Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation

Nature Neuroscience volume 12, pages 857–863 (2009)Cite this article

Abstract

Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47phox subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: NADPH oxidase is the major source of NMDA-induced superoxide formation in neurons.
Figure 2: NMDA-induced oxidative damage was prevented by NADPH oxidase inhibition.
Figure 3: Mitochondria do not substantially contribute to NMDA-induced superoxide production.
Figure 4: NMDA activation of NADPH oxidase is mediated by PKC.
Figure 5: Inhibition of NADPH oxidase prevents NMDA-induced cell death.
Figure 6: NMDA induced superoxide in mouse hippocampus.
Figure 7: NMDA-induced superoxide production in cell bodies and dendrites.

Similar content being viewed by others

References

  1. Lafon-Cazal, M., Pietri, S., Culcasi, M. & Bockaert, J. NMDA-dependent superoxide production and neurotoxicity. Nature 364, 535–537 (1993).

    Article  CAS  Google Scholar 

  2. Mandir, A.S. et al. NMDA, but not non-NMDA, excitotoxicity is mediated by Poly(ADP-ribose) polymerase. J. Neurosci. 20, 8005–8011 (2000).

    Article  CAS  Google Scholar 

  3. MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. & Barker, J.L. NMDA receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones. Nature 321, 519–522 (1986).

    Article  CAS  Google Scholar 

  4. Klann, E. Cell-permeable scavengers of superoxide prevent long-term potentiation in hippocampal area CA1. J. Neurophysiol. 80, 452–457 (1998).

    Article  CAS  Google Scholar 

  5. MacDonald, J.F., Jackson, M.F. & Beazely, M.A. Hippocampal long-term synaptic plasticity and signal amplification of NMDA receptors. Crit. Rev. Neurobiol. 18, 71–84 (2006).

    Article  CAS  Google Scholar 

  6. Aizenman, E., Hartnett, K.A. & Reynolds, I.J. Oxygen free radicals regulate NMDA receptor function via a redox modulatory site. Neuron 5, 841–846 (1990).

    Article  CAS  Google Scholar 

  7. Patel, M., Day, B.J., Crapo, J.D., Fridovich, I. & McNamara, J.O. Requirement for superoxide in excitotoxic cell death. Neuron 16, 345–355 (1996).

    Article  CAS  Google Scholar 

  8. White, R.J. & Reynolds, I.J. Mitochondrial depolarization in glutamate-stimulated neurons: an early signal specific to excitotoxin exposure. J. Neurosci. 16, 5688–5697 (1996).

    Article  CAS  Google Scholar 

  9. Budd, S.L. & Nicholls, D.G. Mitochondria, calcium regulation and acute glutamate excitotoxicity in cultured cerebellar granule cells. J. Neurochem. 67, 2282–2291 (1996).

    Article  CAS  Google Scholar 

  10. Bindokas, V.P., Jordan, J., Lee, C.C. & Miller, R.J. Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine. J. Neurosci. 16, 1324–1336 (1996).

    Article  CAS  Google Scholar 

  11. Dugan, L.L. et al. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate. J. Neurosci. 15, 6377–6388 (1995).

    Article  CAS  Google Scholar 

  12. Andreyev, A.Y., Kushnareva, Y.E. & Starkov, A.A. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc.) 70, 200–214 (2005).

    Article  CAS  Google Scholar 

  13. Adam-Vizi, V. & Chinopoulos, C. Bioenergetics and the formation of mitochondrial reactive oxygen species. Trends Pharmacol. Sci. 27, 639–645 (2006).

    Article  CAS  Google Scholar 

  14. Dykens, J.A. Isolated cerebral and cerebellar mitochondria produce free radicals when exposed to elevated Ca2+ and Na+: implications for neurodegeneration. J. Neurochem. 63, 584–591 (1994).

    Article  CAS  Google Scholar 

  15. Duan, Y., Gross, R.A. & Sheu, S.S. Ca2+-dependent generation of mitochondrial reactive oxygen species serves as a signal for poly(ADP-ribose) polymerase-1 activation during glutamate excitotoxicity. J. Physiol. (Lond.) 585, 741–758 (2007).

    Article  CAS  Google Scholar 

  16. Nicholls, D.G. Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons. J. Biol. Chem. 281, 14864–14874 (2006).

    Article  CAS  Google Scholar 

  17. Bedard, K. & Krause, K.H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313 (2007).

    Article  CAS  Google Scholar 

  18. Tejada-Simon, M.V. et al. Synaptic localization of a functional NADPH oxidase in the mouse hippocampus. Mol. Cell. Neurosci. 29, 97–106 (2005).

    Article  CAS  Google Scholar 

  19. Robinson, K.M. et al. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043 (2006).

    Article  CAS  Google Scholar 

  20. Zhao, H. et al. Superoxide reacts with hydroethidine, but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic. Biol. Med. 34, 1359–1368 (2003).

    Article  CAS  Google Scholar 

  21. Suh, S.W., Gum, E.T., Hamby, A.M., Chan, P.H. & Swanson, R.A. Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase. J. Clin. Invest. 117, 910–918 (2007).

    Article  CAS  Google Scholar 

  22. Suh, S.W. et al. Glucose and NADPH oxidase drive neuronal superoxide formation in stroke. Ann. Neurol. 64, 654–663 (2008).

    Article  CAS  Google Scholar 

  23. Stolk, J., Hiltermann, T.J., Dijkman, J.H. & Verhoeven, A.J. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am. J. Respir. Cell Mol. Biol. 11, 95–102 (1994).

    Article  CAS  Google Scholar 

  24. Bender, J.G. & Van Epps, D.E. Inhibition of human neutrophil function by 6-aminonicotinamide: the role of the hexose monophosphate shunt in cell activation. Immunopharmacology 10, 191–199 (1985).

    Article  CAS  Google Scholar 

  25. Gupte, S.A. et al. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart. J. Mol. Cell. Cardiol. 41, 340–349 (2006).

    Article  CAS  Google Scholar 

  26. Inanami, O. et al. Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47PHOX on serine 303 or 304. J. Biol. Chem. 273, 9539–9543 (1998).

    Article  CAS  Google Scholar 

  27. Uchida, K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog. Lipid Res. 42, 318–343 (2003).

    Article  CAS  Google Scholar 

  28. Lai, B. et al. Inhibition of Qi site of mitochondrial complex III with antimycin A decreases persistent and transient sodium currents via reactive oxygen species and protein kinase C in rat hippocampal CA1 cells. Exp. Neurol. 194, 484–494 (2005).

    Article  CAS  Google Scholar 

  29. Raineri, I. et al. Strain-dependent high-level expression of a transgene for manganese superoxide dismutase is associated with growth retardation and decreased fertility. Free Radic. Biol. Med. 31, 1018–1030 (2001).

    Article  CAS  Google Scholar 

  30. Abramov, A.Y., Scorziello, A. & Duchen, M.R. Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation. J. Neurosci. 27, 1129–1138 (2007).

    Article  CAS  Google Scholar 

  31. Porasuphatana, S., Tsai, P. & Rosen, G.M. The generation of free radicals by nitric oxide synthase. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 134, 281–289 (2003).

    Article  Google Scholar 

  32. Cheng, Y. & Sun, A.Y. Oxidative mechanisms involved in kainate-induced cytotoxicity in cortical neurons. Neurochem. Res. 19, 1557–1564 (1994).

    Article  CAS  Google Scholar 

  33. Atlante, A. et al. Glutamate neurotoxicity in rat cerebellar granule cells: a major role for xanthine oxidase in oxygen radical formation. J. Neurochem. 68, 2038–2045 (1997).

    Article  CAS  Google Scholar 

  34. Hardingham, G.E., Fukunaga, Y. & Bading, H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. Neurosci. 5, 405–414 (2002).

    Article  CAS  Google Scholar 

  35. Favaron, M. et al. Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death. Proc. Natl. Acad. Sci. USA 87, 1983–1987 (1990).

    Article  CAS  Google Scholar 

  36. Koponen, S. et al. Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase C zeta. J. Neurochem. 86, 442–450 (2003).

    Article  CAS  Google Scholar 

  37. Dang, P.M., Fontayne, A., Hakim, J., El Benna, J. & Perianin, A. Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide–mediated neutrophil respiratory burst. J. Immunol. 166, 1206–1213 (2001).

    Article  CAS  Google Scholar 

  38. Reynolds, I.J. & Hastings, T.G. Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation. J. Neurosci. 15, 3318–3327 (1995).

