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

Significance

The deposition of α-synuclein as insoluble amyloid fibrils and the spreading of such species in the brain are two hallmarks of Parkinson disease. It is therefore of great importance to understand in detail the process of aggregation of this protein. We show by a series of in vitro measurements that amyloid fibrils of α-synuclein can grow under a wide range of solution conditions but that they can multiply rapidly only under a much more select set of solution conditions, mimicking those in endosomes and other organelles. The quantitative characterization of α-synuclein aggregation described here provides new insights into the microscopic mechanisms underlying α-synuclein aggregation in the context of Parkinson disease.

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.

The conversion of soluble peptide and protein molecules into insoluble amyloid fibrils is of great interest in fields of science ranging from molecular medicine to nanotechnology (1). The formation of amyloid fibrils is a characteristic feature of a substantial number of increasingly common medical disorders, including neurodegenerative conditions such as Alzheimer’s and Parkinson diseases (2). Elucidating the fundamental mechanistic steps involved in the conversion from the soluble to the fibrillar forms of the peptides and proteins involved in such disorders is crucial for understanding their origin and proliferation, and hence for exploring in a rational manner new and effective therapeutic strategies through which to combat their onset or progression (3).

One general aspect of amyloid diseases is that once the first aggregates are formed it is very difficult to stop or reverse the aggregation process. This implies that aggregation needs to be studied in both the absence and presence of preformed aggregates, commonly known as seeds, to deepen our understanding of the mechanism of the self-assembly process in vivo. It is possible to define from in vitro studies the rate constants for the multiplicity of microscopic steps that increase the number and total mass of the different types of aggregates that are populated during this process. Such an analysis has recently been carried out for the Aβ42 peptide (4) associated with Alzheimer’s disease by combining experimental and theoretical methodologies. A major finding of that study is that the production of toxic oligomeric species, which can also subsequently convert into fibrillar aggregates, does not result solely from primary nucleation, but is catalyzed by the presence of mature amyloid fibrils, a process known as secondary nucleation (5–8). This auto-catalytic mechanism dominates the aggregation process of Aβ42 under quiescent solution conditions (4). These results show that the overall kinetics of aggregation of Aβ42 are determined by a complex combination of primary and secondary processes, as well as by the growth of oligomeric nuclei and fibrils.

It would be highly desirable to achieve for α-synuclein a level of understanding similar to that now obtained for Aβ42. It has been found, however, that it is extremely challenging to obtain reproducible kinetic data for α-synuclein aggregation in vitro (9–11), except at mildly acidic pH values and in the presence of hydrophobic surfaces (12). In the present article, we investigate the aggregation of α-synuclein in the presence of preformed seeds. Our approach is based on the systematic variation in the quantities of seed fibrils, the monomer concentration, and the solution conditions. We find for α-synuclein at neutral pH and in the absence of other factors, such as agitation or surfactants, that the growth of existing aggregates and higher-order assembly of fibrils occur at much greater rates than either primary nucleation or secondary processes. However, at mildly acidic pH values secondary nucleation is strongly accelerated, changing the overall mechanistic character of the aggregation process.

Results

Seeded Experiments Simplify the Kinetic Analysis.

It has been widely established that the rate of conversion of soluble proteins into amyloid fibrils can often be strongly accelerated by the addition of preformed seed fibrils (13, 14); this type of phenomenon is also well known in related processes, notably crystallization (15). The addition of seeds may accelerate the aggregation process by at least two different mechanisms: elongation and surface-catalyzed secondary nucleation. The presence of seeds eliminates the need for primary nucleation, and the length of the lag phase decreases with increasing seed concentration. In the presence of a large number of seeds (compared with the number of species formed by primary nucleation or secondary processes during the time course of the experiment), the aggregation profile is expected to be a single exponential function (14) (SI Appendix, section 2). This behavior is a consequence of the fact that the number of growth-competent fibril ends remains constant throughout the duration of the experiment; the rate of fibril elongation then varies solely with the concentration of soluble protein, which is progressively depleted during the reaction. Primary nucleation is often much slower than elongation, as a consequence of the higher energy barriers for de novo formation of clusters and aggregates compared with the addition onto an existing template (16, 17), and plays no significant role in the strongly seeded regime. If the length distribution, and hence the number of growing ends of the seed fibrils, is defined it is possible to determine the absolute molecular rate constants of fibril elongation.

It has been shown previously that amyloid fibril fragmentation for many proteins can be strongly enhanced by mechanical action, such as shaking or sonication of the solution, and that after prolonged exposure to such mechanical stresses the length distribution tends toward a limit that is solely determined by the mechanical properties of the fibrils (18). We make use of this feature in the present study to manipulate the average length of the fibrils to obtain a narrow distribution of short seed fibrils (Fig. 1 and SI Appendix, section 9). The exceptional reproducibility of aggregation time courses between independent batches of seed fibrils that can be achieved with such an approach is demonstrated in SI Appendix, section 5. The high degree of reproducibility of these experiments allows the effects of changes in internal and external conditions to be measured with great accuracy.

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).

α-Synuclein Fibril Elongation Occurs by Monomer Addition.

We first investigated the dependence of the elongation rate on the concentration of soluble protein molecules and observed a linear relationship at low concentrations and saturation at higher concentrations (Fig. 2 A and B). This overall sublinear behavior is similar to that observed for other proteins, including the Sup35 yeast prion (20), S6 (21), insulin, and α-lactalbumin (22). The saturation of the elongation rate with monomer concentration, reminiscent of two-step Michaelis–Menten behavior observed in enzyme kinetics, has been shown to stem from the diffusive nature of the elongation reaction; the incorporation of a monomer onto the end of a growing fibril can be described as the crossing of a single free energy barrier associated with an intrinsic timescale that is of the order of hundreds of microseconds (22), the inverse of which is the prefactor of the limiting elongation rate (22). If the data obtained in the present study for α-synuclein are fitted to this model, which is described by the equation $r\left(m\right)=r_{max}m/(m_{1/2}+m)$, we obtain a value of 46 μM for $m_{1/2}$, the concentration at half maximal rate, $r_{max}$. In SI Appendix, section 6 we discuss how this quantity displays a dependence on the conditions of the experiment.

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.

It has been proposed that amyloid fibrils of different proteins, including α-synuclein (23, 24), can grow most efficiently by the addition of oligomers to the fibril ends. In our experiments we used α-synuclein isolated by gel filtration whose CD spectrum is completely consistent with an unfolded monomeric protein (SI Appendix, section 3). Therefore, the kinetic analysis in this work as well as similar analyses in earlier studies (20–22) suggests that, at least under the conditions of these experiments, amyloid fibrils of α-synuclein grow primarily by such addition. The molecular rate constant of fibril elongation by monomer addition, k₊, could be determined from the ThT fluorescence time courses of seeded aggregation, together with an analysis of the seed fibril length distribution, and is ∼2 × 10³ M⁻¹⋅s⁻¹, corresponding to an average length increase of ∼1 nm/min (at 20 μM; SI Appendix, section 8).

α-Synuclein Aggregation at Neutral pH Is Dominated by Fibril Elongation.

Having established the highly quantitative and reproducible nature of seeded aggregation of α-synuclein, we designed further experiments in PBS buffer at neutral pH to obtain insights into the relative significance of the other molecular processes that are generally important in filamentous growth, in particular primary nucleation and secondary processes such as fibril fragmentation (7, 25) and monomer-dependent secondary nucleation (4, 6). We systematically decreased the concentration of seed fibrils, and therefore the rate of consumption of monomer by fibril elongation, under quiescent conditions (Fig. 2C). The experiments without added seed fibrils show no detectable increase in ThT fluorescence over the timescale used here (up to 40 h), consistent with data from earlier studies under quiescent conditions (9). Moreover, a global fit of the dataset to a model that considers only the elongation of seed fibrils and has a single free parameter, the elongation rate constant, reproduces very well the overall scaling behavior, as shown in Fig. 2C. In addition, the early time behavior (t ≤ 1 h) is in excellent agreement with elongation being the dominant process, as illustrated by the linear scaling of the initial aggregation rate with the concentration of seed fibrils (Fig. 2C). It is evident, however, that elongation alone is not able to explain every detail of the complete dataset shown in Fig. 2C.

During the experiments involving seeded aggregation, macroscopic gel-like assemblies of fibrils were observed, notably at high protein and high salt concentrations. The fluorescence experiments were performed with an optical fiber positioned below the sample (bottom optics; Materials and Methods) and flocculation of fibrils, followed by aggregation and sedimentation of the flocs, would be expected to enhance the fluorescence signal relative to a spatially homogeneous sample. In addition, the formation of an amyloid gel is likely to affect both the mobility of the soluble protein molecules and the accessibility of the fibril ends, and therefore to decrease the overall rate of conversion from soluble to aggregated protein; these phenomena are likely to be the origin of the deviation from simple exponential behavior observed in the data in Fig. 2.

We investigated these processes further by repeating the experiments shown in Fig. 2 in 20 mM phosphate buffer (PB) without added NaCl (SI Appendix, Fig. S9A), where no macroscopic assemblies of fibrils were visible even at the end of the experimental measurements. In SI Appendix, Fig. S9 C and D we show a comparison of spatially resolved absorbance measurements of aggregated samples in PB and PBS that clearly illustrates a homogeneous distribution of fibrils in the former and an inhomogeneous distribution in the latter case, suggesting that the higher-order assembly of fibrils is less pronounced in the absence of added salt; indeed, the dataset acquired without added NaCl is better described by an elongation-only model than that shown in Fig. 2C. To determine the timescale over which the higher-order assembly of fibrils and subsequent enhanced sedimentation can occur, we performed a seeded aggregation experiment with top and bottom optics simultaneously (SI Appendix, Fig. S9B). In the absence of NaCl, the bottom and top readings yield superimposable curves over timescales of several hours, whereas for PBS the data start to deviate just minutes after the start of the experiments, indicating sample inhomogeneities of the order of hundreds of micrometers. To illustrate this process even more clearly, we monitored seeded aggregation experiments under different conditions in microcapillaries, using an inverted fluorescence microscope, and found that in the confined space of a capillary the flocculation of α-synuclein fibrils into microscopic aggregates is followed by gelation that results from irreversible interaction of the fibril flocs; videos of these experiments can be found in Movies S1–S6.

The conclusion from these experiments is that α-synuclein fibrils can behave under some solution conditions as unstable colloidal suspensions, and so tend to aggregate into larger structures that can subsequently form gels. This process is readily observed under conditions of physiological salt concentrations where the electrostatic repulsion between the fibrils, which are likely to have a similar charge density to the monomer, is strongly screened.

Primary Nucleation and Fragmentation Can Be Selectively Enhanced.

Previous work has shown that the process of nucleation of α-synuclein amyloid fibrils is likely to be heterogeneous and catalyzed by environmental features, such as air–water interfaces (26, 27), lipid bilayers (28), SDS micelles and other anionic surfactants (11, 29, 30), or artificial interfaces such as the coatings of containers or stir bars (31). In addition, mechanical action, such as shaking and stirring, is often used to accelerate the aggregation of α-synuclein (32). For the latter type of conditions, it is likely that the mechanical action influences primary nucleation (33) (e.g., through a disturbance of the air–water interface) and also secondary processes [e.g., through an enhanced rate of fragmentation (4)]. To investigate these phenomena, we conducted a series of seeded experiments in the presence of beads of 1–2 mm in size (with periodic shaking cycles; Materials and Methods).

To test whether or not the effect on aggregation of the addition of beads stems from the additional surface area introduced into the system, we used beads made from two different materials, hydrophobic Teflon and hydrophilic glass (Fig. 3 A and B). To distinguish the effects of the beads on primary nucleation and on fragmentation, we performed control experiments without seed fibrils, as well as carrying out the equivalent experiments without added beads. In the absence of any added beads, we observe behavior analogous to that shown in Fig. 2, namely, no detectable increase in fluorescence without added seeds, and approximately linear behavior for low seed concentrations (0.1% by mass), indicating that the shaking protocol used here is not sufficient to induce aggregation under these conditions. However, in the presence of beads, aggregation is observed even without addition of seed fibrils. Furthermore, the plots from experiments carried out in the presence of 0.1% seed fibrils show pronounced convex behavior, characteristic of an increase with time in the number of growing aggregates. The observation that the seeded aggregation experiments show an acceleration in the fluorescence signal several hours before the unseeded reactions show any significant fluorescence strongly suggests that the dominant effect of the addition of beads is to increase the fibril fragmentation rate (4). Based on these data, it is not, however, possible to exclude some effect of the beads on the process of primary nucleation. Nevertheless, any such effect is likely to be indirect, for example through a disturbance of the air–water interface, rather than the result of a direct surface-catalysis process on the beads, given that the surface hydrophobicity of the beads has little influence on their effects on the aggregation process.

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.

We also performed seeded and unseeded aggregation experiments in the presence of 1 mM SDS under quiescent conditions (Fig. 3D), obtaining exponential and sigmoidal aggregation curves, respectively. These results suggest that the presence of SDS strongly enhances the primary nucleation of α-synuclein amyloid fibrils, potentially through the opening of an alternative nucleation pathway, possibly coaggregation with SDS, whereas both the pathway and the free energy barrier for seed fibril elongation remain unaffected. These findings are in line with earlier studies (11, 30, 34) and show that both primary and secondary processes in aggregating α-synuclein at neutral pH can be strongly influenced by a variety of factors and solution conditions.

