Social deficits in Shank3-deficient mouse models of autism are rescued by histone deacetylase (HDAC) inhibition

Abstract

Haploinsufficiency of the SHANK3 gene is causally linked to autism spectrum disorder (ASD), and ASD-associated genes are also enriched for chromatin remodelers. Here we found that brief treatment with romidepsin, a highly potent class I histone deacetylase (HDAC) inhibitor, alleviated social deficits in Shank3-deficient mice, which persisted for ~3 weeks. HDAC2 transcription was upregulated in these mice, and knockdown of HDAC2 in prefrontal cortex also rescued their social deficits. Nuclear localization of β-catenin, a Shank3-binding protein that regulates cell adhesion and transcription, was increased in Shank3-deficient mice, which induced HDAC2 upregulation and social deficits. At the downstream molecular level, romidepsin treatment elevated the expression and histone acetylation of Grin2a and actin-regulatory genes and restored NMDA-receptor function and actin filaments in Shank3-deficient mice. Taken together, these findings highlight an epigenetic mechanism underlying social deficits linked to Shank3 deficiency, which may suggest potential therapeutic strategies for ASD patients bearing SHANK3 mutations.

Figure 1. Treatment with the HDAC inhibitor romidepsin induces the robust and prolonged rescue of autism-like social deficits in Shank3-deficient mice, while a variety of drugs for psychiatric disorders fail to do so

(a) Immunoblots and quantification analysis of the level of acetylated H3 and total H3 in the nuclear fraction of cortical slices from wild-type (WT) or Shank3^+/ΔC (Het) mice injected (i.p.) with saline or romidepsin (RMD, 0.25 mg/kg, 3×). Immunoblotting was performed at 4-5 days post-injection. F_1,20=11.3, P=0.0031; * P<0.05, two-way ANOVA, n=6 each group. (b, c) Plots showing the time spent investigating either the social (Soc) or nonsocial (NS) stimulus (b) and the social preference index (c) during 3-chamber sociability testing of saline-injected WT (n=18), saline-injected Shank3^+/ΔC (n=17), RMD-treated Shank3^+/ΔC (n=17) and RMD-treated WT (n=12) mice. In (b), F_3,120=11.4, P<0.0001; +++ P<0.001 (Soc vs. NS), ** P<0.01, *** P<0.001, two-way ANOVA. In (c), F_1,60=58.5, P<0.0001; *** P<0.001, two-way ANOVA. (d) Representative heat maps illustrating the time spent in different locations of the 3 chambers from the social preference tests of all groups. Locations of Sol and NS stimuli are labeled with the circles. (e) Plots of social preference index in Shank3^+/ΔC mice treated with romidepsin (n=10) or saline (n=10) at different time points. F_{1,18(treatment)}=124.3, P<0.0001; *** P<0.001 (saline vs. romidepsin), ### P<0.001 (pre- vs. post-injection), two-way rmANOVA. (f) Plots of social preference index in Shank3^+/ΔC mice treated with different doses of romidepsin (0.025 mg/kg, n=9; 1 mg/kg, n=8) at different time points. F_{1,15(treatment)}=51.3, P<0.0001; *** P<0.001 (1 mg/kg vs. 0.025 mg/kg RMD); ### P<0.001 (pre- vs. post-injection), two-way rmANOVA. (g) Scatter plots showing total sniffing time in social approach tests of saline-injected WT (n=12), saline-injected Shank3^+/ΔC (n=14), RMD-treated Shank3^+/ΔC (n=12) and RMD-treated WT (n=12) mice. F_1,46=6.0, P=0.018; * P<0.05, ** P<0.01, two-way ANOVA. (h) Representative heat maps illustrating the time spent in different locations of the apparatus from the social approach tests of all groups. Locations of social stimuli are labeled with the circles. (i-n) Plots of social preference index in Shank3^+/ΔC mice treated with fluoxetine (10 mg/kg, i.p., 14×, i, n=9), clozapine (5 mg/kg, i.p., 3×, j, n=11), valproic acid (VPA, 100 mg/kg, i.p., 3×, k, n=11), aripiprazole (1 mg/kg, i.p., 3×, l, n=9), risperidone (0.1 mg/kg, i.p., 3×, m, n=10), or Trichostatin A (TSA, 0.5 mg/kg, i.p., 3×, n, n=8). F_2,30=19.2 (VPA), P<0.0001; F_2,21=19.7, P<0.0001 (TSA); ### P<0.001 (pre- vs. post-injection), one-way ANOVA. (o) Representative heat maps illustrating the time spent in different locations of the 3 chambers from the social preference tests of a Shank3^e4-9 mouse before and after romidepsin treatment (0.25 mg/kg, i.p., 3×). (p) Box plots showing the time spent investigating either Soc or NS stimulus during sociability testing in WT (n=8) or homozygous Shank3^e4-9 mice (n=10) before and after romidepsin treatment. F_2,50=9.2, P=0.0003; +++ P<0.001 (Soc vs. NS), * P<0.05, *** P<0.001, two-way ANOVA. (q) Scatter plots showing the preference index of the sociability testing in individual Shank3^e4-9 mice before and after romidepsin treatment (n=10). t₉=4.36, ** P=0.0018, paired two-tailed t-test. (r) Plots of social preference index in Shank3^e4-9 mice (n=10) treated with romidepsin at different time points. F_6,63=11.8, P<0.0001; * P<0.05, ** P<0.01, *** P<0.001 (pre- vs. post-injection), one-way ANOVA. Shank3^+/ΔC mice (a-n) and WT mice (a-n,p) are all males (5-6 weeks old); Shank3^e4-9 mice (o-r) are 6 males and 4 females (5-6 weeks old). Data are presented as median with interquartile range (a,b,p) or mean ± SEM (c,e-g,i-n,q,r). Each set of the experiments was replicated for at least 3 times. See Supplementary Fig. 8 for blot source data.

