Function and regulation of P-glycoprotein: implications for amyloid-β export

Abstract

Alzheimer’s disease (AD) is a devastating neurodegenerative condition in which accumulation of amyloid-β (Aβ) peptides as plaques in the brain constitutes a hallmark feature. These peptides have been shown to cause impaired synaptic function, memory impairment, and neurotoxicity. Aβ peptides are routinely synthesised in neurons as normal physiological products. However, impaired clearance mechanisms lead to their accumulation in AD. How these hydrophobic, aggregation-prone and membrane-associated peptides are released from cells is poorly understood. P-glycoprotein (P-gp) is an ATP-dependent efflux transporter, whose expression and function in the brain have been shown to decline with age, Aβ deposition, and AD progression. Whilst P-gp can export Aβ peptides out of the brain at the blood-brain barrier, whether it serves a corresponding role in the neuron, where these peptides are predominantly generated, remains unexplored.

This thesis examines the function and regulation of P-gp in the context of Aβ export. The first section describes the design and optimisation of enzyme-linked immunosorbent assays for the specific, sensitive and efficient quantification of cell-secreted Aβ peptides. Next, the role of P-gp in Aβ export is investigated in vitro utilising an overexpression model and a human neuroblastoma cell line. The final chapter explores the post-translational regulation of P-gp, focusing on its modification via ubiquitination and the subsequent effects on protein expression and functionality. Data presented here support a novel role for P-gp in the export of neuronally-generated Aβ peptides, and moreover implicate the ubiquitin E3 ligase, Nedd4, in the regulation of P-gp expression and activity. By clarifying the metabolic pathway of the Aβ peptide and elucidating the function and regulation of P-gp under physiological circumstances, these findings may assist our understanding of what, how and why disruptions to Aβ clearance occur with ageing and disease.