Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: Antibodies used in the present study

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Recombinant, Immunohistochemistry-IF, Transduction

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: The interaction of BRAG2a with endophilin 3 and PSD-95 is important for the mGluR-dependent decrease in AMPAR surface level. A, B, The effect of BRAG2 knockdown on basal surface GluA1 level. A, Representative immunofluorescence images of dendritic shafts and spines of cultured hippocampal neurons transfected with shBRAG2 (BRAG2-KD) or shControl (control) vectors at DIV14 and subjected to surface AMPAR labeling with anti-GluA1 (extracellular) antibody at DIV16. B, Quantification of surface GluA1 levels. Immunofluorescence intensity of surface GluA1 in BRAG2-KD-transfected neurons was normalized to that in the control-transfected neurons. Note that knockdown of BRAG2 did not affect the basal surface GluA1 levels. C, D, The effect of BRAG2 knockdown on surface GluA1 levels following DHPG treatment. D, Quantification of surface GluA1 levels. Note that BRAG2 knockdown blocked the decrease in surface GluA1 levels following 50 μm DHPG treatment (DHPG 50). Asterisks indicate statistical significance (one-way ANOVA; F(2,61) = 4.507, p = 0.0149; followed by Tukey–Kramer post hoc test, *p < 0.05). E, F, The effect of BRAG2 knockdown on surface GluN2B levels following DHPG treatment. E, Representative immunofluorescence images of dendritic shafts and spines of BRAG2-KD or control hippocampal neurons treated with 50 μm DHPG and subjected to surface NMDAR labeling with anti-GluN2B (extracellular) antibody. F, Quantification of surface GluN2B levels; the immunofluorescence intensities of surface GluN2B in BRAG2-KD and control neurons treated with DHPG were normalized to that in control and DHPG-untreated (DHPG 0) neurons. Note that DHPG treatment did not change surface GluN2B levels in BRAG2-KD or control neurons (one-way ANOVA: F(2,59) = 0.964, p = 0.387). Data for each surface GluN2B were obtained from 20–22 transfected neurons from three plates, and these results were confirmed by three independent experiments. G, H, The effect of coexpression of shBRAG2 and BRAG2a or its mutants on the DHPG-induced decrease in surface GluA1 levels. G, Representative immunofluorescence images of dendritic shafts and spines of neurons transfected with BRAG2-KD alone or with BRAG2-KD and sh-res BRAG2a-WT, BRAG2b-WT, BRAG2a-P956/957A, BRAG2a-ΔSTVV, or BRAG2a-ΔSec7. H, Quantification of surface GluA1 levels. Note that the BRAG2-knock-down phenotype on DHPG-induced surface GluA1 levels could be rescued by coexpression of shRNA-resistant BRAG2a-WT, but not BRAG2b-WT, BRAG2a-P956/957A, BRAG2a-ΔSTVV, or BRAG2a-ΔSec7. Asterisks indicate statistical significance (one-way ANOVA: F(5,138) = 12.822, p < 0.0001; followed by Tukey–Kramer post hoc test, *p < 0.05). Each surface GluA1 data were obtained from three independent experiments, and 20–28 transfected neurons from three plates were analyzed in each experiment. Scale bars, 2 μm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Immunofluorescence, Cell Culture, Transfection, Labeling, shRNA

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: Lateral distribution of BRAG2a, PSD-95, GluA1/2, Arf6, endophilin 3, AP-2, and clathrin along the postsynaptic membrane. A, B, D, E, G, H, J, K, M, N, P, Q, S, and T, Representative postembedding immunoelectron microscopic images of the distribution of BRAG2a (A, B), PSD-95 (D, E), GluA1/2 (G, H), Arf6 (J, K), endophilin 3 (M, N), AP-2/α-adaptin (P, Q), and clathrin (S, T) in excitatory asymmetric synapses of the mouse hippocampal CA1 region. C, F, I, L, O, R, and U, Histograms showing lateral distribution of immunogold particles for BRAG2a (C), PSD-95 (F), GluA1/2 (I), Arf6 (L), endophilin 3 (O), AP-2/α-adaptin (R), and clathrin (U) along the postsynaptic membrane. The edge of the postsynaptic density (arrows) is defined as 0. The bin size of the histogram is 25 nm, and the synaptic and extrasynaptic sites are shown in the left and right sides, respectively. Scale bars, 100 nm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques:

Journal: The Journal of Neuroscience

Article Title: BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3

doi: 10.1523/JNEUROSCI.1645-19.2020

Figure Lengend Snippet: The effect of DHPG treatment on BRAG2a, PSD-95, and GluA1/2 levels in the PSD and extrasynaptic region using acute hippocampal slice cultures. A, B, The effect of the DHPG treatment on the expression of GluA1/2 and Arc/Arg3.1, and the phosphorylation of p38 MAPK in acute hippocampal slice cultures. A, Representative immunoblots of acute hippocampal slices treated with or without DHPG with anti-GluA1/2, anti-Arc/Arg3.1, anti-phospho-p38 MAPK, anti-p38 MAPK, and anti-α-tubulin antibodies. B, Quantification of immunoreactive intensities of GluA1/2, Arc/Arg3.1, and phospho/total p38 MAPKs. Each immunoreactive intensity was normalized by the respective intensity for α-tubulin and expressed as the ratio with the control value. Note that the DHPG treatment induced the significant upregulation of Arc/Arg3.1 and the phosphorylation of p38 MAPK in hippocampal slices. Data for each group were obtained from three culture plates (n = 3). These results were confirmed by three independent experiments. C–T, Representative postembedding immunoelectron microscopic images (C–F, I–L, O–R) and histograms (G, H, M, N, S, T) of the lateral distribution of immunogold particles for BRAG2a (C–H), PSD-95 (I–N), and GluA1/2 (O–T) in axospinous asymmetric synapses in the CA1 stratum radiatum of acute mouse hippocampal slices following 50 μm DHPG treatment. The edge of the postsynaptic density (arrowheads) is defined as 0. The bin size of the histogram is 25 nm. U, V, Quantification of immunogold particles for BRAG2a, PSD-95, and GluA1/2 in the PSD (U) and extrasynaptic (V) region along the postsynaptic membrane. Values in U and V are expressed as the average immunogold particle numbers in the PSD of axospinous synapses and the percentage of immunogold particles in the extrasynaptic region in total immunoreactive particles along the postsynaptic membrane, respectively. Note that the DHPG treatment decreased the immunogold particles for GluA1/2 but not BRAG2a or PSD-95 without any changes in the proportion of BRAG2a, PSD-95, or GluA1/2 in the extrasynaptic region. *p < 0.05 (t test). Data for each group were obtained from three slices (n = 3). These results were confirmed by three independent experiments from different mice. Scale bars, 100 nm.

Article Snippet: Two days after the transfection, neurons were treated with the conditioned medium containing 0.1 μ m TTX for 30 min, followed by incubation with 50 μ m DHPG for 15 min and the conditioned medium for 45 min. To label surface AMPA and NMDA receptors, cultured neurons were incubated with either GluA1 (extracellular) antibody (1.0 μg/ml; Alomone Labs) or GluN2B (extracellular) antibody (1.0 μg/ml; Watanabe et al., 1998 ) at 10°C for 30 min, washed quickly with warmed neuronal maintenance medium, and then subjected to immunostaining.

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Functional validation of a human GLUD2 variant in a murine model of Parkinson’s disease

doi: 10.1038/s41419-020-03043-2

Figure Lengend Snippet: a Expression levels of NR2A, GluA1, GluA2, EAAT1, and EAAT2 in the SN of AAV-GFP, MPTP + AAV-GFP, MPTP + AAV-GLUD2, and MPTP + AAV-GLUD2 T1492G groups of mice were determined by western blotting. n = 6. b , c Immunofluorescent staining of GFP, GFAP, and EAAT1 or EAAT2 in the SNpc of AAV-GFP, MPTP + AAV-GFP, MPTP + AAV-GLUD2, and MPTP + AAV-GLUD2 T1492G groups (scale bars: upper, 40 µm; lower, 8 µm). d Effect of GLUD2 or its mutant on the expression of GDH2, GDH1, GFAP, EAAT1 and EAAT2 in MPP + -treated U251 cells was determined by western blotting. The bottom band (~55 KD) in the GDH2 blot is non-specific. n = 3. Results are expressed as the mean ± SEM. ** p < 0.01, * p < 0.05 vs. AAV-GFP group or untreated U251 cells. ## p < 0.01 vs. MPTP + AAV-GFP or MPP + group. && p < 0.01, & p < 0.05 vs. MPP + + GLUD2 group. Statistical significance was determined by one-way ANOVAs and Tukey tests for post-hoc comparisons.

Article Snippet: Anti-GFAP (#80788), NR2A (#4205), GluA1 (#13185), Bax (#14796), and GluA2 (#13607) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Staining, Mutagenesis

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Effects of PDI knockdown on PP2A bindings to PDI and GluA2 in the hippocampus following KA injection. KA increases PDI:PP2A and GluA2:PP2A bindings in control siRNA-infused animals, but not in PDI siRNA-infused animals. ( a ) Representative Western blot images for the PDI:PP2A and GluA2:PP2A bindings. ( b – c ) Quantitative analyses of the effects of PDI siRNA on PP2A level ( b ) and PDI:PP2A binding ( c ) following KA injection (*, # p < 0.05 vs. control siRNA vs. saline; n = 7, respectively; Kruskal–Wallis test followed by Tukey post-hoc test).

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques: Knockdown, Injection, Control, Western Blot, Binding Assay, Saline

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Effects of PDI knockdown on PICK1 bindings to GluA2 in the hippocampus following KA injection. PDI siRNA does not influence PICK1 level in both saline- and KA-treated groups. As compared to control siRNA, PDI siRNA increases GluA2A:PICK1 binding in saline-treated group. Although KA does not affect GluA2:PICK1 binding in control siRNA-infused group, it increases it in PDI siRNA-infused group. ( a ) Representative Western blot images for the GluA2:PICK1 binding. ( b – c ) Quantitative analyses of the effects of PDI siRNA on PICK1 level ( b ) and GluA2:PICK1 binding ( c ) following KA injection (*, # p < 0.05 vs. control siRNA vs. saline; n = 7, respectively; Kruskal–Wallis test followed by Tukey post-hoc test). ( d ) Scheme of the role of PDI in AMPAR internalization. PDI may reduce lead to reduction-induced PP2A activation, which would abolish PICK1-mediated AMPAR internalization by dephosphorylating GluA2 S880 and CaMKII T286 sites.

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques: Knockdown, Injection, Saline, Control, Binding Assay, Western Blot, Activation Assay

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques:

Journal: Neuroscience research

Article Title: Roles of 5-HT 1A receptor in the expression of AMPA receptor and BDNF in developing mouse cortical neurons.

doi: 10.1016/j.neures.2016.09.008

Figure Lengend Snippet: Fig. 1. Expression of 5-HT1A receptor and AMPA receptor subunit GluR1 in cortical neurons cultured for 3 days (A and B) and 14 days (C and D). (A) Neurons at 3 DIV were stained

Article Snippet: To examine the localization of 5-HT1A receptor and GluR1 receptor, cortical neurons at 3 DIV and 14 DIV were incubated overnight at 4 ◦C with the rat anti-5-HT1A receptor antibody (1:1000 dilution) and rabbit anti-GluR1 antibody (#13185, 1:200 dilution, Cell Signaling Technology, U.S.A.), followed by the incubation with Alexa Fluor 488-conjugated goat anti-rat IgG antibody and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature.

Techniques: Expressing, Cell Culture, Staining

Journal: Neuroscience research

Article Title: Roles of 5-HT 1A receptor in the expression of AMPA receptor and BDNF in developing mouse cortical neurons.

doi: 10.1016/j.neures.2016.09.008

Figure Lengend Snippet: Fig. 2. Effects of 5-HT1A receptor agonist 8-OH-DPAT on the mRNA expression of BDNF and AMPA receptor subunits, GluR1 and GluR2, in cortical neurons in vitro. Neurons were

Article Snippet: To examine the localization of 5-HT1A receptor and GluR1 receptor, cortical neurons at 3 DIV and 14 DIV were incubated overnight at 4 ◦C with the rat anti-5-HT1A receptor antibody (1:1000 dilution) and rabbit anti-GluR1 antibody (#13185, 1:200 dilution, Cell Signaling Technology, U.S.A.), followed by the incubation with Alexa Fluor 488-conjugated goat anti-rat IgG antibody and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature.

Techniques: Expressing, In Vitro

Journal: Neuroscience research

Article Title: Roles of 5-HT 1A receptor in the expression of AMPA receptor and BDNF in developing mouse cortical neurons.

doi: 10.1016/j.neures.2016.09.008

Figure Lengend Snippet: Fig. 3. Effects of 5-HT1A receptor agonist 8-OH-DPAT on the mRNA expression of BDNF and AMPA receptor subunits, GluR1 and GluR2, in the frontal cortex (A), and Tph2 and 5-HTT

Article Snippet: To examine the localization of 5-HT1A receptor and GluR1 receptor, cortical neurons at 3 DIV and 14 DIV were incubated overnight at 4 ◦C with the rat anti-5-HT1A receptor antibody (1:1000 dilution) and rabbit anti-GluR1 antibody (#13185, 1:200 dilution, Cell Signaling Technology, U.S.A.), followed by the incubation with Alexa Fluor 488-conjugated goat anti-rat IgG antibody and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature.

Techniques: Expressing

Journal: Journal of neurochemistry

Article Title: TrkB activation by 7, 8-dihydroxyflavone increases synapse AMPA subunits and ameliorates spatial memory deficits in a mouse model of Alzheimer's disease.

doi: 10.1111/jnc.13432

Figure Lengend Snippet: Fig. 4 7, 8-Dihydroxyflavone (7, 8-DHF) increased the accumulation of both GluA1 and GluA2 subunits at the synapses in Tg2576 mice. (a) Representative blots of GluA1-4 from synaptoneurosomal lysates of hippocampus (n = 8 for each group). (b) 7, 8-DHF increased the levels of GluA1and GluA2, and remained the levels of GluA3 and GluA4 unchanged. (c) Representative blots of GluA1-4 from whole lysates of hippocampus. (d) 7, 8-DHF did not affect the levels of GluA1-4 in whole lysates of hippocampus. (e) Representative blots of Ser818 and Ser831of GluA1, Ser880 of GluA2, and Ser845 of GluA1 from synaptoneurosomal extracts of hippocampus. (f) 7, 8-DHF increased the levels of Ser831, and Ser818, and remained the level of Ser880 and Ser845 unchanged. Error bars show SD in all results. *p < 0.05, **p < 0.01 compared to the wild-type (WT)- vehicle mice. #p < 0.05, ##p < 0.05 compared to Tg-vehicle mice.

Article Snippet: Comparable motor and visual function between various groups were observed in the Table 1 The list of primary antibodies used in this study Product name Host Categories No Companies Concentration AMPA Receptor (GluR1) (D4N9V) Rabbit 13185 Cell Signaling Technology, Beverly, MA, USA 1 : 1000 AMPA Receptor (GluR2) (E1L8U) Rabbit 13607 Cell Signaling Technology 1 : 1000 AMPA Receptor (GluR3) Rabbit 3437 Cell Signaling Technology 1 : 1000 AMPA Receptor (GluR4) (Arg860) Rabbit 3825 Cell Signaling Technology 1 : 1000 Phospho-AMPA Receptor (GluR1) (Ser818) Rabbit Cell Signaling Technology 1 : 1000 Phospho-AMPA Receptor (GluR1) (Ser831) Rabbit A4352 Sigma-Aldrich 1 : 1000 Phospho-AMPA Receptor (GluR1) (Ser845) (D10G5) Rabbit 8084 Cell Signaling Technology 1 : 500 Phospho-AMPA Receptor (GluR2) pS880 Rabbit MABN103 Merck Millipore, Temecula, CA, USA 1 : 200 BDNF Rabbit ab46176 Abcam Cambridage, UK 1 : 100 TrkB Rabbit ab33655 Abcam 1 : 200 Phospho-TrkB (Tyr816) Rabbit Merck Millipore 1 : 100 CaMKII Mouse ab3908 Abcam 1 : 1000 Phospho-Erk1/2 Rabbit 9911 Cell Signaling Technology 1 : 500 Akt Rabbit 9272 Cell Signaling Technology 1 : 500 CREB Rabbit 3360R-100 Biovision, Mountain view, CA, USA 1 : 500 Phospho-CaMKII (Thr286) Mouse 3742 Cell Signaling Technology 1 : 1000 Phospho-Erk1/2 Rabbit 3441-100 BioVision 1 : 1000 Phospho-Akt (Ser473) Rabbit 9271 Cell Signaling Technology 1 : 800 Phospho-CREB (Ser133) Rabbit 9191 Cell Signaling Technology 1 : 500 Amyloid precursor protein Rabbit PA5-16730 Thermo Scientific Pierce, Waltham, MA, USA 1 : 200 Amyloid beta 40 Rabbit PA3-16760 Thermo Scientific Pierce 1 : 500 Amyloid beta 42 Rabbit PA3-16761 Thermo Scientific Pierce 1 : 500 Glyceraldehyde-3-phosphate dehydrogenase Rabbit G2267 Sigma-Aldrich 1 : 10000 © 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 620--636 (b)(a) (d)(c) (f)(e) (h)(g) (j)(i) © 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 620--636 visible-platform test (data not shown).

Techniques: