Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: The D1R–NMDAR interaction bidirectionally regulates the surface distribution and dynamics of D1R and NMDAR. (A) Immunostaining of surface D1R-CFP (green) and GluN1 subunit (red) in hippocampal neurons. The yellow arrow shows overlay. (B) Immunostaining of surface D1R-CFP in control or after D1/5R agonist, TAT-t2, or TAT-[N2A15] application. (Scale bar, 250 nm.) (C) Normalized measures of D1R-CFP clusters intensity in control (n = 32 neuronal fields), D1/5R agonist-treated (n = 24 neuronal fields; *P < 0.05 compared with control), TAT-NSt2–treated (non-sense of TAT-t2, n = 19 neuronal fields), TAT-t2–treated (n = 21 neuronal fields; **P < 0.01 compared with TAT-NSt2), TAT-NSt3–treated (non-sense of TAT-t3, n = 11 neuronal fields), TAT-t3–treated (n = 12 neuronal fields; P > 0.05 compared with TAT-NSt3), TAT-[NS15]–treated (n = 27 neuronal fields; P > 0.05), or TAT-[N2A15]–treated (n = 21 neuronal fields, *P < 0.05 compared with TAT-[NS15]) conditions. (D) Representative trajectories (1,000 frames, 20-Hz acquisition rate) of surface single D1R-CFP (Left) (green) (scale bar, 400 nm) and GluN1-NMDAR (Right) (blue) (scale bar, 300 nm) in the absence and presence of either D1/5R agonist (10 µM, 15 min) or TAT-t2 (10 µM, 15 min). Bold dotted line, perisynaptic area; thin dotted line, PSD area. (E) Plot of the MSD of surface D1R-CFP (Upper) (green) and GluN1-NMDAR (Lower) (blue) versus time in presence of TAT-NS or TAT-t2 peptides (10 µM, 15 min). The SEM is included for each data point (D1R: TAT-NS, n = 986 trajectories, and TAT-t2, n = 1,326; GluN1-NMDAR: TAT-NS, n = 198, and TAT-t2, n = 134). (F and G) Representative surface distributions of single D1R-CFP (green) (F) and GluN1-NMDAR (blue) (G) in the synaptic area (PSD + perisynaptic area) in control, D1/5R agonist, and TAT-t2 conditions. Each dot represents the detection of a single receptor during a frame. Comparisons of the time spent in the synaptic area (dwell time) by single D1R-CFP (control, n = 173 trajectories; D1/5R agonist, n = 142, **P < 0.01; TAT-t2, n = 752, *P < 0.05) (F) and GluN1-NMDAR (control, n = 189 trajectories; D1/5R agonist, n = 157, *P < 0.05; TAT-t2, n = 134, **P < 0.01) (G) and the synaptic fraction of detected single D1R-CFP (control, n = 14 neuronal fields; D1/5R agonist, n = 19, **P < 0.01; D1/5R agonist in the presence of dynasore, n = 47, **P < 0.01; TAT-t2, n = 15, ***P < 0.001) (F), D5R-CFP (n = 16, P > 0.05) (F), and GluN1-NMDAR (control, n = 11; D1/5R agonist, n = 15, *P < 0.05; TAT-t2, n = 14, *P < 0.05) (G). Dyn., dynasore; D1/5 ago., D1/5 receptor agonist SKF-38393.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

doi: 10.1073/pnas.1310145110

Figure Lengend Snippet: D1R activation or D1R/GluN1-NMDAR interaction blockade increases synaptic NMDAR content and favors AMPAR synaptic long-term potentiation. (A) (Left) Excitatory postsynaptic current traces recorded at −70 mV and +40 mV from a representative hippocampal CA1 pyramidal cell, before and 10 min after exposure to D1/5R agonist. (Right) Relative change over time of the AMPA/NMDA ratio at CA1 synapses in the absence or presence of D1/5R agonist (n = 13, *P < 0.05 10 min after agonist) and in the absence or presence of vehicle (n = 7, P > 0.05). (B) Surface imaging of GluN1-SEP in neurons incubated with either TAT-NS or TAT-t2 (10 µM). (Scale bar, 5 µm.) (Right) Average value of GluN1-SEP content in the synaptic area after TAT-NS or TAT-t2 application (n = 8 neurons per group, **P < 0.01). (C) Dendritic fragment of a hippocampal neuron expressing Homer 1c-DsRed (Upper) and GluA1-SEP (Lower). SEP only fluoresces at neutral pH when receptors are inserted at the plasma membrane. Ten minutes after chemical LTP induction (cLTP), the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. (Insets) High magnification of a synaptic GluA1-SEP cluster. (Scale bar, 2 µm.) (D) Comparison of the synaptic GluA1-SEP fluorescence intensity before and after cLTP with prior TAT-NS (n = 198 synapses, *P < 0.05) or TAT-t2 (n = 215 synapses, *P < 0.05) (TAT-NS versus TAT-t2; *P < 0.05) application. (E) Schematic model of the D1R–NMDAR surface interplay in hippocampal neurons. D1Rs are highly diffusive at the neuronal surface and are dynamically retained in clusters in the vicinity of glutamate synapses where they interact with NMDAR. Dopamine release disrupts this interaction and favors the lateral redistribution of both receptors: D1Rs freely explore extrasynaptic areas, whereas NMDARs laterally reach the PSD where they impact on the long-term plasticity of glutamate synapses.

Article Snippet: For single-nanoparticle tracking, QD 655 coupled to goat anti-rabbit F(ab′) 2 or anti-mouse IgG (Invitrogen) was incubated (1:10,000, 10 min) onto neurons previously exposed for 10 min to either mouse monoclonal anti-GFP (1 µg; Invitrogen), rabbit polyclonal anti-D1R (1 µg; Lifespan Biosciences), mouse monoclonal anti-GluA2:00 AMPAR subunit (1 µg; Millipore), or rabbit polyclonal anti–GluN1-NMDAR subunit (1 µg; Alomone Laboratories) antibodies.

Techniques: Activation Assay, Imaging, Incubation, Expressing, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Protective effects of Gryllus bimaculatus (Gb) extract on abnormal expression levels of glutamatergic and GABAergic synaptic proteins in the valproic acid (VPA)-induced autism spectrum disorder (ASD) mouse brain tissues. Immunoblot analyses for GRM5, vGluT1, NMDA R1, GABA R1α, and VGAT proteins were performed on prefrontal cortex (PFC) tissue lysates collected at embryonic day 15 (E15) (A) , postnatal day 3 (P3) (B) , and P40 (C) from mice subjected to various treatment combinations. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Expressing, Western Blot, Saline, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Regulatory effects of Gryllus bimaculatus (Gb) extract on excitatory and inhibitory neuronal activity in primary cortical neurons from valproic acid (VPA)-treated embryonic mice. (A) Schematic representation of primary cortical neuron cultures derived from embryonic mouse brains. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (B,D) Immunoblot analyses of NMDA R1, vGluT1, GRM5, GABA R1α, VGAT, NLGN3, NRXN1, and Tuj-1 in cultured primary cortical neuron lysates. Equal amounts of protein were loaded per lane, with β-tubulin used as a loading control. The bars represent fold-changes in the densitometric values of individual protein bands relative to the corresponding β-tubulin band densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant). (C) Confocal microscopy images of cortical neurons from various experimental groups. Cells were cultured for 7 days, fixed, and subsequently immunostained for vGluT1 (red), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm.

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Activity Assay, Derivative Assay, Saline, Western Blot, Cell Culture, Control, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Crucial role of astrocytes in excitatory and inhibitory (E/I) neurotransporter activities in Gryllus bimaculatus (Gb) extract-treated mixed cultures from valproic acid (VPA)-treated mouse brain. (A) Schematic representation of three different types of mixed culture systems derived from embryonic and postnatal mouse brains: Type 1, astrocytes from each treatment group combined with neurons from untreated mice; Type 2, astrocytes from untreated mice combined with neurons from each treatment group; Type 3, astrocytes and neurons both derived from the same treatment group. Astrocytes from postnatal day 3 mouse brains were seeded for 7 days, followed by the addition of cortical neurons from embryonic day 15 mouse brains onto astrocytes monolayers for an additional 7 days. (B–D) Confocal microscopy images of the different types of mixed cultures. Cells were fixed and immunostained for Tuj-1 (green) and GFAP (purple), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (E) Western blots analysis of type III mixed culture. Cell lysates were immunoblotted for Tuj-1, GFAP, synaptophysin, NMDA receptor 1 (NMDA R1), GABA receptor 1α (GABA R1α), EAAT1, and EAAT2. Equal amounts of protein were loaded per each lane, with β-actin serving as the loading control. Bars represent fold-changes in the densitometric values of the bands relative to the corresponding β-actin densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Derivative Assay, Confocal Microscopy, Saline, Western Blot, Control

