Animals
Male 10-12 weeks-old C57BL/6J (Jackson Laboratory) and CD-1 retired breeders (Charles River) were purchased and used to set up the chronic social defeat stress paradigm. All mice were housed on a 12 h light-dark schedule with food and water available ad libitum. All experiments performed were approved by the Fourth military medical university, Institutional Animal Care and Use Committee. Male littermates were randomly assigned to either experimental or control group. Behavioral tests were done on visibly healthy (i.e., no skin irritation, agile, and no developmental malformation of eyes or teeth) mice in 10-14 weeks of age; electrophysiological slice experiments involving whole-cell patch clamp recordings were performed on P50-60 mice.
Two single guide RNA (sgRNA) sequences targeting sites upstream (sgRNA1) and downstream (sgRNA2) of the Mup gene cluster, respectively, were transcribed in vitro using MEGAshortscript™ Kit (AM1345; Ambion Life Technologies, Carlsbad, USA). A mixture of plasmids encoding hCas9, sgRNA1 and sgRNA2 was microinjected into the fertilized C57BL/6 eggs at a ratio of 100:35:35 (ng/μl), and the transgenic embryos were planted into pseudopregnant ICR recipients. The founder was genotyped with primers spanning the Cas9 cleavage sites. Founder lines of successful deletion of the Mup gene cluster were identified through PCR genotyping of tail DNA. PCR products were further verified through Sanger sequencing. The genotyping primers were detailed below. MupdelF1 (5′-CATAAAGCACTAAAGCAGG-3′) anneals to sequence upstream of Mup gene cluster. MupwtF1 (5′-ATACTTAGCATACTGTCCCTG-3′) anneals to sequence within Mup gene cluster. MupR1 (5′-GAACAGTCCTTGGCCTATC-3′) anneals to sequence downstream of Mup gene cluster. MupdelF1 and MupR1 amplify a fragment of ∼200 bp corresponding to the knockout mutant. MupwtF1 and MupR1 amplify a fragment of ∼485 bp corresponding to the WT allele. The Mup-knockout mice are available upon request.
Mups Camk2a-CKO conditional knockout mice were generated by crossing the floxed Mups allele (Mupsfl/fl) mice with Camk2a-Cre transgenic mice, which expresses Cre recombinase in excitatory neurons under the control of the Camk2a promoter and conditional knockout genes in excitatory neurons. Mupsfl/fl mice were obtained by crossing between Mupsfl/w mice, which were generated by GemPharmatech Co., Ltd. A targeting vector containing the first two exons of the SARM1 gene was created by recombineering. Briefly, transformed ES colonies were screened by long-template PCR with the following primer sets: GJS0219111712-01-Mup-5wt-tF1 (5'-AAATCCTACAAGCCCTCAGCAG-3') and GJS0219111712-01-Mup-5wt-tR1 (5'-TAGCTCCAGGTCTGCATCATTC-3') to generate a fragment of 255 bp corresponding to the wild-type alle and a fragment of 347 bp corresponding to the floxed alle; 10884 (5'-GTTCTCCGTTTGCACTCAGG-3') and oIMR8990 (5'-CAGGTTCTTGCGAACCTCAT-3') to generate a ∼485 bp band for Camk2a-cre positive clones.
Depression contagion
Social defeat was performed with small modifications as described previously11, with the addition of an “depression contagion” component. Briefly, CD-1 mice with consistent attack latencies (30 sec on three consecutive screening tests) were housed in cages fitted with perforated Plexiglas dividers, allowing sensory, but not physical, contact. Naïve C57BL/6J mice were housed in pairs for at least 2 weeks, and then assigned to either observer or defeated mice. Observer mice were placed into the empty compartment adjacent to the CD-1 aggressor, and defeated mice were placed into the compartment containing the aggressor. During this time, the defeated mice was attacked by the CD-1 aggressor and adopted a defensive posture. After 20 min, the defeated mice were put back with observer for social interaction overnight. This process was repeated for 10 consecutive days, such that each day the observer “watched” the defeat of its partner mouse by a novel CD-1. The term“watched” in this model refers to all sensory stimuli associated with the defeated mice experience and not visual stimuli alone. In parallel, control mice were generated by 10 days of repeated daily transfers on each side of a perforated Plexiglas partition with a CD-1 mouse on the other side. Behavioral tests were carried out at 24 hours as the sequence indicated in Figure 1A.
Animal screening
Mice were subjected to depression contagion and then subdivided into groups of high-empathy or low-empathy animals as described previously44. In each of 6 different behavioral tests (open field test, elevated plus maze, tail suspension test, forced swimming test, sucrose preference test, novelty-suppressed feeding test), if their performance were 1 SD worse than mean values of control mice, their depression score was added for 1 point. If their performance were 1 SD better than mean values of control mice, their score was subtracted for 1 point. If their performance were within 1 SD of the mean values for control mice, their score in this test were 0. After summing all their scores, if it was greater than or equal to 1, this mouse was considered high-empathy individual. If it was lower than less than or equal to -1, this mouse was considered low-empathy individual.
Open field test.
The open field test was conducted on day 11 after depression contagion as described in a previous report 45. It was carried out in the open fixed, a square arena with clear Plexiglas walls and floor, and placed inside an isolation chamber with dim illumination and a fan. Mice were placed in the center of the box and allowed to freely explore for a 15 min period. In all behavioral test, mice were videotaped using cameras and analyzed with a DigBehv software (Jiliang, Shanghai, China).
Elevated plus maze
The Elevated plus maze (EPM) was conducted after open field test on the same day as described in the report 46. The apparatus comprised of two open arms (25×8×0.5 cm) and two closed arms (25×8×12 cm) that extended from a common central platform (8×8 cm). The apparatus elevated to a height of 50 cm above the floor. Mice were allowed to habituate to the testing room for 2 days before the test, and pretreated with gentle handling two times per day to eliminate their nervousness. For each test, individual animals were placed in the center square, facing an open arm, and allowed to move freely for 5 min. The number of entries and time spent in each arm were recorded with a DigBehv software (Jiliang, Shanghai, China).
Social interaction test
Social interaction tests were performed on day 12 after depression contagion. We measured the time spent in the interaction zone, corner zones and locomotor activity in an open-field arena. Their movements were monitored and recorded with a DigBehv software (Shanghai Jiliang Software Technology Co. Ltd., China) for 20 minutes each test session.
Tail suspension test (TST)
Tail suspension test was performed in a quiet room as described previously 47. Each mouse was suspended 50 cm above the floor and a small piece of adhesive taped to a wooden stick near the end of the mice tail about 2 cm, and the mice views were surrounded by a barrier. The mice tail test lasted 6 min, and the last 4 min was its immobility time. Mice were considered immobile when they showed hopelessness and were nearly immobile or completely motionless.
Forced swimming test (FST).
Swimming sessions were carried out by placing mice in individual glass cylinders (30 cm height× 10 cm diameter) containing water at 23–25°C, 25 cm deep in such a way that mice could not support themselves by touching the bottom with their paws or tail, modified from instructions reported previously (54). Two swimming sessions were conducted: an initial 15-min pre-test (session inducing behavioral despair) followed 24 h later by a 5-min test but only the last 4 min were analyzed by a software. After every swimming session, each mouse was dried, warmed and returned to its home cage.
Sucrose preference test (SPT).
As reported previously 48, before the first time of the test, all rats were trained to get accustomed to 1% sucrose solution (w/v) in their home cages. Two bottles of 1% sucrose solution were placed on each cage for 24 h. For the following period of 24 h, 1% sucrose in one bottle was replaced with pure water. After adaptation, rats were deprived of food and water for 24 h, followed by the SPT the next morning, in which the rats were fed with two pre-weighed bottles of liquid at the same time: the one containing 1% sucrose solution and the other pure water. The bottles were counterbalanced across the left and right sides of the cages throughout the experiment to avoid position preference effects. After 12 h, the two bottles were re-weighed.
Novelty-suppressed feeding test.
This test was performed as described previously 49. Briefly, the animals were deprived of all food in the home cage 24 h before testing. One food pellet was placed on a piece of round filter paper (12.5 cm in diameter) at the center of the acrylic testing chamber (50 × 50 × 40 cm), with the floor covered with bedding. Then, the animal was placed in a corner of the chamber. The latency to approach and to begin feeding on the food was measured for a maximum of 5 min under 140 lux illumination.
Proteomics.
