title: Conditional deletion of the glutamate transporter GLT-1 reveals that astrocytic GLT-1 protects against fatal epilepsy while neuronal GLT-1 contributes significantly to glutamate uptake into synaptosomes.
abstract: GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAP-CreERT2. Elimination of GLT-1 from astrocytes resulted in loss of ∼80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (Vmax) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake.
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115, Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Geraldine T
authors.initials: GT
authors.lastname: Petr
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115, Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115.
authors.forename: Natalie M
authors.identifier: http://orcid.org/0000-0002-4273-8519
authors.initials: NM
authors.lastname: Frederick
authors.affiliation: Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115.
authors.forename: Sameer C
authors.initials: SC
authors.lastname: Dhamne
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115, Department of Neurosurgery, Children's Hospital Boston, Boston, Massachusetts 02115, and.
authors.forename: Mustafa Q
authors.identifier: http://orcid.org/0000-0003-4004-5564
authors.initials: MQ
authors.lastname: Hameed
authors.affiliation: Center for Neural Science, New York University, New York, New York 10003.
authors.forename: Clive
authors.identifier: http://orcid.org/0000-0002-4119-0053
authors.initials: C
authors.lastname: Miranda
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bedoya
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115, Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Kathryn D
authors.initials: KD
authors.lastname: Fischer
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115, Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Wencke
authors.initials: W
authors.lastname: Armsen
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115.
authors.forename: Jianlin
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway.
authors.forename: Niels C
authors.initials: NC
authors.lastname: Danbolt
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115.
authors.forename: Alexander
authors.initials: A
authors.lastname: Rotenberg
authors.affiliation: Center for Neural Science, New York University, New York, New York 10003.
authors.forename: Chiye J
authors.identifier: http://orcid.org/0000-0003-4010-9425
authors.initials: CJ
authors.lastname: Aoki
authors.affiliation: Department of Neurology and the F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, Massachusetts 02115, Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, paul.rosenberg@childrens.harvard.edu.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Rosenberg
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism* / Q000648:ultrastructure; D001835:Body Weight; D001921:Brain / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D004569:Electroencephalography; D004827:Epilepsy / Q000378:metabolism* / Q000401:mortality / Q000517:prevention & control*; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D017729:Presynaptic Terminals / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism*
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D008081:Liposomes; C493396:Slc1a2 protein, mouse; D018698:Glutamic Acid
keywords: glia; ischemia; plasticity; seizures; spillover; transport
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