How neurons in their native environments respond to localized physical disruptions such as axon severing is poorly understood. Femtosecond laser surgery allows precise cutting of individual axons within living transparent Caenorhabditis elegans such that regeneration can be observed in vivo. We applied this technology to investigate the role of the cell death machinery in the neuronal response to laser severing of ALM touch neurons visualized by Pmec-4GFP in young adult C. elegans. Unexpectedly, we found that CED-3 caspase, extensively characterized as the essential executioner protease in apoptosis, acts to promote early events in neuronal regeneration including outgrowth and reconnection. Time-lapse imaging of ced-3 mutants revealed defects in the early stage of regenerative growth cone formation, i.e., the sprouting of short, often transient, exploratory processes. In addition, CED-3 also functions in early reconnection events as ced-3 neurons are deficient in time to reconnection. The apoptotic caspase activator CED-4/Apaf-1 also is required for efficient regeneration, but the upstream apoptotic regulators CED-9/Bcl2 and the BH3 domain proteins EGL-1 and CED-13 are not required, indicating a regulatory mechanism distinct from the classical C. elegans apoptotic pathway. Regeneration also depends on the calcium-storing ER chaperone calreticulin crt-1, which contributes to the calcium flux that occurs immediately after axotomy. Our initial work revealed an unexpected reconstructive role for proteins known to orchestrate cell death that may be conserved in higher organisms. A key question is how is CED-3 caspase locally activated and controlled to promote regeneration activities rather than acting in cell killing? Epistasis experiments suggest that ced-3, ced-4, and crt-1 function within the same regenerative pathway; therefore, we propose the following regeneration model: Axotomy induces a localized calcium influx amplified by CRT-1, calcium binds to putative CED-4 EF-hand calcium binding domains to activate CED-4 oligomerization, which promotes procaspase CED-3 binding and stimulates activating protease cleavage (proposed CED-4/CED-3 interaction similar to apoptosis). Current work is focused on determining whether CED-4 EF-hand domains bind calcium and whether they are required for CED-4 function in regeneration.

Affiliations:
- Institut de Biologie de l'Ecole Normale Superieure, Paris, France
- Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA.
- Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ.