Adult male C57BL mice were purchased from SPF Biotechnology (Beijing, China). α-Syn knockout mice were provided by Professor Yang Hui of Capital Medical University. All animals were housed at room temperature under a 12/12 h light/dark cycle and had free access to food and water. All animal experiments were approved by the Animal Care and Use Committee of the Institute of Animal Management, Capital Medical University, and conducted in accordance with ethical requirements.
2. Hypoxic treatment
All mice were administered hypoxic treatment in a closed hypoxic chamber (China Innovation Instrument Co., Ltd, Ningbo, Zhejiang, China), which accurately set the desired hypoxic concentration and pattern. For chronic hypoxia, mice were treated continuously with 13% O2 for 1, 3, 7, and 14 days. The hypoxic chamber was opened briefly for food and water additions every 3 days. Intermittent hypoxic mice were treated with 10 cycles of 5-min 13% O2 (hypoxia) and 5-min 21% O2 (normoxia) per day for 14 days, followed by an additional continuous hypoxic treatment. Hypoxic-reoxygenated mice were fed normoxic for 28 days after receiving chronic hypoxia.
3. The siP-Syn peptide administration
The polypeptide we used was based on a previous study. The sequence was 5-FITC-(Acp)-YGRKKRRQRRRYEMPSEEGYQD. The peptide was formed by fusing the HIV Tat (transcriptional counter-activator) signal (YGRKKRRQRRR) with a competing peptide spanning amino acids 125 to 135 (YEMPSEEGYQD), the phosphorylation site (ser129) in α-syn (siP-Syn).
The siP-Syn peptide (Chinapeptides, Shanghai, China) was injected via the tail vein every 3 days for 6 weeks at a dose of 10 mg/kg. The mice were treated in a hypoxia-inducing environment and tested for behavior two weeks after injection. Polypeptides were stored at -20ºC and protected from light. Repeated freezing and thawing were prevented.
4. Behavioral tests
The cognitive function of the mice in each group was evaluated by novel object recognition tests and Morris water maze tests. For the novel object recognition tests, the experimental device was a rectangular box with a length, width, and height of 40 cm. It had no lid, and the camera was placed on the top. The experiment was divided into three stages: adaptation stage, familiarity stage, and experimental stage. Adaptation stage: Each mouse was put into the experimental device and left to explore freely for 5 min to adapt. Familiarity stage: Two identical objects A (old objects) were placed in the experimental device, and the mice were allowed to explore freely for 5 min. Experimental stage: One object A was removed from the experimental device, and a new object B was put into it (the color and shape were different from A). The mice were put into the experimental device and left to move freely for 5 min. The time that the mice explored the old and new objects were recorded. Discrimination index = (new object recognition time - object recognition time)/(new object recognition time + object recognition time).
The water maze device was a circular device with a diameter of 120 cm. It was divided into four quadrants and had a small platform. Each quadrant had a different color and marker shape for spatial memory recognition by the mice. Water was added to the pool until it was 1–2 cm higher than the platform. The water temperature was about 25ºC. Training stage: The mice were placed in the pool at the same position in one of the four quadrants, and the time spent finding the platform was recorded. If the mice could not find the platform within 60 seconds, the time spent finding the platform was recorded as 60 seconds. The mice were then guided to swim to the platform and allowed to stay there for 5 seconds. The training was performed once a day for 5 days in total. Test stage: On day 6, the platform was removed and the mice were placed in the quadrant farthest from the platform. The time at which the mice first found the location of the original platform and the number of times of crossing within 60 seconds were recorded.
5. Nissl staining
Brain tissue from the mice were cut into 10 μm sections on a frozen slicer and pasted on a slide. The sections were then fixed in 70% ethanol and successively dehydrated in 100% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 2 minutes each. After the xylene became transparent, the sections were placed in 1% tar purple (Solarbio, G1430) and incubated for 30 min. The sections were cleaned with distilled water and separated with 70% alcohol for several minutes. The sections were dehydrated with 70% ethanol, 80% ethanol, and 95% ethanol for 2 min each, dehydrated with 100% ethanol, and sealed with neutral gum.
6. Separation of soluble and insoluble components
Single mouse hippocampal tissue was isolated, and 300 μl mixed protein lysis solution with RIPA (Applygen, C1053), a protease inhibitor (Merck, 539134), and a phosphatase inhibitor (Merck, 524628) (=100:1:2) were added. After ultrasonic crushing, ice cracking was performed for 30 min. The preparation was centrifuged at 4ºC at 12,000 rpm for 15 min. The supernatant contained the RIPA-soluble protein components. The precipitate at the bottom of the tube was the insoluble fraction. Urea (8 M) was added to the precipitate to dissolve the RIPA-insoluble protein components.
