Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

Abstract

The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.

Keywords: electrostatic interactions; kinetic analysis; neurodegenerative disease; prion-like behavior; seeding.

Fig. 1.

Atomic force microscopy images, acquired with tapping mode in air, of (A) typical mature seed fibrils (formed at pH 6.5) and (B) of a low concentration of seed fibrils after prolonged exposure to monomeric α-synuclein, deposited onto mica; see Materials and Methods for details on the preparation of the seed fibrils and SI Appendix, section 4 for a discussion of the influence of the solution conditions on the properties of the seeds. The fibrils (A and B) have an average height of ∼7 nm and exhibit the typical dimensions and twist of mature α-synuclein amyloid fibrils that consist of several protofilaments (19).

Fig. 2.

α-Synuclein fibril elongation occurs by monomer addition and is the dominant growth process at neutral pH. (A) Variation of the concentration of soluble (monomeric) protein at constant seed concentration (30 °C, 3.5 μM seed fibrils, PBS buffer, quiescent conditions). (B) The elongation rate as a function of the concentration of soluble protein is initially linear and then starts to saturate. The rates have been extracted through linear fits to the early times of the dataset and the concentration at which the elongation rate has reached half of its maximal value was determined to be 46 μM under these conditions. (C) Variation of seed concentration at constant monomer concentration (50 μM monomer, 37 °C, PBS buffer, quiescent conditions). The seed concentrations are expressed as percentages of the concentration of soluble protein. A global fit is shown that takes into account only elongation (dotted lines); the model reproduces well the overall scaling of the dataset but does not describe all of its features, therefore indicating that other processes, in particular the higher-order assembly of fibrils, are at play (discussed in the text). (D) The initial aggregation rates as a function of the concentration of seed fibrils.

Fig. 3.

Primary nucleation and fragmentation in α-synuclein aggregation can be selectively enhanced. Seeded aggregation (50 μM monomer, 50 nM seeds, 37 °C, PBS, shaking) in the presence of beads made from glass (A) and Teflon (B) in the wells of the plates. (C) At higher seed concentrations (5% by mass), the presence of the beads has little effect on the kinetics. (D) 1 mM SDS induces aggregation under quiescent conditions (PB, 75 μM α-synuclein); the strong fluorescence at the start of the unseeded experiment is likely to be due to an effect of SDS on ThT fluorescence. (Inset) The effect of SDS on the elongation of seed fibrils. The elongation kinetics of fibrils that were formed in the absence of SDS are very similar in the absence or presence of SDS.

Fig. 4.

The rates of the secondary nucleation processes in α-synuclein aggregation exhibit a dramatic pH dependence. (A) Growth of α-synuclein fibrils at very low seed concentrations in PBS buffer (pH 7.4, 45 °C, quiescent conditions). Both the seed and the monomer concentrations vary (Inset). A global fit is shown (continuous lines), considering only elongation, with two free parameters, the elongation rate constant k₊ and the saturation concentration for elongation, m_1/2. The fit yields k₊ = 392 M⁻¹⋅s⁻¹ and m_1/2 = 49.8 μM. (B) Seeded aggregation (50 μM monomer, 50 nM seeds, 10 mM PB, 37 °C, quiescent conditions) as a function of pH. (C) Experiments similar to those shown in A, but at pH 5.2 (10 mM PB, 37 °C, quiescent conditions), where significant secondary nucleation is observed. (D) Comparison of the elongation rate constant k₊ (red) and the maximal rate of fibril production through secondary nucleation (blue) at pH 5.2 (PB) and pH 7.4 (PBS), as well as an approximate indication of the pH dependence of both elongation and secondary nucleation. (E and F) Images (brightfield) of microwells at the end of the seeded experiment shown in C [10 μM monomer added at the beginning of the experiment, 3.5 nM seeds (E) and 35 nM seeds (F)]. Most of the ThT fluorescence is localized in the small assemblies of fibrils, the sizes of which have been determined and their distributions plotted as histograms.