Metabotropic glutamate receptor 5 as a target for the modulation of the reactive astrocyte phenotype in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder due to upper and lower motor neurons (MNs) death. Recognized as a non-cell autonomous disease, ALS is also characterized by damage and degeneration of glial cells, such as astrocytes, microglia, and oligodendrocytes. In particular, astrocytes acquire a reactive and toxic phenotype defined by an abnormal proliferation and by the release of neurotoxic factors. One major cause for MN degeneration in ALS is represented by the glutamate-mediated excitotoxicity, due to the alteration of glutamate transmission mechanisms, including glutamate receptor function. In this context, the Group I metabotropic glutamate receptor 5 (mGluR5) has been proposed to play an important role in ALS, since it is largely overexpressed during disease progression and is involved in the altered neuronal and glial cellular processes. We previously demonstrated that mGluR5 produces abnormal glutamate release in the spinal cord of the SOD1G93A mouse model of ALS and that halving its expression has a positive impact on in-vivo disease progression, including motor neuron survival, astrogliosis and microgliosis. We here investigated the consequences of reducing the mGluR5 expression in SOD1G93A mice on the reactive phenotype of spinal cord astrocytes cultured from late symptomatic (120 days old) SOD1G93A, age matched WT and SOD1G93AGrm5-/+ mice. SOD1G93A astrocytes displayed a higher cytoplasmic calcium concentration respect to WT cells and knocking down of mGluR5 reduced calcium level, both under basal and 3,5-DHPG-stimulated conditions. GFAP and S100β, two markers of astrogliosis, were increased in SOD1G93A astrocytes, whereas their overexpression was reduced in SOD1G93AGrm5-/+ cells. The same positive effect was obtained in the case of NLRP3, a marker strictly related to inflammation, which was upregulated in SOD1G93A astrocytes and less expressed in double mutant astrocytes. The partial ablation of mGluR5 also resulted in a lower cellular presence of misfolded SOD1. Both the expression and secretion of pro-inflammatory cytokines were strongly reduced in SOD1G93AGrm5-/+ respect to SOD1G93A astrocytes. The uncoupling between oxygen consumption and ATP synthesis and the impairment of mitochondria function, present in SOD1G93A astrocytes, were recovered in double mutant astrocytes. Notably, the viability of spinal MNs co-cultured with SOD1G93AGrm5-/+ astrocytes was significantly increased respect to MNs co-cultured with SOD1G93A astrocytes. The acute in-vitro treatment of SOD1G93A astrocytes with an antisense nucleotide (ASO) specific for mGluR5 decreased the mRNA and protein expression of mGluR5 in these cells and led to the reduction of GFAP and S100β. The in-vitro pharmacological treatment with the negative allosteric modulator of mGluR5, CTEP, also reduced the expression of GFAP and S100β in SOD1G93A astrocytes. Altogether, these results indicate that mGluR5 ablation has a positive impact on astrocytes in SOD1G93A mice, supporting the idea that the in-vivo amelioration of the disease progression, registered after mGluR5 genetical or pharmacological silencing, involve astrocyte phenotype improvement. As a whole, it may be outlined that mGluR5 may represent a potential therapeutic target able to preserve MNs from death, also by modulating the reactive astroglia phenotype in ALS. Due to the active contribution of microglia to ALS pathogenesis, the effect of mGluR5 partial ablation in SOD1G93A mice on the balance between the pro- and anti-inflammatory profile of microglia acutely purified from the brain and spinal cord of WT, Grm5-/+, SOD1G93A and SOD1G93AGrm5-/+ mice has been investigated at the early (90 days) and late symptomatic (120 days) stages of ALS by detecting the mRNA and protein levels of some relevant markers involved in neuroinflammation, such as IL-1β, CD86, iNOS, TNF-α (pro-inflammatory), Arginase 1, IL-10, CD206 and IL-4 (anti-inflammatory). Experiments are in progress to complete this part of the project.