Skip to main content
SpringerLink
Log in

Single-Neuron Transcriptome and Methylome Sequencing for Epigenomic Analysis of Aging

  • Protocol
  • First Online:
Book cover Biological Aging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1048))

Abstract

Enormous heterogeneity in transcription and signaling is the feature that slows down progress in our understanding of the mechanisms of normal aging and age-related diseases. This is critical for neurobiology of aging where the enormous diversity of neuronal populations presents a significant challenge in experimental design. Here, we introduce Aplysia as a model for genomic analysis of aging at the single-cell level and provide protocols for integrated transcriptome and methylome profiling of individually identified neurons during the aging process. These single-cell RNA-seq and DNA methylation assays (methyl-capture/methyl enrichment) are compatible with all major next generation sequencing platforms (we used Roche/454 and SOLiD/Life Technologies as illustrative examples) and can be used to integrate an epigenetic signature with transcriptional output. The described sequencing library construction protocol provides both quantitative and directional information from transcriptional profiling of individual cells. Our results also confirm that different copies of DNA in polyploid Aplysia neurons behave similarly with respect to their DNA methylation.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Moroz LL, Kohn AB (2010) Do different neurons age differently? Direct genome-wide analysis of aging in single identified cholinergic neurons. Front Aging Neurosci 2:1–18

    Google Scholar 

  2. Day JJ, Sweatt JD (2011) Epigenetic modifications in neurons are essential for formation and storage of behavioral memory. Neuropsychopharmacology 36:357–358

    Article  PubMed  Google Scholar 

  3. Day JJ, Sweatt JD (2010) DNA methylation and memory formation. Nat Neurosci 13:1319–1323

    Article  PubMed  CAS  Google Scholar 

  4. Penner MR, Roth TL, Barnes CA, Sweatt JD (2010) An epigenetic hypothesis of aging-related cognitive dysfunction. Front Aging Neurosci 2:9

    PubMed  Google Scholar 

  5. Miller CA, Gavin CF, White JA, Parrish RR, Honasoge A, Yancey CR, Rivera IM, Rubio MD, Rumbaugh G, Sweatt JD (2010) Cortical DNA methylation maintains remote memory. Nat Neurosci 13:664–666

    Article  PubMed  CAS  Google Scholar 

  6. Sweatt JD (2010) Neuroscience. Epigenetics and cognitive aging. Science 328:701–702

    Article  PubMed  CAS  Google Scholar 

  7. Feng J, Zhou Y, Campbell SL, Le T, Li E, Sweatt JD, Silva AJ, Fan G (2010) Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat Neurosci 13:423–430

    Article  PubMed  CAS  Google Scholar 

  8. Miller CA, Sweatt JD (2007) Covalent modification of DNA regulates memory formation. Neuron 53:857–869

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  10. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935

    Article  PubMed  CAS  Google Scholar 

  11. Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929–930

    Article  PubMed  CAS  Google Scholar 

  12. Szulwach KE, Li X, Li Y, Song CX, Wu H, Dai Q, Irier H, Upadhyay AK, Gearing M, Levey AI, Vasanthakumar A, Godley LA, Chang Q, Cheng X, He C, Jin P (2011) 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nat Neurosci 14:1607–1616

    Article  PubMed  CAS  Google Scholar 

  13. Wiers CE (2012) Methylation and the human brain: towards a new discipline of imaging epigenetics. Eur Arch Psychiatry Clin Neurosci 262(3):271–273

    Article  PubMed  Google Scholar 

  14. Byrne JH (1987) Cellular analysis of associative learning. Physiol Rev 67:329–339

    PubMed  CAS  Google Scholar 

  15. Carew TJ, Walters ET, Kandel ER (1981) Classical conditioning in a simple withdrawal reflex in Aplysia californica. J Neurosci 1:1426–1437

    PubMed  CAS  Google Scholar 

  16. Carew TJ, Walters ET, Kandel ER (1981) Associative learning in Aplysia: cellular correlates supporting a conditioned fear hypothesis. Science 211:501–504

    Article  PubMed  CAS  Google Scholar 

  17. Hawkins RD, Kandel ER, Bailey CH (2006) Molecular mechanisms of memory storage in Aplysia. Biol Bull 210:174–191

    Article  PubMed  CAS  Google Scholar 

  18. Hawkins RD, Schacher S (1989) Identified facilitator neurons L29 and L28 are excited by cutaneous stimuli used in dishabituation, sensitization, and classical conditioning of Aplysia. J Neurosci 9:4236–4245

    PubMed  CAS  Google Scholar 

  19. Kandel ER (2001) The molecular biology of memory storage: a dialogue between genes and synapses. Science 294:1030–1038

    Article  PubMed  CAS  Google Scholar 

  20. Marinesco S, Kolkman KE, Carew TJ (2004) Serotonergic modulation in Aplysia. I. Distributed serotonergic network persistently activated by sensitizing stimuli. J Neurophysiol 92:2468–2486

    Article  PubMed  CAS  Google Scholar 

  21. Marinesco S, Wickremasinghe N, Carew TJ (2006) Regulation of behavioral and synaptic plasticity by serotonin release within local modulatory fields in the CNS of Aplysia. J Neurosci 26:12682–12693

    Article  PubMed  CAS  Google Scholar 

  22. Kandel ER (1976) Cellular basis of behavior. W.H. Freeman and Company, San Francisco

    Google Scholar 

  23. Kandel ER (1979) Behavioral biology of aplysia. W.H. Freeman and Company, San Francisco

    Google Scholar 

  24. Moroz LL (2011) Aplysia. Curr Biol 21:R60–R61

    Article  PubMed  CAS  Google Scholar 

  25. Iwamoto K, Bundo M, Ueda J, Oldham MC, Ukai W, Hashimoto E, Saito T, Geschwind DH, Kato T (2011) Neurons show distinctive DNA methylation profile and higher interindividual variations compared with non-neurons. Genome Res 21:688–696

    Article  PubMed  CAS  Google Scholar 

  26. Iwamoto K, Ueda J, Bundo M, Kojima T, Kato T (2011) Survey of the effect of genetic variations on gene expression in human prefrontal cortex and its application to genetics of psychiatric disorders. Neurosci Res 70:238–242

    Article  PubMed  CAS  Google Scholar 

  27. Gerdes R, Fieber LA (2006) Life history and aging of captive-reared California sea hares (Aplysia californica). J Am Assoc Lab Anim Sci 45:40–47

    PubMed  CAS  Google Scholar 

  28. Stommes D, Fieber LA, Beno C, Gerdes R, Capo TR (2005) Temperature effects on growth, maturation, and lifespan of the california sea hare (Aplysia californica). Contemp Top Lab Anim Sci 44:31–35

    PubMed  CAS  Google Scholar 

  29. Hirsch HR, Peretz B (1984) Survival and aging of a small laboratory population of a marine mollusc, Aplysia californica. Mech Ageing Dev 27:43–62

    Article  PubMed  CAS  Google Scholar 

  30. Antonov I, Ha T, Antonova I, Moroz LL, Hawkins RD (2007) Role of nitric oxide in classical conditioning of siphon withdrawal in Aplysia. J Neurosci 27:10993–11002

    Article  PubMed  CAS  Google Scholar 

  31. Rajasethupathy P, Fiumara F, Sheridan R, Betel D, Puthanveettil SV, Russo JJ, Sander C, Tuschl T, Kandel E (2009) Characterization of small RNAs in Aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB. Neuron 63:803–817

    Article  PubMed  CAS  Google Scholar 

  32. Bailey CH, Kandel ER (2008) Synaptic remodeling, synaptic growth and the storage of long-term memory in Aplysia. Prog Brain Res 169:179–198

    Article  PubMed  CAS  Google Scholar 

  33. Moroz LL, Kohn AB (2011) Parallel evolution of nitric oxide signaling: diversity of synthesis and memory pathways. Front Biosci 16:2008–2051

    Article  CAS  Google Scholar 

  34. Moroz LL, Edwards JR, Puthanveettil SV, Kohn AB, Ha T, Heyland A, Knudsen B, Sahni A, Yu F, Liu L, Jezzini S, Lovell P, Iannucculli W, Chen M, Nguyen T, Sheng H, Shaw R, Kalachikov S, Panchin YV, Farmerie W, Russo JJ, Ju J, Kandel ER (2006) Neuronal transcriptome of Aplysia: Neuronal compartments and circuitry. Cell 127:1453–1467

