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Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse

Nature Neuroscience volume 9, pages 119–126 (2006)Cite this article

Abstract

Uptake of the neurotransmitter glutamate is effected primarily by transporters expressed on astrocytes, and downregulation of these transporters leads to seizures and neuronal death. Neurons also express a glutamate transporter, termed excitatory amino acid carrier–1 (EAAC1), but the physiological function of this transporter remains uncertain. Here we report that genetically EAAC1-null (Slc1a1−/−) mice have reduced neuronal glutathione levels and, with aging, develop brain atrophy and behavioral changes. EAAC1 can also rapidly transport cysteine, an obligate precursor for neuronal glutathione synthesis. Neurons in the hippocampal slices of EAAC1−/− mice were found to have reduced glutathione content, increased oxidant levels and increased susceptibility to oxidant injury. These changes were reversed by treating the EAAC1−/− mice with N-acetylcysteine, a membrane-permeable cysteine precursor. These findings suggest that EAAC1 is the primary route for neuronal cysteine uptake and that EAAC1 deficiency thereby leads to impaired neuronal glutathione metabolism, oxidative stress and age-dependent neurodegeneration.

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Figure 1: Genotyping and glutamate transporter expression.
Figure 2: Performance on the Morris water maze test.
Figure 3: Brain atrophy in the EAAC1−/− mice.
Figure 4: Oxidative stress in neurons of EAAC1−/− mouse brain.
Figure 5: Increased vulnerability of neurons in EAAC1−/− mice to oxidative stress.
Figure 6: Reduced scavenging of reactive oxygen species in neurons of EAAC1−/− mice.
Figure 7: Bicuculline does not potentiate the oxidant effects of H2O2 or SIN-1.
Figure 8: Neuronal glutathione deficiency and vulnerability to oxidant stress in EAAC1−/− mice are both reversed by NAC.

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References

  1. Danbolt, N.C. Glutamate uptake. Prog. Neurobiol. 65, 1–105 (2001).

    Article  CAS  Google Scholar 

  2. Rothstein, J.D. et al. Localization of neuronal and glial glutamate transporters. Neuron 13, 713–725 (1994).

    Article  CAS  Google Scholar 

  3. Shashidharan, P. et al. Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody. Brain Res. 773, 139–148 (1997).

    Article  CAS  Google Scholar 

  4. Arriza, J.L., Eliasof, S., Kavanaugh, M.P. & Amara, S.G. Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc. Natl. Acad. Sci. USA 94, 4155–4160 (1997).

    Article  CAS  Google Scholar 

  5. Tanaka, K. et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702 (1997).

    Article  CAS  Google Scholar 

  6. Peghini, P., Janzen, J. & Stoffel, W. Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration. EMBO J. 16, 3822–3832 (1997).

    Article  CAS  Google Scholar 

  7. Rothstein, J.D. et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16, 675–686 (1996).

    Article  CAS  Google Scholar 

  8. Coco, S. et al. Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons. Eur. J. Neurosci. 9, 1902–1910 (1997).

    Article  CAS  Google Scholar 

  9. Sepkuty, J.P. et al. A neuronal glutamate transporter contributes to neurotransmitter GABA synthesis and epilepsy. J. Neurosci. 22, 6372–6379 (2002).

    Article  CAS  Google Scholar 

  10. Zerangue, N. & Kavanaugh, M.P. Interaction of L-cysteine with a human excitatory amino acid transporter. J. Physiol. (Lond.) 493, 419–423 (1996).

    Article  CAS  Google Scholar 

  11. Bendahan, A., Armon, A., Madani, N., Kavanaugh, M.P. & Kanner, B.I. Arginine 447 plays a pivotal role in substrate interactions in a neuronal glutamate transporter. J. Biol. Chem. 275, 37436–37442 (2000).

    Article  CAS  Google Scholar 

  12. Wu, G., Fang, Y.Z., Yang, S., Lupton, J.R. & Turner, N.D. Glutathione metabolism and its implications for health. J. Nutr. 134, 489–492 (2004).

    Article  CAS  Google Scholar 

  13. Dringen, R., Pfeiffer, B. & Hamprecht, B. Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J. Neurosci. 19, 562–569 (1999).

    Article  CAS  Google Scholar 

  14. Shanker, G., Allen, J.W., Mutkus, L.A. & Aschner, M. The uptake of cysteine in cultured primary astrocytes and neurons. Brain Res. 902, 156–163 (2001).

    Article  CAS  Google Scholar 

  15. Murphy, T.H., Schnaar, R.L. & Coyle, J.T. Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake. FASEB J. 4, 1624–1633 (1990).

    Article  CAS  Google Scholar 

  16. Sagara, J.I., Miura, K. & Bannai, S. Maintenance of neuronal glutathione by glial cells. J. Neurochem. 61, 1672–1676 (1993).

    Article  CAS  Google Scholar 

  17. Sato, H. et al. Distribution of cystine/glutamate exchange transporter, system x(c)-, in the mouse brain. J. Neurosci. 22, 8028–8033 (2002).

    Article  CAS  Google Scholar 

  18. Baker, D.A. et al. Neuroadaptations in cystine-glutamate exchange underlie cocaine relapse. Nat. Neurosci. 6, 743–749 (2003).

    Article  CAS  Google Scholar 

  19. Wang, X.F. & Cynader, M.S. Astrocytes provide cysteine to neurons by releasing glutathione. J. Neurochem. 74, 1434–1442 (2000).

    Article  CAS  Google Scholar 

  20. Dringen, R., Gutterer, J.M., Gros, C. & Hirrlinger, J. Aminopeptidase N mediates the utilization of the glutathione precursor CysGly by cultured neurons. J. Neurosci. Res. 66, 1003–1008 (2001).

    Article  CAS  Google Scholar 

  21. Chen, Y. & Swanson, R.A. The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures. J. Neurochem. 84, 1332–1339 (2003).

    Article  CAS  Google Scholar 

  22. Himi, T., Ikeda, M., Yasuhara, T., Nishida, M. & Morita, I. Role of neuronal glutamate transporter in the cysteine uptake and intracellular glutathione levels in cultured cortical neurons. J. Neural Transm. 110, 1337–1348 (2003).

    Article  CAS  Google Scholar 

  23. Dringen, R. Metabolism and functions of glutathione in brain. Prog. Neurobiol. 62, 649–671 (2000).

    Article  CAS  Google Scholar 

  24. Jain, A., Martensson, J., Stole, E., Auld, P.A. & Meister, A. Glutathione deficiency leads to mitochondrial damage in brain. Proc. Natl. Acad. Sci. USA 88, 1913–1917 (1991).

    Article  CAS  Google Scholar 

  25. Schulz, J.B., Lindenau, J., Seyfried, J. & Dichgans, J. Glutathione, oxidative stress and neurodegeneration. Eur. J. Biochem. 267, 4904–4911 (2000).

    Article  CAS  Google Scholar 

  26. Yang, Y., Kinney, G.A., Spain, W.J., Breitner, J.C. & Cook, D.G. Presenilin-1 and intracellular calcium stores regulate neuronal glutamate uptake. J. Neurochem. 88, 1361–1372 (2004).

    Article  CAS  Google Scholar 

  27. Morris, R. Development of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60 (1984).

    Article  CAS  Google Scholar 

  28. Hogg, N., Darley-Usmar, V.M., Wilson, M.T. & Moncada, S. Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochem. J. 281, 419–424 (1992).

    Article  CAS  Google Scholar 

  29. Himi, T., Ikeda, M., Yasuhara, T. & Murota, S.I. Oxidative neuronal death caused by glutamate uptake inhibition in cultured hippocampal neurons. J. Neurosci. Res. 71, 679–688 (2003).

    Article  CAS  Google Scholar 

  30. Hogg, N., Singh, R.J. & Kalyanaraman, B. The role of glutathione in the transport and catabolism of nitric oxide. FEBS Lett. 382, 223–228 (1996).

    Article  CAS  Google Scholar 

  31. Beckman, J.S. & Koppenol, W.H. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271, C1424–C1437 (1996).

    Article  CAS  Google Scholar 

  32. Griffith, O.W. & Meister, A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. Biol. Chem. 254, 7558–7560 (1979).

    CAS  PubMed  Google Scholar 

  33. Crow, J.P. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1, 145–157 (1997).

    Article  CAS  Google Scholar 

  34. Avshalumov, M.V. & Rice, M.E. NMDA receptor activation mediates hydrogen peroxide-induced pathophysiology in rat hippocampal slices. J. Neurophysiol. 87, 2896–2903 (2002).

    Article  CAS  Google Scholar 

  35. Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. & Prochiantz, A. Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307, 462–465 (1984).

    Article  CAS  Google Scholar 

  36. Grover, L.M. & Yan, C. Blockade of GABAA receptors facilitates induction of NMDA receptor-independent long-term potentiation. J. Neurophysiol. 81, 2814–2822 (1999).

    Article  CAS  Google Scholar 

  37. Mazor, D. et al. Red blood cell permeability to thiol compounds following oxidative stress. Eur. J. Haematol. 57, 241–246 (1996).

