Endothelial nitric oxide deficiency promotes Alzheimer's disease pathology

Abstract

Aging and the presence of cerebrovascular disease are associated with increased incidence of Alzheimer's disease. A common feature of aging and cerebrovascular disease is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelium derived NO on amyloid precursor protein (APP) related phenotype in late middle aged (LMA) (14-15 month) endothelial nitric oxide synthase deficient (eNOS(-/-) ) mice. APP, β-site APP cleaving enzyme (BACE) 1, and amyloid beta (Aβ) levels were significantly higher in the brains of LMA eNOS(-/-) mice as compared with LMA wild-type controls. APP and Aβ1-40 were increased in hippocampal tissue of eNOS(-/-) mice as compared with wild-type mice. LMA eNOS(-/-) mice displayed an increased inflammatory phenotype as compared with LMA wild-type mice. Importantly, LMA eNOS(-/-) mice performed worse in a radial arm maze test of spatial learning and memory as compared with LMA wild-type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline. Cardiovascular risk factors are associated with increased incidence of Alzheimer's disease (AD). A common feature of these risk factors is decreased endothelial nitric oxide (NO). We observed, in mice deficient in endothelial nitric oxide synthase, increased amyloid precursor protein (APP), β-site APP cleaving enzyme 1, amyloid beta levels, microglial activation, and impaired spatial memory. This suggests chronic loss of endothelial NO may be an important contributor to the pathogenesis of sporadic AD.

Keywords: Alzheimer's disease; amyloid precursor protein; endothelium; memory; microglia; nitric oxide.

Figure 1

Levels of APP, BACE1 and Aβ were increased in the brains of LMA eNOS^−/− mice. (A) Fixed tissue sections from the brains of wild type and eNOS^−/− animals were immunolabeled with anti-APP, anti-BACE1 and anti-Aβ. Representative images of the cortex are shown. Magnification 40x; bar is representative of 20 μm. (B) Brain tissue from eNOS^−/− and wild type animals was analyzed via Western blotting using anti-APP, anti-BACE1, and anti-Actin (loading control) antibodies. Representative image and densitometric analysis is shown (n=12 animals). (C) Aβ_1-40 and (D) Aβ_1-42 levels from 8 individual brain lysates (250 μg total protein/sample) from LMA eNOS^−/− and LMA wild type control were analyzed via commercially available ELISA kits. Data are represented as mean ± SD (*P<0.05 compared to wild type control mice).

Figure 2

Levels of the microglial marker, CD68, are increased in the brains of LMA eNOS^−/− mice. (A) Fixed tissue sections from the brains of LMA eNOS^−/− and LMA wild type mice were immunolabeled with anti-CD68. Representative images of the cortex are shown. Magnification 40x; bar is representative of 20μm. (B) Brain tissue from LMA eNOS^−/− and LMA wild type animals was Western blotted using anti-CD68, anti-GFAP, anti-NeuN, and anti-Actin (loading control) antibodies. Representative image is shown. Densitometric analysis was performed for (C) CD68, (D) GFAP, and (E) NeuN. Data are presented as mean ±SD (*P<0.05 compared to wild type control mice, n=10–12 animals).

Figure 3

Levels of CD68, Iba-1, and MHC II were increased in the brains of eNOS^−/− mice. (A) Brain tissue from 4 month or 15 month old eNOS^−/− and wild type mice was Western blotted using anti-CD68, anti-Iba-1, anti-MHC II, or anti-Actin (loading control) antibodies. Representative image is shown. Densitometric analysis was performed for (B) CD68, (C) Iba-1, and (D) MHC II. Data are presented as mean ± SD (n=8 animals, *P<0.05 compared to age-matched wild type control; #P<0.05 based on genotype: 2-way ANOVA, followed by Tukey-Kramer post hoc tests for individual comparisons).

Figure 4

APP and Aβ_1-40 levels were increased in the hippocampus of LMA eNOS^−/− mice. (A) Fixed tissue sections from the brains of wild type and eNOS^−/− animals were immunolabeled with anti-APP, anti-BACE1 and anti-Aβ. Representative images of the hippocampus are shown. Magnification 40x; bar is representative of 20 μm. (B) Hippocampal tissue from LMA eNOS^−/− and LMA wild type animals was Western blotted using anti-APP, anti-BACE1, and anti-Actin (loading control) antibodies. Representative image and densitometric analysis is shown. (C) Aβ_1-40 and (D) Aβ_1-42 levels from hippocampal lysates (200 μg total protein/sample) from LMA eNOS^−/− and wild type control mice were analyzed via commercially available ELISA kits. Data are represented as mean ± SD (n=6 animals, *P<0.05, **P<0.01 compared to wild type control mice).

Figure 5

Levels of GM-CSF, IL-1α, and MIP-1β are increased in the brains of LMA eNOS^−/− mice. (A) Brain lysates from LMA eNOS^−/− and age-matched wild type control mice were analyzed via a commercially available cytokine array (300μg total protein loaded for each sample). A representative image is shown. Closed arrow depicts GM-CSF, arrowhead depicts IL-1α, and open arrow depicts MIP-1β. (B) GM-CSF (C) IL-1α, and (D) MIP-1β levels from 8 individual brain lysates (500 μg) from 4 month and 15 month old eNOS^−/− and wild type control mice were analyzed via commercially available ELISA kits. Data are represented as mean ± SD (n=7–8 animals, *P<0.05, **P<0.01, and ***P<0.001 compared to age-matched wild type control mice; #P<0.05 based on genotype: 2-way ANOVA, followed by Tukey-Kramer post hoc tests for individual comparisons).

Figure 6

LMA eNOS^−/− animals committed more errors than LMA wild type mice in an 8-arm radial arm maze. The number of (A) incorrect runs, (B) revisiting errors, and (C) time to complete were observed over the course of 7 days of testing. Data are presented as ±SD (*P<0.05, n=8–11 animals).