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. 2004 Jul 14;24(28):6301-6.
doi: 10.1523/JNEUROSCI.1404-04.2004.

Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity

Jianzheng Zhou  1 , Margaret L Sutherland
Affiliations

Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity

Jianzheng Zhou et al. J Neurosci. .

Abstract

Glutamate is the predominant excitatory neurotransmitter in the CNS, and it is removed from the synaptic cleft by sodium-dependent glutamate transport activity. Glutamate transporter-1 (GLT-1) is expressed predominantly in astroglial cells and is responsible for the largest proportion of glutamate transport in the adult forebrain. In the present study, we demonstrate the ability of endogenous and recombinant GLT-1 to form clusters in astrocytic processes and characterize the mobility and physiological importance of these clusters in the regulation of GLT-1 activity in the presence or absence of neurons. At the distal end of C6 glioma cell processes, GLT-1 clusters undergo rapid morphological changes in both shape and size, and these changes are inhibited by cytochalasin D treatment, suggesting that the morphogenesis of GLT-1 clusters is highly dependent on the actin network. Treatment of astrocytes with phorbol 12-myristate 13-acetate (PMA) quickly and preferentially decreases GLT-1 localization on the process membrane, leading to de novo generation of GLT-1 clusters along the process shaft. Pretreatment with the PKC inhibitor bisindolylmaleimide II (Bis II), with sucrose (0.4 m), or through the expression of a dominant-negative form of dynamin prevents PMA-induced GLT-1 internalization and cluster formation. In terms of glutamate transporter function, PMA treatment elicits a significant decrease in GLT-1 activity that is prevented by preexposure to either Bis II or hypertonic treatment. Together, these data indicate that GLT-1 trafficking and cluster formation in glial cell processes are dynamic events that play important roles in regulating glutamate uptake in astrocytes and glioma cells.

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Figures

Figure 1.
Figure 1.
Mobility of GLT-1 clusters in the astrocytic process. Double immunostaining of GLT-1 (A) and GFAP (B) in astrocyte cultures showing clusters of endogenous GLT-1 in the fork or distal end of the process, as indicated by yellow arrowheads in A and the composite image (C), is indicated. Astrocytes (D) and C6 cells (E) transfected with the GFP-tagged GLT-1 expression cassette demonstrate GLT-1 cluster formation, as indicated by yellow arrowheads, whereas expression of GFP alone fails to form clusters (F). Please note that some long and thin processes in the astrocyte (D) are not generated directly from the cell body but instead from a filopodia-thick structure that is linked with the soma. This structure is outlined in yellow and referred to as a secondary process. Time-lapse imaging of live astrocytes reveals the mobility of GLT-1 clusters in astrocytic processes of both astrocyte cultures alone (G-L) and in cocultures with neurons (O-S). A differential interference contrast image (M) shows the neuron-astrocyte coculture in which the neuron grows on top of an astrocyte expressing GFP-tagged GLT-1. The inset (N) outlines the visualized portion of GLT-1 cluster mobility in the neuron-astrocyte coculture. A GLT-1 cluster on the process moves in a retrograde direction, as indicated by the arrowhead, although most of the clusters are stationary, as indicated by arrows. Scale bars: (in C) A-C, (in M) M, N, 15 μm; D, 10 μm; E, 7 μm; F, (in S) O-S, 9 μm; (in L) G-L, 3 μm.
Figure 2.
Figure 2.
Morphogenesis of the GLT-1 cluster is dependent on the actin network. At the distal end of C6 cell processes, GFP-taggedGLT-1 undergoes rapid morphogenesis. A GLT-1 cluster may split into several smaller clusters (A, top), with significant expansion of the membrane area and perimeter (B), or disappear when a new process is produced (A, bottom). The numbers in A and D represent time (in minutes). C, A schematic showing the experimental protocol for the observation of morphological changes in GLT-1 cluster dimensions before and after CytoD treatment. D, Time-lapse images showing morphological changes in the GLT-1 cluster before (top) and after (bottom) CytoD treatment for 60 min. E, The quantitation of area and perimeter of a GLT-1 cluster before and after CytoD treatment (10 μm). Scale bars: A, 1.5 μm; D, 1.0 μm.
Figure 3.
Figure 3.
PMA treatment preferentially reduces the level of GLT-1 protein in astrocytic process. Representative time-series pictures show changes in GLT-1 intensity and clusters in the astrocytic processes after treatments with 400 nm PMA (A), 400 nm PMA and 10 μm Bis II in combination (B), and 400 nm PMA on a dominant-negative dynamin-expressing astrocyte (C). The arrowheads in A and B outline the appearance of new clusters. D, Time-series pictures showing changes in GLT-1 intensity associated with the soma membrane after treatment with PMA (400 nm). The inset to the left outlines the visualized portion of the astrocyte. E, Time-series pictures showing changes in GLT-1 intensity after treatment with PMA (400 nm) on the astrocytic secondary process. Quantitations are based on five to nine independent experiments. The numbers in A-E represent time (in minutes). Scale bars: A, C, 5 μm; B, 10 μm; D, 25 μm (left image) and 15 μm (other 4 images); E, 8 μm.
Figure 4.
Figure 4.
PMA treatment decreases GLT-1 activity. A, A representative immunoblot (top) showing the redistribution of GLT-1 in the membrane after treatments with 400 nm PMA, 10 μm Bis II, or a combination of both. The bar graph (bottom) summarizes changes in GLT-1 immunoreactivity after treatment with PMA, Bis II, or both. *p < 0.01, compared with control. Control values are measured in the absence of PMA or Bis II. Mean ± SE; n = 4. B, Quantitation of glutamate uptake in the presence or absence of DHK (500 nm) (left) and GLT-1-specific (right) glutamate uptake after treatment with PMA, Bis II, or both in C6 cells stably expressing GFP-tagged GLT-1. Mean ± SE; *p < 0.05; **p < 0.01; #p < 0.05, compared with control; n = 9. C, Quantitation of glutamate uptake in the presence or absence of DHK (500 nm) (left) and GLT-1-specific (right) glutamate uptake after treatment with PMA, Bis II, or both in primary astrocyte cultures. Mean ± SE; **p < 0.01, compared with control; ++p < 0.01, compared with Bis II; n = 9. D, Quantitation of GLT-1-specific activity after treatment with PMA (400 nm), high sucrose (0.4 m), or PMA and high sucrose in combination in C6 cells stably expressing GFP-tagged GLT-1. Mean ± SE; **p < 0.01; #p < 0.05, compared with control; n = 6.
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