Lay summary: The brains of Alzheimer’s disease (AD) patients are characterised by the deposition of plaques that lead the spectrum of characteristic neurological problems. The plaques are formed from the normal breakdown of an important nerve cell protein. However, in AD the protein breakdown products are not removed from the brain, thereby forming plaques. The process for removal of the breakdown products has not been fully elucidated; although it is thought to involve a family of transporters (ABC family) located at the blood-brain barrier. The activity of these transporters in AD patients is thought to be impaired, hence the increased accumulation of breakdown products in brain tissue. However, their role in mediating this transport process has not been conclusively proven. This research proposal will provide direct evidence on whether ABC family proteins are able to transport protein breakdown products responsible for plaque formation.
Project description: The brains of Alzheimer’s patients are characterised by several morphological alterations including the presence of insoluble fibrils and plaques. The presence of such fibrils and plaques interferes with neuronal transmission and is associated with memory deficit and cognitive perturbation. The fibrils comprise aggregates or oligomers of Amb-peptides generated from the breakdown of the Amyloid Precursor Protein. The underlying cause of Amb-peptide accumulation in the brain is due to reduced clearance from the CNS, although the precise mechanism remains elusive. Considerable evidence suggests that ABC transporters, in particular ABCB1 and ABCG2, are involved in the process of Amb-peptide clearance. Both proteins are expressed in the endothelial cells of the BBB, which is the exit point for substances from the CNS to the systemic circulation. To date, the experimental evidence is correlative or produced through indirect strategies.
The aim of the current proposal is to ascertain whether the efflux pumps ABCB1 and ABCG2 transport Amb-peptides across biological membranes.
A direct investigative strategy will be used to address this objective and remove the complexity of cellular biological membranes. In particular, this will involve the use of purified ABCB1 or ABCG2 reconstituted into model membrane vesicles. Using these proteoliposomal vesicles it will be possible to directly measure whether the proteins are capable of mediating Amβ-peptide transport across biomembranes. The laboratory has 15 years’ experience with the purification and reconstitution of ABC proteins, in particular the efflux pumps ABCB1 and ABCG2. Methods have been developed for the expression of protein in insect cells, a purification system generated for poly-histidine tagged protein and reconstitution optimised through a detergent adsorption technique. Functional assays for reconstituted pumps have also been developed to validate retention of function following purification and to assess pharmacological interactions.
Demonstration of a direct role in transport of Amb-peptides across biomembranes has a number of significant consequences. First, it will reveal the molecular identity of the transporter mediating clearance of peptides from the brain. Moreover, this finding would render the transporters potential therapeutic targets. Strategies to increase the cellular activity or expression of these transporters could alleviate or prevent the accumulation of harmful Amb-peptide deposits in the brain.