Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth.

During development, a neuron transitions from a state of rapid growth to a stable morphology, and neurons within the adult mammalian CNS lose their ability to effectively regenerate in response to injury. Here, we identify a novel form of neuronal regeneration, which is remarkably independent of DLK-1/DLK, KGB-1/JNK, and other MAPK signaling factors known to mediate regeneration in Caenorhabditis elegans, Drosophila, and mammals. This DLK-independent regeneration in C. elegans has direct genetic and molecular links to a well-studied form of endogenous activity-dependent ectopic axon outgrowth in the same neuron type. Both neuron outgrowth types are triggered by physical lesion of the sensory dendrite or mutations disrupting sensory activity, calcium signaling, or genes that restrict outgrowth during neuronal maturation, such as SAX-1/NDR kinase or UNC-43/CaMKII. These connections suggest that ectopic outgrowth represents a powerful platform for gene discovery in neuronal regeneration. Moreover, we note numerous similarities between C. elegans DLK-independent regeneration and lesion conditioning, a phenomenon producing robust regeneration in the mammalian CNS. Both regeneration types are triggered by lesion of a sensory neurite via reduction of neuronal activity and enhanced by disrupting L-type calcium channels or elevating cAMP. Taken as a whole, our study unites disparate forms of neuronal outgrowth to uncover fresh molecular insights into activity-dependent control of the adult nervous system's intrinsic regenerative capacity.

Neuronal regeneration in C. elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation.

Regulated calcium signals play conserved instructive roles in neuronal repair, but how localized calcium stores are differentially mobilized, or might be directly manipulated, to stimulate regeneration within native contexts is poorly understood. We find here that localized calcium release from the endoplasmic reticulum via ryanodine receptor (RyR) channels is critical in stimulating initial regeneration following traumatic cellular damage in vivo. Using laser axotomy of single neurons in Caenorhabditis elegans, we find that mutation of unc-68/RyR greatly impedes both outgrowth and guidance of the regenerating neuron. Performing extended in vivo calcium imaging, we measure subcellular calcium signals within the immediate vicinity of the regenerating axon end that are sustained for hours following axotomy and completely eliminated within unc-68/RyR mutants. Finally, using a novel optogenetic approach to periodically photo-stimulate the axotomized neuron, we can enhance its regeneration. The enhanced outgrowth depends on both amplitude and temporal pattern of excitation and can be blocked by disruption of UNC-68/RyR. This demonstrates the exciting potential of emerging optogenetic technology to beneficially manipulate cell physiology in the context of neuronal regeneration and indicates a link to the underlying cellular calcium signal. Taken as a whole, our findings define a specific localized calcium signal mediated by RyR channel activity that stimulates regenerative outgrowth, which may be dynamically manipulated for beneficial neurotherapeutic effects.