Since seven ASD-linked mutations in human CTTNBP2 are conserved among human, rat and mouse (Additional file 1: Fig. S1), we used rat cultured hippocampal neurons and knockin mice to analyze how ASD-linked mutations influence the function of CTTNBP2 in terms of dendritic spine density, neuronal activity and mouse behaviors. We primarily overexpressed ASD-linked mutations in wild-type (WT) hippocampal neurons and used heterozygous knockin mice to mimic monoallelic mutations of CTTNBP2 in patients. Based on our analysis of dendritic spine formation in cultured neurons, we then focused on three mutations, i.e. M120I, R533* and D570Y, because they altered the subcellular distribution of CTTNBP2 and impaired dendritic spine formation. A series of biochemical and cell biology approaches were employed to dissect the molecular defects caused by ASD mutations. Neuronal morphology and mouse behaviors of ASD mutant mice were then investigated to elucidate the physiological impact of ASD-linked mutations in the Cttnbp2 gene in vivo. All morphometric, electrophysiological and behavioral analyses were conducted blind by relabeling the samples before analysis by another member in the laboratory. All statistical methods and results are summarized in Additional file 2: Table S1, Additional file 3: Table S2.
All the animal experiments in this study were performed with the approval of the Academia Sinica Institutional Animal Care and Utilization Committee (Protocol # 12-10-414 and 11-12-294). Animal housing and handling were conducted according to the guidelines of the Council of Agriculture Guidebook for the Care and Use of Laboratory Animals. For primary hippocampal neuron culture, pregnant rats were sacrificed by CO2 inhalation. Embryonic day E18.5 fetal pups of both sexes were isolated and sacrificed by decapitation. Male mice at the age of 2-3 months were used for behavioral assays. All animals were housed and bred in the animal facility of the Institute of Molecular Biology, Academia Sinica, under controlled humidity and temperature and a 12 h light/dark cycle (light off at 20:00). Animals accessed water and food (#5K54, LabDiet) ad libitum. All genetically modified mice had been backcrossed to WT C57BL/6 mice for more than six generations to minimize off-target effects of CRISPR/Cas9 editing.
CRISPR/Cas9 technology for introducing ASD-associated mutations into Cttnbp2
A paired-nicking approach [22, 23] was applied to insert mutation sequences into exon 4 of Cttnbp2. For the Cttnbp2 M120I mutation, the paired guide RNAs (5′- AGAAAGGATGTCCGCACAGC and 5′- AGTGGGCCATGACAGCTTCA) and a single-stranded DNA template (ssODN: 5′- TGTGAGCCAGTTCTGCTGTTCCTTGCTATTATGGGAAAAACAGATGACGTCTCAGTACCTTTTTTTGTCTGCTCTCAGCGGCCACCAGCTGTGCGGACATCCTTTCTTGtATTTTTCTaCAGTGGGCCATGACAGCTTCAAGGATGGAGAGTGGATTGGTGCAGACTGGCTTCTCTTTGTCACCAGGACCTGCTTCATAG) were used. Two nucleotides (in lower case) were changed in order to alter the coding sequences of methionine to isoleucine at residue 120 (shown in bold and underlined) and to disrupt the PstI site (underlined) without changing the encoded amino acid.
For the Cttnbp2 D570Y mutation, a guide RNA (5′-ATCAACTTTGGCCCCTGCAT) and a single-stranded DNA template (ssODN: 5′- GGAAATCCTCCTCCTATCCCTCCCAAAAAGCCAGGGCTCTCCCAAACTCCTTCTCCGCCACACCCCCAACTGAGGGCCTCCAATGCAGGGGCCAAAGTTtATAACAAgATTGTGGCTTCACCTCCCTCTACTTTGCCACAAGG) were used. Two nucleotides (lower case) were changed in order to alter the coding sequence from aspartic acid to tyrosine at residue 570, and to create a PsiI site and disrupt the MluCI site without changing the encoded amino acid, respectively.
A T7 promoter sequence (5′- TTAATACGACTCACTATA) was added upstream of gRNA sequences and a partial tracrRNA sequence (5′- GTTTTAGAGCTAGAAATAGC) was added downstream of the gRNA sequence. The oligo was annealed with reverse tracrRNA (5′-TTTAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC) and PCR-amplified using Phusion DNA Polymerase (Thermo-Fisher Scientific) according to the manufacturer’s instructions. The amplified product was purified using QIAquick PCR Purification Kit (28106, Qiagen) and it served as the in vitro transcription template.
The sgRNAs were synthesized using HiScribe™ T7 Quick High Yield RNA Synthesis Kit from NEB (2050S, NEB). Cas9AD10A mRNA was synthesized using mMESSAGE mMACHINE T7 Ultra Kit (AM1345, Thermo-Fisher Scientific) using pCAG-T3-hCasD10A-pA (#51638, Addgene) as template, purified using MEGAclear Transcription Clean-up Kit (AM1908, Thermo-Fisher Scientific), and eluted with injection buffer (10 mM Tris–HCl pH7.2 and 0.1 mM ethylenediaminetetraacetic acid (EDTA)). The quality and quantity of RNAs were analyzed using a NanoDrop ND-1000 (Thermo-Fisher Scientific). To extract genomic DNA, ~ 3 mm of mouse tail was cut and lysed in 600 μl 50 mM NaOH and boiled for 30 min. After complete tissue lysis, samples were cooled down briefly on ice before adding 60 μl of 1 M Tris–Cl pH 8.0 to neutralize the pH. Around 0.5-1 μl of DNA was used for PCR reaction. Primer sequences to amplify the M120I mutant allele by PCR were 5′-ATGGCTTTCCAGGCTTGTCAG-3′ and 5′-AGCCCACTCCCACCAAAACTA-3′. To amplify the D570Y mutant allele by PCR, we designed the primer set: 5′-GCCAAGCAGCTAGCTCGGAATAC-3′ and 5′-GTTCAGTCCAGGGGTTCCAGCAG-3′. To distinguish M120I and D570Y alleles, we digested the PCR product by PstI (NEB) and MluCI (NEB), respectively.
For immunostaining and biochemical analysis, the GW1-HA-CTTNBP2, GW1-myc-CTTNBP2, GW1-myc-NCC, and GW1-myc-Mid constructs were described previously [4, 5, 21]. For mEPSC recording (Fig. 1d), pCAG-GFP-P2A was constructed by inserting SuperfolderGFP and P2A sequences into an empty pCAG vector . The original superfolder GFP was obtained from , the P2A sequence is from , and the empty pCAG vector was ordered from Addgene. HA-tagged Cttnbp2 was PCR-amplified using the primer set 5′- GCGATATCTTAGGGAGGGTG-3′ and 5′- CCAGATATCATGTACCCATATGAC -3′ and cloned into pCAG-GFP-2A vector plasmid at the EcoRV site. Myc-cortactin was a generous gift from Dr. Morgan Sheng at MIT. To construct the GFP-P-rich domain, the P-rich domain was PCR-amplified using the primer set 5′-GGAATTCCGAACCGGTTTAAAG-3′ and 5′-GGAATTCTTAGGGAGGGTG-3′ and cloned into pEGFP-C2 plasmid at the EcoRI site. Site-directed mutagenesis using PCR was performed to construct ASD-associated Cttnbp2 mutant constructs. Sequences of all primer pairs (from 5′ to 3′) are as follows (uppercase letters indicate mutated residues):
The following antibodies and working concentration were used in this study: anti-CTTNBP2 (A5, A7, and 9W, rabbit, homemade, 0.5 μg/ml) [5, 20], anti-Myc tag (9B11, Cell Signaling Technology, 1/1000 for staining; 06-549, Millipore, 1 μg/ml), anti-HA tag (3F10, Roche, 0.5 μg/ml), anti-HA tag (Y-11, Santa Cruz Biotechnology, 0.5 μg/ml), anti-GFP (ab13970, Abcam, 0.5 μg/ml), anti-αtubulin (B-5-1-2, Sigma-Aldrich, 1 μg/ml), anti-acetyl tubulin (6-11B-1, Sigma-Aldrich, 1 μg/ml), anti-βactin (AC-74, Sigma-Aldrich, 1/1000), anti-cortactin (H-191, Santa Cruz Biotechnology, 0.5 μg/ml), anti-FOS (#2250, clone 9F6, Cell Signaling Technology, 1/200), anti-mouse HRP (NA931, GE Healthcare, 1/5000), anti-rabbit HRP (NA934, GE Healthcare, 1/5000), anti-chicken Alexa Fluor 488 (A-11039, Invitrogen, 1 μg/ml), anti-mouse Alexa Fluor 555 (A-21424, Invitrogen, 1 μg/ml), anti-rat Alexa Fluor 594 (A-21209, Invitrogen, 1 μg/ml), and anti-rabbit Alexa Fluor 647 (A-21244, Invitrogen, 2 μg/ml).
