Journal: eLife

Article Title: Microglia TREM2R47H Alzheimer-linked variant enhances excitatory transmission and reduces LTP via increased TNF-α levels

doi: 10.7554/elife.57513

Figure Lengend Snippet: Figure 7. LTP is impaired in RH mutant rats, and the impairment could be rescued by application of anti-TNF-a antibody. (A) The input-output slope is significantly increased in Trem2R47H/R47H rats [two-way ANOVA, stimulation intensity x genotype interaction F(6, 144)=6.745, p<0.0001****; post-hoc Sidak’s multiple comparisons test: 1.2 mV p=0.0175*; 1.4 mV: p=0.0021**; 1.6 mV: p=0.0001***] Representative traces of fEPSPs in response to increasing stimulus from 0.4 to 1.6 mA are shown on the top. (B) LTP is impaired in Trem2R47H/R47H rats. Average traces of the baseline and the last 5mins of LTP are shown on top. (C) Plot of fEPSP slope change of the last 10 min of LTP in B (unpaired t test, p<0.0001****). (D) The increase of input- out slope in Trem2R47H/R47H rats is reversed by application of anti-TNF-a [ANOVA for repeated measures F(18, 300)=6.579, p<0.0001****; post-hoc Tukey’s multiple comparisons test: 0.8 mV RH/RH + Isotype vs. w/w + anti-TNF-a p=0.0479*; 1.0 mV RH/RH + Isotype vs. RH/RH + anti-TNF-a p=0.0061**, RH/RH + Isotype vs. w/w + anti-TNFa p=0.0072**, RH/RH + Isotype vs. w/w + Isotype p=0.0113*; 1.2 mV RH/RH + Isotype vs. RH/RH + anti-TNFa p=0.0009***, RH/RH + Isotype vs. w/w + anti-TNF-a p=0.0009***, RH/RH + Isotype vs. w/w + Isotype p=0.0029**; 1.4 mV RH/RH + Isotype vs. RH/RH + anti-TNF-a p<0.0001****, RH/RH + Isotype vs. w/w + anti-TNF-a p<0.0001****, RH/RH + Isotype vs. w/w + Isotype p=P < 0.0001****; 1.6 mV RH/RH + Isotype vs. RH/RH + anti-TNF-a p<0.0001****, RH/RH + Isotype vs. w/w + anti-TNF-a P p<0.0001****, RH/RH + Isotype vs. w/w + Isotype p<0.0001****]. Representative fEPSP traces are shown on top. (E) The impaired LTP is restored by application of anti-TNF-a antibody. The average traces of the baseline and the last 5mins of LTP are shown on top. (F) Plot of fEPSP slope change of the last 10 min of LTP in E [one-way ANOVA, F(3,

Article Snippet: Aging Cell 18: e13033 Rat App allele with humanize Ab region Genetic reagent (Rattus Norvegicus) Trem2R47H Tambini and D’Adamio, 2020 Sci Rep 10: 4122 Rat Trem2 allele with R47H mutation Commercial assay or kit V-PLEX Plus Ab Peptide Panel 1 Meso Scale Discovery Cat# K15200G Used following manufacturer’s recommendations Commercial assay or kit V-PLEX Proinflammatory Panel 2 Meso Scale Discovery Cat# K15059D Used following manufacturer’s recommendations Commercial assay or kit CD11b/c (Microglia) Micro-Beads, rat antibody Cat# 130-105-634 Miltenyi Biotec RRID:AB_2783886 Used following manufacturer’s recommendations Commercial assay or kit Adult Brain Dissociation Kit Miltenyi Biotec Cat# 130-107-677 Used following manufacturer’s recommendations Commercial assay or kit RNeasy RNA Isolation kit Qiagen Cat# 74106 Used following manufacturer’s recommendations Commercial assay or kit High-Capacity cDNA RT kit Thermo Cat# 4368814) Used following manufacturer’s recommendations Commercial assay or kit TaqMan Fast Advanced Mix Thermo Cat# 4444556 Used following manufacturer’s recommendations Commercial assay or kit Gapdh RealTime PCR Thermo Rn01775763_g1 Used following manufacturer’s recommendations Commercial assay or kit Treml1 RealTime PCR Thermo Rn01511908_g1 Used following manufacturer’s recommendations Antibody Polyclonal Goat IgG anti-Rat TNFa Cat# AF-510-NA R and D Systems RRID:AB_354511 10 ng/ml in ACSF Antibody Polyclonal Goat IgG. antibody Cat# AB-108-C R and D Systems RRID:AB_354267 10 ng/ml in ACSF Software, algorithm LinRegPCR software hartfaalcentrum.nl Software, algorithm pCLAMP10 software Molecular Devices, Software, algorithm Image Lab software Biorad RRID:SCR_014210 Software, algorithm GraphPad Prism RRID:SCR_002798 Ren et al. eLife 2020;9:e57513.

