NOX activity is increased in mild cognitive impairment

Abstract

This study was undertaken to investigate the profile of NADPH oxidase (NOX) in the clinical progression of Alzheimer's disease (AD). Specifically, NOX activity and expression of the regulatory subunit p47phox and the catalytic subunit gp91phox was evaluated in affected (superior and middle temporal gyri) and unaffected (cerebellum) brain regions from a longitudinally followed group of patients. This group included both control and late-stage AD subjects, and also subjects with preclinical AD and with amnestic mild cognitive impairment (MCI) to evaluate the profile of NOX in the earliest stages of dementia. Data show significant elevations in NOX activity and expression in the temporal gyri of MCI patients as compared with controls, but not in preclinical or late-stage AD samples, and not in the cerebellum. Immunohistochemical evaluations of NOX expression indicate that whereas microglia express high levels of gp91phox, moderate levels of gp91phox also are expressed in neurons. Finally, in vitro experiments showed that NOX inhibition blunted the ability of oligomeric amyloid beta peptides to injure cultured neurons. Collectively, these data show that NOX expression and activity are upregulated specifically in a vulnerable brain region of MCI patients, and suggest that increases in NOX-associated redox pathways in neurons might participate in the early pathogenesis of AD.

FIG. 1.

NOX activity in affected and nonaffected brain regions of control, PCAD, MCI, and AD patients. Tissue samples from the (A) superior/middle temporal gyri (SMTG) and (B) cerebellum (CBLM) were collected from normal control subjects, subjects with preclinical Alzheimer's disease (PCAD), subjects with amnestic mild cognitive impairment (MCI), and subjects with definitive, late-stage Alzheimer's disease (AD) and analyzed for NOX activity, as described in Methods. All data were analyzed with one-way ANOVA followed by Tukey's post hoc analyses, as described in Methods. **Significantly increased (p < 0.01) NOX activity in SMTG of MCI samples as compared with control SMTG samples.

FIG. 2.

p47^phox expression in affected and nonaffected brain regions of control, PCAD, MCI, and AD patients. Tissue samples from the (A, B) superior/middle temporal gyri (SMTG) and (C, D) cerebellum (CBLM) were collected from normal control subjects, subjects with preclinical Alzheimer's disease (PCAD), subjects with amnestic mild cognitive impairment (MCI), and subjects with definitive, late-stage Alzheimer's disease (AD) and analyzed for p47^phox expression with Western blot, as described in Methods. (A) Representative blots illustrating p47^phox and tubulin expression in SMTG from control, PCAD, MCI, and AD samples. (B) p47^phox expression in SMTG was quantified with Western blot, and data were analyzed with one-way ANOVA followed by Tukey's post hoc analyses, as described in Methods. *Significantly increased (p < 0.05) p47^phox expression in MCI samples as compared with control samples. (C) Representative blots illustrating p47^phox and tubulin expression in CBLM from control, PCAD, MCI, and AD samples. (D) Quantification of p47^phox expression in cerebellar tissue homogenates.

FIG. 3.

gp91^phox expression in affected and nonaffected brain regions of control, PCAD, MCI, and AD patients. Tissue samples from the (A and B) superior/middle temporal gyri (SMTG) and (C and D) cerebellum (CBLM) were collected from normal control subjects, subjects with preclinical Alzheimer's disease (PCAD), subjects with amnestic mild cognitive impairment (MCI), and subjects with definitive, late-stage Alzheimer's disease (AD) and analyzed for gp91^phox expression with Western blot, as described in Methods. (A) Representative blots illustrating gp91^phox and tubulin expression in SMTG from control, PCAD, MCI, and AD samples. (B) gp91^phox expression in SMTG was quantified with Western blot, and data were analyzed with one-way ANOVA followed by Tukey's post hoc analyses, as described in Methods. *Significantly increased (p < 0.05) gp91^phox expression in MCI samples as compared with control samples. (C) Representative blots illustrating gp91^phox and tubulin expression in CBLM from control, PCAD, MCI, and AD samples. (D) Quantification of gp91^phox expression in cerebellar tissue homogenates.

FIG. 4.

Cell-type–specific expression of cortical gp91^phox in control and MCI patients. Brain sections from the cortex were collected from normal control subjects and subjects with amnestic mild cognitive impairment (MCI) and analyzed for gp91^phox expression with immunocytochemistry, as described in Methods. (Top) Representative low-magnification and high-magnification (insert) images depict the pattern of gp91^phox staining in cortex of control (left) and MCI (right) patients. Arrow, gp91^phox immunoreactivity in a cell with neuronal morphology. (Middle) Representative low-magnification and high-magnification (insert) images depict the pattern of gp91^phox staining (black DAB stain) specifically in Iba-1–positive microglia (NOVAred stain) in cortex of control and MCI patients. Arrow, gp91^phox immunoreactivity not associated with Iba-1 expression. (Bottom) Representative low-magnification and high-magnification (insert) images depict the pattern of gp91^phox staining (black DAB stain) specifically in NeuN-positive neurons (NOVAred stain) in cortex of control and MCI patients. Arrow, gp91^phox immunoreactivity in NeuN-positive cells.

FIG. 5.

Effects of NOX inhibition on oligomeric Aβ-mediated neurotoxicity in vitro. Primary hippocampal neurons were treated with 36 nM oligomeric Aβ (Aβ Olig) in the presence or absence 100 μM apocynin (Apoc) for 24 h, and neuronal injury was determined with MTT and cell counts, as described in Methods. (A) At the end of the exposure period, cells were treated with 0.5 mg/ml MTT for 2 h, after which the medium was aspirated, the formazan precipitates dissolved in DMSO, and the amount of formazan conversion analyzed at 570 nm. Results were generated from three separate experiments with at least four dishes per treatment group in each. Data are presented as percentage of MTT conversion in control cultures and were analyzed with one-way ANOVA. ***Statistically significant decrease (p < 0.001) in neuronal survival after exposure to oligomeric Aβ, as compared with control neurons. ^##Statistically significant increase (p < 0.01) in neuronal survival in neurons treated with oligomeric Aβ in the presence of apocynin, as compared with cells treated with oligomeric Aβ alone. (B) Blinded cell counts based on repeated measures of morphology were conducted to quantify neuronal survival, as described in Methods. Results were generated from three separate experiments with at least four dishes per treatment group in each. Data reflect the number of cells remaining at the end of the exposure period and are expressed as the percentage of cells counted in each field initially. Data were analyzed with one-way ANOVA. ***Statistically significant decrease (p < 0.001) in neuronal survival after exposure to oligomeric Aβ as compared with control neurons. ^##Statistically significant increase (p < 0.01) in neuronal survival in neurons treated with oligomeric Aβ in the presence of apocynin, as compared with cells treated with oligomeric Aβ alone. (C) Representative images of primary hippocampal neurons taken either immediately before (Initial) or after (Final) exposure to oligomeric Aβ in the presence or absence of apocynin. Arrows, Specific neurons that were healthy at the onset of exposure but did not survive exposure to oligomeric Aβ; they illustrate the decrease in cell loss noted in cells co-treated with apocynin.