title: Cleavage of the vesicular glutamate transporters under excitotoxic conditions.
abstract: Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters (VGLUTs), and alterations in the transporters expression directly regulate neurotransmitter release. We investigated changes in VGLUT1 and VGLUT2 protein levels after ischemic and excitotoxic insults. The results show that VGLUT2 is cleaved by calpains after excitotoxic stimulation of hippocampal neurons with glutamate, whereas VGLUT1 is downregulated to a lower extent. VGLUT2 was also cleaved by calpains after oxygen/glucose deprivation (OGD), and downregulated after middle cerebral artery occlusion (MCAO) and intrahippocampal injection of kainate. In contrast, VGLUT1 was not affected after OGD. Incubation of isolated synaptic vesicles with recombinant calpain also induced VGLUT2 cleavage, with a little effect observed for VGLUT1. N-terminal sequencing analysis showed that calpain cleaves VGLUT2 in the C-terminus, at Asn(534) and Lys(542). The truncated GFP-VGLUT2 forms were found to a great extent in non-synaptic regions along neurites, when compared to GFP-VGLUT2. These findings show that excitotoxic and ischemic insults downregulate VGLUT2, which is likely to affect glutamatergic transmission and cell death, especially in the neonatal period when the transporter is expressed at higher levels.
authors.affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
authors.forename: Andrea C
authors.initials: AC
authors.lastname: Lobo
authors.forename: João R
authors.initials: JR
authors.lastname: Gomes
authors.forename: Tatiana
authors.initials: T
authors.lastname: Catarino
authors.forename: Miranda
authors.initials: M
authors.lastname: Mele
authors.forename: Pedro
authors.initials: P
authors.lastname: Fernandez
authors.forename: Ana R
authors.initials: AR
authors.lastname: Inácio
authors.forename: Ben A
authors.initials: BA
authors.lastname: Bahr
authors.forename: Armanda E
authors.initials: AE
authors.lastname: Santos
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Wieloch
authors.forename: Ana Luísa
authors.initials: AL
authors.lastname: Carvalho
authors.forename: Carlos B
authors.initials: CB
authors.lastname: Duarte
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002154:Calpain / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D005947:Glucose / Q000172:deficiency; D018698:Glutamic Acid / Q000494:pharmacology*; D006624:Hippocampus / Q000166:cytology; D000860:Hypoxia / Q000473:pathology; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000187:drug effects*; D051381:Rats; D017208:Rats, Wistar; D013572:Synaptic Vesicles / Q000187:drug effects; D014162:Transfection; D050598:Vesicular Glutamate Transport Protein 1 / Q000235:genetics / Q000378:metabolism*; D050599:Vesicular Glutamate Transport Protein 2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D018690:Excitatory Amino Acid Agonists; D050598:Vesicular Glutamate Transport Protein 1; D050599:Vesicular Glutamate Transport Protein 2; D018698:Glutamic Acid; D002154:Calpain; D053148:Caspase 3; D005947:Glucose