Techniques: Mutagenesis

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) expression increased in human umbilical artery endothelial cells (HUAECs), human umbilical vein endothelial cells (HUVECs), and rat brain microvascular endothelial cells (RBMECs) stimulated with VEGF. (A,B) Endothelial cell (EC) migration distance was evaluated by scratch assay. ECs were treated with a dose curve of RGMa (500–3000 ng/ml), and images were taken at the beginning and 12 h. (C,D) ECs were treated with RGMa (2 µg/ml), and lysates were collected over a time course of 50 min. The amount of phosphorylated focal adhesion kinase (p-FAK) protein was visualized with western blot analysis. (E,F) ECs were treated with VEGF (50 ng/ml), and lysates were collected over a course of 120 min. p-FAK protein levels were visualized with western blot analysis. (G–I) Quantitative real-time polymerase chain reaction showed RGMa mRNA level was upregulated in HUAECs, HUVECs, and RBMECs exposed to VEGF (50 ng/ml) for 30 min. (J–L) RGMa protein levels were visualized in HUAECs, HUVECs, and RBMECs exposed to VEGF at 30 min by western blot analysis. Data in bar graphs represent the means ± SD of ≥4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, # VS 1500 ng/ml, ### P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Migration, Wound Healing Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) suppressed VEGF expression, phosphorylation of focal adhesion kinase (FAK), proliferation, migration, and tube formation in ECs. (A,B) Lysate was collected, and VEGF was detected by western blot in human umbilical artery endothelial cells (HUAECs) treated with RGMa (2 µg/ml); (C–E) ELISA kit assay showed VEGFA decreased in endothelial cell (EC)-culture supernatant exposed to RGMa compared with cell-culture supernatant from control group. (F–H) Cell proliferation was evaluated with cell-counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays (I,J) . RGMa decreased proliferation of ECs stimulated and unstimulated with VEGF. (K,L) FAK (Tyr397) phosphorylation was measured with western blot in HUAECs treated with vehicle, RGMa (2 µg/ml), VEGF (50 ng/ml), or VEGF plus RGMa. (M–P) ECs were grown to 100% confluence, serum-starved overnight, wounded with a sterile pipette tip to remove cells, and treated with control, RGMa, VEGF, or VEGF plus RGMa. Photographs (40×) were taken at 12 h after injury. Wound closure of ≥3 wells was quantified and reported as mean ± SD. (Q–T) Migration activity of ECs treated with RGMa, VEGF, VEGF plus RGMa, or control was measured with transwell assay. Photographs (200×) were taken 18 h after treatment. (U–X) HUAECs were starved overnight, treated as indicated, and seeded into 96-well plates coated with Matrigel. Photographs (40×) were taken at 3 h after treatment. The number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Phospho-proteomics, Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Cell Counting, Sterility, Transferring, Activity Assay, Transwell Assay

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited angiogenesis in vitro via neogenin. (A) Human umbilical artery endothelial cells (HUAECs) were transfected with CRISPR/Cas9 neogenin knockout kit and purified with puromycin, then the result of neogenin knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or neogenin gRNA were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa. The focal adhesion kinase (FAK) (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or neogenin gRNA were determined by scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: In Vitro, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Phospho-proteomics, Migration

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Unc5b is involved in the effect of repulsive guidance molecule a (RGMa) on phosphorylation of focal adhesion kinase (FAK), migration, and tube formation in human umbilical artery endothelial cells (HUAECs). (A) HUAECs were transfected with CRISPR/Cas9 Unc5b knockout kits and purified with puromycin, then the effect of Unc5b knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or Unc5b gRNA were treated with control, RGMa, VEGF, or VEGF plus RGMa. FAK (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or Unc5b gRNA were determined with scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length was analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Phospho-proteomics, Migration, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Control

