Journal: bioRxiv

Article Title: DISC1 regulates N-Methyl-D-Aspartate receptor dynamics: Abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness

doi: 10.1101/349365

Figure Lengend Snippet: DISC1 interacts with GluN1, TRAK1 associates with GluN2B. ( a ) HA-GluN1 co-immunoprecipitates FLAG-DISC1 from transfected COS7 cells. ( b ) Upper, GluN1 cytoplasmic tail (C0-C1-C2) peptide array hybridised with Flag-DISC1. Each spot represents a single peptide. red, black and green text marks the C0, C1 and C2 cassettes respectively. ER retention signals are in bold. ( c ) FLAG-DISC1 co-immunoprecipitates GST-tagged C0-C1-C2. ( d ) DISC1 peptide array probed with FLAG-C0-C1-C2. ( e ) DISC1 361-385 GluN1 binding region alanine scan probed with FLAG-C0-C1-C2. C, unmutated peptide. ( f ) HA-GluN1 co-immunoprecipitates GST-DISC1 amino acids 358-499 from transfected COS7 cells. ( g ) Co-localisation of FLAG-DISC1 and endogenous GluN1 in cultured DIV8 and DIV14 hippocampal neurons. Arrowheads and arrows indicate example sites of colocalisation. ( h ) COS7 cells transfected with GluN1 or HA-GluN2B expression constructs were labelled with antibodies specific for GluN1 or HA, plus the ER marker Calreticulin. ( i ) COS7 cells co-transfected with FLAG-TRAK1 plus GluN1 (left) or HA-GluN2B (right) expression constructs were labelled using antibodies specific for GluN1 or HA, plus antiFLAG and the mitochondrial dye Mitotracker CMXRos. ( j ) COS7 cells triple-transfected with FLAG-TRAK1, GluN1 plus HA-GluN2B expression constructs were labelled using antibodies specific for GluN1, HA and FLAG, plus Mitotracker CMXRos. COS7 cells were used because they are ideal for exogenous protein expression due to their high transfection efficiency, large size and low profile, which facilitate co-immunoprecipitation and colocalisation studies to complement endogenous protein studies in neurons. ( k ) Trak1 immunoprecipitates GluN2B from adult mouse brain synaptosome and light membrane fractions. Scale bars, 50μm in G, otherwise 20μm; white boxes indicate enlarged areas

Article Snippet: The following antibodies were used in the surface NMDAR expression study: GluN1 Mouse monoclonal IgG2a (BD Biosciences, 556308), GluN2A Rabbit polyclonal (Alomone Labs, AGC-002), GluN2B Rabbit polyclonal (Alomone Labs, AGC-003), βIII-Tubulin Rabbit polyclonal (Abcam, Ab18207), βIII-Tubulin/Tuj1 Mouse monoclonal IgG2a (Cambridge Bioscience, 801201) and PSD95 Mouse monoclonal IgG1k (ABR, MA1-046).

Techniques: Transfection, Peptide Microarray, Binding Assay, Cell Culture, Expressing, Construct, Marker, Immunoprecipitation

Journal: bioRxiv

Article Title: DISC1 regulates N-Methyl-D-Aspartate receptor dynamics: Abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness

doi: 10.1101/349365

Figure Lengend Snippet: Trak1/GluN2B co-immunoprecipitation. Trak1 was immunoprecipitated from adult mouse brain synaptosome and light membrane fractions as shown in . Trak1 was detected in the lysates using secondary antibody conjugated to alkaline phosphatase.

Article Snippet: The following antibodies were used in the surface NMDAR expression study: GluN1 Mouse monoclonal IgG2a (BD Biosciences, 556308), GluN2A Rabbit polyclonal (Alomone Labs, AGC-002), GluN2B Rabbit polyclonal (Alomone Labs, AGC-003), βIII-Tubulin Rabbit polyclonal (Abcam, Ab18207), βIII-Tubulin/Tuj1 Mouse monoclonal IgG2a (Cambridge Bioscience, 801201) and PSD95 Mouse monoclonal IgG1k (ABR, MA1-046).

Techniques: Immunoprecipitation

Journal: bioRxiv

Article Title: DISC1 regulates N-Methyl-D-Aspartate receptor dynamics: Abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness

doi: 10.1101/349365

Figure Lengend Snippet: Altered distal dendritic NMDAR trafficking in Disc1 Der1/Der1 hippocampal neurons. ( a ) Green Dendra2 fluorescence in dendrites of a DIV8 mouse hippocampal neuron transfected with GluN1-Dendra2 plus HA-GluN2B. Scale bar, 5μm ( b )( c ) Mean red fluorescence intensity, b, or ROI dendritic length, c, in the central bin in Disc1 wt/wt and Disc1 Der1/Der1 DIV8 neurons was equal at time zero following photoconversion. ( d ) Quantification of fluorescence intensity over time in successive 5μm dendritic bins distal to the centre of the photoconversion ROI. Data analysed by timepoint-paired two tailed t-test. ( e ) Model of dendritic GluN1-Dendra2 motility. Photoconverted GluN1-Dendra2 progresses in a wave-like fashion, with the fastest and slowest moving GluN1-Dendra2 at the leading and trailing edges, respectively, and the bulk travelling as the ‘crest’. ( f ) Fluorescence peak velocity estimates for the 10μm and 15μm bins. Average time to peak fluorescence was converted to velocity, indicated above each bar. Average velocities were determined from the two bins. ( g ) Fast-moving GluN1-Dendra2 maximum velocity estimates for the 25μm-40μm bins. Average time to fluorescence appearance was converted to velocity, indicated above each bar. Average velocities were determined from the four bins. WT, Disc1 wt/wt ; HOM, Disc1 Der1/Der1 ; error bars represent SEM; **** p<0.0001; *** p<0.001; ** p<0.01; *p<0.05; n indicated on graphs

Article Snippet: The following antibodies were used in the surface NMDAR expression study: GluN1 Mouse monoclonal IgG2a (BD Biosciences, 556308), GluN2A Rabbit polyclonal (Alomone Labs, AGC-002), GluN2B Rabbit polyclonal (Alomone Labs, AGC-003), βIII-Tubulin Rabbit polyclonal (Abcam, Ab18207), βIII-Tubulin/Tuj1 Mouse monoclonal IgG2a (Cambridge Bioscience, 801201) and PSD95 Mouse monoclonal IgG1k (ABR, MA1-046).

Techniques: Fluorescence, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: DISC1 regulates N-Methyl-D-Aspartate receptor dynamics: Abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness

doi: 10.1101/349365

Figure Lengend Snippet: Dendritic NMDAR and PSD95 expression in hippocampal neurons. ( a ) 3D-SIM images of surface GluN1, GluN2A or Glun2B, and total PSD95 and βIII-tubulin (Tuj1). WT, Disc1 wt/wt ; HET, Disc1 (wt)/Der1 ; HOM, Disc1 Der1/Der1 ; scale bars, 2μm in main images, 0.6μm in enlarged insets indicated by white boxes ( b ) 3D-SIM image of a dendrite segment showing PSD95 (green) and βIII-tubulin (Tuj1, magenta). Scale bar B-F, 1μm ( c ) Identification of Imaris surfaces for PSD95. These three-dimensional surfaces are counted and their volume is quantified by the software. ( d ) PSD95 surfaces split into individual nanodomains. ( e ) Identification of the centre of each individual nanodomain, and conversion to Imaris spots using a bespoke MATLAB (MathWorks) XTension script. ( f ) Individual nanodomains are assigned to clusters, and the number per cluster is counted using the Imaris spots MATLAB XTension ‘Split into Surface Objects’.

Article Snippet: The following antibodies were used in the surface NMDAR expression study: GluN1 Mouse monoclonal IgG2a (BD Biosciences, 556308), GluN2A Rabbit polyclonal (Alomone Labs, AGC-002), GluN2B Rabbit polyclonal (Alomone Labs, AGC-003), βIII-Tubulin Rabbit polyclonal (Abcam, Ab18207), βIII-Tubulin/Tuj1 Mouse monoclonal IgG2a (Cambridge Bioscience, 801201) and PSD95 Mouse monoclonal IgG1k (ABR, MA1-046).

Techniques: Expressing, Software

Journal: bioRxiv

Article Title: DISC1 regulates N-Methyl-D-Aspartate receptor dynamics: Abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness

doi: 10.1101/349365

Figure Lengend Snippet: Altered dendritic NMDAR surface expression and GluN1 localisation to the post-synaptic density in Disc1 wt/Der1 and Disc1 Der1/Der1 hippocampal neurons. ( a ) Objects in 3D-SIM images visualised using the Imaris Isosurface tool. Touching objects are separated, with boundary lines between touching objects visible in the enlarged images. scale bars, 2μm in the full-size images, 0.6μm in enlarged insets indicated by white boxes ( b ) GluN1, GluN2A or GluN2B surface puncta density, total surface volume and individual surface puncta volume (all normalised to dendritic segment volume) from 3D reconstructions of primary dendrite segments of cultured DIV21 hippocampal neurons. Data analysed by Kruskal-Wallis (puncta density p=0.02, p=0.03, p=0.0004, total volume p=0.0025, p=0.4, p=0.003, puncta volume p=0.003, p<0.0001, p<0.0001 for GluN1, GluN2A and GluN2B respectively) followed by Dunn’s multiple comparison test. ( c ) Reconstructed 3D-SIM images of dendrites. Colocalised voxels, which contain signal from both PSD95 and surface-expressed GluN1 are shown in white. Scale bars, 3μm ( d ) GluN1 co-localisation with the PSD95 was evaluated on three measures. Pearson’s and Mander’s coefficients respectively indicate overall correlation of each signal or amount of GluN1 signal co-localised with PSD95 signal, and vice versa. Data analysed by Kruskal-Wallis (p=0.004, p=0.08, p=0.001 for Pearson’s, Mander’s M1 and Mander’s M2) followed by Dunn’s multiple comparison test. WT, Disc1 wt/wt ; HET, Disc1 wt/Der1 ; HOM, Disc1 Der1/Der1 ; error bars represent SEM; *** p<0.001; ** p<0.01; * p<0.05; n indicated on graphs

Article Snippet: The following antibodies were used in the surface NMDAR expression study: GluN1 Mouse monoclonal IgG2a (BD Biosciences, 556308), GluN2A Rabbit polyclonal (Alomone Labs, AGC-002), GluN2B Rabbit polyclonal (Alomone Labs, AGC-003), βIII-Tubulin Rabbit polyclonal (Abcam, Ab18207), βIII-Tubulin/Tuj1 Mouse monoclonal IgG2a (Cambridge Bioscience, 801201) and PSD95 Mouse monoclonal IgG1k (ABR, MA1-046).

Techniques: Expressing, Cell Culture

Journal: bioRxiv

Article Title: DISC1 regulates N-Methyl-D-Aspartate receptor dynamics: Abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness

doi: 10.1101/349365

Figure Lengend Snippet: NMDAR subunit GluN2A and GluN2B co-localisation with the post-synaptic density marker PSD95. ( a ) Reconstructed 3D-SIM images of dendrites. Colocalised voxels, which contain signal from both PSD95 and surface-expressed GluN1 are shown in white. White boxes indicate enlarged regions. Scale bars, 3μm ( b ) Pearson’s coefficient indicates overall correlation of each signal. Mander’s coefficients measure the amount of subunit fluorescent signal co-localised with total PSD95 signal, and vice versa. Data were analysed by Kruskal-Wallis (p=0.02 for GluN2A Mander’s M1, p=0.06 for GluN2B Mander’s M1) followed by Dunn’s multiple comparison test. WT, Disc1 wt/wt ; HET, Disc1 (wt)/Der1 ; HOM, Disc1 Der1/Der1 ; error bars represent SEM; * p<0.05 ( c ) Immunoblots of hippocampus lysates were prepared from nine week mice, and probed with antibodies specific for GluN1. GluN2A and GluN2B NMDAR subunits, followed by loading controls Gapdh and Vcl. Subunit expression relative to the loading controls was quantified using densitometry. Data were analysed by Kruskal-Wallis test and no significant differences were found WT, Disc1 wt/wt ; HET, Disc1 (wt)/Der(1) ; HOM, Disc1 Der(1)/Der(1) ; error bars represent SEM; n indicated on graphs

Article Snippet: The following antibodies were used in the surface NMDAR expression study: GluN1 Mouse monoclonal IgG2a (BD Biosciences, 556308), GluN2A Rabbit polyclonal (Alomone Labs, AGC-002), GluN2B Rabbit polyclonal (Alomone Labs, AGC-003), βIII-Tubulin Rabbit polyclonal (Abcam, Ab18207), βIII-Tubulin/Tuj1 Mouse monoclonal IgG2a (Cambridge Bioscience, 801201) and PSD95 Mouse monoclonal IgG1k (ABR, MA1-046).

Techniques: Marker, Western Blot, 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: 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: Cell Metabolism

Article Title: A Role for p53 in the Adaptation to Glutamine Starvation through the Expression of SLC1A3

doi: 10.1016/j.cmet.2018.07.005

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-AGC1 (D5I6I) , Cell Signaling Technology , Cat#64169.

Techniques: Recombinant, In Vitro, In Vivo, Cloning, Plasmid Preparation, Software, CRISPR

Journal: iScience

Article Title: GluK2 Q/R editing regulates kainate receptor signaling and long-term potentiation of AMPA receptors

doi: 10.1016/j.isci.2023.107708

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti-GluA3 , Alomone , RRID: AB_2039883.

Techniques: Recombinant, Protein Extraction, Software