ELEVATED GLUTATHIONE LEVELS ACCOUNT FOR HALF THE PROTECTION SEEN AGAINST GLUCOSE DEPRIVATION WITH OVEREXPRESSION OF BCL-XL IN MOUSE ASTROCYTES

Abstract

S237 INTRODUCTION: Bcl-2 and bcl-xL are anti-apoptotic genes which are promising candidate genes for gene therapy to ameliorate brain injury associated with stroke. They are thought to have effects on mitochondrial function. Our previous studies showed that overexpression of bcl-2 protects primary mouse astrocytes from glucose deprivation (GD) and hydrogen peroxide (H2 O2) injury [1]. We tested the ability of bcl-xL to protect astrocytes from injury and determined the effect of overexpressing this gene on cellular glutathione, iron and ferritin levels. METHODS: Primary astrocyte cultures were prepared as previously described [2] according to a protocol approved by the Stanford animal care and use committee. The cultures were infected with retroviral vectors to express either bcl-2, bcl-x (L) or the control gene beta-galactosidase. Mock infected controls were also studied. Experiments were performed at least three times on different cultures. Glucose deprivation was performed by washing into a balanced salt solution, combined oxygen-glucose deprivation by washing into anoxic balanced salt solution, or cells were injured by exposure to 400 [micro sign]M H2 O2. Cell injury was quantitated by lactate dehydrogenase efflux. Glutathione was measured using monochlorobimane [1]. Ferritin protein levels were estimated using Western analysis; bands were quantitated by scanning densitometry. Iron levels were quantitated using the colorimetric procedure of Fish [4]. Statistical significance was determined using ANOVA followed by Student Neuman Keuls for multiple comparisons. RESULTS: Astrocytes overexpressing bcl-xL were significantly protected against H2 O2 exposure and glucose deprivation, but not combined oxygen-glucose deprivation. Bcl-xL and bcl-2 overexpressing astrocytes showed higher glutathione levels, and greater iron and ferritin staining than beta-galactosidase infected and mock infected cells. Glutathione levels in bcl-xL overexpressing cells were 20.1 +/- 1 nmol/mg protein, while in beta-galactosidase expressing controls 17.2 +/- 0.8. Higher levels were maintained in bcl-xL overexpressing cells after 7 hr of glucose deprivation. When glutathione levels were reduced in both astrocyte cultures by preincubation with 0.1 mM buthionine sulfoximine, levels were 4.3 +/- 0.8 and 3.9 +/- 0.5 respectively. When these cultures with equivalent glutathione levels were subjected to glucose deprivation the extent of protection by bcl-xL overexpression was reduced to approximately half. DISCUSSION: These results demonstrate that bcl-xL overexpression in astrocytes provides protection against GD and H2 O2 injury, the same spectrum of injuries previously reported to be decreased by bcl-2. These injuries are also the ones in which the greatest loss of glutathione is seen. When glutathione levels were reduced equally in bcl-xL overexpressing and control astrocytes, protection against GD was reduced but not eliminated. This suggests that the protection afforded by this gene is partially due to the elevation of glutathione levels, but not exclusively to this. The elevated ferritin levels observed may be a second component of the protection seen against injury due to overexpression of bcl-xL. Supported in part by a grant from IARS.