Energy thresholds in brain mitochondria. Potential involvement in neurodegeneration.

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.

Distribution of mRNAs encoding the peroxisome proliferator-activated receptor alpha, beta, and gamma and the retinoid X receptor alpha, beta, and gamma in rat central nervous system.

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.

Molecular cloning of rat mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase and detection of the corresponding mRNA and of those encoding the remaining enzymes comprising the ketogenic 3-hydroxy-3-methylglutaryl-CoA cycle in central nervous system of suckling rat.

We have investigated, by RNase protection assays in rat brain regions and primary cortical astrocyte cultures, the presence of the mRNA species encoding the three mitochondrially located enzymes acetoacetyl-CoA thiolase, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mt. HMG-CoA synthase) and HMG-CoA lyase (HMG-CoA lyase) that together constitute the ketogenic HMG-CoA cycle. As a prerequisite we obtained a full-length cDNA encoding rat HMG-CoA lyase by degenerate oligonucleotide-primed PCR coupled to a modification of PCR-rapid amplification of cDNA ends (PCR-RACE). We report here: (1) the nucleotide sequence of rat mt. HMG-CoA lyase, (2) detection of the mRNA species encoding all three HMG-CoA cycle enzymes in all regions of rat brain during suckling, (3) approximately twice the abundance of mt. HMG-CoA synthase mRNA in cerebellum than in cortex in 11-day-old suckling rat pups, (4) significantly lower abundances of mt. HMG-CoA synthase mRNA in brain regions derived from rats weaned to a high-carbohydrate/low-fat diet compared with the corresponding regions derived from the suckling rat, and (5) the presence of mt. HMG-CoA synthase mRNA in primary cultures of neonatal cortical astrocytes at an abundance similar to that found in liver of weaned animals. These results provide preliminary evidence that certain neural cell types possess ketogenic potential and might thus have a direct role in the provision of fatty acid-derived ketone bodies during the suckling period.

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.

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.

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

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

Energy metabolism of adult astrocytes in vitro.

In this study we established cultures of astrocytes from the forebrain of the adult rat. The homeostatic regulatory mechanisms of the aerobic and anaerobic pathways of energy metabolism in these cells showed that adult astrocytes express many of the regulatory properties previously demonstrated in neonatal astrocytes. Changes in mitochondrial respiration and ATP production were readily evident upon incubation with the relevant substrates. Inhibition of mitochondrial respiration led to a compensatory increase in anaerobic glycolysis as evidenced by an increased release of lactate. We assessed the role of cytosolic calcium in the regulation of the mitochondrial energy metabolism. Increases in cytosolic calcium concentration in response to ATP or stimulation of mechanical receptors were followed by depolarizations of the mitochondrial membrane potential, whose magnitude reflected the amplitude of the cytosolic calcium response. The changes in mitochondrial membrane potential were largely dependent on the presence of external calcium. These results provide the first evidence of a signalling mechanism in astrocytes by which changes in cytosolic calcium mediate changes in respiration, possibly through mitochondrial calcium uptake and subsequent activation of several mitochondrial dehydrogenases. This signalling pathway would thus ensure that energy demands due to changes in cytosolic calcium concentrations are met by increases in energy production through increases in mitochondrial oxidative phosphorylation.

Mechanisms of intracellular calcium regulation in adult astrocytes.

Microfluorimetric techniques were used to measure changes in intracellular calcium in astrocytes cultured from the forebrain of the adult rat. Application of ATP consistently raised intracellular calcium. The response persisted in the absence of extracellular calcium, but then quickly declined upon repeated agonist application. Thapsigargin abolished responses to nucleotides following depletion of the endoplasmic reticular calcium stores. Calcium release was inhibited by caffeine, but was dramatically increased through inositol phosphate receptor sensitization by the sulphydryl reagent thimerosal. Responses to repeated nucleotide applications resulted in a gradual decline of peak calcium concentrations, suggesting a (post)receptor-mediated desensitization or gradual depletion of the internal calcium stores. Subsequent application of ionomycin suggested intracellular calcium depletion as the relevant mechanism. Depletion of the internal calcium stores with ATP, ionomycin or thapsigargin failed to reveal a calcium influx pathway. These results suggest that the capacitative mechanism of calcium entry does not operate in response to nucleotide receptor activation in these cells, and that the immediate refilling of the internal calcium stores is primarily determined by re-uptake of cytosolic calcium into the endoplasmic reticulum. A complete refilling of this calcium store by extracellular calcium may be a much slower process. Control of these signal transduction pathways is crucial to the maintenance of the calcium/energy homeostasis of the adult astrocyte in the central nervous system.

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.

Nitric oxide-mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes.

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.

Identification of a 58-kDa antigen with increased immunoreactivity in the cerebella of multiple sclerosis patients.

An increase in immunoreactivity associated with a 58-kDa antigen was found in a majority of MS cerebellar homogenates examined by Western blot analysis using antisera obtained by selective immunization of rabbits with autopsy cerebella. Two-dimensional immunoblotting demonstrated that the majority of the increased immunoreactivity observed in MS cerebella was associated with the highest apparent pI of three immunoreactive species at 58 kDa. Immuno-crossreaction with rat cerebellar homogenates demonstrated that the 58-kDa antigen was developmentally regulated, showing the greatest immunoreactivity at embryonic day 15. The 58-kDa cerebellar antigen may represent a membrane protein which is re-expressed as part of the onset of MS.