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Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects

Nature Neuroscience volume 20, pages 1247–1259 (2017)Cite this article

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Abstract

Antipsychotic drugs remain the standard for schizophrenia treatment. Despite their effectiveness in treating hallucinations and delusions, prolonged exposure to antipsychotic medications leads to cognitive deficits in both schizophrenia patients and animal models. The molecular mechanisms underlying these negative effects on cognition remain to be elucidated. Here we demonstrate that chronic antipsychotic drug exposure increases nuclear translocation of NF-κB in both mouse and human frontal cortex, a trafficking event triggered via 5-HT2A-receptor-dependent downregulation of the NF-κB repressor IκBα. This upregulation of NF-κB activity led to its increased binding at the Hdac2 promoter, thereby augmenting Hdac2 transcription. Deletion of HDAC2 in forebrain pyramidal neurons prevented the negative effects of antipsychotic treatment on synaptic remodeling and cognition. Conversely, virally mediated activation of NF-κB signaling decreased cortical synaptic plasticity via HDAC2. Together, these observations may aid in developing therapeutic strategies to improve the outcome of schizophrenia treatment.

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Figure 1: Chronic atypical antipsychotic drug treatment up-regulates HDAC2 in cortical pyramidal neurons.
Figure 2: Chronic clozapine treatment negatively regulates synaptic remodeling and cognition via HDAC2.
Figure 3: Serotonin 5-HT2A receptor-dependent increased binding of NF-κB to the Hdac2 promoter in mouse and human cortical neurons after chronic atypical antipsychotic drug treatment.
Figure 4: Serotonin 5-HT2A receptor-dependent increased nuclear translocation of NF-κB in mouse and human cortical neurons after chronic atypical antipsychotic drug treatment.
Figure 5: Serotonin 5-HT2A receptor-dependent downregulation of IκBα transcription after chronic clozapine treatment via MAPK–ERK.
Figure 6: Cortical pyramidal NF-κB is necessary for the effects of chronic atypical antipsychotic drug treatment on HDAC2 expression and cognitive deficits.
Figure 7: Virally mediated augmentation of NF-κB function in frontal cortex pyramidal neurons impairs synaptic remodeling and synaptic plasticity via HDAC2.
Figure 8: Virally mediated augmentation of NF-κB function in frontal cortex pyramidal neurons exacerbates schizophrenia-related behaviors via HDAC2.

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Acknowledgements

The authors thank M. Fribourg, P. Roussos (Icahn School of Medicine at Mount Sinai), P. Bos, S. Bowers, A. Ellaithy (Virginia Commonwealth University School of Medicine), A. Gallitano (The University of Arizona), T. Nabeshima (Fujita Health University), M. Hiramatsu (Meijo University) and K. Yamada (Nagoya University) for their critical review of the manuscript; F. Isoda and C. Mobbs for their help in promoter assays; A. Meredith for his help in evaluating immunohistological assays; S. Morgello and the Manhattan HIV Brain Bank for providing control brain cortex; H. Morishita (Icahn School of Medicine at Mount Sinai) for the donation of CaMKIIα-Cre mice; D. Benson (Icahn School of Medicine at Mount Sinai) for the generous gift of Neuro2A cells; K. Deisseroth (Stanford University) for providing the p2A construct; J. Gingrich (Columbia University) for the donation of 5-HT2A knockout mice; E. Olson (University of Texas Southwestern Medical Center), R. Bassel-Duby (University of Texas Southwestern Medical Center) and E. Nestler (Icahn School of Medicine at Mount Sinai) for their gift of loxP-flanked Hdac2 mice; K. Hideshima and A. Hojati for assistance with biochemical assays; and the staff members of the Basque Institute of Legal Medicine for their cooperation in the study. NIH R01 MH084894 (J.G.M.), NIH R01 MH111940 (J.G.M.), Dainippon Sumitomo Pharma (J.G.M.), NARSAD (J.G.M.), the Japan Society for the Promotion of Science (JSPS) 15H06719 and 16K19786 (D.I.), NIH R01 MH104491 (G.W.H.), NIH R01 MH086509 (S.A.), NIH P50 MH096890 (S.A.), MINECO/ERDF SAF2009-08460 (J.J.M. and L.F.C.), SAF2013-45084R (J.J.M. and L.F.C.), Basque Government IT616-13 (J.J.M.), NIH R21 MH103877 (S.D.) and NIH R01 MH090264 (S.J.R.) participated in the funding of this study. RNA-seq analysis was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai and the NIH infrastructure grant S10OD018522. C.M. and A.G.B. were recipients of a postdoctoral and a predoctoral fellowship from the Basque Government, respectively. D.I. was a recipient of postdoctoral fellowships from JSPS (Young Scientists JSPS 23-3454) and the Uehara Memorial Foundation.

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Author notes

  1. Aintzane García-Bea

    Present address: Department of Psychiatry, University of Oxford, Oxford, UK

  2. Mitsumasa Kurita

    Present address: Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan

Authors and Affiliations

  1. Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

    Daisuke Ibi, Mario de la Fuente Revenga, Justin M Saunders, Supriya A Gaitonde, José L Moreno, Maryum K Ijaz, Vishaka Santosh, Juan F López-Giménez, Carlos R Escalante & Javier González-Maeso

  2. Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Daisuke Ibi, José L Moreno, Alexey Kozlenkov, Terrell Holloway, Jeremy Seto, Aintzane García-Bea, Mitsumasa Kurita, Grace E Mosley, Yan Jiang, Stella Dracheva, Schahram Akbarian & Javier González-Maeso

  3. Department of Chemical Pharmacology, Meijo University, Nagoya, Japan

    Daisuke Ibi

  4. Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Nebojsa Kezunovic, Daniel J Christoffel, Scott J Russo, Schahram Akbarian & George W Huntley

  5. Department of Pharmacology, University of the Basque Country UPV/EHU, Leioa, Bizkaia, Spain

    Carolina Muguruza, Aintzane García-Bea, Luis F Callado & J Javier Meana

  6. Centro de Investigación Biomédica en Red de Salud Mental CIBERSAM, Leioa, Bizkaia, Spain

    Carolina Muguruza, Luis F Callado & J Javier Meana

  7. James J. Peters Virginia Medical Center, Bronx, New York, USA

    Alexey Kozlenkov & Stella Dracheva

  8. Department of Biological Sciences, New York City College of Technology, Brooklyn, New York, USA

    Jeremy Seto

  9. BioCruces Health Research Institute, Bizkaia, Barakaldo, Spain

    Luis F Callado & J Javier Meana

  10. Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Scott J Russo, Stella Dracheva, Schahram Akbarian, George W Huntley & Javier González-Maeso

  11. Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC-CSIC), Santander, Cantabria, Spain

    Juan F López-Giménez

  12. Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Yongchao Ge & Javier González-Maeso

  13. Department of Psychiatry, University of Oxford, Oxford, UK

    Mitsumasa Kurita

  14. Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan

    Aintzane García-Bea

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Contributions

D.I., M.d.l.F.R. and J.G.-M. designed experiments, analyzed data and wrote the manuscript. D.I. and M.d.l.F.R. performed experiments. J.G.-M. supervised the research. S.A.G., J.M.S., M.I., T.H., J.L.M., A.G.-B., M.K., G.E.M. and J.F.L.-G. assisted with experiments. N.K., supervised by G.W.H., performed electrophysiological studies. C.M., supervised by L.F.C. and J.J.M., performed assays in postmortem brain samples. L.F.C. and J.J.M. obtained and classified postmortem human brain samples. V.S., supervised by C.R.E., performed fluorescence anisotropy assays. A.K., supervised by S.D., helped with nuclei separation assays. Y.J., supervised by S.A., helped with experiments in postmortem human brain. J.S. and Y.G. performed biostatistical analyzes. D.J.C., supervised by S.J.R., helped with synaptic structure assays. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Javier González-Maeso.

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Integrated supplementary information

Supplementary Figure 1 HDAC2 immunoreactive levels after chronic antipsychotic treatment in mouse frontal cortex

(a,b) HDAC2 immunoreactivity levels in CaMKIIα-positive (a) and PV-positive (b) neurons in mouse frontal cortex after chronic treatment with clozapine, risperidone or haloperidol. Mice were treated chronically (21 days) with clozapine (10 mg/kg), risperidone (4 mg/kg), or haloperidol (1 mg/kg), or vehicle, and sacrificed one day after the last injection. See also Fig. 1d,e for high-magnification images and Fig. 1f,g for quantitative assessment. Nuclei were stained in blue with DAPI. Scale bars, 20 μm (a,b).

Supplementary Figure 2 HDAC2 immunoreactive levels after chronic or subchronic antipsychotic treatment

(a,b) HDAC2 immunoreactivity levels in CaMKIIα-positive neurons in mouse hippocampus after chronic treatment with clozapine, risperidone or haloperidol. Mice were treated chronically (21 days) with clozapine (10 mg/kg), risperidone (4 mg/kg), or haloperidol (1 mg/kg), or vehicle, and sacrificed one day after the last injection. Representative immunohistochemical images (a). Quantitative assessment (n = 30-70 cells from 5-6 mice per experimental condition, b). (c,d) HDAC2 immunoreactivity levels in CaMKIIα-positive neurons in mouse frontal cortex after sub-chronic treatment with clozapine, risperidone or haloperidol. Mice were treated sub-chronically (2 days) with clozapine (10 mg/kg), risperidone (4 mg/kg), or haloperidol (1 mg/kg), or vehicle, and sacrificed one day after the last injection. Representative immunohistochemical images (c). Quantitative assessment (n = 30-40 cells from 5-6 mice per experimental condition, d). Nuclei were stained in blue with DAPI. Scale bars, 20 μm (a,c). Mean ± s.e.m. n.s., not significant. One-way ANOVA with Bonferroni’s post-hoc test (b, P = 0.16, F3,167 = 1.71; d, P = 0.31, F3,126 = 1.19).

Supplementary Figure 3 Hdac2 expression in Hdac2-cKO mice, and enriched biological pathways after chronic clozapine treatment.

(a-c) Evaluation of Hdac2 mRNA expression by qRT-PCR at different locations along the HDAC2 transcript in frontal cortex (n = 6 mice per experimental condition, a), hippocampus (n = 3 mice per experimental condition, b), and cerebellum (n = 3 mice per experimental condition, c) of HDAC2-cKO mice and control littermates. Note that expression of the Hdac2 coding region flanked by loxP sites was decreased in the frontal cortex and hippocampus, but not cerebellum, of HDAC2loxP/loxP:CaMKIIα-Cre mice (HDAC2-cKO) as compared to control littermates. (d) Expression of Hdac1, Hdac3, Hdac4, Hdac5, Hdac6, Hdac7, Hdac8, and Hdac9 mRNAs assayed by qRT-PCR in the frontal cortex of HDAC2-cKOs and control littermates (n = 6 mice per experimental condition). (e) Representation of the top 5 enriched biological pathways from the gene ontology database for the genes that are regulated by chronic clozapine treatment in HDAC2-cKO mice and control littermates (see also Fig. 1n). **P < 0.01. Mean ± s.e.m. Two-tailed unpaired t-test (a, Exon 1: P = 0.64, t10 = 0.47; Exon 2-3: P = 0.0003, t10 = 5.48; Exon 3: P = 0.0005, t10 = 5.00; Exon 4: P = 0.0003, t10 = 5.43; Exon 8-9: P = 0.82, t10 = 0.22; β-actin: P = 0.80, t10 = 0.25; b, Exon 1: P = 0.31, t4 = 1.14; Exon 2-3: P = 0.0025, t4 = 6.75; Exon 3: P = 0.0048, t4 = 5.67; Exon 4: P = 0.008, t4 = 4.90; Exon 8-9: P = 0.06, t4 = 2.56;b-actin: P = 0.76, t10 = 0.31; c, Exon 1: P = 0.71, t4 = 0.38; Exon 2-3: P = 0.73, t4 = 0.35; Exon 3: P = 0.88, t4 = 0.14; Exon 4: P = 0.53, t4 = 0.68; Exon 8-9: P = 0.86, t4 = 0.18; β-actin: P = 0.18, t10 = 1.61; d, Hdac1: P = 0.49, t10 = 0.71; Hdac3: P = 0.27, t10 = 1.15; Hdac4: P = 0.88, t10 = 0.15; Hdac5: P = 0.73, t10 = 0.34; Hdac6: P = 0.60, t10 = 0.52; Hdac7: P = 0.17, t10 = 1.46; Hdac8: P = 0.13, t10 = 1.63; Hdac9: P = 0.06, t10 = 2.04; β-actin: P = 0.43, t10 = 0.81). The α value was corrected for multiple independent null hypothesis by using the Holm’s sequentially Bonferroni method, Student’s t-test (a-c, α = 0.008; d, α = 0.005).

Supplementary Figure 4 Characterization of AAV vector

(a) Schematic representation of the recombinant AAV vector used to over-express eYFP in cortical pyramidal neurons. (b) Representative image of AAV-mediated transgene expression in mouse frontal cortex. AAV-CaMKIIα::eYFP (AAV-eYFP) was injected into the frontal cortex, and eYFP expression was revealed by immunohistochemistry 3 weeks after surgery. (c,d) Viral-mediated over-expression of eYFP in frontal cortex CaMKIIα-positive (c), but not PV-positive (d), neurons. (e,f) Synaptophysin immunoreactive signal on frontal cortex of mice injected with AAV-eYFP or mock (n = 5 mice per experimental condition). Mean ± s.e.m. n.s., not significant. Two-tailed unpaired t-test (f, P = 0.26, t8 = 0.216). Nuclei were stained in blue with DAPI (c-e). Scale bars, 100 μm (b), 20 μm (c), 50 μm (d,e).

Supplementary Figure 5 Setup of the novel-object recognition test, and findings in heterozygous Hdac2loxP/+:CaMKIIα-Cre mice

(a) Experimental setup of the novel object recognition test. (b,c) Timeline of the experimental strategy (see Fig. 2f-i). (d) Effect of chronic (21 days) clozapine (10 mg/kg) treatment on exploratory preference in heterozygous HDAC2loxP/+:CaMKIIα-Cre mice and controls (HDAC2loxP/+). Note that chronic clozapine treatment impaired novel object recognition in control (HDAC2loxP/+) mice (n = 9 mice per experimental condition), an effect that did not occur in heterozygous HDAC2loxP/+:CaMKIIα-Cre littermates (n = 7 mice per experimental condition). Mean ± s.e.m. ***P < 0.001; n.s., not significant. Two-way ANOVA with Bonferroni’s post-hoc test (c, controls: P = 0.034, F1,32 = 4.90; heterozygous: P < 0.0002, F1,24 = 26.66).

Supplementary Figure 6 [3H]Ketanserin binding displacement curves, and fluorescence anisotropy assay

(a) [3H]Ketanserin binding competition curves by atypical antipsychotic (clozapine, paliperidone [active metabolite of risperidone], quetiapine, norquetiapine [active metabolite of quetiapine] and sulpiride), atypical antipsychotic-like (volinanserin) and typical antipsychotic (haloperidol) drugs in HEK293 cells stably expressing human 5-HT2A receptors (n = 2 experiments performed in triplicate). Binding affinities (log Ki values ± s.e.m.) are shown. (b) Schematic representation of the DNA sequences coding for Rel homology regions (RHR) of p65 (residues 19-291) and of p50 (residues 39-350) that were subcloned into the pET-DUET1 expression vector. (c) Fluorescence anisotropy assay shows physical interaction between NF-κB (p65/p50) and the PRDII element from the interferon-β promoter. Fluorescein-labelled DNA fragment of the PRDII element was titrated with different concentrations of NF-κB (p65/p50). DNA fraction bound was plotted as a function of NF-κB (p65/p50) concentration (n = 2 experiments performed in triplicate). Mean ± s.e.m.

Supplementary Figure 7 Effect of chronic antipsychotic treatment on HDAC2 expression in 5HT2A-KO mice, IκBβ and p50 expression in control mice, and IκBα expression in 5HT2A-KO mice

(a,b) HDAC2 immunoreactivity levels in CaMKIIα-positive neurons in mouse frontal cortex of 5HT2A-KO mice after chronic treatment with clozapine or risperidone. Mice were treated chronically (21 days) with clozapine (10 mg/kg) or risperidone (4 mg/kg), or vehicle, and sacrificed one day after the last injection. Representative immunohistochemical images (a). Quantitative assessment (n = 30-40 cells from each of the 4-5 mice per experimental condition, b). (c) Absence of effect of chronic clozapine treatment on protein levels of IκBβ and p50 (p105) in mouse frontal cortex. Mice were chronically (21 days) injected with clozapine (10 mg/kg) and sacrificed one day after the last injection (n = 12 mice per experimental condition). (d) Absence of effect of chronic clozapine treatment on IκBα mRNA in frontal cortex of 5HT2A-KO mice (n = 18-22 mice per experimental condition; see also Fig. 3g). Mean ± s.e.m. n.s., not significant. One-way ANOVA with Bonferroni’s post-hoc test (b, P = 0.89, F2,107 = 0.10). Two-tailed unpaired t-test (c, IκBβ: P = 0.91, t22 = 0.10; p50: P = 0.32, t22 = 1.01; d, P = 0.59, t38 = 0.53).

Supplementary Figure 8 Validation of nuclear extraction protocol.

(a,b) Validation of the nuclear extraction protocol from mouse frontal cortex (a) and post-mortem human frontal cortex (b) tissue samples. Representative immunoblots are shown.

Supplementary Figure 9 Effect of activation of 5-HT2A receptor on IκBα mRNA expression in Flp-In T-REx HEK293 cells

(a,b) Generation of Flp-In T-REx HEK293 cells that express c-Myc-5-HT2A-eCFP or c-Myc-5-HT2A-I181D-eCFP in an inducible manner. These cells were maintained in the presence of 10 ng/ml doxycycline for 24 h, and then imaged to detect eCFP (a). Membranes were prepared from such cells, and density of c-Myc-5-HT2A-eCFP and c-Myc-5-HT2A-I181D-eCFP was measured by radioligand binding assays with [3H]ketanserin (n = 2 independent experiments per group; b). (c) Time-course of the effect of 5-HT (10 μM) on expression changes of IκBα mRNA in Flp-In T-REx HEK293 cells that harbored the human c-Myc-5-HT2A-eCFP receptor (n = 3 independent experiments per group). (d) The effect of 5-HT on IκBα mRNA expression is blocked by the 5-HT2A antagonist M100907. Cells were treated for 60 min with 5-HT (10 μM) or vehicle after being pre-treated for 15 min with M100907 (10 μM) or vehicle (n = 2-3 independent experiments per group). (e) The effect of 5-HT (10 μM) on IκBα mRNA expression is absent in Flp-In T-REx HEK293 cells harboring c-Myc-5-HT2A-I181D-eCFP at the Flp-In locus—a mutant receptor that does not activate Gq/11 proteins19 (n = 3 independent experiments per group). (f) The effect of 5-HT (10 μM) on IκBα mRNA expression is blocked by the MEK1/2 inhibitor SL-327(10 μM) in Flp-In T-REx HEK293 cells that harbored the human c-Myc-5-HT2A-eCFP receptor at the inducible Flp-In locus (n = 3 independent experiments per group). (g) Chronic treatment with clozapine (10 mg/kg), risperidone (4 mg/kg), quetiapine (10 mg/kg), sulpiride (10 mg/kg), or volinanserin (1 mg/kg), but not with haloperidol (1 mg/kg), down-regulates 5-HT2A receptor density in mouse frontal cortex (see also Fig. 5h). Mice were treated chronically (21 days) with the indicated drug, or vehicle, and sacrificed 24 h after the last injection. [3H]Ketanserin binding was tested in frontal cortex plasma membrane preparations (n = 6 mice per experimental condition; see also Fig. 5h). Mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Extra sum-of-squares test (b, P = 0.53, F1,26 = 0.39). One-way ANOVA with Bonferroni’s post-hoc test (c, P < 0.001, F3,8 = 16.20; d, P < 0.01, F3,7 = 10.20; f, P < 0.001, F3,8 = 139.3). Two-tailed unpaired t-test (e, P = 0.52, t4 = 0.70).

Supplementary Figure 10 p65 and HDAC2 do not form a protein complex in mouse frontal cortex.

(a,b) Immunoprecipitation with anti-p65 antibodies results in co-immunoprecipitation of anti-p50, but not anti-HDAC2, in mouse frontal cortex. Nuclear fractions of mouse frontal cortex were immunoprecipitated (IP) with antibody to p65 and blotted with antibodies to HDAC2 and p50 (b). Cytosolic and nuclear fractions of mouse frontal cortex were subject to immunoblotting analysis with antibodies against histone H1 (a).

Supplementary Figure 11 Characterization of AAV-Flag-dn-IκBα-p2A-eYFP viral vector, HDAC2 promoter assay in HEK293 cells, characterization of AAV-Cre viral vector and effect of chronic clozapine treatment on IκBα in the frontal cortex of Hdac2-cKO mice.

(a) Schematic representation of the recombinant AAV vector used to over-express both Flag-tagged dominant negative IκBα (IκBα-S32A-S36A; Flag-dn-IκBα) and eYFP under the CaMKIIα promoter in mouse frontal cortex pyramidal neurons. The arrowhead indicates p2A cleavage site. (b) Representative immunohistochemical images of mouse frontal cortex injected with the AAV-CaMKIIα::Flag-dn-IκBα-p2A-eYFP construct. Note that signals of Flag-dn-IκBα (red) and eYFP (green) do not overlap, indicating high cleavage efficiency of the p2A site. (c) AAV-CaMKIIα::Flag-dn-IκBα-p2A-eYFP was injected into the frontal cortex, and Flag expression was measured by Western blotting three weeks after surgery (Flag-dn-IκBα: ∼ 40 kDa). (d,e) Viral-mediated over-expression of eYFP in frontal cortex CaMKIIα-positive (b), but not PV-positive (e), neurons. (f) Neuro2A cells, which endogenously express CaMKIIα, were transfected with either AAV-CaMKIIα::eYFP (AAV-eYFP) or AAV-CaMKIIα::Flag-dn-IκBα-p2A-eYFP (AAV-Flag-dn-IκBα-p2A-eYFP). Western blot with anti-Flag antibody showed the expected molecular weight of Flag-dn-IκBα in cells transfected with AAV-Flag-dn-IκBα-p2A-eYFP, indicating high cleavage efficacy of the p2A peptide. (g) Neuro2A cells were transfected with the AAV-CaMKIIα::Flag-dn-IκBα-2A-eYFP construct. Red signal (Flag-dn-IκBα) that does not overlap with green signal (eYFP) indicates high cleavage efficacy of the p2A peptide. (h) Luciferase activity of HEK293 cells co-transfected with Flag-p65, or mock, together with either the HDAC2 promoter—luciferase construct or with the version of this construct in which putative NF-κB binding site (-394 to -384) has been deleted (n = 4-5 independent experiments per group). (i) Luciferase activity of HEK293 cells co-transfected with HDAC2 promoter—luciferase construct in combination with mock (pcDNA3.1), 0.05 and 0.1 μg of the Flag-p65 construct. The effect of Flag-p65 on HDAC2 promoter activity was prevented by co-transfection of proteasome degradation-resistant Flag-dn-IκBα (n = 4 independent experiments per group). (j,k) Representative immunohistochemical images of mouse frontal cortex injected with the AAV-CaMKIIα::mCherry-Cre construct. Note that signals of mCherry (red) co-localizes with CaMKIIα-positive (j), but not PV-positive (k), neurons. (l,m) Chronic clozapine treatment down-regulates protein levels of IκBα in the frontal cortex of HDAC2-cKO mice. Animals were treated chronically (21 days) with clozapine (10 mg/kg), or vehicle, and sacrificed one day after the last injection (n = 7 mice per experimental condition). Representative immunoblots are shown (l). Mean ± s.e.m. *P < 0.05; **P < 0.01; n.s., not significant. Two-way ANOVA with Bonferroni’s post-hoc test (h, P < 0.01, F1,13 = 14.06; i, P < 0.01, F1,18 = 8.05). Two-tailed unpaired t-test (l, P = 0.04, t12 = 2.18). Nuclei were stained in blue with DAPI (b,d,e,g,j,k). Scale bars, 20 μm (b,d,e,g,j,k).

Supplementary Figure 12 Characterization of AAV-HA-caIKK-β-p2A-eYFP viral vector, and effect of AAV-HA-caIKK-eYFP on HDAC1

(a) Schematic representation of the recombinant AAV vector used to over-express both HA-tagged constitutively active IκB-kinase (IKK-β-S177E-S181E; HA-caIKK-β) and eYFP under the CaMKIIα promoter in mouse frontal cortex pyramidal neurons. The arrowhead indicates p2A cleavage site. (b) AAV-CaMKIIα::HA-caIKK-β-p2A-eYFP was injected into the frontal cortex, and HA expression was measured by Western blotting at the indicated time after surgery (HA-caIKK-β: ∼ 85 kDa). (c) Representative immunohistochemical images of mouse frontal cortex injected with the AAV-CaMKIIα::HA-caIKK-β-p2A-eYFP construct. Note that signals of HA-caIKK-β (red) and eYFP (green) do not overlap, indicating high cleavage efficiency of the p2A site. (d) Neuro2A cells were transfected with either AAV-CaMKIIα::eYFP (AAV-eYFP) or AAV-CaMKIIα::HA-caIKK-β-p2A-eYFP (AAV-HA-caIKK-β-p2A-eYFP). Western blot with anti-HA antibody showed the expected molecular weight of HA-caIKK-β in cells transfected with AAV-HA-caIKK-β-p2A-eYFP, indicating high cleavage efficacy of the p2A peptide. (e) Neuro2A cells were transfected with the AAV-CaMKIIα::HA-caIKK-β-p2A-eYFP construct. Red signal (HA-caIKK-β) that does not overlap with green signal (eYFP) indicates high cleavage efficacy of the p2A peptide. (f,g) AAV-mediated over-expression of HA-caIKK-β-eYFP does not affect HDAC1 in eYFP-positive frontal cortex neurons. Representative immunohistochemical images (f). Quantitative assessment (HDAC1, n = 13-21 cells from 4 mice per experimental condition, g). Two-way ANOVA with Bonferroni’s post-hoc test (g, P = 0.84, F1,70 = 0.03). Nuclei were stained in blue with DAPI (b,e,f). Scale bars, 10 μm (b), 20 μm (f).

Supplementary Figure 13 Effect of HA-caIKK-β on MK801-induced hyperlocomotor activity in Hdac2-cKO and control mice

(a,b) AAV-mediated over-expression of HA-caIKK-β increases hyperlocomotor activity induced by the dissociative drug MK801 (0.3 mg/kg) in control mice (n = 4-12 mice per experimental condition), but not in HDAC2-cKO littermates (n = 4-11 mice per experimental condition). Time course of MK801-induced locomotion measured in 5-min blocks (time of injection is indicated by arrows; see also Fig. 8b,c).

Supplementary Figure 14 Effect of AAV-HA-caIKK-β-eYFP on aggressive time and social interaction in Hdac2-cKO and control mice

Effect of AAV-eYFP or AAV-HA-caIKK-β-eYFP on aggressive time and escape time during the social interaction paradigm in control mice and HDAC2-cKO littermates (n = 6-8 mice per group). Time-course of social interaction (a,b,d,e). Quantitative assessment (c,f). Mean ± s.e.m. Two-way ANOVA with Bonferroni’s post-hoc test (c, P = 0.07, F1,24 = 3.43; f, P = 0.13, F1,24 = 2.33).

Supplementary Figure 15 Whole blot images of the immunoblots presented in main and supplementary figures.

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Ibi, D., de la Fuente Revenga, M., Kezunovic, N. et al. Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects. Nat Neurosci 20, 1247–1259 (2017). https://doi.org/10.1038/nn.4616

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