ABSTRACT-Amylold is a 39-to 43-amino-add neurotoxic peptide that aggregtes to form the core of Ahemer disease-sociated senile (amyloid) plaques. No EPR Spectrscopy and Spin Trapping. 8APs were solubilized to 1.0 mg/ml by addition of buffer [150 mM phosphatebuffered saline at pH 7.4 (PBS) or Hepes at pH 7.4] to lyophilized powder. BSA was solubilized similarly, to concentrations of 1-62 mg/ml. Buffer used for spin trapping contained 50 mM N-tert-butyl-a-phenylnitrone (PBN) (Sigma or Aldrich). In studies designed to inhibit putative metalcatalyzed reactions, the chelator deferoxamine mesylate or EDTA (Sigma) was dissolved to 10 mM in PBS/PBN prior to peptide addition. In a further attempt to nullify buffer-borne metals, PBS/PBN was stirred overnight with Chelex 100, a nonspecific metal-chelating agent.Peptide solutions were aliquoted into a 300-A4 aqueous quartz EPR flat cell that was subsequently sealed at both ends and immersed in a 3rC water bath for 0-135 hr and removed periodically for EPR analysis. EPR spectra were acquired on a Bruker (Billerica, MA) 300 EPR spectrometer equipped with computerized data acquisition software. Instrumental parameters were microwave power = 20 mW, modulation amplitude = 0.% G, gain = 1 x i1W, and conversion time =

The relationship between Alzheimer disease (AD) and aging is not currently known. In this study, postmortem frontal-and occipital-pole brain samples were obtained from 16 subjects with AD, 8 age-matched controls, and 5 young controls. These samples were analyzed both for protein oxidation products (carbonyl) and the activities of two enzymes vulnerable to mixed-function oxidation, glutamine synthetase and creatine kinase. Glutamine synthetase is more sensitive to mixed-function oxidation than creatine kinase. Carbonyl content rises exponentially with age, at double the rate in the frontal pole compared with the occipital pole. Compared with young controls, both aged groups (AD and age-matched controls) have increased carbonyl content and decreased glutamine synthetase and creatine kinase activities, which are more marked in the frontal than occipital pole in all instances. We conclude that protein oxidation products accumulate in the brain and that oxidation-vulnerable enzyme activities decrease with aging in the same regional pattern (frontal more affected than occipital). However, only glutamine synthetase activity distinguishes AD from age-matched controls: Because glutamine synthetase activity is differentially reduced in the frontal pole in AD, we suggest that AD may represent a specific brain vulnerability to age-related oxidation.Aging can be defined as the nonfunctional alteration of structure or homeostatic capability in an individual organism as it lives (1). A particular consequence of the aging process, at the cellular level, is the accumulation of proteins covalently modified by specific mechanisms-e.g., mixedfunction oxidation (MFO), deamidation, ubiquitin conjugation, and glycation (2-7). These steps have been reported to mark proteins for subsequent proteolysis.MFO is of particular interest because of the site-specific nature of the modifications (8-13), tending to occur near the metal-containing catalytic site of particular enzymes-e.g., glutamine synthetase. Free-radical-mediated forms of MFO could be one of the important mechanisms in normal aging (14)(15)(16), causing protein structural alterations, loss of enzymatic function, or interference with regulatory protein interactions.In this study, we provide evidence that the level of oxidatively modified protein is increased in the aging brain. We also demonstrate that in Alzheimer disease (AD), a disease strongly associated with aging, there is regional loss of glutamine synthetase activity. Glutamine synthetase is an enzyme particularly sensitive to MFO. We propose the hypothesis that AD is an alteration in the normal aging process in selectively vulnerable brain cells or brain areas that are challenged by increased oxidation with advancing age. MATERIALS AND METHODSBrain Tissue Samples. Brain specimens were obtained at autopsy from 16 patients with AD, 8 age-matched controls (AC), and 5 young controls (YC). Tissues were received from the Kentucky Medical Examiners Program and the University of Kentucky AD Research Center, under approv...

Four biomarkers of neuronal protein oxidation [W/S ratio of MAL‐6 spin‐labeled synaptosomes, phenylhydrazine‐reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)‐demented and age‐matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation‐sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque‐dense regions of the AD brain may represent environments of elevated oxidative stress.

There is increasing evidence that free radical damage to brain lipids, carbohydrates, proteins, and DNA is involved in neuron death in neurodegenerative disorders. The largest number of studies have been performed in Alzheimer's disease (AD) where there is considerable support for the oxidative stress hypothesis in the pathogenesis of neuron degeneration. In autopsied brain there is an increase in lipid peroxidation, a decline in polyunsaturated fatty acids (PUFA) and an increase in 4‐hydroxynonenal (HNE), a neurotoxic aldehyde product of PUFA oxidation. Increased protein oxidation and a marked decline in oxidative‐sensitive enzymes, glutamine synthetase and creatinine kinase, are found in the brain in AD. Increased DNA oxidation, especially 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) is present in the brain in AD. Immunohistochemical studies show the presence of oxidative stress products in neurofibrillary tangles and senile plaques in AD. Markers of lipid peroxidation (HNE, isoprostanes) and DNA (8‐OHdG) are increased in CSF in AD. In addition, inflammatory response markers (the complement cascade, cytokines, acute phase reactants and proteases) are present in the brain in AD. These findings, coupled with epidemiologic studies showing that anti‐inflammatory agents slow the progression or delay the onset of AD, suggest that inflammation plays a role in AD. Overall these studies indicate that oxidative stress and the inflammatory cascade, working in concert, are important in the pathogenetic cascade of neurodegeneration in AD, suggesting that therapeutic efforts aimed at both of these mechanisms may be beneficial.

Oxygen free radicals and oxidative events have been implicated as playing a role in bringing about the changes in cellular function that occur during aging. Brain readily undergoes oxidative damage, so it is important to determine if aging-induced changes in brain may be associated with oxidative events. Previously we demonstrated that brain damage caused by an ischemia/reperfusion insult involved oxidative events. In addition, pretreatment with the spintrapping compound N-tert-butyl-a-phenylnitrone (PBN) diminished the increase in oxidized protein and the loss of glutamine synthetase (GS) activity that accompanied ischemia/reperfusion injury in brain. We report here that aged gerbils had a significantly higher level of oxidized protein as assessed by carbonyl residues and decreased GS and neutral protease activities as compared to young adult gerbils. We also found that chronic treatment with the spin-trapping compound PBN caused a decrease in the level of oxidized protein and an increase in both GS and neutral protease activity in aged Mongolian gerbil brain. In contrast to aged gerbils, PBN treatment of young adult gerbils had no significant effect on brain oxidized protein content or GS activity. Male gerbils, young adults (3 months of age) and retired breeders (15-18 months of age), were treated with PBN for 14 days with twice daily dosages of 32 mg/kg. If PBN administration was ceased after 2 weeks, the significantly decreased level of oxidized protein and increased GS and neutral protease activities in old gerbils changed in a monotonic fashion back to the levels observed in aged gerbils prior to PBN administration. We also report that old gerbils make more errors than young animals and that older gerbils treated with PBN made fewer errors in a radial arm maze test for temporal and spatial memory than the untreated aged controls. These data can be interpreted to indicate that oxidation of cellular proteins may be a critical determinant of brain function. Moreover, it also implies that there is an age-related increase in vulnerability of tissue to oxidation that can be modified by free radical trapping compounds.