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 1mM ONOO−, whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10–1000μM) 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.

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-β 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.

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.

In recent years World Precision Instruments Inc. (WPI) produced for commercial use a selective and sensitive electrochemical sensor for the detection of the important biological free radical nitric oxide (NO). Though many kinds of NO sensors are now commercially available WPI offers a range of sensors of variable size and applicability for the detection of NO in vivo and in in vitro biomedical samples. This article overviews the working characteristics of the sensors and their utility for biomedical research.

The inner mitochondrial membrane is the site of the electron-transport chain (ETC), and is where the process of oxidative phosphorylation (OXPHOS) occurs that facilitates ATP synthesis. In view of the key role the ETC plays in energy metabolism, damage to one or more of the respiratory-chain complexes could lead to an impairment of cellular ATP formation. However, each of the complexes of the ETC appears to exert varying degrees of control over respiration. In vitro studies suggest that substantial loss of activity of an individual complex may be required before ATP synthesis is compromised. However, the degree of control a particular complex exerts over respiration may differ between cell types. The chapter presents descriptions of the predominant metabolic pathways located to mitochondria that are responsible for NADH and FADH2 generation. The optimal mitochondrial function is essential for cell survival. There is an increasing body of evidence to suggest that mitochondrial dysfunction occurs in a number of neurodegenerative disorders.

The level of phosphocreatine (PCr) and the intracellular pH (pH(i)) of superfused cortical brain slices from adult or 10-day-old rats were monitored using P-31 NMR. When the glucose in the superfusing medium was replaced by 3-hydroxybutyrate (3HB), there was a significant reduction in PCr of the adult but not the neonatal slices. The level of PCr of the adult slices was reduced by a greater amount by aglycaemic hypoxia compared with the neonatal brain slices and pH(i) was decreased by the same amount. After aglycaemic hypoxia, the levels of PCr of the neonatal slices recovered to the same extent when perfused with glucose or 3HB alone or a mixture of glucose and 3HB. The recovery of the PCr was significantly more in the neonatal than the adult brain slices with glucose alone after aglycaemic hypoxia, whereas pH(i) returned to control levels in both tissue types and with all substrates. The relative recovery of the PCr of the adult slices after aglycaemic hypoxia was the same with either 3HB or glucose. However, if glucose and 3HB were applied together, recovery of PCr was significantly improved compared with glucose alone.

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.

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.

Abstract: 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.

The role played by external calcium and calcium channels in the recovery from aglycaemic hypoxia in cortical brain slices from 10-day old rats was investigated by1H and31P NMR. 30 minutes of aglycaemic hypoxia significantly decreased the levels of phosphocreatine (PCr), ATP, lactate and intracellular pH (pHi). After a 30 minute recovery period there was incomplete recovery of PCr and ATP with lactate increasing by 50% with pHi normal. When the aglycemic hypoxia was carried out in media which had no added calcium (≈10 μM) the PCr and ATP recovery was significantly greater. Application of diltiazem or verapamil but not nifedipine significantly improved the recovery from the aglycemic hypoxia. These data suggest that calcium influx through L-type voltagegated calcium channels is involved in the ischemic damage in neonatal brain which manifests itself as a decrease in the energy state and an increase in lactate.

The effect of glutathione depletion, in vivo, on rat brain nitric oxide synthase activity has been investigated and compared to the effect observed in vitro with cultured neurones. Using L-buthionine sulfoximine rat brain glutathione was depleted by 62%. This loss of glutathione was accompanied by a significant increase in brain nitric oxide synthase activity by up to 55%. Depletion of glutathione in cultured neurones, by approximately 90%, led to a significant 67% increase in nitric oxide synthase activity, as judged by nitrite formation, and cell death. It is concluded that depletion of neuronal glutathione results in increased nitric oxide synthase activity. These findings may have implications for our understanding of the pathogenesis of neurodegenerative disorders in which loss of brain glutathione is considered to be an early event.

The effect of GTP concentration of forskolin-stimulated adenylyl cyclase activity was examined in synaptosomal membranes from 15-day-old rats that were hypothyroid owing 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.

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.

The abundances of G protein alpha-subunits (Gi1 alpha, Gi2 alpha, G0 alpha and Gq/ll alpha) were measured in synaptosomal membranes isolated from forebrain and hindbrain regions of euthyroid and hypothyroid neonatal rats at 10, 15, 20 and 25 days post-partum. The findings show that hypothyroidism causes a distinct perturbation of the normal developmental profile of these signalling components. It is suggested that these changes may contribute to some of the neurological deficits arising from hypothyroidism in early development.

Abstract: We report the isolation of cDNA clones encoding the somatic form of the E1α 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 E1α sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1α subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1α cDNA, in conjunction with cDNA probes to the E1β, 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.

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.

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.

cDNA clones encoding the testis-specific form of the rat pyruvate dehydrogenase complex E1 alpha subunit have been isolated. Comparison of the predicted amino acid sequence with those of the somatic and testis-specific E1 alpha forms of man and mouse and the somatic E1 alpha form of rat indicates the change of a serine residue, believed to be phosphorylated in vivo by pyruvate dehydrogenase E1 alpha-specific kinase, to an alanine at position 233. The implications of this change are discussed. Northern blot analysis and RNase protection assays indicate that the expression of mRNA encoding testis-specific E1 alpha subunit is restricted to testis whereas mRNA for the somatic form is found in all tissues analyzed, albeit in very small amounts in testis.