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.
Highlights
An unmet clinical need is a reliable premortem diagnostic biomarker for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), two common synucleinopathies [1]
Following a screen of recombinant αSyn (rec-Syn) products from several sources, we found that a commercial product of monomeric human wild-type rec-Syn was most suitable for this real-time quaking induced conversion (RT-QuIC) assay
Ultrasensitive detection of αSynD seeding activity in cerebrospinal fluid (CSF) samples of PD and DLB After we successfully detected αSynD in highly diluted brain homogenate (BH) of PD and DLB cases, we evaluated the sensitivity of our RT-QuIC assay for postmortem CSF samples
Summary
An unmet clinical need is a reliable premortem diagnostic biomarker for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), two common synucleinopathies [1]. Definitive diagnosis of PD and DLB relies mainly on postmortem detection of disease-associated alpha-synuclein (αSynD), deposited as protein aggregates within the Lewy body inclusions in the central nervous system (CNS) [2, 3]. Overwhelming evidence suggests that αSynD aggregates are formed as a consequence of protein misfolding cascades and may spread through prion-like transmission across cells and tissues during disease pathogenesis [4]. These αSynD aggregates may potentially serve as a promising biomarker of PD and DLB for premortem diagnosis and treatment. Immunohistochemical (IHC) staining of phosphorylated αSyn has been observed in peripheral tissues of PD and DLB, but wide variations in analytic performance have been reported by various groups as standardization of IHC staining remains a formidable challenge [8,9,10,11,12]
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