Transcriptional Analysis of SUMOylation Protein
Differentially expression genes (DEGs) were selected on the basis of a false discovery rate (FDR) value < 0.05 from three datasets, including GSE8262 [31], GSE62385 [32], GSE1357 [33] and the genes related to the SUMOylation from the Human Protein Atlas. The bioinformatics analysis was performed with R software (version 4.0.3 GUI 1.73, Catalina build).
Cell Culture
BV-2 microglial cells and HT-22 neuronal cells were cultured in Dulbecco's modified Eagle's medium (Gibco, DMEM) with 1% penicillin and streptomycin and 10% fetal bovine serum (Gibco) in a humidified incubator containing with 5% CO2 at 37°C. The supernatant medium of BV‐2 cells with or without SENP1 overexpression was collected to culture HT-22 cells.
Cih Treatment Of Bv-2 Cells
BV-2 cells were placed into cell incubator, and a gas control chamber was used to regulate the percentage of nitrogen and oxygen in atmosphere. Oxygen levels alternate between 1% and 21% within 400 s, in a cyclic repetitive format. After 12 hours of CIH treatment, BV‐2 cells were collected for subsequent experiments. In the control group, cells were cultured in a normoxic state (21% oxygen content).
Cells Infection With Senp1 Adenovirus For Gene Overexpression
SENP1 overexpressed BV-2 cells were constructed by SENP1 adenovirus infection. Cells were cultured under CIH or normoxic conditions for subsequent experiments. BV‐2 cells were inoculated into a 12‐well plate for 24 h. Two hours after adding 2 µl of SENP1 adenovirus or 1µl of GFP adenovirus control solution, 1 mL of fresh medium was added. After 48 hours, cells were replaced with fresh medium before treatment with CIH or normoxia. Transfection efficiencies of adenovirus was assessed via evaluating GFP fluorescence and were proven to be greater than 80%. qRT‐PCR, western blot and immunofluorescence analysis were used to verify the validity of SENP1 overexpression.
Small Interfering Rna Transfection
The expression of TOM1 in BV-2 cells was knocked down using small interfering RNAs (siRNAs), with a non-targeting siRNA (siRNA-NT) serving as the negative control. siRNA was purchased from Sigma-Aldrich (Shanghai) Trading Co. Ltd. (EMU210641, Sigma-Aldrich) and transfected into BV‐2 cells using Lipofectamine 3000 reagent (Catalog #. L3000015, invitrogen). Four siRNAs were used in this study, one negative control and three positive siRNA-TOM1s. Finally, the siRNA with highest transfection efficiency among the three positive ones was selected as the official siRNA-TOM1 for the experiment, and the target specific sequence was demonstrated in the methods section. The transfection efficiency was detected by western blot analysis.
Elisa Assay
Levels of IL-1β and TNF-α in supernatants were measured by ELISA kits (BioLegend) following the manufacturers’ instructions.
Animals
Specific pathogen-free (SPF) C57BL/6J mice, aged 10–12 weeks old (weight, about 30g), were obtained from the Department of Experimental Animal Science, the School of Medicine, Shanghai Jiao Tong University, which were housed under a 12/12-hour light/dark cycle in a room of controlled humidity of 40%~60% with a constant temperature of 22°C ± 1°C and provided access to standard food and water.
Generation of SENP1 +/- mice [25]: SENP1+/- ES cell line XG001 cells were generated using a gene trap protocol with the trapping construct pGT1Lxf containing the intron from the engrailed-2 gene upstream of the gene encoding the β-galactosidase/neomycin-resistance fusion protein. The vector was inserted into intron 8 of the SENP1 locus. A male chimeric mouse was generated from the ES cell line. SENP1 knockdown (SENP1+/-, KD) mice were generated by mating the chimeric male and SENP1+/+ female mice. To avoid any potential sex-related effect, an equal number of male and female mice were included in each group. Wild-type (WT) and SENP1 KD mice were randomly divided into normoxia and CIH groups, respectively, yielding in four groups (n = 12 per group, sample size was predetermined for overall power of α = 0.05 and β = 0.8, based on existing data, previous literature [34], experimental funds and experimental detection requirements of intermittent hypoxia mouse model): WT-Normoxia, WT-CIH, SENP1 KD-Normoxia and SENP1 KD-CIH. Mice genotype identification was conducted before the experiment.
Cih Treatment Of Mice
Mice from the normoxia and CIH groups were placed in two identical commercially designed chambers (Bio-instruments, Redfield, NY), and the fluctuation of O2 concentration in chambers was regulated by pouring into O2 and N2. During exposure periods, the concentration of O2 was cyclically reduced from 21–10% in the CIH group. Mice were housed in chambers for a total of 4 weeks and exposed to CIH (CIH group) or normoxic conditions (normoxia group) for a total of 8 h a day (08:00–16:00). Chambers were disinfected weekly after 5 pm, and the weight of mice was recorded.
CIH protocol is consisted of 4-min cycles including 2 min of hypoxia (10% O2) and 2 min of normoxia (21% O2). Hypoxia was initiated by injecting N2 into the chamber, resulting in a low point of 10% O2 in 2 min, after which O2 was injected within 2 min, resulting in 21% O2 within 2 min. Each cycle lasted 4 min, for a total of 15 cycles/hour and 120 cycles/day. The mice in the normoxia group were exposed to normoxic conditions in a chamber identical to the CIH group. For the remaining 16 h, O2 concentration was kept at 21% in both CIH and normoxia groups.
Morris Water Maze (Mwm) Trial
Spatial learning and memory were assessed using the MWM trial as described previously[35]. Briefly, water temperature was kept at 22-24 ̊C. Acquisition test was performed four times per day for 5 consecutive days; mice were put into the pool from four different directions and allowed to swim freely until they climbed up to platform under the water surface. if they did not find the platform within 60 sec, they were be guided to the platform and stayed on the platform for 10 sec. Four tests were conducted 20 min apart and their swimming track was recorded. The time of climbing to the hidden platform was defined as the escape latency. The platform was then removed on day 6 before retention tests were performed, mice were put into the pool from the same orientation and allowed to swim freely in the pool for 60 sec and the swimming track was recorded. The time of climbing to the hidden platform, the number of crossings over the platform area and the swimming speed were recorded. experimental parameters were recorded using a Super Maze Morris Water video analysis system (model, XR‐XM101; Shanghai Xinruan information Technology co., ltd.).
Quantitative Reverse Transcription-polymerase Chain Reaction
Total RNA was isolated by Trizol reagent (10296028, Invitrogen), and complementary DNA was generated using the Takara RNA PCR kit (RR420A, Takara) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT‐PCR) was performed with PrimeScript™ RT-PCR kit (RR036A, Takara) using the QuantStudio 3 Real-Time PCR Systems (Applied Biosystems). Quantification of the gene expression was performed using the 2−ΔΔCT method, with GAPDH as an endogenous control.
Western Blot Analysis
Total protein of cells and hippocampus samples was extracted using RIPA solution, and was quantified by BCA assay Kit (P0012S, Beyotime, Shanghai, China). The extracted proteins (30 µg in each lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocked, membranes were then incubated with corresponding primary and secondary antibodies.
Primary antibodies used in this study including: anti-SENP1 (1:1000; Abcam, catalog no. ab236094), anti-p65 (1:1000, Abcam, catalog no. ab239882), IL-1β (1:1000, Abcam, catalog no. ab234437), TNF-α (1:1000, Abcam, catalog no. ab215188), anti‐Aβ42 (1:1000; Abcam, catalog no. ab224275), anti‐Aβ40 (1:1000; Abcam, catalog no. ab203826), anti‐TOM 1 (1:1000/1:100; Abcam, catalog no. ab99356), anti‐β‐actin (1:1000; Abcam, catalog no. ab179467), and anti-SUMO-1 (1:1000; SANTA, catalog no. sc-5308). Goat Anti-Rabbit IgG H&L (1:2000; Abcam, catalog no. ab7090), Goat Anti-Mouse IgG H&L (1: 1000; Abcam, catalog no. ab6789) were used as secondary antibodies, and bands were visualized using the Enhanced Chemiluminescence System (Millipore, CPS1A60-1KT). The density of the bands was quantified with ImageJ software (NCIH) and normalized to that of β-actin.
Co-immunoprecipitation Assay
Cells or fresh hippocampus samples were collected by centrifugation at 300 ⋅ g for 15 min and lysed with ice-cold cell lysis buffer (Beyotime, P0013B). The determination of protein SUMOylation procedure is briefly described as following: for co‐immunoprecipitation (Co‐IP), after pre‐clearing the lysate supernatant with protein A/G PLUS‐agarose, TOM1 antibody and 100 µl of protein A/G PLUS‐agarose were added into the lysate and cultured at 4°C overnight. The mixture was then washed by cold PBS, and the resulting immune complexes were collected after centrifugation at 5,000 ⋅ g and elution with lysis buffer. The complex was then subjected to western blotting using specific antibodies to detect the SUMOylation of proteins.
Immunohistochemical Analysis
Tissue sections were deparaffinized and rehydrated, after which antigen retrieval was achieved in a microwave oven for 15min. Subsequently, endogenous peroxidase activity was blocked for 10 min by 3% hydrogen peroxide, and nonspecific binding sites were blocked for 30min at room temperature by 5% BSA. The primary antibodies (SENP1, Aβ42, Aβ40) were added on sections and incubated overnight at 4°C. The sections were incubated with an appropriate HRP-conjugated secondary antibody and counterstained with hematoxylin.
Immunofluorescence Analysis
Cells: After removing the medium and washing with PBS, BV-2 cells were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.03% Triton X-100 (Beyotime) and blocked with 5% BSA (Beyotime) for 60 min. Cells were then incubated with SENP1 at 4°C overnight. Next day, cells were washed and incubated with Alexa Fluor 647 conjugated anti-mouse IgG (1:100; Abcam, catalog no. ab150115) for 1 h and labeled with DAPI (Thermo Fisher Scientific, Scotts Valley, CA) for 5 min.
Brain tissue: After anesthesia, mice were perfused with PBS and 4% paraformaldehyde as previously described [36].Next, 4-µm-thick brain slices were prepared using a vibratome. After dewaxing and blocking, slices were incubated with primary antibodies as follows: Iba-1 (1:100; Abcam, catalog no. ab178846) at 4°C overnight. After washing, they were incubated with Alexa Fluor 488 conjugated anti-mouse IgG (1:100; Abcam, catalog no. ab150077), and cell nuclei were stained with DAPI for 5 minutes. The fluorescence intensity of SENP1 was analyzed by the fluorescence microscopy (Olympus BX51).
Cell Migration Assay
The migration ability of BV-2 cells was evaluated using a wound-healing assay. Briefly, cells were seeded in six-well plates at a density of 1 × 105 cells/well until confluentce. After creating linear scratch wounds with 200-µl pipette tip, the medium was replaced with serum-free medium and cells were cultured for the indicated times. Images were taken under a × 10 objective lens.
Cell Viability Assay
The viability of BV-2 cells was evaluated by Cell Counting Kit-8 (CCK-8) assay (100 µl; Abcam, catalog no. ab228554), in accordance with the manufacturer's protocol. Briefly, the BV-2 cells were seeded in 96-well plates (1.0 × 105 cells/well), and incubated with CCK-8 solution for 2 hours at 37°C, after which the optical density (OD) of each well was determined at 450 nm.
Cell Transwell Migration Assay
The effect of SENP1 transfection on the migration of BV-2 cells was studied using 8-µm pore size transwell chambers (8mm; Corning, catalog no. 3413). Cells in different groups were transferred to the top chamber at a density of 1 × 105 cells/well and were cultured in a serum-free medium, and 600 µl of complete medium was added to the lower chamber. Cells were allowed to migrate for 24 hours at 37°C in 5% CO2. Cells on the upper surface of each membrane were wiped away with a cotton swab, and the filters were fixed in 4% paraformaldehyde followed by Crystal Violet staining for 15 min. Five random fields were counted per chamber by using an inverted microscope (Olympus, Japan).
Flow Cytometry Analysis
HT-22 neuronal cells were purchased from the cell Bank of Type culture collection of the Chinese academy of sciences to explore the effects of microglia‐mediated inflammatory response on neuronal cells. HT‐22 cells were routinely cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 2 mM L‐glutamine, penicillin (100 u/ml) and streptomycin (100 g/ml; Gibco; Thermo Fisher Scientific, Inc.) at 37 ̊C with 5% CO2. HT‐22 cells (1x105 cells/ml) were cultured with the BV‐2 cell‐conditioned medium under different conditions at 37 ̊C with 5% CO2 for 12 h. Suspended HT‐22 cells were washed twice with PBS containing 2% FBS and stained with annexin V FITC apoptosis Kit (cat. no. 556547; Becton‐dickinson and company). in brief, 100 µl binding buffer before 5 µl FITC‐labelled annexin V (20 µg/ml) and 5 µl PI (50 µg/ml) were added and incubated in the dark at room temperature for 15 min. a tube without annexin V FITC and PI was used as Blank, whereas annexin V or Pi was used the single‐standard control. in total, 400 µl binding buffer was added at the end of the experiment and then assessed by flow cytometry (CytoFlex; Beckman coulter, inc.). The percentage of early‐ and late‐stage
Tunel Assay
Apoptosis was detected using the TUNEL apoptosis assay kit (cat. no. C1088; Beyotime institute of Biotechnology). Briefly, after tissue fixation, embedding, slicing, dewaxing and antigen retrieval as aforementioned, 40-µm tissue sections were treated with 0.1% Triton X‐100 for 10 min, and then incubated with TUNEL reagent at 37 ̊C in the dark for 60 min. The nuclei were stained with 5 µg/ml DAPI at room temperature for 5 min. The morphological changes of apoptotic cells were observed under a fluorescence microscope in three fields of view (Olympus BX51; Olympus Corporation). Green fluorescence was considered to indicate apoptotic cells.
Statistical Analysis
Data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed using the SPSS statistical software program 26.0. Normality and homogeneity of variance were tested before comparing differences between groups. If yes, significant differences were determined by independent two-tailed Student's t-test; if not, non-parametric test was used. Multiple comparisons were assessed by Line Segment Detector (LSD) or Dunnett’s T3 test after one-way analysis of variance (ANOVA). Repeated measures analysis of variance (ANOVA) was performed for the water maze data as appropriate. The experiments and analysis were performed by investigators blinded to the groups’ allocation. P < 0.05 indicated statistical significance.