Group I metabotropic glutamatergic receptors regulating glutamate release and microglia phenotype in a murine model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the death of upper and lower motor neurons. Although the aetiology of the disease is still unclear, glutamate (Glu)-mediated excitotoxicity is a major cause. Our previous studies demonstrated that presynaptic Group-I metabotropic Glu receptors (mGluR1 and mGluR5) are over-expressed in spinal cord synaptosomes of 120-day-old SOD1G93A mice, that represent the late stage of the disease, and that their activation by the selective mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) produced abnormal Glu release. The aim of the present study was to investigate whether mGluR1 and mGluR5 also affect Glu release during the pre- and early-symptomatic time-course of the pathlogy (30, 60 and 90 days), in the same animal model. Our results showed that the mGluR1/5 agonist 3,5-DHPG evoked the release of glutamate in a concentration-dependent way and the effects were almost superimposable between 30/60-day-old WT and SOD1G93A mice. At variance, 0.3 μM 3,5-DHPG significantly increased Glu release (25%, p<0.05) in 90-day-old SOD1G93A mice but not in WT aged controls. The involvement of both metabotropic glutamate receptor subtypes was demonstrated using mGluR1 and mGluR5 selective antagonists/negative allosteric modulators (LY367385, MPEP, respectively). The analysis of the molecular mechanisms underlying the 3,5-DHPG-evoked Glu release revealed that it was of vesicular origin and induced by Ca2+ released from intra terminal stores. Confocal imaging confirmed that both mGluR1 and mGluR5 were co-localized onto glutamatergic nerve terminals and their expression was increased in SOD1G93A mice at the onset of the disease. We have also set up a method to isolate extracellular vesicles enriched in exosomes to investigate whether EVs derived from cultured activated astrocytes, treated with a mGluR5 antagonist, were able to change the the inflammatory pattern of microglia.