Journal: iScience

Article Title: KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner

doi: 10.1016/j.isci.2022.105623

Figure Lengend Snippet: KIBRA regulates the basal expression of extrasynaptic AMPA receptors in the adult hippocampus (A, C, E, and G) Representative western blot images from sub-region CA1 of the adult hippocampus. Samples are normalized to loading control and quantified as % WT in B, D, F, and H (see for details). (B) Adult-onset KIBRA deletion from neurons decreases the total expression GluA2 and GluA1, but not the excitatory synaptic scaffold PSD-95 (unpaired t-tests, corrected for multiple comparisons). GluA2, WT = 100 ± 3%, cKO = 67 ±3; GluA1, WT = 100 ± 2%, cKO = 80 ± 4%; PSD95, WT = 100 ± 3%, cKO = 93 ± 3%. (D) Loss of KIBRA from the adult brain decreases expression of membrane-localized GluA2 (unpaired t-tests with Welch’s correction, corrected for multiple comparisons). GluA2, WT = 100 ± 2%, cKO = 83 ± 5%; GluA1, WT = 100 ± 4%, cKO = 102 ± 2%; PSD95, WT = 100 ± 6%, cKO = 105 ± 4%. (F) Adult-onset KIBRA deletion does not alter basal expression of synaptic AMPA receptors (unpaired Mann-Whitney tests, corrected for multiple comparisons). GluA2, WT = 100 ± 3%, cKO = 98 ± 6%; GluA1, WT = 100 ± 2%, cKO = 94 ± 8%; PSD95, WT = 100 ± 8%, cKO = 106 ± 5%. (H) Decrease in expression of the KIBRA homolog WWC2 following adult-onset deletion of KIBRA (unpaired t-tests, corrected for multiple comparisons). Total, WT = 100 ± 4%, cKO = 70 ± 2%; membrane, WT = 100 ± 5%, cKO = 92 ± 6%; synaptic, WT = 100 ± 5%, cKO = 87 ± 7%. (I) Representative western blot with equal protein loaded for total, cytosolic, membrane and synaptic fractions, demonstrating depletion of the postsynaptic scaffold PSD95 from the cytosolic fraction and enrichment in the membrane fraction with further enrichment in the synaptic fraction. Data shown as mean ± SEM, n on bar graphs = number of animals.

Article Snippet: GluA2, rabbit polyclonal , Alomone Labs , Cat# AGC-005 RRID: AB_2039881.

Techniques: Expressing, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner

doi: 10.1016/j.isci.2022.105623

Figure Lengend Snippet: Juvenile-onset deletion of KIBRA has minimal effect on hippocampal AMPAR expression (B, D, and F) Representative western blot images from sub-region CA1 of the juvenile hippocampus. (A) Acute reduction of KIBRA in the juvenile hippocampus decreases total expression of AMPAR subunit GluA2 but not GluA1 or PSD95 (unpaired t-tests, corrected for multiple comparisons, Welch’s correction for GluA2). GluA2, WT = 100 ± 1%, cKO = 86 ±3; GluA1, WT = 100 ± 3%, cKO = 95 ± 4%; PSD95, WT = 100 ± 4%, cKO = 95 ± 3%. (C) Larger decrease in total AMPAR expression in adult compared to juvenile KIBRA cKO mice (unpaired t-tests, corrected for multiple comparisons). For each group, data is shown as % decrease from respective WT (GluA2, juvenile cKO = −14 ± 4%, adult cKO = −33 ± 4%; GluA1, juvenile cKO = −3 ± 5%, adult cKO = −19 ± 4%). (E) Juvenile-onset deletion of KIBRA does not affect expression of membrane-associated AMPARs or PSD95 in the juvenile hippocampus (unpaired t- (GluA1, GluA2) or Mann-Whitney (PSD95) tests, corrected for multiple comparisons). GluA2, WT = 100 ± 6%, cKO = 95 ± 4%; GluA1, WT = 100 ± 6%, cKO = 101 ± 11%; PSD95, WT = 100 ± 3%, cKO = 98 ± 17%. (G) Loss of KIBRA in the juvenile brain does not alter basal expression of synaptic AMPA receptors or PSD95 (unpaired t- (GluA1, PSD95) or Mann-Whitney (GluA2) tests, corrected for multiple comparisons). GluA2, WT = 100 ± 14%, cKO = 89 ± 12%; GluA1, WT = 100 ± 7%, cKO = 85 ± 6%; PSD95, WT = 100 ± 7%, cKO = 96 ± 11%. Data shown as mean ± SEM, n on bar graphs = number of animals. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: GluA2, rabbit polyclonal , Alomone Labs , Cat# AGC-005 RRID: AB_2039881.

