Phospho-S129 Alpha-Synuclein Is Present in Human Plasma but Not in Cerebrospinal Fluid as Determined by an Ultrasensitive Immunoassay.

Cristina Cariulo,Paola Martufi,Eugenia Scaricamazza,Margherita Verani,Andrea Caricasole,Andreas Weiss,Giulia Maria Sancesario,Lara Petricca,Giuseppe Sancesario,Tommaso Schirinzi,Nicola Biagio Mercuri,Hilal A Lashuel,Sean M Deguire,Giordana Bruni,Lucia Azzollini

doi:10.3389/fnins.2019.00889

Abstract

Accumulation and aggregation of misfolded alpha-synuclein is believed to be a cause of Parkinson’s disease (PD). Phosphorylation of alpha-synuclein at S129 is known to be associated with the pathological misfolding process, but efforts to investigate the relevance of this post-translational modification for pathology have been frustrated by difficulties in detecting and quantifying it in relevant samples. We report novel, ultrasensitive immunoassays based on single-molecule counting technology, useful for detecting alpha-synuclein and its S129 phosphorylated form in clinical samples in the low pg/ml range. Using human CSF and plasma samples, we find levels of alpha-synuclein comparable to those previously reported. However, while alpha-synuclein phosphorylated on S129 could easily be detected in human plasma, where its detection is extremely sensitive to protein phosphatases, its levels in CSF were undetectable, with a possible influence of a matrix effect. In plasma samples from a small test cohort comprising 5 PD individuals and five age-matched control individuals we find that pS129 alpha-synuclein levels are increased in PD plasma samples, in line with previous reports. We conclude that pS129 alpha-synuclein is not detectable in CSF and recommend the addition of phosphatase inhibitors to plasma samples at the time of collection. Moreover, the findings obtained on the small cohort of clinical plasma samples point to plasma pS129 alpha-synuclein levels as a candidate diagnostic biomarker in PD.

Highlights

Alpha-synuclein (SNCA) is a protein implicated in the pathogenesis of synucleopathies, of which Parkinson’s disease (PD) is the most prominent example
With an interest to provide an independent measurement of phosphorylation at S129 (pS129) SNCA in human cerebrospinal fluid (CSF), we applied a quantitative fluorescent sandwich immunoassay coupled to single-molecule counting technology to develop and validate ultrasensitive novel immunoassays for the detection of SNCA and its post-translationally modified variant phosphorylated at S129
In the course of these experiments, we observed a significant sensitivity of plasma pS129 SNCA levels to inhibition of endogenous phosphatase activity, with pS129 SNCA levels which were at least 10-fold higher in plasma samples supplemented with a phosphatase inhibitor cocktail immediately after collection

Summary

Introduction

Alpha-synuclein (SNCA) is a protein implicated in the pathogenesis of synucleopathies, of which Parkinson’s disease (PD) is the most prominent example. Only two immunoassays detecting the native pS129 SNCA protein, based on the Luminex technology (Wang et al, 2012) and on ELISA (Majbour et al, 2016) have been developed and employed to detect and quantify pS129 SNCA in human CSF. These assays have successfully detected pS129 SNCA in human CSF and established its levels from ca. Validation of pS129 SNCA detection in CSF in independent laboratories is still required (Parnetti et al, 2019)

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Conclusion