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Mapping of the cystine–glutamate exchanger in the mouse eye: a role for xCT in controlling extracellular redox balance

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Abstract

The cystine–glutamate exchanger (system xc−) is responsible for the exchange of extracellular cystine for intracellular glutamate. In this study, we mapped the expression of xCT, the light chain subunit of system xc− in the different tissues of 3–6-week-old mouse (C57BL/6J) eye and have used an xCT knockout mouse to verify labelling specificity. Moreover, using the xCT knockout mouse, we investigated whether xCT was involved in maintaining extracellular redox balance in the eye. xCT transcript and protein were present in the cornea, lens and retina of wild-type mice, but not knockout mice. xCT was localised to the corneal epithelium, and the lens epithelium and cortical fibre cells but was absent in the iris. xCT localisation could not be determined in the ciliary body or retina, since xCT labelling was also detected in the knockout indicating a lack of specificity of the xCT antibody in tissues of a neural origin. Intracellular cysteine and cystine concentrations were similar in the wild-type and xCT knockout mouse for the cornea, lens, and retina. While extracellular cysteine levels were similar between the plasma, aqueous humour, and vitreous humour of the wild-type and xCT knockout mouse, extracellular cystine levels in the plasma and aqueous were significantly elevated in the xCT knockout mouse relative to the wild type. This suggests that loss of xCT results in an increased oxidative environment, particularly within the anterior chamber of the eye in which the aqueous humour resides. How this oxidative shift impacts ocular tissues that interface with the aqueous humour over time will be the focus of future work.

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References

  • Atluri H, Talluri RS, Mitra AK (2008) Functional activity of a large neutral amino acid transporter (LAT) in rabbit retina: a study involving the in vivo retinal uptake and vitreal pharmacokinetics of l-phenyl alanine. Int J Pharm 347(1–2):23–30

    Article  CAS  Google Scholar 

  • Baker DA, Xi Z-X, Shen H, Swanson CJ, Kalivas PW (2002) The origin and neuronal function of in vivo nonsynaptic glutamate. J Neurosci 22(20):9134–9141

    Article  CAS  Google Scholar 

  • Baker DA, McFarland K, Lake RW, Shen H, Tang X-C, Toda S, Kalivas PW (2003) Neuroadaptations in cystine–glutamate exchange underlie cocaine relapse. Nat Neurosci 6(7):743

    Article  CAS  Google Scholar 

  • Banjac A, Perisic T, Sato H, Seiler A, Bannai S, Weiss N et al (2008) The cystine/cysteine cycle: a redox cycle regulating susceptibility versus resistance to cell death. Oncogene 27(11):1618–1628. https://doi.org/10.1038/sj.onc.1210796

    Article  CAS  PubMed  Google Scholar 

  • Bannai S, Kitamura E (1980) Transport interaction of L-cystine and L-glutamate in human diploid fibroblasts in culture. J Biol Chem 255(6):2372–2376

    CAS  PubMed  Google Scholar 

  • Bannai S, Sato H, Ishii T, Sugita Y (1989) Induction of cystine transport activity in human fibroblasts by oxygen. J Biol Chem 264(31):18480–18484

    CAS  PubMed  Google Scholar 

  • Bridges CC, Hu H, Miyauchi S, Siddaramappa UN, Ganapathy ME, Ignatowicz L, Maddox DM, Smith SB, Ganapathy V (2004) Induction of cystine-glutamate transporter xc- by human immunodeficiency virus type 1 transactivator protein tat in retinal pigment epithelium. Investig Ophthalmol Vis Sci 45(9):2906–2914

    Article  Google Scholar 

  • Chen Y, Swanson RA (2003) The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures. J Neurochem 84(6):1332–1339

    Article  CAS  Google Scholar 

  • De Bundel D, Schallier A, Loyens E, Fernando R, Miyashita H, Van Liefferinge J, Vermoesen K, Bannai S, Sato H, Michotte Y (2011) Loss of system xc- does not induce oxidative stress but decreases extracellular glutamate in hippocampus and influences spatial working memory and limbic seizure susceptibility. J Neurosci 31(15):5792–5803

    Article  Google Scholar 

  • Delamere NA (2005) Ciliary body and ciliary epithelium. Adv Organ Biol 10:127–148

    Article  Google Scholar 

  • Dun Y, Mysona B, Van Ells T, Amarnath L, Shamsul Ola M, Ganapathy V, Smith SB (2006) Expression of the cystine-glutamate exchanger (xc-) in retinal ganglion cells and regulation by nitric oxide and oxidative stress. Cell Tissue Res 324(2):189–202

    Article  CAS  Google Scholar 

  • Fukushima D, Doi H, Fukushima K, Katsura K, Ogawa N, Sekiguchi S, Fujimori K, Sato A, Satomi S, Ishida K (2010) Glutamate exocrine dynamics augmented by plasma glutamine and the distribution of amino acid transporters of the rats pancreas. J Physiol Pharmacol 61(3):265–271

    CAS  PubMed  Google Scholar 

  • Gu Y, Albuquerque CP, Braas D, Zhang W, Villa GR, Bi J et al (2017) mTORC2 regulates amino acid metabolism in cancer by phosphorylation of the cystine-glutamate antiporter xCT. Mol Cell 67(1):128–138.e7

    Article  CAS  Google Scholar 

  • Guo CX, Nor MNM, Danesh-Meyer HV, Vessey KA, Fletcher EL, O’Carroll SJ, Acosta ML, Green CR (2016) Connexin43 mimetic peptide improves retinal function and reduces inflammation in a light-damaged albino rat model. Investig Ophthalmol Vis Sci 57(10):3961–3973

    Article  CAS  Google Scholar 

  • Hu RG, Lim J, Donaldson PJ, Kalloniatis M (2008) Characterization of the cystine/glutamate transporter in the outer plexiform layer of the vertebrate retina. Eur J Neurosci 28(8):1491–1502

    Article  Google Scholar 

  • Jacobs MD, Donaldson PJ, Cannell MB, Soeller C (2003) Resolving morphology and antibody labeling over large distances in tissue sections. Microsc Res Tech 62(1):83–91

    Article  CAS  Google Scholar 

  • Jones DP, Mody VC Jr, Carlson JL, Lynn MJ, Sternberg P Jr (2002) Redox analysis of human plasma allows separation of pro-oxidant events of aging from decline in antioxidant defenses. Free Radic Biol Med 33(9):1290–1300

    Article  CAS  Google Scholar 

  • Kinoshita H, Okabe H, Beppu T, Chikamoto A, Hayashi H, Imai K, Mima K, Nakagawa S, Ishimoto T, Miyake K (2013) Cystine/glutamic acid transporter is a novel marker for predicting poor survival in patients with hepatocellular carcinoma. Oncol Rep 29(2):685–689

    Article  CAS  Google Scholar 

  • Kunikata H, Ida T, Sato K, Aizawa N, Sawa T, Tawarayama H, Murayama N, Fujii S, Akaike T, Nakazawa T (2017) Metabolomic profiling of reactive persulfides and polysulfides in the aqueous and vitreous humors. Sci Rep 7:1–10

    Article  Google Scholar 

  • La Bella V, Valentino F, Piccoli T, Piccoli F (2007) Expression and developmental regulation of the cystine/glutamate exchanger (xc-) in the rat. Neurochem Res 32(6):1081–1090

    Article  Google Scholar 

  • Lall MM, Ferrell J, Nagar S, Fleisher LN, McGahan MC (2008) Iron regulates L-cystine uptake and glutathione levels in lens epithelial and retinal pigment epithelial cells by its effect on cytosolic aconitase. Investig Ophthalmol Vis Sci 49(1):310–319

    Article  Google Scholar 

  • Langford MP, Redmond P, Chanis R, Misra RP, Redens TB (2010) Glutamate, excitatory amino acid transporters, xc- antiporter, glutamine synthetase, and γ-glutamyltranspeptidase in human corneal epithelium. Curr Eye Res 35(3):202–211

    Article  CAS  Google Scholar 

  • Langford MP, Redens TB, Liang C, Kavanaugh AS, Texada DE (2015a) EAAT and Xc-exchanger inhibition depletes glutathione in the transformed human lens epithelial cell line SRA 01/04. Current Eye Res 41(3):357–366

