BV2 murine microglial cells (provided by Professor Xiao Min Wang, Capital Medical University, Beijing, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Corning, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS, Corning, Manassas, VA, USA) and 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO2.
Human embryonic kidney (HEK) 293T cells were cultured in DMEM (Corning, Manassas, VA, USA) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2. Cells were prepared for experiments at early passages (< 5) and 80% confluency.
Rat primary microglia were extracted from the whole brains of 0 or 1-day-old Sprague-Dawley rats (from Beijing WeitongLihua Laboratory Animal Center, SCXK 2016-0006, Beijing, China) as previously described . Briefly, brain tissues were freed from meninges and blood vessels on the surface carefully, and were dissociated by gentle trituration with a pipette; then, the cell suspension was filtered through a 40-μm pore nylon strainer. The isolated cells were transferred to a poly-l-lysine (PLL; Sigma-Aldrich, St. Louis, MO, USA)-coated 75-cm2 flask and incubated at 37 °C and 5% CO2. After 14 days, primary microglia were harvested by shaking the flasks for 2 h at 180 rpm, 37 °C. The purity of the microglial cells was immunolabeled with an antibody against ionized calcium binding adaptor molecule (Iba)-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan); over 98% of the cells were immunopositive.
Rat primary cortical neurons were cultured as previous described . Briefly, surgeries were performed under sodium pentobarbital anesthesia. Primary cortical neurons were extracted from Sprague-Dawley rats and cultured in PLL-coated 6-well plates (1.0 × 106 cells/well) or 96-well plates (1.0 × 104 cells/well) in neurobasal medium (Gibco, Grand Island, NY, USA) with nerve growth factor (10 ng/mL), l-glutamine (1 mM, Invitrogen, Carlsbad, CA, USA), and B27 supplement (Gibco, Grand Island, NY, USA). The purity of the neuronal cells was immunolabeled with an antibody against neuronal nuclei (NeuN, 1:50; Cell Signaling Technology, Danvers, MA, USA); over 95% of the cells were immunopositive.
For the activity of mGluR5 stimulation or blocking, CHPG (150 μM; Tocris Biosciences, Ellisville, MO, USA) or MTEP (100 μM; Tocris Biosciences, Ellisville, MO, USA) were utilized on microglia for the indicated time points after α-syn transfection. Microglial cells were serum starved for another 18 h prior to exposure to CHPG or MTEP. LPS (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) was applied to trigger α-syn expression and stimulate microglia for 24 h. To block protein synthesis, BV2 cell cultures were treated with cycloheximide (CHX, 2 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) for the indicated time points. The lysosome activity was blocked by treatment with NH4Cl (1, 5, 10 mM; Sigma-Aldrich, St. Louis, MO, USA) for 12 h and the proteasome activity was blocked by treatment with MG132 (1, 5, 10 μM; Sigma-Aldrich, St. Louis, MO, USA) for 12 h. Urate (2, 6, 8-trioxy-purine, 200 μM; Sigma-Aldrich, St. Louis, MO, USA) prepared as a 1000× stock solution by dissolving in 1 M NaOH was used for microglia after lentivirus vector (LV)-α-syn infection for 24 h. Recombinant human α-synuclein protein (1 μM; Abcam, Cambridge, MA, USA) was performed in primary microglia.
Plasmids construction and transfection
The cDNAs of rat FLAG-mGluR5 and FLAG-GFP-mGluR3 were kind gifts from Prof. Jun Qi He (Capital Medical University, Beijing, China). The cDNAs of human pCMV-myc-α-syn (myc-α-syn) and myc-α-syn N terminus (amino acids 1–65, abbreviated to myc-α-syn N) were kindly provided by Prof. Hui Yang (Capital Medical University, Beijing, China). The cDNAs of myc-α-syn N terminus deletion (amino acids 61–140, abbreviated to myc-α-syn delN) were produced by Genechem (Shanghai, China). At approximately 80% confluence, BV2 microglial cells or HEK293T cells were transfected with plasmids using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) followed by further analysis.
Lentivirus generation and recombinant adeno-associated virus (AAV) construction
Lentivirus generation was utilized for primary microglia. For overexpression of α-syn, the sequence of human α-syn cDNA was cloned into the pLVX-Ubi-EGFP lentiviral vector with BamHI and AgeI restriction sites (Shanghai Genechem, Co., Ltd., China), abbreviated to LV-α-syn. The pLVX-Ubi-EGFP vector was used as the control (abbreviated to LV-NC). The lentivirus titer units were 1.0 × 109 TU/mL. Cells were infected with multiplicity of infection (MOI) 100 after 5 μg/mL polybrene supplement and then were treated with CHPG or MTEP for further analysis.
Recombinant AAV was used in animals. For overexpression of α-syn, the full length of human α-syn mRNA was cloned into the AAV9-CMV-betaGlobin-MCS-P2A-EGFP-SV40 Poly A viral vector with AsisI and MluI restriction enzymes constructed by Vigene Biosciences (Shandong, China). The resulting packaged virus was verified by quantitative real-time PCR detection and the AAV9-CMV-betaGlobin-hSNCA-P2A-EGFP-SV40 Poly A (abbreviated to AAV-α-syn) genome titer was 8.17 × 1013 vg/mL and the titer of the control viral AAV9-CMV-betaGlobin-EGFP-SV40 Poly A (abbreviated to AAV-GFP) was 1.02 × 1014 vg/mL.
Cell viability measurement
Viability of primary neurons was detected with the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Cell Tilter 96 Aqueous Assay; Promega, Madison, WI, USA) assay or Cell Counting Kit-8 (CCK-8) assay (SUDGEN, China). Primary neurons (1.0 × 104 cells/well) were seeded in PLL-coated 96-well plates and cultured for 24 h. After 24 h of conditional medium application, for MTS assay, MTS solution was mixed to the cells followed by incubation for 1.5 h at 37 °C, and the absorbance was measured at 490 nm on a microplate reader (Elx800; Bio-Tek Instruments, Winooski, VT, USA); for CCK-8 assay, 10 μl of CCK-8 reagent was applied into the medium. After reaction for 2 h at 37 °C, the absorbance was measured at 450 nm on a microplate reader. The cell viability was determined according to the absorbance.
Co-immunoprecipitation and western blotting
Cells or brain tissues were harvested and lysed in lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 1% Triton X-100, pH 7.4) with 1× protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Extracts (1000 μg) were pre-cleared with proteinA/G agarose (40 μl/tube; Sigma-Aldrich), and then incubated with anti-α-syn, -FLAG, or -myc antibody with constant rotation at 4 °C overnight. The precipitant was harvested by centrifugation at 10,000×g for 2 min and washed 3 times with lysis buffer to remove nonspecific binding proteins followed by elution, and then beads were removed by centrifugation at 10,000×g for 2 min and the supernatants were analyzed by western blotting.
For cells protein extraction, cells were washed twice with ice-cold PBS, and lysed in lysis buffer (1 M Tris-HCl [pH 7.4], 5 M NaCl, 10% NP-40, 10% Na-deoxycholate, 100 mM EDTA). For tissues protein extraction, tissues were lysed with RIPA lysis buffer (Solarbio, Beijing, China). For membranous and cytoplasmic protein extraction, after 3.0 × 107 BV2 cells were transfected with α-syn for 48 h, the cytoplasmic and membrane fractions were isolated and collected using the Membrane and Cytosol Protein Extraction kit (Beyotime Institute of Biotechnology, Shanghai, China) followed by western blotting as previously described . Briefly, samples were separated on SDS polyacrylamide gels, transferred to nitrocellulose membranes, and blocked in 5% nonfat milk in Tris-buffered saline (TBS). Membranes were incubated with the following primary antibodies, including β-tubulin (1:1000), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1000), β-actin (1:1000), normal mouse IgG (1:1000), cyclooxygenase-2 (COX-2, 1:1000), phosphorylated (p-) c-Jun N terminal kinase (JNK, 1:1000), p-extracellular signal-regulated protein kinase (ERK, 1:1000), p-p38 (1:1000), p-NF-κB p65 (1:1000), p-AKT (1:1000), JNK (1:1000), ERK (1:1000), p38 (1:1000), NF-κB p65 (1:1000), and AKT (1:1000) (all from Cell Signaling Technology, Danvers, MA, USA); mGluR5 (1:1000), inducible nitric oxide synthase (iNOS, 1:1000), and TNF-α (1:1000) (all from Abcam, Cambridge, MA, USA); Na+-K+ ATPase (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA); α-syn (1:500, BD Biosciences, Franklin Lakes, NJ, USA); tyrosine hydroxylase (TH, 1:5000, Sigma-Aldrich, St. Louis, MO, USA); IL-1β (1:400, R&D Systems, Minneapolis, MN, USA), c-Myc (1:2000, Clontech, Mountain View, CA, USA); and FLAG (1:1000, EMD Millipore, Temecula, CA, USA) at 4 °C overnight, washed with Tris-buffered saline containing 0.1% Tween 20, incubated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Protein signals were visualized using enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA), quantified by Image J software (NIH), and analyzed by GraphPad Prism 5.0 software (GraphPad Inc., La Jolla, CA, USA).
Nitrite level measurement
BV2 cells (4.0 × 105 cells/well) or rat primary microglia (1.0 × 106 cells/well) were cultured in 6-well plates coated with PLL and then incubated for 24 h with the indicated treatment. NO levels in the culture supernatants were determined using a Griess kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The absorbance was measured at 540 nm on a microplate reader.
Enzyme-linked immunosorbent assay (ELISA) measurement
BV2 cells (4.0 × 105 cells/well), rat primary microglia (1.0 × 106 cells/well), and HEK293T cells (4.0 × 105 cells/well) were seeded in PLL-coated 6-well plates and incubated with indicated treatment. TNF-α and prostaglandin E2 (PGE2) concentrations in the culture medium were measured with ELISA kits (ExCell Bio, Shanghai, China) based on the manufacturer’s procedure. The absorbance at 450 nm and 420 nm was measured for TNF-α and PGE2 on a microplate reader, respectively. α-syn level in the culture medium was tested with Human Alpha-synuclein ELISA Kit (Abcam, Cambridge, MA, USA) based on the manufacturer’s procedure. The absorbance at 450 nm was measured on a microplate reader.
Real-time PCR analysis
Rat brain substantia nigra (SN) section samples were processed for total RNA extraction using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was detected by NanoDrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA), and the quality was checked on agarose gels. One microgram total RNA was used for reverse transcription with ImProm-II Reverse Transcription System (Promega, Madison, WI, USA) in a total volume of 20 μl according to the manufacturer’s procedure. Quantitative SYBR Green PCR measurements for α-syn gene expression were performed by the SYBR FAST qPCR Kit Master Mix (2×) (Kapa Biosystems, Wilmington, MA, USA) with prevalidated primers. For α-syn gene amplification, the forward and reverse primers were 5′-AAGGGTACCCACAAGAGGGA-3′ and 5′-AACTGAGCACTTGTACGCCA-3′, respectively. For housekeeping gene GAPDH, forward and reverse primers were 5′-TGACATCAAGAAGGTGGTGAAGC-3′ and 5′-GGAAGAATGGGAGTTGCTGTTG-3′, respectively. The amplification was performed with 40 cycles of 95 °C for 3 s and 60 °C for 30 s on a CFX96TM real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The fold change of α-syn gene expression was normalized to that of GAPDH and was determined by the 2−ΔΔCt method.
Animals and treatment
Male Sprague-Dawley rats (Beijing WeitongLihua Laboratory Animal Center, SCXK 2016-0006, Beijing, China) weighing 200–250 g were housed under a 12-h light/dark cycle at 22 ± 2 °C with access to food and water ad libitum, and acclimated for 7 days before experiments. All procedures were performed on the basis of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Committee on Animal Care and Usage (Capital Medical University). The sample sizes used in this study were based on estimations from a power analysis.