7. Separation of nuclear components
Nuclear separation was performed using a nuclear separation kit (Applygen, P1202) according to the instructions. Briefly, the mouse hippocampus was put into a pre-cooled glass homogenizer, and 500 μL of Buffer1 reagent was added. After grinding and mixing, the homogenate was placed on ice for 10 min for full lysis. The homogenate was transferred to a new pre-labeled centrifuge tube and centrifuged at 800 x g for 5 min at 4ºC. After centrifugation, the nuclear crude extract was at the bottom of the tube. Then, 500 μL Buffer2 was added to the bottom of the tube containing the crude nuclear extract. The crude nuclear extract was suspended and mixed by shock. Following centrifugation at 4,000 × g at 4ºC for 5 min, the supernatant was discarded. Then, 500 μL Buffer2 was added again after suspension and centrifuged again to obtain relatively pure nuclei. Finally, 50 μL suspension buffer was used to re-suspend and obtain complete and unbroken nuclei.
8. Western blots
Mouse hippocampus protein lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently immunoblotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk at room temperature for 1 h. After TBST washing (three times, 5 min per wash), the membranes were incubated with the indicated primary antibodies at 4°C overnight with shaking. The primary antibodies included β-actin (Sigma-Aldrich, A5316), lamin B1 (HUABIO Antibodies, ET1606-27), HIF-1α (Abcam, ab228649), HIF-2α (Novus, NB100-122), Acer2 (PA5-39016), PP2A (Invitrogen, MA5-32920), p-PP2A (Invitrogen, MA5-32158), α-syn (BD, 8249518), and p-α-syn (Abcam, ab51253). After incubation, the membranes were washed three times and then incubated at room temperature for 1 h with secondary antibodies, including IRDye 680RD goat anti-mouse IgG (H+L) (Licor, 926-68070), IRDye 680RD goat anti-rabbit IgG (H+L) (Licor, 926-68071), IRDye 800CW goat anti-mouse IgG (H+L) (Licor, 926-32210), IRDye 800CW goat anti-rabbit IgG (H+L) (Licor, 926-32211). Membranes were scanned using a detection system (Odyssey, USA), and band intensities were normalized to β-actin. Statistical analyses were performed using ImageJ and GraphPad software.
Mice brain sections were boiled in citric acid buffer (pH = 6.0) for 10 min for antigen repair. Sections were placed in 0.01 M PBS and washed 3 times for 10 min each. Sections were placed in 1% PBST and incubated at room temperature for 30 min. Sections were then placed in 5% BSA and sealed for 2 h at room temperature. The sections were then incubated overnight at 4℃ in primary antibody: p-α-syn (WAKO, 015-25191) or MAP2 (Abcam, ab32454). The sections were washed in 0.01 M PBS 3 times for 10 min each. Sections were placed in fluorescent secondary antibody and incubated at room temperature for 2 h, protected from light. The secondary antibodies were goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (Invitrogen, A11029), and goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 (Invitrogen, A11037). DAPI was used for nuclei staining before sections were mounted (Sigma, D9542). A fluorescence microscope was used to observe and photograph the images.
10. Enzyme activity detection
A PP2A enzyme activity detection kit (Enzyme-Linked Biotechnology, JK56324), CK enzyme activity detection kit (Enzyme-Linked Biotechnology, JK68421), and PLK enzyme activity detection kit (Enzyme-Linked Biotechnology, JK89575) were used according to the instructions to detect enzyme activities. Briefly, a standard hole, blank hole, and sample hole were set. Then, 50 μL standard was added to the standard well, 50 μL sample dilution buffer to the blank well, and 10 μL sample with 40 μL dilution buffer to the sample well. Then, 100 μL horseradish peroxidase (HRP)-labeled antibody was added to each well. The wells were sealed and incubated at 37℃ for 60 min. The plate was washed 5 times before 50 μL substrates A and B were added. The plate was incubated at 37℃ for 15 min away from light. Stop solution (50 μL) was added, and the OD value of each well was measured at 450 nm within 15 min. A standard curve with R2 > 0.99 was drawn to prove the existence of a linear relationship. Finally, the concentrations and activities of PP2A, CK, and PLK were calculated.
11. Targeted metabolomics
The mouse hippocampus homogenate was melted at 4°C for 30–60 minutes. Then, 100 μL homogenate was transferred to a labeled 1.5 mL centrifuge tube, 300 µL methanol added, and the contents mixed on a vortex mixer for 1 h. The precipitate was collected by centrifugation at 12,000 rpm for 10 min. Then, 95 µL supernatant was transferred to a labeled 1.5-mL microcentrifuge tube, and 5 µL of an internal standard of Sph/Cer was added. After mixing well and centrifugation at 12,000 rpm for 10 min, the supernatant was transferred to a 250 µL vial insert for analysis. The ACQUITY UPLC HSS T3 2.1 × 100 mm, 1.8 μm column (Waters, Milford, MA, USA) was used as the stationary phase. It was suited for lipid retention. The mobile phase consisted of solvent A (0.1% formic acid-acetonitrile/water (3:2 v/v)) and solvent B (0.1% formic acid-isopropanol/acetonitrile (9:1 v/v)) with a gradient elution (0–1.5 min, 68–68% A; 1.5–15.5 min, 68–15% A; 15.5–15.6 min, 15–3% A; 15.6–18.0 min, 3–3% A; 18.0–18.1 min, 3–68% A; 18.1–20 min, 68–68% A). The flow rate of the mobile phase was 0.26 mL/min. The column temperature was maintained at 55°C. The injection volume was 4 μL. A Waters TQ-S mass spectrometer equipped with an ESI probe was employed. The positive ESI source temperature was 150℃, the capillary voltage was 3.0 kV, the cone voltage was 20 V, the desolvation temperature was 500℃, and the desolvation gas flow was 1,000 L/h. The LC-MS system was controlled by Masslynx software. Data were collected and processed with the same software.
An RNeasy kit (Qiagen, 74104) was used to extract total RNA from mice hippocampal tissue, and then the Transcriptor High Fidelity cDNA synthesis kit (Roche, 5081963001) was used to reverse transcribe the RNA into cDNA. All operations were according to the instructions. The following primers were used: mouse β-actin, forward: 5´-GGCTGTATTCCCCTCCATCG-3´, reverse: 5´-CCAGTTGGTAACAATGCCATGT-3´; mouse Acer2, forward: 5´-TGGTGCGAGGACAACTACACTATC-3´, reverse 5´-AAGCATCTGACCCAGGAAACTC-3´.
13. Primary neuron culture and processing
A newborn mouse was disinfected and its head was cut off immediately. The cerebral cortex was removed and the hippocampus was separated under a stereomicroscope. The tissue was cut to less than 1 mm3 in DMEM medium (Gibco, C11995500B7). A 1:40 volume ratio of papain solution was added with subsequent digestion at 37℃ for 30 min. The papain was removed, DMEM culture medium was added, and the tissue was blown to make a cell suspension. The cell suspension was passed through a 40 μm filter (Falcon, 352340). The cells were diluted to a certain density and then moved to polylysine (Sigma, P1399)-coated plates for culturing at 37℃ in a 5% CO2 incubator. After 4–8 h, DMEM was replaced with Neurobasal medium (Gibco, 21103049) containing 50×B27 (Gibco, 17504044), 100× glutamine (Invitrogen, 25030081), and 100× penicillin-streptomycin (Thermo, 15140122). Half of the medium was changed every 3 days after cell adhesion.
14. Cell viability detection
Cell viability was detected by the MTT assay kit (Sigma, M2128). MTT (5 mg) was placed in a 5 mL EP tube, and PBS buffer (1 mL) added to dissolve the MTT. After blending, the stock solution was kept at -20℃ and protected from light. After the experimental treatments, the cell supernatant in the 96-well plate were discarded, 10 μL MTT solution and 90 μl culture solution were added to the pre-designed cell wells. The cells were incubated at 37℃ with 5% CO2 for 4 hours. The supernatant was discarded and 100 μl DMSO was added and mixed gently for 10 min while avoiding light. The OD value at 490 nm was detected by an enzyme marker.
15. Cytotoxicity detection
Cytotoxicity was detected by the LDH assay (Roche, 4744926001). The powder was dissolved in ddH2O and mixed thoroughly to make the catalytic solution. Then, 250 μL of the catalytic solution was added to the staining solution (11.25 mL) and mixed thoroughly. Then, 100 μL cell supernatant of each group was added to the new 96-well plate. The LDH reaction solution (100 μL) was added with subsequent incubation at room temperature away from light for 30 min. After the incubation, 50 μL stop solution was added to each well and gently mixed for 10 min. The OD value of each well was measured at 490 nm by a microplate reader.
16. Cell death detection
PI/Hoechst detection was used to detect the cell death rate. Hoechst labels all cells as blue fluorescence, and PI labels only dead cells as red fluorescence. Therefore, the ratio of red to blue can be used to calculate the cell death rate. After the cells were treated, the original medium was discarded, and the cells were rinsed three times with PBS. The PI and Hoechst mixture was added into the cell culture well and incubated at 37℃ for 10 min under dark conditions. The cells were removed from the incubator, the mixture of PI and Hoechst was discarded, and the cells were rinsed three times with PBS. Confocal microscopy was used for observation and imaging.
17. Statistical analysis
Excel and GraphPad Prism 9.0 software were used for data preservation, recording, statistics, and analyses. Image data were analyzed by ImageJ and other software. All results were analyzed using the t-test, one-way ANOVA, and two-way ANOVA as appropriate. Data are expressed as the mean ± standard error (mean ± SEM), with P ≤ 0.05 as a significant difference.