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

ABSTRACT

BACKGROUND: Deposition of the amyloid ß protein (Aß) into neuritic plaques is the neuropathological hallmark of Alzheimer's Disease (AD). Aß is generated through the cleavage of the Amyloid Precursor Protein (APP) by ß-secretase and γ-secretase. Currently, the evaluation of APP cleavage by ß-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 ß-secretase enzymatic assay utilizing a novel chimeric protein that incorporates the natural sequence of APP and more closely replicates its cleavage by ß-secretase under physiological conditions. METHODS: We have developed a chimeric protein construct, ASGß, incorporating the ß-site cleavage sequence of APP targeted by ß-secretase and its intracellular trafficking signal into a Phosphatase-eGFP secreted reporter system. Upon cleavage by ß-secretase, ASGß 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 ASGß that can be utilized to assay ß-secretase in real time. RESULTS: ASGß is specifically targeted by ß-secretase, being cleaved exclusively at the site responsible for the generation of Aß. Dosage response to ß-secretase inhibitors shows that ß-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 ß-secretase activity and Aß production under physiological conditions, as well as evaluating factors that regulate this cleavage.