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 Parkinson's disease, Alzheimer's 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.     

Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture

In order to investigate the potential neuroprotective role played by glucose metabolism during brain oxygen deprivation, the susceptibility of cultured neurones and astrocytes to 1 h of oxygen deprivation (hypoxia) or oxygen and glucose deprivation (OGD) was examined. OGD, but not hypoxia, promotes dihydrorhodamine 123 and glutathione oxidation in neurones but not in astrocytes reflecting free radical generation in the former cells. A specific loss of mitochondrial complex-I activity, mitochondrial membrane potential collapse, ATP depletion and necrosis occurred in the OGD neurones, but not in the OGD astrocytes. Furthermore, superoxide anion but not nitric oxide formation was responsible for these effects. OGD decreased neuronal but not astrocytic NADPH concentrations; this was not observed in hypoxia and was independent of superoxide or nitric oxide formation. These results suggest that glucose metabolism would supply NADPH, through the pentose-phosphate pathway, aimed at preventing oxidative stress, mitochondrial damage and neurotoxicity during oxygen deprivation to neural cells.     

Oxidative stress and mitochondrial dysfunction in neurodegeneration; cardiolipin a critical target?

Oxidative stress and subsequent impairment of mitochondrial function is implicated in the neurodegenerative process and hence in diseases such as Parkinson's and Alzheimer's disease. Within the brain, neuronal and astroglial cells can display a differential susceptibility to oxidant exposure. Thus, astrocytes can up regulate glutathione availability and, in response to mitochondrial damage, glycolytic flux. Whilst neuronal cells do not appear to possess such mechanisms, neuronal glutathione status may be enhanced due to the trafficking of glutathione precursors from the astrocyte. However, when antioxidants reserves are not sufficient or the degree of oxidative stress is particularly great, mitochondrial damage occurs, particularly at the level of complex IV (cytochrome oxidase). Whilst the exact mechanism for the loss of activity of this enzyme complex is not know, it is possible that loss and/or oxidative modification of the phospholipid, cardiolipin is a critical factor. Consequently, in this short article, we also consider (a) cardiolipin metabolism and function, (b) the susceptibility of this molecule to undergo oxidative modification following exposure to oxidants such as peroxynitrite, (c) loss of mitochondrial cardiolipin in neurodegenerative disorders, (d) methods of detecting cardiolipin and (e) possible therapeutic strategies that may protect cardiolipin from oxidative degradation.     

Nitric oxide inhibits mitochondrial movement in forebrain neurons associated with disruption of mitochondrial membrane potential

Nitric oxide (NO) has a number of physiological and pathophysiological effects in the nervous system. One target of NO is the mitochondrion, where it inhibits respiration and ATP synthesis, which may contribute to NO-mediated neuronal injury. Our recent studies suggested that impaired mitochondrial function impairs mitochondrial trafficking, which could also contribute to neuronal injury. Here, we studied the effects of NO on mitochondrial movement and morphology in primary cultures of forebrain neurons using a mitochondrially targeted enhanced yellow fluorescent protein. NO produced by two NO donors, papa non-oate and diethylamine/NO complex, caused a rapid cessation of mitochondrial movement but did not alter morphology. Movement recovered after removal of NO. The effects of NO on movement were associated with dissipation of the mitochondrial membrane potential. Increasing cGMP levels using 8-bromoguanosine 3',5'-cyclic monophosphate, did not mimic the effects on mitochondrial movement. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-induced activation of soluble guanylate cyclase, did not block the effects of NO. Thus, neither increasing nor decreasing cGMP levels had an effect on mitochondrial movement. Based on these data, we conclude that NO is a novel modulator of mitochondrial trafficking in neurons, which may act through the inhibition of mitochondrial function.     

Nitric oxide-mediated mitochondrial damage: A potential neuroprotective role for glutathione

In this study we have investigated the mechanisms leading to mitochondrial damage in cultured neurons following sustained exposure to nitric oxide. Thus, the effects upon neuronal mitochondrial respiratory chain complex activity and reduced glutathione concentration following exposure to either the nitric oxide donor, S-nitroso-N-acetylpenicillamine, or to nitric oxide releasing astrocytes were assessed. Incubation with S-nitroso-N-acetylpenicillamine (1 mM) for 24 h decreased neuronal glutathione concentration by 57%, and this effect was accompanied by a marked decrease of complex I (43%), complex II–III (63%), and complex IV (41%) activities. Incubation of neurons with the glutathione synthesis inhibitor, l-buthionine-[S,r]-sulfoximine caused a major depletion of neuronal glutathione (93%), an effect that was accompanied by a marked loss of complex II–III (60%) and complex IV (41%) activities, although complex I activity was only mildly decreased (34%). In an attempt to approach a more physiological situation, we studied the effects upon glutathione status and mitochondrial respiratory chain activity of neurons incubated in coculture with nitric oxide releasing astrocytes. Astrocytes were activated by incubation with lipopolysaccharide/interferon-γ for 18 h, thereby inducing nitric oxide synthase and, hence, a continuous release of nitric oxide. Coincubation for 24 h of activated astrocytes with neurons caused a limited loss of complex IV activity and had no effect on the activities of complexes I or II–III. However, neurons exposed to astrocytes had a 1.7-fold fold increase in glutathione concentration compared to neurons cultured alone. Under these coculture conditions, the neuronal ATP concentration was modestly reduced (14%). This loss of ATP was prevented by the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine. These results suggest that the neuronal mitochondrial respiratory chain is damaged by sustained exposure to nitric oxide and that reduced glutathione may be an important defence against such damage.     

Astrocyte influences on ischemic neuronal death

Glutamate excitotoxicity, oxidative stress, and acidosis are primary mediators of neuronal death during ischemia and reperfusion. Astrocytes influence these processes in several ways. Glutamate uptake by astrocytes normally prevents excitotoxic glutamate elevations in brain extracellular space, and this process appears to be a critical determinant of neuronal survival in the ischemic penumbra. Conversely, glutamate efflux from astrocytes by reversal of glutamate uptake, volume sensitive organic ion channels, and other routes may contribute to extracellular glutamate elevations. Glutamate activation of neuronal N-methyl-D-aspartate (NMDA) receptors is modulated by glycine and D-serine: both of these neuromodulators are transported by astrocytes, and D-serine production is localized exclusively to astrocytes. Astrocytes influence neuronal antioxidant status through release of ascorbate and uptake of its oxidized form, dehydroascorbate, and by indirectly supporting neuronal glutathione metabolism. In addition, glutathione in astrocytes can serve as a sink for nitric oxide and thereby reduce neuronal oxidant stress during ischemia. Astrocytes probably also influence neuronal survival in the post-ischemic period. Reactive astrocytes secrete nitric oxide, TNFalpha, matrix metalloproteinases, and other factors that can contribute to delayed neuronal death, and facilitate brain edema via aquaporin-4 channels localized to the astrocyte endfoot-endothelial interface. On the other hand erythropoietin, a paracrine messenger in brain, is produced by astrocytes and upregulated after ischemia. Erythropoietin stimulates the Janus kinase-2 (JAK-2) and nuclear factor-kappaB (NF-kB) signaling pathways in neurons to prevent programmed cell death after ischemic or excitotoxic stress. Astrocytes also secrete several angiogenic and neurotrophic factors that are important for vascular and neuronal regeneration after stroke.     

Astrocytes and manganese neurotoxicity

Increasing evidence suggests that astrocytes are the site of early dysfunction and damage in manganese neurotoxicity. Astrocytes accumulate manganese by a high affinity, high capacity, specific transport system. Chronic exposure to manganese leads to increased pallidal signal hyperintensities on T1-weighted magnetic resonance images and selective neuronal loss in basal ganglia structures together with characteristic astrocytic changes known as Alzheimer type II astrocytosis. Manganese is sequestered in mitochondria where it inhibits oxidative phosphorylation. Exposure of astrocytes to manganese results in important changes including (i) decreased uptake of glutamate; (ii) increased densities of binding sites for the "peripheral-type" benzodiazepine receptor (PTBR), a class of receptor localized to mitochondria of astrocytes and involved in oxidative metabolism, mitochondrial proliferation, and neurosteroid synthesis; (iii) increased gene expression and activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), known to be associated with apoptosis; (iv) increased uptake of L-arginine, a precursor of nitric oxide, together with increased expression of the inducible form of nitric oxide synthase (iNOS). Potential consequences of these alterations in astrocytic gene expression include failure of energy metabolism, production of reactive oxygen species (ROS), increased extracellular glutamate concentration and excitotoxicity which could play a key role in manganese-induced neuronal cell death as a direct result of impaired astrocytic-neuronal interactions.     

Inflammatory neurodegeneration mediated by nitric oxide,glutamate, and mitochondria

In inflammatory, infectious, ischemic, and neurodegenerative pathologies of th central nervous system (CNS) glia become “activated” by inflammatory mediators, and express new proteins such as the inducible isoform of nitric oxide synthase (iNOS). Although these activated glia have beneficial roles, in vitro they potently kill cocultured neurons, and there is increasing evidence that they contribute to pathology in vivo. Nitric oxide (NO) from iNOS appears to be a key mediator of such glial-induced neuronal death. The high sensitivity of neurons to NO is partly due to NO causing inhibition of respiration, rapid glutamate release from both astrocytes and neurons, and subsequent excitotoxic death of the neurons. NO is a potent inhibitor of mitochondrial respiration, due to reversible binding of NO to cytochrome oxidase in competition with oxygen, resulting in inhibition of energy production and sensitization to hypoxia. Activated astrocytes or microglia cause a potent inhibition of respiration in cocultured neurons due to glial NO inhibiting cytochrome oxidase within the neurons, resulting in ATP depletion and glutamate release. In some conditions, glutamate-induced neuronal death can itself be mediated by N-methyl-d-aspartate (NMDA)-receptor activation of the neuronal isoform of NO synthase (nNOS) causing mitochondrial damage. In addition NO can be converted to a number of reactive derivatives such as peroxynitrite, NO₂, N₂O₃, and S-nitrosothiols that can kill cells in part by inhibiting mitochondrial respiration or activation of mitochondrial permeability transition, triggering neuronal apoptosis or necrosis.     

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons

The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.     

Metabotropic glutamate receptor 3 activation prevents nitric oxide‐induced death in cultured rat astrocytes

Altered glial function may contribute to the initiation or progression of neuronal death in neurodegenerative diseases. Thus, modulation of astrocyte death may be essential for preventing pathological processes in the CNS. In recent years, metabotropic glutamate receptor (mGluR) activation has emerged as a key target for neuroprotection. We investigated the effect of subtype 3 mGluR (mGluR3) activation on nitric oxide (NO)‐induced astroglial death. A mGluR3 selective agonist, LY379268, reduced inducible NO synthase expression and NO release induced by bacterial lipopolysaccharide and interferon‐γ in cultured rat astrocytes. In turn, a NO donor (diethylenetriamine/NO) induced apoptotic‐like death in cultured astrocytes, which showed apoptotic morphology and DNA fragmentation, but no caspase 3 activation. LY379268 prevented astrocyte death induced by NO exposure, which correlates with a reduction in: phosphatidylserine externalization, p53 and Bax activation and mitochondrial permeability. The reported effects of LY379268 were prevented by the mGluR3 antagonist (s)‐α‐ethylglutamic acid. All together, these findings show the protective effect of mGluR3 activation on astroglial death and provide further evidence of a role of these receptors in preventing CNS injury triggered by several inflammatory processes associated with dysregulated NO production.     

Protective Effect of Carnosine During Nitrosative Stress in Astroglial Cell Cultures

Formation of nitric oxide by astrocytes has been suggested to contribute, via impairment of mitochondrial function, to the neurodegenerative process. Mitochondria under oxidative stress are thought to play a key role in various neurodegenerative disorders; therefore protection by antioxidants against oxidative stress to mitochondria may prove to be beneficial in delaying the onset or progression of these diseases. Carnosine has been recently proposed to act as antioxidant in vivo. In the present study, we demonstrate its neuroprotective effect in astrocytes exposed to LPS- and INFγ-induced nitrosative stress. Carnosine protected against nitric oxide-induced impairment of mitochondrial function. This effect was associated with decreased formation of oxidatively modified proteins and with decreased up-regulation oxidative stress-responsive genes, such as Hsp32, Hsp70 and mt-SOD. Our results sustain the possibility that carnosine might have anti-ageing effects to brain cells under pathophysiological conditions leading to degenerative damage, such as aging and neurodegenerative disorders.     

Astrocytic metabolic and inflammatory changes as a function of age

This study examines age‐dependent metabolic‐inflammatory axis in primary astrocytes isolated from brain cortices of 7‐, 13‐, and 18‐month‐old Sprague–Dawley male rats. Astrocytes showed an age‐dependent increase in mitochondrial oxidative metabolism respiring on glucose and/or pyruvate substrates; this increase in mitochondrial oxidative metabolism was accompanied by increases in COX3/18SrDNA values, thus suggesting an enhanced mitochondrial biogenesis. Enhanced mitochondrial respiration in astrocytes limits the substrate supply from astrocytes to neurons; this may be viewed as an adaptive mechanism to altered cellular inflammatory–redox environment with age. These metabolic changes were associated with an age‐dependent increase in hydrogen peroxide generation (largely ascribed to an enhanced expression of NOX2) and NFκB signaling in the cytosol as well as its translocation to the nucleus. Astrocytes also displayed augmented responses with age to inflammatory cytokines, IL‐1β, and TNFα. Activation of NFκB signaling resulted in increased expression of nitric oxide synthase 2 (inducible nitric oxide synthase), leading to elevated nitric oxide production. IL‐1β and TNFα treatment stimulated mitochondrial oxidative metabolism and mitochondrial biogenesis in astrocytes. It may be surmised that increased mitochondrial aerobic metabolism and inflammatory responses are interconnected and support the functionality switch of astrocytes, from neurotrophic to neurotoxic with age.     

Differential effect of nitric oxide on glutathione metabolism and mitochondrial function in astrocytes and neurones: implications for neuroprotection/neurodegeneration?

Primary culture rat astrocytes exposed to the long acting nitric oxide donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) for 24 h approximately double their concentration of glutathione (GSH) and show no sign of cell death. In contrast, GSH was depleted by 48%, and significant loss of mitochondrial respiratory chain complex activity and cell death were observed in primary culture rat neurones subjected to DETA-NO for 18 h. Northern blot analysis suggested that mRNA amounts of both subunits of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, were elevated in astrocytes following nitric oxide (NO) exposure. This correlated with an increase in astrocytic GCL activity. Neurones on the other hand did not exhibit increased GCL activity when exposed to NO. In addition, the rate of GSH efflux was doubled and gamma-glutamyltranspeptidase (gamma-GT) activity was increased by 42% in astrocytes treated with NO for 24 h. These results suggest that astrocytes, but not neurones, up-regulate GSH synthesis as a defence mechanism against excess NO. It is possible that the increased rate of GSH release and activity of gamma-GT in astrocytes may have important implications for neuroprotection in vivo by optimizing the supply of GSH precursors to neurones in close proximity.     

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.     

Astrocytes aged in vitro show a decreased neuroprotective capacity

Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100beta increased at 90 days when cells were positive for the senescence beta-galactosidase marker. In long-term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn-superoxide dismutase (SOD-1) and haeme oxygenase-1 (HO-1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90-DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H2O2 exposure. Enhanced glutamate uptake was probably because of up-regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age-related neurodegenerative processes.     

