Animals
C57BL6 mice were purchased from Beijing HFK Bioscience (Beijing, China). All the animal work presented has been approved by the Institutional Animal Care and Use Committee of the Tongji Medical College and followed NIH guidelines for the care and use of laboratory animals. All mice were housed in a specific pathogen-free animal facility at the Tongji Medical College on a 12/12 h light/dark cycle.
In vivo mouse model of MPTP
C57BL/6 mice (8–10 weeks old) were treated with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP, 2.5mg/kg, Sigma) by intra-peritoneal injection for 5days. Animals motor behavior were analyzed 1 week after the last injection. Mice were decapitated and the brain SNpc region were removed for WB, qPCR, immunofluorescence analysis or metabolomics related test. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Tongji Medical College following NIH guidelines.
Pole test and Rotaroad test
Behavior tests were evaluated 7 days after MPTP injection. Mice were trained to climb the rotaroad and pole 2 days before the test. For rotaroad test, the angular speed (RPM) is 20 RPM. Animals with impaired motor coordination fall off quickly and the endurance time of each animal was calculated. For pole test, a 50 cm length, 1 cm tip diameter of the vertical wooden with a cork in the top was used. Animals were placed on the cork, and the time for the mouse to climb from the cork to the low end was calculated.
Cell Culture and Treatment
Human SH-SY5Y cells were cultured in 1:1 mixture of Dulbecco’s Modified Eagle Medium and F12 medium (DMEM/F12; Gibco, Thermo Fisher Scientific, Suzhou, China) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, GrandIsland, USA). Cells were treated with MPP+ iodide (MPP+, 2mmol, Sigma, USA) at a final concentration of 2 mmol for 48h. Specific siRNA sequences, targeting human HK2 gene (HK2 siRNA: ACGACAGCATCATTGTTAA), were transfected in SH-SY5Y cells by using the lipofectamine 3000 reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. A non-targeting siRNA sequence (Thermo Fisher Scientific, USA) was used as a negative control. SH-SY5Y was exposed to MPP+ in 6-8h after siRNA transfection. 5nm HK2 inhibitor 3-Bromopyruvic acid (3-Brpa, MedChemExpress, USA) or 2mM MPP+ were used for treatment. SH-SY5Y cells were exposed to 3-Brpa together with MPP+. Cells were harvested after MPP+ treatment for 48h. 15mM L-(+)-lactic acid (L-lac, sigma, USA) treated SH-SY5Y was collected after 24h incubation in the incubator. Cells had been plated 24 h earlier at a concentration of 1×106 cells per well.
Cell Viability Assay
Cell viability was detected using the CCK8 assay system (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). Briefly, SH-SY5Y cells transfected HK2 siRNA, negative control siRNA or intervened with 3-Brpa were treated with or without 2 mmol MPP+ for 48 h. Medium was discarded after incubation for 48 h and replaced with 100 µl of medium containing 10 µl of CCK8 reagent per well and incubated at 37°C for 3 h. The absorbance at 450 nm was measured using an ELX800 microplate reader (Bio-Tek, Winoosk, VT, United States). The values of absorbance were calculated as percentage of the control group.
Flow Cytometry Assay
Cell apoptosis was analyzed using the Annexin V-FITC/PI-Percp apoptosis detection kit (Vazyme, Jiangsu, China). Briefly, SH-SY5Y cells were treated and incubated in incubator. Then, both attached and detached cells were harvested and resuspended with 200 µl of binding buffer, 5µl Annexin V-FITC and 5 µl PI-Percp were added to the mixtures respectively. After staining for 15 min in the dark, flow cytometry was performed with FACS Calibur (Becton, Dickinson and Company, USA). Cells that were negative for PI and positive for Annexin V were identified as early apoptotic cells, and cells that were positive for PI and Annexin V were identified as late apoptotic cells.
Quantitative PCR
Total RNA was extracted from circulating leukocytes using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (+g DNA wiper) (Vazyme, Jiangsu, China)according to the manufacturer’s instructions. Quantitative PCR was performed using ChamQ SYBR Color qPCR Master Mix (Without ROX) (Vazyme, Jiangsu, China) as the instructions in Biorad CFXTM Real-Time PCR detection system. The primer sequences for real-time PCR are listed in Table S1.
Protein extraction, and western immunoblot
Cell lysates were prepared using the radio-immunoprecipitation assay (RIPA) buffer (Servicebio, Wuhan, China) containing a protease inhibitor cocktail (Roche, IN, USA). Western blot analysis of target proteins was conducted as described using appropriate primary antibodies, followed by probing to the corresponding conjugated secondary antibody. The reactive bands were visualized using ECL plus reagents (Servicebio, Wuhan, China), and the relative intensities of each band were analyzed using the Image J 1.46r software. The bands were incubated with following primary antibodies: GAPDH antibody (1:10000, Proteintech, 60004-1-Ig), anti-HK2 antibody (1:1000, abcam, ab209847), anti-TH antibody (1:5000, abcam, ab137869), anti-LDHA antibody (1:1000, Cell signaling technology, 3582S), anti-CD36 antibody (1:500, Proteintech, 18836-1-AP), anti-Ppar-γ antibody (1:500, Proteintech, 16643-1-AP), anti-Bax antibody (1:1000, abcam, ab32503), anti-Bcl2 antibody (1:1000, abcam, ab32124), anti-AMPKα1/2 (H-300) antibody (1:500, Santa cruz Biotechnology, sc-25792), anti-Phospho-AMPKα1/2 (Thr 183/172) antibody (1:500, Santa cruz Biotechnology, sc-101630), anti-Phospho-Akt (Thr308) antibody (1:1000, Cell signaling technology, 4056S), anti-Akt antibody (1:1000, Cell signaling technology, 9272S), anti-Phospho-mTOR (Ser2448) antibody (1:1000, Cell signaling technology, 5336S), anti-mTOR (7C10) antibody (1:1000, Cell signaling technology, 2983S).
