MPTP model of Parkinson’s disease
Six-week-old male C57BL/6 mice (20–22 g; DBL, Korea) were divided into two groups: the control group (CTL) and the MPTP intoxicated group (MPTP). Mice in the CTL were injected with 0.9% (100 µL) saline intraperitoneally once daily for 4 weeks, and mice in the MPTP were injected with MPTP-HCl (20 mg/kg of free base) in 0.9% (100 µL) saline intraperitoneally once daily for 4 weeks to produce a sustained chronic parkinsonism mouse model. On the day after the final MPTP treatment, the mice were anesthetized using Alfaxan and perfused with 0.05 M sodium phosphate buffer (PBS). The Sangji University Animal Experimentation Committee approved all animal protocols used in this study. Guidelines around animal experiments have been followed. All reagents used in the study were purchased from Sigma (USA) unless otherwise stated.
Rna Extraction And Microarray Analysis
Total RNA was extracted from the bilateral SN using an RNeasy Plus Mini Kit (QIAGEN, USA) in. The quality of the isolated RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA). An aliquot of the total RNA was subjected to an Affymetrix GeneChip® Mouse Gene 1.0 ST Array (Affymetrix, USA) according to the GeneChip Whole Transcript Sense Target Labeling Assay manual [7, 8].
Microarray Data Analysis
Quality control of the scanned data was confirmed using the Expression Console software (Affymetrix). Microarray data were analyzed using the GenPlex ver. 3.0 (ISTECH, Korea). The mean signal intensities of the genes were obtained from the two chips for each group. Following normalization, the genes that satisfied the conditions of Student’s t-test significance criterion (p < 0.05) and the fold change cutoff [2] were identified using the differentially expressed genes (DEG)-finding module.
Cell Lines And Cultures
The neuroblastoma cell line SH-SY5Y was maintained under standard cell culture conditions (37°C, 5% CO2) in minimum essential medium (MEM; Welgen, Namcheon-myeon) containing 10% fetal bovine serum (FBS; BioWhittaker), 0.1 mM nonessential amino acids, 100 U/mL of penicillin, and 100 mg/mL streptomycin.
Short Interfering Rna (Sirna) Knockdown
Stealth siRNA against Srpk3 (5- GAA AAC UGC CUG UUU GUU U -3) and negative control duplexes (i.e., scrambled siRNA against Srpk3, 5- UUC UCC GAA CGU GUC ACG UTT -3) were purchased from Bioneer Inc (Korea).
Sirna Transfection
The SH-SY5Y cells were incubated in Opti-MEM medium (Gibco, USA) for at least one day before performing siRNA transfection. The transfection reagent (Promega, USA) and Srpk3 siRNA were used (3.5:1) for transfection when the density of SH-SY5Y cells was 60%. Transfection was continued for 24 h.
Mpp+ Treatment
The SH-SY5Y cells were treated with 500 µM MPP+ iodide (Sigma) for 18 h.
Immunohistochemistry
After four weeks, the brains of mice were resected, fixed in PBS containing 4% paraformaldehyde for 12 h at 4°C, rinsed with PBS, immersed in 30% sucrose solution for 12 h at 4°C, and then cryo-sectioned. Sections of the brains were cut using a cryomicrotome (30-µm thickness). Immunohistochemical analyses were performed using an ABC kit and a Mouse on Mouse (M.O.M) immunodetection kit (Vector Laboratories, CA) by a modification of the avidin-biotin-peroxidase method. Briefly, sections encompassing the entire striatum (ST) and SN were incubated in PBS with 3% H2O2, exposed to 3% bovine serum albumin and 0.3% Triton X-100 for 1 h, and treated with an avidin biotin blocking kit (Vector Laboratories). Brain sections were treated with an M.O.M. mouse Ig-blocking reagent (Vector Laboratories) for 1 h at room temperature prior to incubation with the primary antibody. Each section was stained overnight with anti-TH (1:2000; Santa Cruz Biotechnology, USA), anti-Srpk3 (1:1000; Cloud-clone Corp., USA), and anti-α-syn (1:500; Santa Cruz Biotechnology, USA) antibodies at 4°C. The sections were incubated with a biotinylated anti-mouse IgG, followed by an avidin-biotin-peroxidase complex, and then developed using a diaminobenzidine-hydrogen peroxide solution (0.03% hydrogen peroxide and 0.003% 3,3-diaminobenzidine in 0.05 M Tris, pH 7.0).
Immunofluorescence
The cells were treated using the same procedures as those used for immunohistochemistry. The cells were incubated with primary antibodies and then with biotinylated anti-mouse IgG and anti-rabbit IgG. The brain sections were treated with a fluorescein avidin DCS (Vector Laboratories, USA) for mouse anti-TH (1:2,000) and rabbit anti-Srpk3 (1:1000). Thereafter, cells were treated with an avidin biotin blocking kit and an M.O.M mouse Ig blocking reagent (Vector Laboratories), followed by staining with anti-TH and anti-Srpk3 IgG at 4°C overnight. Each group was treated with a biotinylated anti-mouse IgG and anti-rabbit IgG, followed by incubation with fluorescein avidin D and rhodamine avidin D (Vector Laboratories). Photographic documentation was performed using a Nikon X-Cite-Series 120 Q microscope (Nikon, Japan). Images were collected using an ECLIPSE Ni-U microscope (Nikon) equipped with a digital camera (DS-Fi2; Nikon). Images were acquired using the imaging software NIS-Elements F ver. 4.00.00 (Nikon). Tiff images and quantification data were collected from each image data file using ImageJ software (NIH, USA).
Western Blotting
Western blotting
The ST and SN regions were homogenized using radioimmunoprecipitation assay buffer on ice for 30 min. After centrifugation at 12,000 rpm for 20 min, the soluble supernatant samples were then collected, and equal concentrations of protein samples were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, USA). The membranes were blocked with 5% skim milk in 0.1% Tris-buffered saline (TBS) for 1 h at room temperature and were incubated with anti-TH (1:2,000), anti-α-syn (1:500), anti-Srpk3 (1:2000), and anti-β-actin (1:5,000, Santa Cruz Biotechnology) antibodies. Thereafter, the membranes were washed with 0.1% TBS containing 0.1% Tween-20 (TBST) and incubated with an anti-mouse IgG-peroxidase antibody (1:2,000, Bio-Rad), and the antigen-antibody complexes were visualized using the Pierce ECL western blotting Substrate (Thermo Scientific, USA). The percentage of densitometric values for immunoreactive bands was calculated in each experiment. The density was measured using the ImageJ software (NIH).
Statistical analysis
Statistical analyses were performed using analysis of variance (ANOVA) and Student’s t-test in SPSS 25 (SPSS Inc., USA). All values are presented as mean ± standard error.