Abstract
BackgroundActivation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. In contrast, the consequences of mGluR5 inhibition are less well understood. Here, we used BV-2 cells, retaining key characteristics of primary mouse microglia, to examine whether mGluR5 inhibition by 2-methyl-6-(phenylethynyl)-pyridine (MPEP) enhances cellular stress and production of inflammatory mediators.MethodsBV-2 cells were treated with MPEP, followed by determination of cellular stress using fluorescent dyes and high-content imaging. The expression of inflammatory mediators, endoplasmic reticulum (ER)-stress markers and phosphorylated AMPKα was analyzed by quantitative PCR, ELISA and Western blotting. Additionally, phospholipase C (PLC) activity, cellular ATP content and changes in intracellular free Ca2+ ([Ca2+]i) were measured using luminescence and fluorescence assays.ResultsTreatment of BV-2 microglia with 100 μM MPEP increased intracellular reactive oxygen species (ROS), mitochondrial superoxide, mitochondrial mass as well as inducible nitric oxide synthase (iNOS) and IL-6 expression. Furthermore, MPEP reduced cellular ATP and induced AMPKα phosphorylation and the expression of the ER-stress markers CHOP, GRP78 and GRP96. The MPEP-dependent effects were preceded by a rapid concentration-dependent elevation of [Ca2+]i, following Ca2+ release from the ER, mainly via inositol triphosphate-induced receptors (IP3R). The MPEP-induced ER-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the Ca2+ chelator BAPTA-AM. Pretreatment with the AMPK agonist AICAR partially abolished, whilst the inhibitor compound C potentiated, the MPEP-dependent ER-stress. Importantly, the PLC inhibitor U-73122 and the Gi-protein inhibitor pertussis toxin (PTX) blocked the MPEP-induced increase in [Ca2+]i. Moreover, pretreatment of microglia with AICAR, BAPTA-AM, U-73122 and PTX prevented the MPEP-induced generation of oxidative stress and inflammatory mediators, further supporting a role for Gi-protein-mediated activation of PLC.ConclusionsThe results emphasize the potential pathophysiological role of mGluR5 antagonism in mediating oxidative stress, ER-stress and inflammation through a Ca2+-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca2+ release from the ER.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0190-7) contains supplementary material, which is available to authorized users.
Highlights
Activation of metabotropic glutamate receptor 5 by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators
Inhibition of metabotropic glutamate receptor 5 (mGluR5) induces inflammatory mediators and cellular stress Microglia are known to increase their production of inflammatory mediators as well as reactive oxygen species (ROS) and reactive nitrogen species (RNS) upon activation
In order to investigate the role of mGluR5 on microglial activity, we treated BV-2 mouse microglia with the pharmacological mGluR5 activator CHPG and the inhibitor MPEP and assessed their impact on cellular stress markers and inflammatory mediators
Summary
Activation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. Metabotropic glutamate receptors (mGluRs) are expressed in many different cell types throughout the brain and spinal cord [2]. They have been considered as promising targets for neuro-protective agents in acute and chronic neurodegenerative disorders [3,4]. The specific mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to inhibit microglial activation, oxidative stress and the release of inflammatory mediators both in vitro and in vivo [9,10,11,12,13,14]. A dysregulation resulting in decreased mGluR5 activity may promote the initiation and/or progression of neurodegenerative disorders
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