Potential sources of interference with the highly sensitive detection and quantification of alpha-synuclein seeds by qRT-QuIC.

Viktoria C Ruf,Georg S Nübling,Song Shi,Uwe Ködel,Daniel Weckbecker,Brit Mollenhauer,Armin Giese,Felix Schmidt

doi:10.1002/2211-5463.12844

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disease which is histologically characterized by loss of dopaminergic neurons in the substantia nigra and deposition of aggregated alpha‐synuclein (aSyn) in the brain. The detection of aSyn in well accessible fluids has been one of the central approaches in the development of biomarkers for PD. Recently, real‐time quaking‐induced conversion (RT‐QuIC) has been successfully adapted for use with aSyn seeds. Here, we systematically analysed parameters potentially impacting the reliability of this assay by using quantitative real‐time quaking‐induced conversion (qRT‐QuIC) with in vitro‐formed aSyn seeds. Seeds diluted in cerebrospinal fluid (CSF) accelerated the seeding reaction and slightly increased the sensitivity without affecting specificity. Repeated freeze–thaw cycles decreased the apparent lag times of seeds diluted in ddH2O but did not alter the seeding activity of seeds diluted in CSF. High levels of artificial contamination with blood resulted in prolonged apparent lag times, while sensitivity and specificity were unaffected. Altogether, qRT‐QuIC with aSyn seems to be robust concerning sensitivity and specificity in our model system, but quantitative interpretation might be limited under certain conditions.

Highlights

Parkinson’s disease (PD) is a progressive neurodegenerative disease which is histologically characterized by loss of dopaminergic neurons in the substantia nigra and deposition of aggregated alpha-synuclein in the brain
To adapt qRT-QuIC for use with aSyn seeds and to optimize the assay conditions, we used artificial, in vitroaggregated aSyn as seeds to provide standardized conditions with low interexperimental variation and to save precious and irretrievable patient samples. aSyn seeds were prepared by incubating monomeric aSyn at a concentration of 50 μM for 96 h at 37 °C under vigorous orbital shaking at 1400 r.p.m
thioflavin T (ThT) fluorescence measurements of the seed preparations obtained showed an increased fluorescence intensity compared to buffer or aSyn monomer (Fig. 1A), indicating that the in vitro-formed seed preparations consist at least in part of amyloid fibrils which could be confirmed by electron microscopy (EM) (Fig. 1B)

Summary

Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disease which is histologically characterized by loss of dopaminergic neurons in the substantia nigra and deposition of aggregated alpha-synuclein (aSyn) in the brain. Real-time quaking-induced conversion (RT-QuIC) has been successfully adapted for use with aSyn seeds. We systematically analysed parameters potentially impacting the reliability of this assay by using quantitative real-time quaking-induced conversion (qRT-QuIC) with in vitro-formed aSyn seeds. Real-time quaking-induced conversion (RT-QuIC), referred to as protein misfolding cyclic amplification by some authors and initially developed for the ultrasensitive detection of infectious prions [11,12], has been successfully adapted for use with aSyn seeds and substrate and represents so far one of the most promising approaches [13,14,15,16]. We have used our qRT-QuIC assay with in vitro-formed aSyn seeds and substrate to systematically analyse parameters which in daily practice potentially interfere with the reliability of the assay

Methods

Results

Discussion

Conclusion