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

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 rece...

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Autores principales: Melo, Carlos V., Mele, Miranda, Curcio, Michele, Comprido, Diogo, Silva, Carla G., Duarte, Carlos B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543267/
https://www.ncbi.nlm.nih.gov/pubmed/23326507
http://dx.doi.org/10.1371/journal.pone.0053793
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author Melo, Carlos V.
Mele, Miranda
Curcio, Michele
Comprido, Diogo
Silva, Carla G.
Duarte, Carlos B.
author_facet Melo, Carlos V.
Mele, Miranda
Curcio, Michele
Comprido, Diogo
Silva, Carla G.
Duarte, Carlos B.
author_sort Melo, Carlos V.
collection PubMed
description 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.
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spelling pubmed-35432672013-01-16 BDNF Regulates the Expression and Distribution of Vesicular Glutamate Transporters in Cultured Hippocampal Neurons Melo, Carlos V. Mele, Miranda Curcio, Michele Comprido, Diogo Silva, Carla G. Duarte, Carlos B. PLoS One Research Article 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. Public Library of Science 2013-01-11 /pmc/articles/PMC3543267/ /pubmed/23326507 http://dx.doi.org/10.1371/journal.pone.0053793 Text en © 2013 Melo et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Melo, Carlos V.
Mele, Miranda
Curcio, Michele
Comprido, Diogo
Silva, Carla G.
Duarte, Carlos B.
BDNF Regulates the Expression and Distribution of Vesicular Glutamate Transporters in Cultured Hippocampal Neurons
title BDNF Regulates the Expression and Distribution of Vesicular Glutamate Transporters in Cultured Hippocampal Neurons
title_full BDNF Regulates the Expression and Distribution of Vesicular Glutamate Transporters in Cultured Hippocampal Neurons
title_fullStr BDNF Regulates the Expression and Distribution of Vesicular Glutamate Transporters in Cultured Hippocampal Neurons
title_full_unstemmed BDNF Regulates the Expression and Distribution of Vesicular Glutamate Transporters in Cultured Hippocampal Neurons
title_short BDNF Regulates the Expression and Distribution of Vesicular Glutamate Transporters in Cultured Hippocampal Neurons
title_sort bdnf regulates the expression and distribution of vesicular glutamate transporters in cultured hippocampal neurons
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543267/
https://www.ncbi.nlm.nih.gov/pubmed/23326507
http://dx.doi.org/10.1371/journal.pone.0053793
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