Abstract
Microglial-related factors have been implicated in the signaling cascades that contribute to neuronal cell death in various neurodegenerative disorders. Thus, strategies that reduce microglial activation and associated neurotoxicity may have therapeutic benefit. Group II and III metabotropic glutamate receptors (mGluRs) are expressed in microglia and can modulate microglial activity in primary cell cultures. We demonstrate that the group I receptor member mGluR5 is highly expressed in primary microglial cultures and the BV2 microglial cell line. Activation of mGluR5 using the selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) significantly attenuates microglial activation in response to lipopolysaccharide and interferon-gamma, as indicated by a reduction in the expression of inducible nitric-oxide synthase, production of nitric oxide and tumor necrosis factor-alpha, and intracellular generation of reactive oxygen species. In addition, microglial-induced neurotoxicity is also markedly reduced by CHPG treatment. The anti-inflammatory effects of CHPG are mediated by the mGluR5 receptor, because either a selective mGluR5 antagonist or small interference RNA knockdown attenuated the actions of this drug. CHPG blocked the lipopolysaccharide-induced increase in expression and enzymatic activity of NADPH oxidase. Moreover, the protective effects of CHPG were significantly reduced when the NADPH oxidase subunits p22(phox) or gp91(phox) were knocked down by small interference RNA. These data suggest that mGluR5 represents a novel target for modulating microglial-dependent neuroinflammation, and may have therapeutic relevance for neurological disorders that exhibit microglial-mediated neurodegeneration.
Highlights
Grant R01 5R01NS037313-08 from NINDS. 1 To whom correspondence should be addressed: Dept. of Neuroscience, particular, activated microglia exert neurotoxic effects by releasing inflammatory mediators such as eicosanoids, cytokines, chemokines, and reactive free radicals [3]
In addition to their expression on neurons, Metabotropic glutamate receptors (mGluRs) are expressed on other cells, including microglia, actions of CHPG
CHPG pre-treatment onstrate that mGluR5 is expressed in cultured microglia, in control-siRNA microglia resulted in a 54.52 Ϯ 2.82% reduc- whereas the other group I mGluR, mGluR1, is minimally tion in LPS-stimulated TNF␣ release (Fig. 6C), whereas CHPG expressed; this profile is consistent with the previous report of treatment in p22phox- or gp91phox-siRNA microglia resulted in a mGluR5 mRNA expression in microglia [19]
Summary
Microglial Cultures—Primary cortical microglial were obtained from postnatal day 2 Sprague-Dawley rat pups and cultured as described before [28]. BV2 microglia cultured in 24-well plates were transfected with the appropriate siRNA (100 nM) using Lipofectamine2000 (Invitrogen). After 24 h of transfection, cells were pre-treated with CHPG (4 mM) for 1 h, stimulated with LPS (100 ng/ml), and cultured for an additional 24 h. Cells were incubated in blocking buffer (10% normal goat serum in PBS containing 0.1% Triton X-100) for 2 h, followed by overnight incubation at 4 °C in primary antibody (mGluR5, 1:100 (Abcam, Cambridge, MA); ED1, 1:100 (AbD Serotec, Raleigh, NC); p22phox, 1:50 (Santa Cruz Biotechnology); and gp91phox, 1:100 (BD Transduction, Franklin Lakes, NJ)). Measurement of PI Hydrolysis—BV2 microglia, cultured in 96-well plates, were incubated overnight with 0.625 Ci/well myo-[3H]inositol (PerkinElmer Life Sciences) to label the cell membrane phosphoinositides (PIs). The soluble fraction containing total cell extracts was recovered, the protein concentration was determined, and samples were equalized
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