Human fetal spinal cord samples
6 fetuses with spina bifida at 24-33 weeks of pregnancy and 6 normal fetuses were selected from the Shengjing Birth Cohort (SJBC). All fetuses were matched by maternal and gestational ages. The SJBC study is an ongoing prospective cohort study that has enrolled pregnant women, spouses, and their children living in Northeastern China since April 2017. Gynaecologists and fetal ultrasonologists at our hospital evaluated the classification of malformations and ensured the accuracy of the final diagnosis. Spinal cord samples from spina bifida fetuses were obtained from the edge of the defect area. The control samples were obtained from corresponding segments of normal spinal cord (approval no. 2017PS264K).
Animal models
Outbred Wistar rats (10–12 weeks old, weighing 240–300 g each) were provided by the Animal Center of Shengjing Hospital, China Medical University (Shenyang, Liaoning, China). The female rats were mated with the males overnight. The morning during which the sperm were observed under a microscope was considered gestational day 0 (E0). Pregnant rats were induced by a single intragastric administration of ATRA (4% [wt/vol] in olive oil; 140 mg/kg body weight; Sigma-Aldrich, St. Louis, MO, USA) or olive oil on E10. The posterior spinal cords of embryos were dissected on E11, E12, and E14 and stored at –80 °C for further analysis. All animal experiments were conducted in accordance with the guidelines of the Medical Ethics Committee of the Shengjing Hospital of China Medical University (approval no. 2020PS153K).
Small interfering RNA (siRNA) and plasmid construction
The siRNAs against ApoM, Nnt, and Zic3 were synthesised using RIBOBIP (Guangzhou, China). The sequences of the siRNAs were as follows: ApoM siRNA 5ʹ-CCTCTTGCTTGGACTTCAA-3ʹ; Nnt siRNA 5ʹ-GCTTACCTTGGCACTTACA-3ʹ; and Zic3 siRNA 5ʹ-GAACAACCACGTCTGCTAT-3ʹ. ApoM, Nnt, Zic3, and Elavl2 overexpression plasmids and the control plasmid were purchased from SyngenTech Inc. (Beijing, China).
Cell culture and transfection
Mouse C17.2 neural stem cells were cultured in minimum essential medium (MEM; Gibco, MA, USA) with 10% foetal bovine serum (FBS; Gibco, MA, USA) and 1% MEM with non-essential amino acids (Gibco, MA, USA). All cells were maintained in a humidified 37 °C incubator with 5% CO2. The transfections were performed using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. After specific times of transfection, cells were harvested and stored at –80 °C prior to further analyses.
RNA extraction and real-time quantitative reverse-transcription PCR (qRT-qPCR)
Total RNA was isolated from rat embryonic spinal cords and C17.2 cells using TRIzol reagent (Takara, Ohtsu, Japan) in accordance with the manufacturer’s recommendations. An ultra-micro spectrophotometre (Thermo Scientific Nanodrop 2000, Waltham, MA, USA) was used to measure the total RNA concentration and purity. Genomic DNA was removed, and RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara, Ohtsu, Japan). mRNA expression levels were measured using the SYBR Premix Ex Taq kit (Takara, Ohtsu, Japan) on a 7500 Real-Time PCR System (StepOnePlus, ABI Company, Oyster Bay, NY, USA). Primers were designed and synthesised by Sangon Biotech (Shanghai, China) and are listed in Supplementary Table S3. The classic 2−ΔΔCt method was used to analyse relative gene expression after normalisation to GAPDH.
Protein extraction and immunoblotting
Rat embryonic spinal cords or C17.2 cells were lysed in radioimmunoprecipitation buffer (Solarbio Science & Technology, Beijing, China) to obtain the total protein. MinuteTM Mitochondrial Extraction kits (Invent Biotechnologies, Inc., Plymouth, MN, USA) were used to isolate the mitochondrial and cytosolic fractions, following the manufacturer’s instructions. Nuclear Protein Extraction kits (Abmart, #A10009, Shanghai, China) were used to extract the nuclear and cytoplasmic proteins according to the manufacturer’s instructions. Protein concentrations were determined using bicinchoninic acid assays (Takara, Ohtsu, Japan).
