A Novel Cell-based β-secretase Enzymatic Assay for Alzheimer's Disease

Background: Deposition of the amyloid beta protein (A beta) into neuritic plaques is the neuropathological hallmark of Alzheimer's Disease (AD). A beta is generated through the cleavage of the Amyloid Precursor Protein (APP) by beta-secretase and gamma-secretase. Currently, the evaluation of APP cleavage by beta-secretase in experimental settings has largely depended on models that do not replicate the physiological conditions of this process. Objective: To establish a novel live cell-based beta-secretase enzymatic assay utilizing a novel chimeric protein that incorporates the natural sequence of APP and more closely replicates its cleavage by beta-secretase under physiological conditions. Methods: We have developed a chimeric protein construct, ASG beta, incorporating the beta-site cleavage sequence of APP targeted by beta-secretase and its intracellular trafficking signal into a Phosphatase-eGFP secreted reporter system. Upon cleavage by beta-secretase, ASG beta releases a phosphatase-containing portion that can be measured in the culture medium, and an intracellular fraction that can be detected through Western Blot. Subsequently, we have generated a cell line stably expressing ASG beta that can be utilized to assay beta-secretase in real time. Results: ASG beta is specifically targeted by beta-secretase, being cleaved exclusively at the site responsible for the generation of A beta. Dosage response to beta-secretase inhibitors shows that beta-secretase activity can be positively correlated to phosphatase activity in culture media. Conclusion: Our findings suggest this system could be a high-throughput tool to screen compounds that aim to modulate beta-secretase activity and A beta production under physiological conditions, as well as evaluating factors that regulate this cleavage.