BAX DELETION FURTHER ORDERS THE CELL-DEATH PATHWAY IN CEREBELLAR GRANULE CELLS AND SUGGESTS A CASPASE-INDEPENDENT PATHWAY TO CELL-DEATH

Dissociated cerebellar granule cells maintained in medium containing 2 5 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 mu M NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased gluco se uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control w ithin 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 i ncreased 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 ca spases 2, 3, and 7; in contrast, Bax-deficient granule cells did not c leave DEVD-AMC. These results place BAX downstream of metabolic change s, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is requi red for apoptotic, but not excitotoxic, cell death. In wild-type cells , Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cl eavage 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-indepen dent death pathway downstream of BAX in cerebellar granule cells.