Journal: Glia

Article Title: Alzheimer's disease‐associated R47H TREM2 increases, but wild‐type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss

doi: 10.1002/glia.24318

Figure Lengend Snippet: Summary of qPCR primers

Article Snippet: For detection of human TREM2 on CHME‐3 cells and BV‐2 cells, rat anti‐human/mouse TREM2 (clone: 237920) conjugated to Phycoerythrin (PE) fluorophore (R&D systems, FAB17291P) and isotype control, monoclonal rat IgG2B (clone: 141945) conjugated to PE fluorophore (R&D systems, IC013P) were used.

Techniques:

Journal: Glia

Article Title: Alzheimer's disease‐associated R47H TREM2 increases, but wild‐type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss

doi: 10.1002/glia.24318

Figure Lengend Snippet: Overexpression of R47H hTREM2 increases BV‐2 phagocytic uptake of phosphatidylserine beads and synaptosomes compared with WT hTREM2 and TREM2 KO. (a, b, c) Microscopy images of BV‐2 microglia expressing eGFP (green) co‐cultured with (a) Carboxylated beads (red). (b) Rhodamine‐phosphatidylserine beads (red) and (c) pH‐rodo labeled synaptosomes (red). (d, e, f) Phagocytic uptake of three phagocytic targets was measured using flow cytometry. (d) Phagocytic uptake of sky‐blue 5 um carboxyl‐latex beads analyzed after 2 h co‐culture and shown as % uptake. (e) Rhodamine labeled phosphatidylserine beads analyzed after 1.5 h co‐culture shown as % uptake (f) synaptosomes isolated from rat and stained with pH‐rodo analyzed after 1.5 h co‐culture and shown as mean fluorescence intensity (MFI). (d, e, f) Cells were pre‐treated with the phagocytosis inhibitor cytochalasin D (CytoD) as a negative control. The CytoD negative control values have been removed from the data points presented. Each data point is the mean result from an independent experiment with three technical repeats. Co‐localization of phagocytic target in red with green BV‐2 cells shows target that has most likely been phagocytosed. Phagocytosis was quantified using flow‐cytometry and appropriate controls. Paired data points from the same experiment are represented by the same shape and color within a figure. Error bars are SEM and Tukey's multiple comparisons one‐way ANOVA was performed (d) KO Control versus WT hTREM2 ** P = .0038, KO Control versus R47H hTREM2 ** P = .0066 (e) KO Control versus R47H hTREM2 ** P = .0066, WT hTREM2 versus R47H hTREM2 * P = .0333, KO Control versus WT hTREM2 ns = 0.3663 (f) KO Control versus R47H hTREM2 * P = .0127, WT hTREM2 versus R47H hTREM2 * P = .0209, KO Control versus WT hTREM2 ns = 0.9352.

Article Snippet: For detection of human TREM2 on CHME‐3 cells and BV‐2 cells, rat anti‐human/mouse TREM2 (clone: 237920) conjugated to Phycoerythrin (PE) fluorophore (R&D systems, FAB17291P) and isotype control, monoclonal rat IgG2B (clone: 141945) conjugated to PE fluorophore (R&D systems, IC013P) were used.

Techniques: Over Expression, Microscopy, Expressing, Cell Culture, Labeling, Flow Cytometry, Co-Culture Assay, Isolation, Staining, Fluorescence, Negative Control, Control

Journal: Glia

Article Title: Alzheimer's disease‐associated R47H TREM2 increases, but wild‐type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss

doi: 10.1002/glia.24318

Figure Lengend Snippet: Overexpression of WT TREM2 in CHME‐3 cells reduces phagocytosis of synaptosomes and phosphatidylserine coated beads. Phagocytosis of (a) Synaptosomes isolated from rat and stained with pH‐rodo analyzed after 1 h of treatment and shown as mean fluorescence intensity (MFI) and (B) rhodamine‐labeled phosphatidylserine‐coated beads analyzed after 1 h of treatment and shown as MFI. Overexpression of WT TREM2 decreased uptake of both targets compared to control. R47H expression also decreased uptake of synaptosomes and phosphatidylserine‐coated beads but did so less than WT TREM2. Repeats done on the same day with the different cell lines are depicted by the same color and symbols. (a & b) N = 3, statistics: repeated measures, one way ANOVA, followed by Tukey's post hoc test.* p < .05, ** p < .01.

Article Snippet: For detection of human TREM2 on CHME‐3 cells and BV‐2 cells, rat anti‐human/mouse TREM2 (clone: 237920) conjugated to Phycoerythrin (PE) fluorophore (R&D systems, FAB17291P) and isotype control, monoclonal rat IgG2B (clone: 141945) conjugated to PE fluorophore (R&D systems, IC013P) were used.

Techniques: Over Expression, Isolation, Staining, Fluorescence, Labeling, Control, Expressing

Journal: Glia

Article Title: Alzheimer's disease‐associated R47H TREM2 increases, but wild‐type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss

doi: 10.1002/glia.24318

Figure Lengend Snippet: TREM2 induces an increase in CST7 mRNA expression and knock down of CST7 increases phosphatidylserine coated bead uptake in TREM2 overexpressing CHME‐3 cells. (a) Data is expressed as fold change over non‐target control and all samples were normalized to 18 S rRNA housekeeping gene. CST7 mRNA level is increased when TREM2 is present and when treated with RNAi against CST7 , CST7 is successfully reduced in all cell lines by at least 50%. RNA was extracted from CHME‐3 microglia 48 h after treatment with non‐target (scrambled) or CST7 targeting RNAi. N = 5. Repeats done on the same day with the three variant cell lines are depicted by the same color and symbols. Statistics: repeated measures, one way ANOVA with Tukey's post hoc test. ** p < .01, *** p < .001. (b) Uptake of rhodamine‐labeled, phosphatidylserine‐coated 3 micron beads by control, WT and R47H TREM2 overexpressing CHME‐3 cells 48 h after treatment with non‐target (scrambled) or CST7 targeting RNAi. Phagocytosis was analyzed after 1 h treatment of CHME‐3 cells with phosphatidylserine‐coated beads. N = 5. Statistics: repeated measures, one way ANOVA with Tukey's post hoc test. * p < .05, *** p < .001.

Article Snippet: For detection of human TREM2 on CHME‐3 cells and BV‐2 cells, rat anti‐human/mouse TREM2 (clone: 237920) conjugated to Phycoerythrin (PE) fluorophore (R&D systems, FAB17291P) and isotype control, monoclonal rat IgG2B (clone: 141945) conjugated to PE fluorophore (R&D systems, IC013P) were used.

Techniques: Expressing, Knockdown, Control, Variant Assay, Labeling

Journal: Glia

Article Title: Alzheimer's disease‐associated R47H TREM2 increases, but wild‐type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss

doi: 10.1002/glia.24318

Figure Lengend Snippet: Overexpression of R47H TREM2 mutant in CHME‐3 microglia leads to increased neuron (LUHMES) loss in co‐culture with neurons. (a) Image of differentiated LUHMES cells. (b) Image of CHME‐3 cells. (c) Image of LUHMES (blue) and CHME‐3 (green) co‐culture. (d) Percentage neuron loss in LUHMES:CHME‐3 (100,000: 100,000) co‐culture after treatment ±2 μM Aβ for 48 h. Percentage loss is compared to neuron only control. Number of neurons was identified via blue CellTrace Violet staining and counted using automated software. N = 4. Statistics: two‐way ANOVA, with Sidak's post hoc test. * p < .05, *** p < .001, **** p < .0001.

Article Snippet: For detection of human TREM2 on CHME‐3 cells and BV‐2 cells, rat anti‐human/mouse TREM2 (clone: 237920) conjugated to Phycoerythrin (PE) fluorophore (R&D systems, FAB17291P) and isotype control, monoclonal rat IgG2B (clone: 141945) conjugated to PE fluorophore (R&D systems, IC013P) were used.

Techniques: Over Expression, Mutagenesis, Co-Culture Assay, Control, Staining, Software

Journal: Glia

Article Title: Alzheimer's disease‐associated R47H TREM2 increases, but wild‐type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss

doi: 10.1002/glia.24318

Figure Lengend Snippet: pSYK activation by liposomes & antibodies in hiPSC‐derived microglia. hiPSC‐derived microglia expressing homozygous wild‐type or R47H TREM2 were exposed to the indicated concentrations of (a) phosphatidylserine‐containing liposomes, (b) phosphatidylserine‐containing liposomes or (c) anti‐TREM2 antibodies, and phosphorylated SYK (pSYK) measured by alphaLISA, 2 min after adding liposomes or 5 min after adding antibodies. pSYK is normalized to the level before addition of liposomes or antibodies. The experiment was repeated on 3 separate occasions, of which means and SEM are represented. Significant differences between WT and R47H TREM2 expressing cells were tested by unpaired t test at each concentration, * p < .05.

Article Snippet: For detection of human TREM2 on CHME‐3 cells and BV‐2 cells, rat anti‐human/mouse TREM2 (clone: 237920) conjugated to Phycoerythrin (PE) fluorophore (R&D systems, FAB17291P) and isotype control, monoclonal rat IgG2B (clone: 141945) conjugated to PE fluorophore (R&D systems, IC013P) were used.

Techniques: Activation Assay, Liposomes, Derivative Assay, Expressing, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Schematic representation of TREM2 processing by ADAM10/17. Cleavage occurs C‐terminal of residue His 157. The entire ectodomain (residues 19–171) was used for immunization of rats to generate TREM2 antibodies. CTF, C‐terminal fragment; sTREM2, soluble TREM2. Immunoblot analysis of membrane fractions of HEK293 Flp‐In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane‐bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. Calnexin served as a loading control. Levels of membrane‐bound TREM2 were quantified by MSD ELISA. Data represent the mean ± SEM ( n = 3). One‐way ANOVA, Tukey's post hoc test; P (DMSO vs GM) = 0.0011; P (DMSO vs isotype) = 0.992; P (isotype vs 4D9) = 0.0005; n.s., not significant. Immunoblot analysis of conditioned media from HEK293 Flp‐In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. sAPPα served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated. Levels of sTREM2 were quantified by MSD ELISA. Data represent the mean ± SEM ( n = 3). One‐way ANOVA, Tukey's post hoc test; P (DMSO vs GM) < 0.0001; P (DMSO vs isotype) = 0.6372; P (isotype vs 4D9) < 0.0001; n.s., not significant. 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12. An anti‐HA antibody was used as a positive control, while empty vector‐transfected HEK293 Flp‐In cells were used as a negative control. Scale bar = 10 μm. Peptide ELISAs detect anti‐mouse TREM2 antibody binding to tiled stalk region peptides, full‐length stalk peptide, or a truncated ADAM cleavage site peptide. The binding epitope of 4D9 antibody is located 12‐amino acids N‐terminal of the ADAM cleavage site at His 157. Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope (upper panel). Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 (lower panel). 4D9 binding to the mouse TREM2 ECD is competed off by a stalk region peptide. A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1.3 μM. Data represent the mean ± SEM ( n = 3). ECD, extracellular domain. Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ECD evaluated by Biacore, k on = 5.9 × 10 5 M −1 s −1 , k off = 4.0 × 10 −5 s −1 , K D = 68 pM. 4D9 binding to human TREM2 or mouse TREM1 was undetectable. Data information: Statistical evaluations are displayed as follows: ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Western Blot, Stable Transfection, Inhibition, Negative Control, Enzyme-linked Immunosorbent Assay, Positive Control, Plasmid Preparation, Transfection, Binding Assay, Sequencing, Titration, SPR Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Flow cytometry dose–response curve for cell binding of 4D9 mAb (EC50 = 0.29 nM), 4D9 Fab (EC50 = 0.17 nM), and isotype to HEK cells stably overexpressing mouse TREM2. Data represent the mean ± SEM ( n = 2). In vitro ADAM17 sheddase activity is blocked by 4D9‐effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM‐conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab, and isotype control. Data represent the mean ± SEM ( n = 6). One‐way ANOVA, Tukey's post hoc test; P (4D9 Fab vs 4D9 mAb) = 0.8855; P (4D9 Fab vs uncleaved) < 0.0001; P (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant. ELISA‐mediated quantification of sTREM2 in conditioned media from HEK293 cells stably overexpressing mouse TREM2 treated with a dose titration of 4D9 mAb (EC50 = 2.3 nM), 4D9 Fab, or isotype for 18 h. Data represent the mean ± SEM ( n = 3). AlphaLISA‐mediated quantification of p‐SYK levels in HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb, 4D9 Fab, or isotype for 5 min. Data represent the mean ± SEM ( n = 3). AlphaLISA‐mediated quantification of p‐SYK levels in HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1 mg/ml POPC/POPS liposomes and 20 μg/ml 4D9 mAb, 4D9 Fab, or isotype for 5 min. p‐SYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM ( n = 3). Two‐way ANOVA, Tukey's post hoc test (cell line effect: F 1,16 = 365.7, P ≤ 0.0001; treatment effect: F 3,16 = 39.35, P < 0.0001; cell line × treatment effect: F 3,16 = 38.75, P < 0.0001); P (no Ab vs isotype) = 0.6218; P (no Ab vs 4D9 mAb) < 0.0001; P (no Ab vs 4D9 Fab) = 0.7301; P (isotype vs 4D9 mAb) < 0.0001; P (isotype vs 4D9 Fab) > 0.9999; P (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant. Schematic representation of the proposed mechanism of action of antibody 4D9. Binding of 4D9 to TREM2 leads to receptor clustering on the cell surface, thereby driving downstream p‐SYK signaling. At the same time, cell‐surface levels are enhanced by inhibition of ectodomain shedding, potentially because of the inability of proteases to cleave dimeric substrates. Data information: Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Stable Transfection, In Vitro, Activity Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Titration, Plasmid Preparation, Inhibition