The proteomics analysis was carried out by Beijing Genomics institution (BGI, Beijing, China) as described previously50. Mice were anesthetized by isoflurane and brains were removed. mPFC tissue was quickly hand dissected and frozen with liquid nitrogen. Three pairs of mPFC samples from high-empathy mice and low-empathy mice were used for proteomic analysis. Tissues were extracted with Lysis buffer. The samples were then sonicated on ice and centrifuged at 4°C, 17,000 g for 30 min. After further preparation, the protein (100 µg each) was digested with Trypsin Gold (Promega, Madison, WI, USA) (30:1, protein: trypsin) at 37°C for 16 h and labeled according to the manufacture’s protocol for 4-plex iTRAQ reagent (Applied Biosystems). The labeled peptide mixtures were then under a strong cation exchange chromatography was performed fractionation with a LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan). For LC- ESI-MS/MS analysis, the peptides were separated using a LC-20AD nano HPLC (Shimadzu, Kyoto, Japan). The eluate was subjected to nano electrospray ionization followed by tandem mass spectrometry in a Q EXACTIVE Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Intact peptides were detected at a resolution of 70,000 and then selected for MS/MS using high-energy collision dissociation (HCD) operating with a normalized collision energy setting of 27. Ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure was applied for the 15 most abundant precursor ions above a threshold ion count of 20,000 in the MS survey scan.
Bioinformatics analysis.
Protein identification and quantification were performed in Protein Pilot™ 4.5 software (AB Sciex). Searches were based on the Paragon database search algorithm and an integrated false discovery rate analysis function for peptide identification. According to the standard parameters, a unique protein had to contain at least two unique peptides, and the false discovery rate had to be less than 1%. To strengthen credibility, we considered only the iTRAQ ratio of the proteins within the range 0.5–20. A 95% confidence interval was chosen as the significance threshold for protein identification, and the detected protein threshold (unused ProtScore) was set to ≥1.3 to minimize the false positive identification of proteins. Functional classification and Gene Ontology (GO) enrichment analyses of the DEPs were carried out using DAVID (https://david.ncifcrf.gov/). Proteins were classified by GO category (http://www.geneontology.org), including “biological process,” “cell component,” and “molecular function.” The KEGG (http://www.genome.jp/kegg/) database was employed to identify significantly enriched pathways. The significance was determined with slight modifications as recommended by the authors of DAVID according to the Benjamini-corrected p value <0.05. Functional protein association networks were explored in STRING v.10.5 (http://string-db.org/). The heat map was generated in MeV 4.9 software.
Western blotting
The mPFC proteins were extracted as previously described. Animals were anaesthetized using isoflurane, and mPFC tissue was quickly dissected from the brain and homogenized in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail tablets). After protein concentration measurement by BCA assay, 10–20 μg protein for each lane was separated on a 10% SDS-PAGE gel and transferred for western blot analysis. Mouse anti-MUP1 (1:1,000, R&D Systems), mouse anti-β-actin (1:10,000, Sigma), rabbit anti-GLT1 (1:500, Sigma), rabbit anti-Syt1 (1:500, Proteintech Group, Inc) antibodies, rabbit anti-STXBP1 (1:500, Proteintech Group, Inc), rabbit anti-GLAST (1:500, Proteintech Group, Inc) antibodies, and high-sensitivity ECL reagent (ZATA Life, Inc) were used. All the bands were analysed with Tanon 4200 system (Biotanon, Shanghai, China).
Immunohistochemistry
Mice were anaesthetized using isoflurane, and then perfused transcardially with ice-cold PBS (pH 7.4) followed by 4% paraformaldehyde. After overnight post fix in 4% paraformaldehyde solution, brains were cryoprotected in 30% sucrose for 1 day. Coronal sections (40 μm) were cut on a microtome (Leica) and collected in PBS and stored at 4℃ for further use. The antibodies used were mouse anti-MUP1 (1:50, R&D Systems), rabbit anti-phospho-cfos (Ser32) (1:200, Cell Signaling Technology, Inc.), Alexa Fluor 546 goat anti-mouse IgG (all 1:1,000, Invitrogen), Alexa Fluor 488 goat anti-rabbit IgG (all 1:1,000, Invitrogen). Slices for checking the injection site were counterstained with Hoechst in the final incubation step. Fluorescent image acquisition was performed with an Olympus Fluoview FV1000 confocal microscope.
