Jb Clark

β-Amyloid inhibits integrated mitochondrial respiration and key enzyme activities

Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), α‐ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post‐mortem Alzheimers disease brain. β‐Amyloid is strongly implicated in Alzheimers pathology and can be formed intracellularly in neurones. We have investigated the possibility that β‐amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the β‐amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimers brain. Mitochondrial respiration, electron transport chain complex activities, α‐ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. β‐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, α‐ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by β‐amyloid. The Km of cytochrome oxidase for reduced cytochrome c was raised by β‐amyloid. We conclude that β‐amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimers disease.

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

Abstract: The Ca2+‐independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 µg/ml) plus interferon‐γ (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‐γ 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.

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

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

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.

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.

Astrocyte–neurone communication following oxygen–glucose deprivation

We looked at the possible interactions between astrocytes and neurones during reperfusion using an in vitro model of ischaemia–reperfusion injury, as a controlled environment that lends itself easily to manipulation of the numerous variables involved in such an insult. We constructed a chamber in which O2 can be lowered to a concentration of 1 µm and developed a primary cortical neuronal culture that is 99% pure and can survive to at least 10 days in vitro. We also established a novel system for the co‐culture of astrocytes and neurones in order to study the communication between these cells in a manner that allows the complete separation of one cell type from another. Neurone cultures showed profound cell death following an ischaemic period of only 15 min. We co‐cultured neurones that had been subjected to a 15‐min ischaemic insult with either non‐insulted astrocytes or astrocyte‐conditioned medium during the reperfusion stage. Both astrocytes and astrocyte‐conditioned medium enhanced neuronal survival. Our data also suggest that astrocyte‐sourced neuronal glutathione synthesis may play a role in preventing neuronal death.

REGULATION OF THE KETOGENIC ENZYME MITOCHONDRIAL 3-HYDROXY-3- METHYLGLUTARYL-COA SYNTHASE IN ASTROCYTES AND MENINGEAL FIBROBLASTS Implications in Normal Brain Development and Seizure Neuropathologies

Metabolic fuels may be defined as complex carbon molecules that can be exploited for the energy released upon breakage of their carbon-carbon bonds. Fatty acids and hexoses, principally glucose, represent the two major classes of such complex carbon molecules that are used for fuel by eukaryotes. Typically, glucose is first metabolized anaerobically (glycolysis) to three-carbon molecules (pyruvate, phosphoenolpyruvate) which then provide the precursors of the tricarboxylic acid or Krebs cycle, ie: oxaloacetate and acetyl-CoA. The cycling of such intermediates via the Krebs cycle then effects the con-