    Article  CAS  Google Scholar 

  38. Parsons, J.L. & Chipman, J.K. The role of glutathione in DNA damage by potassium bromate in vitro. Mutagenesis 15, 311–316 (2000).

    Article  CAS  Google Scholar 

  39. Corcoran, G.B. & Wong, B.K. Role of glutathione in prevention of acetaminophen-induced hepatotoxicity by N-acetyl-L-cysteine in vivo: studies with N-acetyl-D-cysteine in mice. J. Pharmacol. Exp. Ther. 238, 54–61 (1986).

    CAS  PubMed  Google Scholar 

  40. Umansky, V. et al. Glutathione is a factor of resistance of Jurkat leukemia cells to nitric oxide-mediated apoptosis. J. Cell. Biochem. 78, 578–587 (2000).

    Article  CAS  Google Scholar 

  41. Zerangue, N. & Kavanaugh, M.P. Flux coupling in a neuronal glutamate transporter. Nature 383, 634–637 (1996).

    Article  CAS  Google Scholar 

  42. Smith, C.P. et al. Assignment of the gene coding for the human high-affinity glutamate transporter EAAC1 to 9p24: potential role in dicarboxylic aminoaciduria and neurodegenerative disorders. Genomics 20, 335–336 (1994).

    Article  CAS  Google Scholar 

  43. Gonzalez, M.I., Kazanietz, M.G. & Robinson, M.B. Regulation of the neuronal glutamate transporter excitatory amino acid carrier-1 (EAAC1) by different protein kinase C subtypes. Mol. Pharmacol. 62, 901–910 (2002).

    Article  CAS  Google Scholar 

  44. Canolle, B. et al. Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol. J. Neurochem. 88, 1521–1532 (2004).

    Article  CAS  Google Scholar 

  45. Aoyama, K. et al. Acidosis causes endoplasmic reticulum stress and caspase-12-mediated astrocyte death. J. Cereb. Blood Flow Metab. 25, 258–370 (2005).

    Article  Google Scholar 

  46. Suh, S.W. et al. Hypoglycemic neuronal death and cognitive impairment are prevented by poly(ADP-ribose) polymerase inhibitors administered after hypoglycemia. J. Neurosci. 23, 10681–10690 (2003).

    Article  CAS  Google Scholar 

  47. Hochman, D.W. & Schwartzkroin, P.A. Chloride-cotransport blockade desynchronizes neuronal discharge in the “epileptic” hippocampal slice. J. Neurophysiol. 83, 406–417 (2000).

    Article  CAS  Google Scholar 

  48. Yin, H.Z., Sensi, S.L., Ogoshi, F. & Weiss, J.H. Blockade of Ca2+-permeable AMPA/kainate channels decreases oxygen-glucose deprivation-induced Zn2+ accumulation and neuronal loss in hippocampal pyramidal neurons. J. Neurosci. 22, 1273–1279 (2002).

    Article  CAS  Google Scholar 

  49. Meister, A., Anderson, M.E. & Hwang, O. Intracellular cysteine and glutathione delivery systems. J. Am. Coll. Nutr. 5, 137–151 (1986).

    Article  CAS  Google Scholar 

  50. Lantz, R.C., Lemus, R., Lange, R.W. & Karol, M.H. Rapid reduction of intracellular glutathione in human bronchial epithelial cells exposed to occupational levels of toluene diisocyanate. Toxicol. Sci. 60, 348–355 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Burns for technical assistance and M. Yenari and S. Massa for critical reading of the manuscript. This work was supported by grants from the US National Institutes of Health and the Department of Veterans Affairs.

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Authors and Affiliations

  1. Department of Neurology, University of California San Francisco, San Francisco, 94143, California, USA

    Koji Aoyama, Sang Won Suh, Aaron M Hamby, Wai Yee Chan, Yongmei Chen & Raymond A Swanson

  2. Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, 94121, California, USA

    Koji Aoyama, Sang Won Suh, Aaron M Hamby, Jialing Liu, Wai Yee Chan, Yongmei Chen & Raymond A Swanson

  3. Department of Neurosurgery, University of California San Francisco, San Francisco, 94143, California, USA

    Jialing Liu

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Supplementary Fig. 1

Markers of oxidant stress are absent from corpus callosum of aged EAAC1−/− mice. (PDF 416 kb)

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Aoyama, K., Suh, S., Hamby, A. et al. Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse. Nat Neurosci 9, 119–126 (2006). https://doi.org/10.1038/nn1609

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