Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson’s disease

Brain tissue was obtained from the Institute of Neuropathology HUB-ICO-IDIBELL Biobank and the Hospital Clinic-IDIBAPS Biobank following the guidelines of the Spanish legislation on this matter and the approval of the local ethics committees. The post-mortem interval between death and tissue processing was between 3 and 20 h. Pathological cases were categorized as having PD pathology (Lewy body disease pathology) stages 1 to 6 according to the nomenclature of Braak et al. [11]. Only typical cases according to the Braak classification were included. Cases with concomitant tauopathies, excepting Alzheimer’s disease-related pathology stages I-II/0-B [12], vascular disease, and metabolic syndrome were excluded from the present study. Middle-aged (MA) cases had not suffered from neurologic, psychiatric, or metabolic diseases (including metabolic syndrome), and did not have abnormalities in the neuropathological examination excepting sporadic Alzheimer’s disease-related pathology stages I-II/0-B of Braak and Braak.

In total, 122 brains including 44 MA and 78 cases with PD-related pathology were included in the present study. Incidental PD (iPD or incidental Lewy Body Disease iLBD) occurred in 13 cases (mostly stages 1, 2, and 3 of Braak). Pre-parkinsonian symptoms in iPD cases were not recorded. Regarding PD cases, all of them had been treated for their motor symptoms. The disease duration ranged from 6 to 16 years. The most common causes of death in the MA and PD cases were infections, neoplasia, and acute cardiac disease.

Five regions were examined for mRNA expression: frontal cortex area 8, substantia nigra, angular gyrus, precuneus, and putamen; the selection of these areas was based on their differing vulnerability to PD and to their accumulative involvement with disease progression. Number of cases, mean ages, and standard deviation for each group are summarized in Table 1. A summary of individual cases and methods used for all cases examined is shown in Additional file 1: Table S1. Most cases here analysed were also the subject of other studies [13‐15].

Purification of RNA from the substantia nigra, right frontal cortex area 8, angular gyrus, precuneus, and putamen was carried out using RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) following the protocol provided by the manufacturer and performing the optional DNase I digest to avoid extraction and later amplification of genomic DNA. The concentration of each sample was obtained from A260 measurements with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and RNA integrity was tested using the Agilent 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA).

Retrotranscription reaction of RNA samples was carried out with the High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) following the guidelines provided by the manufacturer, and using a Gene Amp® 9700 PCR System thermocycler (Applied Biosystems). A parallel reaction for one RNA sample was processed in the absence of reverse transcriptase to rule out DNA contamination.

RT-qPCR was conducted in duplicate on cDNA samples obtained from the retrotranscription reaction using 1,000ηg of RNA, diluted 1:20 in 384-well optical plates (Kisker Biotech, Steinfurt, Germany) utilizing the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). Parallel amplification reactions were carried out using 20x TaqMan Gene Expression Assays and 2x TaqMan Universal PCR Master Mix (Applied Biosystems). TaqMan probes used in the study are shown in Additional file 2: Table S2. The reactions were performed using the following parameters: 50 °C for 2 min, 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. TaqMan PCR data were captured using the Sequence Detection Software (SDS version 2.2, Applied Biosystems). Subsequently, threshold cycle (CT) data for each sample were analysed with the double delta CT (ΔΔCT) method. First, delta CT (ΔCT) values were calculated as the normalized CT values for each target gene in relation to the endogenous controls β-glucuronidase (GUS-β) and X-prolylaminopeptidase (aminopeptidase P) 1 (XPNPEP1) for normalization [16, 17]. Second, ΔΔCT values were obtained with the ΔCT of each sample minus the mean ΔCT of the population of MA samples (calibrator samples). The fold-change was determined using the equation 2^-ΔΔCT. These housekeeping genes were selected because they show no modifications in several neurodegenerative diseases in human post-mortem brain tissue [16, 17]. Similar results were obtained using GUS-β and XPNPEP1 as correctors; GUS-β was selected for representation.

The normality of distribution of the mean fold-change values obtained by RT-qPCR for each region and stage between MA and PD cases were analysed with the Kolmogorov-Smirnov test. The non-parametric Mann–Whitney test was performed to compare each group when the samples did not follow a normal distribution, while the unpaired t test was used for normal variables. t test was used instead of one-way ANOVA when analyzing MA and PD cases in parallel in the same optical plate. Statistical analysis was performed with GraphPad Prism version 5.01 (La Jolla, CA, USA) and Statgraphics Statistical Analysis and Data Visualization Software version 5.1 (Warrenton, VA, USA). Differences between groups were considered statistically significant at P-values: *P < 0.05, **P < 0.01 and ***P < 0.001.

