Elsevier

Biochimie

Volume 94, Issue 3, March 2012, Pages 617-627
Biochimie

Research paper
Extracellular cysteine (Cys)/cystine (CySS) redox regulates metabotropic glutamate receptor 5 activity

https://doi.org/10.1016/j.biochi.2011.09.013Get rights and content

Abstract

Extracellular cysteine (Cys)/cystine (CySS) redox potential (Eh) has been shown to regulate diverse biological processes, including enzyme catalysis, gene expression, and signaling pathways for cell proliferation and apoptosis, and is sensitive to aging, smoking, and other host factors. However, the effects of extracellular Cys/CySS redox on the nervous system remain unknown. In this study, we explored the role of extracellular Cys/CySS Eh in metabotropic glutamate receptor 5 (mGlu5) activation to understand the mechanism of its regulation of nerve cell growth and activation. We showed that the oxidized Cys/CySS redox state (0 mV) in C6 glial cells induced a significant increase in mGlu5-mediated phosphorylation of extracellular signal-regulated kinase (ERK), blocked by an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (MEK), U0126, a nonpermeant alkylating agent, 4-acetamide-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS), and a specific mGlu5 antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), respectively. ERK phosphorylation under oxidized extracellular Cys/CySS Eh was confirmed in mGlu5-overexpressed human embryonic kidney 293 (HEK293) cells. Oxidized extracellular Cys/CySS Eh also stimulated the generation of intracellular reactive oxygen species (ROS) involved in the phosphorylation of ERK by mGlu5. Moreover, activation of mGlu5 by oxidized extracellular Cys/CySS Eh was found to affect expression of NF-κB and inducible nitric oxide synthase (iNOS). The results also showed that extracellular Cys/CySS Eh involved in the activation of mGlu5 controlled cell death and cell activation in neurotoxicity. In addition, plasma Cys/CySS Eh was found to be associated with the process of Parkinson’s disease (PD) in a rotenone-induced rat model of PD together with dietary deficiency and supplementation of sulfur amino acid (SAA). The effects of extracellular Cys/CySS Eh on SAA dietary deficiency in the rotenone-induced rat model of PD was almost blocked by MPEP pretreatment, further indicating that oxidized extracellular Cys/CySS Eh plays a role in mGlu5 activity. Taken together, the results indicate that mGlu5 can be activated by extracellular Cys/CySS redox in nerve cells, which possibly contributes to the process of PD. These in vitro and in vivo findings may aid in the development of potential new nutritional strategies that could assist in slowing the degeneration of PD.

Highlights

► Oxidized extracellular Cys/CySS Eh induces an increase in mGlu5-mediated ERK activity. ► Oxidized extracellular Cys/CySS Eh stimulates ROS involved in ERK activity by mGlu5. ► Oxidized extracellular Cys/CySS Eh affects expression of NF-κB and iNOS by mGlu5. ► Extracellular Cys/CySS Eh controls cell death and cell activation in neurotoxicity. ► Cys/CySS Eh is associated with PD process in rat model of PD together with SAA diets.

Introduction

Reversible redox reactions of thiol/disulfide couples regulate diverse biologic processes, including enzyme catalysis, gene expression, and signaling for cell proliferation and apoptosis [1], [2], [3]. The cysteine (Cys)/cystine (CySS) redox couple represents the predominant low-molecular-weight thiol/disulfide pool found in plasma and is sensitive to aging, smoking, and other host factors [4], [5]. These findings are intriguing particularly when considered in conjunction with data showing that alteration in extracellular Cys/CySS redox potential (Eh) can drive signal transduction [6]. Moreover, extracellular Cys/CySS redox state also has been implicated in cell growth and apoptosis [3], [6], [7]. Depressed thiols levels in plasma and tissues have been implicated in a number of human diseases such as Alzheimer’s and Parkinson’s, diabetes, cystic fibrosis, and HIV infection [8], [9], [10], [11].

Metabotropic glutamate receptors (mGluRs), a type of G-protein-coupled receptor (GPCR), are a large family of surface receptors composed of multiple domains. They possess an extracellular Venus flytrap domain (VFT) where agonists bind and a heptahelical transmembrane domain (HD) is common to all GPCRs that is responsible for G-protein activation. For most of these receptors, a cysteine-rich domain (CRD) is linked to the two domains [12], [13]. CRD is composed of nine highly conserved Cys and is known to be important for signal transduction in mGlu-like receptors [14]. mGlu5, a type of group I mGluR, is a disulfide-linked dimer [15], making it a good candidate for evaluating sensitivity to extracellular thiol/disulfide redox.

mGlu5, widely expressed in astrocytes, has been utilized as a target for pharmacotherapy in Parkinson’s disease (PD) [16], [17]. PD is a complex chronic neurodegenerative disorder primarily involving loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNc), decay of the nigrostriatal tract, symptomatic rigidity, bradykinesia, and resting tremor. As tyrosine hydroxylase (TH) catalyzes the formation of l-3,4-dihydroxyphenylalanine (l-DOPA), the rate-limiting step in the biosynthesis of dopamine, the disease can be considered a TH-deficiency syndrome. Moreover, loss of these neurons is also associated with non-neuronal pathology such as activated microglial cells and reactive astrocytes [18]. Glial cells may be a source of trophic factors and can protect against reactive oxygen species (ROS) and glutamate. They can also mediate a variety of deleterious events related to the production of reactive oxygen and nitrogen species of cytokines that may contribute to neuronal injury and cell death in PD [19].

Based on the above evidence, we hypothesized that extracellular Cys/CySS Eh could affect the activation of mGlu5 and may have a role in the pathogenesis of PD. We first tested this hypothesis by modifying extracellular Cys/CySS Eh and detecting the activation of mGlu5 in C6 glial cells. We found that alteration of extracellular Cys/CySS Eh was sufficient to alter phosphorylation of ERK, with the greatest phosphorylation observed under the oxidized condition (0 mV) through mGlu5 activation. Oxidized extracellular Cys/CySS redox state also promoted the production of ROS, the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and controlled the rotenone-induced cell death and activated the cells via induction of inducible nitric oxide synthase (iNOS). Next, the effects of plasma Cys/CySS Eh in a rotenone-induced rat model of PD were also examined. Our results may provide a novel mechanism by which mGlu5 can be activated by extracellular Cys/CySS redox state and a theoretical basis for understanding the regulation of mGlu5 activity in nerve cells and the pathogenesis of PD.

Section snippets

Preparation of media and measurement of Cys/CySS Eh

Various extracellular thiol/disulfide redox potentials were established by varying the concentrations of Cys and CySS added to Cys-free Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4 mM l-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin (catalog no. 21012; Invitrogen, CA, USA), as previously reported [20]. Stock solutions for Cys and CySS (10 mM; pH 7.4) were made fresh before each experiment and filtered through a 0.2-μm syringe filter. To generate the desired redox state

Effect of extracellular Cys/CySS Eh on ERK phosphorylation

Studies have shown that reduced extracellular Cys/CySS Eh (−150 mV) controls Caco-2 cells proliferation through upregulation of the ERK pathway [6] and that oxidized extracellular Cys/CySS Eh (−46 mV) controls lung fibroblast proliferation and matrix expression through upregulation of transforming growth factor-β [20]. This indicates control of cellular function may be extracellular redox potential dependent in terms of cell type. To determine whether extracellular Cys/CySS Eh also plays a role

Discussion

Cys and CySS constitute the most abundant low-molecular-weight thiol/disulfide couple in human plasma. Studies have shown that extracellular Cys/CySS redox potential controls cells proliferation through regulation of relative factors such as ERK and NF-κB [6], [20] and oxidized thiol/disulfide couples are associated with many diseases [8], [9], [10], [11]. In this study, we examined the role of extracellular Cys/CySS Eh in the activation of mGlu5 to understand its mechanism of regulation of

Acknowledgments

This work was supported by the National Natural Science Foundation of the People’s Republic of China (Nos. 30873087 and 30973406), the Beijing Municipal Natural Science Foundation (Nos. 5102011 and 7082010), the State Key Development Program of Basic Research of China (No. 2009CB522205), and Science and Technology Development Projects of the Beijing Municipal Commission of Education (No. KM 200910025001). The Funding Project for Academic Resource Development in Institutions of Higher Learning

References (45)

  • C. Wersinger et al.

    Bimodal induction of dopamine-mediated striatal neurotoxicity is mediated through both activation of D1 dopamine receptors and autoxidation

    Mol. Cell. Neurosci.

    (2004)
  • Y.M. Go et al.

    A key role for mitochondria in endothelial signaling by plasma cysteine/cystine redox potential

    Free Radic. Biol. Med.

    (2010)
  • Y.S. Nkabyo et al.

    Thiol/disulfide redox status is oxidized in plasma and small intestinal and colonic mucosa of rats with inadequate sulfur amino acid intake

    J. Nutr.

    (2006)
  • Y.M. Lee et al.

    Oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of Parkinson disease

    J. Biol. Chem.

    (2008)
  • D.P. Jones et al.

    Measuring the poise of thiol/disulfide couples in vivo

    Free Radic. Biol. Med.

