Oxidized lipoproteins, beta amyloid peptides and Alzheimer's disease.

Recent studies have provided strong evidence for the involvement of oxidative stress in the pathogenesis of Alzheimer's disease (AD) and beta-amyloid peptides (ABeta) have been implicated to play an important role in mediating these oxidative events. Lipoproteins (LP) in the brain are likely targets of oxidative insult and together enhance ABeta -mediated toxicity to neurons. We hypothesize that uptake of oxidized LP by neuron leads to an acceleration of the intracellular oxidative pathways and exacerbation of neuron cell death. In our previous studies, we demonstrated the ability of oxidized low-density LP from plasma to induce cell death in PC12 cells. In this study, a synthetic LP fraction was prepared using lipids extracted from rat brain and incubated with albumin and apoE. This brain lipid-derived LP (BLP) was subjected to oxidation by incubation with Fe(3+)and subsequently tested with primary cortical neurons in culture. To study uptake of the BLP, native and oxidized BLP containing apoE3 or apoE4 were labeled with [(14)C]cholesterol or the fluorescent probe 3,3-dioctadecylindo-carbocyanine (Di-I) prior to exposing to cultured neurons. Results showed that regardless of the labeling method, oxidized BLP were more effectively taken up by the neurons than the native BLP. Cell viability was assessed by assaying the release of lactate dehydrogenase (LDH) into the medium and by determining the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), an agent depicting mitochondrial activity. While exposure of neurons to oxidized BLP and aggregated ABeta (1-42) alone could result in MTT reduction (24%), greater reduction (40%) could be observed when oxidized LP was added together with ABeta. Neuronal cell death due to oxidized BLP could be ameliorated by resveratrol, a polyphenolic compound known for its antioxidant properties. Taken together, these results are in agreement with the notion that ABeta and oxidized BLP can synergistically enhance oxidative damage in neurons and antioxidants such as resveratrol can ameliorate these damages.

Grape polyphenols protect neurodegenerative changes induced by chronic ethanol administration.

Increased oxidative stress in the brain due to chronic ethanol consumption is known to result in a number of neurodegenerative changes. This study was designed to test whether dietary supplementation of grape polyphenols (GP) can offer protection to the neurodegenerative changes resulting from chronic ethanol consumption. Sprague-Dawley rats were fed a Leiber-DeCarli liquid diet with ethanol or isocaloric amount of maltose, and with or without GP for 2 months. Chronic ethanol caused significant decreases in synaptosomal Na,K-ATPase (20.5%) and dopamine uptake (22.8%) activities compared with pair-fed controls. Although GP alone did not alter activities of these membrane-bound proteins, GP supplementation was able to completely protect the decrease in synaptic protein function elicited by chronic ethanol consumption.

Oxidized lipoproteins may play a role in neuronal cell death in Alzheimer disease.

Oxidative stress in the central nervous system (CNS) may cause oxidation of lipoprotein particles. The oxidized lipoproteins may damage cellular and subcellular membranes, leading to tissue injury and cell death. Human low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) are oxidized by transition metal ions, such as Cu2+. Using PC 12 cells, we tested the cytotoxicity of oxidized LDL and VLDL. Cell death was increased in a dose-dependent manner. Antioxidants added to the incubation medium, such as vitamins E or C, or resveratrol showed some protection. Results indicated that oxidized lipoproteins may serve as an oxidative stressor, which may initiate the neuronal cell death leading to the manifestation of Alzheimer disease (AD).

Oxidized lipoproteins activate NF-kappaB binding activity and apoptosis in PC12 cells.

Oxidative stress in the central nervous system may cause oxidation of lipoproteins. The oxidized lipoproteins may in turn damage cellular and subcellular membranes and other biomolecules, leading to tissue injury and cell death. Recently, we have demonstrated that oxidized LDL and VLDL induced cell death in a dose-dependent manner. The present study examined the possible signal transduction cascade leading to cell death by oxLDL and oxVLDL in PC12 cells. Using the electrophoretic mobility shift assay, we found that both oxLDL and oxVLDL activated the binding of NF-kappaB to the consensus sequence in the promoter region of the target genes, followed by apopototic cell death. Resveratrol protects the cells from both the activation of NF-kappa-B/ DNA binding activity and apoptotic cell death. Results indicated that oxidized lipoproteins may serve as an oxidative mediator and may activate apoptosis through a nuclear signalling pathway contributing to the pathology in Alzheimer's disease.

Amelioration of oxidative stress by antioxidants and resveratrol in PC12 cells.

The goal of this study was to investigate the effect of resveratrol, an active ingredient found in grapes and other plant products, in ameliorating oxidative stress. Oxidative stress was induced by addition of Fe2+ and t-butyl hydroperoxide to the cultured PC12 cell medium. Resveratrol, vitamins C and/or E, were added to the cell culture medium during oxidative stress. The combination of resveratrol and vitamins C and/or E was more effective in protecting the cell than was any of these three antioxidants alone.

Dihydroxy-acid dehydratase, a [4Fe-4S] cluster-containing enzyme in Escherichia coli: effects of intracellular superoxide dismutase on its inactivation by oxidant stress.

Dihydroxy-acid dehydratase (DHAD) has a [4Fe-4S] cluster and is reported to be facilely inactivated by oxidant stress. To directly assess the biological effects in vivo of superoxide dismutase (SOD) on the oxidant sensitivity of DHAD, we used an Escherichia coli K-12 parent strain (CGSC5073) and derived strains OB 1, OB 2, and OB 3 that lacked one of or both FeSOD and MnSOD. In the K-12 parent strain half the cellular DHAD activity was lost in 15 min at 0.8 atm oxygen, less than 10 microM aerobic nitrofurantoin, or about 5 microM aerobic paraquat (PQ) and in about 1 min at 10 microM aerobic PQ. Oxygen and metabolism were required for PQ to inactivate DHAD in cells; adding dithiothreitol to cell-free extracts did not restore DHAD activity. The Km was not appreciably changed for DHAD that was 50 and 70% inactivated in cells, respectively, by hyperbaric oxygen (HBO) and PQ, compared to cells in exponential, aerobic growth. Thus, active site oxidative impairment of individual enzyme molecules apparently was all-or-none. DHAD activity was greatly decreased when measured in extracts made from strains that lacked both SODs unless SOD was added to cell suspensions before extracts were made. DHAD was more sensitive in strains lacking both SODs than in the parent strain to inactivation by aerobic PQ and HBO. Anaerobic (compared to aerobic) growth increased DHAD specific activity by 20% or less in the parent strain and in strains OB 1 and OB 2 (lacking MnSOD and FeSOD, respectively); however, in strain OB 3 (lacking both SODs) DHAD was increased 60%. DHAD was partially inactivated by the oxidant stress of aerobic growth, but remained in a form detectable by DHAD antibody, and the ratio of active to inactive DHAD decreased greatly in cells lacking SOD. Thus, SOD helped maintain DHAD as an active holoenzyme and benefitted cells growing aerobically or when exposed to low levels of PQ.

Asparagine synthetase: an oxidant-sensitive enzyme in Escherichia coli.

Oxidant stress inhibits the growth of Escherichia coli, which is partially relieved by adding asparagine to the culture medium. Asparagine synthetase (AS), assayed using hydroxylamine as an amino donor, was decreased in a concentration-dependent manner by exposure of cultures to oxygen from near-anaerobic to hyperbaric oxygen (HBO) and by aerobic, but not by anaerobic, paraquat. The specific activity of AS was not decreased when cells were exposed to HBO without a carbon and energy source. HBO caused less AS inactivation in cells containing mutations in both superoxide dismutase (SOD) genes and producing no active SOD. Whether or not cells had catalase had no effect on HBO sensitivity of AS. Aerobic paraquat depressed AS less in cells lacking either catalase or superoxide dismutases. Cells which were decompressed following HBO poisoning had AS restored to normal activity whether or not chloramphenicol was present. These results indicate that asparagine synthetase is oxidant-sensitive; paraquat requires aerobic conditions and HBO requires energy metabolism for AS inactivation; and cells can repair oxidatively-damaged enzyme molecules. The failure of superoxide dismutase or catalase to protect AS suggests that its oxidant-inactivation in cells is not a simple effect of superoxide or hydrogen peroxide.

The inactivation of dihydroxy-acid dehydratase in Escherichia coli treated with hyperbaric oxygen occurs because of the destruction of its Fe-S cluster, but the enzyme remains in the cell in a form that can be reactivated.

The enzyme dihydroxy-acid dehydratase previously has been shown to be inactivated in vivo in Escherichia coli within minutes of exposure to hyperbaric O2. In this paper, we show its inactivation is due to the destruction of its catalytically active [4Fe-4S] cluster. The inactivation is not followed by an appreciable decrease in the amount of dihydroxy-acid dehydratase protein as determined by Western blots. Thus, the protein from the inactivated enzyme remains unproteolyzed in the cells. Dihydroxy-acid dehydratase activity recovers after the cells treated with hyperbaric O2 are returned to ambient oxygen. Since this recovery in activity is not accompanied by a significant increase in dihydroxy-acid dehydratase protein and is not prevented by chloramphenicol, it appears primarily to be due to reactivation of the previously inactivated enzyme. The reactivation occurs by reconstitution of the enzyme's Fe-S cluster. These results demonstrate that this enzyme can cycle between forms in which the Fe-S cluster is either present or absent. The facile ability to cycle between these two forms would be compatible with a regulatory role in addition to a catalytic role for this enzyme.

Protein A of quinolinate synthetase is the site of oxygen poisoning of pyridine nucleotide coenzyme synthesis in Escherichia coli.

De novo biosynthesis of pyridine nucleotide coenzymes in Escherichia coli is initiated by an enzyme complex (quinolinate synthetase) containing protein B which converts L-aspartate into iminoaspartate and protein A, which then generates quinolinate on the pathway to the coenzymes. This complex has been shown to be poisoned by hyperbaric oxygen. We performed assays made dependent on both proteins B and A versus only protein A, using cell-free extracts of hyperbaric-oxygen poisoned and aerobically grown cells. The specific activities were reduced by similar amounts of 68% and 60%, respectively, when measured in assays made dependent on enzymes B and A virus only protein A that was derived from oxygen-poisoned extract. Thus, protein A is the oxygen-sensitive component.