br Materials and methods br Results br Discussion Neurotrans

Materials and methods
Results
Discussion Neurotransmitter uptake is one of the most important aspects of synaptic transmission, it fact, the existence of a specific uptake system is one of the requisites that any neuroactive substance has to cover to be considered a neurotransmitter. It has been assumed that its physiological role is mainly restricted to the recycling of the neurotransmitter (Holz and Fisher, 2006). Dysfunction of the various transporter systems result in a plethora of neurological and psychiatry disorders ranging from epilepsy to depression (Kanner, 2011, Wankerl et al., 2010). Glu transporters are not the exemption and have also been described to actively participate in the homeostasis of glutamatergic transmission (Danbolt, 2001). Glial Glu uptake and its rapid transformation into glutamine to complete the Glu/glutamine shuttle provided a biochemical framework for the involvement of these Glyoxalase I inhibitor in glutamatergic neurotransmission (Shank and Campbell, 1984). Thereafter, the expression and characterization of glia neurotransmitter receptors paved the way into the concept of the tripartite synapse (Araque et al., 1999) and more recently the involvement of the extracellular matrix in synaptic signaling has postulated the so called tetrapartite synapse (Dityatev and Rusakov, 2011). In this context, glia cells associated to glutamatergic synapses respond to synaptic activity via receptors through the generation of Ca2+ waves (Muller et al., 1996) and via transporters by the Glu/glutamine shuttle and its associated inward Na+ current (Owe et al., 2006). Of particular interest, has been glial gene expression regulation by Glu (Balazs, 2006, Gallo and Ghiani, 2000, Lopez-Bayghen and Ortega, 2010, Lopez-Bayghen et al., 2007). During our studies on the involvement of mTOR in Glu-dependent translational control in cultured BGC, we realized that part of the response was not sensitive to Glu receptors antagonists and decided to explore whether EAAT-1/GLAST, the unique Glu transporter present in these cells, was involved. A d-Asp mediated mTOR phosphorylation was found although not characterized at that time (Zepeda et al., 2009). In an effort to fully understand these findings and to link a transporter-inward Na+ current to a lasting response, we analyzed d-Asp-dependent translational and transcriptional regulation. We chose mTOR as an index of translational control and AP-1 DNA binding and gene reporter assays as a demonstration of transcriptional regulation. Both levels of gene expression regulation are affected by EAAT-1/GLAST in a Na+ and Ca2+-dependent manner involving PI3-K/PKB/mTOR/4EBP1/AP-1 signaling components (Aguirre et al., 2002). One could argue that neither d-Asp nor THA, TBOA or PDC are specific enough to rule out the involvement of other transporters such as GLT-1 (EAAT-2) in the described d-Asp effects. This suggestion is unlikely since BGC harbor exclusively EAAT-1/GLAST and the reported IC50 for dihidrokainate, a specific GLT-1 blocker is >3mM (Ruiz and Ortega, 1995). What could be the physiological relevance of a transporter-mediated signaling cascade in cells that express functional Glu receptors? It is difficult to have a precise answer to this question at this moment. Nevertheless, it is important to mention that exposure of mouse cerebellar slices to Glu induces inward Na+ currents accompanied by an increase in intracellular Na+ concentration that is marginally sensitive to CNQX but inhibited by TBOA. Furthermore, the same scenario is present upon electrical stimulation of the parallel fibers (Kirischuk et al., 2007). It is tempting to speculate that the signaling triggered by the transporters, having slower inactivation kinetics (Bazille et al., 2005) are linked to sustained biochemical responses involved in the expression of polypeptides that participate in neuron/glia coupling, such as the Na+/K+ ATPase that allows glia cells to clear efficiently the synaptic space. In support of this idea, is the fact that EAAT-1/GLAST associates with this plasma membrane ATPase and even, operates as an unit (Rose et al., 2009). Other glia proteins, which expression might be regulated through the activity of the transporters are those that participate either in the Glu/glutamine shuttle like glutamine synthetase (GS) and the sodium-coupled neutral amino acid transporters (SNAT) or in the astrocyte/neuron lactate shuttle like the monocarboxylate transporters (Pellerin et al., 2007). Work currently in progress in our lab is aimed in this direction.