Astrocytic mitochondrial respiratory chain damage: effect on cellular ATP levels.

72 h The primary function of the mitochondrial respiratory chain is to generate energy in the form of ATP via the four complexes of the electron transport chain and the ATP synthetase. Damage to one or more of the respiratory chain complexes may therefore result in depletion of cellular energy reserves and cell death. There is increasing evidence that defects in mitochondrial energy metabolism may underlie the pathology of some neurodegenerative diseases [ I ] . Furthermore the mitochondrial respiratory chain is susceptible to oxidative damage, and nitric oxide (NO) has been implicated in the development of mitochondrial dysfunction [ I ] . Exposure of astrocytes, a major source of NO within the brain, to interferon (1FN)-y k lipopolysaccharide (LPS) results in induction of nitric oxide synthase (NOS) activity and a corresponding increase in NO release. We have previously shown that induction of NOS in cultured astrocytes by IFN-y k LPS is accompanied by damage to the mitochondrial respiratory chain [2, 31. However this damage occurs in the absence of cell death. We hypothesised that this was due to an up-regulation of glycolysis resulting in maintenance of astrocytic energy homeostasis. To test this hypothesis, astrocytes were exposed to IFN-y+ LPS for up to 72 h. The activities of the mitochondria1 complexes NADH-CoQ, (complex I), succinate-cytochrome c reductase (complex IVIII) and cytochrome c oxidase (complex IV) and citrate synthase (a mitochondrial marker) were determined in cell homogenates; cell death was assessed by lactate dehydrogenase (LDH) release; and ATP was extracted from astrocytes and measured using the luciferin-luciferase method. The exposure of astrocytes to IFN-y + LPS produced a significant time-dependent loss of both complex IMII and complex IV, which was prevented by the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) (Table 1). Both complex I and citrate synthase were unaffected. There was no significant increase in LDH release, indicating that IFN-y + LPS treatment did not cause cell death. In addition, astrocytic ATP levels were also unaffected by IFN-y + LPS treatment (Table 2). We have previously demonstrated that exposure to IFh-y + LPS causes a significant increase in both astrocytic lactate production and glucose consumption, which is prevented by a NOS inhibitor [2]. These findings indicate that although IFN-y + LPS-mediated NOS induction in astrocytes causes marked inhibition of mitochondrial respiratory chain complexes, these events do not affect astrocyte survival due to the ability of these cells to switch to glycolysis and consequently maintain their ATP levels and energy homeostasis. However it has previously been demonstrated that complex activity can be decreased by approximately 70% before major changes in mitochondrial respiration and ATP synthesis occurs [4]. Therefore it is possible that despite the mitochondrial respiratory chain damage observed here, the threshold of decreased complex activity was not reached. y+LPS 10.4+3.2* 0.43M. 19*