Oligomeric and phosphorylated alpha-synuclein as potential CSF biomarkers for Parkinson’s disease

Background Despite decades of intensive research, to date, there is no accepted diagnosis for Parkinson’s disease (PD) based on biochemical analysis of blood or CSF. However, neurodegeneration in the brains of PD patients begins several years before the manifestation of the clinical symptoms, pointing to serious flaw/limitations in this approach. Results To explore the potential use of alpha-synuclein (α-syn) species as candidate biomarkers for PD, we generated specific antibodies directed against wide array of α-syn species, namely total-, oligomeric- and phosphorylated-Ser129-α-syn (t-, o- and p-S129-α-syn). Next we sought to employ our antibodies to develop highly specific ELISA assays to quantify α-syn species in biological samples. Finally we verified the usefulness of our assays in CSF samples from 46 PD patients and 48 age-matched healthy controls. We also assessed the discriminating power of combining multiple CSF α-syn species with classical Alzheimer’s disease biomarkers. The combination of CSF o-/t-α-syn, p-S129-α-syn and p-tau provided the best fitting predictive model for discriminating PD patients from controls. Moreover, CSF o-α-syn levels correlated significantly with the severity of PD motor symptoms (r = -0.37). Conclusion Our new ELISA assays can serve as research tools to address the unmet need for reliable CSF biomarkers for PD and related disorders. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0072-9) contains supplementary material, which is available to authorized users.

neuropathological and clinical information for research purposes was given by either the donor or the next of kin. Extensive neuropathological assessment of disease-relevant brain regions was provided by established and qualified neuropathologists (JMR and WK). The density and distribution of LBs and LNs pathology was determined based on immunohistochemical analysis of α-syn (Clone KM51, Novastra, Bioconnect BV; dilution 1:500, pretreatment in citrate buffer pH 6.0 in the microwave and 80% formic acid) and Braak staging protocol [1]. Furthermore, the density of neurofibrillary tangles (NFT) and Aβ were determined based on Bodian Silver staining and immunohistochemical analysis of hyperphosphorylated tau (Clone AT8, Pierce, Rockford, IL) and Aβ (Clone 6F/3D, DAKO, DakoCytomation BV) throughout the brain using the criteria provided by Braak [2] and Brain Net Europe [3,4].
Substantia nigra and hippocampus tissue of 12 clinically diagnosed and pathologically confirmed idiopathic PD or PD with dementia (PDD) patients (Braak PD stage 4-6) and 10 aged-matched controls were included in this study. Only idiopathic PD cases were included with an age of onset > 45 years and no history of other disorders of the central nervous system. The control group consisted of healthy subjects without neurological or psychiatric disease and without LB pathology (HC; Braak PD stage 0). All donors were carefully selected based on their pathological assessment and clinical records. Furthermore, all subjects were controlled and matched for postmortem delay and cerebrospinal fluid pH value.

Tissue processing and immunohistochemistry
Brain tissue collected at autopsy was immersion-fixed in 4% formaldehyde for four weeks and subsequently disease-relevant areas were dissected and embedded in paraffin. Tissue samples were cut in 10 μm thick sections with a microtome, mounted on positively-charged glass slides (Menzel-Glaser SuperFrost Plus, Braunschweig, Germany) and dried in a stove overnight at 37°C. Antigen retrieval consisted of steaming in citrate buffer pH 6.0 and 5% Bovine albumin (BSA) in TBS -triton (TBS-Tx) served as blocking. Transverse sections of the mesencephalon at the level of the oculomotor nerve including the SN and temporal cortex including the hippocampus and parahippocampal gyrus sections were incubated with the primary antibody Syn-O2 using a dilution of 1:5K overnight at 4°C. EnVisiontm visualisation system (DAKO) was used to detect the primary antibody. EnVisiontm contains both the secondary antibodies for anti-mouse lgG specificity as well as a horseradish peroxidise (HRP)-conjugated polymer backbone. The dark brown chromogenic response was generated using 3, 3'-diaminobenzidine (DAB) peroxidise (Sigma-Aldrich) which reacts with the HRP enzyme label. Sections were counterstained using cresyl violet (Nissl) to visualize neurons and layers. A subset of sections was pretreated with proteinase K which is known to eliminate all non-accumulated, monomeric, soluble α-syn [5]. Adjacent sections were incubated with antibodies against α-syn (Clone KM-51, Novacastra, dilution 1:500; Syn-1, BD Biosciences, dilution 1:2K) for comparison of staining patterns.

Transgenic mice description
Approximately 7 kb of the mouse α-syn promoter was combined with human α-syn cDNA and the SV40 poly-adenylation sequence in a construct used to establish transgenic mouse lines by pronuclear microinjection into oocytes from C57Bl6xD2 hybrids. The subsequent transgenic lines were bred into C57Bl6 mice for at least nine generations. Non-transgenic mice from the same colony served as controls. Expression of the transgene was assessed by Northern blotting, in situ hybridization and Western blotting (Syn-1) using standard methods [6].

SNCA-null mice:
KO mice were created by replacing a 400 bp genomic fragment containing the translocation start with a neocassette. WT mice in which the fragment was not knocked out were also used as controls [6].

Brain lysates preparation
Frozen brain samples (1 g) from transgenic (Tg), wild-type (WT) and knock-out (KO) mice were homogenized in 5 ml of CelLytic buffer comprising mild detergent, bicine and 150 mMNaCl (Sigma-Aldrich) and containing a cocktail of protease inhibitors including AEBSF, aprotinin, E-64, EDTA and leupeptin (Pierce) and then centrifuged at 3000 g for 30 min. The supernatant was collected, and the total protein concentration was measured. All samples were adjusted to 1 mg/ml and then stored at -80°C. The samples were thawed on ice directly prior to analysis.

In vivo microdialysis in freely moving mice to sample extracellular α-syn
Transgenic mice overexpressing human α-syn with 50 weeks of age were used in this study. The mice were housed on a 12-hr light/dark cycle with conditions maintained at standard indoor temperature (21 ± 2°C) and humidity (55 ± 5%). Food and tap water were available ad libitum.
To enable microdialysis in the hippocampus, mice were anaesthetized with sevoflurane (Abbott laboratories, Gentofte, Denmark) and an intracerebral guide cannula (brainlink) was stereotaxically implanted into the brain, positioning the microdialysis probe in the hippocampus sampling regimen was used throughout the experiment period. To avoid tissue damage, the experimental window was set from 16h after probe implantation. The dialysates were stored at -