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Selective epigenetic alteration of layer I GABAergic neurons isolated from prefrontal cortex of schizophrenia patients using laser-assisted microdissection

Abstract

Among the most consistent results of studies of post-mortem brain tissue from schizophrenia patients (SZP) is the finding that in this disease, several genes expressed by GABAergic neurons are downregulated. This downregulation may be caused by hypermethylation of the relevant promoters in affected neurons. Indeed, increased numbers of GABAergic interneurons expressing DNA methyltransferase 1 (DNMT1) mRNA have been demonstrated in the prefrontal cortex (PFC) of SZP using in situ hybridization. The present study expands upon these findings using nested competitive reverse transcription-polymerase chain reaction combined with laser-assisted microdissection to quantitate the extent of DNMT1 mRNA overexpression in distinct populations of GABAergic neurons obtained from either layer I or layer V of the PFC of SZP. In a cohort of eight SZP and eight non-psychiatric subject (NPS) post-mortem BA9 tissue samples, DNMT1 mRNA was found to be selectively expressed in GABAergic interneurons and virtually absent in pyramidal neurons. DNMT1 mRNA expression was approximately threefold higher in GABAergic interneurons microdissected from layer I of SZP relative to the same neurons microdissected from NPS. GABAergic interneurons obtained from layer V of the same samples displayed no difference in DNMT1 mRNA expression between groups. In the same samples, the GABAergic neuron-specific glutamic acid-decarboxylase67 (GAD67) and reelin mRNAs were underexpressed twofold in GABAergic interneurons isolated from layer I of SZP relative to GABAergic interneurons microdissected from layer I of NPS, and unaltered in GABAergic interneurons of layer V. These findings implicate an epigenetically mediated layer I GABAergic dysfunction in the pathogenesis of schizophrenia, and suggest novel strategies for treatment of the disease.

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References

  1. Robertson KD . DNA methylation and human disease. Nat Rev Genet 2005; 6: 597–610.

    Article  CAS  Google Scholar 

  2. Klose RJ, Bird AP . Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci 2006; 31: 89–97.

    Article  CAS  Google Scholar 

  3. Goll MG, Bestor TH . Eukaryotic cytosine methyltransferases. Annu Rev Biochem 2005; 74: 481–514.

    Article  CAS  Google Scholar 

  4. Bird A . DNA methylation patterns and epigenetic memory. Genes Dev 2002; 16: 6–21.

    Article  CAS  Google Scholar 

  5. Robertson KD . DNA methylation, methyltransferases, and cancer. Oncogene 2001; 20: 3139–3155.

    Article  CAS  Google Scholar 

  6. Szyf M . DNA methylation and demethylation as targets for anticancer therapy. Biochemistry (Moscow) 2005; 70: 533–549.

    Article  CAS  Google Scholar 

  7. Yen RW, Vertino PM, Nelkin BD, Yu JJ, el-Deiry W, Cumaraswamy A et al. Isolation and characterization of the cDNA encoding human DNA methyltransferase. Nucleic Acids Res 1992; 20: 2287–2291.

    Article  CAS  Google Scholar 

  8. Goto K, Numata M, Komura J, Ono T, Bestor TH, Kondo K . Expression of DNA methyltransferase gene in mature and immature neurons as well as proliferating cells in mice. Differentiation 1994; 56: 39–44.

    Article  CAS  Google Scholar 

  9. Brooks PJ, Marietta C, Goldman D . DNA mismatch repair and DNA methylation in adult brain neurons. J Neurosci 1996; 16: 939–945.

    Article  CAS  Google Scholar 

  10. Veldic M, Caruncho HJ, Liu WS, Davis J, Satta R, Grayson DR et al. DNA-methyltransferase 1 mRNA is selectively overexpressed in telencephalic GABAergic interneurons of schizophrenia brains. Proc Natl Acad Sci USA 2004; 101: 348–353.

    Article  CAS  Google Scholar 

  11. Veldic M, Guidotti A, Maloku E, Davis JM, Costa E . In psychosis, cortical interneurons overexpress DNA-methyltransferase 1. Proc Natl Acad Sci USA 2005; 102: 2152–2157.

