Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes

Journal name:
Nature
Volume:
468,
Pages:
263–269
Date published:
DOI:
doi:10.1038/nature09582
Received
Accepted
Published online

Abstract

Mutations in the X-linked MECP2 gene, which encodes the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2), cause Rett syndrome and several neurodevelopmental disorders including cognitive disorders, autism, juvenile-onset schizophrenia and encephalopathy with early lethality. Rett syndrome is characterized by apparently normal early development followed by regression, motor abnormalities, seizures and features of autism, especially stereotyped behaviours. The mechanisms mediating these features are poorly understood. Here we show that mice lacking Mecp2 from GABA (γ-aminobutyric acid)-releasing neurons recapitulate numerous Rett syndrome and autistic features, including repetitive behaviours. Loss of MeCP2 from a subset of forebrain GABAergic neurons also recapitulates many features of Rett syndrome. MeCP2-deficient GABAergic neurons show reduced inhibitory quantal size, consistent with a presynaptic reduction in glutamic acid decarboxylase 1 (Gad1) and glutamic acid decarboxylase 2 (Gad2) levels, and GABA immunoreactivity. These data demonstrate that MeCP2 is critical for normal function of GABA-releasing neurons and that subtle dysfunction of GABAergic neurons contributes to numerous neuropsychiatric phenotypes.

At a glance

Figures

  1. Viaat-Mecp2-/y mice lose MeCP2 in GABA+ neurons and develop stereotypies, self-injury and compulsive behaviour.
    Figure 1: Viaat-Mecp2/y mice lose MeCP2 in GABA+ neurons and develop stereotypies, self-injury and compulsive behaviour.

    a, Wild-type cortex layer 2/3 neurons from 17-week-old mice labelled with 4′,6-diamidino-2-phenylindole (DAPI), MeCP2 and GABA reveal 50% higher MeCP2 levels in GABA+ (circled) than in GABA cells (asterisk). Data normalized to MeCP2 level in GABA cells; n = 3 mice. Scale bars, 10μm. b, Viaat–Cre expression as assessed by Rosa26R–eYFP reporter and colocalization of eYFP and GABA in 14-week-old mice. Scale bars, 100μm. c, More than 90% of GABA+ cells in Viaat-Mecp2/y mice lack MeCP2. Data from n = 3 mice per genotype. d, Seven-week-old Viaat-Mecp2/y mice showing forepaw and hindlimb clasping (arrowhead). e, Viaat-Mecp2/y mice showing fur loss at 15 weeks of age and self-injury, including ocular damage, at 24 weeks (arrowhead). f, g, Viaat-Mecp2/y mice show an approximately 300% increase in grooming time (f) and in the number of holes explored with≥2 sequential nose-pokes (seq. pokes, g). Mecp2flox/y, Flox. WT, wild type; wks, weeks. Error bars are mean ± s.e.m. ***P<0.001.

  2. MeCP2 deficiency in GABAergic neurons causes several Rett syndrome-like features.
    Figure 2: MeCP2 deficiency in GABAergic neurons causes several Rett syndrome-like features.

    af, Viaat-Mecp2/y mice show more footslips (a), a reduced number of side touches on a dowel (b), shorter latency to fall on a rotarod (c) and wire (d), reduced forelimb grip strength (e) and pronounced hypoactivity (f). g, Viaat-Mecp2/y mice show intact social recognition but increased social interaction with novel and familiar partners. h, Viaat-Mecp2/y mice spend 60% more time interacting with an unfamiliar mouse than compared to controls. The wire cup served as a familiar inanimate control without social valence. i, Viaat-Mecp2/y mice show a similar interaction time with a novel inanimate Lego object compared to controls. j, Viaat-Mecp2/y mice are poor nest builders. k, l, Viaat-Mecp2/y mice have an impaired maximum acoustic startle response (ASR) to 120dB (k) and increased prepulse inhibition (PPI) at 78 and 82dB prepulses (l). A.U., arbitrary units. m, n, Viaat-Mecp2/y mice show a similar learning rate during training (m) but reduced crossings over the target platform location during the probe test (n). Error bars are mean ± s.e.m. *P<0.05, **P<0.01 and ***P<0.001.

  3. Loss of MeCP2 in inhibitory GABAergic neurons compromises respiration and survival.
    Figure 3: Loss of MeCP2 in inhibitory GABAergic neurons compromises respiration and survival.

    a, Viaat-Mecp2/y mice show premature lethality with 50% survival by 26 weeks. Dlx5/6-Mecp2/y mice survive for at least 80 weeks. b, Representative plethysmography traces from 32-week-old wild-type, Flox, Dlx5/6–Cre, Viaat–Cre, Dlx5/6-Mecp2/y and Viaat-Mecp2/y mice. Only Viaat-Mecp2/y mice show pronounced apnoeas and an abnormal respiratory pattern. ce, Viaat-Mecp2/y mice show a 42% reduction in tidal volume (c), a 45% reduction in minute volume (d) and an increased number of apnoeas longer than 0.4s (e). fh, Dlx5/6-Mecp2/y mice show no alterations in tidal volume or minute volume, and no apnoeas. Error bars are mean ± s.e.m. *P<0.05, ***P<0.001.

  4. MeCP2 deficiency in GABAergic neurons reduces Gad1, Gad2 and GABA levels.
    Figure 4: MeCP2 deficiency in GABAergic neurons reduces Gad1, Gad2 and GABA levels.

    a, b, Somatic GABA immunoreactivity in GABA+ cells from 15–17-week old Flox and Viaat-Mecp2/y mice labelled with DAPI, MeCP2 and GABA. GABA cells are labelled with asterisks. Images show loss of MeCP2 and reduced GABA immunoreactivity in GABA+ cells (circled) of Viaat-Mecp2/y mice by 37% in cortical layer 2/3 neurons (a) and 50% in striatal neurons (b). Data normalized to GABA level in wild type; n = 2–4 mice per genotype. Scale bars, 10μm. c, d, Gad1 and Gad2 mRNA levels are reduced in Viaat-Mecp2/y cortex by 36% and 28%, respectively (c), and in Viaat-Mecp2/y striatum by 54% and 62%, respectively (d). Colour key is the same as that for a and b. e, Gad1 and Gad2 mRNA levels are unaltered in CamKIIα-Mecp2/y cortex. f, ChIP reveals MeCP2 occupancy of Gad1 and Gad2 promoters in wild type, which is absent in IgG and knockout mice. g, h, Mapping MeCP2 occupancy upstream of the transcription start site, after normalization with IgG, reveals increased occupancy in wild type (black line) across Gad1 (g) and Gad2 (h) promoters without enhanced MeCP2 binding in knockout mice (red line). KO, knockout; TSS, transcription start site. Error bars are mean ± s.e.m. *P<0.05, ***P<0.001.

  5. MeCP2 deficiency in GABAergic neurons results in reduced mIPSC quantal size in cortical layer 2/3 and striatal neurons, EEG hyperexcitability and impaired hippocampal LTP.
    Figure 5: MeCP2 deficiency in GABAergic neurons results in reduced mIPSC quantal size in cortical layer 2/3 and striatal neurons, EEG hyperexcitability and impaired hippocampal LTP.

    ad, Data from three mice per genotype and the number of neurons recorded is shown. a, b, mIPSC amplitude and charge are reduced in Viaat-Mecp2/y cortical slices. Average traces for each genotype are overlaid and graphs show mIPSC amplitude and charge (a), and frequency (b). c, d, mEPSC amplitude and charge are unaltered in Viaat-Mecp2/y cortical slices. Average traces for each genotype are overlaid and graphs show mEPSC amplitude and charge (c), and frequency (d). eh, Data from two independent autaptic striatal cultures and the number of neurons recorded are shown. Average traces for each genotype are overlaid and bar graphs show that knockout neurons have reduced mIPSC amplitude and charge (e), but no differences in frequency (f). g, 5μM GABA evokes similarly sized responses from wild-type and knockout neurons. h, Paired pulse ratio is similar between wild-type and knockout neurons. i, EEG recordings from constitutive null and Viaat-Mecp2/y mice compared to wild type. Constitutive null Mecp2/y mice (n = 4) occasionally develop electrographic seizures, but predominantly show hyperexcitability discharges. Viaat-Mecp2/y mice (n = 7) frequently show hyperexcitability discharges, but do not show electrographic seizures. jl, Acute hippocampal slices from 11–13-week old mice, six mice per genotype, reveal reduced magnitude of theta burst stimulation induced LTP (j) and saturating LTP leads to no further increases in synaptic potentiation (k) in Viaat-Mecp2/y slices. Number of slices recorded is shown in the figure. fEPSP, field excitatory postsynaptic potential; TBS, theta-burst stimulation. l, There is a significant increase in potentiation with the second TBS in controls but not in Viaat-Mecp2/y slices. Number of slices recorded shown in k. Error bars are mean ± s.e.m. **P<0.01, ***P<0.001.

