Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Examples of VE-2A, VE-2B, VE-2BΔ7, and VE three hours after exit from the ER immunostained for SAP102 (red), and synaptophysin (Sp; pseudocolored blue), and merged. VE-2B, and VE-2BΔ7 demonstrate significant clustering compared to VE (quantified in E), and targeting to synaptophysin (quantified in D). (B) Higher magnifications of clusters from VE-2B and VE-2BΔ7 seen in A (indicated by boxes) demonstrate roughly equivalent colocalization to SAP102 (quantified in C) and synaptophysin (quantified in D; scale bars 1 µm). (C) VE-2B and VE-2BΔ7 co-localized with postsynaptic SAP102 at 2X background (52–255 inclusive gray scale). By one way Anova (P<0.01) there was a significant group difference. Using Tukey’s post hoc pairwise comparisons, both VE-2B and VE-2BΔ7 pixel overlap with SAP102 was significantly greater than VE (p<0.05), however VE-2B and VE-2BΔ7 were not significantly different than eachother. This relationship between VE-2B and VE-2BΔ7 overlap with SAP102 was only obtained when analyzing pixel overlap at 2X background. (D) The percent of overlap of VE-2B, and VE-2BΔ7 with synaptophysin was significantly greater than VE (one-way Anova with post hoc pairwise comparisons to VE * p<0.05). Surprisingly, VE-2A was not significantly different than VE among the 4 groups in a post hoc comparison. VE-2B targeted to synaptophysin significantly better than VE-2A (** p<0.05) but no differently than VE-2BΔ7. These relative differences were the same regardless of the green or blue threshold. The same results were obtained at this time point after ER release using synapsin as the presynaptic marker (data not shown). (E) Clustering was measured using Zeiss LSM510 image analysis software. Average intensity was calculated from each intensity graph of 20–30 dendrites for a total of 839.5 µm (VE-2B), 750.4 µm (VE-2BΔ7), and 776.0 µm (VE). A cluster was defined as being more than twice the average intensity of each dendrite for equal to or greater than 0.4 µm. The average number of clusters per µm ± SEM is plotted in E. There was a significant effect of group by one-way Anova. Post hoc comparisons indicated Both VE-2B and VE-2BΔ7 showed significantly more clustering than VE (p<0.05), and were not significantly different from each other. (F) Examples of immunogold labeling with i14 α-VSVG antibody (10 nm; arrowheads) and α-NR2A/B antibody (5 nm; arrows) indicate localization of VE-2B at synapses 3 hours after release from the ER (pre, presynaptic terminal; post, postsynaptic process). Scale bar is 100 nm. Quantification of 10 nm gold indicated that 10 of 47 synapses were labeled within 0–100 nm, and 18 of 47 (38.3%) synapses showed immunogold labeling within 0–500 nm of the postsynaptic density.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Marker, Software, Labeling

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B and VE-2BΔ7 were allowed to exit the ER for 45 minutes and then immunostained for surface expression with I1 antibody and presynaptic terminals with anti-synapsin. Yellow arrows in VE-2BΔ7 indicate surface puncta not in the vicinity of synapsin (right panels). At 45 minutes after ER exit, only about 30% of VE-2B and VE-2BΔ7 puncta in dendrites showed any immunostaining with i1 antibody. (B) The VE and VE-2BΔ7 surface puncta more than 1 µm away from synapsin were significantly greater in relative number than VE-2B (left panel). The surface VE-2BΔ7 within 0.3 µm is similar to VE-2B but not significantly different from VE, while VE-2B within 0.3 µm is significantly different than VE (one-way Anova considering VE, VE-2B, and VE-2BΔ7 in the >1.0 micron bins and then in the <0.3 micron bins, then pairwise post hoc comparisons; p<0.05). Centroids and distances were calculated with images thresholded at 2X mean background. Percent pixel overlap of green puncta with synapsin in the same data set showed no difference in the total VE-2B and VE-2BΔ7 at any threshold and trended toward increased synaptic localization at 45 minutes after permissive temperature, but did not reach significance when compared to VE, as was apparent at 3 hours (one-way Anova, p = 0.11; right panel, indicated as ‘total’). Green and red images from the same data set also were merged and color-thresholded for yellow to define the surface population. Percent overlap of yellow puncta with synapsin (blue) was then assessed for VE, VE-2B, and VE-2BΔ7 (right panel, indicated as ‘surface’). VE-2BΔ7 surface pixel overlap with synapsin trended toward a decrease compared to VE-2B at 2X background but not significantly until thresholded at 3X background (one-way Anova, post hoc comparison p<0.05). (C) Model of trafficking of NR2B. NR2B forms hetero-oligomers with NR1 subunits at the level of the ER , but the NR2 distal C-terminus is necessary and sufficient to confer significant synaptic localization , . NR2A/B clusters with SAP102 early in the secretory pathway, and significantly so at the level of the cis-medial- Golgi. PSD-95 is added as part of the NR2B/NR1-SAP102 complex as soon as the TGN. NR2B/NR1-SAP102 complexes may be cotransported to the vicinity of the synapse, and also cotransported at least in-part along dendrites via Kif-17, mLin-2/Cask, mLin7, mLin10, and SAP97 in a poly-protein complex [see ] and added to postsynaptic structures. The NR2B/SAP102/PSD-95 association does not appear to be essential for immediate synaptic targeting, but is required for maintenance of position on the synaptic surface.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Expressing, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Platelet Activating Factor Enhances Synaptic Vesicle Exocytosis Via PKC, Elevated Intracellular Calcium, and Modulation of Synapsin 1 Dynamics and Phosphorylation

doi: 10.3389/fncel.2015.00505

Figure Lengend Snippet: cPAF increases synapsin I phosphorylation and synapsin I dispersion from presynaptic boutons during stimulation. (A–D) Hippocampal neurons expressing synapsin I-GFP and Tdtomato were treated with 1 μM cPAF for 2 min then stimulated with 900 pulses at 10 Hz. (A) Synapsin I-GFP (Syn-GFP) is clustered at presynaptic boutons while tdtomato fills the entire axon. Scale bar = 2 μm. (B) Syn-GFP fluorescence at synapses decreases upon stimulation then reclusters within the bouton. Scale bar = 2 μm. (C) Timelapse measurements of the change in Syn-GFP fluorescence intensity obtained from the boutons numbered in (A) normalized to the fluorescence at time 0. (D) Average Δ F / F 0 in syn-GFP fluorescence from all boutons treated with 1 μM cPAF or vehicle. Error bars ±SEM (cPAF: 141 boutons from three coverslips; vehicle: 240 boutons from four coverslips;). Non-linear regression of decay part of synapsin I dispersion curve followed by testing the null hypothesis of one curve for all data sets has p < 0.0001. Statistical analysis comparing cPAF to vehicle treated boutons at the end of stimulation (1.5 min) was done using the Student’s t -test p = 0.0005. (E) Representative western blots showing phosphorylated synapsin I (pSynapsin) at Site 3 (Ser603) and Site 1 (Ser9) and GAPDH immunoreactivity from cell lysates obtained from primary hippocampal cultures that were treated 0, 2, or 20 min with 1 μM cPAF. Blot on right was additionally treated for a total of 30 min with 50 μM APV and 10 μM CNQX to block glutamatergic neurotransmission. (F) pSynapsin/GAPDH ratios calculated by densitometric scanning of the blots. Data are means ± SEM with n = 4 (two independent experiments each performed in duplicate). Statistical analysis: one-way ANOVA followed by Dunnett’s multiple comparison test to 0 min control. ANOVA: Site 3, p = 0.098; Site 3 with APV/CNQX, p = 0.024; Site 1, p = 0.211; Site 1 with APV/CNQX, p = 0.126. Dunnett’s multiplicity adjusted p -values (compared to 0 min control): ∗ p < 0.1 ∗∗ p < 0.05.

Article Snippet: Membranes were blocked with 5% milk in TBSt for 30 min and probed with primary antibodies (phospho-synapsin Ser9 (Cell Signaling Technologies #2311), phospho-synapsin I Ser603 (Rockland 612-401-C95), and GAPDH (EMD Millipore: Calbiochem CB1001) in 3% BSA in TBSt at 4° overnight.