Figure 2. Romidepsin treatment does not affect locomotor or anxiety-like behaviors or neuronal survival in Shank3-deficient mice

(a-d) Scatter plots showing a variety of behaviors in saline-injected WT (n=15), saline-injected Shank3^+/ΔC (n=15), romidepsin (RMD, 0.25 mg/kg, 3×)-treated Shank3^+/ΔC (n=15) and RMD-treated WT (n=12) mice, including the number of midline crossing in locomotion tests (a), the latency to fall during rotarod tests (b), the time spent in the center during open-field tests (c), and the time spent self-grooming (d). In (d), F_{1,53(treatment)}=2.55, P=0.117; *** P<0.001, two-way ANOVA. (e, f) Confocal images (e) and quantification (f) of layer V prefrontal cortical neurons (immunostained with α-CaMKII and NeuN) in WT or Shank3^+/ΔC mice treated with saline or romidepsin. Slices were collected for immunostaining at 4-5 days post-injection. n=27 images/3 mice each group. All animals used are males (5-6 weeks old). Data are presented as mean ± SEM (a-d) or median with interquartile range (f). Each set of the experiments was replicated for at least 3 times.

Figure 3. Shank3 deficiency induces HDAC2 upregulation, and HDAC2 knockdown in PFC rescues autism-like social deficits

(a) Quantitative real-time RT-PCR data on the mRNA level of class I HDAC family members (HDAC1, 2, 3, 8) in PFC from WT and Shank3^+/ΔC mice. t₁₆=3.75, ** P=0.0017 (HDAC2), n=9 each group, two-tailed t-test. (b) Immunoblots and quantification analysis of the protein level of HDAC1 and HDAC2 in the nuclear fraction of PFC neurons from WT vs. Shank3^+/ΔC mice. t₁₆=3.00, ** P=0.0085 (HDAC2), n=9 each group, two-tailed t-test. (c) qPCR and Western blot data showing HDAC2 mRNA and protein levels in PFC from WT and Shank3^e4-9 mice. t₁₄=3.24, ** P=0.006 (mRNA), n=8 each group; t₁₆=3.79, ** P=0.0016 (protein), n=9 each group, two-tailed t-test. (d) qPCR and Western blot data showing HDAC2 mRNA and protein levels in PFC infected with one of the two different HDAC2 shRNA lentiviruses or a scrambled shRNA lentivirus. shRNA-1: t₁₀=7.28, *** P<0.0001 (HDAC2 mRNA), n=6 each group; t₁₀=10.84, *** P<0.0001 (HDAC2 protein), n=6 each group; shRNA-2: t₁₄=11.33, *** P<0.0001 (HDAC2 mRNA), n=8 each group; t₁₀=12.47, *** P<0.0001 (HDAC2 protein), n=6 each group, two-tailed t-test. (e, f) Plots showing the time spent investigating either the social (Soc) or nonsocial (NS) stimulus (e) and the social preference index (f) during 3-chamber sociability testing of WT or Shank3^+/ΔC mice with PFC injection of one of the two different HDAC2 shRNA lentiviruses or a scrambled shRNA lentivirus (n=8-10 each group). In (e), F_5,92=5.1, P=0.0004; In (f), F_2,46=26.2, P<0.0001; +++ P<0.001 (Soc vs. NS), ** P<0.01, *** P<0.001, two-way ANOVA. (g) Representative heat maps of the 3-chamber sociability tests of WT or Shank3^+/ΔC mice injected with different viruses. All animals used are males (5-8 weeks old). Data are presented as median with interquartile range (a,b,c,d,e) or mean ± SEM (f). Each set of the experiments was replicated for at least 3 times. See Supplementary Fig. 8 for blot source data.

Figure 4. β-catenin is nuclear-translocated to activate HDAC2 transcription in Shank3-deficient mice, and manipulation of β-catenin directly affects HDAC2 transcription and social behaviors

(a) Co-immunoprecipitation data from cortical lysates of WT mice showing the specific binding of β-catenin to Shank3. (b) Immunoblots and quantification analysis of the level of β-catenin in synaptic membrane, cytosol or nuclear fractions of cortical slices from WT and Shank3^+/ΔC (Het) mice. t₁₀=3.35, ** P=0.0074 (synapse), t₁₀=2.78, * P=0.0194 (cytosol), t₁₀=4.01, ** P=0.0025 (nucleus), n=6 each group, two-tailed t-test. (c) Quantification analysis of synaptic and nuclear β-catenin levels in cortical slices from WT and Shank3^e4-9 mice. t₁₀=4.33, ** P=0.0015 (synapse), t₁₀=3.82, ** P=0.0034 (nucleus), n=6 each group, two-tailed t-test. (d) PCR images showing the ChIP (β-catenin-occupied DNA), input (total DNA) and no-template control (NTC) signals with 3 primers (P0, P1, P2) designed against different regions of the HDAC2 gene, including the promoter region containing (P0) or lacking (P1) the TCF/LEF binding motif (labeled with vertical lines) and a down-stream intron (P2). TSS, transcriptional start site. GAPDH was used as a control. (e) ChIP assay data showing the binding of β-catenin at HDAC2 promoter region (containing TCF/LEF binding motif) in PFC lysates from WT and Shank3^+/ΔC mice. t₁₂=2.6, * P=0.02, n=7 each group, two-tailed t-test. (f) Quantitative real-time RT-PCR data on the mRNA level of other β-catenin target genes (Vegf, Jun, Ccnd1, and Neurod1) in PFC from WT and Shank3^+/ΔC mice (n=8 each group). (g) Images showing the β-catenin (GFP-tagged) adenovirus-infected medial PFC region (top) and PFC neurons (bottom). (h) qPCR data on the mRNA level of HDAC2 and other β-catenin target genes in PFC of WT mice with the overexpression of GFP-tagged β-catenin (n=10) or GFP control (n=8). t₁₆=13.44, *** P<0.0001 (HDAC2), two-tailed t-test. (i-k) Representative heat maps (i), plots of social interaction time (j) and social preference (k) in 3-chamber sociability tests of WT mice with the overexpression of β-catenin (n=12) or GFP control (n=10) in PFC. j: F_1,40=20.5, P<0.0001; +++ P<0.001 (Soc vs. NS), ** P<0.01 (GFP vs. β-catenin), two-way ANOVA. k: t₂₀=6.4, *** P<0.0001, two-tailed t-test. (l, m) qPCR and Western blot data showing the mRNA and protein level of β-catenin (l) and HDAC2 (m) in Shank3^+/ΔC mice with the stereotaxic injection of β-catenin shRNA or a scrambled shRNA lentivirus into the PFC. In (I), t₁₀=6.47, *** P<0.0001 (β-catenin mRNA); t₁₀=4.88, *** P=0.0006 (β-catenin protein). In (m), t₁₀=6.20, *** P=0.0001 (HDAC2 mRNA); t₁₀=5.05, *** P=0.0005 (HDAC2 protein), n=6 each group, two-tailed t-test. (n-p) Representative heat maps (n), plots of social interaction time (o) and social preference (p) in 3-chamber sociability tests of Shank3^+/ΔC mice injected with β-catenin shRNA (n=11) or a scrambled shRNA (n=11) lentivirus into the PFC. o: F_1,40=15.0, P=0.0004; +++ P<0.001 (Soc vs. NS), *** P<0.001 (β-catenin shRNA vs. scrambled shRNA), two-way ANOVA. p: t₂₀=4.36, *** P=0.0003, two-tailed t-test. All animals used are males (5-8 weeks old). Data are presented as median with interquartile range (b,c,e,f,h,j,l,m,o) or mean ± SEM (k,p). Each set of the experiments was replicated for at least 3 times. See Supplementary Fig. 8 for blot source data.

Figure 5. Romidepsin treatment increases Grin2a transcription and histone acetylation, and restores NMDAR synaptic function in PFC of Shank3-deficient mice

(a) Quantitative real-time RT-PCR data on the mRNA level of NMDAR and AMPAR subunits and Shank3 in PFC slices from saline-injected WT (n=8), saline-injected Shank3^+/ΔC (n=8), romidepsin (RMD, 0.25 mg/kg, 3×)-treated Shank3^+/ΔC (n=10) and RMD-treated WT (n=6) mice. F_{1,28(treatment)}=5.96, P=0.021 (Grin2a); F_{1,28(treatment)}=0.52, P=0.48 (Shank3); * P<0.05; ns, not significant, two-way ANOVA. (b, c) Quantification analysis and representative immunoblots of the protein level of NMDAR and AMPAR subunits and Shank3 in PFC slices from WT or Shank3^+/ΔC mice injected with saline or romidepsin. F_{1,20(treatment)}=5.58, P=0.030 (Grin2a); F_{1,20(treatment)}=0.62, P=0.44 (Shank3); * P<0.05, two-way ANOVA, n=6 each group. (d) ChIP assay data showing the acetylated histone H3 level at Grin2a and Grin2b promoter regions in PFC lysates from saline-injected WT (n=6), saline-injected Shank3^+/ΔC (n=6), RMD-treated Shank3^+/ΔC (n=6) and RMD-treated WT (n=4) mice. F_{1,18(treatment)}=5.88, P=0.026 (Grin2a); ** P<0.01, two-way ANOVA. (e, f) Input-output curves of NMDAR-EPSC (e) and AMPAR-EPSC (f) in PFC pyramidal neurons from WT vs Shank3^+/ΔC mice treated with romidepsin or saline (n=16 cells/4 mice each group). Recordings were performed at 4-5 days post-injection. In (e), * P<0.05, ** P<0.01, *** P<0.001 (Shank3^+/ΔC+RMD vs. Shank3^+/ΔC+saline), two-way rmANOVA. Inset: representative NMDAR-EPSC and AMPAR-EPSC traces. (g) Box plots showing the NMDAR-EPSC to AMPAR-EPSC ratio in PFC pyramidal neurons from WT vs Shank3^+/ΔC mice treated with romidepsin or saline. Inset: representative EPSC traces. F_1,36=4.8, P=0.036; * P<0.05, two-way ANOVA, n=10 cells/3 mice each group. (h) Input-output curves of NMDAR-EPSC in Shank3^+/ΔC mice treated with fluoxetine (5 mg/kg, i.p., 14×). Inset: representative NMDAR-EPSC traces. * P<0.05, ** P<0.01, *** P<0.001 (Shank3^+/ΔC+fluoxetine vs. WT+saline), two-way rmANOVA, n=12 cells/3 mice each group. (i) Input-output curves of NMDAR-EPSC in PFC pyramidal neurons of WT or Shank3^+/ΔC mice with the PFC injection of a HDAC2 shRNA or a scrambled control shRNA lentivirus. ** P<0.01, *** P<0.001 (Shank3^+/ΔC+HDAC2 shRNA vs. Shank3^+/ΔC+scrambled shRNA), two-way rmANOVA, n=12 cells/3 mice each group. All animals used are males (5-6 weeks old). Data are presented as median with interquartile range (a,b,c,g) or mean ± SEM (e,f,h,i). Each set of the experiments was replicated for at least 3 times. See Supplementary Fig. 9 for blot source data.

