IKK mediates ischemia-induced neuronal death

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Abstract

The IκB kinase complex IKK is a central component of the signaling cascade that controls NF-κB–dependent gene transcription. So far, its function in the brain is largely unknown. Here, we show that IKK is activated in a mouse model of stroke. To investigate the function of IKK in brain ischemia we generated mice that contain a targeted deletion of Ikbkb (which encodes IKK2) in mouse neurons and mice that express a dominant inhibitor of IKK in neurons. In both lines, inhibition of IKK activity markedly reduced infarct size. In contrast, constitutive activation of IKK2 enlarged the infarct size. A selective small-molecule inhibitor of IKK mimicked the effect of genetic IKK inhibition in neurons, reducing the infarct volume and cell death in a therapeutic time window of 4.5 h. These data indicate a key function of IKK in ischemic brain damage and suggest a potential role for IKK inhibitors in stroke therapy.

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Figure 1: Cerebral ischemia activates IKK in neurons.
Figure 2: Deficiency of IKK2 in neurons reduces ischemic brain damage.
Figure 3: Inhibition of IKK in neurons protects against ischemic damage.
Figure 4: Constitutive activation of IKK2 in neurons enhances ischemic damage.
Figure 5: The IKK inhibitor BMS-345541 ameliorates ischemic brain damage.
Figure 6: Inhibition of IKK reduces the expression of genes involved in eicosanoid biosynthesis.

References

  1. 1

    Dirnagl, U., Iadecola, C. & Moskowitz, M.A. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 22, 391–397 (1999).

  2. 2

    Bergeron, M., Yu, A.Y., Solway, K.E., Semenza, G.L. & Sharp, F.R. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur. J. Neurosci. 11, 4159–4170 (1999).

  3. 3

    Iadecola, C. et al. The transcription factor interferon regulatory factor 1 is expressed after cerebral ischemia and contributes to ischemic brain injury. J. Exp. Med. 189, 719–727 (1999).

  4. 4

    Schneider, A. et al. NF-κB is activated and promotes cell death in focal cerebral ischemia. Nat. Med. 5, 554–559 (1999).

  5. 5

    Pizzi, M. et al. Opposing roles for NF-kappa B/Rel factors p65 and c-Rel in the modulation of neuron survival elicited by glutamate and interleukin-1beta. J. Biol. Chem. 277, 20717–20723 (2002).

  6. 6

    Mattson, M.P. & Camandola, S. NF-kappaB in neuronal plasticity and neurodegenerative disorders. J. Clin. Invest. 107, 247–254 (2001).

  7. 7

    Qin, Z.H. et al. Nuclear factor kappaB nuclear translocation upregulates c-Myc and p53 expression during NMDA receptor-mediated apoptosis in rat striatum. J. Neurosci. 19, 4023–4033 (1999).

  8. 8

    Denk, A., Wirth, T. & Baumann, B. NF-kappaB transcription factors: critical regulators of hematopoiesis and neuronal survival. Cytokine Growth Factor Rev. 11, 303–320 (2000).

  9. 9

    Hallenbeck, J.M. The many faces of tumor necrosis factor in stroke. Nat. Med. 8, 1363–1368 (2002).

  10. 10

    Scholzke, M.N., Potrovita, I., Subramaniam, S., Prinz, S. & Schwaninger, M. Glutamate activates NF-kappaB through calpain in neurons. Eur. J. Neurosci. 18, 3305–3310 (2003).

  11. 11

    Bui, N.T., Livolsi, A., Peyron, J.F. & Prehn, J.H. Activation of nuclear factor kappaB and Bcl-x survival gene expression by nerve growth factor requires tyrosine phosphorylation of IkappaBalpha. J. Cell Biol. 152, 753–764 (2001).

  12. 12

    Karin, M., Yamamoto, Y. & Wang, Q.M. The IKK NF-kappa B system: a treasure trove for drug development. Nat. Rev. Drug Discov. 3, 17–26 (2004).

  13. 13

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

  14. 14

    Hayden, M.S. & Ghosh, S. Signaling to NF-kappaB. Genes Dev. 18, 2195–2224 (2004).

  15. 15

    Sakurai, H. et al. Tumor necrosis factor-alpha-induced IKK phosphorylation of NF-kappaB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway. J. Biol. Chem. 278, 36916–36923 (2003).