Preparation and transfection of cultured primary hippocampal neurons
Preparation of primary rat hippocampal culture was described previously [5, 21]. Briefly, hippocampi were carefully collected at embryonic day E18.5 and digested with papain solution [0.6 mg/ml papain, 0.5 mM EDTA, 1.5 mM CaCl2, 0.06% DNase I, 0.2 mg/ml cysteine] at 37 °C for 25 min. The papain solution was removed and the digested hippocampi were gently washed with Hank’s balanced salt solution (HBSS) buffer. To dissociate the cells, the digested hippocampi were gently pipetted. The cell suspension without debris was then transferred to a new tube and centrifuged at 900 rpm for 5 min to collect dissociated cells. The cell pellets were re-suspended and cell density was determined. For a 12-well plate, 2 × 105 cells/well were seeded on a polylysine-coated glass coverslip.
Cresyl violet staining
Fifty-μm-thick brain sections were mounted onto glass slides coated with 0.5% gelatin and air-dried. The sections were then stained with cresyl violet solution (0.1% cresyl violet in 1% acetic acid) and destained several times with 70% ethanol until the signal was clear. The sections were serially dehydrated with 70%, 90% and 100% ethanol and then xylene for mounting using Permount mounting medium (Fisher Scientific).
For neuron morphology analysis, rat primary cultured hippocampal neurons were fixed by 4% paraformaldehyde with 4% sucrose in phosphate-buffered saline (PBS) for 10 min and permeabilized by 0.2% Triton X-100 in PBS for 10 min. After washing with PBS, the sections were blocked with 10% Bovine serum albumin (BSA) for 30 min in room temperature, and incubated with primary antibodies in 3% BSA overnight in 4 °C. After washing with PBS, the coverslips were incubated with secondary antibodies 3% BSA for 1 h in room temperature. For C-FOS staining, two hours after the reciprocal social stimulation test, the brain was fixed with 4% paraformaldehyde in PBS. Fifty μm-thick brain sections were treated with 1% H2O2 in Tris–Cl buffer, pH 7.6, for 30 min and permeabilized with 0.05% Tween-20 in PBS for 15 min. After washing with PBS, the sections were blocked with TNB buffer (0.5% blocking reagent in PBS, TSA Fluorescein System Kit, No.1715186, Perkin Elmer) for 1 h and then incubated overnight with primary antibody in TNB buffer at 4 °C. After washing with 0.05% Tween-20 in PBS, sections were incubated with biotinylated goat anti-rabbit IgG secondary antibody (1/200, vectastain, Vector Laboratories) in TNB buffer for 2 h. The immunoreactivity was developed using Vectastain Elite ABC Kit (Vector Laboratories) based on the manufacturer’s instructions. For immunofluorescence staining, brain sections were incubated with primary antibody as described above, followed by incubation with the secondary antibodies conjugated with Alexa Fluor-488, -555, -594, and/or -647 (Invitrogen) for 2 h.
Microscopy and morphometry analyses
True-color imaging (for C-FOS staining and Cresyl violet stain) was performed using an upright microscope (Microscope Axio Imager M2, Carl Zeiss) equipped with a 10 ×/NA 1.4 oil (Plan-Apochromat, Carl Zeiss) objective lens, and AxioCam (Carl Zeiss) and Zen 2011 software (Carl Zeiss). Shading correction and white balance was applied to correct the signal. For cresyl violet staining, the images were tiled up to acquire entire sections. Fluorescence images were captured using a confocal microscope (LSM700, Carl Zeiss) equipped with a 63 ×/NA 1.4 oil objective lens (Plan-Apochromat, Carl Zeiss) and Zen 2009 (Carl Zeiss) at room temperature. To analyze spine morphology of hippocampal CA1 in vivo, Cttnbp2 mutant mice were crossed with Thy1-YFP transgenic mice (#003782, Thy1-YFP-H, The Jackson Laboratory) . The first branch of the apical dendrite of CA1 pyramidal neurons was selected for analysis. A 15-μm-long dendritic fragment 5 μm distant from the branch point was used to determine the density and length of spines and the width of spine heads. The Z-series images were captured at 0.2 μm intervals with the “Region” function in Zen 2009 (Carl Zeiss) and processed using the “maximum projection” function. Quantifications were performed using ImageJ. The spine density of two or three dendritic segments from the same neuron was averaged to represent the density of each neuron. Ten neurons were imaged from each animal and at least three mice were used for each group of an experiment.
