Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides

Abstract

The two major forms of the amyloid-beta (Aβ) peptide found in plaques in patients suffering from Alzheimer's disease, Aβ40 and Aβ42, only differ by two amino acids in the C-terminal region, yet they display markedly different aggregation behavior. The origins of these differences have remained challenging to connect to specific molecular-level processes underlying the aggregation reaction. In this paper we use a general strategy to apply the conventional workflow of chemical kinetics to the aggregation of the Aβ40 peptide to identify the differences between Aβ40 and Aβ42 in terms of the microscopic determinants of the aggregation reaction. Our results reveal that the major source of aggregates in the case of Aβ40 is a fibril-catalyzed nucleation process, the multistep nature of which is evident through its saturation behavior. Moreover, our results show that the significant differences in the observed behavior of the two proteins originate not simply from a uniform increase in all microscopic rates for Aβ42 compared with Aβ40, but rather are due to a shift of more than one order of magnitude in the relative importance of primary nucleation versus fibril-catalyzed secondary nucleation processes. This analysis sheds light on the microscopic determinants of the aggregation behavior of the principal forms of Aβ and outlines a general approach toward achieving an understanding at the molecular level of the aberrant deposition of insoluble peptides in neurodegenerative disorders.

Keywords: aggregation mechanism; neurodegeneration; protein aggregation; rate law.

Fig. 1.

A schematic depiction of the microscopic steps involved in fibrillar aggregation. (A) The principal processes relevant for filamentous aggregation, their dependence on the concentrations of monomers (m) and fibrils, and how they contribute to the changes in the number (P) and mass concentrations (M) of fibrils. The rate constants are k₊ (elongation at fibril ends), k_n (primary nucleation in solution of order n_c), k₂ (secondary nucleation on the fibril surface of order n₂), and k₋ (fibril fragmentation). (B) A graphical depiction of the proposed reaction scheme for secondary nucleation (Supporting Information, section 1.1) wherein monomers first come together in the presence of an aggregate, then monomer-independent reaction and detachment steps take place to yield the product.

Fig. 2.

Global fit to experimental data. (A) The global fit of Eq. 4 to the unseeded aggregation data with three free parameters: K_M and the combinations k₊k_n and k₊k₂ for the entire dataset (n₂ and n_c were set to 2). The three independent experiments for each monomer concentration are in the same color, with the fit to those three runs being a solid curve in the same color. A zoomed-in version of the high concentration curves and the residuals are shown in Supporting Information, section 1.8 and Figs. S5 and S6. The values of the fitted parameters are given in Fig. 3B. (B) A plot of the average half-time versus the initial monomer concentration. The solid blue line is the theoretical prediction, not a fit, of the half-time, using Eq. 11 and the parameters determined in A. The regions of unsaturated and fully saturated secondary nucleation are marked on the graph, $\sqrt{K_M}$ gives the concentration of half-saturation of secondary nucleation, and the dashed blue line denotes the region where elongation may begin to saturate. (C) Fits to the three possible models with simple secondary pathways (single-step secondary nucleation, fragmentation without secondary nucleation, and a competition of fragmentation and single-step secondary nucleation) were performed to show that neither can achieve a good global fit (see also Fig. S6).

Fig. 3.

Rates and mechanistic differences between Aβ40 and Aβ42 aggregation. (A) Summary of the full reaction scheme. On the left and right, the limiting cases of high and low monomer concentration are shown. In low monomer conditions secondary nucleation is effectively single-step and in the high monomer limit the second step of the secondary nucleation process becomes rate-determining (RDS). Whereas both are relevant for Aβ40, only the low monomer behavior is observed for Aβ42. (B) Table of the combined rate constants obtained from the global fit and the individual microscopic rate constants of aggregation (in both cases n₂ = n_c = 2). The ratio k_n/k₂ describes the aggregate concentration above which secondary nucleation will be producing more nuclei than primary nucleation and shows that secondary nucleation is significantly more important than primary nucleation for Aβ40. The errors in the upper table were estimated by fitting different subsets of the replicates at each concentration; for details see Supporting Information, section 1.6. The elongation rate and hence all parameters in the lower table are only accurate to within a factor of 3 (Supporting Information, section 2.1). (C) Comparison of the individual rate constants of Aβ40 and Aβ42, normalized to the rate in Aβ42 and shown on a logarithmic scale. Note that the relative difference in rate constant is significantly larger for primary nucleation than for the other two processes.

Fig. 4.

Comparison of Aβ40 and Aβ42 aggregation data. Comparison of the experimental kinetic datapoints of Aβ40 (red) and Aβ42 (blue) at a monomer concentration of 3.5 μM. The times for each dataset are rescaled by the corresponding half-times and the steeper increase of the Aβ40 curve is indicative of its stronger autocatalytic behavior, on a relative scale. The solid lines (Aβ40 in red and Aβ42 in blue) are a prediction for the same system in the absence of secondary nucleation but with all of the other rate constants at the values determined from the experiment. The significant difference in these predictions highlights the larger relative contribution of secondary nucleation for Aβ40 relative to Aβ42.