Cell Type-Specific DNA Methylation Analysis in Neurons and Glia

  • Miki BundoEmail author
  • Tadafumi Kato
  • Kazuya Iwamoto
Part of the Neuromethods book series (NM, volume 105)


The brain is a highly heterogeneous tissue with many types of neuronal and glial cells. Each brain cell type is expected to contain distinctive epigenetic profiles, which contribute to the complex regulation of gene expression. Within the fields of neuroscience, biological psychiatry and neurology, many studies based on postmortem human brains have suggested altered epigenetic status in patients with neuropsychiatric disorders. However, because most of these studies were performed using bulk brain tissue, interpretation of the reported data is quite difficult and often inconsistent across studies. To overcome this problem, several groups have performed epigenetic analyses using isolated neuronal and nonneuronal nuclei. Their work has successfully revealed cell type-specific epigenetic profiles. Here we describe a detailed method for isolation of neuronal and nonneuronal, which involves extracting nuclei from frozen brain tissue, staining them with fluorescence-labeled anti-NeuN antibody, and isolating NeuN+ (neuronal) and NeuN+ (nonneuronal) nuclei by flow cytometry.

Key words

Neuron Nonneuron NeuN Nuclei Sorting 



We thank to Kenji Ohtawa at Research Resources Center at the RIKEN BSI for his help in determining the nuclei sorting conditions for flow cytometry.


  1. 1.
    Kato T, Kurokawa M (1967) Isolation of cell nuclei from the mammalian cerebral cortex and their assortment on a morphological basis. J Cell Biol 32:649–662PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Vakakis N, Hearn MT, Veitch B, Austin L (1991) Rapid isolation of rat brain nuclei on percoll gradients. J Neurochem 57:307–317CrossRefPubMedGoogle Scholar
  3. 3.
    Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201–211PubMedGoogle Scholar
  4. 4.
    Kim KK, Adelstein RS, Kawamoto S (2009) Identification of neuronal nuclei (NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors. J Biol Chem 284:31052–31061PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Rehen SK et al (2005) Constitutional aneuploidy in the normal human brain. J Neurosci 25:2176–2180CrossRefPubMedGoogle Scholar
  6. 6.
    Spalding KL, Bhardwaj RD, Buchholz BA, Druid H, Frisen J (2005) Retrospective birth dating of cells in humans. Cell 122:133–143CrossRefPubMedGoogle Scholar
  7. 7.
    Siegmund KD et al (2007) DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons. PLoS One 2:e895PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Jiang Y, Matevossian A, Huang HS, Straubhaar J, Akbarian S (2008) Isolation of neuronal chromatin from brain tissue. BMC Neurosci 9:42PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Iwamoto K et al (2011) Neurons show distinctive DNA methylation profile and higher interindividual variations compared with non-neurons. Genome Res 21:688–696PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Lister R et al (2013) Global epigenomic reconfiguration during mammalian brain development. Science 341:1237905PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Grindberg RV et al (2013) RNA-sequencing from single nuclei. Proc Natl Acad Sci U S A 110:19802–19807PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    McConnell MJ et al (2013) Mosaic copy number variation in human neurons. Science 342:632–637PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Evrony GD et al (2012) Single-neuron sequencing analysis of L1 retrotransposition and somatic mutation in the human brain. Cell 151:483–496PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Bundo M et al (2014) Increased l1 retrotransposition in the neuronal genome in schizophrenia. Neuron 81:306–313CrossRefPubMedGoogle Scholar
  15. 15.
    Graham JM, Rickwood D (1997) Subcellular fractionation: a practical approach. IRL Press at Oxford University Press, OxfordGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Molecular Psychiatry, Graduate School of MedicineThe University of TokyoBunkyo-kuJapan
  2. 2.Laboratory for Molecular Dynamics of Mental DisordersRIKEN Brain Science InstituteWako-cityJapan

Personalised recommendations