Reactive phenotype and oxidative stress in i-Astrocytes from patients affected by Amyotrophic Lateral Sclerosis: how the mGluR5 negative allosteric modulator CTEP affects these pathological features

BACKGROUND. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial non-cell-autonomous neurodegenerative disease, characterized by motor neurons (MNs) death. The metabotropic glutamate receptor type 5 (mGluR5) plays a key role in modulating astrocyte reactivity and glutamate excitotoxicity as major cause of MNs loss. We provided in-vitro and in-vivo evidence showing that modulation of the 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 SOD1^G93A ALS mice. Here we investigated the impact of in-vitro mGluR5 modulation by CTEP on the phenotype of i-Astrocytesdifferentiated from inducible neural progenitor cells (iNPCs) of ALS patients and control subjects.

RESULTS. In-vitro pharmacological modulation with CTEP did not alter the mGluR5 total expression. Confocal microscopy, immunohistochemical experiments and RT-qPCR analyses showed that 5 days in-vitro exposure to 100nM CTEP reduced the over-expression of reactive astrocytes and neuroinflammatory markers (GFAP, S100β, C3, NLRP3), in i-Astrocytes carrying the C9orf72 and SOD^A4V ALS-mutations vs. untreated cells and control i-Astrocytes from healthy donors. Of note, the improved phenotype following mGluR5 negative modulation, was accompanied by increased Nrf2 nuclear translocation, amelioration of the antioxidant enzymes activity (glutathione reductase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, catalase), reduced ROS and malondialdehyde accumulation, compared to untreated and control cells. Preliminary results also showed that CTEP hampers the excessive intracellular calcium mobilization in ALS i-Astrocytes vs. untreated and control cells.

CONCLUSIONS. We here show that the in-vitro pharmacological negative modulation of the mGluR5 by CTEP positively affects the reactive phenotype of human-derived i-Astrocytes from C9orf72 and SOD^A4V ALS patients, mainly by ameliorating the oxidative stress response of these cells. These data extend our previous results in SOD1^G93A in-vitro and in-vivo models, thus further encouraging a potential translational application of mGluR5 modulators in clinical trials.