Mechanisms underlying the loss of mitochondrial membrane potential in glutamate excitotoxicity

Glutamate excitotoxicity amplifies neuronal death following stroke. We have explored the mechanisms underlying the collapse of mitochondrial potential (Δψm) and loss of [Ca2+]c homeostasis in rat hippocampal neurons in culture following toxic glutamate exposure. The collapse of Δψm is multiphasic and Ca2+-dependent. Glutamate induced a decrease in NADH autofluorescence which preceded the loss of Δψm. Both the decrease in NADH signal and the loss of Δψm were suppressed by Ru360 and both were delayed by inhibition of PARP (by 3-AB or DPQ). During this period, addition of mitochondrial substrates (methyl succinate and TMPD-ascorbate) or buffering [Ca2+]i (using BAPTA-AM or EGTA-AM), rescued Δψm. These data suggest that mitochondrial Ca2+ uptake activates PARP which in turn depletes NADH, promoting the initial collapse of Δψm. After > ~ 20 min, buffering Ca2+ or substrate addition failed to restore Δψm. In neurons from cyclophilin D−/− (cypD−/−) mice or in cells treated with cyclosporine A, removal of Ca2+ restored Δψm even after 20 min of glutamate exposure, suggesting involvement of the mPTP in the irreversible depolarisation seen in WT cells. Thus, mitochondrial depolarisation represents two consecutive but distinct processes driving cell death, the first of which is reversible while the second is not.