Techniques: Expressing, Fluorescence, Western Blot, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Mesenchymal Stem Cells Restore Endothelial Integrity and Alleviate Emotional Impairments in a Diabetic Mouse Model via Inhibition of MMP-9 Activity

doi: 10.3390/ijms26073355

Figure Lengend Snippet: Figure 2. DM mice exhibit synaptic deficits and increased neuroinflammation in the HIP. (A) Repre- sentative WB images showing the expression levels of the presynaptic protein synapsin I (SYN1) and the postsynaptic protein postsynaptic density protein 95 (PSD95) in the HIP of CTL and DM mice. β-actin was used as an internal control. (B) Semi-quantitative analysis of PSD95 and SYN1 expression levels from immunoblot experiments. n = 6. (C) Representative images of Iba1 immunostaining in the HIP of CTL and DM mice. The area within the dashed box is magnified and displayed in the insets. Scale bars: 100 µm (main images) and 20 µm (insets). (D) Quantitative analysis of Iba1-positive microglia in the HIP of CTL and DM mice. n = 5. (E) qPCR analysis of iNOS and Arg1 mRNA levels in the HIP of CTL and DM mice. n = 6. (F) qPCR analysis of IL-6, IL-1β, and TNF-α mRNA levels in the HIP of CTL and DM mice. n = 6. (G) ELISA measuring IL-6, IL-1β, and TNF-α levels in the serum of CTL and DM mice. n = 3. Data are presented as mean ± SEM. Statistical significance was determined using a one-tailed unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked and incubated overnight with the following antibodies: PSD95 (SYSN, Toronto, ON, Canada, 124002, 1:1000); SYN1 (SYSN, 106011, 1:1000); Cldn5 (Invitrogen, 35-2500, 1:1000); Ocln (Invitrogen, 71-1500, 1:1000); and MMP9 (Boster, Shanghai, China; PB9669, 1:1000).

Techniques: Expressing, Control, Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Mesenchymal Stem Cells Restore Endothelial Integrity and Alleviate Emotional Impairments in a Diabetic Mouse Model via Inhibition of MMP-9 Activity

doi: 10.3390/ijms26073355

Figure Lengend Snippet: Figure 6. MSC treatment attenuated DM-induced neuroinflammation and synaptic deficits. (A) Representative immunofluorescence images of Iba1 staining in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. The dashed box indicates the magnified region shown in the insets. Scale bars: 100 µm (main images) and 20 µm (insets). (B) Quantitative analysis of Iba1+ microglia in the HIP. n = 4. (C) qPCR analysis of iNOS and Arg1 mRNA levels in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. n = 9. (D) qPCR analysis of mRNA levels of IL-6, IL-1β, and TNF-α in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. n = 9. (E) ELISA results showing serum levels of IL-6, IL-1β, and TNF-α in CTL, MSC, DM, and DM+MSC mice. n = 4. (F) Representative WB images of PSD95 and SYN1 expression in the HIP. β-actin was used as a loading control. (G) Semi-quantitative analysis of PSD95 and SYN1 protein levels. n = 6. Data are presented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test. ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked and incubated overnight with the following antibodies: PSD95 (SYSN, Toronto, ON, Canada, 124002, 1:1000); SYN1 (SYSN, 106011, 1:1000); Cldn5 (Invitrogen, 35-2500, 1:1000); Ocln (Invitrogen, 71-1500, 1:1000); and MMP9 (Boster, Shanghai, China; PB9669, 1:1000).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Evaluation of the C-domain of heparanase during AGE-induced macrophage inflammatory response

doi: 10.3892/etm.2017.4609

Figure Lengend Snippet: The viability of macrophages treated with AGEs, anti-HPA and Syn-1 antibody. The viability of macrophages was measured using MTT. (A) Macrophages were treated with AGEs (0, 25, 50, 100, 200, 400 and 800 mg/l) for 4, 8, 12, 16, 20 and 24 h. (B) Macrophages were pretreated with anti-HPA before culture with 25, 50 and 100 mg/l AGEs for 4, 8, 12, 16, 20 and 24 h. (C) Macrophages were pretreated with anti-HPA plus anti-Syn-1 antibody before culture with 25, 50 and 100 mg/l AGEs for 4, 8, 12, 16, 20 and 24 h. The results are the mean of 6 culture wells (mean ± SEM). *P<0.05, as compared to the control group. All of the experiments were performed independently in triplicate. AGEs, advanced glycation end products; HPA, heparanase; Syn-1, syndecan-1.

Article Snippet: Macrophages were plated at 5×10 4 cells/ml and incubated with or without AGEs (Shanghai Yixin Biotechnology Co., Ltd., Shanghai, China), and antibody against C-domain of HPA (Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) or anti-HPA plus Syn-1 antibody (R&D Systems, Inc., Minneapolis, MN, USA) was applied for various periods of time (4–24 h).

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: Evaluation of the C-domain of heparanase during AGE-induced macrophage inflammatory response

doi: 10.3892/etm.2017.4609

Figure Lengend Snippet: C-domain of HPA mediates AGE-induced macrophage migration via Syn-1. Cells were cultured with 25, 50 and 100 mg/l AGEs for 24 h with or without pre-treatment with anti-HPA or anti-HPA plus anti-Syn-1 antibody, respectively. The migration was measured by Transwell assays. Results were normalized to the number of macrophages that migrated in the control group. The results are the mean of 6 culture wells (mean ± SEM). *P<0.05. All of the experiments were performed independently in triplicate. HPA, heparanase; AGEs, advanced glycation end products; Syn-1, syndecan-1.

Article Snippet: Macrophages were plated at 5×10 4 cells/ml and incubated with or without AGEs (Shanghai Yixin Biotechnology Co., Ltd., Shanghai, China), and antibody against C-domain of HPA (Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) or anti-HPA plus Syn-1 antibody (R&D Systems, Inc., Minneapolis, MN, USA) was applied for various periods of time (4–24 h).

Techniques: Migration, Cell Culture

Journal: Experimental and Therapeutic Medicine

Article Title: Evaluation of the C-domain of heparanase during AGE-induced macrophage inflammatory response

doi: 10.3892/etm.2017.4609

Figure Lengend Snippet: C-domain of HPA mediated release of inflammatory factor TNF-α and IL-1β in AGE-induced macrophages via Syn-1. Cells cultured with 25, 50 and 100 mg/l AGEs for 24 h with or without pre-treatment with anti-HPA antibody recognized C-domain of HPA or anti-HPA plus Syn-1 antibody. (A) The mRNA levels of TNF-α and IL-1β were assessed with RT-qPCR. (B) The secretion of TNF-α and IL-1β in supernatant was measured by ELISA. The results are the mean of 6 culture wells (mean ± SEM). *P<0.05. All of the experiments were performed independently in triplicate. HPA, heparanase; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; AGEs, advanced glycation end products; Syn-1, syndecan-1; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Macrophages were plated at 5×10 4 cells/ml and incubated with or without AGEs (Shanghai Yixin Biotechnology Co., Ltd., Shanghai, China), and antibody against C-domain of HPA (Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) or anti-HPA plus Syn-1 antibody (R&D Systems, Inc., Minneapolis, MN, USA) was applied for various periods of time (4–24 h).

Techniques: Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Evaluation of the C-domain of heparanase during AGE-induced macrophage inflammatory response

doi: 10.3892/etm.2017.4609

Figure Lengend Snippet: Effects of C-domain of HPA on the HPA activity and Syn-1 protein in AGE-induced macrophages. Cells were cultured with 100 mg/l AGEs for 24 h with or without pre-treatment with anti-HPA antibody recognized C-terminal of HPA. (A) The enzyme activity of HPA was evaluated by heparan degrading enzyme assay. (B) The Syn-1 protein expression was performed by western blot analysis. The results are the mean of 6 culture wells (mean ± SEM). All the experiments were performed independently in triplicate.*P<0.05 significantly different from control (0 mg/l) and AGEs at 100 mg/l; # P<0.05 significantly different from AGEs at 100 mg/l. HPA, heparanase; AGEs, advanced glycation end products; Syn-1, syndecan-1.

Article Snippet: Macrophages were plated at 5×10 4 cells/ml and incubated with or without AGEs (Shanghai Yixin Biotechnology Co., Ltd., Shanghai, China), and antibody against C-domain of HPA (Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) or anti-HPA plus Syn-1 antibody (R&D Systems, Inc., Minneapolis, MN, USA) was applied for various periods of time (4–24 h).

Techniques: Activity Assay, Cell Culture, Enzymatic Assay, Expressing, Western Blot