Up-regulation of P-glycoprotein reduces intracellular accumulation of beta amyloid: investigation of P-glycoprotein as a novel therapeutic target for Alzheimer's disease

Abstract

Objectives: Several studies have suggested the efflux transporter P-glycoprotein (P-gp) to play a role in the etiology of Alzheimer's disease through the clearance of amyloid beta (Aβ) from the brain. In this study, we aimed to investigate the possibility of P-gp as a potential therapeutic target for Alzheimer's disease by examining the impact of P-gp up-regulation on the clearance of Aβ, a neuropathological hallmark of Alzheimer's disease.

Methods: Uptake studies for ¹²⁵I-radiolabelled Aβ₁₋₄₀, and fluorescent immunostaining technique for P-gp and fluorescent imaging of Aβ₁₋₄₀ were carried out in LS-180 cells following treatment with drugs known to induce P-gp expression.

Key findings: Approximately 10-35% decrease in ¹²⁵I-Aβ₁₋₄₀ intracellular accumulation was observed in cells treated with rifampicin, dexamethasone, caffeine, verapamil, hyperforin, β-estradiol and pentylenetetrazole compared with control. Also, fluorescent micrographs showed an inverse relationship between levels of P-gp expression and 5-carboxyfluorescein labelled Aβ (FAM-Aβ₁₋₄₀) intracellular accumulation. Quantitative analysis of the micrographs revealed that the results were consistent with those of the uptake studies using ¹²⁵I-Aβ₁₋₄₀.

Conclusions: The investigated drugs were able to improve the efflux of Aβ₁₋₄₀ from the cells via P-gp up-regulation compared with control. Our results elucidate the importance of targeting Aβ clearance via P-gp up-regulation, which will be effective in slowing or halting the progression of Alzheimer's disease.

Figure 1

Representative Western blot and densitometry analysis for the ratio of P-gp to β-actin expression levels in LS-180 cells treated with (a) 50 μm caffeine (CAF) (b) 50 μm dexamethasone (DEX) (c) 100 nm hyperforin (HYP) (d) 50 μm rifampicin (RIF) and (e) 50 μm verapamil (VER) compared with control (CTRL). (f) A representative figure for dose dependent increase in P-gp expression (column bars) and activity (line) following treatment with verapamil. The data are expressed as the mean + SD, n = 3 independent experiments. *P ≤ 0.05 vs control.

Figure 2

Effect of treatment of LS-180 cells with rifampicin (RIF), verapamil (VER), dexamethasone (DEX), b-estradiol (EST), pentylenetetrazole (PTZ), hyperforin (HYP) and caffeine (CAF), on the intracellular accumulation of radiolabelled ¹²⁵I-Aβ_1–40. Percent change in the accumulation of Aβ_1–40 was evaluated in the presence (dashed bars) and absence (solid bars) of verapamil, a specific P-gp inhibitor. The data are expressed as mean ± SD, n = 3 independent experiments, each drug 4 replicate/experiment. *P < 0.05.

Figure 3

Representative fluorescent micrographs of P-gp (a, b and c) and FAM-Aβ_1–40 (d, e and f) for control (a and d) and treated LS-180 cells with rifampicin (b and e) and caffeine (c and f). Quantitative folds change in the P-gp expression and FAM-Aβ_1–40 intracellular accumulation were measured using ImageJ version 1.44. The data are expressed as mean ± SD, n = 3. *P < 0.05. Scale bar = 25 μm.