NEW INSIGHTS INTO THE ROLE OF ABC-TRANSPORTERS IN AMYLOID-BETA CLEARANCE FROM CELLS IN THE BRAIN

Abstract

Accumulation of toxic amyloid-beta peptides in the brain is an established hallmark of Alzheimer's Disease. Several members of the ABC transporter family of proteins implicated in the clearance of amyloid-beta peptides across the blood brain barrier (BBB) endothelium include the drug transporter ABCB1 (or P-glycoprotein) and the lipid transporter ABCG4. However, despite years of research in this area, there are still fundamental gaps in our understanding of whether amyloid-beta peptides are direct substrates for these transporters, and how these transport processes are regulated in various cell types in the brain. We have recently published that ABCG4 is a substrate for an E3-ubiquitin ligase, NEDD4–1 (see Aleidi et al JBC 2015;290:24604), which has also been implicated in regulating ABCB1 activity. Our research aims to investigate whether these transporters may potentially have a combined function in this clearance pathway and how this may be regulated. ABC transporter expression was measured via PCR and Western Blotting in Be(2)-C neuronal and hCMEC/D3 BBB endothelial cells. Cellular ABCB1 activity was measured using Calcein-AM assays while purified recombinant ABCB1, reconstituted in vesicles, was used to measure ATP-consumption in the presence of non-aggregated amyloid-beta (1–40)-peptide. ABCB1 was expressed in BBB endothelial cells as expected but was also surprisingly highly expressed in neuronal cells, a cell type in which ABCB1 function has not been previously characterised. Cellular ABCB1 activity was in line with expression of the transporter, suggesting that ABCB1 is active in neurons. Preliminary data show that amyloid-beta (1–40)-peptide directly stimulates ATP consumption of purified reconstituted ABCB1. In addition, ABCG4 was expressed in neurons as previously described, but also in BBB endothelial cells, where its function is similarly unknown. In both cell types, ABCG4 was not regulated by Liver X receptor (LXR) ligands, as expected from previously published work on the regulation of its close family member, the lipid transporter ABCG1. ABCB1 has the potential to be of importance in the initial export of amyloid-beta peptides from neurons. We are currently investigating a potentially combined function of these two transporters in net amyloid-beta export from endothelial cells and neurons in the brain.