Journal: Journal of Lipid Research

Article Title: The cholesteryl ester transfer protein (CETP) raises cholesterol levels in the brain

doi: 10.1016/j.jlr.2022.100260

Figure Lengend Snippet: CETP promotes ABCA7 and TREM2 expression in the liver. A: Feeding schedule and study design. Wt and CETP tg mice were fed for 3 months starting at the age of 2 months. Biochemical analyses were performed at 5 months. B–D: Normalized RT-qPCR from mouse liver tissue. B: H mgcr , C: L dlr , and D: L rp 1 expression, n = 6–14, mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test. E: Western blot analysis of ABCA7 and TREM2 from liver lysates. Antibodies used: rabbit-anti-GAPDH (14C10; Cell Signaling), anti-TREM2 (Mab1729; R&D Systems), and anti-ABCA7 (polyclonal; Thermo Fisher Scientific), n = 6; mean ± SEM, Student's t -test. F–I: Expression analysis of ABCA7 and TREM2 from liver samples. Normalized RT-qPCR levels of liver F: A bca 7 and H: T rem 2 ; n = 6–14, mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test. Western blot quantification of G: ABCA7 and I: TREM2; n = 6; mean ± SEM, Student's t -test.

Article Snippet: Primary antibodies used were 22C11 (Millipore), anti-GAPDH (14C10; Cell Signaling), TP2 (kind gift of the Ottawa Heart Institute), anti-triggering receptor expressed in myeloid cells 2 (TREM2) (Mab1729; R&D Systems) and anti-ABCA7 (polyclonal; Thermo Fisher Scientific), and horseradish peroxidase-coupled secondary antibodies (Promega).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot

Journal: Brain, behavior, and immunity

Article Title: Early life stress impairs synaptic pruning in the developing hippocampus

doi: 10.1016/j.bbi.2022.09.014

Figure Lengend Snippet: ELS reduces expression of TREM2 and CD11b while increasing CX3CR1 in P17 microglia. (A) Scatter plots for quantifying Trem2 positive microglia in CTL, LB and UPS. (B) Percentage of TREM2-positive microglia. Median fluorescence intensity (MFI) plots and surface expression of CD11b (C-D), CD45 (E-F) and CX3CR1 (G-H). CTL-males: n = 16, CTL-females: n = 14, LB males: n = 10, LB-females: n = 9, UPS-males: n = 12, UPS-females: n = 16. Error bars represent mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Microglia were isolated from the hippocampus as described above and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and then stained with rat-anti CD11b-PeCy7 1:100 (Cat# 25-0112-82, ebiosciences), rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences), APC-rat anti Trem2 antibodies 1:10 (Cat# FAB17291A R & D systems), or APC-mouse anti CX3CR1 antibodies 1:100 (Cat# 149008) for 25 min on ice.

Techniques: Expressing, Fluorescence

Journal: Brain, behavior, and immunity

Article Title: Early life stress impairs synaptic pruning in the developing hippocampus

doi: 10.1016/j.bbi.2022.09.014

Figure Lengend Snippet: Trem2 is essential for normal phagocytic activity. (A-B) Effects of Trem2 genotype (WT, Hets, Ko) on ex vivo phagocytic activity in microglia isolated from the hippocampus of 17-day old pups (n = 10–17 pups per group, 50 % females). (C) Representative confocal images (top row) and Imaris models (middle and lower rows) of microglia from Trem2 wildtype (WT), heterozygous (Hets) and knockout P17 littermates. Staining for Iba1 (green), CD68 (blue), and PSD95 (red). Middle row: reconstruction of Iba1 & CD68 staining. Lower row: reconstruction of CD68 and PSD95 staining inside microglia. Effects of Trem2 genotype on microglial volume (D), CD68 volume inside microglia (E), number of PSD95 puncta in microglia (F) and PSD95 puncta inside CD68 (G), n = 5 cells per mouse and 4–5 mice per group, 50 % females). Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Microglia were isolated from the hippocampus as described above and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and then stained with rat-anti CD11b-PeCy7 1:100 (Cat# 25-0112-82, ebiosciences), rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences), APC-rat anti Trem2 antibodies 1:10 (Cat# FAB17291A R & D systems), or APC-mouse anti CX3CR1 antibodies 1:100 (Cat# 149008) for 25 min on ice.

Techniques: Activity Assay, Ex Vivo, Isolation, Knock-Out, Staining

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Trem2 Y38C variant and Trem2 deficient mice affects microglial morphology. a CRISPR/Cas9 strategy to generate the tyrosine to cystine point mutation (red) in exon 2 of murine Trem2 to create the Trem2 Y38C/Y38C mouse model. The sequences for the reference genome, guide RNA, and homology directed repair (HDR), which indicates the Y38C variant (red) and a silent mutation (blue) to ablate the protospacer adjacent motif (PAM), are shown. b Quantification of Trem2 mRNA levels in cortical lysates showed similar Trem2 expression in WT (black) and Trem2 Y38C/Y38C (green) mice and no expression in Trem2 −/− (red) mice. Data are presented as mean ± SEM fold change normalized gene expression relative to WT. N = 8 (4 males and 4 females for each group) c Immunohistochemistry for TREM2 (green) and Iba-1 (red) is shown in cortices of 6 month WT, Trem2 Y38C/Y38C , and Trem2 −/− mice. Merged imaged indicates colocalization of TREM2 staining with Iba-1+ (red) cell bodies in WT and Trem2 Y38C/Y38C mice. No TREM2 staining was observed in Trem2 −/− mice. (representative images from females). d Quantification of total TREM2 in WT, Trem2 Y38C/Y38C , and Trem2 −/− cortical lysates by ELISA indicates slightly higher levels of TREM2 in Trem2 Y38C/Y38C as compared to the WT mice. e sTREM2 levels, quantified in TBS soluble cortical extracts, were similar in both Trem2 Y38C/Y38C and WT. f Quantification of the number of Iba-1 positive cells showed similar counts in cortices of 6 months old WT, Trem2 Y38C/Y38C and Trem2 −/− mice. g Quantification of Iba-1 immunoreactive area showed increased staining in Trem2 Y38C/Y38C and Trem2 −/− mice. f-g Data represented as mean ± SEM, N = 6 (3 males and 3 females for each group; n = 56 fields for WT, n = 89 fields for Trem2 Y38C/Y38C , and n = 112 fields for Trem2 −/− ). h Skeletonized reconstructions (representative reconstructions from females), i quantification of number of branches and j number of junctions in microglia from the cortices of 6 months old WT, Trem2 Y38C/Y38C and Trem2 −/− mice. h-j Data represented as mean ± SEM, N = 6 (3 males and 3 females for each group, each data point represents microglia analyzed). b,d-i One-way ANOVA with Tukey’s post hoc test. * P < 0.05, *** P < 0.001, **** P < 0.0001

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: Variant Assay, CRISPR, Mutagenesis, Expressing, Gene Expression, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Loss of functional TREM2 alters gene expression associated with oligodendrocyte/myelin and neuronal function. a Volcano plot showing differentially expressed genes in 6 month Trem2 Y38C/Y38C versus WT cortices and Trem2 −/− versus WT cortices. Dashed line represents P < 0.005. Significantly altered genes are shown in green with downregulated and upregulated genes above |logFC| > 0.58 threshold of in green. b-c Enrichr pathway analysis of the shared downregulated genes between Trem2 Y38C/Y38C and Trem2 −/− mice (ranked by P value; Fischer’s exact test, P-adjusted, Benjamini-Hochberg method for correction for multiple hypotheses testing). b Tissue enrichment analysis showing significant enrichment in brain regions. Dashed line represents P < 0.005. c Enrichment analysis for specific cellular compartments showing myelin-related enrichment. Dashed line represents P < 0.005. d Top 10 pathways significantly altered using IPA pathway analysis. Analysis based on significantly differentially expressed genes between Trem2 Y38C/Y38C versus WT mice and Trem2 −/− vs WT mice. Yellow line represents P < 0.05 yellow line (ranked by P value, Fischer’s extact test). e Top 5 predicted upstream regulators of differentially expressed genes in Trem2 Y38C/Y38C versus WT mice and Trem2 −/− vs WT mice using IPA Upstream Regulator analysis. Activated (black) and inhibited (grey) upstream regulators are shown. P value, Fisher’s exact test. Sample sizes: WT mice, N = 8 (4 males, 4 females); Trem2 Y38C/Y38C mice, N = 8 (4 males, 4 females); Trem2 −/− mice, N = 5 (1 male, 4 females)

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: Functional Assay, Gene Expression

