Development of a Biochemical Diagnosis of Parkinson Disease by Detection of α-Synuclein Misfolded Aggregates in Cerebrospinal Fluid

Abstract

IMPORTANCE Parkinson disease (PD) is a highly prevalent and incurable neurodegenerative disease associated with the accumulation of misfolded α-synuclein (αSyn) aggregates. An important problem in this disease is the lack of a sensitive, specific, and noninvasive biochemical diagnosis to help in clinical evaluation, monitoring of disease progression, and early differential diagnosis from related neurodegenerative diseases. OBJECTIVE To develop a novel assay with high sensitivity and specificity to detect small quantities of αSyn aggregates circulating in cerebrospinal fluid (CSF) of patients affected by PD and related synucleinopathies. DESIGN, SETTING, AND PARTICIPANTS The strategy evaluated in this proof-of-concept study uses the protein misfolding cyclic amplification (PMCA) technology that detects minute amounts of misfolded oligomers by taking advantage of their ability to nucleate further aggregation, enabling a very high amplification of the signal. The technology was first adapted with synthetic αSyn oligomers prepared in vitro and used to screen in 2 blinded cohorts of CSF samples from German and Japanese patients with PD (n = 76) and individuals serving as controls affected by other neurologic disorders (n = 65), neurodegenerative diseases (n = 18), and Alzheimer disease (n = 14). The kinetics of αSyn aggregation were measured by αSyn-PMCA in the presence of CSF samples from the participants to detect αSyn oligomeric seeds present in this biological fluid. The assays were conducted from November 15, 2013, to August 28, 2015. MAIN OUTCOMES AND MEASURES Kinetic parameters correlated with disease severity at the time of sample collection, measured by the Hoehn and Yahr scale, with the lowest grade indicating unilateral involvement with minimal or no functional impairment, and the highest grade defining patients with complete confinement to wheelchair or bed. RESULTS Studies with synthetic αSyn aggregates showed that αSyn-PMCA enabled to detect as little as 0.1 pg/mL of αSyn oligomers. The αSyn-PMCA signal was directly proportional to the amount of αSyn oligomers added to the reaction. A blinded study of CSF samples correctly identified patients affected by PD with an overall sensitivity of 88.5% (95% CI, 79.2%-94.6%) and specificity of 96.9% (95% CI, 89.3%-99.6%). The αSyn-PMCA results for different patients correlated with the severity of the clinical symptoms of PD (Japanese cohort: r s = −0.54, P = .006; German cohort: r s = −0.36, P = .02). CONCLUSIONS AND RELEVANCE The findings suggest that detection of αSyn oligomers by αSyn-PMCA in the CSF of patients affected by PD may offer a good opportunity for a sensitive and specific biochemical diagnosis of the disease. Further studies are needed to investigate the usefulness of αSyn-PMCA to monitor disease progression and for preclinical identification of patients who may develop PD.

Current state of Alzheimer’s fluid biomarkers

“…There are reports of increased CSF concentrations of α-synuclein oligomers in CSF of PD patients [132, 233, 364], and recent publications on sensitive assays that appear to detect the minute amounts of putative seeds of α-synuclein oligomers in CSF [86, 325]. …”

Seeding selectivity and ultrasensitive detection of tau aggregate conformers of Alzheimer disease

“…Such testing can provide intra vitam diagnoses that can be virtually 100% sensitive and specific [2, 34]. We and others have recently described α-synuclein RT-QuIC and closely related assays for the CSF-based early diagnosis of Parkinson disease (PD) and Lewy body dementia (LBD) [11, 18, 41]. With respect to AD specifically, a significant development was the report of a seed amplification assay (called Aβ-PMCA) for Aβ oligomers, which are another key feature of AD pathogenesis [38].…”

Absolute Quantification of Amyloid Propagons by Digital Microfluidics

“…26,27,34 To enable a direct comparison between the sensitivity of this method with d-AQuA, we developed an amyloid amplification assay for insulin in a microplate with a digital read-out (Figure 5). As the precision of digital read-outs increases with the number of replicate reactions, we established the assay in a 384-well format.…”

“…24−27 These diseases are associated with the ability of proteins to self-assemble into amyloid fibrils in a nucleation-dependent polymerization reaction. 28,29 This process typically follows a sigmoidal kinetic progression involving primary nucleation, aggregate growth and fibril elongation, along with secondary processes, such as fragmentation and surface-induced nucleation events that serve to amplify the number of aggregates.…”