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A DEVD‐Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K + Deprivation
Author(s) -
D'Mello Santosh R.,
Aglieco Fabio,
Roberts Melanie R.,
Borodezt Kristin,
Haycock John W.
Publication year - 1998
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1998.70051809.x
Subject(s) - apoptosis , caspase , programmed cell death , dna fragmentation , caspase 3 , microbiology and biotechnology , proteolysis , poly adp ribose polymerase , caspase 1 , biology , enzyme , granule (geology) , western blot , biochemistry , chemistry , polymerase , gene , paleontology
Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K + (25 m M KCI) to medium containing lower levels of K + (5 m M KCI). 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 µ M ), 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 µ M ) 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.