title: Exocytosis regulates trafficking of GABA and glycine heterotransporters in spinal cord glutamatergic synapses: a mechanism for the excessive heterotransporter-induced release of glutamate in experimental amyotrophic lateral sclerosis.
abstract: The impact of synaptic vesicle endo-exocytosis on the trafficking of nerve terminal heterotransporters was studied by monitoring membrane expression and function of the GABA transporter-1 (GAT-1) and of type-1/2 glycine (Gly) transporters (GlyT-1/2) at spinal cord glutamatergic synaptic boutons. Experiments were performed by inducing exocytosis in wild-type (WT) mice, in amphiphysin-I knockout (Amph-I KO) mice, which show impaired endocytosis, or in mice expressing high copy number of mutant human SOD1 with a Gly93Ala substitution (SOD1(G93A)), a model of human amyotrophic lateral sclerosis showing constitutively excessive Glu exocytosis. Exposure of spinal cord synaptosomes from WT mice to a 35mM KCl pulse increased the expression of GAT-1 at glutamatergic synaptosomal membranes and enhanced the GAT-1 heterotransporter-induced [(3)H]d-aspartate ([(3)H]d-Asp) release. Similar results were obtained in the case of GlyT-1/2 heterotransporters. Preventing depolarization-induced exocytosis normalized the excessive GAT-1 and GlyT-1/2 heterotransporter-induced [(3)H]d-Asp release in WT mice. Impaired endocytosis in Amph-I KO mice increased GAT-1 membrane expression and [(3)H]GABA uptake in spinal cord synaptosomes. Also the GAT-1 heterotransporter-evoked release of [(3)H]d-Asp was augmented in Amph-I KO mice. The constitutively excessive Glu exocytosis in SOD1(G93A) mice resulted in augmented GAT-1 expression at glutamatergic synaptosomal membranes and GAT-1 or GlyT-1/2 heterotransporter-mediated [(3)H]d-Asp release. Thus, endo-exocytosis regulates the trafficking of GAT-1 and GlyT-1/2 heterotransporters sited at spinal cord glutamatergic nerve terminals. As a consequence, it can be hypothesized that the excessive GAT-1 and GlyT-1/2 heterotransporter-mediated Glu release, in the spinal cord of SOD1(G93A) mice, is due to the heterotransporter over-expression at the nerve terminal membrane, promoted by the excessive Glu exocytosis.
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Milanese
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Bonifacino
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Fedele
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Rebosio
authors.affiliation: Department of Pharmacy, Unit of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Cattaneo
authors.affiliation: Department of Neuroscience and Brain Technologies, Fondazione Istituto Italiano di Tecnologia, 16163 Genoa, Italy; Department of Experimental Medicine, University of Genoa, 16132 Genoa, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Benfenati
authors.affiliation: Institute of Biophysics, National Research Council, 16149 Genoa, Italy.
authors.forename: Cesare
authors.initials: C
authors.lastname: Usai
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: D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism*; D000818:Animals; D004195:Disease Models, Animal; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics; D013116:Spinal Cord / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D013569:Synapses / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D050485:GABA Plasma Membrane Transport Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C000606290:SOD1 protein, human; C493414:Slc6a1 protein, mouse; C493270:Slc6a5 protein, mouse; C493267:Slc6a9 protein, mouse; C075637:amphiphysin; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1
keywords: Amyotrophic lateral sclerosis; GABA heterotransporter; Glutamate excitotoxicity; Glutamate release; Glycine heterotransporter; SOD1(G93A) mice; Transporter trafficking