glua2 n terminal  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction

doi: 10.3390/ijms24043924

Figure Lengend Snippet: Surface GluA2 AMPA receptor in neonatal hippocampal neurons is decreased in A350V neurons and is increased after heat treatment. ( A ) Representative images (20× magnification) of total (T.GluA2) and surface (S.GluA2) GluA2 AMPA receptor in wild-type (WT) and mutant (A350V) IQSEC2 hippocampal neurons. Neurons treated with heat (40 °C) for 1 h are labelled as MUT.H.S or WT.H.S. ( B ) Quantification of relative surface GluA2 expression (ratio: S.GluA2/T.GluA2). Data are reported as the mean ± SEM obtained from at least 4 neuronal culture preparations for each of the 4 groups. ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: To label neuronal surface GluA2 AMPAR, coverslips were incubated at 4 °C overnight in PBS containing 3% normal goat serum and mouse monoclonal extracellular anti-N-terminal GluA2 IgG (Synaptic System, Gottingen, Germany, cat# 182111).

Techniques: Mutagenesis, Expressing

Journal: The EMBO Journal

Article Title: miR ‐124‐dependent tagging of synapses by synaptopodin enables input‐specific homeostatic plasticity

doi: 10.15252/embj.2021109012

Figure Lengend Snippet: Example images of a FRAP experiment: SP − and SP + spines from the same cultured hippocampal neuron transfected with SEP‐GluA2 (gray and color‐coded) + RFP‐SP (red) 10s before, and 0, 370, 750 s after the photobleaching. Scale bars: 1 μm. Dotted lines represent the outline of dendrites. Arrowheads indicate dendritic spines which were targeted for photobleaching. SEP‐GluA2 fluorescence intensity at SP − vs. SP + spines. Each pair of dot plots represents average fluorescence for SP − and SP + spines from a same neuron (four cultures). ** P = 0.0061 (two‐tailed paired Student's t ‐test). Quantification of FRAP dynamics for SP − vs. SP + spines. Recovery curves represent SEP‐GluA2 fluorescence average per cell ( n = 14 cells, from four cultures). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by two divergent models ( P < 0.0001). Quantification of the recovery fraction 750 s after the photobleaching. Each pair of dot plots represents average recovery for SP − and SP + spines from a same neuron (four cultures). ** P = 0.0067 (Wilcoxon matched‐pairs signed rank test). Data information: Data are represented as mean ± SEM.

Article Snippet: For surface staining of AMPAR subunits, cultured hippocampal neurons were incubated live for 10 min at 37°C with a mouse monoclonal antibody raised against the N‐terminal domain of GluA2 that also recognizes GluA1 and GluA3 (Synaptic Systems, 182411 Clone 248B7) and diluted to 1:100 in the culture medium.

Techniques: Cell Culture, Transfection, Fluorescence, Two Tailed Test

Journal: The EMBO Journal

Article Title: miR ‐124‐dependent tagging of synapses by synaptopodin enables input‐specific homeostatic plasticity

doi: 10.15252/embj.2021109012

Figure Lengend Snippet: A, B Expression levels of miR‐124, miR‐92a and miR‐181 (A), and GluA2 and SP mRNAs (B) determined by qRT–PCR in neurons treated with TTX or left untreated (UT). Data are expressed as a percentage of the UT condition (miR‐124: UT, n = 11, TTX, n = 14; miR‐92a: UT, n = 11, TTX, n = 14; miR‐181: UT, n = 11, TTX, n = 14; GluA2 mRNA: UT, n = 11, TTX, n = 14; SP mRNA: UT, n = 11, TTX, n = 14, n indicates the number of experiments). * P < 0.05, ns, P > 0.05 (Mann Whitney test). C Sequence alignment showing complementarity between Gria2 or SP 3'UTR and miR‐124 binding seed region (highlighted in red). D Levels of luciferase activity measured from HEK‐293 expressing miR‐Ctrl or miR‐124 together with the Renilla luciferase coding sequence reporter fused to the Gria2 or SP 3'UTR wild‐type (WT) or mutated (MUT) to prevent miR‐124 binding. Data are expressed as a percentage of miR‐Ctrl condition (GluA2‐3'UTR‐WT: miR‐Ctrl, n = 5, miR‐124, n = 5; GluA2‐3'UTR‐MUT: miR‐Ctrl, n = 5, miR‐124, n = 5; SP‐3'UTR‐WT, miR‐Ctrl, n = 6, miR‐124, n = 6; SP‐3'UTR‐MUT: miR‐Ctrl, n = 6, miR‐124, n = 6, n indicates the number of experiments). * P < 0.05, *** P < 0.001, ns, not significant, P > 0.05 (Mann–Whitney test). Data information: Data are represented as mean ± SEM.

Article Snippet: For surface staining of AMPAR subunits, cultured hippocampal neurons were incubated live for 10 min at 37°C with a mouse monoclonal antibody raised against the N‐terminal domain of GluA2 that also recognizes GluA1 and GluA3 (Synaptic Systems, 182411 Clone 248B7) and diluted to 1:100 in the culture medium.

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Sequencing, Binding Assay, Luciferase, Activity Assay

Journal: The EMBO Journal

Article Title: miR ‐124‐dependent tagging of synapses by synaptopodin enables input‐specific homeostatic plasticity

doi: 10.15252/embj.2021109012

Figure Lengend Snippet: Micrographs showing dendrites from neurons transfected with Homer1c‐DsRed (magenta) and SEP‐GluA2 constructs containing wild‐type (WT) or mutated (MUT) 3'UTR (green) and treated with TTX for 48 h, or left untreated (UT). Scale bar: 10 μm. SEP‐GluA2 synaptic fluorescence intensity for each condition, normalized to GluA2‐3'UTR‐WT untreated neurons (3'UTR‐WT: UT, n = 29, TTX, n = 37; 3'UTR‐MUT: UT, n = 18, TTX, n = 14; n indicates the number of cells, from three cultures). * P < 0.05, ns, not significant, P > 0.05 (Kruskal‐Wallis test followed by Dunn's multiple comparison test). Micrographs showing dendrites from neurons transfected with Homer1c‐BFP (magenta), GFP‐SP‐shRNA (gray) and a rescue RFP‐SP construct containing wild‐type (WT) or mutated (MUT) 3'UTR (geen) and treated with TTX, or left untreated (UT). Scale bar: 10 μm. Percentage of SP + synapses for each condition (3'UTR‐WT: UT, n = 26, TTX, n = 28; 3'UTR‐MUT: UT, n = 26, TTX, n = 24; n indicates the number of cells, from three cultures). ** P < 0.01, *** P < 0.001, ns, not significant, P > 0.05 (two‐way ANOVA test followed by Tukey's multiple comparison test). Micrographs showing Homer1c‐GFP (magenta) and immunostaining for surface AMPARs (cyan) and endogenous SP (green) in neurons transfected with or without 50 nM SP TSB‐LNA and treated with TTX, or left untreated (UT). Scale bar: 10 μm. Percentage of SP + synapses for each condition (Control: UT, n = 36, TTX, n = 52; SP TSB‐LNA: UT, n = 62, TTX, n = 29; n indicates the number of cells from three cultures). * P < 0.05, ** P < 0.01, ns, not significant, P > 0.05 (Kruskal–Wallis test followed by Dunn's multiple comparison test). AMPAR synaptic fluorescence intensity for each condition, normalized to control untreated neurons (Control: UT, n = 30, TTX, n = 36; SP TSB‐LNA: UT, n = 37, TTX, n = 20; n indicates the number of cells from two cultures). * P < 0.05, ns, not significant, P > 0.05 (Kruskal–Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.

Article Snippet: For surface staining of AMPAR subunits, cultured hippocampal neurons were incubated live for 10 min at 37°C with a mouse monoclonal antibody raised against the N‐terminal domain of GluA2 that also recognizes GluA1 and GluA3 (Synaptic Systems, 182411 Clone 248B7) and diluted to 1:100 in the culture medium.

Techniques: Transfection, Construct, Fluorescence, Comparison, shRNA, Immunostaining, Control

Journal: The EMBO Journal

Article Title: miR ‐124‐dependent tagging of synapses by synaptopodin enables input‐specific homeostatic plasticity

doi: 10.15252/embj.2021109012

Figure Lengend Snippet:

Article Snippet: For surface staining of AMPAR subunits, cultured hippocampal neurons were incubated live for 10 min at 37°C with a mouse monoclonal antibody raised against the N‐terminal domain of GluA2 that also recognizes GluA1 and GluA3 (Synaptic Systems, 182411 Clone 248B7) and diluted to 1:100 in the culture medium.

Techniques: Western Blot, Immunofluorescence, Proximity Ligation Assay

Journal: Molecular Psychiatry

Article Title: IQSEC2 mutation associated with epilepsy, intellectual disability, and autism results in hyperexcitability of patient-derived neurons and deficient synaptic transmission

doi: 10.1038/s41380-021-01281-0

Figure Lengend Snippet: a A representative trace of excitatory postsynaptic currents (EPSCs) that were measured in a control neuron at 5 weeks post-differentiation. b A representative trace of EPSCs measured in an IQSEC2 -mutant neuron at 5 weeks post-differentiation. c The average amplitude of synaptic events was increased in the IQSEC2 -mutant neurons ( p = 0.053). d The average rate of synaptic events was not significantly different between control and IQSEC2 -mutant neurons. e The cumulative distribution of the amplitude of synaptic events is right-shifted in the IQSEC2 -mutant neurons indicating higher amplitudes of synaptic events. f A representative trace of synaptic activity measured in a control neuron at 7 weeks of age. g A representative trace of synaptic activity measured in an IQSEC2 -mutant neuron at 7 weeks. h The average amplitude of synaptic events is similar between IQSEC2 -mutant and control neurons at 7 weeks of age. i The average rate of synaptic events was not significantly different between control and IQSEC2 -mutant neurons at 7 weeks. j The cumulative distribution of the amplitude of synaptic events shows a slight right shift in IQSEC2 -mutant neurons. k A representative trace of synaptic activity measured in a control neuron at 11 weeks. l A representative trace of synaptic activity measured in an IQSEC2 -mutant neuron at 11 weeks. m The average amplitude of synaptic events is similar between IQSEC2 -mutant and control neurons at 11 weeks post-differentiation. n The average rate of synaptic events is significantly decreased in IQSEC2 -mutant neurons compared to control neurons at 11 weeks. o The cumulative distribution of the amplitude of synaptic events. p Immunoblots after BS 3 -crosslinking of samples to assess surface (crosslinking “+”) and total protein expression of AMPARs (crosslinking “−”). q Quantification of relative total protein levels of AMPA receptor subunits in samples not cross-linked. r Quantification of relative surface protein levels of AMPA receptor subunits in samples treated with BS 3 (crosslinking “+”). Surface expression is the ratio of s- GluA2 to the total GluA2 signal in the lane. Signal intensities normalized to actin signal and wild type as 100%. s Representative images of labeled total (t GluA2 ) and surface (s GluA2 ) GluA2 in IQSEC2 -mutant and control DG granule neurons. t Quantification of the percentage of s GluA2 to t GluA2 as a measure surface GluA2 expression. u (left). An image of the FACS gating of a control neuronal culture at 7 weeks after the start of differentiation. The live neurons that passed all the gating (GFP positive indicating PROX1 expression and live) were ~55% of the cells in the culture (marked with the red line). u (right). An image of the FACS gating of an IQSEC2 -mutant neuronal culture at 7 weeks. The live neurons that passed all the gating were ~41% of the cells in the culture. v (left). An image of the FACS gating of a control neuronal culture at 11 weeks. The live neurons that passed all the gating were approximately 36% of the cells in the culture. v (right) An image of the FACS gating of an IQSEC2 -mutant neuronal culture at 11 weeks. The live neurons that passed all the gating were approximately 14% of the cells in the culture. w (left) Immunohistochemistry staining for DAPI (blue), MAP2 (green), and Caspase3 (red) in an example control neuronal culture. w (right) Immunohistochemistry staining for DAPI (blue), MAP2 (green), and Caspase3 (red) in an example IQSEC2 -mutant neuronal culture. x An increased average percentage of neurons stained for caspase3 (out of the total MAP2 positive neurons) in the IQSEC2 -mutant neuronal cultures compared to the control neuronal cultures indicate an increased number of apoptotic neurons. In this figure, asterisks indicate statistical significance by the following code: * p value < 0.05, ** p < 0.01. Error bars represent the standard error.

Article Snippet: To label surface GluA2 , sections were incubated at 4 °C overnight in PBS containing 0.5% normal goat serum and a mouse monoclonal Alexa Fluor 488-conjugated extracellular anti-N-terminal GluA2 antibody (Millipore-Sigma, MAB397A4).

Techniques: Control, Mutagenesis, Activity Assay, Western Blot, Expressing, Labeling, Immunohistochemistry, Staining