SN4741 cells derived from embryonic substantia nigra and maintained in Dulbecco’s-modified Eagle’s high-glucose medium (DMEM, Life Technologies, Rockville, MD, USA) supplemented with 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana, CA, USA), 1% glucose (Sigma, St. Louis, MO, USA), 1% penicillin–streptomycin (Gibco™, Invitrogen, China) and 2 mmol/L l-glutamine (Gibco™, Invitrogen, China). SN4741 cells were kindly provided by Prof. Qian-Yang of the Fourth Military Medical University . Cells were grown at 37 °C in 5% CO2 and subcultured every 3 days as described previously .
Cell viability, reactive oxygen species (ROS) and chymotrypsin-like function
CCK-8 (Dojindo, Kumamoto, Japan) was used to analyze cell viability under different treatment conditions according to the manufacturer’s recommendations. Cells were trypsinized, suspended and cultured in 96-well plates at a concentration of 5× 103 cells/well. Each sample was made in triplicate. The plate contained blank, positive and negative control wells. PR-957 (Selleck, Houston, CA, USA) was used as a selective inhibitor of β5i in SN4741 cells . Cells were treated with 6-OHDA or PR-957 overnight or for 48 h, respectively, or with control solution. At different time points, 10 μl cell counting kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) solution dissolved in 100 μl DMEM (Life Technologies, Rockville, MD, USA) replaced the drug in each well. The incubation continued for another 0.5, 1, or 2 h at 37 °C following the manufacturer’s instructions. The optical density (OD) value at 450 nm was measured to calculate cell viability using the formula: cell viability (%) = [OD (Sample)-OD (blank control)]/ [OD (negative control)-OD (blank control)] by an ELISA microplate reader (ELX800, BioTeK, USA).
Rhodamine 123 (Sigma-Aldrich, St Louis, MO, USA) was used to measure the mitochondrial membrane potential disruption. Cells were suspended and cultured in 6-well plates. After overnight incubation, groups were exposed to 6-OHDA, PR-957 or control solution. Cells were washed 3 times with PBS and reincubated with 100 μl DMEM (Life Technologies, Rockville, MD, USA) containing 10 μg/mL rhodamine 123 at 37 °C for 30 mins. The fluorescence of rhodamine 123 was detected by a fluorescence spectrophotometer (Shimadzu, Matsuyama, Japan, RF5000U) at 490 nm excitation (Ex) and 520 nm emission (Em).
2′, 7′-Dichlorofluorescin diacetate (DCFH-DA; Sigma-Aldrich, St Louis, MO, USA) was used to measure ROS level following manufacturer’s recommendations. Cells were treated with 6-OHDA or PR-957 at different concentrations and exposure durations. After the cells were washed 3 times with PBS, DCFH-DA diluted in DMEM to 10 μM was added and incubated at 37 °C for 20 min. Cells were washed 3 times with DMEM, and the resultant optical density was measured at 488 nm excitation and 525 nm emission by a microplate reader (Spectramax Gemini XS, Molecular Devices, Pennsylvania, USA). The amount of generated ROS was calculated using the formula: [OD (Sample)-OD (Negative control)]/ OD (Negative control).
The chymotrypsin-like activity (CTL) of the immunoproteasome was assayed with Suc-LLVY-AMC . Cells were seeded at a concentration of 1× 104 cells/well in 96-well plates. Each test was performed in 4 replicates. After treatment with different concentrations of 6-OHDA, cells were harvested and lysed in proteolysis buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM DTT ± 0.25 mM ATP). Then, 100 μl containing 2 μg cell lysate was mixed with 50 μM Suc-LLVY-AMC (Sigma-Aldrich, St Louis, MO, USA). After 1 h of equilibration, fluorescence was monitored for 3 h using a SpectraMax M5 plate reader (Molecular Devices, Pennsylvania, USA, Ex/Em: 370 nm/460 nm).
Overexpression plasmid and shRNA transfection
The β5i overexpression plasmid was synthesized by GeneCopoeia (Product ID: EX-Mm34282-M29, GeneCopoeia, Guangzhou, China). This sequence was inserted into a p-EZ-M29 vector containing neomycin as a stable selection marker. The insertion was confirmed by sequencing. The mU6 vector contained the mCherryFP gene as a marker to identify transfection efficiency (Product ID: CSHCTR001, GeneCopoeia, Guangzhou, China). PSMB8 was suppressed by specific shRNA in the mU6 vector (Product ID: RSH052242-mU6, GeneCopoeia, Guangzhou, China) with target sequences GGAATGCAGCCCACTGAATTC, GGAAGGTTCAGATTGAAATGG, GCAGGAAGTTACATTGCTACC and GCCAAGGAATGCAGGCTATAC and the hairpin loop sequence TCAAGAG. The mU6-pri vector (Product ID: CSHCTR001-mU6, GeneCopoeia, Guangzhou, China) without the target gene and an empty plasmid were used in the negative control (NC) and mock (M) groups, respectively. First, we detected β5i mRNA by qQT-PCR and then confirmed β5i protein expression by Western blot.