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Flow cytometry detection of cell‐surface binding of 4D9 (blue) and isotype (green) to wild‐type and Trem2 − / − mouse bone marrow‐derived macrophages. Representative histograms are shown as MFI (mean fluorescent intensity). Graphs represent the median signal intensity ± SEM ( n = 3). Student's t ‐test (two‐tailed); Trem2 +/+ : P = 0.0005; Trem2 −/− : P = 0.4435; n.s., not significant. Immunoblot analysis of membrane fractions of bone marrow‐derived macrophages (BMDMs) upon treatment with 4D9 antibody reveals increased levels of membrane‐bound TREM2 relative to an isotype antibody. BMDMs from TREM2 knockout mice were included to show the specificity of the anti‐TREM2 antibody. Calnexin served as a loading control. Levels of membrane‐bound TREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM ( n = 5–6). Unpaired t ‐test (two‐tailed) with Welch's correction; P < 0.0001. No gender‐specific differences could be observed. Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies. BMDMs from TREM2 knockout mice were included to show the specificity of the anti‐TREM2 antibody. Soluble APP served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows: Ab hc, antibody heavy chain; Ab lc, antibody light chain. Levels of sTREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM ( n = 6). Unpaired t ‐test (two‐tailed) with Welch's correction; P < 0.0001. No gender‐specific differences could be observed. Wild‐type, Trem2 +/− , and Trem2 −/− BMDMs were plated in low concentration of M‐CSF on 4D9 or isotype‐coated wells for 5 days, and cellular ATP levels were measured by luminescence detection to indicate cell viability. 4D9 and isotype data for each genotype represent the mean ± SEM ( n = 2 and n = 1, respectively). Data information: Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Derivative Assay, Two Tailed Test, Western Blot, Knock-Out, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: A Flow cytometry detection of cell‐surface 4D9 (red) binding and isotype control (blue) on primary wild‐type and Trem2 − / − mouse microglia. Data are shown as mean fluorescent intensity (MFI). B Uptake assay for fluorescently labeled myelin, Aβ(1–42), and inactivated Escherichia coli particles. The top row shows that myelin and Aβ (pseudocolored in white) accumulate within the plasma membrane of primary microglia (labeled with DyLight 649 isolectin and pseudocolored in red). Overnight, pre‐treatment with antibody 4D9 significantly increased the percentage of substrate‐positive cells (middle row). Pre‐treatment with the isotope control did not affect the uptake rate (bottom row). No changes in the uptake of E. coli particles were observed. Substrate uptake in the absence of antibody is shown in the top row. Hoechst 33342 was used to counter stain the nuclei (in cyan) and to assess cell density. Scale bar = 20 μm. C–E Quantification of the change in uptake of myelin (C), Aβ(1–42) (D), and E. coli (E) upon antibody treatment relative to the uptake of the respective substrate in the absence of antibody. The number of substrate‐positive cells relative to the total number of cells was quantified in each condition. Data represent the mean ± SEM ( n = 3). Two‐way ANOVA (myelin), Sidak's multiple comparisons test (time effect: F 1,4 = 0.01123, P = 0.9207; treatment effect: F 1,4 = 27.98, P = 0.0061; time x treatment effect: F 1,4 = 0.5948, P = 0.4836); P (1 h) = 0.1362; P (2 h) = 0.0185; unpaired t ‐test (two‐tailed) with Welch's correction (Aβ(1–42) and E. coli ); P (Aβ(1–42)) = 0.0151; P ( E. coli ) = 0.5751; n.s., not significant. Data information: Statistical evaluations are displayed as follows: * P < 0.05.