Animals
Shank3f/f(e4-9) mouse line was developed in our previous study16. Tek-Cre (B6.Cg-Tg(Tek-cre)1Ywa/J, stock no. 008863), Ai-14 (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, stock no. 007908), C3H/HeJ (stock no. 000659), and C57BL/6J (stock no. 000664) mice were purchased from Jackson Laboratory. Tek-Cre males were crossed with Shank3f/f females to obtain heterozygous double mutant Shank3 f/wt:Tek-Cre offspring. Conditional Shank3 f/f:Tek-Cre (eShank3-KO) mutants were obtained from breeding between Shank3 f/wt:Tek-Cre males with Shank3 f/wt females. eShank3-KO mutants and littermate control Tek-Cre were obtained from breeding between Shank3 f/wt:Tek-Cre males and Shank3 f/wt females. Ai-14 Cre reporter mice were crossed with Tek-Cre mice and used to visualize endothelial cells. The eShank3-KO:Ai-14f/wt (eShank3-KO:Ai-14) triple mutants were obtained from breeding between eShank3-KO male and Shank3 f/f:Ai-14f/f females. Tek-Cre males were crossed with Ai-14f/f females to obtain Tek-Cre:Ai-14f/wt (Tek-Cre: Ai-14) offspring as a control. Genotyping was conducted using PhireTM Tissue Direct PCR Master Mix (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. To determine the sex of P5 pups, Rbm31x/y genes were examined51. All the primers used for genotyping were described in SI.Table 3. All mice were housed together with their littermates at a maximum of five per cage. The mice were kept on a regular 12 h light/dark cycle and were given ad libitum access to food and water. All tests were performed during the light cycle. All procedures were performed with protocols approved by the University of Tennessee Institutional Animal Care and Use Committee in accordance with U.S. National Institutes of Health guidelines.
Cell culture and transfection
bEnd.3 BEC line (mouse brain endothelial cell line), purchased from ATCC (Manassas, VA, USA, #CRL2299™) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, ThermoFisher Scientific), 100 U/ml penicillin, and 100 mg/ml streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2. The human brain endothelial cell line, hCMEC/D3 cells was purchased from Millipore sigma (Burlington, MA, USA, #SCC066). The hCMEC/D3 cells were maintained in Endothelial Cell Basal Medium MV2 supplemented with SupplementPack (0.05 ml/ml fetal calf serum, 5 ng/ml epidermal growth factor, 10 ng/ml basic fibroblast growth factor, 20 ng/ml insulin-like growth factor, 0.5 ng/ml vascular endothelial growth factor, 1 mg/ml ascorbic acid, and 0.2 mg/ml hydrocortisone) (PromoCell, Heidelberg, Germany), 100 U/mL penicillin, and 100 mg/ml streptomycin on cell culture plates coated with rat tail collagen I (Millipore sigma). The human embryonic kidney cell line, HEK293T cells used for adeno associated virus (AAV) production were maintained in DMEM/F-12 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. HEK293FT cells (ThermoFisher Science) used as a host for lentiviral production were maintained in DMEM containing 10% FBS supplemented with 0.1 mM MEMTM non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. The mouse neuroblastoma cell line, Neuro-2A cells used for surveyor assay of genome editing by Shank3 sgRNA in CRISPR/Cas9 system were maintained in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Transfection was performed using jetOPTIMUS® DNA transfection reagent (Polyplus-transfection, Illkirch, France) for bEnd.3 BECs, PEI Max® (linear polyethylenimine, Mw 40,000) (Polyscience, Inc. Warrington, PA, USA) for HEK293T and Neuro-2A cells, and Lipofectamine 3000 (ThermoFisher Scientific) for HEK293FT cells according to the manufacturer’s instructions.
Primary BEC isolation and enrichment
Mouse brain endothelial cell isolation was performed using Adult Brain Dissociation Kit (Milterny Biotec, Gaithersburg, MD, USA, #130-107-677), CD45 MicroBeads (Milterny Biotec, #130-052-301), and CD31 MicroBeads (Milterny Biotec, #130-097-418) according to the manufacturer’s instructions. Brains dissected from 8-12 weeks old mice or postnatal day 5 pups were washed with cold D-PBS containing calcium, magnesium, glucose, and pyruvate (ThermoFisher Science). The brains were cut into eight sagittal slices using a scalpel and transferred into 15 ml tube containing enzyme mixture 1 and 2 supplied in Adult Brain Dissociation Kit. The minced brain slices were incubated at 37 °C for 1 h, shaking gently every 5 min. The dissociated cells suspension was strained through a 70 mm cell strainer placed on top of a 15 ml centrifuge tube. The collected cell suspension was centrifuged at 300 x g for 10 min at 4 °C. After completely aspirating the supernatant, the cell pellet was resuspended with 8 ml of debris removal solution. The 4 ml of cold D-PBS was gently overlaid on top of the debris removal solution and cell mixture in 15 ml centrifuge tube, forming a clear layer on top. The 15 ml centrifuge tube was centrifuged at 3,000 x g for 10 min at 4 °C. The clear top phase and debris middle layers were aspirated, leaving a milky mixture beneath the debris. After washing with cold D-PBS, cell pellet was resuspended with 1 ml of cold 1X red blood cell removal solution and incubated for 10 min at 4 °C. Then, 10 ml of 0.5% bovine serum albumin diluted in D-PBS (D-PBS/BSA buffer) added and the mixture was centrifuged at 300 x g for 10 min at 4 °C. The supernatant was completely aspirated, and the pellet was resuspended in 80 ml of D-PBS/BSA buffer. For depletion of leukocytes, the resuspended cells were incubated with 20 ml of CD45 MicroBead for 15 min in the dark at 4 °C by gently inverting on a mini-tube rotator. The cells were washed by adding 1 ml of D-PBS/BSA buffer and centrifuging at 300 x g for 5 min. The pellet was resuspended 500 ml of D-PBS/BSA buffer and applied on the pre-wet LD column (Miltenyi Biotech, #130-042-901) placed at QuadroMACS™ Separator. The flow-through containing unlabeled cells was collected and centrifuged at 300 x g for 10 min. Subsequently, cell pellet was resuspended in 90 ml of D-PBS/BSA buffer and incubated with 10 ml of CD31 MicroBead for 15 min in the dark at 4 °C by gently inverting on a mini-tube rotator to magnetically label the CD31+ endothelial cells. The cells were washed by adding 1 ml of D-PBS/BSA buffer and centrifuging at 300 x g for 5 min. Then, the pellet resuspended with 500 ml of D-PBS/BSA buffer was applied on the pre-wet MS column (Miltenyi Biotech, #130-042-201) placed at OctoMACS™ Separator. The CD31+ cells bound MS column was washed three times with 500 ml of D-PBS/BSA buffer. After removing MS column from the separator, magnetically labeled CD31+ endothelial cells were collected by adding 1 ml of D-PBS/BSA buffer. The CD31+ endothelial cells were subjected to RNA extraction and cDNA synthesis or cultured in Endothelial Cell Basal Medium MV2 supplemented with Supplement Pack, 100 U/ml penicillin, 100 mg/ml streptomycin on plates coated with rat tail collagen I.
