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

Abstract

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.

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

“…The diversity in the K m values of GlyT2 for glycine in different preparations is yet unknown. However, in our hands, the K m value for GlyT2 in cortical astrocytes is similar to the one obtained in this work (Aroeira et al, 2015).…”

“…However, the BDNF-dependent inhibition of glycine uptake here described seems to result from TrkB-FL receptors activation. This is sustained by three lines of evidence: 1) the effect of BDNF was prevented in the presence of a tyrosine kinase inhibitor, k252a, 2) the effect of BDNF action required the activation of the PLCγ/PKC, PI3K/Akt and MAPK pathways since it was abolished by drugs that inhibit those signalling cascades and 3) the blockade of Rho-GTPases' activity, which was shown to be the transducing pathway for the TrkB-T mediated action of BDNF in astrocytes to control glycine uptake (Aroeira et al, 2015), did not revert the action of BDNF.…”

“…BDNF decreases glycine uptake by neuronal GlyT2, as shown in this work, but also has an inhibitory effect upon glial GlyT1 (Aroeira et al, 2015), which will increase M A N U S C R I P T…”

“…Truncated TrkB receptors, namely TrkB-T1, TrkB-T2 and TrkB-T-Shc, originated from alternative splicing, lack the intracellular kinase domain (Stoilov et al, 2002;Binder and Scharfman, 2004;Blum and Konnerth, 2005). We recently reported that glycine transport into astrocytes is inhibited by BDNF, which by activating TrkB-T1 receptors, promotes GlyT1 and GlyT2 internalization through a Rho-GTPase activity dependent mechanism (Aroeira et al, 2015). In contrast, BDNF facilitates GABA transport into astrocytes, this action being also mediated by TrkB-T receptors (Vaz et al, 2011).…”

“…TrkB-T receptors in astrocytes can modulate glycine uptake mediated by GlyT through coupling to RhoGTPases (Aroeira et al, 2015). Since synaptosomes, besides expressing TrkB-FL, also express TrkB-T isoforms, we decided to evaluate the involvement of RhoGTPases in the effect of BDNF effect upon glycine uptake.…”