Introduction

α-Synuclein (α-syn) is a pre-synaptic neuronal protein that has been linked to a number of neurodegenerative disorders named as “synucleinopathies”1. However, the role of α-syn in the pathogenesis of Alzheimer’s disease (AD) has been increasingly recognized since Uéda et al. reported the presence of a non-Aβ component in the extracellular plaques found in the brains of AD patients, which was shown to be a fragment of α-syn2. In fact, the role of α-syn became of particular interest when the co-existence of α-syn and tau pathology was observed in the brains of patients with AD, Parkinson’s disease (PD) and dementia with Lewy bodies (DLB)27. The study was approved by the local Ethical Committee (Comitato Etico delle Aziende Sanitarie della Regione Umbria - CEAS Umbria) and informed written consent was signed by all patients enrolled or by their legal representatives. The work was carried out according to the Declaration of Helsinki.

Immunoassays to quantify α-syn species in the CSF

CSF t-, o- and p-S129-α-syn levels were measured using our recently published ELISA assays28. Briefly, for measuring t-α-syn, a 384-well ELISA microplate was coated by overnight incubation at 4 °C with 0.1 μg/ml Syn-140 (sheep anti-α-syn polyclonal antibody) in 200 mM NaHCO3, pH 9.6 (50 μl/well). Similarly, Syn-140 was used for measuring p-S129-α-syn, while the conformation-specific monoclonal antibody (Syn-O2)29, which is specific for α-syn oligomers (0.2 μg/ml), was used as the primary antibody to capture o-α-syn. The plate was then washed with phosphate-buffered saline containing 0.05% Tween-20 (PBST) and incubated with 100 μl/well of blocking buffer (PBST containing 2.5% gelatin) for 2 hours at 37 °C. After washing, 50 μl of the CSF samples (thawed on ice and Tween-20 added to a final concentration of 0.05%) were added to each well, and the plate was incubated at 37 °C for another 2.5 hours. Antibodies included 11D12 (mouse anti-α-syn monoclonal antibody) for measuring t-α-syn, PS129 (mouse anti-pS129-α-syn monoclonal antibody) for measuring pS129-α-syn and FL-140 (rabbit polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for measuring o-α-syn were diluted to the desired concentration (1:5000, 1:1,000 and 1:1,000, respectively) in the blocking buffer before being added to the corresponding wells and incubated at 37 °C for 2 hours. Next, the plate was washed and then incubated for 2 hours at 37 °C with 50 μl/well of species-appropriate secondary antibodies: donkey anti-mouse IgG HRP or goat anti-rabbit IgG HRP (Jackson ImmunoResearch, US), diluted in blocking buffer (1:20,000). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto, Pierce Biotechnology, Rockford, IL). Then, the chemiluminescence (relative light units) was immediately measured using a VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assays was carried out using 50 μl/well of serial dilutions of recombinant human α-syn, p-S129-α-syn or o-α-syn in artificial CSF. The samples were screened in a blinded fashion and tested randomly. All the results were confirmed with at least two independent experiments. A series of internal controls was also run to check for run-to-run variations.

Measurement of AD biomarkers

CSF Aβ42, total tau, and p-tau were measured using an ELISA technique (INNOTEST ß amyloid 1–42, hTAU-Ag, p-TAU 181 Ag, Fujirebio Europe, Gent, Belgium) as previously described30.

Data analysis and statistics

Statistical analysis was performed using R software v. 2.15. Continuous variables were described by the median and interquartile range because data distributions were skewed. Correlations were calculated using Spearman’s Rho (rs). The Mann-Whitney test was used for initial comparisons between the two diagnostic groups (p < 0.05). The accuracy of the diagnostic value of the biomarkers31 was assessed by calculating the area under the curve (AUC) of the receiver operating characteristic (ROC) curve32. Cut-off values were calculated using sensitivity and specificity values that maximized Youden’s index. Because there was a significant difference in age (p < 0.0001) and in the distribution of gender (p < 0.01) between the groups, we corrected for these using a logistic regression approach to adjust for covariates. All CSF samples with an erythrocyte count >500 cells/μl were excluded from further analysis, as traces of blood may influence CSF α-syn levels33,34.

Additional Information

How to cite this article: Majbour, N. K. et al. Increased levels of CSF total but not oligomeric or phosphorylated forms of alpha-synuclein in patients diagnosed with probable Alzheimer’s disease. Sci. Rep. 7, 40263; doi: 10.1038/srep40263 (2017).

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