Article

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

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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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References

  1. 1.

    & Schizophrenia. Lancet 374, 635–645 (2009).

  2. 2.

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

  3. 3.

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

  4. 4.

    , , , & 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).

  5. 5.

    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).

  6. 6.

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

  7. 7.

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

  8. 8.

    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).

  9. 9.

    , , , & Impairing effects of chronic haloperidol and clozapine treatment on recognition memory: possible relation to oxidative stress. Schizophr. Res. 73, 377–378 (2005).

  10. 10.

    & 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).

  11. 11.

    , & Modeling cognitive endophenotypes of schizophrenia in mice. Trends Neurosci. 32, 347–358 (2009).

  12. 12.

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

  13. 13.

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

  14. 14.

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

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

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

  20. 20.

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

  21. 21.

    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).

  22. 22.

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

  23. 23.

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

  24. 24.

    , & Molecular mechanisms contributing to dendritic spine alterations in the prefrontal cortex of subjects with schizophrenia. Mol. Psychiatry 11, 557–566 (2006).

  25. 25.

    , , & Cortical parvalbumin interneurons and cognitive dysfunction in schizophrenia. Trends Neurosci. 35, 57–67 (2012).

  26. 26.

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

  27. 27.

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

  28. 28.

    , , & 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).

  29. 29.

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

  30. 30.

    , , & Clozapine-induced locomotor suppression is mediated by 5-HT2A receptors in the forebrain. Neuropsychopharmacology 37, 2747–2755 (2012).

  31. 31.

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

  32. 32.

    , , , & JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 32, D91–D94 (2004).

  33. 33.

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

  34. 34.

    , , & Neurotrophic factors and structural plasticity in addiction. Neuropharmacology 56 (Suppl 1): 73–82 (2009).

  35. 35.

    , , , & Nuclear factor-kappaB is a critical mediator of stress-impaired neurogenesis and depressive behavior. Proc. Natl. Acad. Sci. USA 107, 2669–2674 (2010).

  36. 36.

    & MAPK cascade signalling and synaptic plasticity. Nat. Rev. Neurosci. 5, 173–183 (2004).

  37. 37.

    , & 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).

  38. 38.

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

  39. 39.

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

  40. 40.

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

  41. 41.

    , & Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb. Protoc. 2012, 199–204 (2012).

  42. 42.

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

  43. 43.

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

  44. 44.

    , , , & Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action. Neuroreport 9, 3897–3902 (1998).

  45. 45.

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

  46. 46.

    , & PCP: from pharmacology to modelling schizophrenia. Curr. Opin. Pharmacol. 5, 101–106 (2005).

  47. 47.

    , , & NMDA receptors and schizophrenia. Curr. Opin. Pharmacol. 7, 48–55 (2007).

  48. 48.

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

  49. 49.

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

  50. 50.

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

  51. 51.

    , & 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).

  52. 52.

    , , & Structure of NF-kappaB p50/p65 heterodimer bound to the PRDII DNA element from the interferon-beta promoter. Structure 10, 383–391 (2002).

  53. 53.

    , , & Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  54. 54.

    , , , & NPDock: a web server for protein-nucleic acid docking. Nucleic Acids Res. 43, W425-W430 (2015).

  55. 55.

    , , , & Loss of histone deacetylase 2 improves working memory and accelerates extinction learning. J. Neurosci. 33, 6401–6411 (2013).

  56. 56.

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

  57. 57.

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

  58. 58.

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

  59. 59.

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

  60. 60.

    , & featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

  61. 61.

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

  62. 62.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  63. 63.

    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).

  64. 64.

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

  65. 65.

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

  66. 66.

    , , & 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).

  67. 67.

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

  68. 68.

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

  69. 69.

    & 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).

  70. 70.

    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).

  71. 71.

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

  72. 72.

    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).

  73. 73.

    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).

  74. 74.

    , , , & 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).

  75. 75.

    , & 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).

  76. 76.

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

  77. 77.

    , , , & Reduced dendritic spine density in auditory cortex of subjects with schizophrenia. Neuropsychopharmacology 34, 374–389 (2009).

  78. 78.

    , , & Review of pathological hallmarks of schizophrenia: comparison of genetic models with patients and nongenetic models. Schizophr. Bull. 36, 301–313 (2010).

  79. 79.

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

  80. 80.

    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).

  81. 81.

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

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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.

Author information

Author notes

    • Aintzane García-Bea
    •  & Mitsumasa Kurita

    Present addresses: Department of Psychiatry, University of Oxford, Oxford, UK (A.G.-B.) and Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan (M.K.)

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, Barakaldo, Bizkaia, 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

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Javier González-Maeso.

Integrated supplementary information

Supplementary figures

  1. 1.

    HDAC2 immunoreactive levels after chronic antipsychotic treatment in mouse frontal cortex

  2. 2.

    HDAC2 immunoreactive levels after chronic or subchronic antipsychotic treatment

  3. 3.

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

  4. 4.

    Characterization of AAV vector

  5. 5.

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

  6. 6.

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

  7. 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

  8. 8.

    Validation of nuclear extraction protocol.

  9. 9.

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

  10. 10.

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

  11. 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.

  12. 12.

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

  13. 13.

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

  14. 14.

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

  15. 15.

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

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Tables 1–6

  2. 2.

    Life Sciences Reporting Summary