Direct evidence for glutathione as mediator of apoptosis in neuronal cells

Recent evidence has focused attention on the role of oxidative stress in va rious acute and chronic neurodegenerative diseases. Particularly, a decreas e in the level of the powerful antioxidant glutathione (GSH) and death of d opaminergic neurons in substantia nigra are prominent features in Parkinson 's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals vi a oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by meta bolic manipulations in a clonal cell line of neuronal origin (mouse neurobl astoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosy nthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship betwe en GSH depletion and apoptosis was evidenced in this neuronal cell type. GS H depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation afte r 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose ge l. These results suggested that redox desequilibrium induced by GSH depleti on may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal de ath in Parkinson's disease. (C) 1998 Elsevier, Paris.