Journal: The Journal of Neuroscience

Article Title: Vezatin Is Essential for Dendritic Spine Morphogenesis and Functional Synaptic Maturation

doi: 10.1523/JNEUROSCI.3084-11.2012

Figure Lengend Snippet: Vezatin concentrates in spines during neuronal differentiation. A, Developmental distribution of Vezatin showing its concentration in growth cone and dendrites early during neuronal differentiation (inset, left) and in dendrites and spines in maturing and mature neurons (insets, middle and right). B, At the early stage of neuronal differentiation (left) and later, at the onset of dendritic spine development, Vezatin does not colocalize with presynaptic Syb2 (right, arrow). C, Postsynaptic Vezatin is partially codistributed with transsynaptic N-cadherin. C1, The arrowhead and the arrow are pointing to a spine where Vezatin is (yellow) and is not (red aside green) clearly overlapping the N-cadherin signal, respectively. D, Vezatin is expressed in the soma of interneurons stained with GAD67 (left). Vezatin does not codistribute with GAD65 at the axonal compartment (right). MAP2 is used to label dendrite. E, Snapshot of a time-lapse image of a living cortical neuron transfected with Vezatin-GFP and RFP-actin showing codistribution in a hemicircumferential ring in a growth cone. Top, inset, DIV 3, fixed cultured hippocampal neuron immunostained for Vezatin and GluA2, a glutamate receptor, showing that Vezatin expression is restricted to an arc-shaped region of the growth cone. F, Vezatin colocalizes with phalloidin (F-actin) in dendrites and in developing spines at early synaptic sites (DIV 14). Scale bars, 10 μm. G, Protein expression in mouse hippocampi extracts. The anti-Vezatin antibody recognized the two major Vezatin isoforms (see text) with band intensities approximately 2× weaker in the heterozygous floxed/null (fl/null) compared to the floxed/floxed (fl/fl) extract. Other few alternatively spliced variants are expressed in minor quantities. Loading control, GAPDH.

Article Snippet: Rabbit antibody anti-GluA2 was used at 1:200 (Alomone Labs).

Techniques: Concentration Assay, Staining, Transfection, Cell Culture, Expressing

Journal: The Journal of Neuroscience

Article Title: Vezatin Is Essential for Dendritic Spine Morphogenesis and Functional Synaptic Maturation

doi: 10.1523/JNEUROSCI.3084-11.2012

Figure Lengend Snippet: Subcellular spine Vezatin expression in vitro (A–E) and in vivo (F–G). A, Vezatin labels all stages of spine differentiation. Insets, Higher magnifications of a filopodium (arrowhead) and a thin spine (left). A stubby (arrow) and a mushroom-like (arrowhead) are shown on the right (top and bottom insets). B, Confocal images showing that Vezatin (green) and PSD95 (red) colocalize (yellow) at spine heads. Asterisks and arrowheads point to representative mushroom and stubby spines, respectively. Bottom right, Reconstructed view using the Imaris software package. C, Vezatin extends beyond the PSD95/PSD margin toward the neck of the spine: snapshot of a confocal spine 3D reconstruction (21 DIV). Overlay (yellow) between Vezatin (green) and PSD95 (red) staining. Both markers colocalize at the tip of the spine head, but PSD95 is more central than Vezatin. D, Vezatin is in the synaptosomal membrane fraction as PSD95 or GluA2 and GluN1 (P2). Vezatin is also in the cytosolic fraction (S2). Crude protein extract was obtained by clearing the lysate by gentle centrifugation (S1) followed by high-speed centrifugation (S2 and P2). E, High-magnification images of a 21 DIV spine whose cup-shaped base is colabeled (overlay) with Vezatin (green), phalloidin (red), and PSD95 (blue). Scale bars: A, 10 μm; B (left), E, 2 μm; B (right), 1 μm. F, Vezatin is expressed in hippocampus, cortex, and MHb. Other regions including striatum [caudate putamen (CPu) and cerebral peduncle (CP)], amygdala, and thalamus [ventral postero-medial thalamic nucleus (VPM) and ventral postero-lateral thalamic nucleus (VPL)] expressed Vezatin at a low level (objective 1.6 ×). Scale bar: 1000 μm. G, Confocal microscopy at low-power resolution indicates that Vezatin is expressed in soma (objective 20×) [SP in CA1/CA3 and interior/exterior (int/ext) in DG]. No staining is observed in the hilus (DG), except in cells attributed to astrocytes and/or inhibitory neurons. DAPI staining is used to label nuclei. G1, Higher-power resolution (objective 40×, zoom 2.67). Left, Vezatin is in dendrites (SR in CA1). Middle, Vezatin is in spines partially codistributing with the transsynaptic N-cadherin marker (CA1 SR region). Inset, Arrowhead and arrow are pointing to spines where postsynaptic Vezatin (green) is (yellow) and is not (green aside red) overlapping N-cadherin (red). Right, Vezatin is in postsynaptic thorny excrescences (in SL in CA3) that mark the location of mossy fiber synaptic terminations (arrow). Scale bars: G, 400 μm; G1, 100 μm. SP, stratum pyramidale; SL, stratum lacunosum.

