THE ROLE OF CED-3-RELATED CYSTEINE PROTEASES IN APOPTOSIS OF CEREBELLAR GRANULE CELLS

The CED-3-related cysteine proteases (CRCPs) have been implicated as m ediators of apoptosis, primarily in hematogenous cell systems, but the ir role in neuronal apoptosis remains unclear. The present study exami ned the role of two CRCP families-CPP32- and interleukin-lp converting enzyme (ICE)-like cysteine proteases-in apoptosis of cerebellar granu le cells (CGCs) caused by withdrawal of serum and/or potassium (K+). S erum deprivation potentiated apoptosis caused by K+ withdrawal, reduci ng cell viability by approximately one half of control values after 12 hr as measured by calcein fluorescence. Cell death after serum/K+ dep rivation was significantly attenuated by the CPP32-like inhibitor z-DE VD-fmk; however, the ICE-like inhibitor z-YVAD-fmk had only slightly p rotective effects at the highest concentration used. Both inhibitors r educed CPP32-like activity directly in an in vitro fluorometric assay system, although z-DEVD-fmk showed much greater potency. K+ and serum/ K+ deprivation each were accompanied by increased CPP32-like activity; however, ICE-like activity was absent after 12 hr of serum and/or Kdeprivation. CPP32 mRNA levels were unchanged after K+ deprivation but increased after serum and combined serum/K+ withdrawal as measured by reverse transcription-PCR (RT-PCR), with peak values at 4 hr reaching 210 +/- 37% and 269 +/- 42% of control levels, respectively. In contr ast, ICE mRNA was undetectable by RT-PCR. These results are consistent with the hypothesis that CPP32-like proteases play an important role in apoptosis of CGCs caused by deprivation of K+ or serum/K+.