For cultured hippocampal neurons, we focused on a 20-μm-long segment of the primary dendrite starting 20 μm away from the soma to measure dendritic spine density and the synaptic distribution of CTTNBP2 and cortactin. At least two clearly recognized dendrites were quantified and averaged (using blinded sample relabeling) for each neuron to represent the spine density or protein distribution of each neuron. The sample sizes of examined neurons and dendritic segments are summarized in Additional file 2: Table S1. To quantify the synaptic enrichment of cortactin, we quantified the cortactin signal in individual spines and normalized them with the average signal of cortactin in the soma of the same neuron. To quantify synaptic CTTNBP2, we performed line scanning from the tip of the dendritic spine to the dendritic shaft. The CTTNBP2 signals along the lines were determined using the line scanning method in ImageJ (NIH). The signal within the range of 0-0.5 μm from the spine tip indicated the synaptic region, whereas signals within the range 1-1.5 μm from the tip represented the base of the dendritic spine and the dendritic shaft. Five spines of each neuron were randomly sampled blindly from thirty-five neurons for each group.
To quantify the acetyl-tubulin/tubulin ratio in neurons, we measured a 5-μm segment of primary dendrite within the range of 5–20 μm away from the soma, and used the α-tubulin signal to normalize the acetyl-tubulin signal. This ratio represents microtubule stability in proximal dendrites.
Detailed information on morphometry sampling (number of examined mice, neurons, dendrites, and spines) is presented in Additional file 2: Table S1.
Cultured rat hippocampal neurons were transfected at 14 DIV, and whole-cell patch-clamps were performed at 18 DIV to record miniature EPSCs (mEPSCs). Neurons were incubated in extracellular solution containing 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2.2H2O, 0.53 mM MgCl2.6H2O, 5.56 mM Glucose, 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH7.4(NaOH), 0.001 mM tetrodotoxin, and 0.02 mM bicuculline. The intracellular solution contained 140 mM K-gluconate, 5 mM NaCl, 2 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid), 10 mM HEPES, 4 mM Mg-ATP, 0.3 mM Na-GTP pH 7.3(KOH). Neurons were voltage-clamped at −70 mV, and mEPSCs were recorded with an Axon Axopatch 200B amplifier (Molecular Devices) and filtered at 1 kHz. Clampfit software (10.4; Molecular Devices) was used to detect mEPSCs from the raw data with an amplitude threshold of 5 pA.
The antibody-protein A complex was first prepared by incubating 20 μl of myc antibody (9B11, Cell Signaling) with 20 μl of Protein A beads (17046901, GE Healthcare) overnight and washing with PBS to remove unbound antibody. To prepare protein extract for cortactin/oligomerization coimmunoprecipitation experiments, COS1 cell lysates were extracted with RIPA buffer [1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 50 mM Tris–Cl pH 7.4, 150 mM NaCl, 2 mM EDTA and protease inhibitors] and the debris was removed by centrifugation (16,000 × g for 20 min at 4 °C using a table-top microcentrifuge, Heraeus Biofuge Fresco). Lysate was incubated with myc tag antibody-coated Protein A beads for 4 h at 4 °C and washed once with each of the following buffers: (1) RIPA buffer, (2) 10 mM Tris–Cl, 1% Triton × 100, pH7.4, (3) 10 mM Tris–Cl, 0.1% Triton × 100, 0.5 M LiCl, pH7.4, and (4) 10 mM Tris–Cl, pH7.4. For GFP-P-rich coimmunoprecipitation, lysates were extracted with 1% Triton × 100 in PBS and incubated with myc tag antibody-coated Protein A beads for 4 h at 4 °C and washed three times with PBS. After removing the final wash buffer, 2 × SDS-PAGE sample buffer (4% SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 200 mM β-mercaptoethanol) was added and boiled for 10 min.
Microtubule spin down assay
A Microtubule Binding Protein Spin-down Assay Biochem Kit (BK029, Cytoskeleton, Denver, CO) was used to test the interaction of CTTNBP2 with microtubule according to the manufacturer’s instructions. Tubulin solution was added into General Tubulin Buffer with Taxol (GTB; 80 mM PIPES pH 7, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP and 20 mM Taxol) at 35 ˚C for 20 min to induce microtubule formation. To prepare CTTNBP2 proteins, COS1 cells were transfected with either WT or D570Y mutant expression plasmid. One day later, soluble total protein lysates containing CTTNBP2 proteins were collected from the supernatants after centrifugation at 16,000 × g for 20 min at 4 °C (table-top microcentrifuge, Heraeus Biofuge Fresco). Different amounts of protein lysates were incubated with or without microtubule at room temperature for 30 min. The samples were then placed onto 100 μl cushion buffer (60% glycerol in GTB) and centrifuged at 100,000 × g for 40 min at room temperature. The pellet fraction is the microtubule binding fraction and the supernatant is the unbound fraction. The sample was then boiled in 2 × sample buffer for 10 min and analyzed by immunoblotting.
Mouse behavioral assays
The open field test was conducted as described previously [6, 12] to monitor locomotor activity and anxiety. Briefly, the test animal was placed in the center of a transparent acrylic box (40 × 40 × 30 cm) and allowed to freely explore the environment. The experiment was videotaped for 10 min from above the box. The central zone of the box was defined by a square (20 × 20 cm) equidistant from the walls. The area of the defined central zone is equal to the sum of the four corners. To track and analyze the movement of the mice, the Smart Video Tracking System (Panlab) was employed. Total moving distance (to indicate locomotor activity) and the ratio of time spent at the center to that at the corner (to represent the degree of anxiety) were measured.
Elevated plus maze
A plus maze composed of two open arms and two closed arms (30 × 5 cm) extending from a small central platform (5 × 5 cm) was elevated from the floor to a height of 45.5 cm for the test. The test mouse was placed at the center of the platform and allowed to freely explore the environment for 10 min. The Smart Video Tracking System (Panlab) was used to track the movement of the animal. The percentages of time spent in open arms and closed arms were assessed.
Reciprocal social interaction (RSI)
Test mice were individually housed for approximately one week before the experiment. A stranger adult male mouse of the same age as test mice or one week younger was placed into the home cage of the test mouse for 10 min. During the entire session, the lid of the cage was kept open to limit aggressive behaviors. Mouse behaviors were recorded by videotaping from above. The total time the test mouse spent sniffing the stranger mouse (head-to-head, head-to-body and head-to-anogenital) was manually recorded to represent social interaction.
The three-chamber was performed as described previously [10, 12]. In brief, the apparatus was a rectangular transparent plastic box (17.5 × 41.4 × 22 cm), with two dividing walls that separate the box into three equal chambers. Each dividing wall had a sliding entrance to connect different chambers. Two cylindrical wire cages (10.5 cm in diameter and 11 cm in height) were put in the left and right chambers. The experiment comprised three 10-min sessions (habituation, sociability and novelty preference), with 5 min intervals between each session. During intervals, test mice were placed back in their home cage. In every session, the test mouse was placed into the central chamber and the two sliding doors were then simultaneously opened to allow the mouse to freely explore the whole environment. In the habituation session, both cylindrical wire cages were empty. In the sociability test session, an object (Ob) was placed in one of the wire cages and a stranger mouse (S1) was placed in the other wire cage. In the social novelty preference session, the object was replaced by another stranger mouse (S2). Mouse behaviors were recorded by videotaping from above. Sniffing toward the cylindrical wire cages was considered as social interaction, which was quantified manually without knowing the genotype of mice. The value of (TS1-TOb)/(TS1 + TOb) was defined as the preference index of sociability. The value of (TS2-TS1)/(TS2 + TS1) was defined as novelty preference. TOb is the interaction time with the object, TS1 is the interaction time with S1, and TS2 is the interaction time with S2.
Statistical analysis and graphical outputs were performed using PRISM 5.03 or 8.3 (Graphpad software). All sample sizes, statistical data and corresponding statistical methods are summarized in Tables S1 and S2. In brief, to compare multiple groups with one variant, one-way ANOVA with Bonferroni multiple comparison post hoc test was performed for normally distributed data and Kruskal–Wallis test with Dunn’s multiple comparison test was performed for nonparametric distributed data. To compare multiple groups with two variants, two-way ANOVA was employed. To compare two groups of unrelated datasets, two-tailed Mann–Whitney test or unpaired t test was employed. In this report, P < 0.05 is regarded as significant. For cumulative distribution analysis, a Kolmogorov–Smirnov (K-S) test was performed (https://www.aatbio.com/tools/kolmogorov–smirnov-k-s-test-calculator). Outliers in the dataset were excluded using the “Identify outlier” tool in PRISM. All image analyses, including immunoblotting and morphometric analyses, were conducted using ImageJ (NIH). All morphometric and behavioral assay data were analyzed blind to minimize personal bias by relabeling the samples before being analyzed by another member of the laboratory. All statistical methods and results are summarized in Additional file 2: Table S1, Additional file 3: Table S2.