Axon Regeneration Pathways Identified by Systematic Genetic Screening in C. elegans

Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.
Neuron (Impact Factor: 15.05). 09/2011; 71(6):1043-57. DOI: 10.1016/j.neuron.2011.07.009
Source: PubMed


The mechanisms underlying the ability of axons to regrow after injury remain poorly explored at the molecular genetic level. We used a laser injury model in Caenorhabditis elegans mechanosensory neurons to screen 654 conserved genes for regulators of axonal regrowth. We uncover several functional clusters of genes that promote or repress regrowth, including genes classically known to affect axon guidance, membrane excitability, neurotransmission, and synaptic vesicle endocytosis. The conserved Arf Guanine nucleotide Exchange Factor (GEF), EFA-6, acts as an intrinsic inhibitor of regrowth. By combining genetics and in vivo imaging, we show that EFA-6 inhibits regrowth via microtubule dynamics, independent of its Arf GEF activity. Among newly identified regrowth inhibitors, only loss of function in EFA-6 partially bypasses the requirement for DLK-1 kinase. Identification of these pathways significantly expands our understanding of the genetic basis of axonal injury responses and repair.


Available from: Bruce Bowerman, May 08, 2014
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    • "Previous studies suggest that disorganized microtubules occupy the retraction bulbs in the adult CNS where regeneration is abortive (Erturk et al., 2007), highlighting the importance of proper cytoskeletal re-organization for growth cone formation. In support of this, application of taxol, a MTstabilizing agent, facilitates growth cone formation and promotes axon regeneration (Hellal et al., 2011; Sengottuvel et al., 2011; Chen et al., 2011; Ruschel et al., 2015). However, axon growth involves coordination of microtubule and actin structures (Gomez and Letourneau, 2014; Vitriol and Zheng, 2012; Coles and Bradke, 2015). "
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    ABSTRACT: After axotomy, neuronal survival and growth cone re-formation are required for axon regeneration. We discovered that doublecortin-like kinases (DCLKs), members of the doublecortin (DCX) family expressed in adult retinal ganglion cells (RGCs), play critical roles in both processes, through distinct mechanisms. Overexpression of DCLK2 accelerated growth cone re-formation in vitro and enhanced the initiation and elongation of axon re-growth after optic nerve injury. These effects depended on both the microtubule (MT)-binding domain and the serine-proline-rich (S/P-rich) region of DCXs in-cis in the same molecules. While the MT-binding domain is known to stabilize MT structures, we show that the S/P-rich region prevents F-actin destabilization in injured axon stumps. Additionally, while DCXs synergize with mTOR to stimulate axon regeneration, alone they can promote neuronal survival possibly by regulating the retrograde propagation of injury signals. Multifunctional DCXs thus represent potential targets for promoting both survival and regeneration of injured neurons.
    Neuron 11/2015; 88(4). DOI:10.1016/j.neuron.2015.10.005 · 15.05 Impact Factor
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    • "Unexpectedly, the regrowth-inhibitory function of EFA-6 is independent of its GEF activity, and instead is mediated by its N-terminal domain (Chen et al., 2011). The EFA-6 N-terminal domain inhibits MT growth at the cell cortex of C. elegans embryos via a conserved motif of 18 amino acids (O'Rourke et al., 2010). "
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    ABSTRACT: Axon injury triggers a series of changes in the axonal cytoskeleton that are prerequisites for effective axon regeneration. In C. elegans the signaling protein EFA-6 is a potent intrinsic inhibitor of axon regrowth. Here we show that axon injury triggers rapid EFA-6-dependent inhibition of axonal microtubule (MT) dynamics, concomitant with relocalization of EFA-6. EFA-6 relocalization and axon regrowth inhibition require a conserved 18-aa motif in its otherwise intrinsically disordered N-terminal domain. The EFA-6 N-terminus binds the MT-associated proteins TAC-1/Transforming-Acidic-Coiled-Coil, and ZYG-8/Doublecortin-Like-Kinase, both of which are required for regenerative growth cone formation, and which act downstream of EFA-6. After injury TAC-1 and EFA-6 transiently relocalize to sites marked by the MT minus end binding protein PTRN-1/Patronin. We propose that EFA-6 acts as a bifunctional injury-responsive regulator of axonal MT dynamics, acting at the cell cortex in the steady state and at MT minus ends after injury.
    eLife Sciences 09/2015; 4. DOI:10.7554/eLife.08695 · 9.32 Impact Factor
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    • "Statistics: ANOVA; ***p < 0.01. studies of CAMSAPs (see below) and because an increase in dynamic MTs correlates with enhanced regrowth in mutants such as efa-6 (Chen et al., 2011). These observations suggest the absolute level of dynamic MTs may not be the critical determinant of regrowth capacity and that, instead, the change in the number of dynamic MTs after injury may be key. "
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    ABSTRACT: Precise regulation of microtubule (MT) dynamics is increasingly recognized as a critical determinant of axon regeneration. In contrast to developing neurons, mature axons exhibit noncentrosomal microtubule nucleation. The factors regulating noncentrosomal MT architecture in axon regeneration remain poorly understood. We report that PTRN-1, the C. elegans member of the Patronin/Nezha/calmodulin- and spectrin-associated protein (CAM-SAP) family of microtubule minus-end-binding proteins, is critical for efficient axon regeneration in vivo. ptrn-1-null mutants display generally normal developmental axon outgrowth but significantly impaired regenerative regrowth after laser axotomy. Unexpectedly, mature axons in ptrn-1 mutants display elevated numbers of dynamic axonal MTs before and after injury, suggesting that PTRN-1 inhibits MT dynamics. The CKK domain of PTRN-1 is necessary and sufficient for its functions in axon regeneration and MT dynamics and appears to stabilize MTs independent of minus-end localization. Whereas in developing neurons, PTRN-1 inhibits activity of the DLK-1 mitogen-activated protein kinase (MAPK) cascade, we find that, in regeneration, PTRN-1 and DLK-1 function together to promote axonal regrowth.
    Cell Reports 11/2014; 9(3):874-883. DOI:10.1016/j.celrep.2014.09.054 · 8.36 Impact Factor
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