Article Snippet: Rabbit antibody anti-GluA2 was used at 1:200 (Alomone Labs).

Techniques: Expressing, In Vitro, In Vivo, Software, Staining, Centrifugation, Confocal Microscopy, Marker

Journal: Neural Plasticity

Article Title: Seizure-Induced Regulations of Amyloid- β , STEP 61 , and STEP 61 Substrates Involved in Hippocampal Synaptic Plasticity

doi: 10.1155/2016/2123748

Figure Lengend Snippet: A single ECS but not chronic ECS increases the level of Tyr 876 -phosphorylated GluA2 in the hippocampus. Immunoblot analysis for phosphorylation of GluA2 at Tyr 876 (Y 876 ) or 3Tyr (3Y: Tyr 869 , Tyr 873 , and Tyr 876 ) and total GluA2 expression in the hippocampal crude membrane (P2) fraction following a single ECS ( n = 5 rats per time point) (a–c) and chronic ECS ( n = 5 rats for 72 h time point and n = 6 rats per all other time points) (d–f). The ratio of the phosphorylated GluA2 band intensity over the β -actin band intensity (a-b, d-e) and the ratio of total GluA2 band intensity over the β -actin band intensity (c, f) were calculated per each time point and normalized to that of “no seizure” (NS) sham group. Data shown represent the mean band intensity ± SEM. (a–c) A single ECS increases the level of Tyr 876 -phosphorylated GluA2 at 96 h time point ((b) ∗ p < 0.05) but does not alter the level of 3Tyr-phosphorylated GluA2 and total GluA2. (d–f) Chronic ECS does not change Tyr-phosphorylation of GluA2 (d-e) and total GluA2 expression (f).

Article Snippet: Phosphorylation site specific antibodies used include anti-GluN2B-pTyr 1472 which recognizes phosphorylated Tyr-1472 of GluN2B (P1516-1472, PhosphoSolutions), anti-ERK1/2-pThr 202 /Tyr 204 which recognizes phosphorylated Thr 202 /Tyr 204 of ERK1 and Thr 185 /Tyr 187 of ERK2 (#9106, Cell Signaling), anti-GluA2-p3Y which recognizes phosphorylated Tyr 869 , Tyr 873 , and Tyr 876 (3Y) of GluA2 (#3921S, Cell Signaling), and anti-GluA2-pY 876 which recognizes phosphorylated Tyr 876 of GluA2 (#4027S, Cell Signaling).

Techniques: Western Blot, Phospho-proteomics, Expressing, Membrane

Journal: Neural Plasticity

Article Title: Seizure-Induced Regulations of Amyloid- β , STEP 61 , and STEP 61 Substrates Involved in Hippocampal Synaptic Plasticity

doi: 10.1155/2016/2123748

Figure Lengend Snippet: A single ECS but not chronic ECS increases the level of Tyr 876 -phosphorylated GluA2 in the hippocampus. Immunoblot analysis for phosphorylation of GluA2 at Tyr 876 (Y 876 ) or 3Tyr (3Y: Tyr 869 , Tyr 873 , and Tyr 876 ) and total GluA2 expression in the hippocampal crude membrane (P2) fraction following a single ECS ( n = 5 rats per time point) (a–c) and chronic ECS ( n = 5 rats for 72 h time point and n = 6 rats per all other time points) (d–f). The ratio of the phosphorylated GluA2 band intensity over the β -actin band intensity (a-b, d-e) and the ratio of total GluA2 band intensity over the β -actin band intensity (c, f) were calculated per each time point and normalized to that of “no seizure” (NS) sham group. Data shown represent the mean band intensity ± SEM. (a–c) A single ECS increases the level of Tyr 876 -phosphorylated GluA2 at 96 h time point ((b) ∗ p < 0.05) but does not alter the level of 3Tyr-phosphorylated GluA2 and total GluA2. (d–f) Chronic ECS does not change Tyr-phosphorylation of GluA2 (d-e) and total GluA2 expression (f).

Article Snippet: Phosphorylation site specific antibodies used include anti-GluN2B-pTyr 1472 which recognizes phosphorylated Tyr-1472 of GluN2B (P1516-1472, PhosphoSolutions), anti-ERK1/2-pThr 202 /Tyr 204 which recognizes phosphorylated Thr 202 /Tyr 204 of ERK1 and Thr 185 /Tyr 187 of ERK2 (#9106, Cell Signaling), anti-GluA2-p3Y which recognizes phosphorylated Tyr 869 , Tyr 873 , and Tyr 876 (3Y) of GluA2 (#3921S, Cell Signaling), and anti-GluA2-pY 876 which recognizes phosphorylated Tyr 876 of GluA2 (#4027S, Cell Signaling).

Techniques: Western Blot, Phospho-proteomics, Expressing, Membrane

Journal: Nature Communications

Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: a Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled for surface GluA2 (under non-permeabilizing conditions). Graph showing the number of surface GluA2 labeling, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA, F (2, 15) = 38.28) (Tukey’s test P control-NMDAR < 0.0001, P control-mGluR < 0.0001, P NMDAR-mGluR = 0.8438). b Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled with an antibody against endogenous LC3 (autophagic structures) and MAP2 (dendrites). Graph showing the number of dendritic LC3-positive puncta in secondary dendrites, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F2,24) = 15.11, P < 0.0001) (Tukey’s test Pcontrol-NMDA = 0.0005, P control-mGluR = 0.0001). c Same as in b , but neurons were pretreated for 1 h before, during and after the pulse with wortmannin (500 nM) or SBI-0206965 (500 nM). Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions (U: untreated, W: wortmannin, S: SBI-0206965). Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(8,45) = 33.83, P < 0.0001) (Tukey’s test P control/S-NMDA/S = 0.3677, P control/W-NMDA/W = 0.9986, P NMDA/U-NMDA/W < 0.0001, P NMDA/U-NMDA/S < 0.0001, P control/S-DHPG/S = 0.9674, P control/W-DHPG/W = 0.9989, P DHPG/U-DHPG/W < 0.0001, P DHPG/U-DHPG/S < 0.0001). d Same as in b with neurons that were infected with AAV plasmids carrying 4 shRNA sequences against atg5 ( sh-atg5 ) or scrambled control ( sh-scramble ), under the CamK2a promoter. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(5,30) = 16.94, P < 0.0001) (Tukey’s test P control/scr-control/atg5 = 0.9999, P NMDA/scr-NMDA/atg5 = 0.0025, P DHPG/scr-DHPG/atg5 < 0.0001, P control/scr-NMDA/scr < 0.0001, P control/scr-DGPG/scr < 0.0001, P control/atg5-NMDA/atg5 = 0.8959, P control/atg5-DHPG/atg5 = 0.9637). e Same as in b , but neurons were immunolabeled 15 min after NMDAR- and mGluR-LTD and treated for 1 h before, during and after the pulse with Ifenprodil (10 μM) or MTEP (10 μM) and JNJ16259685 (10 μM) to pharmacologically inhibit NR2B and mGluR1/5 receptors, respectively. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F (3,32) = 74.46, P < 0.0001) (Tukey’s test, P NMDA-NMDA+IFE < 0.0001, P DHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 μm for all panels.

