Journal: Frontiers in Aging Neuroscience

Article Title: Effects of Aging on the Structure and Expression of NMDA Receptors of Somatostatin Expressing Neurons in the Mouse Hippocampus

doi: 10.3389/fnagi.2021.782737

Figure Lengend Snippet: Summary of the results of the expression of NMDAR.

Article Snippet: Controls were performed omitting the anti-GluN1 or anti-GluN2B antibody, as well as incubating with these antibodies previously pre-absorbed overnight with an excess of its immunogenic peptide (GluN1 blocking peptide, Alomone, Jerusalem, Israel) or (GluN2B blocking peptide, Alomone, Jerusalem, Israel), respectively.

Techniques: Expressing

Journal: Frontiers in Aging Neuroscience

Article Title: Effects of Aging on the Structure and Expression of NMDA Receptors of Somatostatin Expressing Neurons in the Mouse Hippocampus

doi: 10.3389/fnagi.2021.782737

Figure Lengend Snippet: Distribution of GluN2B immunoreactivity in young and aged GIN mice hippocampus. Panoramic confocal plane showing the distribution of O-LM cells (green) and GluN2B immunoreactivity (red) in the hippocampus of 3-month-old (A1) and 16-month-old (B1) mice. Different regions and strata are indicated with dotted lines. (A2,B2) High magnification view from the different CA1 strata in 3-month-old (A2) and 16-month-old (B2) mice. (A3,B3) Enlarged view of the squared regions in panels (A2,B2) , showing double immunofluorescence for GFP/GluN2B, in strata oriens , and pyramidale. Note the presence of GluN2B + clusters in pyramidal neurons in 16-month-old (B3) , but not in 3-month-old (A3) mice. Scale bar: 150 μm for panels (A1,B1 ), 67 μm for panels (A2,B2) , and 21 μm for panels (A3,B3) .

Article Snippet: Controls were performed omitting the anti-GluN1 or anti-GluN2B antibody, as well as incubating with these antibodies previously pre-absorbed overnight with an excess of its immunogenic peptide (GluN1 blocking peptide, Alomone, Jerusalem, Israel) or (GluN2B blocking peptide, Alomone, Jerusalem, Israel), respectively.

Techniques: Immunofluorescence

Journal: Frontiers in Aging Neuroscience

Article Title: Effects of Aging on the Structure and Expression of NMDA Receptors of Somatostatin Expressing Neurons in the Mouse Hippocampus

doi: 10.3389/fnagi.2021.782737

Figure Lengend Snippet: Analysis of the density and percentage of area covered with GluN2B immunoreactive puncta in the somata and in the periphery of O-LM cells during aging. (A–F) Double GFP/GluN2B immunohistochemistry in 3-month-old (A) , 9-month-old (B) , 16-month-old (C) female mice and in 3-month-old (D) , 9-month-old (E) , 16-month-old (F) male mice. In panels (C2,F2) , a detailed view of the GluN2B clustering in aged mice can be observed. (G–I) Graphs showing the density and percentage of area covered with GluN2B immunoreactive puncta in the somata (G1,G2–I2) and in its periphery (G3,G4–I4) in animals segregated by sex (G1–4) , pooled females (H1–4) and males (I1–4) (all graphs represent mean ± SEM., * p -value <0.05, ** p -value <0.01, **** p -value <0.0001). Scale bar: 5 μm.

Article Snippet: Controls were performed omitting the anti-GluN1 or anti-GluN2B antibody, as well as incubating with these antibodies previously pre-absorbed overnight with an excess of its immunogenic peptide (GluN1 blocking peptide, Alomone, Jerusalem, Israel) or (GluN2B blocking peptide, Alomone, Jerusalem, Israel), respectively.

