MDR1-P-Glycoprotein (ABCB1) Mediates Transport of Alzheimer's amyloid-beta peptides--implications for the mechanisms of Abeta clearance at the blood-brain barrier

Abstract

Amyloid-beta (Abeta) is the major component of the insoluble amyloid plaques that accumulate intracerebrally in patients with Alzheimer's disease (AD). It has been suggested that MDR1-P-glycoprotein (ABCB1, P-gp) plays a substantial role in the elimination of Abeta from the brain. In the present study, MDR1-transfected LLC cells growing in a polarized cell layer were used to characterize the interaction of Abeta1-40/1-42 with P-gp. In this system, P-gp-mediated transport can be followed by the efflux of the fluorescent dye rhodamine-123, or of Abeta itself from the cells into the apical extracellular space. Abeta significantly decreased the apical efflux of rhodamine-123, and the transcellular transport of Abeta1-40 and Abeta1-42 into the apical chamber could be demonstrated using both ELISA and fluorescence (FITC)-labeled peptides. This transport was inhibited by a P-gp modulator. Furthermore, ATP-dependent, P-gp-mediated transport of the fluorescence-labeled peptides could be demonstrated in isolated, inside-out membrane vesicles. Our data support the concept that P-gp is important for the clearance of Abeta from brain, and thus may represent a target protein for the prevention and/or treatment of neurodegenerative disorders such as AD.

Figure 1

Expression, localization and function of human P‐gp in MDR1‐transfected LLC cells. A. Immunoblot analysis: Membrane vesicles (50 µg protein) from MDR1‐transfected (LLC‐MDR1) and control (LLC) cells were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis as described in the Methods section. Protein levels were analyzed with the C219 monoclonal antibody against P‐gp. B. Confocal laser scanning immunofluorescence microscopy: P‐gp (green fluorescence) was stained with the primary antibody C219 in MDR1‐transfected (upper and middle panel) and control (lower panel) cells. The upper and lower panels show optical sections in the xy‐plane, the middle panel a vertical section in the xz‐plane. C. Transcellular transport (upper panel) and intracellular accumulation (lower panel) of Rh123: LLC (□) and LLC‐MDR1 (▪) cells were grown on Transwell membrane inserts. Upper panel: Rh123 (10 µM) was delivered either to the basal compartments (B→A) or to the apical compartments (A→B). After 60 minutes at 37°C, fluorescence in the opposite compartments was measured. Lower panel: Rh123 (10 µM) was delivered to the basal compartments. At the time points indicated, fluorescence inside the cells was determined. Data represent means ± SD (n = 6). *Significant difference between the two cell lines according to Student’s t‐test (P < 0.05).

Figure 2

Determination of transcellular transport and fibril formation of Aβ peptides. A. LLC‐MDR1 and LLC control cells were grown on Transwell membrane inserts. After incubation with different concentrations of Aβ1‐40 in the basal compartments at 37°C for 2 h, Aβ peptides were determined in the apical compartments by ELISA. Data are presented as the ratio between LLC‐MDR1 and LLC cells (means ± SD for n = 3). *Significant difference between the two cell lines according to Student’s t‐test (P < 0.05). B. Aβ1‐40 or Aβ1‐42 fibril formation was followed using the thioflavin T assay. Aβ1‐40 or Aβ1‐42 peptides (5 µM) were dissolved either in the Transwell assay (TAB‐40 or TAB‐42) or the membrane vesicle (VAB‐40 or VAB‐42) buffer and incubated at 37°C with (for VAB) or without (for TAB) shaking at 37°C. After the indicated time, samples were mixed with thioflavin T (5 µM final concentration) and the normalized fluorescence intensity determined (excitation 460 nm, emission 485 nm). Data represent means ± SD (n = 3).

Figure 3

Transcellular Transport of fluorescein‐(FITC‐)‐conjugated Aβ1‐40 and Aβ1‐42. LLC cells (□) and LLC‐MDR1 cells (▪) were grown on Transwell membrane inserts. (A,C) FITC‐Aβ1‐40 and FITC‐Aβ1‐42 (5 µM) were delivered either to the basal compartments (B→A) or to the apical compartments (A→B). After 60 minutes at 37°C, fluorescence in the opposite compartments was measured. (B,D) FITC‐Aβ1‐40 and FITC‐Aβ1‐42 (5 µM) were delivered to the basal compartments in the absence or presence of 10 µM cyclosporine A (+CsA). After 60 minutes at 37°C, fluorescence in the apical compartments was measured. Data represent means ± SD (n = 4). *Significant difference, Student’s t‐test (P < 0.05).

Figure 4

Transport of Rh123 into membrane vesicles from LLC cells and LLC‐MDR1 cells and its inhibition by Aβ peptides. A. Membrane vesicles were incubated with 10 µM Rh123 in the presence of 4 mM ATP (▴) or 4 mM 5′‐AMP (▾). Vesicle‐associated fluorescence was determined by size‐exclusion centrifugation. B. Net ATP‐dependent transport into the vesicles from LLC‐MDR1 (▪) and from LLC (□) cells was calculated by subtracting values obtained in the presence of 5′‐AMP from those in the presence of ATP. C. ATP‐dependent transport (10 µM Rh123) in the presence of 5 µM Aβ1‐40 and Aβ1‐42 and 100 µM verapamil, respectively. Data represent means ± SD (n = 4). *Significant difference from control as determined by Student’s t‐test (P < 0.05).

Figure 5

Transport of fluorescein (FITC‐)‐conjugated Aβ1‐40 and Aβ1‐42 into membrane vesicles from LLC‐MDR1 and control cells. (A,C) Membrane vesicles were incubated with 5 µM FITC‐Aβ1‐40 (A) and Aβ1‐42 (C) in the presence of 4 mM ATP (▴) or 4 mM 5′‐AMP (▾). Vesicle‐associated fluorescence was determined by size‐exclusion centrifugation. (B,D) Net ATP‐dependent transport into the vesicles from LLC‐MDR1 (▪) and from LLC (□) cells was calculated by subtracting values obtained in the presence of 5′‐AMP from those obtained in the presence of ATP. Data represent means ± SD (n = 4).