(190n) Transport of Amyloid-? across the Blood Brain Barrier By P-Glycoprotein: A Novel Therapeutic Target in Alzheimer’s Disease

Authors: 
Holt, H., University of South Carolina
Moore, E., University of South Carolina
Riese, M., University of South Carolina
Faucett, M., University of South Carolina
González, F., Biomedical Engineering
Moss, M. A., University of South Carolina
Alzheimer’s disease (AD), the most common form of neurodegenerative disorder, is the 6th leading cause of death in the US. AD is characterized by an accumulation of aggregated amyloid-β protein (Aβ) in the brain. Currently under study as a therapeutic approach is the transport of excess Aβ across the blood-brain barrier (BBB). P-glycoprotein (P-gp), an ATP binding cassette transporter located on the apical side of the BBB, has been shown to transport Aβ. However, the aggregation state of transported Ab has not been explored. While monomeric Aβ is inert, oligomeric Aβ exhibits neurotoxicity and initiates the formation of Aβ fibrils that accumulate as plaques in the brain. Even so, formation of oligomeric Aβ may be important to its clearance from the brain. To explore this hypothesis, this study sought to determine the Aβ assembly state (monomer, oligomer, fibril) that most effectively interacts with P-gp.

An ATPase activity assay was used to quantify Aβ binding by P-gp. When a ligand binds P-gp, ATP is hydrolyzed to release inorganic phosphate (Pi). The concentration of Pi, measured via absorbance, correlates with binding activity. Inverted vesicles, which allow Ab to bind P-gp on the outer vesicle surface, are incubated alone (negative control), in the presence of verapamil (positive control), or in the presence of Aβ prepared to optimize different assembly states. Results demonstrate that Aβ oligomers preferentially bind P-gp compared to Aβ monomer or larger aggregates, including fibril and sonicated fibril.

Uniquely, P-gp contains two distinct binding sites. To determine the P-gp binding site with which oligomeric Aβ interacts, a competition binding assay was performed. Rhodamine 123 (R123) and Hoechst 33342 (H33342) are fluorescent compounds with well-described alternate binding sites on P-gp. The transport of each R123 and H33342 by P-gp into a vesicle is evidenced by a quenching of fluorescence allowing for the calculation of each compound’s transport rate; this transport is impeded by the presence of any compound that binds to the same site on P-gp. Here, inverted vesicles and MgATP are incubated in the presence of both oligomeric Aβ and either R123 or H33342 for identification of the binding site. The rate of fluorescence quenching shows oligomeric Aβ’s competitive binding relationship to a specific binding site on P-gp.

Together, this study demonstrates the size of the Aβ aggregate species plays a crucial role in the binding of Aβ to P-gp for transport. In addition, through the competitive binding assay, the probable P-gp binding site of Aβ has been identified. Future studies will confirm these relationships between oligomeric Aβ and P-gp in a cellular transport assay.