Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Expression of GluN2A (A) and GluN2B (B) and basal extracellular levels of L‐glutamate (C) and D‐serine (D) in 4‐weeks and 8‐weeks of age S286L‐TG and wild‐type littermate. Ordinates indicate mean ± SD ( n = 6) of (A) expression levels of GluN2A relative to GAPDH in the plasma membrane fraction (B) expression levels of GluN2B relative to GAPDH in the plasma membrane fraction, (C) basal extracellular L‐glutamate level (μM) and (D) basal extracellular D‐serine level (μM) in the frontal cortex of wild‐type (gray column) and S286L‐TG (blue column). The lower‐side panels in A and B indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to 4‐weeks of age (4 W) and # p < 0.05 relative to wild‐type using two‐way ANOVA with Scheffe's post hoc test. F ‐values were in (A) expression of GluN2A ( F age [1, 20] = 46.7 [ p < 0.05], F genotype [1, 20] = 5.34 [ p < 0.05], F age*genotype [1, 20] = 1.1 [ p > 0.05]), (B) expression of GluN2B ( F age [1, 20] = 22.4 [ p < 0.05], F genotype [1, 20] = 8.3 [ p < 0.05], F age*genotype [1, 20] = 2.0 [ p > 0.05]), (C) L‐glutamate level ( F age [1, 20] = 3.2 [ p > 0.05], F genotype [1, 20] = 21.2 [ p < 0.05], F age*genotype [1, 20] = 1.9 [ p > 0.05]) and (D) D‐serine level ( F age [1, 20] = 8.4 [ p < 0.05], F genotype [1, 20] = 21.6 [ p < 0.05], F age*genotype [1, 20] = 2.8 [ p > 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Clinical Proteomics, Membrane, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Effects of chronic administration of probenecid, MK‐801, memantine, and FP802 on expression of GluN2A and GluN2B in S286L‐TG and wild‐type littermates. All rats were chronically administered by vehicle (control), probenecid (PBN: 100 mg/kg/day), MK‐801 (0.1 mg/kg/day), memantine (MEM: 10 mg/kg/day) and FP802 (40 mg/kg/day) for 2‐weeks (from 6‐weeks to 8‐weeks of age). Ordinates indicate mean ± SD ( n = 6) of expression levels of GluN2A (A1‐A4) and GluN2B (B1‐B4) relative to GAPDH in wild‐type (A1‐A2, B1‐B2) and S286L‐TG (A3‐A4, B3‐B4). The right‐side panels indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to control using one‐way ANOVA with Scheffe's post hoc test. F ‐values regarding effects of probenecid and MK‐801 on GluN2A expression in wild‐type (A1) ( F [2, 15] = 7.8 [ p < 0.05]), GluN2A in S286L (A3) ( F [2, 15] = 19.4 [ p < 0.05]), GluN2B in wild‐type (B1) ( F [2, 15] = 12.1 [ p < 0.05]) and GluN2B in S286L‐TG (B3) ( F [2, 15] = 18.2 [ p < 0.05]). F ‐values regarding effects of memantine and FP802 on GluN2A in wild‐type (A2) ( F [2, 15] = 0.4 [ p > 0.05]), GluN2A in S286L‐TG (A4) ( F [2, 15] = 7.1 [ p < 0.05]), GluN2B in wild‐type (B2) ( F [2, 15] = 0.2 [ p > 0.05]) and GluN2B in S286L‐TG (B4) ( F [2, 15] = 4.8 [ p < 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Control, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Effects of chronic combined administration of memantine with FP802 on ADSHE seizure frequency (A), sucrose preference (B), expression of GluN2A (C1) and GluN2B (C2), and basal extracellular levels of L‐glutamate (D) and D‐serine (E) in S286L‐TG and wild‐type littermate. All rats were chronically administered by vehicle (control) and combined of memantine (MEM: 10 mg/kg/day) with FP802 (40 mg/kg/day) for 2‐weeks (from 6‐weeks to 8‐weeks of age). Ordinates indicate mean ± SD ( n = 6) of (A) ADSHE seizure frequency (count h −1 ), (B) consumption of sucrose preference (%), (C1) expression levels of GluN2A relative to GAPDH, (C2) expression levels of GluN2B relative to GAPDH, (D) basal extracellular L‐glutamate level (μM) and (E) basal extracellular D‐serine level (μM). The right‐side panels in C1‐C2 indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to control and # p < 0.05 relative to wild‐type using student T ‐test or one‐way or two‐way ANOVA with Scheffe's post hoc test. F ‐values were in (B) sucrose preference: MEM ( F memantine+FP802 [1, 20] = 21.3 [ p < 0.05], F genotype [1, 20] = 5.1 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 5.3 [ p < 0.05]), (D) L‐glutamate level: ( F memantine+FP802 [1, 20] = 5.3 [ p < 0.05], F genotype [1, 20] = 15.9 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 4.8 [ p < 0.05]), (E) D‐serine level: ( F memantine+FP802 [1, 20] = 7.6 [ p < 0.05], F genotype [1, 20] = 22.4 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 10.4 [ p < 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Control, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP1 and ZIP3 localization in the DCN. A . Immunofluorescent imaging of DCN slices labeled with ZnT3 (red) and the postsynaptic markers GluN2A or CaMKII (green). B . Immunofluorescent staining of ZnT3, ZIP1 or ZIP3 (red) with the synaptic terminal marker VGLUT1 (green). C . GluN2A or CaMKII (green) immunofluorescent co-labeling with ZnT1, ZIP1 or ZIP3 (red), bottom panels show colocalization analyses, represented by Mander’s colocalization coefficient, calculated between the postsynaptic marker and each of the Zn 2+ transporters. For GluN2A colocalization, Tukey’s multiple comparisons test analysis shows a significant difference between ZnT3 and ZIP3 p-value: 0.016, as well as between ZnT3 and ZnT1 p-value: 0.031, F (1.669, 4.451) = 22.32. In case of CaMKII, Tukey’s multiple comparisons test shows significance for ZIP3 p-value: 0.009 and ZnT1 p-value: 0.016, F (3, 15) = 7.202. D . Immunofluorescent staining of PAX6 or Tbr2 (green) with ZIP1 or ZIP3 (red). The colocalization analyses for PAX6, bottom panel, shows Mander’s coefficients of the two Zn 2+ transporters (Mann Whitney test, p-value: 0.003). White arrows indicate examples of cells that show co-localization of the markers

