Mice were housed according to Federation of Laboratory Animal Science Associations (FELASA) guidelines. All mice were bred and maintained in a vivarium at 22 C in a 12-hr light/dark cycle, with food and water available ad libitum. The Shank3B KO line was purchased from Jackson Laboratories. Shank3B KO and wild type littermate mice were produced through crosses of heterozygote males and females. The genetic background for the Shank3B mouse lines are C57BL/6J. Experiments were performed with 8- to 10-week-old male mice. All experimental protocols were approved by the Animal Care and Use Committee of Bar Ilan University.
To determine the Shank3B genotypes, DNA was extracted from ear samples notched at the time of weaning using the Kapa mouse genotyping kit. The following primers were used to determine Shank3B mice genotype: common Fw 5'-GAGCTCTACTCCCTTAGGACTT-3'; Rv mutant 5'-TCAGGGTTATTGTCTCATGAGC-3' (~330bp) and for wild type: Rv 5'- TCCCCCTTTCACTGGACACCC-3' (~250bp).
Reciprocal dyadic social interaction test. The reciprocal dyadic social interaction test was done as previously described14,9. Prior to the test, each mouse was separated for social isolation of 1-2 hours. 5-week-old male RCF white stranger mouse was used as the social stimulus. Both the stranger and the tested mouse were placed in a 40 × 40 × 20 cm cage. During the interaction, the mice were recorded for 20 min, with the last 10 min quantified by an observer blind to treatment. Cowlog V3 software was used to score the social contact initiated by the test mouse (Helsinki University, Helsinki, Finland).
3-chambered social interaction test. The test took place in a Non-Glare Perspex box (60X40cm) with two partitions that divide the box to three chambers, left, center and right (20X40cm). The mouse is placed in the middle chamber for habituation (5 min) when the entry for both side chambers is barred. Test mouse was then allowed to explore the whole arena (10 min), where they freely choose between interacting with a novel mouse in one chamber, or stay in an empty chamber (social test). After 10 min ended, a second stranger mouse is introduced to the empty chamber, and the test mouse is allowed ten minutes to freely choose between interacting with the novel or familiar mouse.
Ultrasonic vocalizations. The ultrasonic vocalization test was done as previously described10,14. Both Shank3B KO and WT males met WT females, all sexually naive. Prior to the test, each mouse was placed in separate cages for social isolation for 1-2 hours, the female was placed in the cage of the male. UVs were recorded for the first five minutes of encounter to prevent extremely high sexual arousal and mating behaviors. The females were in the same cage in order to synchronize their estrus cycle and had met the males on the same day. UVs were recorded with Avisoft-RECORDER v. 4.2.21 recording program. The settings included a sampling rate of 250 kHz and a format of 16 bit. For spectrogram generation, recordings were transferred to Avisoft-SASLab Pro Version 5.2.07 and a fast Fourier transformation (FFT) was conducted. Spectrograms were generated with an FFT length of 256 points and a time window overlap of 50% (100% Frame, FlatTop window).
Mesenchymal stem cells preparation
Human MSC were purchased from Lonza (cat:PT-2501, Basel, Switzerland) and were cultured as previously described35. Before the exosome collection, the cells were cultured in exosome-free platelets medium for 3 days and this medium was then collected.
Exosomes Purification protocol.
Purification of exosomes was done using differential centrifugation protocol. First, the conditioned medium was centrifuge for 10 minutes at 300g. The supernatant was recovered and centrifuged for 10 minutes at 2,000g. Once again, the supernatant was centrifuged for 30 minutes at 10,000g. The supernatant was filtrated through a 0.22 µm filter and centrifuged for 70 minutes at 100,000g. The pellet containing the exosomes and proteins was washed in PBS and then centrifuged for 70 minutes at 100,000g. The pellet containing the purified exosomes was re-suspended in 200 µm of sterilized PBS. MSC-exo were characterized using Nanosight technology, TEM, western blotting as previously described14–16,41.
In-vivo treatment protocol
At the age of 4 weeks, 10 Shank3B KO male mice were treated with a total of 20ul MSC-exo at a concentration of 107particals/ul. The treatment was performed as previously described14. In general, each Shank3B KO treated mouse received 5ul of exosomes via intranasal administration for 4 days, every other day (all together 8 days of treatment). The behavioral experiment was done three weeks after the last treatment.
FACS analysis of exosomes
For FACS analysis, exosomes were coated onto 4-μm-diameter aldehyde/sulfate latex beads. 50μl exosomes were incubated with 12.5μl 4-μm-diameter aldehyde/sulfate latex beads (cat# A37304, Invitrogen) for 15 min at room temperature. 700μl sterile PBS was added, and the mixture was then transferred to 4C° and gentle shaking over-night. After centrifugation, the pellet was blocked by incubation with 200μl 100 mM glycine for 30 min at room temperature. Exosome-coated beads were washed in PBS and resuspended in 100μl sterile PBS. Afterwards, beads were incubated with CD63-APC (cat#130-118-078, Miltenyi biotec), CD81-APC (cat# 130-119-787 Miltenyi biotec) or IgG1 Isotype control (cat#130-113-434, Miltenyi biotec) fluorescent Abs for 15 min on ice in the dark. Beads were analyzed by flow cytometry using Gallios flow analyzer FACS (Beckman Coulter). Data was analyzed using the Kaluza Analysis Software (Beckman Coulter).
Exosomes were labeled with PKH26 (Sigma-Aldrich). PKH26 (2µL) in 500 µL diluent was then added to 50 µL exosomes in PBS for 5 minutes of incubation. Exosomes were suspended in 70ml PBS and were centrifuged for 90 minutes at 100,000g at 4˚C. The pellet was suspended in 200 µL of PBS15,16.
Proteomic analysis of MSC-exo
The samples were subjected to lysis and in solution tryptic digestion using the S-Trap method (by Protifi). The resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high resolution, high mass accuracy mass spectrometry (Q Exactive HF). Each sample was analyzed in the instrument separately in a random order in discovery mode and the DATA processing was done by MaxQuant v184.108.40.206. The data was searched with the Andromeda search engine against the human proteome database appended with common lab protein contaminants and the following modifications: Fixed modification-cysteine carbamidomethylation, variable modifications- methionine oxidation, asparagine and glutamine deamidation, protein N-terminal acetylation. The quantitative comparisons were calculated using Perseus v220.127.116.11. Decoy hits were filtered out. The resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high resolution, high mass accuracy mass spectrometry (Q Exactive HF). Each sample was analyzed on the instrument separately in a random order in discovery mode. Raw data was processed with MaxQuant v18.104.22.168. The data was searched with the Andromeda search engine against the human proteome database appended with common lab protein contaminants and the following modifications: Fixed modification-cysteine carbamidomethylation. Variable modifications- methionine oxidation, asparagine and glutamine deamidation, protein N-terminal acetylation. The quantitative comparisons were calculated using Perseus v22.214.171.124. Decoy hits were filtered out. Gene onthology was performed by using the ToppGene Suite42. Presented GO terms met a p-value of <0.05 at Benjamini–Yekutieli False Detection Rate (FDR).
For immunostaining Shank3B KO male mice (n=2) received intranasal treatment of 5ul of PKH26-lebeled MSC-exo and were sacrificed 24 hours post administration. Mice were perfused and fixated with PBS and 4% paraformaldehyde (PFA). The brains were incubated in PFA for 24 h followed by 30% sucrose for 48 h and stored at 4 °C. The brains were frozen in chilled 2-methylbutane (Sigma-Aldrich), stored at 4 °C, and subsequently sectioned into slices at 10 lm. Slides were incubated with blocking solution (5% goat/ donkey serum, 1% BSA, 0.5% Triton X-100 in PBS) for 1 h. Thereafter, slides were incubated overnight at 4 °C with primary antibody in blocking solution (mouse anti-CD11b, 1:500, Abcam) and secondary antibody in blocking solution (goat anti-mouse Alexa 488, 1:500, Molecular Probes, Invitrogen) for 1–2 h at room temperature. Next, nuclei were counterstained with DAPI (1:500; Sigma-Aldrich). Sections were ultimately mounted with fluorescent mounting solution (Fluoro- mount-G, Southern Biotech), covered with a cover slide, and sealed with nail polish.
Brain sample dissection
MSC-exo and saline treated Shank3B KO (n=5) and WT (n=5) Brain samples were removed from mice that had not been subjected to any behavioral testing and were kept at normal light cycle facilities (not reverse light cycle). The entire mouse brain was removed at approximately 12:00pm (light cycle is 7:00am to 7:00pm) and placed in an adult mouse brain matrix (Zivic Industries, Pittsburgh, USA). Brain slices (bregma 2.8 – 1.42) were removed and prefrontal cortex regions were obtained by using a 13-gauge biopsy punch needle (VGC, New Delhi, India). The cerebellum was removed using a scalpel. Brain samples were frozen with dry ice and kept in -80º until mRNA extraction.
Real-time PCR was performed on an ABI ViiA™ 7 RealTime PCR detection system in 10 μl volume containing FastStart Universal SYBR Green Master (Roche, Basel, Switzerland) and primers (Supplementary Table 6) at a concentration of 0.5 μM each. 10 ng of cDNA was dispersed in each well, and all samples were tested in triplicates. PCR program consists of 15-minute activation phase at 95 degree Celsius, followed by 40 cycles at the following temperatures: 10s of 94 degrees, 30s of 60 degrees. Real-Time PCR data were normalized to the housekeeping gene HPRT.
All behavioral and molecular experiments were analyzed with GraphPad (Prism). UV was analysed by SASLab Pro (Avisoft.). On-way ANOVA followed by Bonferroni correction was used for social behavioral tests and UV. Power calculation for the real-time PCR was calculated with an online calculator, (http://onlinestatbook.com/2/calculators/power_calc.html). The two-tailed power value was 0.985 which met out requirement for five samples. One-way ANOVA followed by Tukey’s post hoc was used for real-time PCR analysis. The number of samples chosen to perform