References

  1. Chahrour, M. & Zoghbi, H. Y. The story of Rett syndrome: from clinic to neurobiology. Neuron 56, 422437 (2007)
  2. Lam, C. W. et al. Spectrum of mutations in the MECP2 gene in patients with infantile autism and Rett syndrome. J. Med. Genet. 37, e41 (2000)
  3. Klauck, S. M. et al. A mutation hot spot for nonspecific X-linked mental retardation in the MECP2 gene causes the PPM-X syndrome. Am. J. Hum. Genet. 70, 10341037 (2002)
  4. Cohen, D. et al. MECP2 mutation in a boy with language disorder and schizophrenia. Am. J. Psychiatry 159, 148149 (2002)
  5. Carney, R. M. et al. Identification of MeCP2 mutations in a series of females with autistic disorder. Pediatr. Neurol. 28, 205211 (2003)
  6. Amir, R. E. et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature Genet. 23, 185188 (1999)
  7. Hagberg, B., Aicardi, J., Dias, K. & Ramos, O. A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett’s syndrome: report of 35 cases. Ann. Neurol. 14, 471479 (1983)
  8. Weese-Mayer, D. E. et al. Autonomic nervous system dysregulation: breathing and heart rate perturbation during wakefulness in young girls with Rett syndrome. Pediatr. Res. 60, 443449 (2006)
  9. Weese-Mayer, D. E. et al. Autonomic dysregulation in young girls with Rett syndrome during nighttime in-home recordings. Pediatr. Pulmonol. 43, 10451060 (2008)
  10. Deidrick, K. M., Percy, A. K., Schanen, N. C., Mamounas, L. & Maria, B. L. Rett syndrome: pathogenesis, diagnosis, strategies, therapies, and future research directions. J. Child Neurol. 20, 708717 (2005)
  11. Jedele, K. B. The overlapping spectrum of Rett and Angelman syndromes: a clinical review. Semin. Pediatr. Neurol. 14, 108117 (2007)
  12. Hagberg, B. Clinical manifestations and stages of Rett syndrome. Ment. Retard. Dev. Disabil. Res. Rev. 8, 6165 (2002)
  13. Neul, J. L. et al. Specific mutations in methyl-CpG-binding protein 2 confer different severity in Rett syndrome. Neurology 70, 13131321 (2008)
  14. Guy, J., Hendrich, B., Holmes, M., Martin, J. E. & Bird, A. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nature Genet. 27, 322326 (2001)
  15. Chen, R. Z., Akbarian, S., Tudor, M. & Jaenisch, R. Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice. Nature Genet. 27, 327331 (2001)
  16. Shahbazian, M. et al. Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3. Neuron 35, 243254 (2002)
  17. Gemelli, T. et al. Postnatal loss of methyl-CpG binding protein 2 in the forebrain is sufficient to mediate behavioral aspects of Rett syndrome in mice. Biol. Psychiatry 59, 468476 (2006)
  18. Fyffe, S. L. et al. Deletion of Mecp2 in Sim1-expressing neurons reveals a critical role for MeCP2 in feeding behavior, aggression, and the response to stress. Neuron 59, 947958 (2008)
  19. Samaco, R. C. et al. Loss of MeCP2 in aminergic neurons causes cell-autonomous defects in neurotransmitter synthesis and specific behavioral abnormalities. Proc. Natl Acad. Sci. USA 106, 2196621971 (2009)
  20. Adachi, M., Autry, A. E., Covington, H. E., III & Monteggia, L. M. MeCP2-mediated transcription repression in the basolateral amygdala may underlie heightened anxiety in a mouse model of Rett syndrome. J. Neurosci. 29, 42184227 (2009)
  21. Ballas, N., Lioy, D. T., Grunseich, C. & Mandel, G. Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology. Nature Neurosci. 12, 311317 (2009)
  22. Maezawa, I., Swanberg, S., Harvey, D., LaSalle, J. M. & Jin, L. W. Rett syndrome astrocytes are abnormal and spread MeCP2 deficiency through gap junctions. J. Neurosci. 29, 50515061 (2009)
  23. Gong, S. et al. Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs. J. Neurosci. 27, 98179823 (2007)
  24. Chaudhry, F. A. et al. The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons. J. Neurosci. 18, 97339750 (1998)
  25. Wojcik, S. M. et al. A shared vesicular carrier allows synaptic corelease of GABA and glycine. Neuron 50, 575587 (2006)
  26. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001)
  27. Samaco, R. C. et al. A partial loss of function allele of methyl-CpG-binding protein 2 predicts a human neurodevelopmental syndrome. Hum. Mol. Genet. 17, 17181727 (2008)
  28. Swerdlow, N. R., Geyer, M. A. & Braff, D. L. Neural circuit regulation of prepulse inhibition of startle in the rat: current knowledge and future challenges. Psychopharmacology (Berl.) 156, 194215 (2001)
  29. Monory, K. et al. The endocannabinoid system controls key epileptogenic circuits in the hippocampus. Neuron 51, 455466 (2006)
  30. Kohwi, M. et al. A subpopulation of olfactory bulb GABAergic interneurons is derived from Emx1- and Dlx5/6-expressing progenitors. J. Neurosci. 27, 68786891 (2007)
  31. Martin, D. L. & Rimvall, K. Regulation of γ-aminobutyric acid synthesis in the brain. J. Neurochem. 60, 395407 (1993)
  32. Tsien, J. Z. et al. Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87, 13171326 (1996)
  33. Chao, H. T., Zoghbi, H. Y. & Rosenmund, C. MeCP2 controls excitatory synaptic strength by regulating glutamatergic synapse number. Neuron 56, 5865 (2007)
  34. Dani, V. S. & Nelson, S. B. Intact long-term potentiation but reduced connectivity between neocortical layer 5 pyramidal neurons in a mouse model of Rett Syndrome. J. Neurosci. 29, 1126311270 (2009)
  35. Dani, V. S. et al. Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome. Proc. Natl Acad. Sci. USA 102, 1256012565 (2005)
  36. Medrihan, L. et al. Early defects of GABAergic synapses in the brain stem of a MeCP2 mouse model of Rett syndrome. J. Neurophysiol. 99, 112121 (2008)
  37. Zhang, L., He, J., Jugloff, D. G. & Eubanks, J. H. The MeCP2-null mouse hippocampus displays altered basal inhibitory rhythms and is prone to hyperexcitability. Hippocampus 18, 294309 (2008)
  38. Cui, Y. et al. Neurofibromin regulation of ERK signaling modulates GABA release and learning. Cell 135, 549560 (2008)
  39. Fernandez, F. et al. Pharmacotherapy for cognitive impairment in a mouse model of Down syndrome. Nature Neurosci. 10, 411413 (2007)
  40. Tabuchi, K. et al. A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice. Science 318, 7176 (2007)
  41. Chadman, K. K. et al. Minimal aberrant behavioral phenotypes of neuroligin-3 R451C knockin mice. Autism Res. 1, 147158 (2008)
  42. Skene, P. J. et al. Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state. Mol. Cell 37, 457468 (2010)
  43. Yasui, D. H. et al. Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes. Proc. Natl Acad. Sci. USA 104, 1941619421 (2007)
  44. Chen, W. G. et al. Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science 302, 885889 (2003)
  45. Martinowich, K. et al. DNA methylation-related chromatin remodeling in activity-dependent Bdnf gene regulation. Science 302, 890893 (2003)
  46. Akbarian, S. et al. Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics. Arch. Gen. Psychiatry 52, 258266 (1995)
  47. Fatemi, S. H. et al. Glutamic acid decarboxylase 65 and 67kDa proteins are reduced in autistic parietal and cerebellar cortices. Biol. Psychiatry 52, 805810 (2002)
  48. Addington, A. M. et al. GAD1 (2q31.1), which encodes glutamic acid decarboxylase (GAD67), is associated with childhood-onset schizophrenia and cortical gray matter volume loss. Mol. Psychiatry 10, 581588 (2005)
  49. Lundorf, M. D. et al. Mutational screening and association study of glutamate decarboxylase 1 as a candidate susceptibility gene for bipolar affective disorder and schizophrenia. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 135B, 94101 (2005)
  50. Fatemi, S. H., Stary, J. M., Earle, J. A., Araghi-Niknam, M. & Eagan, E. GABAergic dysfunction in schizophrenia and mood disorders as reflected by decreased levels of glutamic acid decarboxylase 65 and 67kDa and Reelin proteins in cerebellum. Schizophr. Res. 72, 109122 (2005)

