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The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO − ). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1 mM ONOO − , whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10–1000 μM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II–III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO − exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.

The purpose of this study was to determine the extracellular concentrations of N-acetylaspartate (NAA) in the rat cerebral cortex, striatum, and hippocampus of halothane-anaesthetised rats by intracerebral microdialysis, and to examine the effects of high K(+)-induced local depolarisation, which provokes synchronous neurotransmitter release, cell swelling, and acid-base changes. Basal levels of NAA in the extracellular fluid (ECF) were determined by the zero net flux method. Tissue levels of NAA in the cortex, striatum, and hippocampus were 8.4, 5.7, and 7.2 mmol/kg, respectively. The corresponding extracellular concentrations of NAA were much lower (35.1, 83.7, and 23.0 microM). High tissue/ECF concentration ratios may suggest little release or leakage of NAA under basal conditions, and potent reuptake mechanisms for NAA in the cellular membrane of CNS cells. There was no change in ECF NAA during K(+)-induced local depolarising stimuli produced in the striatum, but NAA levels consistently increased after the K+ stimuli, irrespective of whether or not Ca2+ was present in the perfusion medium. These data confirm that NAA is not a neurotransmitter and suggest strongly that NAA is not directly involved in the release and reuptake or metabolism of neuroactive compounds. The increase of NAA in the ECF immediately after K+ stimulation may reflect an involvement in brain osmoregulation and/or acid-base homeostasis.

We report the isolation of cDNA clones encoding the somatic form of the E1 alpha subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1 alpha sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1 alpha subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1 alpha cDNA, in conjunction with cDNA probes to the E1 beta, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5-fold increase in the concentration of each of the subunit mRNAs and a 1.2-fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six- and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational mechanisms.

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by approximately 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2*- generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4-6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.

The antioxidant glutathione (GSH) plays an important role in protecting the mitochondrial electron transport chain (ETC) from damage by oxidative stress in astrocytes and neurones. Neurones co-cultured with astrocytes have greater GSH levels, compared to neurones cultured alone, leading to the hypothesis that astrocytes play a key role in brain GSH metabolism by supplying essential GSH precursors to neurones. A previous study has postulated that damage to the ETC following exposure to reactive nitrogen species (RNS) is less in co-cultured neurones, compared to neurones cultured alone, because of the greater GSH levels in the former cells. To investigate this further, primary culture rat neurones were co-cultured with either rat astrocytes activated with IFN-gamma and LPS to produce NO, or NO-generating astrocytes that had been depleted of intracellular GSH by 87% following incubation with the GSH synthesis inhibitor L-buthionine-S,R-sulfoximine (L-BSO). Neurones incubated with NO-generating astrocytes depleted of GSH were unable to elevate GSH levels, unlike neurones co-cultured with NO-generating astrocytes. Complexes II + III and IV of the neuronal ETC were significantly inhibited following exposure to NO-generating astrocytes depleted of GSH. No ETC damage was observed in neurones co-cultured with NO-generating astrocytes. Although neurones co-cultured with GSH depleted astrocytes did not increase cellular GSH levels, the activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, was increased by 218%, compared to neurones cultured with control astrocytes. This suggests that neuronal GCL activity could be modulated when GSH metabolism is inhibited in neighboring astrocytes.

Apoptosis may be initiated in neurons via mitochondrial release of the respiratory protein, cytochrome c. The mechanism of cytochrome c release has been studied extensively, but little is known about its dynamics. It has been claimed that release is all-or-none, however, this is not consistent with accumulating evidence of cytosolic mechanisms for 'buffering' cytochrome c. This study has attempted to model an underlying disease pathology, rather than inducing apoptosis directly. The model adopted was diminished activity of the mitochondrial respiratory chain complex I, a recognized feature of Parkinson's disease. Titration of rat brain mitochondrial respiratory function, with the specific complex I inhibitor rotenone, caused proportional release of cytochrome c from isolated synaptic and non-synaptic mitochondria. The mechanism of release was mediated, at least in part, by the mitochondrial outer membrane component Bak and voltage-dependent anion channel rather than non-specific membrane rupture. Furthermore, preliminary data were obtained demonstrating that in primary cortical neurons, titration with rotenone induced cytochrome c release that was subthreshold for the induction of apoptosis. Implications for the therapy of neurodegenerative diseases are discussed.

We have examined the action of nitric oxide (NO) on the ability of Fenton's reagent (ferrous iron and hydrogen peroxide), to oxidize a number of organic optical probes. We found that NO is able to arrest the oxidation of organic compounds at concentrations of NO found in brain, in vivo. We present evidence that Fenton's reagent proceeds via a ferryl intermediate ([Fe[double bond]O]2+), before the generation of hydroxyl radical *OH. NO reacts rapidly with this ferryl, blocking the production of *OH. We propose that NO has an important role in protecting biological tissues, and the brain in particular, from Fenton chemistry.

