All animal experiments were conducted under the National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57BL/6 mice were obtained from the Experimental Animal Center of Chongqing Medical University. Ctnnd2 KO (Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$) mice in a C57BL/6 background were generated by CRISPR/Cas-mediated genome engineering, in collaboration with Nanjing Institute of Biomedicine, Nanjing University. Briefly, both Cas9 mRNA and sgRNA were microinjected into the zygotes, where sgRNA directed Cas9 endonuclease to cleave within intron 1-2 and intron 2-3 of mouse Ctnnd2 gene, yielding a double-strand break (DSB). Such breaks were then repaired by non-homologous end joining and proposed to result in disruption of Ctnnd2 gene expression in vivo. We bred the homozygous mutant mice from heterozygotes and their ups were genotyped by PCR as usual. For genotyping, mouse tail DNA fragments were amplified by PCR using the following primers: 5${}^{\prime }$-TTCTGTATTTCACAGTACCAAC-3${}^{\prime }$/5${}^{\prime }$-AACTCATCATAAGAAACACCTG-3${}^{\prime }$, and 5${}^{\prime }$-ACAGAATTATATCACACTTGTCC-3${}^{\prime }$/5${}^{\prime }$-ACTGTCACCCTACTTTAGTGTTA-3${}^{\prime }$.

All mice were housed at (25 $\pm$ 3 ${}^{\circ }$C) and relative humidity (55% $\pm$ 5%) under a 12 h/12 h light/dark cycle. The corresponding adenovirus was conducted on experimental male mice on a postnatal day (PND) 21 and behavior tests on PND49. Mice were then sacrificed for further analyses. No animals were excluded from the analysis.

The adeno-associated virus (AAV)-based Rictor RNA interference vector pAKD-CMV-bGlobin-GPR-H1-shRNA (AAV-Rictor) were constructed by Obio Technology, Shanghai China, containing the target sequences (GCCAGTAAGATGGGAATCA, sense; TGATTCCCATCTTACTGGC, antisense). The verification of the inhibitory efficiency on Rictor expression was conducted 4 weeks later after the AAV injection.

Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg, i.p.) and then fixed in a stereotaxic frame (Stoelting Instruments, Wood Dale, IL, USA). AAV (3.61 $\times$ 10${}^{12}$ TU/mL, 0.5 $\mu$L total volume per side) was injected bilaterally into the dorsal hippocampus (AP: –1.7 mm, ML: $\pm$ 1.3 mm, DV: –1.0 mm) over 5 min (injection rate of 0.1 $\mu$L per min). The syringe was maintained in situ for at least 5 min after every injection, to limit reflux along the injection tracks. Behavioral tests were conducted 4 weeks later.

Golgi-Cox staining was performed to examine the neuronal spine density of the hippocampus using an FD Rapid Golgi Stain Kit (PK401; FD Neuro-Technologies, Columbia, USA), according to the manufacturer’s instructions. After anesthetized, the brains of the mice were rapidly removed. After rising in distilled water, brains were immersed in a 1:1 (v/v) mixture of Solutions A and B for 2 days, and then kept at room temperature for 2 weeks. Brains were then immersed in Solution C and stored at 4 ${}^{\circ }$C for another 3 days. After that, brains were cut into 100-$\mu$m thick coronal sections, which were mounted on gelatin-coated slides and dried naturally. Then slides were stained with a mixture of Solution D, Solution E, and distilled water (1:1:2) for 10 min. After another wash with distilled water, the slides were dehydrated by 4 cycles in gradient ethanol (50%, 75%, 90%, 100%, and 100%, each for 4 min) and then cleared with xylene. The hippocampal areas were imaged using an Olympus microscope system. The number of dendritic spines along the secondary branches of CA1 pyramidal neuron dendrites were counted from 6 cells per hippocampus and the mean number for each 10-$\mu$m dendritic length measured in a double-blinded manner by ImageJ software (1.6.0, National Institutes of Health, New York, USA).

Transmission electron microscopy (TEM) was used to evaluate the density of spine synapses and the thickness of postsynaptic density (PSD) within the mouse hippocampus of each group. Briefly, mice were deeply anesthetized, and both hippocampi isolated. The hippocampi were transferred into 2.5% glutaraldehyde and kept at 4 ${}^{\circ }$C for 24 h, cut into slices, and fixed in glutaraldehyde for further 3 days. The slices were then washed in PBS, fixed in 1% osmium tetroxide, stained with an aqueous solution of 2% uranyl acetate, dehydrated in a gradient of alcohol and acetone, embedded in an embedding medium, incubated in an oven, and cut into ultrathin sections. The sections were stained with 4% uranyl acetate and citrate and examined under a transmission electron microscope (JEM-1400 Plus, JEOL, Japan) at 40,000$\times$ and 100,000$\times$ magnification. Both the synaptic density and the PSD thickness were analyzed using Image-Pro Plus 6.0 (Media Cybernetics Inc.). The number of synapses per unit volume of tissue (Nv) was calculated from the number of synapses per unit of area (Na) according to the formula Nv = 8ENa / $\pi$${}^2$ (where E is the mean of the reciprocals of the observed PSD lengths for each synaptic profile category), as described previously [25].

Western blot analyses were performed to examine the expression of targeted proteins within the hippocampus. Hippocampi were isolated rapidly and then lysed in RIPA buffer (P0013B; Beyotime, Shanghai, China) with a 15% protease inhibitor cocktail (4693116001, Roche, Basel, Switzerland). Protein concentration was measured with an Enhanced BCA assay kit (P0012, Beyotime), according to the manufacturer’s instructions. Extracted proteins were then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked by incubation for 1.5 h at room temperature in freshly prepared TBST containing 5% skim milk, and then incubated with the indicated primary antibodies at 4 ${}^{\circ }$C overnight. Immunoblotted membranes were washed with TBST, and incubated with horseradish peroxidase (HRP)-conjugated anti-goat, anti-rabbit, and anti-mouse secondary antibodies for 1 h at 37 ${}^{\circ }$C, respectively. The protein bands were visualized with Western Lighting-ECL and quantified using ImageJ (1.6.0, National Institutes of Health, New York, USA). All experiments were repeated at least three times on independently prepared lysates from different mice.

The F-actin/G-actin ratio analysis was according to the previous studies [26]. Briefly, the hippocampi were homogenized in cold lysis buffer and centrifuged at 15,000g for 30 min to obtain soluble actin (G-actin) in the supernatant. The pellet was re-suspended in the same volume of lysis buffer and incubated at 4 ${}^{\circ }$C for 1 h to depolymerize the insoluble F-actin. This suspension was then centrifuged at 15,000 rpm for 30 min to obtain F-actin in the supernatant. Equal amounts of G-actin and F-actin were loaded onto gels and analyzed by western blot.

The primary antibodies and dilutions were as follows: rabbit anti-Rictor (1:1000, 2140, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKTSer473 (1:1000, 9271, Cell Signaling), rabbit anti-AKT (1:1000, 9272, Cell Signaling) rabbit anti-actin (1:1000, GTX61452, Gentex, Zeeland, MI, USA), rabbit anti-cofilin (1:1000, 3312, Cell Signaling), rabbit anti-profilin-1 (1:1000, GTX63456, Gentex), rabbit anti-GluR1 (1:1000, 2452486, Millipore, Burlington, MA, USA), rabbit anti-synapsin 1 (1:30000, AB1543, Millipore), rabbit anti-ELKS (1:5000, 180507, Abcam, Cambridge, UK), and mouse anti-$\beta$-actin (1:2000, AA128, Beyotime Biotech) as the loading control. The secondary antibodies included HRP-conjugated goat anti-rabbit lgG and HRP-conjugated goat anti-mouse IgG (1:2000, ZB-2301 and ZB-2305, Zhongshan Biotech).

