Alzheimer’s disease (AD) is the most common form of neurodegenerative disorder, affects over 5 million people, and is the 6th leading cause of death in the United States. AD is characterized by an accumulation of amyloid-β protein (Aβ) in the brain. Currently under study as a therapeutic approach is the transport of excess Aβ out of the brain through the single endothelial cell thickness of 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 monomeric and oligomeric forms of Aβ. While monomeric Aβ is inert, oligomeric Aβ exhibits neurotoxicity and leads to the formation of Aβ fibrils that accumulate as plaques in the brain. However, 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 interact with P-gp.
An ATPase activity assay was used to quantify Aβ binding by P-gp. In this assay, when ligand binds to P-gp, ATP is hydrolyzed and inorganic phosphate (Pi) is released. Inverted vesicles containing P-gp (BD Biosciences) are incubated alone (negative control), in the presence of verapamil (positive control), or in the presence of Aβ prepared with different assembly states. Addition of MgATP initiates binding, which is halted for analysis by the addition of SDS. The concentration of Pi is then measured via absorbance to quantify binding relative to the negative control.
To study monomeric Aβ, Aβ1-40 (Anaspec) is purified using SEC. Aβ fibrils are formed by incubating the purified Aβ monomer under continuous agitation and in the presence of a high salt concentration. Sonicated Aβ fibrils are prepared by sonicating the prepared Aβ fibrils for 5 min. Aβ oligomers are prepared from Aβ1-42(Anaspec) solubilized in DMSO and diluted into aqueous buffer. Oligomer sizes are then determined using SDS-PAGE and Western blot.
10 μM Aβ monomer and Aβ fibril exhibited negligible binding of P-gp, while 1 mM Aβ oligomer and 10 µM sonicated Aβ fibril effectively bound P-gp, as observed by Pi concentrations elevated over the negative control. Moreover, Aβ oligomer exhibited the most pronounced binding, even at a 10-fold lower concentration. These results demonstrate that the size of the Aβ aggregate species plays a crucial role in the binding of Aβ to P-gp for transport. These experiments will be continued in MDR1 MDCK cells to ensure that this correlation is upheld in a cell culture model.
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