title: Neuronal regeneration in C. elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation.
abstract: 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.
authors.affiliation: Department of Physiology and Biophysics, Photonics Center, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Lin
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Physiology and Biophysics, Photonics Center, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: James
authors.identifier: http://orcid.org/0000-0002-0832-6377
authors.initials: J
authors.lastname: Shay
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118, and.
authors.forename: Melissa
authors.identifier: http://orcid.org/0000-0002-5290-439X
authors.initials: M
authors.lastname: McLoed
authors.affiliation: Department of Physiology and Biophysics, Photonics Center, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Kevin
authors.initials: K
authors.lastname: Roodhouse
authors.affiliation: Department of Physiology and Biophysics, Photonics Center, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Chung
authors.affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worchester, Massachusetts 01605.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Clark
authors.affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worchester, Massachusetts 01605.
authors.forename: Jennifer K
authors.identifier: http://orcid.org/0000-0003-3526-2302
authors.initials: JK
authors.lastname: Pirri
authors.affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worchester, Massachusetts 01605.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Alkema
authors.affiliation: Department of Physiology and Biophysics, Photonics Center, Boston University School of Medicine, Boston, Massachusetts 02118, cvgabel@bu.edu.
authors.forename: Christopher V
authors.identifier: http://orcid.org/0000-0002-2763-3938
authors.initials: CV
authors.lastname: Gabel
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans; D002118:Calcium / Q000378:metabolism*; D000075402:Channelrhodopsins; D040542:Mechanotransduction, Cellular / Q000502:physiology; D009416:Nerve Regeneration / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D062308:Optogenetics / Q000379:methods*; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D013347:Subcellular Fractions / Q000502:physiology
chemical_list: D000075402:Channelrhodopsins; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium
keywords: C. elegans; calcium; optogenetics; regeneration; ryanodine receptor