Regulation of beta-amyloid levels in the brain of cholesterol-fed rabbit, a model system for sporadic Alzheimer's disease

Abstract

Accumulation of beta-amyloid (Abeta) peptide in the brain is a major hallmark of Alzheimer's disease (AD). Hypercholesterolemia is a risk factor for AD and has been shown by laboratory studies to cause Abeta accumulation. Abeta levels in the brain are governed by its generation from amyloid precursor protein by beta-secretase (BACE1), degradation by the insulin degrading enzyme (IDE), clearance from the brain by the low density lipoprotein receptor-related protein (LRP-1), and transport from circulation into the brain by receptor for advanced glycation end products (RAGE). However, the mechanisms by which hypercholesterolemia causes Abeta accumulation in the brain and contributes to the pathogenesis of AD are still to be determined. In the present study, we determined the extent to which hypercholesterolemia-induced Abeta accumulation is associated with alterations in BACE1, IDE, LRP-1, and RAGE expression levels. We show that hypercholesterolemia increases Abeta production, an effect that is associated with increased levels of BACE1 and RAGE and reduced levels of IDE and LRP-1. These results suggest that reducing Abeta accumulation in the brain may require strategies that combine reduction of generation and transport of Abeta in addition to acceleration of degradation and clearance of this peptide.

Fig. 1

ELISA assay demonstrated that the 2% cholesterol-enriched diet significantly increased Aβ40and Aβ42 in the cortex (A) and in the hippocampus (B); Aggregated Aβ levels were significantly increased in cortex and unchanged in hippocampus. Values from control (n=6) and cholesterol-fed (n=6) rabbits were expressed as mean ± standard error: Aβ1–40 (p = 0.005), Aβ1–42 (p = 0.002), and aggregated Aβ (p = 0.004) in cortex. Aβ1–40 (p =0.0461) and Aβ1–42 (p =0.0431) in hippocampus tissue. *p<0.05; **p<0.01 (Student t test)

Fig. 2

Immunofluorescence staining showing increased expression of Aβ levels (green) as detected by 6E10 antibody in the CA1 region of the hippocampus and in the adjacent cortex of a cholesterol-fed rabbit brain compared to a control rabbit. DAPI (blue) was used to stain nuclei. Bar= 20μm.

Fig. 3

Representative Western blots (A) and densitometric analysis (B) showing increased levels of BACE1 and RAGE in hippocampus and cortex from cholesterol-fed rabbits in comparison to control animals. *p<0.05; **p<0.01 (Student t test).

Fig. 4

(A) Immunofluorescence staining demonstrating a dramatic increase in expression of BACE1 (green) in the CA1 region of the hippocampus and adjacent cortex in sections from a cholesterol-fed rabbit brain compared to section from a control rabbit. DAPI (blue) was used as nuclear counterstain. Bar= 20μm. (B) Immunostaining for RAGE is low in sections from CA1 region of hippocampus or adjacent cortex in a control rabbit. In section from CA1 and adjacent cortex from a cholesterol-fed rabbit, immunoreactivity to RAGE (green) is increased in hippocampus and in cortex. Nuclei were stained with DAPI (blue). Bar= 20μm

Fig. 4

(A) Immunofluorescence staining demonstrating a dramatic increase in expression of BACE1 (green) in the CA1 region of the hippocampus and adjacent cortex in sections from a cholesterol-fed rabbit brain compared to section from a control rabbit. DAPI (blue) was used as nuclear counterstain. Bar= 20μm. (B) Immunostaining for RAGE is low in sections from CA1 region of hippocampus or adjacent cortex in a control rabbit. In section from CA1 and adjacent cortex from a cholesterol-fed rabbit, immunoreactivity to RAGE (green) is increased in hippocampus and in cortex. Nuclei were stained with DAPI (blue). Bar= 20μm

Fig. 5

Representative Western blots (A) and densitometric analysis (B) showing decreased levels of IDE and LRP-1 in hippocampus and cortex from cholesterol-fed rabbits in comparison to control animals. *p<0.05; **p<0.01; ***p<0.001 (Student t test).

Fig. 6

Immunofluorescence staining for LRP-1 (green) showing a marked immunoreactivity in the CA1 region of the hippocampus and a lesser reactivity in cortex from a control rabbit. In a cholesterol-fed rabbit, the immunoreactivity to LRP-1 antibody is greatly reduced in both hippocampus and cortex. DAPI (blue) was used as nuclear marker. Bar=20μm.

Fig. 7

Immunofluorescence staining for IDE (green) is higher in the CA1 region of the hippocampus than the cortex of a control rabbit. IDE immunostaining is greatly reduced in both hippocampus and cortex from a cholesterol-fed rabbit. DAPI (blue) was used as nuclear marker. Bar=20μm.

Fig. 8

A schema showing the possible mechanisms involved in hypercholesterolemia-induced Aβ accumulation. A high cholesterol diet increases blood cholesterol levels (hypercholesterolemia) and leads to increased levels of BACE1 and RAGE, and decreased levels of IDE and LRP-1. Increased levels of BACE1 accelerate processing of APP to Aβ, and increased levels of RAGE result in increased transport of Aβ from the circulation into the brain. Decreased levels of IDE in the brain lead to a decreased cleavage of Aβ and decreased levels of LRP-1 result in reduced clearance of Aβ from the brain out to the circulation. All these changes triggered by hypercholesterolemia may contribute to increased levels of Aβ in the brain. Accumulation of Aβ potentially causes neurodegeneration and may ultimately contribute to the pathogenesis of Alzheimer’s disease. Reducing hypercholesterolemia or maintaining normal levels of cholesterol in the blood would prevent changes in BACE1, RAGE, IDE and LRP-1, thereby precluding Aβ accumulation and subsequent deleterious effects.