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: 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: Scientific Reports

Article Title: Molecular mechanism of nur77 gene expression and downstream target genes in the early stage of forskolin-induced differentiation in PC12 cells

doi: 10.1038/s41598-020-62968-y

Figure Lengend Snippet: The Nur77-Synapsin1 signaling pathway has an important role in differentiation of forskolin-treated PC12 cells. ( A ) Cells were treated with 10 μM forskolin. synapsin1, β-tubulin III, neurod and gapdh mRNA were detected by qPCR. mRNAs were normalized against gapdh mRNA. **P < 0.01 compared with 0 hr. ( B ) Immunoblot analysis of forskolin-induced Synapsin1, β-tubulin III and NeuroD. Cells were treated with 10 μM forskolin for 24 hr. ( C ) Quantification of B, comparing the protein levels of Synapsin1, β-tubulin III, NeuroD and GAPDH protein in the indicated times. The amount of protein was quantified and normalized to that of GAPDH. **P < 0.01 compared with 0 hr., N.S. denotes not significant for statistical analysis. ( D ) Cells transfected with nur77 siRNA were incubated in the presence or absence of 10 μM forskolin for 24 hr. Synapsin1, β-tubulin III and gapdh mRNA were detected by qPCR. mRNAs were normalized against gapdh mRNA. **P < 0.01 compared with untreated. ( E ) Cells transfected with nur77 siRNA were incubated in 10 μM forskolin for 24 hr. Synapsin1, β-tubulin III and GAPDH were detected by immunoblot analysis. These bands were representatives of three independent experiments. ( F ) Quantification analysis of the protein levels of Synapsin1 and β-Tubulin III was performed (n = 3). The amount of these protein was quantified and normalized to that of GAPDH. *P < 0.05 compared with untreated. ( G ) After co-transfection with synapsin1 siRNA or ncRNA containing a GFP-expression plasmid for 48 hr, cells were treated with 10 μM forskolin for 24 hr. Phase contrast (Phase) and GFP-expression (GFP) images were detected and photographed. Scale bar: 50 μm. ( H ) Histograms of neurite lengths of forskolin-treated PC12 cells (open bars) pretreated with synapsin1 siRNA (gray bars) and ncRNA (hatched bars). Cells pretreated with siRNA or ncRNA in the presence of a GFP-expression plasmid for 48 hr were then treated with forskolin for a further 24 hr. Cells were cultured without (closed bars: untreated) or with 10 μM forskolin (open, hatched and gray bars). For analysis of neurite outgrowth, cells (more than 200 cells /well) were randomly photographed. ***P < 0.001 ( synapsin1 siRNA vs. ncRNA).

Article Snippet: PCR primers used were those of TaqMan® gene expression assay kits for nur77 (Assay ID: Rn00666994_g1), creb (Assay ID: Rn06140207_g1), synapsin1 (Assay ID: Rn00569468_m1), tubulin, beta 3 class III (Assay ID: Rn01431594_m1), neurod1 (Assay ID: Rn00824571_s1), and gapdh (Assay ID: Rn01775763_g1).

Techniques: Western Blot, Transfection, Incubation, Cotransfection, Expressing, Plasmid Preparation, Cell Culture

Journal: Scientific Reports

Article Title: Molecular mechanism of nur77 gene expression and downstream target genes in the early stage of forskolin-induced differentiation in PC12 cells

doi: 10.1038/s41598-020-62968-y

Figure Lengend Snippet: The nur77 gene expression-dependent molecular pathway to forskolin-induced neuronal outgrowth. ( A ) In the forskolin-untreated (basal) condition, CREB binds to the −78 CRE site (black closed box in the nucleus) in the nur77 promoter region, causing expression of Nur77. ( B ) After stimulation with forskolin, the PKA-CREB pathway is activated through adenylate cyclase production. Then, CREB is phosphorylated (P-CREB) and the P-CREB binds to the three CRE sites in the nur77 gene upstream of the TSS. The binding sites (−242, −222 and −78) are shown as black closed boxes in the nucleus. Binding of phosphorylated CREB to the nur77 gene promoter region strongly induces Nur77 expression, after which Nur77 upregulates expression of Synapsin1 to promote differentiation of PC12 cells.

Article Snippet: PCR primers used were those of TaqMan® gene expression assay kits for nur77 (Assay ID: Rn00666994_g1), creb (Assay ID: Rn06140207_g1), synapsin1 (Assay ID: Rn00569468_m1), tubulin, beta 3 class III (Assay ID: Rn01431594_m1), neurod1 (Assay ID: Rn00824571_s1), and gapdh (Assay ID: Rn01775763_g1).

Techniques: Gene Expression, Expressing, Binding Assay