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Cell Culture, Immunolabeling, Labeling, Infection, shRNA

Journal: Nature Communications

Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: a Confocal images of dendrites immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction and in the absence or presence of Dynamin-1 inhibitory peptide (50 µM) or SBI-0206965 (500 nM), a selective inhibitor of the ULK1 kinase activity. Inhibitors were applied 25 min before, during and 15 min after the pulses. Scale bar: 10 µm. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 9 independent experiments. Statistical analysis was performed using one-way ANOVA (F (8, 72) = 7.411, P < 0.0001) (Tukey’s test P control-control/D > 0.99, P control-control/S = 0.9971, P NMDA-NMDA/D = 0.0451, P NMDA-NMDA/S = 0.0008, P DHPG-DHPG/D = 0.0017, P DHPG-DHPG/S = 0.0002). b Confocal images of dendrites of neurons expressing 4 scrambled sequences ( sh-scramble ), or 4 sh-RNAs against atg5 ( sh-atg5 ), immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 10 independent experiments. Statistical analysis was performed using one-way ANOVA (F (5, 54) = 30.02, P < 0.0001) (Tukey’s test, P control/scr-control/atg5 = 0.0626, P NMDA/scr-NMDA/atg5 < 0.0001, P DHPG/scr-DHPG/atg5 < 0.0001, P control/atg5-NMDA/atg5 > 0.99, P control/atg5-DHPG/atg5 = 0.8602, P control/scr-NMDA/scr = 0.0008, P control/scr-DHPG/scr < 0.0001). c Representative images of consecutive confocal z-planes of cultured neurons immunostained with antibodies against PSD95, LC3, and MAP2 to label the dendrites, 15 min after cLTD. Note the colocalization of PSD95 and LC3 in dendritic spines (yellow arrows) and in the dendritic shaft (white arrows), in consecutive z-planes. Scale bar: 10 µm. Graph showing the percentage of PSD95 puncta co-localizing with LC3 in consecutive confocal z-planes in dendritic spines and shafts in control neurons or 15 min after chemically induced NMDAR- or mGluR-LTD. Bars represent mean values ± SEM. N = 8 independent experiments. Statistical analysis was performed by one-way ANOVA (F(5,42) = 48.43, P < 0.0001) (Tukey’s test for dendritic shaft, P control-NMDA = 0.0569, P control-DHPG = 0.1948, for dendritic spines, P control-NMDA < 0.0001, P control-DHPG < 0.0001). d Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of Bafilomycin A1 (50 µM) for 15 min before, during, and 15 min after the NMDA and DHPG pulses. e Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of SBI-0206965 (500 nM) for 30 min before, during, and 15 min after the NMDA and DHPG pulses. f Western blot analysis for GluA2 and PSD95 in lysates of cultured shscrambled or sh-atg5 expressing neurons in control conditions or 15 min after NMDAR- and mGluR-LTD. d – f Graphs showing the levels of PSD95 and GluA2 levels in the indicated conditions, normalized to total protein levels. Bars represent mean values ± SEM. Statistical analysis was performed by one-way ANOVA. d (N = 9 independent experiments) PSD95: F(5,48) = 15.08, P < 0.0001 (Tukey’s test P control-control/Baf = 0.7566, P control-NMDA = 0.0016, P control-DHPG = 0.0081, P NMDA-NMDA/Baf < 0.0001, P DHPG-DHPG/Baf = 0.0013. GluA2: F(5,48)=6.627, P < 0.0001 (Tukey’s test P control-control/Baf = 0.9692, P control-NMDA = 0.0014, P control-DHPG = 0.0067, P NMDA-NMDA/Baf = 0.0421, P DHPG-DHPG/Baf = 0.0127. e ( N = 7 independent experiments) PSD95: F(5,36) = 23.80, P < 0.0001. (Tukey’s test P control-control/SBI > 0.99, P NMDA-NMDA/SBI < 0.0001, P DHPG-DHPG/SBI < 0.0001, P control-NMDA < 0.0001, P control-DHPG < 0.0001, P control/SBI-NMDA/SBI = 0.9764, P control/SBI-DHPG/SBI = 0.6286). Panel e, GluA2: F(5,36)=11.73, P < 0.0001. (Tukey’s test P control-control/SBI = 0.9179, P NMDA-NMDA/SBI = 0.0001, P DHPG-DHPG/SBI = 0.0002, P control-NMDA = 0.0099, P control-DHPG = 0.0323, P control/SBI-NMDA/SBI = 0.9959, P control/SBI-DHPG/SBI = 0.9407). f ( N = 7 independent experiments) PSD95: F(5,36) = 10.93, P < 0.0001. (Tukey’s test P control/scr-control/atg5 = 0.7927, P NMDA/scr-NMDA/atg5 = 0.0045, P DHPG/scr-DHPG/atg5 = 0.0003, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0030, P control/atg5-NMDA/atg5 = 0.9488, P control/atg5-DHPG/atg5 = 0.9976). GluA2: F(5,36) = 10.79, P < 0.0001. (Tukey’s test P control/scr-control/atg5 > 0.99, P NMDA/scr-NMDA/atg5 = 0,0001, P DHPG/scr-DHPG/atg5 = 0.0019, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0021, P control/atg5-NMDA/atg5 = 0.5844, P control/atg5-DHPG/atg5 > 0.99).

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Immunolabeling, Activity Assay, Labeling, Expressing, Cell Culture, Western Blot

Journal: Nature Communications

Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: a – d Western blot analyses of different fractions along the autophagic vesicle purification procedure, using antibodies against a autophagosomal markers (LC3B, p62, Atg16L1, and Atg9A), b ER-Golgi markers (TGN, LMAN1, SAR1a), c endosomal markers (Rab11b, EEA1), and d markers of the plasma-membrane (Stx4), extracellular vesicles (Alix) and nuclear extracts (TBP). N = 3 independent experiments. e Graph showing the cell component analysis, as false discovery rate (FDR)-corrected p -values, of the dynamic cargo (total of 393 proteins) that is enriched (up) or less abundant (down) in AVs after LTD, compared to control. f Graphical representation of proteins enriched in AVs upon LTD, with relation to the synapse. g Western blot analysis of PK-treated control and LTD-AVs, validating the presence of the proteins identified by the proteomic analyses in the autophagic vesicles. Postsynaptic density (PSD) fraction was used as reference. Graph showing the fold change of the normalized levels of the proteins validated by western blot, as a ratio of LTD to control. Cargo proteins were normalized to the levels of p62, which remains unaffected at the early phase of LTD. N = 3 independent AV preparations. Bars represent mean values ± SEM. Statistical analysis was performed using paired, two-tailed Student’s t -test (GluA1, N = 6, P = 0.0002; GluA2, N = 6, P = 0.0039; Pick1, N = 5, P = 0.011; SAP97, N = 5, P = 0.0179; FYN, N = 8, P < 0.0001; CamKIIa, N = 8, P < 0.0001; IL1RAPL1, N = 8, P = 0.0004; Adam22, N = 4, P = 0.0018; INA, N = 3, P = 0.0287; MYH10, N = 8, P < 0.0001; ITPKA, N = 6, P = 0.0006; KCC2, N = 4, P = 0. 0352; cofilin-1, N = 6, P = 0.005; dynamin, N = 6, P = 0.0005; p62, N = 6, P = 0.9809). All indicated molecular weights in a – d and g are in kDaltons (kD).

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Western Blot, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet:

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Concentration Assay, Activity Assay, Plasmid Preparation, Avidin-Biotin Assay, Infection, In Vivo