Equal amounts of protein were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk solution for 1 h and subsequently incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used in the experiment: anti-ApoM (1:1000, Cell Signaling Technology, #5709, MA, USA), anti-Nnt (1:1000, Proteintech, 13442-2-AP, Wuhan, China), anti-Cytochrome c (1:1000, Cell Signaling Technology, #11940, MA, USA), anti-AIF (1:1000, Cell Signaling Technology, #5318, MA, USA), anti-VDAC1 (1:2 000, Proteintech, 55259-1-AP, Wuhan, China), anti-Bcl2 (1:1000, Proteintech, 26593-1-AP, Wuhan, China), anti-Bax (1:1000, Cell Signaling Technology, #14796, MA, USA), anti-Caspase3 (1:1000, Novus Biologicals, NB100-56708, CO, USA), anti-Caspase9 (1:1000, Proteintech, 10380-1-AP, Wuhan, China), anti-PINK1 (1:1000, Proteintech, 23274-1-AP, Wuhan, China), anti-PRKN (1:2000, Proteintech, 14060-1-AP, Wuhan, China), anti-LC3 (1:1000, Cell Signaling Technology, #4108, MA, USA), anti-Elavl2 (1:3000, Proteintech, 14008-1-AP, Wuhan, China), anti-Lamin B1 (1:50000, Proteintech, 66095-1-Ig, Wuhan, China), anti-Zic3 (1:1000, Novus Biologicals, NBP1-33207, CO, USA), and anti-GAPDH (1:5000, Proteintech, 60004-1-Ig, Wuhan, China). After incubation with the corresponding secondary antibody (1:5000, Proteintech, Wuhan, China), the protein bands were detected using a chemiluminescent substrate (Millipore, MA, USA) and quantified using ImageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA).
Mitochondrial permeability transition pore (mPTP) opening assay
The opening of mPTP was assessed using the cobalt quenching of calcein-AM fluorescence (Beyotime, C2009S, Shanghai, China). After being transfected with ApoM-siRNA, C17.2 cells were harvested using trypsin and loaded with calcein-AM (1 μM) at 37 °C for 30 min in the dark. CoCl2 (1 mM) was then added, and the cells were incubated for another 30 min. The fluorescence of the cells in each experiment was measured using a Tecan Infinite 200 Pro Microplate reader (Tecan, NSW, Australia). For the imaging experiments, the cells were cultured in 12-well plates and treated according to the protocol described above after transfection. Cell images were captured using an ECLIPSE 80i fluorescence microscope (Nikon, Kyoto, Japan). Data were analysed using ImageJ software.
Determination of reactive oxygen species (ROS)
A Reactive Oxygen Species Assay kit (Beyotime, S0033, Shanghai, China) was used to determine the ROS levels. After being transfected, C17.2 cells were harvested using trypsin and resuspended in MEM without FBS. The cells were then incubated with DCFH-DA (diluted 1:1000) in a 37 °C incubator in the dark for 30 min. We used a Tecan Infinite 200 Pro Microplate reader (Tecan, NSW, Australia) to measure the fluorescence intensity of DCFH and set the excitation and emission wavelengths to 488 and 525 nm, respectively. For the imaging experiments, the cells were cultured in 12-well plates and observed under an ECLIPSE 80i fluorescence microscope (Nikon, Kyoto, Japan). The data were analysed using ImageJ software.
Detection of mitochondrial membrane potential (MMP)
The abundance of MMP was measured using a JC-1 fluorescent probe (Beyotime, C2006, Shanghai, China). The transfected C17.2 cells were collected using trypsin and incubated with JC-1 working solution at 37 °C for 30 min. The abundance of MMP was determined using a Tecan Infinite 200 Pro Microplate reader (Tecan, NSW, Australia). For the imaging experiments, the cells were cultured in 12-well plates and treated according to the protocol described above after transfection. The cells were observed under a laser scanning confocal microscope (Zeiss, Oberkochen, Germany), and the data were analysed using ImageJ software.
Transmission electron microscopy (TEM)
C17.2 cells were seeded onto 10-centimetre plates and transfected with ApoM siRNA for 48 h. The cells were treated with carbonyl cyanide m-chlorophenylhydrazine (CCCP, Beyotime, Shanghai, China) for the last 12 h. The cells were then trypsinised, suspended, and fixed at room temperature in the dark for 30 min in 0.01 M phosphate buffer (pH 7.2) containing 2.5% glutaraldehyde. The samples were incubated overnight at 4 °C. A TEM analysis was performed following the fixation, dehydration, infiltration, and embedding of the samples. Using a ultramicrotome (Leica Microsystems, EM UC7, Wetzlar, Germany), ultra-thin sections (70-nanometre thick) were collected on 150-mesh copper grids and post-stained with 2% uranyl acetate and 2.6% lead citrate. The sections were observed using TEM (Hitachi High-Tech Co., HT 7800, Tokyo, Japan) at 80 kV.
RNA stability measurement
Nnt mRNA stability was measured using an actinomycin D (Act D, Sigma-Aldrich, MO, USA) assay. In brief, 20 µg/mL of Act D was added into the cell media at 48-h post-transfection with siRNAs or plasmids. After 0, 1, 2, 4, and 8 h of the Act D treatment, RNA was isolated from the cells, and the Nnt mRNA levels were tested using qRT-PCR. The ‘relative one phase decay model’ of nonlinear regression was used to fit the RNA expression value decay curve over time and calculate the half-life time (t1/2).
Co-immunoprecipitation
C17.2 cells were lysed in IP/western lysing solution (Beyotime, P0013, Shanghai, China) for 60 min on ice and then centrifuged at 12000 × g for 15 min to remove debris. For immunoprecipitation, 4 µg of ApoM antibody (Cell Signaling Technology, #5709, MA, USA) or Elavl2 antibody (Proteintech, 14008-1-AP, Wuhan, China) was bound to Protein G Magnetic Beads (Cell Signaling Technology, #70024, MA, USA) on a rotator at room temperature for 45 min, with the corresponding IgG (Cell Signaling Technology, MA, USA) used as a negative control. Lysates were incubated with the antibody-crosslinked resin overnight at 4 °C to accomplish antigen immunoprecipitation. The beads were washed five times with lysis buffer before being reconstituted with 1× SDS-PAGE sample buffer and analysed by immunoblotting.
RNA immunoprecipitation-qPCR assay
The RIP assay was performed using the EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, 17-700, MA, USA) according to the manufacturer’s recommendations. The cells (1.0 × 107) were washed with cold PBS and lysed on ice using the harsh lysis buffer. The supernatants were collected by centrifugation at 15 000 × g and 4 °C. Magnetic beads pre-incubated with IgG or antibodies specific to ApoM (Cell Signaling Technology, #5709, MA, USA) or Elavl2 (Cell Signaling Technology, #5709, MA, USA) were incubated with lysates overnight at 4 °C. An aliquot of the lysate was used as the input control. Immunoprecipitated and input RNAs were isolated and analysed by qRT-PCR. The immunoprecipitated Nnt mRNA was normalised to the input control. The primers used are listed in Supplementary Table S3.
RNA pull-down assay
An RNA pull-down assay was performed using an RNA pull-down kit (Bersin Bio, Guangzhou, China) according to the manufacturer’s protocol. Biotin-labelled Nnt and LacZ probes were constructed by Biosense Bioscience Co., Ltd. (Guangzhou, China). Briefly, biotin-labelled RNAs were captured by streptavidin-conjugated magnetic beads for 2 h at 25 °C. Next, the complex comprising biotin-labelled RNA and lysates of C17.2 cells was purified using streptavidin-agarose for 2 h at 4 °C. Finally, the RNA-conjugated proteins were eluted, and the protein level of Elavl2 was determined by western blotting.
Fluorescence in situ hybridisation (FISH) and immunofluorescence (IF) staining
FISH probes and a FISH kit were provided by RiboBio (Guangzhou, China). C17.2 cells were seeded onto coverslips in 24-well plates and transfected for 48 h. The cells were then fixed with 4% paraformaldehyde for 30 min and permeabilised with 0.5% Triton X-100 for 30 min. The cells were washed thrice with PBS and treated with pre-hybridisation buffer containing a 1% blocking solution. The Cy3-labelled Nnt probe was diluted in hybridisation buffer containing a 1% blocking solution (100 µL). The surface of the coverslip was covered for incubation at 37 °C for 12 h in a dark and humid chamber. Next, the cells were washed five times with gradient standard saline citrate at 42 °C and treated with Elavl2 antibody (1:200, Proteintech, 14008-1-AP, Wuhan, China) at 4 °C overnight, followed by incubation with Alexa Fluor 488 (1:500, Cell Signaling Technology, #4412, MA, USA) for 2 h at room temperature in the dark. Nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI), and images were acquired using a laser scanning confocal microscope (Zeiss, Oberkochen, Germany).
Chromatin immunoprecipitation-qPCR assay
A ChIP assay was conducted using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, #9002, MA, USA), following the manufacturer’s instructions. Briefly, C17.2 cells were cultured until there were approximately 1 × 107 cells and cross-linked with 1% formaldehyde. Samples were then harvested, and chromatin was digested with micrococcal nuclease and sonicated to a length of approximately 150–900 bp. An aliquot of each ChIP sample was prepared as an input control, and the rest of the chromatin was immunoprecipitated with anti-Zic3 (Novus Biologicals, NBP1-33207, CO, USA) or anti-IgG as a negative control overnight at 4 °C. After being washed and reverse cross-linked, the eluted DNA was purified using columns and quantified by qPCR. The precipitated genomic DNA was amplified using the primers for the ApoM promoter region listed in Supplementary Table S3.
Luciferase reporter assay
The pGL4.18 plasmid containing the wild-type (WT) promoter sequence of Nnt mRNA (Nnt-WT) was cotransfected into C17.2 cells with or without the ApoM plasmid. Another pGL4.18 plasmid containing the WT (ApoM-WT) or mutant-type (ApoM-Mut) promoter sequences of ApoM mRNA was cotransfected into C17.2 cells with or without the Zic3 plasmid. Luciferase reporter plasmids were cotransfected into C17.2 cells. After 48 h, luciferase activity was detected using a Dual-Luciferase Reporter Assay System (Promega, WI, USA) and measured using a Tecan Infinite 200 Pro Microplate reader (Tecan, NSW, Australia). Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity.
Recombinant adenoviruses and intra-amniotic injections
GFP, rat Zic3 cDNA, and rat ApoM cDNA were cloned into E1- and E3-deleted human Ad type 5 vectors under the control of a cytomegalovirus (CMV) promoter, generating recombinant Ad (i.e., Ad-GFP-Zic3 or Ad-GFP-ApoM). Control GFP (Ad-GFP)-expressing adenoviral vectors were constructed using the CMV promoter. The viral titres were 2.51 × 1011 pfu/mL for Ad-GFP-Zic3, 1.99 × 1011 pfu/mL for Ad-GFP-ApoM, and 2.51 × 1011 pfu/mL for Ad-GFP. The adenoviruses used in this study were obtained from Hanbio Biotechnology (Shanghai, China). Intra-amniotic Ad injections were administered to E16 embryos as previously described (32, 33). Pregnant rats were anaesthetised with pentobarbitone sodium (40 mg/kg of body weight). An incision was made in the abdominal wall, and the uterus was exteriorised. Under an operating microscope, foetuses with a uniform position and defective-sized spina bifida were chosen and randomly divided into PBS-, Ad-GFP-, Ad-GFP-Zic3-, and Ad-GFP-ApoM-injected groups. Microinjections were performed with transuterine injections of 5 μL of solution using a glass micropipette (internal tip diameter, 100 μm) connected to a Hamilton syringe. The micropipettes for the injections were made from borosilicate glass capillaries (model GD-1; Narishige Scientific Instruments, Tokyo, Japan) with a micropipette puller (model PB-7; Narishige Scientific Instruments). After the injection, the uterus was returned to the abdomen, and the abdominal wall was closed. The pregnant rats recovered from anaesthesia within 1 h and were returned to their home cages. They were euthanised on E21 with an overdose of pentobarbitone sodium, and the injected foetuses were harvested for analysis.
Fluorescence imaging and tissue preparation
On E21, the injected foetuses were harvested, and a fluorescence stereomicroscope (Leica, M165FC, Germany) fitted with a Nikon DS-Qi2 digital camera (Nikon, NY-1S35, Japan) was used to capture in vivo fluorescence images of the foetuses. The fetuses were then transcardially perfused with 15 mL of physiological saline, followed by 25 mL of 4% paraformaldehyde. The lumbosacral spinal column containing muscle, spinal cord, and subcutaneous tissue was dissected and post-fixed in 4% paraformaldehyde overnight. The tissues were then cryoprotected in 20% sucrose for 24 h, embedded with the optimal cutting temperature compound (SAKURA, Japan), and sectioned into 30-micrometre-thick slices using a freezing microtome (Thermo, Microm HM525, Germany). GFP-positive cells were observed using an ECLIPSE 80i fluorescence microscope (Nikon, Kyoto, Japan). All of the sections were stored at –80 °C for subsequent experiments.
TUNEL staining
The TUNEL assay was performed using the One Step TUNEL Apoptosis Assay Kit (Beyotime, C1090, Shanghai, China). Based on the manufacturer’s protocol, the sections were fixed with 4% paraformaldehyde for 1 h and 0.5% Triton x-100 for 5 min at room temperature. The sections were then washed and incubated with TUNEL reaction mixture at 37 °C for 1 h in the dark. After being washed, the sections were incubated for 5 min with DAPI (Beyotime, C1002, Shanghai, China) and mounted using anti-fade mounting media. Images were acquired using an ECLIPSE 80i fluorescence microscope (Nikon, Tokyo, Japan).
Immunofluorescence staining
C17.2 cells were seeded onto coverslips in 12-well plates and transfected for 48 h. The cells were then fixed with 4% paraformaldehyde and permeabilised with 0.5% Triton for 30 min. After being blocked with 5% bovine serum albumin (BSA) for 45 min, the coverslips were transferred to a humidified chamber and incubated with the primary antibodies mouse anti-Tomm20 (1:1000, Abcam, ab56783, MA, USA) and rabbit anti-LC3 (1:100, Cell Signaling Technology, #4108, MA, USA) overnight at 4 °C, followed by incubation with Alexa Fluor 488 (1:500, Cell Signaling Technology, #4412, MA, USA) and Alexa Fluor 555 (1:500, Cell Signaling Technology, #4409, MA, USA) for 2 h at room temperature in the dark.
The frozen sections were fixed with 4% paraformaldehyde and permeabilised with 0.5% Triton for 1 h. They were then blocked with 10% BSA for 1 h. The primary antibodies used were mouse anti-Tomm20 (1:1000, Abcam, ab56783, MA, USA) and rabbit anti-LC3 (1:100, Cell Signaling Technology, #4108, MA, USA), and the secondary antibodies included Alexa Fluor 647 (1:1000, Invitrogen, A32795, MA, USA) and Alexa Fluor 555 (1:500, Cell Signaling Technology, #4409, MA, USA). Nuclei were counterstained with DAPI, and images were acquired using a laser scanning confocal microscope (Zeiss, Oberkochen, Germany).
Statistical analyses
Statistical analyses and graph generation were performed using GraphPad Prism 7.0 software (GraphPad Software). All data are presented as the mean ± standard deviation. A two-tailed Student’s t test was used to analyse the differences between two groups, and a one-way analysis of variance (ANOVA) was used to analyse the data among multiple groups. All the measurements were repeated at least three times, with consistent trends and differences, and the results were considered to be statistically significant at *P < 0.05 and **P < 0.01, with “ns” representing no significance.