Mouse neuron isolation and enrichment
Neuron isolation was performed using Adult Brain Dissociation Kit (Milterny Biotec, #130-107-677) and Neuron isolation Kit (Milterny Biotec, #130-115-389) according to the manufacturer’s instructions. The 8-9 weeks aged adult mice brains were dissected, washed with cold D-PBS containing calcium, magnesium, glucose, and pyruvate (ThermoFisher Science), cut into eight pieces, and dissociated with enzyme mixture 1 and 2 supplied in Adult Brain Dissociation Kit. The procedures for brain dissociation, and debris and red blood cell removal were same as for mouse brain endothelial cells isolation. After removing red blood cells, the pelleted cells were resuspended in 80 ml of D-PBS/BSA buffer. The cell mixture was incubated with 20 ml of Non-Neuronal Cells Biotin-Antibody Cocktail for 5 min in the dark at 4 °C. The cells were washed by adding 1 ml of D-PBS/BSA buffer and centrifuging at 300 x g for 5 min. The pellet was resuspended 80 ml of D-PBS/BSA buffer, added 20 ml of Anti-Biotin MicroBeads, and incubated for 5 min in the dark at 4 °C. The volume was adjusted to 500 ml with D-PBS/BSA buffer. Subsequently, the pre-wet LS columns (Miltenyi Biotech, #130-042-401) was placed at QuadroMACS™ Separator and the cell mixture was applied on the columns. The flow-through containing neurons was collected and centrifuged at 300 x g for 10 min. The pelleted neurons were subjected to RNA extraction and cDNA synthesis.
Cloning
The full-length rat Shank3 plasmids containing HA epitope (pcDNA3.1-HA-rShank3) were kindly provided by Dr. Kihoon Han (Korea University). The synthesized DNA fragment, excluding the 24 nucleotides of exon 18 of Shank3, was digested with SfiI and PspXI restriction enzymes, followed by cloning into same vector to generate the pcDNA3.1-HA-Shank3De18. DNA fragments were synthesized from Integrated DNA Technologies (Coralville, IA, USA).
HA-tagged BioID2 (BioID2-HA) in phSyn1-WASH1-BioID2-HA33 was amplified by the PCR using the primers described in SI.Table 3. Subsequently, BioID2-HA digested with HindIII and BamHI was cloned into pcDNA3 vector to generate the pcDNA3-BioID2-HA plasmid. To generate the pcDNA3-Shank3DE18-BioID2-HA plasmid, the Shank3De18 fragment lacking the stop codon (TGA) from pcDNA3.1-HA-Shank3DE18 was inserted into pcDNA3-BioID2-HA plasmid using NdeI and HindIII restriction enzyme site.
The HA-tagged human GSK3b plasmid with a serine 9 to alanine mutation (HA-GSK3βS9A-pcDNA3) was purchased from Addgene (Watertown, MA, USA, #14754)52. The DNA fragment of hGSK3βS9A in HA-GSK3βS9A-pcDNA3 was amplified by the PCR using the primers described in SI.Table 3. Subsequently, the amplified GSK3βS9A was digested with NotI and NheI and cloned into CSII backbone plasmid of lentivirus construct with EF1α promoter, triple HA epitopes and blasticidin resistance gene to generate CSII-EF1α-3X HA-GSK3βS9A-BSD plasmid. Additionally, the fragment of hGSK3βS9A in HA-GSK3βS9A-pcDNA3 was amplified by the PCR using the primers described in SI.Table 3. The fragments of hGSK3βS9A cut with NotI were cloned by replacing the iCre of the AAV-CLDN5 (Ple261)-iCre (pEMS1982, Addgene, #49113)50 plasmid to generate the AAV-CLDN5-hGSK3βS9A-HA plasmid. A DNA fragment encoding the HA tag was synthesized and inserted into AAV-CLDN5 (Ple261)-iCre (pEMS1982) by replacing the iCre to make an AAV-CLDN5-HA plasmid as a MOCK plasmid.
The FLAG-tagged mouse C/EBPα plasmid (pcDNA3-Flag-C/EBPα) was purchased from Addgene (# 66978)53. The DNA fragment of mouse C/EBPα plasmid in pcDNA3 Flag-C/EBPα was amplified by the PCR using the primers described in SI.Table 3. Subsequently, the amplified C/EBPα PCR products were digested with NotI and BamHI and cloned into CSII backbone plasmid of lentivirus construct with EF1α promoter, triple HA epitopes and blasticidin resistance gene to generate CSII-EF1α-3X HA-C/EBPα-BSD plasmid.
The pL-CRISPR-EFS-GFP plasmid containing sgRNA and CRISPR associated protein 9 (Cas9) expression cassette was purchased from Addgene (#57818)54. This plasmid simultaneously expresses Cas9 enzyme and green fluorescent protein (GFP) via P2A cleavage site under control of elongation factor 1α short (EFS) promoter. The CMV promoter of the pCS3+MT vector digested with SalI and BamH1 was cloned into the pL-CRISPR-EFS-GFP vector digested with XhoI and BamHI to replace the EFS promoter with the CMV promoter and named pL-CRISPR-CMV-GFP.
pcDNA3-human β-Catenin (#16828)55, pcDNA3-S33Y β-Catenin (#19286)55, Tag5Amyc-GSK3β WT (#16260)56, pCAGImC-V5-CSNK1A1 (#92014)57, pAdDeltaF6 (#112867), pUCmini-iCAP-PHP.eB (#103005)22, pUCmini-iCAP-PHP.V1 (#127847)49, pAAV-CaMKIIa-EGFP (#50469), pMD2.G (#12259), pMDLg/pRRE (#12251)58, and pRSV-Rev (#12253)58 were purchased from Addgene.
Generation of stable eShank3-KO bEnd.3 BEC line using CRISPR/Cas9
The CRISPR guide sequence for mouse Shank3-KO was designed using the CRISPR design tool (CRISPOR) which provided the following sequences: 5’- TGG ACC CAA CCG CGC CCG TG-3’, which targets exon 2 of Shank3. The sgRNA oligonucleotides 5’-CAC CGT GGA CCC AAC CGC GCC CGT G-3’ and 5’-CAC CTG GGT TGG CGC GGG CAC CAA A-3’ were annealed and cloned into the BsmBI site of the pL-CRISPR-CMV-GFP vector. The construct was verified by sequencing analysis. After transfecting into Neuro-2A, the genomic DNA was subjected to the surveyor assay using GeneArt Gemonic Cleavage Detection kit (ThermoFisher Scientific) according to the manufacturer’s instructions.
To generate a stable eShank3-KO bEnd.3 BEC line, the pL-CRISPR-CMV-GFP-Shank3 sgRNA plasmid was transfected into bEnd.3 BECs, followed by collection of bEnd.3 BECs expressing GFP using BD FACSAria II cell sorter (BD, Franklin Lakes, NJ, USA). The sorted bEnd.3 BECs were seeded at a single cell/well in 96 well plate. The eShank3-KO bEnd.3 BECs were selected by sequencing the PCR product of Shank3 gene performed with following primers; 5’-GCT AGC ACC GGG ATG GAC -3’ and 5’- CAA AGA TCA CAC CCT GAG CG -3’. Off-target genes of Shank3 sgRNA were predicted using CRISPOR and Off-Spotter under conditions allowing up to five or fewer mismatched nucleotides. Five potential off-target genes (Ahi1, Mylk, Alox12, Col7a1, and Mppe1) were identified and verified by sequencing analysis. The primer information for the sequencing analysis is provided in SI.Table 3.
Generation of a stable 3X HA-GSK3βS9A / eShank3-KO and a 3X HA-C/EBPa / eShank3-KO double mutant bEnd.3 BEC lines
To produce the lentiviral particles of 3X HA-GSK3βS9A or 3X HA-C/EBPα, a 3rd generation lentiviral packaging system was utilized. HEK293FT cells were seeded at 4 x 106 cells in a 100 mm dish and co-transfected with 10 μg of CSII-EF1α-3X HA-GSK3βS9A (or C/EBPα)-BSD, 2.5 μg of pMD2.G, 2.5 μg of pMDLg/pRRE, and 2.5 μg of pRSV-Rev using 40 μl of lipofectamine 3000. On day 3 post-transfection, the supernatant containing lentiviral particles from the culture media was collected, centrifuged at 500 x g for 10 min at 4 °C and filtered through a 0.45 mm cellulose acetate membrane syringe filter (Millipore sigma, #CLS431220). The lentiviral particles were subsequently concentrated using polyethylene glycol (PEG) 6000 as previously described59. Lentiviral titer was determined using qPCR Lentivirus Titer Kit (Abm, Richmond, BC, Canada) according to the manufacturer’s instruction.
To generate stable cell line expressing 3X HA-GSK3βS9A (or 3X HA-C/EBPα) in eShank3-KO bEnd.3 BECs, the eShank3-KO bEnd.3 BECs seeded at 1 x 105 cells/well on a 6 well plate were incubated for 16-24 h. After changing the medium, the lentiviral particles of 3X HA-GSK3βS9A (or 3X HA-C/EBPα) (MOI, 50) together with 8 ml/ml of polybrene (Millipore sigma) were added into the wells. Twenty-four hours later, the culture medium containing the lentiviral particles was replaced with a fresh medium and the cells were selected with 8 m/ml of blasticidin (ThermoFisher Scientific) for 10 days.
AAV production and titration
To produce AAV-PHP.eB-CaMKIIa-EGFP, HEK293T cells were co-transfected with pAdDeltaF6 as a helper plasmid, pUCmini-iCAP-PHP.eB plasmid expressing AAV capsid, and AAV-CaMKIIa-EGFP plasmid expressing EGFP under control of CaMKIIa promoter. To produce AAV-PHP.v1-CLDN5-GSK3βS9A-HA and AAV-PHP.v1-CLDN5-HA, HEK293T cells were co-transfected with pAdDeltaF6, pUCmini-iCAP-PHP.V1 plasmid, and AAV-CLDN5-GSK3βS9A-HA or AAV-CLDN5-HA plasmid expressing GSK3βS9A-HA or HA under control of CLDN5 promoter. AAV purification was performed as previously described23,24,60,61. After 72 h incubation, HEK293T cells were collected and lysed with lysis buffer [20 mM Tris-HCl (pH8.5), 150 mM NaCl] through four times of freeze-thaw process in dry ice/ethanol bath and 37 °C water bath. Cell lysates were incubated with benzonase (50 U/ml, Millipore sigma) for 30 min at 37 °C, followed by centrifugation with 4,500 rpm for 30 min at 4 °C. To purify the viral particles, supernatants were loaded onto 60, 40, 25, and 15% of iodixanol (OptiPrepTM, Cosmo Bio USA, Inc., Carlsbad, CA, USA) solution layers and ultracentrifuged at 67,000 x g for 1 h at 18 °C. The virus particles collected in 40% of iodixanol solution layer were purified and concentrated by exchanging with ice-cold PBS using Amicon filter unit (100K MWCO, Millipore sigma). AAV titration was measured through quantitative real-time PCR (qRT-PCR) method using SYBR™ Select Master Mix for CFX (ThermoFisher Scientific). For the titration of AAV-PHP.eB-CaMKIIa-EGFP, an AAV-CaMKIIa-EGFP plasmid containing the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) region was diluted from 106 to 1011 copies/ml and used to generate a standard curve. The primer pair targeting WPRE was described in the SI.Table 3. For the titration of AAV-PHP.v1-CLDN5-GSK3βS9A-HA and AAV-PHP.v1-CLDN5-HA, an AAV-CLDN5 (Ple261)-iCre (pEMS1982) plasmid was used for generating a standard curve. The primer pair targeting CLDN5 was described in the SI.Table 3. AAV samples and standard plasmids were denatured in 50 ml of PCR alkaline digestion buffer [25 mM NaOH, 0.2 mM EDTA] for 10 min at 100 °C, followed by neutralization by adding 50 ml of 40 mM Tris-HCl (pH5.0). Subsequently AAV sample and standard plasmid were subjected to qRT-PCR using IQ™5 (BioRad, Hercules, CA, USA) real-time PCR equipment. Titration of AAV samples was determined based on the standard curve.
Subcellular fractionation
The cell fractionation was performed with Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Scientific, #78840) according to the manufacturer’s instructions. Briefly, cell pellets resuspended in 150 ml of CEB containing the protease inhibitor cocktail were incubated for 10 min at 4 °C, followed by centrifugation at 500 x g for 5 min. The supernatant was transferred to a new microtube and used as cytoplasm fraction. To harvest the nuclear fraction, the cell pellet was resuspended with 150 ml of ice-cold MEB and centrifugated at 3,000 x g for 5 min. The resulting pellet was incubated in 75 ml of ice-cold NEB containing protease inhibitor cocktail for 30 min at 4 °C. After centrifugation at 5,000 x g for 5 min, the supernatant was transferred to a new microfuge tube (supernatant 1). Further, the pellet was resuspended in 75 ml of NEB containing protease inhibitor cocktail, CaCl2 (5mM) and micrococcal nuclease (300 unit/100 µl) for 15 min at RT. After centrifugation at 16,000 x g for 5 min, the supernatant was transferred to the microtube containing supernatant 1 and used as nucleus fraction.
RNA isolation, cDNA production, and qRT-PCR
Total RNA was isolated using PureLink™ RNA Mini Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The cDNAs were obtained through reverse transcription PCR with the RNAs from each BEC using M-MLV reverse transcriptase (ThermoFisher Scientific) and random hexamers. The cDNA library of primary human brain microvascular endothelial cell (HBMEC) was purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). PCR was performed with DreamTaq™ Hot Start Green PCR Master Mix (ThermoFisher Scientific). The qRT-PCR was performed with SYBR™ Select Master Mix for CFX (ThermoFisher Scientific) containing SYBR® GreenER™ dye using IQ™5 (BioRad) real-time PCR equipment. The primers for qRT-PCR were described in SI.Table 3. The specificity of PCR products was analyzed by agarose gel electrophoresis. All relative gene expressions from results obtained via qRT-PCR were analyzed using 2-DDCt values.
Western blot analysis
Cells were homogenized and sonicated in lysis buffer [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100] supplemented with protease inhibitor cocktail (IBI Scientific, Dubuque, IA, USA), 1 mM PMSF, and 1 mM Na3VO4. Protein concentration was determined with Pierce™ BCA Protein Assay kit (ThermoFisher Scientific). Thirty micrograms of lysate per sample was denatured and reduced by Laemmli sample buffer (Bio-Rad) containing sodium dodecyl sulfate (SDS) and β-mercaptoethanol, respectively. The protein samples were separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using Bolt™ 4 - 12% bis-tris gradient gel (ThermoFisher Scientific) and then electrophoretically transferred to nitrocellulose membranes (ThermoFisher Scientific). The membranes were blocked with 5% skim milk in phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS-T) for 1 h at RT and further incubated with 5% skim milk in PBS-T containing primary antibodies overnight at 4 °C. After washing three times, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-2357) or horseradish peroxidase-conjugated anti-mouse IgG kappa binding protein (m-IgGκ BP) (Santa Cruz Biotechnology, #sc-516102) for 1 h at RT and washed three times with PBS-T. The HRP-conjugated streptavidin (Cell Signaling Technology, Danvers, MA, USA, #3999) was used for detection of biotinylated proteins. Immunoreactive bands were visualized with SuperSignal™ West Dura Extended Duration Substrate (ThermoFisher Scientific). Images were acquired with Azure C300 (azure biosystems, Dublin, CA, USA) and western blot (WB) bands were quantified using ImageJ software (NIH). The primary antibodies used in this study are as follows: mouse monoclonal anti-SHANK3 (Cell Signaling Technology, #64555), rabbit monoclonal anti-ZO1 (Cell Signaling Technology, #8193), mouse monoclonal anti-Claudin5 (ThermoFisher Scientific, #35-2500), rabbit polyclonal anti-ZO2 (ThermoFisher Scientific, #71-1400), rabbit polyclonal anti-Afadin (Millipore sigma, #A0224), mouse monoclonal anti-Cingulin-like protein 1 (Santa Cruz Biotechnology, #sc-377525), mouse monoclonal anti-Cortactin (Abcam, Cambridge, UK, #ab33333), rabbit monoclonal anti- β-Catenin (Abcam, #ab32572), rabbit monoclonal anti-phospho-β-Catenin (Ser552) (Cell Signaling Technology, #5651), rabbit monoclonal anti-Histone H3 (Cell Signaling Technology, #4499), mouse monoclonal anti-C/EBPα (ThermoFisher Scientific, #MA1-825), mouse monoclonal anti-HA tag (Abcam, #ab1424), rabbit polyclonal anti-HA tag (Abcam, #ab9110), mouse monoclonal anti-FLAG-M2 (Millipore sigma, #F1804), rabbit monoclonal V5-Tag (Cell Signaling Technology, #13202), mouse anti-MYC Tag (Millipore sigma, #05-419), rabbit monoclonal anti-α-Tubulin (Abcam, #ab52866), and mouse monoclonal anti-b-Actin (Abcam, #ab8226).
Immunohistochemistry and immunocytochemistry
Immunohistochemistry (IHC) was conducted as previously described23,24,60,62-64. Pups at P5 and adult mice were anaesthetized with hypothermia and isoflurane, respectively, and transcardially perfused with PBS containing 25U/ml heparin, and then perfused with 4 % paraformaldehyde (PFA) solution. The brains were dissected and fixed by immersion in 4% PFA overnight at 4 °C, and then cryopreserved in 30% sucrose in PBS. Frozen brains embedded in Tissue-Tek O.C.T compound (Sakura® Finetek, Torrance, CA, USA) were sectioned 50 μm thick using Leica CM1950 (Leica Biosystems, Wetzlar, Germany) cryostat equipment. Brain sections were permeabilized with PBS containing 0.2% Triton X-100 for 15 min.
