Neurotoxicity from glutathione depletion is dependent on extracellular trace copper

Glutathione (GSH) is an important antioxidant, and its depletion in neurons has been implicated in several neurodegenerative disorders. Aberrant copper metabolism is also implicated in neurodegeneration and may result in the generation of toxic free radicals. However, little is known about the relationship between GSH depletion and copper homeostasis. In the present study, we examined the role of extracellular trace biometals in neuronal cell death induced by GSH depletion. Treatment of primary cortical neurons with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, induced a rapid loss of intracellular GSH, leading to decreased neuronal cell viability. Neuronal cell death induced by GSH depletion was dependent on trace levels of extracellular copper in the culture medium (1.6 microM). Neurons were protected against GSH depletion-mediated toxicity when cultured in Chelex 100-treated medium containing tenfold less copper (0.16 microM) than normal medium. The addition of copper, but not iron or zinc, to Chelex 100-treated medium restored the neurotoxicity induced by GSH depletion. Moreover, BSO toxicity in normal medium was inhibited by copper chelators. The neurotoxic effects of copper in GSH-depleted neurons involved generation of copper(I) and subsequent free radical-mediated oxidative stress. These studies demonstrate a critical role for extracellular trace copper in neuronal cell death caused by GSH depletion and may have important implications for the understanding of toxic processes in neurodegenerative diseases.