Caffeine protects against oxidative stress and Alzheimer's disease-like pathology in rabbit hippocampus induced by cholesterol-enriched diet

Abstract

Cholesterol has been linked to the pathogenesis of sporadic Alzheimer's disease (AD) as a risk factor increasing beta-amyloid (Abeta) and oxidative stress levels. Caffeine has antioxidant properties and has been demonstrated to reduce Abeta levels in transgenic mouse models of familial AD. However, the effects of caffeine on cholesterol-induced sporadic AD pathology have not been determined. In this study, we determined the effects of caffeine on Abeta levels, tau phosphorylation, oxidative stress generation, and caffeine-target receptors in rabbits fed a 2% cholesterol-enriched diet, a model system for sporadic AD. Our results showed that the cholesterol-enriched diet increased levels of Abeta, tau phosphorylation, and oxidative stress measured as increased levels of reactive oxygen species and isoprostanes, glutathione depletion, and increased levels of endoplasmic reticulum stress marker proteins. Additionally, the cholesterol-enriched diet reduced the levels of adenosine A(1) receptors (A(1)R) but not ryanodine or adenosine A(2A) receptors. Caffeine, administered at 0.5 and 30mg/day in the drinking water, reduced the cholesterol-induced increase in Abeta, phosphorylated tau, and oxidative stress levels and reversed the cholesterol-induced decrease in A(1)R levels. Our results suggest that even very low doses of caffeine might protect against sporadic AD-like pathology.

Keywords: Adenosine receptors; Alzheimer’s disease; Caffeine; Cholesterol; Endoplasmic reticulum stress; Oxidative stress; Tau; β-Amyloid.

Fig. 1

Caffeine reduces cholesterol-enriched diet-induced increase in Aβ40 (a) and Aβ42 (b) levels in rabbit hippocampus. Caffeine was administered at 0.5 mg and 30 mg/day in drinking water to rabbits fed normal chow or a 2% cholesterol-enriched diet for 12 weeks. The amount of Aβ measured by ELISA was normalized with the protein concentration in the samples and expressed as pg/mg of protein. Values expressed are mean value ± SEM from three different experiments. ***p<0.001 versus controls, #p<0.05, # #p<0.01, # # #p<0.001 versus rabbits fed with the 2% cholesterol-enriched diet (cholesterol).

Fig. 2

Caffeine dose-dependently regulates Aβ production and degradation in hippocampus from cholesterol-fed rabbits. Representative Western blots (a) and densitometric analysis (b) showing that while both 0.5 and 30 mg/day caffeine reduce the cholesterol-enriched diet-induced increase in AβPP, caffeine reduces BACE1 at 30 mg/day and increases IDE levels at 0.5 mg/day. Values are expressed as mean value ± SEM from three different experiments. *p<0.05, **p<0.01 compared to controls, #p<0.05, # #p<0.01, # # #p<0.001 compared to rabbits fed with 2% cholesterol diet.

Fig. 3

Caffeine regulates tau phosphorylation in cholesterol-fed rabbits in a dose-dependent manner. Western blots (a) and densitometric analyses (b) show that caffeine at 30 mg/day but not at 0.5 mg/day reduces tau phosphorylation in hippocampus from rabbits fed a 2% cholesterol-enriched diet. Tau phosphorylation is detected by PHF-1 and CP13 antibodies. **p<0.01 versus controls, #p<0.05 versus rabbits fed with 2% cholesterol diet.

Fig.4

Caffeine, at 0.5 and 30 mg/day reduces levels of p–GSK-3β (a,b) enzyme that is involved in tau phosphorylation. Although it reduced p–GSK-3β levels, 0.5 mg/day caffeine didn’t reduce tau phosphorylation, suggesting that reduction of p–GSK-3β is not sufficient to prevent tau phosphorylation in cholesterol-fed rabbits. *p<0.05 versus controls, # #p<0.01 versus rabbits fed with 2% cholesterol diet.

Fig. 5

Caffeine dose-dependently reduces reactive oxygen species (ROS) generation, glutathione depletion and isoprostane formation. ROS generation was measured with increased fluorescence with the DCFH-DA assay as well as H₂O₂ production, and isoprostane level is detected with formation of 8-iso-PGF2α. Caffeine reduces the cholesterol-enriched diet-induced increase in ROS generation (a, b) at 30 mg/day and reduced isoprostane formation at both 0.5 and 30 mg/day (c). **p<0.01, ***p<0.001 versus controls, # # #p<0.001 versus rabbits fed with 2% cholesterol-enriched diet.

Fig. 6

Caffeine dose-dependently reduces endoplasmic reticulum (ER) stress induced by cholesterol-enriched diet in rabbit hippocampus. Representative Western blots (a) and densitometric analysis (b) showing increased levels of the ER-resident proteins calreticulin, grp78 and grp94 and of the ER stress marker protein gadd153. Caffeine administered at 30 mg/day of caffeine/day in drinking water decreased the cholesterol-induced increase in calreticulin, grp78, grpP94, and gadd153 levels. At 0.5 mg/day, caffeine only reduces grp78 and calreticulin levels. *p<0.05, **p<0.01 versus controls, #p<0.05, # #p<0.01 versus rabbits fed with 2% cholesterol-enriched diet.

Fig. 7

Caffeine restores adenosine A1 receptor (A₁R) levels. Representative Western blots (a) and densitometric analysis (b) showing that the 2% cholesterol-enriched diet doesn’t affect levels of ryanodine receptor (RyR) or adenosine 2A receptor (A_2AR) but reduces and adenosine A1 receptor (A₁R) levels. Caffeine, at 0.5 mg/day > 30 mg/day, restores A₁R levels. **p<0.01 versus controls, #p<0.05, # #p<0.01 versus rabbits fed with 2% cholesterol diet.