Immunocytochemistry (ICC) was performed as described in our previous studies23,24,60,61,64. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 15 min. Subsequently, cells and brain sections were blocked with serum-free blocking buffer (Abcam) for 1 h at RT and incubated overnight at 4 °C with primary antibodies in blocking buffer.
In this study, we used mouse monoclonal anti-SHANK3 (Santa Cruz Biotechnology, #sc-377470), rat monoclonal anti-mouse CD31 (BD/Pharmingen™, #553370), rabbit monoclonal anti-HA-Tag (Cell Signaling Technology, #3724), mouse monoclonal anti-HA (Abcam, #ab130275), rabbit polyclonal anti-ZO1 (ThermoFisher Scientific, #61-7300), rabbit polyclonal anti-Claudin5 (Abcam, #ab131259), and rat monoclonal anti-GFAP (ThermoFisher Scientific, #13-0300). After washing with PBS-T, cells and brain sections were subsequently incubated with blocking buffer containing species-specific Alexa Fluor secondary antibodies for 2 h at RT. Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-rat IgG, Alexa Fluor 555-conjugated goat anti-mouse IgG, Alexa Fluor 555-conjugated goat anti-rabbit IgG, or Alexa Fluor 555-conjugated goat anti-rat IgG was used as a secondary antibody. Alexa Fluor 488-conjugated streptavidin (ThermoFisher Scientific) was used for detecting biotinylated proteins. Nuclei were co-stained with 4’,6-diamidino-2-phenylindole (DAPI, Millipore sigma). The cells and brain sections were mounted with ProLong™ Glass Antifade Mountant (ThermoFisher Scientific). Images were acquired with a Zeiss LSM 710 confocal microscope under the control of Zen software (Carl Zeiss AG, Oberkochen, Germany). To compare the relative expression between two groups, images were captured under identical conditions with the same laser power and gain of the detector for both groups. Intensity of fluorescence was quantified using ImageJ software (NIH).
Immunoprecipitation
The bEnd.3 BEC lysates were prepared with immunoprecipitation (IP) buffer [25 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol] containing protease inhibitor cocktail, 1 mM PMSF, and 1 mM Na3VO4. 10 mg of cell lysates were incubated with mouse monoclonal anti-SHANK3 antibody (Santa Cruz Biotechnology, #sc-377470) or control mouse IgG (ThermoFischer Scientific, #31903) for 3 h at 4 °C with gently inverting rotation, followed by further incubation with Dynabeads® Protein G (ThermoFischer Scientific) for 2 h at 4 °C with gently inverting rotation. The immune complexes were washed five times with IP buffer and resuspended in 4X Laemmli sample buffer (BioRad, #1610747). The immune complexes were subjected to SDS-PAGE and WB analysis. Mouse IgG TrueBlot® ULTRA (Rockland immunochemicals, Pottstown, PA, USA, #18-8817-31) was used as a secondary antibody to detect immunoblotted target protein bands without interference from immunoprecipitated mouse IgG heavy and light chains. For the co-immunoprecipitation experiments, HEK293T cells co-transfected with plasmids expressing HA-eSHANK3, GSK3β-MYC, β-CateninS33Y-FLAG, and V5-CK1α were lysed with the IP buffer [20 mM Tris (pH 8.0), 137 mM NaCl, 10% Glycerol, 1% NP-40, and 2 mM EDTA] containing protease inhibitor cocktail (ThermoFisher Scientific, USA, 78440). After centrifugation, the resulting cell lysates (1 mg/700 µl/IP) were pre-cleared with 30 µl of protein A/G bead (50% slurry, Santa Cruz Biotechnology, #sc-2003) for 4 h at 4 °C, followed by centrifugation at 2,500 x g, 4 °C for 1 min. The cleared cell lysates were incubated with anti-MYC antibody overnight at 4 °C with rotation at 20 rpm (Thomas scientific, Swedesboro, NJ, USA). Next day, the cell lysates were further incubated with 30 µl of protein A/G bead (50% slurry) for 4 h at 4 °C with rotation 20 rpm, followed by centrifugation at 2,500 x g, 4 °C for 1 min. The resulting pellet was washed five times with the IP buffer and then resuspended in 4X Laemmli sample buffer for WB analysis.
Whole-brain clearing, light-sheet imaging, and analysis
Brain clearing and light-sheet imaging were conducted as described in our previous study24. To clear the whole brain tissue, the pup at P5 was anaesthetized with hypothermia by placing it on wet ice for 30 sec and perfused transcardially with PBS containing 25 U/ml heparin followed by 4% PFA solution. The dissected brain was fixed overnight in 4% PFA at 4 °C and then washed three times in PBS with gentle shaking. The PFA-fixed brain was subjected to SHIELD post-fixation using a modified SHIELD protocol, with incubation for 2 days in the SHIELD OFF solution at 4°C, followed by 3 hours in 2.5% SHIELD Epoxy in SHIELD ON buffer at 37°C, and an overnight incubation in SHIELD ON buffer at 37°C. The brain sample was cleared for 2 days at room temperature in Clear+ delipidation buffer. For imaging, the brain sample was then refractive index matched through incubation in EasyIndex (RI = 1.52). Transparent brain was imaged in the LifeCanvas Technologies (Cambridge, MA, USA) using SmartSPIM light-sheet microscope at 3.6x magnification with a pixel size of 1.8 mm X 1.8 mm, and Z-step size of 4 mm. The brain-wide tdTomato signal, originating from endothelial cells, and autofluorescence signal were detected using a 561 nm laser and a 488 nm laser, respectively. 3D reconstruction of vascular networks was performed with SyGlass software (SyGlass, Morgantown, WV, USA) using Oculus Rift S VR headsets and controllers (Meta Platforms Technologies, Menlo Park, CA, USA) as described in our previous study24.
