Human subjects
Thirty-six unrelated MDD patients (20 females and 16 males) aged 39.9 ± 12.5 (SD) years and twenty unrelated healthy subjects (10 females and 10 males) aged 36.6 ± 11.1 (SD) years were recruited from the DBPR (manuscript NCOMMS-21-04052). All subjects provided written informed consent prior to participating in this study, which was approved by the Ethics Committee of the DBPR (manuscript NCOMMS-21-04052). None of the MDD patients or healthy controls had taken any psychotropic medications within 4 weeks. All patients were clinically diagnosed by at least two psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV) criteria and the 17-item Hamilton Rating Scale for Depression (HAMD-17). Hippocampal tissues of the human brain were provided by the Human Brain Bank, DBPR (manuscript NCOMMS-21-04052). The postmortem intervals between the death of donors and brain collection were less than 40 h, and detailed information on the donors is provided in Supplementary Table 1.
Microarray
Total blood RNA was isolated with a PAXgene Blood RNA Kit (Qiagen, 762174). The optical density values at 260/280 were approximately 1.9-2.0, and the quality of the RNA was also confirmed with an Agilent 2100 (Agilent Technologies, Santa Clara, CA). RNA samples underwent reverse transcription to synthesize double-stranded complementary DNA (cDNA), and the cDNA was then transcribed into cRNA in vitro. The cRNA was then reverse-transcribed into cDNA. The cDNA was fluorescently labeled and hybridized using a 4 × 180K lncRNA+mRNA Human Gene Expression Microarray V3.0 (CapitalBio Technology, Beijing, China). Agilent Feature Extraction (v10.7) software (Santa Clara, CA, USA) was used to analyze the hybridization map and extract the data. The chip fluorescence scanning images were saved as DAT files for analysis by the Agilent G2565CA Microarray Scanner. The data normalization and difference analysis were performed with Agilent GeneSpring software. Two groups of sample data underwent a t-test analysis to obtain the corrected P-values and fold change values. The microarray data were validated by qPCR, and the results for this microarray data have not been published. The expression of genes encoding RNA methylation-modifying enzymes was derived from the microarray data (in house data).
Animals
C57BL/6J mice at the age of 4-6 weeks were purchased from DBPR (manuscript NCOMMS-21-04052) and bred for 4-5 weeks to perform experiments. C57BL/6J, Ftofl/fl mice (Stock No. 027830) were provided by the Jackson Laboratory. These mice possess loxP sites on either side of exon 3 of Fto. Removal of the floxed sequence by Cre recombinase creates a null Fto allele. Mice were housed under a 12-h light/dark cycle at 23 ± 2 °C. All animal work was in accordance with the institutional guidelines of the Beijing Administration Office of Laboratory Animals and approved by the DBPR (manuscript NCOMMS-21-04052). This study was approved by the DBPR (manuscript NCOMMS-21-04052).
Cell culture
The Neuro-2a cell line, which was provided by the Cell Resource Center (IBMS, CAMS/PUMC), was cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (04-001-1A, Biological Industries), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in 5% CO2. Plasmids were transfected into Neuro-2a cells with Neofect™ DNA transfection reagent (TF201201, China). siRNA targeting Ythdf2 (sc-155424, Santa Cruz) was transfected into Neuro-2a cells with RNAi MAX (13778150, Thermo).
Drug administration
Formoterol (F9552, Sigma) and ICI 118,551 (HY-13951, MedChemExpress) were diluted in saline and administered via i.p. at dosages of 2 mg/kg and 10 mg/kg, respectively. To block the effects of formoterol, ICI 118,551 was administered 30 min before formoterol.
Mouse models of depression
Chronic unpredictable mild stress (CUMS). The CUMS model was established referring to a previous work46. Animals were subjected to 11 stressors at random for 35 days (three stressors per day from 1 to 21 d and two stressors per day from 22 to 35 d), which included a hot plate (45 °C/10 min), leaving the lights on overnight, turning the lights off in the daytime, bedding deprivation overnight, crowding (12 h), swimming stress (16 °C/3 min), food and water deprivation (12 h), wet bedding (12 h), an elevated platform (30 min), tilting of the cage at 45 degrees (12 h), and restraint stress (1 h).
Chronic restraint stress (CRS). CRS exposure was performed as previously described47. Mice were placed in polypropylene conical tubes (50 ml) with pores at the bottom enabling air flow. Animals had no access to food and water during restraint stress. Four hours later, the mice were returned to their home cages. The same stressor was administered once at a random time each day, which was continued for 21 days.
Social defeat stress (SDS). Social defeat stress exposure was performed according to the published protocol48. Briefly, C57BL/6J mice were placed into a cage in the presence of CD-1 aggressor mice for 5-10 min per day for 10 consecutive days. Then, the C57BL/6J and CD-1 mice were separated by a perforated Plexiglas plate for 24 h, which blocked all physical contact. Each C57BL/6J mouse was exposed to different CD-1 mice every day.
Behavioral test
Animals were transported and acclimated to the testing room 1 h before each behavioral test.
Forced swimming test (FST). Mice were individually placed in a beaker (height: 19 cm; diameter: 14 cm) containing 14 cm of water (23 ± 2 °C). The total test duration was 6 min. The process was videotaped, and the immobility time in the last 4 min was scored by an experienced observer who did not know the experimental treatment. Floating or only slight movement to maintain balance was considered immobility.
Tail suspension test (TST). The TST was performed with tail suspension hardware (Med Associates, USA). The mouse was suspended by its tail with a short adhesive tape connected to a load cell that transmits a signal corresponding to activity. The total test time was 6 min. After setting a lower threshold, the duration of immobility was recorded and analyzed by tail suspension software (SOF-821, Med Associates).
Sucrose consumption test (SCT). Mice were acclimatized to 1% sucrose solution for 48 h before the test. Then, each mouse was individually housed and deprived of water for 12 h. SCT was performed for 1 h, in which the amount of the 1% sucrose solution consumed was recorded.
Sucrose preference test (SPT). Mice were acclimatized to two-bottle drinking (one bottle contained water, and the other contained a 1.5% sucrose solution) for 48 h, during which the position of each bottle was switched at 24 h. Then, the mice were deprived of drinking water for 24 h. SPT was performed by providing the mice once again with two bottles for drinking for 2 h, during which the position of each bottle was switched after 1 h. The ratio of the sucrose solution consumed to the total fluid intake in the SPT was determined as the sucrose preference.
Social interaction test (SIT). The SIT was performed according to a previous report. It consists of two 2.5 min phases: the first is performed without a CD-1 mouse, and the second is performed with a CD-1 mouse. The two phases were separated by a pause of 30 sec. In both phases, the C57BL/6J mouse was placed into the rear corner of the open field opposite the wire-mesh compartment. The time spent in the interaction zone (14 cm × 24 cm) was recorded and analyzed by the EthoVision video tracking system (Noldus, the Netherlands). The social interaction ratio was calculated as the time spent in the interaction zone in the presence of the CD-1 mouse to the time spent in the absence of a CD-1 mouse. Brains of susceptible and control mice were harvested for gene expression analysis.
Novel suppressed feeding test (NSFT). We performed the NSFT by referring to a previous study. Mice were deprived of food for 24 h. During the NSFT, each mouse was placed into the corner of a square chamber (50 cm × 50 cm× 20 cm) opposite a food pellet that was placed in the center of the chamber. The latency to start eating the pellet was recorded. If the mouse did not eat the pellet within 6 min, the latency was recorded as 6 min.
Open field test (OFT). Each mouse was individually placed in one corner of a square Plexiglas box (50 cm × 50 cm × 40 cm) in a brightly lit room, allowing for free exploration of the open field arena for 10 min. The time spent in the center area of the box (12.5 cm × 12.5 cm) and the distance traveled in the box during the testing period were recorded and analyzed by the EthoVision video tracking system (Noldus, the Netherlands).
Elevated zero maze test (EZM). The EZM was performed in a circular elevated maze with two open and two enclosed arms (height: 43.5 cm; diameter: 45 cm; track width: 6.5 cm). Each mouse was individually placed in the cross area of the maze facing the closed arms and allowed to freely explore the maze for 5 min. The time spent in the open arms and the distance traveled in the maze were recorded and analyzed by the EthoVision video tracking system (Noldus, the Netherlands).
Vector construction, rAAV packaging and administration
The mouse Fto and Sirt1 cDNAs were cloned into the rAAV expression vector with a CAG promoter. The Fto shRNA targeting 5’-GCAGCTGAAATACCCTAAACT -3’ was cloned into the rAAV expression vector with a U6 promoter. AAV-CAG-Fto-2A-EGFP (Cat. # AAV2/9-XT071), AAV-CAG-Sirt1 (Cat. # AAV2/9-XT393), AAV-U6-Fto shRNA-CMV-ZsGreen (Cat. # AAV2/9-XT071), AAV-CMV-bGlobin-Cre-eGFP (Cat. # AAV2/9-S0231-9-H50), and AAV-CMV-bGlobin-eGFP-WPRE (Cat. # AAV2/9-S0263-9-H50) were packaged by Taitool Bioscience (Shanghai Taitool Bioscience Co.Ltd., China). The final titer of each rAAV was 3×1012 - 4×1012 vector genome (v.g.)/ml.
RNA preparation for quantitative real-time PCR
Total RNA from the hippocampus was extracted using TRIzol (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions, and the samples were stored at -80 °C. One microgram of total RNA was used to synthesize cDNA by the Transcriptor First Strand cDNA Synthesis kit (04897030001, Roche). Quantitative PCRs were performed with FastStart Essential DNA Green Master Mix (06924204001, Roche) on a LightCycler 96 Real-Time System (Roche). The primers used in the qPCR analysis are shown in Supplementary Table 4. Each reaction was performed in triplicate. A fragment of Gapdh was amplified as the internal control. Differences in gene expression were calculated by the 2-ΔΔCT method and are presented as the relative fold change.
Western blot analysis
Brain samples were lysed in RIPA lysis buffer with protease inhibitor cocktail (B14001, Bimake) by a tissue homogenizer, which was followed by ultrasonication and centrifugation. The bicinchoninic acid method was used to determine the protein concentrations, and proteins were mixed with 5× Laemmli sample buffer and denatured for 5 min at 95 °C. A total of 30 µg of each sample was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. Then, the membrane was incubated in blocking buffer (TBST buffer containing 5% skim milk powder) for 60 min at room temperature. Next, the membranes were incubated overnight at 4 °C with the primary antibodies. Primary antibodies were as follows: anti-FTO (1:1000, ab92821, Abcam), anti-Adrb2 (1:1000, ab182136, Abcam), anti-Sirt1 (1:1000, 8469S, Cell Signaling Technology), anti-c-Myc (9402S, 1:1000, Cell Signaling Technology), and anti-GAPDH (1:10000, GTX100118, GeneTex), anti Rabbit (1:5000, GTX213110-01,GeneTex), and anti-Mouse (1:5000, GTX213111-01 , GeneTex). After incubation for 1 h with the corresponding secondary antibodies, the protein bands were detected by chemiluminescence using an ECL reagent.
Stereotaxic surgery
Ten-week-old male mice were stereotaxically injected with rAAVs. Mice were anesthetized with 0.7% pentobarbital sodium via i.p. One microliter of rAAV per side was microinjected bilaterally into the hippocampus (2.2 mm posterior to bregma, 2.1 mm lateral to midline, and 1.7 mm dorsal to bregma) at a rate of 300 nanoliters/min with a Micro4 Syringe Pump Controller (World Precision Instruments, USA). Four weeks later, further experiments were performed.
m6A Dot blot
Total RNA in 3 sample volumes of RNA incubation solution was denatured at 65 ℃ for 5 min and immediately chilled on ice. RNA samples were spotted in triplicate onto an Amersham Hybond-N+ membrane (RPN119B, GE Healthcare) with a Bio-Dot Apparatus (#170-6545, Bio-Rad). Next, they were crossed-linked to the membrane by UV light and then blocked with blocking buffer (PBST buffer containing 5% nonfat milk). The membrane was incubated overnight at 4 °C with anti-m6A antibody (1:5000, 202003, Synaptic Systems) followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:5000, sc-2030, Santa Cruz Biotechnology) for one hour at room temperature. Finally, the signal was detected by a chemiluminescent reaction using an ECL reagent.
MeRIP-seq
A refined MeRIP-Seq (N6-methyladenosine (m6A) RNA immunoprecipitation sequencing) method was performed based on a previously described protocol49,50. The hippocampus was dissected from 3 mice, and the tissues were pooled together to obtain a sufficient quantity of mRNA for immunoprecipitation followed by RNA sequencing. Briefly, poly-A-purified RNA was extracted and randomly fragmented into approximately 100-200 nucleotide-long fragments. After enriching for methylated RNA fragments with a specific anti-m6A antibody (1:100, ABE572, Millipore), the m6A-seq library was constructed using the SMARTer Stranded Total RNA-Seq Kit-v2 (Pico Input Mammalian, 634413, Takara/Clontech, Japan) and sequenced on Illumina Novaseq 6000.
