Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid~3-peptide (A/3). A~-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~3and 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 p~MHNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque-and A/3-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 HNEcaused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 /2M). The results obtained are discussed with relevance to the hypothesis of A/3-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.

The free radical theory of aging proposes that reactive oxygen species (ROS) cause oxidative damage over the lifetime of the subject. It is the cumulative and potentially increasing amount of accumulated damage that accounts for the dysfunctions and pathologies seen in normal aging. We have previously demonstrated that both normal rodent brain aging and normal human brain aging are associated with an increase in oxidative modification of proteins and in changes in plasma membrane lipids. Several lines of investigation indicate that one of the likely sources of ROS is the mitochondria. There is an increase in oxidative damage to the mitochondrial genome in aging and a decreased expression of mitochondrial mRNA in aging. We have used a multidisciplinary approach to the characterization of the changes that occur in aging and in the modeling of brain aging, both in vitro and in vivo. Exposure of rodents to acute normobaric hyperoxia for up to 24 h results in oxidative modifications in cytosolic proteins and loss of activity for the oxidation-sensitive enzymes glutamine synthetase and creatine kinase. Cytoskeletal protein spin labeling also reveals synaptosomal membrane protein oxidation following hyperoxia. These changes are similar to the changes seen in senescent brains, compared to young adult controls. The antioxidant spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) was effective in preventing all of these changes. In a related study, we characterized the changes in brain protein spin labeling and cytosolic enzyme activity in a series of phenotypically selected senescence-accelerated mice (SAMP), compared to a resistant line (SAMR1) that was derived from the same original parents. In general, the SAM mice demonstrated greater oxidative changes in brain proteins. In a sequel study, a group of mice from the SAMP8-sensitive line were compared to the SAMR1-resistant mice following 14 days of daily PBN treatment at a dose of 30 mg/kg. PBN treatment resulted in an improvement in the cytoskeletal protein labeling toward that of the normal control line (SAMR1). The results of these and related studies indicate that the changes in brain function seen in several different studies may be related to the progressive oxidation of critical brain proteins and lipids. These components may be critical targets for the beneficial effects of gerontotherapeutics both in normal aging and in disease of aging.

The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the secondorder reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated ßamyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.

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