Transmission electron microscopy
Pups at P5 were anesthetized by hypothermia and intracardially perfused with PBS containing 25 U/ml heparin for 5 min, followed by perfusion with 10 ml of fixative solution [4% PFA and 2% glutaraldehyde in 0.13 M sodium cacodylate buffer (pH7.2)]. Perfusion was performed by gravity-fed method to minimize the rupture of cerebral vasculature. After dissecting the brains, the PFC region was cut at a thickness of 2 mm coronally, and then fixed overnight at 4 °C in fixative solution. Tissues were washed in 0.13 M sodium cacodylate buffer, and post-fixed with 1% osmium tetroxide in 0.13 M sodium cacodylate buffer for 2 h. Tissues were washed in 0.13 M sodium cacodylate buffer and distilled water followed by dehydration in a series of ethanol dilutions. Infiltration was performed with 1:1 ratio mixture of Embed812 resin and Acetone overnight at RT followed by incubation with 100% Embed812 resin three times for 2 h. Tissues were embedded in resin at 65°C overnight. Subsequently, tissue blocks were trimmed and sectioned in a Leica EM UC 7 ultramicrotome at 60-65 nm thick and mounted on copper 200 mesh grids. Sections were stained using uranyless and lead citrate and visualized at 60kV in JEM 2000EX II Transmission Electron Microscope (JEOL Co, Tokyo, Japan). For the quantification of the electron densities between BECs, the images of 15~30 cortical vessels, ranging in diameter between 3 and 6 mm, were randomly obtained from 3 mice per group. The electron density scan across adjoined BEC membranes was performed using ImageJ software (NIH).
Proteomics
BioID2 method for eSHANK3 interactome analysis: Protein identification using BioID2 was performed according to previously published method33,65. bEnd.3 BECs were transfected with Shank3De18-BioID2-HA, BioID2-HA, or HA-Shank3De18 plasmid. After 48 h transfection, the media were replaced by fresh media containing 50 mM biotin (Millipore sigma) and further incubated for 24 h. After three times washing with PBS for removal of biotin remaining in media, cells were extracted with high-salt RIPA buffer [25 mM Tris-HCl (pH 7.4), 500 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA] containing protease inhibitor cocktail, 1 mM PMSF, and 1 mM Na3VO4. The cell lysates were incubated for 30 min on ice and sonicated three times for 10 s, followed by centrifugation at 13,000 rpm for 20 min at 4 °C. The supernatants were subjected to filtration using Amicon® Ultra-4 Centrifugal Filter Units (Millipore sigma) with 10 kDa molecular weight cutoff to prevent the binding of any residual free biotin in the cell lysate to NeutrAvidin agarose. Biotin-free and concentrated cell lysates obtained through filtration were incubated with 15 ml of NeutrAvidin agarose (ThermoFisher Scientific) overnight at 4 °C by gently inverting on a mini-tube rotator. NeutrAvidin agarose beads were collected by centrifugation at 800 x g for 1 min at 4 °C and washed sequentially with the following washing buffers; twice with high-salt RIPA buffer, twice with wash buffer A (2% SDS in D.W), once with wash buffer B [50 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, and 1% Triton X-100], once with wash buffer C [10 mM Tris-HCl (pH 8.1), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% sodium deoxycholate], twice with wash buffer D [50 mM Tris-HCl (pH 7.4) and 50 mM NaCl], and once with wash buffer E [50 mM ammonium bicarbonate (pH8.3)] at 4 °C. After wash, the NeutrAvidin agarose beads were resuspended with 100 ml of 100 mM ammonium bicarbonate. 5% of the samples were used for WB analysis and 95% of the samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
LC-MS/MS sample preparation and analysis: Sample processing, LC-MS/MS analysis, and data acquisition for proteins identification were performed at Proteomics and Metabolomics Core, the mass spectrometry facility at UTHSC. The samples, bound to NeutrAvidin agarose beads, were reduced with 1 mM dithiothreitol (DTT) for 45 min at 4°C and alkylated with 5 mM iodoacetamide for 20 min at room temperature, and further reduced with 5 mM DTT for 15 min at RT. Subsequently, LysC/Trypsin (Promega, Madison, WI, USA) was added to the samples and the digests were incubated at 37 °C overnight with shaking at 500 rpm using an Eppendorf ThermoMixer F1.5. Supernatant containing peptide digest was collected, and NeutrAvidin agarose beads were extracted with 100 ml of 50% acetonitrile/0.5% trifluoroacetic acid (TFA) for 10 min at 4 °C. The extracts were combined with their corresponding peptide digests and subjected to vacuum drying using a SpeedVac concentrator for 3 h. The peptide digests were desalted using Pierce C-18 spin tips (ThermoFisher Scientific) according to the manufacturer’s instructions, and then subjected to vacuum drying. Each sample was dissolved in 100 ml of 3% acetonitrile/0.05% TFA.
Quantitative LC-MS/MS was performed with 5 ml of each sample using an Ultimate 3000RSLCnano HPLC system (ThermoFisher Scientific) coupled with a Orbitrap Fusion Lumos Mass Spectrometer (ThermoFisher Scientific) via a nanoelectrospray ionization source. The sample was first trapped on C18 Acclaim PepMap 100 (75 ml X 20 mm) column (ThermoFisher Scientific), and then the analytical separation was performed using C18 Acclaim PepMap RSLC (75 ml X 500 mm) column (ThermoFisher Scientific). The mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. Peptides were separated at a flow rate of 300 nl/min with a column temperature of 40 °C using the following linear gradient parameters: 3% B over 4 min, 3% B to 5% B over 1 min, 5% B to 25% B over 50 min, 25% B to 30% B over 5 min, 30% B to 90% B over 3 min, held at 90% B for 10 min, 90% B to 3% B over 3 min, and then 3% B was held for 24 min. Data collection was performed in a data-dependent acquisition (DDA) mode. The full MS scan (full) was performed using an Orbitrap analyzer with a resolution of 120,000 (FWHM, at m/z=200). The MS2 scan were performed by 0.7 m/z isolation with the quadrupole, normalized HCD collision energy of 30%, and analyzed with Orbitrap with a resolution of 30,000 (FWHM, at m/z=200). Dynamic exclusion was set to 30 seconds, and Monoisotopic Precursor Selection (MIPS) was set to Peptide. An intensity threshold of ≥10,000 was applied, and charge states was set to between 2 and 6. The raw MS data was searched on Proteome Discoverer 2.4 (ThermoFisher Scientific) software using SequestHT as a search engine with following parameters: digestion reagent, trypsin; dynamic modification, oxidation of Met, acetylation of the protein N-terminus; static modification, carbamidomethylation of Cys; precursor ion mass tolerance, 10 ppm; fragment ion mass tolerance; 0.02 Da. The database for protein identification used SwissProt ver.2017-10-25 (25,097 entries) with TaxID 10090 (Mus musculus). For each sample, four biological replicates were analyzed.
The data generated by the mass spectrometry analysis was transferred to the UTHSC Molecular Bioinformatics Core using SFTP. The data was normalized using normalizeCylclicLoess function of R/Bioconductor-package limma66 after log2 transformation. The normalized data matrix was loaded into R to gather statistics and determine differential expression. The mean, variance, and standard deviation were calculated for each protein across each condition. The fold change was then calculated for all proteins. One-way ANOVA was implemented to determine significance for each protein. The p values were then adjusted for multiplicity using the Benjamini Hochberg method67. Only proteins with an adjusted p value < 0.05 were considered differentially expressed. Analysis for the protein-protein interaction mapping and the bioinformatic Gene ontology (GO) was performed using StringDB68 and Gorilla69,70, respectively. Protein networks figure was visualized using Cytoscape (v3.10), with nodes representing the gene names of proteins identified in the proteomic analysis. The node positions were manually adjusted to visualize the module more concisely. The node size is proportional to log2 (fold change) over control groups. From the GO analysis, cell component result was visualized in a tree and a bar plots.
Transwell permeability (TWP) assay
bEnd.3 BECs and eShank3-KO bEnd.3 BECs ± IWR-1-endo (1 mM) or bEnd.3 BECs, eShank3-KO bEnd.3 BECs and GSK3βS9A-eShank3-KO bEnd.3 BECs were seeded on cell culture inserts (ThermoFisher Scientific, 08-770) in a 24-well plate (1 x 104 cells/insert/well) and incubated for 2~3 days until the monolayer of the cells in each insert was formed. Then, 100 ml of 300 mg/ml of 4 kDa FITC-Dextran (Millipore sigma, #46944) was added to each insert after removing 100 ml of medium from each insert to make 100 mg/ml of 4 kDa FITC-Dextran as a final concentration. After 4 h incubation at RT, media from the receiver plates were collected and subjected to the measurement of fluorescence at excitation/ emission = 485 nm/535 nm using SpectraMax M2e (Molecular device, San Jose, CA, USA). IWR-1-endo was purchased from Apexbio Technology LLC (Houston, TX, USA) and treated for 48 h.
Trans-endothelial electrical resistance (TEER) assay
TEER assays were performed with Maestro Z platform and software (Axion Biosystems, Atlanta, GA, USA) using CytoView Z plates containing electrodes in each well. Before seeding cells into the CytoView Z 96 well impedance plate (Axion BioSystems, # Z96-IMP-96B), the plate added with 100 ml of cell culture media was inserted into Maestro Z instrument to measure baseline impedance for the electrode. The cells were seeded at 5,000 cells/well on the CytoView Z 96 well impedance plate, and the plate was then inserted into Maestro Z instrument preset with the cell culture condition (37 °C and 5% CO2). The electrical impedance across each monolayer was measured every minute at a low frequency of 1 Hz for a duration of the experiments up to 95-120 hr.
In vivo BBB permeability assay
BBB permeability assay was performed to assess BBB leakage. For P5 pups, 30 ml of sodium fluorescein (Millipore sigma) dissolved in PBS at a concentration of 50 mg/ml was intraperitoneally injected. After 1 h of circulation, pups were anesthetized by hypothermia, and blood was collected by right heart needle aspiration, followed by transcardial perfusion with 10 ml of PBS containing 25 U/ml heparin at a rate of 1 ml/min to remove blood from the intravascular compartment. To induce the expression of HA-GSK3βS9A in the brain endothelium, pups at P0 were anesthetized by placing them on the wet ice for 30 sec, and then injected 20 ml of the mixture of AAV-PHP.v1-CLDN5-GSK3βS9A-HA (1 X 1011 vg) and 0.05% trypan blue dye in PBS into the superficial temporal vein using a syringe with a 30-gauge needle under the surgical stereoscope. As a control, AAV-PHP.v1-CLDN5-HA was injected in the same way instead of AAV-PHP.v1-CLDN5-GSK3βS9A-HA. In vivo BBB permeability of GSK3βS9A-induced pups at P5 was assessed using the same method with sodium fluorescein.
For adults, 100 ml sodium fluorescein dissolved in PBS at a concentration of 200 mg/ml was intraperitoneally injected. After 1 h circulation, mice were anesthetized with isoflurane, and blood was collected from right heart. And then these mice were transcardially perfused with 50 ml of PBS/ heparin at a rate of 5 ml /min. Brains were collected and individually weighed. Brains homogenized with 200 ml of PBS, and the lysate was centrifuged at 13,000 rpm for 10 min at 4 °C. Two hundred microliters of the supernatant was mixed with 200 ml of 15% trichloroacetic acid (TCA) and centrifuged at 13,000 rpm for 10 min at 4 °C. Three hundred microliters of the supernatant was mixed with 75 ml of 5N NaOH, and fluorescence was measured in 100 ml of the mixture using SpectraMax M2e fluorescence spectrophotometer (Molecular Devices, San Jose, CA) at ex/em = 485 nm/ 535 nm. Serum was obtained by centrifugation at 13,000 rpm for 10 min at 4 °C and then mixed with 15 % TCA of ten times volume followed by centrifugation. The supernatants were neutralized with 5 N NaOH at a 4:1 ratio and diluted with PBS, and then fluorescence was measured. The amount of fluorescein for each sample was determined using standards ranging from 0.1 to 3 μg/ml. The BBB leakage of sodium fluorescein was normalized with the amount measured in serum.
Electrophysiology
AAV-PHP.eB-CaMKIIa-EGFP with the ability to cross the BBB22 was used to select pyramidal neurons adjacent to the BBB in PFC. Pups at P0 were anesthetized by placing them on the wet ice for 30 sec. Twenty microliters of the mixture of AAV-PHP.eB-CaMKIIa-EGFP (5 X 1010 vg) and 0.05% trypan blue dye in PBS was injected into the superficial temporal vein using syringe with a 30-gauge needle under the surgical stereoscope. Mouse at P5 or adult age (17-19 weeks) was anesthetized and rapidly decapitated. Brain was quickly removed and submerged in N-methly-D-glutamine (NMDG)-artificial CSF containing 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM D-glucose, 2 mM thiourea, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 0.5 mM CaCl2, and 10 mM MgSO4 with pH 7.4 and osmolarity ~315 mOsm at RT for 1 min. All solutions used for electrophysiological experiment were bubbled with 95% O2 and 5% CO2. Brain was cut with thickness of 400 µm in oxygenated NMDG-aCSF solution using Leica VT1200S vibratome. The slices were placed in oxygenated NMDG-aCSF at 30-32 °C for 12 min, then allowed to recover at RT in oxygenated HEPES-aCSF containing 92 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM D-glucose, 2 mM thiourea, 5 mM sodium ascorbate, 3 mM sodium pyruvate, 2 mM CaCl2, and 2 mM MgSO4 with pH 7.4 and osmolarity ~315 mOsm for a minimum of 50 minutes. During the recording, slices were maintained in oxygenated external bath solution containing 119 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 24 mM NaHCO3, 12.5 mM D-glucose, 2 mM CaCl2, and 2 mM MgSO4 with pH 7.4 and osmolarity ~310 mOsm at 28-30 °C. Current-clamp recordings of pyramidal neurons in PFC region were performed using patch pipettes (3.5-5.5 MΩ resistance) filled with the internal solution containing 150 mM potassium gluconate, 2 mM MgCl2, 1.1 mM EGTA, 10 mM HEPES, 3 mM sodium ATP, and 0.2 mM sodium GTP with pH 7.2 and osmolarity ~290 mOsm. Pyramidal neurons were identified with EGFP signals. The synaptic responses were amplified with Multiclamp 700B Microelectrode Amplifier (Molecular Devices) and digitized with Digidata 1440A Digitizer (Molecular Devices). To elicit action potentials, depolarizing currents (-300 pA to 400 pA, 100 pA/step, current injection time; 1s, inter-injection interval; 9s) were injected in a stepwise manner. During recording, resting membrane potentials were simultaneously measured. The action potentials were analyzed using pCLAMP10 software (Molecular Devices).
Behavioral tests
Ultrasonic vocalization test: The Ultrasonic vocalization test in pups at P5 was performed in isolation container placed in a sound-proof box as described previously71. The microphone (M500-384 USB Ultrasound Microphone, Pettersson Elektronik, Uppsala, Sweden) was placed on the top through a hole in the top of the box and adjusted the 12-15 cm from the bottom of the recording box. Pups were isolated from their mother and placed in the recording box, followed by recording for 5 min. After recording, pup was returned to the mother. Ultrasonic vocalization was recorded using the BatSound® Touch Lite (Pettersson Elektronik) software. Acoustic frequencies below 40 kHz and above 150 kHz were filtered away to reduce background noise interference. The number and duration of calls were analyzed using Adobe Audition (Audio recording and editing software) software.
Round social arena (RSA) test: RSA paradigm was developed in our previous study for precise analysis of social behaviors24,30. This test was performed in a round-shaped chamber (49 (D) X 45 (H) cm) under 35 lx illumination. The transparent inner cage (8 (D) X 10.5 (H) cm) was placed in the center of the arena. A camera was placed on the ceiling to track the test animal’s overall behaviors, while wide-angle (180 degree) fish-eye lens camera installed on the top of the inner cage monitored the detailed social interactions between the test mouse and social stimulus. First, mouse was given full access to empty inner cage for 10 min. After returning the test mouse to its home cage, juvenile female C3H/HeJ (P30-P50) social stimulus was placed into empty cage. After the test mouse was brought back into the chamber, test mouse was allowed free access to social stimulus for 10 min. Behaviors including times spent in S zone, and frequency of entry into S zone were analyzed using Ethovision XT (Noldus, Wageningen, Netherlands) software. A circular zone of 3 cm around the inner cage was named as a S zone. The duration in the S zone was measured as the time the mouse's nose stayed within the S zone. The duration of sniffing was measured by analyzing video recorded with a fish-eye lens camera using Ethovision XT software. Sniffing was defined as the test mouse actively exploring the inner cage with its nose directed towards it.
Grooming behavior test: Grooming behavior test was performed in a standard mouse cage filled with 1-2 cm thick beddings under 35 lx illumination. The mouse was placed in the cage and the video was recorded for 30 min. The bottom of the cage was replaced with new bedding for the next mouse recording. The total duration of grooming behavior was manually scored by the experimenters in a double-blinded manner.
Marble-burying test: The marble-burying test was performed in a standard rat cage (48 X 27 X 20 cm) filled with 5 cm thick beddings. Twenty glass marbles were placed on the surface of the bedding in 5 rows of 4 marbles. Each mouse was placed into a corner of the cage. During the test, the cage was covered with a filter-top, and the mouse was allowed to explore for 30 min. The number of buried marbles was counted both fully buried and at least two-thirds covered by bedding in a double-blinded manner.
Open field test: Since some of the global Shank3-KO mice have shown various other behavioral abnormalities, including reduced motor performance and anxiety-like behaviors20,72-74, we assessed the behaviors using open field and light-dark box tests at adult age. Exploratory behavior in a novel environment was performed in an open field square chamber (37 X 37 X 36 cm) for 15 min under 180 lx illumination. Mouse was placed in the arena and recorded the distance traveled, mean velocity, and the time spent in the center (23 x 23 cm) and the margin of the arena using Ethovision XT software.
Light dark box test: The box for the light dark box test was divided into two compartments having a small removable door (6 X 6 cm) for entry of mouse. The illumination of light on the bottom of the center of the light-side and dark-side was set at 600 lx and 5 lx, respectively. The mouse was placed in the dark-side for 1min with the door closed. After removing the door to allow access to the other side, the mouse was placed in the center of the dark-side of the box and the video was recorded for 5 min. The time spent in light-side and the number of entries into the light-side was analyzed with Ethovision XT software.
Statistical analysis
All statistical analyses were performed using GraphPad Prism software version 9.1 (GraphPad Software, Inc., CA, US). Two-tailed unpaired t-test was used for two group comparisons between bEnd.3 BECs and eShank3-KO bEnd.3 BECs or between eShank3-KO and Tek-Cre control mice. One-way ANOVA followed by Dunnett’s multiple comparisons test was used for three group comparisons of bEnd.3 BECs, eShank3-KO BECs, and IWR-1-endo treated or GSK3βS9A and C/EBP1α induced eShank3-KO BECs. Two-way ANOVA with repeated measure and following Šídák’s multiple comparisons test was used for the time (or stage)-dependent behavior tests. Two-way ANOVA with repeated measures followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli was used for current-clamp recordings. Mixed-effects analysis and Šídák’s multiple comparisons test were used for analyzing electron density of TJs. All the detailed statistical methods and results are described in SI.Table 1. Data are represented as mean ± SEM. p value < 0.05 was considered statistically significant.
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