Single-Molecule Counting Coupled to Rapid Amplification Enables Detection of α-Synuclein Aggregates in Cerebrospinal Fluid of Parkinson's Disease Patients

Abstract

α-Synuclein aggregation is a hallmark of Parkinson's disease and a promising biomarker for early detection and assessment of disease progression. The prospect of a molecular test for Parkinson's disease is materializing with the recent developments of detection methods based on amplification of synuclein seeds (e.g. RT-QuIC or PMCA). Here we adapted single-molecule counting methods for the detection of α-synuclein aggregates in cerebrospinal fluid (CSF), using a simple 3D printed microscope. Single-molecule methods enable to probe the early events in the amplification process used in RT-QuIC and a precise counting of ThT-positive aggregates. Importantly, the use of single-molecule counting also allows a refined characterization of the samples and fingerprinting of the protein aggregates present in CSF of patients. The fingerprinting of size and reactivity of individual aggregate shows a unique signature for each PD patients compared to controls and may provide new insights on synucleinopathies in the future.

Keywords: Parkinson's disease; confocal spectroscopy; isothermal amplification; single-molecule counting; α-synuclein.

Figure 1

Detection of individual ThT‐positive aggregates using single molecule counting. A) Principle of single molecule detection: a very small (femtolitre) detection volume is coupled to single photon detectors, in order to measure freely diffusing single particles with high sensitivity. As fluorescently labelled particles diffuse in and out of the confocal volume, bursts of photons are collected by the detectors. B) In single molecule experiments, bulk ThT is deconvoluted into baseline and individual peaks. C) Principle of fingerprinting of each individual peak; the number of photons detected correlates with the number of fluorophores and the residence time of the aggregates. D) Total ThT fluorescence as a function of time. αSyn WT aggregation was detected by increased ThT fluorescence in the absence (red) and presence (blue) of 1 nM seed, using either a plate reader (light red/light blue) or AttoBright (bright red/ bright blue). E) ThT signal measured on a plate reader for the first 10 h, as in (A). No difference is visible between unseeded (light pink) and seeded (light blue) reactions in this time frame. F) The same experiment as in (B) performed on AttoBright shows a marked difference between seeded (bright blue) and unseeded (bright red) experiments; note that at t=0, the presence of seeds (1 nM) is already detectable. Error bars are mean ± s.d of 3× 400 s measurements.

Figure 2

Detection limit upon seeding conditions. A) Kinetics of amplification at different concentrations of αSyn fibrils analysed using the Brightness parameter (B parameter). Each sample was incubated with 20 μM αSyn K23Q monomer in the presence of ThT (10 μM) at 55 °C without shaking and analysed on AttoBright. Seeds concentrations varied between 100 nM and 100 fM as indicated. Error bars are mean ± s.d of 3 × 300 s measurements. B) Increase of sensitivity upon amplification, using the B parameter. B was measured as a function of fibrils concentration, before (0 h, red squares) and after 5 h amplification (black triangles). Different concentrations of αSyn seeds were amplified and the aggregates were detected by ThT fluorescence. Data are averaged over three independent dilutions on separate days, each sample was measured 3×300 s. C) Detection limit using the number of events per minute as a parameter. Box plot representing the results for 4 separate experiments (3 technical replicates each) for unseeded (circles), and seeded experiments using 10 fM (squares), 1 fM (upright triangles) and 100 aM seeds (downward triangles).

Figure 3

Comparison between amplification protocols. A) Typical fluorescence time traces obtained before (grey) and after amplification at 37 °C in the absence (blue) or with orbital shaking (500 rpm, green), or at 55 °C without shaking (red). 2 pM seeds were incubated with 20 μM monomeric αsyn K23Q and 10 μM ThT and amplification was carried out as indicated. 100 sec fluorescence time traces were obtained before and after 5 h amplification. B) Average number of ThT‐positive events per traces as a function of reaction conditions, as in (A). Fluorescence time traces obtained as in (A) were analysed to detect individual ThT‐positive events. All amplification protocols lead to an increase in the number of events detected. C) Total ThT fluorescence as a function of reaction conditions, as in (A). Fluorescence time traces obtained as in (A) were analysed to detect individual ThT‐positive events. Total ThT fluorescence for the trace was calculated as the sum of the area under the curve for all individual events. All amplification protocols lead to an increase in the total ThT fluorescence. D) Fingerprinting of the different reaction conditions. For each reaction conditions as in (A), the intensity (calculated as the area under the curve, AUC) was plotted as the function of diffusion time for each individual event. Note the increased presence of high but sharp events (AUC>10³ photons, diffusion time <200 ms) when amplification occurred at either 37 °C with orbital shaking or 55 °C without shaking. All experiments were carried out as a biological triplicate with 2 successive measurements for each sample. (Average ± SE) are plotted where relevant. Student's t‐tests were performed to analyse statistical significance. In all cases, ** indicates p<0.01 and *, p<0.1.

Figure 4

Direct detection and amplification of biological samples. A) Typical fluorescence time traces obtained for a patient's sample (S4) before (left) and after (right) amplification. 2 μL of CSF were diluted 10 times in 20 μM αSyn K23Q monomer and 10 μM ThT. Amplification was carried out at 55 °C, without shaking, for 5 hours. B) Comparison between PD patients (dark grey) and healthy controls (dotted) samples, before (left) and after (right) amplification. Fluorescence time traces were acquired as in (A) and analysed for the presence of ThT‐positive events. The percentage of positive reads was calculated as the number of traces presenting at least 1 ThT‐positive event over the total number of traces. C) Comparison of the number of ThT‐positive events detected between PD patients (dark grey) and healthy controls (dotted) samples, before (left) and after (right) amplification. Fluorescence time traces were acquired as in (A) and analysed for the presence of ThT‐positive events. The number of events was calculated for each time trace. The graph shows (average ± SE) number of ThT‐positive events per minute. Each sample was analysed at least 4 times (4 biological repeats, individual amplifications on separate days). The unpaired t‐test with Welch's correction (which does not assume that data have the same standard deviation) indicates a significant difference between the group of PD patients and the group of controls, with P value <0.0001.

Figure 5

Characterisation of ThT‐positive events from biological samples. A) Physical characterisation of ThT‐positive events in biological fluids, detected after amplification. The fluorescence time traces collected as in Figure 5 were analysed to detect individual ThT‐positive events. These events were analysed based on maximal intensity (left), diffusion time (centre) or total intensity (area under the curve, right), comparing healthy controls (grey) and PD patients (red) samples. The percentages of events presenting left: a maximal intensity <300 photons (light dotted pattern) or >300 photons (heavy dotted pattern), ‐centre) diffusion time <250 ms (light color) or >250 ms (darker color), and right: AUC<2000 photons (light upward stripes) or >2000 photons (dark downward stripes) were calculated and plotted. B) Maximal intensity as a function of diffusion time is represented for each individual ThT‐positive event, detected following amplification of PD patients (red circles) and healthy controls (grey triangles) samples. 4 types of events can be identified: small (S), high (H), long (L) and neutral (N) events, as depicted. Limits are set at 250 ms and 300 photons, on the x‐ and y‐axis, respectively. C) Comparison of the number of ThT‐positive events belonging to the categories described in (B), between healthy controls (grey) and PD patients (red) samples. Each category (S, H, L and N) is represented by a different shade, as indicated.