Transfection was performed based on manufacturer’s instructions (Invitrogen, Grand Island, NY, USA). Cells were suspended and seeded in 24-well plates at a 50% cell density after counting. After 24 h of culture, transfection was performed as follows. Solution A contained 20 pmol shRNA dissolved in 50 μl Opti-MEM without serum, and B solution contained 1 μl lipofectamine 3000 (Invitrogen, Grand Island, NY, USA) dissolved in 50 μl Opti-MEM without serum. Solution A and B were mixed and kept at room temperature for 20 min. The culture medium for each well was replaced with 400 μl serum-free medium. Cells were incubated in this mixture (serum-free medium containing solutions A and B) for 6 h for transfection, which was then replaced with serum medium. Transfection efficiency was assessed by fluorescence on the following day.
Partial 6-OHDA lesion and behavioral test
Forty male Sprague Dawley (SD) rats, ranging from 280 to 300 g in weight, were bred and maintained in the Specific Pathogen-Free Laboratory Animal Center at Guangzhou Medical University (Guangzhou, China). Weight-matched rats were randomly assigned to four groups: the sham group, 6-OHDA (Sigma-Aldrich, St Louis, MO, USA) group, PR-957 (Adooq Bioscience, CA, USA) group and 6-OHDA plus PR-957 group. Rats were anesthetized with ketamine (10%) /xylazine (2%) (Sigma Aldrich, St Louis, MO, USA) and injected with 8 μg 6-OHDA in 4 μl solvent [0.9% w/v NaCl with 0.1% ascorbic acid (Sigma-Aldrich, St Louis, MO, USA)] into the left anterior medial bundle (Coordinates: AP: - 4.0 mm, ML: - 1.5 mm, DV: - 7.8 mm). Animals in the 6-OHDA plus PR-957 group were given the same dose of 6-OHDA followed by 4 μl PR-957 (50 nM) injected into the lateral ventricle. The sham group was given the same volume of solvent [0.9% w/v NaCl with 0.1% ascorbic acid]. At 4 weeks after the 6-OHDA injection, rats were tested in the rotation test. Rotation asymmetry was calculated for 30 min after intraperitoneal injection of 0.6 mg/kg apomorphine (Sigma-Aldrich, St Louis, MO, USA) as described previously . All animal studies followed the institutional guidelines for animal experiments of Guangzhou Medical University. All procedures were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University.
After electrophoresis of proteins from SN4141 cells or the midbrain of rats and blocking with 0.5% BSA in PBS, the PVDF membranes (Pall Corporation, Pensacola, FL, USA) were incubated with primary antibodies such as anti-β5i (1:800, Abcam, Cambridge, MA, USA), anti-β5 (1:1000, Abcam, Cambridge, MA, USA) or anti-β-actin (1:2000, CST, Danvers, MA, USA) at 4 °C overnight. The primary antibodies were diluted in blocking solution (LI-COR Biosciences, Lincoln, NE, USA). After the membranes were washed, they were incubated with fluorescent-conjugated secondary antibodies (1: 15000; LI-COR Biosciences, Lincoln, NE, USA) for 1 h in the dark. The Odyssey infrared fluorescence detection system (LI-COR Biosciences, Lincoln, NE, USA) was used for scanning and analysis. For traditional Western blot, secondary antibodies conjugated with horseradish peroxidase (HRP, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the chemical luminescence detection method (ECL, Pierce Biotechnology, Rockford, IL, USA) were used. Data were scanned and analyzed using the GE 600 system (GE Healthcare, Piscataway, NJ, USA).
Following the protocol used by Goyal et al. , 1.0 × 104 cells were inoculated in 96-well plates (Corning, Sigma-Aldrich, Dorset, UK) for the in-cell western assay. Cells were cultured in DMEM with 10% FCS (Irvine Scientific, Santa Ana, CA, USA) for 48 h, which was then replaced with 6-OHDA dissolved in FCS-free DMEM, but the control group was cultured in FCS-free DMEM. Then, each well was washed with PBS and fixed in 4% formaldehyde for 1 h. Formaldehyde was washed away with PBS, and cells were incubated with 0.1% Triton X-100 in PBS (3 times, 5 min each). Then, cells were treated with blocking solution (LI-COR Biosciences, Lincoln, NE, USA) and incubated with mouse anti-β5i (1:800, Abcam, Cambridge, MA, USA) and rabbit anti-β5i (1:800, Abcam, Cambridge, MA, USA) overnight at 4 °C. After the cells were washed, fluorescent-conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA), diluted at 1: 1000 in PBS, were added, and the cells were incubated for 1 h in the dark at room temperature. Cells were with PBS three times in the dark. Then, plates were imaged on an Odyssey infrared scanner (LI-COR Biosciences, Lincoln, NE, USA).
Immunofluorescence staining and immunohistochemistry
Brain tissue was cut at a thickness of 15 μm and stored at − 20 °C. Primary antibodies used for immunohistochemistry included mouse monoclonal anti-tyrosine hydroxylase (TH) (1:500, MAB318, Merck Millipore, Billerica, MA, USA), anti-β5i (1:500, Abcam, Cambridge, MA, USA) and anti-TAP-1 (1:500, ab10356; Abcam, Cambridge, MA, USA). TAP-1 is a downstream protein that receives peptides provided by the immunoproteasome . After overnight incubation with primary antibodies, the tissue or cells were incubated with secondary antibodies such as Cy3-conjugated anti-mouse IgG (1:400, Jackson Immuno-research laboratory, PA, USA) and/or Alexa 488-conjugated anti-rabbit IgG (1:400, Molecular Probes, Eugene, OR, USA). Images were acquired using a fluorescence microscope (BX51, Olympus, Fujinon, Japan). For immunohistochemistry, the secondary antibody used was a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:1000, Kangcheng, Shanghai, China). Sections were stained with 3, 3′-diaminobenzidine (DAB) kits (Wuhan Boster Bioengineering Co., Ltd., Wuhan, China). Images were acquired under a microscope (Olympus AX70; Olympus, Tokyo, Japan). Four images at 200× magnification were taken, with each image covering an area of the SN or striatum, and combined into one figure. Images were analyzed by ImageJ software (version 1.45; National Institutes of Health, Bethesda, Maryland, USA).
Fast TH staining and laser capture microdissection (LCM)
To reduce RNA degradation, we used fast TH staining to detect DA neurons. Slices were fixed in acetone-methanol solution at − 20 °C for 10 min, washed with PBS containing 1% Triton X-100, incubated with the TH antibody (MAB318, Merck Millipore, Billerica, MA, USA) at a 1:100 dilution for 10 min, rinsed in PBS with Triton twice, and incubated with the goat antirabbit antibody with HRP (1:100, Kangcheng, Shanghai, China) for 5 min. Immunohistochemistry staining was done by DAB kits (Wuhan Boster Bioengineering Co., Ltd., Wuhan, China). The stained slices were dehydrated in RNase-free solutions as follows: 100% acetone for 5 min, 75% ethanol, 95 and 100% ethanol for 1 min each, and then xylene twice for 1 min and 5 min.
As described previously [26, 27], nonfixed fresh brain tissue was rapidly frozen and cut into 8-μm-thick slices. Slices were collected on to polyethylene naphthalate membrane-coated glass slides (Life Technologies, Grand Island, NY, USA). After fast tyrosine hydroxylase staining, TH+ neurons in the substantia nigra were captured by the Arcturus XT system (Life Technologies, CA, USA). Laser power was set at 70 mW and 150 mV. Approximately 300–450 TH+ neurons were collected, and total RNA was extracted using the mirVana PARIS Kit (PN AM1556, Austin, TX, Ambion, USA) and converted into cDNA by a Reverse Transcription Kit (Takara, Shiga, Japan). RT1A (rat monomorphic MHC class I antigen) binds the peptide or antigens translocated by TAP into the ER, and its mRNA level in DA neurons was detected by qRT-PCR. The PCR primers (TIANGEN Biotech, China) used were as follows: GAPDH-F: 5’-TACTAGCGGTTTTACGGGCG-3′ and GAPDH-R: 5’-TCG-AACAGGAGGAGCAGAGAGCGA-3′; TAP-1-F: 5’-GGCAGACTCAGTTC-CTCTCAC-3′ and TAP-1-R: 5’-CAGAACGGGTTGGGGATCAA-3′; RT1A (Rat monomorphic MHC class I antigen) -F: 5’-GCTCACACTCGCTGCGGTAT-3′ and RT1A-R: 5’-GCCATACATCTCCTGGATGG-3′. GAPDH was used as an internal control, and mRNA expression was analyzed using the 2−ΔΔCT method .
All experiments were repeated at least 3 times. Data are shown as the mean ± SD. ANOVA was followed by Tukey’s or Student-Newman-Keuls (SNK) post hoc testing. P < 0.05 was considered statistically significant. All analyses were performed using SPSS.13 and STATA software (Version 14; StataCorp, College Station, TX, USA).