For the LPS-induced model, the rats (n = 45) were randomly divided into 3 groups: sham group (PBS + intraperitoneal (i.p.) injection of vehicle, n = 14), LPS group (LPS + i.p. injection of vehicle, n = 15), and LPS + urate group (LPS + i.p. injection of urate, n = 16). Rats received stereotaxic injection of LPS (Sigma-Aldrich, 5 μg/μl, dissolved in PBS, for a total dose of 10 μg /300 g) and i.p. injection with 200 mg/kg urate (40 mg/mL, dissolved in 0.9% NaCl solution) or its vehicle twice per day, with 1 h between two injections . Rats were anesthetized by pentobarbital sodium i.p. injection and positioned in a stereotaxic apparatus. LPS was injected into the right SN (AP = − 5.5; ML = − 1.5; DV = − 8.3) at a rate of 0.5 μl/min. The needle was remained in place for over 5 min before slow retraction to prevent reflux along the injection tract. The mortality rate of rats in the LPS injection group was < 10%. Rats were killed by anesthetization with pentobarbital sodium followed by decapitation 4 weeks after LPS administration for further analysis.
For the AAV-α-syn-induced model, to observe the progression of PD degeneration with α-syn overexpression, the animals (n = 100) were divided into 2 groups: AAV-GFP (AAV-GFP virus vectors + i.p. injection of vehicle, n = 50) and AAV-α-syn (AAV-α-syn virus vectors + i.p. injection of vehicle, n = 50). To further investigate the mGluR5 mediation on the α-syn-induced inflammatory effect, the animals (n = 60) were divided into 5 groups: AAV-GFP (n = 10), AAV-α-syn (n = 10), AAV-α-syn + MTEP (AAV-α-syn virus vectors + i.p. injection of MTEP, n = 15), AAV-α-syn + urate (AAV-α-syn virus vectors + i.p. injection of urate, n = 15), and AAV-GFP + urate (AAV-GFP virus vectors + i.p. injection of urate, n = 10). The rats were deeply anesthetized by intraperitoneal injection of pentobarbital sodium and fixed in a stereotaxic apparatus. Animals were injected with 3 μl of viral solution (AAV-GFP or AAV-α-syn) into the right SN pars compacta (AP = − 5.5; ML = − 1.5; DV = − 8.3) at a flow rate of 0.2 μl/min through the hole in the skull. The needle was kept for an additional 5 min before withdrawal. In AAV-α-syn + MTEP group, animals received MTEP (1.5 mg/kg/day) i.p. injection at 1 week before virus injection for continuous 3 weeks. In AAV-α-syn + urate group or AAV-GFP + urate group, rats received i.p. injection with 200 mg/kg urate (40 mg/mL, dissolved in 0.9% NaCl solution) or vehicle twice per day, with 1 h between two injections. The death rate after surgery was < 10%. After the indicated time of virus delivery, the animals were sacrificed and brain tissues were harvested for further analysis.
Rats were tested at indicated periods post-surgery with the rotation test, rota-rod behavioral test, and open field test. In apomorphine (Sigma-Aldrich)-induced rotation behavior test, the rotational rate (number of rotations during the 30-min testing period divided by 30) was calculated. Motor function was determined by a rota-rod treadmill for rats under an accelerating rotor mode. Animals training consisted of four trials daily for 3 days (with 15 min rests between trials). Rats were placed on the rotating rod at 5 rpm and accelerating gradually to 30 rpm for 5 min, and the interval times from the beginning until rats fell off was recorded as performance time. The performance on the rota-rod test was measured 5 times per rat. Open field test was also used to judge locomotor activity as previously described [24, 25]. Briefly, the equipment consisted of a 100 cm × 100 cm × 100 cm square arena that was divided into 9 equal squares. A single rat was placed in the center of the square, and the distance of traveling in the area of the apparatus was counted for 30 min.
