Journal of Neuropathology & Experimental Neurology:
Oxidative Stress in the Progression of Alzheimer Disease in the Frontal Cortex
Ansari, Mubeen A. PhD; Scheff, Stephen W. PhD
From the Sanders-Brown Center on Aging (MAA, SWS), and Spinal Cord Brain Injury Research Center (SWS), University of Kentucky, Lexington, Kentucky.
Send correspondence and reprint requests to: Stephen W. Scheff, PhD, 101 Sanders-Brown, Center on Aging, University of Kentucky, Lexington, KY 40536-0230; E-mail: email@example.com
This work was supported by Grants AG27219, PO1 AG14449, and AG028383 from the National Institutes of Health.The authors declare that they have no competing financial interests.
We investigated oxidative stress in human postmortem frontal cortexfrom individuals characterized as mild cognitive impairment (n= 8), mild/moderate Alzheimer disease (n = 4), and late-stage Alzheimer disease (n = 9). Samples from subjects with no cognitive impairment (n = 10) that were age- and postmortem interval-matched with these cases were used as controls. The short postmortem intervalbrain samples were processed for postmitochondrial supernatant, nonsynaptic mitochondria, and synaptosome fractions. Samples were analyzed for several antioxidants (glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, superoxide dismutase, catalase) and the oxidative marker, thiobarbituric acid reactive substances. The tissue was also analyzed for possible changes in protein damage using neurochemical markers for protein carbonyls, 3-nitrotyrosine, 4-hydroxynonenal, andacrolein. All 3 neuropil fractions (postmitochondrial supernatant, mitochondrial, and synaptosomal) demonstrated significant disease-dependent increases in oxidative markers. The highest changes were observed in the synaptosomal fraction. Both mitochondrial and synaptosomal fractions had significant declines in antioxidants (glutathione, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase). Levels of oxidative markers significantly correlated with Mini-Mental Status Examination scores. Oxidative stress was more localized to the synapses, with levels increasing in a disease-dependent fashion. These correlations implicate an involvement of oxidative stress in Alzheimer disease-related synaptic loss.
Oxidative stress is defined as a marked imbalance between the reactive oxygen species (ROS) and its removal by the antioxidant system. This may originate from an overproduction of ROS or from a reduction in antioxidant defenses (1, 2). Brain tissue has multiple potential sources of ROS (3) and a large oxidative capacity, but its ability to combat oxidative stress is limited (4). Increasing evidence suggests that oxidative stress that is normally associated with aging is a prominent and early feature of Alzheimer disease (AD) and plays a role in its pathogenesis (5-7).
Glutathione (GSH) is a well-known antioxidant that is synthesized in the cytoplasm and is present in higher concentrations in the mitochondrial matrix. Because most oxidized GSH (GSSG) forms are under oxidative stress (8), several different enzymes are necessary to reduce GSSG to maintain the GSH/GSSG ratio. Under severe conditions of oxidative stress, cells are not able to maintain the appropriate GSH/GSSG ratio, causing an accumulation of GSSG and resulting in protein modifications. The level of GSSG is a key factor in determining neuronal susceptibility to ROS/reactive nitrogen species (RNS)-mediated neuronal injury (9-11). The cytosolic GSH pool is critical for maintaining plasma membrane integrity and adenosine triphosphate levels in synaptosomes (12).
The widespread occurrence of protein nitration in neurons is caused by increased oxidative stress (13-15). Lipid peroxidation (LPO) generates various reactive aldehydes, such as acrolein, which rapidly incorporates into proteins to generate carbonyl derivatives. Protein-bound acrolein is a powerful marker of oxidative protein damage that plays an important role in the formation of neurofibrillary tangles (NFTs) in AD (16). Both acrolein and 4-hydroxynonenal (4-HNE), another product of LPO, can alter phospholipid asymmetry of membrane lipid bilayer, initiating apoptotic neuronal loss in mild cognitive impairment (MCI) and AD (17). The reactivity of LPO with key mitochondrial enzymes (18) increases free radical release into the cytoplasm (19), resulting in elevated oxidative stress that can affect synaptic function and neuronal death in AD (20, 21).
It is now clear that there are regional variations in the levels of oxidative stress in AD brain resulting from differences in the levels of antioxidant defenses and rates of oxygen consumption (22-25). Higher levels of oxidative DNA damage in mitochondria of the frontal, parietal, and temporal lobes suggest that mitochondrial oxidative stress may be an important contributor to the pathogenesis of AD (25). Transient or sustained mitochondrial dysfunction can deplete adenosine triphosphate levels, increase ROS generation, and initiate apoptosis leading to neurodegeneration (22, 26).
When neuronal DNA damage resulting from oxidative stress is not completely repaired, it can cause accumulated synaptic protein alterations (27,28). Postsynaptic regions are subjected to particularly high levels of calcium influx and oxidative stress as a result of local activation of glutamate receptors; therefore, they are likely sites at which neurodegenerative processes are initiated in AD (29). Increased oxidative stress precedes the loss of several synaptic proteins (10,30), and this may contribute to synaptic degeneration (31). It is well known that synaptic mitochondria differ enzymatically (32, 33) and may be more sensitive to ROS than nonsynaptic mitochondria (34, 35). Defects in synaptic mitochondria can alter synaptic functions, leading to alterations in cognitive function. It is unclear whether levels of oxidative stress are high in mitochondria as a function of disease progression and whether the levels are similar to those observed in synaptic terminals.
Alzheimer disease is a progressive dementing disorder characterized by both a loss of working memory and episodic memory. Although senile plaques and neurofibrillary tangles are considered hallmarks of the disease, many nondemented elderly individuals have these lesions, often at levels suggestive of early AD (36-38). There are also elderly individuals who manifest some cognitive deficits that are greater than expected of those observed in normal aging but are not clinically demented. These individuals are thought to be in a transitional stage between normal aging and dementia and have been termed MCI (39-41). The conversion rate from MCI to mild AD (mAD) is 10% to 15% per year (42).
Here, we analyzed multiple biochemical markers of oxidative stress and antioxidant defenses in postmitochondrial supernatant (PMS), mitochondrial, and synaptic fractions in age-matched noncognitively impaired (NCI), MCI, mAD, and AD subjects in the frontal cortex (FC), a cortical region known to be involved in AD.
MATERIALS AND METHODS
All chemicals and reagents used were purchased from Sigma (St Louis, MO) unless stated otherwise.
Frozen FC samples from NCI (n = 10), MCI (n = 8), mAD (n = 4), and AD (n = 9) subjects were obtained from the University of Kentucky Alzheimer's Disease Center Rapid Autopsy Program. The diagnosis of probable AD was made according to criteria developed by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (43). The diagnosis of MCI was identical to that used previously (44). The Human Investigations Committee of the University of Kentucky College of Medicine approved the studies. Individuals included in these studies agreed to an annual clinical evaluation and brain donation at the time of death. The NCI subjects were without a history of dementia or other neurological disorders and underwent annual mental status testing and semiannual physical and neurological examinations as part of the University of Kentucky Alzheimer's Disease Center normal volunteer longitudinal aging study (38). Additional demographic parameters of NCI, MCI, mAD, and AD patients available from medical records are provided in the Table. When selecting subjects, agonal state before death was taken into consideration. Standard criteria for exclusion were the presence of 1) significant cerebral stroke regardless of antemortem date, 2) large cortical infarcts identified in the postmortem neuropathologic evaluation, 3) significant head trauma within 12 months before autopsy, 4) individuals on a respirator longer than 12 hours before death, 5) individuals in coma longer than 12 hours immediately before death, 6) individuals who had been under excessive medication for periods longer than 6 months before death, or 7) individuals currently undergoing radiation therapy for CNS tumor. As part of the rapid autopsy protocol, we asked the individuals who informed us of a subject's death what the conditions were for the 24 hours preceding death and if any significant events may have dramatically influenced the process. Questionable tissue was not included in the study.
TABLE. Clinical and ...Image Tools
Isolation of Tissue Fractions
Subcellular fractions (synaptosome, mitochondria, and PMS) were isolated from FC using standard procedures with some modifications (45). Briefly, the brain tissue was gently homogenized with a Wheaton tissue homogenizer in ice-cold sucrose isolation buffer (250 mmol/L sucrose, 10 mmol/L HEPES, 1.0 mmol/L EDTA, pH 7.2, 4.0 μg/mL leupeptin, 4.0 μg/mL pepstatin, 5.0 μg/mL aprotinin, 20.0 μg/mL trypsin inhibitor). The homogenates were subjected to centrifugation for 5 minutes at 1330 × g at 4°C. The collected supernatants were spun at 21,200 × g at 4°C for 10 minutes. The PMS fraction was collected. Pellets were resuspended and layered over a discontinuous sucrose gradient (1.0 mol/L, pH 8.0; and 1.18 mol/L, pH 8.5) and spun at 85,500 × g for 1 hour at 4°C. The purified synaptosomes were collected from the sucrose gradient interface and the mitochondrial fraction was collected from the bottom layer. Synaptosomes and mitochondrial fractions were diluted with isolation buffer and centrifuged at 32,000 × g for 20 minutes, and the collected pellet was used for the analysis.
Determination of antioxidants and associated enzymes were performed as previously described (9, 10, 30). For GSH and GSSG, changes in fluorescence at emission 420 nm were recorded by excitation at 350 nm. For the estimation of glutathione peroxidase (GPx), glutathione reductase (GR) enzyme activity, the disappearance of nicotinamide adenosine dinucleotide phosphate (NADPH) and glucose-6-phosphate dehydrogenase (G-6PD) activity with formation of NADPH at 340 nm was recorded at room temperature. The enzyme activity was calculated as nmol NADPH oxidized per minute per milligram protein using a molar extinction coefficient of 6.22 × 1.03 M−1 cm−1. Glutathione-S-transferase (GST) (EC 184.108.40.206) activity was measured by the method as previously described (10, 30). The reaction mixture consisted of 0.1 mol/L phosphate buffer (pH 6.5), 1.0 mmol/L reduced GSH, 1.0 mmol/L 1-chloro-2,4-dinitrobenzene, and 0.03 mL previously mentioned sample in a final volume of 0.3 mL. The changes in absorbance were recorded at 340 nm, and the enzyme activity was calculated as nmol 1-chloro-2,4-dinitrobenzene conjugate formed per minute per milligram protein using a molar extinction coefficient of 9.6 × 103 M−1 cm−1. For GST activity, the changes in absorbance were recorded at 340 nm, and the enzyme activity was calculated as nmol 1-chloro-2,4-dinitrobenzene conjugate formed per minute per milligram protein, using a molar extinction coefficient of 9.6 × 103 M−1 cm−1.
Superoxide Dismutase and Catalase Activity
Superoxide dismutase ([SOD] EC 220.127.116.11) and catalase (CAT) activities were measured as previously described (10, 30, 46). Briefly, for SOD, triethylenetetramine was used as a copper/zinc (Cu/Zn)-SOD inhibitor to measure manganese-SOD activity. The absorbance was monitored for 5 minutes at 480 nm, and activity was calculated using a molar extinction coefficient of 4.02 × 103 M−1 cm−1 expressed as nanomoles of epinephrine protected from oxidation per minute per milligram protein. For CAT, changes in absorbance were recorded at 240 nm, and activity was calculated in terms of nanomoles H2O2 consumed per minute per milligram protein using a molar extinction coefficient of 43.6 M−1 cm−1.
Thiobarbituric Acid Reactive Substances
The estimation of thiobarbituric acid reactive substances (TBARS) was performed as previously described (30, 46). The rate of LPO was expressed as nanomoles of TBARS formed per 30 minutes per milligram protein using a molar extinction coefficient of 1.56 × 105 M−1 cm−1.
Protein Carbonyls, 3-Nitrotyrosine, 4-HNE, and Acrolein
Protein carbonyls (PCs), 3-nitrotyrosine (3-NT), 4-HNE, and acrolein were assessed by following a standard previously described protocol (9, 10). For PCs, 5-μL samples (normalized to 4 mg/mL) were incubated for 20 minutes at room temperature with 5 μL of 12% sodium dodecyl sulfate and 10 μL of 20 mmol/L 2,4-dinitrophenyl hydrazine, and neutralized with 7.5 μL neutralization solution (2 mol/L Tris in 30% glycerol). For 3-NT, 4-HNE, and acrolein, 5-μL samples were incubated for 20 minutes at room temperature with 5 μL of 12% sodium dodecyl sulfate and 5 μL of modified Laemmli buffer containing 0.125 mol/L Tris base, pH 6.8, 4% (vol/vol) sodium dodecyl sulfate, and 20% (vol/vol) glycerol. Each sample (250 ng) was loaded into a well on a nitrocellulose membrane in a slot-blot apparatus under vacuum. The membrane was blocked in buffer (3% bovine serum albumin) in PBS/Tween for 1 hour and incubated with a 1:100 anti-DNP, 1:2000 anti-3-NT, 1:5000 anti-4-HNE dilution of polyclonal antibody, and 1:5000 monoclonal ant-acrolein in PBS/Tween for 90 minutes. The membrane was washed in PBS/Tween for 5 minutes 3 times after incubation. The membrane was incubated for 1 hour after washing with an anti-rabbit, anti-goat, and anti-mouse IgG alkaline phosphatase secondary antibody diluted 1:8000 in PBS/Tween. The membrane was washed 3 times in PBS/Tween for 5 minutes and developed in Sigma Fast tablets. Blots were dried, scanned with Adobe PhotoShop, and quantified with Scion Image as previously described. No nonspecific binding of antibody to the membrane was observed.
Total protein concentrations were measured using the Pierce BCA method (Sigma).
Ultrastructural Analysis of Mitochondrial Preparations
Random samples of both synaptosomal and nonsynaptic mitochondrial preparations were analyzed with ultrastructural techniques to verify the relative purity of the preparations. Mitochondrial samples were fixed with 1% glutaraldehyde/4% paraformaldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4). Samples were postfixed in 1% osmium tetroxide (OsO4), stained en bloc with 0.5% uranyl acetate, dehydrated in a graded series of ethanol, treated with propylene oxide, and embedded in epoxy resin as previously described (44). Cured blocks were sectioned with an ultramicrotome and imaged with a Zeiss 902 electron microscope. Pictures were taken at an initial magnification of 4,400× and photographically enlarged to approximately 18,000×. Random pictures (n = 5) were taken from each preparation.
Enzymatic and nonenzymatic oxidative stress markers are reported as group mean ± SD. Group means were evaluated with a 1-way analysis of variance coupled with a Student Newman-Keuls post hoc test. Correlations were calculated using the Pearson product-moment correlation coefficient test (StatView 5.0, SAS Institute). For significance, α = 0.05 was set.
The Table shows the characteristics of the sample population by diagnostic group. The NCI, MCI, mAD, and AD groups were similar in age, sex, brain weight, and postmortem interval; they did not show any significant differences (p > 0.05) for any of these variables. An analysis of variance revealed a significant difference in the Mini Mental State Examination (MMSE) between groups (F3,27 = 61.922, p < 0.0001). Post hoc analysis revealed a statistically significant change between NCI and MCI and between MCI and AD but not with mAD. The AD and mAD groups were also significantly different. The AD group had the highest incidence of Braak stage VI subjects. Subjects in all groups were highly educated with a mean education level of 16 ± 3 years and failed to reveal a significant difference between groups (F3,27 = 2.277, p > 0.1).
As shown in Figure 1, the synaptosomal preparation contained primarily synaptosomes, and the nonsynaptic fraction contained primarily mitochondria and no synaptosomes. No precise quantitative measurements were made on either preparation, or grading of relative purity was attempted. All assessments were qualitative.
The level of internal antioxidant tripeptide molecule GSH was measured in the PMS, mitochondrial, and synaptosomal fractions of the FC in NCI, MCI, mAD, and AD subjects. Glutathione levels demonstrated a significant change in the PMS (F3, 27 = 6.335, p < 0.005), mitochondria (F3,27 = 7.761, p < 0.001), and synaptosome (F3,27 = 12.935, p < 0.0001) fractions. In all fractions, levels decreased with a decline in cognitive status. The greatest change was observed in the synaptosomal fraction (MCI, 77.7%; mAD, 51.3%; AD, 51.2%) compared with NCI (Fig. 2A). Both AD groups were significantly lower (p < 0.05) than the MCI group but not significantly different from each other. Although a similar trend was observed in the PMS and mitochondrial fractions, the cognitively impaired groups were not significantly (p > 0.05) different from each other. These results indicate that GSH decline is an early event in the progression of the disease. There was a significant correlation (p < 0.0005) between the subjects' MMSE and the GSH levels in all 3 fractions (data not shown). Thus, individuals with lower MMSE scores had lower levels of GSH.
Analysis of GSSG levels in the PMS, mitochondrial, and synaptosomal fractions demonstrated significant differences between the NCI, MCI, mAD, and AD groups (Fig. 2B). The GSSG increased significantly in the PMS (F3,27 = 33.736, p < 0.0001), mitochondrial (F3,27 = 34.544, p < 0.0001) and synaptosomal (F3,27 = 10.622, p < 0.0001) fractions. In all fractions, the levels of GSSG increased with increased cognitive impairment; AD subjects had significantly higher levels than the MCI and mAD groups (p < 0.05) in the PMS and mitochondrial fractions. The greatest change was observed in the synaptosomes with increases correlating with decreased cognitive status: 257% (MCI), 343% (mAD), and 366% (AD).
The GSH/GSSG ratios significantly declined in the PMS (F3,27 = 69.409, p < 0.0001), mitochondrial (F3,27 = 48.169, p < 0.0001), and synaptosomal (F3,27 = 32.125, p < 0.0001) fractions compared with the NCI group (Fig. 2C). In the PMS and mitochondrial fractions, the levels of the AD group were significantly lower than MCI (p < 0.01) and mAD (p < 0.05) groups. The synaptosomal fraction was significantly decreased to about the same level in all cognitively impaired groups. These data suggest that a decline in antioxidants occurs early in the progression of the disease and that the free radical burden continues to increase with a decline in cognitive status. There was a significant correlation (p < 0.0001) between the subjects; MMSE and GSSG ratios in all 3 fractions (not shown), that is, individuals with lower MMSE scores had lower GSH/GSSG ratios.
The activity of GPx in the mitochondrial fraction was significantly (F3,27 = 5.164, p < 0.01) altered only in the mAD (61%) and AD (57%) groups. Although in synaptosomes, it was significantly (F3,27 = 6.342, p < 0.005) depleted in MCI (70%), mAD (58.39%), and AD (58%) compared with the NCI group. The PMS fraction (F3,27 = 0.769, p > 0.1) failed to show any significant change in GPx activity (Fig. 3A). The mAD group had levels equivalent to that seen in the more advanced AD group in the mitochondrial, synaptosomal, and PMS fractions.
The GR reduces GSSG into GSH to continue the antioxidant cycle during detoxification of oxidants. The activity of GR in synaptosomes was significantly (F3,27 = 7.126, p < 0.001) depleted in the MCI (66%), mAD (63.71%), and AD (60%) groups compared with the NCI cohort. These cognitively impaired groups were not significantly different from each other. The PMS (F3,7 = 0.781, p > 0.1) and mitochondrial (F3,27 = 2.030, p > 0.1) fractions failed to show any significant change in GR activity (Fig. 3B).
The GST is used for detoxification of external or internal toxicants. The activity of GST was significantly lower in the mitochondrial (F3,27 = 4.868, p < 0.01) and synaptosomal (F3,27 = 12.760, p < 0.0001) fractions but failed to reach significance in the PMS fraction (F3,27 = 2.810, p > 0.1). The MCI group was only significantly different from the NCI group in the synaptosomal fraction (p < 0.01), whereas the AD groups were significantly lower in the mitochondrial and synaptosomal fractions (Fig. 3C). The mAD and AD groups demonstrated similar degrees of decline.
The G-6PD provides electrons for the reduction of oxidized nicotinamide adenine dinucleotides (NAD+/NADP+). The activity of G-6PD in synaptosomes was significantly (F3,27 = 5.297, p < 0.01) depleted in the MCI (67%), mAD (59%), and AD (65%) groups compared with the NCI subjects, and these groups were not significantly different from each other. The PMS fractions of the MCI, mAD, and AD groups failed to show any significant (F3,27 = 0.899, p > 0.1) change in G-6PD activity (Fig. 3D).
The SOD is an important enzyme that reduces the superoxide burden in tissue. Analysis of the SOD activity in the PMS (F3,27 = 4.927, p < 0.01), mitochondria (F3,27 = 5.322, p < 0.01), and synaptosomal (F3,27 = 5.612, p < 0.005) fractions demonstrated a significant decline in FC (Fig. 4A). The activity of SOD in PMS was significantly depleted in the mAD (62%) and AD (59%) groups compared with NCI. Analysis of the synaptosomal fraction revealed that SOD activity was significantly depleted in the MCI (70%), mAD (66%), and AD (61%) groups compared with the NCI subjects. The activity of manganese-SOD in the mitochondrial fraction was also significantly (F3,27 = 5.243, p < 0.01) reduced in the MCI (74%), mAD (65%), and AD (63%) groups compared with the NCI cohorts. In all fractions, the MCI, mAD, and AD cases were not significantly different from each other (p > 0.1). There was a significant correlation (p < 0.005) between the subjects' MMSE and SOD levels in all 3 fractions (not shown), that is, individuals with lower MMSE scores had lower levels of SOD.
Catalase is an important enzyme that breaks down H2O2 into H2O and O2. Analysis of synaptosomes revealed a significant decline in CAT activity (F3,27 = 13.652, p < 0.0001) (Fig. 4B). The decline mirrored the change in cognitive status with the MCI group (68%) demonstrating a loss in activity that was not as severe as that observed in the mAD (44%) and AD (29%) groups. The mAD and AD groups were not significantly different. The PMS fraction also showed significant depletion in CAT activity (F3,27 = 30.792, p < 0.0001) with the loss in the AD group (31%) significantly greater (p < 0.01) compared with MCI (69%) and mAD (66%) subjects. Catalase levels in the PMS and synaptosomal fractions revealed a significant (p < 0.0001) correlation with the subjects' MMSE scores (not shown), that is, lower levels of CAT were associated with lower MMSE scores.
Thiobarbituric Acid Reactive Substances
The TBARS levels were evaluated as a measure of total LPO. The analysis demonstrated a significant increase in TBARS in the PMS (F3,27 = 21.067, p < 0.0001), mitochondria (F3,27 = 8.657, p < 0.0005), and synaptosomes (F3,27 = 19.038, p < 0.0001) fractions. In all fractions, the MCI, mAD, and AD groups were significantly elevated (p < 0.01) compared with NCI subjects. In the PMS and synaptosome fractions, the AD group showed the highest TBARS value and was significantly elevated compared with both the MCI and mAD groups. In the mitochondrial fraction, all 3 cognitively impaired groups showed similar increases compared with the NCI cohorts (Fig. 5). The subjects' MMSE and TBARS levels were significantly correlated (p < 0.0001) in all 3 fractions (data not shown), that is, individuals with lower MMSE scores had higher TBARS levels. Furthermore, when the subjects were grouped according to Braak scores (I-II, III-IV, V-VI), individuals with the highest scores showed the highest levels of TBARS in both the synaptosomal and PMS fractions (p < 0.05). The mitochondrial fraction did not show this relationship.
Protein Oxidation, Nitration, and LPO Levels
Levels of protein oxidation and nitration products (PC and 3-NT) were evaluated using slot blots (Fig. 6). Protein carbonyls demonstrated a significant increase in the PMS (F3,27 = 10.356, p < 0.0005), mitochondria (F3,27 = 8.261, p < 0.0005), and synaptosomal (F3,27 = 9.738, p < 0.0005) fractions (Fig. 7A). Although the PC values were significantly elevated in all fractions as a function of the degree of cognitive impairment, there were no significant differences between the cognitively impaired groups, suggesting that PC formation is an early event in the disease process.
The 3-NT levels were also significantly elevated in the PMS (F3,27 = 8.953, p < 0.0005), mitochondrial (F3,27 = 8.543, p < 0.0005), and synaptosomal fractions (F3,27 = 8.123, p < 0.0005). Changes in individual cognitive groups closely mirrored those observed in the PC analysis and were not significantly different from each other (Fig. 7B).
Analysis of 4-HNE demonstrated a significant increase in the PMS (F3,27 = 11.390, p < 0.0001), mitochondria (F3,27 = 5.206, p < 0. 01), and synaptosomes (F3,27 = 20.672, p < 0.0001) fractions (Fig. 7C). Post hoc analysis revealed that for all fractions, the levels of 4-HNE were significantly elevated in cognitively impaired groups compared with NCI cohorts but were not significantly different from each other, with the exception of the mitochondrial fraction in the AD group that was significantly higher than the MCI group. Together, these data indicate that the increase in 4-HNE is an early and persistent event.
Significantly increased acrolein levels were also found in the PMS (F3,27 = 9.978, p < 0.0001), mitochondria (F3,27 = 6.594, p < 0.005), and synaptosomal fractions (F3,27 = 28.410, p < 0.0001). Changes as a function of cognitive status mirrored that observed in the 4-HNE analysis with the greatest increases observed in the AD groups (Fig. 7D). Both the mAD and AD groups were significantly elevated compared with the MCI group in the synaptosomal fraction. The levels of protein oxidation, nitration, and LPO in the synaptosomal fraction were analyzed for possible association with each individual's MMSE score. There was a significant positive correlation with PCs (r = 0.536, p < 0.005), 3-NT (r = 0.560, p < 0.005), 4-HNE (r = 0.675, p < 0.0001), and acrolein (r = 0.786, p < 0.0001) (Fig. 8).
With one exception, no significant relationship between protein oxidation, nitration, and LPO of the PMS, mitochondrial, and synaptosomal fractions with Braak scores was identified (p > 0.05). In the mitochondrial fraction, levels of PCs were significantly elevated in the subjects with the highest Braak scores (p < 0.05) (not shown).
Our results support and extend previous findings of enhanced oxidative stress in MCI and mAD. Numerous studies have established that MCI is a transition state between normal aging and dementia (39, 40, 47). There is also compelling evidence that oxidative stress is elevated in the brains of individuals with MCI (15, 48-51). We analyzed FC samples not only from subjects with MCI but also mild and late-stage AD. For all cellular oxidative markers studied, there was a stepwise increase in each of the subcellular fractions as a function of cognitive change, reinforcing the idea that oxidative stress is not only an early event, but also an active persistent process.
Oxidative stress has been implicated in many neurodegenerative disorders, including AD (24, 52-54), and is associated with ROS formation in neurons and synaptosomes (52, 55). Although neuronal membranes are rich in polyunsaturated fatty acids, a source of LPO reactions, their ability to combat oxidative stress is limited (4). The cortex from individuals with AD has increased polyunsaturated fatty acid oxidation, altered antioxidant defenses, high rates of oxygen consumption, and oxidative stress (22), with regional variations (23-25). Levels of oxidative DNA damage in the brains of individuals with AD are significantly elevated in the mitochondria from frontal, parietal, and temporal lobes (25). The attention on mitochondria is not only caused by its adenosine triphosphate production, but also because of the many proapoptotic and antiapoptotic molecules associated with these organelles (56). Mitochondria regulate intracellular Ca2+ homeostasis and ROS formation (57, 58) and contribute to neurodegeneration (22, 25, 26).
All samples used were obtained from the superior FC in cases with relatively short postmortem intervals. Although the AD-related involvement of this region is well documented, the literature reporting possible oxidative stress is mixed. Early studies reported both significant elevations in TBARS (59, 60) or no change in late-stage AD compared with age-matched controls (61-63). Lipid peroxidation changes as determined by F2-isoprostanes were only recently reported in the FC (50) in AD and in MCI. The levels were moderately elevated in AD compared with MCI. A more recent study using F2-isoprostanes reported significant FC increases in mAD but no significant differences in MCI (64). The levels also failed to correlate with the clinical diagnosis but were strongly correlated with AD pathology. In the present study, significant increases in all markers of oxidative stress were observed in the MCI and in both mAD and AD groups. The greatest changes were observed in the synaptosomal fraction, with AD subjects having significantly greater LPO than both MCI and mAD cases. In all fractions, there were no significant differences between the MCI and mAD groups, supporting the idea that MCI is an early and perhaps progressive transition from a cognitively normal state.
A decrease in GSH levels and subsequent GSSG production has been linked to neuronal loss in AD (11, 65). The GSH/GSSG ratio is an indicator of oxidative and/or nitrosative burden in the system, which was decreased significantly in FC (all 3 fractions) of the MCI, mAD, and AD groups. The low levels of GSH may be directly related to increased ROS/RNS, lipid peroxides, and highly reactive hydroxyl radicals (9, 46, 66, 67). A low GSH/GSSG ratio could contribute to promoting free radical load and oxidative stress (10, 65).
To eliminate the peroxides, GSH works in conjunction with GPx and produces GSSG, which is reconverted to GSH by GR at the consumption of NADPH. A reduction in GSH may impair H2O2 clearance and promote OH formation, thus increasing the free radical load, which triggers oxidative stress (9, 10, 30, 46) and pathological pathways in AD (68). Glutathione also acts as a peroxynitrite reductant, thereby providing enzymatic defense against peroxynitrites. Reduced GPx activity in mitochondria and synaptosomes could directly affect the clearance of ROS and lipid peroxides, as indicated by elevated levels of oxidative markers. Glutathione-S-transferase, another detoxification enzyme (9, 46, 69), can alleviate damage from 4-HNE and acrolein by catalyzing its conjugation with GSH (70). The depletion of GST observed in all 3 FC fractions could result in increased protein modification/dysfunction, leading to further oxidative stress and decline of GSH. Balancing the GSH/GSSG level requires an important cofactor, NADPH, which is produced from reactions catalyzed by G-6PD and 6-phosphogluconate dehydrogenase. Thus, GST, GR, and G-6PD are secondary antioxidant enzymes that play an important role in detoxifying ROS/RNS by maintaining a ready supply of intermediates such as GSH and NADPH (9, 10, 30, 71). In the present study, these 3 enzymes were significantly depleted in the synaptosomal fraction. Decreased activity of GR would directly affect GSH as would a reduction in the levels of GST, resulting in overall low levels of antioxidant defense, thus predicting the overpowering influences of ROS/RNS in AD (9, 72). Prior studies have reported increased levels of G-6PD in end-stage AD subjects (73, 74), suggesting a complex role for some antioxidant enzymes.
The most abundant ROS superoxide radical (O2−•) has been implicated in memory function and synaptic plasticity (75, 76) and also in neuronal death (77, 78). The first enzymatic reaction in the reduction pathway is dismutation of 2 molecules of O2−• when they are converted into H2O2 and O2. The enzyme Cu/Zn-SOD and/or manganese-SOD protect neurons from high O2−• environment (78, 79). Moreover, GPx and CAT participate in the elimination of H2O2, which is one of the most toxic molecules in the brain (9, 46, 80). Depletion in CAT in synaptosomes and PMS fractions represents the loss of one of the major defense against ROS. Decreased activity of manganese-SOD in mitochondrial and synaptosomal fractions could lead to further oxidative stress and progressively enhance peroxynitrite production as part of the secondary damage cascade (78, 79, 81). Decreased activity of Cu/Zn-SOD also compromises defense capacities against oxidative stress (10, 30) in PMS fractions. In the 3 FC fractions analyzed, CAT and SOD levels were significantly decreased in MCI, mAD, and AD. It is unclear if this decline precedes the onset of oxidative stress or is in response to increased free radical production.
During oxidative stress, formation of carbonyls derived from proteins and lipids such as PCs, 4-HNE, and acrolein causes damage to biomembranes and participate in the formation of neurofibrillary tangles in AD (16, 82, 83). These carbonyls (alkenals) form the immediate substrate for GSH and are involved in neuronal apoptosis in MCI and AD (17), which is seen as a consequence of GSH depletion. Elevated oxidative stress resulting from PC, 3-NT, 4-HNE, or acrolein can lead to delayed neuronal death in FC of MCI, mAD, and AD. Cortical synaptosomes show decreased ability to detoxify lipid peroxides, including 4-HNE (84), suggesting that decreases in antioxidants play a role in AD-related pathology (4). The present findings support the idea that the deleterious effects of increased oxidative stress may be the consequence of diminished antioxidant defenses. Levels of 4-HNE and acrolein were especially elevated in synaptosomal fractions of the FC for MCI, mAD, and AD, suggesting that the antioxidant defenses were not able to offset oxidative stress. The reactivity of 4-HNE with mitochondrial key enzymes (18) increases free radical release into the cytoplasm (19) and downstream effects of mitochondrial dysfunction yield to loss of synaptic function and neuronal death in AD (20, 21). Protein carbonyl, 3-NT, 4-HNE, and acrolein levels were elevated throughout the MCI, mAD, and AD samples in this study signaling protein modification via oxidation, nitration, and binding with the lipid by-products acrolein and 4-HNE. Synaptic proteins also might be affected through these modifications. These results provide further insight into the relationship of synaptic and mitochondrial oxidative stress and the progression of cognitive change associated with AD. Oxidative stress-related damage to nucleic acids has been suggested as a prime contributing factor in the disease progression as a result of mistakes in base pairings resulting in compromised transcription and translation (85).
Relatively recent studies have shown that β-amyloid plays a central role in AD and may be key in disease-related synaptic change (86). The β-amyloid oligomers can induce AD-like pathology including oxidative stress (87) and play an important role in synaptic loss, which is linked to increased synaptic activity (88-90).
The nature of the terminal illness and the rapidity of death, as well as the methods used to harvest, prepare, and store tissue samples can play a critical role in determining postmortem changes, especially neurochemical parameters (91-93). Hypoxia is one of the key agonal events that can have a major impact on neurochemical analyses and tissue morphology. Prolonged agonal state with marked hypoxia increases tissue lactate levels, which lowers the brain pH, thus affecting protein integrity. Although we cannot control the agonal state or the various conditions of each patient in the period before death, we screened each case before including it in this study; cases in which there is an obvious event that would affect the tissue analyses were excluded. Therefore, we feel confident that our results were not influenced by differences in agonal states between groups.
This is the first study to report a strong correlation between levels of synaptic LPO, protein oxidation, and nitration, and the subjects' global cognitive status (Fig. 8). Changes in antioxidant levels (GSH, SOD, CAT) also strongly correlated with the MMSE score, supporting the idea that significant changes in oxidative stress is an early event that plays an important role in the progression of the disease. Although it is unclear whether the decline in antioxidant level precedes the increase in oxidants, the levels certainly are not capable of neutralizing them. These finding suggests that efforts aimed at increasing brain antioxidants should have beneficial values if enacted early in the disease process or as a prophylactic measure.
1. Halliwell B. Reactive oxygen species and the central nervous system. J Neurochem 1992;59:1609-23
2. Halliwell B. Role of free radicals in the neurodegenerative diseases: Therapeutic implications for antioxidant treatment. Drugs Aging 2001;18:685-716
3. Faraci FM. Reactive oxygen species: Influence on cerebral vascular tone. J Appl Physiol 2006;100:739-43
4. Mantha AK, Moorthy K, Cowsik SM, et al. Neuroprotective role of neurokinin B (NKB) on beta-amyloid (25-35) induced toxicity in aging rat brain synaptosomes: Involvement in oxidative stress and excitotoxicity. Biogerontology 2006;7:1-17
5. Markesbery WR, Lovell MA. DNA oxidation in Alzheimer's disease. Antioxid Redox Signal 2006;8:2039-45
6. Markesbery WR, Lovell MA. Damage to lipids, proteins, DNA, and RNA in mild cognitive impairment. Arch Neurol 2007;64:954-56
7. Moreira PI, Nunomura A, Nakamura M, et al. Nucleic acid oxidation in Alzheimer disease. Free Radic Biol Med 2008;44:1493-505
8. Starke DW, Chen Y, Bapna CP, et al. Sensitivity of protein sulfhydryl repair enzymes to oxidative stress. Free Radic Biol Med 1997;23:373-84
9. Ansari MA, Joshi G, Huang Q, et al. In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: Relevance to Alzheimer's disease and other oxidative stress-related neurodegenerative disorders. Free Radic Biol Med 2006;41:1694-703
10. Ansari MA, Roberts KN, Scheff SW. Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury. Free Radic Biol Med 2008;45:443-52
11. Benzi G, Moretti A. Age- and peroxidative stress-related modifications of the cerebral enzymatic activities linked to mitochondria and the glutathione system. Free Radic Biol Med 1995;19:77-101
12. Martinez M, Ferrandiz ML, Diez A, et al. Depletion of cytosolic GSH decreases the ATP levels and viability of synaptosomes from aged mice but not from young mice. Mech Ageing Dev 1995;84:77-81
13. Good PF, Werner P, Hsu A, et al. Evidence of neuronal oxidative damage in Alzheimer's disease. Am J Pathol 1996;149:21-8
14. Smith MA, Richey Harris PL, Sayre LM, et al. Widespread peroxynitrite-mediated damage in Alzheimer's disease. J Neurosci 1997;17:2653-57
15. Williams TI, Lynn BC, Markesbery WR, et al. Increased levels of 4-hydroxynonenal and acrolein, neurotoxic markers of lipid peroxidation, in the brain in mild cognitive impairment and early Alzheimer's disease. Neurobiol Aging 2006;27:1094-99
16. Calingasan NY, Uchida K, Gibson GE. Protein-bound acrolein: A novel marker of oxidative stress in Alzheimer's disease. J Neurochem 1999;72:751-56
17. Bader Lange ML, Cenini G, Piroddi M, et al. Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease. Neurobiol Dis 2008;29:456-64
18. Floyd RA, Hensley K. Oxidative stress in brain aging. Implications for therapeutics of neurodegenerative diseases. Neurobiol Aging 2002;23:795-807
19. Cash AD, Perry G, Ogawa O, et al. Is Alzheimer's disease a mitochondrial disorder? Neuroscientist 2002;8:489-96
20. Onyango I, Khan S, Miller B, et al. Mitochondrial genomic contribution to mitochondrial dysfunction in Alzheimer's disease. J Alzheimer's Dis 2006;9:183-93
21. Reddy PH, Beal MF. Are mitochondria critical in the pathogenesis of Alzheimer's disease? Brain Res Brain Res Rev 2005;49:618-32
22. Cardoso SM, Santana I, Swerdlow RH, et al. Mitochondria dysfunction of Alzheimer's disease cybrids enhances Aβ toxicity. J Neurochem 2004;89:1417-26
23. Culmsee C, Landshamer S. Molecular insights into mechanisms of the cell death program: Role in the progression of neurodegenerative disorders. Curr Alzheimer Res 2006;3:269-83
24. Hensley K, Hall N, Subramaniam R, et al. Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation. J Neurochem 1995;65:2146-56
25. Wang J, Xiong S, Xie C, et al. Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease. J Neurochem 2005;93:953-62
26. Zhu X, Smith MA, Perry G, et al. Mitochondrial failures in Alzheimer's disease. Am J Alzheimer's Dis Other Demen 2004;19:345-52
27. Forero DA, Casadesus G, Perry G, et al. Synaptic dysfunction and oxidative stress in Alzheimer's disease: Emerging mechanisms. J Cell Mol Med 2006;10:796-805
28. Urano S, Asai Y, Makabe S, et al. Oxidative injury of synapse and alteration of antioxidative defense systems in rats, and its prevention by vitamin E. Eur J Biochem 1997;245:64-70
29. Mattson MP, Partin J, Begley JG. Amyloid beta-peptide induces apoptosis-related events in synapses and dendrites. Brain Res 1998;807:167-76
30. Ansari MA, Roberts KN, Scheff SW. A time course of contusion-induced oxidative stress and synaptic proteins in cortex in a rat model of TBI. J Neurotrauma 2008;25:513-26
31. Gilman CP, Chan SL, Guo Z, et al. p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage, and oxidative and excitotoxic insults. Neuromolecular Med 2003;3:159-72
32. Faff-Michalak L, Albrecht J. The two catalytic components of the 2-oxoglutarate dehydrogenase complex in rat cerebral synaptic and nonsynaptic mitochondria: Comparison of the response to in vitro treatment with ammonia, hyperammonemia, and hepatic encephalopathy. Neurochem Res 1993;18:119-23
33. Gillardon F, Rist W, Kussmaul L, et al. Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque deposition. Proteomics 2007;7:605-16
34. Brown MR, Sullivan PG, Geddes JW. Synaptic mitochondria are more susceptible to Ca2+overload than nonsynaptic mitochondria. J Biol Chem 2006;281:11658-68
35. Naga KK, Sullivan PG, Geddes JW. High cyclophilin D content of synaptic mitochondria results in increased vulnerability to permeability transition. J Neurosci 2007;27:7469-75
36. Goldman WP, Price JL, Storandt M, et al. Absence of cognitive impairment or decline in preclinical Alzheimer's disease. Neurology 2001;56:361-67
37. Haroutunian V, Perl DP, Purohit DP, et al. Regional distribution of neuritic plaques in the nondemented elderly and subjects with very mild Alzheimer disease. Arch Neurol 1998;55:1185-91
38. Schmitt FA, Davis DG, Wekstein DR, et al. "Preclinical" AD revisited: Neuropathology of cognitively normal older adults. Neurology 2000;55:370-76
39. Flicker C, Ferris SH, Reisberg B. Mild cognitive impairment in the elderly: Predictors of dementia. Neurology 1991;41:1006-9
40. Morris JC, Storandt M, Miller JP, et al. Mild cognitive impairment represents early-stage Alzheimer disease. Arch Neurol 2001;58:397-405
41. Petersen RC. Mild cognitive impairment: Transition between aging and Alzheimer's disease. Neurologia 2000;15:93-101
42. Petersen RC, Doody R, Kurz A, et al. Current concepts in mild cognitive impairment. Arch Neurol 2001;58:1985-92
43. McKhann G, Drachman D, Folstein M, et al. Clinical diagnosis of Alzheimer's disease: Report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 1984;34:939-44
44. Scheff SW, Price DA, Schmitt FA, et al. Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology 2007;68:1501-8
45. Garey RE, Harper JW, Heath RG. Postmortem isolation of synaptosomes from human brain. Brain Res 1974;82:151-62
46. Ahmad AS, Ansari MA, Ahmad M, et al. Neuroprotection by crocetin in a hemi-parkinsonian rat model. Pharmacol Biochem Behav 2005;81:805-13
47. Luis CA, Loewenstein DA, Acevedo A, et al. Mild cognitive impairment: Directions for future research. Neurology 2003;61:438-44
48. Butterfield DA, Reed T, Perluigi M, et al. Elevated protein-bound levels of the lipid peroxidation product, 4-hydroxy-2-nonenal, in brain from persons with mild cognitive impairment. Neurosci Lett 2006;397:170-73
49. Keller JN, Schmitt FA, Scheff SW, et al. Evidence of increased oxidative damage in subjects with mild cognitive impairment. Neurology 2005;64:1152-56
50. Markesbery WR, Kryscio RJ, Lovell MA, et al. Lipid peroxidation is an early event in the brain in amnestic mild cognitive impairment. Ann Neurol 2005;58:730-35
51. Pratico D, Sung S. Lipid peroxidation and oxidative imbalance: Early functional events in Alzheimer's disease. J Alzheimers Dis 2004;6:171-75
52. Butterfield DA, Boyd-Kimball D. The critical role of methionine 35 in Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity. Biochim Biophys Acta 2005;1703:149-56
53. Perry G, Castellani RJ, Smith MA, et al. Oxidative damage in the olfactory system in Alzheimer's disease. Acta Neuropathol 2003;106:552-56
54. Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Chem Res Toxicol 1997;10:485-94
55. Cardoso SM, Pereira C, Oliveira CR. The protective effect of vitamin E, idebenone and reduced glutathione on free radical mediated injury in rat brain synaptosomes. Biochem Biophys Res Commun 1998;246:703-10
56. Antonsson B. Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways. Mol Cell Biochem 2004;256-257:141-55
57. Marlatt M, Lee HG, Perry G, et al. Sources and mechanisms of cytoplasmic oxidative damage in Alzheimer's disease. Acta Neurobiol Exp (Wars) 2004;64:81-87
58. Sheehan JP, Swerdlow RH, Miller SW, et al. Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer's disease. J Neurosci 1997;17:4612-22
59. Balazs L, Leon M. Evidence of an oxidative challenge in the Alzheimer's brain. Neurochem Res 1994;19:1131-37
60. Subbarao KV, Richardson JS, Ang LC. Autopsy samples of Alzheimer's cortex show increased peroxidation in vitro. J Neurochem 1990;55:342-45
61. Lovell MA, Ehmann WD, Butler SM, et al. Elevated thiobarbituric acid-reactive substances and antioxidant enzyme activity in the brain in Alzheimer's disease. Neurology 1995;45:1594-601
62. Lyras L, Cairns NJ, Jenner A, et al. An assessment of oxidative damage to proteins, lipids, and DNA in brain from patients with Alzheimer's disease. J Neurochem 1997;68:2061-69
63. Palmer AM, Burns MA. Selective increase in lipid peroxidation in the inferior temporal cortex in Alzheimer's disease. Brain Res 1994;645:338-42
64. Forman MS, Mufson EJ, Leurgans S, et al. Cortical biochemistry in MCI and Alzheimer disease: Lack of correlation with clinical diagnosis. Neurology 2007;68:757-63
65. Bains JS, Shaw CA. Neurodegenerative disorders in humans: The role of glutathione in oxidative stress-mediated neuronal death. Brain Res Brain Res Rev 1997;25:335-58
66. Ansari MA, Ahmad AS, Ahmad M, et al. Selenium protects cerebral ischemia in rat brain mitochondria. Biol Trace Elem Res 2004;101:73-86
67. Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A 1990;87:1620-24
68. Perez-De La Cruz V, Gonzalez-Cortes C, Galvan-Arzate S, et al. Excitotoxic brain damage involves early peroxynitrite formation in a model of Huntington's disease in rats: Protective role of iron porphyrinate 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinate iron (III). Neuroscience 2005;135:463-74
69. Xie C, Lovell MA, Markesbery WR. Glutathione transferase protects neuronal cultures against four hydroxynonenal toxicity. Free Radic Biol Med 1998;25:979-88
70. Baez S, Segura-Aguilar J, Widersten M, et al. Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes. Biochem J 1997;324:25-28
71. Shah ZA, Vohora SB. Antioxidant/restorative effects of calcined gold preparations used in Indian systems of medicine against global and focal models of ischaemia. Pharmacol Toxicol 2002;90:254-59
72. Ishrat T, Khan MB, Hoda MN, et al. Coenzyme Q10 modulates cognitive impairment against intracerebroventricular injection of streptozotocin in rats. Behav Brain Res 2006;171:9-16
73. Russell RL, Siedlak SL, Raina AK, et al. Increased neuronal glucose-6-phosphate dehydrogenase and sulfhydryl levels indicate reductive compensation to oxidative stress in Alzheimer disease. Arch Biochem Biophys 1999;370:236-39
74. Martins RN, Harper CG, Stokes GB, et al. Increased cerebral glucose-6-phosphate dehydrogenase activity in Alzheimer's disease may reflect oxidative stress. J Neurochem 1986;46:1042-45
75. Kishida KT, Klann E. Sources and targets of reactive oxygen species in synaptic plasticity and memory. Antioxid Redox Signal 2007;9:233-44
76. Tejada-Simon MV, Serrano F, Villasana LE, et al. Synaptic localization of a functional NADPH oxidase in the mouse hippocampus. Mol Cell Neurosci 2005;29:97-106
77. Harman D. Alzheimer's disease: Role of aging in pathogenesis. Ann N Y Acad Sci 2002;959:384-95; >[discussion 463-65]>
78. Resende R, Moreira PI, Proenca T, et al. Brain oxidative stress in a triple-transgenic mouse model of Alzheimer disease. Free Radic Biol Med 2008;44:2051-57
79. Callio J, Oury TD, Chu CT. Manganese superoxide dismutase protects against 6-hydroxydopamine injury in mouse brains. J Biol Chem 2005;280:18536-42
80. Dringen R, Pawlowski PG, Hirrlinger J. Peroxide detoxification by brain cells. J Neurosci Res 2005;79:157-65
81. Bayir H, Kagan VE, Clark RS, et al. Neuronal NOS-mediated nitration and inactivation of manganese superoxide dismutase in brain after experimental and human brain injury. J Neurochem 2007;101:168-81
82. Sayre LM, Zelasko DA, Harris PL, et al. 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer's disease. J Neurochem 1997;68:2092-97
83. Smith MA, Sayre LM, Anderson VE, et al. Cytochemical demonstration of oxidative damage in Alzheimer disease by immunochemical enhancement of the carbonyl reaction with 2,4-dinitrophenylhydrazine. J Histochem Cytochem 1998;46:731-35
84. Sidell KR, Montine KS, Picklo MJ Sr, et al. Mercapturate metabolism of 4-hydroxy-2-nonenal in rat and human cerebrum. J Neuropathol Exp Neurol 2003;62:146-53
85. Nunomura A, Perry G, Aliev G, et al. Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol 2001;60:759-67
86. Marcello E, Epis R, Di Luca M. Amyloid flirting with synaptic failure: Towards a comprehensive view of Alzheimer's disease pathogenesis. Eur J Pharmacol 2008;585:109-18
87. De Felice FG, Velasco PT, Lambert MP, et al. Abeta oligomers induce neuronal oxidative stress through an N
-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine. J Biol Chem 2007;282:11590-601
88. Cirrito JR, Yamada KA, Finn MB, et al. Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron 2005;48:913-22
89. Selkoe DJ. Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior. Behav Brain Res 2008;192:106-13
90. Knobloch M, Mansuy IM. Dendritic spine loss and synaptic alterations in Alzheimer's disease. Mol Neurobiol 2008;37:73-82
91. Arranz B, Blennow K, Ekman R, et al. Brain monoaminergic and neuropeptidergic variations in human aging. J Neural Transm 1996;103:101-15
92. Dodd PR, Hambley JW, Cowburn RF, et al. A comparison of methodologies for the study of functional transmitter neurochemistry in human brain. J Neurochem 1988;50:1333-45
93. Palmer AM, Lowe SL, Francis PT, et al. Are post-mortem biochemical studies of human brain worthwhile? Biochem Soc Trans 1988;16:472-75
This article has been cited 25 time(s).
Journal of Alzheimers DiseaseEvidence for Early Cognitive Impairment Related to Frontal Cortex in the 5XFAD Mouse Model of Alzheimer's DiseaseJournal of Alzheimers Disease
Experimental Biology and MedicineOxidation and ubiquitination in neurodegenerationExperimental Biology and Medicine
Applied Physiology Nutrition and Metabolism-Physiologie Appliquee Nutrition Et MetabolismeOophorectomy hinders antioxidant adaptation promoted by swimming in Wistar ratsApplied Physiology Nutrition and Metabolism-Physiologie Appliquee Nutrition Et Metabolisme
Free Radical Biology and MedicineLipid peroxidation triggers neurodegeneration: A redox proteomics view into the Alzheimer disease brainFree Radical Biology and Medicine
Archives of Medical ResearchUnderstanding Risk Factors for Alzheimer's Disease: Interplay of Neuroinflammation, Connexin-based Communication and Oxidative StressArchives of Medical Research
Oxidative Medicine and Cellular LongevityOxidative Stress and the Pathogenesis of Alzheimer's DiseaseOxidative Medicine and Cellular Longevity
Frontiers in Aging NeuroscienceMolecular mechanisms of cognitive dysfunction following traumatic brain injuryFrontiers in Aging Neuroscience
Journal of Alzheimers DiseaseOxidative Modification of Brain Proteins in Alzheimer's Disease: Perspective on Future Studies Based on Results of Redox Proteomics StudiesJournal of Alzheimers Disease
Neuromolecular MedicineTelomere Shortening and Alzheimer's DiseaseNeuromolecular Medicine
Antioxidants & Redox SignalingMitochondrial DNA Oxidative Damage and Repair in Aging and Alzheimer's DiseaseAntioxidants & Redox Signaling
Neurological SciencesCan urinary excretion rate of 8-isoprostrane and malonaldehyde predict postoperative cognitive dysfunction in aging?Neurological Sciences
Redox ReportOxidative stress in Alzheimer's disease: Primary villain or physiological by-product?Redox Report
European Journal of Medicinal ChemistrySynthesis and evaluation of 7,8-dehydrorutaecarpine derivatives as potential multifunctional agents for the treatment of Alzheimer's diseaseEuropean Journal of Medicinal Chemistry
Current Alzheimer Research
Prodromal Metabolic Phenotype in MCI Cybrids: Implications for Alzheimer's Disease
Current Alzheimer Research, 10(2):
Current Alzheimer Research
Synaptic Aging is Associated with Mitochondrial Dysfunction, Reduced Antioxidant Contents and Increased Vulnerability to Amyloid-beta Toxicity
Current Alzheimer Research, 10(3):
Journal of NeuroimmunologyThe neurotoxic effect of astrocytes activated with toll-like receptor ligandsJournal of Neuroimmunology
Bioorganic & Medicinal ChemistryDesign, synthesis and neuroprotective evaluation of novel tacrine-benzothiazole hybrids as multi-targeted compounds against Alzheimer's diseaseBioorganic & Medicinal Chemistry
Neurochemical ResearchAlteration in Glutathione Content and Associated Enzyme Activities in the Synaptic Terminals but not in the Non-synaptic Mitochondria from the Frontal Cortex of Parkinson's Disease BrainsNeurochemical Research
CellIntegrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer's DiseaseCell
Current Neurovascular Research
Computational Insights into the Role of Glutathione in Oxidative Stress
Current Neurovascular Research, 10(2):
Neurochemistry InternationalH2O2 and PAF mediate A beta 1-42-induced Ca2+ dyshomeostasis that is blocked by EGb761Neurochemistry International
Journal of NeuroscienceA Calorie-Restricted Diet Decreases Brain Iron Accumulation and Preserves Motor Performance in Old Rhesus MonkeysJournal of Neuroscience
Expert Review of NeurotherapeuticsOxidative stress and Alzheimer's disease: dietary polyphenols as potential therapeutic agentsExpert Review of Neurotherapeutics
Journal of Medicinal ChemistryNovel Tacrine-8-Hydroxyquinoline Hybrids as Multifunctional Agents for the Treatment of Alzheimer's Disease, with Neuroprotective, Cholinergic, Antioxidant, and Copper-Complexing PropertiesJournal of Medicinal Chemistry
Environmental Health PerspectivesMolecular Mechanism of Acrylamide Neurotoxicity: Lessons Learned from Organic ChemistryEnvironmental Health Perspectives
Alzheimer disease; Free radicals; Mild cognitive impairment; Mitochondria; Neurodegeneration; Oxidative stress; Synapses
© 2010 American Association of Neuropathologists, Inc
Highlight selected keywords in the article text.