Distinct types of cell death underlie the pathology of many human diseases, yet very little is understood of non-apoptotic cell death mechanisms. Analysis of non-apopotic death mechanisms in C. elegans is likely to provide mechanistic insights relevant to critical problems in neuronal injury and pathology. Gain-of-function mutations in specific C. elegans ion channel genes encode hyperactive channels that induce necrotic-like death of the neurons that express these mutant genes (Chalfie and Wolinsky, 1990; Driscoll and Chalfie, 1991). For example, dominant mutations affecting the
mec-4 degenerin channel induce degeneration of six touch receptor neurons.
mec-4(d )-induced cell death occurs independently of all known regulators and executors of programmed cell death in C. elegans , and is necrotic-like in that it is characterized by dramatic cellular swelling. We are investigating the requirements for the progression and execution of degenerative cell death inflicted by
mec-4(d) and other insults in C. elegans . Essentially nothing is known of the molecules that execute necrotic cell death, although it is clear that, like in apoptosis, proteolysis is likely to be a major component of the death mechanism. We have found that specific aspartyl proteases of the Cathepsin D family are required for the efficient execution of necrotic-like cell death in C. elegans . Several mutant nematode strains have been found to exhibit up to 90% reduced Cathepsin D protease activity (Jacobson et al., 1988). We assessed the requirement for Cathepsin D activity in necrotic-like cell death induced by
mec-4(d) by testing
cad-1 alleles
j1 and
j14 for the ability to block cell death. Necrotic-like cell death induced by
mec-4(d) was significantly reduced in these genetic backgrounds. In addition, several mutations that have been reported to cause 80-90% reduced cathepsinD activity (
daf-4(
e1364) ,
mec-8(
e398) and
unc-52(
su250) also suppress
mec-4(d)- induced degeneration. Starvation is another condition that leads to significant reduction of Cathepsin D activity in C. elegans (Sarkis et al. 1988). We have observed that starvation also interferes with necrotic-like cell death induced by
mec-4(d) . To further demonstrate the involvement of Cathepsin D proteolytic activity in neurodegeneration, we assayed the effects of Pepstatin A, a potent and specific inhibitor of Cathepsin D on
mec-4(d) induced necrotic-like cell death. Pepstatin A significantly blocked neurodegeneration. Thus, one or more cathepsin D proteases play a significant role in
mec-4(d)- induced degeneration. There are several genes in the C. elegans genome that encode putative Cathepsin D proteases. The most highly similar to biochemically characterized mammalian aspartyl proteases has been designated
asp-4 (Tcherepanova et al., 2000). We employed dsRNA interference to quench
asp-4 expression and to determine the effects on neurodegeneration. Results indicate that ASP-4 activity is required for necrotic-like cell death induced by
mec-4(d ). Conversely, overexpression of
asp-4 in a
mec-4(d);
cad-1(
j1) mutant completely reversed the suppressing effects of
cad-1(
j1) on
mec-4(d) induced neurodegeneration. We tested all other Cathepsin D proteases in the C. elegans genome for similar effects on death when inactivated via RNAi or when over-expressed. Our analysis revealed that ASP-3 is the only other aspartyl protease required for necrotic-like cell death. This is the first clear genetic demonstration of a requirement for a protease in the execution of necrotic-like cell death in C. elegans . Aspartyl proteases such as Cathepsin D are mostly lysosomal enzymes but they have also been found to function in the cytoplasm. Interestingly, Cathepsin D has been implicated in endocytic degradation of proteins and
mec-4(d)- induced cell death involves extensive plasma membrane internalization that resembles endocytosis. Such a process may require the function of Cathepsin D. Our results suggest that Cathepsin D activity plays a pivotal role in the destruction of the cellular contents during
mec-4(d)- induced neurodegeneration. Preliminary results suggest that cathepsin D may also play a role in necrotic-like cell death induced by insults other than
mec-4(d) in the nematode. Establishment of a central role for cathepsin D in the execution of degenerative cell death would make cathepsin proteases attractive targets for therapeutic intervention in human injury and disease.