Knocking down metabotropic glutamate receptor 1 improves survival and disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1(G93A)), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1(G93A) background (SOD1(G93A)Grm1(crv4/+)), by crossing the SOD1(G93A) mutant mouse with the Grm1(crv4/+) mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1(G93A)Grm1(crv4/+) mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1(G93A) mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1(G93A)Grm1(crv4/+)compared to SOD1(G93A) mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact on mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors.

Milanese M ，Giribaldi F ，Melone M ，Bonifacino T ，Musante I ，Carminati E ，Rossi PI ，Vergani L ，Voci A ，Conti F ，Puliti A ，Bonanno G ... -  《-》

In-vivo effects of knocking-down metabotropic glutamate receptor 5 in the SOD1(G93A) 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.

Bonifacino T ，Cattaneo L ，Gallia E ，Puliti A ，Melone M ，Provenzano F ，Bossi S ，Musante I ，Usai C ，Conti F ，Bonanno G ，Milanese M ... -  《-》

In-vivo genetic ablation of metabotropic glutamate receptor type 5 slows down disease progression in the SOD1(G93A) 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.

Bonifacino T ，Provenzano F ，Gallia E ，Ravera S ，Torazza C ，Bossi S ，Ferrando S ，Puliti A ，Van Den Bosch L ，Bonanno G ，Milanese M ... -  《-》

Enhanced Function and Overexpression of Metabotropic Glutamate Receptors 1 and 5 in the Spinal Cord of the SOD1(G93A) Mouse Model of Amyotrophic Lateral Sclerosis during Disease Progression.

Bonifacino T ，Rebosio C ，Provenzano F ，Torazza C ，Balbi M ，Milanese M ，Raiteri L ，Usai C ，Fedele E ，Bonanno G ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Group I metabotropic glutamate autoreceptors induce abnormal glutamate exocytosis in a mouse model of amyotrophic lateral sclerosis.

Glutamate-mediated excitotoxicity plays a major role in ALS and reduced astrocytic glutamate transport was suggested as a cause. Based on previous work we have proposed that abnormal release may represent another source of excessive glutamate. In this line, here we studied the modulation of glutamate release in ALS by Group I metabotropic glutamate (mGlu) receptors, that comprise mGlu1 and mGlu5 members. Synaptosomes from the lumbar spinal cord of SOD1/G93A mice, a widely used murine model for human ALS, and controls were used in release, confocal or electron microscopy and Western blot experiments. Concentrations of the mGlu1/5 receptor agonist 3,5-DHPG >0.3 μM stimulated the release of [(3)H]d- aspartate, used to label the releasing pools of glutamate, both in control and SOD1/G93A mice. At variance, ≤0.3 μM 3,5-DHPG increased [(3)H]d-aspartate release in SOD1/G93A mice only. Experiments with selective antagonists indicated the involvement of both mGlu1 and mGlu5 receptors, mGlu5 being preferentially involved in the high potency effects of 3,5-DHPG. High 3,5-DHPG concentrations increased IP3 formation in both mouse strains, whereas low 3,5-DHPG did it in SOD1/G93A mice only. Release experiments confirmed that 3,5-DHPG elicited [(3)H]d-aspartate exocytosis involving intra-terminal Ca(2+) release through IP3-sensitive channels. Confocal microscopy indicated the co-existence of both receptors presynaptically in the same glutamatergic nerve terminal in SOD1/G93A mice. To conclude, activation of mGlu1/5 receptors produced abnormal glutamate release in SOD1/G93A mice, suggesting that these receptors are implicated in ALS and that selective antagonists may be predicted for new therapeutic approaches. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Giribaldi F ，Milanese M ，Bonifacino T ，Anna Rossi PI ，Di Prisco S ，Pittaluga A ，Tacchetti C ，Puliti A ，Usai C ，Bonanno G ... -  《-》