Research paperExtracellular cysteine (Cys)/cystine (CySS) redox regulates metabotropic glutamate receptor 5 activity
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
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These authors contributed equally to this study.