Affiliation:
1. Department of Psychiatry, Townsend Family Laboratories, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada
Abstract
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.
Funder
Canadian Institutes of Health Research (CIHR)
Publisher
Bentham Science Publishers Ltd.
Subject
Neurology (clinical),Neurology
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