Identifying molecules critical for necrotic cell death is a major objective for therapeutic intervention with deleterious consequences of stroke, ischemia and degenerative disease. Unlike apoptotic cell death, little is understood of molecular mechanisms of necrosis. Our interest is in elaborating necrotic-like cell death mechanisms in C. elegans . A variety of different insults can initiate necrotic-like cell death in this nematode, leading to similar morphological changes in the affected cells. Necrosis-inducing genes include dominant alleles of the
mec-4 and
deg-1 degenerins which encode ion-channel subunits with similarity to mammalian epithelial Na + -ion channels (ENaCs), a dominant allele of the
deg-3 acetylcholine receptor a -subunit, and a transgenicially expressed constitutively activated mutant of the G-protein subunit G a S . That channel hyperactivating mutations induce necrosis is reminiscent of the initiation of excitotoxic cell death in humans. We study necrotic neurodegeneration with a focus on channel-hyperactivating mutations in the
mec-4 gene as the death-initiating event. The MEC-4 protein is expressed in the six touch neurons in C. elegans , where it normally participates in the formation of mechanically gated ion channels and is involved in the transduction of mechanical stimuli. The dominant
mec-4(d ) allele encodes a hyperactive channel that leads to necrotic demise of the touch receptor neurons. We established a behavioral assay that allowed us to isolate suppressor mutations that block
mec-4(d) -induced neurodegeneration. Ectopic expression of
mec-4(d) in the ventral nerve cord leads to the appearance of a number of swelling and degenerating cells visible as vacuoles during the larval stage L1. As a consequence of this cell death, worms are severely paralyzed. We mutagenized 45,000 haploid genomes and isolated 24 suppressors with reconstituted locomotion. Such strains harbor candidate suppressors of
mec-4(d)- induced neurodegeneration. One strong suppressor locus maps to chromosome V and is represented by 4 alleles. Mutations affecting this locus can also suppress defects associated with hyperactivated mutants of degenerins
unc-8 ,
unc-105 and
deg-1 . Interestingly, strong alleles also strongly suppress paralysis consequent to expression of human A b 1-42 fragment in C. elegans muscle (see Link, 1995, PNAS 92: 9368). We observe modest suppressor effects on G a S -induced degeneration. We cloned this locus and found it to encode calreticulin. The strongest suppressor alleles are nonsense alleles that appear to be null (antibody staining performed by the lab of J. Ahnn, Kwangju Institute of Sci. and Tech., Korea). Calreticulin is a Ca 2+ binding protein of the ER that plays critical roles in chaperone function and in maintenance of Ca 2+ homeostasis. Both functions may contribute to suppression of neurodegeneration. Prompted by an interest in the contributions of Ca 2+ release in neurotoxicity, we found that mutations in ER Ca 2+ release channels
itr-1 (IP3 receptor) and
unc-68 (ryanodine receptor) also significantly suppress
mec-4(d)- induced degeneration. Conversely, pharmacologically-induced release of ER Ca 2+ in the
crt-1;
mec-4(d) mutant can bypass the
crt-1 block of cell death. Our data suggest release of ER Ca 2+ stores is a critical step in necrotic cell death in C. elegans . Since ER Ca 2+ release has been implicated in excitotoxic cell death, our data provides one of the first molecular indications that necrotic death mechanisms may be conserved from nematodes to humans.