Bruno De Araujo Herculano, Zhe Wang and Weihong Song* Pages 128 - 134 ( 7 )
Background: Deposition of the amyloid β protein (Aβ) into neuritic plaques is the neuropathological hallmark of Alzheimer’s Disease (AD). Aβ is generated through the cleavage of the Amyloid Precursor Protein (APP) by β-secretase and γ-secretase. Currently, the evaluation of APP cleavage by β-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 β-secretase enzymatic assay utilizing a novel chimeric protein that incorporates the natural sequence of APP and more closely replicates its cleavage by β-secretase under physiological conditions.
Methods: We have developed a chimeric protein construct, ASGβ, incorporating the β-site cleavage sequence of APP targeted by β-secretase and its intracellular trafficking signal into a Phosphatase-eGFP secreted reporter system. Upon cleavage by β-secretase, ASGβ 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 ASGβ that can be utilized to assay β-secretase in real time.
Results: ASGβ is specifically targeted by β-secretase, being cleaved exclusively at the site responsible for the generation of Aβ. Dosage response to β-secretase inhibitors shows that β-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 β-secretase activity and Aβ production under physiological conditions, as well as evaluating factors that regulate this cleavage.
Alzheimer's disease, BACE1, β-secretase, secreted alkaline phosphatase, enzymatic assay, Amyloid Precursor Protein (APP).
Department of Psychiatry, Townsend Family Laboratories, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Department of Psychiatry, Townsend Family Laboratories, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Department of Psychiatry, Townsend Family Laboratories, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3