BDNF Regulates the Expression and Distribution of Vesicular Glutamate Transporters in Cultured Hippocampal Neurons

Abstract

BDNF is a pro-survival protein involved in neuronal development and synaptic plasticity. BDNF strengthens excitatory synapses and contributes to LTP, presynaptically, through enhancement of glutamate release, and postsynaptically, via phosphorylation of neurotransmitter receptors, modulation of receptor traffic and activation of the translation machinery. We examined whether BDNF upregulated vesicular glutamate receptor (VGLUT) 1 and 2 expression, which would partly account for the increased glutamate release in LTP. Cultured rat hippocampal neurons were incubated with 100 ng/ml BDNF, for different periods of time, and VGLUT gene and protein expression were assessed by real-time PCR and immunoblotting, respectively. At DIV7, exogenous application of BDNF rapidly increased VGLUT2 mRNA and protein levels, in a dose-dependent manner. VGLUT1 expression also increased but only transiently. However, at DIV14, BDNF stably increased VGLUT1 expression, whilst VGLUT2 levels remained low. Transcription inhibition with actinomycin-D or α-amanitine, and translation inhibition with emetine or anisomycin, fully blocked BDNF-induced VGLUT upregulation. Fluorescence microscopy imaging showed that BDNF stimulation upregulates the number, integrated density and intensity of VGLUT1 and VGLUT2 puncta in neurites of cultured hippocampal neurons (DIV7), indicating that the neurotrophin also affects the subcellular distribution of the transporter in developing neurons. Increased VGLUT1 somatic signals were also found 3 h after stimulation with BDNF, further suggesting an increased de novo transcription and translation. BDNF regulation of VGLUT expression was specifically mediated by BDNF, as no effect was found upon application of IGF-1 or bFGF, which activate other receptor tyrosine kinases. Moreover, inhibition of TrkB receptors with K252a and PLCγ signaling with U-73122 precluded BDNF-induced VGLUT upregulation. Hippocampal neurons express both isoforms during embryonic and neonatal development in contrast to adult tissue expressing only VGLUT1. These results suggest that BDNF regulates VGLUT expression during development and its effect on VGLUT1 may contribute to enhance glutamate release in LTP.

MeCP2 is required for activity-dependent refinement of olfactory circuits

“…Treatment with ampakines during early postnatal age may maintain the proper E/I balance, and improve the expression of guidance and adhesion molecules important for glomerular maturation, as well as BDNF (Lauterborn et al, 2000; Mackowiak et al, 2002). BDNF has proven to be important for glomerular maturation (Tisay et al, 2000), as well as it regulates vesicular glutamate transporters expression (Melo et al, 2013). Future work will explore the mechanism underlying the effect of ampakine treatment on the olfactory circuit maturation in Mecp2 KO mice.…”

BDNF-induced local protein synthesis and synaptic plasticity

“…Synaptic vesicle proteins and proteins related to their traffic were also shown to be upregulated by BDNF (Melo et al, 2013;Tartaglia et al, 2001;Thakker-Varia et al, 2001) (see Section 4.6), but additional studies are required to determine whether some of these alterations are extended to the synapse. Taken together, the BDNF-induced changes in the neuronal proteome described above suggest several putative mechanisms that may contribute to synaptic potentiation by BDNF.…”

“…This hypothesis is based on studies showing induction of LTP by exogenously applied BDNF at the medial perforant path-granule cell synapses in vivo (Messaoudi et al, 1998), an effect that is blocked by the transcription inhibitor actinomycin D (Messaoudi et al, 2002). Furthermore, BDNF induces the expression of genes coding for regulators of synaptic activity, such as Arc Zheng et al, 2009) and several synaptic vesicle proteins, including vesicular glutamate transporters (Melo et al, 2013;Tartaglia et al, 2001). However, the role of BDNF in transcription regulation has been addressed mainly using bath application of the neurotrophin, which does not allow distinguishing the localization of the TrkB receptors involved in the control of gene expression.…”

“…Furthermore, incubation of cultured hippocampal neurons with the neurotrophin upregulates the vesicular glutamate transporter protein levels, as well as the clustering of the transporters along neurites (Melo et al, 2013). However, at this point it is not possible to predict whether these effects may play a role in synaptic potentiation by BDNF because, as discussed above, no attempt has been made to specifically address the effect of axonal stimulation with BDNF.…”

“I’ve a Grand Memory for Forgetting”

“…One of these mechanisms is an increase in the expression of vesicular glutamate receptor 1 (VGLUT1). VGULT1 loads glutamate into the presynaptic vesicles carrying glutamate to the presynaptic membrane, and also increases the number of synaptic vesicles that dock at the active area of the presynaptic membrane (Melo et al, 2013).…”