Abstract

Excessive Ca²⁺ entry during glutamate receptor overactivation (“excitotoxicity”) induces acute or delayed neuronal death. We report here that deficiency in bax exerted broad neuroprotection against excitotoxic injury and oxygen/glucose deprivation in mouse neocortical neuron cultures and reduced infarct size, necrotic injury, and cerebral edema formation after middle cerebral artery occlusion in mice. Neuronal Ca²⁺ and mitochondrial membrane potential (Δψ_m) analysis during excitotoxic injury revealed that bax-deficient neurons showed significantly reduced Ca²⁺ transients during the NMDA excitation period and did not exhibit the deregulation of Δψ_m that was observed in their wild-type (WT) counterparts. Reintroduction of bax or a bax mutant incapable of proapoptotic oligomerization equally restored neuronal Ca²⁺ dynamics during NMDA excitation, suggesting that Bax controlled Ca²⁺ signaling independently of its role in apoptosis execution. Quantitative confocal imaging of intracellular ATP or mitochondrial Ca²⁺ levels using FRET-based sensors indicated that the effects of bax deficiency on Ca²⁺ handling were not due to enhanced cellular bioenergetics or increased Ca²⁺ uptake into mitochondria. We also observed that mitochondria isolated from WT or bax-deficient cells similarly underwent Ca²⁺-induced permeability transition. However, when Ca²⁺ uptake into the sarco/endoplasmic reticulum was blocked with the Ca²⁺-ATPase inhibitor thapsigargin, bax-deficient neurons showed strongly elevated cytosolic Ca²⁺ levels during NMDA excitation, suggesting that the ability of Bax to support dynamic ER Ca²⁺ handling is critical for cell death signaling during periods of neuronal overexcitation.