(228er) Maximizing P-Glycoprotein Expression in the Presence of Certain Therapeutic Compounds

Alzheimerâ??s disease (AD), 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. P-glycoprotein (P-gp), an ATP binding cassette transporter located on the apical side of the blood-brain barrier, has been shown to transport the oligomeric aggregates of Aβ. While monomeric Aβ is inert, oligomeric Aβ exhibits neurotoxicity and leads to the formation of Aβ fibrils that deposit as amyloid plaques, which are characteristic of AD brain. However, formation of oligomeric Aβ may be important to its clearance from the brain.

Previously, a correlation was demonstrated between low levels of P-gp expression at the blood-brain barrier and high levels of Aβ aggregate deposits around the cerebrovascular. Also, it has been recently discovered that the presence of certain forms of Aβ in the cells of the blood-brain barrier can reduce P-gp levels through the ubiquitin-proteasome degradation pathway. These findings identify P-gp as a novel therapeutic target for AD. This study seeks to identify therapeutic compounds that will increase the expression levels of P-gp at the blood-brain barrier in order to mitigate the accumulation of Aβ by allowing for increased clearance of Aβ from the brain.

Madin-Darby Canine Kidney (MDCK) epithelial cells continuously express the multidrug resistance 1 (MDR1) gene responsible for the presence of P-gp. These cells are often used in the study of P-gp due to the availability of a MDCK-MDR1 cell line, a MDCK cell line transfected to express additional P-gp via the MDR1 gene, that can be used for comparison when up-regulating P-gp expression in standard MDCK cells. Cells were treated with three prospective therapeutic compounds: verapamil, caffeine, and rifampicin. Verapamil is a known P-gp substrate recently suggested to up-regulate P-gp expression. Rifampicin, an antibiotic used to treat tuberculosis and leprosy, and caffeine have been shown in epidemiological studies to reduce dementia symptoms. Following treatment, alteration of P-gp expression was determined using confocal microscopy to visualize cell membrane location of P-gp and SDS-PAGE with Western blotting to quantify P-gp expression. Results highlight the prospective therapeutic potential of these compounds.