Journal: bioRxiv

Article Title: Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

doi: 10.1101/2024.11.21.624499

Figure Lengend Snippet: Lactate-induced NMDAR potentiation in HEK cells hinges on the expression of CaMKII and its interaction with the GluN2B subunit. (A) Representative isolated I NMDAR traces recorded from CaMKII-expressing HEK cells transiently transfected with expressing constructs encoding the NMDAR subunits GluN1 and Glu2B. Currents were evoked by puffs of glutamate/glycine at holding voltages ranging from +60 mV to -20 mM (in 20 mV decrement steps), recorded at 30-sec intervals during baseline (control), after 5-6 min in the presence of 10 mM lactate, during the washout period, or in the presence of 50 µM AP5. (B) Lactate stimulates peak I NMDAR in HEK cells expressing functional NMDAR (GluN1::GluN2B) in CaMKIIα-expressing HEK cells (right panels) but not in cells lacking CaMKIIα (WT, left panels). This effect is noticeable at holding potentials above 20 mV. In contrast, lactate prolongs decay times in both types of HEK cells, independent of CaMKIIα expression (bottom panels). (C) Summary bar charts of I NMDAR amplitudes (top) and decay times (bottom) ± SEM in response to puffs of co-agonists at a holding potential of +30 mV, in the presence of lactate or pyruvate in NMDAR-expressing HEK cells with or without CaMKIIα expression. (D) Time-course of peak I NMDAR in the absence and presence of 10 mM lactate for CaMKIIα-expressing HEK cells transfected with GluN1 and either WT or mutant GluN2B subunits. The two GluN2B variants (L1298/R1300Q and R1300Q/S1203D) are known to disrupt the interaction between GluN2B and CaMKIIα. (E, F) Quantitative summaries of the I NMDAR amplitudes (E) and decay times (F). Bar charts are expressed as percentages of averaged baseline values ± SEM. Statistical significance was determined as described in .

Article Snippet: Cells were incubated overnight at 4°C with mouse anti-PSD-95 (1:1000, Abcam ab2723) and rabbit anti-GluN2B (1:1000, Cell Signaling Technology 14544) antibodies in 5% BSA in PBS.

Techniques: Expressing, Isolation, Transfection, Construct, Control, Functional Assay, Mutagenesis

Journal: bioRxiv

Article Title: Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

doi: 10.1101/2024.11.21.624499

Figure Lengend Snippet: Lactate potentiation of NMDAR responses depends on two redox-sensitive cysteine-rich sequences in the intrinsically disordered CTD of GluN2B subunit (A, left panels) Representative traces of I NMDAR evoked by puffs of glutamate/glycine at the soma, recorded in cultured neurons at a holding potential of +30 mV. Traces during the baseline period are marked with a white circle, and those 5 and 8 minutes after the onset of the lactate treatment with a colored circle. Top example traces include 4 mM NADH in the intracellular patch pipette solution, while bottom traces do not. The representative traces with lactate were recorded 15 and 18 minutes after going whole-cell. (A, right panel) The time- course of I NMDAR amplitudes induced by co-agonists is shown before, during, and after lactate exposure, with (purple dots) and without (dashed line) intracellular NADH. The dashed line illustrates the time-course of I NMDAR amplitudes without NADH, as shown in . (B, left panels) Representative I NMDAR traces evoked by brief applications of NMDAR co-agonists were recorded in cultured neurons at a holding potential of +30 mV with a patch pipette solution containing the reductant DTT (top) or the oxidizer DTNB (bottom) during treatment with lactate (orange circle) or the baseline control period (white circle). (B, right panel) Bar chart summarizing the peak I NMDAR responses ± SEM in neurons exposed to lactate (10mM) with or without intracellular infusion of reductants (DTT, 1 mM and βME, 300 µM) or the DNTB (200 µM). (C) Prediction of intrinsic disorder by residue position in mouse GluN2B in reducing (plus, dark purple line) or oxidizing/normal (minus, red line) conditions. Redox- sensitive stretches are identified by the divergence of the red and dark purple lines across the disorder score threshold of 0.5. These regions are highlighted in light purple and marked by dashed rectangles that form areas highlighted in light purple delineated by dashed rectangles. The beige background indicates the C-terminal domain (CTD) of the protein. (D, top) Representative I NMDAR traces evoked by brief applications of NMDAR co-agonists, recorded at a holding potential of +30 mV in HEK cells in the presence of lactate (colored circles) or during the baseline control period (white circle). (D) Quantitative summaries of the effect of 10 mM lactate (expressed as percentage of baseline) on I NMDAR amplitudes in CaMKIIα- expressing HEK cells transfected with GluN1 and WT Glu2B (WT), C946-C954S mutant GluN2B (Δredox 1-CTD GluN2B), or C1215S-C1218S-C1239-C1242S-C1245S GluN2B (Δredox 2-CTD GluN2B) subunits. Summary data are expressed as percentages of averaged baseline values ± SEM. Statistical significance was determined as described in .

Article Snippet: Cells were incubated overnight at 4°C with mouse anti-PSD-95 (1:1000, Abcam ab2723) and rabbit anti-GluN2B (1:1000, Cell Signaling Technology 14544) antibodies in 5% BSA in PBS.

Techniques: Cell Culture, Transferring, Control, Residue, Expressing, Transfection, Mutagenesis

Journal: bioRxiv

Article Title: Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

doi: 10.1101/2024.11.21.624499

Figure Lengend Snippet: Lactate potentiates the binding between CaMKII and GluN2B and drives a CaMKII-dependent increase of GluN2B-PSD-95 clustering. (A) Lactate increases the association between GluN2B and CaMKII. Representative western blots showing expression of CaMKII and GluN2B in total protein extract (input), CaMKII, GluN2B, p1303-GluN2B, and PSD-95 in synaptosome extract (synaptosomes), and CaMKII and GluN2B in synaptosome extracts immunoprecipitated with GluN2B antibody (IP: GluN2B) treated or not with 10 mM of lactate (Lac) or pyruvate (Pyr). (B) Quantitative summary plot for the synaptosome-enriched proteins immunoprecipitated with a GluN2B antibody expressed as a ratio of their respective control (Ctl) ± SEM. Statistical significance: * p < 0.05, Two-Way ANOVA followed by Dunnett’s multiple comparisons test. (C) Representative images of proximity ligation assays (PLA) for GluN2B/PSD-95 (green) in cultured cortical neurons treated with pyruvate (Pyr, 10 mM) or lactate (Lac, 10 mM) in the presence or absence of the CaMKII inhibitor peptide myr-AIP-2 (1 µM) or its scrambled control (scr, 1 µM). MAP2 immunostaining (blue) was used to delineate the dendrites. Scale bar, 5 µM. (D) Quantitative summary with data expressed as the mean ± SD. PLA cluster numbers per 10 µM of dendrite. ***p<0.001, One-Way ANOVA followed by Bonferroni’s multiple comparison tests.

Article Snippet: Cells were incubated overnight at 4°C with mouse anti-PSD-95 (1:1000, Abcam ab2723) and rabbit anti-GluN2B (1:1000, Cell Signaling Technology 14544) antibodies in 5% BSA in PBS.

Techniques: Binding Assay, Western Blot, Expressing, Immunoprecipitation, Control, Ligation, Cell Culture, Immunostaining, Comparison

Journal: bioRxiv

Article Title: Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

doi: 10.1101/2024.11.21.624499

Figure Lengend Snippet: Schematic representation of the cellular mechanisms involved in the modulation of NMDAR responses and facilitation of synaptic plasticity by lactate. (A) Extracellular lactate is transported passively along its concentration gradient through the monocarboxylate transporters 2 (MCT-2) into neurons (1) where it is converted by the lactate dehydrogenase (LDH) in presence of the oxidized form of nicotinamide adenine dinucleotide (NAD + ) to pyruvate and the reduced form of nicotinamide adenine dinucleotide (NADH) (2). This metabolic process increases the NADH/NAD+ ratio, leading to a more reduced intracellular milieu. Such a change in the internal redox state, in turn, affects cysteine-rich redox-sensitive sites on the C-terminal domain of the NMDAR GluN2B subunits favoring CaMKII binding (3) and modulates the activity of ryanodine receptors (RyRs), causing, together with the activation of NMDAR, an increase in intracellular Ca 2+ levels (4). Elevated intracellular Ca 2+ levels activate CaMKII which allow its binding to NMDAR (5). Pyruvate is imported into the mitochondria and metabolized by the tricyclic acid cycle and the oxidative phosphorylation to produce ATP to sustain the energy needs of neurons (6). (B) A detailed view of the molecular interaction between NMDARs and CaMKII under the influence of lactate. Lactate-induced NADH production alters the intracellular redox environment, promoting the rearrangement of the intrinsically disordered structure of redox-sensitive cysteine-rich sites within the C-terminal domain of GluN2B. These modifications, along with the activation of CaMKII by Ca 2+ , promote the binding of CaMKII to NMDARs, stimulating the accumulation of NMDAR at synapses, illustrated by an increase in the association between GluN2B and PSD-95, ultimately enhancing synaptic NMDAR function and synaptic plasticity (C).

Article Snippet: Cells were incubated overnight at 4°C with mouse anti-PSD-95 (1:1000, Abcam ab2723) and rabbit anti-GluN2B (1:1000, Cell Signaling Technology 14544) antibodies in 5% BSA in PBS.

Techniques: Concentration Assay, Binding Assay, Activity Assay, Activation Assay

Journal: Biological psychiatry

Article Title: Type 1 Equilibrative Nucleoside Transporter Regulates Ethanol Drinking through Accumbal N -Methyl-D-Aspartate Receptor Signaling

doi: 10.1016/j.biopsych.2011.02.013

Figure Lengend Snippet: Effects of NMDAR antagonist CGP37849 in molecular signaling and ethanol drinking. (A) NMDA glutamate receptor antagonist CGP37849 (10 mg/kg, i.p.) normalized the expression of altered phosphorylated signaling molecules in the NAc of ENT1−/− mice. Representative blots and expression levels are expressed as fold change compared to ENT1+/+ mice after normalization with GAPDH. Representation of phosphoprotein: pNg (Ser36) [t(14) = 4.2, p < 0.001], pPKCγ (Thr514), pCaMKII (Thr286), pCREB (Ser133). *p < 0.05 compared to ENT1+/+ mice after normalization by GAPDH by unpaired, two-tailed t-test. (B) Normalization of PP1/PP2A activity in ENT1−/− mice after the treatment of CGP37849. n = 8 for each genotype. (C) Ethanol consumption (g/kg/day) is reduced in both genotypes with CGP37849 treatment. (D) Ethanol preference (%) is also reduced in both genotypes with CGP37849 treatment compared to saline-treated control (S). n = 16 for each genotype. All data are presented as mean ± SEM.

Article Snippet: To examine the effect of {"type":"entrez-protein","attrs":{"text":"CGP37849","term_id":"875309805","term_text":"CGP37849"}} CGP37849 (NMDA glutamate receptor-specific antagonist, TOCRIS, Ellisville, MO) on ethanol drinking, a new group of mice was given saline, 1.0, 5.0 and 10 mg/kg {"type":"entrez-protein","attrs":{"text":"CGP37849","term_id":"875309805","term_text":"CGP37849"}} CGP37849 ( i.p. ) for 4 d during the 10% ethanol drinking session.

Techniques: Expressing, Two Tailed Test, Activity Assay, Saline, Control

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Regional expression changes in GluN2A, GluN2B, and GABA A R in WT and AD mice with aging. (A) Representative immunoblots showing GluN2A, GluN2B, and GABA A R expression across the hippocampus, prefrontal cortex, cortex, midbrain, and cerebellum in young and old WT mice. GluN2A expression remained largely stable with aging, except for an increase in the prefrontal cortex. GluN2B expression decreased with age across most regions, while GABA A R expression was reduced in the hippocampus and cortex in old WT mice. (B) Representative immunoblots from young and old AD mice showing GluN2A, GluN2B, and GABA A R levels across the same regions. GluN2A expression increased with aging in nearly all regions, while GluN2B and GABA A R levels decreased broadly.

Article Snippet: GluN2B , AB_1264223 , NMDA Receptor 2B (GluN2B) Antibody #4207 , H, M, R , (Cell Signaling Technology Cat# 4207, RRID: AB_1264223) , polyclonal , rabbit.

