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: 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: bioRxiv

Article Title: The proteasome regulator PI31 is required for protein homeostasis, synapse maintenance and neuronal survival in mice

doi: 10.1101/711515

Figure Lengend Snippet: A. PI31 fl/fl Hb9:Cre mice develop motor defects, kyphosis of the spine and muscle atrophy. Representative picture of PI31 fl/fl Hb9:Cre mice and their WT controls at 6 months of age. A representative H&E staining of thoracic cross sections of 5-month old PI31 fl/fl Hb9 cre mice and control littermates (PI31 fl/fl without Cre) showing atrophied musculature of KO mice. B. Relative body weight of 1 and 5 months old PI31 fl/fl Hb9:Cre mice and their controls littermate. PI31 fl/fl Hb9:Cre mice suffer progressive weight loss. 1MO – control, n=8, KO=4, 5MO - control, n=8, KO=4. Weight of WT controls at each age was set to 1. Statistical analysis was performed with a two-tailed paired t test, ** stands for p-value < 0.01. C . Inactivation of PI31 in motor neurons disrupts the structure of the Neuro-Muscular Junction (NMJ). Innervation of the Triangularis Sterni muscle was visualized by staining for □3-Tubullin/Synapsin (green), and post-synaptic muscle end plates were visualized with □-Bungarotoxin (red). 5 month old PI31 fl/fl Hb9:Cre mutant mice have fragmented NMJs, axonal swellings (indicated with white arrowheads) and massive axonal sprouting D . Loss of PI31 in motor neurons results in a progressive pathology, illustrated by the increase in axonal tip swellings with age. A bar diagram of the average number of axonal swelling per area unit (AU = 850 m 2 ) at different ages as annotated. ** stands for p-value < 0.01, *** stands for p-value < 0.001 E. P62 granules accumulate at the NMJ of PI31 fl/fl Hb9:Cre, but not in control littermates. Representative image of NMJ in the Triangularis Sterni muscle of PI31 fl/fl and PI31 fl/fl Hb9:Cre mice (4MO females). P62 granules in magenta (marked with white arrowheads), motor neurons in green (stained by anti □3-Tubullin and Synapsin antibodies).

Article Snippet: After blocking, tissue was incubated with Alexa488 conjugated primary antibodies for Tuj1 (mouse monoclonal, 1:200; BD Pharmingen #560339) and Synapsin (Rabbit monoclonal, 1:200; CST #13197) over night at 4c.

Techniques: Staining, Control, Two Tailed Test, Mutagenesis