Download references

Author information

Affiliations

  1. Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA

    • Hsiao-Tuan Chao,
    • Mingshan Xue,
    • Jeffrey L. Noebels,
    • Christian Rosenmund &
    • Huda Y. Zoghbi
  2. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA

    • Hsiao-Tuan Chao,
    • Hongmei Chen,
    • Rodney C. Samaco,
    • Maria Chahrour,
    • Jeffrey L. Noebels,
    • Christian Rosenmund &
    • Huda Y. Zoghbi
  3. Department of Neurology, Baylor College of Medicine, Houston, Texas 77030, USA

    • Jong Yoo,
    • Jeffrey L. Noebels &
    • Huda Y. Zoghbi
  4. Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA

    • Jeffrey L. Neul,
    • Hui-Chen Lu &
    • Huda Y. Zoghbi
  5. Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas 77030, USA

    • Jeffrey L. Neul,
    • Hui-Chen Lu &
    • Huda Y. Zoghbi
  6. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA

    • Huda Y. Zoghbi
  7. The Rockefeller University and Howard Hughes Medical Institute, New York, New York 10021, USA

    • Shiaoching Gong &
    • Nathaniel Heintz
  8. Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ontario K1N 6N5, Canada

    • Marc Ekker
  9. Department of Psychiatry, University of California, San Francisco, California 94158, USA

    • John L. R. Rubenstein
  10. Present addresses: Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093, USA (M.X.); Neurocure, Neuroscience Research Center, Charite Universitaetsmedizin Berlin, 10117, Germany (C.R.).

    • Mingshan Xue &
    • Christian Rosenmund

Contributions

H.-T.C. and H.Y.Z. conceived the study. H.-T.C., M.X., C.R. and H.Y.Z. designed experiments with input from H.C., R.C.S., J. L. Neul, H.-C.L. and J. L. Noebels. H.-T.C., H.C., R.C.S., M.X., M.C., J.Y. and J. L. Neul performed experiments. H.-T.C., H.C., M.X., J.Y. and J. L. Neul analysed data; H.-T.C., M.X., C.R. and H.Y.Z. interpreted data with input from H.C., R.C.S., J.Y., J. L. Neul, H.-C.L. and J. L. Noebels. S.G. and N.H. provided reagents for generation of Viaat–Cre; J.L.R.R. and M.E. provided Dlx5/6–Cre mice. H.-T.C., M.X. and H.Y.Z. wrote the manuscript and H.C., R.C.S., M.C., J.L. Neul, S.G., J.L.R.R, J. L. Noebels and C.R. provided input.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Information (1.1M)

    This file contains information on additional experimental procedures, Supplementary Figures 1-13 with legends, Supplementary Table 1 and additional references.

  2. Supplementary Table (207K)

    The file contains Supplementary Table

  3. Supplementary Table (168K)

    The file contains Supplementary Table 3

Zip files

  1. Supplementary Movies (54.1M)

    This zipped file contains Supplementary Movies 1-5

Additional data