Journal: Frontiers in Molecular Neuroscience

Article Title: A developmental delay linked missense mutation in Kalirin-7 disrupts protein function and neuronal morphology

doi: 10.3389/fnmol.2022.994513

Figure Lengend Snippet: E1577K impairs surface expression of NR2B . (A) Representative regions of secondary dendrites with GFP-cell fill and NR2B surface stain. Insets show a single spine from each condition. Scale bar -10 μm. (B) Number of NR2B surface puncta normalized to total dendrite (GFP) area measured. (C) Average surface NR2B puncta size. (D) Average intensity of NR2B surface puncta. N = 2, 5 cells per replicate. (E) Regions of dendrites stained with Kalirin-7 and total NR2B to assess colocalization following overexpression of indicated constructs. Deconvoluted insets indicate Kalirin-7 and NR2B puncta adjacent within indicated spines. * p < 0.05. ** p < 0.01. *** p < 0.005.

Article Snippet: Proteins were separated by SDS PAGE, immobilized on PVDF by Western blot, and visualized [Flag M2 (Sigma #F1804), NR2B (Novus Biologicals, #NB100-74475), Kalirin-spectrin (Sigma #02122)].

Techniques: Expressing, Staining, Over Expression, Construct

Journal: Frontiers in Molecular Neuroscience

Article Title: A developmental delay linked missense mutation in Kalirin-7 disrupts protein function and neuronal morphology

doi: 10.3389/fnmol.2022.994513

Figure Lengend Snippet: E1577K shows impaired Rac1-GEF activity. (A) Western Blot of anti-FLAG pulldown of Kalirin-7 and E1577K from HEK293T, with co-immunoprecipitation of transfected NR2B (Mock = non-transfected). (B) HEK293T cells were transfected with the indicated conditions and active-Rac1 was pulled down from lysates with PAK-PBD coated Sepharose beads. Positive (+ ve) and Negative (-ve) samples indicate Mock transfected lysates Loaded with GTP-γ-S and GDP, respectively. Blots were probed with anti-FLAG and anti-Rac1. (C) Quantification of Active-RAC1 pulldown under the indicated conditions. * p < 0.05.

Article Snippet: Proteins were separated by SDS PAGE, immobilized on PVDF by Western blot, and visualized [Flag M2 (Sigma #F1804), NR2B (Novus Biologicals, #NB100-74475), Kalirin-spectrin (Sigma #02122)].

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Transfection

Journal: Scientific Reports

Article Title: Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches

doi: 10.1038/s41598-017-00519-8

Figure Lengend Snippet: Design of lentiviral vectors for NR2B manipulation in LMAN. ( a ) Diagram of the eGFPhNR2B vector to overexpress human NR2B . ( b ) Diagram of the eGFP vector for control injections. ( c ) Western blots showing no human NR2B protein in HEK293FT cells without transfection (Lane 1), and greater NR2B protein (* ~180 kD band) in cells transfected with the eGFPhNR2B plasmid (possible degraded or unfinished NR2B protein product is detected as an additional lower band) (Lane 2). ( d ) HEK293FT cells transfected with the eGFP plasmid show GFP-expressing cells labeled using a GFP antibody (green). ( e ) Cells transfected with the eGFP plasmid stained with an anti-NR2B antibody show no labeled cells. ( f ) Merged image of ( d ) and ( e ). ( g ) Cells transfected with the eGFPhNR2B plasmid show GFP-expressing cells (localized in cell membranes) labeled using a GFP antibody (green). ( h ) Cells transfected with the eGFPhNR2B plasmid show NR2B-expressing cells (localized in cell membranes) stained with anti-NR2B antibody (red). ( i ) Merged image of ( g ) and ( h ) showing complete overlap of the human NR2B tagged with eGFP (yellow). ( j ) Darkfield view of a brain section showing the injection site in LMAN. ( k ) Cells expressing GFP-tagged (green) human NR2B ~3 weeks after injection of the eGFPhNR2B lentivirus in LMAN. ( l ) Darkfield photograph of coronal sections showing drawings of LMAN and part of nidopallium that was microdissected for protein analysis. ( m ) Western blots showing NR2B protein and actin protein from microdissected tissues of nidopallium and LMAN in juveniles and adult animals (3 animals shown as examples). ( n ) Western blot comparing endogenous NR2B protein and actin in LMAN of an adult bird (Lane 1) with additional NR2B overexpression in birds with eGFPhNR2B lentivirus in LMAN ~35–40 days after surgery (Lanes 3–7; * ~180 kD NR2B protein). Protein Standard is included in Lane 2. ( o ) Quantification of NR2B protein levels normalized to actin levels in Western blots. Even though the values were higher in all 4 animals, protein levels were not significantly higher in nidopallium relative to LMAN in juveniles (p = 0.125) possibly due to low sample size, but were higher in nidopallium compared to LMAN in adults (p = 0.05). Band density was quantified using ImageJ and results analyzed using two sample Wilcoxon tests for comparisons among groups, or paired Wilcoxon signed-rank tests for within-group comparisons. Scale bars, 10 μm ( d–i ).

Article Snippet: This construct design was made based off of the pEGFP-NR2B plasmid containing the rat NR2B sequence that is commercially available from Addgene (Addgene plasmid #17925).

Techniques: Plasmid Preparation, Control, Western Blot, Transfection, Expressing, Labeling, Staining, Injection, Over Expression

Journal: Scientific Reports

Article Title: Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches

doi: 10.1038/s41598-017-00519-8

Figure Lengend Snippet: Immunostaining of NR2B protein in LMAN cells. ( a ) Quantification showing higher levels of labeled cells in birds injected with the eGFPhNR2B lentivirus in LMAN (blue) compared to control birds with injections of the eGFPhNR2B lentivirus in nidopallium or the eGFP lentivirus in LMAN (red). Control-injected animals did not differ in number of labeled cells (p = 0.73, two sample Wilcoxon test). ( b ) Examples of labeled LMAN cells in eGFP-injected animals (i), and in eGFPhNR2B-injected animals (ii). Scale bar, 200 μm.

Article Snippet: This construct design was made based off of the pEGFP-NR2B plasmid containing the rat NR2B sequence that is commercially available from Addgene (Addgene plasmid #17925).

Techniques: Immunostaining, Labeling, Injection, Control

Journal: Scientific Reports

Article Title: Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches

doi: 10.1038/s41598-017-00519-8

Figure Lengend Snippet: Representative sonograms of songs and mean end syllable repetitions before (presurgery) and after (postsurgery) injections. ( a ) Presurgery song of a control bird Pink 124. ( b ) Postsurgery song of Pink 124 injected with eGFPhNR2B lentivirus in the motor nidopallium showing no change in song after injections. ( c ) Presurgery song motif of a bird Green 04. ( d ) Postsurgery song motif of Green 04 showing end motif syllable stuttering after overexpression of human NR2B in LMAN. Black letters indicate syllable types in song motifs. Sonograms were generated using Avisoft-SASLab Pro software (Avisoft Bioacoustics, Germany). ( e ) Mean number of end syllable repetitions in control and NR2B groups of birds. ( f ) Time course of mean end syllable stuttering in birds (n = 8) of the NR2B overexpression group that were injected with the eGFPhNR2B lentivirus in LMAN. Pre indicates end syllable repetitions before injections. Each time point is an average of 10 song bouts; weeks 2–12 were compared to presurgery levels using paired t tests; asterisk represents p value < 0.01. ( g ) Percentage of motifs (mean ± SE) showing end syllable and introductory note repetitions in birds of the controls and NR2B groups. We used a cut-off of more than 3 repeats of introductory notes at the beginning of each motif of a song bout to be included as repetitions in our analyses for quantification. P values were determined using paired Wilcoxon signed-rank tests.

Article Snippet: This construct design was made based off of the pEGFP-NR2B plasmid containing the rat NR2B sequence that is commercially available from Addgene (Addgene plasmid #17925).

Techniques: Control, Injection, Over Expression, Generated, Software

Journal: Scientific Reports

Article Title: Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches

doi: 10.1038/s41598-017-00519-8

Figure Lengend Snippet: Overexpression of human NR2B induced changes in song motif structure. ( a ) Average number of syllables per song motif. ( b ) Mean number of introductory notes per song motif. ( c ) Song motif duration. ( d ) Intersyllable gap durations. ( e ) Average of all syllable durations (bird Red08 of the control group appeared to be an outlier, which was confirmed by a Dixon’s test in R in both presurgery (p = 0.02) and postsurgery (p < 0.0001) conditions, and thus not included in the statistical analyses of the entire group. Each line is one animal presurgery and postsurgery, mean ± SE, with p values determined using paired Wilcoxon signed-rank tests.

Article Snippet: This construct design was made based off of the pEGFP-NR2B plasmid containing the rat NR2B sequence that is commercially available from Addgene (Addgene plasmid #17925).

