2.1 Reagents
Rotenone (R8875), peptidoglycan (69554) and DMSO (C6164) were purchased from Sigma-Aldrich. Rapamycin was purchased from MCE (HY-10219, USA). Fetal bovine serum (FBS) was purchased from Invigentech (A6901, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Cytiva (SH30022.01, USA). Penicillin–streptomycin solution (CR2102024) and trypsin (G4004) were purchased from Servicebio (Wuhan, China). DAPI (ab104139) was purchased from Abcam.
2.2 Animals and experimental design
Male C57BL/6J mice aged 10 weeks (22–24g) were purchased from Huaxing Laboratory Animal Research Center, Zhengzhou. The mice were housed four per cage and kept on a standard environmental condition (temperature 22 ± 2°C, humidity 50–60%,12h light/dark cycle) with free access to food and water. All the procedures were performed following the guide to care and use laboratory animals. The animal experiments were approved by the Medical Ethics Committee of the Second Affiliated Hospital of Zhengzhou University (No. 2022142).
The schematic illustration of the animal experimental design is shown as Fig. 3a. A total of 48 mice were randomly and equally divided into four groups: the control group, the rotenone group (i.p. rotenone 0.75mg/kg, every other day for 4 consecutive weeks)[25], the rotenone plus rapamycin group (i.p. rotenone 0.75mg/kg, every other day for 4 consecutive weeks, o.g. rapamycin 2mg/kg, every other day for 4 consecutive weeks begin at week 5)[26–29] and the rapamycin group (o.g. rapamycin 2mg/kg, every other day for 4 consecutive weeks begin at week 5). Rotenone was freshly prepared in a solution of 1% DMSO + 5%Tween-20 (diluted with PBS), rapamycin was diluted in the same way. Control mice received an equivalent volume of vehicle (1% DMSO + 5%Tween-20 diluted with PBS). These mice were weighed weekly for 8 weeks. Behavioral tests were performed at the last four days. All mice were sacrificed after behavioral tests for further analysis.
2.3 Cell culture and treatment
BV-2 murine microglia cells were obtained from Mingjing Biology, shanghai. Cells were cultured in DMEM/high glucose medium contain 10% FBS, penicillin (100 units/mL), and streptomycin (100 µg/mL) and maintained at standard condition (37℃, 5% CO2). Rapamycin (15nM)[24, 30, 31] and/or peptidoglycan(12.5µM)[32] were added to the cells coculture for 24 hours, then samples were collected for further analysis after washed with PBS for 3 times.
2.4 Behavioral tests
Rotarod and open field tests were employed to evaluate Rotenone-induced motor deficits in mice.
2.4.1 Rotarod test
Mice were placed on the rotarod, and then the rod revolved at a speed of 5–15 rpm /min for 5 minutes to train them. After the training, mice were allowed to rest for 1 hour, then they were placed on the rotating rod revolved at a gradual increasing speed of 5–40 rpm/min for 3 minutes, then 40 rpm/min for 2 minutes. The experiment was ended when the animal slid off the spinner or grabbed the spinner and spun more than 2 turns and the time was defined as latency. Each mouse tested twice a day with an interval of 1 h for 3 consecutive days.
2.4.2 Open field test
Locomotor activities were evaluated in the open-field apparatus consisting of a light-yellow plastic board (45 × 45 cm) surrounded by light yellow walls (40 cm in height). each mouse was gently placed in the center of the open field area to explore it for the next 5 minutes. The movements were recorded by a camera tracking system (Smart5. System, the Netherlands). Total distances moved were calculated.