Samples of the substantia nigra including 14 MA and 14 PD cases of frontal cortex area 8 (0.1 g of tissue) were homogenized with a glass homogenizer in Mila lysis buffer (0.5 M Tris at pH 7.4 containing 0.5 methylenediaminetetracetic acid at pH 8.0, 5 M NaCl, 0.5 % Na doxicholic, 0.5 % Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, bi-distilled water) with protease and phosphatase inhibitor cocktails (Roche Molecular Systems, Pleasanton, CA, USA), and then centrifuged at 4 °C for 15 min at 13,000 rpm (ultracentrifuge Beckman with 70Ti rotor, CA, USA). Protein concentration was measured by Smartspect™plus spectrophotometer (Bio-Rad, CA, USA) using the Bradford method (Merck, Darmstadt, Germany). Samples containing 20 μg of protein and the standard Precision Plus Protein™ Dual Color (Bio-Rad) were loaded onto 10 % and 12 % acrylamide gels. Proteins were separated in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membranes using the Trans-Blot®Turbo™ transfer system (Bio-Rad) at 200 mA/membrane for 20 min. Non-specific bindings were blocked by incubation in 5 % albumin in Tris-buffered saline (TBS) containing 0.2 % Tween for 1 h at room temperature. After washing, the membranes were incubated at 4 °C overnight with several antibodies in TBS containing 5 % albumin and 0.2 % Tween. A list of the antibodies used is shown in Additional file 3: Table S3. Monoclonal antibody anti-β-actin diluted 1:30,000 (β-Actin, A5316; Sigma-Aldrich, St. Louis, MO, USA) was blotted for the control of protein loading. Afterwards, the membranes were incubated for 1 h with the appropriate HRP-conjugated secondary antibody (1:1,000, Dako, Glostrup, Denmark), and the immune complexes were visualized with a chemiluminescence reagent (ECL, Amersham, GE Healthcare, Buckinghamshire, UK). Densitometry of western blot bands was assessed with the TotalLab program (TotalLab Quant, Newcastle, UK) and subsequently analysed with GraphPad Prism by one-way ANOVA with post hoc Tukey’s student range test for multiple comparisons. We used one-way ANOVA instead of t test because each gel contained MA and different stages of PD cases. Differences were considered statistically significant with P-values: *P <0.05; **P <0.01; ***P <0.001.

Immunohistochemical study of selected nucleolar proteins was performed on 4 μm-thick dewaxed paraffin sections of the substantia nigra. PD cases were analysed including 2 stage 1 cases (1 male and 1 female), 3 stage 3 males, 2 stage 4 males, and 3 stage 5 males. Tissue sections were boiled in citrate buffer for 20 min to retrieve antigenicity. Endogenous peroxidases were blocked with peroxidase (Dako, Glostrup, Denmark) followed by 10 % normal goat serum. The primary antibodies were mouse monoclonal anti-nucleophosmin (NPM1) and rabbit polyclonal anti-nucleoplasmin-3 (NPM3). A few sections of the substantia nigra and frontal cortex area 8 were incubated with anti-α-synuclein oligomer-specific antibody (Agrisera, Vännäs, Sweden) at a dilution of 1:1,000. Following incubation with the primary antibody at room temperature overnight, the sections were incubated with EnVision + system peroxidase (Dako) at room temperature for 15 min. The peroxidise reaction was visualized with diaminobenzidine (DAB) and H₂O₂. The omission of the primary antibody in some sections was used as a control for the immunostaining; no signal was obtained with the incubation only of the secondary antibody. No immunogenic peptides were available for any antibody used. Sections were slightly counterstained with haematoxylin.

Double-labelling immunofluorescence was carried out on de-waxed sections, 4 μm-thick, which were stained with a saturated solution of Sudan black B (Merck, DE) for 15 min to block the autofluorescence of lipofuscin granules present in cell bodies, and then rinsed in 70 % ethanol and washed in distilled water. The sections were boiled in citrate buffer to enhance antigenicity and blocked for 30 min at room temperature with 10 % foetal bovine serum diluted in PBS. Then, the sections were incubated at 4 °C overnight with combinations of primary antibodies. After washing, the sections were incubated with Alexa488 or Alexa546 (1:400, Molecular Probes, US) fluorescence secondary antibodies against the corresponding host species. Nuclei were stained with DRAQ5™ (1:2,000, Biostatus, UK). After washing, the sections were mounted on Immuno-Fluore mounting medium (ICN Biomedicals, US), sealed, and dried overnight. Sections were examined with a Leica TCS-SL confocal microscope. Again, omission of the primary antibody in some sections was used as a control for the immunostaining.

Quantitative studies were carried out in the substantia nigra on serial non-consecutive sections stained with haematoxylin and eosin or processed for NPM1 and NPM3 immunohistochemistry. Nucleolar counts were performed directly under the ocular of the microscope at a magnification x200 in three areas (0.48 mm²) selected at random in every one of the eleven cases (2 PD1, 3 PD3, 3 PD4, 3 PD5). Results of NPM1 and NPM3-stained nucleoli per section were expressed as percentage of the total nucleoli visualized in haematoxylin and eosin-stained. Sections of PD at stage 1, stained with haematoxylin and eosin, were used to quantify neurons in which the nucleolus was visualized in that section.

To learn whether reduced NPM1 immunoreactivity in substantia nigra dopaminergic neurons was linked to α-synuclein inclusions, double-labelling immunofluorescence and confocal microscopy using anti-NPM1 and anti-α-synuclein antibodies was used to analyze six cases (1 tissue section per case) of PD at stages 4–5.

Brain samples (0.1 g) of substantia nigra pars compacta, frontal cortex area 8 and putamen from MA (n = 3 per group) and stage 5 PD cases (n = 3 per group) were homogenized in a glass homogenizer, in 750 μl of ice-cold PBS+ (sodium phosphate buffer pH 7.0, plus protease inhibitors), sonicated, and centrifuged at 2,700 g at 4 °C for 10 min. The pellet was discarded and the resulting supernatant was ultra-centrifuged at 133,000 g at 4 °C for one hour. The supernatant (S2) was kept as the PBS-soluble fraction. The resulting pellet was re-suspended in a solution of PBS, pH 7.0, containing 0.5 % sodium deoxycholate, 1 % Triton, and 0.1 % SDS, and this was ultra-centrifuged at 133,000 g at 4 °C for one hour. The resulting supernatant (S3) was kept as the deoxycholate-soluble fraction. The corresponding pellet was re-suspended in a solution of 2 % SDS in PBS and maintained at room temperature for 30 min. Afterwards, the samples were centrifuged at 133,000 g at 25 °C for one hour and the resulting supernatant (S4) was the SDS-soluble fraction. Equal amounts of each fraction were mixed with reducing sample buffer and processed in parallel for 10 % SDS-PAGE electrophoresis and western blotting. Membranes were incubated with anti-α-synuclein oligomer-specific antibody (Agrisera, Vännäs, Sweden) at a dilution of 1:1,000. The protein bands were visualized with the ECL method (Amersham).

Small pieces of frozen brain samples (0.2 g) of frontal cortex area 8 from MA (n = 2, cases 4 and 15 of the Additional file 1: Table S1) and 2 PD cases stages 5–6 (n = 2, cases 113 and 120 of the Additional file 1: Table S1) were homogenized in a 6 ml ice-cold Solution D buffer (0.25 M sucrose, 25 mM potassium chloride (KCl), 5 mM magnesium chloride (MgCl₂), and 20 mM Tris–HCl pH 7.5) with 0.1 % Triton, and then centrifuged at 1,000 g for 10 minutes at 4 °C (Ultracentrifuge Beckman with 70Ti rotor). The supernatant obtained was discarded and the pellet was re-suspended in 2 ml of Optiprep (D1556, Sigma, St Louis, MO, USA) to allow better separation by density gradient, and centrifuged at 3,200 g for 20 minutes at 4 °C. The new supernatant obtained was discarded again whereas the pellet was re-suspended in 500 μl ice-cold PBS buffer. Isolated nuclei were stained with mouse antibody to NeuN (see Additional file 3: Table S3). Primary antibody was visualized with appropriate secondary antibodies conjugated with Alexa 488. DNA content was determined using DAPI (4',6-diamidino-2-phenylindole). Subsequently, samples were centrifuged at 1,000 g for 10 min at 4 °C, and the solution obtained was re-suspended in 800 μl ice-cold PBS buffer.

Flow cytometry sorting was performed with a Beckman Coulter MoFlo Astrios. Nuclei were sorted at 25 PSI (pounds per square inch) through a 100 micron nozzle. Sample and collection tubes were kept at 10 °C for the duration of the sorting. Afterwards, NeuN+ (neuronal nuclei) and NeuN- (assumed glial nuclei) were collected separately in Optiprep and centrifuged at 3,200 g for 20 minutes at 4 °C. The pellet obtained was mixed with Laemmli buffer (2 % SDS, 10 % Glycerol, 0.002 % bromophenol blue, 6.25 mM Tris–HCl pH 6.8, bidistilled H₂O, 2 % β-mercaptoethanol and phosphatase inhibitor cocktails), and pellets containing NeuN+ and NeuN- nuclei were processed in parallel for 10 % SDS-PAGE electrophoresis and western blotting. Demonstration that this fraction contained only nuclei without cytoplasmic contamination was carried out by western blotting with SOD-1 and histone H3 antibodies as indicated in Additional file 3: Table S3. Anti-α-synuclein oligomer-specific antibody (Agrisera, Vännäs, Sweden) was used to identify the presence of oligomers; the bands were visualized with the ECL method (Amersham).