title: Gephyrin Cleavage in In Vitro Brain Ischemia Decreases GABAA Receptor Clustering and Contributes to Neuronal Death.
abstract: GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the central nervous system, and changes in GABAergic neurotransmission modulate the activity of neuronal networks. Gephyrin is a scaffold protein responsible for the traffic and synaptic anchoring of GABAA receptors (GABAAR); therefore, changes in gephyrin expression and oligomerization may affect the activity of GABAergic synapses. In this work, we investigated the changes in gephyrin protein levels during brain ischemia and in excitotoxic conditions, which may affect synaptic clustering of GABAAR. We found that gephyrin is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, as well as after intrahippocampal injection of kainate, giving rise to a stable cleavage product. Gephyrin cleavage was also observed in cultured hippocampal neurons subjected to transient oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia, and after transient middle cerebral artery occlusion (MCAO) in mice, a model of focal brain ischemia. Furthermore, a truncated form of gephyrin decreased the synaptic clustering of the protein, reduced the synaptic pool of GABAAR containing γ2 subunits and upregulated OGD-induced cell death in hippocampal cultures. Our results show that excitotoxicity and brain ischemia downregulate full-length gephyrin with a concomitant generation of truncated products, which affect synaptic clustering of GABAAR and cell death.
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal.
authors.forename: João T
authors.initials: JT
authors.lastname: Costa
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal.|Institute for Interdisciplinary Research, University of Coimbra (IIIUC), Coimbra, 3030-789, Portugal.
authors.forename: Miranda
authors.initials: M
authors.lastname: Mele
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal.
authors.forename: Márcio S
authors.initials: MS
authors.lastname: Baptista
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal.
authors.forename: João R
authors.initials: JR
authors.lastname: Gomes
authors.affiliation: Laboratory for Experimental Brain Research, Division of Neurosurgery, Department of Clinical Sciences, Lund University, BMC A13, S-22184, Lund, Sweden.
authors.forename: Karsten
authors.initials: K
authors.lastname: Ruscher
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal.|Institute for Interdisciplinary Research, University of Coimbra (IIIUC), Coimbra, 3030-789, Portugal.
authors.forename: Rui J
authors.initials: RJ
authors.lastname: Nobre
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal.|Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.
authors.forename: Luís Pereira
authors.initials: LP
authors.lastname: de Almeida
authors.affiliation: Laboratory for Experimental Brain Research, Division of Neurosurgery, Department of Clinical Sciences, Lund University, BMC A13, S-22184, Lund, Sweden.
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Wieloch
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal. cbduarte@ci.uc.pt.|Department of Life Sciences, University of Coimbra, 3004-517, Coimbra, Portugal. cbduarte@ci.uc.pt.|Center for Neuroscience and Cell Biology, Faculty of Medicine, Polo I, Rua Larga, University of Coimbra, 3004-504, Coimbra, Portugal. cbduarte@ci.uc.pt.
authors.forename: Carlos B
authors.initials: CB
authors.lastname: Duarte
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology*; D002154:Calpain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D016923:Cell Death; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D005947:Glucose / Q000172:deficiency; D018698:Glutamic Acid / Q000633:toxicity; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism* / Q000473:pathology*; D009498:Neurotoxins / Q000633:toxicity; D010100:Oxygen; D017208:Rats, Wistar; D011963:Receptors, GABA-A / Q000378:metabolism*; D013569:Synapses / Q000187:drug effects / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D009498:Neurotoxins; D011963:Receptors, GABA-A; C073441:gephyrin; D018698:Glutamic Acid; D002154:Calpain; D005947:Glucose; D010100:Oxygen
keywords: Brain ischemia; Calpains; Excitotoxicity; GABAergic synapses; Gephyrin