This study was carried out in accordance with the principles of the Basel Declaration and recommendations of Dalian Medical University for laboratory animals. The protocol was approved by the Animal Ethics Committee of Dalian Medical University.
Construction of the MPTP-treated PD mouse model
Fifty-two C57BL/6 male mice, weighting 20~25g and 8 to 10-week-old, were randomly divided into two groups: control (n=26) and MPTP (n=26). The intraperitoneal injection with MPTP (25 mg/kg or 10ml/kg, dissolved in physiological saline) was preformed onto mice 10 times at intervals of 3.5 days in MPTP group. Meanwhile, the mice were treated with the same volume of physiological saline (10 ml/kg) via intraperitoneal injection in control group. Locomotor activity was examined and the parkinsonian biological markers including 5-HT and DA were detected by RP-HPLC, and TH was tested by immunohistochemistry (IHC) as described previously.
Cell culture, treatment and cell viability assay
Neuroblastoma SH-SY5Y cells were routinely grown in Dulbecco’s modified Eagle’s medium/F12 nutrient mixture (DMEM:F12) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 100 units/ml of penicillin/streptomycin. Cells were cultured at 37°C under a humidified atmospheric condition containing 5% carbon dioxide. To investigate PD-like neurotoxicity induced by MPP+, SH-SY5Y cells were normally grown for 24 h followed by incubation with MPP+ at various concentrations for another 24 h. The morphology of cells was examined under an inverted microscope. The optimum MPP+ concentration was determined by plotting cell viability against MPP+ contents. Cell viability was evaluated by MTT assay in 96-well plates. After treatment with MPP+, the SH-SY5Y cells were incubated with 100 μl of MTT solution (0.5 mg/mL in PBS) at 37 °C for another 4 h. Then, DMSO was added into cells in order to dissolve formazan crystals, and the absorbance at 490 nm was read on a microplate ELISA reader (Thermo Scientific). All experiments were performed independently in triplicate.
Preparation of protein lysates
For mice, on the 7th day after the last MPTP injection, the mice in each group were sacrificed by cervical dislocation. The mouse brains were separated and washed with ice-cold 0.9 % physiological saline. The different specialized structures of mouse brain, including striatum, midbrain, cerebellum, cortex, hippocampus, brain stem were dissected carefully, and homogenized in ice cold RIPA lysis buffer [50 mmol/L Tris (pH7.4) containing 150 mmol/L NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS and 1 mmol/L PMSF] followed by clearance at 14000 rpm for 30 min twice. For cells, the cultured SH-SY5Y cells in a 10 cm dish were digested with trypsin, and collected by centrifugation at 1000 rpm for 5 min. Then, the SH-SY5Y cells were broken by ultrasonication (5S on, 3S off) followed by removal of insoluble fragments through centrifugation at 14000 rpm for 30 min twice. Subsequently, the protein concentrations of the supernatants were determined using a BCA kit (keyGEN BioTECH, China). Thus, the protein lysates were obtained and frozen at –80°C for a further investigation.
Processing of fixed brain tissues
On the 7th day after the last MPTP injection, the mice were anesthetized by inhalation of diethyl ether, and mechanically fixed to expose the heart completely via opening the chest. The mice were intracardially perfused with 0.9 % physiological saline and 4 % paraformaldehyde in phosphate-buffered saline (PBS; 50 mmol/L of NaH2PO4; 5 mmol/L of KCl; 1.5 mmol/L of MgCl2; and 80.1 mmol/L of NaCl; pH 7.4) for 30 min respectively. On the one hand, the different specialized structures including striatum, midbrain, cerebellum, cortex, hippocampus, brain stem were dissected carefully, post-fixed in the above fixative, and stored at 4°C for IHC examination. On the other hand, the whole mouse brains were saturated in 15 % picric acid in PBS followed by storage in 20 % sucrose in PBS at 4°C for immunofluorescence staining.
Reversed phase High Performance Liquid Chromatography (RP-HPLC) Analysis of DA and 5-HT Content
The equal volume of cold acetone was added for removing high-abundance proteins from the striatal homogenate of mice and cell lysate of neuroblastoma SH-SY5Y away. The level of dopamine(DA) and 5-hydroxytryptamine (5-HT) in SH-SY5Y cells and striatum of mouse brains were measured by performing RP-HPLC respectively. The samples were injected into HPLC analysis, and eluted through a C18 column (3.9 mm × 150 mm, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase was a mixture of acetic acid buffer (pH 3.5, containing 12 mmol/L acetic acid, 0.26 mmol/L EDTA disodium): methanol = 86:14; the flow rate was constant at 0.5 ml/min over the course of HPLC separation, and the eluted compounds were detected by monitoring the absorbance at the wavelength of 280nm.
Western blot analysis (WB)
Eighty microgram of soluble proteins for each different specialized structures and neuroblastoma SH-SY5Y cells was separated by running a 15 % SDS-acrylamide gel electrophoresis (SDS-PAGE) in a vertical electrophoresis apparatus and transferred to a PVDF membrane in blotting buffer (20 mmol/L Tris-base, 150 mmol/L glycine and 20 % methanol) to make protein accessible to monoclonal antibody detection using anti-β-actin antibody (Abcam), anti-ADP/ATP translocase 1 antibody (anti-ANT1, Abcam) and anti-GAPDH antibody (Abcam). The protein bands were visualized by an enhanced chemiluminescent (ECL) system (GE Healthcare Bio-Sciences Corp.).
The different fixed specialized structures were routinely processed for embedding in a paraffin block followed by dehydration using a gradient concentration of alcohol (70%, 80%, 90%, 95%, 100% respectively). Then, the samples were diaphanized by immersion in xylol, and embedded in paraffin. The paraffin-embedded sections were sliced to 10 μm thickness and mounted on glass slides for IHC analysis using the primary antibody of anti-ANT1 and anti-TH (tyrosine hydroxylase, Santa Cruz Biotechnology, Santa Cruz, CA). Diaminobenzidine was used for visualization of immunolabeling.
Detection and quantification of intracellular ROSs
Measurement of the intracellular ROSs was performed using the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA; Sigma‑Aldrich), which can be oxidized to the highly fluorescent dichlorofluorscein (DCF). Cells were seeded and cultured onto black 96-well plates with a clear bottom. After exposure to MPP+ for another 24 h, SH-SY5Y cells were incubated with DCFH-DA (10 μmol/L) for 30 minutes. After washing with PBS twice, the fluorescence intensity was determined using a fluorescence microplate reader (Beckman) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
After the whole mouse brain was frozen in Milli-Q water in freezing microtome/cryostat (-40 °C), six micron-thick sections were cut and mounted on glass slides for immunofluorescence analyses using the monoclonal antibodies including anti-ANT1 and anti-α-synuclein as the probes. SH-SY5Y cells were seeded on poly-L-lysine-coated coverslips in 6-wells plates for 12 h. Then, the cells were fixed with 4% paraformaldehyde followed by permeabilization with 0.5% Triton X-100. After blocked with 1% normal serum in PBS for 30 min at room temperature, the SH-SY5Y cells were incubated with monoclonal antibodies, including anti-ANT1 and anti-α-synuclein respectively at 4 °C overnight. The secondary antibodies conjugated to rhodamine/FITC were used in this study. All samples were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA). The images were acquired with an Olympus inverted fluorescence microscope (Olympus Corporation of the Americas, Center Valley, PA, USA).
Expression, purification and detection of α-synuclein expressed in Escherichia coli BL21 (DE3)
α-synuclein gene was chemically synthesized and sequenced followed by ligation with pCold II to yield the expression vector of pCold II-α-synuclein in Takara company. The pCold II-α-synuclein was transformed into E. coli BL21 (DE3) cells for over-expression of the α-synuclein fusion protein. A single colony of E. coli BL21 (DE3) carrying pCold II-α-synuclein was grown in 5 ml of LB medium overnight, and the resulting culture was used to inoculate 200 ml of LB medium. When the optical density of the culture was 0.5 at 600 nm, 0.6 mmol/L isopropyl-D-thiogalactopyranoside (IPTG) was added to induce α-synuclein expression for another 20 h at 16°C. The cells harboring pCold II-α-synuclein were lysed by sonication (30s pulse with 30s interval for 20cycles) in pre-chilled lysis buffer (pH8.0, 50 mmol/L NaH2PO4, 300 mmol/L NaCl, 20 mmol/L imidazole, 1 mmol/L phenylmethyl sulphonyl fluoride [PMSF]). The cytoplasmic fractions were applied to a pre-equilibrated Ni-NTA column (Sigma, St. Louis, MO, USA) of 1.0 mL column volume after they were clarified by centrifugation at 27,000 g at 4 °C for 40 min. The purification was performed according to the manufacturer’s instruction. The column was washed with 20 ml wash buffer (pH8.0, 50 mmol/L NaH2PO4, 300 mmol/L NaCl, 40 mmol/L imidazole, 1 mmol/L PMSF) to remove unbound protein, and eluted with 10 ml elute buffer (pH8.0, 50 mmol/L NaH2PO4, 300 mmol/L NaCl, 250 mmol/L imidazole, 1 mmol/L PMSF). The eluted fractions were collected, and their protein concentration was determined using a BCA kit. α-synuclein in the eluted fractions was verified by running a 15% SDS-PAGE followed by Western blot analysis. The probing of the membrane with antibody of (anti)-polyhistidine monoclonal HIS-1 (Sigma, St. Louis, MO, USA) was conducted manually, and the colorimetric detection of protein bands was developed by ECL solution .
Identification of putative α-synuclein interaction partners
Pull-down assay for α-synuclein interaction partners was performed using Ni-NTA magnetic agarose beads (Qiagen, Hong Kong, PRC). The purified His6-tagged α-synuclein (bait, 8μg) was allowed to bind to the Ni-NTA magnetic agarose bead suspension (40 μl) in 500 μl pull-down reactions under gentle rotation at 4°C for 2 h. Then, the supernatants were removed using a magnetic MagRack6™ (Qiagen, Hong Kong, PRC) whereas the magnetic beads bound with α-synuclein protein were remained in a microcentrifuge tube. Five hundred microliters of whole-cell lysate of mouse brains with protease inhibitor cocktail (Sigma, St. Louis, MO, USA) (prey) was added to the α-synuclein-bound Ni-NTA beads and incubated at 4°C for 2 h with gentle rotation. After incubation, unbound proteins were removed by washing using wash buffer (pH8.0, 50 mmol/L NaH2PO4, 300 mmol/L NaCl, 40 mmol/L imidazole, 1 mmol/L PMSF). The beads were then suspended in 50 μl elute buffer (pH8.0, 50 mmol/L NaH2PO4, 300 mmol/L NaCl, 250 mmol/L imidazole, 1 mmol/L PMSF), and the eluted fractions were collected. The bound proteins were run on a 15% SDS-PAGE gel stained with a silver staining kit (Sigma, St. Louis, MO, USA), and analyzed by Western blot analysis coupled with a 15% SDS-PAGE separation. The probing of the membrane with (anti)-ANT1 monoclonal antibody and DBA was conducted manually, and the colorimetric detection of protein bands was developed by ECL solution.
Silver-stained gel lane was excised into small pieces followed by decolorization in 400μl mixed solution containing 100mmol/L Na2S2O3: 300mm K3Fe(CN)6 (1:1). The reduction of the decolorized gel was performed in 100mmol/L NH4HCO3 solution, and the digestion of the gel was conducted in 50mmol/L NH4HCO3 solution containing 12.5 ng/μl of sequencing-grade trypsin for 20 h at 37°C according to a modified in-gel trypsin digestion procedure. Then, the trypsin-digested peptide fragments were desalted using C18 StageTip column followed by lyophilization. The lyophilized peptide fragments were dissolved in 0.1% formic acid for subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
The LC-MS/MS analysis was performed using shotgun separation of peptide mixtures on a reversed phase C18 column (75µm*150mm, 3µm, C18, Thermo Scientific, Waltham, USA) by EasynLC1200 chromatographic system (Thermo Scientific, Waltham, USA). Optimum separation was achieved with a binary mobile phase at a ﬂow rate of 300nl/min. Solvent A: 0.1% formic acid aqueous solution; solvent B: 0.1% formic acid, 95% acetonitrile and water. The gradient elution program was: 0–3min, ramping to 7% from 2% of solution B; 3–48min, ramping to 35% from 7% of solution B; 48–53min, ramping to 90% from 35% of solution B; 53–60min, 90% of solution B. The MS analysis was conducted by Q-Exactive Plus mass spectrometer in 60min. The Q-Exactive Plus MS analysis was set to data-dependent acquisition, and the MS instrument was operated in the positive mode. Working conditions of MS were as follows: mass range 300–1800 m/z at 70000 @ m/z200 resolution for triggering MS/MS events; automated gain control (AGC) at 1e6; maximum IT at 50ms. The data of tandem MS was acquired under the following conditions: 20 ions with the highest intensity triggered by each full-scan of MS; the resolution of the tandem mass spectrum: 17500 @ m/z200; AGC: 1e5; maximum IT: 50ms; MS2 Activation Type: HCD; Isolation window: 2.0Th, Normalized collision energy: 27. The MS data retrieval was performed by MaxQuant126.96.36.199 software against Uniprot Mus musculus Database.
Image Quantification and Statistical Analyses
Quantification of the protein bands was conducted using Image J (version 1.42). The color images were converted to gray-level intensity for quantification. Slight variations in background staining were corrected by subtracting background density. Digital analysis on the immunohistochemistry images was performed using Image-Pro Plus software (version 6.0).
Statistical analyses were conducted using GraphPad Prism 6 and SPSS (version 19). The statistical difference between the MPTP-treated mice and the control mice was evaluated using a two-tailed equal variance Student’s t-test. WB and IHC data were analyzed in triplicate. A threshold of P values <0.05 was considered statistically significant.