Rapid assembly of amyloid-β peptide at a liquid/liquid interface produces unstable β-sheet fibers

Accumulation of aggregated amyloid-β peptide (Aβ) in the brain is a pathological hallmark of Alzheimer's disease (AD). In vitro studies indicate that the 40- to 42-residue Aβ peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Aβ peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air - water interfaces have been shown previously to promote Aβ aggregation, but detailed characterization of these aggregates has not been presented. In this study Aβ(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Aβ in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Aβ aggregates had a β-sheet secondary structure. However, these aggregates were far less stable than Aβ(1-40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Aβ protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Aβ with β-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic β-sheet structure of an in vitro Aβ aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo.