4-maleimido-2-2-6-6-tetramethylpiperidinooxyl and Alzheimer-Disease

There is increasing evidence of DNA oxidation and altered DNA repair mechanisms in Alzheimer's disease (AD) brain. Histones, which interact with DNA, conceivably could provide a protective shield for DNA against oxidative stress. However, because of their abundant lysine residues, histones may be a target for 4-hydroxynonenal (HNE) modification. In this study, we have shown that HNE binds to histones and that this binding affects the conformation of the histone, measured by electron paramagnetic resonance in conjunction with a protein-specific spin label. The covalent modification to the histone by HNE affects the ability of the histone to bind DNA. Interestingly, acetylated histones appear to be more susceptible to HNE modifications than control histones. Conceivably, altered DNA-histone interactions, subsequent to oxidative modification of histones by the lipid peroxidation product HNE, may contribute to the vulnerability of DNA to oxidation in AD brain.

Topics: Acetylation; Aldehydes; Alzheimer Disease; Animals; Cattle; Cyclic N-Oxides; Cysteine Proteinase Inhibitors; DNA; Dose-Response Relationship, Drug; Drug Interactions; Electron Spin Resonance Spectroscopy; Histones; Oxidative Stress; Protein Binding; Protein Conformation; Sodium Chloride; Thymus Gland; Time Factors

Several functional differences have been reported among the three human e2, e3, and e4 alleles of apolipoprotein E (apoE). One functional difference lies in the antioxidant potential of these alleles; e4 has the poorest potential. Interestingly, e4 also correlates with increased oxidative damage in the Alzheimer's disease (AD) brain, which may explain why the inheritance of the e4 allele is a risk factor for the onset of AD. Beta-amyloid (Abeta) is also intimately involved in AD and promotes oxidative damage in vitro; therefore, we have examined the role of the different apoE alleles in modulating Abeta(1-42)-induced oxidation to synaptosomes. Measurement of specific markers of oxidation in synaptosomes isolated from mice that express one of the human apoE alleles indicates that Abeta-induced increases of these markers can be modulated by apoE in an allele-dependent manner (e2>e3>e4). Increases in reactive oxygen species formation and protein and lipid oxidation were always greatest in e4 synaptosomes as compared to e2 and e3 synaptosomes. Our data support the role of apoE as a modulator of Abeta toxicity and, consistent with the antioxidant potentials of the three alleles, suggest that the e4 allele may not be as effective in this role as the e2 or e3 alleles of apoE. These results are discussed with reference to mechanistic implications for neurodegeneration in the AD brain.

Topics: Aldehydes; Alleles; Alzheimer Disease; Amyloid beta-Peptides; Animals; Apolipoproteins E; Brain; Cyclic N-Oxides; Lipid Peroxidation; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neurons; Oxidative Stress; Peptide Fragments; Reactive Oxygen Species; Synaptosomes; Thiobarbiturates

Peroxynitrite (ONOO ) is a highly reactive, oxidizing anion with a half-life of <1 s that is formed by reaction of superoxide radical anion with nitric oxide. Several reports of ONOO--induced oxidation of lipids, proteins, DNA, sulfhydryls, and inactivation of key enzymes have appeared. ONOO- has also been implicated as playing a role in the pathology of several neurodegenerative disorders, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis, among others. Continuing our laboratory's interest in free radical oxidative stress in brain cells in AD, the present study was designed to investigate the damage to brain neocortical synaptosomal membrane proteins and the oxidation-sensitive enzyme glutamine synthetase (GS) caused by exposure to ONOO-. These synaptosomal proteins and GS have previously been shown by us and others to have been oxidatively damaged in AD brain and also following treatment of synaptosomes with amyloid beta-peptide. The results of the current study showed that exposure to physiological levels of ONOO- induced significant protein conformational changes, demonstrated using electron paramagnetic resonance in conjunction with a protein-specific spin label, and caused oxidation of proteins, measured by the increase in protein carbonyls. ONOO- also caused inactivation of GS and led to neuronal cell death examined in a hippocampal cell culture system. All these detrimental effects of ONOO- were successfully attenuated by the thiol-containing antioxidant tripeptide glutathione. This research shows that ONOO- can oxidatively modify both membranous and cytosolic proteins, affecting both their physical and chemical nature. These findings are discussed with reference to the potential involvement of ONOO- in AD neurodegeneration.

Topics: Alzheimer Disease; Animals; Cell Survival; Cells, Cultured; Cyclic N-Oxides; Cytosol; Gerbillinae; Glutathione; Hippocampus; Male; Membrane Proteins; Neurons; Nitrates; Nitric Oxide; Oxidants; Oxidation-Reduction; Oxidative Stress; Spin Labels; Synaptosomes

Given the increasing evidence of oxidative stress in AD brain and studies from different perspectives that appear to show a converging, central role for A beta in the pathogenesis and etiology of AD, insight into A beta-associated free radical oxidative stress will likely lead to a greater understanding of AD and, potentially, to better therapeutic strategies in this disorder. This article outlined methods to investigate markers of oxidative stress induced by A beta in brain membrane systems. Especially important are markers for protein oxidation, lipid peroxidation, and ROS generation by A beta. Oxidative stress and its sequelae are likely related to both necrotic and apoptotic mechanisms of neurotoxicity, and A beta-associated free radical oxidative stress may be of fundamental importance in Alzheimer's disease etiology and pathogenesis. The methods described here provide some means for investigating this possibility.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Biomarkers; Cell Count; Cerebral Cortex; Creatine Kinase; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radicals; Glutamate-Ammonia Ligase; Hippocampus; Humans; Mitochondria; Neurons; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Spin Labels; Synaptosomes; Tetrazolium Salts; Thiazoles; Trypan Blue

The oral administration of 9-amino-1,2,3,4-tetrahydroacridine (THA) is purported to increase the mental function of Alzheimer's disease patients (Summers et al. (1986) N. Engl. J. Med. 315, 1241-1245). Numerous erythrocyte membrane proteins are known to be identical or highly similar to neuronal proteins. In a previous study (Butterfield and Palmieri [1990) Free Radical Res. Commun., in press), we showed that THA greatly increased skeletal protein-protein interactions in erythrocyte membranes as monitored by a spin label specifically bound to membrane proteins. In this report, a structure-activity study has been performed to determine which THA structural components are involved in its effect on the physical state of human erythrocyte membrane skeletal proteins. The results imply that both the planarity of the molecule and the amino group at the 9-position of the parent acridine molecule are important in the mechanism of interaction with membrane proteins.

Topics: Alzheimer Disease; Aminoacridines; Cyclic N-Oxides; Erythrocyte Membrane; Humans; Membrane Proteins; Spin Labels; Structure-Activity Relationship; Tacrine