  16. 16

    Pasparakis, M. et al. TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2. Nature 417, 861–866 (2002).

  17. 17

    Minichiello, L. et al. Essential role for TrkB receptors in hippocampus-mediated learning. Neuron 24, 401–414 (1999).

  18. 18

    Tronche, F. et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat. Genet. 23, 99–103 (1999).

  19. 19

    Kaltschmidt, C., Kaltschmidt, B., Henkel, T., Stockinger, H. & P.A., B. Selective recognition of the activated form of transcription factor NF-κB by a monoclonal antibody. Biol. Chem. Hoppe-Seyler 376, 9–16 (1995).

  20. 20

    Endres, M. et al. Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family. J. Cereb. Blood Flow Metab. 18, 238–247 (1998).

  21. 21

    Gabriel, C., Justicia, C., Camins, A. & Planas, A.M. Activation of nuclear factor-kappaB in the rat brain after transient focal ischemia. Brain Res. Mol. Brain Res. 65, 61–69 (1999).

  22. 22

    Li, Q., Estepa, G., Memet, S., Israel, A. & Verma, I.M. Complete lack of NF-kappaB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation. Genes Dev. 14, 1729–1733 (2000).

  23. 23

    Yin, M.J., Yamamoto, Y. & Gaynor, R.B. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature 396, 77–80 (1998).

  24. 24

    Vartiainen, N., Goldsteins, G., Keksa-Goldsteine, V., Chan, P.H. & Koistinaho, J. Aspirin inhibits p44/42 mitogen-activated protein kinase and is protective against hypoxia/reoxygenation neuronal damage. Stroke 34, 752–757 (2003).

  25. 25

    Burke, J.R. et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J. Biol. Chem. 278, 1450–1456 (2003).

  26. 26

    Pieper, A.A., Verma, A., Zhang, J. & Snyder, S.H. Poly (ADP-ribose) polymerase, nitric oxide and cell death. Trends Pharmacol. Sci. 20, 171–181 (1999).

  27. 27

    Bonventre, J.V. et al. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 390, 622–625 (1997).

  28. 28

    Iadecola, C. et al. Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc. Natl. Acad. Sci. USA 98, 1294–1299 (2001).

  29. 29

    Kaltschmidt, C., Kaltschmidt, B., Neumann, H., Wekerle, H. & Baeuerle, P.A. Constitutive NF-κB activity in neurons. Mol. Cell. Biol. 14, 3981–3992 (1994).

  30. 30

    Han, H.S., Karabiyikoglu, M., Kelly, S., Sobel, R.A. & Yenari, M.A. Mild hypothermia inhibits nuclear factor-kappaB translocation in experimental stroke. J. Cereb. Blood Flow Metab. 23, 589–598 (2003).

  31. 31

    Song, Y.S., Lee, Y.S. & Chan, P.H. Oxidative stress transiently decreases the IKK complex (IKKα, β, and γ), an upstream component of NF-κB signaling, after transient focal cerebral ischemia in mice. J. Cereb. Blood Flow Metab. 25, 1301–1311 (2005).

  32. 32

    Khoshnan, A. et al. Activation of the IκB kinase complex and nuclear factor-kappaB contributes to mutant huntingtin neurotoxicity. J. Neurosci. 24, 7999–8008 (2004).

  33. 33

    Zhang, W. et al. Neuronal activation of NF-κB contributes to cell death in cerebral ischemia. J. Cereb. Blood Flow Metab. 25, 30–40 (2005).

  34. 34

    Chen, L.W. et al. The two faces of IKK and NF-kappaB inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia-reperfusion. Nat. Med. 9, 575–581 (2003).

  35. 35

    Luedde, T. et al. Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion-injury. J. Clin. Invest. 115, 849–859 (2005).

  36. 36

    Staub, F. et al. Swelling, acidosis, and irreversible damage of glial cells from exposure to arachidonic acid in vitro. J. Cereb. Blood Flow Metab. 14, 1030–1039 (1994).

  37. 37

    Aguirre, V. et al. Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J. Biol. Chem. 277, 1531–1537 (2002).

  38. 38

    Hu, M.C. et al. IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 117, 225–237 (2004).

  39. 39

    Waterfield, M., Jin, W., Reiley, W., Zhang, M. & Sun, S.C. IkappaB kinase is an essential component of the Tpl2 signaling pathway. Mol. Cell. Biol. 24, 6040–6048 (2004).