Immunohistochemistry and immunofluorescence
Rats were deeply anesthetized with pentobarbital sodium and transcardially perfused with saline followed by 4% paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO, USA) dissolving in 0.1 M PBS (pH 7.4). The brains were dehydrated in 20% and 30% sucrose solutions and then coronally sectioned at 40-μm thickness on a freezing microtome (Leica, Solms, Germany). For the immunohistochemistry, sections in SN and striatum (STR) regions were incubated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. After washing with PBS, sections were permeabilized with 0.3% Triton X-100 in PBS for 30 min followed by incubation with 10% normal horse serum for 1 h at room temperature and then incubated in the primary antibody anti-TH (1:2000, Sigma-Aldrich) overnight at 4 °C. After 3 washes with PBS, biotinylated anti-mouse secondary antibody (Vector Stain ABC kit, Burlingame, CA, USA) and diaminobenzidine (DAB, Zhongshan Golden bridge Biotechnology, Beijing, China) were utilized to visualize immunoreactivity. Consecutive sections for 20 slices were selected from each brain for detection. Unbiased stereology was applied to count the number of TH+ neurons in SN region on a DM5000B microscope (Leica Microsystems, Bannockburn, IL, USA) with Stereo Investigator software (MBF Bioscience, Williston, VT, USA). The intensity of TH+ fibers in the STR was quantified using Image Pro Plus v5.0 image analysis software (Datacell, London, UK).
For immunofluorescence, cultured cells or tissues from SN regions were fixed with 4% PFA for 15 min followed by permeabilization with 0.3% Triton X-100 in PBS for 30 min, and then incubation with 10% normal horse serum for 1 h at room temperature. Primary antibodies were added at 4 °C overnight. After washing in PBS, the sections were incubated with Alexa Fluor 594 donkey anti-rabbit (1:200, Invitrogen), Alexa Fluor 647 donkey anti-goat (1:200, Invitrogen), Alexa Fluor 488 donkey anti-mouse (1:200, Invitrogen), Alexa Fluor 405 donkey anti-rat (1:100, Abcam), Alexa Fluor 488 donkey anti-rabbit (1:200, Invitrogen), and Alexa Fluor 594 donkey anti-mouse (1:200, Invitrogen) for 1 h in the dark. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Cell Signaling Technology) for 5 min. Primary antibodies included rabbit polyclonal anti-mGluR5 (1:200, EMD Millipore), goat monoclonal anti-Iba-1 (1:200; Abcam), mouse monoclonal anti-α-syn (1:100, BD Biosciences), rat monoclonal anti-lysosome-associated membrane protein (LAMP)-1 (1:50, Santa Cruz Biotechnology), rabbit polyclonal anti-NeuN (1:50; Cell Signaling Technology). and mouse monoclonal anti-TH (1:2000, Sigma-Aldrich). Images were captured using a confocal microscope (TCS SP8; Leica, Solms, Germany).
Protein-protein modeling simulation
The crystal structures of the protein α-syn for docking process were obtained from the Protein Data Bank database (PDB ID: 2NOA); the 3D (three-dimensional) structures of protein mGluR5 were constructed from ab initio modeling utilizing the software I-TASSER. Protein-protein docking simulations for mGluR5-α-syn complex were based on professional software Rosetta (http://robetta.bakerlab.org/). In docking processes, the protein α-syn and mGluR5 were assigned as “ligand” and “receptor”, respectively, and global scanning of the rotational and translational space were performed. Molecular docking results were scored in Rosetta with the default values of 1-Å grid step and 4-Å surface-layers used for clustering. Finally, a maximum number of 50 conformers were considered, and the conformation with the lowest binding energy was selected for final molecular dynamics simulations.
Molecular dynamics simulations were performed by using the AMBER16 package (http://ambermd.org/) with default scoring parameters utilizing the GAFF and AMBERff 14SB force field. All the simulations were carried out at constant temperature (310 K) and pressure (1 bar), and simulations modeling results were recorded at each 2 fs time-scale step. Van der Waals energy and short range electrostatic energy calculations radius cutoff were set as 10 Å, and the long-range electrostatic energy was calculated with default configurations by the PME method.
Data are expressed as the mean ± SD or mean ± SEM and analyzed using Prism 5.0 software (GraphPad Inc., La Jolla, CA, USA). Where parametric tests were used, we checked normal distribution and difference in variance by the Shapiro-Wilk test and an F test, respectively. A two sample unpaired Student’s t test was used for two-group comparisons. One-way ANOVA followed by Dunnett’s test was used for multiple-group comparisons. Two-way repeated ANOVA was used to determine the statistical changes in each measure. At least three independent experiments were performed for each assay. p < 0.05 were considered significant throughout the study.