4-O-Methylhonokiol attenuates memory impairment in presenilin 2 mutant mice through reduction of oxidative damage and inactivation of astrocytes and the ERK pathway

Presenilin 2 (PS2) mutation increases Aβ generation and neuronal cell death in the brains of Alzheimer disease (AD) patients. In a previous study, we showed that increased oxidative damage and activation of extracellular signal-regulated kinase (ERK) were associated with Aβ generation and neuronal cell death in neuronal cells expressing mutant PS2. In this study, we show that oral treatment with 4-O-methylhonokiol, a novel compound isolated from Magnolia officinalis, for 3 months (1.0mg/kg) prevented PS2 mutation-induced memory impairment and neuronal cell death accompanied by a reduction in Aβ(1-42) accumulation. We also found that 4-O-methylhonokiol inhibited PS2 mutation-induced activation of ERK and β-secretase, and oxidative protein and lipid damage, but recovered glutathione levels in the cortex and hippocampus of PS2 mutant mice. Additionally, 4-O-methylhonokiol prevented PS2 mutation-induced activation of astrocytes as well as production of TNF-α, IL-1β, reactive oxygen species (ROS), and nitric oxide (NO) in neurons. Generation of TNF-α, IL-1β, ROS, and NO and ERK activation in cultured astrocytes treated with lipopolysaccharide (1μg/ml) were also prevented by 4-O-methylhonokiol in a dose-dependent manner. These results suggest that the improving effects of 4-O-methylhonokiol on memory function may be associated with a suppression of the activation of ERK and astrocytes as well as a reduction in oxidative damage. Thus, 4-O-methylhonokiol may be useful in the prevention and treatment of AD.     

Mitochondrial Glutathione: A Modulator of Brain Cell Death

The small fraction of glutathione in mitochondria in nonneural tissues is an important contributor to cell survival under some conditions. However, there has been only limited characterization of the properties and function of mitochondrial glutathione in cells from the brain. In astrocytes in culture, highly selective depletion of this glutathione pool does not affect cell viability, at least in the first 24 h, but does greatly increase susceptibility to exposure to nitric oxide or peroxynitrite. In vivo, a selective partial loss of glutathione develops during focal cerebral ischemia and persists during reperfusion. The timing and distribution of glutathione loss shows an apparent association with the likelihood that tissue infarction will subsequently develop. Furthermore, infarct volume is greatly decreased by intracerebroventricular infusion of glutathione monoethylester, a compound that can increase mitochondrial glutathione. Together these recent findings indicate that alterations in mitochondrial glutathione are likely to contribute to the severity of tissue damage in stroke and possibly other neurological disorders. Thus, this antioxidant pool provides a potentially useful target for therapeutic intervention.     

Nitric oxide modulation in neuroinflammation and the role of mesenchymal stem cells

Nitric oxide is a versatile mediator formed by enzymes called nitric oxide synthases. It has numerous homeostatic functions and important roles in inflammation. Within the inflamed brain, microglia and astrocytes produce large amounts of nitric oxide during inflammation. Excessive nitric oxide causes neuronal toxicity and death and mesenchymal stem cells can be used as an approach to limit the neuronal damage caused by neuroinflammation. Mesenchymal stem cell therapy ameliorates inflammation and neuronal damage in disease models of Alzheimer’s disease, Parkinson’s disease, and other neuroinflammatory disorders. Interestingly, we have reported that in vitro, mesenchymal stem cells themselves contribute to a rise in nitric oxide levels through microglial cues. This may be an undesirable effect and highlights a possible need to explore acellular approaches for mesenchymal stem cell therapy in the central nervous system.     

Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and sysemic lupus erythematosus

Nitric oxide (NO) has been shown to regulate T cell functions under physiological conditions, but overproduction of NO may contribute to T lymphocyte dysfunction. NO-dependent tissue injury has been implicated in a variety of rheumatic diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Several studies reported increased endogenous NO synthesis in both SLE and RA, and recent evidence suggests that NO contributes to T cell dysfunction in both autoimmune diseases. The depletion of intracellular glutathione may be a key factor predisposing patients with SLE to mitochondrial dysfunction, characterized by mitochondrial hyperpolarization, ATP depletion and predisposition to death by necrosis. Thus, changes in glutathione metabolism may influence the effect of increased NO production in the pathogenesis of autoimmunity.