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Oligodendrocyte/Myelin impairments in adult Trem2 Y38C/Y38C and Trem2 −/− mice. a-c Mbp, Mobp and Olig2 transcript levels were determined by quantitative-PCR. a Mbp transcripts were reduced in both Trem2 Y38C/Y38C and Trem2 −/− cortical lysates and b-c Mobp and Olig2 mRNA reduced significantly in Trem2 −/− cortical lysates. d Western blot analysis and e Quantification of myelin proteins CNPase and MBP. CNPase levels were reduced significantly in Trem2 −/− mice and Mbp levels were significantly reduced in both Trem2 Y38C/Y38C and Trem2 −/− mice. a-e Data represented as mean ± SEM. N = 4 (2 males, 2 females); One-way ANOVA with Tukey’s post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Decreased synaptic elements in adult Trem2 Y38C/Y38C and Trem2 −/− mice. Microdissected a,b cortices and c,d hippocampi of WT, Trem2 Y38C/Y38C and Trem2 −/− mice were analyzed using western blot with antibodies against PSD95 and synaptophysin. a,b Trem2 Y38C/Y38C and Trem2 −/− mice showed significant reduction in synaptophysin but not in PSD-95 protein levels. c,d PSD95 and synaptophysin protein levels were significantly reduced in hippocampi of Trem2 Y38C/Y38C and Trem2 −/− mice. e Comparison of PSD95 and synaptophysin protein expression in hippocampal lysates of WT, Trem2 Y38C/Y38C and Trem2 −/− mice at P20 and 6 months. Comparing P20 to 6 months, PSD95 levels increase significantly (black asterisk) in WT mice (black), while both Trem2 Y38C/Y38C (green) and Trem2 −/− (red) mice show comparatively less increase resulting in reduced PSD95 levels at 6 months as compared to WT. Synaptophysin levels were maintained in WT mice (black) and Trem2 Y38C/Y38C (green), while Trem2 −/− (red) mice showed significantly lower levels at 6 months as compared to P20 (red asterisk) indicating loss of presynaptic elements in adult mice. Sample sizes for each group: N = 6–8 (3–4 each, males and females). Data are shown as mean ± SEM. b,d One-way ANOVA with Tukey’s post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. e Two-way ANOVA with Bonferroni’s post hoc test; * P < 0.05, WT mice at 6 months versus P20 (black) and Trem2 −/− mice at 6 months versus P20 (red)

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: Western Blot, Comparison, Expressing

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Hippocampal synaptic plasticity is reduced in Trem2 Y38C/Y38C and Trem2 −/− mice. a Input-output curves were similar in Trem2 Y38C/Y38C , Trem2 −/− , and WT mice. b,c Hippocampal Schaffer collateral-CA1 LTP is significantly reduced in Trem2 Y38C/Y38C (green) and Trem2 −/− (red) mice as compared to WT mice (black) at 6 months of age. b Superimposed representative traces before (black) and 60 min after high-frequency stimulation (gray) in each genotype. c Mean time course of fEPSP slope in hippocampal slices from WT (black), Trem2 Y38C/Y38C (green) and Trem2 −/− (red). Arrow indicates time of high frequency stimulation. d Average of normalized fEPSP slope for final 10 min of recording (60–70 min) relative to 10-min baseline average (dotted line). Data are shown as mean ± SEM. Sample sizes: WT mice, N = 15 (7 females and 8 males, n = 33 recordings); Trem2 Y38C/Y38C mice, N = 7 (3 females and 4 males, n = 20 recordings); Trem2 −/− mice, N = 8 (4 males and 4 females, n = 15 recordings). One-way ANOVA with Tukey’s post hoc test. *** P < 0.001, **** P < 0.0001

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: Characterization of BMDM from wild-type, TREM2-IPD, TREM2-sol and TREM2-KO mice. a Schematic representation of the TREM2 receptor. Black, red and yellow asterisks indicate the cleavage site in WT, the mutated cleavage site in TREM2-IPD and in TREM2-sol, respectively. b Flow cytometry analysis of murine cell surface TREM2 on BMDM. MFI: median fluorescence intensity. Sheddase inhibitor: DCP333 (DPC), sheddase activator PMA. c Analysis of supernatants from b of murine soluble TREM2 from BMDM. d ATP-based cell survival assay of BMDM upon M-CSF deprivation for 2 and 3.5 days. ATP levels of cells cultured with M-CSF ( n = 7) were set as 100% survival and compared to the ATP concentration after 2 ( n = 4) and 3.5 days ( n = 3) without M-CSF for each genotype. Statistics: Holm–Sidak’s two-way ANOVA multiple comparisons (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). e In vitro phagocytosis capacity of BMDM over 12 h (area-under-the curve) with 5 µg pHrodo-myelin per well ( n = 3). Fluorescence measurements in wells without prey were used as controls (data not shown). f Representative images of the Cathepsin B activity assay taken by the In-Cell Analyzer. The nuclei are stained with DAPI (blue), and the red fluorescence signals are derived from cleaved Magic red. g Quantification of the Cathepsin B assay images. The fluorescence integrated density of the Magic red signal was measured and normalized to the nuclei count. A significant ( p < 0.05) increase in normalized fluorescence between the DMSO control and K-18 within one genotype is marked by #. Statistics for d and g : Holm–Sidak’s two-way ANOVA with multiple comparisons (* p ≤ 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001). Statistics for e : One-way ANOVA test with Holm–Sidak’s multiple comparisons test (*** p < 0.001; **** p < 0.0001). All data are presented as means ± SEM

