Journal: bioRxiv

Article Title: Phenotype of mice carrying an NMDA receptor GluN2B protein-truncating variant associated with intellectual disability

doi: 10.1101/2025.10.02.680004

Figure Lengend Snippet: Relative mRNA expression levels of Grin1 , Grin2a , and Grin2b in the hippocampus of adult male (A) and female (B) Grin2b +/+ and Grin2b ⁺ /Δ mice, as determined by RT-qPCR. Data were normalized to the corresponding mean Grin2b +/+ value and are presented as mean ± SEM and analyzed using Student’s t-test to compare the expression in Grin2b ⁺ /Δ versus Grin2b +/+ mice; asterisks (*) indicate statistically significant differences. (C) Representative chromatograms of Sanger sequencing of PCR amplicons spanning the region of the frameshift mutation (gray box) in male and female Grin2b ⁺ /Δ mice and the plasmid DNA mixture of Grin2b ⁺: Grin2b Δ at a 1:1 ratio; yellow arrowheads indicate two of the five analyzed nucleotides. (D) The relative expression levels of Grin2b mRNA show comparable expression of wild-type and mutant alleles. Data are presented as mean ± SEM and were analyzed using one-way ANOVA (no significant differences were found).

Article Snippet: At DIV 14, surface GluN2A and GluN2B subunits were labeled in primary hippocampal neurons from Grin2b +/+ or Grin2b +/Δ mice by live staining with a rabbit anti-GluN2A antibody (1:250; AGC-002; Alomone) or a rabbit anti-GluN2B (1:250; AGC-003; Alomone), both targeting extracellular epitopes.

Techniques: Expressing, Quantitative RT-PCR, Sequencing, Mutagenesis, Plasmid Preparation

Journal: bioRxiv

Article Title: Phenotype of mice carrying an NMDA receptor GluN2B protein-truncating variant associated with intellectual disability

doi: 10.1101/2025.10.02.680004

Figure Lengend Snippet: (A) Volcano plot of proteomics analysis comparing protein levels in hippocampal tissue from adult Grin2b +/+ and Grin2b ⁺ /Δ mice. The x-axis shows log₂ fold-change (negative: downregulated; positive: upregulated), and the y-axis shows –log₁₀ p-values. The horizontal change of color marks the p = 0.05 significance threshold, and vertical dashed lines indicate a two-fold change. Proteins significantly downregulated in Grin2b ⁺ /Δ mice are shown in blue; significantly upregulated proteins are shown in yellow; non-significant changes are in gray. Statistical significance was determined using a two-sample test (see Methods). (B) Protein abundance levels of GluN1, GluN2A, GluN2B-no-CTD, and GluN2B-CTD in Grin2b +/+ ( 2b +/+ ), Grin2b ⁺ /Δ ( 2b ⁺ /Δ ), and Grin2b ⁺ /L825V ( 2b ⁺ /LV ) mice, normalized to the wild-type Grin2b +/+ reference. Data are presented as mean ± SEM; asterisks (*) indicate statistically significant differences (one-way ANOVA followed by the Dunnett method). (C) Relative CN calculated as the CN of GluN1, GluN2A, and GluN2B subunits in Grin2b +/+ , Grin2b ⁺ /Δ , and Grin2b ⁺ /L825V mice, normalized to the CN of GluN1 subunit in Grin2b +/+ mice. Arrows indicate the reference GluN subunit levels in Grin2b +/+ mice. Data are presented as mean ± SEM; asterisks (*) indicate significant differences in CN between genotypes (one-way ANOVA followed by the Dunnett method). (D) Estimated relative abundance of GluN1/GluN2B diheteromers (1-1-2B-2B), GluN1/GluN2A/GluN2B triheteromers (1-1-2A-2B), and GluN1/GluN2A diheteromers (1-1-2A-2A) in Grin2b +/+ , Grin2b ⁺ /Δ , and Grin2b ⁺ /L825V mice, based on the CN data in (C) and calculation methods described Methods. Dotted lines indicate total NMDAR content.

Article Snippet: At DIV 14, surface GluN2A and GluN2B subunits were labeled in primary hippocampal neurons from Grin2b +/+ or Grin2b +/Δ mice by live staining with a rabbit anti-GluN2A antibody (1:250; AGC-002; Alomone) or a rabbit anti-GluN2B (1:250; AGC-003; Alomone), both targeting extracellular epitopes.

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Phenotype of mice carrying an NMDA receptor GluN2B protein-truncating variant associated with intellectual disability

doi: 10.1101/2025.10.02.680004

Figure Lengend Snippet: (A) Left: Representative immunofluorescence images showing surface and intracellular expression of GluN2B-GFP subunits in HEK293T cells co-transfected with GluN1 and either wild-type (2B-GFP) or mutant (2B Δ -GFP) constructs. DAPI staining was used as a cell viability marker. Right: Quantification of the normalized surface/intracellular fluorescence ratio in HEK293T cells ( n ≥ 100 cells per group, four independent experiments). (B) Left: Representative images of surface and intracellular expression of GluN2B-GFP or GluN2B Δ -GFP in HEK293T cells also co-transfected with GluN1 and GluN2A (2A). Right: Quantification of the normalized surface/intracellular fluorescence ratio ( n ≥ 180 cells per group, four independent experiments). (C) Left: Representative fluorescence images showing surface and intracellular localization of GluN2A-GFP (2A-GFP) in HEK293T cells also co-transfected with GluN1 and either GluN2B (2B) or GluN2B Δ (2B Δ ). Right: Quantification of the normalized surface/intracellular fluorescence ratio for GluN2A-GFP ( n ≥ 100 cells per group, four independent experiments). (D) Left: Representative images of surface and intracellular immunostaining of GluN2B-GFP and GluN2B Δ -GFP in hippocampal neurons from Grin2b +/+ mice. Yellow outlines indicate neuronal somata used for quantification. Right: Normalized surface/intracellular fluorescence ratios for GluN2B-GFP and GluN2B Δ -GFP in the somata of hippocampal neurons ( n ≥ 20 cells per group, four independent experiments). Data are shown as mean ± SEM. Asterisks (*) indicate statistically significant differences ( p ≤ 0.05, Student’s t-test).

Article Snippet: At DIV 14, surface GluN2A and GluN2B subunits were labeled in primary hippocampal neurons from Grin2b +/+ or Grin2b +/Δ mice by live staining with a rabbit anti-GluN2A antibody (1:250; AGC-002; Alomone) or a rabbit anti-GluN2B (1:250; AGC-003; Alomone), both targeting extracellular epitopes.

Techniques: Immunofluorescence, Expressing, Transfection, Mutagenesis, Construct, Staining, Marker, Fluorescence, Immunostaining

Journal: bioRxiv

Article Title: Phenotype of mice carrying an NMDA receptor GluN2B protein-truncating variant associated with intellectual disability

doi: 10.1101/2025.10.02.680004

Figure Lengend Snippet: (A) Primary hippocampal microisland cultures stained with β3-tubulin. Representative images show neuronal morphology at 7 DIV (left) and 21 DIV (right). Insets display the same microislands at a lower magnification, with neurons labeled by β3-tubulin (red), astrocytes by glial fibrillary acidic protein (GFAP) (green), and nuclei by DAPI (blue). Scatter plots show the current density distribution in individual hippocampal neurons by DIV. Currents were induced by 100 µM kainate (B) or 100 µM NMDA in the presence of 10 µM glycine (C). Insets show representative whole-cell responses evoked by kainate or NMDA in neurons prepared from Grin2b +/+ (black) or Grin2b +/Δ (red) animals and cultured for 21 DIV. (D) The scatter plot shows the distribution of ifenprodil (3 µM) inhibition of NMDA-evoked currents recorded in individual neurons by DIV. Inset shows the effect of 3 µM ifenprodil on responses to 100 μM NMDA in neurons prepared from Grin2b +/+ (black) or Grin2b +/Δ (red) animals cultured for 21 DIV. Blue lines indicate the expected sum of voltage-dependent and voltage-independent inhibition of diheteromeric GluN1/GluN2B (1-1-2B-2B; 85.3%), and GluN1/GluN2A (1-1-2A-2A; 7.9%), and triheteromeric GluN1/GluN2A/GluN2B (1-1-2A-2B; 31.7%) receptors by 3 µM ifenprodil at -60 mV (with IC 50 values and Hill slopes from ) Asterisks (*) Indicate significant differences with respect to: Gen, genotype; Age, age (DIV); Int, genotype × age interaction. Data were power transformed and tested using ANOVA, followed by pairwise comparisons (LSD method).

Article Snippet: At DIV 14, surface GluN2A and GluN2B subunits were labeled in primary hippocampal neurons from Grin2b +/+ or Grin2b +/Δ mice by live staining with a rabbit anti-GluN2A antibody (1:250; AGC-002; Alomone) or a rabbit anti-GluN2B (1:250; AGC-003; Alomone), both targeting extracellular epitopes.

Techniques: Staining, Labeling, Cell Culture, Inhibition, Transformation Assay

Journal: bioRxiv

Article Title: Phenotype of mice carrying an NMDA receptor GluN2B protein-truncating variant associated with intellectual disability

doi: 10.1101/2025.10.02.680004

Figure Lengend Snippet: (A–B) Scatter plots show the distribution of peak current densities for AMPAR-eEPSCs (A) and NMDAR-eEPSCs (B) recorded from individual Grin2b +/+ (gray) and Grin2b ⁺ /Δ (red) neurons, plotted as a function of DIV. Insets: representative traces from Grin2b +/+ (black) and Grin2b ⁺ /Δ (red) neurons at 8 DIV. (C) Weighted deactivation time constants of NMDAR-eEPSCs plotted by DIV. Inset: scaled NMDAR-eEPSCs from Grin2b +/+ (black) and Grin2b ⁺ /Δ (red) neurons at 8 DIV. (D) Ifenprodil (3 µM) inhibition of NMDAR-eEPSC peak amplitudes plotted by DIV. (C) Weighted deactivation time constants of NMDAR-eEPSCs recorded in the presence of ifenprodil (3 µM) and plotted by DIV. Inset: example traces from Grin2b +/+ (black) and Grin2b ⁺ /Δ (red) neurons recorded at 15 DIV, showing the effect of ifenprodil (gray). The green lines in C, E, and D indicate mean weighted deactivation time constants of NMDAR-eEPSCs recorded from mouse hippocampal pyramidal neurons at -60 mV (C), mean ifenprodil (3 µM) inhibition of NMDAR-eEPSCs at +40 mV(D), and mean weighted deactivation time constants of NMDAR-eEPSCs recorded in the presence of ifenprodil (3 µM) after conditional knockout of GluN2A (Δ2A) or GluN2B (Δ2B) subunits . Asterisks (*) indicate significant differences for: Gen, genotype; Age, age (DIV); Int, genotype × age interaction. Data were power transformed and analyzed by ANOVA followed by pairwise comparisons (LSD method).

Article Snippet: At DIV 14, surface GluN2A and GluN2B subunits were labeled in primary hippocampal neurons from Grin2b +/+ or Grin2b +/Δ mice by live staining with a rabbit anti-GluN2A antibody (1:250; AGC-002; Alomone) or a rabbit anti-GluN2B (1:250; AGC-003; Alomone), both targeting extracellular epitopes.

Techniques: Inhibition, Knock-Out, Transformation Assay

Journal: Frontiers in Endocrinology

Article Title: Dorzagliatin shows potential in preventing cognitive impairment in diabetes: evidence from Mendelian randomization analysis and animal study

doi: 10.3389/fendo.2025.1755359

Figure Lengend Snippet: Dorzagliatin prevented diabetes-induced downregulation of synaptic proteins in Goto Kakizaki rats. (A) Western blot analysis of selected glutamate receptors and postsynaptic density protein 95 (PSD-95) in hippocampus of each group. GAPDH was used as an internal control. (B–D) Statistics of GluN1 (B) , GluN2A (C) and PSD-95 (D) protein levels in the hippocampus of T2D (Goto Kakizaki-vehicle) and control (Wistar-vehicle). (E) Western blot analysis of selected glutamate receptors and postsynaptic density protein 95 (PSD-95) in the hippocampus of Goto Kakizaki-vehicle and Goto Kakizaki-dorzagliatin rats. (F–H) Statistics of GluN1 (F) , GluN2A (G) and PSD-95 (H) protein levels in the hippocampus of Goto Kakizaki-vehicle group and Goto Kakizaki-dorzagliatin group. Data are expressed as mean ± SEM. Student’s t test, two tailed. *P < 0.05, **P < 0.01. (n=4 per group). hippo, hippocampus; dorza, dorzagliatin.

Article Snippet: The membranes were blocked with 1×TBST and 5% BSA (tank blotting) or 5% skim milk (Semi-dry blotting) for 1 hour at room temperature and then incubated overnight at 4°C with following primary antibodies, respectively: mouse monoclonal anti-GluN1 antibody (Millipore, Cat. No: 05-432); rabbit polyclonal anti-GluN2A antibody (NOVUS, Cat. No: NB300-105); rabbit monoclonal anti-GLUT1 antibody (Abcam, Cat. No: ab115730); rabbit polyclonal anti-GLUT3 antibody (Bioss, Cat. No: bs-1207R); rabbit polyclonal anti-IR antibody (Abcam, Cat. No: ab137747); rabbit monoclonal anti-PSD95 antibody (Abcam, Cat. No: ab238135).

Techniques: Western Blot, Control, Two Tailed Test