Measurement of cellular beta-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems.
Amyloid-beta peptide (Abeta), a putatively causative agent of Alzheimer's disease (AD), is proteolytically derived from beta-amyloid precursor protein (APP). Here we describe cellular assays to detect the activity of the key protease beta-site of APP cleaving enzyme 1 (BACE1) based on an artificial reporter construct containing the BACE1 cleavage site of APP. These methods allow identification of inhibitors and indirect modulators of BACE1. In primary neuronal cultures transfected with human APP constructs (huAPP), Abeta production was modified by BACE1 inhibitors similarly to the production of endogenous murine Abeta in wild-type cells and to that of different transgenic neurons. To further improve the assay, we substituted the extracellular domain of APP by secreted alkaline phosphatase (SEAP). SEAP was easily quantified in the cell culture supernatants after cleavage of SEAP-APP by BACE1 or alpha-secretases. To render the assay specific for BACE1, the alpha-secretase cleavage site of SEAP-APP was eliminated either by site-directed mutagenesis or by substituting the transmembrane part of APP by the membrane domain of the erythropoietin receptor (EpoR). The pharmacology of these constructs was characterized in detail in HEK293 cells (human embryonic kidney cell line), and the SEAP-APP-EpoR construct was also introduced into primary murine neurons and there allowed specific measurement of BACE1 activity.
Volbracht
Christiane
C
H. Lundbeck A/S, 2500 Valby, Denmark.
Penzkofer
Stephan
S
Mansson
David
D
Christensen
Kenneth Vielsted
KV
Fog
Karina
K
Schildknecht
Stefan
S
Leist
Marcel
M
Nielsen
Jacob
J
D000469:Alkaline Phosphatase / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000201:enzymology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D009154:Mutation; D009474:Neurons / Q000201:enzymology*
D016564:Amyloid beta-Protein Precursor; D000469:Alkaline Phosphatase; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse