GLYCINERGIC NEUROTRANSMISSION IN RAT HIPPOCAMPUS: A ROLE IN EPILEPTIC STATUS

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BDNF, via truncated TrkB receptor, modulates GlyT1 and GlyT2 in astrocytes

Publication . Aroeira, Rita I.; Sebastião, Ana M; Valente, Cláudia A.

Glycine transporters (GlyT), GlyT1 and GlyT2, are responsible for the termination of glycine-mediated synaptic activity through removal of neurotransmitter from synaptic cleft. Brain-derived neurotrophic factor (BDNF) activates its high affinity tropomyosin-related kinase (Trk) receptors, namely TrkB, which includes full length (TrkB-FL) and truncated (TrkB-T) isoforms. In this article we evaluated the influence of BDNF upon the activity of glycine transporters in astrocytes. We report that BDNF decreases GlyT1- and GlyT2- mediated [(3) H]glycine transport in primary cultures of astrocytes from rat cerebral cortex. BDNF decreased Vmax but not Km values of transport, which suggests that BDNF induces transporter internalization. Accordingly, dynasore, an inhibitor of dynamin/clathrin-dependent endocytosis, prevented the influence of BDNF upon GlyT-mediated transport. While quantifying mRNA and protein levels, we detected a predominance of truncated isoforms over the TrkB-FL receptor. The effect of BDNF was not abolished by specific inhibitors of PLCγ, PI3K and MAPK, indicating that it did not occur through TrkB-FL canonical pathways. However, BDNF action was lost in the presence of a Rho family-specific blocker (toxin B), a signaling pathway that has been associated to TrkB-T1. Furthermore, the effect of BDNF was abolished upon TrkB-T knockdown in astrocytes by RNA interference. Immunofluorescence assays confirmed an increased GlyT expression in endosomes upon BDNF incubation, which was prevented in the presence of either dynasore or toxin B. We conclude that BDNF, acting on TrkB-T1 receptors, inhibits glycine uptake in astrocytes by promoting GlyT internalization through a Rho-GTPase activity dependent mechanism.

2015Journal article

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BDNF modulates glycine uptake in hippocampal synaptosomes by decreasing membrane insertion of glycine transporter 2

Publication . Aroeira, Rita I.; Vaz, Sandra H.; Sebastião, Ana M; Valente, Cláudia A.

Glycine transporter 2 (GlyT2) is localized in the nerve terminals of glycinergic neurons, promoting glycine uptake and ensuring the refilling of glycinergic vesicles. Brain-derived neurotrophic factor (BDNF) activates its high affinity TrkB receptors, which occur in two isoforms, full length (TrkB-FL) and truncated (TrkB-T1/T2). After BDNF binding to TrkB receptor, several intracellular cascades are triggered, specifically PLC, Akt and MAPK signalling pathways. We herein show that BDNF decreases [(3)H]glycine uptake mediated by GlyT2 in isolated nerve endings (synaptosomes) obtained from rat hippocampus, by reducing the maximum velocity (Vmax) of transport while not influencing the transporter affinity constant (Km) for glycine. Western Blot analysis detected both TrkB receptor isoforms in the synaptosomes but the BDNF effect seems to be mediated by TrkB-FL since: 1) the tyrosine kinase inhibitor, k252a, prevented the effect of BDNF, and 2) the effect of BDNF was lost in the presence of specific inhibitors of TrkB signalling pathways, namely U73122, LY294002 and U0126 (inhibitors of PLC, Akt and MAPK pathways, respectively). Monensin, a transporter recycling inhibitor, prevented the BDNF action upon glycine uptake, suggesting that BDNF reduces GlyT2 insertion in the plasma membrane. It is concluded that BDNF effect upon glycine uptake into glycinergic nerve terminals requires the activation of the TrkB-FL receptor and its canonical signalling pathways and occurs by inhibiting GlyT2 membrane incorporation.

2016Journal article

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