Techniques: Immunohistochemistry

Journal: EBioMedicine

Article Title: NMDA receptor modulation of glutamate release in activated neutrophils

doi: 10.1016/j.ebiom.2019.08.004

Figure Lengend Snippet: GluN2B NMDAR subunit modulates neutrophil ROS production. (A) Histograms illustrating effect of GluN2B receptor antagonist Co 101244 (100 μM) on ROS generation (quantified by 123-dihydrorhodamine) triggered by lipopolysaccharide (100 ng·ml −1 ), fMLP, and opsonised E coli (3 × 10 6 ·ml −1 ) in whole blood preparation and direct PKC stimulation using PMA. (B) Population data for GLuN2B antagonist Co 101244 on ROS generation (quantified by 123-dihydrorhodamine) in primary human neutrophils after ligands shown in panel A, presented as %PMA response (i.e. PMA alone with no GluN2B antagonist present; *p < 0.05, ANOVA; n = 5–8). (C) Co 101244 in submicromolar concentrations reduces ROS release (quantified by 123-dihydrorhodamine) from CD16+ neutrophils in whole blood samples following E Coli and PMA, standardized as % of PMA response (i.e. PMA alone with no GluN2B antagonist present; mean ± sem, n = 3–7/group; *p < 0.01, ANOVA). (D) Oxygen consumption (measured by direct respirometry (Seahorse XF96)) before, and following, PMA-stimulation in isolated highly purified (>98%) primary human neutrophils pretreated with Co 101244 (25–250 μM) or phosphate-buffered saline control (mean ± sem, *p < 0.05; n = 5, one-way ANOVA, (drug × time)). (E) Primary human neutrophil phagocytosis of opsonized FITC-labeled E coli is reduced by GluN2B antagonist Co 101244 (100 μM; *p < 0.05; n = 5 patient samples, paired t -test). (F) ROS generation in microbead purified peritoneal neutrophils (Ly6G+) obtained 3 h after intraperitoneal injection of 1 g/g zymosan in C57B/6 mice. GluN2B antagonist Co 101244 was incubated with isolated cells for 30 minutes ex-vivo in presence/absence of PMA. (G) Graph shows summary data for peritoneal neutrophil ROS (quantified by 123-dihydrorhodamine) expressed as %PMA response (i.e. PMA alone with no GluN2B antagonist present) after IP zymosan alone and PMA post-peritoneal harvest. Co 101244 reduced ROS in lavage neutrophils and after stimulation with PMA (p < 0.01, paired t -Test; n = 10 mice; mean ± sem).

Article Snippet: Numbers above histograms indicate days of DMSO treatment. (C) Confocal images (Zeiss LSM 510) showing cell surface staining for GluN2B, in absence or presence of specific blocking peptide anti- GluN2B Receptor (extracellular), conjugated to ATTO-594 (1:500; Alomone antibodies, Israel).

Techniques: Isolation, Purification, Labeling, Injection, Incubation, Ex Vivo

Journal: EBioMedicine

Article Title: NMDA receptor modulation of glutamate release in activated neutrophils

doi: 10.1016/j.ebiom.2019.08.004

Figure Lengend Snippet: Maturation of GluN2B NMDAR subunit expression in primary human granulocytes and neutrophil-like HL60 cells. (A) RT-PCR showing GluN2B mRNA in primary human neutrophils, undifferentiated (designated as HL60-) and HL60 cells differentiated with DMSO for 6 days (designated as HL60+), referenced to human neuroblastoma LN229 cell line (which expresses NMDAR in low quantities). GluN2B mRNA was higher in DMSO-differentiated cells (p < 0.01; n = 5; mean ± sem) (B) GluN2B expression in differentiated neutrophil-like HL60 cells following 3 days (blue line) or 6 (red line) days treatment with 1.25% DMSO (dimethyl sulfoxide). Isotype control is shown in grey for both timepoints. Numbers above histograms indicate days of DMSO treatment. (C) Confocal images (Zeiss LSM 510) showing cell surface staining for GluN2B, in absence or presence of specific blocking peptide anti- GluN2B Receptor (extracellular), conjugated to ATTO-594 (1:500; Alomone antibodies, Israel). Lower panel shows extracellular co-staining of GluN2B and CD16. (D) Differentiation of HL60 cells with 1.25% DMSO into neutrophil-like cells for up to 6 days is associated with higher increase in ROS (quantified by 123-dihydrorhodamine) following PMA stimulation, compared to HL60 cells differentiated for 3 days with 1.25% DMSO. n = 3–5 group; 3 independent experiments. (E) shRNA knockdown of GluN1 in HL60-neutrophil like cell line. (F) Effect of shRNA knockdown of GluN1 in HL60-neutrophil like cell line PMA-induced ROS production, as quantified by 123-dihydrorhodamine (*p < 0.05; n = 3 independent experiments). (G) Oxygen consumption (measured by direct respirometry (Seahorse XF96)) following PMA-stimulation in scrambled (red circles) or GluN1 deficient (blue circles) HL60 cells (mean ± sem, *p < 0.01; n = 5, ANOVA). White and black circles represent unstimulated control cells for each scrambled or GluN1 deficient genotype, respectively. (H) Confocal microscopy expression of GluN2B 24 h after transfection of primary human neutrophils with either scrambled siRNA, or siRNA targeted at knocking down GluN1 or GluN2B NMDAR subunits. (I) Representative histograms of ROS production (quantified by 123-dihydrorhodamine) following PMA activation of primary human neutrophils 24 h after transfection. Black areas indicate Alexa-647 transfected cells (mean (SD)) 22 ± 3% neutrophils were transfected, as determined by flow cytometric analysis of Alexa-647 conjugated to siRNA. (J) Summary ROS data for siRNA knockdown experiments in human primary cells, before (baseline) and after PMA stimulation (mean ± sem, *p < 0.05; n = 3, comparing ROS generation in scrambled versus GluN1 or GluN2B siRNA after PMA stimulation; ANOVA).

Article Snippet: Numbers above histograms indicate days of DMSO treatment. (C) Confocal images (Zeiss LSM 510) showing cell surface staining for GluN2B, in absence or presence of specific blocking peptide anti- GluN2B Receptor (extracellular), conjugated to ATTO-594 (1:500; Alomone antibodies, Israel).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Blocking Assay, shRNA, Confocal Microscopy, Transfection, Activation Assay

Journal: EBioMedicine

Article Title: NMDA receptor modulation of glutamate release in activated neutrophils

doi: 10.1016/j.ebiom.2019.08.004

Figure Lengend Snippet: GluN2B-MDAR scaffolding proteins modulate neutrophil ROS generation. (A) Histograms illustrating ROS generation (quantified by 123-dihydrorhodamine) following stimulation with E. coli and PMA-which is reduced by ZL006 (1–50 μM) in purified primary human neutrophils (n = 7). (B) E. coli -induced ROS in whole blood samples is reduced by ZL006 in a dose-dependent manner (10–50 μM; n = 7). (C) Summary data showing effect of ZL006 on PMA-induced ROS generation in neutrophils, as quantified by 123-dihydrorhodamine. Data expressed as % respective control (mean ± sem; n = 7 subjects; *p = 0.01, ANOVA). (D) shRNA knockdown of GluN1B is associated with reduced nNOS protein expression in differentiated HL60 neutrophil-like cells. (E) PMA-induced ROS production (quantified by 123-dihydrorhodamine) is attenuated in bone-marrow derived neutrophils from wild-type and homozygous PSD-95 deficient. (F) Population data for ROS in wild-type and homozygous PSD-95 deficient bone marrow derived neutrophils (mean ± sem; n = 6–7 mice/group; *p = 0.02 unpaired t -test). (G) ERK phosphorylation in primary human neutrophils is reduced by GluN2B antagonist Co 101244 following PMA-stimulation phospho-flow cytometry in CD16+ cells, human whole blood (mean ± sem; n = 5 experiments; *p < 0.05; two-way (drug × time) ANOVA with Tukey posthoc comparison). Immunoblot above graph shows similar results obtained in five separate experiments. Numbers below123-dihydrorhodamine immunoblot denote minutes after PMA stimulation (in the presence/absence of GluN2B subunit antagonist Co 101244) or phosphate-buffered saline control. (H) PMA-induced ROS production is attenuated in bone-marrow derived neutrophils from wild-type and homozygous SAP102 deficient mice. (I) Population data for wild-type and homozygous SAP-102 deficient bone marrow derived neutrophils (mean ± sem; n = 6–7 mice/group; *p = 0.04 unpaired t -test).

Article Snippet: Numbers above histograms indicate days of DMSO treatment. (C) Confocal images (Zeiss LSM 510) showing cell surface staining for GluN2B, in absence or presence of specific blocking peptide anti- GluN2B Receptor (extracellular), conjugated to ATTO-594 (1:500; Alomone antibodies, Israel).