Techniques: Over Expression, Control

Journal: Scientific Reports

Article Title: Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches

doi: 10.1038/s41598-017-00519-8

Figure Lengend Snippet: Song sequencing changes after overexpression of human NR2B in LMAN. ( a ) Song motif linearity. ( b ) Song motif consistency. ( c ) Song motif stereotypy. ( d ) Percentage sequential match. Each line is one animal presurgery and postsurgery, mean ± SE, with p values determined using paired Wilcoxon signed-rank tests.

Article Snippet: This construct design was made based off of the pEGFP-NR2B plasmid containing the rat NR2B sequence that is commercially available from Addgene (Addgene plasmid #17925).

Techniques: Sequencing, Over Expression

Journal: Scientific Reports

Article Title: Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches

doi: 10.1038/s41598-017-00519-8

Figure Lengend Snippet: Song similarity changes after overexpression of human NR2B in LMAN. ( a ) Percentage similarity before and after injections in control or NR2B groups. ( b ) Percentage accuracy. Each line is one animal presurgery and postsurgery, mean ± SE, with p values determined using paired Wilcoxon signed-rank tests.

Article Snippet: This construct design was made based off of the pEGFP-NR2B plasmid containing the rat NR2B sequence that is commercially available from Addgene (Addgene plasmid #17925).

Techniques: Over Expression, Control

Journal: Scientific Reports

Article Title: Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches

doi: 10.1038/s41598-017-00519-8

Figure Lengend Snippet: Correlations between number of cells expressing high levels of NR2B protein in LMAN and song features in animals injected with the eGFPhNR2B lentivirus in LMAN. ( a ) Protein levels of NR2B in LMAN are correlated with variability in song motif duration after injections. ( b ) Protein levels of NR2B in LMAN are also correlated with variability in percentage sequential match after injections. P and R 2 values were determined by regression analyses. Red boxes represent the control groups mean variability in song motif duration ( a ), and percentage sequential match ( b ) that were not included in the regression analyses and are shown for comparison only. ( c ) Protein levels of NR2B in LMAN are not correlated with mean number of syllable repetitions.

Article Snippet: This construct design was made based off of the pEGFP-NR2B plasmid containing the rat NR2B sequence that is commercially available from Addgene (Addgene plasmid #17925).

Techniques: Expressing, Injection, Control, Comparison

Journal: Journal of Neuroscience

Article Title: ANKS1B Gene Product AIDA-1 Controls Hippocampal Synaptic Transmission by Regulating GluN2B Subunit Localization

doi: 10.1523/jneurosci.4029-14.2015

Figure Lengend Snippet: Figure 4. Altered GluN2A-mediated and GluN2B-mediated synaptic transmission and syn- apticexpressioninAIDA-1cKOmice.A,AveragedNMDAR-EPSCs(top)andsummarydata(bot- tom)showinglesssensitivitytotheselectiveGluN2BantagonistRo25-6981(500nM)inAIDA-1

Article Snippet: Transfection of primary neuronal cultures [at 7–11 d in vitro (DIV)] or HEK cells was performed with vectors for the following: mKATE2-AIDA-1 (57 kDa isoform), eGFP-tagged GluN2A (Addgene plasmid 45445), GluN2B (Addgene plasmid 45447; Barria and Malinow, 2002), CASK (gift from Brett Abrahams, Albert Einstein College of Medicine), or YFP-KIF17 (gift from William Green, University of Chicago) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.

Techniques: Transmission Assay

Journal: Journal of Neuroscience

Article Title: ANKS1B Gene Product AIDA-1 Controls Hippocampal Synaptic Transmission by Regulating GluN2B Subunit Localization

