Glutamate Transporter Cluster Formation in Astrocytic Processes Regulates Glutamate Uptake Activity

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.

Cooperative Astrocyte and Dendritic Spine Dynamics at Hippocampal Excitatory Synapses

“…It further suggests that the targeted astrocytes in our system may be of the subtype that is important for glutamate reuptake (Volterra and Meldolesi, 2005). GLT-1 was found to be clustered on the surface of these cells as has been reported previously using immunostaining of cultured astrocytes (Zhou and Sutherland, 2004). The EGFPf labeled protoplasmic astrocytes were negative for other markers including neurofilament, NG2, CNP, and MBP (data not shown).…”

Ciliary Neurotrophic Factor Activates Astrocytes, Redistributes Their Glutamate Transporters GLAST and GLT-1 to Raft Microdomains, and Improves Glutamate HandlingIn Vivo

“…In purified raft fractions, glutamate uptake is more than 10 times higher than in nonraft membranes and when these rafts are disrupted after cholesterol removal, uptake is significantly reduced both in cortical culture and purified plasma membrane vesicles (Butchbach et al, 2004). In addition, recent data indicate that the formation of glutamate transporter clusters in glial cell processes, as we observed in vitro under CNTF treatment, plays important roles in regulating glutamate uptake in astrocytes (Zhou and Sutherland, 2004). Glutamate transporter enrichment in rafts of CNTFactivated astrocytes could therefore have strong functional consequences on the efficiency of glutamate homeostasis.…”

Oxidative and excitotoxic insults exert differential effects on spinal motoneurons and astrocytic glutamate transporters: Implications for the role of astrogliosis in amyotrophic lateral sclerosis

“…Both the oxidative and excitotoxic treatments mediated changes in astrocytic phenotype, which were much more prominent following oxidative insult, typical of reactive astrocytes (Pekny and Nilsson, 2005), and likely to improve handling of extracellular Glu (Escartin et al, 2006). The early maintenance of Glu uptake after SIN-1 treatment was not due to either altered overall or cell surface expression of EAAT1/2, indicating astrocytes possess mechanisms to preserve net EAAT activity, and which here was accomplished by a kinetic compensation (increased K m despite decreased V max ) normalizing transport for 2 h. Given the changes in astrocytic phenotype and EAAT distribution, and evidence that GFAP modulates EAAT function and is co-localized with EAAT2 (Hughes et al, 2004;Shobha et al, 2007;Zhou and Sutherland, 2004), the early increase in GFAP may be involved in the initial maintenance of Glu transport in the face of oxidative stressors. In the context of GFAP-EAAT2 interactions, this relationship may be relevant to the progression of ALS (Boillee et al, 2006) as the increase in GFAP immunoreactivity continued for 8 h and may represent a homeostatic response, which was unable to prevent the major loss of EAAT2.…”