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.
Highlights
Address correspondence and reprint requests to Professor Marcus Rattray, Bradford School of Pharmacy, School of Life Sciences, University of Bradford, Bradford BD7 1DP, UK
We used confocal microscopy to investigate the association between Glutamate Transporter 1 (GLT-1) and mitochondria using Volocity software to calculate the level of co-localization between these two proteins
We found no association between GLT-1 and mitochondria in pure primary astrocytes, whereas in primary astrocytes cultured with neurons, there was an increased distribution of mitochondria into the thicker processes, as well as a small meaningful positive correlation between GLT-1 and mitochondria, that is a greater degree of overlap than expected by chance
Summary
An emerging theory is that GLT-1 is part of an activity-dependent macromolecular complex in astrocytes which efficiently couples energy provision to demand to maintain effective neurotransmission (Genda et al 2011; Jackson et al 2014). This theory predicts that GLT1: mitochondrial co-localization at the plasma membrane of astrocytes should become elevated under conditions where demand is increased. It is notable that TRAK2 is involved in the trafficking of a number of transmembrane ion channels and receptors including Kir2.1, and GABAA receptors in neurons, and it is possible that this protein has a role in astrocytes
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
