Genetic Downregulation of the Metabotropic Glutamate Receptor Type 5 Dampens the Reactive and Neurotoxic Phenotype of Adult ALS Astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100ß and vimentin. In vitro exposure to an anti-mGluR5 antisense oligonucleotide or to the negative allosteric modulator CTEP also ameliorated the altered reactive astrocyte phenotype. Downregulating mGluR5 in SOD1G93A mice reduced the synthesis and release of the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α and ameliorated the cellular bioenergetic profile by improving the diminished oxygen consumption and ATP synthesis and by lowering the excessive lactate dehydrogenase activity. Most relevantly, mGluR5 downregulation hampered the neurotoxicity of SOD1G93A astrocytes co-cultured with spinal cord MNs. We conclude that selective reduction in mGluR5 expression in SOD1G93A astrocytes positively modulates the astrocyte reactive phenotype and neurotoxicity towards MNs, further supporting mGluR5 as a promising therapeutic target in ALS.

In-vivo genetic ablation of metabotropic glutamate receptor type 5 slows down disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease due to motor neuron (MN) loss. The mechanisms causing selective MN death are largely unknown, thus prejudicing successful pharmacological treatments. Major causes of MN damage are effects downstream of the abnormal glutamate (Glu) neurotransmission. Group I metabotropic Glu receptors (mGluR1, mGluR5) actively contribute to the excitotoxicity in ALS and represent druggable molecular targets. We previously demonstrated that halving mGluR1 or mGluR5 expression in the widely studied SOD1G93A mouse model of ALS had a positive impact on disease onset, clinical progression and survival, as well as on cellular and biochemical parameters altered in ALS. Whereas these effects were similar in female and male mGluR1 heterozygous SOD1G93Amice, only male mGluR5 heterozygous SOD1G93A mice showed improved motor skills during disease progression. To further validate the role of Group I mGluRs in ALS, we generated in this study mGluR1 or mGluR5 null mice expressing the SOD1G93A mutation (SOD1G93AGrm1crv4/crv4 or SOD1G93AGrm5-/-, respectively). SOD1G93AGrm1crv4/crv4 mice showed early and progressive motor impairments and died even before SOD1G93A mice, while SOD1G93AGrm5-/- mice exhibited delayed disease onset, longer survival, and ameliorated motor skills than SOD1G93A mice. No difference between female and male SOD1G93AGrm5-/- mice were observed. These effects were associated with enhanced MN preservation and decreased astrocytic and microglial activation. Our results strongly support the assumption that constitutively lowering of mGluR5 expression has a positive impact in mice with ALS by counteracting the abnormal Glu transmission and this could be a potentially effective pharmacological target in ALS.

Characterization of the Mitochondrial Aerobic Metabolism in the Pre- and Perisynaptic Districts of the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disease characterized by muscle wasting, weakness, and spasticity due to a progressive degeneration of cortical, brainstem, and spinal motor neurons. The etiopathological causes are still largely obscure, although astrocytes definitely play a role in neuronal damage. Several mechanisms have been proposed to concur to neurodegeneration in ALS, including mitochondrial dysfunction. We have previously shown profound modifications of glutamate release and presynaptic plasticity in the spinal cord of the SOD1G93A mouse model of ALS. In this work, we characterized, for the first time, the aerobic metabolism in two specific compartments actively involved in neurotransmission (i.e. the presynaptic district, using purified synaptosomes, and the perisynaptic astrocyte processes, using purified gliosomes) in SOD1G93A mice at different stages of the disease. ATP/AMP ratio was lower in synaptosomes isolated from the spinal cord, but not from other brain areas, of SOD1G93A vs. control mice. The energy impairment was linked to altered oxidative phosphorylation (OxPhos) and increment of lipid peroxidation. These metabolic dysfunctions were present during disease progression, starting at the very pre-symptomatic stages, and did not depend on a different number of mitochondria or a different expression of OxPhos proteins. Conversely, gliosomes showed a reduction of the ATP/AMP ratio only at the late stages of the disease and an increment of oxidative stress also in the absence of a significant decrement in OxPhos activity. Data suggest that the presynaptic neuronal moiety plays a pivotal role for synaptic energy metabolism dysfunctions in ALS. Changes in the perisynaptic compartment seem subordinated to neuronal damage.

In-vivo effects of knocking-down metabotropic glutamate receptor 5 in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1G93A mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1G93A mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1G93A mice with reduced expression of mGluR5 (SOD1G93AGrm5-/+) by crossing the SOD1G93A mutant mouse with the mGluR5 heterozigous Grm5-/+ mouse. SOD1G93AGrm5-/+ mice showed prolonged survival probability and delayed pathology onset. These effects were associated to enhanced number of preserved MNs, decreased astrocyte and microglia activation, reduced cytosolic free Ca2+ concentration, and regularization of abnormal Glu release in the spinal cord of SOD1G93AGrm5-/+ mice. Unexpectedly, only male SOD1G93AGrm5-/+ mice showed improved motor skills during disease progression vs. SOD1G93A mice, while SOD1G93AGrm5-/+ females did not. These results demonstrate that a lower constitutive level of mGluR5 has a significant positive impact in mice with ALS and support the idea that blocking Group I mGluRs may represent a potentially effective pharmacological approach to the disease.

Altered mechanisms underlying the abnormal glutamate release in amyotrophic lateral sclerosis at a pre-symptomatic stage of the disease.

Abnormal Glu release occurs in the spinal cord of SOD1(G93A) mice, a transgenic animal model for human ALS. Here we studied the mechanisms underlying Glu release in spinal cord nerve terminals of SOD1(G93A) mice at a pre-symptomatic disease stage (30days) and found that the basal release of Glu was more elevated in SOD1(G93A) with respect to SOD1 mice, and that the surplus of release relies on synaptic vesicle exocytosis. Exposure to high KCl or ionomycin provoked Ca(2+)-dependent Glu release that was likewise augmented in SOD1(G93A) mice. Equally, the Ca(2+)-independent hypertonic sucrose-induced Glu release was abnormally elevated in SOD1(G93A) mice. Also in this case, the surplus of Glu release was exocytotic in nature. We could determine elevated cytosolic Ca(2+) levels, increased phosphorylation of Synapsin-I, which was causally related to the abnormal Glu release measured in spinal cord synaptosomes of pre-symptomatic SOD1(G93A) mice, and increased phosphorylation of glycogen synthase kinase-3 at the inhibitory sites, an event that favours SNARE protein assembly. Western blot experiments revealed an increased number of SNARE protein complexes at the nerve terminal membrane, with no changes of the three SNARE proteins and increased expression of synaptotagmin-1 and ß-Actin, but not of an array of other release-related presynaptic proteins. These results indicate that the abnormal exocytotic Glu release in spinal cord of pre-symptomatic SOD1(G93A) mice is mainly based on the increased size of the readily releasable pool of vesicles and release facilitation, supported by plastic changes of specific presynaptic mechanisms.