    Google Scholar 

  • Langford MP, Redens TB, Texada DE (2015b) Excitatory amino acid transporters, xc- antiporter, γ-glutamyl transpeptidase, glutamine synthetase, and glutathione in human corneal epithelial cells. In: Studies on the cornea and lens. Springer, New York, pp 67–82

    Google Scholar 

  • Lewerenz J, Hewett SJ, Huang Y, Lambros M, Gout PW, Kalivas PW, Massie A, Smolders I, Methner A, Pergande M (2013) The cystine/glutamate antiporter system xc- in health and disease: from molecular mechanisms to novel therapeutic opportunities. Antioxid Redox Signal 18(5):522–555

    Article  CAS  Google Scholar 

  • Li B, Lee MS, Lee RS, Donaldson PJ, Lim JC (2012) Characterization of glutathione uptake, synthesis, and efflux pathways in the epithelium and endothelium of the rat cornea. Cornea 31(11):1304–1312

    Article  Google Scholar 

  • Li B, Umapathy A, Tran LU, Donaldson PJ, Lim JC (2013) Molecular identification and cellular localisation of GSH synthesis, uptake, efflux and degradation pathways in the rat ciliary body. Histochem Cell Biol 139(4):559–571

    Article  CAS  Google Scholar 

  • Lim J, Lam YC, Kistler J, Donaldson PJ (2005) Molecular characterization of the cystine/glutamate exchanger and the excitatory amino acid transporters in the rat lens. Investig Ophthalmol Vis Sci 46(8):2869–2877

    Article  Google Scholar 

  • Lim J, Lorentzen KA, Kistler J, Donaldson PJ (2006) Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens. Exp Eye Res 83(2):447–455

    Article  CAS  Google Scholar 

  • Lim JC, Lam L, Li B, Donaldson PJ (2013) Molecular identification and cellular localization of a potential transport system involved in cystine/cysteine uptake in human lenses. Exp Eye Res 116:219–226

    Article  CAS  Google Scholar 

  • Mackic JB, Kannan RAM, Kaplowitz N, Zlokovic BV (1997) Low de novo glutathione synthesis from circulating sulfur amino acids in the lens epithelium. Exp Eye Res 64(4):615–626

    Article  CAS  Google Scholar 

  • Massie A, Schallier A, Kim SW, Fernando R, Kobayashi S, Beck H, De Bundel D, Vermoesen K, Bannai S, Smolders I, Conrad M, Plesnila N, Sato H, Michotte Y (2011) Dopaminergic neurons of system xc- deficient mice are highly protected against 6-hydroxydopamine-induced toxicity. FASEB J 25(4):1359–1369

    Article  CAS  Google Scholar 

  • Merckx E, Albertini G, Paterka M, Jensen C, Albrecht P, Dietrich M, Van Liefferinge J, Bentea E, Verbruggen L, Demuyser T (2017) Absence of system xc- on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis. J Neuroinflammation 14(1):9

    Article  Google Scholar 

  • Mysona B, Dun Y, Duplantier J, Ganapathy V, Smith SB (2009) Effects of hyperglycemia and oxidative stress on the glutamate transporters GLAST and system xc- in mouse retinal Müller glial cells. Cell Tissue Res 335(3):477–488

    Article  CAS  Google Scholar 

  • O’Brien KB, Miller RF, Bowser MT (2005) D-Serine uptake by isolated retinas is consistent with ASCT-mediated transport. Neurosci Lett 385(1):58–63

    Article  Google Scholar 

  • Persa C, Pierce A, Ma Z, Kabil O, Lou MF (2004) The presence of a transsulfuration pathway in the lens: a new oxidative stress defense system. Exp Eye Res 79(6):875–886

    Article  CAS  Google Scholar 

  • Persa C, Osmotherly K, Chen KC-W, Moon S, Lou MF (2006) The distribution of cystathionine β-synthase (CBS) in the eye: implication of the presence of a trans-sulfuration pathway for oxidative stress defense. Exp Eye Res 83(4):817–823

    Article  CAS  Google Scholar 

  • Pow DV (2001) Visualising the activity of the cystine-glutamate antiporter in glial cells using antibodies to aminoadipic acid, a selectively transported substrate. Glia 34(1):27–38

    Article  CAS  Google Scholar 

  • Sato H, Tamba M, Ishii T, Bannai S (1999) Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem 274(17):11455–11458

