Cloning and expression of a rat brain interleukin-1β-converting enzyme (ICE)-related protease (IRP) and its possible role in apoptosis of cultured cerebellar granule neurons

Several members of the IL1β-converting enzyme (ICE) family of proteases recently have been implicated in the intracellular cascade mediating the apoptotic death of various cell types. It is unclear, however, whether ICE- related proteases are involved in apoptosis of mammalian neurons and, if so, how they are activated. Here we report the cloning of an ICE-related protease (IRP) from rat brain, which displays strong sequence identity to human CPP32. In situ hybridization histochemistry reveals that this IRP mRNA is expressed in neuron-enriched regions of the developing and adult rat brain but is profoundly downregulated in the adult (compared with developing) brain. To investigate whether this IRP is involved in the death of neurons in the developing brain, we studied IRP expression in cultured cerebellar granule neurons. In cultured cerebellar granule neurons, reduction of extracellular K⁺ reliably induces apoptosis and stimulates overexpression of IRP mRNA. The latter is especially prominent 4 hr after switching from high K⁺ to low K⁺ medium. The expression of IRP mRNA was maintained at this level for at least 8 hr and was followed by apoptotic death of these neurons. Induction of IRP mRNA and cell death are blocked completely by adding depolarizing concentrations of K⁺ ≤90 min after switching to low K⁺ medium (i.e., before the commitment point for apoptosis) and partially blocked by brain- derived neurotrophic factor (BDNF), which also partially rescues granule neurons from low K⁺-induced apoptosis. In addition, overexpression of IRP cDNA in HeLa cells results in cell death accompanied by strong internucleosomal cleavage of DNA, a typical feature of apoptosis. Finally, we detected cleavage of the putative death substrate poly (ADP-ribose) polymerase (PARP), beginning 8 hr after changing from high K⁺ to low K⁺ medium, coinciding with the time course of induced expression of the IRP gene. Our data suggest that transcriptional activation of IRP could be one of the mechanisms involved in the apoptotic death of cerebellar granule neurons.