doi: 10.1523/jneurosci.4029-14.2015

Figure Lengend Snippet: Figure 5. shRNA-mediated knockdown of AIDA-1 alters synaptic NMDAR subunit composition in rat primary neurons. A, Left, WesternblotshowingreductionofAIDA-1proteinlevelsinculturedratcorticalneuronsbytwodifferentAIDA-1-targetingshRNAs (shAIDA#1 and shAIDA#2) compared with a nontargeting control (shNT). Neurons were transduced with lentiviruses expressing shRNAs at 8 DIV for 7–12 d. Right, Quantification of shRNA-induced knockdown of AIDA-1 splice variants (57 and 50 kDa) and normalizedtocontrol(shNT):(shAIDA#1:[57kDa],12.25.2%;[50kDa],29.710.2%;shAIDA#2:[57kDa],28.16.0%;[50 kDa], 27.5 7.1%; n 6–8 samples from 3 different cultures). Statistical significance was determined using one-way ANOVA and post hoc t test (2-tailed, unpaired) with Tukey’s correction for multiple comparisons. B, Quantification for the mean intensity ofGluN2Bpuncta(shAIDA#1,81.61.0%ofshNT;shAIDA#2,62.71.0%ofshNT),orGluN2Apuncta(shAIDA#1,156.41.7% ofshNT;shAIDA#2,152.11.5%ofshNT)thatcolocalizedwiththesynapticmarkerShank(n6–11cellsfrom3independent cultures).NeuronsweretransducedonDIV7andimagedonDIV17–22.C,RepresentativeimagesshowingthatAIDA-1knockdown reduces synaptic levels of GluN2B and increases synaptic levels of GluN2A in DIV 21 rat primary hippocampal cultures. GluN2 subunit is red, shank is green, and MAP2 is blue in the merged image. D, Representative normalized NMDAR-EPSCs (top) and summary data (bottom) showing that shRNA-mediated knockdown of AIDA-1 accelerates the decay kinetics of NMDAR-EPSCs (shNT,201.09.4ms;shAIDA#2,163.56.9ms,n24cellsfrom6independentcultures).E,Averagedsampletraces(top)and summarydata(bottom)showingthatshRNA-mediatedknockdownofAIDA-1decreasedNMDAR-EPSCsensitivitytotheGluN2B- specificantagonistRo25-6981(200nM;shNT,49.33.2%;shAIDA#2,68.54.0%;n24cellsfrom6independentcultures). NeuronsweretransducedatDIV7andrecordedatDIV12–21.SummarygraphsrepresentthemeanSEM.**p0.01,***p 0.001.

Article Snippet: Transfection of primary neuronal cultures [at 7–11 d in vitro (DIV)] or HEK cells was performed with vectors for the following: mKATE2-AIDA-1 (57 kDa isoform), eGFP-tagged GluN2A (Addgene plasmid 45445), GluN2B (Addgene plasmid 45447; Barria and Malinow, 2002), CASK (gift from Brett Abrahams, Albert Einstein College of Medicine), or YFP-KIF17 (gift from William Green, University of Chicago) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.

Techniques: shRNA, Knockdown, Control, Transduction, Expressing

Journal: Journal of Neuroscience

Article Title: ANKS1B Gene Product AIDA-1 Controls Hippocampal Synaptic Transmission by Regulating GluN2B Subunit Localization

doi: 10.1523/jneurosci.4029-14.2015

Figure Lengend Snippet: Figure7. AIDA-1preferentiallyassociateswithGluN2B-containingNMDARs.A,CoimmunoprecipitationsfrommousewholehippocampallysatespreparedatP42showthatAIDA-1interactswith GluN1,withPSD95,andpreferentiallywithGluN2BoverGluN2A.TheextentofGluN2BorGluN2AbindingtoAIDA-1wasdeterminedbynormalizingtheimmunoprecipitation(IP)signalbytheinput (n 5 independent coimmunoprecipitations; input, 15 g; GluN2B, 69.5 3.3%; GluN2A, 10.5 1.2%). B, Primary rat hippocampal neurons at DIV 21 were immunostained for the dendritic marker MAP2, synaptic marker Shank, AIDA-1, and GluN2A or GluN2B. C, The extent of colocalization of AIDA-1 with GluN2A or GluN2B was quantified by Mander’s coefficients, and shows that AIDA-1colocalizespreferentiallywithGluN2B(n6–8cellsfrom4independentcultures;GluN2A-AIDA,10.51.2%;GluN2B-AIDA,50.61.8%).D,CoexpressionofGluN2B(eGFP-GluN2B)or GluN2A(eGFP-GluN2A)withAIDA-1(57kDaisoform;mKATE2,aredfluorescentprotein)inratprimaryhippocampalneuronsatDIV14for3dshowsextensivecolocalizationofAIDA-1andGluN2B indendrites,andmuchlesswithGluN2A.E,RepresentativeWesternblotshowingAIDA-1isoformandGluN2BdonotcoimmunoprecipitatewhencoexpressedinHEK293FTcells.Datapresentedas mean SEM. ***p 0.001.

Article Snippet: Transfection of primary neuronal cultures [at 7–11 d in vitro (DIV)] or HEK cells was performed with vectors for the following: mKATE2-AIDA-1 (57 kDa isoform), eGFP-tagged GluN2A (Addgene plasmid 45445), GluN2B (Addgene plasmid 45447; Barria and Malinow, 2002), CASK (gift from Brett Abrahams, Albert Einstein College of Medicine), or YFP-KIF17 (gift from William Green, University of Chicago) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.