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Labeling, Staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: 4D9 demonstrates standard IgG pharmacokinetics in vivo . Peripheral clearance rates of 4D9 on a human IgG‐effectorless backbone compared with an isotype control were determined in wild‐type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 h, and 4 and 7 days after 10 mg/kg intravenous injection of antibody. Brain antibody concentration was measured 24 h post‐intravenous dosing of isotype hIgG at 100 mg/kg, and 4D9‐hIgG dosed intravenously at 100, 50, and 10 mg/kg. Detection of hIgG levels by ELISA demonstrated dose‐dependent brain concentrations of 4D9 in the single digit nM range. Animals were perfused to minimize IgG contribution from the plasma. Data represent the mean ± SEM ( n = 5). Schematic depicting the TE assay setup for bound and total sTREM2 detection. For the bound assay, a secondary anti‐human IgG detects 4D9 antibody bound to soluble TREM2 in CSF and plasma. For the total assay, a saturating amount of 4D9 antibody is added to eliminate unbound sTREM2. The ratio of 4D9‐bound sTREM2 to total sTREM2 is calculated to determine the level of TE achieved by the concentration of antibody present. Target engagement time course demonstrated near 100% 4D9‐bound sTREM2 in CSF of wild‐type mice at 24 h post‐dose. Over a 10‐day time course with time points at days 1, 2, 4, and 7 and study termination at day 10, the bound/total sTREM2 reduces gradually to reach ˜ 50% by day 10. Animals were dosed intravenously with 50 mg/kg of isotype and 4D9 antibodies. Data represent the mean ± SEM ( n = 5). Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10 mg/kg with > 50% bound sTREM2 at 1 mg/kg of antibody. 4D9‐bound sTREM2 was undetectable at 0.1 mg/kg and lower. 4D9 was IV‐dosed at 50, 10, 1, 0.1, 0.01, 0.001, and 0.0001 mg/kg, and isotype‐dosed at 50 mg/kg. CSF bound: total sTREM2 in wild‐type mice was measured at 24 h. Data represent the mean ± SEM ( n = 5). 4D9 antibody demonstrates a dose‐dependent increase in total brain TREM2 levels. Quantification of total TREM2 in brain lysates from wild‐type mice dosed with 4D9 or isotype control was performed by a MSD‐platform‐based ELISA. Data represent the mean ± SEM ( n = 5) and are shown as pg TREM2 per ug of total protein. One‐way ANOVA, Dunnett's post hoc test, P (isotype vs 4D9 [100 mg/kg]) < 0.0001; P (isotype vs 4D9 [50 mg/kg]) = 0.0007. Data information: Only male mice were used. Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Schematic outlining study design and timeline of intraperitoneal injections of either isotype control of 4D9 antibody in 6‐month‐old APP‐NL‐G‐F and age‐matched WT mice. Immunohistochemical costainings of TREM2 (magenta) and IBA1 (green) microglia in the cortex of isotype control and 4D9‐injected WT and APP‐NL‐G‐F mice. Side panel shows images from each staining at a larger magnification indicated by the dotted white boxes. Scale bar = 10 μm; scale bar (inset) = 2.5 μm. Quantification of cortical TREM2 stainings shown in (B). Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,23 = 70.63, P ≤ 0.0001; treatment effect: F 1,23 = 14.33, P = 0.0010; genotype × treatment effect: F 1,23 = 14.51, P = 0.0009); P (WT isotype vs WT 4D9) > 0.9999; P (WT isotype vs APP isotype) = 0.02; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.0001; n.s., not significant. Quantification of cortical IBA1 staining shown in B. Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,21 = 215.4, P ≤ 0.0001; treatment effect: F 1,21 = 0.5994, P = 0.4475; genotype × treatment effect: F 1,21 = 0.2992, P = 0.5902); P (WT isotype vs APP isotype) < 0.0001; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.9986; n.s., not significant. Confocal images of P2RY12 (red), IBA1 (green), and Aβ (gray) costainings from cortex. Top panel: Dotted white boxes indicate the areas in P2RY12 and IBA1 costainings that are magnified as inset. Bottom panel: Insets show P2RY12, IBA1, and Aβ costainings at a larger magnification. Of note, we did not observe a complete co‐localization of P2RY12 and IBA1 suggesting different microglial populations. Scale bar = 10 μm; scale bar (insets) = 5 μm. Quantification of P2RY12‐stained cells in the cortex shown in (E). Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,23 = 71.99, P ≤ 0.0001; treatment effect: F 1,23 = 9.029, P = 0.0063; genotype × treatment effect: F 1,23 = 5.93, P = 0.0231); P (WT isotype vs APP isotype) = 0.0018; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.0051. Data information: Each data point for all quantifications represents an average of n = 3 replicates per mouse. All data represent the mean ± SEM ( n = 6–7). No gender‐specific differences could be observed. Statistical evaluations are displayed as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Immunohistochemical staining, Injection, Staining