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Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells

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Abstract

Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor–mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells.

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Abbreviations

AMPA:

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid

EAAT:

Excitatory amino acid transporter

EPSC:

Excitatory postsynaptic current

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GTRAP48:

Glutamate transporter-4-associated protein

LTD:

Long-term depression

mGluR:

Metabotropic glutamate receptors

mTOR:

Mammalian target of rapamycin

mTORC1:

mTOR complex 1

NF-кB:

Nuclear factor-кB

PSD95:

Postsynaptic density protein 95

Rheb:

Ras homolog enriched in brain

TSC:

Tuberous sclerosis complex

References

  1. Billups B, Rossi D, Oshima T, Warr O, Takahashi M, Sarantis M, et al. Physiological and pathological operation of glutamate transporters. Prog Brain Res. 1998;116:45–57.

    Article  CAS  PubMed  Google Scholar 

  2. Yi JH, Hazell AS. Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury. Neurochem Int. 2006;48:394–403.

    Article  CAS  PubMed  Google Scholar 

  3. Huang YH, Bergles DE. Glutamate transporters bring competition to the synapse. Curr Opin Neurobiol. 2004;14:346–52.

    Article  CAS  PubMed  Google Scholar 

  4. Halassa MM, Fellin T, Haydon PG. The tripartite synapse: roles for gliotransmission in health and disease. Trends Mol Med. 2007;13:54–63.

    Article  CAS  PubMed  Google Scholar 

  5. Seal RP, Amara SG. Excitatory amino acid transporters: a family in flux. Ann Rev Pharmacol Toxicol. 1999;39:431–56.

    Article  CAS  Google Scholar 

  6. Dehnes Y, Chaudhry FA, Ullensvang K, Lehre KP, Storm-Mathisen J, Danbolt NC. The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia. J Neurosci. 1998;18:3606–19.

    CAS  PubMed  Google Scholar 

  7. Fairman WA, Sonders MS, Murdoch GH, Amara SG. Arachidonic acid elicits a substrate-gated proton current associated with the glutamate transporter EAAT4. Nat Neurosci. 1998;1:105–13.

    Article  CAS  PubMed  Google Scholar 

  8. Massie A, Cnops L, Smolders I, McCullumsmith R, Kooijman R, Kwak S, et al. High-affinity Na+/K+-dependent glutamate transporter EAAT4 is expressed throughout the rat fore- and midbrain. J Comp Neurol. 2008;511:155–72.

    Article  CAS  PubMed  Google Scholar 

  9. Su LD, Shen Y. Blockade of glutamate transporters facilitates cerebellar synaptic long-term depression. Neuroreport. 2009;20:502–7.

    Article  PubMed  Google Scholar 

  10. Brasnjo G, Otis TS. Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression. Neuron. 2001;31:607–16.

    Article  CAS  PubMed  Google Scholar 

  11. Aspuria PJ, Tamanoi F. The Rheb family of GTP-binding proteins. Cell Signal. 2004;16:1105–12.

    Article  CAS  PubMed  Google Scholar 

  12. Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 2003;17:1829–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, Pan D. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol. 2003;5:578–81.

    Article  CAS  PubMed  Google Scholar 

  14. Manning BD, Cantley LC. Rheb fills a GAP between TSC and TOR. Trends Biochem Sci. 2003;28:573–6.

    Article  CAS  PubMed  Google Scholar 

  15. Stocker H, Radimerski T, Schindelholz B, Wittwer F, Belawat P, Daram P, et al. Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat Cell Biol. 2003;5:559–65.

    Article  CAS  PubMed  Google Scholar 

  16. Ji YF, Zhou L, Xie YJ, Xu SM, Zhu J, Teng P, et al. Upregulation of glutamate transporter GLT-1 by mTOR-Akt-NF-κB cascade in astrocytic oxygen-glucose deprivation. Glia. 2013;61:1959–75.

    Article  PubMed  Google Scholar 

  17. Zou J, Zhou L, Du XX, Ji Y, Xu J, Tian J, et al. Rheb1 is required for mTORC1 and myelination in postnatal brain development. Dev Cell. 2011;20:97–108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Barski JJ, Dethleffsen K, Meyer M. Cre recombinase expression in cerebellar Purkinje cells. Genesis. 2000;28:93–8.

    Article  CAS  PubMed  Google Scholar 

  19. Wu ZY, Zhu LJ, Zou N, Bombek LK, Shao CY, Wang N, et al. AMPA receptors regulate exocytosis and insulin release in pancreatic β cells. Traffic. 2012;13:1124–39.

    Article  CAS  PubMed  Google Scholar 

  20. Fukata Y, Lovero KL, Iwanaga T, Watanabe A, Yokoi N, Tabuchi K, et al. Disruption of LGI1-linked synaptic complex causes abnormal synaptic transmission and epilepsy. Proc Natl Acad Sci U S A. 2010;107:3799–804.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Shao CY, Zhu J, Xie YJ, Wang Z, Wang YN, Wang Y, et al. Distinct functions of nuclear distribution proteins LIS1, Ndel1 and NudCL in regulating axonal mitochondrial transport. Traffic. 2013;14:785–97.

    Article  CAS  PubMed  Google Scholar 

  22. Xie YJ, Zhou L, Jiang N, Zhang N, Zou N, Zhou L, et al. Essential roles of leucine-rich glioma inactivated 1 in the development of embryonic and postnatal cerebellum. Sci Rep. 2015;5:7827.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang DJ, Su LD, Wang YN, Yang D, Sun CL, Zhou L, et al. Long-term potentiation at cerebellar parallel fiber-Purkinje cell synapses requires presynaptic and postsynaptic signaling cascades. J Neurosci. 2014;34:2355–64.

    Article  CAS  PubMed  Google Scholar 

  24. Jackson M, Song W, Liu MY, Jin L, Dykes-Hoberg M, Lin CI, et al. Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins. Nature. 2001;410:89–93.

    Article  CAS  PubMed  Google Scholar 

  25. Harrington LS, Findlay GM, Gray A, Tolkacheva T, Wigfield S, Rebholz H, et al. The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J Cell Biol. 2004;166:213–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shah OJ, Hunter T. Critical role of T-loop and H-motif phosphorylation in the regulation of S6 kinase 1 by the tuberous sclerosis complex. J Biol Chem. 2004;279:20816–23.

    Article  CAS  PubMed  Google Scholar 

  27. O’Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006;66:1500–8.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell. 2006;22:159–68.

    Article  CAS  PubMed  Google Scholar 

  29. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005;307:1098–101.

    Article  CAS  PubMed  Google Scholar 

  30. Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H, et al. Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res. 2005;65:7052–8.

    Article  CAS  PubMed  Google Scholar 

  31. Shen Y, Linden DJ. Long-term potentiation of neuronal glutamate transporters. Neuron. 2005;46:715–22.

    Article  CAS  PubMed  Google Scholar 

  32. Shimamoto K, Lebrun B, Yasuda-Kamatani Y, Sakaitani M, Shigeri Y, Yumoto N, et al. DL-threo-beta-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. Mol Pharmacol. 1998;53:195–201.

    CAS  PubMed  Google Scholar 

  33. Huang YH, Dykes-Hoberg M, Tanaka K, Rothstein JD, Bergles DE. Climbing fiber activation of EAAT4 transporters and kainate receptors in cerebellar Purkinje cells. J Neurosci. 2004;24:103–11.

    Article  CAS  PubMed  Google Scholar 

  34. Nikkuni O, Takayasu Y, Iino M, Tanaka K, Ozawa S. Facilitated activation of metabotropic glutamate receptors in cerebellar Purkinje cells in glutamate transporter EAAT4-deficient mice. Neurosci Res. 2007;59:296–303.

    Article  CAS  PubMed  Google Scholar 

  35. Hansel C, Linden DJ, D'Angelo E. Beyond parallel fiber LTD: the diversity of synaptic and non-synaptic plasticity in the cerebellum. Nat Neurosci. 2001;4:467–75.

    CAS  PubMed  Google Scholar 

  36. Takayasu Y, Iino M, Kakegawa W, Maeno H, Watase K, Wada K, et al. Differential roles of glial and neuronal glutamate transporters in Purkinje cell synapses. J Neurosci. 2005;25:8788–93.

    Article  CAS  PubMed  Google Scholar 

  37. Watase K, Hashimoto K, Kano M, Yamada K, Watanabe M, Inoue Y, et al. Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. Eur J Neurosci. 1998;10:976–88.

    Article  CAS  PubMed  Google Scholar 

  38. Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science. 1997;276:1699–702.

    Article  CAS  PubMed  Google Scholar 

  39. Matsugami TR, Tanemura K, Mieda M, Nakatomi R, Yamada K, Kondo T, et al. From the Cover: Indispensability of the glutamate transporters GLAST and GLT1 to brain development. Proc Natl Acad Sci U S A. 2006;103:12161–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wadiche JI, Amara SG, Kavanaugh MP. Ion fluxes associated with excitatory amino acid transport. Neuron. 1995;15:721–8.

    Article  CAS  PubMed  Google Scholar 

  41. Wadiche JI, Kavanaugh MP. Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel. J Neurosci. 1998;18:7650–61.

    CAS  PubMed  Google Scholar 

  42. Auger C, Attwell D. Fast removal of synaptic glutamate by postsynaptic transporters. Neuron. 2000;28:547–58.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Dr. Bo Xiao (Sichuang University, Chengdu, China) for providing Rheb1f/f mice and Dr. Xiaobing Yuan (Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China) for providing L7-Cre mice. We are grateful for helpful advice from members of Shen lab. We thank the Core Facilities of Zhejiang University Institute of Neuroscience for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (31471024, 31271148, 81471398, and 31200818), Zhejiang Provincial Natural Science Foundation (LY15C090001), Seeds Fund for Interdisciplinary Research at Zhejiang University (JCZZ-2013037), Public Benefit Research Project of Science and Technology Department of Zhejiang Province (2013C33233), and Shenzhen Committee for Technological Renovation (ZDSY20120617112838879).

Conflict of Interest

All authors declare that there are no conflicts of interest on the paper.

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    Authors and Affiliations

    1. Ningbo Key Laboratory of Behavioral Neuroscience, Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Physiology and Pharmacology, Ningbo University School of Medicine, Ningbo, 315211, China

      Nan-Wei Jiang & Qin-Wen Wang

    2. Department of Neurobiology, Zhejiang University School of Medicine, 866 Yu Hang Tang Road, Hangzhou, 310058, China

      De-Juan Wang, Ya-Jun Xie, Liang Zhou, Li-Da Su & Ying Shen

    3. Neuroscience Care Unit, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China

      Li-Da Su

    4. Shenzhen Key laboratory for Molecular Biology of Neural Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

      Huashun Li

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    Correspondence to Qin-Wen Wang or Ying Shen.

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    Nan-Wei Jiang, De-Juan Wang and Ya-Jun Xie contributed equally to this work.

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    Jiang, NW., Wang, DJ., Xie, YJ. et al. Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells. Cerebellum 15, 314–321 (2016). https://doi.org/10.1007/s12311-015-0701-9

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