Glutamate leakage from a compartmentalized intracellular metabolic pool and activation of the lipoxygenase pathway mediate oxidative astrocyte death by reversed glutamate transport

Astrocytes have essential roles for neuron survival and function, so that their demise in neurodegenerative insults, such as ischemia, deserves attention. A major event of the cell death cascade in ischemia is the reversed operation of excitatory amino acid transporters (EAAT), releasing glutamate. Cytotoxicity is conventionally attributed to extracellular glutamate accumulation. We previously reported that mimicking such dysfunction by EAAT substrate inhibitors, whose uptake induces glutamate release by heteroexchange, triggers glutathione (GSH) depletion and oxidative death of differentiated astrocytes in culture. Here we demonstrate that astrocyte death, although correlated with glutamate release, is not resulting from high extracellular glutamate-mediated toxicity. L-glutamate per se was gliotoxic only at concentrations much higher than the maximum reached with the potent EAAT substrate inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), and toxicity was lower. Moreover, high glutamate concentrations offered protection against PDC. Protection was also provided by L-aspartate, which is both transported by EAAT and metabolized into glutamate, and by inhibiting glutamine synthetase, which uses transported glutamate to synthesize glutamine. Neither D-aspartate, a metabolically inert EAAT substrate, nor compounds that can provide glutamate intracellularly but are not EAAT substrates offered protection. Interestingly, only the compounds providing protection prevented PDC-induced GSH depletion. These data strongly suggest that reversed uptake-mediated astrocyte death results from the leakage of glutamate from a compartmentalized intracellular metabolic pool specifically fuelled by EAAT, crucial for preserving GSH contents. In addition, we provide evidence for a minor contribution of the cystine-glutamate antiporter x(c) (-) but a major role of the 5-lipoxygenase pathway in this death mechanism.