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Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium-dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na+-dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na+-dependent glutamate/aspartate transporter and Na+-dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover.
l-Glutamate (l-Glu) is the major excitatory amino acid neurotransmitter in the vertebrate brain. Membrane-specific l-Glu receptors expressed in neurons and glial cell mediate most, albeit not all, of its effects. Ionotropic receptors of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), N-methyl-D-aspartate (NMDA), and kainate (KA) subtypes are proteins composed of different subunits (Sager et al. 2009). Metabotropic Glu receptors have been subdivided in terms of sequence similarity and signaling mechanisms in three groups. Signaling through these receptors is carried out by the phospholipase C and Ca2+ for Group I and inhibition of adenylate cyclase for Groups II and III (Wieronska and Pilc 2009).
l-Glu extracellular levels are tightly regulated through its uptake, mostly into glial cells, by a family of Na+-dependent l-Glu transporters known as excitatory amino acid transporters of which EAAT-1, also known as Na+-dependent glu/aspartate transporter (GLAST) and EAAT-2 or Glu transporter-1 (Glt-1) are mainly expressed in glia cells (Danbolt 2001). Once this amino acid has been removed, it is rapidly converted to l-glutamine (l-Gln) through the action of Gln synthetase (GS) (Shank and Campbell 1984). Neutral amino acid transporters mediate both the glial release and the neuronal uptake of l-Gln. Several transporter systems have been described for these amino acids, based not only in sequence identity but also on their kinetic properties (Dolinska et al. 2000, 2003; Hagglund et al. 2011). In general terms, it has been assumed that l-Gln release from astrocytes (Albrecht 1989). One of the transporters that carries out this release is the Na+-dependent neutral amino acid transporter 3 (SNAT3), a member of System N, a family of l-Gln transporters capable of acting in a reversed fashion (Broer et al. 2004). In contrast, neuronal l-Gln uptake has been postulated to be carried out by members of System A transporters (SNAT2) (Jenstad et al. 2009), although this proposal has been questioned at least for neocortical neurons (Grewal et al. 2009).
Bergmann glial cells (BGC) extend their processes through the molecular layer of the cerebellar cortex surrounding excitatory and inhibitory synapses (Somogyi et al. 1990). The tripartite relationship between pre-synapse, post-synapse, and surrounding glia as a source of neuroactive substances like ATP and D-serine has also been acknowledged (Henneberger et al. 2010). Therefore, it has been postulated that BGCs are involved in neuronal communication and that they might even constitute a neuronal reservoir (Hansson and Ronnback 1995; Malatesta et al. 2003; Anthony et al. 2004). When cultured, BGC become an excellent model in which the molecular and cellular basis of glial–neuronal signaling can be analyzed in the context of glutamatergic transmission (Lopez-Bayghen et al. 2007). In such preparations, l-Glu acting through its receptors, changes gene expression at the transcriptional and translational levels (Gonzalez-Mejia et al. 2006; Rosas et al. 2007). In addition, it has become evident that plasma membrane l-Glu transporters are also involved in glutamatergic signaling. For example, it has been demonstrated that l-Glu uptake activity triggers glucose influx (Magistretti 2009) and increases GS activity (Lehmann et al. 2009) in astrocytes. Furthermore, in BGC cultures, GLAST/EAAT-1, the sole l-Glu transporter expressed in these cells (Regan et al. 2007) is linked to the mammalian target of rapamycin (mTOR) activation and thus protein expression regulation (Martinez-Lozada et al. 2011). With these evidences in mind, we decided to explore the putative role of l-Glu transporters in the regulation of l-Gln release. To reach this goal, we first characterized the L-[3H] Gln uptake activity in our culture system and evaluated if GLAST/EAAT-1 substrates such as D-Aspartate (D-Asp) would induce a significant l-Gln release. Once we were able to detect such an effect, we hypothesized that both transporters are present in a macromolecular complex and that D-Asp treatment would be linked to the appearance of such complex. Indeed, we were able to detect a coupling between GLAST and SNAT3.
These results suggest that glia cells associated to glutamatergic synapses behave as l-Glu sensors and that these cells may control, to a greater extent than assumed before, excitatory transmission. Moreover, our findings strengthen the notion of the active participation of glia cells in synaptic transmission.
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Over the last decade, the concept of the tripartite synapse has been widely supported (Araque et al. 1999; Volterra and Meldolesi 2005; Halassa et al. 2007; Haydon et al. 2009). Glia cells express a battery of neurotransmitter receptors and transporters that enable them to respond to neuronal activity. Within glutamatergic synapses, the fundamental role of astrocytes in the recycling of the neurotransmitter has long been acknowledged (Bak et al. 2006). The l-Glu/Gln shuttle provides good evidence about the capacity of glial cells to respond to synaptic activity through the modification of their cellular functions such as GS expression and activity (Lehmann et al. 2009). Another example of a tight coupling of glia cells to glutamatergic synaptic activity is the astrocyte/neuron lactate shuttle (Magistretti 2009). Furthermore, depolarization of the cerebellar parallel fibers activates Bergmann glial l-Glu receptors and transporters (Balakrishnan and Bellamy 2009).
Using as a model system cultured BGC from chick cerebellum, we characterized several signaling cascades involved in l-Glu-dependent transcriptional regulation (Lopez-Bayghen et al. 2007). We have been able to demonstrate that l-Glu signaling is also supported by the unique l-Glu transporter expressed in these cells, GLAST/EAAT1 (Martinez-Lozada et al. 2011). All these findings, as well as the lack of a complete biochemical characterization of the l-Glu/Gln shuttle, led us to explore a plausible interaction between l-Glu uptake and l-Gln release.
The first issue that we had to tackle before any biochemical experiment could even be designed, was the identification of the expression of l-Gln transporters that function in reverse mode within BGC. Since System N members could fulfill this requirement, and SNAT3 expression is prominent in the days just before birth and in the adulthood in the rat, we restricted our approach to SNAT3. However, we must mention that SNAT5 has been shown to be present in rat Bergmann glia cells (Cubelos et al. 2005). The results presented in Fig. 1, demonstrate, in the one hand, the specificity of our anti-peptide anti-GLAST/EAAT1 antibodies (Fig. 1a) as well as that SNAT3 is expressed in chick cerebellar BGC, both in situ as well as in our culture system. It is important to note the similar labeling of the Bergmann glia processes with anti-GLAST, anti-KBP, and anti-SNAT3 antibodies. Unfortunately, all these antibodies were generated in goat, preventing us to do double immunolabeling experiments. Nevertheless, if one compares the decoration with anti-calbindin antibodies, a Purkinje cells marker, it remains clear that SNAT3 co-localizes with KBP and GLAST.
The fact that the kinetic parameters detected for SNAT3 in our culture system are in the mM range as has been reported for other glia cells, made us confident that indeed, Bergmann glia express SNAT3 transporters. One could argue that several reports settle a Km value of approximately 1 mM (Kilberg et al. 1980; Chaudhry et al. 2001; Broer et al. 2004) for SNAT3 and that the ones we report here are higher (2.9 mM). It should be noted, however, that the reported constants were obtained in heterologous glial cultures, whereas we use an enriched BGC culture (Ortega et al. 1991). Besides, our model system is avian origin in contrast to rodent cells used in the referred studies. Whether the apparent discrepancy is related to the cell population examined or the species used, it is not known at this moment. At this stage, we cannot rule out a plausible involvement of SNAT5 in our measurements of L-[3H] Gln uptake or release. A pertinent observation could also be that if we expect that this carrier would function in a reverse manner, why we would be interested in the characterization of the uptake activity. The answer is simple: it is easier to measure uptake than release, and for kinetic purposes it is valid approach (Broer et al. 2004).
In any event, it became important to demonstrate a l-Glu-induced l-Gln release. Given the fact that in our experimental conditions, > 70% of the L-[3H] Gln loaded in the cells remains as L-[3H] Gln as judged by ion exchange chromatography, the experiments described in Fig. 4 show that D-Asp is capable to trigger l-Gln release in a more efficient manner than l-Glu. A plausible explanation to this finding is that l-Glu binds to receptors and transporters, whereas D-Asp would only binds to GLAST/EAAT1. These results strengthen our confidence in the hypothesis of a complex between these transporters. A detectable immunoreactive SNAT3 polypeptide is present in anti-GLAST immunoprecipitates and vice versa (Fig. 5a). Whether this is a direct protein–protein interaction between these two transporters, it is not known at this moment, work in progress in our lab is aimed at that direction. In support to our hypothesis, it is important to mention that recently the group of Billups, using electrophysiological recordings, has reported that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body release l-Gln as a consequence of l-Glu transport activation (Uwechue et al. 2012).
What could it be the purpose of a GLAST/SNAT3 association? One could postulate that in order for SNAT3 to work in reverse mode a significant rise in intracellular Na+ within the vicinity of the transporter should be taking place. A functional coupling with GLAST would provide that local rise in Na+ concentration, if this interpretation is correct then, first of all, D-Asp influx should prevent any L-[3H] Gln uptake, and as demonstrated in Fig. 3, this is exactly the case. The fact that this prevention in L-[3H] Gln uptake is dose dependent suggests that this is an specific effect. A valid argument in the interpretation of these results is that the reported Km of BGC GLAST/EAAT1 is 62 μM and the IC50 values for the prevention in L-[3H] Gln uptake is of 0.873 μM. A plausible explanation for this apparent discrepancy lies in the signal amplification inherent to cell signaling. For example, in BGC, a 1 mM l-Glu has to be applied to record a Na+ inward current (Bennay et al. 2008), whereas the EC50 for l-Glu obtained through Oct-2/DNA binding activity is 164 μM (Mendez et al. 2004). Furthermore, it could be expected that D-Asp should increase GLAST/SNAT3 association whereas l-Gln not. Indeed, the results presented in Fig. 5c support this vision. A model of our interpretation of the results is shown in Fig. 6.
Figure 6. Schematic representation of the Na+-dependent glutamate/aspartate transporter (GLAST)/Na+-dependent neutral amino acid transporter 3 (SNAT3) complex. l-Glu released from the parallel fibers is taken up into Bergmann glia cells through GLAST. The increase in [Na+]i favors the operation, in reverse mode, of SNAT3 with the consequent release of l-Gln. Glu, l-glutamate; Gln, glutamine; GLU-R, Glutamate receptors; GLAST, sodium-dependent glutamate/aspartate transporter; SNAT, sodium-dependent neutral amino acid transporter.
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Taken together, our results provide a support to the critical involvement of glia cells in the modulation of l-Glu-mediated neurotransmission in what is nowadays known as the tripartite synapse.