A sensitive and quantitative assay for measuring cleavage of presenilin substrates.
The presenilin (PS) proteins are components of the gamma-secretase activity, which is central in the pathogenesis of Alzheimer's disease. Here we present a novel cell-based reporter gene assay for the quantification of PS-controlled gamma-secretase cleavage of the Alzheimer amyloid precursor protein (APP). We show that this assay offers several advantages, including increased sensitivity and specificity, improved quantification of cleavage, and simultaneous detection of all gamma-secretase cleavages in APP. Furthermore, the APP assay can be used in parallel with a similar assay that records gamma-secretase cleavage of a Notch receptor. The use of these assays to analyze the effects of two known gamma-secretase inhibitors and postulated PS active site mutants on APP and Notch processing demonstrated that inhibitors and mutants that differently affect Notch and APP cleavage can be identified rapidly. The possibility in using these assays for high throughput screening of candidate gamma-secretase inhibitors for APP and Notch in parallel opens up new vistas to systematically search for novel inhibitors that selectively block APP cleavage while not affecting Notch signaling.
Karlström
Helena
H
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Von Eulers vag 3, SE-171 77 Stockholm, Sweden.
Bergman
Anna
A
Lendahl
Urban
U
Näslund
Jan
J
Lundkvist
Johan
J
D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000032:analysis / Q000737:chemistry; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins; D004622:Embryo, Mammalian / Q000166:cytology; D004625:Embryo, Nonmammalian; D010450:Endopeptidases / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D053764:Presenilin-1; D051880:Receptors, Notch; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D029721:Drosophila Proteins; D008024:Ligands; D008565:Membrane Proteins; C497759:N protein, Drosophila; C508853:PSEN1 protein, human; D053764:Presenilin-1; D051880:Receptors, Notch; D004247:DNA; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human