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited cytoskeleton reassembly, filopodia, and lamellipodia formation in human umbilical artery endothelial cells (HUAECs) via neogenin and Unc5b. Before the immunofluorescence experiment, HUAECs were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa for 40 min. F-actin was stained with phalloidin conjugated with FITC and phosphorylated focal adhesion kinase (p-FAK) connected with primary antibody was labeled with Alexa Fluor 555 donkey anti-rabbit (H + L) secondary antibody. (A) Immunofluorescence showed the cytoskeleton (green) change and p-FAK (red) distribution. (B) Immunofluorescence showing the cytoskeleton change of HUAECs transfected with SgRNA or neogenin gRNA. (C) Immunofluorescence showed the cytoskeleton change of HUAECs transfected with SgRNA or Unc5b gRNA. The filopodia are indicated as sharp spikes (arrowhead), and lamellipodia (arrow) are indicated as flat intensive staining. The merged images are shown in the upper panels, and the amplified indicated areas are shown in the lower panel for different groups. Photographs were obtained with laser scanning confocal microscopy (Nikon, A1 + R, magnification 400×). Results shown are representative images of ≥4 independent experiments.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Immunofluorescence, Staining, Labeling, Transfection, Amplification, Confocal Microscopy

Journal: Journal of neuroinflammation

Article Title: Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

doi: 10.1186/s12974-016-0531-9

Figure Lengend Snippet: Fig. 3 Expression of ROS-related molecules and contribution of NOX enzymes to myelin phagocytosis and ROS production. a Expression of ROS-related molecules. Rat microglia were stimulated with cytokines for 24 h with or without a subsequent 6-h exposure to myelin (plus or minus sign indicates presence/absence of myelin), as in Figs. 1a and 2a. b Effect of NOX inhibition on phagocytosis of myelin fragments. The activation treatments, assay, and normalization of data were the same as Fig. 2b, but now comparing the pan-NOX inhibitor, 5 μM VAS2870. c Effect of NOX inhibition on ROS levels in microglia that were treated as in Fig. 2b. As above, ROS levels were normalized to unstimulated microglia without myelin (dashed line), data are expressed as mean ± SEM (n = 6 individual cultures), and results were analyzed by two-way ANOVA with Bonferroni’s post hoc test. The comparisons are as follows: asterisk unstimulated (CTL) versus different activation states in the absence of myelin, dagger sign unstimulated (CTL) versus different activation states in the presence of myelin, number sign effects of myelin (a) or VAS2870 (b, c) within a particular activation state. One symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001

Article Snippet: To induce different activation states, microglia were exposed to recombinant rat cytokines (R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Inhibition, Activation Assay

Journal: Journal of neuroinflammation

Article Title: Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

doi: 10.1186/s12974-016-0531-9

Figure Lengend Snippet: Fig. 8 Transcript expression of K+ channels and SOCE-related genes is affected by the microglial activation state and myelin phagocytosis. a Single cytokines with or without myelin. Microglia were stimulated for 24 h with 20 ng/mL IFN-γ + 50 ng/mL TNF-α (I + T) or 20 ng/mL IL-4, and then treated with 25 μg/mL myelin for 6 h. b M2a→M1 paradigm. Microglia were treated with 20 ng/mL IL-4 for 2 h followed by I + T for 22 h. c M1→M2 paradigm. Cells were treated with I + T for 2 h followed by IL-4 (M1→M2a) or 20 ng/mL IL-10 (M1→M2c) for 22 h. All data are expressed as mRNA counts in 200 ng total RNA (mean ± SEM) for six individual cultures. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. The dashed lines in b and c indicate expression levels in unstimulated (control) microglia. Comparisons are as follows: asterisk differences from control microglia, dagger sign CTL versus different activation states in the presence of myelin, number sign effects of myelin within a particular activation state (a) or effects of a second stimulus on the first stimulus (b and c). One symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001

Article Snippet: To induce different activation states, microglia were exposed to recombinant rat cytokines (R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Activation Assay, Control