Critical role of interfaces and agitation on the nucleation of Aβ amyloid fibrils at low concentrations of Aβ monomers

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid beta-peptide (A beta) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence A beta amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20 mu M A beta in the presence or absence of air-water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all A beta concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher A beta concentrations (10 and 20 mu M). Importantly, when the 5 mu M A beta solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5 mu M A beta solutions, were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5 mu M A beta solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro. (C) 2010 Elsevier B.V. All rights reserved.