High content analysis of γ-secretase activity reveals variable dominance of presenilin mutations linked to familial Alzheimer’s disease

Cristina Florean, Enrico Zampese, Marion Zanese, Lucia Brunello, François Ichas, Francesca De Giorgi, Paola Pizzo
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


Auteurs Bordeaux Neurocampus