The Core Apoptotic Executioner Proteins CED-3 and CED-4 Promote Initiation of Neuronal Regeneration in Caenorhabditis elegans

Laser severing of individual axons in the nematode Caenorhabditis elegans revealed that the apoptotic executioner caspase CED-3 and its regulator CED-4/Apaf-1 play an unexpected beneficial role in promoting axonal regeneration. 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. Author Summary: Clinical success in reconnecting neurons damaged by injury will require detailed molecular understanding of how mature axons respond to being severed. To decipher intrinsic molecular pathways that stimulate axon regeneration, we use the small transparent model, Caenorhabditis elegans, in which individual labeled axons can be laser-severed without damage to neighboring tissue, and regrowing axons can be observed directly in the living animal. We find that the apoptotic protein CED-3, well known for its developmental roles in cell death, also unexpectedly acts in a beneficial role to promote regeneration of severed axons. Initial post-surgery outgrowth is impaired in a ced-3 mutant, suggesting that CED-3 is involved in the early steps of axonal regeneration. The activation of CED-3 caspase in this context occurs independently of major cell death regulatory pathways, but efficient regeneration does require the caspase activator CED-4/Apaf-1, the conserved regeneration kinase DLK-1, and calreticulin-dependent calcium fluxes within the severed neuron. Our data suggest a novel conserved pathway for neuronal reconstruction, and call into question the practice of blocking caspases to treat neuronal injury in the clinic.