    Article  CAS  Google Scholar 

  12. Grayson DR, Jia X, Chen Y, Sharma RP, Mitchell CP, Guidotti A et al. Reelin promoter hypermethylation in schizophrenia. Proc Natl Acad Sci USA 2005; 102: 9341–9346.

    Article  CAS  Google Scholar 

  13. Abdolmaleky HM, Cheng K, Russo A, Smith CL, Faraone SV, Wilcox M et al. Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: a preliminary report. Am J Med Genet B Neuropsychiatr Genet 2005; 134: 60–66.

    Article  Google Scholar 

  14. Impagnatiello F, Guidotti AR, Pesold C, Dwivedi Y, Caruncho H, Pisu MG et al. A decrease of reelin expression as a putative vulnerability factor in schizophrenia. Proc Natl Acad Sci USA 1998; 95: 15718–15723.

    Article  CAS  Google Scholar 

  15. Guidotti A, Auta J, Davis J, DiGiorgi Gerevini V, Dwivedi Y, Grayson DR et al. Decrease in reelin and glutamic acid decarboxylase67 (GAD67) expression in schizophrenia and bipolar disorder: a postmortem brain study. Arch Gen Psychiatry 2000; 57: 1061–1069.

    Article  CAS  Google Scholar 

  16. Fatemi SH, Earle JA, McMenomy T . Reduction in reelin immunoreactivity in hippocampus of subjects with schizophrenia, bipolar disorder and major depression. Mol Psychiatry 2000; 5: 654–663.

    Article  CAS  Google Scholar 

  17. Eastwood SL, Harrison PJ . Cellular basis of reduced cortical reelin expression in schizophrenia. Am J Psychiatry 2006; 163: 540–542.

    Article  Google Scholar 

  18. Grayson DR, Chen Y, Costa E, Dong E, Guidotti A, Kundakovic M et al. The human reelin gene: transcription factors (+), repressors (−) and the methylation switch (+/−) in schizophrenia. Pharmacol Ther 2006; 111: 272–286.

    Article  CAS  Google Scholar 

  19. Tremolizzo L, Doueiri MS, Dong E, Grayson DR, Davis J, Pinna G et al. Valproate corrects the schizophrenia-like epigenetic behavioral modifications induced by methionine in mice. Biol Psychiatry 2005; 57: 500–509.

    Article  CAS  Google Scholar 

  20. Costa E, Davis J, Grayson DR, Guidotti A, Pappas GD, Pesold C . Dendritic spine hypoplasticity and downregulation of reelin and GABAergic tone in schizophrenia vulnerability. Neurobiol Dis 2001; 8: 723–742.

    Article  CAS  Google Scholar 

  21. Torrey EF, Barci BM, Webster MJ, Bartko JJ, Meador-Woodruff JH, Knable MB . Neurochemical markers for schizophrenia, bipolar disorder, and major depression in postmortem brains. Biol Psychiatry 2005; 57: 252–260.

    Article  CAS  Google Scholar 

  22. Guidotti A, Auta J, Davis JM, Dong E, Grayson DR, Veldic M et al. GABAergic dysfunction in schizophrenia: new treatment strategies on the horizon. Psychopharmacology (Berlin) 2005; 180: 191–205.

    Article  CAS  Google Scholar 

  23. Lewis DA, Hashimoto T, Volk DW . Cortical inhibitory neurons and schizophrenia. Nat Rev Neurosci 2005; 6: 312–324.

    Article  CAS  Google Scholar 

  24. Liu WS, Pesold C, Rodriguez MA, Carboni G, Auta J, Lacor P et al. Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse. Proc Natl Acad Sci USA 2001; 98: 3477–3482.

    Article  CAS  Google Scholar 

  25. Niu S, Renfro A, Quattrocchi CC, Sheldon M, D'Arcangelo G . Reelin promotes hippocampal dendrite development through the VLDLR/ApoER2-Dab1 pathway. Neuron 2004; 41: 71–84.

    Article  CAS  Google Scholar 

  26. Weeber EJ, Beffert U, Jones C, Christian JM, Forster E, Sweatt JD et al. Reelin and ApoE receptors cooperate to enhance hippocampal synaptic plasticity and learning. J Biol Chem 2002; 277: 39944–39952.

    Article  CAS  Google Scholar 

  27. Dong E, Caruncho H, Liu WS, Smalheiser NR, Grayson DR, Costa E et al. A reelin–integrin receptor interaction regulates Arc mRNA translation in synaptoneurosomes. Proc Natl Acad Sci USA 2003; 100: 5479–5484.

    Article  CAS  Google Scholar 

  28. Levenson JM, Sweatt JD . Epigenetic mechanisms in memory formation. Nat Rev Neurosci 2005; 6: 108–118.

    Article  CAS  Google Scholar 

  29. Qiu S, Korwek KM, Pratt-Davis AR, Peters M, Bergman MY, Weeber EJ . Cognitive disruption and altered hippocampus synaptic function in reelin haploinsufficient mice. Neurobiol Learn Mem 2006; 85: 228–242.

    Article  CAS  Google Scholar 

  30. Carboni G, Tueting P, Tremolizzo L, Sugaya I, Davis J, Costa E et al. Enhanced dizocilpine efficacy in heterozygous reeler mice relates to GABA turnover downregulation. Neuropharmacology 2004; 46: 1070–1081.

    Article  CAS  Google Scholar 

  31. Larson J, Hoffman JS, Guidotti A, Costa E . Olfactory discrimination learning deficit in heterozygous reeler mice. Brain Res 2003; 971: 40–46.

    Article  CAS  Google Scholar 

  32. Levenson JM, Roth TL, Lubin FD, Miller CA, Huang IC, Desai P et al. Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus. J Biol Chem 2006; 281: 15763–15773.

    Article  CAS  Google Scholar 

  33. Torrey EF, Webster M, Knable M, Johnston N, Yolken RH . The Stanley Foundation Brain Collection and Neuropathology Consortium. Schizophr Res 2000; 44: 151–155.

    Article  CAS  Google Scholar 

  34. Benes FM, Davidson J, Bird ED . Quantitative cytoarchitectural studies of the cerebral cortex of schizophrenics. Arch Gen Psychiatry 1986; 43: 31–35.

    Article  CAS  Google Scholar 

  35. Selemon LD, Rajkowska G, Goldman-Rakic PS . Elevated neuronal density in prefrontal area 46 in brains from shizophrenic patients: application of a three-dimensional, sterologic counting method. J Comp Neurol 1998; 392: 402–412.

    Article  CAS  Google Scholar 

  36. Grayson DR, Ikonomovic S . Competitive RT-PCR to quantitate steady-state mRNA levels. In: Boulton AA, Baker GB, Bateson AN (eds). Neuromethods, vol. 34: In vitro Neurochemical Techniques. Humana Press: Tatowa, NJ, 1999, pp 127–151.

    Google Scholar 

  37. Gabbott PL, Somogyi P . Quantitative distribution of GABA-immunoreactive neurons in the visual cortex (area 17) of the cat. Exp Brain Res 1986; 61: 323–331.

    CAS  PubMed  Google Scholar 

  38. Gabbott PL, Bacon SJ . Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: II. Quantitative areal and laminar distributions. J Comp Neurol 1996; 364: 609–636.

    Article  CAS  Google Scholar 

  39. Beaulieu C . Numerical data on neocortical neurons in adult rat, with special reference to the GABA population. Brain Res 1993; 609: 284–292.

    Article  CAS  Google Scholar 

  40. Rodriguez MA, Caruncho HJ, Costa E, Pesold C, Liu WS, Guidotti A . In Patas monkey, glutamic acid decarboxylase-67 and reelin mRNA coexpression varies in a manner dependent on layers and cortical areas. J Comp Neurol 2002; 451: 279–288.

    Article  CAS  Google Scholar 

  41. MacDonald JL, Gin CS, Roskams AJ . Stage-specific induction of DNA methyltransferases in olfactory receptor neuron development. Dev Biol 2005; 288: 461–473.

    Article  CAS  Google Scholar 

  42. Fan G, Beard C, Chen RZ, Csankovski G, Sun Y, Siniaia M et al. DNA hypomethylation perturbs the function and survival of CNS neurons in postnatal animals. J Neurosci 2001; 21: 788–797.

    Article  CAS  Google Scholar 

  43. Deng J, Szyf M . Downregulation of DNA (cytosine-5-)methyltransferase is a late event in NGF-induced PC12 cell differentiation. Brain Res Mol Brain Res 1999; 71: 23–31.

    Article  CAS  Google Scholar 

  44. Hansen RS, Wijmenga C, Luo P, Stanek AM, Canfield TK, Weemaes CM et al. The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc Natl Acad Sci USA 1999; 96: 14412–14417.

    Article  CAS  Google Scholar 

  45. Wong AH, Gottesman II, Petronis A . Phenotypic differences in genetically identical organisms: the epigenetic perspective. Hum Mol Genet 2005; 14: R11–R18.

    Article  CAS  Google Scholar 

  46. Petronis A . Epigenetics and twins: three variations on the theme. Trends Genet 2006; 22: 347–350.

    Article  CAS  Google Scholar 

  47. Singer-Sam J, Grant M, LeBon JM, Okuyama K, Chapman V, Monk M et al. Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation. Mol Cell Biol 1990; 10: 4987–4989.

    Article  CAS  Google Scholar 

  48. Ramon y Cajal S . Recuerdos de mi vida. English. Garland: New York, 1988.

    Google Scholar 

  49. Tanibuchi I, Goldman-Rakic PS . Dissociation of spatial-, object-, and sound-coding neurons in the mediodorsal nucleus of the primate thalamus. J Neurophysiol 2003; 89: 1067–1077.

    Article  Google Scholar 

  50. Steriade M . Synchronized activities of coupled oscillators in the cerebral cortex and thalamus at different levels of vigilance. Cereb Cortex 1997; 7: 583–604.

    Article  CAS  Google Scholar 

  51. Tamminga C, Hashimoto T, Volk DW, Lewis DA . GABA neurons in the human prefrontal cortex. Am J Psychiatry 2004; 161: 1764.

    Article  Google Scholar 

  52. Mountcastle VB . Perceptual Neuroscience: The Cerebral Cortex. Harvard University Press: Cambridge, MA, 1998.

    Google Scholar 

  53. Rotaru DC, Barrionuevo G, Sesack SR . Mediodorsal thalamic afferents to layer III of the rat prefrontal cortex: synaptic relationships to subclasses of interneurons. J Comp Neurol 2005; 490: 220–238.

    Article  Google Scholar 

  54. Hendry SH, Jones EG . GABA neuronal subpopulations in cat primary auditory cortex: co-localization with calcium binding proteins. Brain Res 1991; 543: 45–55.

    Article  CAS  Google Scholar 

  55. Gabbott PL, Jays PR, Bacon SJ . Calretinin neurons in human medial prefrontal cortex (areas 24a,b,c, 32′, and 25). J Comp Neurol 1997; 381: 389–410.

    Article  CAS  Google Scholar 

  56. Kawaguchi Y, Kondo S . Parvalbumin, somatostatin and cholecystokinin as chemical markers for specific GABAergic interneuron types in the rat frontal cortex. J Neurocytol 2002; 31: 277–287.

    Article  Google Scholar 

  57. Hamshere ML, Bennett P, Williams N, Segurado R, Cardno A, Norton N et al. Genomewide linkage scan in schizoaffective disorder: significant evidence for linkage at 1q42 close to DISC1, and suggestive evidence at 22q11 and 19p13. Arch Gen Psychiatry 2005; 62: 1081–1088.

    Article  CAS  Google Scholar 

  58. Benes FM . Emerging principles of altered neural circuitry in schizophrenia. Brain Res Brain Res Rev 2000; 31: 251–269.

    Article  CAS  Google Scholar 

  59. Berretta S, Munno DW, Benes FM . Amygdalar activation alters the hippocampal GABA system: ‘partial’ modelling for postmortem changes in schizophrenia. J Comp Neurol 2001; 431: 129–138.

    Article  CAS  Google Scholar 

  60. Nakao K, Matsuyama K, Matsuki N, Ikegaya Y . Amygdala stimulation modulates hippocampal synaptic plasticity. Proc Natl Acad Sci USA 2004; 101: 14270–14275.

    Article  CAS  Google Scholar 

  61. Gisabella B, Bolshakov VY, Benes FM . Regulation of synaptic plasticity in a schizophrenia model. Proc Natl Acad Sci USA 2005; 102: 13301–13306.

    Article  CAS  Google Scholar 

  62. Tremolizzo L, Carboni G, Ruzicka WB, Mitchell CP, Sugaya I, Tueting P et al. An epigenetic mouse model for molecular and behavioral neuropathologies related to schizophrenia vulnerability. Proc Natl Acad Sci USA 2002; 99: 17095–17100.

    Article  CAS  Google Scholar 

  63. Dong E, Agis-Balboa RC, Simonini MV, Grayson DR, Costa E, Guidotti A . Reelin and glutamic acid decarboxylase67 promoter remodeling in an epigenetic methionine-induced mouse model of schizophrenia. Proc Natl Acad Sci USA 2005; 102: 12578–12583.

    Article  CAS  Google Scholar 

  64. Chen Y, Sharma RP, Costa RH, Costa E, Grayson DR . On the epigenetic regulation of the human reelin promoter. Nucleic Acids Res 2002; 30: 2930–2939.

    Article  CAS  Google Scholar 

  65. Mitchell CP, Chen Y, Kundakovic M, Costa E, Grayson DR . Histone deacetylase inhibitors decrease reelin promoter methylation in vitro. J Neurochem 2005; 93: 483–492.

    Article  CAS  Google Scholar 

  66. Noh JS, Sharma RP, Veldic M, Salvacion AA, Jia X, Chen Y et al. DNA methyltransferase 1 regulates reelin mRNA expression in mouse primary cortical cultures. Proc Natl Acad Sci USA 2005; 102: 1749–1754.

    Article  CAS  Google Scholar 

  67. Miles R, Toth K, Gulyas AI, Hajos N, Freund TF . Differences between somatic and dendritic inhibition in the hippocampus. Neuron 1996; 16: 815–823.

    Article  CAS  Google Scholar 

  68. Wassef AA, Hafiz NG, Hampton D, Molloy M . Divalproex sodium augmentation of haloperidol in hospitalized patients with schizophrenia: clinical and economic implications. J Clin Psychopharmacol 2001; 21: 21–26.

    Article  CAS  Google Scholar 

  69. Casey DE, Daniel DG, Wassef AA, Tracy KA, Wozniak P, Sommerville KW . Effect of divalproex combined with olanzapine or risperidone in patients with an acute exacerbation of schizophrenia. Neuropsychopharmacology 2003; 28: 182–192.

    Article  CAS  Google Scholar 

  70. Simonini MV, Camargo LM, Dong E, Maloku E, Veldic M, Costa E et al. The benzamide MS-275 is a potent, long-lasting brain region-selective inhibitor of histone deacetylases. Proc Natl Acad Sci USA 2006; 103: 1587–1592.

    Article  CAS  Google Scholar 

  71. Costa E, Auta J, Grayson DR, Matsumoto K, Pappas GD, Zhang X et al. GABAA receptors and benzodiazepines: a role for dendritic resident subunit mRNAs. Neuropharmacology 2002; 43: 925–937.

    Article  CAS  Google Scholar 

  72. Wolkowitz OM, Pickar D . Benzodiazepines in the treatment of schizophrenia: a review and reappraisal. Am J Psychiatry 1991; 148: 714–726.

    Article  CAS  Google Scholar 

  73. Carpenter WTJ, Buchanan RW, Kirkpatrick B, Breier AF . Diazepam treatment of early signs of exacerbation in schizophrenia. Am J Psychiatry 1999; 156: 299–303.

    PubMed  Google Scholar 

  74. Akbarian S, Huntsman MM, Kim JJ, Tafazzoli A, Potkin SG, Bunney Jr WE et al. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls. Cereb Cortex 1995; 5: 550–560.

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by National Institutes of Health Grant R01 MH62682 (to DG) and National Institute of Mental Health Grants MH 071667 (to EC) and MH 070855 (to AG).

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Ruzicka, W., Zhubi, A., Veldic, M. et al. Selective epigenetic alteration of layer I GABAergic neurons isolated from prefrontal cortex of schizophrenia patients using laser-assisted microdissection. Mol Psychiatry 12, 385–397 (2007). https://doi.org/10.1038/sj.mp.4001954

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