All graphs show the means $\pm$ S.E.M. from at least 3 separate experiments. Statistical analyses were performed using SPSS 21.0 (IBM, Armonk, NY, USA). In experiments with two treatment groups, means were compared by two-tailed independent-samples Student’s t-test. Multiple groups mean with normal distributions and equality of variance were compared by one- or two-way ANOVA with post hoc Tukey’s tests for multiple pair-wise comparisons. Morris water maze test data were analyzed by two-way repeated mixed measures ANOVA with post hoc Tukey’s tests. Values of p 0.05 were considered to be significant.

Using a CRISPR/Cas9-mediated gene-targeting strategy, we generated mutant mice with exon 2 deleted within the endogenous murine Ctnnd2 gene (Fig. 1A). Heterozygous mice were used to produce the homozygotes and their wild-type (WT) control littermates. Genotyping was applied to identify the mutant mice by two different pairs of primers, yielding a 1074 bp/421 bp fragment pattern for wild-type and 459 bp/- for Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (Fig. 1B). In three-chamber sociability tests, the total entries showed a no difference between WT and Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (Fig. 1D,G) but Ctnnd2${}^{\mathrm{–}\mathrm{/}\mathrm{–}}$ mice spent less time interacting with stranger 1 (p 0.05) and more time staying in the center chamber (p 0.01) in social interaction test, compared with the WT mice (Fig. 1E). Meanwhile, Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice spent more time staying in the center chamber (p 0.05) and less time interacting with the stranger 2 (p 0.05) in the novelty preference test (Fig. 1H). In the open field test, Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice showed stereotypic and repetitive behaviors as we observed more grooming frequency of them than the WT ones (p 0.001; Fig. 1J). Moreover, Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice exhibited anxiety-like behaviors, as evidence by the reduced number of center grid crossings (p 0.01; Fig. 1K) and vertical (p 0.01; Fig. 1L). Additionally, Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice showed abnormal exploratory behaviors, as evidenced by decreased numbers in climbing (p 0.01; Fig. 1M). The total cross grid number showed no difference between WT and Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (Fig. 1N). These data suggest that Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice showed decreased social interaction and novelty preference, stereotypic and anxiety-like behaviors, decreased exploratory behaviors.

Fig. 1.

Autism-like behavioral phenotypes of Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice. (A) The strategy of generation of Ctnnd2 mutant mice. Both Cas9 mRNA and sgRNA were co-injected into mouse zygotes; the sgRNA directed Cas9 nuclease to cleavage the exon 2 of mouse Ctnnd2 gene and thus resulted in disruption of CTNND2 protein. (B) Genotyping results of the mutant mice and their WT control littermates. (C–H) Three chamber test of Ctnnd2-mutant mice. Left panel, schematic presentation (C, F); middle panel, statistical analysis of the total entries in the social interaction test (D) and novelty preference (G), respectively; right panel, the time spent in each test (E, H). S1, stranger mouse 1; IS1, interaction with stranger mouse 1; C, center chamber; O, object; IO, interaction with the object; S2, stranger mouse 2; IS2, interaction with stranger mouse 2. (I–N) Open-field test of the mice. I, schematic presentation of the test; grooming time (J), number of center grid crossing (K), frequency of vertical rearing (L), and climbing times (M) were measured and analyzed in the open-field test. Statistical analysis was performed using a t-test. n = 8 mice per group for a behavioral test. Data are the means $\pm$ SEM. *, p 0.05; **, p 0.01; ***, p 0.001.

