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: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Synapse volumes increase correlatively during synaptic maturation. (A–D) Representative distribution of RIM1/2 and GluA1 under stochastic optical reconstruction microscopy (STORM). Scale 2 μm in top panels and 500 nm in lower panels. (E–G) Volumes of identified synaptic RIM1/2, GluA1, and PSD-95 clusters across different developmental stages. Numbers in bars denote the synapse numbers. (H,I) Correlations between the volumes of GluA1 and RIM1/2 clusters (H) and the volumes of PSD-95 and RIM1/2 (I) within the same synapses. Linear regressions were conducted on synapses of DIV7 (gray circles and line) and DIV18 (dark blue circles and line). Data from synapses of DIV10 and 14 were plotted with dark yellow and dark red crosses. Also refer to , and for more details on correlations and statistics. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques: Microscopy

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: The heterogeneity of synaptic protein distribution increases with development. (A–D) Representative density maps of synaptic GluA1 at different developmental stages. Scale bars, 200 nm. (E–G) Normalized autocorrelation functions of RIM1/2 (E) , GluA1 (F) , and PSD-95 (G) . g a above 1 suggests a significant non-uniform distribution. (H–J) Developmental changes of nanocluster number (left), normalized density within nanocluster (middle), and nanocluster volume of RIM1/2 (H) , GluA1 (I) , and PSD-95 (J) . Numbers in bars denote the synapse numbers. Also refer to for more statistical details. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Correlation between presynaptic and postsynaptic protein heterogeneity in mature synapses. (A,B) Scatter plots for heterogeneity of GluA1 (A) and PSD-95 (B) against that of RIM1/2. All data points across all developmental stages could be fitted with linear functions as shown with lines. (C,D) Linear regressions of the relationships between heterogeneity of GluA1/PSD-95 and RIM1/2 at DIV7 (gray) and DIV18 (dark blue). (E) Relationship between heterogeneity and cluster volume of RIM1/2 at DIV7 (gray) and DIV18 (dark blue). Data points with cluster volume >1 × 10 7 nm 3 were fitted with linear functions. It is noted that g a of immature synapses was significantly lower than that of matured synapses. (F) Averaged g a of synapses with cluster volume of 1–4 × 10 7 nm 3 for immature (DIV7-10) and mature synapses (DIV18). Also refer to for more details. *** p < 0.001, t-test. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Evolvement of trans-synaptic nano-alignment during synaptic maturation. (A–D) Representative examples of synapses with RIM1/2 (red) and GluA1 (blue) co-labeled and imaged with STORM. Thick color denotes detected nanoclusters. Scale bar, 200 nm. (E) Normalized local density of GluA1 along with distances from RIM1/2 nanoclusters for synapses at different developmental stages. (F) , Averaged enrichment of GluA1 within 50 nm from peaks of RIM1/2 nanoclusters ( n = 85, 60, 30, 54, and 54 nanoclusters). The open bar represents the enrichment indices of synapses with the position of nanoclusters randomized within synaptic clusters. (G) Fraction of nanoclusters that were enriched with protein across the cleft. (H–J) Enrichment between RIM1/2 and PSD-95 for synapses at different developmental stages. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey's multiple comparisons test in (F,I) , z -test in (G,J) . Also refer to for more statistical details. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques: Labeling

Journal: Life Science Alliance

Article Title: Loss of PLA2G4E compromises synaptic structure and cognitive outcomes in mice

doi: 10.26508/lsa.202503323

Figure Lengend Snippet: (A, B) Representative Western blot and quantification of the expression of different synaptic proteins in mouse cortex along the first postnatal month (n = 3 mice per group and time point). Two-way ANOVA followed by Tukey’s post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001. (C) At PND10, the percentage of hippocampal synaptosomes expressing GluA1 is significantly lower in Pla2g4e −/− mice with respect to their WT littermates (n = 4). Data are shown as the mean ± SEM. *** P < 0.001. PND, postnatal day. Source data are available for this figure.

Article Snippet: After blocking with 4 ml of ice-cold blocking buffer (5% FBS in PBS), resuspended synaptosomes were incubated for 30 min at 4°C with primary antibodies against GluA1 (#13185, 1:1,500; Cell Signaling) and Nrx1β (75-216, 1:400; NeuroMab), and calcein AM (100 nM, #65-0853-39; eBioscience).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: SYNPLA: A synapse-specific method for identifying learning-induced synaptic plasticity loci

doi: 10.1101/473314

Figure Lengend Snippet: a ) Diagram of SYNPLA targeting presynaptic myc-NRXN and. cLTP induces mobilization of GluA1 from an extrasynaptic pool and insertion into the post-synaptic density, decreasing the distance between the targeted proteins and permitting PLA. b ) Representative images of SYNPLA reactions performed on myc-NRXN expressing cultured rat hippocampal neurons at 14 days in vitro for the indicated conditions; PLA shown in gray. Scale bar: 10 μm. c ) Quantification of a single SYNPLA experiment (as in f ). The number of SYNPLA puncta detected in each field of view (round) and the average (square) ± SEM across all fields of view are shown; *** p<0.001, 1-way ANOVA followed by Tukey-Kramer post-hoc test. d ) Quantification of six independent experiments (indicated by different colors); the average number of SYNPLA puncta across 10-12 fields of view for each experiment (circles, normalized to CTRL) and the average PLA signal across experiments (square) ± SEM are shown; ** p<0.01, 1-way ANOVA (with Tukey-Kramer post-hoc test). e ) Diagram of SYNPLA in rat organotypic hippocampal slices. Sindbis virus expressing myc-NRXN is injected in the presynaptic CA3 region SYNPLA between myc-NRXN and endogenous GluA1 is measured in the postsynaptic CA1 region. f ) Representative images of SYNPLA under the indicated conditions in region CA1 of organotypic slice cultures. Scale bar: 10 μm. g ) Quantification of SYNPLA puncta in organotypic slices, from three independent experiments (indicated with different colors, 4 slices per condition (one circle per slice)); squares indicate average ± SEM across experiments; *** p<0.001, paired t-test.

Article Snippet: For Supplementary Fig. 2 different primary antibodies were used as negative controls: guinea pig anti-GluA1 (AGP-009, Alomone labs), rabbit anti-cFos (Cell signaling, #2250); all 1:100 dilution.

Techniques: Expressing, Cell Culture, In Vitro, Injection

Journal: bioRxiv

Article Title: SYNPLA: A synapse-specific method for identifying learning-induced synaptic plasticity loci

doi: 10.1101/473314

Figure Lengend Snippet: a ) Diagram of a general PLA reaction. Note that A and B must be sufficiently close to permit complementary binding and ligation of ABo1 and ABo2 and subsequent amplification to make a rolony. b ) Diagram of PLA targeting recombinant presynaptic myc-NRXN and postsynaptic HA-NLGN. c ) PLA rolonies (white dots, right) formed between postsynaptic cultured mouse hippocampal neuron expressing HA-NLGN + mCherry (red; middle) and co-cultured presynaptic neurons expressing myc-NRXN + GFP (green; left). Scale bar: 10 μm. d ) PLA puncta in each sample (circles) and average (squares) ± SEM for indicated expression and days in vitro (color indicates data acquired on the same day). *** p<0.001, 2-way ANOVA (see Methods); * p<0.05, 1-way ANOVA (with Tukey-Kramer post-hoc test). e ) PLA reaction between endogenous GluA1 and recombinant myc in cultured rat hippocampal neurons (exposed to cLTP, see ) expressing myc-NRXN and cytosolic fluorescent protein (green), subsequently immunostained for endogenous PSD-95 (red). Scale bars: 10 μm (left); 5 μm (right). f ) PLA puncta localize to synapses. In PSD-95 and myc-NRXN channels (from e , right), pixels were set to zero (masked) for values below a progressively increasing threshold (x axis). Puncta (PLA or randomly placed) display colocalization if non-zero pixels exist within 0.14 μm in both thresholded channels. For indicated threshold, ∼95% of PLA, while only ∼50% of randomly placed (mean ± SEM of 30 placements) puncta colocalized with pre- and post-synaptic markers. See Supplementary Fig. 3 and Methods for details.

Article Snippet: For Supplementary Fig. 2 different primary antibodies were used as negative controls: guinea pig anti-GluA1 (AGP-009, Alomone labs), rabbit anti-cFos (Cell signaling, #2250); all 1:100 dilution.

Techniques: Binding Assay, Ligation, Amplification, Recombinant, Cell Culture, Expressing, In Vitro

Journal: eNeuro

Article Title: Proteasomal-Mediated Degradation of AKAP150 Accompanies AMPAR Endocytosis during cLTD

doi: 10.1523/ENEURO.0218-19.2020

Figure Lengend Snippet: Model for LTD-mediated AKAP150 degradation through the proteasome system. Under basal conditions, a pool of palmitoylated AKAP150 is anchoring and regulating PKA and CaN function in dendritic spines. After LTD induction, a weak and extended Ca 2+ influx depalmitoylate and remove AKAP150 from spines. This removal promotes CaN-mediated dephosphorylation and consequent endocytosis of GluA1-AMPARs. During this process AKAP150 is ubiquitinated and degraded via proteasome. The presence of 20S proteasome inhibitor MG132 blocks not only LTD-mediated AKAP150 degradation but also its removal from spines. Maintenance of AKAP150 levels is sufficient to prevent LTD-induced GluA1-AMPARs endocytosis suggesting that AKAP150 degradation by the proteasome could be an important factor in the regulation of surface GluA1-AMPARs at spines.

Article Snippet: Primary antibodies were: anti-phospho-Ser-845-GluA1 (1:1000; clone EPR2148; 04–1073; RRID: AB_1977219 ; Merck-Millipore), anti-GluA1 (1:1000; AB1504; RRID: AB_2113602 ; Merck-Millipore), anti-AKAP150 (1:1000; 07–210; RRID: AB_310430 ; Merck-Millipore), anti-PSD95 (1:1000; clone 6G6-1C9; ab2723; RRID: AB_303248 ; Abcam), anti-SAP97 (1:1000; clone K64/15; 75–030; RRID: AB_2091920 ; UC Davis/NIH NeuroMab), anti-Ubiquitin (1:5000; U5379; RRID: AB_477667 ), anti-β-tubulin (1:20,000; clone 5H1; 556321; RRID: AB_396360 ; BD Biosciences), anti-β-actin (1:20,000; clone AC-74; A2228; RRID: AB_476697 ; Sigma), and anti-GAPDH (1:40 000; clone 6C5; AM4300; RRID: AB_437392 ; Thermo Fisher Scientific).

Techniques: De-Phosphorylation Assay

Journal: Journal of Neuroinflammation

Article Title: Thymosin β4 reverses phenotypic polarization of glial cells and cognitive impairment via negative regulation of NF-κB signaling axis in APP/PS1 mice

doi: 10.1186/s12974-021-02166-3

Figure Lengend Snippet: The effects of Tβ4 intervention and inflammatory pathway inhibition on neuronal survival and neurotransmitter release-related proteins. a Representative photomicrographs of Nissl staining. b Histograms are the quantitative analysis of the number of survival neurons in the PtA and hippocampus. The data are presented as mean ± SEM ( n = 3/group) in the different experimental groups. White dashed line implicates separated cortex layers, and white dotted circle refers to neuronal loss, with black arrow pointing to the location. c Representative photomicrographs of Map2 DAB staining and d Map2 immunoreactivity in the PtA and CA1 region. The data are presented as mean ± SEM ( n = 4/group) in the different experimental groups. e The protein levels of Synapsin1, PSD95, GLUR1, 5-HT 1A R, and CREB in the cortex and hippocampus were measured by western blotting. f – j The measurement of gray intensity of these proteins in the cortex and hippocampus. The data are presented as mean ± SEM ( n = 5/group) in the different experimental groups. # p < 0.05, ## p < 0.01 vs WT mice; * p < 0.05, ** p < 0.01 vs APP/PS1 mice

Article Snippet: The membrane were incubated overnight at 4 °C with the following primary antibodies: GluR1, Synapsin1, PSD95, TLR4, TLR2, TNFR2, MyD88, IKK-β, IKB-α, NF-κB p65, phospho-NF-κB p65(Ser536), NF-κB p52 (Cell Signaling Technologies), 5-HT 1A R (GeneTex), GAPDH, β-actin, and α-tubulin(Proteintech group), followed by incubation with an HRP-conjugated secondary antibodies for 2 h at room temperature.

Techniques: Inhibition, Staining, Western Blot