title: Neuronal influences are necessary to produce mitochondrial co-localization with glutamate transporters in astrocytes.
abstract: Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K(+). In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial:GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments. Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity.
authors.affiliation: Reading School of Pharmacy, University of Reading, Reading, UK; Bradford School of Pharmacy, University of Bradford, Bradford, UK.
authors.forename: Christopher I
authors.initials: CI
authors.lastname: Ugbode
authors.forename: Warren D
authors.initials: WD
authors.lastname: Hirst
authors.forename: Marcus
authors.initials: M
authors.lastname: Rattray
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D015153:Blotting, Western; D002450:Cell Communication; D002478:Cells, Cultured; D018920:Coculture Techniques; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D027342:Excitatory Amino Acid Transporter 2
keywords: EAAT2; Glial cell; TRAK2; co-culture; glutamate transporter; rho kinase