Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC

Abstract

The diagnosis and treatment of synucleinopathies such as Parkinson disease and dementia with Lewy bodies would be aided by the availability of assays for the pathogenic disease-associated forms of α-synuclein (αSyn^D) that are sufficiently sensitive, specific, and practical for analysis of accessible diagnostic specimens. Two recent αSyn^D seed amplification tests have provided the first prototypes for ultrasensitive and specific detection of αSyn^D in patients' cerebrospinal fluid. These prototypic assays require 5-13 days to perform. Here, we describe an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay that has similar sensitivity and specificity to the prior assays, but can be performed in 1-2 days with quantitation. Blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases [12 Parkinson's and 17 dementia with Lewy bodies] and 31 non-synucleinopathy controls, including 16 Alzheimer's cases, yielded 93% diagnostic sensitivity and 100% specificity for this test so far. End-point dilution analyses allowed quantitation of relative amounts of αSyn^D seeding activity in cerebrospinal fluid samples, and detection in as little as 0.2 μL. These results confirm that αSyn^D seeding activity is present in cerebrospinal fluid. We also demonstrate that it can be rapidly detected, and quantitated, even in early symptomatic stages of synucleinopathy.

Keywords: Alzheimer; Amplification; Cerebrospinal fluid; Diagnosis; Lewy body; PMCA; Parkinson; Prion; RT-QuIC; Synuclein.

Fig. 1

Detection of αSyn seeding activity in BH and CSF using K23Q (blue), WT (red) and WT* (green; commercial wild-type rαSyn lacking a 6× histidine tag [7]) substrates. Reactions were seeded in quadruplicate with brain homogenate (BH) (top panel) or CSF (bottom panel) from Parkinson’s disease (PD) or non-synucleinopathy (NS) including corticobasal degeneration cases (CBD BH) and healthy donors (NS CSF). For the BH assays, 10^− 3 (closed symbols) or 10^− 4 (open symbols) brain tissue dilutions from a single PD or NS case were used. For the CSF assays, 15 μl (undiluted) from two PD cases and one NS case was used. Each sample trace represents the average ThT signal of quadruplicate wells. For clarity only every other data point is plotted. The vertical dashed line designates the assay cutoff time used in subsequent analyses

Fig. 2

Detection of αSyn seeding activity in BH (a) and CSF (b) from cases with DLB but not non-synucleinopathy cases using K23Q rαSyn. Two μl of 10⁻³ dilutions of DLB (red; n = 3) or CBD (gray; n = 3) BH, or 15 μl (undiluted) CSF from DLB (red; n = 3) or healthy donors (NS CSF, gray; n = 3) were used per reaction. Each trace represents the average ThT signal of the four replicate wells

Fig. 3

Blinded testing of CSF samples by α-synuclein RT-QuIC. Samples from non-synucleinopathy (NS), Alzheimer’s disease (AD), dementia with Lewy bodies (DLB) or Parkinson’s disease (PD) patients, were tested blinded using the K23Q substrate. Quadruplicate reactions were seeded with 15 μL of CSF. Each sample trace represents the average ThT signal of the four wells. Panel a shows the average fluorescence enhancement kinetics for the AD, DLB and PD patients over time along with the associated standard deviation at each time point. Data points in Panel b indicate the average fluorescence obtained for each individual case at 48 h. Bars show the average +/− SD for type of case. The dashed line shows the fluorescence threshold for a positive result. Data points in Panel c show the hours required for the average fluorescence to exceed the threshold for individual cases. Bars show the average +/− SD for type of case. The dashed line indicates the end of the reaction at 48-h. Blue x symbol indicates sample 15/044 which was tested twice and both times had only one well crossing fluorescence threshold out of the four replicates. This sample was considered negative, as it did not meet our criteria for overall sample positivity (see Materials and Methods)

Fig. 4

End-point dilutions of synucleinopathy BH (a; sample # 081017) or CSF (b; sample # 10/005) samples by αSyn RT-QuIC. Each sample trace represents the average ThT signal of quadruplicate wells. Tables to the right of each graph indicate the concentration of SD₅₀ units calculated by Spearman-Kärber analysis for these, and additional, cases. End-point dilution experiments used for the additional calculated values shown in the upper and lower panels are provided in Additional files 4 and 5, respectively

Fig. 5

End-point dilutions of synthetic seeds spiked into CSF. Synthetic rαSyn fibrils were generated by continuous shaking at 1000 rpm at 37 °C for 3 days in a 1.5 mL tube containing 100 μL of 1 mg/ml WT rαSyn. Samples were monitored by ThT fluorescence. Following fibrilization the samples were spiked into non-synucleinopathy CSF and diluted in 10-fold serial dilutions. Each sample trace represents the average ± SEM ThT signal of quadruplicate wells. For clarity, data points were plotted every fourth point and negative controls, which were all below the positivity threshold, are not displayed