Synaptic activity increases hnRNP A2/B1 in dendrites and induces the delivery of hnRNP A2/B1 to the synapse by a BDNF-dependent mechanism.

<p>Cultured hippocampal neurons were stimulated or not with bicuculline (50 µM), 4-AP (2.5 mM) and glycine (10 µM), for 30 min. Where indicated, neurons were treated with TrkB-Fc (1 µg/ml) for 30 min and then stimulated or not with bicuculline in the presence of the BDNF scavenger. The cells were immunostained for hnRNP A2/B1 (red), MAP2 (blue), and PSD95 (green) (A). White arrows indicate PSD95-positive clusters that also contain hnRNP A2/B1 (A). The integrated fluorescence intensity, area and number of hnRNP A2/B1 puncta in dendrites (B, C, and D) and at the synapse (as defined by the signal of hnRNP A2/B1 that overlaps with PSD95) (E, F, and G) was analysed using ImageJ software and represented per dendritic area. The percentage of dendritic hnRNP A2/B1 signal that colocalizes with PSD95 was also analysed (H). Results are normalized to control and are averaged of 3–7 different experiments performed in independent preparations. The following number of cells was used in the analysis of the dendritic-localized hnRNP A2/B1 puncta: Ctr (n = 87 cells), Bic (n = 64 cells), TrkB-Fc (n = 47 cells), TrkB-Fc+Bic (n = 48 cells). For the analysis of synaptic-localized hnRNP A2/B1 puncta the following number of cells was analysed: Ctr (n = 76 cells), Bic (n = 54 cells), TrkB-Fc (n = 35 cells), TrkB-Fc+Bic (n = 35 cells). Error bars, mean ± SEM. Statistical analysis was performed by one-way ANOVA, followed by Bonferroni's test. n.s. Not significant, * P<0.05; ** P<0.01; *** P<0.001. Scale bar = 4 µm.</p

BDNF upregulates hnRNP A2/B1 mRNA in the cell body compartment of hippocampal neurons.

<p>Cultured hippocampal neurons were stimulated or not with 100 ng/ml BDNF, for 30 min or 2 h. The cell body mRNA was mechanically separated from the transcripts of neurites and 500–1000 ng of RNA from each compartment was used in the reverse transcription reaction. The analysis of hnRNP A2/B1 mRNA levels was performed by qRT-PCR using <i>Ppia</i> as internal control gene. The results are the average ± SEM of five (cell body compartment) or seven (neurite compartment) independent transcription reactions, performed in distinct preparations. Statistical analysis of logtransformed expression data was performed by one-way ANOVA, followed by Dunnet's test. * P<0.05.</p

Synaptic activity and the neurotrophin BDNF increase the abundance of hnRNP A2/B1 protein in the soma of hippocampal neurons, outside the nucleus.

<p>(A) Cultured hippocampal neurons were stimulated or not with bicuculline (50 µM), 4-AP (2.5 mM) and glycine (10 µM) for 30 min and then the cells immunostained for MAP2 (blue), hnRNP A2/B1 (red). Scale bar = 20 µm and 4 µm for low and high magnification images, respectively. (B) Cultured hippocampal neurons were stimulated or not with BDNF (100 µg/ml), for 30 min and then the cells immunostained for MAP2 (blue), hnRNP A2/B1 (red). Scale bar = 20 µm and 4 µm for low and high magnification images, respectively (C) The intensity of hnRNP A2/B1 protein in the cell body was analysed in similar regions of interest within the soma, away from the nucleus, using ImageJ software. Results are normalized to control and are averaged of 4–7 different experiments performed in independent preparations. The following number of cells was used in the analysis of the somatic-localized hnRNP A2/B1 Ctr (n = 71 cells), Bic (n = 40 cells), BDNF (n = 61 cells). Error bars, mean ± SEM. Statistical analysis was performed by one-way ANOVA, followed by Dunnet's test.* P<0.05; *** P<0.001.</p

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

<div><p>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.</p> </div

TrkB receptor inhibition blocks BDNF upregulation of VGLUT1 and VGLUT2.

<p>(A–C) Cultured hippocampal neurons at DIV7 (A, B) and DIV14 (C) were stimulated with BDNF (100 ng/ml), for the indicated periods of time, in the presence or absence of a selective inhibitor of tyrosine kinase activity, K252a (200 nM), and VGLUT1 (A, C) and VGLUT2 (B) protein levels were determined by western blot. Quantification of the indicated number of experiments, performed in independent preparations, is presented as mean percentage ± SEM compared to the control (unstimulated neurons). (D–F) DIV7 hippocampal neurons were stimulated with IGF-1 or bFGF, for 4 or 20 h, and VGLUT1 (D) and VGLUT2 (E) protein levels were determined by western blot. Quantification of 4 different experiments, performed in independent preparations, is presented as mean percentage ± SEM compared to the control. Statistical significance was determined by One Way ANOVA followed by Bonferronís multiple comparison test with a confidence interval of 99% (*p<0.05, **p<0.01, ***p<0.001). (F) DIV7 hippocampal neurons were stimulated with IGF-1 or bFGF, for 15 or 30 min, and the levels of ERK1/2 phosphorylation were determined by western blot. The antibody used specifically recognizes the phosphorylated isoforms 1 and 2 of ERK, but not the nonphosphorylated (presumably inactive) proteins.</p

Effect of BDNF on the subcellular distribution of VGLUT2 in cultured rat hippocampal neurons.

<p>Hippocampal neurons at 7 DIV were stimulated with BDNF (100 ng/ml) for 30 min or 6 h. Neurons were immunolabeled with specific antibodies for total VGLUT2 (green) and β-tubulin I (red) (A) (Scale bar: 10 µm). Arrowheads show the location of VGLUT2 puncta. The acquired fluorescence images were analysed to assess the number (B), integrated density (mean intensity×puncta area) (D) and intensity (E) of VGLUT2 puncta in neurites, as well as for VGLUT1 immunoreactivity in the soma (C). Results were normalized for neuritic length (B, D and E) or soma area (C). The protein localization was visualized using a Zeiss Axio Observer 2.1 fluorescence microscope (63x Objective). Quantitative particle analysis was performed using ImageJ software. Results are shown as mean percentage of control of at least three independent experiments (n ≥30 cells per condition). **p<0.01; ***p<0.001, significantly different in comparison to the respective control (unpaired Student’s <i>t</i>-test).</p

Inhibition of the PI3-K/Akt and Ras-ERK signaling pathways has no significant effect on BDNF-induced upregulation of VGLUT1 and VGLUT2 protein levels.

<p>(A–D) DIV7 cultured hippocampal neurons were stimulated with BDNF (100 ng/ml), for the indicated periods of time, in the presence or absence of Ras-ERK pathway inhibitors PD098059 (20 µM) or U0126 (10 µM) (A, B), or PI3K/Akt inhibitors LY294002 (30 µM) or Wortmannin (300 nM) (C, D), and VGLUT1 (A, C) and VGLUT2 (B, D) protein levels were determined by western blot. Quantification of the indicated number of experiments, performed in independent preparations, is presented as mean percentage ± SEM compared to the control (unstimulated neurons). Statistical significance was determined by One Way ANOVA followed by Bonferronís multiple comparison test with a confidence interval of 99% (*p<0.05, **p<0.01, ***p<0.001). (E) Schematic representation of BDNF-induced TrkB receptor trans-activation and downstream effectors and inhibitors of the PI3-K/Akt and Ras-ERK signaling pathways.</p

BDNF upregulates VGLUT1 and VGLUT2 protein expression through a translation-dependent mechanism.

<p>(A–B) Cultured hippocampal neurons at DIV7 (A) and DIV14 (B) were incubated with 100 ng/ml BDNF for different time periods and total VLGUT1 and VGLUT2 protein levels were compared to control (without BDNF) expression, upon normalization with β-actin I levels. (C–D) Cultured hippocampal neurons at DIV7 were pre-incubated or not with the translation inhibitors emetine or anisomycin (2 µM) for 30 min before BDNF stimulation during 30 min or 3 h and VGLUT1 (C) and VGLUT2 (D) protein levels were compared to control expression. When the effect of translation inhibitors was tested, the cells were incubated with the compounds during stimulation with BDNF. (A–D) Quantification of 3–5 different experiments, performed in independent preparations, is presented as mean percentage ± SEM compared to the control (unstimulated neurons). Statistical significance was determined by One Way ANOVA followed by Bonferronís multiple comparison test with a confidence interval of 99% (*p<0.05, **p<0.01, ***p<0.001).</p

Inhibition of the PLCγ signaling pathway blocks BDNF-induced upregulation of VGLUT1 and VGLUT2 protein levels.

<p>(A–D) DIV7 cultured hippocampal neurons were stimulated with BDNF (100 ng/ml) for the indicated periods of time, in the presence or absence of U73122 (PLCγ inhibitor; 5 µM) (A, B), chelerytrine (PKC inhibitor; 5 µM) or KN-93 (CAMKII inhibitor; 1 µM) (C, D), and VGLUT1 (A, C) and VGLUT2 (B, D) protein levels were determined by western blot. Quantification of the indicated number of experiments, performed in independent preparations, is presented as mean percentage ± SEM compared to the control (unstimulated neurons). Statistical significance was determined by One Way ANOVA followed by Bonferronís multiple comparison test with a confidence interval of 99% (*p<0.05, **p<0.01, ***p<0.001). (E) Schematic representation of BDNF-induced TrkB receptor trans-activation and downstream PLCγ signaling pathway effectors and inhibitors.</p

The effect of BDNF on VGLUT1 and VGLUT2 protein levels is dependent on gene expression.

<p>(A–B) Hippocampal neurons were stimulated with BDNF (100 ng/ml) for the indicated periods of time, in the presence or in the absence of the transcription inhibitors α-amanitin (1.5 µM) or actinomycin-D (1.5 µM), and VGLUT1 (A) and VGLUT2 (B) protein levels were determined by western blot. (C) The variation of <i>Slc17a7</i> (VGLUT1) and <i>Slc17a6</i> (VGLUT2) mRNA levels was assayed by real-time PCR, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053793#s2" target="_blank">methods</a> section. The neurons were stimulated with 100 ng/ml BDNF during 30 minutes (grey) or 3 hours (red). (A–C) Quantification of 4–5 experiments, performed in independent preparations, is presented as mean percentage ± SEM compared to the control (unstimulated neurons), and normalized to <i>18S</i> reference gene. Statistical significance was determined by One Way ANOVA followed by Bonferronís multiple comparison test with a confidence interval of 99% (*p<0.05, **p<0.01, ***p<0.001).</p