SUPPLEMENTARY INFORMATION Articles https://doi.org/10.1038/s41593-018-0110-8 In the format provided by the authors and unedited. Social deficits in Shank3-deficient mouse models of autism are rescued by histone deacetylase (HDAC) inhibition Luye Qin 1, Kaijie Ma 1, Zi-Jun Wang 1, Zihua Hu 2, Emmanuel Matas 1, Jing Wei 1 and Zhen Yan 1 * 1 Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY, USA. 2 Center for Computational Research, New York State Center of Excellence in Bioinformatics & Life Sciences, State University of New York at Buffalo, Buffalo, NY, USA. These authors contributed equally: Luye Qin and Kaijie Ma. *e-mail: zhenyan@buffalo.edu Nature Neuroscience www.nature.com/natureneuroscience 2018 Nature America Inc., part of Springer Nature. All rights reserved.
Supplementary Figure 1 Romidepsin treatment leads to the sustained increase of social interaction time and social preference in young Shank3- deficient mice. (a) Box plots showing the time spent investigating either the social (Soc) or nonsocial (NS) stimulus during sociability testing in young (5-6 weeks old) male Shank3 +/ΔC mice with a brief treatment of romidepsin (RMD, 0.25 mg/kg, i.p., 3x, n=10) or saline (n=10) prior to and at different days after the injection. F 3,36(treatment) =137.4, P<0.0001; +++ P<0.001, (Soc vs. NS), ** P<0.01, *** P<0.001 (saline vs. romidepsin), two-way rmanova. (b) Representative heat maps illustrating the time spent in different locations of the 3 chambers from the social preference tests in Shank3 +/ΔC mice treated with RMD or saline. Locations of Soc and NS stimuli are labeled with the circles. (c) Bar graphs (mean ± SEM) and scatter plots showing the preference index of the sociability testing in adult (10-11 weeks old) Shank3 +/ΔC (n=13) or WT (n=10) mice before and after romidepsin (0.25 mg/kg, i.p., 3x) treatment. F 3,45 =10.5, P<0.0001; * P<0.05,*** P<0.001, one-way ANOVA. (d) Bar graphs (mean ± SEM) and scatter plots showing the social preference index in Shank3 +/ΔC mice (n=9, ~10 weeks old) before and after the second-round romidepsin treatment. F 2,24 =2.6, ^ P=0.09, one-way ANOVA.
Supplementary Figure 2 Current antipsychotics fail to increase social interaction time, while the pan-hdac inhibitor trichostatin A (TSA) transiently improves social behaviors in Shank3-deficient mice. (a-e) Box plots showing the time spent investigating either the social (Soc) or nonsocial (NS) stimulus during sociability testing in young (5-6 weeks old) male Shank3 +/ΔC mice treated with fluoxetine (10 mg/kg, i.p., 14x, a, n=9), clozapine (5 mg/kg, i.p., 3x, b, n=11), valproic acid (VPA, 100 mg/kg, 3x, c, n=11), aripiprazole (1 mg/kg, 3x, d, n=9) or risperidone (0.1 mg/kg, 3x, e, n=10). In (c), F 2,40(treatment) =0.71, P=0.5; +++ P<0.001 (Soc vs. NS), two-way rmanova. (f, g) Plots showing the preference index (f) and the time spent investigating either the Soc or NS stimulus (g) during sociability testing in Shank3 +/ΔC mice before and after TSA treatment (0.5 mg/kg, i.p., 3x, n=8). In (f), F 2,21 =19.7, P<0.0001, one-way ANOVA. In (g), F 2,28(treatment) =4.15, P=0.026; ++ P<0.01, +++ P<0.001 (Soc vs. NS), ### P<0.001 (pre- vs. post-injection), two-way rmanova. Inset (d,e,g): Representative heat maps illustrating the time spent in different locations of the 3 chambers (blue: 0 sec; red: ~20 sec) from the social preference tests of drug-treated Shank3 +/ΔC mice.
Supplementary Figure 3 Histone acetylation sites are identified on Grin2a and Grin2b promoters. (a) PCR images showing the ChIP (AcetyH3-occupied DNA), input (total DNA) and no-template control (NTC) signals with 3 primers (P1, P2, P3) designed against the promoter regions of Grin2a and Grin2b. The expected sizes of PCR products are labeled on the gels. The final primers used in the quantitative ChIP experiments are labeled by red circles. Top: diagram showing the primer locations on the 5' upstream sequence of Grin2a and Grin2b. TSS, transcriptional start site. (b) The amplification curve and melt curve for AcetyH3- ChIP, input, and IgG control samples.
Supplementary Figure 4 Romidepsin treatment restores NMDAR synaptic function and global histone acetylation at 16 18 d, but not 30 32 d, postinjection. (a,b) Input-output curves of NMDAR-EPSC in PFC pyramidal neurons from Shank3 +/ΔC mice (Het, male) receiving treatment of romidepsin (RMD, i.p., 0.25 mg/kg, 3x) or saline. Recordings were performed at 16-18 days (a) or 30-32 days (b) post-injection. n=12 cells/3 mice each group. In (a), F 1,22(treatment) =13.56, P=0.0013; * P<0.05, *** P<0.001, two-way rmanova. Data are mean ± SEM. Inset: representative NMDAR-EPSC traces. (c,d) Immunoblots and quantification analysis of the level of acetylated H3 and total H3 in the nuclear fraction of cortical slices from WT or Shank3 +/ΔC mice (male) treated with saline or romidepsin at 16-18 days or 30-32 days post-injection. n=6 each group. In (d), F 2,15 =12.55, P=0.0006 (16-18 days); F 2,15 =27.72, P<0.0001 (30-32 days); ** P<0.01, *** P<0.001, ns, not significant, one-way ANOVA.
Supplementary Figure 5 Histone acetylation sites are identified on Arhgef7 and Limk1 promoters. (a,b) PCR images showing the ChIP (AcetyH3-occupied DNA), input (total DNA) and no-template control (NTC) signals with 3 primers (P1, P2, P3) designed against the promoter regions of Arhgef7 and Limk1. The expected sizes of PCR products are labeled on the gels. Top Inset: diagram showing the primer locations on the 5' upstream sequence of Arhgef7 and Limk1. TSS, transcriptional start site. Right Inset: The amplification curve and melt curve for AcetyH3-ChIP, input, and NTC samples. The final primers used in the quantitative ChIP experiments are labeled by red circles.
Supplementary Figure 6 Romidepsin treatment restores actin filaments in PFC of Shank3-deficient mice. (a) High magnification confocal images (40x) of F-actin staining with phalloidin (co-stained with PSD-95 and DAPI) in PFC slices of WT vs. Shank3 +/ΔC mice (5-6 weeks old, male) with i.p. injections of saline or romidepsin (0.25 mg/kg, 3x). (b) Quantification of PSD-95 levels (integrated densities) in PFC slices of different animal groups. n=27 images/3 mice each group.
Supplementary Figure 7 Romidepsin treatment has no effect on the expression of genes encoding NMDAR subunits or actin regulators in other brain regions and peripheral organs of Shank3-deficient mice. (a,b) Quantitative real-time RT-PCR data on the mrna level of Grin1, Grin2a, Grin2b, Arhgef7 and Limk1 in striatum, VTA, kidney and heart from WT or Shank3 +/ΔC mice (5-6 weeks old, male) injected with saline or romidepsin (0.25 mg/kg, i.p., 3x). n=6 each group.
Supplementary Figure 8 Full blots for Figures 1, 3 and 4. -
Supplementary Figure 9 Full blots for Figures 5 and 6 and Supplementary Figure 4. -