Open AccessUltrasensitive Detection of Aggregated α-Synuclein in Glial Cells, Human Cerebrospinal Fluid, and Brain Tissue Using the RT-QuIC Assay: New High-Throughput Neuroimmune Biomarker Assay for Parkinsonian Disorders
Author(s) -
Sireesha Manne,
Naveen Kondru,
Monica Hepker,
Huajun Jin,
Vellareddy Anantharam,
Mechelle M. Lewis,
Xuemei Huang,
Anumantha G. Kanthasamy,
Anumantha G. Kanthasamy
Publication year - 2019
Publication title -
journal of neuroimmune pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.219
H-Index - 70
eISSN - 1557-1904
pISSN - 1557-1890
DOI - 10.1007/s11481-019-09835-4
Subject(s) - synucleinopathies , dementia with lewy bodies , microglia , progressive supranuclear palsy , cerebrospinal fluid , biomarker , pathology , alpha synuclein , microbiology and biotechnology , biology , chemistry , parkinson's disease , medicine , immunology , dementia , biochemistry , inflammation , disease
Adult-onset neurodegenerative disorders, like Parkinson's disease (PD) and dementia with Lewy bodies (DLB), that share the accumulation of aggregated α-synuclein (αSyn agg ) as their hallmark molecular pathology are collectively known as α-synucleinopathies. Diagnosing α-synucleinopathies requires the post-mortem detection of αSyn agg in various brain regions. Recent efforts to measure αSyn agg in living patients include quantifying αSyn agg in different biofluids as a biomarker for PD. We adopted the real-time quaking-induced conversion (RT-QuIC) assay to detect very low levels of αSyn agg . We first optimized RT-QuIC for sensitivity, specificity, and reproducibility by using monomeric recombinant human wild-type αSyn as a substrate and αSyn agg as the seed. Next, we exposed mouse microglia to αSyn pre-formed fibrils (αSyn PFF ) for 24 h. RT-QuIC assay revealed that the αSyn PFF is taken up rapidly by mouse microglia, within 30 min, and cleared within 24 h. We then evaluated the αSyn RT-QuIC assay for detecting αSyn agg in human PD, DLB, and Alzheimer's disease (AD) post-mortem brain homogenates (BH) along with PD and progressive supranuclear palsy (PSP) cerebrospinal fluid (CSF) samples and then determined protein aggregation rate (PAR) for αSyn agg . The PD and DLB BH samples not only showed significantly higher αSyn agg PAR compared to age-matched healthy controls and AD, but RT-QuIC was also highly reproducible with 94% sensitivity and 100% specificity. Similarly, PD CSF samples demonstrated significantly higher αSyn agg PAR compared to age-matched healthy controls, with 100% sensitivity and specificity. Overall, the RT-QuIC assay accurately detects αSyn agg seeding activity, offering a potential tool for antemortem diagnosis of α-synucleinopathies and other protein-misfolding disorders. Graphical Abstract A schematic representation of αSyn RT-QuIC assay.