Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to upper and lower motor neuron (MNs) death. Recognized as a non-cell-autonomous disease, ALS is also characterized by damage and degeneration of glial cells, such as astrocytes, microglia, and oligodendrocytes. Astrocytes acquire a reactive and toxic phenotype defined by an abnormal proliferation and by the release of neurotoxic factors. Recent studies reported that the uptake of [18F]-fluorodeoxyglucose (FDG) is increased in the spinal cord (SC) and decreased in the motor cortex (MC) of patients with ALS, suggesting that the disease might differently affect the two nervous districts with different time sequence or with different mechanisms. Here we show that MC and SC astrocytes harvested from newborn B6SJL-Tg (SOD1G93A) 1Gur (SOD1G93A) mice could play different roles in the pathogenesis of the disease. Spectrophotometric and cytofluorimetric analyses showed an increase in redox stress, a decrease in antioxidant capacity, and a relative mitochondria respiratory uncoupling in MC SOD1G93A astrocytes. By contrast, SC mutated cells showed a higher endurance against oxidative damage, through the increase in antioxidant defense and a preserved respiratory function. Thus, SOD1G93A mutation differently impaired MC and SC astrocyte biology in a very early stage of life. One major cause for MN degeneration in ALS is represented by glutamate-mediated excitotoxicity, due to the alteration of glutamate transmission mechanisms, including glutamate receptor function. In this context, the Group I metabotropic glutamate receptor 5 (mGluR5) has been proposed to play an important role in ALS, since it is largely overexpressed during disease progression and is involved in the altered neuronal and glial cellular processes. My research group previously demonstrated that mGluR5 produces abnormal glutamate release in the spinal cord of the SOD1G93A mouse model of ALS and that halving its expression has a positive impact on in-vivo disease progression, including motor neuron survival, astrogliosis, and microgliosis. They also investigated the consequences of reducing the mGluR5 expression in SOD1G93A mice on the reactive phenotype of spinal cord astrocytes cultured from late symptomatic (120 days old) SOD1G93A mice. Also in this model, reducing the mGluR5 expression ameliorated the astrocyte phenotype. UNIVERSITY OF GENOVA 8 Here, I translated this study to human astrocytes derived from healthy donors and ALS patients. We investigated the in-vitro pharmacological treatment effect of chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), a negative allosteric modulator of mGluR5 on i-astrocytes differentiated from the inducible neural progenitor cells (iNPCs) obtained from the skin fibroblast (i-astrocytes) of two ALS patients and two healthy donors. The overexpression of anti-glial fibrillary acid protein (GFAP), S100 calcium-binding protein β (S100β), and Complement component 3 (C3), three markers of astrogliosis, was reduced in CTEP-treated i-astrocytes. The same positive effect was obtained in the case of NLR family pyrin domain containing 3 (NLRP3) and nuclear factor erythroid 2-related factor 2 (NRF2), markers strictly related to inflammation and oxidative stress respectively, which are upregulated in ALS astrocytes. In-vitro pharmacological treatment with CTEP also reduced the expression of mGluR5 in mutated i-astrocytes. In addition, the CTEP treatment caused a decrement in antioxidant enzymatic activity such as malondialdehyde (MDA), glucose-6-phosphate dehydrogenase (G6PD), Glutathione reductase (GR), Glutathione peroxidase (GP), and catalase compared to the untreated samples, suggesting that the drug could cause a reduction of oxidative stress. Altogether, these results indicate that reduction of mGluR5 activation has a positive impact on i-astrocytes in ALS patients supporting the idea that the in-vivo amelioration of the disease progression, registered after mGluR5 genetical or pharmacological silencing, involve an astrocyte phenotype improvement also in humans. As a whole, mGluR5 may represent a potential therapeutic target to preserve MNs from death, also by modulating the reactive astroglial phenotype in ALS.

Characterization of Mouse and Human Astrocytes in Amyotrophic Lateral Sclerosis: Effects of Oxidative Stress and Blockade of the Metabotropic Glutamate Receptor 5

KUMAR, MANDEEP
2022

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to upper and lower motor neuron (MNs) death. Recognized as a non-cell-autonomous disease, ALS is also characterized by damage and degeneration of glial cells, such as astrocytes, microglia, and oligodendrocytes. Astrocytes acquire a reactive and toxic phenotype defined by an abnormal proliferation and by the release of neurotoxic factors. Recent studies reported that the uptake of [18F]-fluorodeoxyglucose (FDG) is increased in the spinal cord (SC) and decreased in the motor cortex (MC) of patients with ALS, suggesting that the disease might differently affect the two nervous districts with different time sequence or with different mechanisms. Here we show that MC and SC astrocytes harvested from newborn B6SJL-Tg (SOD1G93A) 1Gur (SOD1G93A) mice could play different roles in the pathogenesis of the disease. Spectrophotometric and cytofluorimetric analyses showed an increase in redox stress, a decrease in antioxidant capacity, and a relative mitochondria respiratory uncoupling in MC SOD1G93A astrocytes. By contrast, SC mutated cells showed a higher endurance against oxidative damage, through the increase in antioxidant defense and a preserved respiratory function. Thus, SOD1G93A mutation differently impaired MC and SC astrocyte biology in a very early stage of life. One major cause for MN degeneration in ALS is represented by glutamate-mediated excitotoxicity, due to the alteration of glutamate transmission mechanisms, including glutamate receptor function. In this context, the Group I metabotropic glutamate receptor 5 (mGluR5) has been proposed to play an important role in ALS, since it is largely overexpressed during disease progression and is involved in the altered neuronal and glial cellular processes. My research group previously demonstrated that mGluR5 produces abnormal glutamate release in the spinal cord of the SOD1G93A mouse model of ALS and that halving its expression has a positive impact on in-vivo disease progression, including motor neuron survival, astrogliosis, and microgliosis. They also investigated the consequences of reducing the mGluR5 expression in SOD1G93A mice on the reactive phenotype of spinal cord astrocytes cultured from late symptomatic (120 days old) SOD1G93A mice. Also in this model, reducing the mGluR5 expression ameliorated the astrocyte phenotype. UNIVERSITY OF GENOVA 8 Here, I translated this study to human astrocytes derived from healthy donors and ALS patients. We investigated the in-vitro pharmacological treatment effect of chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), a negative allosteric modulator of mGluR5 on i-astrocytes differentiated from the inducible neural progenitor cells (iNPCs) obtained from the skin fibroblast (i-astrocytes) of two ALS patients and two healthy donors. The overexpression of anti-glial fibrillary acid protein (GFAP), S100 calcium-binding protein β (S100β), and Complement component 3 (C3), three markers of astrogliosis, was reduced in CTEP-treated i-astrocytes. The same positive effect was obtained in the case of NLR family pyrin domain containing 3 (NLRP3) and nuclear factor erythroid 2-related factor 2 (NRF2), markers strictly related to inflammation and oxidative stress respectively, which are upregulated in ALS astrocytes. In-vitro pharmacological treatment with CTEP also reduced the expression of mGluR5 in mutated i-astrocytes. In addition, the CTEP treatment caused a decrement in antioxidant enzymatic activity such as malondialdehyde (MDA), glucose-6-phosphate dehydrogenase (G6PD), Glutathione reductase (GR), Glutathione peroxidase (GP), and catalase compared to the untreated samples, suggesting that the drug could cause a reduction of oxidative stress. Altogether, these results indicate that reduction of mGluR5 activation has a positive impact on i-astrocytes in ALS patients supporting the idea that the in-vivo amelioration of the disease progression, registered after mGluR5 genetical or pharmacological silencing, involve an astrocyte phenotype improvement also in humans. As a whole, mGluR5 may represent a potential therapeutic target to preserve MNs from death, also by modulating the reactive astroglial phenotype in ALS.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1081384
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