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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.

Peroxidation of membrane lipids results in release of the aldehyde 4‐hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid β‐peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25‐35‐induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein‐bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time‐ and concentration‐dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na+,K+‐ATPase activity and induced an increase of neuronal intracellular free Ca2+ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ‐induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.

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Exposure of cultured rat hippocampal neurons to glutamate resulted in accumulation of cellular peroxides (measured using the dye 2,7‐dichlorofluorescein). Peroxide accumulation was prevented by an N‐methyl‐d‐aspartate (NMDA) receptor antagonist and by removal of extracellular Ca2+, indicating the involvement of NMDA receptor‐induced Ca2+ influx in peroxide accumulation. Glutamate‐induced reactive oxygen species contributed to loss of Ca2+ homeostasis and excitotoxic injury because antioxidants (vitamin E, propyl gallate, and N‐tert‐butyl‐α‐phenylnitrone) suppressed glutamate‐induced elevation of intracellular Ca2+ concentration ([Ca2+]i) and cell death. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain‐derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor, each suppressed accumulation of peroxides induced by glutamate and protected neurons against excitotoxicity. bFGF, NGF, and BDNF each increased (to varying degrees) activity levels of superoxide dismutases and glutathione reductase. NGF increased catalase activity, and BDNF increased glutathione peroxidase activity. The ability of the neurotrophic factors to suppress glutamate toxicity and glutamate‐induced peroxide accumulation was attenuated by the tyrosine kinase inhibitor genistein, indicating the requirement for tyrosine phosphorylation in the neuroprotective signal transduction mechanism. The data suggest that glutamate toxicity involves peroxide production, which contributes to loss of Ca2+ homeostasis, and that induction of antioxidant defense systems is a mechanism underlying the [Ca2+]i‐stabilizing and excitoprotective actions of neurotrophic factors.

We determined levels of thiobarbituric acid-reactive substances (TBARS), a measure of lipid peroxidation, and the activity of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), and catalase (CAT) in the amygdala, hippocampus, pyriform cortex, superior and middle temporal gyri, inferior parietal lobule, middle frontal gyrus, occipital pole, and cerebellum of 13 Alzheimer's disease (AD) and 10 control brains. Levels of TBARS were elevated in all AD brain regions except the middle frontal gyrus, and elevation levels reached statistical significance in the hippocampus and pyriform cortex and marginal significance in the amygdala of AD subjects compared with age-matched controls. Significant elevation of GSH-Px activity was present in AD hippocampus compared with control. Moderate but statistically insignificant elevations of GSH-Px activity also were present in the amygdala and pyriform cortex in AD. GSSG-R activity was significantly elevated in the amygdala and hippocampus in AD subjects compared with controls. CAT activity was significantly elevated in AD hippocampus and superior and middle temporal gyri. SOD levels were elevated in all brain regions in AD patients compared with controls, although none of these elevations reached statistical significance. Antioxidant enzyme activities were significantly elevated where lipid peroxidation was most pronounced, suggesting a compensatory rise in antioxidant activity in response to increased free radical formation. This study supports the concept that the brain in AD is under increased oxidative stress and demonstrates that the oxidative changes are most pronounced in the medial temporal lobe, where histopathologic alterations are most severe.

Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD. Keywords: Alzheimer's disease, gas chromatography/ mass spectrometry, 8-hydroxyguanine, mitochondrial DNA, nuclear DNA, oxidative stress. Alzheimer's disease (AD) is a progressive, irreversible, neurodegenerative disorder. The key to understanding AD is to elucidate the pathogenesis of neuron degeneration in specific brain regions. AD is associated with several risk factors, including age, genetic factors, presence of apolipoprotein E-4 alleles, educational attainment, head injury, hyperhomocysteinemia and diet (Schofield and Mayeux 1998;Seshadri et al. 2002).Increasing evidence suggests that oxidative stress and damage are associated with aging. The free radical theory of aging suggests that aging is due to the cumulative damage from free radical mediated reactions (Harman 1973; Smith et al. 2000a,b;Harman 2003). Mitochondria are primary sites of free radical production and their DNA and protein may be more easily oxidized than in other organelles (Wallace 1992(Wallace , 1999. Mitochondria produce ATP at the inner membrane through coupling of oxidative phosphorylation with respiration (Eckert et al. 2003), which is the major endogenous source of reactive oxygen species (ROS) (Sampson et al. 1998;Smith et al. 1998). During respiration, 2% of total oxygen consumed by the cell is converted into superoxide radicals by leaked electrons (Halliwell and Gutteridge 1989).Because of the proximity to ROS, lack of protective histones, and limited repair mechanisms (Wallace 1992;Ames et al. 1993), mitochondrial DNA (mtDNA) is susceptible to oxidative damage. These alterations could theoretically cause functional consequences because it has no noncoding sequences (Wallace 1992). As a consequence of ox...