Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: TREM2 agonism induces PLC activity in iPSC- derived macrophages. ( A ) Representative Western blot showing phosphorylation of SYK after TREM2 stimulation for 5 min using a TREM2-specific antibody (AF1828, R&D Systems) in Parent and TREM2 Ko cells. Full immunoblot images are presented in Supplementary Fig. 6. ( B ) Ca 2+ flux induced by TREM2 ligation in Parent cells is absent in TREM2 Ko cells. n = 4. ( C) SYK inhibitor BIIB-057 (SYKi) reduces TREM2 antibody-evoked Ca 2+ signal in a dose-dependent manner in Parent cells. n = 3. ( D) TREM2-induced IP1 accumulation determined by HTRF assay is prevented in TREM2 Ko cells. n = 3, data shown represent mean ± SEM, ( B , D ) two-way ANOVA followed by Bonferroni’s multiple comparison test, ( C ) one-way ANOVA followed by Bonferroni’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To induce TREM2 signalling, cells were stimulated with a goat polyclonal human TREM2 antibody (AF1828, R&D systems) at 1, 2, 5 or 10 μg/ml for 5 min at 37 °C.

Techniques: Activity Assay, Derivative Assay, Western Blot, Phospho-proteomics, Ligation, HTRF Assay, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: PLCγ2 Ko leads to dysregulation of cell surface marker expression and morphological changes in iPSC-derived macrophages. ( A ) Western blot confirms lack of PLCγ2 protein in PLCγ2 Ko and shows reduction in TREM2 expression in PLCγ2 KO macrophages compared to Parent. n = 4. Full immunoblot images are presented in Supplementary Fig. 7. ( B ) Levels of soluble TREM2 detected in cell supernatant 8 days after plating are lower in PLCγ2 Ko cells compared to Parent. n = 4, ( C ) Macrophage surface markers CD11b, CD14, and CD45 were measured by flow cytometry, compared to relevant isotype IgG. Annotations indicate frequency of marker positivity in the Parent line. ( D) Morphology of macrophage lines was determined by phalloidin staining and analysis of cell roundness and cell area. Scale bar 50 μm. n = 4, data shown represent mean ± SEM, One-way ANOVA followed by Bonferroni’s multiple comparison test. * p < 0.05, ** p < 0.01.

Article Snippet: To induce TREM2 signalling, cells were stimulated with a goat polyclonal human TREM2 antibody (AF1828, R&D systems) at 1, 2, 5 or 10 μg/ml for 5 min at 37 °C.

Techniques: Marker, Expressing, Derivative Assay, Western Blot, Flow Cytometry, Staining, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: PLCγ2 regulates TREM2-mediated signalling in iPSC- derived macrophages. ( A ) Representative Western blots showing phospho-SYK after TREM2 ligation using AF1828 in Parent and PLCγ2 Ko cells. ( B ) Quantification of SYK phosphorylation after TREM2 ligation shows no effect of PLCγ2 deficiency, n = 4. Full immunoblot images are presented in Supplementary Fig. 9. ( C ) pSYK HTRF assay confirms similar phosphorylation levels upstream of PLCγ2 upon TREM2 stimulation in Parent and PLCγ2 Ko cells, n = 3. ( D ) Ca 2+ flux induced by TREM2 ligation is prevented in PLCγ2 Ko cells. n = 3 ( E ) PLCγ2 deficiency abolishes TREM2-induced IP1 production as determined by HTRF assay. n = 3 ( F ) Representative Western blots and ( G ) quantification of ERK1/2 phosphorylation shows no major difference between Parent and PLCγ2 Ko cells. Full immunoblot images are presented in Supplementary Fig. 10. Data shown represent mean ± SEM, two-way ANOVA followed by Bonferroni’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To induce TREM2 signalling, cells were stimulated with a goat polyclonal human TREM2 antibody (AF1828, R&D systems) at 1, 2, 5 or 10 μg/ml for 5 min at 37 °C.

Techniques: Derivative Assay, Western Blot, Ligation, Phospho-proteomics, HTRF Assay, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: Primary antibodies used for Western blot.

Article Snippet: To induce TREM2 signalling, cells were stimulated with a goat polyclonal human TREM2 antibody (AF1828, R&D systems) at 1, 2, 5 or 10 μg/ml for 5 min at 37 °C.

Techniques: Western Blot

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: (a) Antibody schematic comparing human specific ATV:TREM2 and mouse specific ATV:4D9 with high affinity TREM2 binding. (b) Epitope map of overlapping stalk binding regions for ATV:TREM2 and ATV:4D9 Fabs (space filled model of TREM2 ECD ). The binding epitope of ATV:4D9 antibody is located 12-amino acids N-terminal of the ADAM cleavage site at His157. (c) FACS analysis of cell binding of ATV:TREM2 to hTREM2-DAP12 HEK293 or parental cells. Endogenous TfR expression on HEK293 cells drives weak binding observed for ATV:ISO and ATV:TREM2 (n = 3 independent experiment, mean ± SEM). ( d ) FACS detection of ATV:TREM2 (100 nM) binding to WT and TREM2 KO iMG with isotype control (ATV:ISO). (e) Soluble TREM2 measured in the supernatant of hTREM2-DAP12 HEK293 cells dosed with ATV:TREM2 for 24 h shows dose-dependent reduction of sTREM2 to levels similar to 1 uM GM6001 (n = 3 independent experiment, mean ± SEM). ( f ) ATV:TREM2 and lipid ligands induce pSyk signaling in iMG 24 h post antibody exposure (n = 3 independent experiments, Tukey’s multiple comparisons test, mean ± SEM). (g) Human monocytes cultured in limited M-CSF with plate coated ATV:TREM2 or ATV:ISO shows dose-dependent activity of ATV:TREM2 (EC50 0.95 +/− 0.45 nM). Representative data from one out of four human donors are shown.

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Binding Assay, Expressing, Control, Cell Culture, Activity Assay

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: Comparative TREM2 antibody binding affinity and cellular activity

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Binding Assay

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: a , Antibody schematic of ATV:TREM2 with human TREM2 Fab affinity and ATV binding site in the Fc domain and effectorless Fc mutations. b , Fluorescence polarization (FP)-based detection of TREM2 stalk peptide cleavage by recombinant ADAM17 ( n = 4 independent experiments; two-tailed unpaired t -test, mean ± s.e.m.). c , ATV:TREM2 and PS liposome co-treatment enhanced pSyk in WT iMG ( n = 3 independent experiments; Tukey’s multiple comparisons test, mean ± s.e.m.). d , Schematic illustrating ATV and TREM2 Fab valency effects on pSyk signaling. Antibodies include anti-TREM2 and ATV:TREM2 with MV and BV Fabs. e , hTREM2-DAP12 HEK293 cells treated with a dose response of antibodies for 5 minutes, followed by pSyk detection ( n = 3 independent experiments; mean ± s.e.m.). f , pSyk is blocked by co-treatment of ATV:TREM2 and anti-TfR. hTREM2-DAP12 HEK293 cells were dosed with 100 nM TREM2 antibodies and a titration anti-TfR. pSyk was detetced 5 minutes after treatment ( n = 3 independent experiments; mean ± s.e.m.). g , TfR and TREM2 co-IP. hTREM2-DAP12 HEK293 cells were treated with 100 nM per antibody for 5 minutes, followed by IP on cell lysates with anti-TREM2. TfR was detected by western blot. h , Co-IP quantification of western blot data in g ( n = 6 independent experiments; Wilcoxon test for ISO versus anti-TREM2, two-tailed paired t -test for ATV:ISO versus ATV:TREM2, mean ± s.e.m.). i , Schematic of BioID TREM2 receptor clustering assay strategy. j , Representative western blot detection of biotinylated TREM2 after streptavidin IP. TREM2-BioID expression was induced 24 hours before the assay with 2 ng ml −1 of Dox. Cells were treated with 100 nM antibody and 2 µM biotin. k , Quantification of western blot from j ( n = 4 independent experiments; ratio of two-tailed paired t -test, mean ± s.e.m.). l , Representative immunofluorescence images of hTREM2-DAP12 HEK293 cells stained for IgG (green), pSyk (yellow) and EEA1 (red). Cells were treated with 10 nM per antibody for 10 min. m , Quantification of spot intensity for IgG and pSyk immunofluorescence per cell ( n = 3 independent experiments with 3,000–5,000 cells per condition; Tukey’s multiple comparisons test, mean ± s.e.m.). n , Quantification of percent of IgG or pSyk spots localized within EEA1 + endosomes ( n = 3 independent experiments with 3,000–5,000 cells per condition; Tukey’s multiple comparisons test, mean ± s.e.m.).

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Binding Assay, Fluorescence, Recombinant, Two Tailed Test, Titration, Co-Immunoprecipitation Assay, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: ( a ) Representative Western blot of co-IP of TREM2 with TfR. hTREM2-DAP12 HEK293 cells were treated with 100 nM ATV:TREM2, anti-TREM2, or isotype controls for 5 min at 37 °C. ( b ) Co-IP quantification of Western blot from (A) (n = 6 independent experiments; two-tailed paired t-test for ISO vs anti-TREM2; two tailed Wilcoxon test ATV:ISO vs ATV:TREM2, mean ± SEM). ( c ) Schematic illustration of cis- and trans- activation models that could mediate pSyk enhancement by ATV:TREM2. ( d ) Western blot validation of TfR knockdown in the TfR RNAi cell line. ( e ) Cell based cis/trans assay indicates ATV:TREM2 enhances pSyk activity in cis. Relative pSYK signal is expressed as raw pSYK AlphaLisa value normalized to ATV:TREM2 treated control (n = 3 independent experiment, mean ± SEM). (f) Normalized pSyk signal measured by AlphaLisa assay. TfR RNAi cells were treated with 10 nM anti-TREM2 or ATV:TREM2 pre-incubated with a dose titration of recombinant TfR protein for 5 min at 37 °C (n = 3 independent experiment, mean ± SEM). ( g ) Normalized pSyk signal detected by AlphaLisa. TfR RNAi cells were treated with 10 nM anti-TREM2 or ATV:TREM2 pre-incubated with a dose titration of a secondary anti-human IgG Fc antibody for 5 min at 37 °C (n = 3 independent experiment, mean ± SEM) (h) Immunofluorescence microscopy of hTREM2-DAP12 HEK293 cells demonstrates reduction of surface TREM2 levels with ATV:TREM2 vs anti-TREM2, no changes in total TREM2 levels, consistent with re-distribution of the receptor from the plasma membrane to endosomes (n = 4 independent experiments except for anti-TREM2 MV (n = 3), Tukey’s multiple comparisons test, mean ± SEM). (i) hTREM2-DAP12 HEK293 cells dosed with antibody for 10 minutes shows that at similar amounts of bound antibody detected by anti-IgG (representing 5 nM of ATV:TREM2 and 10 nM of anti-TREM2, n = 4 independent experiments, Tukey’s multiple comparisons test, mean ± SEM (j) Images depicting masking algorithm used to identify whether TfR-Alexa-647 labeled recycling endosomes (rainbow spots in middle images) either contain (green spots in right-most images) or do not contain (red spots in right-most images) IgG spots (white spots in left-most image) upon dosing with anti-TREM2 (top row) or ATV:TREM2 (bottom row) for 10 minutes with 10 nM antibody. (k) Representative images for hTREM2-DAP HEK293 cells dosed with 10 nM antibody for 10 minutes including 20 ug mL −1 TfR-Alexa-647, fixed, permeabilized, and stained with anti-IgG and anti-pSyk. IF shows ATV increased colocalization of antibody with pSyk in early endosomes. (l) Quantification of percent of IgG or pSyk spots localized within Tf-positive endosomes (n = 3 independent experiments, Tukey’s multiple comparisons test, mean ± SEM).

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Two Tailed Test, Activation Assay, Biomarker Discovery, Knockdown, Activity Assay, Control, Incubation, Titration, Recombinant, Immunofluorescence, Microscopy, Clinical Proteomics, Membrane, Labeling, Staining

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: a , Representative western blot images for p-mTOR (S2448), pAKT (S473), pGSK3b (S9) and pRPS6 (S235/236) in WT iMG treated with 100 nM ATV:TREM2 or isotype control. b – e , Quantification of mTOR-S2448 ( b ), AKT-S473 ( c ), RPS6-S235/236 ( d ) and GSK3b-S9 ( e ) phosphorylation levels were normalized to actin. Relative expression was calculated by normalizing to vehicle control (PBS) for each experiment ( n = 10 independent experiments ( b – d ) and n = 9 independent experiments ( e ); two-tailed paired t -test, mean ± s.e.m.). f , ATV:TREM2 induces proliferation in WT iMG but not TREM2 KO iMG. WT or TREM2 KO iMG were treated with 100 nM ATV:TREM2 or isotype control for 96 hours. Forty-eight hours after dose, 20 µM EdU was added to media. The proliferation index was calculated as percentage of EdU + cells normalized to vehicle control (PBS) ( n = 3 independent experiments; two-tailed multiple-paired t -test, mean ± s.e.m.). g , Quantification of WT and PLCG2 KO iMG proliferation. iMG were treated with 100 nM ATV:TREM2 or ATV:ISO ( n = 4 independent experiments (WT) and n = 3 independent experiments ( TREM2 KO); two-tailed multiple-paired t -test, mean ± s.e.m.). h , Representative images of WT iMG proliferation. Cells were treated with vehicle (DMSO) or mTOR inhibitor AZD8055 (AZD). EdU + iMG are marked with open arrow. i , Quantification of WT iMG proliferation treated with mTOR inhibitor AZD8055. 20 nM AZD8055 was co-dosed with 100 nM ATV:TREM2 for 96 hours ( n = 5 independent experiments (DMSO) and n = 4 independent experiments (AZD); two-tailed multiple-paired t -test, mean ± s.e.m.). j , Nuclei quantification of iMG co-treated with ATV:TREM2 and AZD. Relative nuclei count was normalized to vehicle control (PBS) for each experiment ( n = 5 independent experiments (DMSO) and n = 4 independent experiments (AZD); two-tailed unpaired t -test, mean ± s.e.m.). k , RNA-seq of iMG treated for 4 days with PBS, 100 nM ATV:ISO or ATV:TREM2 or 10 ng ml −1 LPS, 20 ng ml −1 TGFβ or 20 ng ml −1 IFNγ. Relative expression (z-scores) of the top-most upregulated or downregulated genes selected from pathways of interest. Pathway definitions were taken from the hallmark MSigDB collection; genes shown are a subset of those found in the leading edge of the gene set for each category.

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Western Blot, Control, Phospho-proteomics, Expressing, Two Tailed Test, RNA Sequencing

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: ( a ) Representative Western blot images of phosphorylation of 4EBP1 (T37/46) and ERK1/2 (T202/Y204) in WT iMGs treated for 96 h with 100 nM ATV:TREM2 or an isotype control. ( b ) Quantification of p4EBP1 (T37/46) and pERK1/2 (T202/Y204) normalized to actin. Relative expression was calculated by normalizing to PBS vehicle control for each experiment (n = 4 independent experiments, two-tailed paired t-test, mean ± SEM). ( c ) Representative Western blot images of total protein levels of mTOR and AKT in WT iMG treated for 96 h with 100 nM ATV:TREM2 or an isotype control. ( d ) Quantification of total mTOR and AKT protein normalized to actin. Relative expression was calculated by normalizing to PBS control for each experiment (n = 4 independent experiments, two-tailed paired t-test, mean ± SEM). e ) Representative Western blot images for p-mTOR (S2448), pAKT (S473), pGSK3b (S9) pRPS6 (S235/236), p4EBP1 (T37/46) and pERK1/2 (T202/Y204) showing inhibition of mTOR pathway activation in WT iMG co-treated with 20 nM AZD8055 and 100 nM ATV:TREM2 after 96 h. ( f ) Quantification of phosphorylation targets shown in (E). Phosphorylation signals were normalized to actin. Relative expression was calculated by normalizing to PBS vehicle control for each experiment (n = 4 independent experiments, two-tailed paired t-test, mean ± SEM)). ( g ) Heatmap of human cytokine profiling in supernatant from WT iMG treated with 100 nM ATV:TREM2 for 96 h. Media collected from iMG treated with 10 ng mL −1 LPS for 24 h was used for comparison.

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Western Blot, Phospho-proteomics, Control, Expressing, Two Tailed Test, Inhibition, Activation Assay, Comparison

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: a – e , Four-day single-dose study. a , ELISA detection of antibodies in whole brain lysates for mice dosed with ATV:TREM2 (1, 3, 10 or 30 mg kg −1 ) and anti-TREM2 (30 mg kg −1 ) 1 day after dose ( n = 5 mice per group). b , Microglia detected by IBA1 staining (purple), and proliferative cells were detected by EdU labeling (green) 4 days after dose. c , Quantification of IBA1/EdU staining ( n = 5 mice per group; Dunnett’s multiple comparisons test, mean ± s.e.m.). d , CSF1R detected by ELISA in whole brain lysate ( n = 5 mice per group; Kruskal–Wallis test for day 1 and Dunnett’s multiple comparisons test for day 4). e , CSF1R detected by ELISA in CSF ( n = 5 mice per group for day 1 (30 mg kg −1 ATV:ISO); day 1 (3 mg kg −1 ATV:TREM2); day 1 (10 mg kg −1 ATV:TREM2); day 1 (30 mg kg −1 ATV:TREM2); day 4 (30 mg kg −1 ATV:ISO). n = 4 mice per group for rest and Dunnett’s multiple comparisons test for day 1; Kruskal–Wallis test for day 4, mean ± s.e.m.). f , g , Ex vivo microglial phagocytosis for different substrates. Myelin debris ( f ) ( n = 8 mice per group; Dunnett’s multiple comparisons test) and Aβ ( g ) ( n = 8 mice per group; two-tailed unpaired t -test, mean ± s.e.m.). a – g , Circle represents male mice, and triangle represents female mice.

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Labeling, Ex Vivo, Two Tailed Test

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: (a) Antibody levels detected in brain lysates by ELISA shows increased brain exposure of ATV:TREM2 (30 mg kg −1 ) compared anti-TREM2 (30 mg kg −1 ) and comparable brain exposure for ATV:TREM2 at 10 mg kg -1 and anti-TREM2 at 30 mg kg −1 at day 4 post-dose. (n = 5 mice/group except for ATV:TREM2 3 mg kg −1 (n = 4)). (b) ATV:TREM2 increases CSF1R in the brain compared to brain exposure matched anti-TREM2 day 2 post dose (n = [8, 9, 8, 9, 10]mice/group, Kruskal-Wallis test, mean ± SEM). ( c ) CSF1R analysis in CSF same as in B. (n = [8, 9, 8, 9, 10]mice/group, Tukey’s multiple comparisons test, mean ± SEM) ( b - c ) Circles represents male mice and triangle represents female mice. (d) Detection of antibody concentrations show matched brain exposure of three ATV:TREM2 molecules with different ATV affinities 1-day post-dose (n = 5 mice/group). (e-f) High and mid-affinity ATV:TREM2 molecules induce comparable increase of CSF1R in the brain ( e) n = 5 mice/group except for Vehicle (Veh) and day 4 10 mg kg −1 8,000 nM (n = 4, Kruskal-Wallis test, compared to vehicle group ) and CSF (f) (n = 5 mice/group, except for Veh and day 4 5 mg kg −1 110 nM n = 4), Dunnett’s multiple comparisons test for day 1, Kruskal-Wallis test for day 4) day 1 and 4 post dose whereas low-affinity ATV:TREM2 induces a weak elevation of CSF1R in CSF at day 4 (n = 4-5 mice/group, mean ± SEM). (g) Schematic of experimental approach to evaluate in vivo dosed antibody impact to microglial phagocytosis ex vivo. A single dose of ATV:TREM2, anti-TREM2, or ATV:ISO was administrated to human TREM2 tg; TfR mu/hu KI mice and brain microglia were isolated 2 days post dose for ex vivo myelin phagocytosis analysis. The same method was used to assess amyloid phagocytosis. (h) FACS gating strategy to quantify pHrodo-myelin positive microglia. After treatment with pHrodo-green myelin and staining, cell suspensions containing microglia were analyzed using BD FACS Aria III. Single cells were separated from debris by FSC and SSC characteristics. Live microglia were identified as a population of CD11b + and propidium iodide negative cells. pHrodo-myelin uptake was then quantified in 20,000 microglia recorded from each sample. (i) Antibody concentrations were detected in brain lysates by ELISA which shows comparable levels of ATV:TREM2 and anti-TREM2 day 2 post dose (n = [9, 10, 10, 9, 10] mice/group). (j) ATV:TREM2 induces transcription of genes associated with phagocytosis . Axl, Itgax, Lgals3 mRNA levels were detected in isolated brain microglia after peripheral administration of 10 mg kg −1 ATV:TREM2 1-, 4-, and 7-days post dose. Graphs represent bulk mRNA measured by RNA-seq. Data shown as log 2 counts per million (with a pseudocount of 1 added) in each biological replicate (n = 8 mice/group, each group compared to ATV:ISO group, Kruskal-Wallis test for Axl, Dunnett’s multiple comparisons for Itgax and Lgals3 , mean ± SEM).

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Enzyme-linked Immunosorbent Assay, In Vivo, Ex Vivo, Isolation, Staining, RNA Sequencing

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: (a) Representative autoradiography images of sagittal brain sections from WT; hTREM2 tg; TfR mu/hu mice 96 h after administration of ATV:TREM2, anti-TREM2, and ATV:ISO radiolabeled with [ 111 In]DOTA or [ 125 I]SIB. (b,c) Longitudinal SPECT/CT imaging quantification of whole-brain uptake of single dose (100 µCi, 200 µL, 1.5 mg kg −1 , IV) ATV:TREM2, anti-TREM2, and ATV:ISO, radiolabeled with [ 125 I]SIB (b) or [ 111 In]DOTA (c) in WT; hTREM2 tg; TfR mu/hu mice. Whole brain %ID/g was corrected for contribution from cerebral blood volume. Data are represented as mean +/− SEM (n = 3 mice/group). (d) Percent of catabolized ATV:TREM2 in several brain regions 96 h after single dose exceeds that of ATV:ISO control. [ 111 In]DOTA and [ 125 I]SIB signal was quantified by ex vivo gamma counting in resected brain regions, and percent of catabolized antibody was estimated by subtracting %ID/g for [ 125 I]SIB signal from that of [ 111 In]DOTA signal (n = 4 mice/group, one-way ANOVA with Dunnett’s multiple comparison test for each region, except for cerebellum which applied Brown-Forsythe and Welch ANOVA tests, mean ± SEM).

