Victoria C. Stewart

Nitric oxide-mediated mitochondrial damage in the brain: Mechanisms and implications for neurodegenerative diseases

Abstract: Within the CNS and under normal conditions, nitric oxide (•NO) appears to be an important physiological signalling molecule. Its ability to increase cyclic GMP concentration suggests that •NO is implicated in the regulation of important metabolic pathways in the brain. Under certain circumstances •NO synthesis may be excessive and •NO may become neurotoxic. Excessive glutamate‐receptor stimulation may lead to neuronal death through a mechanism implicating synthesis of both •NO and superoxide (O2•−) and hence peroxynitrite (ONOO−) formation. In response to lipopolysaccharide and cytokines, glial cells may also be induced to synthesize large amounts of •NO, which may be deleterious to the neighbouring neurones and oligodendrocytes. The precise mechanism of •NO neurotoxicity is not fully understood. One possibility is that it may involve neuronal energy deficiency. This may occur by ONOO− interfering with key enzymes of the tricarboxylic acid cycle, the mitochondrial respiratory chain, mitochondrial calcium metabolism, or DNA damage with subsequent activation of the energy‐consuming pathway involving poly(ADP‐ribose) synthetase. Possible mechanisms whereby ONOO− impairs the mitochondrial respiratory chain and the relevance for neurotoxicity are discussed. The intracellular content of reduced glutathione also appears important in determining the sensitivity of cells to ONOO− production. It is concluded that neurotoxicity elicited by excessive •NO production may be mediated by mitochondrial dysfunction leading to an energy deficiency state.

Nitric oxide, mitochondria and neurological disease.

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurological disorders, such as Parkinsons disease, Alzheimers disease, multiple sclerosis, stroke and amyotrophic lateral sclerosis. There is also a growing body of evidence to implicate excessive or inappropriate generation of nitric oxide (NO) in these disorders. It is now well documented that NO and its toxic metabolite, peroxynitrite (ONOO-), can inhibit components of the mitochondrial respiratory chain leading, if damage is severe enough, to a cellular energy deficiency state. Within the brain, the susceptibility of different brain cell types to NO and ONOO- exposure may be dependent on factors such as the intracellular reduced glutathione (GSH) concentration and an ability to increase glycolytic flux in the face of mitochondrial damage. Thus neurones, in contrast to astrocytes, appear particularly vulnerable to the action of these molecules. Following cytokine exposure, astrocytes can increase NO generation, due to de novo synthesis of the inducible form of nitric oxide synthase (NOS). Whilst the NO/ONOO- so formed may not affect astrocyte survival, these molecules may diffuse out to cause mitochondrial damage, and possibly cell death, to other cells, such as neurones, in close proximity. Evidence is now available to support this scenario for neurological disorders, such as multiple sclerosis. In other conditions, such as ischaemia, increased availability of glutamate may lead to an activation of a calcium-dependent nitric oxide synthase associated with neurones. Such increased/inappropriate NO formation may contribute to energy depletion and neuronal cell death. The evidence available for NO/ONOO--mediated mitochondrial damage in various neurological disorders is considered and potential therapeutic strategies are proposed.

Astrocyte-Derived Nitric Oxide Causes Both Reversible and Irreversible Damage to the Neuronal Mitochondrial Respiratory Chain

Cytokine‐stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO‐), can inhibit components of the mitochondrial respiratory chain. We used astrocytes as a source of NO/ONOO‐ and monitored the effects on neurons in coculture. We previously demonstrated that astrocytic NO/ONOO‐ causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24‐h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte‐derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO‐producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO‐producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48‐h coculture with NO‐producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short‐term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic‐derived NO for longer periods causes permanent damage to neuronal complex IV.

Pretreatment of Astrocytes with Interferon‐α/β Prevents Neuronal Mitochondrial Respiratory Chain Damage

Abstract: Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Interferon‐β is used for the treatment of multiple sclerosis, but currently little is known regarding its mode of action. Exposure of astrocytes in culture to interferon‐γ plus lipopolysaccharide results in stimulation of nitric oxide release. Using a coculture system, we have been able to use astrocytes as a source of nitric oxide/peroxynitrite in an attempt to “model” the effects of raised cytokine levels observed in multiple sclerosis and to monitor the effect on neurones. Our results indicate that stimulation of astrocytic nitric oxide synthase activity causes significant damage to the mitochondrial activities of complexes II/III and IV of neighbouring neurones. This damage was prevented by a nitric oxide synthase inhibitor, suggesting that the damage was nitric oxide‐mediated. Furthermore, interferon‐α/β also prevented this damage. In view of these results, we suggest that a possible mechanism of action of interferon‐β in the treatment of multiple sclerosis is that it prevents astrocytic nitric oxide production, thereby limiting damage to neighbouring cells, such as neurones.

Pretreatment of Astrocytes with Interferon‐α/β Impairs Interferon‐γ Induction of Nitric Oxide Synthase

Abstract: Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Exposure of astrocytes, in primary culture, to interferon‐γ results in stimulation of nitric oxide synthase activity and increased nitric oxide release. In contrast to interferon‐γ, interferon‐α/β had a minimal effect on astrocytic nitric oxide formation. Furthermore, pretreatment of astrocytes with interferon‐α/β inhibited (∼65%) stimulation by interferon‐γ of nitric oxide synthase activity and nitric oxide release. Treatment with interferon‐α/β at a concentration as low as 10 U/ml caused inhibition of mitochondrial cytochrome c oxidase. Furthermore, the damage to cytochrome c oxidase was prevented by the putative interferon‐α/β receptor antagonist oxyphenylbutazone. In view of these observations, our current hypothesis is that the mitochondrial damage caused by exposure to interferon‐α/β may impair the ability of astrocytes to induce nitric oxide synthase activity on subsequent interferon‐γ exposure. These results may have implications for our understanding of the mechanisms responsible for the therapeutic effects of interferon‐α/β preparations in multiple sclerosis.

Preservation of extracellular glutathione by an astrocyte derived factor with properties comparable to extracellular superoxide dismutase

Cultured rat and human astrocytes and rat neurones were shown to release reduced glutathione (GSH). In addition, GSH oxidation was retarded by the concomitant release of a factor from the cells. One possibility is that this factor is extracellular superoxide dismutase (SOD). In support of this, the factor was found to bind heparin, have a molecular mass estimated to be between 50 and 100 kDa, and CuZn‐type SOD protein and cyanide sensitive enzyme activity were demonstrated in the cell‐conditioned medium. In addition, supplementation of native medium with exogenous CuZn‐type SOD suppressed GSH oxidation. We propose that preservation of released GSH is essential to allow for maximal up‐regulation of GSH metabolism in neurones. Furthermore, cytokine stimulation of astrocytes increased release of the extracellular SOD, and enhanced stability of GSH. This may be a protective strategy occurring in vivo under conditions of oxidative stress, and suggests that SOD mimetics may be of therapeutic use.