Rates of Secondary Nucleation Processes in α-Synuclein Aggregation Show a Dramatic pH Dependence.

We next investigated the kinetics of seeded aggregation in PBS buffer in a regime of very low seed concentrations (Fig. 4A). We decreased the concentration of seed fibrils to a situation where, in the absence of primary nucleation and secondary processes, each seed fibril would in theory have to grow to a final length of 10³ to 10⁴ times its initial length to convert all of the available soluble protein molecules into fibril mass (i.e., nanomolar seed concentration vs. micromolar monomer concentration). In these experiments, we varied the concentrations of both soluble protein and of fibrils. Fig. 4A shows a global fit to a kinetic model where the elongation rate varies linearly with the concentration of seed fibrils (as in Fig. 2D) and sublinearly with the concentration of soluble monomer (as in Fig. 2B), using the saturation concentration of elongation, m_1/2, and the elongation rate constant k₊ as fitting parameters. This model gives a better global fit than in the case of the higher seed concentrations and the value of m_1/2 determined here is in remarkable agreement with that determined from the experimental data shown in Fig. 2B (49.8 μM vs. 45.8 μM), given the well-known difficulties of determining such asymptotic quantities from hyperbolic relationships (35). At these low seed concentrations, the depletion of monomer by elongation of the fibrils is slow compared with the higher-order assembly of fibrils that can interfere with elongation. Interestingly, the global fit in Fig. 4 yields a lower elongation rate constant k₊ (∼4 × 10² M⁻¹⋅s⁻¹) compared with that from the fit in Fig. 2C (2.2 × 10³ M⁻¹⋅s⁻¹; see SI Appendix, sections 8 and 11 for details on the fits), even though the experiments have been carried out under very similar solution conditions. Indeed, a detailed look at the data reveals that the rates of increase in fluorescence are slightly higher at the beginning, compared with at the end, of the experiment, leading to a small deviation between the fit and the data at the earlier times. These results are in agreement with the hypothesis put forward above that the higher-order assembly effectively decreases the overall seeding efficiency over time.

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.

We then performed seeded aggregation experiments in PB at low ionic strength (10 mM) as a function of pH, ranging from 4.8 to 6.2, at low seed concentrations (0.04% by mass, Fig. 4B). We observed that some of the kinetic traces, in the pH range 4.8–5.6, show clear signs of positive curvature, a finding that suggests that secondary processes are much more pronounced at mildly acidic compared with neutral pH. To investigate this phenomenon further, we carried out kinetic experiments with systematic variations in the concentrations of both soluble and fibrillar protein, similar to those shown in Fig. 4A, at pH 5.2, where the positive curvature observed in Fig. 4B is maximal. We see very strong positive curvature throughout the entire dataset, confirming the contribution of a secondary process to the aggregation mechanism.

Because of the current lack of a mathematical model of secondary nucleation processes of the type described here, the datasets in Fig. 4 A and C that were acquired at low concentrations of seed fibrils were analyzed numerically (SI Appendix, section 13) and the maximal values for the quantity $dP/dt$, the rate of formation of new fibrils through secondary nucleation, was determined under those conditions. Fig. 4D shows a comparison of this quantity, as well as of the elongation rate constant, k₊, as a function of pH. Whereas the elongation rate constant changes by approximately one order of magnitude from pH 5.2 to pH 7.4 (SI Appendix, section 9), the rate of fibril production through secondary nucleation changes, quite remarkably, by at least four orders of magnitude.

Discussion

In this work, we have used aggregation assays in the presence of preformed seed fibrils to gain insight into various aspects of α-synuclein aggregation. First, we have measured the average elongation rate of mature α-synuclein amyloid fibrils under different conditions. From these experiments we have been able to determine the second-order rate constant for growth by monomer addition, k₊, to be ca. 2 × 10³ M⁻¹⋅s⁻¹ at 37 °C in PBS buffer. This quantity has so far been determined for only a very few other amyloidogenic proteins in bulk solution; prominent examples include Aβ42 [k₊ ∼3 × 10⁶ M⁻¹⋅s⁻¹ (4)] and a polyQ peptide with 23 glutamine residues [k₊ ∼10⁴ M⁻¹⋅s⁻¹ (36)]. This rate constant has also been determined from surface-based biosensor experiments for a range of proteins (37) and the results are generally in good agreement with the ones from the corresponding bulk solution experiments.

In addition to being able to elongate, α-synuclein fibrils are also subject to higher-order assembly processes that can be best described as flocculation followed by gelation of the protein aggregates (Movies S1–S10 show seeded experiments carried out in microcapillaries). These processes are strongly influenced by the salt concentration and therefore they are likely to be controlled by electrostatic interactions. Interestingly, the higher-order assembly of the growing fibrils strongly decreases their ability to seed aggregation. This effect most likely stems from a decrease in the effective diffusion rate of the soluble protein molecules and of the accessibility of the growth-competent ends under such conditions. Most of the experiments described in this paper were performed under quiescent conditions, which is likely to be more relevant to physiological behavior than experiments carried out under agitation, and is also simpler to interpret. We have found that under these conditions and at neutral pH values α-synuclein fibrils are not able to multiply at a significant rate. In agreement with other studies, however, we have shown that mechanical agitation can induce fragmentation of the fibrils (4, 38). By contrast, at mildly acidic pH (below pH 5.8), we have found that the same seed fibrils that were unable to multiply at neutral pH show strong signs of a secondary process giving rise to fibril proliferation even under quiescent conditions. Although such a change of ∼2 pH units increases the elongation rate constant by only approximately one order of magnitude, the production of new growing fibrils through secondary nucleation is enhanced by at least four orders of magnitude (Fig. 4D). The finding that the secondary process can be attributed to a surface-catalyzed process (SI Appendix, section 14 and Fig. 4 E and F) is consistent with the pH behavior that we have detected; its sharp pH dependence is most likely to be related to the titration of carboxylate groups in the acidic C terminus of the protein. SI Appendix, Fig. S12 shows the net charge of the protein, calculated as a function of pH, based on the pK_a values and Hill coefficients reported for 250 μM α-syn in 20 mM PB (39). Whereas the single histidine residue (H50) has a pK_a value of 6.8, the ca. 4.4 units calculated change in net charge between pH 6.0 and 5.0 is mainly due to the partial protonation of several carboxylate groups in the protein, especially in the acidic C terminus, which contains 16 carboxylate groups, 13 of which have elevated pK_a values (39). At lower protein concentrations and buffer strengths, as in the present study, as well as in fibrils with many negative groups close together, these pK_a values are likely to be increased even more, in line with the salt dependence of α-synuclein pK_a values (39) and similar studies (40).

We conclude that the findings presented in this article may have significant implications for understanding the aggregation process of α-synuclein in vivo. It has been shown that the specific chemical microenvironments of cellular compartments, such as endosomes, can enhance protein aggregation by several orders of magnitude (41). Our study presents a physicochemical rationale for such effects in the case of the aggregation of α-synuclein. Our findings concerning the seeded aggregation of α-synuclein are likely to be of significant physiological importance, not just in the context of the primary growth of fibrils within cells that gives rise to Lewy bodies, but also in the context of the finding that healthy cells can be invaded by α-synuclein aggregates in a “prion-like” manner (42, 43), where aggregates spread to neighboring cells and accelerate the conversion of soluble protein molecules into fibrils and ultimately additional Lewy bodies. Such prion-like behavior can be explained by the existence of a secondary mechanism that is able to multiply existing aggregates, which can then be transmitted via diffusion or other means to neighboring cells. Our finding that such processes do indeed exist under quiescent conditions, and that they are very strongly influenced by the solution conditions, may help to explain the cellular localization as well as the kinetics and the mechanism of the spread of pathological aggregation in Parkinson disease.

Materials and Methods

Details can be found in SI Appendix. Wild-type human α-synuclein was recombinantly expressed and purified as described previously (12, 44). Seed fibrils were produced by incubating 500-μL solutions of α-synuclein at concentrations between 300 and 800 μM in 20 mM phosphate buffer at pH values between 6.3 and 7.4 for 48 to 72 h at ca. 40 °C. The increase in ThT fluorescence was monitored in low-binding, clear-bottomed half-area 96-well plates. Most experiments were performed under quiescent conditions, except for the experiments with added beads. Atomic force microscopy images were taken using a Nanowizard II atomic force microscope using tapping mode in air. The capillary experiments were performed in square borosilicate glass capillaries, which were monitored with an Observer D.1 inverted fluorescence microscope through a Filter set 47 using an Evolve 512 camera. The images of the wells after the aggregation were taken with an Olympus SZ61 stereomicroscope.