Overexpression of bcl-x(L) protects astrocyte from glucose deprivation andis associated with higher glutathione, ferritin, and iron levels

Background: The possibility of altering outcome from ischemia-like injury b y overexpressing the anti-cell death gene bcl-x(L) was studied. Cells are k nown to die by different pathways including apoptosis, or programmed cell d eath, and necrosis, The bcl-x(L) gene Is a member of a family of apoptosis regulating genes and often displays the death-inhibiting properties of the prototype of this family, bcl-2. It is of special interest to study bcl-x(L ) for possible brain protection, because, unlike bcl-2, it is important for normal brain development, Methods: Overexpression of bcl-x(L), was achieved in primary astrocyte cult ures using a retroviral vector. Cultures of astrocytes overexpressing bcl-x (L), or a control gene were injured by hydrogen peroxide, glucose deprivati on, or combined oxygen and glucose deprivation. Outcome was assessed morpho logically and by release of lactate dehydrogenase. We assessed antioxidant effects by measuring glutathione using monochlorobimane, ferritin by immuno blotting, the level of iron spectrophotometrically, and superoxide using io donitrotetra-zolium violet and dihydroethidium. Results: Protection by bcl-x(L), was found against glucose deprivation and hydrogen peroxide exposure but not combined oxygen and glucose deprivation. Higher levels of superoxide were found, without increased levels of lipid peroxidation. Overexpression of bcl-x(L), was associated with elevated glut athione levels, elevated ferritin levels, and increased amounts of Icon. Th e increased glutathione contributed to the protection from glucose deprivat ion. Conclusions: Overexpression of bcl-x(L), protects astrocytes from oxidative injury with the same spectrum of protection seen previously for bcl-2. The Increased antioxidant defense observed should be beneficial against both a poptotic and necrotic cell death. The effects on levels of ferritin and iro n are novel and identify a new area of interest for this gene family. Wheth er this relates to the effects of these genes on mitochondrial function rem ains to be elucidated.