Journal: The Journal of Neuroscience

Article Title: Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABA A Receptors

doi: 10.1523/JNEUROSCI.2069-14.2014

Figure Lengend Snippet: Endogenous proBDNF decreases the colocalization of postsynaptic GABAAR β2/3 clusters with presynaptic VAMP1 puncta. A, proBDNF protein levels in hippocampal culture supernatants in control and aprotinin conditions. Treatment with aprotinin (Apro; 3 μg/ml, 12 h) increases significantly the concentration of proBDNF in the supernatant compared with control (CTR). n = 3; ***p < 0.001. B, Hippocampal cultures (14 DIV) were treated for 12 h with vehicle (CTR), aprotinin (Apro), aprotinin plus TAT-pep5 (Apro + TATpep5), or TAT-pep5 alone, and stained for GABAAR β2/3 subunit (green), VAMP1 (red), and MAP2 (blue). Scale bars: top, 20 μm; bottom, 3 μm. C, The percentage of β2/3 subunit-positive clusters that colocalized with VAMP1 puncta/intracellular MAP2 fluorescence ratio was significantly reduced in cultures treated with aprotinin (3 μg/ml, 12 h). D, E, Aprotinin (Apro) significantly decreased the average β2/3 subunit-positive area fraction/intracellular MAP2 fluorescence ratio (D) and the average perimeter of GABAAR β2/3 clusters (E) at the cell surface of MAP2-positive neurons, in comparison with the vehicle-treated control (CTR), in the presence of TAT-pep5 (Apro + TATpep5) or TAT-pep5 alone (TATpep5). Thirty-five neurons analyzed per condition, n = 4; ***p < 0.001. F, Quantification of surface VAMP1/MAP2 fluorescence ratio, in neurons treated with aprotinin alone (Apro), aprotinin plus TAT-pep5 (Apro + TATpep5), or TAT-pep5 alone (Tatpep5; 2 μm), in comparison with the vehicle-treated control (CTR). Thirty-five neurons analyzed per condition, per experiment; n = 4; ***p < 0.001, **p < 0.01. G, Cell-surface levels of GABAARs measured using ELISA assays with GABAAR β2/3 subunit-specific antibody in neurons treated with aprotinin alone (Apro; 3 μg/ml, 12 h), with Apro in the presence of TAT-pep5 (Apro + TAT-pep5; 2 μm), with Apro and nifedipine (Apro + nifedipine; 10 μm), and only in the presence TAT-pep5 (TAT-pep5; 10 μm), and expressed as a percentage of vehicle-treated controls (CTR). H, Total GABAAR β2/3 subunit expression normalized to control nontreated conditions. n = 6; ***p < 0.001. I, Relative expression of GABAAR β3 mRNA transcripts in neurons treated with aprotinin (Apro; 3 μg/ml, 12 h), with aprotinin and TAT-pep5 (Apro + TATpep5; 2 μm), with K252a alone (200 nm), or with TAT-pep5 alone (2 μm), and expressed as a percentage of vehicle-treated controls (CTR). GABAAR β3 subunit gene expression was normalized to the expression of the housekeeping gene, GAPDH, in the same RNA preparation. n = 6; ***p < 0.001.

Article Snippet: TTX was purchased from Abcam; ROCKi (Y-27632) was purchased from Millipore; tPA-Stop was purchased from American Diagnostica; k252a, TAT-pep5, and Stattic were purchased from Calbiochem; nifedipine, aprotinin, leupeptin, and AG490 were purchased from Sigma-Aldrich; CR-proBDNF recombinant ( Escherichia coli ) mutated human and mBDNF were purchased from Alomone Labs. NBQX, CNQX, and d -APV were obtained from the Molecular, Cellular, and Genomic Neuroscience Research Branch of the National Institute of Mental Health.

Techniques: Concentration Assay, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Neuroscience

Article Title: Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABA A Receptors

doi: 10.1523/JNEUROSCI.2069-14.2014

Figure Lengend Snippet: Apoptosis is not triggered by proBDNF. A, Image of hippocampal neurons transfected at 10 DIV with the CR-proBDNF Cherry (in red) and/or Cherry (in red), and costained at 14 DIV with the cleaved caspase-3 (in blue). B, Cultured hippocampal neurons (14 DIV) in control conditions (CTR), or treated with CR-proBDNF (25 ng, 30 min), or CR-proBDNF plus TAT-pep5 (CR-proBDNF + TATpep5; 2 μm), or aprotinin (Apro; 3 μg/ml, 12 h), or aprotinin plus TAT-pep5 (Apro + TATpep5; 2 μm), and immunolabeled with cleaved caspase-3 antibody (red), and costained with anti-MAP2 antibody (green). C, Ratio of cleaved caspase-3-positive (apoptotic) neuronal number to total MAP2-positive cells, and expressed as a percentage of vehicle-treated controls (CTR). Overexpression of CR-proBDNF (CR-proBDNF Cherry), or exogenous application of CR-proBDNF (CR-proBDNF) or treatment with aprotinin (Apro) did not increase the number of apoptotic neurons compared with control (CTR). Fifty neurons analyzed per condition, per experiment; n = 4; p > 0.05.