The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV ), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by approximately 72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by approximately 70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.

The Ca(2+)-independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 microgram/ml) plus interferon-gamma (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH-ubiquinone-1 reductase (complex I) and succinate-cytochrome c reductase (complex II-III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate-cytochrome c reductase activity, but NADH-ubiquinone-1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The lipopolysaccharide/interferon-gamma treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide-mediated neurotoxicity.

The effect of reperfusion following 30 min of cerebral ischaemia on brain mitochondrial respiratory chain activity has been studied in the gerbil. The state 3 respiration rates with both FAD- and NAD-linked substrates were reduced after ischaemia. After 5 min of reperfusion, state 3 respiration with FAD-linked substrates was restored, but levels of NAD-linked substrates did not return to control values until 30 min of reperfusion. By 120 min of reperfusion state 3 respiration decreased relative to control values with all substrates studied. Measurement of the individual respiratory chain complexes showed that complex I, complex II-III, and complex V activities were reduced after ischaemia. By 5 min of reperfusion complex II-III activity was restored, but the activities of complexes I and V did not return to control values until 30 min of reperfusion. In contrast, complex IV activity was unaffected by ischaemia or 5 and 30 min of reperfusion but was significantly reduced after 120 min of reperfusion, possibly owing to free radical production and lipid peroxidation.

We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPAR alpha, PPAR beta, and PPAR gamma and the rat retinoid X receptor (RXR) isoforms RXR alpha, RXR beta, and RXR gamma. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPAR alpha, PPAR beta, RXR alpha, and RXR beta mRNAs are ubiquitously present in different brain regions during development, PPAR gamma mRNA is essentially undetectable, and RXR gamma mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPAR alpha, PPAR beta, RXR alpha, and RXR beta mRNAs are present in all cell types, albeit that PPAR alpha and RXR alpha mRNAs are at levels near the limit of detection in CGNs. PPAR gamma mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPAR alpha mRNA in adult astrocytes. RXR gamma mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.

Peroxynitrite has been reported to inhibit irreversibly mitochondrial respiration. Here we show that three sequential additions of 200 microM peroxynitrite (initial concentration) to rat brain mitochondria (0.2 mg of protein/ml) significantly stimulated state 4 respiration and that further additions progressively inhibited it. No stimulation of state 3 respiration or of the maximal enzymatic activities of the respiratory chain complexes was observed on identical peroxynitrite exposure. State 4 respiration is a consequence of the proton permeability of the mitochondrial inner membrane, and we demonstrate that the peroxynitrite-induced stimulation of state 4 respiration is accompanied by a decreased mitochondrial membrane potential, suggesting an increase in this proton leak. Cyclosporin A did not affect the stimulation, suggesting no involvement of the mitochondrial permeability transition pore. The stimulation was prevented by the lipid-soluble vitamin E analogue Trolox, suggesting the involvement of lipid peroxidation, a proposed mechanism of peroxynitrite cytotoxicity. Lipid peroxidation has previously been reported to increase membrane bilayer proton permeability. The high polyunsaturate content of brain mitochondrial phospholipids may predispose them to peroxidation, and thus a peroxynitrite-induced, lipid peroxidation-mediated increase in proton leak may apply particularly to brain mitochondria and to certain neurodegenerative disorders thought to proceed via mechanisms of mitochondrial oxidative damage.

We have previously identified cerebellum to contain significantly higher levels, compared with other brain regions, of the mRNA encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). In this report, we extend these observations, using primary cultures of cerebellar astrocytes and cerebellar granule neurons, and show that mHS mRNA was not readily detected in these cell types, suggesting that other cerebellar cell types account for mHS mRNA abundances observed in cerebellum. In contrast, we report, for the first time, the ready detection of mHS mRNA together with the mRNAs encoding the remaining enzymes of the 3-hydroxy-3-methylglutaryl-CoA cycle, namely, mitochondrial acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA lyase, in primary cultures of neonatal meningeal fibroblasts. Based on observations of the effects of fetal calf serum in the culture medium and the documented effects of various hormones on mHS mRNA levels in liver, we show that the glucocorticoid hydrocortisone effects a selective fourfold increase in mHS mRNA abundances in both neonatal meningeal fibroblasts and neonatal cortical astrocytes cultured in a serum-free/hormone-free medium.

Manganese-salen complexes (Mn-Salen), including EUK-8 [manganese N,N'-bis(salicylidene)ethylenediamine chloride] and EUK-134 [manganese 3-methoxy N,N'-bis(salicylidene)ethylenediamine chloride], have been reported to possess combined superoxide dismutase (SOD) and catalase mimetic functions. Because of this SOD/catalase mimicry, EUK-8 and EUK-134 have been investigated as possible therapeutic agents in neurological disorders resulting from oxidative stress, including Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis. These actions have been explained by the ability of the Mn-Salen to remove deleterious superoxide (O(2)(-)) and H(2)O(2). However, in addition to oxidative stress, cells in models for neurodegenerative diseases may also be subjected to damage from reactive nitrogen oxides (nitrosative stress), resulting from elevated levels of NO and sister compounds, including peroxynitrite (ONOO(-)). We have been examining the interaction of EUK-8 and EUK-134 with NO and ONOO(-). We find that in the presence of a per-species (H(2)O(2), ONOO(-), peracetate and persulphate), the Mn-Salen complexes are oxidized to the corresponding oxo-species (oxoMn-Salen). OxoMn-Salens are potent oxidants, and we demonstrate that they can rapidly oxidize NO to NO(2) and also oxidize nitrite (NO(2)(-) to nitrate (NO(2)(-)). Thus these Mn-Salens have the potential to ameliorate cellular damage caused by both oxidative and nitrosative stresses, by the catalytic breakdown of O(2)(-), H(2)O(2), ONOO(-) and NO to benign species: O(2), H(2)O, NO(2)(-) and NO(3)(-).

Beta-amyloid deposition and compromised energy metabolism both occur in vulnerable brain regions in Alzheimer's disease. It is not known whether beta-amyloid is the cause of impairment of energy metabolism, nor whether impaired energy metabolism is specific to neurons. Our results, using primary neuronal cultures, show that 24-h incubation with A beta(25-35) caused a generalized decrease in the specific activity of mitochondrial enzymes per milligram of cellular protein, induced mitochondrial swelling, and decreased total mitochondrial number. Incubation with A beta(25-35) decreased ATP concentration to 58% of control in neurons and 71% of control in astrocytes. Levels of reduced glutathione were also lowered by A beta(25-35) in both neurons (from 5.1 to 2.9 nmol/mg protein) and astrocytes (from 25.2 to 14.9 nmol/mg protein). We conclude that 24-h treatment with extracellular A beta(25-35) causes mitochondrial dysfunction in both astrocytes and neurons, the latter being more seriously affected. In astrocytes mitochondrial impairment was confined to complex I inhibition, whereas in neurons a generalized loss of mitochondria was seen.

Cytokine-stimulated astrocytes produce nitric oxide, which can inhibit components of the mitochondrial respiratory chain. We have previously demonstrated that prolonged exposure (48 h) to rat astrocytic nitric oxide damages complexes II--III and IV of neighbouring rat neurons in coculture, resulting in neuronal death. Expanding on these observations, we have now shown that the NMDA receptor antagonist, MK-801, prevents this damage, suggesting involvement of glutamate. We postulate that astrocyte-derived nitric oxide stimulates release of neuronal glutamate. Indeed we demonstrate that neurons incubated with nitric oxide-generating astrocytes display enhanced glutamate release. Furthermore, direct exposure to the nitric oxide donor, DETA-NONOate resulted in a loss of activity of all the neuronal mitochondrial complexes, which was again prevented by MK-801. Thus, nitric oxide, generated by both cytokine-stimulated astrocytes and by a nitric oxide donor, causes activation of the NMDA receptor leading to damage to the neuronal mitochondrial respiratory chain. Glutamate exposure is known to damage the neuronal mitochondrial respiratory chain via neuronal nitric oxide synthase. Therefore, we propose that astrocyte-derived nitric oxide is capable of eliciting neuronal glutamate release, which in turn activates the neuronal NMDA receptor and stimulates further formation of reactive nitrogen species via neuronal nitric oxide synthases, leading to mitochondrial damage and neuronal death. Our findings support the hypothesis that glutamate, reactive nitrogen species and mitochondrial dysfunction may have a role in the neurodegenerative process.

Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post-mortem Alzheimer's disease brain. beta-Amyloid is strongly implicated in Alzheimer's pathology and can be formed intracellularly in neurones. We have investigated the possibility that beta-amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the beta-amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimer's brain. Mitochondrial respiration, electron transport chain complex activities, alpha-ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. Beta-amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by beta-amyloid. The K(m) of cytochrome oxidase for reduced cytochrome c was raised by beta-amyloid. We conclude that beta-amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease.

The process of nitric-oxide (NO)-induced cellular toxicity may involve energy deprivation since the radical is reported to prevent both mitochondrial oxidative phosphorylation and glycolysis. In order to determine whether these processes are important in NO-induced blood-brain barrier (BBB) dysfunction, we used a cell culture model of the BBB and compared the effects of gaseous NO, potassium cyanide (KCN, a mitochondrial respiratory chain inhibitor) and iodoacetate [IA, an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] on endothelial cell ATP content, GAPDH activity and barrier integrity. NO lead to a rapid breakdown in model barrier integrity and resulted in a reduction in endothelial cell ATP content and GAPDH activity. KCN had no effect on endothelial cell ATP content or barrier integrity, while IA, at a concentration that completely blocked endothelial cell GAPDH activity, resulted in a rapid decline in ATP content but did not lead to a decline in barrier integrity until at least 2 h of exposure. These results indicate that inhibition of endothelial cell GAPDH activity rather than mitochondrial respiration causes an energy deficiency and delayed barrier dysfunction. However, the rapid detrimental effects of gaseous NO on barrier integrity cannot be fully explained by endothelial cell energy depletion and may be related to the actions of the free radical and its products on cellular lipids.

It is becoming increasingly evident that the mitochondrial genome may play a key role in neurodegenerative diseases. Mitochondrial dysfunction is characteristic of several neurodegenerative disorders, and evidence for mitochondria being a site of damage in neurodegenerative disorders is partially based on decreases in respiratory chain complex activities in Parkinson's disease, Alzheimer's disease, and Huntington's disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant balance perturbation, are thought to underlie defects in energy metabolism and induce cellular degeneration. Efficient functioning of maintenance and repair process seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of genes termed vitagenes. A promising approach for the identification of critical gerontogenic processes is represented by the hormesis-like positive effect of stress. In the present review, we discuss the role of energy thresholds in brain mitochondria and their implications in neurodegeneration. We then review the evidence for the role of oxidative stress in modulating the effects of mitochondrial DNA mutations on brain age-related disorders and also discuss new approaches for investigating the mechanisms of lifetime survival and longevity.

Background The mitochondrial myopathies typically affect many organ systems and are associated with mutations in mitochondrial DNA (mtDNA) that are maternally inherited. However, there is also a sporadic form of mitochondrial myopathy in which exercise intolerance is the predominant symptom. We studied the biochemical and molecular characteristics of this sporadic myopathy.Methods We sequenced the mtDNA cytochrome b gene in blood and muscle specimens from five patients with severe exercise intolerance, lactic acidosis in the resting state (in four patients), and biochemical evidence of complex III deficiency. We compared the clinical and molecular features of these patients with those previously described in four other patients with mutations in the cytochrome b gene.Results We found a total of three different nonsense mutations (G15084A, G15168A, and G15723A), one missense mutation (G14846A), and a 24-bp deletion (nucleotides 15498 to 15521) in the cytochrome b gene in the five patients. Each of these mutations impairs the enzymatic function of the cytochrome b protein. In these patients and those previously described, the clinical manifestations included progressive exercise intolerance, proximal limb weakness, and in some cases, attacks of myoglobinuria. There was no maternal inheritance and there were no mutations in tissues other than muscle. The absence of these findings suggests that the disorder is due to somatic mutations in myogenic stem cells after germ-layer differentiation. All the point mutations involved the substitution of adenine for guanine, but all were in different locations.Conclusions The sporadic form of mitochondrial myopathy is associated with somatic mutations in the cytochrome b gene of mtDNA. This myopathy is one cause of the common and often elusive syndrome of exercise intolerance. (N Engl J Med 1999; 341:1037-44.) (C)1999, Massachusetts Medical Society.

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been demonstrated in Alzheimer's disease, and after the expression of the amyloid precursor protein (APP) in cultured cells, suggesting that mitochondria might be involved in beta-amyloid toxicity. Recent evidence suggests that the proteolysis of APP to generate beta-amyloid is at least in part intracellular, preceding the deposition of extracellular fibrils. We have therefore investigated the effect of incubation of isolated rat brain mitochondria with the beta-amyloid fragment 25-35 (100 microM) on the activities of the mitochondrial respiratory chain complexes I, II-III, IV (cytochrome c oxidase) and citrate synthase. The peptide caused a rapid, dose-dependent decrease in the activity of complex IV, white it had no effect on the activities on any of the other enzymes tested. The reverse sequence peptide (35-25) had no effect on any of the activities measured. We conclude that inhibition of mitochondrial complex IV might be a contributing factor to the pathogenesis of Alzheimer's disease.

In order to test the effect of hypothermia on mitochondrial function damage following cerebral ischaemia/reperfusion, Mongolian gerbils were submitted to 30 min bilateral carotid occlusion and 2 h of reperfusion at 37 degreesC or 30 degreesC. After normothermic (37 degreesC) ischaemia/reperfusion, significant decreases in mitochondrial state 3 (+ADP) oxygen consumption (-42.2%), complex II-III activity in synaptosomes (-31.7%) and complex IV were measured, in both free mitochondria and synaptosomes (-30.3% and -27. 8% respectively). However, following hypothermic (30 degreesC) reperfusion, both respiration rates and all enzyme activities remained at levels not significantly different from those in the sham operated controls.

Using a cell culture model of the blood–brain barrier (BBB) we have evaluated the role of endothelial cell glutathione in protecting barrier integrity against nitric oxide (NO)-induced oxidative stress. The co-culture of human umbilical vein endothelial cells (ECV304) with rat (C6) glioma cells, or incubation with glioma cell or primary astrocytic conditioned medium, resulted in a decline in endothelial cell glutathione. Exposure to a single addition of NO gas induced a rapid breakdown in model barrier integrity in endothelial/glioma co-cultures. Addition of NO gas or tumour necrosis factor-α (TNF-α) also resulted in a loss of membrane integrity, as measured by an enhanced release of lactate dehydrogenase, only from endothelial cells treated with glioma conditioned medium. Furthermore, assessment of viability in endothelial cells grown alone or treated with glioma conditioned medium, by propidium iodide labelled flow cytometry, demonstrated no difference in the number of positively stained cells after NO exposure. These results indicate that when enhanced endothelial monolayer barrier formation occurs via astrocytic–endothelial interactions, cellular glutathione levels are decreased. This renders the barrier cells, under these conditions, more susceptible to oxidative stress but does not necessarily lead to greater cell death.

Purpose To determine the profile of blood flow velocities and resistive indices in the ocular vessels of patients with atherosclerotic carotid disease and characterise the effect of endarterectomy on these parameters. Methods Following a preliminary study on healthy volunteers, ophthalmic colour Doppler ultrasound examinations were performed on 27 male and 11 female patients with carotid disease. These measurements were compared with central retinal artery perfusion pressures and intraoperative internal carotid artery stump pressures. Results Significant changes were seen on the endarterectomised side. The peak systolic velocity in the ophthalmic artery, and resistive indices in the ophthalmic artery, central retinal artery and nasal posterior ciliary artery, rose from pre-operative values. No correlation between colour Doppler ultrasound measurements and intraoperative internal carotid artery stump pressures was present. When compared with ophthalmodynamometry readings, a relationship was noted with maximum velocities in the central retinal vein. Conclusion Carotid endarterectomy alters the haemodynamics in selected vessels of the ocular circulation as measured by colour Doppler ultrasound, but more work is required to determine the clinical utility of this investigative modality.

Decreases in mitochondrial respiratory chain complex activities have been implicated in neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and Alzheimer's disease. However, the extent to which these decreases cause a disturbance in oxidative phosphorylation and energy homeostasis in the brain is not known. We therefore examined the relative contribution of individual mitochondrial respiratory chain complexes to the control of NAD-linked substrate oxidative phosphorylation in synaptic mitochondria. Titration of complex I, III, and IV activities with specific inhibitors generated threshold curves that showed the extent to which a complex activity could be inhibited before causing impairment of mitochondrial energy metabolism. Complex I, III, and IV activities were decreased by approximately 25, 80, and 70%, respectively, before major changes in rates of oxygen consumption and ATP synthesis were observed. These results suggest that, in mitochondria of synaptic origin, complex I activity has a major control of oxidative phosphorylation, such that when a threshold of 25% inhibition is exceeded, energy metabolism is severely impaired, resulting in a reduced synthesis of ATP. Additionally, depletion of glutathione, which has been reported to be a primary event in idiopathic Parkinson's disease, eliminated the complex I threshold in PC12 cells, suggesting that antioxidant status is important in maintaining energy thresholds in mitochondria. The implications of these findings are discussed with respect to neurodegenerative disorders and energy metabolism in the synapse.

We have previously reported that the co-culture of endothelial and glioma cell lines provides an in vitro model for investigating properties of the blood-brain barrier (BBB). To characterise the model system further we have investigated the effects of vasoactive substances implicated in increases in BBB permeability. Additionally, we have also examined whether activation of cyclic AMP signalling pathways, which elevate cerebral endothelial cell barrier function, similarly modulate our model system. ATP, histamine, bradykinin, and serotonin significantly decreased model BBB transendothelial electrical resistance and manipulations which elevate cyclic AMP enhanced culture resistance. These data indicate that our model BBB system responds in a manner characteristic of cerebral microvascular endothelial cells and the BBB in vivo. These data further emphasize the usefulness of our model 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.

The effect of GTP concentration on forskolin-stimulated adenylyl cyclase activity was examined in synaptosomal membranes from 15-day-old rats that were hypothyroid due to administration of propylthiouracil and a low-iodine diet to the mothers during pregnancy and suckling. In membranes from the forebrain hypothyroidism abolished the overall stimulatory effect of GTP, which was seen in the euthyroid case. In membranes from the hindbrain hypothyroidism had the opposite effect in that there was an enhancement of an overall stimulatory effect of GTP. It is suggested that these findings reflect changes during early development of the brain in the expression of various G-proteins and/or the expression of different isoforms of adenylyl cyclase.

Nitric oxide (NO.) has been implicated in the process of cerebral ischemia/reperfusion injury. We have examined the production of NO., as reflected by nitrite (NO2-) + nitrate (NO3-) accumulation, from synaptosomes isolated from neonatal or adult rat brain and subjected to a period of glucose and oxygen deprivation. There was a significant increase in the amount of NO2- + NO3- production from adult synaptosomes under these conditions, whereas there was no difference compared to control in the production of NO2- + NO3- from the neonatal synaptosomes. The total antioxidant status of the synaptosomes at these different stages of brain development was found to be the same. These data suggest that the vulnerability of the adult brain to ischemia/reperfusion injury may be associated with the production of NO. from nerve terminals. The ratios of antioxidant capacity to NO. production under such conditions have been shown here to be different between the neonatal and adult nerve terminals. Thus the well documented resistance of neonatal brain to ischemia/reperfusion injury may involve the neonatal nerve terminal being under less oxidative stress than the adult.

The effect of specific irreversible inhibitors of complexes I, III, IV and V of the mitochondrial respiratory chain, (rotenone, myxothiazol, cyanide and oligomycin, respectively) on mitochondrial N-acetylaspartate production, and its relationship to oxidative phosphorylation (ATP production and oxygen consumption) were investigated in isolated rat brain mitochondria. Mitochondrial N-acetylaspartate production, ATP production and oxygen consumption were all significantly decreased in the presence of each of the inhibitors used compared with control incubations, and correlated positively with each other. It is postulated that decreased N-acetylaspartate levels seen in disease states by 1H NMR spectroscopy in vivo may reflect primarily an impaired mitochondrial energy production rather than neuronal cell loss.

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 form (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 − .

In this article we demonstrate the immunocytochemical localization of nitric oxide synthase in mitochondria isolated from heart, skeletal muscle, and kidney, using a monoclonal antibody directed against the endothelial form of nitric oxide synthase. The possibility that mitochondrially located nitric oxide synthase is a ubiquitous regulator of mitochondrial oxidative phosphorylation in mammalian cells is discussed.

The accumulation of subunit c of the mitochondrial ATP synthase in late-infantile neuronal lipofuscinosis (LINCL) and juvenile neuronal lipofuscinosis (JNCL) is well documented. The purification of the subunit from diverse sources has been reported previously, although not from the brain of Batten disease patients. This proteolipid has now been purified from late-infantile Batten disease brain. The procedures used were an original combination of the conventional solubilisation, differential centrifugation, organic solvent extractions, preparative gel electrophoresis, and FPLC. Gel filtration of the purified protein indicated molecular mass equal to or greater than 2 X 10 6 Da ; however, electrophoresis of this pure protein suggested a molecular mass of approximately 3,500 Da, which is a characteristic of subunit c. The pure protein may be solubilised in aqueous buffer containing < lib lithium dodecyl sulphate (LDS). The protein binds dicyclohexylcarbodiimide (DCCD) and shows immunoreactivity to antibodies raised against ovine storage bodies.

This study examines the relationship of genotype to phenotype in 14 unselected patients who were found to harbour the A3243G transition in the mitochondrial transfer RNALeu(UUR) gene commonly associated with the syndrome of mitochondrial encephalopathy, lactic acidosis and strokes (MELAS). Only 6 of the 14 cases (43%) had seizures and recurrent strokes, the core clinical features of the MELAS phenotype. Of the remaining cases, four had an encephalomyopathy with deafness, ataxia and dementia, two had syndromes with progressive external ophthalmoplegia and two had limb weakness alone. Even within the MELAS subgroup, the majority of patients had one or more clinical manifestations considered to be atypical of the MELAS syndrome. They included developmental delay, ophthalmoparesis, pigmentary retinopathy and intestinal pseudo-obstruction. The proportion of mutant mitochondrial DNA (mtDNA) in muscle was generally higher in patients with recurrent strokes than in those without strokes, the highest levels being observed in MELAS cases with early onset disease. Studies of isolated muscle mitochondria identified a range of respiratory chain abnormalities mostly involving Complex I; immunoblots of Complex I in 3 of 10 cases showed selective loss of specific subunits encoded by nuclear genes. In the group as a whole, however, no clear correlations were observed between the severity or extent of the respiratory chain abnormality and clinical phenotype or the proportion of mutant mtDNA in biopsied skeletal muscle. These discrepancies suggest that, in patients harbouring the common MELAS3243 mutation, differences in heteroplasmy and the proportions of mutant mtDNA may not be the sole determinants of disease expression and that additional genetic mechanisms are involved in defining the range of clinical and biochemical phenotypes associated with this aberrant mitochondrial genome.

