 determined using a scintillation counter. After incubation, slides were dipped in ice-cold tris-buffer for 5 seconds. Slices were then washed again 2x 15 minutes in ice-cold tris-buffer and dipped in ice-cold dH2O for 20 seconds. Sections were left to dry under a fume hood overnight. After drying, sections were exposed for 5 weeks to a BAS IP-TR2040 Fuji Imaging Plate (Science Imaging, Sweden) together with [3H] specific microscales at 4°C. Imaging plates were scanned on the BAS-2500 Phosphor Image Scanner: FLA-9000, Starion. ImageJ was used for densitometric analysis of signal intensity. Concentrations were expressed as nCi/mg. Quantification was performed blindly in separate regions of interest (ROIs) according to Paxinos and Franklin’s atlas of the mouse brain [14].
RNA was extracted from fresh frontal cortex sections that were scraped off from parallel slides to the ones allocated for the receptor autoradiography analysis. For each animal, a total of 12 coronal frontal sections of 15 μm thickness were pooled together. Total RNA was purified using the PureLink RNA Mini Kit (Ambion) following the manufacturer’s instructions. All RNA samples were treated once with DNase using TURBO DNA-free Kit (Ambion) according to the manufacturer’s instructions. RNA samples were suspended in RNase-free water and were quantified using Agilent 2100 Bioanalyzer (Agilent Technologies). Only samples with RIN ≥ 6 were included in the analysis. RNA samples were stored at minus 80°C until further use.
A two-step real-time PCR was subsequently performed: RNA samples matched on concentration were reverse transcribed into cDNA with qScript cDNA SuperMix Kit (Quanta BioSciences) according to manufacturer’s instructions. SuperMix reaction mixture consisted of 5x reaction buffer containing optimized concentrations of MgCl2, dNTPs (dATP, dCTP, dGTP, and dTTP), recombinant RNase inhibitor protein, qScript reverse transcriptase, random primers, oligo(dT) primers, and stabilizers. cDNA synthesis step was performed following incubation times of 5 minutes at 25°C, 30 minutes at 42°C, 5 minutes at 85°C, and 5 minutes at 25°C. Thereafter cDNA product was diluted 5-fold in nuclease-free water and kept at minus 20°C until further use.
To check RNA samples for contamination with nuclear DNA a negative control PCR was done on all samples prior to cDNA synthesis omitting the reverse transcription step.
Real-time PCR reactions were performed using PerfeCTa SYBR Green FastMix (2x) Kit (Quanta BioSciences) with forward and reverse primers specific for the receptor (mouse), Rpl13a (mouse reference gene), and GAPDH (mouse reference gene) (TAG Copenhagen). Primer sequences were as follows: : F = 5′-GCA GTC CAT CAG CAA TGA GC-3′ and R = 5′-GCA GTG GCT TTC TGT TCT CC-3′; Rpl13a: F = 5′-GGA GGG GCA GGT TCT GGT AT-3′ and R = 5′-TGT TGA TGC CTT CAC AGC GT-3′; GAPDH: F = 5′-CAT CAA GAA GGT GGT GAA GCA-3′ and R = 5′-CTG TTG AAG TCA CAG GAG ACA-3′. qPCR was performed with 30 seconds for initial activation at 95°C, then 40 cycles of 5 seconds at 95°C for denaturation, 15 seconds at 60°C for annealing (END read), and 10 seconds at 72°C for elongation. To verify product a melting curve analysis between 55 and 95°C was performed after each run. Relative quantification (target/reference) was made using the ΔCt method [15].
All statistical analyses were performed in GraphPad Prism 6. Significance level was set at a value ≤ 0.05. All values are presented as mean ± standard error of the mean (SEM). Student’s -test was used for comparing mRNA levels for the receptors analyzed, AS protein levels, and and values between the PD and control brain samples. Multiple -tests were used for comparing receptor binding levels in h-SNCA tg and wt mice for the different ROIs.
The binding assay, performed on human brain tissue from the frontal cortex, showed a significant difference in of the receptor between PD () and control () brain samples, with higher maximum binding level in the PD group (PD: ; controls: ; Student’s test ) (Figure 1(a)). There was no significant difference between the PD and control group when comparing (affinity) (PD: ; controls ; ). Mean PMI (postmortem interval) was different between the two groups (PD and controls). We controlled for a potential effect of the PMI on binding results, and no correlation between and PMI (, ) or between and PMI (, ) was found.
