Elucidating the Oligomerization and Cellular Interactions of a Trimer Derived from Aβ through Fluorescence and Mass Spectrometric Studies

Abstract

Aβ oligomers play a central role in the neurodegeneration observed with Alzheimer's disease. Our laboratory has developed covalently stabilized trimers derived from residues 17−36 of Aβ as model systems for studying Aβ oligomers. In the current study, we apply the emerging techniques of fluorescence lifetime imaging microscopy (FLIM) and native mass spectrometry (native MS) to better understand the assembly and interactions of the oligomer model system 2AT-L in aqueous solutions and with cells. 2AT-L and fluorescently labeled 2AT-L analogues assemble in the membrane-like environment of SDS-PAGE, showing diffuse bands of oligomers in equilibrium. Native ion mobility-mass spectrometry (native IM-MS) of 2AT-L allows for the identification of discrete oligomers in solution and shows similar patterns of oligomer formation between 2AT-L and fluorescently labeled analogues.Fluorescence microscopy with SH-SY5Y cells reveals that fluorescently labeled 2AT-L analogues colocalize within lysosomes. FLIM studies with phasor analysis further elucidate the assembly of 2AT-L within cells and establish the occurrence of FRET, indicating the presence of oligomers within cells. Collectively, these multiple complementary techniques help better understand the complex behavior of the 2AT-L model system.

A β-barrel-like tetramer formed by a β-hairpin derived from Aβ

“…50,51 The caspase 3/7, dye-leakage, and simulation data suggest that peptides 1a and 2a , and other peptides stabilized in the β-hairpin alignment described by Tycko and coworkers can interact with lipid membranes in a manner that our laboratory is beginning to explore. 52,53…”

“…50,51 The caspase 3/7, dyeleakage, and simulation data suggest that peptides 1a and 2a, and other peptides stabilized in the b-hairpin alignment described by Tycko and coworkers can interact with lipid membranes in a manner that our laboratory is beginning to explore. 52,53 Conclusion b-Hairpins are a key structural component of Ab oligomers and may contribute directly to oligomer heterogeneity through variation in hairpin alignment and topology. In this investigation, we studied the assembly and structure of oligomers formed by peptides derived from Ab 12-40 that were stabilized in the b-hairpin alignment reported by Tycko and coworkers.…”