Techniques: Expressing, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner

doi: 10.1016/j.isci.2022.105623

Figure Lengend Snippet: KIBRA is required for LTP-induced increases in AMPAR expression in the adult hippocampus (A) Experimental design. Transverse hippocampal slices were collected from adult mice following basal stimulation (0.033 Hz) or LTP (TBS). The stimulated region of CA1 was microdissected 30 or 120 min after LTP or basal stimulation. Data from both time points was combined as no difference in AMPAR induction was observed between 30 and 120min post LTP. (B–D) Representative GluA2 and GluA1 immunoblot images after baseline stimulation or LTP from adult WT (cre-negative tamoxifen treated) or KIBRA cKO (cre-positive tamoxifen treated) mice. LTP increases GluA2 (C) and GluA1 (E) expression in WT but not KIBRA cKO mice. Data plotted as % increase over baseline stimulation, mean ± SEM (GluA2, WT mean= 151 ± 15%, cKO = 100 ± 8%; GluA1, WT mean = 119 ± 8%, cKO = 101 ± 4%). LTP-stimulated slices were compared to baseline-stimulated slices from the same animal. Number of slices is indicated on each bar. One sample t-test, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: GluA2, rabbit polyclonal , Alomone Labs , Cat# AGC-005 RRID: AB_2039881.

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner

doi: 10.1016/j.isci.2022.105623

Figure Lengend Snippet: LTP does not increase AMPAR expression in juvenile WT or KIBRA cKO mice Transverse hippocampal slices were collected from juvenile mice following basal stimulation (0.033 Hz) or LTP (TBS). The stimulated region of CA1 was microdissected 30 min after LTP or basal stimulation. (A and C) Representative GluA2 and GluA1 immunoblot images after baseline stimulation or LTP from juvenile WT (cre-negative tamoxifen treated) or KIBRA cKO (cre-positive tamoxifen treated) mice. LTP did not induce increases in total GluA2 (B) or GluA1 (D) expression in juvenile WT or KIBRA cKO mice. Data plotted as % increase over baseline stimulation, mean ± SEM (GluA2, WT mean = 108 ± 11%, cKO = 107 ± 7 %; GluA1, WT mean = 106 ± 6%, cKO = 114 ± 7%). LTP-stimulated slices were compared to baseline-stimulated slices from the same animal. For each group, n = 18 slices from 10 mice. One sample Wilcoxon test, p > 0.05 for all comparisons shown.

Article Snippet: GluA2, rabbit polyclonal , Alomone Labs , Cat# AGC-005 RRID: AB_2039881.

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner

doi: 10.1016/j.isci.2022.105623

Figure Lengend Snippet:

Article Snippet: GluA2, rabbit polyclonal , Alomone Labs , Cat# AGC-005 RRID: AB_2039881.

Techniques: Recombinant, Blocking Assay, 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: 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, Co-Immunoprecipitation Assay, Membrane, Immunofluorescence, Western Blot, GST Pulldown Assay

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: Co-Immunoprecipitation Assay, Immunoprecipitation, Membrane

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, Immunofluorescence

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, Comparison, Immunofluorescence

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, Comparison, Western Blot, Expressing, Immunofluorescence

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