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IKK Kinase Assay for Assessment of Canonical NF-κB Activation in Neurons

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NF-kappa B

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1280))

Abstract

Nuclear factor kappa B (NF-κB) is a potent transcription factor highly expressed in the central nervous system (CNS) where it has been shown to be required for multiple behavioral paradigms of learning and memory in both mammalian and invertebrate systems. NF-κB dimers are found in neuronal cell bodies, are also present at synapses, and can participate in the activity-dependent regulation of gene expression in response to excitatory neurotransmission. Multiple serine-directed phosphorylation events are critical in the canonical NF-κB activation pathway, including activation of the IκB kinase complex (IKK) and phosphorylation and degradation of the inhibitor of NF-κB (IκB). In this chapter, we describe methods for immunoprecipitation (IP) of the IKK complex from dissociated cultured murine hippocampal neurons, followed by in vitro kinase assay to evaluate excitatory neurotransmission-induced IKK activation by monitoring phosphorylation of a GST-IκBα substrate. These methods can also be successfully implemented in subcellular-reduced brain preparations, such as biochemically isolated synapses.

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References

  1. Bonizzi G, Karin M (2004) The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 25:280–288

    Article  CAS  PubMed  Google Scholar 

  2. Li Q, Verma IM (2002) NF-kappaB regulation in the immune system. Nat Rev Immunol 2:725–734

    Article  CAS  PubMed  Google Scholar 

  3. Hayden MS, Ghosh S (2011) NF-kappaB in immunobiology. Cell Res 21:223–244

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Sen R, Baltimore D (1986) Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46:705–716

    Article  CAS  PubMed  Google Scholar 

  5. Verhelst K, Carpentier I, Beyaert R (2011) Regulation of TNF-induced NF-kappaB activation by different cytoplasmic ubiquitination events. Cytokine Growth Factor Rev 22:277–286

    Article  CAS  PubMed  Google Scholar 

  6. Courtois G, Gilmore TD (2006) Mutations in the NF-kappaB signaling pathway: implications for human disease. Oncogene 25:6831–6843

    Article  CAS  PubMed  Google Scholar 

  7. Karin M (2006) Nuclear factor-kappaB in cancer development and progression. Nature 441:431–436

    Article  CAS  PubMed  Google Scholar 

  8. Lawrence T (2009) The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol 1:a001651

    Article  PubMed Central  PubMed  Google Scholar 

  9. DiDonato JA, Mercurio F, Karin M (2012) NF-kappaB and the link between inflammation and cancer. Immunol Rev 246:379–400

    Article  PubMed  Google Scholar 

  10. Albensi BC, Mattson MP (2000) Evidence for the involvement of TNF and NF-kappaB in hippocampal synaptic plasticity. Synapse 35:151–159

    Article  CAS  PubMed  Google Scholar 

  11. Burrone J, O'Byrne M, Murthy VN (2002) Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature 420:414–418

    Article  CAS  PubMed  Google Scholar 

  12. Christoffel DJ et al (2011) IkappaB kinase regulates social defeat stress-induced synaptic and behavioral plasticity. J Neurosci 31:314–321

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Freudenthal R, Romano A (2000) Participation of Rel/NF-kappaB transcription factors in long-term memory in the crab Chasmagnathus. Brain Res 855:274–281

    Article  CAS  PubMed  Google Scholar 

  14. Freudenthal R, Romano A, Routtenberg A (2004) Transcription factor NF-kappaB activation after in vivo perforant path LTP in mouse hippocampus. Hippocampus 14:677–683

    Article  CAS  PubMed  Google Scholar 

  15. Kaltschmidt B et al (2006) NF-kappaB regulates spatial memory formation and synaptic plasticity through protein kinase A/CREB signaling. Mol Cell Biol 26:2936–2946

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Meffert MK, Baltimore D (2005) Physiological functions for brain NF-kappaB. Trends Neurosci 28:37–43

    Article  CAS  PubMed  Google Scholar 

  17. Meffert MK et al (2003) NF-kappa B functions in synaptic signaling and behavior. Nat Neurosci 6:1072–1078

    Article  CAS  PubMed  Google Scholar 

  18. O'Mahony A et al (2006) NF-kappaB/Rel regulates inhibitory and excitatory neuronal function and synaptic plasticity. Mol Cell Biol 26:7283–7298

    Article  PubMed Central  PubMed  Google Scholar 

  19. O'Riordan KJ et al (2006) Regulation of nuclear factor kappaB in the hippocampus by group I metabotropic glutamate receptors. J Neurosci 26:4870–4879

    Article  PubMed  Google Scholar 

  20. Russo SJ et al (2009) Nuclear factor kappa B signaling regulates neuronal morphology and cocaine reward. J Neurosci 29:3529–3537

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Schmeisser MJ et al (2012) IkappaB kinase/nuclear factor kappaB-dependent insulin-like growth factor 2 (Igf2) expression regulates synapse formation and spine maturation via Igf2 receptor signaling. J Neurosci 32:5688–5703

    Article  CAS  PubMed  Google Scholar 

  22. Boersma MC et al (2011) A requirement for nuclear factor-kappaB in developmental and plasticity-associated synaptogenesis. J Neurosci 31:5414–5425

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Kaltschmidt C, Kaltschmidt B, Baeuerle PA (1993) Brain synapses contain inducible forms of the transcription factor NF-kappa B. Mech Dev 43:135–147

    Article  CAS  PubMed  Google Scholar 

  24. Guerrini L, Blasi F, Denis-Donini S (1995) Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro. Proc Natl Acad Sci U S A 92:9077–9081

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Kaltschmidt C, Kaltschmidt B, Baeuerle PA (1995) Stimulation of ionotropic glutamate receptors activates transcription factor NF-kappa B in primary neurons. Proc Natl Acad Sci U S A 92:9618–9622

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Wajant H, Scheurich P (2011) TNFR1-induced activation of the classical NF-kappaB pathway. FEBS J 278:862–876

    Article  CAS  PubMed  Google Scholar 

  27. Carter BD et al (1996) Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75. Science 272:542–545

    Article  CAS  PubMed  Google Scholar 

  28. Yeiser EC et al (2004) Neurotrophin signaling through the p75 receptor is deficient in traf6-/- mice. J Neurosci 24:10521–10529

    Article  CAS  PubMed  Google Scholar 

  29. Vallabhapurapu S, Karin M (2009) Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol 27:693–733

    Article  CAS  PubMed  Google Scholar 

  30. Kaltschmidt C et al (1994) Constitutive NF-kappa B activity in neurons. Mol Cell Biol 14:3981–3992

    PubMed Central  CAS  PubMed  Google Scholar 

  31. Kanarek N, Ben-Neriah Y (2012) Regulation of NF-kappaB by ubiquitination and degradation of the IkappaBs. Immunol Rev 246:77–94

    Article  PubMed  Google Scholar 

  32. Lubin FD, Sweatt JD (2007) The IkappaB kinase regulates chromatin structure during reconsolidation of conditioned fear memories. Neuron 55:942–957

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Yamamoto Y et al (2003) Histone H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene expression. Nature 423:655–659

    Article  CAS  PubMed  Google Scholar 

  34. DiDonato JA et al (1997) A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. Nature 388:548–554

    Article  CAS  PubMed  Google Scholar 

  35. Pomerantz JL, Denny EM, Baltimore D (2002) CARD11 mediates factor-specific activation of NF-kappaB by the T cell receptor complex. EMBO J 21:5184–5194

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Zandi E et al (1997) The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell 91:243–252

    Article  CAS  PubMed  Google Scholar 

  37. Beaudoin GM 3rd et al (2012) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7:1741–1754

    Article  CAS  PubMed  Google Scholar 

  38. Nunez J (2008) Primary culture of hippocampal neurons from p0 newborn rats. J Vis Exp 19:e895

    Google Scholar 

  39. Geleziunas R et al (1998) Human T-cell leukemia virus type 1 Tax induction of NF-kappaB involves activation of the IkappaB kinase alpha (IKKalpha) and IKKbeta cellular kinases. Mol Cell Biol 18:5157–5165

    PubMed Central  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Anca B. Mihalas & Mollie K. Meffert

  2. Seattle Children’s Research Institute, Seattle, WA, 98101, USA

    Anca B. Mihalas

  3. Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Mollie K. Meffert

Authors
  1. Anca B. Mihalas
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  2. Mollie K. Meffert
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Correspondence to Mollie K. Meffert .

Editor information

Editors and Affiliations

  1. Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA

    Michael J. May

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Mihalas, A.B., Meffert, M.K. (2015). IKK Kinase Assay for Assessment of Canonical NF-κB Activation in Neurons. In: May, M. (eds) NF-kappa B. Methods in Molecular Biology, vol 1280. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2422-6_5

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  • DOI: https://doi.org/10.1007/978-1-4939-2422-6_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2421-9

  • Online ISBN: 978-1-4939-2422-6

  • eBook Packages: Springer Protocols

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