Amyloid Beta Transport and Effects on Permeability in a Human Brain Endothelial Cell Line

Tai, Leon M. (2009). Amyloid Beta Transport and Effects on Permeability in a Human Brain Endothelial Cell Line. PhD thesis The Open University.



The clearance of neurotoxic amyloid beta (Aβ) from the brain represents a novel therapeutic target for Alzheimer's disease (AD). The ability of two blood-brain barrier (BBB) drug transporters, P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP), to transport Aβ was investigated using a human brain endothelial cell (BEC) line, hCMEC/D3. P-gp expression by hCMEC/D3 cells was stable over a high passage number, polarised on the apical membrane, consistent with the blood side in vivo, and comparable, albeit slightly reduced, to primary isolated human BECs. The P-gp inhibitors tariquidar and vinblastine prevented the efflux of rhodamine 123 from hCMEC/D3 cells, indicative of functional P-gp expression. hCMEC/D3 cells therefore constituted a suitable model to investigate P-gp substrate interactions in vitro. P-gp, and to a lesser extent BCRP, inhibition, increased the net influx and decreased the efflux of 0.1 nM 125I Aβ 1-40 in hCMEC/D3 cells. Both P-gp and BCRP inhibition increased the apical-to-basolateral but not the basolateral-to-apical permeability of hCMEC/D3 cells to 125I Aβ 1-40. This data is consistent with P-gp and BCRP, acting in vivo to prevent blood-borne Aβ peptides entering the brain but not to clear Aβ load from the brain.

Vascular dysfunction is emerging as a key pathological hallmark in AD, including increased BBB permeability. The effect of Aβ on the permeability of hCMEC/D3 cells was therefore investigated. Aβ 1-40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kDa FITC-dextran. Increased permeability was associated with a specific decrease in the tight junction protein occludin, but not claudin-5 or ZO-1, both at the protein and mRNA levels. JNK and p38MAPK inhibition prevented Aβ 1-40-mediated occludin down-regulation and increased paracellular permeability of hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing vascular dysfunction in AD.

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