A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K+ deprivation

Abstract

Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCl) to medium containing lower levels of K+ (5 mM KCl). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp · (O-methyl) · fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp · fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10-25 μM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin- 1β converting enzyme-preferring inhibitor, and N-acetyl ASp-Glu-Val- Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD · cmk provided only modest (<20%) protection and only at the highest concentration (100 1/4 mM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD · fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD · fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+- deprived granule neurons.