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Autoradiography, Single Photon Emission Computed Tomography, Imaging, Control, Ex Vivo, Comparison

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: a , Representative microscopy images from iMG treated with 10 µM oleic acid and then 100 nM ATV:ISO or ATV:TREM2 for 48 hours. BODIPY fluorescence (shown in green) was quantified ( n = 5 independent experiments; two-tailed paired t -test, mean ± s.e.m.). b , Heat map of LC–MS analysis for TG species and acyl carnitines modulated by ATV:TREM2 in iMG treated with myelin. Plotted values are log 2 -transformed raw counts and scaled by row. c , d , ATV:TREM2 reduces TGs and increases acyl carnitines in WT iMG treated with myelin ( c ) ( n = 6 independent experiments (TG) and n = 3 independent experiments (acyl carnitines); two-tailed paired t -test, mean ± s.e.m.) but not PLCG2 KO iMG treated with myelin ( d ) ( n = 3 independent experiments; two-tailed paired t -test, mean ± s.e.m.). e , Seahorse fatty acid oxidation OCR respiration measurements in TREM2 KO and PLCG2 KO iMG ( n = 3 independent experiments; mean ± s.e.m.). f , ATV:TREM2 increases maximal respiration in WT iMG detected with Seahorse fatty acid oxidation OCR measurements ( n = 7 independent experiments; two-tailed paired t -test, mean ± s.e.m.). The CPT-1 inhibitor etomoxir blocks the effect of ATV:TREM2 on respiration ( n = 5 independent experiments; two-tailed paired t -test, mean ± s.e.m.). g , h , Seahorse analysis for glucose oxidation. ATV:TREM2 was treated at 100 nM for 3 days. The ATV:TREM2 effect is blocked by an MPC inhibitor, UK5099 ( n = 4 independent experiments; two-tailed paired t -test, mean ± s.e.m.). i , ATV:TREM2 increases average TMRE intensity in iMG after 3 days with 100 nM antibody ( n = 6 independent experiments; two-tailed paired t -test, mean ± s.e.m.). j , Representative images of super-resolution microscopy of TMRE staining in iMG. Mitochondria were segmented into networked and punctate morphologies. k , Morphometric analysis of the prevalence of networked mitochondria ( n = 3 independent experiments, two-tailed paired t -test). l , Volcano plots of RNA-seq analysis of microglia isolated from mice dosed with 10 mg kg −1 of ATV:ISO or ATV:TREM2 for 1 day, 4 days or 7 days. Red or blue indicate significantly upregulated or downregulated genes, respectively. The x axis represents log 2 fold change in expression compared to vehicle-treated mice, and the y axis represents –log 10 adjusted P value. m , Relative expression (z-scores) of the top-most upregulated or downregulated genes at day 1 after dose selected from oxidative phosphorylation and glycolysis pathways. OCR, oxygen consumption rate.

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Microscopy, Fluorescence, Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Super-Resolution Microscopy, Staining, RNA Sequencing, Isolation, Expressing, Phospho-proteomics

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: (a) Additional species of triglycerides (TG) and short chain carnitines modulated in iMG with ATV:TREM2 treatment (n = 3-5 independent experiment, two-tailed paired t-test, mean ± SEM). (b) ATV:TREM2 does not modulate TG in PLCG2 KO iMG (n = 3 independent experiment, two-tailed paired t-test, mean ± SEM). (c) Maximal respiration measured by Seahorse fatty acid oxidation kit is reduced in both TREM2 KO and PLCG2 KO iMG (n = 3-4 independent experiment, two-tailed paired t-test, mean ± SEM). (d) ATV:TREM2 increased spare capacity measured by Seahorse fatty acid oxidation kit (n = 8 independent experiment, two-tailed paired t-test, mean ± SEM). (e) ATV:TREM2 does not modulate maximal respiration or spare capacity in TREM2 KO iMG (n = 4 independent experiment, two-tailed paired t-test, mean ± SEM). (f) ATV:TREM2 does not modulate maximal respiration and spare capacity PLCG2 KO iMG (n = 4 independent experiment, two-tailed paired t-test, mean ± SEM). (g) RNAseq analysis of brain microglia isolated from hTREM2 tg; TfR mu/hu mice dosed with 10 mg kg −1 ATV:TREM2. GSEA for top pathways based on a p-value cutoff of 0.05 for up- or downregulated gene sets 1 day post ATV:TREM2 dose.

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Two Tailed Test, Isolation

Journal: Nature Neuroscience

Article Title: A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

doi: 10.1038/s41593-022-01240-0

Figure Lengend Snippet: a , Coronal and axial slices show cold scaled group average images of TSPO-PET (SUV H ) projected upon a standard MRI T1-weighted atlas from 5×FAD;hTREM2 tg;TfR mu/hu mice (top row) or WT;hTREM2 tg;TfR mu/hu mice (bottom row) mice dosed with 10 mg kg −1 of antibody. b , c , Quantification of TSPO-PET 1, 4, and 8 days after dose in 5×FAD;hTREM2 tg;TfR mu/hu mice ( b ) and WT;hTREM2 tg;TfR mu/hu mice ( c ). Scatter plot of individual TSPO-PET (SUV H ) values. Dotted lines represent linear associations between interval after antibody dosing and TSPO-PET quantification per group and with a 95% confidence interval ( n = 6 mice per group; two-tailed unpaired t -test for each timepoint, except for day 8, which used the two-tailed unpaired t -test with Welch’s correction). d , Coronal and axial slices show cold scaled group average images of FDG (SUV) projected upon a standard MRI T1-weighted atlas from 5×FAD;hTREM2 tg;TfR mu/hu mice (top row) or WT;hTREM2 tg;TfR mu/hu mice (bottom row) after 10 mg kg −1 of antibody. e , f , Quantification of cortical glucose uptake measured by FDG-PET 1, 4, and 8 days after dose of ATV:ISO or ATV:TREM2 for 5×FAD;hTREM2 tg;TfR mu/hu mice ( e ) and WT;hTREM2 tg;TfR mu/hu mice ( f ). Scatter plot of individual FDG (SUV) values. Dotted lines represent linear associations between interval after antibody dosing and FDG-PET quantification per group with a 95% confidence interval ( n = 6 mice per group; two-tailed unpaired t -test for each timepoint). g , h , Regional correlation of biomarker alterations (5×FAD;hTREM2 tg;TfR mu/hu versus WT;hTREM2 tg;TfR mu/hu mice) between FBB-PET at 5 months and TSPO-PET (SUV H ) ( g ) and FDG-PET (SUV) ( h ) at the group level.

Article Snippet: For TREM2 IP, goat anti-TREM2 antibody (R&D Systems, AF1828) and normal goat IgG (R&D Systems, AB-108-C) was used as capturing antibody.

Techniques: Two Tailed Test, Biomarker Discovery

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A, B) Metabolic analysis of oxygen consumption rate (OCR) and (F, G) proton efflux rate (PER) in Common variant (Cv), TREM2 R47H and TREM2 −/− iPS-Mg after 24 h treatment with succinate (10 mM) or citrate (10 mM) . (C) Basal respiration, (D) maximal respiration, (E) spare respiratory capacity , (H, J) basal glycolysis, (I, K) compensatory glycolysis, and (L) mitoOCR/GlycoPER ratio in basal conditions and upon LPS/IFNγ stimulation in iPS-Mg expressing Cv, TREM2 R47H or TREM2 −/− after incubations with citrate or succinate. Data are presented as mean ± SEM. Statistical significance was addressed using One-way ANOVA or Two-way ANOVA with Tukey post-hoc analysis to compare controls and treated groups respectively; *p<0.05 (n=5-6)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay, Expressing

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Representative images and ( B, C ) quantification of LipidSpot TM 488 staining of lipid droplets in Common variant (Cv), TREM2 R47H and TREM2 −/− variant iPS-Mg after medium supplementation with citrate (10mM) or succinate (10mM) for 24h. (D) Basal lactate production and (E) lactate production upon LPS/IFN-γ stimulation were determined in the iPS-Mg cell culture medium after supplementation with citrate or succinate. (F) Representative histogram and (G) mitochondrial superoxide (MitoSOX) production as determined by FACS in Cv, TREM2 R47H and TREM2 −/− variant iPS-Mg after succinate or citrate supplementation or rotenone treatment (positive control). Data are presented as mean ± SEM. Statistical significance was addressed using One-way ANOVA or Two-way ANOVA with Tukey post-hoc analysis to compare controls and treated groups respectively; *p<0.05 (n=5-6)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Staining, Variant Assay, Cell Culture, Positive Control

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Metabolic analysis of oxygen consumption rate (OCR) (B) Maximal respiration, and (C) spare respiratory capacity in Common variant (Cv), TREM2 R47H and TREM2 −/− iPS-Mg after 24 h LPS/IFN-γ stimulation and treatment with succinate (10 mM) or citrate (10 mM). Data are presented as mean ± SEM. Statistical significance was addressed using One-way ANOVA or Two-way ANOVA with Tukey post-hoc analysis to compare controls and treated groups respectively; *p<0.05 (n=5-6)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Representative histograms of Aβ 1-42 uptake in iPS-Mg upon citrate supplementation. (B) The ability of common variant (Cv), TREM2 R47H and/or TREM2 −/− variant iPS-Mg to phagocytose Aβ 1-42 oligomers was assessed after citrate and succinate supplementation in basal conditions and (C) upon inhibition of glycolysis by 2-Deoxyglucose (2-DG). (D, E) LPS/IFN-γ evoked pro-inflammatory release of TNFα and (F, G) IL-1β were assessed by ELISA in the culture medium of iPS-Mg treated with citrate or succinate in basal conditions and after glycolytic inhibition by 2-DG. Data are presented as mean ± SEM. Statistical significance was addressed using One-way ANOVA or Two-way ANOVA with Tukey post-hoc analysis to compare controls and treated groups respectively; *p<0.05 (n=2-12)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Basal and LPS/IFN-γ evoked pro-inflammatory release of TNFα and (B) IL-1β were assessed by ELISA in the culture medium of iPS-Mg expressing common variant TREM2, TREM2 R47H and TREM2 −/− . Data are presented as mean ± SEM. Statistical significance was addressed using two-way ANOVA with Tukey post-hoc analysis *p<0.05 (n=4-10)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Shed soluble TREM2 (sTREM2) was determined in the culture medium of unstimulated common variant (Cv), TREM2 R47H and TREM2 −/− iPS-Mg after treatments with citrate or succinate and/or (B) 2-DG mediated inhibition of glycolysis in basal conditions and upon LPS/IFNγ stimulation. Data are presented as mean ± SEM. Statistical significance was addressed using Two-way ANOVA with Tukey post-hoc analysis; *p<0.05 (n=3-8)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay, Inhibition

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Representative cellular stress proteome array dot blots and heatmaps from (B) Common variant (Cv), (C) TREM2 R47H , (D) TREM2 −/− variant iPS-Mg lysates after 24h incubations with citrate (10mM) or succinate (10mM). (I) Representative western blots and quantifications for HSP70 (E) at basal and (G) upon LPS/IFN-γ stimulation, and ratio p-NFκB/NFκB (F) at basal and (H) following LPS/IFN-γ treatment, in Cv, TREM2 R47H and TREM2 −/− variant iPS-Mg after treatments with citrate or succinate. Citrate supplemented Cv iPS-Mg group is not included in the selected representative blots (I ). Data are presented as mean ± SEM. Statistical significance was addressed using Two-way ANOVA with Tukey post-hoc analysis; *p<0.05.

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay, Western Blot

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: Graphical summary of changes in TREM2 R47H iPS-Mg after supplementation with citrate or succinate

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques:

Journal: The FASEB Journal

Article Title: Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages

doi: 10.1096/fj.201902183r

Figure Lengend Snippet: FIGURE 1 TREM2 is shed by meprin β in HEK293_ADAM10/17−/− cells. (A) Illustration of membrane bound TREM2 and DAP12. Upon shedding by ADAM10, the remaining C-terminal fragment (CTF) of TREM2 is cleaved within the membrane by γ-secretase, thereby releasing the intracellular domain (ICD). (B) Co-expression of TREM2 with meprin β or ADAM10 in HEK293_ADAM10/17−/− cells and Western blot analyses of TREM2 fragments in membrane-enriched fractions and cell supernatants. Anti-TFR antibody was used as loading control. (C) Conditioned media from TREM2 and ADAM10 transfected HEK293_ADAM10/17−/− cells were incubated with the recombinant ectodomains of meprin β and ADAM10 at 37°C for 4 hours. sTREM2 levels were analyzed by Western blot. (D) HEK293_ADAM10/17−/− cells transfected with TREM2 were incubated with recombinant meprin β and proteins from membrane-enriched fractions and cell supernatants were analyzed by Western blot. (E) Time series showing the cleavage of membrane bound TREM2 in HEK293_ADAM10/17−/− cells by recombinant soluble meprin β analyzed by Western blot. (F) Incubation of TREM2 overexpressing HEK293_ADAM10/17−/− cells with recombinant meprin α or meprin β and analysis of TREM2 by Western blot. All transient cell transfections and Western blot analyses were at least performed in biological triplicates

Article Snippet: For immunoblot detection, the following antibodies were used: anti-human TREM2 (AF1828; R&D Systems; 1:1000), anti-murine TREM2 (clone 5F4)34; anti-flag (F1804; Sigma; 1:2000), anti-human meprin β (polyclonal, Pineda; 1:1000), anti-myc (9B11; Cell Signaling), anti-his (34660; Qiagen; 1:1000), anti-GAPDH (14C10; Cell Signaling; 1:2000), anti-TFR (ab84036; Abcam; 1:1000).

Techniques: Membrane, Expressing, Western Blot, Control, Transfection, Incubation, Recombinant

Journal: The FASEB Journal

Article Title: Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages

doi: 10.1096/fj.201902183r

Figure Lengend Snippet: FIGURE 2 Meprin β cleaves TREM2 between arginine 136 and aspartate 137. (A) Incubation of sTREM2 with recombinant soluble meprin β (smeprin β) led to the generation of TREM2 fragments analyzed by Western blot (AF1828; R&D Systems; 1:1.000). (B) sTREM2 was incubated with smeprin β for 2 hours at 37°C and subsequently digested with PNGaseF. The meprin β generated TREM2 fragment is about 10 kDa smaller than the uncleaved TREM2 as analyzed by SDS-PAGE. (C) Coomassie stained gel of sTREM2 incubated with meprin β (2 hours at 37°C) that was used for determination of cleavage sites. Gel bands were excised, split into two samples and digested with either chymotrypsin or LysargiNase in heavy water. (E) Illustration of the TREM2 ectodomain construct used for cleavage site identification (sTREM2). While TREM2 is cleaved by ADAM10 and ADAM17 between histidine 157 and serine 158, the major meprin β cleavage site is further N-terminal between arginine 136 and aspartate 137. (F) Co-expression of TREM2 WT and AD-associated variants with meprin β in HEK293_ADAM10/17−/− cells and Western blot analyses of TREM2 fragments in membrane-enriched fractions and cell supernatants. Anti-GAPDH antibody was used as loading control. All transient cell transfections and Western blot analyses were at least performed in biological triplicates

Article Snippet: For immunoblot detection, the following antibodies were used: anti-human TREM2 (AF1828; R&D Systems; 1:1000), anti-murine TREM2 (clone 5F4)34; anti-flag (F1804; Sigma; 1:2000), anti-human meprin β (polyclonal, Pineda; 1:1000), anti-myc (9B11; Cell Signaling), anti-his (34660; Qiagen; 1:1000), anti-GAPDH (14C10; Cell Signaling; 1:2000), anti-TFR (ab84036; Abcam; 1:1000).

Techniques: Incubation, Recombinant, Western Blot, Generated, SDS Page, Staining, Construct, Expressing, Membrane, Control, Transfection

Journal: The FASEB Journal

Article Title: Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages

doi: 10.1096/fj.201902183r

Figure Lengend Snippet: FIGURE 3 Impaired phagocytosis of fluorogenic E coli particles in cells co-expressing meprin β and TREM2. (A) Illustration of the TREM2/DAP12 fusion construct used in the phagocytosis assays. (B) Proteolytic cleavage of the overexpressed fusion constructs in HEK293_ ADAM10/17−/− cells transfected with meprin β or ADAM10. Proteins from cell supernatants and membrane-enriched fractions were analyzed by Western blot. (C) Phagocytosis assay with transfected HEK293_ADAM10/17−/− cells. Relative particle uptake analyzed by FACS is indicated for differentially transfected cells. Data are represented as means of median fluorescence intensity ± SEM from two independent experiments and expressed relative to empty vector transfected control (n = 8 and 9, respectively). (D) Relative particle uptake of THP-1 cells transfected with wild- type TREM2 and meprin β (n = 10) was analyzed using a Tecan fluorescent reader measuring mean fluorescence intensity of a defined number of cells. (E) Relative particle uptake of U937 cells transfected with wild-type TREM2 and meprin β (n = 6) was analyzed using a Tecan fluorescent reader measuring mean fluorescence intensity of a defined number of cells. All transient cell transfections and Western blot analyses were at least performed in biological triplicates. Statistical differences were calculated by One-way ANOVA followed by a Newman-Keuls multiple comparison test. Comparison of two test groups was performed using an unpaired two-tailed t test (*P < .05; **P < .01; ****P < .0001)

Article Snippet: For immunoblot detection, the following antibodies were used: anti-human TREM2 (AF1828; R&D Systems; 1:1000), anti-murine TREM2 (clone 5F4)34; anti-flag (F1804; Sigma; 1:2000), anti-human meprin β (polyclonal, Pineda; 1:1000), anti-myc (9B11; Cell Signaling), anti-his (34660; Qiagen; 1:1000), anti-GAPDH (14C10; Cell Signaling; 1:2000), anti-TFR (ab84036; Abcam; 1:1000).

Techniques: Expressing, Construct, Transfection, Membrane, Western Blot, Phagocytosis Assay, Fluorescence, Plasmid Preparation, Control, Comparison, Two Tailed Test

Journal: The FASEB Journal

Article Title: Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages

doi: 10.1096/fj.201902183r

Figure Lengend Snippet: FIGURE 4 Cleavage of TREM2 on macrophages by meprin β. (A) Membrane-enriched fractions from BMDMs of Mep1b−/− mice showed higher levels of mature TREM2 than the wild-type controls visualized by Western blot. Enhanced levels of mature TREM2 at 45 kDa were detected (murine-specific anti-TREM2, clone 5F4) in samples of Mep1b−/− mice, which was decreased upon LPS stimulation. Anti-STAT3 was used as loading control. (B) Densitometric analysis of TREM2 band intensities in non LPS stimulated BMDMs from Mep1b−/− and wild-type mice as seen in (A). The comparison of two test groups (n = 5) was performed using an unpaired two-tailed t test. (*P < .05). (C) Phagocytosis assay with BMDMs from wild-type and Mep1b−/− mice. Cytochalasin D (CytoD) was used to block endocytosis. Relative particle uptake measured by FACS is indicated for different cells. Data are represented as means of median fluorescence intensity ± SD (n = 3). Statistical differences were calculated by One-way ANOVA followed by a Newman-Keuls multiple comparison test (*P < .05). (D) Determination of sTREM2 levels in serum reveals significantly lower levels in Mep1b−/− (n = 7, all male) than in wild-type mice (n = 5, all male). Data are represented as means ± SEM. (E) Cartoon illustrating the interaction of TREM2 with soluble and membrane bound meprin β resulting in the degradation of the TREM2 ectodomain

Article Snippet: For immunoblot detection, the following antibodies were used: anti-human TREM2 (AF1828; R&D Systems; 1:1000), anti-murine TREM2 (clone 5F4)34; anti-flag (F1804; Sigma; 1:2000), anti-human meprin β (polyclonal, Pineda; 1:1000), anti-myc (9B11; Cell Signaling), anti-his (34660; Qiagen; 1:1000), anti-GAPDH (14C10; Cell Signaling; 1:2000), anti-TFR (ab84036; Abcam; 1:1000).

Techniques: Membrane, Western Blot, Control, Comparison, Two Tailed Test, Phagocytosis Assay, Blocking Assay, Fluorescence

Journal: Acta neuropathologica

Article Title: TREM2 expression in the brain and biological fluids in prion diseases.

doi: 10.1007/s00401-021-02296-1

Figure Lengend Snippet: Fig. 3 mRNA Trem2 expression in the brain of sCJD MM1 and VV2 mouse models. RT-qPCR analysis Trem2 expression in mouse models of sCJD MM1 (tg340-CJD MM1) (left panels) and sCJD VV2 (tg361-CJD VV2) (right panels) subtypes. Trem2 expression was analysed in five brain regions: anterior and posterior cortex, cer- ebellum, olfactory bulb, and brainstem, at three time points: pre-clinical (80 days post inoculation (dpi)), early-clinical (120 dpi), and clinical (180 dpi). CJD-inoculated animals (black columns) were compared to their corresponding control- inoculated animals (white columns). Four to eight animals per time point and condition were used. Normalization was performed using the house- keeping gene Hprt; *p < 0.05, **p < 0.01

Article Snippet: Human TREM2 goat polyclonal antibody (RD Systems, AF1828) was used diluted 1:20 for immunohistochemistry and at a dilution of 1:200 for western blotting.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Neuron

Article Title: TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function.

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: Figure 1. TREM2 Binds to Ab (A and B) The binding of Ab1-42 monomers or oligomers with Fc, TREM2-Fc, or TREM1-Fc. Representative images (A) and quantified densi- tometric analysis (B). All values were normalized to input. M, marker; n = 5 independent experiments; ***p < 0.001 by unpaired t test. (C) Levels of biotin-oAb1-42 bound to immobilized Fc/Fc-fusion proteins were normalized to maximal Ab/TREM2-Fc interactions. n = 3 independent experiments. (D) Levels of Fc/Fc-fusion proteins bound to immobilized biotin-oAb1-42 were normalized to maximal TREM2-Fc/Ab binding. n = 3 indepen- dent experiments. (E and F) Bio-layer interferometry-binding kinetics of TREM2-His (E) or TREM1-His (F) incubated with immobilized biotin-oAb1-42 (curves correspond to His-tagged proteins with the following concen- trations: 222.2 nM [top] and 74.1, 24.7, 8.23, and 2.74 nM [bottom]; in E and F). (G) Maximal levels of biotin-oAb1-42 bound to im- mobilized wild-type (WT) TREM2-Fc and R47H and R62H mutants (normalized to WT). n = 3 in- dependent experiments; **p < 0.01, one-way ANOVA followed by Dunnett’s post hoc test. (H) Representative images of biotin-oAb1-42 (red) bound to BV2 cells overexpressing TREM2 or BV2 control (DAPI, blue; scale bar, 10 mm). n = 5 independent experiments; **p < 0.01, unpaired t test. (I and J) Levels of Ab1-42 co-precipitated with TREM2 in lysates from APP transgenic TgCRND8 mice (I) or human AD brains (J) were determined by ELISA. Bound Ab was normalized to the IgG control from each sample (n = 5 for TgCRND8, n = 6 for human AD). All values represent mean ± SD. See also Figure S1 and Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Ab1-12 (B436) (Huang et al., 2016) N/A Rabbit anti-human TREM2 Cell Signaling Technology Cat# 91068 RRID:AB_2721119 Goat anti-human TREM2 R&D Systems Cat# AF1828 RRID:AB_2208689 Sheep anti-mouse TREM2 R&D Systems Cat# AF1729 RRID:AB_354956) Mouse anti-Myc Thermo Fisher Scientific Cat# 13-2500 RRID:AB_2533008 Mouse anti-Gas6 Santa Cruz Biotechnology Cat# sc-376087 RRID:AB_10989223 Rabbit anti-phospho-SYK Thermo Fisher Scientific Cat# MA5-14918 RRID:AB_10989513 Rabbit anti-SYK Cell Signaling Technology Cat# 13198 RRID:AB_2687924 Rabbit anti-DAP12 Cell Signaling Technology Cat# 12492 RRID:AB_2721120 Mouse anti-b-actin Sigma Cat# A2228 RRID:AB_476697 Mouse anti-LAMP1 Santa Cruz Biotechnology Cat# sc-20011 RRID:AB_626853 Rabbit anti-phospho-GSK3b Cell Signaling Technology Cat# 5558 RRID:AB_10013750 Rabbit anti-GSK3b Cell Signaling Technology Cat# 12456 RRID:AB_2636978 Rabbit anti-PCNA Cell Signaling Technology Cat# 13110 RRID:AB_2636979 Rabbit anti-cleaved-caspase 3 Cell Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab5076 RRID:AB_2224402 Anti-Ab antibody MOAB-2 Biosensis Cat# M-1586-100 RRID:AB_2492497 Biological Samples For patient information, see Table S1 N/A Chemicals, Peptides, and Recombinant Proteins MG132 EMD Millipore Cat# 474790 Chloroquine Cell Signaling Technology Cat# 14774 Margatoxin Alomone Labs Cat# STM-325 Tertiapin Alomone Labs Cat# STT-250 Ab1-42 peptides Anaspec Cat# AS-20276 Biotin-Ab1-42 peptides Anaspec Cat# AS-23523 FAM-Ab1-42 peptides Anaspec Cat# AS-23525 Recombinant human IgG1 Fc R&D Systems Cat# 110-HG Recombinant human TREM2-Fc protein R&D Systems Cat# 1828-T2 Recombinant human TREM1-Fc protein R&D Systems Cat# 1278-TR Recombinant human CD36-Fc protein R&D Systems Cat# 1955-CD Recombinant human RAGE-Fc protein R&D Systems Cat# 1145-RG Recombinant human Gas6 protein R&D Systems Cat# 885-GSB Recombinant human MerTK-Fc protein R&D Systems Cat# 891-MR Recombinant human TREM2-His protein Sino Biological Cat# 11084-H08H Recombinant human TREM1-His protein Sino Biological Cat# 10511-H08H Critical Commercial Assays Human Ab1-42 ELISA kit Thermo Fisher Scientific Cat# KHB3441 Dynabeads protein G Thermo Fisher Scientific Cat# 10004D QuikChange II Site-Directed Mutagenesis kit Agilent Technologies Cat# 200523 Lipojet SignaGen Laboratories Cat# SL100468 (Continued on next page) e1 Neuron 97, 1023–1031.e1–e7, March 7, 2018

Techniques: Binding Assay, Marker, Incubation, Control, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Journal: Neuron

Article Title: TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function.

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: Figure 2. TREM2 Deficiency Modulates Microglia-Mediated Ab Degradation and Ab-Induced Depolarization (A–D) TREM2 deficiency attenuates microglia- mediated Ab degradation. Cultured microglia from WT or TREM2 KO mice were incubated with 200 nM FAM-Ab1-42 aggregates at 37C for the time indicated in (A), and they were washed/re- incubated in Ab-free media with or without MG132 (B–D) or chloroquine (CQ) (C and D). FAM-Ab in WT or TREM2 KO microglia at different time points was determined by flow cytometry (A and B) or confocal microscopy (C). All values were normal- ized to WT FAM-Ab levels 2 hr after uptake. Quantification of FAM-Ab fluorescence is shown in (D). Scale bar, 20 mm. n = 3 independent mea- surements; *p < 0.05, **p < 0.01, and ***p < 0.001, two-way ANOVA followed by Tukey’s post hoc test. (E) RMP in WT and TREM2 KO microglia with or without 6-hr exposure to oAb1-42 (5 mM). WT-control, n = 7; WT-Ab, n = 8; KO-control, n = 8; KO-Ab, n = 7; *p < 0.05, **p < 0.01, and ***p < 0.001, two-way ANOVA followed by Tukey’s post hoc test. (F) Representative inward currents obtained from primary microglia. (G) TREM2 KO microglia showed significantly reduced peak inward currents (n = 18 cells) when compared to WT (n = 9 cells). ***p < 0.001, unpaired t test. (H) At -150mV, Ab1-42 oligomers (10 min) reduce current amplitude in WT, but not in TREM2 KO microglia. All values were normalized to baseline for each cell. n = 5 cells for each group; *p < 0.05, paired t test. Electrophysiological recordings were taken from 4 independent microglial cultures. All values represent mean ± SD. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Ab1-12 (B436) (Huang et al., 2016) N/A Rabbit anti-human TREM2 Cell Signaling Technology Cat# 91068 RRID:AB_2721119 Goat anti-human TREM2 R&D Systems Cat# AF1828 RRID:AB_2208689 Sheep anti-mouse TREM2 R&D Systems Cat# AF1729 RRID:AB_354956) Mouse anti-Myc Thermo Fisher Scientific Cat# 13-2500 RRID:AB_2533008 Mouse anti-Gas6 Santa Cruz Biotechnology Cat# sc-376087 RRID:AB_10989223 Rabbit anti-phospho-SYK Thermo Fisher Scientific Cat# MA5-14918 RRID:AB_10989513 Rabbit anti-SYK Cell Signaling Technology Cat# 13198 RRID:AB_2687924 Rabbit anti-DAP12 Cell Signaling Technology Cat# 12492 RRID:AB_2721120 Mouse anti-b-actin Sigma Cat# A2228 RRID:AB_476697 Mouse anti-LAMP1 Santa Cruz Biotechnology Cat# sc-20011 RRID:AB_626853 Rabbit anti-phospho-GSK3b Cell Signaling Technology Cat# 5558 RRID:AB_10013750 Rabbit anti-GSK3b Cell Signaling Technology Cat# 12456 RRID:AB_2636978 Rabbit anti-PCNA Cell Signaling Technology Cat# 13110 RRID:AB_2636979 Rabbit anti-cleaved-caspase 3 Cell Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab5076 RRID:AB_2224402 Anti-Ab antibody MOAB-2 Biosensis Cat# M-1586-100 RRID:AB_2492497 Biological Samples For patient information, see Table S1 N/A Chemicals, Peptides, and Recombinant Proteins MG132 EMD Millipore Cat# 474790 Chloroquine Cell Signaling Technology Cat# 14774 Margatoxin Alomone Labs Cat# STM-325 Tertiapin Alomone Labs Cat# STT-250 Ab1-42 peptides Anaspec Cat# AS-20276 Biotin-Ab1-42 peptides Anaspec Cat# AS-23523 FAM-Ab1-42 peptides Anaspec Cat# AS-23525 Recombinant human IgG1 Fc R&D Systems Cat# 110-HG Recombinant human TREM2-Fc protein R&D Systems Cat# 1828-T2 Recombinant human TREM1-Fc protein R&D Systems Cat# 1278-TR Recombinant human CD36-Fc protein R&D Systems Cat# 1955-CD Recombinant human RAGE-Fc protein R&D Systems Cat# 1145-RG Recombinant human Gas6 protein R&D Systems Cat# 885-GSB Recombinant human MerTK-Fc protein R&D Systems Cat# 891-MR Recombinant human TREM2-His protein Sino Biological Cat# 11084-H08H Recombinant human TREM1-His protein Sino Biological Cat# 10511-H08H Critical Commercial Assays Human Ab1-42 ELISA kit Thermo Fisher Scientific Cat# KHB3441 Dynabeads protein G Thermo Fisher Scientific Cat# 10004D QuikChange II Site-Directed Mutagenesis kit Agilent Technologies Cat# 200523 Lipojet SignaGen Laboratories Cat# SL100468 (Continued on next page) e1 Neuron 97, 1023–1031.e1–e7, March 7, 2018

Techniques: Cell Culture, Incubation, Cytometry, Confocal Microscopy, Control

Journal: Neuron

Article Title: TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function.