Receptor binding analysis and alpha-synuclein levels in postmortem prefrontal cortex of Parkinson’s disease (PD) patients. (a) Binding assays with [3H] MDL100.907 comparing membrane suspensions from postmortem prefrontal cortex of Parkinson’s disease (PD) patients and controls show higher maximum binding levels () of the receptor in the PD group. (b) Western blot showing higher levels of alpha-synuclein proteins in the PD brains included in the binding assay. Control = normal controls; PD = Parkinson’s disease patients. Significant. Student’s -test with mean ± SEM.
Detergent soluble monomeric AS protein levels, as measured by electrophoresis and immunoblotting, were increased in the PD brains compared to controls (PD: ; controls: ; Student’s test ) (Figure 1(b)). No correlation was observed between AS protein and receptor binding levels in the PD brains (Spearman ; ).
The regions of interest (ROIs) included FrA (frontal association cortex), PrL + Cg1/2 (prelimbic cortex and cingulate cortex), MO (medial orbital cortex), DLO (dorsolateral orbital cortex), M1 and M2 (primary and secondary motor cortex), and AI (agranular insular cortex). Results from the [3H]-MDL 100.907 autoradiography binding analysis showed significantly lower receptor binding in FrA of h-SNCA mice (h-SNCA: fmol/mg; wt fmol/mg; ). There were no significant differences in receptor binding in the other ROI measured (Figure 2(a)).
receptor mouse prefrontal cortex autoradiography binding analysis and gene expression levels. (a) Results from [3H] MDL 100.907 () binding. FrA [3H] MDL 100.907 binding is significantly lower in alpha-synuclein overexpressing (SNCA) mice compared to wt mice. Bars: grey: wild-type mice (); black: SNCA mice ().. Multiple -test. Data illustrated with mean ± SEM. (b) Results from RT-qPCR show CT ratios (calculated using CT-ratio (receptor/reference) = ) comparing alpha-synuclein overexpressing (SNCA) and wt mice. Reference genes are Rpl13a and GAPDH. No difference in receptor gene expression was found. Bars show mean ± SEM.
Results from RT-qPCR show no difference in (h-SNCA: ; wt ; ) receptor gene expression in the frontal cortex of h-SNCA mice when compared to wt mice (Figure 2(b)).
In this study we looked at alterations in receptor binding levels in the frontal cortex from PD patients, and using a tg h-SNCA mouse model, we furthermore looked for whether AS overexpression could be associated with changes in receptor binding and gene expression. In vitro receptor binding studies on postmortem brain tissue from PD patients with increased AS levels indeed revealed significantly higher receptor binding levels, as indicated by higher maximum binding () in the PD brains. Results obtained using the well validated human AS overexpressing mouse model [12, 13] showed lower receptor binding levels in FrA cortex in the h-SNCA transgenic mice. Differences in receptor binding levels were not accompanied by decreased gene expression.
To our knowledge this is one of the first studies directly looking at receptor binding levels in postmortem tissue samples from the frontal cortex of PD brains in relation to Parkinson’s disease associated pathology of AS overexpression. Previously, at least two studies had addressed the question of receptor alterations in the frontal cortex of PD brains. A study from 1998 examined postsynaptic receptors in the neocortex of eight PD patients [16]. It too found an increase in receptor binding in PFC of postmortem PD tissue, using ligands other than those in this study (8-OH-DPAT and ketanserin). More recently, a Single-Positron-Emission-Tomography (SPECT) study, using the [123I]-5-I-R91150 ligand, reported lower receptor binding in the anterior striatum and premotor cortex and increased receptor binding in the occipital cortex of untreated PD patients [17]. However, no significant differences in receptor binding were detected in the prefrontal and parietal cortices. This discrepancy with our findings could be explained by the fact that patients included in the latter study were in early stages of the disease and therefore not directly comparable to our patient group consisting of end stage PD patients. According to Braak staging of PD, the raphe nuclei, the origin of the serotonergic projection to the substantia nigra, striatum, globus pallidus, subthalamic nucleus, thalamus, and neocortex with connections to cortical regions including frontal cortex and PFC areas—core structures of the cortico-basal ganglia-thalamo-