GPR50 regulates neuronal development as a mitophagy receptor

Plasmids and siRNA

GFP-LC3 was described previously [65]. MitoDsRed was described [19]. The cDNA encoding WT GPR50 (human) or mutant GPR50 was inserted into the FLAG sequence at the C terminal and cloned into the pcDNA3.1 (+) vector between the BamHI and NotI sites. The mutation information of GPR50 mutants is as follows: GPR50^T532A, a.a. T⁵³² were mutated into A⁵³², GPR50^Δ502-505, a.a. 502-505 were deleted. All constructs were confirmed by sequencing. The sequence of siRNA was as follows: GPR50 siRNA (5’-GGAUCUUCAGUGUGCGCAATT-3’), scrambled siRNA (5’-UUCUCCGAACGUGUCACGUTT-3’) (Genepharma).

Mice

Gpr50^−/− mice were generated by replacing the second exon with the Lacz-neo cassette and maintained on a C57BL/6 N background by crossing heterozygous transgenic mice with C57BL/6 N breeders. Gpr50^−/y mice were obtained by hybridization between Gpr50^−/− mice and Gpr50^+/y mice. For PCR genotyping, the following primers were used (F1: 5’-ATCCGGGGGTACCGCGTCGAG-3’; R1: 5’-TACCTCCACCTCCTCCAGCAT-3’; F2: 5’-CAGAGTCACCTGGGACTTGCT-3’; R2: 5’-CACAACGGGTTCTTCTGTTAGTCC-3’; F3: 5’-CAGAGTCACCTGGGACTTGCT-3; R3: 5’-GTAGCAGTAACGGTTGATGGCAATG-3’). A product size of 390 bp was obtained for the WT mouse. The product sizes of 353 bp and 357 bp were identified for GPR50 knockout mice together. All mice were group-housed with 3-5 same-sex cage mates in standard mouse cages in a pathogen-free barrier facility on a 12-h light-dark cycle with lights on at 07:00 and a controlled temperature range of 22–25 °C. Food and water were provided ad lobitum. Behavioral tests were performed during the light phase. All experimental procedures were preapproved by the Ethics Committee of Soochow University and conformed to the Institutional Animal Care and Use Committee guidelines of Soochow University (reference number: 202211A0519).

Generation of GPR50 CRISPR-Cas9 KO cell lines

For the generation of GPR50 KO HEK293T cells, single guide RNAs (sgRNAs) were designed using the online CRISPR design tool (Red CottonTM, Guangzhou, China, The exon region of GPR50 was selected to be targeted by CRISPR/Cas9 genome editing. A ranked list of sgRNAs was generated with specificity and efficiency scores. The pairs of oligos for two targeting sites were annealed and ligated to the YKO-RP006 vector (Ubigene Bioscience Co. Ltd., Guangzhou, China). The YKO-RP006-hRABL6 [gRNA] plasmids containing each target sgRNA sequence were transfected into cells with Lipofectamine 3000 (Thermo Fisher Scientific). 24–48 h after the transfection, puromycin was added to screen the cells. After antibiotic selection, cells were diluted using the limited dilution method and inoculated into a 96-well plate. Single GPR50 KO clones were performed after being cultured for 2–4 weeks and validated by PCR, western blotting, and Sanger sequencing.

Cell culture and transfection

HEK293T (female), HEK293, HeLa (female), and MEF cells were obtained from the American Type Culture Collection (ATCC). Cells were maintained at 37 °C with 5% CO₂ in DMEM-GlutaMAX (Hyclone, SH30243.01) medium supplemented with 10% FBS (Gibco, 005) and 100 U ml⁻¹ penicillin/streptomycin (Gibco, 15140163). Primary neurons were isolated from the hippocampus and cortex of newborn mouse pubs. The hippocampus and cortex were dissected from the whole brain in ice-cold PBS. The tissues were digested with 0.25% trypsin for 15 min at 37 °C, followed by gentle mechanical trituration. Neurons were cultured in neurobasal-A (Gibco, 10888022) medium supplemented with 2% B27 (Gibco, 17504044), 1% Glumax (Gibco, 35050061) and 1% Penicillin-Streptomycin (P/S) at 37 °C. Primary cultured neurons were transfected with WT GPR50 and GPR50 ASD mutant plasmids using Lipofectamine 2000 (Thermo Fisher Scientific).

Drug treatment

The gpr50^−/y and gpr50^-/+ mice were allowed to mate at 5 pm. The vaginal plugs were checked at 8 am the next day. The mice with vaginal plugs were supposed to be in pregnancy for 0.5 days. The gpr50^-/+ mice that were pregnant for 14 days were administrated with mitoQ (New Zealand, 70443110246) orally at 5 mg/kg/d, which were mixed into the food chaw. The pubs were genotyped after birth, and gpr50^−/y pups were fed food containing mitoQ until they were subjected to behavioral tests at 2 months old. Gpr50^+/y and gpr50^−/y mice, fed food with normal saline, served as controls.

Cells were treated with 400 nM bafilomycin A1 (Selleck, S1413) for 12–24 h to block the fusion of autophagosomes to lysosomes. Cells were treated with 50 μM CCCP (Yeasen, China, 40333ES60) for 2 h, 6 h, and 12 h to induce mitochondrial damage.

Analysis of colocalization of LC3 and MitoDsRed and fragmented mitochondria

The quantification was performed as described previously [19]. HeLa cells co-transfected with LC3-GFP and MitoDsRed plasmid were imaged with a confocal microscope. The colocalization ratio of LC3-GFP and MitoDsRed were analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD) as described. After correcting the background, Pearson’s correlation coefficient was calculated as the colocalization ratio. The mitochondrial fluorescent signals display as network or baculiform shape in normal conditions. We used Image J to measure normal and fragmented mitochondria. Considering the network structure formed through mitochondrial fusion and fission, we assessed a minimum of 15 mitochondria per cell and computed their average length. Mitochondria with an average length equal to or less than 5 μm were categorized as undergoing fragmentation, whereas those exceeding 5 μm were deemed normal. The percentage of cells with fragmented mitochondria was counted [66, 67].

Proximity ligation assay (PLA)

Cells were transfected with GPR50 WT, GPR50 LIR mutant, and GPR50 ASD mutant plasmids for 30 h. Cells were then fixed with 4% paraformaldehyde for 20 min at RT. Cells were permeabilized in PBS containing 0.3% triton for 5 min, then washed with PBS twice. After being blocked in Duolink block solution for 2 h at RT, cells were incubated with anti-LC3 (Novus, NB100-2220) and mouse anti-FLAG (Sigma-Aldrich, F3165) antibodies which were diluted in Duolink antibody dilution buffer overnight at 4 °C. The following morning, cells were washed for 10 min in washing buffer A, followed by adding the appropriate Duolink secondary antibodies (Sigma-Aldrich, DUO92002, DUO92004) diluted and mixed according to the manufacturer’s instruction. Cells were incubated for 30 min at 37 °C, after which cells were washed with washing buffer A twice, each 5 min. Ligation and amplification steps of the PLA were performed using the Duolink in situ Green Starter kit (Sigma-Aldrich, DUO92014) according to the manufacturer’s instructions. Cells were then mounted in a Prolong Gold mounting medium with DAPI (Invitrogen, P36941). Images were acquired on a Zeiss LSM900 Confocal microscope. PLA spots were counted using Image J software (NIH, Bethesda, MA, USA).

Quantification of dendritic complexity

Primary neurons were isolated from the hippocampus and cortex of newborn mouse pubs and cultured in neurobasal-A (Gibco, 10888022) medium supplemented with 2% B27 (Gibco, 17504044), 1% Glumax (Gibco, 35050061) and 1% Penicillin-Streptomycin (P/S) at 37 °C. The cultured neurons were transfected with GPR50-FLAG and immunostained for MAP2. The images were captured by an LSM780 confocal microscope (Zeiss, Jena). Sholl analysis was performed using the Image J software under the following parameters (start radius: 2 μm, end radius: 30 μm, radius step: 5 μm). The numbers of intersections at radial intervals of 2 μm starting from the central point of the soma were identified and averaged to create the mean sholl curve.

Co-immunoprecipitation (Co-IP)

Cultured cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP40, 10% glycerol) containing protease inhibitor at 4 °C for 30 min and centrifuged at 16,000 × g for 15 min at 4 °C. The supernatants were collected and subjected to protein quantification using a BCA protein assay kit (Thermo Fisher Scientific, 23225). Cell lysates were incubated with FLAG-conjugated beads (Bimake, B26102) for 12 h at 4 °C. The beads were washed with ice-cold RIPA buffer containing protease inhibitors. Input and co-precipitated fractions were analyzed using SDS–PAGE and immunoblotting. For co-immunoprecipitation of endogenous GPR50 and LC3, brain homogenates were extracted with lysis buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 10% glycerol) containing protease inhibitor and phosphatase inhibitor. The lysates were incubated with an anti-GPR50 antibody (Proteintech, 21514-1-AP) or anti-LC3 antibody (Novus, NB100-2220) overnight at 4°C and precipitated with Protein A/G agarose beads (Santa Cruz, sc-2003). Supernatants were subjected to SDS–PAGE and immunoblotting.

BiCAP interactome analysis and sample preparation for MS

HEK 293 T cells co-transfected with the constructs V1–GPR50 WT and V2–GPR50 WT. After 30 h, cells were lysed with RIPA Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 12000 rpm1% NP-40, 0.25% sodium deoxycholate, Roche EDTA-free protease inhibitor cocktail and NEM), followed by incubation with GFP-trap beads (Proteintech) overnight at 4 °C on a rotating platform. Briefly, beads were washed and using 8 M urea with 100 mM Tris-Cl (pH 8.5) to denature proteins, then, 10 mM Tris(2-Carboxyethyl)-Phosphine HCl (TCEP, Thermo Scientific) for reduction and 15 mM iodoacetamide (IAA, Sigma) for alkylation were added for reduction and alkylation, respectively. The sample was digested with Trypsin at 1:50 (w/w) (Promega) overnight and stopped by 5% Formic Acid (FA, Thermo Scientific), the peptide mixture was desalted by MonoSpinTM C18 column (GL Science). Desalted mixture was dried with a SpeedVac and resuspened in 0.1% FA for MS analysis.

HPLC-tandem MS (MS/MS) analysis of peptides

The peptide mixture was analyzed by a home-made 30 cm-long pulled-tip analytical column (75 μm ID packed with ReproSil-Pur C18-AQ 1.9 μm resin, Dr. Maisch GmbH), the column was then placed in-line with an Easy-nLC 1200 nano HPLC (Thermo Scientific) for mass spectrometry analysis. The analytical column temperature was set at 55 °C during the experiments. The mobile phase and elution gradient used for peptide separation were as follows: 0.1% formic acid in water as buffer A and 0.1% formic acid in 80% acetonitrile as buffer B, 0–1 min, 5%–10% B; 1–96 min, 10–40% B; 96–104 min, 40%–60% B, 104–105 min, 60%–100% B, 105–120 min, 100% B. The flow rate was set as 300 nL/min.

Data-dependent MS/MS analysis was performed with a Q Exactive Orbitrap mass spectrometer (Thermo Scientific). Peptides eluted from the LC column were directly electrosprayed into the mass spectrometer with the application of a distal 2.5-kV spray voltage. A cycle of one full-scan MS spectrum (m/z 300-1800) was acquired followed by top 20 MS/MS events, sequentially generated on the first to the twentith most intense ions selected from the full MS spectrum at a 30% normalized collision energy. Full scan resolution was set to 70,000 with automated gain control (AGC) target of 3e6. MS/MS scan resolution was set to 17,500 with isolation window of 1.8 m/z and AGC target of 1e5. The number of microscans was one for both MS and MS/MS scans and the maximum ion injection time was 50 and 100 ms, respectively. The dynamic exclusion settings used were as follows: charge exclusion, 1 and >8; exclude isotopes, on; and exclusion duration, 30 seconds. MS scan functions and LC solvent gradients were controlled by the Xcalibur data system (Thermo Scientific).

Metabolic extracellular flux analysis

The cellular extracellular acidification rate (ECAR) was determined using the Seahorse XF Glycolytic Rate Assay Kit (Agilent, Santa Clara, 103344-100) on the Seahorse XFe-24 Extracellular Flux Analyzer (Agilent, Santa Clara, USA), following the manufacturer’s protocol. Briefly, cultured cells were plated into a Seahorse XF-24 cell culture microplate (Agilent, 102342-100) overnight. One day before the experiment, the XFe24 sensor cartridge was hydrated overnight at 37 °C in a non-CO₂ incubator with Seahorse XF calibrant (Agilent, 100840-000). Cells were rinsed and incubated in the Seahorse XF DMEM assay medium (Agilent, 103575-100) supplemented with 1 mM pyruvate sodium, 2 mM glutamine, and 10 mM glucose at 37 °C in a non-CO₂ incubator for 1 h. Cell culture microplates were then loaded into the Seahorse XFe24 Analyzer. The test of glycolysis rate was performed as follows: baseline, 3 cycles; injection of Rotenone/Antimycin A (final concentration: 0.5 μM), 3 cycles; injection of 2-DG (final concentration: 50 mM), 5 cycles. Each cycle is composed of mix 3 min, wait 2 min, and measure 3 min. After completion of the measurements, the amount of protein within the plate was determined by BCA. Data are presented as pH changes per min in the medium using the Seahorse Wave 2.6.0 version (Agilent), normalized to total protein content. Glycolic Proton Efflux Rate (glycoPER), referring to the proton efflux rate brought by the production of lactic acid during glycolysis, was calculated as the differences between the total PER and the mitochondrial PER. Mitochondrial PER refers to the proton efflux rate from respiratory sources (contribution of mitochondrial/TCA cycle-derived CO₂ to extracellular acidification). Mitochondrial PER was calculated by measuring OCR before and after adding Rot/AA using the Buffer Factor (BF, 2.8) and CO₂ contribution factor (CCF).

Enrichment of mitochondria

HEK293T cells were treated with 50 μM CCCP (Yeasen, China, 40333ES60) for 2, 6, and 12 h and collected. Mitochondria were isolated using the Cell Mitochondria Separation Kit (Beyotime Co., Nantong, China, C3601) as instructed by the manufacturer. Briefly, cells were collected and homogenized on ice in mitochondrial separation reagent A containing 1 mM PMSF (Beyotime Co., Nantong, China, ST506). Cell homogenates were centrifuged at 600 g at 4 °C for 10 min, and then the supernatant was collected and centrifuged at 11,000 g at 4 °C for 10 min. The precipitate was collected and resuspended in mitochondrial buffer A. The supernatant was collected as the cytoplasmic fraction depleted mitochondria.

High-resolution respirometry

After washing with pre-cooled PBS, the region of the cortex and hippocampus were dissected and put into a tube containing MIR05 respiratory buffer (20 mM HEPES, 110 mM sucrose,10 mM KH₂PO4, 20 mM taurine, 60 mM lactobionic acid, 3 mM MgCl₂, 0.5 mM EGTA [pH 7.1], 1 mg/ml fatty acid-free BSA, catalase 280 U/ml). The tissues were homogenized on ice, and the mitochondrial oxygen consumption rate (OCR) was measured via O₂K (Oroboros instruments). Briefly, the tissue homogenates were placed in the respiration chamber in MIR05. After the baseline recording, the substrate pyruvate (5 mM), glutamate (10 mM), and malate (2 mM) as the substrates of complex I. When the respiration was stabilized, the leak value of complex I was obtained. The maximum oxidative phosphorylation value of complex I was obtained by adding ADP (2.5 mM). Cytochrome C (10 μM) was added to verify the integrity of the mitochondrial membrane. Succinate (10 mM) was added to obtain complexes I and II’s maximum oxidative phosphorylation value. The maximum electron transport capacity was obtained by adding uncoupling agent CCCP (1.5 μM). The maximum electron transport capacity of complex II was obtained by adding rotenone (complex I inhibitor) (0.5 μM). Antimycin A (inhibitor of complex III) (2.5 μM) was added to obtain ROX non-mitochondrial respiration. Add Ascorbate (2 mM) and TMPD (0.5 mM) to detect the respiratory function of complex IV. All measurements were performed at 37 °C.

Detection of Reactive oxygen (ROS)

Cells transfected with GPR50 WT or GPR50 mutant plasmids were treated for 50 μM CCCP for 2 h. Following the manufacturer’s instructions, the ROS test was determined using a ROS assay kit (Beyotime, S0033S). 10⁵ cells were incubated with CellROX (5 μM) in a cell incubator for 30 mins at 37 °C and washed three times with PBS. Cells were then fixed with 4% PFA for 15 min. The immunofluorescent density was determined under the Zeiss confocal microscope.

The mitochondrial superoxide was detected by using MitoSOX Red (Invitrogen). Brain slices isolated acutely were incubated for 4 h in carboxygenated ACSF at 32 °C. 5 μM MitoSOX Red was added to the brain slices and allowed for incubation for 10 min. Slices were then fixed with ice-cold 4% PFA in PBS overnight at 4 °C and cut into 30 μm sections. Brain slices were mounted onto slides with a mounting medium containing DAPI (Vector Laboratories). Slices were imaged using a Zeiss confocal microscope. All parameters (pinhole, gain, contrast, offset) were held constants for all sections from the same experiment.

Mitochondrial membrane potential (ΔΨm) assay

The ΔΨm was measured using the Mitochondrial Membrane Potential Assay Kit with TMRM (Beyotime, C2001S) following the manufacturer’s instructions. The TMRE probe was diluted with neurobasal A culture medium and incubated with cultured cells for 30 min. Cells were imaged using a Zeiss fluorescence microscope. All parameters (pinhole, gain, contrast, offset) were held constant for all sections from the same experiment.

Analysis of ATP levels

ATP levels were analyzed using the ATP detection kit (Beyotime, S0027). The cultured cells were washed with PBS and lysed with 100 μl ATP lysate buffer on ice. The cell lysates were centrifuged at 12,000 g at 4 °C for 5 min, and the supernatants were collected. The ATP detection solution was diluted with ATP detection diluent in a 1:4 ratio. Sample supernatants were mixed with ATP detection solution and standard solution in a 96-well plate following a ratio at 10 μl: 100 μl: 10 μl and analyzed under a luminometer. The ATP levels were calculated based on the standard curve.

Transmission electron microscopy (TEM)

TEM was conducted in collaboration with the Electron Microscope Facility at Soochow University. Mice were anesthetized with isoflurane and transcardially perfused with ice-cold PBS, followed by a fixing solution comprising 2% paraformaldehyde (PFA) and 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Brain specimens were then immersed in this fixing solution at 4 °C overnight. The corpus callosum was dissected into 1 mm³ blocks and further post-fixed overnight in the same solution at 4 °C. Subsequently, the tissues underwent two 15 min washes with 0.1 M phosphoric acid, followed by a 1 h post-fixation step in 1% OsO4 in cacodylate buffer at room temperature. After dehydration with a series of diluted acetone solutions, the specimens were embedded in Lx-112 (Ladd Research Industries). Following polymerization, ultrathin sections ranging from 40 to 60 nm in thickness were prepared and stained with 3% uranyl acetate and lead citrate. Imaging was performed using a Tecnai G2 Spirit BioTwin transmission electron microscope (USA). The normal morphology of mitochondrial inner membranes forms cristae, which increase membrane surface area to accommodate more biochemical reactions. Consequently, mitochondrial cristae are often arranged in uniform and orderly lamellar structures. In summary, we assessed the width of at least 10 cristae per mitochondrion and calculated their average width. Mitochondria with an average width equal to or less than 20 nm were classified as normal, while those exceeding 20 nm were classified as swollen. Mitochondrial morphology within neurons in the hippocampal CA1 region was analyzed from three mice per group using Image J software (NIH, Bethesda, MA, USA).

Behavioral tests

2-month-old Gpr50^+/y, Gpr50^−/y, and Gpr50^−/y mice were subjected to behavioral tests. The experimenter was blinded to the genotype during testing. All behavioral tests were performed in a dimly lit room without noise interference. The experimental apparatus used in every test was cleaned with 30% alcohol after each tested subject.

Three-chamber social preference test

Three-chamber social preference test was described previously [68]. The tested mice were placed in a transparent three-chamber box and allowed to shuttle freely for 10 min. Then, two empty metal cages were placed on both sides of the transparent box. The mice were allowed to explore the three chambers freely for 10 min. Then, one of the metal cages was placed in a stranger mouse of the same age and sex. The test mice were allowed to explore for 10 min among the three chambers, and the time spent by the test mice in the lateral chamber containing either stranger mice or empty cages and the time spent interacting with either stranger mice or empty cages were recorded. In the final stage, another unfamiliar mouse was placed in a metal cage on the other side, and the time spent in the lateral chamber containing either the unfamiliar or familiar mouse and the time spent interacting with the unfamiliar or familiar mouse was recorded.

Buried food test

The tested mice were individually housed for more than 24 h, followed by a three-day acclimatization period. All food was removed for 24 h before testing, but water was freely available. After this period, the mice were placed in a clean rat cage for 5 min of acclimatization. Following this, the mice were removed, the bedding was replaced, and food was buried 3 cm under the bedding in the lower right corner of the cage (the same corner was used in every trial). The tested mice were then placed into the cage with the buried food, and the cage was covered. An observer moved 2 meters away from the cage. The mouse was placed back in the center of the rat cage and given a maximum of 15 min to find the food. The time the mouse took to find the food (indicated by the mouse uncovering it, picking it up, or starting to eat it) was recorded in seconds. If the mouse didn’t find the food within 15 min, the time was recorded as 15 min.

Statistical analysis

All data analyses were performed using GraphPad Prism 8.0 to produce graph values. All graph values are presented as mean ± SEM. All statistical analyses were performed using SPSS 26.0 or GraphPad Prism 8.0. The number of mice included in each experiment was based on standards established in the literature rather than being predetermined by statistical methods. Tests for normality and equal variances were used to determine the appropriate statistical test to employ. Data were analyzed using Student’s t-test (a comparison of the difference between the two groups) after confirming the homogeneity of variance and normal distribution and one-way ANOVA (a comparison between multiple groups) followed by LSD or Dunnett T3 post hoc tests. Statistical details of experiments such as statistical tests, statistical values, and the information related to n can be found in the figure legends. Significance in differences was accepted at p p p p