RNAscope in situ hybridization
Coronal brain sections (20 μm) were prepared as described above. Using an RNAscope multiplex fluorescent reagent kit and appropriately designed probes (ACDbio), we performed fluorescence in situ hybridization according to the manufacturer’s standard protocols. Tissue sections were heated for 1 h at 60°C with a hydrophobic barrier around the perimeter of each section before further processing. Slides showing poor staining were not analyzed.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was carried out as manfacture using commercially available kit used to quantify mouse MUP1 (catlog No. MBS733914, MyBioSource), human MUP1 (catlog No. MBS040679, MyBioSource), Epinephrine (catlog No. E-EL-0045c, Elabscience), Thyroxine (catlog No. EIAT4CX10, Thermofisher), Corticosterone (catlog No. EIACORT, Thermofisher) in plasma, urinary and mPFC according to the manufacturer’s instruction. Plasma used for human MUP1 detection was gathered from clinically diagnosed autism patients and stored at -80°C.
Stereotaxic virus injection and optical fiber implantation
Mice (6-8 weeks old) were anesthetized with gaseous isoflurane and placed on a stereotaxic apparatus (RWD Life Technology Co. Ltd., China). The eyes were covered by a drop of ophthalmic ointment to prevent drying. A heating pad was used to keep the body temperature at 35-37 °C. After shaving the hair and cleaning the incision site with iodine and medical alcohol, the scalp was incised to expose the skull. The connective tissue was gently removed from the skull surface with cotton swabs. Small craniotomy holes (~1 mm diameter) were drilled with the help of microscope for virus injection. Virus was injected stereotaxically into bilateral mPFC (AP, +2.3 mm; LM, ± 0.3 mm; DV, -2.0 mm), Pn at a 15o angle (AP –2.7 mm; LM ± 0.3 mm; DV, -5.7 mm), AOB (AP, +3.5 mm; LM, ± 0.9 mm; DV, -2.0 mm), BLA (AP –1.5 mm; LM ± 2.8 mm; DV, -5.0 mm) using 34 gauge needles controlled a syringe pump (ALC-IP600, Shanghai Alcott Biotech Co., LTD. China) at a speed of 30 nl/min. After injection, the pipette was left in place for an additional 5 min and then slowly retracted. The optic fiber (Thinker Tech Nanjing Biotech Co., Ltd., 100 μm in diameter, 0.22 NA, 3 mm or 7 mm in length) was targeted to the same position as that used for the virus injection, dental cement was applied to cover the skull, and was allowed to harden for 10 min. Behavioral experiments were performed at least 3 weeks post-surgery. Mice were used for further experiments three weeks after injection. Virus injection sites and optical fiber placements were confirmed postmortem in all animals.
To indicate Mup1 expression in different cell types, AAV2/5 virus expressing Cre recombinase under Mup1 promotor control was injected into CAG-loxp-ZsGreen-Stop-loxp-tdTomato mice. In Mup1 expressing cells, ZsGreen expression will be stopped and tdTomato will be expressed, resulting cell color changed from green to red.
For MUP1 over expression, AAVs were generated from the AAV9-CMV-EGFP-P2A-MUP1-3Flag plasmid and produced commercially (300 nl, Obio Technology Corp. Ltd., Shanghai, China). For AAV-mediated knockdown of endogenous MUP1, GLT1, STXBP1 expression, engineered AAV2/9 coexpressing mCherry fluorescence protein under the CMV promoter or control shRNA under the U6 promoter (500nl, Obio Technology Corp. Ltd., Shanghai, China). For every gene, three targets were designed and the most effective target was selected through western blot. Following shRNA sequences were used shRNA: MUP1 (5’-GCAGAAGAAGCTAGTTCTA-3’); GLT1 (5’-GCATTGGTGCAGCCAGTAT-3’); STXBP1 (5’-CCAAGGCACTGTAGACAAA-3’).
To regulate mPFC neurons activity, AAV9-Ef1a-ChR2-EGFP or AAV9-Ef1a-eNpHR-EGFP or AAV9-Ef1a-hM4D (Gi) -EGFP or AAV9-Ef1a- hM3D (Gq) -EGFP or AAV9-Ef1a-EGFP was injected into mPFC. Virus plasmids were purchased from Obio (Shanghai, China). To illustrate AOB-mPFC-Pn circuit, we used a combination of a retrograde RV-N2C-△G-EGFP injected into the Pn, and an anterograde VSV-△G-mCherry (100 nl, from Brain Case Wuhan, China) injected into the AOB. To selectively regulate mPFC-Pn circuit, we used a combination of an anterograde AAV2/1-hSyn-mCherry-2A-CRE injected into the mPFC, and Cre inducible virus AAV9-Ef1a-DIO-ChR2-EGFP or AAV9-Ef1a-DIO-eNpHR-EGFP or AAV9-Ef1a-DIO-hM4D (Gi) -EGFP or AAV9-Ef1a-DIO-hM3D (Gq) -EGFP or AAV9-Ef1a-DIO-EGFP injected into the Pn. Therefore, ChR2 or eNpHR was expressed selectively in the Pn neurons that receiving mPFC projection.
DDREAD experiment
Clozapine-N-Oxide (CNO) was dissolved in Dimethyl sulfoxide (DMSO, D2650, Sigma) to a stocking solution of 0.4g/mL and diluted with saline (0.9% NaCl solution) to a working concentration of 0.2 mg/mL. Stocking solution was stored at 4℃ and fresh CNO solution was prepared each day before experiments. Saline or CNO (1 mg/kg b.w.) was administered intraperitoneal (i.p.) daily to the mice during depression contagion process, in the blind design like that of optogenetic experiments.
fMOST
For whole-brain precise imaging, the GMA embedded mouse brains were imaged by our homemade fMOST system. The imaging system has been described previously51. Briefly, the system used a mercury lamp (X-Cite exacte, Lumen Dynamics) as light source, a digital micro-mirror device (DMD, XD-ED01N, X-digit) to generate the illumination grid pattern and a water immersion objective (1.0 NA, XLUMPLFLN 20XW, Olympus) for imaging. Two scientific complementary metal-oxide-semiconductor cameras (ORCA-Flash4.0, Hamamatsu Photonics K.K.) were used for signal detection. A piezoelectric translational stage (P-725 PIFOC Long-Travel Objective Scanner, E-753 Digital Piezo Controller, PI GmbH) moved the objective for axial scanning. The sample box was screwed onto a high-precision 3D translation stage (ABL20020-ANT130-AVL125, Aerotech Inc.). The 3D translation stage moved the sample for mosaic scanning and sectioning. A diamond knife (Diatome AG) was used for sample sectioning. During imaging, the sample was immersed in a water bath containing PI (1 µg ml-1, wt/vol) and 0.05 M Na2CO3. The objective scanned the surface of the sample in mosaic mode at a step of 2 µm. After one surface was finished, the diamond knife removed the imaged surface and exposed the smooth fresh surface for imaging. The mosaic imaging process was repeated until the entire coronal section was acquired. After data acquisition, the images were preprocessed to generate a series of coronal images. The datasets for complete neuron morphology reconstructions were converted to other format for further process (Please see Visualization and reconstruction part). For whole-brain rabies virus labeled neuron counting, the coronal images were used to generate 50 μm maximum projection images. Then, the labeled neurons were manually counted with ImageJ according to the Allen brain atlas101.
Image preprocessing.
The raw data acquired by the brain positioning system needed image preprocessing for mosaic stitching and illumination correction. This process has been described before23. Briefly, the mosaics of each coronal section were stitched to obtain an entire section based on accurate spatial orientation and adjacent overlap. Lateral illumination correction was performed section by section. Image preprocessing was implemented in C + + and optimized in parallel using the Intel MPI Library (v.3.2.2.006, Intel). The whole data set were executed on a computing server (72 cores, 2 GHz per core) within 6 h. Visualization and reconstruction. We visualized the data set using Amira software (v.5.2.2, FEI) to generate the figures and videos. The data set acquired by the dual-color precise imaging system was separated into the GFP channel and PI channel. The PI-labeled data set was sampled to 3.2 × 3.2 × 50 μm3 and imported into Amira to generate the outline of the mouse brain. To trace the morphology of the input neurons, we transformed the data format of GFP-labeled data from TIFF to TData via the algorithm developed by our lab102. A homemade software Gtree was used to trace the morphology of GFP-labeled neurons at the whole-brain level by human-machine interactions103. Briefly, we loaded the data block of interests into Amira and assigned the initial and terminal points of the fibers in the block, so that Amira could automatically calculate the pathway between initial and terminal points. We repeated this procedure until the reconstruction was finished. The reconstructed neurons were checked back-to-back by three people. The tracing results were saved in SWC format. We loaded the outline of the mouse brain and the tracing results into Amira simultaneously and used the moviemaker module of Amira to generate figures and videos51.
Blue light stimulation.