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: Figure 3. TREM2 Deficiency Alters Microglia Cytokine Expression, Secretion, and Downstream Signaling in Response to Ab (A) IL-6, MIP-1a, or Arg1 expression was determined by qRT-PCR in WT or TREM2 KO microglia in the presence or absence of 1 mM Ab. n = 3 independent experiments; *p < 0.05 and **p < 0.01, two-way ANOVA followed by Tukey’s post hoc test. (B and C) Cytokine levels in conditioned media from WT or TREM2 KO microglial cultures treated with oAb1-42 for 24 (B) or 48 hr (C) were quantified by ELISA. All values were normalized to WT-Ab (0 mM) levels for each time point. n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, two-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Ab1-12 (B436) (Huang et al., 2016) N/A Rabbit anti-human TREM2 Cell Signaling Technology Cat# 91068 RRID:AB_2721119 Goat anti-human TREM2 R&D Systems Cat# AF1828 RRID:AB_2208689 Sheep anti-mouse TREM2 R&D Systems Cat# AF1729 RRID:AB_354956) Mouse anti-Myc Thermo Fisher Scientific Cat# 13-2500 RRID:AB_2533008 Mouse anti-Gas6 Santa Cruz Biotechnology Cat# sc-376087 RRID:AB_10989223 Rabbit anti-phospho-SYK Thermo Fisher Scientific Cat# MA5-14918 RRID:AB_10989513 Rabbit anti-SYK Cell Signaling Technology Cat# 13198 RRID:AB_2687924 Rabbit anti-DAP12 Cell Signaling Technology Cat# 12492 RRID:AB_2721120 Mouse anti-b-actin Sigma Cat# A2228 RRID:AB_476697 Mouse anti-LAMP1 Santa Cruz Biotechnology Cat# sc-20011 RRID:AB_626853 Rabbit anti-phospho-GSK3b Cell Signaling Technology Cat# 5558 RRID:AB_10013750 Rabbit anti-GSK3b Cell Signaling Technology Cat# 12456 RRID:AB_2636978 Rabbit anti-PCNA Cell Signaling Technology Cat# 13110 RRID:AB_2636979 Rabbit anti-cleaved-caspase 3 Cell Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab5076 RRID:AB_2224402 Anti-Ab antibody MOAB-2 Biosensis Cat# M-1586-100 RRID:AB_2492497 Biological Samples For patient information, see Table S1 N/A Chemicals, Peptides, and Recombinant Proteins MG132 EMD Millipore Cat# 474790 Chloroquine Cell Signaling Technology Cat# 14774 Margatoxin Alomone Labs Cat# STM-325 Tertiapin Alomone Labs Cat# STT-250 Ab1-42 peptides Anaspec Cat# AS-20276 Biotin-Ab1-42 peptides Anaspec Cat# AS-23523 FAM-Ab1-42 peptides Anaspec Cat# AS-23525 Recombinant human IgG1 Fc R&D Systems Cat# 110-HG Recombinant human TREM2-Fc protein R&D Systems Cat# 1828-T2 Recombinant human TREM1-Fc protein R&D Systems Cat# 1278-TR Recombinant human CD36-Fc protein R&D Systems Cat# 1955-CD Recombinant human RAGE-Fc protein R&D Systems Cat# 1145-RG Recombinant human Gas6 protein R&D Systems Cat# 885-GSB Recombinant human MerTK-Fc protein R&D Systems Cat# 891-MR Recombinant human TREM2-His protein Sino Biological Cat# 11084-H08H Recombinant human TREM1-His protein Sino Biological Cat# 10511-H08H Critical Commercial Assays Human Ab1-42 ELISA kit Thermo Fisher Scientific Cat# KHB3441 Dynabeads protein G Thermo Fisher Scientific Cat# 10004D QuikChange II Site-Directed Mutagenesis kit Agilent Technologies Cat# 200523 Lipojet SignaGen Laboratories Cat# SL100468 (Continued on next page) e1 Neuron 97, 1023–1031.e1–e7, March 7, 2018

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Neuron

Article Title: TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function.

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: Figure 4. Microglial Migration, Ab Degrada- tion, and Downstream Effects on Microglial Proliferation and Apoptosis Are Altered in TREM2 KO Mice (A–C) Analysis of WT or TREM2 KO brain at 16 hr after FAM-Ab injection. n = 8 for WT, n = 9 for KO mice. (A) Representative images of Ab-injected regions. Scale bar, 20 mm. (B) Quantified average values for total Ab/IBA1- positive (IBA1+) cells. **p < 0.01, nonparametric t test. (C) Quantified Ab deposition area. (D and E) Ab staining analysis in WT or TREM2 KO mouse hippocampus 3 days after oAb1-42 injection. (D) Representative images (scale bar, 300 mm for 83 magnification; scale bar, 60 mm for 403 magnification). (E) Quantified values of the deposition area. n = 9 for WT, n = 8 for KO mice; *p < 0.05, nonparametric t test. (F and G) Immunostaining of PCNA (red) (F), cleaved-caspase-3 (C-caspase-3, red) (G), IBA1 microglial markers (green), and DAPI (blue) in the hippocampus of WT or TREM2 KO mice with con- trol vehicle or oAb injection as indicated. PCNA or c-caspase-3-positive microglial cells are indicated by white circles. Adjacent graphs represent quan- tified overlapping IBA1/PCNA- (F) or IBA1/C-cas- pase-3- (G) stained cells from 8–12 mice per group. Scale bar, 20 mm. *p < 0.05 and **p < 0.01, two-way ANOVA followed by Tukey’s post hoc test. (H) Reconstructed 3D microscopy images of IBA1-stained (white) microglia. Average process length per microglia was quantified and shown in the adjacent graph. n = 9–12 mice/group. Scale bar, 10 mm. ***p < 0.001, two-way ANOVA followed by Tukey’s post hoc test. All graphs represent mean ± SD. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Ab1-12 (B436) (Huang et al., 2016) N/A Rabbit anti-human TREM2 Cell Signaling Technology Cat# 91068 RRID:AB_2721119 Goat anti-human TREM2 R&D Systems Cat# AF1828 RRID:AB_2208689 Sheep anti-mouse TREM2 R&D Systems Cat# AF1729 RRID:AB_354956) Mouse anti-Myc Thermo Fisher Scientific Cat# 13-2500 RRID:AB_2533008 Mouse anti-Gas6 Santa Cruz Biotechnology Cat# sc-376087 RRID:AB_10989223 Rabbit anti-phospho-SYK Thermo Fisher Scientific Cat# MA5-14918 RRID:AB_10989513 Rabbit anti-SYK Cell Signaling Technology Cat# 13198 RRID:AB_2687924 Rabbit anti-DAP12 Cell Signaling Technology Cat# 12492 RRID:AB_2721120 Mouse anti-b-actin Sigma Cat# A2228 RRID:AB_476697 Mouse anti-LAMP1 Santa Cruz Biotechnology Cat# sc-20011 RRID:AB_626853 Rabbit anti-phospho-GSK3b Cell Signaling Technology Cat# 5558 RRID:AB_10013750 Rabbit anti-GSK3b Cell Signaling Technology Cat# 12456 RRID:AB_2636978 Rabbit anti-PCNA Cell Signaling Technology Cat# 13110 RRID:AB_2636979 Rabbit anti-cleaved-caspase 3 Cell Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab5076 RRID:AB_2224402 Anti-Ab antibody MOAB-2 Biosensis Cat# M-1586-100 RRID:AB_2492497 Biological Samples For patient information, see Table S1 N/A Chemicals, Peptides, and Recombinant Proteins MG132 EMD Millipore Cat# 474790 Chloroquine Cell Signaling Technology Cat# 14774 Margatoxin Alomone Labs Cat# STM-325 Tertiapin Alomone Labs Cat# STT-250 Ab1-42 peptides Anaspec Cat# AS-20276 Biotin-Ab1-42 peptides Anaspec Cat# AS-23523 FAM-Ab1-42 peptides Anaspec Cat# AS-23525 Recombinant human IgG1 Fc R&D Systems Cat# 110-HG Recombinant human TREM2-Fc protein R&D Systems Cat# 1828-T2 Recombinant human TREM1-Fc protein R&D Systems Cat# 1278-TR Recombinant human CD36-Fc protein R&D Systems Cat# 1955-CD Recombinant human RAGE-Fc protein R&D Systems Cat# 1145-RG Recombinant human Gas6 protein R&D Systems Cat# 885-GSB Recombinant human MerTK-Fc protein R&D Systems Cat# 891-MR Recombinant human TREM2-His protein Sino Biological Cat# 11084-H08H Recombinant human TREM1-His protein Sino Biological Cat# 10511-H08H Critical Commercial Assays Human Ab1-42 ELISA kit Thermo Fisher Scientific Cat# KHB3441 Dynabeads protein G Thermo Fisher Scientific Cat# 10004D QuikChange II Site-Directed Mutagenesis kit Agilent Technologies Cat# 200523 Lipojet SignaGen Laboratories Cat# SL100468 (Continued on next page) e1 Neuron 97, 1023–1031.e1–e7, March 7, 2018

Techniques: Migration, Injection, Staining, Immunostaining, Microscopy