title: High throughput monitoring of amyloid-β(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays.
abstract: Accumulation of small soluble assemblies of amyloid-β (Aβ)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aβ(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aβ(42). Modeling suggests that oligomerized Aβ(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in Aβ(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aβ(42) oligomerization. Using ELISA and AlphaLISA, we found that Aβ(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aβ(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aβ(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aβ(42) oligomerization.
authors.affiliation: Department of Neurology, Merck Research Laboratories, West Point, PA 19486, USA. wei-qin_zhao@merck.com
authors.forename: Wei-Qin
authors.initials: WQ
authors.lastname: Zhao
authors.forename: Dawn
authors.initials: D
authors.lastname: Toolan
authors.forename: Robert W
authors.initials: RW
authors.lastname: Hepler
authors.forename: Abigail L
authors.initials: AL
authors.lastname: Wolfe
authors.forename: Yuanjiang
authors.initials: Y
authors.lastname: Yu
authors.forename: Eric
authors.initials: E
authors.lastname: Price
authors.forename: Victor N
authors.initials: VN
authors.lastname: Uebele
authors.forename: Joel B
authors.initials: JB
authors.lastname: Schachter
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Reynolds
authors.forename: John J
authors.initials: JJ
authors.lastname: Renger
authors.forename: Alexander
authors.initials: A
authors.lastname: McCampbell
authors.forename: William J
authors.initials: WJ
authors.lastname: Ray
mesh_terms: D000544:Alzheimer Disease / Q000276:immunology; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000096:biosynthesis*; D000818:Animals; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008027:Light; D018625:Microscopy, Atomic Force; D009474:Neurons / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000096:biosynthesis*; D011487:Protein Conformation; D051381:Rats; D015203:Reproducibility of Results; D012542:Scattering, Radiation
chemical_list: D016229:Amyloid beta-Peptides; D000939:Epitopes; D010446:Peptide Fragments; C075222:amyloid beta-protein (1-42)