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Imaging, Labeling, Staining, Marker, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP3 and ZnT1 are in direct contact, which enhances ZnT1 efflux rates. A. Co-immunoprecipitation assay in SH-SY5Y cells over expressing ZnT1 and ZIP3, with or without the murine subunit GluN2A (mGluN2A) or the human glutamate receptor (hGluN2A) subunit. The samples were immunoprecipitated with an antibody against ZnT1. The lysates were then separated on SDS-PAGE and subjected to immunoblotting with antibodies against ZIP3, representative of 3 repetitions. Lysis buffer shown as control. B. Co-immunoprecipitation in HEK293 cells expressing ZnT1 and ZIP3. Samples were immunoprecipitated either with an antibody against ZnT1 (left) or ZIP3 (right). Lysates were then separated on SDS-PAGE and subjected to immunoblotting with antibodies against ZIP3 or ZnT1, representative of 3 repetitions. Lysisbuffer shown as control. Note that expected MW for ZIP3 is 37kDa, identified MW likelyrepresents its dimerization and glycosylation; full blots shown in Supplementary Information. C. Representative traces of FluoZin-3 fluorescence changes in SH-SY5Y cells expressing ZnT1+ZIP3 (orange) or ZnT1+PCDNA as control (blue). Cells were perfused with 200μM Zn2+ in Ringer’s solution added with or without pyrithione (see Methods) at the indicated time. The initial Zn 2+ influx and efflux rates were monitored and compared between the two conditions. Unpaired ttest analysis, *** p-value: 0.0003, t(28)=4.131

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, SDS Page, Western Blot, Lysis, Control, Glycoproteomics, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP3- ZnT1 physical interaction in DCN cartwheel cells. A . Proximity ligation assay (PLA) performed on DCN slices using probes for GluN2A and either ZnT1, ZIP3, or ZIP1. Analysis of PLA puncta (red) was done on the molecular layer (outlined in white) to assess protein–protein interactions in postsynaptic cells of the parallel fibers. The graph (right panel) quantifies PLA puncta normalized to ZnT1-GluN2A puncta. Statistical analysis Dunnett’s multiple comparisons test, p-value: 0.0014, F (2, 11) = 11. B . Co-immunoprecipitation assay using DCN and hippocampal lysates. Protein samples were immunoprecipitated with antibodies against GluN2A, followed by SDS-PAGE separation and immunoblotting with antibodies against ZIP3 to confirm physical interactions. Note that expected MW for ZIP3 is 37 kDa, identified MW likely represents its dimerization and glycosylation; full blots shown in Supplementary Information. C . Schematic representation of Zn 2+ transporters in DCN, indicating the ZIP3-ZnT1-GluN2A complex on the cartwheel cells of the DCN

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Proximity Ligation Assay, Protein-Protein interactions, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Western Blot, Glycoproteomics

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ZIP-ZnT1 complexes mediate a local Zn 2+ -cycle regulating neuronal Zn²⁺ transport

doi: 10.1007/s00018-026-06137-w

Figure Lengend Snippet: ZIP1- ZnT1 physical interaction in the hippocampus. A . Co-immunoprecipitation assay using SH-SY5Y cells that were immunoprecipitated with ZnT1 and then exposed to ZIP1 (left panel) or ZIP3 (right panel) antibodies. Note that the right panel is taken from the gel presented in Fig. , the right lane for mGluN2A-ZIP3-ZnT1 expressing cells overlaps with that figure. Note that expected MW for ZIP3 is 37 kDa, identified MW likely represents its dimerization and glycosylation; full blots shown in Supplementary Information. B . Representative traces of fluorescent imaging of FluoZin-3 changes in SH-SY5Y cells expressing ZnT1 + ZIP1 (green) or ZnT1 + PCDNA as control (blue). Cells were perfused with 200 µM Zn 2+ in Ringer’s solution added at the indicated time, with or without pyrithione (see methods). The initial Zn 2+ influx (middle panel) and efflux (right panel) rates are shown in the bar graphs. Unpaired t-test analysis, *** p-value: 0.0001, t(23) = 5.613. C . Co-immunoprecipitation assay using DCN and hippocampal lysates. Protein samples were immunoprecipitated with antibodies against ZIP1, followed by SDS-PAGE separation and immunoblotting with antibodies against GluN2A. D . Proximity ligation assay (PLA) performed on CA3 hippocampal slices using probes for GluN2A and either ZnT1 or ZIP1. Red puncta represent the GluN2A-ZnT1 or ZIP1 interaction in the CA3 pyramidal cell layer (marked by white lines). Analysis of PLA puncta, performed with the GluN2A and either ZnT1, ZIP1 or ZIP3, on CA3 pyramidal layer to assess protein–protein interactions in postsynaptic cells. The graph (bottom panel) quantifies PLA puncta normalized to ZnT1-GluN2A puncta. Statistical analysis Dunnett’s multiple comparisons test, p-value: 0.013, F (2, 6) = 9.81. E . Schematic presentation of the Zn 2+ -cycle proteins, ZIP1-ZnT1-GluN2A, expressed on the postsynaptic CA3 pyramidal cells that are adjacent to the ZnT-3 and ZIP3 expressing mossy fiber terminals, consistent with previous work

Article Snippet: GluN2A , NeuroMab , 75,288 , 1:300.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing, Glycoproteomics, Imaging, Control, SDS Page, Western Blot, Proximity Ligation Assay, Protein-Protein interactions

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

Journal: International Journal of Biological Sciences

Article Title: Disrupted astrocyte-neuron glutamine-glutamate cycling in the medial prefrontal cortex contributes to depression-like behaviors

doi: 10.7150/ijbs.123740

Figure Lengend Snippet: Changes of phenotype in glutamine synthetase and glutamate transporter-1 (GLT-1) conditional knockout (cKO) mouse (GS - and GLT-1 - ). (A and N) Experimental scheme. The dotted and solid boxes indicate the target proteins of cKO and the expected changes, respectively. The two viruses for Cre recombinase and small guide RNA (sgRNA) were injected into the prelimbic cortex of CRISPR/CAS9-EGFP mice. The blue sequences indicate the sgRNA nucleotide sequences. (B and O) EGFP (green), GS or GLT-1 (red) signals in the prelimbic cortex. Blue signals are DAPI. Scale bars = 50 μm. (C and D, P and Q) Duration in center (C, t=3.151, df=16, p=0.006 ; P, t=2.126, df=16, p=0.049 ) and moved distance measured in open field test. (E and R) Immobile duration measured in tail suspension test (E, t=2.479, df=15, p=0.026 ; R, t=2.784, df=16, p=0.013 ). (F and S) Sucrose preference measured in sucrose preference test (F, t=3.298, df=15, p=0. 005; S, t=2.348, df=15, p=0.033 ). (G and T) Plasma corticosterone (CORT) level. (H and I, U and V) Reactive oxygen/nitrogen species level in the plasma and medial prefrontal cortex (mPFC, I, t=1.941, df=12, p=0.038 ; V, t=5.400, df=12, p<0.001 ). DCF, 2', 7'-dichlorodihydrofluorescein. (J and W) GS activity in the mPFC (J, t=3.962, df=12, p=0.002 ). (K-M, X-Z) Glutamate, glutamine, and γ-aminobutyric acid (GABA) levels in mPFC (K-M, Glu, t=4.817, df=12, p<0.001 ; Gln, t=3.053, df=12, p=0.010 ). (a-d) Expression of GluA1 and GluA2 (b, t=1.959, df=12, p=0.037 ), subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, GluN2A (c, t=3.509, df=12, p=0.002 ) and GluN2B (d, t=2.045, df=12, p=0.032 ), subunits of N -methyl-D-aspartate receptor in the mPFC of GLT-1 cKO mice. All data were represented as mean ± SEM and statistically analyzed by unpaired, two-tailed t-test ( * p<0.05 , ** p<0.01 , and *** p<0.001 ).

Article Snippet: The membrane was then blocked in 5% skim milk (BD Bioscience, Franklin Lakes, NJ, USA), followed by incubation with primary antibodies: GluA1 (MA5-27694, ThermoFisher Scientific), GluA2 (32-0300, ThermoFisher Scientific), GluN2A (sc-390094, Santa Cruz Biotechnology), GluN2B (sc-365597, Santa Cruz Biotechnology), and β-actin (MA1-140, ThermoFisher Scientific) and subsequently with goat anti-mouse IgG (H+L) secondary antibody (31430, ThermoFisher Scientific).

Techniques: Knock-Out, Injection, CRISPR, Suspension, Clinical Proteomics, Activity Assay, Expressing, Two Tailed Test