Abstract
Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD.
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Abbreviations
- ABD-F:
-
4-Flouro-7-aminosulfonylbenzofurazan
- AD:
-
Alzheimer’s disease
- ANOVA:
-
Analysis of variance
- ARE:
-
Antioxidant response element
- CSF:
-
Cerebral spinal fluid
- DMEM:
-
Dulbecco’s modified eagle medium
- DTNB:
-
5,5′-Dithio-bis(2-nitrobenzoic acid)
- EDTA:
-
Ethylenediaminetetraacetic acid
- ELISA:
-
Enzyme-linked immunosorbent sandwich assay
- FBS:
-
Foetal bovine serum
- GCL:
-
Glutamate cysteine ligase
- GSH:
-
Glutathione
- GSSG:
-
Glutathione disulfide
- HPLC:
-
High performance liquid chromatography
- IL-1β:
-
Interleukin-1 beta
- IL-6:
-
Interleukin-6
- Keap1:
-
Kelch like-ECH-associated protein 1
- MRP1:
-
Multidrug resistance protein 1
- NADPH:
-
Reduced nicotinamide adenine dinucleotide phosphate
- NMDA:
-
N-Methyl-D-aspartate
- Nrf2:
-
Nuclear factor erythroid-2-related factor 2
- PD:
-
Parkinson’s disease
- ROS:
-
Reactive oxygen species
- TNB:
-
5-Thio-2-nitrobenzoic acid
- TNF-α:
-
Tumour necrosis factor-alpha
- γ-GT:
-
γ-Glutamyltranspeptidase
- γ-GCL-C:
-
Catalytic glutamate cysteine ligase subunit
- γGCL-M:
-
Modulatory glutamate cysteine ligase subunit
References
Althausen S, Paschen W (2000) Homocysteine-induced changes in mRNA levels of genes coding for cytoplasmic- and endoplasmic reticulum-resident stress proteins in neuronal cell cultures. Brain Res Mol Brain Res 84:32–40
Aschner M (2000) Neuron-astrocyte interactions: implications for cellular energetics and antioxidant levels. Neurotoxicology 21:1101–1107
Bannai S (1984) Transport of cystine and cysteine in mammalian cells. Biochim Biophys Acta 779:289–306
Bannai S, Kitamura E (1980) Transport interaction of l-cystine and l-glutamate in human diploid fibroblasts in culture. J Biol Chem 255:2372–2376
Bezzi P, Domercq M, Brambilla L, Galli R, Schols D, De Clercq E, Vescovi A, Bagetta G, Kollias G, Meldolesi J, Volterra A (2001) CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity. Nat Neurosci 4:702–710
Bolin LM, Zhaung A, Strychkarska-Orczyk I, Nelson E, Huang I, Malit M, Nguyen Q (2005) Differential inflammatory activation of IL-6 (−/−) astrocytes. Cytokine 30:47–55
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brannan TS, Maker HS, Weiss C, Cohen G (1980) Regional distribution of glutathione peroxidase in the adult rat brain. J Neurochem 35:1013–1014
Buranrat B, Prawan A, Kukongviriyapan V (2008) Optimization of resazurin-based assay for cytotoxicity test in cholangiocarcinoma cells. KKU Res J 8:73–80
Chan JY, Kwong M (2000) Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein. Biochim Biophys Acta 1517:19–26
Chanas SA, Jiang Q, McMahon M, McWalter GK, McLellan LI, Elcombe CR, Henderson CJ, Wolf CR, Moffat GJ, Itoh K, Yamamoto M, Hayes JD (2002) Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. Biochem J 365:405–416
Chen Y, Swanson RA (2003) The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures. J Neurochem 84:1332–1339
Chen Y, Shertzer HG, Schneider SN, Nebert DW, Dalton TP (2005) Glutamate cysteine ligase catalysis: dependence on ATP and modifier subunit for regulation of tissue glutathione levels. J Biol Chem 280:33766–33774
Copple IM, Goldring CE, Kitteringham NR, Park BK (2008) The Nrf2-Keap1 defence pathway: role in protection against drug-induced toxicity. Toxicology 246:24–33
Correa F, Ljunggren E, Mallard C, Nilsson M, Weber SG, Sandberg M (2011) The Nrf2-inducible antioxidant defense in astrocytes can be both up- and down-regulated by activated microglia: involvement of p38 MAPK. Glia 59:785–799
Dedon PC, Tannenbaum SR (2004) Reactive nitrogen species in the chemical biology of inflammation. Arch Biochem Biophys 423:12–22
Dringen R (2000) Metabolism and functions of glutathione in brain. Prog Neurobiol 62:649–671
Dringen R, Hamprecht B (1997) Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells. Brain Res 759:67–75
Dringen R, Hamprecht B (1999) N-Acetylcysteine, but not methionine or 2-oxothiazolidine-4-carboxylate, serves as cysteine donor for the synthesis of glutathione in cultured neurons derived from embryonal rat brain. Neurosci Lett 259:79–82
Dringen R, Pfeiffer B, Hamprecht B (1999) Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J Neurosci 19:562–569
Dwyer BE, Raina AK, Perry G, Smith MA (2004) Homocysteine and Alzheimer’s disease: a modifiable risk? Free Radic Biol Med 36:1471–1475
Gavillet M, Allaman I, Magistretti P (2008) Modulation of astrocytic metabolic phenotype by proinflammatory cytokines. Glia 56:975–989
Harvey CJ, Thimmulappa RK, Singh A, Blake DJ, Ling G, Wakabayashi N, Fujii J, Myers A, Biswal S (2009) Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress. Free Radic Biol Med 46:443–453
Hernanz A, De la Fuente M, Navarro M, Frank A (2007) Plasma aminothiol compounds, but not serum tumor necrosis factor receptor II and soluble receptor for advanced glycation end products, are related to the cognitive impairment in Alzheimer’s disease and mild cognitive impairment patients. NeuroImmunoModulation 14:163–167
Ho PI, Ortiz D, Rogers E, Shea TB (2002) Multiple aspects of homocysteine neurotoxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. J Neurosci Res 70:694–702
Huang G, Dragan M, Freeman D, Wilson JX (2005) Activation of catechol-O-methyltransferase in astrocytes stimulates homocysteine synthesis and export to neurons. Glia 51:47–55
Jackman NA, Uliasz TF, Hewett JA, Hewett SJ (2010) Regulation of system x(c)(−)activity and expression in astrocytes by interleukin-1beta: implications for hypoxic neuronal injury. Glia 58:1806–1815
Jin Y, Brennan L (2008) Effects of homocysteine on metabolic pathways in cultured astrocytes. Neurochem Int 52:1410–1415
Jones DP, Carlson JL, Mody VC, Cai J, Lynn MJ, Sternberg P (2000) Redox state of glutathione in human plasma. Free Radic Biol Med 28:625–635
Kohl RL, Quay WB (1979) Cystathionine synthase in rat brain: regional and time-of-day differences and their metabolic implications. J Neurosci Res 4:189–196
Kranich O, Hamprecht B, Dringen R (1996) Different preferences in the utilization of amino acids for glutathione synthesis in cultured neurons and astroglial cells derived from rat brain. Neurosci Lett 219:211–214
Kruman II, Culmsee C, Chan SL, Kruman Y, Guo Z, Penix L, Mattson MP (2000) Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J Neurosci 20:6920–6926
Li Z, Sun L, Zhang H, Liao Y, Wang D, Zhao B, Zhu Z, Zhao J, Ma A, Han Y, Wang Y, Shi Y, Ye J, Hui R (2003) Elevated plasma homocysteine was associated with hemorrhagic and ischemic stroke, but methylenetetrahydrofolate reductase gene C677T polymorphism was a risk factor for thrombotic stroke: a multicenter case-control study in China. Stroke 34:2085–2090
Lipton SA, Kim WK, Choi YB, Kumar S, D’Emilia DM, Rayudu PV, Arnelle DR, Stamler JS (1997) Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA 94:5923–5928
Malaplate-Armand C, Gueguen Y, Bertrand P, Ferrari L, Batt AM (2000) U373-MG response to interleukin-1beta-induced oxidative stress. Cell Biol Toxicol 16:155–163
Mandal PK, Tripathi M, Sugunan S (2012) Brain oxidative stress: detection and mapping of anti-oxidant marker ‘glutathione’ in different brain regions of healthy male/female, MCI and Alzheimer patients using non-invasive magnetic resonance spectroscopy. Biochem Biophys Res Commun 417:43–48
McCaddon A, Hudson P, Hill D, Barber J, Lloyd A, Davies G, Regland B (2003) Alzheimer’s disease and total plasma aminothiols. Biol Psychiatry 53:254–260
Meister A (1988) Glutathione metabolism and its selective modification. J Biol Chem 263:17205–17208
Minich T, Riemer J, Schulz JB, Wielinga P, Wijnholds J, Dringen R (2006) The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulfide from brain astrocytes. J Neurochem 97:373–384
Morris MS (2003) Homocysteine and Alzheimer’s disease. Lancet Neurol 2:425–428
Nicolaou A, Kenyon SH, Gibbons JM, Ast T, Gibbons WA (1996) In vitro inactivation of mammalian methionine synthase by nitric oxide. Eur J Clin Invest 26:167–170
Park SK, Murphy S (1994) Duration of expression of inducible nitric oxide synthase in glial cells. J Neurosci Res 39:405–411
Park Y, Ziegler TR, Gletsu-Miller N, Liang Y, Yu T, Accardi CJ, Jones DP (2010) Postprandial cysteine/cystine redox potential in human plasma varies with meal content of sulfur amino acids. J Nutr 140:760–765
Pietrzik K (2006) Homocysteine as a cardiovascular marker and risk factor. Clin Res Cardiol 95(Suppl 6):VI28–VI33
Popp J, Lewczuk P, Linnebank M, Cvetanovska G, Smulders Y, Kolsch H, Frommann I, Kornhuber J, Maier W, Jessen F (2009) Homocysteine metabolism and cerebrospinal fluid markers for Alzheimer’s disease. J Alzheimers Dis 18:819–828
Robert K, Vialard F, Thiery E, Toyama K, Sinet PM, Janel N, London J (2003) Expression of the cystathionine beta synthase (CBS) gene during mouse development and immunolocalization in adult brain. J Histochem Cytochem 51:363–371
Rushworth SA, Shah S, MacEwan DJ (2011) TNF mediates the sustained activation of Nrf2 in human monocytes. J Immunol 187:702–707
Sagara JI, Miura K, Bannai S (1993) Maintenance of neuronal glutathione by glial cells. J Neurochem 61:1672–1676
Sato H, Fujiwara K, Sagara J, Bannai S (1995) Induction of cystine transport activity in mouse peritoneal macrophages by bacterial lipopolysaccharide. Biochem J 310(Pt 2):547–551
Seshadri S (2006) Elevated plasma homocysteine levels: risk factor or risk marker for the development of dementia and Alzheimer’s disease? J Alzheimers Dis 9:393–398
Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D’Agostino RB, Wilson PW, Wolf PA (2002) Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 346:476–483
Shanker G, Aschner M (2001) Identification and characterization of uptake systems for cystine and cysteine in cultured astrocytes and neurons: evidence for methylmercury-targeted disruption of astrocyte transport. J Neurosci Res 66:998–1002
Shanker G, Allen JW, Mutkus LA, Aschner M (2001) The uptake of cysteine in cultured primary astrocytes and neurons. Brain Res 902:156–163
Steele ML, Robinson SR (2011) Reactive astrocytes give neurons less support: implications for Alzheimer’s disease. Neurobiol Aging 33: 423.e421–413
Steele M, Stuchbury G, Munch G (2007) The molecular basis of the prevention of Alzheimer’s disease through healthy nutrition. Exp Gerontol 42:28–36
Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27:502–522
Tsitsiou E, Sibley CP, D’Souza SW, Catanescu O, Jacobsen DW, Glazier JD (2009) Homocysteine transport by systems L, A and y + L across the microvillous plasma membrane of human placenta. J Physiol 587:4001–4013
Vitvitsky V, Thomas M, Ghorpade A, Gendelman HE, Banerjee R (2006) A functional transsulfuration pathway in the brain links to glutathione homeostasis. J Biol Chem 281:35785–35793
Wild AC, Moinova HR, Mulcahy RT (1999) Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. J Biol Chem 274:33627–33636
Winrow VR, Winyard PG, Morris CJ, Blake DR (1993) Free radicals in inflammation: second messengers and mediators of tissue destruction. Br Med Bull 49:506–522
Yoshiba-Suzuki S, Sagara J, Bannai S, Makino N (2011) The dynamics of cysteine, glutathione and their disulphides in astrocyte culture medium. J Biochem 150:95–102
Acknowledgments
We thank UWS for support through the College Research Grant scheme and Alzheimer’s Australia for their support through the Dementia Research grants program. Megan L. Steele was supported by the Hunter Postgraduate Research Scholarship from Alzheimer’s Australia.
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Steele, M.L., Fuller, S., Maczurek, A.E. et al. Chronic Inflammation Alters Production and Release of Glutathione and Related Thiols in Human U373 Astroglial Cells. Cell Mol Neurobiol 33, 19–30 (2013). https://doi.org/10.1007/s10571-012-9867-6
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DOI: https://doi.org/10.1007/s10571-012-9867-6