    (2009)
  • J.L. Albasanz et al.

    Characterization of metabotropic glutamate receptors in rat C6 glioma cells

    Eur. J. Pharmacol.

    (1997)
  • Y. Luo et al.

    Opposing roles for ERK1/2 in neuronal oxidative toxicity: distinct mechanisms of ERK1/2 action at early versus late phases of oxidative stress

    J. Biol. Chem.

    (2006)
  • V. Martin et al.

    Signaling pathways involved in antioxidant control of glioma cell proliferation

    Free Radic. Biol. Med.

    (2007)
  • J. Hwang et al.

    NF-κB as a common signaling pathway in ganglioside-induced autophagic cell death and activation of astrocytes

    J. Neuroimmunol.

    (2010)
  • O.S. Gardner et al.

    Dependence of peroxisome proliferators-activated receptor ligand-induced mitogen-activated protein kinase signaling on epidermal growth factor receptor transactivation

    J. Biol. Chem.

    (2003)
  • C. Grothe et al.

    The physiological and pharmacological role of basic fibroblast growth factor in the dopaminergic nigrostriatal system

    Brain Res. Rev.

    (2007)
  • A. Saavedra et al.

    Selective injury to dopaminergic neurons up-regulates GDNF in substantia nigra postnatal cell cultures: role of neuron-glia crosstalk

    Neurobiol. Dis.

    (2006)
  • Cited by (13)

    • Blockade of metabotropic glutamate receptor 5 protects against DNA damage in a rotenone-induced Parkinson's disease model

      2015, Free Radical Biology and Medicine
      Citation Excerpt :

      Total protein samples for western blotting were prepared by immediately freezing the tissue in dry ice and storing at −80 °C until use. The rotarod test described previously [24], was used to evaluate the effects of MPEP on rotenone-induced PD symptoms. Rats were tested at three time points: before drug treatment, and at 3 and 4 weeks after the surgery.

    • Thiol redox homeostasis in neurodegenerative disease

      2015, Redox Biology
      Citation Excerpt :

      The extracellular cysteine/cystine ratio shifts towards a more oxidised value during ageing in humans and is viewed as a significant risk factor for disease [10]. For example, a more oxidised cysteine/cystine redox potential significantly increases metabotropic glutamate receptor 5 (mGluR5)-mediated phosphorylation of extracellular signal-regulated kinase (ERK) in astrocytes, leading to increased expression of the transcription factor, nuclear factor-ΚB (NF-ΚB), and inducible nitric oxide synthase, release of reactive oxygen and nitrogen species and increased neurotoxicity [11]. It is believed that the extracellular portion of mGluR5, which contains a cysteine-rich domain incorporating many disulphide bridges, alters to facilitate interaction between glutamate and the receptor at the more oxidised potential.

    • The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate

      2015, Biochimica et Biophysica Acta - General Subjects
      Citation Excerpt :

      Different reduction potential values (Eh) were obtained varying the relative concentrations of both species, maintaining the total concentration fixed at 1 mM, with the addition of 100 mM MOPS-HCl pH 7.4. Values of Eh were calculated by using the Nernst equation: Eh = Eo − RT/nF ln [Cys]2/[CySS], where Eo is the Eh for Cys/CySS at pH 7.4 (− 0.250 V) [31], R is the universal gas constant (8.314 J K− 1 mol− 1), T is the absolute temperature (298 K), n is the number of moles of electrons transferred in the reaction (2), F is the Faraday constant (96,485 J mol− 1 V− 1), and [Cys]2/[CySS] is the ratio between the concentrations of both redox species [32]. GlaUDP-Glc PPase and GlaUDP-GlcNAc PPase were incubated in a final concentration of 0.5 μM during 2 h at room temperature in different redox buffers (to reach redox equilibrium).

    • Amino Acids as biomarkers in the SOD1<sup>G93A</sup> mouse model of ALS

      2014, Biochimica et Biophysica Acta - Molecular Basis of Disease
      Citation Excerpt :

      Reduced estrogen therefore is correlated to lowered activity of two enzymes that incorporate ammonia into other molecules, and increased activity of an ammonia producing enzyme. Cysteine and cystine play multiple biological roles, including immune cell function, synthesis of glutathione (GSH), the major antioxidant of the brain, and cell signaling through its Cys/CySS redox ratio [39,40]. Cystine is exchanged for cellular glutamate through the cystine/glutamate antiporter followed by its reduction to cysteine; this exchange plays an important role in glutamateric signaling [41].

    View all citing articles on Scopus
    1

    These authors contributed equally to this study.

    View full text