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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

γ-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 γ-secretase activity in living cells by flow cytometry using two membrane-bound fluorescent probes, APP-GFP or C99-GFP, as substrates for γ-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 γ-secretase cleavage. Using this feature, enzymatic activity of γ-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 γ-secretase inhibitors (DAPT and L-685,458), we validated our assay showing that the positional GFP-based probes for γ-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 γ-secretase complex - in cells devoid of any endogenous γ-secretase, our method allowed us to evaluate in situ the contribution of different presenilin variants to the modulation of the enzyme.

Original languageEnglish (US)
Pages (from-to)1551-1560
Number of pages10
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1783
Issue number8
DOIs
StatePublished - Aug 2008

Keywords

  • γ-Secretase assay
  • Alzheimer's disease
  • Automated flow cytometry
  • High content analysis
  • Presenilin
  • Recombinant probe

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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