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

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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References

  1. 1

    van Os, J. & Kapur, S. Schizophrenia. Lancet 374, 635–645 (2009).

    CAS  Google Scholar 

  2. 2

    Crilly, J. The history of clozapine and its emergence in the US market: a review and analysis. Hist. Psychiatry 18, 39–60 (2007).

    PubMed  Google Scholar 

  3. 3

    Meltzer, H.Y. Update on typical and atypical antipsychotic drugs. Annu. Rev. Med. 64, 393–406 (2013).

    CAS  PubMed  Google Scholar 

  4. 4

    Miyamoto, S., Miyake, N., Jarskog, L.F., Fleischhacker, W.W. & Lieberman, J.A. Pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical effects of current and future therapeutic agents. Mol. Psychiatry 17, 1206–1227 (2012).

    CAS  PubMed  Google Scholar 

  5. 5

    Husa, A.P. et al. Lifetime use of antipsychotic medication and its relation to change of verbal learning and memory in midlife schizophrenia - an observational 9-year follow-up study. Schizophr. Res. 158, 134–141 (2014).

    PubMed  Google Scholar 

  6. 6

    Fervaha, G. et al. Antipsychotics and amotivation. Neuropsychopharmacology 40, 1539–1548 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Goldberg, T.E. et al. The effect of clozapine on cognition and psychiatric symptoms in patients with schizophrenia. Br. J. Psychiatry 162, 43–48 (1993).

    CAS  PubMed  Google Scholar 

  8. 8

    Nielsen, R.E. et al. Second-generation antipsychotic effect on cognition in patients with schizophrenia--a meta-analysis of randomized clinical trials. Acta Psychiatr. Scand. 131, 185–196 (2015).

    CAS  PubMed  Google Scholar 

  9. 9

    Schröder, N., de Lima, M.N., Quevedo, J., Dal Pizzol, F. & Roesler, R. Impairing effects of chronic haloperidol and clozapine treatment on recognition memory: possible relation to oxidative stress. Schizophr. Res. 73, 377–378 (2005).

    PubMed  Google Scholar 

  10. 10

    Rosengarten, H. & Quartermain, D. The effect of chronic treatment with typical and atypical antipsychotics on working memory and jaw movements in three- and eighteen-month-old rats. Prog. Neuropsychopharmacol. Biol. Psychiatry 26, 1047–1054 (2002).

    CAS  PubMed  Google Scholar 

  11. 11

    Kellendonk, C., Simpson, E.H. & Kandel, E.R. Modeling cognitive endophenotypes of schizophrenia in mice. Trends Neurosci. 32, 347–358 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Millan, M.J. et al. Cognitive dysfunction in psychiatric disorders: characteristics, causes and the quest for improved therapy. Nat. Rev. Drug Discov. 11, 141–168 (2012).

    CAS  PubMed  Google Scholar 

  13. 13

    Gräff, J. & Tsai, L.H. The potential of HDAC inhibitors as cognitive enhancers. Annu. Rev. Pharmacol. Toxicol. 53, 311–330 (2013).

    PubMed  Google Scholar 

  14. 14

    Gräff, J. et al. Epigenetic priming of memory updating during reconsolidation to attenuate remote fear memories. Cell 156, 261–276 (2014).

    PubMed  PubMed Central  Google Scholar 

  15. 15

    Guan, J.S. et al. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature 459, 55–60 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Kurita, M. et al. HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity. Nat. Neurosci. 15, 1245–1254 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Kurita, M. et al. Repressive epigenetic changes at the mGlu2 promoter in frontal cortex of 5-HT2A knockout mice. Mol. Pharmacol. 83, 1166–1175 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    González-Maeso, J. et al. Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior. Neuron 53, 439–452 (2007).

    PubMed  Google Scholar 

  19. 19

    Moreno, J.L. et al. Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia. Sci. Signal. 9, ra5 (2016).

    PubMed  PubMed Central  Google Scholar 

  20. 20

    González-Maeso, J. et al. Identification of a serotonin/glutamate receptor complex implicated in psychosis. Nature 452, 93–97 (2008).

    PubMed  PubMed Central  Google Scholar 

  21. 21

    Moreno, J.L. et al. Identification of three residues essential for 5-hydroxytryptamine 2A-metabotropic glutamate 2 (5-HT2A·mGlu2) receptor heteromerization and its psychoactive behavioral function. J. Biol. Chem. 287, 44301–44319 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Glantz, L.A. & Lewis, D.A. Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia. Arch. Gen. Psychiatry 57, 65–73 (2000).

    CAS  PubMed  Google Scholar 

  23. 23

    Black, J.E. et al. Pathology of layer V pyramidal neurons in the prefrontal cortex of patients with schizophrenia. Am. J. Psychiatry 161, 742–744 (2004).

    PubMed  Google Scholar 

  24. 24

    Hill, J.J., Hashimoto, T. & Lewis, D.A. Molecular mechanisms contributing to dendritic spine alterations in the prefrontal cortex of subjects with schizophrenia. Mol. Psychiatry 11, 557–566 (2006).

    CAS  PubMed  Google Scholar 

  25. 25

    Lewis, D.A., Curley, A.A., Glausier, J.R. & Volk, D.W. Cortical parvalbumin interneurons and cognitive dysfunction in schizophrenia. Trends Neurosci. 35, 57–67 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Montgomery, R.L. et al. Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis, growth, and contractility. Genes Dev. 21, 1790–1802 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Moreno, J.L. et al. Persistent effects of chronic clozapine on the cellular and behavioral responses to LSD in mice. Psychopharmacology (Berl.) 225, 217–226 (2013).

    CAS  Google Scholar 

  28. 28

    Yadav, P.N., Kroeze, W.K., Farrell, M.S. & Roth, B.L. Antagonist functional selectivity: 5-HT2A serotonin receptor antagonists differentially regulate 5-HT2A receptor protein level in vivo. J. Pharmacol. Exp. Ther. 339, 99–105 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Williams, A.A. et al. Reduced levels of serotonin 2A receptors underlie resistance of Egr3-deficient mice to locomotor suppression by clozapine. Neuropsychopharmacology 37, 2285–2298 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    McOmish, C.E., Lira, A., Hanks, J.B. & Gingrich, J.A. Clozapine-induced locomotor suppression is mediated by 5-HT2A receptors in the forebrain. Neuropsychopharmacology 37, 2747–2755 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    González-Maeso, J. et al. Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex. J. Neurosci. 23, 8836–8843 (2003).

    PubMed  PubMed Central  Google Scholar 

  32. 32

    Sandelin, A., Alkema, W., Engström, P., Wasserman, W.W. & Lenhard, B. JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 32, D91–D94 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Gutierrez, H. & Davies, A.M. Regulation of neural process growth, elaboration and structural plasticity by NF-κB. Trends Neurosci. 34, 316–325 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Russo, S.J., Mazei-Robison, M.S., Ables, J.L. & Nestler, E.J. Neurotrophic factors and structural plasticity in addiction. Neuropharmacology 56 (Suppl 1): 73–82 (2009).

    CAS  Google Scholar 

  35. 35

    Koo, J.W., Russo, S.J., Ferguson, D., Nestler, E.J. & Duman, R.S. Nuclear factor-kappaB is a critical mediator of stress-impaired neurogenesis and depressive behavior. Proc. Natl. Acad. Sci. USA 107, 2669–2674 (2010).

    CAS  PubMed  Google Scholar 

  36. 36

    Thomas, G.M. & Huganir, R.L. MAPK cascade signalling and synaptic plasticity. Nat. Rev. Neurosci. 5, 173–183 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Ashburner, B.P., Westerheide, S.D. & Baldwin, A.S. Jr. The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol. Cell. Biol. 21, 7065–7077 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Ito, K. et al. Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression. J. Exp. Med. 203, 7–13 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Chen, Y. et al. HDAC-mediated deacetylation of NF-κB is critical for Schwann cell myelination. Nat. Neurosci. 14, 437–441 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Zhang, G. et al. Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH. Nature 497, 211–216 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Szymczak-Workman, A.L., Vignali, K.M. & Vignali, D.A. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb. Protoc. 2012, 199–204 (2012).

    PubMed  Google Scholar 

  42. 42

    Huynh, Q.K. et al. Characterization of the recombinant IKK1/IKK2 heterodimer. Mechanisms regulating kinase activity. J. Biol. Chem. 275, 25883–25891 (2000).

    CAS  PubMed  Google Scholar 

  43. 43

    Feldman, D.E. Timing-based LTP and LTD at vertical inputs to layer II/III pyramidal cells in rat barrel cortex. Neuron 27, 45–56 (2000).

    CAS  PubMed  Google Scholar 

  44. 44

    Vollenweider, F.X., Vollenweider-Scherpenhuyzen, M.F., Bäbler, A., Vogel, H. & Hell, D. Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action. Neuroreport 9, 3897–3902 (1998).

    CAS  PubMed  Google Scholar 

  45. 45

    Schmid, Y. et al. Acute effects of lysergic acid diethylamide in healthy subjects. Biol. Psychiatry 78, 544–553 (2015).

    CAS  PubMed  Google Scholar 

  46. 46

    Morris, B.J., Cochran, S.M. & Pratt, J.A. PCP: from pharmacology to modelling schizophrenia. Curr. Opin. Pharmacol. 5, 101–106 (2005).

    CAS  PubMed  Google Scholar 

  47. 47

    Kristiansen, L.V., Huerta, I., Beneyto, M. & Meador-Woodruff, J.H. NMDA receptors and schizophrenia. Curr. Opin. Pharmacol. 7, 48–55 (2007).

    CAS  PubMed  Google Scholar 

  48. 48

    Nichols, D.E. Psychedelics. Pharmacol. Rev. 68, 264–355 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Muguruza, C. et al. Dysregulated 5-HT(2A) receptor binding in postmortem frontal cortex of schizophrenic subjects. Eur. Neuropsychopharmacol. 23, 852–864 (2013).

    CAS  PubMed  Google Scholar 

  50. 50

    Marder, E. & Goaillard, J.M. Variability, compensation and homeostasis in neuron and network function. Nat. Rev. Neurosci. 7, 563–574 (2006).

    CAS  Google Scholar 

  51. 51

    Lopez-Gimenez, J.F., Vilaró, M.T. & Milligan, G. Morphine desensitization, internalization, and down-regulation of the mu opioid receptor is facilitated by serotonin 5-hydroxytryptamine2A receptor coactivation. Mol. Pharmacol. 74, 1278–1291 (2008).

    CAS  PubMed  Google Scholar 

  52. 52

    Escalante, C.R., Shen, L., Thanos, D. & Aggarwal, A.K. Structure of NF-kappaB p50/p65 heterodimer bound to the PRDII DNA element from the interferon-beta promoter. Structure 10, 383–391 (2002).

    CAS  PubMed  Google Scholar 

  53. 53

    Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Tuszynska, I., Magnus, M., Jonak, K., Dawson, W. & Bujnicki, J.M. NPDock: a web server for protein-nucleic acid docking. Nucleic Acids Res. 43, W425-W430 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Morris, M.J., Mahgoub, M., Na, E.S., Pranav, H. & Monteggia, L.M. Loss of histone deacetylase 2 improves working memory and accelerates extinction learning. J. Neurosci. 33, 6401–6411 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Cabungcal, J.H. et al. Perineuronal nets protect fast-spiking interneurons against oxidative stress. Proc. Natl. Acad. Sci. USA 110, 9130–9135 (2013).

    CAS  PubMed  Google Scholar 

  57. 57

    Hof, P.R. et al. Comparative Cytoarchitectonic Atlas of the C57BL/6 and 129/Sv Mouse Brains (Elsevier, Amsterdam, 2000).

  58. 58

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Harrow, J. et al. GENCODE: producing a reference annotation for ENCODE. Genome Biol. 7 (Suppl. 1), 1–9 (2006).

    PubMed  Google Scholar 

  60. 60

    Liao, Y., Smyth, G.K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Gentleman, R.C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

    PubMed  PubMed Central  Google Scholar 

  62. 62

    Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Google Scholar 

  63. 63

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Renthal, W. et al. Histone deacetylase 5 epigenetically controls behavioral adaptations to chronic emotional stimuli. Neuron 56, 517–529 (2007).

    CAS  PubMed  Google Scholar 

  65. 65

    Covington, H.E. III et al. Antidepressant actions of histone deacetylase inhibitors. J. Neurosci. 29, 11451–11460 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    Baumann, M.H., Williams, Z., Zolkowska, D. & Rothman, R.B. Serotonin (5-HT) precursor loading with 5-hydroxy-l-tryptophan (5-HTP) reduces locomotor activation produced by (+)-amphetamine in the rat. Drug Alcohol Depend. 114, 147–152 (2011).

    CAS  PubMed  Google Scholar 

  67. 67

    American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders: DSM-IV 4th edn. (Washington, DC, 1994).

  68. 68

    Stan, A.D. et al. Human postmortem tissue: what quality markers matter? Brain Res. 1123, 1–11 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69

    Preece, P. & Cairns, N.J. Quantifying mRNA in postmortem human brain: influence of gender, age at death, postmortem interval, brain pH, agonal state and inter-lobe mRNA variance. Brain Res. Mol. Brain Res. 118, 60–71 (2003).

    CAS  PubMed  Google Scholar 

  70. 70

    Li, J.Z. et al. Systematic changes in gene expression in postmortem human brains associated with tissue pH and terminal medical conditions. Hum. Mol. Genet. 13, 609–616 (2004).

    CAS  PubMed  Google Scholar 

  71. 71

    Imbeaud, S. et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 33, e56 (2005).

    PubMed  PubMed Central  Google Scholar 

  72. 72

    García-Sevilla, J.A. et al. Reduced platelet G protein-coupled receptor kinase 2 in major depressive disorder: antidepressant treatment-induced upregulation of GRK2 protein discriminates between responder and non-responder patients. Eur. Neuropsychopharmacol. 20, 721–730 (2010).

    PubMed  Google Scholar 

  73. 73

    Kozlenkov, A. et al. Differences in DNA methylation between human neuronal and glial cells are concentrated in enhancers and non-CpG sites. Nucleic Acids Res. 42, 109–127 (2014).

    CAS  PubMed  Google Scholar 

  74. 74

    González-Maeso, J., Rodríguez-Puertas, R., Meana, J.J., García-Sevilla, J.A. & Guimón, J. Neurotransmitter receptor-mediated activation of G-proteins in brains of suicide victims with mood disorders: selective supersensitivity of alpha(2A)-adrenoceptors. Mol. Psychiatry 7, 755–767 (2002).

    PubMed  Google Scholar 

  75. 75

    Aschauer, D.F., Kreuz, S. & Rumpel, S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8, e76310 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 76

    Chan, P. et al. Epsilon-sarcoglycan immunoreactivity and mRNA expression in mouse brain. J. Comp. Neurol. 482, 50–73 (2005).

    CAS  PubMed  Google Scholar 

  77. 77

    Sweet, R.A., Henteleff, R.A., Zhang, W., Sampson, A.R. & Lewis, D.A. Reduced dendritic spine density in auditory cortex of subjects with schizophrenia. Neuropsychopharmacology 34, 374–389 (2009).

    PubMed  Google Scholar 

  78. 78

    Jaaro-Peled, H., Ayhan, Y., Pletnikov, M.V. & Sawa, A. Review of pathological hallmarks of schizophrenia: comparison of genetic models with patients and nongenetic models. Schizophr. Bull. 36, 301–313 (2010).

    PubMed  Google Scholar 

  79. 79

    Hughes, R.N. The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory. Neurosci. Biobehav. Rev. 28, 497–505 (2004).

    CAS  PubMed  Google Scholar 

  80. 80

    González-Maeso, J. et al. Effects of age, postmortem delay and storage time on receptor-mediated activation of G-proteins in human brain. Neuropsychopharmacology 26, 468–478 (2002).

    PubMed  Google Scholar 

  81. 81

    Shaffer, J.P. Multiple hypothesis testing. Annu. Rev. Psychol. 46, 561–584 (1995).

    Google Scholar 

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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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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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The authors declare no competing financial interests.

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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