Ultra‐sensitive detection of pathological α‐synuclein in human tissues and biofluids using the RT‐QuIC Assay: Relevance to development of circulating and peripheral biomarkers for diagnosing α‐synucleinopathies and other protein misfolding diseases

Abstract

Parkinson's disease (PD), dementia with Lewy bodies (DLB) and other related adult‐onset neurodegenerative disorders are characterized by accumulation of the pathological form of aggregated α‐synuclein (αSynagg) and are collectively known as α‐synucleinopathies. The identification of α‐synucleinopathies requires the detection of αSynagg, typically postmortem, as a molecular hallmark in various brain regions. Currently, no early diagnostic biomarkers exist for PD or related α‐synucleinopathies. Detection of αSynagg in living patients has promising diagnostic biomarker potential for α‐synucleinopathies. Therefore, we strategically adopted the real‐time quaking‐induced conversion (RT‐QuIC) assay to detect ultra‐low levels of αSynagg. First, we purified monomeric recombinant human wild‐type αSyn and evaluated the validity of the αSyn RT‐QuIC assay for detecting αSynagg as a biomarker in brain homogenates from human subjects with PD, DLB, Alzheimer's disease (AD) along with age‐matched controls. Next, we tested cerebrospinal fluid (CSF) samples from clinically confirmed PD and progressive supranuclear palsy (PSP) subjects. Additionally, we quantified the protein aggregation rate (PAR) for αSynagg based on RT‐QuIC assay kinetics by calculating the time to cross threshold fluorescence. The PD and DLB BH samples showed significantly higher αSynagg PAR compared to age‐matched healthy controls and AD. Importantly, the RT‐QuIC assay was highly reproducible with 100% specificity and 94% sensitivity. Correspondingly, CSF samples from PD subjects demonstrated significantly higher αSynagg PAR compared to age‐matched healthy controls, with 100% sensitivity and specificity. In addition to brain and biofluids, we also tested peripherally accessible submandibular gland (SMG) tissues from PD and controls with 100% sensitivity and 94% specificity using the αSyn RT‐QuIC assay. In addition to demonstrating bonafide α‐synucleinopathies, we tested metal‐induced neurotoxicity in human subjects because exposure to metals such as Mn and other transition metals has been implicated in environmentally linked Parkinsonism. Therefore, we examined the serum and plasma exosomes of welders occupationally exposed to welding fumes and subsequent metal‐induced neurotoxicity. We were able to differentiate the welders from age‐matched controls based on the αSyn RT‐QuIC assay with a high degree of sensitivity and specificity. Collectively, our results demonstrate the potential diagnostic utility of the αSyn RT‐QuIC assay on clinically useful and easily accessible samples, such as CSF, SMG tissues as well as exosomes isolated from serum and plasma, thereby demonstrating its immediate translational potential in biomarker discovery efforts for early diagnosis of neurodegenerative diseases.Support or Funding InformationGrant support‐NIH ES026892, NS100090, NS088206; DoD PD170118; Lloyd and Armbrust endowments to AGK.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.