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.

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

“…The prominence of each event was plotted against its respective residence time to examine population heterogeneity and provide a single-molecule profile (fingerprint) of the sample, as previously described. 34 Briefly, ThT + particles were classed based on their distribution in each quadrant (Figure 2B) where events were defined as small (S, low prominence, short residence time), high (H, high prominence, short residence time), long (L, low prominence, long residence time), or neutral (N, high prominence, long residence time) with thresholds set at 300 photons and 250 ms, respectively. Our data showed that S type particles were the first to be synthesized during the in vitro oligomer-inducing protocol.…”

“…We have established that the detection limit for PFFs using AttoBright was 1 pM monomer equivalent. 34 Our highly sensitive single molecule method was used here to track the aggregation at different steps and to compare the size and reactivity of the different α-syn species (monomers, oligomers and fibrils). After low-speed centrifugation of a 5 h aggregation reaction (18000g for 10 min), many fibrils were still present in the supernatant and dominated the fluorescence time trace (Supporting Figure 2).…”

“…We and others have previously demonstrated that single molecule fluorescence assays were compatible with the use of thioflavin T (ThT) to observe and characterize human α-syn oligomers and larger soluble aggregates (Figure A). ,, We have shown that single molecule detection enables a >100000-fold increase in sensitivity over traditional plate reader measurements for ThT . Traditionally, single molecule measurements are performed on expensive commercial microscopes or on homemade setups that are difficult to replicate between laboratories.…”

“…In this assay adapted for sensitive single molecule detection in small volumes, we use a single incubation step at 55 °C in nonshaking conditions and measure the same sample 5 h later (Supporting Figure 4). We observed that amplification greatly enhanced the number of ThT + species in purified oligomers. Incubation at 37 °C produced a similar increase in ThT + species when seeded with oligomers (Supporting Figure 5).…”

“…SEC eluted fractions (in PBS) were mixed with filtered monomeric α-syn WT (30 μM) and ThT (10 μM) and were incubated at 55 or 37 °C for 5 h in a PCR machine (Biorad, C1000) for amplification. 34 Fractions comprised of ≥76% of its original volume were mixed with α-syn monomers and ThT to reach a final volume of 20 μL per reaction. Samples were loaded without dilution onto the PDMS plate to record fluorescence on AttoBright.…”