The pre-synaptic vesicle protein synaptotagmin is a novel biomarker for Alzheimer’s disease

The reproducibility of the analytical method was investigated in pooled left-over aliquots from decoded CSF samples supplied by the clinical routine section at the Clinical Neurochemistry Laboratory, The Sahlgrenska University Hospital, Mölndal, Sweden, following procedures approved by the Ethical Committee at University of Gothenburg. The quality control CSF pool 1 (QC1 sample) had an amyloid-β_1–42 level of 586 ng/L and a total tau level below 75 ng/L. The QC2 sample had an amyloid-β_1–42 level of 129 ng/L, a total tau level below 75 ng/L, and a phosphorylated tau level below 15 ng/L.

CSF samples from subjects with either dementia due to Alzheimer’s disease or MCI due to Alzheimer’s disease or from a neurological control group were obtained from the Research Memory Center at Lariboisière Fernand-Widal University Hospital APHP. This department is highly experienced in the care management of patients with cognitive disorders and neurodegenerative disease, and has used CSF biomarkers for a long period of time [25‐27]. Patients underwent a comprehensive clinical examination including personal medical and family histories, neurological examination, neuropsychological assessment, lumbar puncture with CSF biomarker analysis, and a brain structural imaging study with MRI. A consensus diagnosis was made by several clinicians (neurologists, geriatricians), neuropsychologists, and a biologist who are experts in CSF biomarkers. For each patient, diagnosis was made considering CSF results and according to validated clinical diagnostic criteria for dementia due to Alzheimer’s disease [28], MCI due to Alzheimer’s disease [29, 30], subjective cognitive impairment [31], and psychiatric disorder (DSM-IV). For all patients, diagnoses were validated in a second step by a neurologist (CP) and a biochemist (EB-A) before selecting CSF samples. In the absence of consensus diagnosis and in cases of disagreement about the final diagnosis, patients were not included in the study. According to this method, CSF from subjects with dementia due to Alzheimer’s disease, subjects with MCI due to Alzheimer’s disease, and neurological controls (no neurodegenerative disorders) was selected. Two sample sets were explored. Recently, the Alzheimer’s disease core CSF biomarkers have been included in the research criteria for the diagnosis of both early and manifest AD by the International Working Group [29] and in the diagnostic guidelines from the National Institute on Aging–Alzheimer’s Association [28], respectively. The following cutoff values were used to define a biochemical Alzheimer’s disease signature as supportive criteria for dementia due to Alzheimer’s disease [28]: amyloid-β_1–42 (<550 ng/L), total tau (>400 ng/L), and phosphorylated tau (>50 ng/L). These criteria was also applied to exclude other diseases and to specify the controls. Sample set I included patients who underwent a lumbar puncture between 2010 and 2012, while sample set II included patients from 2013 to 2015 (Table 1).

CSF was obtained by lumbar puncture between the L3/L4 or L4/L5 intervertebral space, using an atraumatic 24-gauge needle, collected in 10-mL polypropylene tubes. Samples were centrifuged at 1800 × g for 10 minutes at +4 °C, were aliquoted in 500 μL polypropylene tubes, and were stored at –80 °C pending analysis. Samples were frozen at −80 °C within 1 h after collection according to a standardized protocol described in a previous report [26]. A small amount of CSF was used for routine analysis, including total cell count, bacteriologic examination, and total protein and glucose levels.

Amyloid-β_1–42, total tau, and tau phosphorylated at threonine 181 (phosphorylated tau) protein measurements were performed using commercially available assays from Fujirebio (INNOTEST® β-AMYLOID(1-42), INNOTEST® hTAU Ag, and INNOTEST® PHOSPHO-TAU(181P)) according to the manufacturer’s instructions. For the two sample sets, the analysis of these biomarkers was performed in a single hospital laboratory (Lariboisère Hospital Paris) in two runs and averaged results were used for statistical analyses. The quality of CSF evaluations was validated by the Alzheimer’s Association quality control program for CSF biomarkers [32].

The monoclonal antibody clone 41.1 recognizing the cytoplasmic portion of synaptotagmin-1 was purchased from Synaptic Systems (Göttingen, Germany). Recombinant protein of synaptotagmin-1 (catalog number TP327252), transcript variant 3, was from OriGene Technologies, Inc. (Rockville, MD, USA).

The immunoprecipitation method for CSF samples was performed according to Brinkmalm et al. [13] with minor modifications. Briefly, an aliquot (1 μg) of the mouse monoclonal synaptotagmin-1 antibody clone 41.1 (1 g/L) or IgG from murine serum (1 g/L, a negative control; Sigma-Aldrich) was separately added to 50 μL magnetic Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen Corporation) and incubated for 1 h on a rocking platform at room temperature. The beads were washed three times with 1 ml of phosphate-buffered saline (PBS). The antibodies were cross-linked using 20 mM dimethyl pimelimidate dihydrochloride (Sigma-Aldrich) and 0.2 M triethanolamine (pH 8.2; Sigma-Aldrich) according to the manufacturer’s product description. The cross-linked beads were washed twice in PBS and were blocked with Roti-Block (Carl Roth) for 1 h on a rocking platform at room temperature. CSF samples (250 μL) were adjusted with 5 % Tween 20 and PBS to a final concentration of 0.05 % Tween 20 and a final volume of 1 mL. Samples and magnetic beads were incubated overnight on a rocking platform at +4 °C. The magnetic beads/sample solution was transferred to the KingFisher magnetic particle processor (Thermo Fisher Scientific), tube 1. The following three wash steps (tubes 2–4) were conducted for 10 s in 1 mL of each washing buffer: (tube 2) 0.025 % Tween 20 in PBS, (tube 3) PBS, and (tube 4) 50 mM ammonium hydrogen carbonate (NH₄HCO₃, pH 8.0). Synaptotagmin-1 was then eluted from the beads by adding 100 μL of 0.5 % formic acid (tube 5) for 4 min. The eluted fractions were transferred to 0.65 mL prelubricated Costar Microcentrifuge Tube (Fisher Scientific) and dried in a vacuum centrifuge.

Two isotopically labeled custom-made peptides containing U-13C6, U-15 N2-lysine[K] (aa 215–223, VPYSELGG[K] and aa 238–245, HDIIGEFK HeavyPeptide FasTrack 1 standards; Thermo Fisher Scientific) were dissolved in MilliQ water (~500 pmol/μL), mixed, and diluted in 50 mM NH₄HCO₃ to a final concentration of ~10 fmol/μL. The dried immunoprecipitated CSF samples were dissolved in 25 μL of a mixture of trypsin and labeled peptides (1 μg Sequencing Grade Modified Trypsin (Promega) dissolved in 0.01 % aqueous HCl (0.1 g/L) and diluted to 5 mg/L in isotopically labeled peptide mixture in 50 mM NH₄HCO₃, pH ≈ 7.8 (see earlier)), vortexed carefully, and incubated at +37 °C overnight. To stop the enzymatic activity, 5 μL of 10 % aqueous FA was added. The samples were centrifuged (16,900 × g, 10 min, +4 °C) and 27 μL of each sample was transferred to LC-vials (SUN-SRi).

The intra-day variation of synaptotagmin was determined using two QC samples (QC1 and QC2). The immunoprecipitation of synaptotagmin-1 from the QC samples were performed on different days but analyzed on the same occasion. Assessment of reproducibility was performed on two different occasions, and QC samples were randomized in between samples from sample set I (first occasion) and sample set II (second occasion).

Because most of the analytes were not normally distributed (Shapiro–Wilk test, P < 0.05), nonparametric statistics were used for analysis. Data are given as the median (interquartile range). Differences between more than two groups were assessed with Kruskal–Wallis test. Statistically significant results (P < 0.05) were followed by Mann–Whitney U tests to investigate group differences. Receiver operating characteristic (ROC) curves were performed on each subject group on the tryptic peptides of synaptotagmin-1 in order to assess their diagnostic value. For each tryptic peptide of synaptotagmin, the area under the curve and a 95 % confidence interval was calculated using GraphPad Prism 5. The correlation coefficients (rho) were calculated using the Spearman two-tailed correlation test. SPSS 20.0 was employed for most of the statistical analyses.