Tetrahydrobiopterin (BH4) is the cofactor for the aromatic amino acid monoxygenase group of enzymes and for all known isoforms of nitric oxide synthase (NOS). Inborn errors of BH4 metabolism lead to hyperphenylalaninaemia and impaired catecholamine and serotonin turnover. The effects of BH4 deficiency on brain nitric oxide (NO) metabolism are not known. In this study we have used the hph-1 mouse, which displays GTP cyclohydrolase deficiency, to study the effects of BH4 deficiency on brain NOS. In the presence of exogenous BH4, NOS specific activity was virtually identical in the control and hph-1 preparations. However, omission of BH4 from the reaction buffer led to a significant 20% loss of activity in the hph-1 preparations only. The Km for arginine was virtually identical for the control and hph-1 NOS when BH4 was present in the reaction buffer. In the absence of cofactor, the Km for arginine was 3-fold greater for control and 5-fold greater for hph-1 preparations. It is concluded that (a) BH4 does not regulate the intracellular concentration of brain NOS; (b) less binding of BH4 to NOS occurs in BH4 deficiency states; (c) BH4 has a potent effect on the affinity of NOS for arginine; and (d) the availability of arginine for NOS activity may become severely limiting in BH4 deficiency states. Since, in the presence of suboptimal concentrations of BH4 or arginine, NOS may additionally form oxygen free-radicals, it is postulated that in severe BH4 deficiency states NO formation is impaired and the central nervous system is subjected to increased oxidative stress.

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-beta 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-gamma 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-alpha/beta also prevented this damage. In view of these results, we suggest that a possible mechanism of action of interferon-beta in the treatment of multiple sclerosis is that it prevents astrocytic nitric oxide production, thereby limiting damage to neighbouring cells, such as neurones.

Polypeptides of beef heart mitochondrial complex I were isolated from 15 mg of solubilized beef heart mitochondria using antibodies immobilized on an agarose chromatography column. The preparation was examined by SDS electrophoresis and Western blotting using affinity-purified antibodies to complex I and compared to beef heart complex I purified according to the conventional method of Hatefi and Rieske. There was a high degree of homology between the two preparations as judged by SDS-polyacrylamide electrophoresis and by immunoblotting with seven affinity-purified antibodies to various complex I subunits. This method could be applied to the preparation of complex I subunits from small samples such as human muscle biopsy specimens.

The utilization of lactate, glucose, 3-hydroxybutyrate, and glutamine has been studied in isolated brain cells from early newborn rats. Isolated brain cells actively utilized these substrates, showing saturation at concentrations near physiological levels during the perinatal period. The rate of lactate utilization was 2.5-fold greater than that observed for glucose, 3-hydroxybutyrate, or glutamine, suggesting that lactate is the main metabolic substrate for the brain immediately after birth. The apparent Km for glucose utilization suggested that this process is limited by the activity of hexokinase. However, lactate, 3-hydroxybutyrate, and glutamine utilization seems to be limited by their transport through the plasma membrane. The presence of fatty acid-free bovine serum albumin (BSA) in the incubation medium significantly increased the rate of lipogenesis from lactate or 3-hydroxybutyrate, although this was balanced by the decrease in their rates of oxidation in the same circumstances. BSA did not affect the rate of glucose utilization. The effect of BSA was due not to the removal of free fatty acid, but possibly to the binding of long-chain acyl-CoA, resulting in the disinhibition of acetyl-CoA carboxylase and citrate carrier.

