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© The Rockefeller University Press, 0021-9525/1997//205 $5.00
The Journal of Cell Biology, Volume 139, Number 1, , 1997 205-217

Article

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



Timothy M. Miller*, Krista L. Moulder*, C. Michael Knudson{ddagger}, Douglas J. Creedon*, Mohanish Deshmukh*, Stanley J. Korsmeyer{ddagger}, and Eugene M. Johnson, Jr.*

* Department of Neurology and Department of Molecular Biology and Pharmacology, {ddagger} Department of Medicine and Department of Pathology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

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–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.

Abbreviations used in this paper: BAF, Boc-aspartyl(OMe)-fluoromethylketone; NMDA, N-methyl-D-aspartic acid; PI-3-K, phosphatidylinositol-3 kinase; PCD, programmed cell death; TUNEL, TdT-mediated dUTP nick end labeling.

Address all correspondence to Eugene M. Johnson, Jr., Washington University School of Medicine, Department of Molecular Biology and Pharmacology, 660 South Euclid Avenue, Box 8103, St. Louis, MO 63110. Tel.: (314) 362-3926. Fax: (314) 362-7058. E-mail: ejohnson{at}pharmdec.wustl.edu


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