Alternate Aggregation Pathways of the Alzheimer β-Amyloid Peptide: Aβ Association Kinetics at Endosomal pH

The deposition of β-amyloid peptide (Aβ) fibrils around neurons is an invariable feature of Alzheimerʹs disease and there is increasing evidence that fibrillar deposits and/or prefibrillar intermediates play a central role in the observed neurodegeneration. One site of Aβ generation is the endosomes, and we have investigated the kinetics of Aβ association at endosomal pH over physiologically relevant time frames. We have identified three distinct Aβ association phases that occur at rates comparable to endosomal transit times. Rapid formation of burst phase aggregates, larger than 200 nm, was observed within 15 seconds. Two slower association phases were detected by fluorescence resonance energy transfer and termed phase 1 and phase 2 aggregation reactions. At 20 μM Aβ, pH 6, the half lives of the phase 1 and phase 2 aggregation phases were 3.15 minutes and 17.66 minutes, respectively. Atomic force microscopy and dynamic light scattering studies indicate that the burst phase aggregate is large and amorphous, while phase 1 and 2 aggregates are spherical with hydrodynamic radii around 30 nm. There is an apparent equilibrium, potentially mediated through a soluble Aβ intermediate, between the large burst phase aggregates and phase 1 and 2 spherical particles. The large burst phase aggregates form quickly, however, they disappear as the equilibrium shifts toward the spherical aggregates. These aggregated species do not contain α-helical or β-structure as determined by circular dichroism spectroscopy. However, after two weeks β-structure is observed and is attributable to the insoluble portion of the sample. After two months, mature amyloid fibrils appear and the spherical aggregates are significantly diminished.