Interaction of drugs and amyloid beta with blood-brain barrier transporter proteins

Abstract

The blood-brain barrier (BBB) is a unique physical and enzymatic barrier, formed by the endothelial cells of the cerebral capillaries. The BBB regulates movement of endogenous and exogenous compounds into, and out of, the central nervous system (CNS) and protects the brain from exposure to xenobiotics. A number of ATP-binding cassette (ABC) transporters are expressed in brain endothelial cells and actively efflux an array of substances from the cell into the systemic circulation, thereby limiting CNS penetration. Alzheimer’s disease is a progressive, fatal neurodegenerative disorder and the most common form of dementia. One of the key pathological characteristics of Alzheimer’s disease is aggregation of amyloid-β (Aβ(1-42)) peptide within the brain, leading to production of cytotoxic oligomers, plaque formation, inflammation and subsequent neurological deficit. Aβ(1-42) is transported across the BBB, with influx into the brain mediated by the receptor for advanced glycation end products (RAGE) and clearance via the low density lipoprotein-related protein (LRP) receptor 1 (LRP1) and it has been postulated that reduced Aβ(1-42) clearance from the brain may be associated with accumulation of Aβ(1-42) in Alzheimer’s disease. ABC transporter-mediated transport of amyloid-β has also been reported. The overall aims of this thesis were (i) to investigate the roles of ABCB1 (MDR1, P-glycoprotein), ABCC5 (multidrug resistance-associated protein 5, MRP5) and ABCG2 (breast cancer resistance protein, BCRP) in Aβ(1-42) transport, (ii) determine the effects of Aβ(1-42) exposure on ABC transporter expression and function and (iii) determine the effects of Aβ(1-42) exposure on expression of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR), key nuclear receptors governing ABC transporter expression (iv) determine the ability of the ABCB1, ABCC5 and ABCG2 inhibitors and inducers to modify transcellular Aβ(1–42) transport, (v) determine the effect of inflammatory conditions on ABCB1 and ABCG2 activity and expression. In this thesis, primary porcine brain endothelial cells (PBECs) were used as an in vitro BBB model since this model retains phenotypic features of the in vivo BBB, including expression of marker enzymes, ABC transporters, PXR and CAR. Aβ(1-42) significantly inhibited ABCB1, ABCG2 and ABCC5 activity, as determined by increased intracellular accumulation of fluorescent probe substrates, and expression in PBECs, as measured densitometrically by Western blotting, by up to 55%. The inhibitory effect of Aβ(1-42) was both concentration- and time-dependent. Aβ(1-42) treatment also significantly reduced expression of both PXR and CAR in PBECs up to 18 % and 30 %, respectively. Further studies investigated the effects of therapeutic drugs classed as ABC transporter inducers (dexamethasone, doxorubicin, ritonavir and rifampicin) or inhibitors (lovastatin, indomethacin, olanzapine, glibenclamide, chlorpromazine, telmisartan, pantoprazole, quinidine, sulfasalazine and nefazodone) on Aβ(1-42) transport. When PBECs were co-treated with inhibitors and Aβ(1-42) there was a significant increase in intracellular accumulation of Aβ(1-42). Furthermore, when PBECs were pre-loaded with Aβ(1-42), and treated with inhibitors, Aβ(1-42) efflux was significantly reduced. In a Transwell® model of the BBB, treatment of PBECs with inducer therapeutic drugs significantly reduced Aβ(1-42) apical to basal penetration (i.e. uptake) up to 1.75-fold, but significantly increased basal to apical penetration (i.e. efflux clearance) up to 2-fold. Treatment of PBECs with the pro-inflammatory cytokine interleukin-1β significantly increased ABCB1 expression and activity by 32% and 48% respectively, but no significant effect on ABCG2 expression and activity was observed. Under conditions of oxidative stress (i.e. treatment of PBECs with hydrogen peroxide) no significant change in ABCB1 and ABCG2 expression and activity were o

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Berk (Supervisor) & Jeffrey Penny (Supervisor)