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.

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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.
    Full-text · Article · Nov 2015 · Neuron
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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: eLife digest In the nervous system, cells called neurons carry information around the body. These cells have long thin projections called axons that allow the information to pass very quickly along the cell to junctions with other neurons. Neurons in adult mammals are limited in their ability to regenerate, so any damage to axons, for example, due to a stroke or a brain injury, tends to be permanent. Therefore, an important goal in neuroscience research is to discover the genes and proteins that are involved in regenerating axons as this may make it possible to develop new therapies. An internal scaffold called the cytoskeleton supports the three-dimensional shape of the axons. Changes in the cytoskeleton are required to allow neurons to regenerate axons after injury, and drugs that stabilize filaments called microtubules in the cytoskeleton can promote these changes. Chen et al. used a technique called laser microsurgery to sever individual axons in a roundworm known as C. elegans and then observed whether these axons could regenerate. The experiments reveal that a protein called EFA-6 blocks the regeneration of neurons by preventing rearrangements in the cytoskeleton. EFA-6 is normally found at the membrane that surrounds the neuron. However, Chen et al. show that when the axon is damaged, this protein rapidly moves to areas near the ends of microtubule filaments. EFA-6 interacts with two other proteins that are associated with microtubules and are required for axons to be able to regenerate. Chen et al.'s findings demonstrate that several proteins that regulate microtubule filaments play a key role in regenerating axons. All three of these proteins are found in humans and other animals so they have the potential to be targeted by drug therapies in future. The next challenge is to understand the details of how EFA-6 activity is affected by axon injury, and how this alters the cytoskeleton. DOI:
    Full-text · Article · Sep 2015 · eLife Sciences
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    • "For example, screening 654 conserved genes in an axotomy model of mechanosensory neurons in C. elegans identified clusters of genes that promote or repress axon growth. Many of these are components of pathways critical for neuronal plasticity of both development and regeneration; however, some clusters are only required for regeneration (Chen et al., 2011). This highlights how the repair process utilizes plasticity mechanisms important for neuronal development, but also has members unique to the repair process. "
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    ABSTRACT: Regenerative failure remains a significant barrier for functional recovery after central nervous system (CNS) injury. As such, understanding the physiological processes that regulate axon regeneration is a central focus of regenerative medicine. Studying the gene transcription responses to axon injury of regeneration competent neurons, such as those of the peripheral nervous system (PNS), has provided insight into the genes associated with regeneration. Though several individual "regeneration-associated genes" (RAGs) have been identified from these studies, the response to injury likely regulates the expression of functionally coordinated and complementary gene groups. For instance, successful regeneration would require the induction of genes that drive the intrinsic growth capacity of neurons, while simultaneously downregulating the genes that convey environmental inhibitory cues. Thus, this view emphasizes the transcriptional regulation of gene "programs" that contribute to the overall goal of axonal regeneration. Here, we review the known RAGs, focusing on how their transcriptional regulation can reveal the underlying gene programs that drive a regenerative phenotype. Finally, we will discuss paradigms under which we can determine whether these genes are injury-associated, or indeed necessary for regeneration.
    Full-text · Article · Aug 2015 · Frontiers in Molecular Neuroscience
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