The use of knock-out mice unravels distinct roles for mGlu2 and mGlu3 metabotropic glutamate receptors in mechanisms of neurodegeneration/neuroprotection

Abstract

Dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonists have been examined with success in the clinic with positive proof of efficacy in several tests of anxiety and schizophrenia. Moreover, a large body of evidence has accumulated that these drugs have significant neuroprotective potential. An important discussion in the field deals with dissecting effects on mGlu2 versus effects on mGlu3 receptors, which is relevant for the potential use of subtype-selective agonists or allosteric activators. We addressed this issue using mGlu2 and mGlu3 receptor knock-out mice. We used mixed cultures of cortical cells in which astrocytes and neurons were plated at different times and could therefore originate from different mice. Cultures were challenged with NMDA for the induction of excitotoxic neuronal death. The mGlu2/3 receptor agonist, (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), was equally neuroprotective in cultures containing neurons from wild-type, mGlu2-/-, or mGlu3-/- mice. Neuroprotection was instead abolished when astrocytes lacked mGlu3 receptors, unless neuronal mGlu2 receptors were also absent. The latter condition partially restored the protective activity of LY379268. Cultures in which neurons originated from mGlu2-/- mice were also intrinsically resistant to NMDA toxicity. In in vivo experiments, systemic administration of LY379268 protected striatal neurons against NMDA toxicity in wild-type and mGlu2-/- mice but not in mGlu3-/- mice. In addition, LY379268 was protective against nigrostriatal degeneration induced by low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine only in mice lacking mGlu2 receptors. We conclude that neuroprotection by mGlu2/3 receptor agonists requires the activation of astrocytic mGlu3 receptors, whereas, unexpectedly, activation of mGlu2 receptors might be harmful to neurons exposed to toxic insults.

Figure 1.

A, A diagram with the mGlu3 gene targeting vector used to interrupt the mGlu3 gene. The LacZ–Neo cassette was inserted downstream of the NsiI site of grm3 exon (ex) II; the grm3 genomic sequence, from NsiI site in exon II up to HindIII site in grm3 exon III, was inserted downstream of the Neo cassette. B, Gene disruption was confirmed by RT-PCR analysis from total-brain RNA of wild-type and mGlu3^−/− mice. Primers were designed to amplify fragments spanning grm3 exon II–exon III. Lanes 1–4, RT-PCR from wild-type total-brain RNA; lanes 5–8, RT-PCR from mGlu3^−/− total-brain RNA. The following primer combinations were used: for lanes 1 and 5, grm3b and grm3c; for lanes 2 and 6, grm3b and grm3d; for lanes 3 and 7, grm3a and grm3c; for lanes 4 and 8, grm3a and grm3d. C, Distribution of mGlu3 receptor mRNA in the brain of wild-type and mGlu3^−/− mice. Coronal brain sections were processed for in situ hybridization with a ³³P-labeled oligonucleotide specific for the mouse mGlu3 receptor transcript, as described in Materials and Methods. The signal was absent in the brain of knock-out mice.

Figure 2.

Neuroprotection by the mGlu2/3 receptor agonist LY379268 in mixed cortical cultures differentially expressing mGlu2 and mGlu3 receptors in neurons or astrocytes. Cultures were challenged with 60 μm NMDA for 10 min in the absence or presence of LY379268 (1 μm) or the mGlu5 receptor antagonist MPEP (1 μm; used as a control neuroprotective drug). NMDA toxicity was set as 100%. Values are means ± SEM of 9–15 determinations from three to five independent experiments. iGluRs, Ionotropic glutamate receptors. *p < 0.05 (1-way ANOVA and Fisher's PLSD) versus NMDA alone.

Figure 3.

Concentration-dependent neurotoxicity by NMDA in mixed cortical cultures from wild-type mice (a-wt/n-wt), in cultures containing astrocytes from mGlu3 knock-out mice (a-mGlu3^−/−/n-wt), and in cultures containing neurons from mGlu2^−/− knock-out mice (a-wt/n-mGlu2^−/−). Values are means ± SEM of six to nine determinations from two to three independent experiments. *p < 0.05 (1-way ANOVA and Fisher's PLSD) versus the corresponding NMDA values obtained in a-wt/n-wt cultures.

Figure 4.

Neuroprotection by the GCM collected from pure astrocyte cultures treated with LY379268 requires the presence of mGlu3 receptors. A, GCM was collected from cultured astrocytes from wild-type mice (a-wt) or from mGlu3^−/− mice (a-mGlu3^−/−) 20 h after a 10 min exposure to LY379268 (1 μm) and transferred to recipient cultures challenged with NMDA. B, Recipient mixed cultures contained astrocytes lacking mGlu3 receptor (a-mGlu3^−/−/n-wt). Values are means ± SEM of six to nine determinations from two to three independent experiments. iGluRs, Ionotropic glutamate receptors. *p < 0.05 (1-way ANOVA and Fisher's PLSD) versus NMDA alone.

Figure 5.

Depolarization-induced [³H]-GABA release by LY379268 in control mixed cortical cultures or in cultures lacking neuronal mGlu2 (a-wt/n-mGlu2^−/−) or mGlu3 (a-wt/n-mGlu3^−/−) receptors. Values are means ± SEM of nine determinations from three independent experiments. *p < 0.05 (1-way ANOVA and Fisher's PLSD) versus the respective values obtained with KCl alone. Ctrl, Control.

Figure 6.

GAD activity in wild-type, mGlu2^−/−, or mGlu3^−/− mice infused with NMDA in the left striatum and treated 30 min before with LY379268 (10 mg/kg, i.p.). Results are expressed as a percentage of the respective contralateral unlesioned side for each individual determination. In the striatum of control animals, GAD activity was 211 ± 12 cpm of [³H]-GABA/μg prot. *p < 0.05 versus NMDA toxicity in saline-treated mice.

Figure 7.

A, Striatal dopamine, DOPAC, and HVA levels in wild-type, mGlu2^−/−, or mGlu3^−/− mice injected with MPTP (30 mg/kg, i.p., single injection) alone or in combination with LY379268 (1 mg/kg) injected intraperitoneally 30 min before MPTP. Values are mean ± SEM of 8–10 determinations; p < 0.05 (1-way ANOVA and Fisher's PLSD) versus saline-treated mice (*) or versus mice treated with MPTP alone (#). B, C, Immunohistochemical analysis of TH in the corpus striatum (B) and pars compacta of substantia nigra (C) of mice treated with 30 mg/kg MPTP alone or combined with 1 mg/kg LY379268 injected intraperitoneally. Scale bars: B, 400 μm; C, 100 μm. Densitometric data for striatal TH and TH-positive cell counts are also shown. Values (means ± SEM) were calculated from five to six mice per group (10 sections, 10 μm thick, cut every 100 μm, per animal were used for the calculation of the density of TH-positive neurons in the pars compacta of the substantia nigra). p < 0.05 (1-way ANOVA and Fisher's PLSD) versus saline-treated mice (*) or mice treated with MPTP alone (#).

Figure 8.

Western blot analysis of TGF-β1 expression in wild-type, mGlu2^−/−, or mGlu3^−/− mice in basal conditions (A) and after treatment with LY379268 (2 mg/kg, i.p.; B). Animals were killed 3 d later. Densitometric data of TGF-β1 are shown and are the mean ± SEM of three animals performed two times.*p < 0.05 (1-way ANOVA and Fisher's PLSD) versus saline-treated mice.

Figure 9.

Effects of LY379268 on the release of endogenous glutamate (A) and GABA (B) induced by veratridine in striatal synaptosomes prepared from wild type, mGlu2^−/−, and mGlu3^−/− mice. Line scatter plots, Time course of the veratridine-evoked release of neurotransmitters in the absence (open symbols) or presence (filled symbols) of 100 nm LY379268. The minute of superfusion at which collection of every fraction began is shown in the abscissa. LY379268 was added at the end of the first collected fraction (arrows). Results are expressed as nmol/mg prot/fraction. Data are means ± SEM of four experiments run in triplicate (three superfusion chambers for each experimental condition). *p < 0.05 versus control (i.e., no drug added). Bar plots, Concentration–effect relationship of LY379268 on veratridine-evoked release of endogenous neurotransmitters. Open bars, veratridine (10 μm)-evoked release of endogenous neurotransmitters; gray bars, veratridine plus LY379268-evoked release of endogenous neurotransmitters. Results of the induced overflow are expressed as nmol/mg prot. Data are means ± SEM of three to four experiments run in triplicate. *p < 0.05 versus control (i.e., no drug added).