Immunofluorescence analysis
The brain was fixed by intra-heart infusion of 4% aqueous buffered formalin. Midsagittal slices from the SNpc were processed for paraffin embedding and sliced into 4-µm sections. After dewaxing and repairing the antigen, the slides were co-incubated with primary antibodies against anti-HK2 antibody (1:100, abcam, ab209847), anti-TH antibody (1:100, Santa cruz Biotechnology, sc-25269), anti-LDHA antibody (1:200, Cell signaling technology, 3582S), anti-GFAP antibody (1:100, Cell signaling technology, 80788S), anti-Iba1 antibody (1:100, Santa cruz Biotechnology, sc-32725), followed by probing with Alexa 594-labelled or Alexa 488-labelled secondary antibodies (Invitrogen). The tissue slides were assessed by fluorescence microscope (Olympus) in a blinded fashion. Negative controls were prepared by omitting the primary antibody. The percentage of HK2+TH/GFAP/IBA1+ double positive cells was calculated by the amount of HK2+TH/GFAP/IBA1+ double positive cells compared to the TH/GFAP/IBA1+ single positive cells in each section. The percentage of LDHA+TH+ double positive cells was calculated by LDHA+TH+/TH+ ratio in each section.
TUNEL assay
One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, Shanghai, China) was used to determine the apoptosis in tissue section according to the manufacturer’s instructions. The assay is based on the green fluorescent probe fluorescein (FITC) labeled dUTP (fluorescein-dUTP). After dewaxing and repairing the antigen, the slides were treated with 20μg/ml proteinase K without DNase for 15min in 37°C. 2.5μl TdT enzyme, 22.5μl fluorescent labeling solution, 25μl Tunel test solution were added in each tissue sample in the section and incubated for 1h in the dark in 37°C. The tissue slides were assessed by using fluorescence microscope (Olympus) in a blinded fashion. The FITC labeled positive cells were calculated as apoptotic cells.
Lactate and pyruvate production assay:
Lactate and pyruvate fluorometric assay kit (Jiancheng Bioengineering Institute, Nanjing, China) was used to determine lactate and pyruvate levels according to the manufacturer’s instructions. The assay is based on the reduction of NAD+. Briefly, the culture media of SH-SY5Y was collected. 10 μl medium was collected from each sample and incubated with reaction mix for 30 min at 37°C. The fluorescence was detected at 510nm (Lactate assay) or 530nm (Pyruvate assay) using a plate reader (BioTek). Lactate and pyruvate levels were normalized to protein content in each sample.
Hexokinase activity
Hexokinase activity assay was performed as manufacturer’s instructions. The assay is based upon the reduction of nicotinamide adenine dinucleotide (NADH) through a coupled reaction with glucose-6-phosphate dehydrogenase and is determined spectrophotometrically by measuring the increase in absorbance at 340 nm. 150µl reaction mixes was loaded in each well of 96-well microplate. The plate was incubated at room temperature for 15 min to achieve temperature equilibration. 20µl whole-cell lysates sample or positive control were added to start the reaction. Hexokinase solution prepared by dissolving hexokinase (Sigma-Aldrich) in 0.05 M Tris-HCl buffer, pH 8.0, served as positive control. Assay buffer without sample served as negative control. Absorbance (A) at 450 nm was recorded for 30 min and 1 hour. The absorbance from initial linear portion of the curve was used to calculate the enzyme activity. The protein concentration was test by BCA assay (Beyotime Biotechnology, Shanghai, China) to normalize the results.
ATP assay
SH-SY5Y cells are cultured in 12-well plate, treated with MPP+ and\or 3-Brpa, incubated for 48h. Medium was discarded after incubation, and placed with 100μl lysate (Beyotime Biotechnology, Shanghai, China) to lyse the cells. Repeatedly pipetting or shaking the culture plate when lysing the cells to make the lysate fully contact and lyse the cells. After lysis, cell lysates were centrifuged at 12000g at 4°C for 5 minutes, and the supernatant was collected for subsequent determination. And 100μl ATP detection working solution was added to the detect in each hole, then leave it at room temperature for 5 minutes, so that all the background ATP is consumed, thereby reducing the background. After 5 minutes, 20μl of sample or standard sample was added to the detect the concentration in each hole, at least 2 seconds later, the RLU value was measured by using an ELX800 microplate reader (Bio-Tek, Winoosk, VT, United States).
Statistical analysis
All in vitro experiments were conducted with at least three independent biological replications. When only two experimental groups were compared, the unpaired t-test was used. Multiple comparisons were treated by one-way ANOVA followed by Tukey’s test. Statistical analysis of the data was conducted using the GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA), and the results were expressed as mean ± SEM. In all cases, a P value < 0.05 was considered statistically significant.