Single-Molecule Fingerprinting Reveals Different Growth Mechanisms in Seed Amplification Assays for Different Polymorphs of α-Synuclein Fibrils

Abstract

α-Synuclein (αSyn) aggregates, detected in the biofluids of patients with Parkinson's disease (PD), have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinson's disease and turns the presence of αSyn aggregates into a biomarker of the disease. It has become evident that αSyn can form fibrils with slightly different structures, called "strains" or polymorphs, but little is known about their differential reactivity in diagnostic assays. Here, we compared the properties of two well-described αSyn polymorphs. Using single-molecule techniques, we observed that one of the polymorphs had an increased tendency to undergo secondary nucleation and we showed that this could explain the differences in reactivity observed in in vitro seed amplification assay and cellular assays. Simulations and high-resolution microscopy suggest that a 100-fold difference in the apparent rate of growth can be generated by a surprisingly low number of secondary nucleation "points" (1 every 2000 monomers added by elongation). When both strains are present in the same seeded reaction, secondary nucleation displaces proportions dramatically and causes a single strain to dominate the reaction as the major end product.

Keywords: PMCA; Parkinson’s disease; RT-QuIC; preformed fibrils; seed amplification assay; single-molecule detection; strains; α-synuclein.

Figure 1

Characterization of in vitro recombinant αSyn assemblies. (A) Schematic of αSyn aggregation and the different mechanisms at play: The initial formation of aggregates by primary nucleation is followed by the growth of the fibrils by two mechanisms: pure elongation (linear growth) or secondary nucleation (creation of a new fibril on the surface of the first fibril). (B) Schematic of the generation and characterization of recombinant αSyn polymorphs from monomers aggregated in low-salt (sky blue) and high-salt buffer (HSB) (vermilion), using different assays. (C) Transmission electron micrographs showing assemblies of recombinant αSyn under low-salt and high-salt buffer conditions, producing low-salt ribbons and high-salt fibrils, respectively. Scale bar 200 nm. (D) Histogram plot of the diameter of negatively stained low-salt ribbons (17.1 ± 3.3 nm, mean ± standard deviation (SD), N = 308) and high-salt fibrils (15.5 ± 1.9 nm, mean ± standard deviation, N = 233). (E) Proteinase K digestion profile of ribbons, fibrils, and αSyn monomers. Substrates were digested by the addition of proteinase K (1.5 μg/mL), quenched at 5, 15, and 30 min with loading buffer, heated at 90 °C prior to resolving using reducing SDS-PAGE. (F) Typical data obtained from a real-time quaking-induced conversion (RT-QuIC) assay on ribbons (LS) and fibrils (HS) at two different concentrations of seeds. Amplification was carried out in phosphate buffer saline (PBS), in the presence of 7 μM wild-type (WT) αSyn monomer, 10 μM thioflavin (ThT), and 6 glass beads. Fluorescence was recorded every 45 min. Control is an unseeded reaction.

Figure 2

Characterization of recombinant αSyn assembly incubation on SH-SY5Y cells. (A) SH-SY5Y neuroblastoma cell culture scheme. Cells were seeded on coverslips at the bottom of a 12-well plate and imaged using brightfield microscopy (inset images). SH-SY5Y cells were differentiated for 7 days before incubation with αSyn ribbons or fibrils in differentiation media. Effects of αSyn assemblies on cells were assessed by immunostaining and confocal microscopy at 4, 7, and 10 days postaddition of the assemblies. (B) Differentiated SH-SY5Y cells were treated with or without 5 μg/mL different αSyn polymorphs and subsequently fixed for immunofluorescence staining of total αSyn (green punctae) at indicated time points. DAPI staining is represented in blue. Scale bar is 50 μm. (C) Bar graphs quantifying the average intensity of immunostained αSyn per cell. NT, no treatment, ribbons (sky blue) and fibrils (vermilion), respectively. Each symbol represents a field of view (FOV). Total αSyn intensity was normalized by the number of cells based on the DAPI signal. Error bars represent mean ± standard deviation. Statistics used ordinary one-way ANOVA Tukey’s comparison test with a single pooled variance, p ≤ 0.0001 (****), nonsignificant (n.s.).

Figure 3

Single-molecule measurement on recombinant αSyn polymorphs. (A) Schematic of the single-molecule seed amplification assay (smSAA). (1) αSyn polymorphs are seeded in a PBS solution containing thioflavin T (ThT, 10 μM) and human αSyn K23Q (20 μM). (2) The solution is mixed, and half of the reaction is measured using single-molecule spectroscopy to quantify the types of assemblies before an amplification step. (3) Sample is then amplified and measured again. Amplification enhances the signal of existing seeds by promoting αSyn assemblies. (4) Schematic of the microscope setup. The inset shows ThT stained αSyn assemblies diffusing across the confocal volume. (B) Typical fluorescence traces. Fluorescence traces are analyzed to report the total number of ThT⁺ peaks (events) per measurement and (inset) 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. (C) Typical raw trace obtained for amplified ribbons shows sharp peaks (fast-diffusing) (D) The amplified fibrils show a very different profile at the single-molecule level, with very intense and slow-diffusing species. (E) Logarithmic scatter plot comparing peak prominence, residence time, and area under the curve before (− sign, light) and after amplification (+ sign, dark) of LS ribbons and HS fibrils. Gray horizontal line is the median value. Each symbol represents an individual event and statistics used the Kruskall–Wallis test, p ≤ 0.0001 (****), nonsignificant (n.s.).

Figure 4

High-resolution confocal imaging of the amplified fibrils. Confocal microscopy was performed on the fibrils after 5 h of amplification, on a Zeiss LSM880 microscope with an AiryScan unit (see the SI for details). Z-stacks were recorded and analyzed using Zen software to generate the final high-resolution images. In the same setup, amplified ribbons could not be detected, as expected for smaller, fast-diffusing particles.

Figure 5

Mathematical modeling of seed amplification and competition between strains. (A) Visual representation of the different parameters used for simulation: M—the polymer mass concentration, N—the concentration of growth points, and m—the concentration of free monomers. The diagram on the right shows the elementary reactions that we considered in this model. k_e (respectively, k_s, k_f) is the rate of the elongation (respectively, secondary nucleation, fragmentation) reaction. (B) Numerical fit of experimental RT-QuIC data seeded with ribbons or fibrils at 5, 50, or 500 nM using the numerical solution derived from panel (A). (C) Numerical solutions for the time evolution of the relative polymer mass concentration, with different secondary nucleation to elongation ratios denoted by λ where λ = 10^–3 means that one new branch is created for 1000 monomers added through elongation. The relative polymer mass reaches 1 when all monomers have been incorporated into the polymers. The fragmentation rate is set to 0. (D) Numerical modeling of competition between different strains seeded in the same solution. Fragmentation and elongation rates were set to the same value, and a rate of secondary nucleation was applied only for fibrils. The rate constants were extracted from the fit in panel (B). The initial composition of polymer mass was set to 100, 99.9999999, 99.99999, 99.999, 99.9, and 99% of ribbons. (E) Final content of ribbons in the polymer mass at the end of simulation (80 h) is displayed for low seed concentration (0.1% of monomer equivalent) or high seed concentration (1% monomer equivalent). (F) Experimental competition between ribbons and fibrils. The 4 curves correspond to 4 different ratios of ribbons and fibrils in the seeds. Each curve is obtained by averaging the results of 2 RT-QuIC experiments with the same parameters.

Figure 6

Single-molecule profiling of ribbons and fibrils amplified in different conditions. (A) Table of the different conditions used: A (32 °C, 7 μM αSyn WT), B (37 °C, 21 μM αSyn WT), (42 °C, 7 μM αSyn K23Q), D (55 °C, 20 μM αSyn K23Q), and E (55 °C, 20 μM αSyn WT). (B) Bar graph showing the total fluorescence intensity of the trace for low-salt ribbons (blue) and high-salt fibrils (vermilion) before (light) and after 5 h of amplification (bright) in different conditions. Error bar is the standard deviation. (C) Logarithmic scatter plot comparing the residence time of ThT⁺ peaks (events) detected in fluorescence traces before (light) and after amplification (bright), for ribbons (blue) and fibrils (vermilion). Each symbol represents an individual event and statistics used the Kruskall–Wallis test, p ≤ 0.05 (*), p ≤ 0.0001 (****), nonsignificant (n.s.). (D) The events before (black) and after (vermilion) amplification (here for fibrils amplified in conditions D) can be visualized on a scatter plot of prominence vs residence time. We defined 4 quadrants to separate fast-diffusing and slow-diffusing species, and high-intensity vs low-intensity species. The four species are defined as S (dark gray, <300 photons, <250 ms), H (light gray, >300 photons, <250 ms), L (hashed red, <300 photons, >250 ms), and N (red, >300 photons, >250 ms). (E) Distribution of the different species detected after amplification for the different buffers. Results for the ribbons and fibrils are presented in the blue and vermilion frame, respectively.

Figure 7

Correlation between RT-QuIC and single-molecule profiling and effect of salt concentration. (A) RT-QuIC curves obtained for reactions seeded with fibrils in phosphate buffer (PB, pH 7.5) with different concentrations of NaCl. The curves show an increase in reactivity with increasing salt concentrations. (B) RT-QuIC curves obtained for reactions seeded with fibrils in MES buffer (pH 6.0) with different NaCl concentrations; the reactions appear slower when [NaCl] increases. (C) Schematic of the RT-QuIC curve and definition of different parameters for quantification: slope of the reaction (calculated around the midpoint), time-to-threshold (TTT, time point when reactivity reaches 10% of the final value), and time-to-midpoint (TTM, time point when ThT signal has increased to 50% of the final value). (D) From experiments conducted in quiescent conditions and analyzed using our single-molecule method, we determined the smSAA average peak width. The peak width correlates strongly with the slope of the RT-QuIC reaction, as shown here, for fibrils in PB (round) and MES (diamond) buffers. Data for ribbons are color-coded in blue, and in red for fibrils; data are displayed as round dots for phosphate buffer, and diamonds for MES buffer. These colors are used for panels (E–H). (E) smSAA diffusion time as a function of salt concentration for ribbons (blue) and fibrils (vermilion) in phosphate buffer pH 7.5. The red dotted line indicates the arbitrary threshold for slow-diffusing particles (250 ms). (F) SmSAA diffusion times for ribbons (blue) and fibrils (vermilion) in MES buffer pH 6.0; the data show that both fibrils and ribbons have been converted to aggregates with high diffusion times. (G) Scatter plot of smSAA average peak width vs time-to-threshold (TTT), showing that low pH leads to much faster reactions for ribbons and higher diffusion times. (H) Scatter plot of time-to-mid (TTM) vs smSAA average peak width for ribbons and fibrils in all conditions of pH and salt, showing a general trend of decreasing diffusion times with increasing reaction times.

Figure 8

Characterization of αSyn polymorphs obtained by cross-buffer seeded reactions. (A) Ribbons (LS) were seeded in low-salt buffer (LSB) or high-salt buffer (HSB) yielding the products LSxL and LSxH, respectively. Similarly, fibrils (HS) were seeded in low-salt buffer (LSB) or high-salt buffer (HSB) giving rise to HSxL and HSxH products. Representative electron micrographs of negatively stained assemblies after amplification. (B) Box plot quantifying the diameter of filaments from electron micrographs; LSxL-1 (16.4 ± 2.7 nm, mean ± standard deviation, N = 244), LSxL-2 (13.0 ± 2.2 nm, N = 42), LSxH (21.9 ± 6.0 nm, N = 40), HSxL (15.8 ± 1.6 nm, N = 76), HSxH (13.7 ± 2.0, N = 28). Whiskers represent min-max values. (C) Proteinase K digestion profile of seeded products from recombinant αSyn polymorphs in the absence (top) and after the addition of proteinase K (bottom, PK). Black arrow points at a band that discriminates ribbons from fibrils. (D) Logarithmic scatter plot comparing the residence time of events in seeded reaction before (black) and after (vermilion) amplification with αSyn K23Q. The median value is highlighted in blue. Each symbol represents an individual event and statistics used the Kruskall–Wallis test, p ≤ 0.0001 (****), nonsignificant (n.s.). (E) Distribution of ThT⁺ events detected after amplification in cross-seeded reactions. S (dark gray, <300 photons, <250 ms), H (light gray, >300 photons, <250 ms), L (hashed red, <300 photons, >250 ms) and N (red, >300 photons, >250 ms). The number of events detected and used for quantification is indicated above each bar.

Figure 9

Temporal changes of cytoplasmic αSyn inclusions following treatment of SH-SY5Y cells with cross-seeded αSyn aggregates. (A) Confocal images of differentiated SH-SY5Y cells treated without (NT, no treatment) or with 5 μg/mL of diverse αSyn aggregates: ribbons seeded in low-salt buffer (LSxL), ribbons seeded in high-salt buffer (LSxH), fibrils in low-salt buffer (HSxL) and fibrils seeded in high-salt buffer (HSxH). Cells were subsequently fixed for immunofluorescence staining to quantify total αSyn (green) at day 4, 7, and 10 post-incubation with the aggregates. DAPI staining is represented in blue. Scale bar of 50 μm. (B) Bar graphs quantifying the average intensity of immunostained αSyn per cell upon treatment with different αSyn aggregates (NT: not treated, light blue: LSxL, dark blue: LSxH, light vermilion: HSxL, dark vermilion: HSxH). Each symbol represents a FOV. Total αSyn intensity was normalized by the number of cells based on a DAPI signal. Error bars represent mean ± standard deviation. Statistics used ordinary one-way ANOVA Tukey’s comparison test with a single pooled variance, p ≤ 0.0001 (****), nonsignificant (n.s.).