In optogenetic experiments, an external optical fiber (100 μm in diameter, NA: 0.22) was coupled to an implanted optic fiber through a ceramic sleeve. Laser power at the end of an external fiber was measured with a laser power meter and was adjusted to meet experimental requirement. Laser illumination was provided with blue (473 nm) or green (532 nm) diode pumped solid state laser (50 mW) and controlled by a microcontroller. For those with ChR2, laser power was 0.8 mW, and for NpHR experiments, laser power was 3 mW. Animals were anaesthetized using isoflurane, then were connected to the patch cable, and allowed to recover from handling for 1–3 min before the 20-min test was initiated. Photostimulations were controlled by the mouse activity tracking software (Shanghai Jiliang Software Technology Co. Ltd., China), in which once the mouse entered the target zone, photostimulation turned on; and once the mouse left the target zone, photostimulation turned off. During the laser on stage 473 nm (20 Hz, 40 ms; 80% duty cycle, in every 3 s) or 532 nm (continuous light) was given. Activation of ACC cell bodies was induced by 10-Hz light trains with 5-ms pulses of blue light generated by a 473-nm diode-pumped solid-state laser. Before all laser on design, a laser off design was carried out to reveal whether optogenetic manipulation specifically influence the laser on trials instead of general influence of performance across all trials. It also controls for variation across individual mice better.
Co-immunoprecipitation and liquid chromatography-mass spectrometry
Affinity chromatography for mass spectrometric identification of MUP1 receptors were performed as described previously52. Mice mPFCs were lysed in ice-cold immunoprecipitation lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% deoxycholate) containing proteinase inhibitor mixture (Roche, UK). The lysates were centrifuged at 12,000 × g for 15 min at 4◦C and the concentration of the protein was estimated using the BCA kit. The resulting supernatant was adjusted to 1 mg/mL by adding lysis buffer. The lysates were pre-cleared with the mouse IgG (R&D Systems, USA) and anti-His magnetic beads (Catlog. 10104D, Invitrogen, USA), followed by overnight incubation with 20 µg recombinant mouse MUP1-His (LifeSpan BioSciences, Inc, USA) or normal IgG at 4°C. Then, the immuno-complexes were pulled down by incubation with anti-His magnetic beads for 2 h at 4°C on a rotating platform. The beads were washed with lysis buffer three times, mixed with SDS sample buffer, and denatured at 100°C for 5 min. Finally, the samples were centrifuged at 12,000 g at 4◦C for 30 s, and the supernatants were electrophoresed on SDS-PAGE gels. The gels were stained with coomassie blue and each lane containing visible protein bands were excised from the gels, destained in ammonium bicarbonate/acetonitrile, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin.
The samples were then transferred to new vials for LC-MS/MS and analyzed by Beijing Genomics institution (BGI, Beijing, China) as previously described. Briefly, peptides generated from tryptic digestion were injected into a Dionex LC-Packings Ultimate3000 HPLC (Amsterdam, Netherlands) mounted with a C18 column (75 µm × 150 mm) and run with a linear acetonitrile gradient (0–60%) at a flow rate of 400 nl/min. The eluted peptides were injected directly into a Q Exactive hybrid quadrupole-Orbitrap MS (Thermo Fisher, Bremen, Germany) using nanoESI spray at an ion spray of 1800 V. Intact masses were measured at a resolution of 70,000 (at m/z 200) in the Orbitrap using an AGC target value of 3 × 106 charges. The output files from the LC-MS/MS analyses were converted to MGF files using Protein Discovery 1.3 (Thermo Fisher, Bremen, Germany) and delivered to the Mascot 2.3 search engine (Matrix Science, Boston, MA), in which the data were queried against the Swiss-Prot database for mouse proteins with the following search parameters: peptide mass tolerance, 15 ppm; fragment mass tolerance, 20 mmu; methionine oxidation and the samples were separated by NanoLC using an elution that consisted of either water/0.1% formic acid and acetonitrile/0.1% formic acid as the mobile phases A and B, respectively. Finally, the dataset was filtered to only include transmembrane, cell-surface proteins using Panther and Uniprot databases.
Preparation of recombinant mouse proteins and peptides
As previously reported53, cDNAs encoding full length recombinant mouse MUP1 (rmMUP1) or full length STXBP1 (rmSTXBP1) or amino acids 143-234 of GLT1 (rmGLT1aa143-234) was cloned into pMAL-c5x (New England BioLabs) for expression of N-terminal hexahistidine-tag binding protein fusion proteins. rmMUP1, rmSTXBP1 and rmGLT1(aa143-234) were expressed in BL21-Gold (DE3) pLysS E. coli cells (Agilent Technologies). 80 mL of overnight culture was added to 800 mL of 2×TY medium containing ampicillin (100 mg/L) and cells were grown to an optical density of OD600 = 0.6 in an incubator/shaker at 37°C and 210 revolutions per minute (rpm). Protein expression was induced by adding 0.1 M IPTG to the culture and growing for another 3 hr at 30°C. Cells were harvested by centrifuging at 4,500 rpm for 15 min and the pellets resuspended in PBS pH 7.4, 1 complete protease inhibitor cocktail tablet (Roche) per 50 mL and 0.2 mM PMSF. The resuspended pellets were flash frozen in liquid nitrogen and stored at −80°C until purification. The cells were thawed slowly on ice and the cells were lysed by sonication at 4°C. The bacterial lysates were cleared by centrifugation at 35,000 × g at 4°C for 60 min, filtered through a 0.45 μm filter and loaded onto a 5 ml HisTrap HP column (GE Healthcare) pre-equilibrated with PBS pH 7.4, 20 mM imidazole. The eluate was then onto a 5 ml HiTrap Q HP column (GE Healthcare), followed by size exclusion chromatography on a HiLoad Superdex 200 PG 16/600 (GE Healthcare). The proteins were analyzed on Bolt SDS-PAGE 4%–12% Bis-Tris gels (ThermoFisher Scientific) stained with Comus Bright Blue R250. Samples containing pure proteins were pooled, dialysed against PBS pH 7.4, concentrated and stored at −80°C in small aliquots.
GLAST (aa146-236) and SYT1(aa1-57) peptide was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) using the standard solid phase peptide synthesis (SPPS) procedure. Briefly, the syntheses were performed using a Protein Technologies PS3 automated peptide synthesizer. Wang resin preloaded with Fmoc-L-Tyrosine (Novabiochem) at 0.1 mmoL scale was used as a solid-phase support. The coupling of standard Fmoc (9-fluorenylmethoxy-carbonyl)-protected amino acids (Chem-Impex) was achieved with HBTU (O-benzotriazole-N, N, N′, N′-tetramethyluronium hexafluorophosphate; Novabiochem) in the presence of N-methylmorpholine (NMM) in N, N′-dimethylformamide (DMF) for 20-minute cycles. Fmoc deprotection was achieved using 20% piperidine in DMF ( 2×5 minutes). The N-terminus of the peptide was acetylated with acetic anhydride and NMM. Side-chain deprotection and peptide cleavage from the resin were achieved by treating the resin-bound peptide with 5 mL of 100% trifluoroacetic acid (TFA) for 2 hours under N2. After evaporation of TFA under N2, the peptide was washed three times with cold diethyl ether, air-dried, and purified by Agilent ZORBAX EclipsePlus C18 column (1.8 µm, 2.1 × 50 mm) (Agilent, Santa Clara, CA, USA) with a linear 40-minute gradient from 3 to 70% acetonitrile in water with 0.1% TFA. The purified peptides were subsequently characterized by LC–MS.
Biolayer Interferometry Assay (BLI).
rmMUP1 binding to rmSTXBP1, rmGLT1(aa143-234), GLAST(aa146-236) and SYT1(aa1-57) was measured using an Octet RED 96 system (Pall forteBIO Corp, Menlo park, CA, USA)54. Samples of buffer were dispensed into polypropylene 96-well black flat-bottom plates (Greiner Bio-One, Frickenhausen, Germany) at a volume of 180–200 µl per well, and all measurements were performed at 30°C with agitation at 1000 rpm. Amine Reactive 2nd-Generation (AR2G) biosensors were used to immobilized rmMUP1 for rmSTXBP1 and rmGLT1 (aa143-234) binding detection. Sartorius Streptavidin (SA) Biosensors were used to immobilized GLAST (aa146-236) and SYT1 (aa1-57), and rmMUP1 was added in mobile phase. The biosensors were detected in OctetRED for kinetic screening of other protein binding. Briefly, biosensors were dipped in assay buffer containing wells for 1 min to remove any nonspecific protein or unbound protein. Biosensors were then transferred into fresh assay buffer for 100 s to collect a baseline read. Kinetic measurements for proteins binding were performed by dipping pre-coated biosensors into wells containing different concentrations of ligands for 180 s, followed by a 300-s dissociation time by transferring the biosensors into buffer-containing wells. All sensor grams were referenced for buffer effects and then fitted using the OctetRED user software (Pall ForteBio). The binding profile of each sample was summarized as an “nm shift” (the wavelength or spectral shift in nanometers), which represented the difference between the start and end of the kinetic cycle. Kinetic responses were fitted to a 1-site binding model to obtain values for association (kon), dissociation (koff) rate constants, and the equilibrium dissociation constant (Kd). Curves that could not be reliably fitted with the software (R2 < 0.90), usually caused by heterogeneous binding, were excluded from further analysis.
Virtual screening
The dock module in MOE1 was used for structure-based VS (SBVS). The Chemdiv compounds library was selected as VS small molecule library. Figure 1 shown the flowchart of SBVS on small molecules. All compounds were prepared with the Wash module in MOE. The binding site of GLT1 was set around the following residues: Ala198, Thr217, Ala221, Glu223 and Glu224. That of STXBP1 were Arg275, Lys277 and Glu278. After that all compounds were docked to proteins, ranked by high throughput rigid docking with London △G scoring. Then the ranked top 15K compounds were further selected to dock with proteins again, ranked by flexible docking with the “induced fit” protocol. Prior to docking, the force field of AMBER12: EHT and the implicit solvation model of Reaction Field (R-field) were selected. The protonation state of the proteins and the orientation of the hydrogens were optimized by Quickprep module at the PH of 7 and temperature of 300 K. For flexible docking, the docked poses were ranked by London dG scoring first, then a force field refinement was carried out on the top 10 poses followed by a rescoring of GBVI/WSA dG, and the best ranked pose was selected for each compound. After flexible docking, the 15K compounds were divided into structural clusters through fingerprint-based clustering (the distance parameter among clusters was set to 0.5), and the best ranked compound in each cluster was set as cluster center. The best ranked 100 cluster centers were finally identified as potential hits.
Molecular docking
Protein-protein docking in ClusPro server1 was used for molecular docking simulations. The crystal structures for MUP1 (PDB ID: 1I04) and STXBP1(PDB ID: 3C98) were downloaded from RCSB Protein Data Bank respectively. The crystal structures for GLT1, SYN1, SYT1, SYT7 and GLAST were predicated by I-TASSER (Iterative Threading ASSEmbly Refinement). For protein docking, the smaller protein (a smaller number of residues) usually is set as ligand and the other as receptor. The ligand was rotated with 70,000 rotations. For each rotation, the ligand was translated in x, y, and z axis relative to the receptor on a grid. One translation with the best score was chosen from each rotation. Of the 70,000 rotations, 1000 rotation/translation combinations that have the lowest score was chosen. Then, a greedy clustering of these 1000 ligand positions with a 9 Å C-alpha RMSD radius was performed to find the ligand positions with the most “neighbors” in 9 Å, i.e., cluster centers. The top ten cluster centers with most cluster members were then retrieved and inspected visually one by one. The intermolecular contacts from the most probable poses were further evaluated.
Cell culture
The primary astrocytes were isolated from either C57BL/6J one- (P1) to two-day-old (P2) pups using the differential adhesion method The dissociated cells were placed in a 25 cm2tissue culture flask in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific; Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific), penicillin (10 U/mL), and streptomycin (100 mg/mL). When confluent (after 6 to 7 days), the flasks were sealed and shaken at 250 rpm at 37℃ for 16 h. Over 95% of the adherent cells were astrocytes as demonstrated by the anti-glial fibrillary acidic protein (anti-GFAP) immunostaining. Human GBM cell line U251 was obtained from ATCC and were cultured in the same condition. All cells are tested for mycoplasma contamination each month. All cells used in this paper are mycoplasma free. Acquisition and use of these tissues were approved by the Ethics Committee of Fourth Military Medical University.
Human brain tissue sample collection
Brain tissue specimens were collected from a 64 aged woman who underwent brain abscess removing surgical operation in left prefrontal cortex. During the surgery, about 1.2 cm brain abscess tissue was removed and stored immediately in -80℃ for further analyses. All study protocols were complied with the guidelines for the conduct of research involving human subjects as established by the Ethics Committee of Fourth Military Medical University. All procedures were approved by the Ethics Committee of Fourth Military Medical University.
Statistical analysis.
All analyses were performed with Prism (GraphPad) and data sets were assessed for normality and group variance before statistical testing. Values are expressed as mean ± s.e.m. unless otherwise stated. Two-tailed statistical tests are indicated in each figure (*P < 0.05; **P < 0.01); post-test corrections were used every time multiple comparisons were performed (as indicated in each figure).