of dlk-1 improves regeneration, suggesting that activation of this pathway promotes neuronal regrowth. We are currently testing this hypothesis by additional cell biological and biochemical methods.