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

Definitive diagnosis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (alpha Syn(D)) 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 alpha Syn(D) aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current alpha Syn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of alpha Syn(D) across a wide range of biospecimens and in different laboratories. We have streamlined alpha Syn 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 alpha Syn(D) in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of alpha Syn(D) 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 alpha Syn(D) 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 alpha Syn(D) 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 alpha Syn 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 alpha Syn(D) in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of alpha Syn(D) 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.

Automated summary

The development of a streamlined RT-QuIC assay based on commercial reagents allows for uniform analysis of alpha Syn(D) in various biospecimens from PD and DLB cases, achieving high sensitivity and specificity in detecting disease-associated aggregates. The use of this assay can improve diagnostic applications and facilitate efficient utilization of scarce biospecimens for understanding disease pathogenesis and monitoring therapeutic efficacies.