Reactive oxygen species, cell signaling, and cell injury.

Oxidative stress has traditionally been viewed as a stochastic process of cell damage resulting from aerobic metabolism, and antioxidants have been viewed simply as free radical scavengers. Only recently has it been recognized that reactive oxygen species (ROS) are widely used as second messengers to propagate proinflammatory or growth-stimulatory signals. With this knowledge has come the corollary realization that oxidative stress and chronic inflammation are related, perhaps inseparable phenomena. New pharmacological strategies aimed at supplementing antioxidant defense systems while antagonizing redox-sensitive signal transduction may allow improved clinical management of chronic inflammatory or degenerative conditions, including Alzheimer's disease. Introduction of antioxidant therapies into mainstream medicine is possible and promising, but will require significant advances in basic cell biology, pharmacology, and clinical bioanalysis.

Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer's disease brain.

Increasing evidence supports the role of reactive oxygen species (ROS) in the pathogenesis of Alzheimer's disease (AD). Both in vivo and in vitro studies demonstrate that thioredoxin (Trx) and thioredoxin reductase (TR), the enzyme responsible for reduction of oxidized Trx, have protective roles against cytotoxicity mediated by the generation of ROS. The present study measured levels of Trx protein and activities of TR in the brain in AD compared with control subjects, and evaluated the possible protective role of TR and Trx against amyloid beta-peptide (Abeta) toxicity in neuronal cultures. Analysis of Trx protein levels in 10 AD and 10 control subjects demonstrated a general decrease in all AD brain regions studied, with statistically significant decreases in the amygdala (p <.05), hippocampus/parahippocampal gyrus (p <.05), and marginally significant (p <.10) depletions in the superior and middle temporal gryi. Thioredoxin reductase activity levels were increased in all AD brain regions studied with statistically significant increases occurring in AD amygdala (p =.01) and cerebellum (p =.007). To investigate the protective effects of Trx and TR against Abeta-induced toxicity, primary hippocampal cultures were treated with Trx or TR in combination with toxic doses of Abeta. Treatment of cultures with Trx led to a statistically significant concentration-dependent enhancement in cell survival against Abeta-mediated toxicity as did treatment with TR. Together, these data suggest that, although TR is protective against Abeta-mediated toxicity, the increase observed in AD brain offers no protection due to the significant decrease in Trx levels. This decrease in the antioxidant Trx-TR system may contribute to the increased oxidative stress and subsequent neurodegeneration observed in the brain in AD.

Decrease in peptide methionine sulfoxide reductase in Alzheimer's disease brain.

Previous studies have shown that the pathophysiology of Alzheimer's disease (AD) is linked to oxidative stress. Oxidative damage to different biomolecular components of the brain is a characteristic feature of AD. Recent evidence suggests that methionine may act as an antioxidant defense molecule in proteins by its ability to scavenge oxidants and, in the process, undergo oxidation to form methionine sulfoxide. The enzyme peptide, methionine sulfoxide reductase (MsrA), reverses methionine sulfoxide back to methionine, which once again is able to scavenge oxidants. The purpose of this study was to measure the activity of MsrA in the brain of AD patients compared with control subjects. Our results showed that there was a decline in MsrA activity in all brain regions studied in AD and this decline reached statistical significance in the superior and middle temporal gyri (p < 0.001), inferior parietal lobule (p < 0.05), and the hippocampus (p < 0.05) in AD. An elevation of protein carbonyl content was found in all brain regions except the cerebellum in AD and reached statistical significance in the superior and middle temporal gyri and hippocampus. Messenger RNA analysis suggests that the loss in enzyme activity may be the result of a posttranslational modification of MsrA or a defect of translation resulting in inferior processing of the MsrA mRNA. Our results suggest that a decline in MsrA activity could reduce the antioxidant defenses and increase the oxidation of critical proteins in neurons in the brain in AD.

Oxidized lipoproteins increase reactive oxygen species formation in microglia and astrocyte cell lines.

Lipoproteins exist in the central nervous system and surrounding vasculature possibly mediating effects upon cells in the brain during times of oxidative stress or compromised blood-brain barrier. The focus of the present study was to determine the effect of unmodified and oxidatively modified lipoproteins on astrocytes and microglia. Application of oxidized low-density lipoprotein resulted in an increase in DCF fluorescence, which was inhibited by pretreatment with antioxidants, consistent with the formation of reactive oxygen species (ROS). Low-density at concentrations below 20 microg/ml likewise increased ROS formation. Because ROS are associated with numerous astrocyte and microglia activities including proliferation, activation, and cytokine production it is possible that lipoproteins may mediate such effects on glial cells in the central nervous system.

Opposing actions of native and oxidized lipoprotein on motor neuron-like cells.

Lipoproteins are present in the central nervous system and surrounding vasculature and possibly mediate effects relevant to neuronal physiology and pathology. To determine the effects of lipoproteins on motor neurons, native low density lipoproteins (LDL) and oxidized LDL (oxLDL) were applied to a motor neuron cell line. Oxidized LDL, but not native LDL, resulted in a dose- and time-dependent increase in reactive oxygen species and neuron death. Oxidized LDL-induced toxicity was attenuated by a calcium chelator, antioxidants, caspase inhibitors, and inhibitors of macromolecular synthesis. In addition to being nontoxic, application of native LDL attenuated reactive oxygen species formation and neuron loss following glucose deprivation injury. Together, these data demonstrate a possible neuroprotective role for unmodified lipoproteins and suggest oxidized lipoproteins may amplify oxidative stress and neuron loss.

Increased DNA oxidation and decreased levels of repair products in Alzheimer's disease ventricular CSF.

One of the leading etiologic hypotheses regarding Alzheimer's disease (AD) is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Although several recent studies show an increase in levels of brain DNA oxidation in both aging and AD, there have been no studies of levels of markers of DNA oxidation in ventricular CSF. This is a study of levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, in intact DNA and as the "free" repair product that results from repair mechanisms. Free 8-OHdG was isolated from CSF from nine AD and five age-matched control subjects using solid-phase extraction columns and measured using gas chromatography/mass spectrometry with selective ion monitoring. Intact DNA was isolated from the same samples and the levels of 8-OHdG determined in the intact structures. Quantification of results was carried out using stable isotope-labeled 8-OHdG. By using this sensitive methodology, statistically significant elevations (p < 0.05) of 8-OHdG were observed in intact DNA in AD subjects compared with age-matched control subjects. In contrast, levels of free 8-OHdG, removed via repair mechanisms, were depleted significantly in AD samples (p < 0.05). Our results demonstrate an increase in unrepaired oxygen radical-mediated damage in AD DNA as evidenced by the increased presence of 8-OHdG in intact DNA and decreased concentrations of the free repair product. These data suggest that the brain in AD may be subject to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for removal of oxidized bases.

Increased nuclear DNA oxidation in the brain in Alzheimer's disease.

Multiple lines of evidence indicate that oxidative stress is a contributor to neuronal death in Alzheimer's disease (AD). The oxidative damage that occurs to DNA may play a role in both normal aging and neurodegenerative diseases, including AD. This is a study of the oxidative damage that occurs in nuclear DNA in the brains of AD patients and cognitively intact, prospectively evaluated, age-matched control subjects. Nuclear DNA from frontal, temporal, and parietal lobes and cerebellum was isolated from 11 control subjects and 9 AD subjects, and oxidized purine and pyrimidine bases were quantitated using gas chromatography/mass spectrometry. Stable isotope-labeled oxidized base analogues were used as internal standards to measure 5-hydroxyuracil, 5-hydroxycytosine, 8-hydroxyadenine, 4,6-diamino-5-formamidopyrimidine (Fapy-adenine), 8-hydroxyguanine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-guanine). Statistically significant elevations of 5-hydroxycytosine, 5-hydroxyuracil, 8-hydroxyadenine, and 8-hydroxyguanine were found in AD brain compared with control subjects (p < 0.05). There was an increased trend in the levels of Fapy-adenine in the AD brain, and Fapy-guanine showed a trend toward higher levels in control brains compared with AD. A generally higher level of oxidative DNA damage was present in neocortical regions than cerebellum. No significant correlation was observed between the oxidized bases and neurofibrillary tangle and senile plaque counts. Our results demonstrate that nuclear DNA damage by oxygen-derived radicals is increased in AD and support the concept that the brain is under increased oxidative stress in AD.

Effects of mitochondrial respiratory stimulation on membrane lipids and proteins: an electron paramagnetic resonance investigation.

Previous studies have implicated mitochondria-derived reactive oxygen species (ROS) in both the aging process and age-related diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease etc. The current study, utilizing electron paramagnetic resonance (EPR) spectrometry, was designed to determine if mitochondrial respiratory stimulation, under state 4 conditions, caused extensive oxidative modifications to membrane cytoskeletal proteins and lipids in the brain. A mixed population of cortical synaptosomes and mitochondria, prepared by centrifugation techniques using rat brain cortex from adult (4-6 months) female Brown Norway rat brains, were labeled with the lipid-specific spin probe, 5-nitroxyl stearate (5-NS). Stimulation of the mitochondrial electron transport chain was accomplished using 20 mM succinate at 25 degrees C for 3 h. Mitochondrially derived free radicals, when reacted with the paramagnetic center of the spin probe, result in a loss of paramagnetism resulting in loss of intensity. A significant lowering (23%, P<0.0001) in the signal amplitude (B0) of 5-NS, indicative of generation of oxyradicals, was found. The order parameter, an inverse EPR-measure of membrane fluidity of the 5-NS spin labeled mitochondrial and synaptosomal membranes, also decreased following mitochondrial respiratory stimulation (P<0.005). Changes in the physical state of cytoskeletal and transmembrane proteins due to succinate oxidation were measured using MAL-6 (2,2,6, 6,-tetramethyl-4 maleimidopiperdin-1-oxyl), a thiol-specific nitroxide spin label. The ratio of the amplitudes of the weakly to strongly immobilized spin label reaction sites (W/S ratio) in the low-field region of the spectrum was used to determine any alteration in protein conformation. Previous studies in our laboratory have established that increased protein oxidation is associated with a decreased W/S ratio. In the current study, our results indicated significant lowering of the W/S ratio in cortex (30%, P<0.0001) upon stimulation of the mitochondria with 20 mM succinate. Thus, we conclude that respiratory stimulation of mitochondria, due to a hypermetabolic stress with succinate, caused significant oxidative modifications of cortical membrane lipids and proteins.

Aging and caloric restriction affect mitochondrial respiration and lipid membrane status: an electron paramagnetic resonance investigation.

Previous studies have indicated that reactive oxygen species (ROS) are likely involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). ROS, generated by succinate-stimulated mitochondria, have been reported to be spin trapped and detected by electron paramagnetic resonance (EPR). Our aim in the current study was to study the impact of aging on the effect of increased metabolic stimuli on mitochondrial respiration in terms of oxy-radical generation and possible lipid peroxidative changes in brain neocortical membranes. A mixed population of brain synaptosomes and mitochondria from brown norway male rats of differing ages being fed either ad lib (AL) or a caloric-restricted diet (DR) was prepared and labeled with 5-nitroxyl stearate (5-NS), a membrane lipid-specific spin label. The changes in anisotropic motion of the intercalated 5-NS spin probe also allows one to evaluate the status of the membrane fluidity in the lipid microenvironment via the order parameter. Upon succinate stimulation of mitochondria, the ROS generated resulted in a decrease in the EPR signal amplitude of the 5-NS reporter molecule indicative of the flux of oxy-radicals produced and possible peroxidation-induced changes in the synaptosomal lipid membrane. The line width remained constant, indicating that the overall intensity was reduced. The results showed a significant overall age effect in the ability to generate oxygen-derived radicals following metabolic stimulation (p < .0001). Stimulation of state 4 mitochondrial respiration with 20 mM succinate resulted in greater oxy-radical production in 25-month-old animals as compared to younger animals, suggesting increased mitochondrial leakage with age. Free radical stress induced by metabolic stimulation also causes a concomitant increase in membrane fluidity (p < .0001). There was also a significant age effect (p < .0007) on the order parameter of the mixed population of membranes. Although caloric restriction attenuated the membrane rigidization caused by aging, it was found to play a role in limiting the oxy-radical production following metabolic stimulation of mitochondria. The overall effect of age on membrane spin-label intensities EPR signal upon succinate stimulation suggests that progressive mitochondrial dysfunction may be a key factor in the aging process and in development of age-associated diseases.

Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation.

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.