Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old) incubated with BDNF (50 ng/mL). Synaptoneurosomes were immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1, PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( A ) were analyzed for the GluN2B integrated density ( B ) and GluN2B mean gray value ( C ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, in at least three independent experiments performed in different preparations. ****p < 0.0001, by Kruskal-Wallis’s test and Dunn’s multiple comparisons test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation, Immunofluorescence

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) that were stimulated with BDNF (50 ng/ml for 10 or 30 min), as indicated. Neurons were then live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A) , were analyzed for the total number ( B ), intensity ( C ), and area ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per density of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the means of the control and are the means ± SEM of 43-45 cells per condition, from at least three independent experiments performed in different preparations. *p < 0.05, **p < 0.01 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Control, Analysis, Immunofluorescence

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) pre-incubated with GÖ 6983 (100 nM) or vehicle (DMSO; 1:1000 dilution for 40 min) and then either maintained under the same conditions or stimulated with BDNF (50 ng/ml for 30 min), as indicated. Neurons were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A ) were analyzed for the total number ( B ), area ( C ), and intensity ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per µm of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the mean of the DMSO control and are the means ± SEM of 28 - 30 cells per condition, in at least three independent experiments performed in different preparations. ***p < 0.001, ****p < 0.0001 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation, Control, Analysis, Immunofluorescence

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Experimental design for the lithium-pilocarpine model of Status Epilepticus. ( B ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old, treated with saline, saline and ANA-12, Pilocarpine or Pilocarpine and ANA-12). Synaptoneurosomes were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1 and PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( B ) were analyzed for the GluN2B integrated density ( C ) and GluN2B mean gray value ( D ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, from at least four animals for each experimental condition. ****p < 0.0001, **p<0.01 as determined by Kruskal Wallis’s test and Dunn’s multiple comparisons test. Representative western blot and analysis ( E-G ). Proteins were extracted from synaptoneurosomes prepared from the same animals used in the immunocytochemistry experiments. For the immunoblot, antibodies against pTrkB, total TrKB and β-Tubulin were used. In this analysis, pTrkB levels were normalized to total TrkB levels and total TrkB levels were normalized to β-tubulin. Data are means ± SEM of at least four animals for each experimental condition. *p < 0.05, by one-way analysis of variance (ANOVA) followed by Dunnett’s post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Saline, Western Blot, Analysis, Immunocytochemistry, Immunofluorescence

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . ( left ) Representative traces of the evoked fEPSP from control (black), control+AP5 [50µM] (green), nIH (red) and nIH +AP5 [50µM] (blue) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in control, control+AP5, nIH and nIH+AP5. ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in control slices vs control+AP5 [50 µM]. B . ( top ) Representative image for GluN1. (bottom) The graph shows GluN1 did not change protein expression after nIH exposure compared with control. (two tailed t-test, t=0.63; df=4.1; P=0.55). C . (top) Western blot picture for GluN2A. (bottom) Comparison for both conditions show GluN2A decreased the protein content levels after nIH. (two tailed t-test, t=4.017; df=6.43; P=0.006). D . ( top ) Representative immunoblot image for GluN2B. (bottom) Comparison of both conditions show increased GluN2B levels after nIH (two tailed t-test, t=3.43; df=5.78; P=0.014). E . GluN2B/GluN2A comparison ratio. F . ( left ) Representative traces of the evoked fEPSP control +TCN [5 µM] (orange), control + ifenprodil [5 µM] (cyan) and control + both drugs (olive) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS and ( right ) Comparison of fEPSP slope represented as percent change from baseline at 60 min after TBS (one way ANOVA, F (3,20) =43.52; P<0.001). Black dashed line represents the mean slope of the fEPSP 60 min following TBS in control slices from . G . ( left ) Representative traces of the evoked fEPSP from nIH+TCN [5 µM] (dark yellow) and nIH +ifenprodil [5 µM] (light blue) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time and relative to slope before TBS ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS (two tailed t-test, t=12.46; df=4.59; P=0.001). Red dashed line represents the mean slope of the fEPSP 60 min following TBS in nIH slices from . Scale bars for A, F and G: 10 msec x 0.2 mV. The box-plot parameters indicate mean ± S.E. The analysis was performed for A to E using unpaired two-tailed t-test with Welch’s correction and for F and G the analysis was performed using one-way ANOVA followed by Bonferroni post hoc. **P<0.01, ***P<0.001 and ****P<0.0001).

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Expressing, Two Tailed Test, Western Blot, Comparison

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . Malondialdehyde (MDA) content was measured in hippocampal homogenates from control, nIH Saline and 10-Mn. (one way ANOVA, F (2,12) =11.53; P=0.0016). B . (top) Representative blot of nuclear HIF1a performed from control, nIH Saline and IH 10-Mn mice. (bottom) Quantification of HIF1a expression (one way ANOVA, F (2,12) =5.76; P=0.017). C . (top) Immunoblot of NOX2. (bottom) Significant differences was found in hippocampal homogenate from nIH Saline compared to control and nIH Mn (one way ANOVA, F (2,15) =9.26; P=0.0024). D . (top) Representative image of NOX4. ( bottom ) Comparison of NOX4 expression between control, nIH Saline and nIH Mn . (one way ANOVA, F (2,9) =11.46; P=0.0034). E . ( left ) Representative blot of GluN2A from control, nIH Saline and nIH Mn mice. ( right ). Quantification of GluN2A expression. (one way ANOVA, F (2,15) =6.63; P=0.0086). F . ( left ) Immunoblot of GluN2B. ( right ) Significant differences were found in hippocampal homogenate from nIH Saline vs control and nIH Mn . (one way ANOVA, F (2,12) =6.88; P=0.01). G . (top) Representative traces of evoked fEPSP from nIH Mn (purple) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in nIH Mn . ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in nIH vs nIH Mn slices. (two tailed t-test, t=3.41; df=10.61; P=0.0061). Dashed lines represent the mean slope of the fEPSP 60 min following TBS in control (black dashed line) and nIH (red dashed line) slices from . H . (top) Representative traces of the evoked fEPSP from nIH Mn in presence the ifenprodil [5 µM] (light purple) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in nIH Mn in presence the ifenprodil. ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in IH+ifenprodil vs nIH Mn in presence of ifenprodil. (two tailed t-test, t=3.99; df=6.99; P=0.019). Blue dashed line represents the mean slope of the fEPSP 60 min following TBS in ifenprodil treated nIH slices from . For G and H, Scale bars 10 msec x 0.2 mV. The analysis was performed for A to F using one-way ANOVA followed by Bonferroni post hoc and for G and F, the analysis was performed using unpaired two-tailed t-test with Welch’s correction. *P<0.05, **P<0.01, and ***P<0.001.

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Saline, Expressing, Western Blot, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . (top) Representative traces of the evoked fEPSP from adult control (black) and adult mice were exposed to neonatal IH (red) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to baseline prior to TBS. ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min following TBS in adult control vs Adult nIH (two tailed t-test, t=5.70; df=9.04; P=0.003). B . (top) Representative trace of the evoked from control+ TCN-213 [5 µM] and control + ifenprodil [5 µM] in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to baseline prior to TBS. ( right bottom ) fEPSP comparison at 60 min following TBS in control +TCN and control+ ifenprodil (two tailed t-test, t=7.43; df=4.65; P=0.0009). Black dashed line represents the mean slope of the fEPSP 60 min following TBS in control slices from . C . (top) Representative trace of the evoked response from Adult nIH + TCN-213 [5 µM] and Adult nIH + ifenprodil [5 µM] in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to baseline prior to TBS. ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min following TBS (two tailed t-test, t=4.72; df=4.06; P=0.0064). Red dashed line represents the mean slope of the fEPSP after 60 min following TBS in control slices from . D . (top) Representative traces of the evoked fEPSP from adult mice was exposure to neonatal IH+10 days of MnTMPyP (Adult nIH-Mn ) and adult mice receive MnTMPyP after exposure to neonatal IH (Adult REC-Mn ) in baseline conditions before TBS (1) and after TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to slope before TBS in Adult nIH-Mn vs Adult REC-Mn ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min after TBS Adult nIH-Mn vs Adult REC-Mn (two tailed t-test, t=3.79; df=8.16; P=0.0051). E. Representative image of GluN1. ( bottom ) Quantification shows GluN1 protein expression is not changed in Adult nIH , Adult nIH-Mn or 10+Mn exposures compared to control (one way ANOVA, F (3,16)=1.14 ; P=0.93; N=5). F . (top) Representative blot of GluN2A performed from adult mice unexposed, Adult nIH , Adult nIH-Mn or Adult REC-Mn . ( bottom ). Quantification of GluN2A expression from adult control, Adult nIH , Adult nIH-Mn or 10+Mn. (one way ANOVA, F (3,12)=8.31 ; P=0.0029, N=4). G . (top) Immunoblot of GluN2B. ( bottom ) Significant differences were found in hippocampal homogenates from adult control, Adult nIH , Adult nIH-Mn or Adult REC-Mn (one way ANOVA, F (3,12)=4.91 ; P=0.018, N=4). The box plot parameters indicate mean ± S.E. The analysis was performed for A-D using unpaired two-tailed t-test with Welch’s correction. The analysis was performed for E-G using one-way ANOVA followed by Bonferroni post hoc. *P=0.05, **P=0.01, ***P=0.001 and N.S= no significant. Scale bars for A,B, F and G= 10 msec x 0.2 mV

