Skip to main content
SpringerLink
Log in

Monitoring the ATM-Mediated DNA Damage Response in the Cerebellum Using Organotypic Cultures

  • Protocol
  • First Online:
ATM Kinase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1599))

Abstract

The ATM gene and its protein product, the ATM protein kinase, were identified as a result of attempts to understand the molecular basis of the genetic disorder, ataxia-telangiectasia (A-T). The cardinal symptom of A-T is neurodegeneration expressed primarily as progressive cerebellar atrophy. A major tool in the investigation of ATM functions in the cerebellum is cerebellar organotypic cultures, which allow cerebellar slices to live in culture for several weeks without losing their viability and organization. These cultures are amenable to various treatments and manipulations and provide a close look at Purkinje cells in their almost natural environment. We optimized the protocol for establishing and maintaining these cultures and provide here examples of readouts of the DNA damage response in cerebellar organotypic cultures treated with a DNA-damaging agent.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
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. Perlman SL, Boder Deceased E, Sedgewick RP, Gatti RA (2012) Ataxia-telangiectasia. Handb Clin Neurol 103:307–332. doi:10.1016/B978-0-444-51892-7.00019-X

    Article  PubMed  Google Scholar 

  2. Lavin MF (2008) Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nat Rev Mol Cell Biol 9(10):759–769

    Article  CAS  PubMed  Google Scholar 

  3. Crawford TO (1998) Ataxia telangiectasia. Semin Pediatr Neurol 5(4):287–294

    Article  CAS  PubMed  Google Scholar 

  4. Crawford TO, Mandir AS, Lefton-Greif MA, Goodman SN, Goodman BK, Sengul H, Lederman HM (2000) Quantitative neurologic assessment of ataxia-telangiectasia. Neurology 54(7):1505–1509

    Article  CAS  PubMed  Google Scholar 

  5. Hoche F, Seidel K, Theis M, Vlaho S, Schubert R, Zielen S, Kieslich M (2012) Neurodegeneration in ataxia telangiectasia: what is new? What is evident? Neuropediatrics 43(3):119–129. doi:10.1055/s-0032-1313915

    Article  CAS  PubMed  Google Scholar 

  6. Verhagen MM, Martin JJ, van Deuren M, Ceuterick-de Groote C, Weemaes CM, Kremer BH, Taylor MA, Willemsen MA, Lammens M (2012) Neuropathology in classical and variant ataxia-telangiectasia. Neuropathology 32(3):234–244. doi:10.1111/j.1440-1789.2011.01263.x

    Article  PubMed  Google Scholar 

  7. Sahama I, Sinclair K, Pannek K, Lavin M, Rose S (2014) Radiological imaging in ataxia telangiectasia: a review. Cerebellum 13(4):521–530. doi:10.1007/s12311-014-0557-4

    Article  PubMed  Google Scholar 

  8. Taylor AM, Lam Z, Last JI, Byrd PJ (2014) Ataxia telangiectasia: more variation at clinical and cellular levels. Clin Genet. doi:10.1111/cge.12453

    Google Scholar 

  9. Savitsky K, Bar-Shira A, Gilad S, Rotman G, Ziv Y, Vanagaite L, Tagle DA, Smith S, Uziel T, Sfez S, Ashkenazi M, Pecker I, Frydman M, Harnik R, Patanjali SR, Simmons A, Clines GA, Sartiel A, Gatti RA, Chessa L, Sanal O, Lavin MF, Jaspers NG, Taylor AM, Arlett CF, Miki T, Weissman SM, Lovett M, Collins FS, Shiloh Y (1995) A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 268(5218):1749–1753

    Article  CAS  PubMed  Google Scholar 

  10. Savitsky K, Sfez S, Tagle DA, Ziv Y, Sartiel A, Collins FS, Shiloh Y, Rotman G (1995) The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species. Hum Mol Genet 4(11):2025–2032

    Article  CAS  PubMed  Google Scholar 

  11. Ziv Y, Bar-Shira A, Pecker I, Russell P, Jorgensen TJ, Tsarfati I, Shiloh Y (1997) Recombinant ATM protein complements the cellular A-T phenotype. Oncogene 15(2):159–167. doi:10.1038/sj.onc.1201319

    Article  CAS  PubMed  Google Scholar 

  12. Shiloh Y, Ziv Y (2013) The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14(4):197–210

    Article  CAS  Google Scholar 

  13. McKinnon PJ (2012) ATM and the molecular pathogenesis of ataxia telangiectasia. Annu Rev Pathol 7:303–321. doi:10.1146/annurev-pathol-011811-132509

    Article  CAS  PubMed  Google Scholar 

  14. Bhatti S, Kozlov S, Farooqi AA, Naqi A, Lavin M, Khanna KK (2011) ATM protein kinase: the linchpin of cellular defenses to stress. Cell Mol Life Sci 68(18):2977–3006. doi:10.1007/s00018-011-0683-9

    Article  CAS  PubMed  Google Scholar 

  15. Biton S, Barzilai A, Shiloh Y (2008) The neurological phenotype of ataxia-telangiectasia: solving a persistent puzzle. DNA Repair 7(7):1028–1038. doi:10.1016/j.dnarep.2008.03.006

    Article  CAS  PubMed  Google Scholar 

  16. Yang Y, Hui CW, Li J, Herrup K (2014) The interaction of the atm genotype with inflammation and oxidative stress. PLoS One 9(1):e85863. doi:10.1371/journal.pone.0085863

    Article  PubMed  PubMed Central  Google Scholar 

  17. Ditch S, Paull TT (2012) The ATM protein kinase and cellular redox signaling: beyond the DNA damage response. Trends Biochem Sci 37(1):15–22. doi:10.1016/j.tibs.2011.10.002

    Article  CAS  PubMed  Google Scholar 

  18. Tzur-Gilat A, Ziv Y, Mittelman L, Barzilai A, Shiloh Y (2013) Studying the cerebellar DNA damage response in the tissue culture dish. Mech Ageing Dev 134(10):496–505. doi:10.1016/j.mad.2013.04.001

    Article  CAS  PubMed  Google Scholar 

  19. Dupont JL, Fourcaudot E, Beekenkamp H, Poulain B, Bossu JL (2006) Synaptic organization of the mouse cerebellar cortex in organotypic slice cultures. Cerebellum 5(4):243–256. doi:10.1080/14734220600905317

    Article  CAS  PubMed  Google Scholar 

  20. Padmanabhan J, Brown K, Shelanski ML (2007) Cell cycle inhibition and retinoblastoma protein overexpression prevent Purkinje cell death in organotypic slice cultures. Dev Neurobiol 67(6):818–826. doi:10.1002/dneu.20394

    Article  CAS  PubMed  Google Scholar 

  21. Julien S, Schnichels S, Teng H, Tassew N, Henke-Fahle S, Mueller BK, Monnier PP (2008) Purkinje cell survival in organotypic cultures: implication of Rho and its downstream effector ROCK. J Neurosci Res 86(3):531–536. doi:10.1002/jnr.21511

    Article  CAS  PubMed  Google Scholar 

  22. Kessler M, Kiliman B, Humes C, Arai AC (2008) Spontaneous activity in Purkinje cells: multi-electrode recording from organotypic cerebellar slice cultures. Brain Res 1218:54–69. doi:10.1016/j.brainres.2008.04.063

    Article  CAS  PubMed  Google Scholar 

  23. Lu HX, Levis H, Liu Y, Parker T (2011) Organotypic slices culture model for cerebellar ataxia: potential use to study Purkinje cell induction from neural stem cells. Brain Res Bull 84(2):169–173. doi:10.1016/j.brainresbull.2010.12.001

    Article  PubMed  Google Scholar 

  24. Hurtado de Mendoza T, Balana B, Slesinger PA, Verma IM (2011) Organotypic cerebellar cultures: apoptotic challenges and detection. J Vis Exp 51. doi:10.3791/2564

  25. Kapfhammer JP, Gugger OS (2012) The analysis of purkinje cell dendritic morphology in organotypic slice cultures. J Vis Exp 61. doi:10.3791/3637

  26. Zanjani HS, Lohof AM, McFarland R, Vogel MW, Mariani J (2012) Enhanced survival of wild-type and lurcher purkinje cells in vitro following inhibition of conventional PKCs or stress-activated MAP kinase pathways. Cerebellum. doi:10.1007/s12311-012-0427-x

    Google Scholar 

  27. Lebrun C, Avci HX, Wehrle R, Doulazmi M, Jaudon F, Morel MP, Rivals I, Ema M, Schmidt S, Sotelo C, Vodjdani G, Dusart I (2012) Klf9 is necessary and sufficient for Purkinje cell survival in organotypic culture. Mol Cell Neurosci. doi:10.1016/j.mcn.2012.11.010

    PubMed  Google Scholar 

  28. Lonchamp E, Dupont JL, Beekenkamp H, Poulain B, Bossu JL (2006) The mouse cerebellar cortex in organotypic slice cultures: an in vitro model to analyze the consequences of mutations and pathologies on neuronal survival, development, and function. Crit Rev Neurobiol 18(1–2):179–186

    Article  CAS  PubMed  Google Scholar 

  29. Hill RA, Medved J, Patel KD, Nishiyama A (2014) Organotypic slice cultures to study oligodendrocyte dynamics and myelination. J Vis Exp 90:e51835. doi:10.3791/51835

    Google Scholar 

  30. Campeau JL, Wu G, Bell JR, Rasmussen J, Sim VL (2013) Early increase and late decrease of purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures. PLoS One 8(12):e81776. doi:10.1371/journal.pone.0081776

    Article  PubMed  PubMed Central  Google Scholar 

  31. Dar I, Biton S, Shiloh Y, Barzilai A (2006) Analysis of the ataxia telangiectasia mutated-mediated DNA damage response in murine cerebellar neurons. J Neurosci 26(29):7767–7774. doi:10.1523/JNEUROSCI.2055-06.2006

    Article  CAS  PubMed  Google Scholar 

  32. Dar I, Yosha G, Elfassy R, Galron R, Wang ZQ, Shiloh Y, Barzilai A (2011) Investigation of the functional link between ATM and NBS1 in the DNA damage response in the mouse cerebellum. J Biol Chem 286(17):15361–15376. doi:10.1074/jbc.M110.204172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

Work in our lab is funded by the A-T Children’s Project, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, The A-T Ease Foundation, The Israel Cancer Research Fund, The Israel Science Foundation, and the I-CORE Program of the Planning and Budgeting Committee of the Israel Ministry of Education. Y.S. is a Research Professor of the Israel Cancer Research Fund.

Author information

Authors and Affiliations

  1. The David and Inez Myers Laboratory for Cancer Research, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel

    Efrat Tal & Yosef Shiloh

Authors
  1. Efrat Tal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yosef Shiloh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yosef Shiloh .

Editor information

Editors and Affiliations

  1. University of Queensland Centre for Clinical Research (UQCCR), University of Queensland, Herston, Queensland, Australia

    Sergei V. Kozlov

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Tal, E., Shiloh, Y. (2017). Monitoring the ATM-Mediated DNA Damage Response in the Cerebellum Using Organotypic Cultures. In: Kozlov, S. (eds) ATM Kinase. Methods in Molecular Biology, vol 1599. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6955-5_30

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6955-5_30

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6953-1

  • Online ISBN: 978-1-4939-6955-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation