A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K+ deprivation.
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 x (O-methyl) x fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp x fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10-25 microM), 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 x chloromethyl ketone (YVAD x cmk), an interleukin-1beta converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp x fluoromethyl ketone (DEVD x fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD x cmk provided only modest (<20%) protection and only at the highest concentration (100 microM) tested, suggesting that interleukin-1beta converting enzyme and/or closely related caspases were not involved. In comparison, DEVD x 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 x 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.
D'Mello
S R
SR
Department of Physiology and Neurobiology, University of Connecticut, Storrs 06269, USA.
Aglieco
F
F
Roberts
M R
MR
Borodezt
K
K
Haycock
J W
JW
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002531:Cerebellum / Q000166:cytology / Q000502:physiology*; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D009842:Oligopeptides / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011191:Potassium Deficiency / Q000473:pathology / Q000503:physiopathology*; D051381:Rats; D017208:Rats, Wistar
D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D011065:Poly(ADP-ribose) Polymerases; C490359:Casp2 protein, rat; D003546:Cysteine Endopeptidases