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