    Article  PubMed  CAS  Google Scholar 

  35. Moroz LL, Dahlgren RL, Boudko D, Sweedler JV, Lovell P (2005) Direct single cell determination of nitric oxide synthase related metabolites in identified nitrergic neurons. J Inorg Biochem 99:929–939

    Article  PubMed  CAS  Google Scholar 

  36. Moroz LL, Gillette R, Sweedler JV (1999) Single-cell analyses of nitrergic neurons in simple nervous systems. J Exp Biol 202(Pt 4):333–341

    PubMed  CAS  Google Scholar 

  37. Li L, Garden RW, Sweedler JV (2000) Single-cell MALDI: a new tool for direct peptide profiling. Trends Biotechnol 18:151–160

    Article  PubMed  CAS  Google Scholar 

  38. Li L, Sweedler JV (2008) Peptides in the brain: mass spectrometry based measurement approaches and challenges. Annu Rev Anal Chem 1:451–493

    Article  CAS  Google Scholar 

  39. Fuller RR, Moroz LL, Gillette R, Sweedler JV (1998) Single neuron analysis by capillary electrophoresis with fluorescence spectroscopy. Neuron 20:173–181

    Article  PubMed  CAS  Google Scholar 

  40. Liu L, van Groen T, Kadish I, Tollefsbol TO (2009) DNA methylation impacts on learning and memory in aging. Neurobiol Aging 30:549–560

    Article  PubMed  CAS  Google Scholar 

  41. Lovell P, Moroz LL (2006) The largest growth cones in the animal kingdom and dynamics of neuronal growth in cell culture of Aplysia. Integr Comp Biol 46:847–870

    Article  PubMed  CAS  Google Scholar 

  42. Jezzini SH, Bodnarova M, Moroz LL (2005) Two-color in situ hybridization in the CNS of Aplysia californica. J Neurosci Methods 149:15–25

    Article  PubMed  CAS  Google Scholar 

  43. Walters ET, Moroz LL (2009) Molluscan memory of injury: evolutionary insights into chronic pain and neurological disorders. Brain Behav Evol 74:206–218

    Article  PubMed  Google Scholar 

  44. Tang F, Barbacioru C, Nordman E, Li B, Xu N, Bashkirov VI, Lao K, Surani MA (2010) RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat Protoc 5:516–535

    Article  PubMed  CAS  Google Scholar 

  45. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA (2009) mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods 6(5):377–382

    Article  PubMed  CAS  Google Scholar 

  46. Islam S, Kjallquist U, Moliner A, Zajac P, Fan JB, Lonnerberg P, Linnarsson S (2011) Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res 21:1160–1167

    Article  PubMed  CAS  Google Scholar 

  47. Moroz LL, Kohn AB (2010) Do different neurons age differently? Direct genome-wide analysis of aging in single identified cholinergic neurons. Front Aging Neurosci 2:6. doi:10.3389/neuro.24.006.2010

  48. Evrony GD, Cai X, Lee E, Hills LB, Elhosary PC, Lehmann HS, Parker JJ, Atabay KD, Gilmore EC, Poduri A, Park PJ, Walsh CA (2012) Single-neuron sequencing analysis of l1 retrotransposition and somatic mutation in the human brain. Cell 151:483–496

    Article  PubMed  CAS  Google Scholar 

  49. Kohn AB, Moroz TP, Barnes JP, Netherton M, Moroz LL (2013) Single-cell semiconductor sequencing. Trygve O, Tollefsbol (eds.). Biological Aging: Methods and Protocols, Methods in Mol Biol: Methods and Protocols, vol. 1048, doi:10.1007/978-1-62703-556-9_18, # Springer, New York, This volume

  50. Nielsen C (2012) Animal Evolution: Interrelationships of the living phyla. Oxford University Press, Oxford

    Google Scholar 

  51. Valentine JW (2004) On the origin of phyla. The University of Chicago Press, Chicago

    Google Scholar 

  52. Srivastava M, Begovic E, Chapman J, Putnam NH, Hellsten U, Kawashima T, Kuo A, Mitros T, Salamov A, Carpenter ML, Signorovitch AY, Moreno MA, Kamm K, Grimwood J, Schmutz J, Shapiro H, Grigoriev IV, Buss LW, Schierwater B, Dellaporta SL, Rokhsar DS (2008) The Trichoplax genome and the nature of placozoans. Nature 454:955–960

    Article  PubMed  CAS  Google Scholar 

  53. Srivastava M, Simakov O, Chapman J, Fahey B, Gauthier ME, Mitros T, Richards GS, Conaco C, Dacre M, Hellsten U, Larroux C, Putnam NH, Stanke M, Adamska M, Darling A, Degnan SM, Oakley TH, Plachetzki DC, Zhai Y, Adamski M, Calcino A, Cummins SF, Goodstein DM, Harris C, Jackson DJ, Leys SP, Shu S, Woodcroft BJ, Vervoort M, Kosik KS, Manning G, Degnan BM, Rokhsar DS (2010) The Amphimedon queenslandica genome and the evolution of animal complexity. Nature 466:720–726

    Article  PubMed  CAS  Google Scholar 

  54. Zhang Y, Brown MR, Hyland C, Chen Y, Kronengold J, Fleming MR, Kohn AB, Moroz LL, Kaczmarek LK (2012) Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels. J Neurosci 32:15318–15327

    Article  PubMed  CAS  Google Scholar 

  55. Moroz LL, Citarella M, Kohn AB (2012) Genome-wide analysis of plasticity-induced active DNA demethylation in memory-forming circuits: Insights from the Aplysia genome. Society for Neuroscience Meeting, Abstracts, New Orleans, 294.208/CCC233

    Google Scholar 

  56. Guan Z, Giustetto M, Lomvardas S, Kim JH, Miniaci MC, Schwartz JH, Thanos D, Kandel ER (2002) Integration of long-term-memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure. Cell 111:483–493

    Article  PubMed  CAS  Google Scholar 

  57. Lasek RJ, Dower WJ (1971) Aplysia californica: analysis of nuclear DNA in individual nuclei of giant neurons. Science 172:278–280

    Article  PubMed  CAS  Google Scholar 

  58. Guo JU, Ma DK, Mo H, Ball MP, Jang MH, Bonaguidi MA, Balazer JA, Eaves HL, Xie B, Ford E, Zhang K, Ming GL, Gao Y, Song H (2011) Neuronal activity modifies the DNA methylation landscape in the adult brain. Nat Neurosci 14:1345–1351

    Article  PubMed  CAS  Google Scholar 

  59. Heyland A, Moroz LL (2006) Signaling mechanisms underlying metamorphic transitions in animals. Integr Comp Biol 46:743–759

    Article  PubMed  CAS  Google Scholar 

  60. Heyland A, Vue Z, Voolstra CR, Medina M, Moroz LL (2011) Developmental transcriptome of Aplysia californica. J Exp Zool B Mol Dev Evol 316B:113–134

    Article  PubMed  Google Scholar 

  61. Waddington CH (1956) Principles of Embryology. Allen & Unwin, New York

    Book  Google Scholar 

Download references

Acknowledgements

We thank Mr. James Netherton for reading and commenting on the manuscript. We would like to thank Dr. Clarence Lee for help with the MethylMiner libraries. We also thank Dr. Manfred Lee for his technical advice and guidance with the Ion PGM sequencing process as well as anonymous reviewers for their critical comments and suggestions. We also thank Dr. Thomas Ha, Dr. Sami Jezzini, and Mrs. Yelena Bobkova for help with tissue preparations and RNA/DNA quality assays. This work is supported by NIH grants 1R01GM097502, R21RR025699, 5R21DA030118, R01MH097062, McKnight Brain Research Foundation, as well as NSF-0744649, NSF CNS-0821622, and UF Opportunity Fund awards to LLM.

Author information

Authors and Affiliations

  1. The Whitney Laboratory for Marine Biosciences, University of Florida, Saint Augustine, FL, USA

    Leonid L. Moroz & Andrea B. Kohn

Authors
  1. Leonid L. Moroz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrea B. Kohn
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Dept. Biology, University of Alabama, Birmingham Dept. Biology, Birmingham, Alabama, USA

    Trygve O. Tollefsbol

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Moroz, L.L., Kohn, A.B. (2013). Single-Neuron Transcriptome and Methylome Sequencing for Epigenomic Analysis of Aging. In: Tollefsbol, T. (eds) Biological Aging. Methods in Molecular Biology, vol 1048. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-556-9_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-556-9_21

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-555-2

  • Online ISBN: 978-1-62703-556-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation