title: Altered mechanisms underlying the abnormal glutamate release in amyotrophic lateral sclerosis at a pre-symptomatic stage of the disease.
abstract: 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 β-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.
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology, and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy. Electronic address: bonifacino@difar.unige.it.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Bonifacino
authors.affiliation: Department of Pharmacological and Biomolecular Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy. Electronic address: laura.musazzi@unimi.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Musazzi
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology, and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy. Electronic address: milanese@difar.unige.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Milanese
authors.affiliation: Department of Pharmacological and Biomolecular Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy. Electronic address: mara.seguini@unimi.it.
authors.forename: Mara
authors.initials: M
authors.lastname: Seguini
authors.affiliation: Department of Experimental Medicine, Unit of Human Physiology, University of Genoa, Viale Benedetto XV, 16132 Genoa, Italy. Electronic address: antonella.marte@unige.it.
authors.forename: Antonella
authors.initials: A
authors.lastname: Marte
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology, and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy. Electronic address: gallia@difar.unige.it.
authors.forename: Elena
authors.initials: E
authors.lastname: Gallia
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology, and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy. Electronic address: cattaneo@difar.unige.it.
authors.forename: Luca
authors.initials: L
authors.lastname: Cattaneo
authors.affiliation: Department of Experimental Medicine, Unit of Human Physiology, University of Genoa, Viale Benedetto XV, 16132 Genoa, Italy. Electronic address: franco.onofri@unige.it.
authors.forename: Franco
authors.initials: F
authors.lastname: Onofri
authors.affiliation: Department of Pharmacological and Biomolecular Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy. Electronic address: maurizio.popoli@unimi.it.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Popoli
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology, and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy. Electronic address: bonanno@difar.unige.it.
authors.forename: Giambattista
authors.initials: G
authors.lastname: Bonanno
mesh_terms: D000199:Actins / Q000378:metabolism; D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism*; D000818:Animals; D004195:Disease Models, Animal; D005089:Exocytosis / Q000502:physiology; D018698:Glutamic Acid / Q000378:metabolism*; D008822:Mice, Transgenic; D017661:Receptors, Presynaptic / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism*
chemical_list: D000199:Actins; D017661:Receptors, Presynaptic; D018698:Glutamic Acid; C109415:SOD1 G93A protein; D013482:Superoxide Dismutase
keywords: Amyotrophic lateral sclerosis; Excessive glutamate release; Glutamate excitotoxicity; Glutamate release mechanisms; Glycogen synthase kinase 3; Pre-symptomatic SOD1(G93A) mice; Presynaptic proteins; Synapsin-I