    Article  CAS  Google Scholar 

  39. Johnson-Cadwell, L.I., Jekabsons, M.B., Wang, A., Polster, B.M. & Nicholls, D.G. 'Mild Uncoupling' does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons, but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress. J. Neurochem. 101, 1619–1631 (2007).

    Article  CAS  Google Scholar 

  40. Brandes, R.P. Triggering mitochondrial radical release: a new function for NADPH oxidases. Hypertension 45, 847–848 (2005).

    Article  CAS  Google Scholar 

  41. Chan, P.H. et al. Overexpression of SOD1 in transgenic rats protects vulnerable neurons against ischemic damage after global cerebral ischemia and reperfusion. J. Neurosci. 18, 8292–8299 (1998).

    Article  CAS  Google Scholar 

  42. Fernandes, D.C. et al. Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems. Am. J. Physiol. Cell Physiol. 292, C413–C422 (2007).

    Article  CAS  Google Scholar 

  43. Hyrc, K., Handran, S.D., Rothman, S.M. & Goldberg, M.P. Ionized intracellular calcium concentration predicts excitotoxic neuronal death: observations with low-affinity fluorescent calcium indicators. J. Neurosci. 17, 6669–6677 (1997).

    Article  CAS  Google Scholar 

  44. Clark, R.A., Volpp, B.D., Leidal, K.G. & Nauseef, W.M. Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J. Clin. Invest. 85, 714–721 (1990).

    Article  CAS  Google Scholar 

  45. Bey, E.A. et al. Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes. J. Immunol. 173, 5730–5738 (2004).

    Article  CAS  Google Scholar 

  46. Bright, R. et al. Protein kinase C delta mediates cerebral reperfusion injury in vivo. J. Neurosci. 24, 6880–6888 (2004).

    Article  CAS  Google Scholar 

  47. Crisanti, P., Leon, A., Lim, D.M. & Omri, B. Aspirin prevention of NMDA-induced neuronal death by direct protein kinase Czeta inhibition. J. Neurochem. 93, 1587–1593 (2005).

    Article  CAS  Google Scholar 

  48. Sacktor, T.C. PKMzeta, LTP maintenance and the dynamic molecular biology of memory storage. Prog. Brain Res. 169, 27–40 (2008).

    Article  CAS  Google Scholar 

  49. Ying, W. et al. Differing effects of copper, zinc superoxide dismutase overexpression on neurotoxicity elicited by nitric oxide, reactive oxygen species and excitotoxins. J. Cereb. Blood Flow Metab. 20, 359–368 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Mochly-Rosen for advice with the TAT-conjugated peptides, C. Alano and C. Escartin for their careful reviews of the manuscript, and C. Hefner for technical assistance. This work was supported by the Department of Veterans Affairs Merit Review program (R.A.S.) and by the US National Institutes of Health (grants NS14543 to P.H.C. and NS051855 and NS051855 to R.A.S.).

Author information

Authors and Affiliations

  1. Department of Neurology, University of California, San Francisco and Veterans Affairs Medical Center, San Francisco, California, USA

    Angela M Brennan, Sang Won Suh, Seok Joon Won, Tiina M Kauppinen, Hokyou Lee, Ylva Edling & Raymond A Swanson

  2. Departments of Neurosurgery, Neurology and Neurological Sciences, and the Program in Neurosciences, Stanford University School of Medicine, Stanford, California, USA

    Purnima Narasimhan & Pak H Chan

Authors
  1. Angela M Brennan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sang Won Suh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Seok Joon Won
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Purnima Narasimhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tiina M Kauppinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hokyou Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ylva Edling
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pak H Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Raymond A Swanson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.M.B. carried out the cell culture studies and data analysis and prepared the manuscript drafts. S.W.S. supervised the mouse surgical studies and analyzed these data. S.J.W. performed mouse surgery studies and mouse brain histology. P.N. maintained the Sod2+ mouse colony and prepared the Sod2+ cell cultures. T.M.K. and Y.E. assisted with the p47phox translocation studies and data analysis. H.L. assisted in the analysis of the cell culture ethidium fluorescence results. P.H.C. assisted with the studies involving Sod2+ neurons. R.A.S. organized the studies and prepared the final manuscript.

Corresponding author

Correspondence to Raymond A Swanson.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 216 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brennan, A., Won Suh, S., Joon Won, S. et al. NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation. Nat Neurosci 12, 857–863 (2009). https://doi.org/10.1038/nn.2334

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.2334

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing