mGlu5 receptor negative allosteric modulation reduces the aberrant cellular reactivity and neurotoxicity of iNPCs-derived human astrocytes differentiated from ALS patients

BACKGROUND. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial and non-cell-autonomous neurodegenerative disease, characterized by brainstem and spinal cord motor neuron (MN) death. The etiopathological mechanisms are numerous and among them, glutamate excitotoxicity plays a major role. The metabotropic glutamate receptor type 5 (mGluR5) fine modulates glutamate neurotransmission other than astrocyte reactivity, and this mechanism can be exploited as potential target to counteract MN loss in ALS. We provided in-vitro and in-vivo evidence showing that genetic ablation or the pharmacological modulation of mGluR5, by the selective negative allosteric modulator CTEP, positively affects the reactive phenotype and neurotoxicity of ALS astrocytes and significantly improved the life span and disease progression in SOD1G93A ALS mice. Here we investigated in-vitro the impact of mGluR5 modulation by CTEP on human i-Astrocytes differentiated from inducible neural progenitor cells (iNPCs) obtained from skin fibroblasts of ALS patients and healthy donors. With this aim, we monitored the cellular phenotype of i-Astrocytes when exposed to 100nM CTEP by exploiting molecular biology and immunohistochemical studies. We monitored the intracellular calcium level by live imaging and fluorometric detection, moreover we quantified the secretion of neurotoxic factors by ELISA assay. The accumulation of reactive oxygen species, as well as oxidative stress markers have been investigated by immunohistochemical and biochemical studies, moreover we measured the activity of antioxidant response enzymes. As an indirect, but very important readout, we finally investigated the neurotoxicity of the astrocyte conditioned medium on human-derived motor neurons differentiated from iPSCs of healthy donors and ALS patients. RESULTS. In-vitro pharmacological modulation with CTEP did not alter the mGluR5 total expression in i-Astrocytes. RT-qPCR analyses, western blot and immunohistochemical experiments showed that 5 days in-vitro exposure to 100nM CTEP reduced the over-expression of astrogliosis (GFAP, S100β, C3) and neuroinflammation (NLRP3) markers (p<0.05; two-way ANOVA) in i-Astrocytes from ALS-patients vs. untreated or control cells. CTEP treatment was also able to restore oxidative stress conditions by promoting Nrf2 nuclear translocation, enhancement of the antioxidant enzymes activity (glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, catalase), and consequent reduction of ROS and direct oxidative stress markers (p<0.001; two-way ANOVA) in ALS i-Astrocytes. Acute CTEP in-vitro treatment significantly reduces the intracellular calcium mobilization upon mGlu5 receptor activation, in ALS and control i-Astrocytes. On the other hand, the reduced astrocyte reactivity by CTEP treatment translates into a beneficial effect towards iPSCs-derived MNs exposed to the conditioned medium collected from i-Astrocytes. CONCLUSIONS. We here show that the in-vitro pharmacological allosteric blockade of the mGluR5 by CTEP positively affects the reactive phenotype of human-derived i-Astrocytes differentiated form from C9orf72 and SOD1A4V ALS patients, mainly by ameliorating the general oxidative stress response and reducing the release of toxic factors. These data extend our previous results in SOD1G93A in-vitro and in-vivo models, thus further encouraging a translational application of mGluR5 modulators in clinical trials.