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Flow Cytometry, Fluorescence, Clonogenic Cell Survival Assay, Cell Culture, Concentration Assay, In Vitro, Activity Assay, Staining, Derivative Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: MRI indicated myelination deficits in TREM2-sol and TREM2-KO in the acute cuprizone model. a Schematic diagram of the experimental setup for the cuprizone treatment and recovery. Groups consisted of mice treated for 5 weeks with control food or 0.2% cuprizone in food and then switched back to control food (normal food) for the 4-week recovery. MRI measurements were performed at week 0 (baseline), week 3 (except for TREM2-KO and wt2) and week 5 of cuprizone intoxication, at week 7 (2 weeks of recovery on control food, except for TREM2-KO and wt2) and at week 9 (4 weeks of recovery on control food). Mice were culled at week 9 immediately after the last MRI measurement. b Representative MRI images acquired from three mice at baseline, at maximal pathology (5 weeks of receiving 0.2% cuprizone) and at recovery (4 weeks after switching to control food) for the different genotypes. c Corresponding T2-weighted MRI signal intensity (relative to the signal intensity at baseline) in external capsule (EC). Group sizes: n = 7–9 for all genotypes and timepoints. Data are shown as means ± SEM. Statistics: ANOVA with random effects comparisons indicated significant differences with respect to WT mice: *0.01 < p < 0.05, ***0.0001 < p < 0.001, **** p < 0.0001. For each group examined, T2-weighted signals were significantly increased with respect to baseline values (significances not shown). d Analysis of TREM2 levels in the brain for mice receiving control food (ctrl), at peak of cuprizone intoxication (week 5, cpz) and after 4-week recovery (rec). n.d. not detected. Statistics: Holm–Sidak’s multiple comparison test one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, ++++ p < 0.0001 to the respective wt group). Wt1, as well as wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt2 is the wild-type group for the TREM2-KO study

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Control, Comparison

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: TREM2-sol and TREM2-KO display myelin debris, lack of remyelination and axonal pathology in the EC. Representative pictures for the different genotypes and timepoints from histological stainings detecting a myelin with Luxol Fast Blue (LFB) and corresponding quantitative optical density (OD) analysis of LFB in the EC (normalized to WT at control food), b mature oligodendrocytes (GST-π) and corresponding image analysis in EC (GST-π soma area in %), c myelin basic protein debris (dMBP) and corresponding image analysis in EC (dMBP-stained area in %), d neurofilament (SMI312) and corresponding image analysis in EC (SMI312-stained area in %). Group sizes: n = 3-7 for all genotypes and timepoints. Data shown as means ± SEM. wt: wild-type, TREM2-IPD: TREM2 cleavage-reduced, TREM2-sol: TREM2 soluble-only, TREM2-KO: TREM2 knockout. Ctrl: control food, cpz: cuprizone food for 5 weeks, rec: recovery on control food for 4 weeks. EC: external capsule, CC: corpus callosum. Scale bars: 300 µm (overview), 50 µm (close-up). Statistics: Holm–Sidak`s multiple comparison test one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Comparisons not indicated are non-significant. Wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, Wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed. For c the analysis of the respective wt group for TREM2-KO was omitted as no dMBP signal was observed

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Control, Staining, Knock-Out, Comparison

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: Increase of NF-L in plasma from TREM2-sol and TREM2-KO in the acute cuprizone model. NF-L measurements in plasma of WT, TREM2-IPD, TREM2-sol and TREM2-KO mice receiving control food (ctrl), at 5 weeks of cuprizone intoxication (cpz) and at 4-week recovery on normal food (rec). Group sizes: wt ctrl ( n = 4), wt cpz ( n = 4), wt rec ( n = 4), TREM2-IPD ctrl ( n = 3), TREM2-IPD cpz ( n = 7), TREM2-IPD rec ( n = 7), TREM2-sol ctrl ( n = 4), TREM2-sol cpz ( n = 7), TREM2-sol rec ( n = 4), TREM2-KO cpz ( n = 4). One-way ANOVA Holm–Šídák's multiple comparisons test, * p < 0.05, *** p < 0.001, **** p < 0.0001. Comparisons not indicated are non-significant. Wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Clinical Proteomics, Control

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: TREM2-IPD and TREM2-sol mice show both sustained microglia/astrocyte activation and enhanced LAMP-1 in the EC. Representative images for the different genotypes and timepoints from histological stainings detecting a Iba1 and corresponding image analysis of Iba1-positive soma numbers (normalized to WT at week 5 cuprizone), b astrocytes (GFAP) and corresponding image analysis (GFAP-stained area in %), c LAMP-1 (lysosomal-associated membrane protein 1) and corresponding image analysis (LAMP1-stained area in %), as well as d TMEM119 (homeostatic marker) and corresponding image analysis (TMEM119-stained area in %). Group sizes: n = 2-7 for all genotypes and timepoints. Data are shown as means ± SEM. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced, TREM2-sol: TREM2 soluble-only, TREM2-KO: TREM2 knockout. Ctrl: control food, cpz: cuprizone food for 5 weeks, rec: recovery on control food for 4 weeks. EC: external capsule. CC: corpus callosum. Scale bars: 300 µm (overview), 50 µm (close-up). Statistics: Holm–Sidak’s multiple comparison test one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Comparisons not indicated are non-significant. wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed. For d the analysis of the respective wt group for TREM2-KO was omitted as no relevant TMEM119 signal was observed

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Activation Assay, Staining, Membrane, Marker, Knock-Out, Control, Comparison

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: Brain cytokine/chemokine response is reduced in TREM2-sol and TREM2-KO, but enhanced in TREM2-IPD. Cytokine/chemokine measurements in brain detecting MIP-1a, MIP-1b, IP-10 and MCP-1 in WT, TREM2-IPD, TREM2-sol and TREM2-KO with control food (ctrl), at 5 weeks of cuprizone intoxication (cpz) and at 4-week recovery on normal food (rec). Measurements are normalized to wt cpz. Group sizes: wt ctrl ( n = 4), wt cpz ( n = 4), wt rec ( n = 4), TREM2-IPD ctrl ( n = 4), TREM2-IPD cpz ( n = 7), TREM2-IPD rec ( n = 7), TREM2-sol ctrl ( n = 4), TREM2-sol cpz ( n = 7), TREM2-sol rec ( n = 4), TREM2-KO ctrl ( n = 7), TREM2-KO cpz ( n = 4), TREM2-KO rec ( n = 7). Statistics: ordinary one-way ANOVA Holm–Šídák's multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Comparisons not indicated are non-significant. wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Control

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: TREM2-IPD showed enhanced myelination in the chronic cuprizone model. a Schematic diagram of the experimental setup for the cuprizone treatment and recovery. Groups consisted of mice treated for 12 weeks with control food (normal food) or 0.2% cuprizone in food and then switched back to control food for the 3-week recovery. MRI measurements were performed at timepoints indicated. Mice were culled at week 15 immediately after the last MRI measurement. b T2-weighted signals in the CC and EC during the 12-week intoxication period and the recovery phase were significantly increased with respect to baseline values and compared to analyses for mice receiving control diet throughout the experiment. The significance levels # 0.01 < p < 0.05 and ### p < 0.001 correspond to ANOVA with random effects comparisons between WT and TREM2-IPD animals. Representative images for the different genotypes and at week 15 from histological stainings detecting c myelin with Luxol Fast Blue (LFB) and corresponding quantitative optical density analysis of LFB in the EC and CC, and d mature oligodendrocytes (GST-π) and corresponding image analysis in EC and CC (GST-π soma area in %). Group sizes: n = 5–7 for all genotypes and timepoints. Male mice were used for the cuprizone groups. Data are shown as means ± SEM. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced. Ctrl: control food, rec: recovery on control food for 3 weeks. Control refers to TREM2-IPD mice receiving normal food throughout the study. EC: external capsule, CC: corpus callosum. Scale bars: 500 µm. Statistics: ordinary one-way ANOVA Holm–Šídák’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Control

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation