Journal: EMBO Molecular Medicine

Article Title: Impaired gephyrin G-domain trimerization and phase separation in a patient with developmental epileptic encephalopathy

doi: 10.1038/s44321-026-00474-w

Figure Lengend Snippet: ( A ) Cartoon depicting GCN4-based receptor models (Bai et al, ) and experimental design of liquid–liquid phase separation (LLPS) sedimentation assay. ( B ) Representative Coomassie stained SDS-PAGE of LLPS sedimentation assay from WT-gephyrin high oligomers with GCN4-based receptor models for GlyR, GABA A R-α1, and GABA A R-α3 showing the amount of protein present in pellet (P) and supernatant (S) fractions. ( C ) The band intensities in the pellet fraction were measured and are shown as mean ± SD from three independent experiments ( n = 3) and statistical comparisons were performed using a two-way ANOVA (F(15,22) = 25.69, p = 2.39 × 10 −10 ) with Tukey’s multiple comparisons test (p WToligomer vs WTtrimer (GlyR) = 0.000123 (***), p WToligomer vs WTtrimer (α3) = 0.000061 (****)). ( D , E ) Representative Coomassie stained SDS-PAGE of LLPS sedimentation assay from G134R-gephyrin low oligomers ( D ) and WT-gephyrin low oligomers ( E ) with GCN4-based receptor models for GlyR, GABA A R-α1 and GABA A R-α3 showing the amount of protein present in pellet (P) and supernatant (S) fractions. .

Article Snippet: Anti-vesicular GABA transporter (vGAT) , Synaptic Systems , #131003.

Techniques: Sedimentation, Staining, SDS Page

Journal: EMBO Molecular Medicine

Article Title: Impaired gephyrin G-domain trimerization and phase separation in a patient with developmental epileptic encephalopathy

doi: 10.1038/s44321-026-00474-w

Figure Lengend Snippet: ( A ) Representative Western blot analysis of lysates from gephyrin floxed/floxed murine hippocampal neurons infected with moxBFP (-) or moxBFP-P2A-Cre (+) together with either mScarlet-WT-Geph or mScarlet-G134R-Geph. ( B ) Representative confocal images of gephyrin floxed/floxed murine hippocampal neurons transfected with moxBFP-P2A-Cre and infected with either mScarlet-WT-Geph or mScarlet-G134R-Geph and immunostained against vesicular GABA transporter (vGAT, green) and GABA A R γ2 subunit (yellow). ( C ) Quantification of mScarlet-tagged gephyrin clusters was carried out using automated synapse analyses. Each data point represents synaptic cluster density (# per cell area µm −2 ) per acquired cell; n = 20 cells per condition from five independent cultures, mean ± SD are indicated. Data were analyzed by Mann–Whitney U test U = 391, p = 0.00000023 (***), effect size r = 0.820 ( D ) Representative confocal images of gephyrin floxed/floxed murine hippocampal neurons transfected with moxBFP-P2A-Cre and infected with mEGFP-WT-Geph and either mScarlet-WT-Geph or mScarlet-G134R-Geph and immunostained against vesicular GABA transporter (vGAT, yellow). ( E ) Quantification of mScarlet-tagged gephyrin clusters was carried out using automated synapse analyses. Each data point represents synaptic cluster density (# per cell area µm −2 ) per acquired cell; n = 20 cells per condition from five independent cultures, mean ± SD are indicated. Data were analyzed by one-way ANOVA (F 2,57 = 5.139, p = 0.009), Tukey’s post hoc test; p WT + WT vs. WT + G134R = 0.0176 (*); p WT + WT vs. WT + G375D = 0.0228 (*); p WT + G134R vs. WT + G375D = 0.995 (ns). .

Article Snippet: Anti-vesicular GABA transporter (vGAT) , Synaptic Systems , #131003.

Techniques: Western Blot, Infection, Transfection, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Neuroligin-2-Dependent Adhesion Defines a Molecular Checkpoint for Inhibitory Synaptic Plasticity

doi: 10.1523/JNEUROSCI.1746-25.2026

Figure Lengend Snippet: Conceptual model of neuroligin-2-dependent inhibitory synaptic plasticity in hippocampal CA1. A , Molecular mechanisms of iLTP at GABAergic synapses. Postsynaptic Ca 2+ influx, mediated by NMDA receptors and voltage-gated Ca 2+ channels, activates CaMKII signaling, which promotes the stabilization and synaptic retention of GABA A receptors via clustering with gephyrin scaffold. Concurrently, Matrix Metalloproteinase-3 (MMP-3) cleaves extracellular matrix substrates (such as chondroitin sulfate proteoglycans), thereby activating β1-integrin-containing complexes. This cascade modulates synaptic adhesion and receptor confinement to induce iLTP. Neuroligin-2, via interactions with presynaptic partners such as neurexins, orchestrates the reorganization of the synaptic adhesion apparatus and consolidates the plastic change. B , Model of heterosynaptic iLTP at somatostatin-expressing interneuron (SST IN) inputs onto CA1 pyramidal cells (CA1 PC) in the stratum oriens with emphasis on involvement of Neuroligin2-neurexin complexes. Potentiation involves the increased postsynaptic accumulation of neuroligin-2, gephyrin, and GABA A receptors at inhibitory postsynaptic densities (iPSD), independent of presynaptic modifications. This framework establishes neuroligin-2 as a central organizer coupling extracellular cues to postsynaptic scaffold remodeling during the consolidation of inhibitory synaptic strengthening.

Article Snippet: The following primary antibodies were used: mouse anti-gephyrin (Synaptic Systems, catalog #147 111; 1:800), rabbit anti-vesicular GABA transporter (vGAT; Synaptic Systems, catalog #131 002; 1:500), mouse anti-neuroligin-2 (Synaptic Systems, catalog #129 511; 1:200), and mouse anti-active form of integrin β1 (Chemicon, catalog #MAB2079Z; 1:250).

Techniques: Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Neurosteroid withdrawal disrupts GABAergic system development in human cortical organoids: implications for preterm birth

doi: 10.3389/fncel.2025.1715823

Figure Lengend Snippet: Cortical organoids as a model to study ALLO exposure and withdrawal (A–C) GABA-A receptor subunit gene expression is enriched in interneuron populations compared to principal cells. (A) The GABA-A receptor subunits present in both organoid subtypes allow GABA and neurosteroid binding (ALLO). (B) Average expression of GABA-A receptor subunits, in VCOs (ventral cortical organoids, blue) and DCOs (dorsal cortical organoids, brown) treated with DMSO, during the period of ALLO treatment (DIV28-DIV67), expressed as scaled relative abundance to GAPDH expression, and multiplied by 10,000. n = 9. Two-way ANOVAs with BH correction compared organoid subtypes and subunits. #organoid subtype effect. Multiple comparisons: * p < 0.05. (C) Transcriptional expression of GABA-A subunits in human fetal tissues between gestational weeks 13 and 17, after re-analysis of GSE156793 for cerebrum inhibitory and excitatory neurons, expressed as average of normalized read counts. (D–G) ALLO does not modulate AKR1Cs expression in cortical organoids. Transcriptional profile of ALLO-producing enzyme, in (D,E) VCOs (blue) and (F,G) DCOs (orange) treated with DMSO (dark) or ALLO (125 nM, light; from DIV28 to 67, highlighted in green). (D,F) AKR1C2 (Aldo-keto reductase family 1 member C2) and (E,G) AKR1C3 (Aldo-keto reductase family 1 member C3) at each time point (DIV0, 26, 55, 78, 95, 138). n = 3. Two-way ANOVAs with BH correction compared treatments and days.