Within the central nervous system (CNS), tetrahydrobiopterin (BH 4 ) is an essential cofactor for the aromatic amino acid monoxygenases. Consequently, inborn errors of BH 4 metabolism result in hyperphenylalaninaemia and impaired dopamine and serotonin metabolism (Blau et al 1993). Furthermore, the intracellular BH 4 concentration may regulate the rate of aromatic amino acid hydroxylation due to the relatively high K m (μmol/L) of the aromatic amino acid monoxygenases for BH 4 . Evidence for this in man comes from our observation that homovanillic acid, a dopamine metabolite, and 5-hydroxyindoleacetic acid, a serotonin metabolite, both exhibit a strong positive correlation with BH 4 in cerebrospinal fluid (CSF) (Hyland et al 1993). Current treatment regimes for the inborn errors of BH 4 metabolism often focus on phenylalanine restriction and monoamine replacement therapy (Blau et al 1993). While this strategy is beneficial, it may also be incomplete since in a significant number of patients neurological impairment is still apparent (Blau et al 1993). The exact reason for this failure of treatment is not known but may be related to the other cofactor roles of BH 4 . BH 4 is also an obligatory cofactor for all isoforms of nitric oxide synthase (Knowles and Moncada 1994). In view of the key biochemical roles now attributed to nitric oxide (NO), e.g. cGMP formation and regulation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Garthwaite and Boulton 1995; Heales et al 1997), we have postulated that an impaired ability to generate NO may be an important contributing factor to the neurological disturbances associated with BH 4 deficiency. To test this hypothesis, we have utilized the hph-1 mouse, which displays a well-characterized deficiency (-50%) of BH 4 (Brand et al 1995). Using this model, we have obtained data that suggest that NO generation is profoundly disturbed in the brains of these animal, i.e. cerebellar cGMP synthesis is impaired and whole-brain GAPDH activity is increased 4-fold (Brand et al 1996; Heales et al 1997). However, in the presence of an exogenous source of NO, cerebellar cGMP synthesis is restored, suggesting impaired NO generation via NOS (Brand et al 1996). In order to ascertain whether BH 4 availability also influences NO metabolism in man, we have measured, in cerebrospinal fluid (CSF), the NO degradation products nitrite and nitrate (Clelland et al 1996) and tested for a correlation with BH 4 concentration.

L-Lactate (4–32 mM) added exogenously to resting or depolarised rat forebrain synaptosomes led to a significant decrease in intrasynaptosomal pH. Similarly depolarisation-induced increases in intrasynaptosomal calcium, calcium uptake, and acetylcholine release were all inhibited. These effects mimicked those previously observed in synaptosomes under anoxic conditions and suggest that lactate may be involved in limiting the damage due to calcium accumulation occurring during ischaemia. D-Lactate (added exogenously up to 32 mM) did not produce similar effects on these parameters even though the concentrations of intrasynaptosomal D-lactate reached levels comparable to those obtained with L-lactate (at 8–16 mM exogenous concentration). The results suggest that the mechanism of action of lactate on these parameters is stereospecific for the L-enantiomer. The effect of glucose availability on lactate production was assessed to explore the role of substrate availability on ischaemia/anoxic events. When exogenous glucose was increased (10–60 mM), there was no further increase in lactate production in normoxic synaptosomes, which suggests that glucose is not limiting under these conditions. When glucose was removed, as may occur in complete ischaemia, there was a significant decrease in lactate production after 60 min under anoxic or normoxic conditions. It would seem likely therefore that the mechanism underlying the changes observed in synaptosomes incubated under conditions reflecting complete ischaemia does not involve lactate.

We have recently reported that maternal dietary imbalance during pregnancy and lactation can reduce the lifespan of offspring. Rats that were growth restricted in utero by maternal protein restriction and underwent rapid weight gain when suckled by control fed dams died earlier than animals whose mothers were fed a control diet throughout pregnancy and lactation. We demonstrate here that mitochondrial abnormalities and DNA damage occur in the kidney of offspring who die prematurely. We have established by direct measurement and by in vitro supplementation that mitochondrial abnormalities occur because of a functional deficit of the mitochondrial cofactor coenzyme Q9 (CoQ9). These data provide molecular insight into the association between maternal nutrition and determination of offspring lifespan, and identify, a potential dietary intervention to prevent detrimental consequences of imbalanced maternal nutrition.

This study has found that mitochondrial NADH-CoQ1 reductase (complex I) activity is significantly lower in C57 mice astrocytes compared with Wistar and Sprague-Dawley rat astrocytes, and a human astrocytoma cell line. In addition, complex I activity is 4-fold greater in Sprague-Dawley neurones when compared to Wistar or C57 neurones. These findings have important implications for mitochondrial studies involving rodent or human cell line systems, and in particular, indicate the importance of choosing an appropriate model when investigating the mitochondrial respiratory chain.

Since the discovery of the significance of the cholesterol-carrying apolipoprotein E and cholesterolaemia as major risk factors for Alzheimer's Disease (AD) there has been a mounting interest in the role of this lipid as a possible pathogenic agent. In this review we analyse the current evidence linking cholesterol metabolism and regulation in the CNS with the known mechanisms underlying the development of Alzheimer's Disease. Cholesterol is known to affect amyloid-beta generation and toxicity, although it must be considered that the results studies using the statin class of drugs to lower plasma cholesterol may be affected by other effects associated with these drugs. Finally, we report some of our results pointing at the interplay between neurons and astrocytes and NADPH oxidase activation as a new candidate mechanism linking cholesterol and AD pathology.