Techniques: Marker

Journal: Journal of Neuroscience

Article Title: ANKS1B Gene Product AIDA-1 Controls Hippocampal Synaptic Transmission by Regulating GluN2B Subunit Localization

doi: 10.1523/jneurosci.4029-14.2015

Figure Lengend Snippet: Figure8. AIDA-1associateswiththeGluN2BtransportmachineryandregulatesGluN2BexportfromtheER.A,Coimmunopre- cipitationsfromwholemousehippocampusshowingthatAIDA-1associateswithcomponentsoftheGluN2Btransportmachinery KIF17,CASK,andSAP97,butnotKIF1b.B,mKATE2-taggedAIDA-1wascoexpressedwitheitherYFP-taggedKIF17orCASKinHEK cells.CoimmunoprecipitationsshowthatAIDA-1caninteractwithbothKIF17andCASK.C,LossofAIDA-1doesnotalterthetotal levelsofKIF17andCASKinhippocampallysates(n4mice).SummarygraphsrepresentthemeanSEM.D,Westernblotsfor GluN2B,calnexin(ERmarker),and58KGolgiprotein(Golgimarker)showingdifferentbiochemicalfractions(top)fromhippocam- paltissueofWTandAIDA-1cKOmice.Hrepresentswholelysate.Below,AplotoftherelativeabundanceofGluN2Binconsecutive iodixanolgradientfractionsnormalizedtoH(bottomleft)andquantificationofGluN2BinpooledERfractions(bottomright).Bar graph shows quantification (n 3 mice) of total GluN2B in ER fractions (area under the curve; cKO, 132.1 1.5% of WT, p 0.024). Results show GluN2B accumulates in the ER fractions in AIDA-1 cKO mice. Summary graphs represent the mean SEM. *p 0.05.

Article Snippet: Transfection of primary neuronal cultures [at 7–11 d in vitro (DIV)] or HEK cells was performed with vectors for the following: mKATE2-AIDA-1 (57 kDa isoform), eGFP-tagged GluN2A (Addgene plasmid 45445), GluN2B (Addgene plasmid 45447; Barria and Malinow, 2002), CASK (gift from Brett Abrahams, Albert Einstein College of Medicine), or YFP-KIF17 (gift from William Green, University of Chicago) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.

Techniques:

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: ( A ) The chemical structure of ergotamine. ( B ) The H 2 O-injected oocytes did not cause any change with the treatment of 100 µM glutamate ( n = 6–8 oocytes from four different frogs). ( C – F ) Glutamate induced inward currents with or without ergotamine (30 µM and 10 µM). For each subunit of NMDARs, the responses after treating with either glutamate (100 µM) alone or together with ergotamine (30 and 10 µM). Voltage clamp recording was conducted at a holding potential of −80 mV. The coapplication of ergotamine with glutamate resulted in modulation of the recombinant receptors ( C ) NR1a/NR2A, ( D ) NR1a/NR2B, ( E ) NR1a/NR2C, and ( F ) NR1a/NR2D, which in turn reduced glutamate-evoked inward current in a reversible manner ( n = 6–8 oocytes from four different frogs).

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Injection, Recombinant

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: The value of I max , IC 50 , and n H (Hill coefficient) of ergotamine for the glutamate-evoked current in each recombinant receptor. Values represent means ± S.E.M. ( n = 6–8/group). IC 50 , Hill’s coefficient; I max value as determined as described in Materials and methods.

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Recombinant

Journal: Cancers

Article Title: NMDA Receptor Signaling Mediates cFos Expression via Top2β-Induced DSBs in Glioblastoma Cells

doi: 10.3390/cancers11030306

Figure Lengend Snippet: NMDAR signaling impacts cFos expression in primary glioblastoma multiforme (GBM) cells. ( a ) Immunofluorescence staining of the GluN1 and GluN2B subunits of the NMDAR in the G1702 cells. Notably, GluN2B subunits are localized at the end of cellular protrusions (GluN1/GluN2B = green, Hoechst 33342 = blue; scale bar: 25 µm). ( b ) Immunofluorescence staining of 53BP1 (red) and Top2β (green) in G1702 cells. Top2β and 53BP1 form foci which partly co-localize (scale bar: 20 µm). ( c ) Relative cFos/GAPDH expression in G1702 cells treated with 1 mM Glu and 20 µM MK801, 20 µM ifenprodil or 1 µM ICRF193 overnight, analyzed through western blotting (error bars show SD, n = 3, one sample t -test, p > 0.05 (ns), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)).

Article Snippet: The following primary antibodies were used: Rabbit anti-GluN1 (1:100, D65B7 Cell Signaling), mouse anti-GluN2B (1:200, S59-20 Stress Marq), rabbit anti-53BP1 (1:1000; H-300 Santa Cruz) and mouse anti-Top2β (1:100; A-12 Santa Cruz).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot