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 =

No abstract

No abstract

No abstract

Oxidative stress, manifested by protein oxidation, lipid peroxidation, DNA oxidation and 3-nitrotyrosine formation, among other indices, is observed in Alzheimer's disease (AD) brain. Amyloid beta-peptide (1-42) [Abeta(1-42)] may be central to the pathogenesis of AD. Our laboratory and others have implicated Abeta(1-42)-induced free radical oxidative stress in the neurodegeneration observed in AD brain. This paper reviews some of these studies from our laboratory. Recently, we showed both in-vitro and in-vivo that methionine residue 35 (Met-35) of Abeta(1-42) was critical to its oxidative stress and neurotoxic properties. Because the C-terminal region of Abeta(1-42) is helical, and invoking the i + 4 rule of helices, we hypothesized that the carboxyl oxygen of lle-31, known to be within a van der Waals distance of the S atom of Met-35, would interact with the latter. This interaction could alter the susceptibility for oxidation of Met-35, i.e. free radical formation. Consistent with this hypothesis, substitution of lle-31 by the helix-breaking amino acid, proline, completely abrogated the oxidative stress and neurotoxic properties of Abeta(1-42). Removal of the Met-35 residue from the lipid bilayer by substitution of the negatively charged Asp for Gly-37 abrogated oxidative stress and neurotoxic properties of Abeta(1-42). The free radical scavenger vitamin E prevented A(beta (1-42)-induced ROS formation, protein oxidation, lipid peroxidation, and neurotoxicity in hippocampal neurons, consistent with our model for Abeta-associated free radical oxidative stress induced neurodegeneration in AD. ApoE, allele 4, is a risk factor for AD. Synaptosomes from apoE knock-out mice are more vulnerable to Abeta-induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. We also studied synaptosomes from allele-specific human apoE knock-in mice. Brain membranes from human apoE4 mice have greater vulnerability to Abeta(1-42)-induced oxidative stress than brain membranes from apoE2 or E3, assessed by the same indices, consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Using immunoprecipitation of proteins from AD and control brain obtained no longer than 4h PMI, selective oxidized proteins were identified in the AD brain. Creatine kinase (CK) and beta-actin have increased carbonyl groups, an index of protein oxidation, and Glt-1, the principal glutamate transporter, has increased binding of the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE). Abeta inhibits CK and causes lipid peroxidation, leading to HNE formation. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. Other oxidatively modified proteins have been identified in AD brain by proteomics analysis, and these oxidatively-modified proteins may be related to increased excitotoxicity (glutamine s...

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.

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O À:2 leads to formation of peroxynitrite ONOO -, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration. Keywords: Alzheimer's disease, neurodegeneration, 3-nitrotyrosine, proteomics, reactive nitrogen species. 2 leads to formation of peroxynitrate ONOO ) , which, following protonation, generates cytotoxic species that oxidize and nitrate proteins (Beckman 1996). (Formally, the NO 2 added to tyrosine is a nitrite, not nitrate, but the literature has consistently used the latter term, so we shall as well in this paper.)The more common amino acidic targets of oxidation are lysine, histidine, cysteine and methionine (Butterfield and Stadtman 1997), whereas tyrosine is the commonly nitrated amino acid (Souza et al. 2001). In particular, nitration of tyrosine residues is a formal oxidation (Butterfield and Stadtman 1997), a chemical modification that has been used to investigate the mechanism by which tyrosine residues Received November 11, 2002; revised manuscript received February 19, 2003; accepted February 20, 2003. Address correspondence and reprint requests to Professor D. Allan Butterfield, Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506-0055, USA. E-mail: dabcns@uky.eduAbbreviations used: AD, Alzheimer's disease; HCNP, hippocampal cholinergic neurostimulating peptide; IPL, inferior parietal lobule; 3NT, 3-nitrotyrosine; PEBP, phosphatidylethanolamine-binding protein; PBST, phosphate-buffered saline with 0.01% sodium azide and 0.2% Twe...

Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation-related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for laborintensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy-terminal hydrolase L)1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP-2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme a-enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC-71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain.