2.1 Chemicals
Alpha mangostin was isolated from the pericarp of Garcinia mangostana L. and characterized in our Chemical Biology laboratory at NIPER Ahmedabad, as reported previously(Parkhe et al., 2020). Rotenone, Acrylamide, Sodium dodecyl sulfate (SDS), Tween-20, Formaldehyde were procured from Sigma Aldrich. 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), Ammonium persulfate (APS), Tetramethyl-ethylenediamine (TEMED), Sodium chloride, Sodium lauryl sulfate (SLS), Bis-acrylamide, Sodium deoxycholate, Triton-X 100, Phenylmethyl sulfonyl fluoride (PMSF), Glycerol, Carboxymethylcellulose (CMC) were procured from Hi-Media Laboratories Pvt. Ltd. Β-mercaptoethanol was procured from Alfa Aesar. Glacial acetic acid, Potassium chloride, Hydrochloric acid, Sodium hydroxide, chloroform were procured from Fischer scientific. Polyethylene glycol-400 was purchased from Merck. Tris-HCl was purchased from Thermo Fischer. Ladder and polyvinylidene difluoride (PVDF) membrane were procured from Bio-rad. HRP conjugated Enhanced Chemiluminescent Substrate Reagent kit was procured from Invitrogen. BCA (Bicinchoninic acid) reagent was purchased from Thermo Scientific. Primary antibody (TFEB) was procured from Sigma-Aldrich, while primary antibodies (LC3B, α-Syn, phospho-α-Syn, phospho-AMPK, total-AMPK, Beclin-1, β-actin) and secondary antibodies were purchased from Abcam. For immunohistochemistry, the Vectastain ABC kit was purchased from Vector Laboratories.
2.2 Animals
The Institutional Animal Ethics Committee approved all experimental procedures (IAEC: IAEC No.- IAEC/2018/033), and all the experiments were conducted as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India for the Care and Use of Laboratory Animals. Male C57BL/6 mice (7–8 weeks old, 20-28 g) were procured from the Zydus Research Center, Ahmedabad, and housed in a climate-controlled room at temperature: 20 ± 10 °C and humidity: 50 ± 10% (12 hours light/dark cycle), with free access to food and drinking water ad libitum. Behavioral experiments were performed in the light phase.
2.3 Study plan
Animals were habituated to the laboratory conditions for a week before starting the experiment. Behavioral parameters were recorded till 9 weeks at every two weeks. Animals were distributed blindly into 4 groups, namely: Group I: Vehicle control -Animals were administered with 0.5% CMC and a combination of Polyethylene glycol (PEG)-400 and H2O (6:4), Group II: Rotenone group- Animals received rotenone daily (10mg/kg, suspended in 0.5% CMC, Oral) for nine consecutive weeks. Group III: Rotenone plus alpha mangostin (R+AM) – Animals received co-treatment of rotenone (10mg/kg, suspended in 0.5% CMC, Oral) and alpha-mangostin (40mg/kg, suspended in PEG: H2O; 6:4, Oral) for nine consecutive weeks. Group IV: Alpha-mangostin (AM) alone - Animals in these groups were administered with alpha mangostin (40mg/kg, suspended in PEG: H20, Oral) for nine consecutive weeks.
Rotenone is freely soluble in chloroform. A stock solution of 50 mg of rotenone in 1 ml of chloroform was prepared and stored at -20°C for not more than two weeks. For the model induction, rotenone emulsion was prepared in 0.5% CMC(Parkhe et al., 2020). AM was solubilized in the solution of PEG-400, and then water was added. (PEG:H20; 6:4). In all experiments, AM was co-administered daily for nine weeks before 1 hr of rotenone exposure. The dose and route of administration of AM used in the present experiment were chosen based on the previous results(Herrera-Aco et al., 2019). All the animals were sacrificed by decapitation using isoflurane at the end of the 9th week, and brains were quickly removed. Subsequently, striatum and cortex were isolated in ice-plate and processed for western blotting.
2.4 Behavioral assessments
2.4.1 Motor activity
Motor activity was measured using the round beam walk (RBW) test. The beam test was set up as described in a previous study(Suidan et al., 2013). Mice received the two training trials on two beams with different widths. In the first training trial, a wider beam (25mm in diameter) was placed 50cm above the tabletop with one end as the start platform, and another end is placed on the home cage. Mice were placed on the start platform and directed to move towards the home cage. For the second training trial, the wider beam was replaced by medium (15mm in diameter), and mice were placed on the start platform and then moved towards the home cage. After every training trial apparatus was clean with 1% acetic acid. In the test trial case, a medium beam was replaced by a narrow beam (10mm in diameter). To visualize the animal’s feet on the beam, a video camera was placed at the end of the beam opposite the home cage. There was no interval between training and testing trials. Time is taken by the animal to cross the beam and the number of slips was analyzed from the recording by a blind evaluator to the groups.
2.4.2 Muscle strength
Muscle strength was measured using a grip strength meter. The grip strength meter is comprised of a grasping grid linked to a force transducer and digital display unit (IITC Life Science). To assess the grip strength, each mouse was placed on the grid, and the mouse was allowed to grasp the grid with all four paws. Once the mouse grasped the grid and then the mouse was slowly pulled back by the tail base until the grasp was overcome. The maximum force (in grams) required to hold the grid was recorded. Three readings were taken with the time interval of 30 mins, and the mean of the obtained readings was used as representative grip force of the animal at that specific time(Meyer et al., 1979).
2.4.3 Memory impairment
Y-maze assists in evaluating short-term spatial memory based on rodents' inherent nature to explore a novel environment. Y-maze used was made up of wooden material with acrylic accessory and having three arms of equal length were interconnected at 120° angle with each other. Arms were defined as A, B, and C. Every time mice were placed in the center of the Y-maze facing A-arm, they freely explored the maze for 7 mins while being monitored by an experimenter. Mice were considered to visit the particular arm if they entered the arm with all the four-limbs inside. The sequence of entries in each arm by animal and spontaneous alternation was calculated. In the case of spontaneous alternation, we have allowed mice to explore the maze for 7 mins and considered made alternation if mice visited three arms sequentially without repeating any and counted the linear entries. We have also counted repeated entries, and further percent alternation was calculated by using the following equation(Garcia and Esquivel, 2018).
% Alternations = Linear entries / Total entries * 100.
The maze was wiped with 1% acetic acid before testing the next animal for memory impairment.
2.5 Western blotting
Frozen striatum and cortex samples were thawed, homogenized in RIPA buffer (25mM Tris, pH 7.8, 150mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate, 1% Triton X-100, PMSF, and protease inhibitor cocktail), and centrifuged at 16,000g and 4˚C for 20 min. Further, the supernatants were collected, and the protein estimation was performed using the bicinchoninic acid (BCA) method. For Western blotting, the samples were loaded and resolved in 12% and 15% polyacrylamide gels based on the desired molecular weights of proteins. After that, electrophoresis was carried out at constant voltage. The resolved proteins were transferred onto the PVDF membrane followed by blocking with 3% BSA for 2 hours. Further, the incubation of membrane was carried out by using primary antibodies of LC3B (rabbit mAb, Abcam, AB192890 1:2000), TFEB (rabbit pAb, Merck, SAB4503154, 2:1000), Beclin-1 (rabbit mAb, Abcam, ab207612, 1:2000) ,p-AMPK (rabbit pAb, Merck, SAB4503754,1:1000), T-AMPK (rabbit pAb, Abcam, AB131512, 1:1000), alpha-Synuclein (rabbit mAb, Abcam, AB212184,1:1000), Phospho-alpha-Synuclein (rabbit mAb, Abcam, AB51253, 1:1000) and β-actin (mouse mAb, Abcam, AB6276, 0.5:5000) overnight at 4°C with gentle shaking. The next day, the antigen- primary complexed membranes were washed with TBST and further incubated for 1 hour with their respective horseradish peroxidase (HRP)-conjugated secondary antibodies (rabbit pAb, AB6721, 0.5:5000; mouse pAb, AB6789, 0.5:5000). β-actin was used as an internal control and used to normalize the protein levels. The expression level was quantified by densitometric analysis with the help of ImageJ software (NIH, USA)(Parkhe et al., 2020).
2.6. Immunohistochemistry for TH+-fibres in striatum and TH+-cells in SNc:
Mice were anesthetized using a ketamine-xylazine cocktail, accompanied by transcardial perfusion initially with PBS (phosphate buffer saline), followed by 4% paraformaldehyde in 0.05 M PBS, pH 7.4. Before processing, brains were rinsed with PBS for 1 hr. The brains were then cryoprotected with 10%, 20%, and 30% sucrose solution (dissolved in 0.1 M Phosphate buffer). Coronal sections (30 µm-thickness) were cut on a cryostat (Thermo Fisher Scientific) and immersed in 0.05M PBS. Mouse brain sections were taken at three different levels from bregma to A= +1.34, M= +1.18, P= +1.1 (for striatum) and A= -2.92, M= -3.16, P= -3.64 (for SNc) according to Paxinos and Watson atlas mouse brain. Immunohistochemistry was carried out as reported previously(Khairnar et al., 2010), with minor modifications.
Briefly, free-floating sections were pre-treated with 1% hydrogen peroxide and 0.1% Triton-X-100 for 20min to remove endogenous peroxidase activity followed by incubation in blocking buffer (Diluted blocking solution, Vectastain kit, Vector Laboratories) for 1 hr. Further, sections were incubated at 4°C overnight with anti-tyrosine hydroxylase (1:500 dilution, AB152, Merck) in a Blocking solution. The sections were then incubated with a biotinylated secondary antibody (Vectastain kit, Vector Laboratories) at 4 °C for 90 mins and avidin-biotin-peroxidase (Diluted ABC solution, Vector Laboratories) at 4°C for 30 min. The visualization was done by incubation with SIGMAFAST 3,3′-diaminobenzidine tablets (D4293, Sigma-Aldrich) for 8-15 mins. Finally, the sections were mounted on slides, air-dried and dehydrated in increasing grades of ethanol, coverslipped, and imaged using a bright-field microscope at 10X (Leica ICC50 E).
2.7. Quantitative Analysis
The striatal TH+-fibers density was quantified by using image densitometry analysis tool provided in the Image J software (https://imagej.nih.gov NIH, USA) and represented in terms of optical density (OD), as described previously(Altarche-Xifro et al., 2016; Jewett et al., 2017; Tian et al., 2016; Wang et al., 2018). To normalize the gray-scale range (0–255) into OD values, the Rodbard calibration function was employed given in the software. Each image was then converted into an 8-bit image. The OD values were normalized by eliminating OD values of background. At each level of the striatum, the obtained value was first normalized to the vehicle, and values from different levels were averaged after that. Here, OD values represent the strength of the TH+-fibers.
The number of dopaminergic neurons was determined as previously described (Kühn et al., 2003). Briefly, we manually counted TH‑positive cells under bright‑field illumination in the SNc using an x10 objective. Cell counts were determined blindly and it should be noted that the analyses of the TH-immunoreactive profiles were restricted to the SNpc and thus excluded the ventral tegmental area. In addition, neurons were only counted if they contained a nucleus that was surrounded by cytoplasm.
2.8. Statistical analysis:
Data were represented as mean ± SEM. Means of multiple groups were compared using Two-way and One-way ANOVA (analysis of variance), followed by Tukey's multiple comparison test. Differences were considered statistically significant if p<0.05. Statistical software used for analyzing the data was GraphPad Prism, version 5.01, GraphPad Software, Inc.