Oxidative stress in the progression of Alzheimer disease in the frontal cortex

Abstract

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.

Figure 1

Electron photomicrographs of representative isolated mitochondrial (A) and (B) synaptosomal fractions from the frontal cortex of a human postmortem brain sample. Calibration bars: A, 2.0 µm, B, 0.5 µm.

Figure 2

Changes in the glutathione (GSH) system (GSH, oxidized GSH [GSSG] and GSH/GSSG) analyzed in post mitochondrial supernatant (PMS), mitochondrial, and synaptosomal fractions of the frontal cortex from non-cognitively impaired (NCI), mild cognitively impaired (MCI) mild Alzheimer disease (mAD), and AD subjects. All 3 neuropil fractions showed significant declines in GSH (A) and significant increases in GSSG levels (B) with increased cognitive impairment. The changes in GSH and GSSG also resulted in significant changes in the GSH/GSSG ratios with disease progression (C). The synaptosomal fraction showed the greatest changes. Each bar represents the group mean ± SD. **p < 0.01, *p < 0.05 vs. NCI; ^αp < 0.05 vs. MCI.

Figure 3

Different antioxidant enzymes were assessed in post mitochondrial supernatant (PMS), mitochondrial, and synaptosomal fractions of the frontal cortex from non-cognitively impaired (NCI), mild cognitive impairment (MCI,) mild Alzheimer disease (mAD), and AD subjects. (A) Glutathione peroxidase (GPx) activity was significantly changed in mitochondrial and synaptosomal fractions only, not in the PMS. (B) Glutathione reductase (GR) activity showed significant decline only in synaptosomes,. (C) Glutathione-S-transferase (GST) activity showed a significant decline in the mitochondrial and synaptosomal fractions from mAD, and AD, and a significant decline in MCI only in the synaptosomal fraction. (D) Significant alterations in glucose-6-phosphate dehydrogenase (G-6PD) activity were only detected in synaptosomes. Each bar represents the group mean ± SD. **p < 0.01, *p < 0.05 vs. NCI.

Figure 4

Superoxide dismutase (SOD) and catalase (CAT) activities in the post mitochondrial supernatant (PMS), mitochondrial, and synaptosomal fractions of frontal cortex in mild cognitive impairment (MCI) mild Alzheimer disease (mAD), and AD subjects. (A) There was significant depletion in SOD activity in mitochondrial and synaptosomal fractions of MCI, mAD, and AD; the PMS fraction of MCI was not significantly depleted. (B) CAT activity in the PMS and synaptosomal fractions was significantly decreased in MCI, mAD, and AD. Each bar represents the group mean ± SD. **p < 0.01, *p < 0.05 vs. NCI; ^αp < 0.05 vs. MCI; #p < 0.05 vs. mAD.

Figure 5

Thiobarbituric acid reactive substances (TBARS) demonstrated significant increases in all fractions of the frontal cortex from mild cognitive impairment (MCI) mild Alzheimer disease (mAD), and AD subjects. TBARS formation was higher in synaptosomal fractions. Each bar represents the group mean ± SD. *p < 0.01 vs. NCI; ^αp < 0.05 vs. MCI; #p < 0.05 vs. mAD.

Figure 6

Representative Slot-blot for protein carbonyl (PCs) shows half of the synaptosomal fractions of the frontal cortex from non-cognitively impaired (NCI), mild cognitive impairment (MCI) mild Alzheimer disease (mAD), and AD subjects. The slot-blot shows prominent alterations in staining for the different cognitively impaired groups.

Figure 7

Levels of protein modification were analyzed in post mitochondrial supernatant (PMS), mitochondrial, and synaptosomal fractions of the frontal cortex from non-cognitively impaired (NCI), mild cognitive impairment (MCI) mild Alzheimer disease (mAD), and AD subjects. (A) Protein carbonyl (PC) levels showed significant increases in all fractions of the frontal cortex with disease progression. (B) Significant changes in 3-nitrotyrosine levels mirrored the PC changes in all the fractions from MCI, mAD, and AD subjects. (C) 4-hydroxynonenal (4-HNE) and (D) acrolein levels showed significant increases in protein modification via lipid peroxidation. The significant increases in 4-HNE and acrolein were already present in MCI subjects, with the greatest elevations in the synaptosomal fractions. Each bar represents the group mean ± SD. **p < 0.01, *p < 0.05 vs. NCI; ^αp < 0.05 vs. MCI.

Figure 8

Correlations of protein oxidation, nitration and lipid peroxidation in the synaptosomal fraction with the subjects’ Mini Mental State Examination (MMSE) scores. (A–D) There was a positive correlation for protein carbonyls (A), 3-nitrotyrosiine (B), 5-hydroxynonenal (C) and acrolein (D) of increasing levels of oxidative marker with MMSE decline. All cognitive test scores were obtained within 12 months prior to death. **p < 0.0001, *p < 0.005.