2.5 Western blotting.
The midbrain and caudate putamen tissues of mice were dissected in cold PBS. All the samples were stored at -80℃ refrigerator. The tissues and cells were respectively homogenized in cytoplasmic lysate containing 1mM sucrose, 100nM DTT, 5mM EGTA, 10mM Tris-HCl (pH 7.4), 5mM MgCl2, protease inhibitors and phosphatase inhibitor cocktails. The homogenate was centrifuged at 2,500 × rpm for 10 min at 4°C. The supernatant obtained after centrifugation used to detect the expression of cytoplasmic and membrane proteins. The precipitate was dissolved in nucleus lysate containing cytoplasmic lysate,0.1% TritonX-100, 1.5% SDS. The mixture was further ultrasonic crushed on ice for detect the expression of nucleus proteins. Equal amounts of proteins were electrophoresed in 10% or 12% SDS-PAGE and transferred onto a Poly-vinylidene fluoride (PVDF) membrane. We blocked the membranes with 5% BSA at room temperature for 2 hours and then probed them with the following primary antibodies: anti-P-α-synuclein(Ser129 phosphorylated, 1:2000, Abcam, #ab51253), Anti-LC3B (1:1000, Abcam, #ab48394), Anti-Tyrosine hydroxylase (TH, 1:2000, Proteintech, #25859-1-AP), anti-TNF-α (1:2000, Proteintech, #60291-1-Ig), anti-TLR2 (1:1500, Invitrogen, #14-9922-82), anti-NFATc2 (1:1000, CST, #5861), anti-P-NF-κB p65(Ser536 phosphorylated, 1:500, #WL02169) and H3/β-actin (1:5000, ab1791, Abcam;1:2000, GB11001, Serviebio,) at 4°C overnight. The membranes were then washed 6 minutes for four times with TBST and probed with horseradish peroxidase-linked anti-rabbit IgG (1:5000, Abbkine) antibody or horseradish peroxidase-linked anti-mouse IgG (1:5000, Abbkine) antibody at room temperature for 2 hours. The blot signals were detected by ECL reagents (biosharp, BL520A). Band intensities were quantified using ImageJ software (USA), All blots were compared to H3 or β-actin.
2.6 Real-time PCR.
Total RNA was extracted from midbrain/cells using the Universal RNA kit (Cofitt, LBD9515) following the manufacturer’s protocol. Then, the RNA was reverse transcribed with the High speed-strand cDNA Synthesis Plus Kit (Cofitt, LBQ7513). Real-time PCR was carried out in QuantStudio sequence detection system with the SYBR qPCR Master Mix obtained from Vazyme (Q511-00) based on the manufacturer’s protocols. Use the glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) gene as the internal control. All datas were normalization of GAPDH using the 2−ΔΔCt method.
The following primer sequences were used:
α-synuclein: forward: 5′-TGGCTTTGTCAAGAAGGACCAGATG-3′
reverse: 5′-CCACAGGCATGTCTTCCAGGATTC-3′
TLR2: forward: 5′-CTCCCAGATGCTTCGTTGTTCCC-3′
reverse: 5′-GTTGTCGCCTGCTTCCAGAGTC-3′
TNF-α: forward: 5′-CACCACGCTCTTCTGTCTACTGAAC-3′
reverse: 5′-TGACGGCAGAGAGGAGGTTGAC-3′
GAPDH: forward: 5′-GGTTGTCTCCTGCGACTTCA-3′
reverse: 5′-TGGTCCAGGGTTTCTTACTCC-3′.
2.7 Immunofluorescence.
After anesthesia, mice were perfused transcardially with normal saline(NS) and then with cold 4% paraformaldehyde (PFA). The brains were then kept in 4% PFA at 4°C for 4 hours. The brains were dehydrated with 20% sucrose for 1 day then dehydrated with 30% sucrose for an additional 2 days. 25µm coronal sections were cut from the the brain in a cryotome and blocked with 0.3% Triton-100/PBS containing 10% goat serum for 1 hour. In the case of cell samples (BV-2), these were washed 3 times with cold PBS and then fixed with 4% paraformaldehyde for 20 minutes. Samples were permeated in PBS containing 1% Triton X-100 for 10 minutes then blocked with 5% goat serum in PBS for 1 hour at room temperature. After that, probed them with the following primary antibodies: anti-P-α-syn (1:500), Anti-TH (1:500), anti-TNF-α (1:500), anti-TLR2 (1:250), anti-NFATc2 (1:50), anti-glial fibrillary acidic protein (GFAP, 1:500, Proteintech, #16825-1-AP) and anti-ionized calcium binding adaptor molecule-1 (Iba-1,1;500, Affinity, #DF6442) at 25°C overnight. The next day, the samples were washed 5 minutes for four times with PBS and further probed with Cy3- or 488-labeled secondary antibodies (all 1:300, Servicebio) for 2 hours at room temperature. All immunofluorescence-stained images were examined using a Leica DMI4000 fluorescence microscope and captured with a DFC365FX camera. The amounts of TH immunoreactive neurons and Iba-1 immunoreactive microglial in the region of SN were calculated under a microscope (100×). ImageJ software to measure the fluorescence intensity of each target protein in the SN/striatum region or each group of cells.