title: Multiplex assay for live-cell monitoring of cellular fates of amyloid-β precursor protein (APP).
abstract: Amyloid-β precursor protein (APP) plays a central role in pathogenesis of Alzheimer's disease. APP has a short half-life and undergoes complex proteolytic processing that is highly responsive to various stimuli such as changes in cellular lipid or energy homeostasis. Cellular trafficking of APP is controlled by its large protein interactome, including dozens of cytosolic adaptor proteins, and also by interactions with lipids. Currently, cellular regulation of APP is mostly studied based on appearance of APP-derived proteolytic fragments to conditioned media and cellular extracts. Here, we have developed a novel live-cell assay system based on several indirect measures that reflect altered APP trafficking and processing in cells. Protein-fragment complementation assay technology for detection of APP-BACE1 protein-protein interaction forms the core of the new assay. In a multiplex form, the assay can measure four endpoints: total cellular APP level, total secreted sAPP level in media, APP-BACE1 interaction in cells and in exosomes released by the cells. Functional validation of the assay with pharmacological and genetic tools revealed distinct patterns of cellular fates of APP, with immediate mechanistic implications. This new technology will facilitate functional genomics studies of late-onset Alzheimer's disease, drug discovery efforts targeting APP and characterization of the physiological functions of APP and its proteolytic fragments.
authors.affiliation: Neuroscience Center, University of Helsinki, Helsinki, Finland.
authors.forename: Maria
authors.initials: M
authors.lastname: Merezhko
authors.affiliation: Neuroscience Center, University of Helsinki, Helsinki, Finland.
authors.forename: Pranuthi
authors.initials: P
authors.lastname: Muggalla
authors.affiliation: Neuroscience Center, University of Helsinki, Helsinki, Finland.
authors.forename: Niko-Petteri
authors.initials: NP
authors.lastname: Nykänen
authors.affiliation: Neuroscience Center, University of Helsinki, Helsinki, Finland.
authors.forename: Xu
authors.initials: X
authors.lastname: Yan
authors.affiliation: Neuroscience Center, University of Helsinki, Helsinki, Finland.
authors.forename: Prasanna
authors.initials: P
authors.lastname: Sakha
authors.affiliation: Neuroscience Center, University of Helsinki, Helsinki, Finland.
authors.forename: Henri J
authors.initials: HJ
authors.lastname: Huttunen
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002460:Cell Line; D017077:Culture Media, Conditioned; D055354:Exosomes / Q000378:metabolism; D006207:Half-Life; D057166:High-Throughput Screening Assays / Q000379:methods*; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D016564:Amyloid beta-Protein Precursor; D017077:Culture Media, Conditioned; D010446:Peptide Fragments; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509282:Bace1 protein, mouse