Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson's disease

Abstract

Extracellular α-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum α-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived α-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled α-synuclein into mouse brain, that CSF α-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of α-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF α-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal α-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF α-synuclein, a significant correlation between plasma exosomal α-synuclein and disease severity (r = 0.176, p = 0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal α-synuclein were comparable to those determined by CSF α-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS α-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically.

Fig. 1. Transportation of α-synuclein from the brain to blood

Mice were intracerebroventricularly injected with ¹²⁵I-labeled α-synuclein. (a) Brain and blood plasma were then collected at 2, 5, 10, 20 and 60 min after injection. Levels of radioactivity in the brain and the plasma were determined using a gamma counter. (b) Blood was collected at 60 min after injection, followed by L1CAM-containing exosome extraction from platelet-free plasma. Levels of radioactivity were measured in the whole plasma, the exosome fraction (Exo), and the exosome-less fraction (supernatant after immunoaffinity capture; Super). Data shown are mean ± S.D. from 5 mice. CPM, counts per minute.

Fig. 2. Characterization of immunoaffinity-captured exosomes from human blood plasma

(a) Electron micrograph of anti-L1CAM-captured plasma exosomes (inset: immunogold labeling of L1CAM). (b) Western blot showing that Alix, a common exosome marker, and L1CAM were enriched with anti-L1cam capture, but not with normal IgG capture. (c) α-Synuclein (syn) levels in anti-L1CAM-captured plasma exosomes were measured using a Luminex immunoassay, compared to the levels in normal mouse IgG-captured (mIgG) or “Empty” (no bead “capture”) samples. (d) Specificity was also confirmed by using exosome-poor plasma (supernatant after ultracentrifugation). Aliquots from the same pooled samples were used in these experiments (b-d) for comparison.

Fig. 3. Evaluation of α-synuclein concentrations in clinical samples

α-Synuclein (α-syn) concentrations were measured using Luminex and comparisons were performed for α-syn in L1CAM-containing exosomes isolated from plasma (a), total α-syn in plasma (b), or the plasma exosomal α-syn/total α-syn ratio (c) in patients with Parkinson disease (PD; n=267) and healthy controls (Con; n=215). CSF total α-syn concentrations were also evaluated in a subset of the subjects (100 PD and 100 Con) with CSF samples available (d). Data shown are mean ± S.D. **, p<0.001.

Fig. 4. ROC analysis of biomarker candidates for PD diagnosis and correlation with disease severity

(a) In the whole cohort (267 patients with PD and 215 healthy controls), the plasma exosomal α-syn provided an AUC of 0.654 (sensitivity=70.1%, specificity=52.9%) for PD versus controls. (b) The exosomal α-syn/total α-syn ratio in plasma performed similarly (AUC=0.657, sensitivity=71.2%, specificity=50.0%) in the whole cohort. (c) A significant correlation between the plasma exosomal α-syn and the disease severity indexed by the UPDRS motor score was observed in PD patients (r=0.176, p=0.004, Pearson correlation). (d) In a subset of subjects with CSF samples available (100 PD and 100 controls), the CSF total α-syn could produce an AUC of 0.724 (sensitivity=76.8%, specificity=53.5%). No correlation between CSF total α-syn and the UPDRS motor scores could be found (not shown). AUC, area under curve; CSF, cerebrospinal fluid; exo, exosomal; MDS-UPDRS, the Movement Disorder Society (MDS) Unified Parkinson’s Disease Rating Scale; PD, Parkinson disease; ROC, receiver operating characteristic; α-syn, α-synuclein.