MPTP mouse model of Parkinson’s Disease
Six-week-old male inbred C57BL/6J mice (20–22 g; DBL, Korea) were injected with MPTP-HCL (20 mg/kg of free base; Sigma, USA) in 100 µL phosphate-buffered saline (PBS) intraperitoneally once a day for 4 weeks in MPTP group (n = 6) whereas mice were injected with 100 µL PBS in control group (n = 6). The day after the final injections, mice were anesthetized using Alfaxan (4 mL/kg; Careside, Korea) and perfused transcardially with cold PBS. In this study, all animal experiments were approved from Sang Ji University Animal Experimentation Committee.
RNA extraction and microarray analysis
RNA extraction and microarray analysis proceeded in the same procedure described in the previous study [11, 12]. The differentially expressed genes (DEGs) that satisfied the conditions of the fold change cutoff (1.5) and the Student t-test significance criterion (p < 0.05) were identified using the DEG-finding module .
The procedures to obtain brains of 4-week MPTP Parkinson’s disease mouse models for immunohistochemical analysis were described well in previous research report . The procedures were followed to investigate TH in SN regions. Mouse anti-TH antibody (1:200; Santa Cruz Biotechnology, USA) were used for incubation overnight at 4 °C. On the other hand, the procedures were followed until blocking step in observation of TRDN in SN. When observing TRDN, sections encompassing the SN regions were incubated in blocking buffer (1% bovine serum albumin [BSA] and 10% goat serum in PBS) for 1 h. Rabbit anti-TRDN antibody (1:200; MybioSource) were used for incubation overnight at 4 °C. And then, the sections were treated with biotinylated anti-rabbit IgG and avidin–biotin–peroxidase complex and were observed through reaction with diaminobenzidine–hydrogen peroxide solution.
Cell lines and cultures
Cells from the SH-SY5Y cell line were cultured in standard culture conditions (5% CO2, 37 °C). Minimum essential medium (MEM; Welgen, Namcheon-myeon, South Korea) containing 10% fetal bovine serum (BioWest) , 100-U/mL penicillin-streptomycin (Gibco; USA), and 0.1 mM nonessential amino acids (Gibco, USA).
Small interfering (si)RNA knockdown
To downregulate the expression of TRDN and examine the effects, siRNA against TRDN (5′-UC AUG UGG GUA GAC UCA GU-3′) and negative control duplexes (siRNA, 5′-UUC UCC GAA CGU GUC ACG UTT-3′) were used , and the siRNAs were obtained from Bioneer Inc., Korea. SH-SY5Y cells incubated in Opti-MEM medium at least 1 day before siRNA transfection. When transfection starts, the density of SH-SY5Y cells was 70%. Transfection reagent (Promega, USA) was used in a 3.5:1 transfection reagent-to-duplex RNA ratio in Opti-MEM (Gibco) medium during 24 h transfection.
The SH-SY5Y cells were homogenized in 20 mM radioimmunoprecipitation assay buffer (RIPA) on ice for 20 min after PBS washing briefly [8, 11]. When the SN tissues (n = 3) were homogenized in 20 mM RIPA using a sonicator (Qsonica Q55; USA) and incubated on ice for 20 min. After centrifugation at 12,000 rpm at 4 °C for 15 min, supernatant samples of equal protein concentration were separated using 4–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Pall Life Science; USA) . The membranes were blocked with 3% BSA in 0.1% Tris-buffered saline (TBS; 20-mM Tris–HCl [pH 7.5] and 150 mM NaCl containing 0.1% Tween-20, TBST) at room temperature for 1 h and then incubated with primary antibody overnight and then washed with 0.1% TBST [11, 13]. The membrane was also incubated with secondary antibody for 1 h and washed with 0.1% TBST. Rabbit anti-TRDN (1:2000; MyBioSource), rabbit anti-TH (1:5000; Abcam), rabbit anti-β actin (1:5000; Cloud Clone), rabbit anti-Bax (1:1000; Abcam), rabbit anti-Bcl-2 (1:5000; Abcam), and mouse anti-β actin (1:5000; Santa Cruz Biotechnology) were used as primary antibodies. Goat anti-mouse IgG (HRP; suitable ratios; Abcam) and goat anti-rabbit IgG H&L (HRP; suitable ratios; Santa Cruz Biotechnology) were used as secondary antibodies. The antigen-antibody complexes were visualized using ECL detection reagents (GE Healthcare; USA) by Amersham Imager 680 (GE Healthcare).
The cytotoxicity assay was performed to know how decrease TRDN level affects cells. Cell viability and cytotoxicity were examined using EZ-CYTOX (DoGenBio, Korea). The recommended protocol was followed, and cell viability was measured after TRDN siRNA(siTRDN) treatment for 24 h. SH-SY5Y cells were treated with various concentration of siTRDN (5, 10, 25, 50, and 100 nM) and 100-nM negative control siRNA.
Fluorescence-activated cell sorting analysis
Fluorescence-activated cell sorting (FACS) analysis was conducted to do analysis of apoptosis in cells, according to siTRDN treatment. siTRDN treatment was performed in a same way with Cytotoxicity assay. Control group was added to do compensation and nothing was treated in the control group cells. Cells were collected with cell scraper and washed with PBS twice in 2500 rpm, 4 °C for 5 min. Cells were resuspended with binding buffer in 106 cells/mL and 100 µL (105 cells) was aliquot. Fluorescein(FITC)-annexin V and propidium iodide(PI) was treated following suggested protocol of manufacturer (BD ParmingenTM). Samples were diluted 5 times with binding buffer and detection was performed with cytoFLEX (Beckman Coulter Life Sciences; US).
For statistical analysis, Student’s t-test and analysis of variance (ANOVA) in SPSS 25 (Released 2017, PASW statistics for Windows, version 25.0, Chicago: SPSS Inc.) were used. P values less than 0.05 are regarded as significant values and P < 0.05 values are expressed in figures. All values expressed as mean ± SEM.
ImageJ software was used for image processing.