In-vitro and in-vivo studies depict metabotropic glutamate receptor 5 as a potential pharmacological target to modulate disease progression in ALS

BACKGROUND Amyotrophic lateral sclerosis (ALS) is fatal neurodegenerative disease due to a progressive degeneration of motor neurons (MNs). The aetiology is still largely obscure, and several mechanisms have been proposed, including glutamate (Glu)-mediated excitotoxicity. In this context, group I metabotropic Glu receptors (mGluR1/5) play an active role, since their expression and functions are altered, especially in glial cells. Moreover, we previously showed that group I mGluRs sustains the excessive Glu release in the spinal cord of the SOD1G93A mouse model of ALS, and this alteration is already present at the onset of the pathology. METHODS We first investigated in-vivo the effect of mGlu5 receptor genetic ablation in SOD1G93A mice (SOD1G93A-mGluR5-/-). We then tested in-vivo the pharmacological blockade of mGluR5 by oral administration of the mGluR5 negative allosteric modulator CTEP (4 mg/kg every 24h, from 90 days of life). Finally, we studied in-vitro the effects of the mGluR5 genetic downregulation, on the reactive phenotype of spinal cord astrocytes cultured from symptomatic SOD1G93A mice. Histological, functional and biochemical experiments have been performed to characterise astrocytes and their cytotoxicity towards MNs. RESULTS The in-vivo genetic ablation of mGluR5 demonstrates that SOD1G93A-mGluR5-/- mice showed a delayed disease onset and a significant prolonged survival probability. These effects were paralleled by a significant MNs preservation, a decreased astrocyte and microglia activation and by a normalization of the excessive Glu release, compared to age matched SOD1G93A mice. Subsequently, we tested the pharmacological modulation of mGluR5 by CTEP. Behavioural studies showed that, as for the genetic ablation, also CTEP in-vivo treatment significantly slowdown the clinical progression of the pathology and increased the survival probability in SOD1G93A mice. Moreover, we also observed a reduced glial activation and a significant MNs preservation. In-vitro experiments with primary spinal cord astrocytes showed that the elevated cytosolic calcium concentration as well as the over expression of astrogliosis markers (GFAP, S100, Vimentin, NLRP3) were significantly reduced in cells genetically lacking the mGluR5 (SOD1G93A_mGluR5+/-) compared to SOD1G93A astrocytes. Most importantly, the modulation of the reactive phenotype in SOD1G93A_mGluR5+/-astrocytes translates into a reduced release of neuroinflammatory cytokines (IL1b, IL-6, TNFa), improved bioenergetics and a less toxic effect toward co-cultured MNs. CONCLUSIONS Our results demonstrate that a lower constitutive level of mGluR5, or the pharmacological blockade of the receptor by CTEP, has a positive clinical outcome in SOD1G93A ALS mice. The in-vivo effects can be mainly ascribed to a reduced reactive astrogliosis, supporting the role of mGluR5 as a therapeutic target to obtain a shift from a pathological toward a less noxious phenotype of astrocytes in SOD1G93A mice.