Figure 6. Romidepsin treatment elevates the expression of actin regulators and normalizes actin filaments in PFC of Shank3-deficient mice

(a, b) Quantitative RT-PCR data on the mRNA level of actin regulators in the Rac1 signaling pathway (a) and those identified as ASD risk factors (b) in PFC slices from saline-injected WT (n=8), saline-injected Shank3^+/ΔC (n=8), romidepsin (RMD, 0.25 mg/kg, 3×)-treated Shank3^+/ΔC (n=10) and RMD-treated WT (n=6) mice. F_1,28=6.42, P=0.017 (Arhgef7); F_1,28=8.04, P=0.0086 (Limk1); F_{1,28(tretment)}=5.93, P=0.022 (Cttnbp2); F_{1,28(treatment)}=7.71, P=0.01 (Ank2); * P<0.05, two-way ANOVA. (c) Immunoblots and quantification analysis of the protein level of selective actin regulators in PFC slices from WT or Shank3^+/ΔC mice injected with saline or romidepsin. F_1,20=6.87, P=0.016 (βPIX); F_1,20=10.87, P=0.0036 (LIMK1); F_{1,20(treatment)}=4.97, P=0.038 (CTTNBP2); F_{1,20(treatment)}=9.12, P=0.0068 (ANK2); * P<0.05, ** P<0.01, two-way ANOVA, n=6 each group. (d) ChIP assay data showing the acetylated histone H3 level at Arhgef7 and Limk1 promoter regions in PFC lysates from saline-injected WT (n=8), saline-injected Shank3^+/ΔC (n=7), RMD-treated Shank3^+/ΔC (n=9) and RMD-treated WT (n=6) mice. F_{1,26(treatment)}=14.6, P=0.0007 (Arhgef7); F_1,26=10.0, P=0.004 (Limk1); * P<0.05, ** P<0.01, two-way ANOVA. (e) Merged confocal images (40×) of F-actin (phalloidin, red) co-stained with PSD-95 (green) and DAPI (blue) in PFC slices of WT and Shank3^+/ΔC mice treated with romidepsin or saline. Immunohistochemistry was performed at 4-5 days post-injection. (f) Quantification of F-actin (integrated densities) in PFC slices of different animal groups. F_1,104=54.49, P<0.0001; *** P<0.001, two-way ANOVA, n=27 images/3 mice each group. All animals used are males (5-6 weeks old). Data are presented as median with interquartile range (a,b,c,d,f). Each set of the experiments was replicated for at least 3 times. See Supplementary Fig. 9 for blot source data.

Figure 7. Romidepsin treatment induces genome-wide restoration or elevation of genes involved in neural signaling in PFC of Shank3-deficient mice

(a) Heat map representing expression (row z-score) of 187 genes that were downregulated in saline-treated Shank3^+/ΔC mice (SAL, n=3) and normalized in romidepsin (RMD, 0.25 mg/kg, 3×)-treated Shank3^+/ΔC mice (ROM, n=2), compared to wild-type mice (WT, n=3). All animals used are males (5-6 weeks old). (b) Enrichment analysis using gene sets derived from the Biological Process Ontology for the 187 genes that were suppressed in Shank3^+/ΔC mice and restored by romidepsin treatment. AFBM: Actin filament-based movement; AFBP: Actin filament-based process; CSKIT: Cytoskeleton-dependent intracellular transport; ELR: Enzyme-linked receptor protein signaling pathway; TMR: Transmembrane receptor protein signaling pathway; ST: Signal transduction; PSB: Regulation of protein stability; CMOR: Regulation of cell morphogenesis; ASMOR: Anatomical structure morphogenesis; DEVP: Regulation of developmental process. (c) Enrichment analysis for the 41 genes that were unchanged in Shank3^+/ΔC mice but were elevated by romidepsin treatment. C-C Sig: Cell-cell signaling; SYTM: Synaptic transmission; GPCR: G protein-coupled receptor protein signaling pathway; 2^nd Mess: 2^nd messenger signaling; CSR: Cell surface receptor-linked signal transduction; ST: Signal transduction; NTS: Neurotransmitter secretion; RSES: Response to external stimulus. (d) A schematic model showing the potential mechanism underlying the therapeutic effect of romidepsin in Shank3-deficient mice. Normally, Shank3 crosslinks NMDARs to actin cytoskeleton, and binds to the adhesive junction-associated protein β-catenin. Loss of Shank3 leads to the translocation of β-catenin from synapses to nucleus, inducing the upregulation of HDAC2 and chromatin remodeling. The ensuing transcriptional suppression of actin regulators results in the disruption of actin filaments and the synaptic delivery of NMDARs. Treatment with the HDAC inhibitor romidepsin restores or elevates many target genes, including NMDAR subunits, key actin regulators and others involved in neuronal signaling, leading to the normalization of actin cytoskeleton and NMDAR synaptic function. Consequently, the autism-like social deficits are rescued.