Next, we used the Morris water maze (MWM) to evaluate the hippocampus-dependent spatial learning and memory. In the learning phase, a two-way repeated-measures ANOVA revealed that both day (F${}_{(4,32)}$ = 23.537, p 0.01) and group (F${}_{(1,14)}$ = 9.425, p 0.01) could affect the escape latencies, but no detectable day $\times$ group interaction (F${}_{(4,32)}$ = 1.371, p $>$ 0.05). A post hoc analysis showed that there was significance between WT and Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (p $>$ 0.05 for Day 1 and Day 2, p 0.05 for Day 3, Day 4, Day 5; Fig. 2A). Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice spent significantly more time finding the hidden platform than the WT mice (p 0.05) from day 3 to day 5 (p 0.05, Fig. 2A,J). The swim speed showed no difference during the learning stage (Fig. 2B). For the memory test on day 6, we observed a representative swimming pathway (Fig. 2D) and quantified the time spent in each quadrant. We then found that Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice crossed the platform less often (p 0.05; Fig. 2C) and spent less time in the target quadrant (p 0.05; Fig. 2E). No significant differences between WT and Ctnnd2${}^{\mathrm{–}\mathrm{/}\mathrm{–}}$ mice in swim speed and movement distance (Fig. 2F,G). Collectively, these data suggest that spatial learning and memory are impaired in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice.

Fig. 2.

The spatial learning and memory impairment in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice. (A–G) Morris water maze test of the mice. Escape latency time to find the submerged platform and swim speed was observed from day 1 to day 5 of the learning phase (A, B). Statistical analysis was performed using two-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. Representative swimming traces were shown for probe test (D): T, target quadrant; L, left of target quadrant; R, right of target quadrant; O, opposite of target quadrant. The number of platform crossings (C) and time in the target zone (E) during the probe trial on day 6 were analyzed to evaluate the mice’s spatial memory. Two genotypes in swim speed (F) and travel distance (G) during the memory test. Statistical analysis was performed using a t-test. n = 8 mice per group. Data are the means $\pm$ SEM. *, p 0.05.

As Rictor plays an important role in synapse development and synaptic plasticity in the hippocampus, we firstly explored the expression levels of Rictor protein in the hippocampus at different developing stages, using both WT and mutant mice. As shown in Fig. 3, hippocampal Rictor expression was gradually decreased with age and keep stable from post-natal days (PND) 21. Of note, Rictor protein levels in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice were significantly lower than that in WT mice (p 0.05; Fig. 3A,B).

Given the significant changes in Rictor expression, we further conducted AAV-mediated Rictor knockdown induced by specific shRNA (Fig. 3D), with high efficiency (Fig. 3E). The expression of hippocampal Rictor was significantly downregulated (p 0.05) after injection of virus carrying specific shRNA, both in WT and mutant mice (Fig. 3F). There was no significant difference between sham group and vector group (Fig. 3E). Western blotting confirmed decreased hippocampal Rictor expression in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (p 0.05, KO + Vector vs WT + Vector) and further reduction in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice receiving ShRictor-virus infection (p 0.01, KO + ShRictor vs KO + Vector; Fig. 3F). In Morris water maze test, a two-way repeated measures ANOVA revealed that both day (F${}_{(4,28)}$ = 15.64, p 0.01) and group (F${}_{(3,21)}$ = 7.438, p 0.01) could affect the escape latencies, but no detectable day $\times$x group interaction (F${}_{(12,84)}$ = 1.218, p $>$ 0.05). One-way ANOVA showed that there were significance among WT + Vector, WT + ShRictor, WT + Vector and KO + ShRictor (p $>$ 0.05, WT + Vector vs WT + ShRictor for Day 1 and Day 2, p 0.05 for Day 3, Day 4, Day 5; p $>$ 0.05, WT + Vector vs KO + Vector for Day 1 and Day 2, p 0.05 for Day 3, Day 4, Day 5; p $>$ 0.05, KO + Vector vs KO + ShRictor for Day 1 and Day 2, p 0.05 for Day 3, Day 4, Day 5; Fig. 4A). The Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mutant mice needed much increased time to find the hidden platform after Rictor knockdown (p 0.05; Fig. 4A). The escape latency was also delayed in both WT and Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice after Rictor knockdown (p 0.05, WT + ShRictor vs WT + Vector; p 0.05, KO + ShRictor vs KO + Vector, Fig. 4A). The swim speed showed no different during learning stage both day (F${}_{(4,28)}$ = 0.1562, p $>$ 0.05) and group (F${}_{(3,21)}$ = 0.1780, p $>$ 0.05) and day $\times$ group interaction (F${}_{(12,84)}$ = 0.07183, p $>$ 0.05) (Fig. 4B). Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice with shRictor-mediated knockdown made significantly fewer passes over the former platform location (Fig. 4C) and spent less time in the target quadrant (Fig. 4D). Certainly, we found no significant differences in travel distance within the target quadrant among groups (Fig. 4E). The above results indicated that virus-mediated Rictor knockdown deteriorated the impairment in learning and memory in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice.

Fig. 3.

Alteration of Rictor expression in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice. (A) Changes in hippocampal Rictor expression of mice at different developmental stages as indicated. (B) Quantification of Rictor expression in the hippocampus of Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice and their control littermates. (C) The timeline of the WT. KO and/or ShRictor treatments and subsequent examinations. Treatments started from P21. Infections continued for 4 weeks until P49. Behavioral tests started from P49; n = 8 for only Morris water maze, n = 8 for 3-chamber and open field tests. The mice were then sacrificed at P58 for subsequent analyses, (D) Image of GFP expressed by the virus-carrying shRNA-Rictor construct were shown after infected the hippocampus of Ctnnd2${}^{\mathrm{–}\mathrm{/}\mathrm{–}}$ mice. (E, F) Verification of the efficiency of Rictor knockdown by Western blot in C57/BL6 mice (E) and the KO mice (F), respectively. Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey’s test for multiple comparisons. n = 3–4 mice per group. Data are the means $\pm$ SEM.*, p 0.05; **, p 0.01; ${}^{\mathrm{\#}}$, p 0.05.

Fig. 4.

Alteration of Rictor expression affects the spatial learning and memory in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice. (A–E) Morris water maze test of the mice. (A) Escape latency time to find the submerged platform and swim speed was observed from day 1 to day 5 of the learning phase (A, B). Statistical analysis was performed using two-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. (C–E) The number of platform crossing, time in the target zone, and travel distance were analyzed during the probe trial for spatial memory. n = 8 mice per group. Data are the means $\pm$ SEM. *, p 0.05.

We also examined whether ShRictor could affect autism-like behaviors of mutant mice in our experiments. However, when comparing these mice in the three-chamber test, the open field test, we found no significant difference between Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice with or without virus-mediated shRNA knockdown of Rictor, as well as that between WT mice (Fig. 5). These results indicated that Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice showed no changes in autism-like behavior after ShRictor-induced knockdown, suggesting that Rictor was not involved in $\delta$-catenin-related regulating of autism-like behaviors.

Fig. 5.

No effects of Rictor knockdown on autism-like behavior of Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice. (A–D) Social interaction and novelty preference were examined in the 3-chamber test. (E–H) Grooming times and numbers of center grid crossing, vertical rearing, climbing were measured in the open-field test in turn. Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey’s test for multiple comparisons. n = 8 per group. Data are the means $\pm$ SEM. *, p 0.05; **, p 0.01; ***, p 0.001.

With less expression of Rictor in the hippocampus after shRNA-mediated knockdown both in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice and WT controls (Fig. 3E,F), one of its downstream Akt protein showed dramatically reduced phosphorylation at Ser473 in mutant mice (p 0.05, KO + Vector vs WT + Vector) and a further decrease after ShRictor-virus infection (p 0.05, KO + ShRictor vs KO + Vector), as revealed by Western blotting experiments (Fig. 6A). Importantly, the F-actin/G-actin ratio, an important biomarker of actin cytoskeleton polymerization in neurons, was greatly reduced in both Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (p 0.05, KO + Vector vs WT + Vector) and Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice with ShRictor (p 0.05, KO + ShRictor vs KO + Vector) (Fig. 6B). Moreover, the expression of Profilin-1, a polymerization regulator, was also significantly reduced in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (p 0.05, KO + Vector vs WT + Vector), that was exaggerated by ShRictor-mediated knockdown in the mutant mice (p 0.05, KO + ShRictor vs KO + Vector) (Fig. 6C). However, the expression of cofilin protein, a de-polymerization regulator, showed no significant changes among all groups (p $>$ 0.05; Fig. 6D). Taken together, mTORC2/Akt signaling is thus implicated in the regulation of Rictor-induced actin dynamics in hippocampal neurons of Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice, implying a neuronal basis for their behavioral changes.

Fig. 6.

Effects of Rictor knockdown on Akt phosphorylation and actin polymerization within the hippocampus of Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice. (A) Expression levels of AKT and p-AKT (Ser473) were measured (upper panel) and analyzed (lower panel) by Western blot. (B) The ratio of F-actin/G-actin were analyzed by Western blot. (C, D) Expression levels of both Profilin-1 and Cofilin protein in each group were measured by Western blot, using WT and Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. n = 3–4 mice per group. Data are the means $\pm$ SEM. *, p 0.05; **, p 0.01.

As actin dynamics is key to neuronal dendritogenesis, we subsequently detect the dendritic spine density using classic Golgi-like staining (Fig. 7A, left panel). We found that mutant mice exhibited less spine density than the WT littermates (p 0.05), and this reduction was further exacerbated by ShRictor in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (p 0.05, (Fig. 7A, right panel)). TEM was further performed to assess differences in synaptic ultrastructure of hippocampal neurons (Fig. 7B, left panel). The number of synapse density in CA1 area was greatly reduced in mutant mice (p 0.05, KO + Vector vs WT + Vector) that was further decreased by ShRictor (p 0.05, KO + ShRictor vs KO + Vector; Fig. 7B, right panel). Furthermore, PSD thickness was remarkably reduced in the hippocampus of Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (p 0.05, KO + Vector vs WT + Vector) that was also affected by shRNA-mediated Rictor knockdown (p 0.05, KO + ShRictor vs KO + Vector) (Fig. 7C). Together, these data indicate that $\delta$-catenin can regulate spine morphology and synaptic plasticity of neurons in brain, which is may contributed by Rictor-related signaling during synaptic development.

Furthermore, Western blot showed that the expression levels of postsynaptic proteins, such as ELKS and GluR1, were both markedly decreased in Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice (p 0.05, KO + Vector vs WT + Vector). ShRictor further deteriorated the reduction in expression levels of these two synaptic proteins (p 0.01, KO + ShRictor vs KO + Vector) (Fig. 8A,B). In contrast, the expression of synapsin-1, a presynaptic marker protein, was neither changed by Ctnnd2 mutation nor affected by ShRictor in mutant hippocampus (Fig. 6C). These results provide novel cues of synaptic basis for the pathogenesis of Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice.

Fig. 7.

Effects of Rictor knockdown on synaptic morphology of the mutant mice. (A) Spine density in hippocampal CA1 was assessed by Golgi-impregnation. Scale bar, 5 $\mu$m. (B, C) Synapse density (number of synapses per $\mu$m${}^3$) and thickness of post-synaptic density (PSD) in hippocampal CA1 was determined by electron microscopy. Scale bars, 0.5 $\mu$m for B; 200 nm for C. Results was quantified as shown. Left, typical images; right, quantification of the results. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. n = 3–4 mice per group. Data are the means $\pm$ SEM. *, p 0.05.

Fig. 8.

Effects of Rictor on the expression of some typical synaptic proteins in the hippocampus of Ctnnd2${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$ mice. Expression levels of the synaptic proteins ELKS (A), GluR1 (B), and Synapsin-1 (C) were measured by Western blot, respectively. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test for multiple comparisons. n = 3–4 mice per group. Data are the means $\pm$ SEM. *, p 0.05; **, p 0.01.