Presynaptic, release-regulating mGlu2 -preferring and mGlu3 -preferring autoreceptors in CNS: pharmacological profiles and functional roles in demyelinating disease

Abstract

Background and purpose: Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in the CNS. They represent suitable targets for therapeutic approaches to central diseases that are typified by hyperglutamatergicity. The availability of specific ligands able to differentiate between mGlu2 and mGlu3 subunits allows us to further characterize these autoreceptors. In this study we investigated the pharmacological profile of mGlu2/3 receptors in selected CNS regions and evaluated their functions in mice with experimental autoimmune encephalomyelitis (EAE).

Experimental approach: The comparative analysis of presynaptic mGlu2/3 autoreceptors was performed by determining the effect of selective mGlu2/3 receptor agonist(s) and antagonist(s) on the release of [(3)H]-D-aspartate from cortical and spinal cord synaptosomes in superfusion. In EAE mice, mGlu2/3 autoreceptor-mediated release functions were investigated and effects of in vivo LY379268 administration on impaired glutamate release examined ex vivo.

Key results: Western blot analysis and confocal microscopy confirmed the presence of presynaptic mGlu2/3 receptor proteins. Cortical synaptosomes possessed LY541850-sensitive, NAAG-insensitive autoreceptors having low affinity for LY379268, while LY541850-insensitive, NAAG-sensitive autoreceptors with high affinity for LY379268 existed in spinal cord terminals. In EAE mice, mGlu2/3 autoreceptors completely lost their inhibitory activity in cortical, but not in spinal cord synaptosomes. In vivo LY379268 administration restored the glutamate exocytosis capability in spinal cord but not in cortical terminals in EAE mice.

Conclusions and implications: We propose the existence of mGlu2-preferring and mGlu3-preferring autoreceptors in mouse cortex and spinal cord respectively. The mGlu3 -preferring autoreceptors could represent a target for new pharmacological approaches for treating demyelinating diseases.

Figure 1

Presynaptic release‐regulating mGlu_2/3 autoreceptors in cortical nerve terminals of adult mice. (A) Concentration‐dependent inhibition of the 12 mM KCl‐evoked [³H]‐d‐Asp exocytosis by LY379268. Synaptosomes were transiently exposed to the depolarizing stimulus. When indicated, LY379268 was added concomitantly to the depolarizing stimulus. Results are expressed as percentage of the 12 mM KCl‐induced overflow (% of control). The [³H]‐d‐Asp overflow elicited by 12 mM KCl corresponded to 1.13 ± 0.09% of the total synaptosomal radioactivity, and it amounted to 1.5 ± 0.3 nCi. Data are the means ± SEM of four to seven experiments run in triplicate (three superfusion chambers for each experimental condition). *P < 0.05 versus the 12 mM KCl‐evoked tritium overflow. (A, inset) Western blot analysis unveiled the presence of mGlu_2/3 receptor protein dimers in mouse cortical synaptosomes. Percoll‐purified synaptosomes were lysed as described in the Methods section, and lysates (5 μg protein, left lane; 10 μg protein, right lane) were immunoblotted and probed with anti‐mGlu_2/3 antibody and with anti β‐tubulin antibodies. The figure shows a representative Western blot of five analyses carried out on different days. (B) Antagonism by LY341495 of the inhibitory effect of LY379268 (10 nM) on the 12 mM KCl‐evoked [³H]‐d‐Asp overflow from mouse cortical synaptosomes. Results are expressed as percentage of the 12 mM KCl‐evoked tritium overflow (% of control). Data are the means ± SEM of seven experiments run in triplicate. *P < 0.05 versus the 12 mM KCl‐evoked tritium overflow. ^# P < 0.05 versus the 12 mM KCl/10 nM LY379268‐evoked tritium overflow. (C) mGlu_2/3 receptor proteins are present in Stx‐1A and VGLUT1‐positive nerve terminals isolated from the cortex of adult mice. Percoll‐purified synaptosomes were processed for immunocytochemistry and visualized by fluorescence microscopy as described in the Methods section. The figure shows representative images of four to seven independent experiments carried out in different days.

Figure 2

Presynaptic release‐regulating mGlu_2/3 autoreceptors in spinal cord nerve terminals of adult mice. (A) Concentration‐dependent inhibition of the 15 mM KCl‐evoked [³H]‐d‐Asp exocytosis by LY379268. Synaptosomes were stimulated as previously described. Results are expressed as percentage of the 15 mM KCl‐induced overflow (% of control). The [³H]‐d‐Asp overflow elicited by 15 mM KCl corresponded to 2.18 ± 0.21% of the total synaptosomal radioactivity, and it amounted to 2.63 ± 0.5 nCi. Data are the means ± SEM of six to nine experiments run in triplicate (three superfusion chambers for each experimental condition). *P < 0.05 versus the 15 mM KCl‐evoked tritium overflow. (A, inset) mGlu_2/3 receptor protein dimers exist in mouse spinal cord synaptosomes. Synaptosomal lysates were immunoblotted (10 μg protein, left lane; 20 μg protein, right lane) and probed with anti‐mGlu_2/3 antibody and with anti β‐tubulin antibodies. The figure shows a representative Western blot of six analyses. (B) Antagonism by LY341495 of the inhibitory effect of LY379268 (10 nM) on the 15 mM KCl‐evoked [³H]‐d‐Asp overflow from mouse spinal cord synaptosomes. Results are expressed as percentage of the 15 mM KCl‐evoked tritium overflow. Data are the means ± SEM of six experiments run in triplicate. *P < 0.05 versus the 15 mM KCl‐evoked tritium overflow; ⁺ P < 0.05 versus the 15 mM KCl/10 nM LY379268‐evoked tritium overflow.

