Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-Independent Pathway to Cell Death

• Published in: The Journal of cell biology Vol. 139; no. 1; pp. 205 - 217
• Main Authors: Miller, Timothy M., Moulder, Krista L., Knudson, C. Michael, Creedon, Douglas J., Deshmukh, Mohanish, Korsmeyer, Stanley J., Johnson, Eugene M.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 06.10.1997; The Rockefeller University Press
• Subjects: Animals; Apoptosis; Apoptosis - drug effects; Apoptosis - genetics; bcl-2-Associated X Protein; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Cell death; Cell Death - drug effects; Cell Death - genetics; Cells; Cells, Cultured; Cellular biology; Cerebellum - cytology; Cerebellum - enzymology; Cerebellum - metabolism; Cerebellum - physiology; Cultured cells; Cysteine Endopeptidases - metabolism; Cysteine Endopeptidases - physiology; Cysteine Proteinase Inhibitors - pharmacology; Enzyme Activation - drug effects; Gene Deletion; JNK Mitogen-Activated Protein Kinases; Messenger RNA; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; N-Methylaspartate - toxicity; Neurons; Neurons - physiology; PC12 cells; Phosphorylation; Potassium; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - physiology; Proto-Oncogene Proteins c-bcl-2; Purkinje cells; RNA, Messenger - metabolism; T lymphocytes
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.139.1.205

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 μM 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-aminomethyl-coumarin (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.