Bioinformatics analysis of high-throughput sequencing data
Alignment. Sequencing data were analyzed according to the optimized pipeline for refined MeRIP-Seq described in a previous study50. The quality control of raw data was performed by FASTQC (version 0.11.5). The clean reads from the m6A IP (m6A-seq) were aligned to the mouse genome (mm10) using the STAR aligner (version 2.7) with the default parameters after removing reads containing adapters and low-quality reads with cutadapt (version 1.18). Only uniquely mapped and unduplicated reads remained for the subsequent analysis.
m6A peak calling and motif analysis. MeTPeak (version 1.1) with the default settings was used for the transcriptome-wide detection of m6A sites in each sample based on the alignment files generated by STAR aligner. The R package Guitar was used to illustrate the distribution of m6A peaks in mRNA51. The top 3000 peaks were selected for motif analysis with DREME (version 5.1.1), which used 100-nt-long peak summit-centered sense sequences as input. Peaks were annotated to overlapping genomic features and visualized with ChIPseeker (version 1.22.1) using annotations from TxDb.Mmusculus.UCSC.mm10.knownGene. Peaks were assigned to seven regions, including the promoter, 5’ UTR, 3’ UTR, exon, intron, and downstream and distal intergenic regions.
Detection of differential methylation peaks (DMPs). DMPs were identified according to the pipeline described in the four groups included in our study, including wild-type (WT), KD, cKO and OE mice. The following steps were taken to identify DMPs between three groups based on the expression levels in Fto-modified and control samples (WT mice). First, the overlapping m6A peaks from two samples were defined as common peaks. The common peaks between samples from the same group were merged and combined with the common peaks from another group to form the m6A union peaks reference list. Then, the read counts for the union peaks for the IP and input samples were calculated separately and corrected based on the total number of ERCC spike-in reads. The enrichment score of each sample for each peak was obtained by dividing the read counts for IP by the input. High confidence DMPs meeting the criteria of an average enrichment score higher than 6 in the groups and a log2 fold change > 1 or < −1 were selected for further analysis. The DMPs were annotated according to the genomic features with ChIPseeker (version 1.22.1)52. Functional enrichment analysis of the gene ontology (GO) and pathways was performed using ChIPseeker (version 1.22.1) to identify the predominant biological functions of the annotated genes nearest to the DMPs. Fisher’s exact test was applied to identify the significant GO categories and pathways, and FDR correction was performed with a threshold of 0.05.
Gene-specific m6A qPCR
The Magna MeRIP m6A kit (17-10499, Millipore) was used to perform m6A RNA immunoprecipitation according to the manufacturer’s instructions. A QuantiTect SYBR Green RT-PCR Kit (204243, Qiagen) was used to perform reverse transcription and real-time qPCR. The relative expression of the m6A-modified target gene was determined as the Cq value of the m6A IP portion divided by the Cq value of the input portion.
RNA stability
Neuro-2a cells were transfected with Fto-shRNA and Fto overexpression and control vectors. Forty-eight hours later, the cells were treated with 5 μg/ml actinomycin D (A9415, Sigma) and harvested at 0, 3 and 6 h. Total RNA was extracted using TRIzol, and 1 μg of total RNA was used for reverse transcription and qPCR.
Golgi staining
Mice were anesthetized with chloral hydrate (10%, wt/vol, i.p.) and sequentially perfused transcardially with saline. The whole brain was freshly harvested and was immediately immersed in one well of a 12-well plate containing 5 ml of solution A (superGolgi Kit, Cat. # 003010, Bioenno Tech, LLC). According to the manufacturer’s instructions, the solution was renewed two days later, and the impregnation continued for another 9 days. Next, the brain was transferred to postimpregnation buffer for 2 days with renewal of the solution after one day of immersion. Sections that were 100 μm in thickness were cut using a vibratome (VT1200S, Leica). The sections were mounted on adhesive microscope slides (SUPERFROST® PLUS, Cat. # 4951 PLUS-100E, Thermo Scientific). The staining time in solutions C and D was 20 min. Then, the sections were dehydrated in 100% ethanol for 7 min 4 times each, which was followed by vitrification with xylene for 8 min 10 times each. Finally, the sections were coverslipped with neutral resins. For spine density measurement, we imaged one terminal dendrite from the CA1 region of the hippocampus using a 100× objective on a microscope (DM6 B, Leica). A Z-stack of images with an interval of 0.22 μm was acquired for each dendritic segment, which was followed by an extended depth of field algorithm calculation to obtain in focus images using LAS X software (Leica Application Suite X, Leica). The diameters of the selected segments were comparable. A total of 120 segments from 20 mouse brains (6 segments per animal) were used to determine the spine number. The dendritic spines, including thin, stubby, and mushroom types, within a 15-μm length segment were counted manually by an investigator blinded to the experimental treatments.
Statistical analysis
Statistical significance was analyzed with Prism software (GraphPad Prism 8). Two-tailed student’s t test was used for comparisons between the two groups, and one-way or two-way ANOVA was performed for more than two groups. P < 0.05 was considered statistically significant. All the data were exported into Adobe Photoshop CS6 for the preparation of figures. All data are shown as the mean ± s.e.m.