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Two Tailed Test, Comparison, Expressing, Western Blot

Journal: Neurobiology of disease

Article Title: Ethanol exposure alters Alzheimer’s-related pathology, behavior, and metabolism in APP/PS1 mice

doi: 10.1016/j.nbd.2022.105967

Figure Lengend Snippet: Chronic moderate drinking differentially alters NMDA and GABAA receptors in the cortex and hippocampus of APP/PS1 mice. a) Ethanol treatment did not alter cortical Grin2a expression in wildtype or APP/PS1 mice. b) Ethanol-treated APP/PS1 mice had higher cortical Grin2b expression compared to EtOH-treated wildtype mice. 2-way ANOVA revealed a significant treatment × genotype interaction ( p = 0.0319). c) H 2 O-treated APP/PS1 mice showed increased cortical Gabra5 expression compared to H 2 O-exposed wildtype mice (p < 0.05). This effect was lost in EtOH-exposed APP/PS1 Gabra5 mRNA levels. 2-way ANOVA revealed a significant treatment × genotype interaction ( p = 0.0249) and a trend in genotype effects ( p = 0.0723). d) Synaptic GluN2A levels was unaltered in the hippocampus of H 2 O- or EtOH-treated wildtype or APP/PS1 mice. e) Synaptic GluN2B levels was unaltered in the hippocampus of H 2 O- or EtOH-treated wildtype or APP/PS1 mice. f) Ethanol-treated wildtype mice showed increased synaptic GABA A R α5 subunit levels compared to H 2 O-treated wildtype mice. Ethanol treatment had no effect on GABAAR α5 subunit levels in APP/PS1 mice. 2-way ANOVA revealed a significant treatment × genotype effect ( p = 0.0347) and a trend in treatment effects ( p = 0.0644). Wildtype + H2O, n = 10; APP/PS1 + H2O, n = 9; Wildtype + EtOH, n = 7; APP/PS1 + EtOH, n = 8. * p < 0.05.

Article Snippet: The following primary and secondary antibodies were used for this study: APP (including CTFβ and CTFα; Invitrogen; CT695; 1:1000), BACE1 (Cell Signaling; 5606S; 1:1000), ADAM10 (Millipore; AB19026; 1:1000), IDE (Abcam; ab232216; 1:1000), GluN2A (Cell Signaling; 4025; 1:1000), GluN2B (Cell Signaling; 4212; 1:1000), GABAAR α5 (Santa Cruz; Sc393921; 1:1000), and β-actin (Millipore; MAB1501; 1:50,000), anti-mouse (Cell Signaling; 7076S; 1:5000), anti-rabbit (Cell Signaling; 7074S; 1:5000).

Techniques: Expressing

Journal: Antioxidants (Basel, Switzerland)

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors.

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Figure 2. (A) The chemical structure of ergotamine. (B) The H2O-injected oocytes did not cause any change with the treatment of 100 µM glutamate (n = 6–8 oocytes from four different frogs). (C–F) Glutamate induced inward currents with or without ergotamine (30 µM and 10 µM). For each subunit of NMDARs, the responses after treating with either glutamate (100 µM) alone or together with ergotamine (30 and 10 µM). Voltage clamp recording was conducted at a holding potential of −80 mV. The coapplication of ergotamine with glutamate resulted in modulation of the recombinant receptors (C) NR1a/NR2A, (D) NR1a/NR2B, (E) NR1a/NR2C, and (F) NR1a/NR2D, which in turn reduced glutamate-evoked inward current in a reversible manner (n = 6–8 oocytes from four different frogs).

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Injection, Recombinant

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: 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: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

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: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

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: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

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: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

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: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

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: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

Techniques: Expressing