4-hydroxy-2-nonenal and 4-maleimido-2-2-6-6-tetramethylpiperidinooxyl

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

Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid beta-peptide (A beta). A beta-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2-trans-nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of A beta and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1-10 microM HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and A beta-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 microM). The results obtained are discussed with relevance to the hypothesis of A beta-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.

Topics: Aldehydes; Animals; Cerebral Cortex; Cross-Linking Reagents; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Free Radicals; Gerbillinae; Intracellular Membranes; Kinetics; Lipid Peroxidation; Male; Membrane Lipids; Membrane Proteins; Models, Chemical; Phospholipids; Protein Conformation; Spin Labels; Synaptosomes