High content analysis of gamma-secretase activity reveals variable dominance of presenilin mutations linked to familial Alzheimer's disease.
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.
Florean
Cristina
C
Department Biomedical Sciences and CNR Institute of Neuroscience, University of Padua, Via G. Colombo, 3, 35121 Padua, Italy.
Zampese
Enrico
E
Zanese
Marion
M
Brunello
Lucia
L
Ichas
François
F
De Giorgi
Francesca
F
Pizzo
Paola
P
D000544:Alzheimer Disease / Q000235:genetics*; D053829:Amyloid Precursor Protein Secretases / Q000032:analysis* / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D051379:Mice; D009154:Mutation; D053763:Presenilins / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis
D016564:Amyloid beta-Protein Precursor; D005456:Fluorescent Dyes; D053763:Presenilins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D053829:Amyloid Precursor Protein Secretases