Techniques: Expressing, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Comparative Analysis of GluN2A, GluN2B, and GABA A R Receptor Expression in Young and Old WT vs AD Mice. (A,B) Western blot analysis comparing GluN2A levels between WT and AD mice showed increased GluN2A expression in hippocampal and cortical regions of old WT mice compared to AD and a slight decrease in the prefrontal cortex of young AD mice. (C,D) No significant change region-wide between ages, except for an observed decrease in the prefrontal cortex of young AD mice. (E,F) Downregulated GABA A R expression is seen in old AD mice across all regions compared to WT. For each brain region, n = 4–5 mice per group. Bar graphs depict the mean ± SD normalized to β-actin. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: GluN2B , AB_1264223 , NMDA Receptor 2B (GluN2B) Antibody #4207 , H, M, R , (Cell Signaling Technology Cat# 4207, RRID: AB_1264223) , polyclonal , rabbit.

Techniques: Expressing, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Increased extrasynaptic localization of GluN2B subunits with disease progression in AD mice and comparative analysis of ES-GluN2B and synaptic GluN2B expression in midbrain and cortex between WT and AD mice. (A,B) Immunoblot analysis showing significant increases in ES-GluN2B levels across brain regions of AD mice. Quantification reveals a marked increase in ES-GluN2B expression in these regions, indicating a shift from synaptic to extrasynaptic localization with disease. No significant changes in the cerebellum of AD mice. (C,D) Results demonstrate significant increases in ES-GluN2B levels in old AD mice compared to old WT mice, emphasizing the disease-dependent shift toward extrasynaptic localization, which becomes more pronounced with aging in these brain regions. Bar graphs represent the mean ± SD of ES-GluN2B levels normalized to Synaptic GluN2B. For each brain region, n = 4–5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: GluN2B , AB_1264223 , NMDA Receptor 2B (GluN2B) Antibody #4207 , H, M, R , (Cell Signaling Technology Cat# 4207, RRID: AB_1264223) , polyclonal , rabbit.

Techniques: Biomarker Discovery, Expressing, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Region-specific increases in GluN2B phosphorylation and casein kinase IIα (CK2α) expression in AD brain regions. (A,B) Immunoblot and quantification of CK2α protein levels across brain regions (hippocampus, prefrontal cortex, cortex, and midbrain) of aged AD mice compared to aged WT mice. CK2α expression significantly increases with age in AD mice, whereas WT mice exhibit stable or reduced levels. n = 4–5 per group. (C,D) Western blot and quantification showing significant increases in phosphorylation of GluN2B at Ser1480 (pGluN2B) across brain regions (hippocampus, prefrontal cortex, cortex, and midbrain) of aged AD mice compared to aged WT mice. Old AD mice also showed higher pGluN2B levels than their WT counterparts in all regions. The ratio of pGluN2B to total GluN2B is markedly elevated in old AD mice, supporting increased extrasynaptic signaling activity with disease progression. n = 4–5 per group. All bar graphs depict mean ± SD of protein levels normalized to loading controls (GluN2B or β-actin). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: GluN2B , AB_1264223 , NMDA Receptor 2B (GluN2B) Antibody #4207 , H, M, R , (Cell Signaling Technology Cat# 4207, RRID: AB_1264223) , polyclonal , rabbit.

Techniques: Phospho-proteomics, Expressing, Western Blot, Activity Assay, Biomarker Discovery

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Region-specific expression of polysialylation enzymes ST8Sia2 and ST8Sia4 in AD and normal aging. (A,B) Western blot images and quantitative analysis of ST8Sia4 protein expression in the hippocampus, prefrontal cortex, cortex, and midbrain of young and aged WT and AD mice. ST8Sia4 expression significantly increased with age in WT mice but declined in aged AD mice across all regions, notably in the prefrontal cortex and cortex. Similarly, ST8Sia4 showed a significant decrease in aged AD mice compared to aged WT mice. (C,D) ST8Sia2 protein levels showed no significant regional differences. No significant changes were observed across age groups region-wide. (E) Parental IMR32 cells are unmodified. In gRNA-ST conditions, IMR32 cells are transfected with plasmid DNA that expresses dCas9-VP64 and the guide RNA targeting the ST8sia4 promoter, enabling CRISPR-mediated transcriptional activation of ST8sia4. (F) Western blot analysis revealed significantly higher GluN2B phosphorylation at Ser1480 in parental cells compared to PSA–NCAM–overexpressing cells (gRNA-ST), (G) increased PSA-NCAM expression in gRNA-ST cells, (H) with no significant difference in CKIIα levels. Bar graphs depict mean ± SD of protein levels normalized to loading controls (GluN2B or β-actin), n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: GluN2B , AB_1264223 , NMDA Receptor 2B (GluN2B) Antibody #4207 , H, M, R , (Cell Signaling Technology Cat# 4207, RRID: AB_1264223) , polyclonal , rabbit.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, CRISPR, Activation Assay, Phospho-proteomics

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Summary of NMDA-GABA receptor-mediated E/I Balance in Normal Aging and AD. The figure illustrates the shift in E/I balance during normal aging (top) and Alzheimer’s disease (bottom). In normal aging, the balance is maintained by decreased total GluN2B level and increasing GluN2A subunit expression. In contrast, AD shows a significant decrease in GluN2B expression and an upregulation of GluN2A, alongside a marked reduction in GABA A R expression, reflecting diminished inhibitory signaling and overall E/I imbalance. Additionally, increased ES-GluN2B localization further contributes to synaptic dysfunction and excitotoxicity in AD.

Article Snippet: GluN2B , AB_1264223 , NMDA Receptor 2B (GluN2B) Antibody #4207 , H, M, R , (Cell Signaling Technology Cat# 4207, RRID: AB_1264223) , polyclonal , rabbit.

Techniques: Expressing

Journal: OncoTargets and Therapy

Article Title:

Novel mTOR Inhibitor Enhances the Sensitivity of Hepatocellular Carcinoma Cells to Molecular Targeting Agents

doi: 10.2147/ott.s244474

Figure Lengend Snippet: Figure 2 The specificity of 4 on activation of mTOR. MHCC97-H cells were treated by 4, LY294002, GSK2118436 and CP690550. Then, cells were harvested for Western blot and the expression level or the phosphorylation level of P70S6K1, AKT or ERK was examined by their antibodies. β-actin was chosen as the loading control.

Article Snippet: Rabbit antibodies against human phospho-p70S6K (Thr389), p70S6K, phospho-AKT (Ser473), phospho-AKT (Thr308), AKT, phospho-ERK, ERK, phospho-rpS6 (Ser235/6), rpS6, β-actin and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).53–55 In vivo Bioactivity Assessment of the Lead Compounds The nude mice model was used to test the bioactivity of candidate compounds in vivo.

Techniques: Activation Assay, Western Blot, Expressing, Phospho-proteomics, Control

Journal: Nature Communications

Article Title: Haploinsufficiency in the ANKS1B gene encoding AIDA-1 leads to a neurodevelopmental syndrome

doi: 10.1038/s41467-019-11437-w

Figure Lengend Snippet: Analysis of the AIDA-1 interactome yields functional pathways and mechanisms of disease. a Hierarchical analysis of the most significant diseases and functions in IPA reveals the top disorders, physiological systems, and cellular processes regulated by the AIDA-1 interactome ( p -values are given as a range for the diseases and functions annotated in each category). b The top network identified using Ingenuity Pathway Analysis (IPA) revealed known interactors and novel pathways associated with AIDA-1 (network score = 49, number of focus molecules = 25). Solid lines = direct interaction, dashed lines = indirect interaction, filled arrows = activation, open arrows = translocation, dash = inhibition. c (Top) Western blot (Family EIN-1 and EIN-2) and quantitation (Family EIN-2) of NMDAR subunits GluN2A and GluN2B in iPSC-derived neurons show no changes in probands (10 μg lysate). N = 3 biological replicates. (Bottom) Sample images (GluN2B) and quantitation of GluN2A and GluN2B surface expression in neurons from proband EIN-2-1 and unaffected mother EIN-2-M reveal a significant increase in GluN2A, but no change in GluN2B. N = 3 biological replicates based on 60–99 neurons. Scale bar = 10 μm. Bar graphs show mean ± SEM, two-sided Student’s t -test, * p < 0.05, ** p < 0.01

Article Snippet: Antibodies for immunocytochemistry were Oct-4 (1:1000, Abcam), Sox-2 (1:500, Cell Signaling Tech), MAP-2 (1:1000, EnCor), GluN2B (1:500, Alomone), and GluN2A (1:500, Alomone).

Techniques: Functional Assay, Activation Assay, Translocation Assay, Inhibition, Western Blot, Quantitation Assay, Derivative Assay, Expressing