Genetic Downregulation of the Metabotropic Glutamate Receptor Type 5 Dampens the Reactive and Neurotoxic Phenotype of Adult ALS Astrocytes

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100β and vimentin. In vitro exposure to an anti-mGluR5 antisense oligonucleotide or to the negative allosteric modulator CTEP also ameliorated the altered reactive astrocyte phenotype. Downregulating mGluR5 in SOD1G93A mice reduced the synthesis and release of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and ameliorated the cellular bioenergetic profile by improving the diminished oxygen consumption and ATP synthesis and by lowering the excessive lactate dehydrogenase activity. Most relevantly, mGluR5 downregulation hampered the neurotoxicity of SOD1G93A astrocytes co-cultured with spinal cord MNs. We conclude that selective reduction in mGluR5 expression in SOD1G93A astrocytes positively modulates the astrocyte reactive phenotype and neurotoxicity towards MNs, further supporting mGluR5 as a promising therapeutic target in ALS.

The Key Role of Astrocytes in Amyotrophic Lateral Sclerosis and Their Commitment to Glutamate Excitotoxicity

“…The selective toxicity was triggered by activating specific astrocytic mGluRs [320], suggesting a possible mechanism for the glutamate-induced excitotoxicity in astrocytes. This evidence has also been recently confirmed by our research group [78].…”

“…Genetic and pharmacological in vivo approaches have confirmed this hypothesis [198,200,320,337,341,342]. The beneficial effect of mGluR 5 blockade was also confirmed in vitro since the genetic reduction or the pharmacological negative modulation of the receptor in late symptomatic SOD1 G93A mouse-derived spinal cord astrocytes ameliorates their reactive, inflammatory, bioenergetic, and neurotoxic phenotype [78]. These data support glutamate as an enhancer of the astrocyte reactive, proinflammatory, and neurotoxic phenotype in ALS in response to extracellular glutamate via mGluR 5 .…”

“…Indeed, primary astrocyte cultures from the neonatal SOD1 G93A mouse brain did not show upregulation of the classical pro-reactive astrocyte genes, such as inflammatory genes and the reactive factor lipocalin 2 (Lcn2). On the contrary, astrocytes prepared from 2-month-old or late symptomatic stage SOD1 G93A mice recapitulated the typical phenotype observed in post-mortem astrocytes from ALS patients [76][77][78]. This evidence also highlights the importance of in vitro studies focusing on the in vivo maturation of astrocytes during disease progression, spanning from the pre-symptomatic/low-progressing to the symptomatic/fast-progressing stages, which determine their activation upon in vivo exposure to an authentic pathological environment.…”