Apoptosis proteins CED-3 and CED-4 are needed for efficient neuronal regeneration or axonal fusion [1]. In apoptosis, CED-4/APAF1 is an essential activator of the death executor caspase CED-3. In the regrowth consequent to laser axotomy, however, positive roles for
ced-3 and
ced-4 in repair are evident. Calcium changes at the cut site and later throughout the neuron also influence regeneration. Since modest CED-3 overexpression can counteract the deficiency of axonal regeneration in the calreticulin mutant
crt-1(
ok948), calcium is inferred to act upstream of CED-3/CED-4. The relationship between apoptotic executioner proteins and Ca2+-signaling pathways is still not well understood, but it is interesting that the CED-4 protein features two consensus sequences for the Ca2+-binding EF-hand domain (EF-hand I (EFH-I) and EF-hand II (EFH-II)). To test the potential role of Ca2+-binding in CED-4-mediated axonal regeneration, we mutated the potential Ca2+-binding residues of CED-4 via genome editing. Interestingly, substitutions engineered into the EFH-I and EFH-II sites did not alter apoptosis affecting the touch neuron lineages. In contrast, we find that the EFH-I mutant exhibits a significant increase in axonal regrowth, but neither the EFH-II mutant nor EFH-I&II double mutant is effected for regeneration. Using the caspase activated green fluorescent protein (Pmec-4CA-GFP) we can optically measure caspase activity in response to laser-induced neuron damage. We find that while
ced-4/ced-3 mutations reduce caspase activity, the specific
ced-4 EFH-I mutation does not. We examined the two predicted EF-hand domains in the published CED-4 crystal structure [2], and found that only the EFH-I (but not EFH-II) has a helix-loop-helix structure that is similar to a typical EF-hand Ca2+-binding domain. We suggest a model in which the CED-4 EFH-I Ca2+-binding domain functions to negatively regulate regeneration, possibly by turning off CED-3 caspase. Alternatively, the EFH-I disruption might induce a structural change that enhances CED-3 activation and subsequent elevation of axonal regrowth. We are investigating the role of CED-3 in the enhanced regeneration of EFH-I mutant, and seeking an in vitro method to test the Ca2+-binding properties of EFH-I. References: 1. Pinan-Lucarre, B., et al., The core apoptotic executioner proteins CED-3 and CED-4 promote initiation of neuronal regeneration in Caenorhabditis elegans. PLoS Biol, 2012. 10(5): p.
e100133 2. Qi, S., et al., Crystal structure of the Caenorhabditis elegans apoptosome reveals an octameric assembly of CED-4. Cell, 2010. 141(3): p. 446-57.