Article Snippet: Tissue sections were rinsed in PBS-Triton 0.3% (PBS-T) then blocked in PBS-T with 10% normal donkey serum (NDS), followed by overnight incubation at 4 °C in PBS-T-10% NDS with the following primary antibodies: Beta-3 tubulin (B3TUB; 1:500, Cell Signaling, D71G9), Calretinin (CALB2; 1:1000, Millipore Sigma, AB1550), Doublecortin (DCX; 1:500, Abcam, ab113435), GABA transporter type 1 (GAT1; 1:200, AbClonal, A15099), GABA transporter type 3 (GAT3; 1:100, Santa Cruz Biotechnology, sc-376001), Glutamate decarboxylase 65–67 (GAD65-67; 1:100, Santa Cruz Biotechnology, sc-365180), Glial fibrillary acidic protein (GFAP; 1:1000, Dako, Z0334), HuC/HuD (HU; 1:500, Invitrogen, A-21271), Ki67 (MKI67; 1:500, Abcam, ab156956), Nestin (NES; 1:250, Novus Biologicals, NB100-1604), NeuN (NEUN; 1:500, Abcam, ab177487), Somatostatin (SST; 1:300, Santa Cruz Biotechnology, sc-7819), SRY-box 2 (1:500, SOX2; Millipore, AB5603), Vesicular GABA transporter (VGAT; 1:300, AbClonal, A3129), Vimentin (VIM; 1:500, Santa Cruz Biotechnology, sc-373717).

Techniques: Gene Expression, Binding Assay, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Neurosteroid withdrawal disrupts GABAergic system development in human cortical organoids: implications for preterm birth

doi: 10.3389/fncel.2025.1715823

Figure Lengend Snippet: ALLO withdrawal downregulates elements of the GABAergic system in VCOs. (A) Heatmap representing the fold change of GABA-A receptor subunits transcripts of ALLO-exposed organoids (125 nM; from DIV28 to 67) over DMSO-exposed organoids at each time point (DIV55, 78, 95, 138) in VCOs (ventral cortical organoids), ###treatment effect, ###treatment:day effect. (B) Heatmap representing the fold change of eight transcripts involved in GABAergic signaling of ALLO-exposed over DMSO-exposed VCOs at each time point (DIV55, 78, 95, 138), #treatment:day. n = 3. Two-way ANOVAs with BH correction compared treatments and days. Multiple comparisons: * p < 0.05, *** p < 0.001. (C–E) Cumulative effect of ALLO exposure and withdrawal at DIV138 on markers of GABAergic signaling. (C) Representative illustration of whole organoid staining, scale = 100 μm. (D) Representative high magnification illustrations of positive cells, scale = 10 μm, arrows. (E) Quantification in VCOs treated with DMSO (dark) or ALLO (light), normalized as percentage of DAPI+ cells. #treatment effect. VGAT, Vesicular GABA transporter; GAT1, GABA transporter 1; GAT3, GABA transporter 3. n = 5–6. Two-way ANOVAs with BH correction compared treatments and markers. Multiple comparisons: * p < 0.05.

Article Snippet: Tissue sections were rinsed in PBS-Triton 0.3% (PBS-T) then blocked in PBS-T with 10% normal donkey serum (NDS), followed by overnight incubation at 4 °C in PBS-T-10% NDS with the following primary antibodies: Beta-3 tubulin (B3TUB; 1:500, Cell Signaling, D71G9), Calretinin (CALB2; 1:1000, Millipore Sigma, AB1550), Doublecortin (DCX; 1:500, Abcam, ab113435), GABA transporter type 1 (GAT1; 1:200, AbClonal, A15099), GABA transporter type 3 (GAT3; 1:100, Santa Cruz Biotechnology, sc-376001), Glutamate decarboxylase 65–67 (GAD65-67; 1:100, Santa Cruz Biotechnology, sc-365180), Glial fibrillary acidic protein (GFAP; 1:1000, Dako, Z0334), HuC/HuD (HU; 1:500, Invitrogen, A-21271), Ki67 (MKI67; 1:500, Abcam, ab156956), Nestin (NES; 1:250, Novus Biologicals, NB100-1604), NeuN (NEUN; 1:500, Abcam, ab177487), Somatostatin (SST; 1:300, Santa Cruz Biotechnology, sc-7819), SRY-box 2 (1:500, SOX2; Millipore, AB5603), Vesicular GABA transporter (VGAT; 1:300, AbClonal, A3129), Vimentin (VIM; 1:500, Santa Cruz Biotechnology, sc-373717).

Techniques: Staining