Regulation of beta-amyloid secretion in vitro through p-glycoprotein

The ~4 kDa β-amyloid (Aβ) peptide is believed to be central to the pathogenesis of Alzheimer's disease (AD). Aβ is formed by cleavage of the amyloid precursor protein (APP) through what is known as the amyloidogenic pathway. An alternate nonamyloidogenic pathway cleaves within the Aβ sequence of APP to form a secreted soluble fragment called APPs. Initial experiments studying involvement of second messenger pathways in catabolism of APP led to the unexpected observation that the antiprogestin mefipristone (RU486) increased the secretion of APPS from rat pheochromocytoma (PC 12) cells. The effect was both rapid and potent, manifesting within 15 minutes of treating cells with RU486 at nanomolar concentrations. This led to the hypothesis that RU486 was acting through steroid hormone receptors to cause upregulation of APPs secretion. However, treatment of mouse (E82) fibroblasts devoid of steroid hormone receptors with RU486 still increased APPs secretion, suggesting that RU486 was acting through a steroid receptor-independent mechanism. RU486 is also a known antagonist of p-glycoprotein (p-gp), a member of the ATP-binding cassette (ABC) transporter superfamily. P-gp is a transporter of many structurally unrelated lipophilic substrates, most noted for its ability to confer the multidrug resistance phenomenon in cancerous cells. Treatment of human carcinoma (KB-3.1) cells expressing low levels of p-gp with RU486 did not lead to an increase in APPS secretion, however, the effect was restored in KB-T10 cells which overexpressed p-gp. The ability of p-gp inhibitors to increase APPs secretion was also seen in rat primary cortical cultures. The ability of p-gp inhibitors to increase APPs secretion led to a new hypothesis that inhibition of p-gp decreased Aβ release. Transient transfection of HEK293 cells which were stably transfected with a Swedish familial double mutation of APP (K269sw) with a plasmid that encoded human p-gp under the influence of a retroviral promoter (pHaMDRl) increased Aβ secretion approximately 2-fold above control untransfectants. When these cells were treated with the p-gp inhibitors RU486 and RU49953, Af3 secretion was decreased in a dose-dependent manner. Binding studies using synthetic A(3 peptides and reconstituted, purified hamster p-gp vesicles also showed competitive substrate kinetics between Aβ and known p-gp substrates. This was followed by demonstrating direct transport of Aβ through these vesicles in an ATP- and p-gpdependent manner. These data suggest that Aβ is a bona fide p-gp substrate. APP is expressed throughout the body and thus is the production of Aβ. P-gp, however, has a limited expression profile and while being enriched at the blood-brain barrier, its expression within the central nervous system is low. Given the overlapping substrate profiles of several ABC transporters, it is hypothesized that there may exist a brain-expressed ABC transporter that is able to transport Aβ. Several known ABC transporters are expressed in the brain. Generation of an expression profile of brainexpressed ABC transporters in the AD brain may allow for identification of a brainexpressed Aβ transporter, with the hypothesis that increased expression of a brainexpressed Aβ transporter may lead to increased amyloid burden. Using quantitative competitive RT-PCR, the goal is to generate an expression profile of genes in normal aged versus AD brain. Data show an approximately 3-fold increase in gene expression of ABCC5 in Alzheimer frontal cortex compared to age-matched controls. Efforts to create expression profiles for four other ABC transporters are underway in hopes of providing candidate transporters for functional analysis in the future such that a solid bridge between ABC transporters and AD may be forged.Medicine, Faculty ofGraduat