The core apoptotic executioner proteins CED-3 and CED-4 promote initiation of neuronal regeneration in Caenorhabditis elegans.
A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. In C. elegans, individual visualized axons can be laser-cut in vivo and neuronal responses to damage can be monitored to decipher genetic requirements for regeneration. With an initial interest in how local environments manage cellular debris, we performed femtosecond laser axotomies in genetic backgrounds lacking cell death gene activities. Unexpectedly, we found that the CED-3 caspase, well known as the core apoptotic cell death executioner, acts in early responses to neuronal injury to promote rapid regeneration of dissociated axons. In ced-3 mutants, initial regenerative outgrowth dynamics are impaired and axon repair through reconnection of the two dissociated ends is delayed. The CED-3 activator, CED-4/Apaf-1, similarly promotes regeneration, but the upstream regulators of apoptosis CED-9/Bcl2 and BH3-domain proteins EGL-1 and CED-13 are not essential. Thus, a novel regulatory mechanism must be utilized to activate core apoptotic proteins for neuronal repair. Since calcium plays a conserved modulatory role in regeneration, we hypothesized calcium might play a critical regulatory role in the CED-3/CED-4 repair pathway. We used the calcium reporter cameleon to track in vivo calcium fluxes in the axotomized neuron. We show that when the endoplasmic reticulum calcium-storing chaperone calreticulin, CRT-1, is deleted, both calcium dynamics and initial regenerative outgrowth are impaired. Genetic data suggest that CED-3, CED-4, and CRT-1 act in the same pathway to promote early events in regeneration and that CED-3 might act downstream of CRT-1, but upstream of the conserved DLK-1 kinase implicated in regeneration across species. This study documents reconstructive roles for proteins known to orchestrate apoptotic death and links previously unconnected observations in the vertebrate literature to suggest a similar pathway may be conserved in higher organisms.
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, USA.
Berangere
B
Pinan-Lucarre
Christopher V
CV
Gabel
Christopher P
CP
Reina
S Elizabeth
SE
Hulme
Sergey S
SS
Shevkoplyas
R Daniel
RD
Slone
Jian
J
Xue
Yujie
Y
Qiao
Sarah
S
Weisberg
Kevin
K
Roodhouse
Lin
L
Sun
George M
GM
Whitesides
Aravinthan
A
Samuel
Monica
M
Driscoll
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017209:Apoptosis; D001369:Axons / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D019771:Axotomy; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D037282:Calreticulin / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D009416:Nerve Regeneration; D009474:Neurons / Q000378:metabolism / Q000473:pathology / Q000502:physiology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D059008:Time-Lapse Imaging
D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; D037282:Calreticulin; C089224:Ced-4 protein, C elegans; C087233:Ced-9 protein, C elegans; D019253:Proto-Oncogene Proteins c-bcl-2; C498050:DLK-1 protein, C elegans; D020930:MAP Kinase Kinase Kinases; D020169:Caspases; C089223:ced-3 protein, C elegans; D002118:Calcium