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

Abstract

BACKGROUND AND PURPOSEPresynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu 2/3 ) 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 mGlu 2 and mGlu 3 subunits allows us to further characterize these autoreceptors. In this study we investigated the pharmacological profile of mGlu 2/3 receptors in selected CNS regions and evaluated their functions in mice with experimental autoimmune encephalomyelitis (EAE). EXPERIMENTAL APPROACHThe comparative analysis of presynaptic mGlu 2/3 autoreceptors was performed by determining the effect of selective mGlu 2/3 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, mGlu 2/3 autoreceptor-mediated release functions were investigated and effects of in vivo LY379268 administration on impaired glutamate release examined ex vivo. KEY RESULTSWestern blot analysis and confocal microscopy confirmed the presence of presynaptic mGlu 2/3 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, mGlu 2/3 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 IMPLICATIONSWe propose the existence of mGlu 2 -preferring and mGlu 3 -preferring autoreceptors in mouse cortex and spinal cord respectively. The mGlu 3 -preferring autoreceptors could represent a target for new pharmacological approaches for treating demyelinating diseases.

Immuno‐pharmacological characterization of group II metabotropic glutamate receptors controlling glutamate exocytosis in mouse cortex and spinal cord

“…Experiments were carried out to quantify the effects of antagonists alone and of pretreatment of the synaptosomes with antibodies on the KCl‐evoked release of [ 3 H]‐D‐Asp. In accord with our previous results (Di Prisco et al, ) and consistent with Curtis et al . (), at least n = 5 replicates were carried out for each experimental condition.…”

“…The recent discovery of new ligands able to separate the role of the two receptors has allowed the pharmacological characterization of the native mGlu 2/3 receptors. Consistently, we recently proposed the existence of presynaptic mGlu 3 ‐preferring autoreceptors in spinal cord glutamate terminals and presynaptic mGlu 2 ‐preferring autoreceptors in the cortex of adult mice (Di Prisco et al, ). These conclusions originated from the results obtained in release studies with N‐acetyl‐aspartyl‐glutamate (NAAG), which has been shown to selectively activate the mGlu 3 receptor subtype (Neale et al, ; Romei et al, ; Di Prisco et al, ) and LY541850, a selective mGlu 2 receptor agonist with mGlu 3 receptor antagonist activity (Hanna et al, ; Sanger et al, ).…”

“…Purified synaptosomes isolated from the cortex of adult male mice were preloaded with [ 3 H]‐D‐Asp and exposed to a mild (12 mM KCl) depolarizing stimulus (Grilli et al, ; Di Prisco et al, ) in the absence or in the presence of the mGlu 2/3 receptor agonist LY379268. Previous studies showed that 3 nM LY379268 causes a significant, submaximal, inhibition of the [ 3 H]‐D‐Asp exocytosis (Figure , see also Di Prisco et al, ). LY566332 (1 μM), unable on its own to modify the 12 mM KCl‐evoked [ 3 H]‐D‐Asp overflow, significantly enhanced the inhibitory effect of LY379268 on tritium exocytosis.…”

“…Exposure of spinal cord synaptosomes to 15 mM KCl‐enriched solution causes the Ca 2+ ‐dependent release of preloaded [ 3 H]‐D‐Asp from these terminals (Di Prisco et al, ; ). The concomitant presence of LY379268 (30 pM) significantly reduced this release (Figure ; see also Di Prisco et al, ; ). LY566332 (0.1–1 μM) failed to significantly modify the 30 pM LY379268‐induced inhibition of tritium exocytosis (Figure ).…”

Presynaptic Release-Regulating mGlu1 Receptors in Central Nervous System

“…It is the case of the double-faced mGlu2/3 ligand the compound LY541850, which activates mGlu2 receptor subtypes but inhibits mGlu3 receptors (Di Prisco et al, 2016). …”