Techniques: Scaffolding, Purification, shRNA, Expressing, Derivative Assay, Flow Cytometry, Western Blot

Journal: EBioMedicine

Article Title: NMDA receptor modulation of glutamate release in activated neutrophils

doi: 10.1016/j.ebiom.2019.08.004

Figure Lengend Snippet: GluN2B subunit in systemic inflammation and neurologic-related bacterial infection. (A) Serial changes in GluN2B subunit expression in CD16+ primary human neutrophils obtained from surgical patients preoperatively and 48 h after surgery, quantified using flow cytometry. (B) Population data for CD16+ GluN2B expression in the perioperative period. (mean ± sem; p < 0.01, t -test; n = 6 patients assessed serially, preoperative versus postoperative). (C) Neutrophil ROS generation after E coli is added to whole blood from the same surgical patient preoperatively and 2 days after surgery, quantified by 123-dihydrorhodamine. PBS control, Co101244 at 10 μM and 100 μM was added to whole blood sample 10 min before E. coli stimulation. (D) Neutrophil ROS generation following addition of PMA to whole blood, from the same surgical patient preoperatively and 2 days after surgery, quantified by 123-dihydrorhodamine. PBS control, Co101244 at 10 μM and 100 μM was added to whole blood sample 10 min before E. coli stimulation. (E) Summary data (median, interquartile range) for preoperative and postoperative neutrophil responses to E coli in whole blood from 4 surgical patients following pre-incubation with PBS control, Co 101244 at 10 μM and 100 μM for 10 min prior to stimulation. * denotes p = 0.001 for drug versus saline control comparison; p = 0.035, ** denotes comparison between pre versus postoperative values; by repeated measures ANOVA (time × drug treatment). (F) Summary data (median, interquartile range) for preoperative and postoperative neutrophil responses to PMA in whole blood from 4 surgical patients following pre-incubation with PBS control, Co 101244 at 10 μM and 100 μM for 10 min prior to stimulation. * denotes p = 0.02 for drug versus saline control comparison; p = 0.07, comparison between pre versus postoperative values; by repeated measures ANOVA (time × drug treatment). (G) 10 6 purified neutrophils (purity 98 ± 1%) from 4 healthy volunteers were incubated with GluN2B antagonist Co 101244 (0–100 μM shown) and Pseudomonas fluorescens (10 6 cfu·ml −1 ). Asterisk denotes increasing doses of Co 101244 reduce ROS (quantified by 123-dihydrorhodamine fluorescence) in primary human neutrophils following co-culture with 10 6 Pseudomonas fluorescens (mean ± sem, p = 0.011; ANOVA, F (3, 12) = 5.8). (H) Example data from one volunteer showing increasing doses of Co 101244 reduce ROS fluorescence in primary human neutrophils following co-culture with 10 6 Pseudomonas fluorescens.

Article Snippet: Numbers above histograms indicate days of DMSO treatment. (C) Confocal images (Zeiss LSM 510) showing cell surface staining for GluN2B, in absence or presence of specific blocking peptide anti- GluN2B Receptor (extracellular), conjugated to ATTO-594 (1:500; Alomone antibodies, Israel).

Techniques: Infection, Expressing, Flow Cytometry, Incubation, Purification, Fluorescence, Co-Culture Assay

Journal: EBioMedicine

Article Title: NMDA receptor modulation of glutamate release in activated neutrophils

doi: 10.1016/j.ebiom.2019.08.004

Figure Lengend Snippet: Summary of potential role for NMDAR/GluN2B signalling in modifying degree of ROS generation in activated neutrophils. Release of glutamate and co-agonist D-serine after activation by DAMPs/PAMPs promotes calcium influx to augment neutrophil ROS generation. Tonic levels of extracellular glutamate may play a protective role, since GluN2B antagonism promotes apoptosis in non-activated cells.

Article Snippet: Numbers above histograms indicate days of DMSO treatment. (C) Confocal images (Zeiss LSM 510) showing cell surface staining for GluN2B, in absence or presence of specific blocking peptide anti- GluN2B Receptor (extracellular), conjugated to ATTO-594 (1:500; Alomone antibodies, Israel).

Techniques: Activation Assay