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Neuronal influences are necessary to produce mitochondrial co-localization with glutamate transporters in astrocytes.
Ugbode, Christopher I. Hirst, W.D. Rattray, Marcus
Ugbode, Christopher I.
Hirst, W.D.
Rattray, Marcus
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2014-09
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© 2014 The Authors. Published Open Access by Wiley. Reproduced in accordance with the publisher's self-archiving policy.
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Abstract
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.
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Ugbode C, Hirst WD and Rattray M (2014) Neuronal influences are necessary to produce mitochondrial co-localization with glutamate transporters in astrocytes. Journal of Neurochemistry, 130 (5): 668-77.
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