Single Molecule Fingerprinting Reveals Different Amplification Properties of α-Synuclein Oligomers and Preformed Fibrils in Seeding Assay

Abstract

The quantification of α-synuclein aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson's disease. However, multiple species, such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein; these species are likely to coexist in biological samples, and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify individual contributions to the amplification process, recombinant oligomers and preformed fibrils were produced and purified to characterize their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent, but our results reveal that their seeding behavior is very different compared to that of preformed fibrils, in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples is likely to dominate the response in seeding assays.

Keywords: PMCA; Parkinson’s disease; RT-QuIC; oligomers; preformed fibrils; protein misfolding cyclic amplification assay; real time quaking induced conversion; seed amplification assay; single molecule detection; α-Synuclein.

Figure 1

Single molecule fingerprinting for the characterization of α-synuclein species. (A) (left) Schematic of the microscope setup. The inset shows ThT stained and unstained (dark) α-syn oligomers or fibrils (green and white colored particles) diffusing across the confocal volume. Monomeric α-syn and some assemblies do not bind ThT. (right) Photograph of the microscope for recording the fluorescence traces. (B) Characterization of fluorescence traces. A fluorescence trace is analyzed to report the total number of peaks (events) and the prominence of individual peaks, residence time (full width half-maximum), and area under the curve (yellow). The inset shows a region denoted by (*) in the trace.

Figure 2

Single molecule aggregation kinetics and fingerprinting of species. (A) Number of ThT⁺ species detected during the time course of α-syn aggregation. Each curve corresponds to an independent aggregation experiment. Error bars represent the standard deviation. (B) Single molecule fingerprinting of ThT⁺ peaks detected. Peak prominence is plotted against residence time from 2.5 h. Four types of particles can be defined: small (S), high (H), long (L), and neutral (N). The cut-offs were set at 250 ms and 300 photons, on the x- and y-axis, respectively. (C–E) Scatter plots comparing prominence (C), residence time (D), and total intensity (E). Blue line represents the median values. Each symbol represents an individual peak detected in the fluorescence traces generated from five independent experiments in panel A. Statistics used Kruskal–Wallis one-way ANOVA, p ≤ 0.0001 (****).

Figure 3

Isolation of α-syn oligomers. (A) Schematic of the experimental protocol to isolate and detect ThT⁺ α-syn oligomers. After 5 h of aggregation, the reaction sample was centrifuged. The supernatant was further purified using size exclusion chromatography. Collected fractions were supplemented with monomers for amplification reactions and single molecule detection with ThT. (B) Representative fluorescence traces of supernatant and pellet fraction with corresponding electron micrographs of each fraction shown in panel D. (C) Gel filtration chromatogram identifying the elution peaks containing α-syn oligomers and monomers. A representative fluorescence trace corresponding to a fraction containing oligomers is shown. (D) Electron micrographs of the pellet, supernatant, and a SEC fraction containing oligomers. Scale bar is 200 nm. Histogram plot of the diameters of negatively stained particles from the [9.2–9.7 mL] SEC fraction; 12.6 ± 2.5 nm; 1544/2 (mean ± standard deviation; number of particles/fractions stained).

Figure 4

Single molecule characterization of α-syn oligomers. (A) Quantification of ThT⁺ events detected in each fraction after size exclusion, before (black) and after an amplification step (red) in PBS. Negative control is monomeric α-syn and ThT in PBS. A gel filtration profile is shown in the inset for reference. Error bars represent the standard deviation. This color scheme is applied across all figures in this study. (B) Bar graph of the number of ThT⁺ events detected in fractions containing oligomers (elution volume 7.7–10.2 mL) collected from four assembly reactions (R1–R4). (C) Single molecule fingerprinting of oligomers, before (left) and after amplification (right), is obtained by plotting the prominence against residence time. Each symbol represents an individual event with the shape corresponding to R1–R4 coded in panel B. Quadrant numbers report the number of particles in each quadrant and abundance. (D) Logarithmic scatter plot comparing peak prominence, residence time, and area under the curve before and after amplification. Each symbol represents an individual event (N = 34/34 traces before/after amplification). The median value is highlighted in blue. Statistics used Mann–Whitney t test, p ≤ 0.0001 (****), nonsignificant (n.s.).

Figure 5

Single molecule fingerprinting of sonicated α-syn fibrils. (A) Negatively stained EM images of human α-syn fibrils before and after sonication. White arrows point at filamentous twists. Violin plot of tubular diameters of the fibrils prior to sonication and length of fibrils after sonication. Scale bar is 200 nm. (B) Representative fluorescence traces of sonicated fibrils before (black) and after a 5 h amplification (red). (C) Single molecule fingerprinting of sonicated mature human α-syn fibrils before and after amplification (red). Each symbol represents an individual event in the fluorescence traces. Quadrant numbers represent the number of ThT⁺ events. Data are from three independent experiments.

Figure 6

Time course amplification assay comparing α-syn oligomers and sonicated preformed fibrils. (A) Time course measurements of oligomers or fibrils amplified for 2.5, 5, 7.5, and 24 h (orange to red) in the presence of excess α-syn monomers and ThT in PBS. Scatter dot plots comparing the prominence and residence time. Each symbol represents an individual ThT⁺ event. Data were collected from two independent experiments. The median value is highlighted in blue. (B) Bar graphs comparing the fold-change in median AUC of fibrils and oligomers of panel A. Value were normalized to the median total intensity collected at time 0. (C) Time comparison of the number of ThT⁺ species of panel A detected from seeding experiment using α-syn oligomers or sonicated PFFs before and after 2.5 h incubation. Each type of ThT⁺ species is coded by different shades of red.

Figure 7

Single molecule amplification assay at different concentration of seeds. (A) Bar graphs showing the number of ThT⁺ events detected before (black) and after 5 h incubation at 55 °C (red) seeded with oligomers (0.59–2100 nM) and fibrils (0.52 and 2.6 nM). Negative control (no seed) was included. (B) Scatter dot plot of residence time of the ThT⁺ events detected in panel A showing no change in residence time for oligomers but with significant increase in apparent molecular weight when seeded with fibrils. Blue bar represents median and is omitted when there is <2 data points. Each symbol represents an individual event. (C) Scatter dot plot of total intensity (AUC) for oligomers and PFFs, before (black) and after (red) amplification.