In-vitro study on human-derived astrocytes showed the effects of mGluR5 negative allosteric modulator CTEP in reducing the aberrant activation and neurotoxicity of astroglial cells in ALS

BACKGROUND & RATIONALE. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial and non-cell-autonomous neurodegenerative disease, characterized by motor neuron (MN) death. The metabotropic glutamate receptor type 5 (mGluR5) plays a key role in modulating downstream phenomena triggered by excessive or altered glutamatergic neurotransmission in ALS [PMID: 18617894, 22072391]. Glial activation and in particular astrocyte aberrant phenotype shift represent a major cause that sustains MNs loss. We provided in-vitro and in-vivo evidence showing that genetic ablation or pharmacological modulation of mGluR5 by the selective negative allosteric modulator CTEP, counteracts the reactive phenotype and neurotoxicity of astrocytes and significantly improves the life span and disease progression in SOD1G93A ALS mice [PMID:31102766, 33931856, 37566031]. RATIONALE. We here investigated in-vitro the impact of the mGluR5 negative modulation by CTEP on i-Astrocytes differentiated from inducible neural progenitor cells (iNPCs) of SOD1 and C9orf72 ALS patients [PMID: 24379375] and respective healthy donors. RESULTS. In-vitro pharmacological modulation with CTEP did not alter the mGluR5 total expression in i-Astrocytes. RT-qPCR, western blot and immunohistochemical experiments showed that 24h in-vitro exposure to 100nM CTEP reduced the over-expression of markers linked to aberrant activation and neuroinflammation (GFAP, S100β, C3, NLRP3; p<0.05; two-way ANOVA) in i-Astrocytes from ALS-patients vs. untreated cells or controls. Of note, the phenotype shift induced by mGluR5 negative modulation, was accompanied by increased Nrf2 nuclear translocation, enhancement of the antioxidant enzymes (glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, catalase), reduced ROS and lower malondialdehyde accumulation (p<0.05; two-way ANOVA). Acute CTEP in-vitro treatments did not significantly changed the intracellular calcium mobilization in ALS and control i-Astrocytes. On the other hand, the reduced astrocyte reactivity translates into a beneficial effect towards iPSCs-derived MNs exposed to the conditioned medium of i-Astrocytes after CTEP in-vitro exposure (p<0.05; one-way ANOVA). CONCLUSIONS. The in-vitro pharmacological negative modulation of mGluR5 by CTEP positively affects the reactive phenotype and neurotoxicity of i-Astrocytes derived from C9orf72 and SOD1 ALS patients, mainly by ameliorating the oxidative stress response of these cells. The results confirm our previous results in the SOD1G93A mouse model, further encouraging a translational application of mGluR5 modulators in clinical trials. Project funded under the National Recovery and Resilience Plan (NRRP), Italian Ministry of Research funded by the European Union – NextGenerationEU project MNESYS (PE0000006) – A Multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022).