Urea modulation of beta-amyloid fibril growth: experimental studies and kinetic models

Abstract

Aggregation of beta-amyloid (Abeta) into fibrillar deposits is widely believed to initiate a cascade of adverse biological responses associated with Alzheimer's disease. Although it was once assumed that the mature fibril was the toxic form of Abeta, recent evidence supports the hypothesis that Abeta oligomers, intermediates in the fibrillogenic pathway, are the dominant toxic species. In this work we used urea to reduce the driving force for Abeta aggregation, in an effort to isolate stable intermediate species. The effect of urea on secondary structure, size distribution, aggregation kinetics, and aggregate morphology was examined. With increasing urea concentration, beta-sheet content and the fraction of aggregated peptide decreased, the average size of aggregates was reduced, and the morphology of aggregates changed from linear to a globular/linear mixture and then to globular. The data were analyzed using a previously published model of Abeta aggregation kinetics. The model and data were consistent with the hypothesis that the globular aggregates were intermediates in the amyloidogenesis pathway rather than alternatively aggregated species. Increasing the urea concentration from 0.4 M to 2 M decreased the rate of filament initiation the most; between 2 M and 4 M urea the largest change was in partitioning between the nonamyloid and amyloid pathways, and between 4 M and 6 M urea, the most significant change was a reduction in the rate of filament elongation.

Figure 1.

CD spectra of Aβ in 0.4 M, 2 M, 4 M, and 6 M urea (from bottom to top). All the samples contained 140 μM of Aβ and were incubated for ~22–24 h before CD measurements. Due to residual urea, the minimum wavelength was limited to 210–214 nm.

Figure 2.

Representative size exclusion chromatograms of 140 μM of Aβ in 0.4 M, 2 M, 4 M, and 6 M urea (solid line, from top to bottom). The mobile phase was matched to that of the sample buffer. Molecular weight of Aβ peak was determined by calibration of column at each urea concentration using insulin chain B (3.5 kDa), ubiquitin (8.5 kDa), ribonuclease A (13.7 kDa), ovalbumin (43 kDa), and BSA (67 kDa). The letters M and D represent monomer and dimer, respectively. Arrows represent elution times of ubiquitin at different urea concentrations. The shift in elution times with different urea concentrations in mobile phase is likely due to urea-induced volume expansion. In 4 M urea, dimeric species were observed in four of six replicates; in the remaining two samples, initially only monomer was observed, but there was subsequently complete conversion to dimer within ~2–3 h. In 6 M urea, seven of nine replicates showed a purely monomeric peak initially, shown as a dotted line, followed by a complete switch to purely dimer within ~2–3 h. Only dimer was detected in remaining 2/9 replicates.

Figure 3.

(A) Hydrodynamic diameter d_sph and (B) scattering intensity at 90° angle I(90°) as determined by dynamic light scattering for Aβ in 0.4 M (○), 2 M (□), 4 M (♦), and 6 M (×) urea after sample preparation. Light scattering data are shown along with the fits to the kinetic model (see text).

Figure 4.

Size distribution of Aβ aggregates as determined by CONTIN analysis of DLS data. Urea concentration is 0.4 M, 2 M, 4 M, and 6 M reading from top to bottom. Left and right panels for each urea concentration showed Aβ distributions with detectable intensities after ~0.5–3 and ~22–24 h, respectively. Distributions from at least seven data points were collected and averaged for each panel.

Figure 5.

AFM images of Aβ in (A) 0.4 M urea, fourfold diluted; (B) 0.4 M urea, 10-fold diluted; (C) 2 M urea, fourfold diluted; (D) 2 M urea, undiluted; (E) 4 M urea, fourfold diluted; and (F) 6 M urea, fourfold diluted. Aβ in each urea concentration was incubated for 20–22 h before taking AFM images in solution tapping mode. Samples were diluted as indicated just before imaging. Scale bar represents 100 nm.

Figure 6.

Aβ aggregation model schematic.