Ultrasensitive 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

Adult-onset neurodegenerative disorders, like Parkinson's disease (PD) and dementia with Lewy bodies (DLB), that share the accumulation of aggregated alpha-synuclein (alpha Syn(agg)) as their hallmark molecular pathology are collectively known as alpha-synucleinopathies. Diagnosing alpha-synucleinopathies requires the post-mortem detection of alpha Syn(agg) in various brain regions. Recent efforts to measure alpha Syn(agg) in living patients include quantifying alpha Syn(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 alpha Syn(agg). We first optimized RT-QuIC for sensitivity, specificity, and reproducibility by using monomeric recombinant human wild-type alpha Syn as a substrate and alpha Syn(agg) as the seed. Next, we exposed mouse microglia to alpha Syn pre-formed fibrils (alpha Syn(PFF)) for 24 h. RT-QuIC assay revealed that the alpha Syn(PFF) is taken up rapidly by mouse microglia, within 30 min, and cleared within 24 h. We then evaluated the alpha Syn RT-QuIC assay for detecting alpha Syn(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 alpha Syn(agg). The PD and DLB BH samples not only showed significantly higher alpha Syn(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 alpha Syn(agg) PAR compared to age-matched healthy controls, with 100% sensitivity and specificity. Overall, the RT-QuIC assay accurately detects alpha Syn(agg) seeding activity, offering a potential tool for antemortem diagnosis of alpha-synucleinopathies and other protein-misfolding disorders.