SH-SY5Y cells (American type culture collection, gift from Dr. Jianguo Chen) were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) at 37°C with 5% CO2 and 95% air (vol/vol). Rotenone (Sigma-Aldrich, St. louis, MO, USA) was dissolved in dimethyl sulfoxide before dilution with the culture medium. The final concentration of dimethyl sulfoxide (DMSO) per well was 0.2%. DMSO alone was added to the culture medium in control group (“Con-group”). For the dose-dependent study, rotenone was given at a concentration of 0.1, 0.5, 1, 2.5, 5, 10 and 20 μM for 24 hours. For the time-dependent study, rotenone (2.5μM) was given for 3, 6, 12, 24, 36 or 48 hours to induce cell damage.
Cell viability was assessed by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [27, 28]. The MTT assay is a colorimetric assay of the activity of cellular enzymes that reduce the tetrazolium dye, MTT, into insoluble formazan, giving a purple color. Briefly, SH-SY5Y cells were plated at a density of 1 × 104 cells per well in 96-well plates. After exposure to rotenone and vehicle, 20 μl of MTT (5 mg/ml, Sigma-Aldrich) was added into each well before incubation in a humidified incubator at 37°C for 4 hours to allow the formation of purple formazan crystal. Then, 100 μl of the solubilization reagent (0.1 N HCl in anhydrous isopropanol, Sigma-Aldrich) was added into each well and lysate spectrophotometrically measured for absorption at λ 570 nm with background subtraction at 690 nm. Cell viability was expressed as a percentage of the value in untreated control cells.
Detection of apoptosis, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) in SH-SY5Y cells
Annexin V was used to probe phosphatidylserine expression on the cell surface, an event found in apoptosis as well as other forms of cell death . In this study, staurosporine (50nM, Sigma-Aldrich) treatment for 24 hours was employed as a positive control to induce cell apoptosis. SH-SY5Y cells were harvested after treatment with 0.25% trypsin, washed with phosphate buffered solution (PBS) and incubated in PBS containing the Annexin V-fluorescein isothiocyanate (Annexin V, 5 μl in 100 μl PBS) and Propidium Iodide (PI, 100 μg/ml working solution, 1 μl in 100 μl) at 37°C in darkness for 15 minutes. The apoptosis rate = [Annexin V(+)PI(−) cells + Annexin V(+)PI(+) cells] /total cell × 100%. The specific fluorescence of 10,000 cells was analyzed on FACScalibur (BD Biosciences, Franklin Lakes, NJ, USA) within 1 hour after antigen antibody reaction [27–29]. Data were analyzed by using FSC express version 3.0 (De Novo Software, Los Angeles, CA, USA).
It was reported that a decrease in MMP was one of the earliest events in apoptosis . When stained with JC-1, red fluorescence of mitochondria was due to the formation of J-aggregates at high MMP, and green fluorescence of mitochondria to the formation of JC-1 monomers at low MMP . ROS detection was based on ROS-catalyzed formation of fluorescent compound DCF. The nonfluorescent probe DCFH-DA could diffuse passively through the cellular membrane. With intracellular esterase activity, DCFH-DA formed a nonfluorescent compound DCFH, which was oxidized into the fluorescent compound DCF by ROS . For the analysis of MMP and ROS, cells were harvested, resuspended in PBS and immediately stained with JC-1 (1 mg/ml in DMSO, Molecular Probes, Eugene, OR, USA)  or DCFH-DA (10 μM, Invitrogen) , and incubated at 37°C for 30 minutes in the darkness. After washing with ice-cold PBS twice, the samples were subject to FACScan flow Cytometry. Data were analyzed again by using FSC express version 3.0 (De Novo Software).
The microtubule-associated protein1 light chain 3 (LC3) was a marker for all types of autophagic vacuolar organelles. A higher LC3 expression level meant more autophagic vacuolar organelles in the cells. It was reported that LC3 expression level could be related to the induction of autophagy or a block of autophagy and subsequent accumulation of LC3 [33–35]. The mammalian proteins p62 and NBR1 were selectively degraded by autophagy and could act as cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates [36, 37]. The conversion of LC3-I into LC3-II (LC3-II level compared to LC3-I level) and the expression of P62 (which was degraded by autophagy) were indicative of autophagic activity. Higher ratio of LC3-II/LC3-I and lower p62 expression means higher autophagic activity. In this study, LC3 and p62 levels were measured by an immunoblotting method [27, 28, 38]. Cells were rinsed twice with cold PBS and lysed in buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,1 mM EDTA, 1 mM sodium orthovanadate,10 mM sodium fluoride, and 100 mg/ml PMSF). After incubation on ice for 30 minutes, cell lysates were then clarified by centrifugation at 12,000 × g and 4°C for 10 minutes and the supernatant saved for protein analysis and Western blotting. Total protein concentration was determined by the BCA kit (Sigma-Aldrich). Equal amounts of proteins (30 μg) were fractionated by 15% SDS-PAGE, and transferred to nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in Tris-buffered saline (TBS) for 1 hour at room temperature, followed by incubation with primary antibodies against LC3, P62 (Sigma-Aldrich, St. Louis, MO, USA) and β-actin (Santa Cruz, Santa Cruz, CA, USA) overnight at 4°C. The membranes were then washed twice with TBS tween-20 and probed with the corresponding secondary antibodies conjugated with HRP at room temperature for 1 hour. Detection was carried out using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA), followed by autoradiography. The relative intensity of bands was quantified using Quantity One analysis system (Quantity One, Hercules, CA, USA). All data from three independent experiments were expressed as the ratio to optical density values of the corresponding controls for statistical analyses.
SH-SY5Y cells grown on cover slips were fixed with 4% paraformaldehyde at 4°C for 30 minutes, washed with PBS and permeabilized with 0.1% Triton-X100 and 5% bovine serum albumin (Invitrogen) in PBS , followed by incubation at 4°C overnight with the LC3 antibody (1:100, rabbit polyclonal antibody, Sigma-Aldrich, St. Louis, MO, USA) without or with alpha-synuclein (SNCA) antibody (1:100, mouse monoclonal antibody, Billerica, MA, USA). The corresponding secondary FITC-conjugated donkey-anti-rabbit IgG (1:200, vol/vol, Proteintech, Chicago, IL, USA) without or with Cy3-conjugated goat-anti-mouse IgG (1:500, vol/vol, Proteintech, Chicago, IL, USA) diluted in 10 μg/ml Hoechst 33258 (Sigma-Aldrich) was applied at room temperature for 1 hour. Cells were observed by using a confocal microscope (Olympas, Tokyo, Japan) and the images (50 per group, repeat for 3 times) were analyzed by a design-based unbiased method and a morphometry/image analysis system (Image-Pro Plus 6.0 software package, Bethesda, MD, USA; Edit-Convert to-Gray Scale- Enhance-Invert contrast-Apply contrast –Measure-Count/Size-Measure-Density Mean).
Rotenone-induced hemiparkinsonian rat model
This study was approved by the Ethical Committee on Animal Experimentation of Tongji Medical College, Huazhong University of Science and Technology, China. The rotenone-induced stereotaxical hemiparkinsonian rat (inbred adult female Sprague–Dawley rats, 220–260 g) model was used in this study . Briefly, animals were anesthetized with chloral hydrate (400 mg/kg in 0.9% NaCl, i.p.) and fastened on a cotton bed over a stereotaxic frame (RWD Life Science, Shenzhen, China). Rotenone dissolved in DMSO (3 μg/μl) was infused into the right ventral tegmental area (AP: 5.0 mm; L: 1.0 mm; DV: 7.8 mm) at a flow rate of 0.2 μl/minute. The needle was left in place for additional 5 minutes for complete diffusion of the drug. Rotenone was infused into the right SNc (AP: 5.0 mm; L: 2.0 mm; DV: 8.0 mm) at a flow rate of 0.2 μl/minute, with a 5-minute needle retention. After needle withdrawal, proper postoperative care was given until the animals recovered completely. The animals were administrated with ibuprofen and penicillin in the drinking water for 24 hours to alleviate potential postsurgical discomfort and to prevent infection.
The preparation for electron microscopy (EM) was described previously . Harvested by detaching with 0.25% trypsin, SH-SY5Y were washed twice in PBS, and then fixed in 0.01 M PBS containing 2.5% glutaraldehyde. For the brain tissues, the animals were sacrificed 1 day, 2 days, 1 week, 2 weeks or 4 weeks after the stereological surgery. A 1-mm3 tissue block from the left and right SNc regions (−4.5 to −6.2 mm caudal to the bregma) was micro-punched, fixed in PBS containing 2.5% glutaraldehyde, and preserved at 4°C for further processing. The fragments were post-fixed in 1% osmium tetroxide in the same buffer, dehydrated in graded alcohols, embedded in Epon 812, sectioned with an ultramicrotome, and stained with uranyl acetate and lead citrate. The sections were examined with a transmission electron microscope (TEM; Technai 10, Philips, the Netherlands). For the SH-SY5Y cell-based study, the ultrastructural images were quantified by randomly counting of 100 cells and assessing the percentage of the cells with one or more autophagic vacuoles . For the animals study, three blocks of SNc from each group were sectioned discontinuously for 5 slices, and 50 cell profiles (with a nucleus) were examined on each grid for counting the autophagic vacuoles per cell profile at X 13,500 magnification [40, 41]. Each experiment was conducted in triplicate.
Statistical analysis was carried out by using SPSS version 12.0 for Windows software (SPSS, Chicago, IL, USA). Given a normal distribution in all groups, intergroup differences were assessed by one-way analysis of variance (ANOVA) followed by Least square difference's post hoc test . Results are presented as mean ± SEM, with P value of < 0.05 as statistically significant.