Glial metabotropic glutamate receptor-4 increases maturation and survival of oligodendrocytes

Abstract

Group III metabotropic glutamate (mGlu) receptors mediate important neuroprotective and anti-inflammatory effects. Stimulation of mGlu4 receptor reduces neuroinflammation in a mouse model of experimental autoimmune encephalomyelitis (EAE) whereas mGlu4 knockout mice display exacerbated EAE clinical scores. We now show that mGlu4 receptors are expressed in oligodendrocytes, astrocytes and microglia in culture. Oligodendrocytes express mGlu4 receptors only at early stages of maturation (O4 positive), but not when more differentiated (myelin basic protein, MBP positive). Treatment of immature oligodendrocytes with the mGlu4 receptor agonist L-2-Amino-4-phosphonobutyrate (L-AP4; 50 μM for 48 h) accelerates differentiation with enhanced branching and earlier appearance of MBP staining. Oligodendrocyte death induced by exposure to 1 mM kainic acid for 24 h is significantly reduced by a 30-min pretreatment with L-AP4 (50 μM), an effect observed only in the presence of astrocytes, mimicked by the specific mGlu4 receptor positive allosteric modulator N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) (30 μM) and prevented by pretreatment with the mGlu4 receptor antagonist, cyclopropyl-4-phosphonophenylglycine (CPPG) (100 μM). In astrocytes, mGlu4 receptor is the most expressed among group III mGlu receptors, as by Quantitative real time PCR (QRT-PCR), and its silencing prevents protective effects. Protection is also observed when conditioned medium (CM) from L-AP4-pretreated astrocytes is transferred to oligodendrocytes challenged with kainic acid. Transforming growth factor β (TGF-β) mediates the increased oligodendrocyte survival as the effect of L-AP4 is mimicked by addition of 10 ng/ml TGF-β and prevented by incubation with a neutralizing anti-TGF-β antibody. In contrast, despite the expression of mGlu4 receptor in resting and activated microglia, CM from L-AP4-stimulated microglia does not modify kainate-induced oligodendrocyte toxicity. Our results suggest that mGlu4 receptors expressed in astrocytes mediate enhanced survival of oligodendrocytes under conditions of excitotoxicity.

Keywords: astrocytes; experimental autoimmune encephalomyelitis; multiple sclerosis; oligodendrocytes; transforming growth factor β.

Figure 1

Oligodendrocytes express mGlu4 receptor and respond to L-AP4 with enhanced differentiation. Oligodendrocytes that stain positively for O4 (red; A) express also mGlu4 receptors (green; A). Co-labeling of mGlu4 receptor and MBP (B, right panel) reveals that more mature oligodendrocytes (MBP immunopositive (B), left panel) do not express mGlu4 receptor (B, middle panel). Treatment with the mGlu4 receptor agonist, L-AP4 (50 μM, for 48 h), accelerates oligodendrocyte differentiation as revealed by their morphology (D), with enhanced initial branching and increased labeling with MBP, as compared to control conditions (C). Scale bar = 10 μm. Quantification of branching is reported and expressed as number of branches (E) and length of branches (F). Both values are expressed as % of control and each bar represents mean ± SE of six different fields analyzed in three samples from two independent experiments. *p < 0.05 vs. control by Student’s t-test.

Figure 2

The effect of L-AP4 on kainic acid-induced oligodendrocyte death depends on astrocytes. Panel (A) reports the different response of a 24 h treatment with 1 mM kainic acid (KA) in pure oligodendrocyte cultures (− astrocytes) or in oligodendrocyte cultures containing a low percentage of astrocytes (+ astrocytes). Group III mGlu receptor agonist, L-AP4 (50 μM), was added, 30 min before treatment with kainic acid (A). Oligodendrocyte viability was measured by the MTT proliferation assay. In (B), representative plots of the characterization of cell population showing 1.8% (upper panel) and 7.9% (lower panel) GFAP immunopositive cells and more than 90 and 80%, respectively, PDGF-R expressing cells, as by flow cytometric analysis. In (C), the effect of a 15 min pre-treatment with the selective mGlu4 receptor antagonist CPPG (100 μM) on L-AP4 reduction of kainic acid-induced oligodendrocyte death. The last bar on the right shows the effect of a 24 h treatment with the positive allosteric modulator of mGlu4 receptor, PHCCC (30 μM for 24 h), on kainate toxicity. Data in (A) and (C) are mean ± SE of three to four independent experiments, with four replicates per treatment group. *p < 0.05 vs. control, #p < 0.05 between indicated groups and §p < 0.05 vs. kainic acid by one way ANOVA, followed by Newman-Keuls multiple comparison test.