Figure 3

mGlu_2/3 receptor proteins are present in Stx‐1A and VGLUT1‐positive nerve terminals isolated from the spinal cord of adult mice. Percoll‐purified synaptosomes were processed for immunocytochemistry and visualized by fluorescence microscopy as described in the Methods section. The figure shows representative images of eight independent experiments carried out on different days.

Figure 4

Effects of NAAG and of LY541850 on the KCl‐evoked [³H]‐d‐Asp overflow from mouse cortical and spinal cord synaptosomes. (A) Concentration‐dependent inhibition of the 12 mm KCl‐evoked [³H]‐d‐Asp exocytosis by LY541850. Results are expressed as % of control. Data are the means ± SEM of five experiments run in triplicate. *P < 0.05 versus the 12 mM KCl‐evoked tritium overflow. (B) Effect of NAAG (100 nM) on the [³H]‐d‐Asp exocytosis elicited by 12 mM KCl from mouse cortical synaptosomes. Results are expressed as % of control. Data are the means ± SEM of six experiments run in triplicate. (C) Effect of LY541850 (100–1000 nM) on the [³H]‐d‐Asp exocytosis elicited by 15 mM KCl from mouse spinal cord synaptosomes. Results are expressed as % of control. Data are the means ± SEM of six experiments run in triplicate. (D) Concentration‐dependent inhibition of the [³H]‐d‐Asp exocytosis elicited by 15 mM KCl from spinal cord synaptosomes by NAAG. Results are expressed as % of control. Data are the means ± SEM of eight experiments run in triplicate. *P < 0.05 versus the 15 mM KCl‐evoked tritium overflow.

Figure 5

Effects of agonists and antagonists on the KCl‐evoked [³H]‐d‐Asp overflow from mouse cortical and spinal cord synaptosomes. (A) Antagonism by LY341495 of the LY541850‐induced inhibition of the 12 mM KCl‐evoked [³H]‐d‐Asp overflow from mouse cortical synaptosomes. Synaptosomes were exposed to 12 mM KCl in the presence and absence of 100 nM LY379268. When indicated, LY341495 (10–100 nM) was added concomitantly with the mGlu_2/3 agonist. Results are expressed as % of control. Data are the means ± SEM of five experiments run in triplicate. *P < 0.05 versus the 12 mM KCl‐evoked tritium overflow; ^# P < 0.05 versus the 12 mM KCl/100 nM LY341495‐evoked tritium overflow; ^## P < 0.01 versus the 12 mM KCl/100 nM LY341495‐evoked tritium overflow. (B) Antagonism by LY341495 of the NAAG‐induced inhibition of the 15 mM KCl‐evoked [³H]‐d‐Asp overflow from mouse spinal cord synaptosomes. Synaptosomes were exposed to 15 mM KCl in the presence and absence of 10 pM NAAG. When indicated, 100 nM LY341495 was added concomitantly with the mGlu_2/3 agonist. Results are expressed as % of control. Data are the means ± SEM of six experiments run in triplicate. *P < 0.05 versus the 15 mM KCl‐evoked tritium overflow; ⁺ P < 0.05 versus the 15 mM KCl/0.1 pM NAAG‐evoked tritium overflow. (C) Antagonism by LY541850 of the LY379268‐induced inhibition of the 15 mM KCl‐evoked [³H]‐d‐Asp overflow from mouse spinal cord synaptosomes. Synaptosomes were exposed to 15 mM KCl in the presence and absence of 100 nM LY379268. When indicated, LY341495 (0.1–100 nM) was added concomitantly with the mGlu_2/3 agonist. Results are expressed as % of control. Data are the means ± SEM of seven experiments run in triplicate. *P < 0.05 versus the 15 mM KCl‐evoked tritium overflow; ⁺ P < 0.05 versus the 15 mM KCl/0.1 nM LY379268‐evoked tritium overflow.

Figure 6

Clinical scores in EAE mice at different stages of disease. Clinical signs were monitored daily in EAE mice and are expressed as average (median ± SEM). *P < 0.05 versus 1 d.p.i.

Figure 7

Effect of acute in vivo administration of LY379268 on the 15 mM KCl‐evoked [³H]‐d‐Asp overflow from synaptosomes isolated from the spinal cord of control (non‐immunized) and EAE mice. Control (untreated) mice and EAE (grey bar) mice at the acute stage of disease (21 d.p.i.) were acutely injected i.p. (doses as indicated) and then killed within 3 h. The spinal cord was rapidly removed, and synaptosomes were prepared to monitor the 15 mM KCl‐evoked [³H]‐d‐Asp overflow in in vitro experiments. Results are expressed as 15 mM KCl‐induced overflow; data are means of nine experiments run in triplicate. *P < 0.05 versus the 15 mM KCl‐evoked tritium overflow from untreated mice; ^# P < 0.05 versus the 15 mM KCl‐evoked tritium overflow from untreated EAE mice.