A Novel Cell-based β-secretase Enzymatic Assay for Alzheimer's Disease.
| Autor: | De Araujo Herculano B; Department of Psychiatry, Townsend Family Laboratories, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada., Wang Z; Department of Psychiatry, Townsend Family Laboratories, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada., Song W; Department of Psychiatry, Townsend Family Laboratories, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada. |
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| Jazyk: | angličtina |
| Zdroj: | Current Alzheimer research [Curr Alzheimer Res] 2019; Vol. 16 (2), pp. 128-134. |
| DOI: | 10.2174/1567205016666181212151540 |
| Abstrakt: | 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. (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.) |
| Databáze: | MEDLINE |
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