title: Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.
abstract: Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.
authors.affiliation: Department of Neurology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Miller
authors.forename: K L
authors.initials: KL
authors.lastname: Moulder
authors.forename: C M
authors.initials: CM
authors.lastname: Knudson
authors.forename: D J
authors.initials: DJ
authors.lastname: Creedon
authors.forename: M
authors.initials: M
authors.lastname: Deshmukh
authors.forename: S J
authors.initials: SJ
authors.lastname: Korsmeyer
authors.forename: E M
authors.initials: EM
authors.lastname: Johnson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology*; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D017353:Gene Deletion; D048031:JNK Mitogen-Activated Protein Kinases; D051379:Mice; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases; D016202:N-Methylaspartate / Q000633:toxicity; D009474:Neurons / Q000502:physiology; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000378:metabolism; D051028:bcl-2-Associated X Protein
chemical_list: C494827:Bax protein, mouse; D015853:Cysteine Proteinase Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D016202:N-Methylaspartate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D003546:Cysteine Endopeptidases