Figure 3

mGlu4 receptor is expressed in astrocytes and mediates enhanced oligodendrocyte viability. mGlu4 receptor expression in cultured astrocytes is enhanced by exposure for 48 h to LPS (1 μg/ml), as revealed by western blot analysis (A) and immunocytochemistry (B). Representative bands of mGlu4 and α-tubulin are reported and bars showing densitometry result from three independent determinations (A). Co-immunostaining for mGlu4 receptor (green) and GFAP (red) is shown (B). In (C), assessment of the viability of oligodendrocytes challenged with kainic acid (1 mM for 24 h) after transferring of conditioned medium (CM) obtained from astrocytes, under basal or LPS-stimulated conditions, exposed for 2 h, prior to washing and medium collection for the following 18 h, to L-AP4 (50 μM) and the mGlu4 receptor antagonist CPPG (100 μM). Data are mean ± SE of three independent experiments, each run in quadruplicates. *p < 0.05 vs. all other conditions by Student’s t-test (A) and one-way ANOVA followed by Newman-Keuls test (C). Scale bar = 20 μm.

Figure 4

L-AP4 acts on astrocytic mGlu4 receptor to protect oligodendrocyte against kainate toxicity. Expression of group III mGlu receptors, mGlu4, mGlu6, mGlu7 and mGlu8 receptors, was analyzed in astrocytes by QRT-PCR (A). Expression of mGlu4 receptor after transfection of cultured astrocytes with mGlu4 receptor siRNA (siRNA mGluR4) is shown and compared to expression in astrocytes exposed to siRNA negative control (siRNA Neg; (B)). In (C), the effect of CM from astrocytes treated with L-AP4 following silencing of mGlu4 receptor (siRNA mGlu4) or its negative control (siRNA Neg) on kainate-induced oligodendrocyte death. In (A), data are representative of several independent assays each run in triplicates. Data in (B) and (C) are mean ± SE from four independent determinations. * p < 0.05 vs. respective control; § < 0.05 vs. kainate alone. A t-test and one-way ANOVA plus Newman-Keuls test were applied to detect statistically significant differences in (B) and (C), respectively.

Figure 5

TGF-β1 is involved in the protective effect of L-AP4 against kainate-induced oligodendrocyte death. Oligodendrocytes were treated with TGF-β1 (10 ng/ml), L-AP4 (50 μM) or both, 30 min prior to exposure to kainic acid (1 mM for 24 h). Cell viability was assessed by the MTT assay (A). TGF-β1 expression in astrocytes assessed by western blot analysis under basal and L-AP4-stimulated conditions (B). The intracellular content of TGF-β1, assessed by flow cytometry in astrocytes exposed to LPS (0.1 μg/ml for 24 h) and treated with 50 μM L-AP4 for 6 h in the presence of brefeldin (C). Mean fluorescence intensity of all positive events is reported. A representative plot showing basal (empty histogram) and L-AP4-treated (filled histogram), TGF-β1 positive cells is also shown (C). In (D), oligodendrocyte viability after exposure to kainic acid (1 mM for 24 h) assessed in the presence of anti-TGF-β1 antibody (5 μg/ml), after transferring of CM obtained from astrocytes activated with LPS (1 μg/ml for 24 h) and treated with L-AP4 (50 μM) for 2 h prior to medium collection for 18 h. Data are mean ± SE of 4–6 independent experiments, each with three to four replicates. *p < 0.05 vs. respective control and **p < 0.05 vs. kainic acid (vhl) in a and vs. all other kainic acid-treated conditions (vhl; aTGF-β1; L-AP4 + aTGF-β1) in (D). Significance was assessed by ANOVA and Newman-Keuls test. In (B) and (C) a Student’s t-test was applied.

Figure 6

Lack of effect of L-AP4-treated microglia CM on oligodendrocyte survival. mGlu4 receptor expression in microglia under resting conditions (vhl) or upon exposure to LPS (0.1 μg/ml) for 48 h as revealed by immunocytochemistry (A). Microglia cells were labeled with an antibody against the specific microglia marker integrin. In (B), oligodendrocyte survival after 24 h exposure to kainic acid (1 mM for 24 h) is not modified by transferring of CM from microglia, under resting or LPS-activated conditions, treated or not with L-AP4, 50 μM for 2 h, prior to medium collection for additional 18 h. Bars represent mean ± SE from two separate experiments. Scale bar = 10 μm.