Article Snippet: TTX was purchased from Abcam; ROCKi (Y-27632) was purchased from Millipore; tPA-Stop was purchased from American Diagnostica; k252a, TAT-pep5, and Stattic were purchased from Calbiochem; nifedipine, aprotinin, leupeptin, and AG490 were purchased from Sigma-Aldrich; CR-proBDNF recombinant ( Escherichia coli ) mutated human and mBDNF were purchased from Alomone Labs. NBQX, CNQX, and d -APV were obtained from the Molecular, Cellular, and Genomic Neuroscience Research Branch of the National Institute of Mental Health.

Techniques: Transfection, Cell Culture, Immunolabeling, Over Expression

Journal: The Journal of Neuroscience

Article Title: Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABA A Receptors

doi: 10.1523/JNEUROSCI.2069-14.2014

Figure Lengend Snippet: ProBDNF activates the RhoA-ROCK-PTEN pathway and promotes GABAAR dephosphorylation and endocytosis. A, CR-proBDNF decreases the phosphorylation of Ser408/409 in the GABAAR β3 subunit (pGABAAR β3). Total cellular proteins were analyzed using Western blot with polyclonal GABAAR β3 subunit and pGABAAR β3 subunit antibodies. Hippocampal neurons were treated with vehicle (CTR), CR-proBDNF (25 ng, 30 min), CR-proBDNF plus TAT-pep5 (CR-proBDNF + TATpep5; 2 μm), CR-proBDNF plus ROCKi (CR-proBDNF + ROCKi; 50 μm), CR-proBDNF plus Ag490 (CR-proBDNF + Ag490; 50 μm), or CR-proBDNF plus Stattic (CR-proBDNF + Stattic; 10 μm). Proteins were visualized using ECL following incubation with HRP-conjugated secondary antibody. Normalized data (pGABAAR β3/GABAAR β3) are expressed as percentage change with respect to vehicle-treated cultures (defined as 100%). Levels of total GABAAR β3 subunits were used as internal control. n = 4–6 experiments; *p < 0.05, **p < 0.01. B, Cell-surface levels of GABAARs measured using ELISA assays with GABAAR β2/3 subunit-specific antibody in neurons treated with CR-proBDNF alone (25 ng, 30 min) with CR-proBDNF in the presence of ROCKi (CR-proBDNF + ROCKi; 50 μm), or with ROCKi alone (50 μm), and expressed as a percentage of vehicle-treated controls. C, CR-proBDNF decreases the phosphorylation of T366 in the PTEN (pPTEN). Total cellular proteins were analyzed using Western blot with polyclonal PTEN and pPTEN antibodies. Hippocampal neurons were treated with vehicle (CTR), CR-proBDNF (25 ng, 30 min), CR-proBDNF plus ROCKi (CR-proBDNF + ROCKi; 50 μm), or ROCKi alone (50 μm). Proteins were visualized using ECL following incubation with HRP-conjugated secondary antibody. Normalized data (pPTEN/PTEN) are expressed as percentage change with respect to vehicle-treated cultures (defined as 100%). Levels of total PTEN were used as internal control. n = 5 experiments; *p < 0.05. D, Neurons were permeabilized and immunolabeled with anti-GABAAR β2/3 subunit-specific antibody (green), anti-EEA1 rabbit polyclonal antibody (red), and anti-MAP2 chicken polyclonal antibody (blue) to reveal the intracellular distribution of these proteins. GABAAR β2/3 subunits and EEA1 colocalization appear yellow in merge panels (right). Scale bar, 20 μm. E, The percentage of β2/3 subunit-positive clusters that colocalized with EEA1 puncta/intracellular MAP2 fluorescence ratio was significantly increased in cultures treated with CR-proBDNF (25 ng, 30 min). Thirty-five neurons analyzed per condition, n = 4; ***p < 0.001. F, Western blot analysis with the antibody to EEA1. EEA1 expression was upregulated after treatment with CR-proBDNF (25 ng, 30 min). Proteins were visualized using ECL following incubation with HRP-conjugated secondary antibody. Normalized data (EEA1/β-Tubulin) are expressed as percentage change with respect to vehicle-treated cultures (defined as 100%). n = 5 experiments; *p < 0.05. G, Cell-surface levels of GABAARs measured using ELISA assays with GABAAR β2/3 subunit-specific antibody in neurons treated with aprotinin (Apro; 3 μg/ml, 12 h) in the presence or absence of leupeptine (5 μg/ml), or with leupeptine alone (5 μg/ml), and expressed as a percentage of vehicle-treated controls (CTR). H, Total GABAAR β2/3 subunit expression normalized to control nontreated conditions. n = 9; **p < 0.01, ***p < 0.001.

Article Snippet: TTX was purchased from Abcam; ROCKi (Y-27632) was purchased from Millipore; tPA-Stop was purchased from American Diagnostica; k252a, TAT-pep5, and Stattic were purchased from Calbiochem; nifedipine, aprotinin, leupeptin, and AG490 were purchased from Sigma-Aldrich; CR-proBDNF recombinant ( Escherichia coli ) mutated human and mBDNF were purchased from Alomone Labs. NBQX, CNQX, and d -APV were obtained from the Molecular, Cellular, and Genomic Neuroscience Research Branch of the National Institute of Mental Health.

Techniques: De-Phosphorylation Assay, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Immunolabeling, Fluorescence, Expressing

Journal: The Journal of Neuroscience

Article Title: Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABA A Receptors

doi: 10.1523/JNEUROSCI.2069-14.2014

Figure Lengend Snippet: ProBDNF has no effect on phospho-CREB activation but induces ICER expression via the JAK2/STAT3 signaling cascade. A, Immunofluorescence signal of CREB (red, top), pCREB (green, middle) in hippocampal neurons in control conditions (CTR), or treated with CR-proBDNF alone (25 ng; CR-proBDNF), or CR-proBDNF plus mBDNF (CR-proBDNF + mBDNF; 25 ng), or CR-proBDNF plus mBDNF in the presence of K252a (CR-proBDNF + mBDNF + K252a; 200 nm). Right, Merged images with MAP2 staining (blue). Scale bar, 40 μm. B, Average ratio of pCREB/CREB in the different conditions normalized to control. n = 15 neurons analyzed per treatment in n = 3 independent experiments; ***p < 0.001. C, Immunofluorescence signal of ICER (red; top) in hippocampal neurons in control conditions (CTR), or treated with CR-proBDNF (25 ng) for 30 min (CR-proBDNF 30 min), for 12 h (CR-proBDNF 12 h), or for 30 min in the presence of TAT-pep5 (CR-pro-BDNF 30 min + TATpep5; 2 μm), following a costaining with anti-MAP2 chicken polyclonal antibody (green; bottom) to reveal neuronal cells. Scale bar: 20 μm. D, Average fluorescence intensity ratio of ICER/MAP2 in the different conditions normalized to control. Forty-five neurons analyzed per condition, n = 4; ***p < 0.001. E, Western blot analysis with the antibody to ICER. ICER expression was upregulated after acute treatment with CR-proBDNF (25 ng, 30 min) but not with chronic application of CR-proBDNF (25 ng, 12 h). The increase of ICER was antagonized by TAT-pep5 (2 μm), AG490 (50 μm), and Stattic (10 μm). This increase was insensitive to ROCKi (50 μm). Proteins were visualized using ECL following incubation with HRP-conjugated secondary antibody. Normalized data (ICER/β-Tubulin) are expressed as percentage change with respect to vehicle-treated cultures (defined as 100%). n = 4–6 experiments; **p < 0.01, ***p < 0.001. F, Relative expression of ICER mRNA transcripts in neurons treated with CR-proBDNF for 30 min (CR-proBDNF 30 min), with CR-proBDNF for 12 h (CR-proBDNF 12 h; 25 ng), or with CR-proBDNF plus TAT-pep5 for 30 min (CR-proBDNF 30 min + TATpep5; 2 μm), and expressed as a percentage of vehicle-treated controls (CTR). ICER gene expression was normalized to the expression of the housekeeping gene, GAPDH, in the same RNA preparation. n = 6; ***p < 0.001.

Article Snippet: TTX was purchased from Abcam; ROCKi (Y-27632) was purchased from Millipore; tPA-Stop was purchased from American Diagnostica; k252a, TAT-pep5, and Stattic were purchased from Calbiochem; nifedipine, aprotinin, leupeptin, and AG490 were purchased from Sigma-Aldrich; CR-proBDNF recombinant ( Escherichia coli ) mutated human and mBDNF were purchased from Alomone Labs. NBQX, CNQX, and d -APV were obtained from the Molecular, Cellular, and Genomic Neuroscience Research Branch of the National Institute of Mental Health.

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot, Incubation