Poly(ADP-ribose) polymerase-1 protects neurons against apoptosis induced by oxidative stress.

In neurons, DNA is prone to free radical damage, although repair mechanisms preserve the genomic integrity. However, activation of the DNA repair system, poly(ADP-ribose) polymerase (PARP-1), is thought to cause neuronal death through NAD+ depletion and mitochondrial membrane potential (delta psi(m)) depolarization. Here, we show that abolishing PARP-1 activity in primary cortical neurons can either enhance or prevent apoptotic death, depending on the intensity of an oxidative stress. Only in severe oxidative stress does PARP-1 activation result in NAD+ and ATP depletion and neuronal death. To investigate the role of PARP-1 in an endogenous model of oxidative stress, we used an RNA interference (RNAi) strategy to specifically knock down glutamate-cysteine ligase (GCL), the rate-limiting enzyme of glutathione biosynthesis. GCL RNAi spontaneously elicited a mild type of oxidative stress that was enough to stimulate PARP-1 in a Ca2+-calmodulin kinase II-dependent manner. GCL RNAi-mediated PARP-1 activation facilitated DNA repair, although neurons underwent delta psi(m) loss followed by some apoptotic death. PARP-1 inhibition did not prevent delta psi(m) loss, but enhanced the vulnerability of neurons to apoptosis upon GCL silencing. Conversely, mild expression of PARP-1 partially prevented to GCL RNAi-dependent apoptosis. Thus, in the mild progressive damage likely occur in neurodegenerative diseases, PARP-1 activation plays a neuroprotective role that should be taken into account when considering therapeutic strategies.

Interrelationships between astrocyte function, oxidative stress and antioxidant status within the central nervous system.

Astrocytes have, until recently, been thought of as the passive supporting elements of the central nervous system. However, recent developments suggest that these cells actually play a crucial and vital role in the overall physiology of the brain. Astrocytes selectively express a host of cell membrane and nuclear receptors that are responsive to various neuroactive compounds. In addition, the cell membrane has a number of important transporters for these compounds. Direct evidence for the selective co-expression of neurotransmitters, transporters on both neurons and astrocytes, provides additional evidence for metabolic compartmentation within the central nervous system. Oxidative stress as defined by the excessive production of free radicals can alter dramatically the function of the cell. The free radical nitric oxide has attracted a considerable amount of attention recently, due to its role as a physiological second messenger but also because of its neurotoxic potential when produced in excess. We provide, therefore, an in-depth discussion on how this free radical and its metabolites affect the intra and intercellular physiology of the astrocyte(s) and surrounding neurons. Finally, we look at the ways in which astrocytes can counteract the production of free radicals in general by using their antioxidant pathways. The glutathione antioxidant system will be the focus of attention, since astrocytes have an enormous capacity for, and efficiency built into this particular system.

Roles of nitric oxide in brain hypoxia-ischemia.

A large body of evidence has appeared over the last 6 years suggesting that nitric oxide biosynthesis is a key factor in the pathophysiological response of the brain to hypoxia-ischemia. Whilst studies on the influence of nitric oxide in this phenomenon initially offered conflicting conclusions, the use of better biochemical tools, such as selective inhibition of nitric oxide synthase (NOS) isoforms or transgenic animals, is progressively clarifying the precise role of nitric oxide in brain ischemia. Brain ischemia triggers a cascade of events, possibly mediated by excitatory amino acids, yielding the activation of the Ca2+-dependent NOS isoforms, i.e. neuronal NOS (nNOS) and endothelial NOS (eNOS). However, whereas the selective inhibition of nNOS is neuroprotective, selective inhibition of eNOS is neurotoxic. Furthermore, mainly in glial cells, delayed ischemia or reperfusion after an ischemic episode induces the expression of Ca2+-independent inducible NOS (iNOS), and its selective inhibition is neuroprotective. In conclusion, it appears that activation of nNOS or induction of iNOS mediates ischemic brain damage, possibly by mitochondrial dysfunction and energy depletion. However, there is a simultaneous compensatory response through eNOS activation within the endothelium of blood vessels, which mediates vasodilation and hence increases blood flow to the damaged brain area.

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.

Astrocyte NMDA receptors' activity sustains neuronal survival through a Cdk5-Nrf2 pathway.

Neurotransmission unavoidably increases mitochondrial reactive oxygen species. However, the intrinsic antioxidant defense of neurons is weak and hence the mechanism whereby these cells are physiologically protected against oxidative damage is unknown. Here we found that the antioxidant defense of neurons is repressed owing to the continuous protein destabilization of the master antioxidant transcriptional activator, nuclear factor-erythroid 2-related factor-2 (Nrf2). By contrast, Nrf2 is highly stable in neighbor astrocytes explaining their robust antioxidant defense and resistance against oxidative stress. We also show that subtle and persistent stimulation of N-methyl-d-aspartate receptors (NMDAR) in astrocytes, through a mechanism not requiring extracellular Ca²âº influx, upregulates a signal transduction pathway involving phospholipase C-mediated endoplasmic reticulum release of Ca²âº and protein kinase Cδ activation. Active protein kinase Cδ promotes, by phosphorylation, the stabilization of p35, a cyclin-dependent kinase-5 (Cdk5) cofactor. Active p35/Cdk5 complex in the cytosol phosphorylates Nrf2 at Thr(395), Ser(433) and Thr(439) that is sufficient to promote Nrf2 translocation to the nucleus and induce the expression of antioxidant genes. Furthermore, this Cdk5-Nrf2 transduction pathway boosts glutathione metabolism in astrocytes efficiently protecting closely spaced neurons against oxidative damage. Thus, intercellular communication through NMDAR couples neurotransmission with neuronal survival.

Nitric oxide mediates brain mitochondrial maturation immediately after birth.

The possible role of nitric oxide (*NO) in brain mitochondrial maturation was studied. Within the first 5 min after birth, a sharp increase in ATP concentrations was observed, coinciding with an increase in mitochondrial complex II-III (succinate-cytochrome c reductase) activity, while complex I (NADH-CoQ1 reductase) and complex IV (cytochrome c oxidase) activities remained unchanged. Under the same circumstances, cGMP concentrations were increased by 5 min after birth, correlating significantly with ATP concentrations. Since ATP concentrations also correlated significantly with mitochondrial complex II-III activity, these three parameters may be associated. Inhibition of *NO synthase activity brought about by the administration of N(omega)-nitro-L-arginine monomethyl ester to mothers prevented the postnatal increase in cGMP and ATP levels and complex II-III activity. These results suggest that early postnatal mitochondrial maturation in the brain is a *NO-mediated process.

The assumption that nitric oxide inhibits mitochondrial ATP synthesis is correct.

The assumption that reversible inhibition of mitochondrial respiration by nitric oxide (NO.) represents inhibition of ATP synthesis is unproven. NO. could theoretically inhibit the oxygen consumption with continued ATP synthesis, by acting as an electron acceptor from cytochrome c or as a terminal electron acceptor in stead of oxygen. We report here that NO. does reversibly inhibit brain mitochondrial ATP synthesis with a time course similar to its inhibition of respiration. Whilst such inhibition was largely reversible, there appeared to be a small irreversible component which may theoretically be due to peroxynitrite formation, i.e. as a result of the reaction between NO. and superoxide, generated by the mitochondrial respiratory chain.

Inhibition of astrocyte gap junctional communication by ATP depletion is reversed by calcium sequestration.

We have studied the possible role of cellular energy status in the regulation of gap junction permeability in rat astrocytes in primary culture. Incubation with the mitochondrial respiratory chain inhibitor antimycin (5 ng/ml) for 16 h caused a significant decrease in ATP concentrations. This effect was accompanied by a dose-dependent inhibition of gap junction permeability as assessed by the scrape-loading/Lucifer yellow transfer technique. No cell death was observed following this treatment. Restoration of cellular ATP levels by a further 24 h incubation in antimycin-free medium reversed the inhibition of Lucifer yellow transfer caused by antimycin. The inhibition of Lucifer yellow transfer brought about by antimycin treatment was also reversed by a short incubation of the cells with the calcium chelator EGTA plus the calcium ionophore A23187. These results suggest that ATP depiction causes a reversible inhibition of gap junction permeability through a calcium-mediated mechanism.

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-gamma 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, NG-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.

Effect of valproate on lipogenesis in neonatal rat brain.

The effect of valproate on lipogenesis in brain slices from early neonatal rats was studied. The rate of lipid synthesis from lactate and 3-hydroxybutyrate, but not from glucose, was decreased significantly by 1 mM valproate. Separation by high performance liquid chromatography of brain lipids showed that valproate inhibited the synthesis of major phospholipids (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine) from lactate and major sterols (desmosterol, cholesterol and lanosterol) from lactate and 3-hydroxybutyrate. Valproate did not affect sterol synthesis but slightly enhanced phospholipid synthesis from glucose. However, the ratio of phosphatidylserine/phosphatidylethanolamine synthesis was decreased from lactate, glucose and 3-hydroxybutyrate, suggesting that valproate changes phospholipid composition of brain structures. These changes may contribute to the pharmacological action of the drug.

Nitric oxide mediates glutamate-induced mitochondrial depolarization in rat cortical neurons.

Mitochondria have been considered to be a target for glutamate neurotoxicity. The aim of the present work was to investigate the mechanisms leading to glutamate-mediated mitochondrial deenergization, as measured by mitochondrial membrane potential and cell respiration in cultured neurons. Glutamate exposure to cells induced pronounced mitochondrial depolarization associated with an impairment in neuronal respiration, leading to neuronal ATP depletion. These effects were prevented by both the nitric oxide (. NO) synthase inhibitor Nomega-nitro-l-arginine methyl ester and by the N-methyl-d-aspartate glutamate-subtype receptor inhibitor d-(-)-2-amino-5-phosphopentanoate. Our results suggest that glutamate causes ATP depletion by collapsing mitochondrial membrane potential through a.NO-mediated mechanism.

Nitric oxide mediates brain mitochondrial damage during perinatal anoxia.

The possible role of nitric oxide (.NO) in brain energy metabolism during perinatal asphyxia in the rat was studied. Exposure of early neonates to 5 min of anoxia significantly inhibited brain mitochondrial complex II-III activity by 25%, without affecting complex I, complex IV or citrate synthase activities. This insult was accompanied by ATP depletion (54%) and increased concentration of nitrites plus nitrates (1.4-fold), suggesting enhanced .NO synthesis. Administration of Nomega-nitro-L-arginine monomethyl ester (L-NAME) to the mothers inhibited neonatal brain .NO synthase activity, as reflected by the decreased (23%) cyclic GMP concentration. These L-NAME-treated neonates showed complete resistance to anoxic-mediated brain mitochondrial complex II-III damage. Our results suggest that brain mitochondrial dysfunction leading to energy deficiency during perinatal asphyxia is a .NO-mediated process.

Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion.

The role of mitochondrial energy metabolism in glutamate mediated neurotoxicity was studied in rat neurones in primary culture. A brief (15 min) exposure of the neurones to glutamate caused a dose-dependent (0.01-1 mM) increase in cyclic GMP levels together with delayed (24 h) neurotoxicity and ATP depletion. These effects were prevented by either the nitric oxide (.NO) synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (NAME; 1 mM) or by the N-methyl-D-aspartate (NMDA) glutamate-subtype receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV; 0.1 mM). Glutamate exposure (0.1 mM and 1 mM) followed by 24 h of incubation caused the inhibition of succinate-cytochrome c reductase (20-25%) and cytochrome c oxidase (31%) activities in the surviving neurones, without affecting NADH-coenzyme-Q1 reductase activity. The rate of oxygen consumption was impaired in neurones exposed to 1 mM glutamate, either with glucose (by 26%) or succinate (by 39%) as substrates. These effects on the mitochondrial respiratory chain and neuronal respiration, together with the observed glutathione depletion (20%) by glutamate exposure were completely prevented by NAME or APV. Our results suggest that mitochondrial dysfunction and impairment of antioxidant status may account for glutamate-mediated neurotoxicity via a mechanism involving .NO biosynthesis.

Trolox protects mitochondrial complex IV from nitric oxide-mediated damage in astrocytes.

The efficacy of cystine, ascorbate and trolox, a vitamin E analogue, at protecting against nitric oxide-mediated mitochondrial complex IV damage has been investigated in cultured astrocytes. Of these compounds, only trolox afforded protection. It is suggested that lipid peroxidation is responsible for nitric oxide-mediated mitochondrial damage and that inhibitors of this process may be of therapeutic benefit in conditions where excessive nitric oxide production is implicated.

Depletion of brain glutathione results in a decrease of glutathione reductase activity; an enzyme susceptible to oxidative damage.

Loss of the intracellular antioxidant glutathione (GSH) from the substantia nigra is considered to be an early event in the pathogenesis of Parkinson's disease (PD). While the cause of the loss is unclear, an imbalance in the enzymes associated with the synthesis, utilisation, degradation and translocation of GSH has been implicated. The enzyme glutathione reductase is also important in GSH homeostasis: it regenerates GSH from the oxidised from (GSSG). However, to date the activity and regulation of glutathione reductase in conditions such as PD have not been explored. In view of this we have measured the effects of GSH depletion on glutathione reductase activity of the rat brain. Other glutathione related enzymes were also measured. Using pre-weanling rats, brain GSH was depleted by up to 60% by subcutaneous administration of L-buthionine sulfoximine. The only enzyme affected by GSH depletion was glutathione reductase; its activity being reduced by approximately 40%. As GSH inactivates a number of oxidising species including peroxynitrite (ONOO-), we additionally investigated the susceptibility of glutathione reductase to ONOO- in vitro, using purified enzyme. ONOO- decreased glutathione reductase activity in a concentration dependent manner with an apparent 50% inhibition occurring at an initial concentration of 0.09 mM. These data suggest that GSH is important in the maintenance glutathione reductase activity. This may arise in part from its ability to inactivate oxidising agents such as ONOO-.

Nitric oxide produced by activated astrocytes rapidly and reversibly inhibits cellular respiration.

Cultured astrocytes, activated to express the inducible form of nitric oxide synthase, produced up to 1 microM nitric oxide (NO) measured by a NO-selective electrode, while non-activated cells produced no detectable NO. The production of NO was associated with an inhibition of cellular respiration, measured simultaneously by an oxygen electrode. The inhibition of respiration was rapidly reversed by inhibiting the NO synthase or by binding the NO with haemoglobin. The respiratory inhibition had an NO, oxygen and substrate dependence consistent with NO-inhibition at cytochrome oxidase. This is the first demonstration that cells can reversibly inhibit mitochondrial respiration via NO production. This inhibition is large and potentially important in a range of pathophysiological conditions.

Evaluation of the efficacy of potential therapeutic agents at protecting against nitric oxide synthase-mediated mitochondrial damage in activated astrocytes.

Within the central nervous system, nitric oxide is an important physiological messenger. However, when synthesized excessively in neurones, cell death may occur. An impairment of mitochondrial cytochrome oxidase and subsequent cellular energy depletion seems to be a likely mechanism for this neurotoxicity. Within neurones, nitric oxide is synthesized by the constitutive, Ca(2+)-dependent form of nitric oxide synthase (nNOS). Astrocytes, however, possess both the constitutive and the inducible Ca(2+)-independent NOS (iNOS), which is expressed by endotoxin and/or cytokines. In vitro, activation of nNOS rapidly produces neuronal cell death. In contrast to neurones, following induction of iNOS, astrocytes synthesize large quantities of nitric oxide, but cell death is not apparent despite marked damage to mitochondrial cytochrome oxidase. The resistance of astrocytes to nitric oxide synthase-mediated cell damage may be due to their ability to increase their glycolytic rate when mitochondrial ATP synthesis is compromised. On the basis of this phenomenon, we propose that activated astrocytes represent a suitable system for studying the efficacy of potential therapeutic agents at protecting from nitric oxide synthase-mediated mitochondrial damage.

Effect of valproate on the metabolism of the central nervous system.

The effects of valproate on brain energy and lipid metabolism is reviewed. Increasing evidence suggests that valproate uses the monocarboxylic acid carrier in order to cross the blood brain barrier (BBB) and the neural cell plasma membranes. The uptake of valproate into the brain through this mechanism would compete with the uptake of energy precursors, such as the monocarboxylic acids 3-hydroxybutyrate, lactate or pyruvate and with some amino acids, but not with glucose. This could impair brain fuel utilization, specially during the neonatal period or childhood, when lactate or 3-hydroxybutyrate furnishes alternative substrates to glucose for the brain. It is concluded that valproate interference with energy metabolism may have implications for the therapeutic action of the drug, stressing the possibility that valproate-mediated alterations in brain lipid synthesis may contribute to the pharmacological action of the drug.

Inhibition of neonatal brain fuel utilization by valproate and E-delta 2-valproate is not a consequence of the stimulation of the gamma-aminobutyric acid shunt.

Stimulation of the gamma-aminobutyric acid (GABA) shunt by valproate and its major metabolite, E-delta 2-valproate, has been proposed to decrease brain energy metabolism. In order to elucidate this hypothesis, the effect of these drugs on substrate utilization in neonatal rat brain slices was studied. The overall rate of lactate utilization was dose-dependently inhibited by both drugs. Valproate and E-delta 2-valproate inhibited both sterol and fatty acid syntheses from 3-hydroxybutyrate. The rate of glucose utilization was not affected by valproate nor E-delta 2-valproate. The inhibition of the GABA aminotransferase by aminooxyacetate decreased lipogenesis from lactate, 3-hydroxybutyrate and glucose. The inhibitor of the mitochondrial pyruvate carrier, alpha-cyano-4-hydroxycinnamate, strongly decreased the rate of lactate, 3-hydroxybutyrate and glucose utilization, suggesting that the inhibition of pyruvate mitochondrial carrier is not the mode of action of these drugs. It is suggested that inhibition of plasma membrane monocarboxylate carrier by valproate and E-delta 2-valproate, but not the activation of the GABA shunt, is responsible for the inhibition of the brain fuel utilization.

Excitotoxic stimulus stabilizes PFKFB3 causing pentose-phosphate pathway to glycolysis switch and neurodegeneration.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a master regulator of glycolysis by its ability to synthesize fructose-2,6-bisphosphate, a potent allosteric activator of 6-phosphofructo-1-kinase. Being a substrate of the E3 ubiquitin ligase anaphase-promoting complex-Cdh1 (APC(Cdh1)), PFKFB3 is targeted to proteasomal degradation in neurons. Here, we show that activation of N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) stabilized PFKFB3 protein in cortical neurons. Expressed PFKFB3 was found to be mainly localized in the nucleus, where it is subjected to degradation; however, expression of PFKFB3 lacking the APC(Cdh1)-targeting KEN motif, or following NMDAR stimulation, promoted accumulation of PFKFB3 and its release from the nucleus to the cytosol through an excess Cdh1-inhibitable process. NMDAR-mediated increase in PFKFB3 yielded neurons having a higher glycolysis and lower pentose-phosphate pathway (PPP); this led to oxidative stress and apoptotic neuronal death that was counteracted by overexpressing glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Furthermore, expression of the mutant form of PFKFB3 lacking the KEN motif was sufficient to trigger oxidative stress and apoptotic death of neurons. These results reveal that, by inhibition of APC(Cdh1), glutamate receptors activation stabilizes PFKFB3 thus switching neuronal metabolism leading to oxidative damage and neurodegeneration.