THE mitochondrial uniporter modulates neuronal regenerative outgrowth and calcium dynamics following axotomy in C. elegans

Following neuronal injury, calcium signaling plays a critical role in promoting repair processes. Injury produces an initial cytosolic calcium elevation mediated by calcium entry from the cut site, plasma membrane channels, and intracellular storage compartments. Subsequently, a variety of signaling factors are involved in promoting growth cone formation and axon outgrowth and guidance, some of which include DLK-1, CaMP, CED-3, CED-4, and calreticulin. Specific proteins mediating calcium transport have also been reported to significantly affect regenerative outgrowth, particularly inositol triphosphate receptors, voltage-gated calcium channels, and ryanodine receptors. Given that mitochondria can store intracellular calcium and regulate cytosolic calcium levels, we hypothesized that the mitochondrial uniporter (MCU) may play a significant role in neuronal regeneration. We found that inhibiting calcium entry into the mitochondria via a loss of function mutation in MCU significantly enhances axonal outgrowth following laser axotomy of single neurons in C. elegans. This effect is calcium-dependent, with the MCU mutant regenerative phenotype reverting to baseline levels when mutants are chronically treated with the calcium chelator EGTA. We also find that sub-cellular calcium signals at the axon cut site are significantly reduced in MCU mutants, while basal levels of calcium and axon guidance remain unaffected. These findings suggest that mitochondrial calcium regulation plays a significant role in the regeneration of single neurons, and that inhibition of MCU activity may be a promising avenue for the treatment of clinical syndromes derived from axonal injury, such as spinal cord injury.2017-11-03T00:00:00

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