In-vivo and in-vitro evidence supporting the role of mGlu5 receptor as a key regulator of glutamate downstream effects in ALS

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INTRODUCTION. Amyotrophic lateral sclerosis (ALS) is fatal neurodegenerative disease characterised by 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, even in an early stage of the disease. METHODS. Behavioural, histological, and functional experiments have been performed to characterise the effects of mgluR5 modulation in both in-vivo and in-vitro ALS experimental models. We first investigated in-vivo the effect of a partial or total genetic ablation of mGlu5 receptor in the SOD1G93A mouse model of ALS. We then tested in-vivo the pharmacological blockade of mGluR5 by oral administration of the selective negative allosteric modulator CTEP (4 mg/kg every 24h, from 90 days of life). Finally, we studied the effects of mGluR5 genetic or pharmacological modulation, on the reactive phenotype of astrocytes or microglia cells isolated from SOD1G93A mice. RESULTS. The in-vivo genetic ablation of mGluR5 translates into a delayed disease onset and a significant prolonged survival probability, in the SOD1G93A mouse model of ALS. This effect was paralleled by a significant MNs preservation, a decreased activation of astrocytes and microglia, and by a normalization of the excessive Glu release, respect to SOD1G93A naïve mice. Subsequently, we tested the pharmacological modulation of mGluR5 by CTEP. Behavioural studies showed that, as for the genetic ablation, also in-vivo treatment with CTEP significantly slowdown the clinical progression of the pathology and increased the survival probability in SOD1G93A mice. Moreover, we observed a reduced glial activation and a significant MNs preservation. In-vitro experiments with primary spinal cord astrocytes cultured from SOD1G93A mice genetically lacking the mGlu5 receptor showed a reduced cytosolic calcium mobilisation as well as a reduced expression of astrogliosis markers. Most importantly, the modulation of astrocyte' reactive phenotype, reduced the neurotoxic effect toward co-cultured MNs, by a diminished release of neuroinflammatory factors. Microglia cells isolated from the spinal cord of SOD1G93A mice lacking the mgluR5, also showed a shift of their pathological phenotype. CONCLUSIONS. Our results demonstrate that a constitutive genetic downregulation, or the pharmacological blockade of mGlu5 receptor, have a positive clinical outcome in SOD1G93A mice. The in-vivo effects can be mainly ascribed to a reduced glial activation, supporting the role of mGluR5 as a multifactorial therapeutic target to counteract the progression of ALS pathology.

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