    Article  CAS  Google Scholar 

  • Sato H, Shiiya A, Kimata M, Maebara K, Tamba M, Sakakura Y, Makino N, Sugiyama F, Yagami K, Moriguchi T, Takahashi S, Bannai S (2005) Redox imbalance in cystine/glutamate transporter-deficient mice. J Biol Chem 280(45):37423–37429

    Article  CAS  Google Scholar 

  • Shanker G, Allen JW, Mutkus LA, Aschner M (2001) The uptake of cysteine in cultured primary astrocytes and neurons. Brain Res 902(2):156–163

    Article  CAS  Google Scholar 

  • Shih AY, Erb H, Sun X, Toda S, Kalivas PW, Murphy TH (2006) Cystine/glutamate exchange modulates glutathione supply for neuroprotection from oxidative stress and cell proliferation. J Neurosci 26(41):10514–10523

    Article  CAS  Google Scholar 

  • Smith SL, Trachtenberg JT (2007) Experience-dependent binocular competition in the visual cortex begins at eye opening. Nat Neurosci 10(3):370–375

    Article  CAS  Google Scholar 

  • Tomi M, Funaki T, Abukawa H, Katayama K, Kondo T, Ohtsuki S, Ueda M, Obinata M, Terasaki T, Hosoya KI (2003) Expression and regulation of L-cystine transporter, system xc-, in the newly developed rat retinal Müller cell line (TR-MUL). Glia 43(3):208–217

    Article  Google Scholar 

  • Van Liefferinge J, Bentea E, Demuyser T, Albertini G, Follin-Arbelet V, Holmseth S, Merckx E, Sato H, Aerts JL, Smolders I (2016) Comparative analysis of antibodies to xCT (Slc7a11): forewarned is forearmed. J Comp Neurol 2016:1015–1032

    Article  Google Scholar 

  • Wagner CA, Lang F, Bröer S (2001) Function and structure of heterodimeric amino acid transporters. Am J Physiol Cell Physiol 281(4504):1077–1093

    Article  Google Scholar 

  • Watanabe H, Bannai S (1987) Induction of cystine transport activity in mouse peritoneal macrophages. J Exp Med 165(3):628–640

    Article  CAS  Google Scholar 

  • Yoganandarajah V, Li B, Umapathy A, Donaldson PJ, Lim JC (2017) Regional differences in glutathione accumulation pathways in the rat cornea: mapping of amino acid transporters involved in glutathione synthesis. Exp Eye Res 161:89–100

    Article  CAS  Google Scholar 

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Acknowledgements

The researchers wish to acknowledge funding from Auckland Medical Research Fund, the Faculty Research Development Fund from the University of Auckland, the Maurice and Phyllis Paykel Trust, the New Zealand Optometric Vision Research Foundation, the Hope Foundation for Research on Ageing and the New Zealand Association of Optometrists. In addition, we would also like to thank Professor Hideyo Sato for providing us with the xCT KO mice, Associate Professor Robb de Iongh for the mouse embryos, and Ms Satya Amirapu for her assistance with paraffin embedding of P3 lenses and sectioning and deparaffinising of the P3 and E16 tissues.

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Authors and Affiliations

  1. Department of Physiology, School of Medical and Health Sciences, University of Auckland, Auckland, 1023, New Zealand

    Renita M. Martis, Paul J. Donaldson, Bo Li & Julie C. Lim

  2. Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand

    Prasanna K. Kallingappa

  3. School of Medical Sciences, University of Auckland, Auckland, New Zealand

    Renita M. Martis, Paul J. Donaldson, Bo Li, Prasanna K. Kallingappa & Julie C. Lim

  4. NZ National Eye Centre, University of Auckland, Auckland, New Zealand

    Renita M. Martis, Paul J. Donaldson, Bo Li & Julie C. Lim

  5. School of Biological Sciences, University of Auckland, Auckland, New Zealand

    Martin Middleditch

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Correspondence to Julie C. Lim.

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Martis, R.M., Donaldson, P.J., Li, B. et al. Mapping of the cystine–glutamate exchanger in the mouse eye: a role for xCT in controlling extracellular redox balance. Histochem Cell Biol 152, 293–310 (2019). https://doi.org/10.1007/s00418-019-01805-4

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