Streamlined alpha-synuclein RT-QuIC assay for various biospecimens in Parkinson’s disease and dementia with Lewy bodies

Abstract

Definitive diagnosis of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (αSynD) as misfolded protein aggregates in the central nervous system (CNS). The recent development of the real-time quaking induced conversion (RT-QuIC) assay for ultrasensitive detection of αSynD aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current αSyn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of αSynD across a wide range of biospecimens and in different laboratories. We have streamlined αSyn RT-QuIC assay based on a second generation assay platform that was assembled entirely with commercial reagents. The streamlined RT-QuIC method consisted of a simplified protocol requiring minimal hands-on time, and allowing for a uniform analysis of αSynD in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of αSynD aggregates was achieved in million-fold diluted brain homogenates and in nanoliters of CSF from PD and DLB cases but not from controls. Comparative analysis revealed higher seeding activity of αSynD in DLB than PD in both brain homogenates and CSF. Our assay was further validated with CSF samples of 214 neuropathologically confirmed cases from tissue repositories (88 PD, 58 DLB, and 68 controls), yielding a sensitivity of 98% and a specificity of 100%. Finally, a single RT-QuIC assay protocol was employed uniformly to detect seeding activity of αSynD in PD samples across different types of tissues including the brain, skin, salivary gland, and colon. We anticipate that our streamlined protocol will enable interested laboratories to easily and rapidly implement the αSyn RT-QuIC assay for various clinical specimens from PD and DLB. The utilization of commercial products for all assay components will improve the robustness and standardization of the RT-QuIC assay for diagnostic applications across different sites. Due to ultralow sample consumption, the ultrasensitive RT-QuIC assay will facilitate efficient use and sharing of scarce resources of biospecimens. Our streamlined RT-QuIC assay is suitable to track the distribution of αSynD in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of αSynD as a potential biomarker for PD and DLB in clinically accessible biospecimens has broad implications for understanding disease pathogenesis, improving early and differential diagnosis, and monitoring therapeutic efficacies in clinical trials.

High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson’s disease

“…The in vitro generated αSyn fibrils are rich in β-sheets, enabling detection by the amyloid-specific dye Thioflavin T (ThT). αSyn-SAAs are capable of detecting and amplifying αSyn seeds from multiple biospecimens, including brain [11][12][13], cerebrospinal fluid (CSF) [6][7][8][9][10][13][14][15][16], skin [17][18][19], olfactory mucosa [20,21], submandibular gland [22], and gut [23]. Moreover, they have demonstrated accurate detection of αSyn seeds in clinically and pathologically validated cohorts of PD patients [6,7,9,10,13,17,18,24].…”

The Neural Gut–Brain Axis of Pathological Protein Aggregation in Parkinson’s Disease and Its Counterpart in Peroral Prion Infections

“…A quantitative mapping of the temporal–spatial course of the distribution of αSyn PD seeding activity in humans using highly sensitive real-time quaking induced conversion (RT-QuIC) [ 112 , 113 , 114 , 115 ] or protein misfolding cyclic amplification (PMCA) [ 116 ] assays could also provide important contributions to the elucidation of neuroanatomical spreading pathways of αSyn pathology in PD. αSyn PD seeding activity is assumed to constitute the molecular driver underlying the propagation and spatial spread of pathological αSyn aggregation in PD.…”

“…This would possibly also help to settle the controversy on reports claiming the detection of pathological αSyn PD deposits in colonic and other gastro-intestinal biopsies years before the onset of PD motor symptoms [ 27 ]. That detection of αSyn PD seeding activity by RT-QuIC assay in GI tissue from the stomach or colon of PD patients is feasible has been recently demonstrated in two independent studies [ 99 , 115 ]. Thus, using the relatively new and powerful analytical tools of RT-QuIC or PMCA for the quantitative detection of αSyn PD -associated seeding activity in human postmortem tissues and bioptic samples may open new perspectives on both the neural gut–brain axis of pathological protein aggregation and the ultrasensitive diagnostic detection of αSyn PD seeds in PD.…”

Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans

“…A modified version of the α-synuclein RT-QuIC assay described by Barger et al [ 31 ] was used. Dilution of rat CSF (1:10) was performed with 1x N2 supplement (Thermo Fisher) diluted with PBS.…”