  40. 40

    Lamberti, C. et al. Regulation of beta-catenin function by the IkappaB kinases. J. Biol. Chem. 276, 42276–42286 (2001).

  41. 41

    Nurmi, A. et al. Nuclear factor-kappaB contributes to infarction after permanent focal ischemia. Stroke 35, 987–991 (2004).

  42. 42

    Williams, A.J. et al. Delayed treatment with MLN519 reduces infarction and associated neurologic deficit caused by focal ischemic brain injury in rats via antiinflammatory mechanisms involving nuclear factor-kappaB activation, gliosis, and leukocyte infiltration. J. Cereb. Blood Flow Metab. 23, 75–87 (2003).

  43. 43

    Chan, P.H. et al. Brain infarction is not reduced in SOD-1 transgenic mice after a permanent focal cerebral ischemia. Neuroreport 5, 293–296 (1993).

  44. 44

    Azoitei, N., Wirth, T. & Baumann, B. Activation of the IκB kinase complex is sufficient for neuronal differentiation of PC12 cells. J. Neurochem. 93, 1487–1501 (2005).

  45. 45

    Yamaoka, S. et al. Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation. Cell 93, 1231–1240 (1998).

  46. 46

    Ma, J., Qiu, J., Hirt, L., Dalkara, T. & Moskowitz, M.A. Synergistic protective effect of caspase inhibitors and bFGF against brain injury induced by transient focal ischaemia. Br. J. Pharmacol. 133, 345–350 (2001).

  47. 47

    Maier, H.J., Marienfeld, R., Wirth, T. & Baumann, B. Critical role of RelB serine 368 for dimerization and p100 stabilization. J. Biol. Chem. 278, 39242–39250 (2003).

  48. 48

    Wang, X. et al. Inhibition of tumor necrosis factor-alpha-converting enzyme by a selective antagonist protects brain from focal ischemic injury in rats. Mol. Pharmacol. 65, 890–896 (2004).

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Acknowledgements

This study was supported by Deutsche Forschungsgemeinschaft grants SCHW 416/4-2 (to M.S.) and SFB 497/B1 (to B.B.), Bundesministerium für Bildung und Forschung grant NGFN2 (to M.S.), and European Union grant QLG1-CT-1999-00202 (to M.P.). We thank H. Schröck (Heidelberg) for help with physiological parameters, R. Kühn (Neuherberg, Germany) for providing CamKII-Cre mice, R. Klein (Munich) for providing Nestin-Cre mice and Y. Qiu (Bristol-Myers Squibb) for his work toward synthesis of BMS-345541.

Author information

Correspondence to Markus Schwaninger.

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Competing interests

James R. Burke is employed by Bristol-Meyers Squibb. Bristol-Meyers Squibb is interested in the development and commercialization of IKK2 inhibitors as therapeutics for the treatment of stroke.

Supplementary information

Supplementary Fig. 1

Nissl staining of coronal brain sections showed no gross anatomical differences between IKK2nKO and control mice. (PDF 203 kb)

Supplementary Fig. 2

Immunohistochemistry of IKK1/2 in sagittal brain slices of IKK2nDN animals. (PDF 277 kb)

Supplementary Fig. 3

Luciferase activity was measured in brain regions identical to Figure 3c. (PDF 78 kb)

Supplementary Fig. 4

Junb expression in IKK2nCA mice and littermate controls was determined by semi-quantitative RT-PCR in brain regions identical to Figure 4. (PDF 54 kb)

Supplementary Fig. 5

Induction of the proapoptotic genes Fas and Myc and of the antiapoptotic genes Birc3, Bdnf, Epo and Csf3 by MCAO was not inhibited by BMS-345541 treatment. (PDF 80 kb)

Supplementary Table 1

Physiological parameters of IKK2CNSKO mice and control mice. (PDF 23 kb)

Supplementary Table 2

Physiological parameteres of C57Bl/6 mice 20 min after intracerebroventricular injection of 2 μl saline or 20 mM BMS-345541 and 15 min after MCAO. (PDF 19 kb)

Supplementary Methods (PDF 34 kb)

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Herrmann, O., Baumann, B., de Lorenzi, R. et al. IKK mediates ischemia-induced neuronal death. Nat Med 11, 1322–1329 (2005) doi:10.1038/nm1323

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