Ultrasensitive RT-QuIC assay with high sensitivity and specificity for Lewy body-associated synucleinopathies

Abstract

The clinical diagnosis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), is challenging, especially at an early disease stage, due to the heterogeneous and often non-specific clinical manifestations. The discovery of reliable specific markers for synucleinopathies would consequently be of great aid to the diagnosis and management of these disorders. Real-Time Quaking-Induced Conversion (RT-QuIC) is an ultrasensitive technique that has been previously used to detect self-templating amyloidogenic proteins in the cerebrospinal fluid (CSF) and other biospecimens in prion disease and synucleinopathies. Using a wild-type recombinant α-synuclein as a substrate, we applied RT-QuIC to a large cohort of 439 CSF samples from clinically well-characterized, or post-mortem verified patients with parkinsonism or dementia. Of significance, we also studied patients with isolated REM sleep behavior disorder (iRBD) (n = 18) and pure autonomic failure (PAF) (n = 28), representing clinical syndromes that are often caused by a synucleinopathy, and may precede the appearance of parkinsonism or cognitive decline. The results show that our RT-QuIC assay can accurately detect α-synuclein seeding activity across the spectrum of Lewy Body (LB)-related disorders (LBD), including DLB, PD, iRBD, and PAF, with an overall sensitivity of 95.3%. In contrast, all but two patients with MSA showed no α-synuclein seeding activity in the applied experimental setting. The analysis of the fluorescence response reflecting the amount of α-synuclein seeds revealed no significant differences between the clinical syndromes associated with LB pathology. Finally, the assay demonstrated 98% specificity in a neuropathological cohort of 101 cases lacking LB pathology. In conclusion, α-synuclein RT-QuIC provides an accurate marker of synucleinopathies linked to LB pathology and may have a pivotal role in the early discrimination and management of affected patients. The finding of no α-synuclein seeding activity in MSA seems to support the current view that MSA and LBD are associated with different conformational strains of α-synuclein.

Keywords: Biomarker; Diagnosis; Multiple system atrophy; Parkinson’s disease; Prion disease; α-Synuclein.

Fig. 1

Distinctive α-Syn pathology in patients with LBD and MSA. a Moderate number of LBs and Lewy neurites in the medulla of a case with incidental LB pathology (case #18); b numerous pathological aggregates of α-Syn in the locus coeruleus (case #8); c single and multiple LB inclusions in the neurons of substantia nigra (case #8); d numerous LBs and positive dendrites in the amygdala (case #3); e moderate number of LBs and positive dendrites in the temporal neocortex (case #1); f numerous glial cytoplasmic inclusions (GCIs) in oligodendroglia in the internal capsule of a patient with MSA-P; the two boxes on the left show high magnifications of two GCIs (basal ganglia, upper box) and one neuronal cytoplasmic inclusion (pons, lower box)

Fig. 2

Kinetic curves of α-Syn seeding activity measured by RT-QuIC. a Seeding activity of neuropathologically confirmed LB α-Syn + cases (n = 21). Each curve depicts the average of quadruplicates. Standard deviation (SD) was hidden to make the image more readable; b comparison among PD (purple line, n = 71), MSA (yellow line, n = 33), and PSP/CBS (pale blue line, n = 31) clinical cases; c comparison among iRBD (blue line, n = 18), PAF (red line, n = 28), and narcolepsy type 1 plus RBD (gray line, n = 11) clinical cases; d comparison between DLB (green line, n = 48) and AD (dark gray line, n = 60). Clinical and neuropathologically confirmed cases are grouped together. Each curve represents the average of the group, error bars indicate the SD, and the black dashed line indicates the threshold. RFU values are normalized to percentage against the maximum intensity of fluorescence of the respective experimental plate

Fig. 3

Parameters describing the kinetics of α-Syn aggregation in the RT-QuIC assay. a Comparison among the I_max of each group; b comparison of the lag phase of the positive groups; c comparison of the AUC of each diagnostic group analyzed. Box plot colors match those represented in the kinetic curves. Box plots show the range and the average of the distribution. Symbols indicate the outlier according to Tukey test. Statistical analyses were conducted using one-way ANOVA, resulting in a significance of p < 0.0001 (****) between each of the positive groups against the negatives