Abstract
High levels of total α-synuclein (t-α-synuclein) in the cerebrospinal fluid (CSF) were reported in sporadic Creutzfeldt–Jakob disease (sCJD). The potential use of t-α-synuclein in the discrimination of Lewy body dementias (i.e., Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB)) is still under investigation. In addition, phospho-serine-129 α-synuclein (p-α-synuclein) has been described to be slightly increased in the CSF of synucleinopathies. Here, we analyzed t-α-synuclein and p-α-synuclein concentrations and their ratio in the context of differential diagnosis of neurodegenerative diseases. We quantified the levels of CSF t-α-synuclein and p-α-synuclein in a cohort of samples composed of neurological controls (NC), sCJD, PDD, and DLB by means of newly developed specific enzyme-linked immunosorbent assays. T-α-synuclein and p-α-synuclein were specifically elevated in sCJD compared to other disease groups. The area under the curve (AUC) values for t-α-synuclein were higher for the discrimination of sCJD from dementias associated to Lewy bodies as compared to the use of p-α-synuclein. A combination of both markers even increased the diagnostic accuracy. An inverse correlation was observed in CSF between t-α-synuclein and p-α-synuclein, especially in the DLB group, indicating a disease-relevant association between both markers. In conclusion, our data confirm t-α-synuclein and p-α-synuclein as robust biomarkers for sCJD and indicate the potential use of colorimetric t-α-synuclein ELISAs for differential diagnosis of dementia types.
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Abbreviations
- PD:
-
Parkinson’s disease
- ROC:
-
receiver operating characteristic
- AUC:
-
area under the curve
- sCJD:
-
sporadic Creutzfeldt–Jakob disease
- CSF:
-
cerebrospinal fluid
- DLB:
-
dementia with LBs
- ELISA:
-
enzyme-linked immunosorbent assay
- LBs:
-
Lewy bodies
- NC:
-
neurological controls
- p-α-synuclein:
-
phospho-serine-129 α-synuclein
- PDD:
-
Parkinson’s disease dementia
- t-α-synuclein:
-
total α-synuclein
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Acknowledgements
We thank Silja Köchy for indispensable technical assistance.
Funding
AV-P was funded by Georg-August University of Göttingen (Dorothea Schlözer Programme). FL was funded by the Spanish Ministry of Health–Instituto Carlos III (Miguel Servet–CP16/00041). The assay development work has been supported by the Michael J. Fox Foundation (Grant No. 10147). JQT and VM-YL were supported by NIA Grant AG-10124.
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Authors and Affiliations
Contributions
Matthias Schmitz: designed the study, analyzed data, and wrote the manuscript
Anna Villar-Piqué: performed experiments, analyzed data, and wrote the manuscript
Franc Llorens: performed experiments, analyzed data, and critically revised the manuscript
Karin Gmitterová, Daniela Varges, and Peter Hermann: provided samples
Saima Zafar: critically revised the manuscript
Paul Lingor: critically revised the manuscript
Leentje Demeyer: development and qualification of the assay
Erik Stoops: development and qualification of the assay, delivery of materials for the study, and critically reviewed the manuscript
Hugo Vanderstichele: data interpretation and critically reviewed of the manuscript
John Trojanowski: contributed antibody reagents and critically reviewed the manuscript
Virginia M-Y Lee: contributed antibody reagents and critically reviewed the manuscript
Inga Zerr: analyzed and interpreted data and wrote the manuscript
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The present study was conducted according to the revised Declaration of Helsinki and Good Clinical Practice guidelines and has been approved by the local ethics committee in the University Medical Center, Göttingen (No. 9/6/08, 19/11/09, and 18/8/15). Informed consent was given by all study participants or their legal next of kin.
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Not applicable.
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The full databank of the data is available to each co-author on the paper.
Competing Interest
Leentje Demeyer and Erik Stoops are employees of ADx NeuroSciences. Hugo Vanderstichele is a co-founder of ADx NeuroSciences and a founder of Biomarkable. All other authors declare there are no competing interests.
Electronic supplementary material
Suppl. Figure 1
Specificity of the t-α-synuclein ELISA towards synuclein isoforms. α-, β-, and γ-synuclein were used as sample in the α-synuclein ELISA. Serial dilutions of each protein were prepared in assay diluent. β- (Code: S-1003) and γ-synuclein (Code: S-1007) were purchased from rPeptide (Watkinsville, GA, US). The quality for use in the immunoassays of the β- and γ-synuclein protein was confirmed using an ELISA with FL-140 antibody (Santa Cruz) (data not shown). (PNG 506 kb)
Suppl. Figure 2
Characterization of the ADx301 mAb. a) List of synthetic peptides from ADx that were used for the characterization of the mAbs. The biotinylation of the peptides is done at the amino-terminus. Evaluation of the phospho-dependency of mAb ADx301 and polyclonal FL-140 antibody using biotinylated peptides and proteins. b) Test procedure: The experiments are done at room temperature. SV-coated microplates were incubated with biotinylated full-length synuclein or synthetic peptides, with or without phosphorylation, at a concentration of 100 ng/mL. After 5 wash steps, several concentrations of ADx301 and FL-140 antibody (Santa Cruz) were incubated for 1 h. Excess of antibody was removed by an extra wash step. Detection was done using peroxidase-labeled goat anti rabbit antibody (incubation time: 30 min). c) Data of the previous experiment were recalculated. The relative difference in OD values was calculated against values obtained for the non-phosphorylated peptide Pt143. When using secondary modified peptides with an identical sequence, the impact of the modification on the binding capacity of the antibody can then be visualized. Results for antibody FL140 are considered as the control situation (median relative difference was in the range of 100%). For mAb ADx301, it was shown that pY125 showed a decrease in relative difference. So, phosphorylation at a site close to the epitope of ADx301 might have an effect on the binding of the antibody, although phosphorylation is not part of the epitope of the antibody. (PNG 867 kb)
Suppl. Figure 3
Evaluation of parallelism in the t- α -synuclein ELISA. A dilution series was prepared with assay diluent with 5 neat individual (not pooled) CSF samples (Dilution factor: 1–10). Concentrations were recalculated taken into account the applied dilution factor (= concentration x dilution factor). The %CV on all concentrations is calculated as a measure of the parallelism performance, as described before [33]. (PNG 574 kb)
Suppl. Figure 4
ROC curve analysis for the differentiation of sCJD using CSF t- α -synuclein, p- α -synuclein, or their ratio. (PNG 129 kb)
Suppl. Figure 5
Influence of age and gender on the t- α -synuclein and p- α -synuclein levels in sCJD patients and controls. a, b) The sCJD cohort was divided in patients younger than 60 years (n = 13) and older than 60 years (n = 29), as well as in function of gender (males (n = 21), females (n = 21)). Neither age nor gender had a significant effect on the CSF concentrations of t- α -synuclein and p- α -synuclein. c, d) The cohort was divided in patients younger than 60 years (n = 14) and older than 60 years (n = 28), as well as in function of gender (males (n = 20), females (n = 22)). Neither age nor gender had a significant effect on the CSF concentrations of t- α -synuclein and p- α -synuclein. (PNG 237 kb)
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Schmitz, M., Villar-Piqué, A., Llorens, F. et al. Cerebrospinal Fluid Total and Phosphorylated α-Synuclein in Patients with Creutzfeldt–Jakob Disease and Synucleinopathy. Mol Neurobiol 56, 3476–3483 (2019). https://doi.org/10.1007/s12035-018-1313-4
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DOI: https://doi.org/10.1007/s12035-018-1313-4