The mGluR5 negative allosteric modulator CTEP reduces the aberrant activation and neurotoxicity of human-derived astrocytes differentiated from fibroblasts of ALS patients

BACKGROUND & RATIONALE. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial and non-cell-autonomous neurodegenerative disease, characterized by motor neurons (MNs) 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]. astrocyte reactivity as major cause of 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 ALS astrocytes and significantly improved the life span and disease progression in SOD1G93A ALS mice [PMID:31102766, 33931856, 37566031]. We now investigated in-vitro the impact of mGluR5 modulation by CTEP on i-Astrocytes differentiated from inducible neural progenitor cells (iNPCs) of SODA4V and C9orf72 ALS patients [PMID: 24379375]. 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 24h in-vitro exposure to 100nM CTEP reduced the over-expression of markers linked to aberrant activation and neuroinflammation (GFAP, S100β, C3, NLRP3) in i-Astrocytes from ALS-patients vs. untreated cells or i-Astrocytes from healthy donors. Of note, the phenotype shift induced by mGluR5 negative modulation, was accompanied by increased Nrf2 nuclear translocation, enhancement of the antioxidant enzymes activity (glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, catalase), reduced ROS and lower malondialdehyde accumulation. Preliminary results also showed that CTEP hampers the excessive intracellular calcium mobilization in ALS reactive i-Astrocytes. Of note, the reduced astrocyte reactivity translates into a beneficial effect towards iPSCs-derived MNs exposed to the astrocytes conditioned medium after CTEP in-vitro treatments. CONCLUSIONS. The in-vitro pharmacological negative modulation of mGluR5 by CTEP positively affects the reactive phenotype and neurotoxicity of human-derived i-Astrocytes from C9orf72 and SODA4V 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.