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:

Glutamate-induced neuron death requires mitochondrial calcium uptake

Nature Neuroscience volume 1, pages 366–373 (1998)Cite this article

Abstract

We have investigated the role of mitochondrial calcium buffering in excitotoxic cell death. Glutamate acts at NMDA receptors in cultured rat forebrain neurons to increase the intracellular free calcium concentration. Although concurrent inhibition of mitochondrial calcium uptake substantially enhanced this cytoplasmic calcium increase, it significantly reduced glutamate-stimulated neuronal cell death. Mitochondrial inhibition did not affect nitric oxide production or MAP kinase phosphorylation, which have been proposed to mediate excitotoxicity. These results indicate that very high levels of cytoplasmic calcium are not necessarily toxic to forebrain neurons, and that potential-driven uptake of calcium into mitochondria is required to trigger NMDA-receptor-stimulated neuronal death.

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: Mitochondrial inhibitors enhance glutamate-stimulated mitochondrial membrane depolarization.
Figure 2: Mitochondrial inhibitors enhance glutamate-stimulated increases in [Ca2+]i measured using Magfura 2.
Figure 3: Transient inhibition of mitochondrial calcium uptake protects against glutamate toxicity.
Figure 4: Mitochondrial inhibitors and elevated extracellular pH inhibit glutamate-stimulated intracellular acidification.
Figure 5: FCCP does not block glutamate-stimulated increases in NOS activity.
Figure 6: FCCP does not alter glutamate-stimulated increases in MAPK activity.

Similar content being viewed by others

References

  1. Choi, D. W. Ionic dependence of glutamate neurotoxicity. J. Neurosci. 7, 369–379 (1987).

    Article  CAS  Google Scholar 

  2. Tymianski, M., Charlton, M. P., Carlen, P. L. & Tator, C. H. Source specificity of early calcium neurotoxicity in cultured embryonic spinal neurons. J. Neurosci. 13, 2085–2104 (1993).

    Article  CAS  Google Scholar 

  3. Hartley, D. M., Kurth, M. C., Bjerkness, L., Weiss, J. H. & Choi, D. W. Glutamate receptor-induced 45Ca2+ accumulation in cortical culture correlates with subsequent neuronal degeneration. J. Neurosci. 13, 1993–2000 (1993).

    Article  CAS  Google Scholar 

  4. Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S. & Snyder, S. H. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. Natl Acad. Sci. USA 88, 6368–6371 (1991).

    Article  CAS  Google Scholar 

  5. Siman, R., Noszek, J. C. & Kegerise, C. Calpain I activation is specifically related to excitatory amino acid induction of hippocampal damage. J. Neurosci. 9, 1579–1590 (1989).

    Article  CAS  Google Scholar 

  6. Monyer, H., Hartley, D. M. & Choi, D. W. 21-aminosteroids attenuate excitotoxic neuronal injury in cortical cell cultures. Neuron 5, 121–126 (1990).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Coyle, J. T. & Puttfarcken, P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 262, 689–695 (1993).

    Article  CAS  Google Scholar 

  9. 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 

  10. 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 

  11. Nicholls, D. G. & Akerman, K. E. O. Mitochondrial calcium transport. Biochim. Biophys. Acta 683, 57–88 (1982).

    Article  CAS  Google Scholar 

  12. Gunter, T. E., Gunter, K. K., Sheu, S.-S. & Gavin, C. E. Mitochondrial calcium transport: Physiological and pathological relevance. Am. J. Physiol. Cell Physiol. 267, C313–C339 (1994).

    Article  CAS  Google Scholar 

  13. Ankarcrona, M. et al. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961–973 (1995).

    Article  CAS  Google Scholar 

  14. 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 

  15. Schinder, A. F., Olson, E. C., Spitzer, N. C. & Montal, M. Mitochondrial dysfunction is a primary event in glutamate neurotoxicity. J. Neurosci. 16, 6125–6133 (1996).

    Article  CAS  Google Scholar 

  16. Gunter, T. E. & Pfeiffer, D. R. Mechanisms by which mitochondria transport calcium. Am. J. Physiol. Cell Physiol. 258, C755–C786 (1990).

    Article  CAS  Google Scholar 

  17. Nicholls, D. G. in Molecular Mechanisms of Ischemic Brain Damage (eds Kogure, K., Hossman, K. A., Siesjö, B. K. & Welsh, F. A.) 97–106 (Elsevier, Amsterdam, 1985).

    Book  Google Scholar 

  18. 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 

  19. 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 

  20. 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 

  21. Arkles, B. & Brinigar, W. S. Respiratory properties of rat liver mitochondria immobilized on an alkylsilyated glass surface. J. Biol. Chem. 250, 8856–8862 (1975).

    CAS  Google Scholar 

  22. Wang, G. J., Richardson, S. R. & Thayer, S. A. Intracellular acidification is not a prerequisite for glutamate-triggered death of cultured hippocampal neurons. Neurosci. Lett. 186, 139–144 (1995).

    Article  CAS  Google Scholar 

  23. Hoyt, K. R. & Reynolds, I. J. Alkalinization prolongs recovery from glutamate-induced increases in [Ca2+]i by enhancing Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger in cultured rat forebrain neurons. J. Neurochem. (in press).

  24. Favaron, M. et al. Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal culture of neonatal rat cerebellum and cortex. Proc. Natl Acad. Sci. USA 85, 7351–7355 (1988).

    Article  CAS  Google Scholar 

  25. Dykens, J. A., Stern, A. & Trenkner, E. Mechanism of kainate toxicity to cerebellar neurons in vitro is analogous to reperfusion tissue injury. J. Neurochem. 49, 1222–1228 (1987).

    Article  CAS  Google Scholar 

  26. Bading, H. & Greenberg, M. E. Stimulation of protein tyrosine phosphorylation by NMDA receptor activation. Science 253, 912–914 (1991).

    Article  CAS  Google Scholar 

  27. Kawasaki, H. et al. Activation and involvement of p38 mitogen-activated protein kinase in glutamate-induced apoptosis in rat cerebellar granule cells. J. Biol. Chem. 272, 18518–18521 (1997).

    Article  CAS  Google Scholar 

  28. Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q. & Holbrook, N. J. Activation of mitogen-activated protein kinase by H2O2: role in cell survival following oxidant injury. J. Biol. Chem. 271, 4138–4142 (1996).

    Article  CAS  Google Scholar 

  29. 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 

  30. Budd, S. L. & Nicholls, D. G. A reevaluation of the role of mitochondria in neuronal Ca2+ homeostasis. J. Neurochem. 66, 403–411 (1996).

    Article  CAS  Google Scholar 

  31. Wang, G. J., Randall, R. D. & Thayer, S. A. Glutamate-induced intracellular acidification of cultured hippocampal neurons demonstrates altered energy metabolism resulting from Ca2+ loads. J. Neurophysiol. 72, 2563–2569 (1994).

    Article  CAS  Google Scholar 

  32. 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 

  33. Stout, A. K. & Reynolds, I. J. High-affinity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations. Neuroscience (in press).

  34. Brocard, J. B., Rajdev, S. & Reynolds, I. J. Glutamate induced increases in intracellular free Mg2+ in cultured cortical neurons. Neuron 11, 751–757 (1993).

    Article  CAS  Google Scholar 

  35. Rajdev, S. & Reynolds, I. J. Calcium green 5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons. Neurosci. Lett. 162, 149–152 (1993).

    Article  CAS  Google Scholar 

  36. White, R. J. & Reynolds, I. J. Mitochondria accumulate Ca2+ following intense glutamate stimulation of cultured rat forebrain neurones. J. Physiol. (Lond.) 498, 31–47 (1997).

    Article  CAS  Google Scholar 

  37. Hoyt, K. R., Stout, A. K., Cardman, J. M. & Reynolds, I. J. An evaluation of intracellular sodium and mitochondria in the buffering of kainate-induced intracellular free calcium changes in rat forebrain neurons. J. Physiol. (Lond.) 509, 103–116 (1998).

    Article  CAS  Google Scholar 

  38. Dawson, V. L., Kizushi, V. M., Huang, P. L., Snyder, S. H. & Dawson, T. L. Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide deficient mice. J. Neurosci. 16, 2479–2487 (1996).

    Article  CAS  Google Scholar 

  39. Hewett, S. J., Corbett, J. A., McDaniel, M. L. & Choi, D. W. Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-D-aspartate neurotoxicity. Brain Res. 625, 337–341 (1993).

    Article  CAS  Google Scholar 

  40. Pauwels, P. J. & Leysen, J. E. Blockade of nitric oxide formation does not prevent glutamate-induced neurotoxicity in neuronal cultures from rat hippocampus. Neurosci. Lett. 143, 27–30 (1992).

    Article  CAS  Google Scholar 

  41. Demerlé-Pallardy, C., Lonchampt, M.-O., Chabrier, P.-E. & Braquet, P. Absence of implication of L-arginine/nitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia. Biochem. Biophys. Res. Commun. 181, 456–464 (1991).

    Article  Google Scholar 

  42. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A. & Freeman, B. A. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl Acad. Sci. USA 87, 1620–1624 (1990).

    Article  CAS  Google Scholar 

  43. Newmeyer, D. D., Farschon, D. M. & Reed, J. C. Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell 79, 189–192 (1994).

    Article  Google Scholar 

  44. Yang, J. et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129–1132 (1997).

    Article  CAS  Google Scholar 

  45. Susin, S.A. et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184, 1331–1341 (1996).

    Article  CAS  Google Scholar 

  46. Kluck, R. M., Bossy-Wetzel, E., Green, D. R. & Newmeyer, D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136 (1997).

    Article  CAS  Google Scholar 

  47. Kim, C. N. et al. Overexpression of Bcl-X(L) inhibits ara-C-induced mitochondrial loss of cytochrome c and other pertubations that activate the molecular cascade of apoptosis. Cancer Res. 57, 3115–3120 (1997).

    CAS  Google Scholar 

  48. White, R. J. & Reynolds, I. J. Mitochondria and Na+/Ca2+ exchange buffer glutamate-induced calcium loads in cultured cortical neurons. J. Neurosci. 15, 1318–1328 (1995).

    Article  CAS  Google Scholar 

  49. Bredt, D. S., Mourey, R. J. & Snyder, S. H. A simple, sensitive, and specific radioreceptor assay for inositol 1,4,5-trisphosphate in biological tissues. Biochem. Biophys. Res. Comm. 159, 976–982 (1989).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grants NS 34138 (I.J.R.), NS 09998 (A.K.S.) and NS 34007 (E.K.). I.J.R. is an Established Investigator of the American Heart Association.

Author information

Authors and Affiliations

  1. Department of Pharmacology, E1354 Biomedical Science Tower, University of Pittsburgh, Pittsburgh, 15261, Pennsylvania, USA

    Amy K. Stout, Heather M. Raphael & Ian J. Reynolds

  2. Department of Neuroscience, 446 Crawford Hall, University of Pittsburgh, Pittsburgh, 15260, Pennsylvania, USA

    Beatriz I. Kanterewicz & Eric Klann

Authors
  1. Amy K. Stout
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Heather M. Raphael
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Beatriz I. Kanterewicz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eric Klann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ian J. Reynolds
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ian J. Reynolds.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stout, A., Raphael, H., Kanterewicz, B. et al. Glutamate-induced neuron death requires mitochondrial calcium uptake. Nat Neurosci 1, 366–373 (1998). https://doi.org/10.1038/1577

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/1577

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