High content analysis of γ-secretase activity reveals variable dominance of presenilin mutations linked to familial Alzheimer’s disease
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2008-08-01; 1783(8): 1551-1560
DOI: 10.1016/j.bbamcr.2008.03.012
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gamma-Secretase mediates the intramembranous proteolysis of amyloid precursor
protein (APP), Notch and other cellular substrates and is considered a prime
pharmacological target in the development of therapeutics for Alzheimer’s disease
(AD). We describe here an efficient, new, simple, sensitive and rapid assay to
quantify gamma-secretase activity in living cells by flow cytometry using two
membrane-bound fluorescent probes, APP-GFP or C99-GFP, as substrates for
gamma-secretase. The principle of the assay is based on the fact that the soluble
intracellular domain of GFP-tagged APP (AICD-GFP) is released from the membrane
into the cytosol following gamma-secretase cleavage. Using this feature,
enzymatic activity of gamma-secretase could be deduced from the extent of the
membrane retention of the probe observed after plasma membrane permeabilization
and washout of the cleaved fraction. By applying two well-known gamma-secretase
inhibitors (DAPT and L-685,458), we validated our assay showing that the
positional GFP-based probes for gamma-secretase activity behave properly when
expressed in different cell lines, providing the basis for the further
development of a high-throughput and high content screening for AD targeted drug
discovery. Moreover, by co-expression of different familial AD-linked mutated
forms of presenilin–the key component of the gamma-secretase complex–in cells
devoid of any endogenous gamma-secretase, our method allowed us to evaluate in
situ the contribution of different presenilin variants to the modulation of the
enzyme.