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.

FRMD4A–cytohesin signaling modulates the cellular release of tau

“…We have previously used a PCA based on humanized Gaussia princeps luciferase (GLuc) (Remy and Michnick, 2006) for studying cellular regulation of both APP and tau (Martiskainen et al, 2015;Merezhko et al, 2014;Nykänen et al, 2012). Here, we used a tau dimer PCA reporter based on the tau 0N4R isoform carrying complementary GLuc fragments (Fig.…”

Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation

“…To develop a sensitive and quantitative assay to monitor trafficking of proteins to membrane rafts in live cells, we utilized a PCA based on the codon-optimized for mammalian expression form of G. princeps luciferase (GLuc) [18]. Previously, we have successfully used PCAs based on GLuc for studying cellular regulation of both APP and Tau [22,23,[27][28][29][30].…”

Microbial Sources of Amyloid and Relevance to Amyloidogenesis and AlzheimerÂ’s Disease (AD)

“…The Aβ peptides of AD are derived from a polytopic transmembrane β-amyloid precursor protein (βAPP) though tandem beta- and gamma-secretase cleavage events [ 39 – 41 ]. Cellular trafficking of the ~770 amino acid βAPP precursor is regulated by a large βAPP interactome that includes membrane integral and membrane peripheral adaptor proteins, and also by interactions with membrane-associated glycolipids and phospholipids [ 34 , 42 ]. Aβ40 peptides associate with endothelial cells that line the cerebral vasculature, and the more neurotoxic, albeit less abundant, hydrophobic Aβ42 peptides form the central core of the senile plaque (SP) of the parenchymal lesions that characterize AD [ 40 , 43 ].…”

“…The recognition of Aβ42 peptides and their misfolded aggregates by microglial surveillance systems, and the inability of microglial cells to deal with these toxic, pro-inflammatory inclusions, especially in their multimeric and aggregated form are thought to form the molecular basis for the aberrant immune activation, chronic inflammation and elevated oxidative stress that is characteristic of AD neuropathology [ 8 , 34 , 39 , 46 – 51 ]. Notably, (i) Aβ42 peptides as monomers, dimers and fibrils induce patterns of inflammatory gene expression typical of the classical innate-immune and inflammatory response induced by infectious agents such as bacterial LPS, a common endotoxin of the outer membrane of gram-negative bacteria [ 49 , 52 ; see below]; (ii) the presence of bacterial LPS or endotoxin-mediated inflammation strongly contributes to amyloid neurotoxicity [ 19 , 24 – 27 , 32 , 42 , 50 – 55 ]; and (iii) AD amyloids, like prion amyloids, once formed, may induce a self-perpetuating process leading to amplification, aggregation and spreading of pathological protein assemblies, and serial propagation of distinct strains of Aβ prion-like amyloids from AD patients have been recently observed [ 56 , 57 ]. Indeed, an increasing number of studies support the idea (i) that certain self-propagating protein conformations feature in the pathogenesis of several common neurodegenerative diseases including AD; (ii) that pro-inflammatory and immunogenic aggregates of Aβ peptides may become self-propagating in AD brain; and (iii) that certain forms of Aβ peptides are serially transmissible and hence important in the propagation of neurological disease [ 57 ].…”