sheep anti trem2  (R&D Systems)

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT enhances TREM2 recycling and Aβ endocytosis in Aβ-exposed microglia. ( A ) Flowchart of TREM2 internalization test. ( B ) After treatment with IHT, the TREM2 internalization assay was conducted in Aβ-exposed microglia, followed by the fixation of the cells and counterstaining with DAPI. Scale bar = 10 μm. ( C ) TREM2 intensity in Aβ-exposed microglia in panel B ( n > 80). ( D ) Flowchart of TREM2 recycling test. ( E ) After treatment with IHT, the TREM2 recycling assay was performed in Aβ-exposed microglia. The cells were then fixed and counterstained with DAPI. Scale bar = 10 μm. ( F ) TREM2 intensity in Aβ-exposed microglia in panel E ( n > 80). ( G ) After treatment with IHT, membrane TREM2 was labeled with anti-TREM2 antibodies in Aβ-exposed microglia and allowed to undergo internalization for 60 min. Subsequently, the cells were fixed and probed with anti-LC3 antibodies. Scale bar = 5 μm. ( H ) Colocalization ratio of TREM2 with LC3 in single cells in panel H via Manders’ colocalization coefficients ( n > 80). ( I ) After treatment with IHT, membrane TREM2 was labeled with anti-TREM2 antibodies in Aβ-exposed microglia and allowed to undergo internalization for 60 min. Subsequently, the cells were fixed and probed with anti-LAMP1 antibodies. Scale bar = 5 μm. ( J ) Colocalization ratio of TREM2 with LAMP1 in single cells in panel I via Manders’ colocalization coefficients ( n > 80. * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-way ANOVA. Nor, Normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Membrane, Labeling

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: Antibody information

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques:

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: Primer information

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Sequencing

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT downregulates TREM2 in DAM and reduces Aβ load in brains of 9-month-old APP/PS1 mice. ( A ) Escape latency of the mice to reach the target platform during the MWM training period. ( B ) The ratio of dwelling time in the target quadrant compared to all quadrants during the MWM probe test. ( C ) The escape latency of mice to reach the target quadrant during the MWM probe period. ( D ) The average swimming speed of mice during the MWM probe test ( n = 7). ( E ) Brain sections were probed with 6E10 antibodies, and microscopy images of the Aβ plaques were obtained. White arrows indicate plaques. Scale bar = 1 mm. ( F ) Brain sections were incubated with anti-TREM2 and anti-Iba1 antibodies, and microscopy images of DAM in the CA1 region were acquired. Scale bar = 10 μm. ( G ) Number of Aβ plaques in the hippocampus in panel E ( n = 6). ( H ) Number of Aβ plaques in the cortex in panel E ( n = 6). ( I ) Trem2 expression in the CA1 region was measured using qRT-PCR ( n = 3). ( J ) TREM2 intensity in the DAM in panel F (six mice in each group, with 15 cells per mouse). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test. Nor, normoxia; IHT, intermittent hypoxia training; WT, wild type; TG, APP/PS1

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Microscopy, Incubation, Expressing, Quantitative RT-PCR

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT upregulates VPS35 in DAM and Aβ-exposed microglia. ( A ) VPS35 expression in IHT-treated mice brain cortex was detected using Western blotting. (B) Grayscale values of the protein bands in panel A ( n = 6). ( C ) Vps35 expression in the CA1 regions was estimated via qRT-PCR ( n = 3). ( D and E ) Brain sections were labeled with anti-VPS35 and anti-Iba1 antibodies, and microscopy images of DAM ( D ) and microglia not associated with plaques in CA1 region were captured ( E ). Scale bar = 10 μm. ( F ) The average fluorescence intensity of VPS35 in the Iba1 + cells of DAM in panel D ( n = 7, each data point represents the average intensity in DAM of 6 plaques in each mouse brain). ( G ) VPS35 intensity in the Iba1 + cells in panel E ( n = 4). ( H ) After treatment with IHT, VPS35 expression in Aβ-exposed microglia was detected using Western blotting. ( I ) Grayscale values of the protein bands in panel H ( n = 4). ( J ) After treatment with IHT, membrane TREM2 was labeled with anti-TREM2 antibodies in Aβ-exposed microglia and allowed to undergo internalization for 30 min. Subsequently, the cells were fixed and probed with anti-VPS35 antibodies. Scale bar = 3 μm. ( K ) Colocalization ratio of TREM2 with VPS35 in single cells via Manders’ colocalization coefficients ( n > 100). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( B , C and F ) or two-way ANOVA ( G , I , and K ). n.s. indicates no significant difference. Nor, normoxia; IHT, intermittent hypoxia training; WT, wild type; TG, APP/PS1

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Labeling, Microscopy, Fluorescence, Membrane

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: VPS35 chaperone R55 augments TREM2 recycling and Aβ endocytosis by Aβ-exposed microglia. Primary microglia were co-treated with oAβ and R55. ( A ) After R55 treatment, Aβ-exposed microglia were incubated with Aβ-555 for 30 min. The cells were then fixed and probed with anti-Rab5 antibodies, followed by counterstaining using DAPI. Scale bar = 10 μm. ( B ) Colocalization ratio of Aβ-555 with Rab5 in single cells via Manders’ colocalization coefficients. ( C ) After R55 treatment, the TREM2 internalization assay was performed in Aβ-exposed microglia. Subsequently, the cells were fixed and counterstained with DAPI. Scale bar = 20 μm. ( D ) TREM2 intensity in the Aβ-exposed microglia in panel C ( n > 50). ( E ) After R55 treatment, the TREM2 recycling assay was conducted in Aβ-exposed microglia. The cells were further fixed and counterstained with DAPI. Scale bar = 20 μm. ( F ) TREM2 intensity in the Aβ-exposed microglia in panel E ( n > 80). ( G ) After R55 treatment, membrane TREM2 in Aβ-exposed microglia was labeled with anti-TREM2 antibodies and allowed to undergo internalization for 60 min. The cells were then fixed and probed with anti-LAMP1 antibodies. Scale bar = 10 μm. ( H ) Colocalization ratio of TREM2 with LAMP1 in single cells via Manders’ colocalization coefficients ( n > 100). ( I ) Vps35 expression in R55-treated Aβ-exposed microglia was estimated via qRT-PCR ( n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( H ) or two-way ANOVA ( B , D , F and I ). n.s. indicates no significant difference

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Incubation, Membrane, Labeling, Expressing, Quantitative RT-PCR

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT-induced upregulation of Aβ endocytosis by Aβ-exposed microglia depends on VPS35. Primary microglia were transfected with lentivirus expressing Cre-GFP. ( A ) Cells were labeled with anti-VPS35 antibodies. Scale bar = 20 μm. ( B ) VPS35 intensity in GFP − and GFP + cells in panel A ( n > 50). ( C ) Cells were incubated with Aβ-555 for 30 min and then fixed. Scale bar = 10 μm. ( D ) Aβ-555 intensity in GFP - or GFP + cells in panel C ( n > 80). ( E ) Cells were labeled with anti-LAMP1 and anti-TREM2 antibodies, followed by counterstaining using DAPI. Scale bar = 5 μm. ( F ) Colocalization ratio of TREM2 with LAMP1 in single cells via Manders’ colocalization coefficients ( n > 100). ( G to J ) Cells were treated with oAβ to construct Aβ-exposed microglia, followed by treatment with IHT. TREM2 internalization ( G ), TREM2 recycling ( I ), and Aβ-555 uptake ( K ) assays were conducted in the Aβ-exposed microglia (Scale bar = 20 μm). Endocytosed TREM2 ( H ), recycled TREM2 ( J ), and internalized Aβ-555 ( L ) were quantified from panels G, I , and K , respectively ( n > 50). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( B , D and F ) or two-way ANOVA ( H , J and L ). n.s. indicates no significant difference. Nor, normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Transfection, Expressing, Labeling, Incubation, Construct

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT exhibits no significant improvement in Aβ pathology in 7-months microglial VPS35-deficient APP/PS1 mice. ( A ) Labeling information for different groups. ( B ) Flowchart of the development of IHT-treated MG VPS35 KO: APP/PS1 mice. ( C ) Escape latency of the IHT-treated MG VPS35 KO: APP/PS1 mice to find the platform for the first during the MWM training period. ( D ) Swimming trajectories of the mice in the MWM probe period. ( E ) The dwelling time in the target quadrant was calculated as the percentage of the total time in the MWM probe test. ( F ) Total moving distance in the target quadrant was calculated in the MWM probe test. ( G ) The escape latency of mice to archive the target quadrant during the MWM probe period. ( H ) The average swimming speed of mice during the MWM probe test ( n = 5). ( I ) Brain sections of the IHT-treated MG VPS35 KO: APP/PS1 mice were labeled with 6E10 antibodies. White arrows indicate plaques. Scale bar = 2 mm. ( J) Brain sections of the IHT-treated MG VPS35 KO: APP/PS1 mice were probed with anti-PSD95 antibodies to label synapses in the CA1 region. Scale bar = 150 μm. ( K ) Brain sections of the IHT-treated MG VPS35 KO: APP/PS1 mice were stained with anti-Iba1 and anti-TREM2 antibodies. Scale bar = 20 μm. ( L ) Number of Aβ plaques in the hippocampus and cortex regions in panel I . ( M ) PSD95 intensity in the CA1 region in panel J . ( N ) TREM2 intensity in the DAM of CA1 region in panel K (five mice in each group, with five to eight cells per mouse). * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-way ANOVA. n.s. indicates no significant difference. Nor, normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Labeling, Staining

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: IHT-induced attenuation of Aβ pathology and amelioration of Aβ endocytosis by DAM depends on TFEB. 9-month-old APP/PS1 mice were treated with TA1. ( A ) Brain sections of TA1-treated APP/PS1 mice were stained with anti-TFEB/anti-VPS35/anti-TREM2 and anti-Iba1 antibodies, and microscopy images of DAM in CA1 region were captured. Scale bar = 20 μm. ( B to D ) TFEB ( B ), VPS35 ( C ), and TREM2 ( D ) intensities in the DAM in panel A (six mice in each group, with 10 cells per mouse). ( E ) Sh Tfeb BV2 cells were used to construct Aβ-exposed microglia and then treated with IHT. TFEB and VPS35 expression levels were detected via Western blotting. ( F ) Grayscale values of the protein bands in panel E ( n = 3). ( G ) After treatment with IHT, sh Tfeb Aβ-exposed microglia were incubated with Aβ-555 for 30 min, followed by fixation and counterstaining with DAPI. Scale bar = 10 μm. ( H ) Aβ-555 intensity in the GFP + cells in panel G ( n > 50). * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( B to D ) or two-way ANOVA ( F , H ). n.s. indicates no significant difference. Nor, normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Staining, Microscopy, Construct, Expressing, Western Blot, Incubation

Journal: Alzheimer's Research & Therapy

Article Title: Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

doi: 10.1186/s13195-024-01489-6

Figure Lengend Snippet: TFEB inhibition reduces the IHT-induced upregulation of VPS35 in DAM 9-months APP/PS1 mice brains. ( A ) Brain sections of APP/PS1 mice treated with IHT or EO were probed with 6E10 antibodies, and microscopy images of the Aβ plaques were acquired. White arrows indicate plaques. Scale bar = 2 mm. ( B ) Number of Aβ plaques in the hippocampus in panel A ( n = 9). ( C to H ) Brain sections of IHT or EO-treated APP/PS1 mice were stained with anti-TFEB ( C ), anti-VPS35 ( E ), or anti-TREM2 ( G ) and anti-Iba1 antibodies, and microscopy images of DAM in CA1 region were captured. Scale bar = 20 μm. TFEB ( D ), VPS35 ( F ), and TREM2 ( H ) intensities in the DAM cells in panels C, E , and G , respectively. * p < 0.05, ** p < 0.01, and *** p < 0.001 by Student’s t -test ( D , F and H : four mice in each group, with three to five cells per mouse). Nor, normoxia; IHT, intermittent hypoxia training

Article Snippet: After IHT treatment, primary microglia were incubated in DMEM-F12 medium containing sheep anti-TREM2 antibodies (R&D Systems, AF1729) or mouse anti-TFR1 antibodies (Thermo Fisher, 13-6800) for 1 h at 4 °C.

Techniques: Inhibition, Microscopy, Staining

Journal: Journal of Translational Medicine

Article Title: Modulation of hippocampal protein expression by a brain penetrant biologic TNF-α inhibitor in the 3xTg Alzheimer’s disease mice

doi: 10.1186/s12967-024-05008-x

Figure Lengend Snippet: Iba1, TREM2, and β-sheet rich Aβ plaque in the hippocampus of 3xTg-AD mice with TfRMAb-TNFR treatment. Representative confocal images of Iba1-positive microglia (green) and TREM2-positive microglia (red) of the 3xTg-AD mice with or without TfRMAb-TNFR-treatment ( Ai ). Representative 3D rendering of z-stack images generated using Imaris software ( Aii ) and an orthogonal view ( Aiii ) showing Iba1 (green) and TREM2 (red) colocalization. Scale bar = 5–10 µm in A as indicated. Plaque-bearing (subiculum) black-boxed region and plaque-free (CA2) red-boxed region in the thumbnail brain section image in B were imaged to quantify Iba1-positive area % ( B ) and TREM2-positive area % ( C ). The thumbnail image in B was taken from the Allen Institute. Representative images of β-sheet-rich Aβ plaques (blue) and microglia (Iba1, green) staining in the plaque-bearing subiculum of Tg-Saline or Tg-TfRMAb-TNFR mice and the resulting β-sheet-rich Aβ plaque-positive area % ( D ). Representative confocal images showing Iba1 (green), TREM2 (red), and β-sheet-rich Aβ (blue) fluorescence staining in the plaque-bearing subiculum of 3xTg-AD mice with or without TfRMAb-TNFR treatment ( E ). Scale bar = 25 μm in D – E . Mature Aβ plaque-associated microglial MFI ( F ) and mature Aβ plaque-associated TREM2 MFI ( G ). Quantifications in F and G are based on the circular regions of interest outlined in E . The data are shown as Mean ± SEM for WT-Saline (n = 9), Tg-Saline (n = 11), and Tg-TfRMAb-TNFR (n = 11) mice. Data were analyzed using the two-way repeated measures ANOVA with Holm Sidak’s post-hoc test in B – D , and unpaired t-test and Mann–Whitney U test in F , G , respectively. Outliers have been detailed in Additional file : Table. S1. *p < 0.05, **p < 0.01, and ***p < 0.001 for the indicated comparisons

Article Snippet: The sections were washed with deionized water and subjected to permeabilization with 0.3% Triton X-100 in PBS for 60 min at RT and blocked with 0.5% BSA with 0.3% TritonX-100 in PBS for 2 h. The sections were then incubated with anti-Iba1 rabbit antibody (1:1000 Wako, #019-19741) and anti-TREM2 sheep antibody (1:250, R&D system, #AF1729) in 0.5% BSA with 0.3% TritonX-100 in PBS overnight at 4 °C with gentle shaking.

Techniques: Generated, Software, Staining, Saline, Fluorescence, MANN-WHITNEY

Journal: Journal of Translational Medicine

Article Title: Modulation of hippocampal protein expression by a brain penetrant biologic TNF-α inhibitor in the 3xTg Alzheimer’s disease mice

doi: 10.1186/s12967-024-05008-x

Figure Lengend Snippet: Schematic showing the mechanism of action of TfRMAb-TNFR in Aβ plaque reduction. TfRMAb-TNFR increases TREM2-positive microglial clustering around Aβ plaques, which is associated with a reduction in mature Aβ plaque load in the 3xTg-AD mice. No change in tau pathology is observed with TfRMAb-TNFR in this mouse model. Figure was prepared using BioRender.com

Article Snippet: The sections were washed with deionized water and subjected to permeabilization with 0.3% Triton X-100 in PBS for 60 min at RT and blocked with 0.5% BSA with 0.3% TritonX-100 in PBS for 2 h. The sections were then incubated with anti-Iba1 rabbit antibody (1:1000 Wako, #019-19741) and anti-TREM2 sheep antibody (1:250, R&D system, #AF1729) in 0.5% BSA with 0.3% TritonX-100 in PBS overnight at 4 °C with gentle shaking.

Techniques:

Journal: bioRxiv

Article Title: Lesion-remote astrocytes govern microglia-mediated white matter repair

doi: 10.1101/2024.03.15.585251

Figure Lengend Snippet: a , iSCI lesion-remote microglia lipidomics schematic. A parasagittal spinal cord dissection enriched for microglia from Wallerian degenerating white matter. b , Comparison of lipidomic profiles of healthy and iSCI microglia from WT and Ccn1 cKO in principle component space. iSCI microglia from WT and Ccn1 cKO are clearly separable. c , Comparison of WT and Ccn1 cKO significant injury-reactive alterations in microglia lipid profile, including lipid droplet- and myelin-associated lipid subtypes (Log 2 fold-change, iSCI vs healthy, FDR P ≤ 0.01). Non-significantly altered lipid species show in white. d , e , Comparison of differentially expressed CE and PE lipid species in iSCI vs healthy microglia, from WT and Ccn1 cKO spinal cords. f-i , High magnification 3D image and quantification of PLIN2 + lipid droplets and Abca1 in WT and Ccn1 cKO WDM from Wallerian degenerating white matter (PLIN2: n=3-6 mice/group; Abca1 : n=3-4 mice/group). j , Cholesterol efflux measured from cultured primary mouse microglia following stimulation with CCN1 or vehicle (BSA) (n= 3 replicates from independent cultures conditions run in triplicate; Students t-test, ** P ≤ 0.05) . k-p , High magnification 3D image and quantification of TREM2, Gpnmb and Igf1 in WT and Ccn1 cKO WDM from Wallerian degenerating white matter (TREM2: n=3-6 mice/group; Gpnmb and Igf1 n=3-4 mice/group) q , Quantification of spontaneous recovery of left hind-paw cold thermoception over time after iSCI (n= 7-8 mice per group). CE: Cholesterol Esters, TAG: Triacylglycerol, PC: Phosphatidylcholine, PE: Phosphatidylethanolamines, EM: Sphingomyelin, CER: Ceramides, CAR: Carnitine, PS: Phosphatidylserine, PI: Phosphatidylinositol, PG: Phosphatidylglycerols, FA: Fatty Acids. Graphs show mean ± SEM. Opaque and transparent data points illustrate experimental replicate mean and counts from individual tissue sections/cells, respectively. Unless stated otherwise, * P ≤ 0.05, ** P ≤ 0.002, *** P ≤ 0.0002, **** P ≤ 0.0001, Two-way ANOVA with Tukeys. Scale bars: 10μm.

Article Snippet: Primary antibodies include: Rat-CD18 (1:100, Invitrogen), Rat-GFAP (1:1000, Thermofisher), Rabbit-GFAP (1:1000, Dako), Goat-IBA1 (1:1000, Abcam), Rabbit-IBA1 (1:1000, Wako), Rabbit-LPL (1:50, Abcam), Rabbit-PLIN2 (1:500, Progen), Goat-SOX9 (1:200, R&D system), Mouse-SMI32 (1:3000, Biolegend), Sheep-TREM2 (1:250, R&D systems), Rabbit-YAP1 (1:200, Protintech).

Techniques: Dissection, Comparison, Cell Culture

Journal: bioRxiv

Article Title: Lesion-remote astrocytes govern microglia-mediated white matter repair

doi: 10.1101/2024.03.15.585251

Figure Lengend Snippet: a , Microglia lipid species distribution across treatment groups. Note that in both WT and Ccn1 cKO animals, the total microglia lipidome is reduced at 28 dpi. b , Heat map of Z-scores showing relative levels of all significant lipid subtypes detected by unbiased microglia lipidomic analysis . c , d , Lipid pathway enrichment of the WT and Ccn1 cKO injury response highlighting a shift in the predominant lipid pathways employed after iSCI. e , Direct pairwise comparison of WT and Ccn1 cKO microglia lipid profile for healthy and iSCI, including lipid droplet- and myelin-associated lipid subtypes (Log 2 fold-change FDP P ≤ 0.01). f , Schematic of ceramide to sphingomyelin conversion mediated by sphingomyelin synthase predicted by Biopan lipid pathway analysis comparing the iSCI and healthy Ccn1 cKO animals (z-score 1.921). g , Plots show percentage maximum summed intensity for sphingomyelin (SM) and ceramide (CER) in both WT and Ccn1 cKO mice. h , Quantification of the proportion of PLIN2 containing microglia from WT or Ccn1 cKO Ccn1 cKO Wallerian degenerating dorsal column white matter. i-k , High magnification 3D image and quantitative comparison of BODIPY + lipid droplets within IBA1 + WDM nodules from WT and Ccn1 cKO Wallerian degenerating dorsal column white matter (n=3-6 mice/group). l , Relative proportions of IBA1 + microglia that are TREM2 + from WT and Ccn1 cKO Wallerian degenerating dorsal column white matter. m-o , High magnification and 3D image and quantitative comparisons of IBA1 + microglia that contain LPL (n=3-6 mice/group). p , Line graph illustrating spontaneous recovery of locomotor behavior in the left hindlimb after iSCI in WT vs Ccn1 cKO mice by modified Basso mouse scale scoring. Graphs show mean ± SEM. Opaque and transparent data points illustrate experimental replicate mean and counts from individual tissue sections/cells, respectively. Unless stated otherwise, * P ≤ 0.05, ** P ≤ 0.002, *** P ≤ 0.0002, **** P ≤ 0.0001, Two-way ANOVA with Tukeys. Scale bars, 10 µm

Article Snippet: Primary antibodies include: Rat-CD18 (1:100, Invitrogen), Rat-GFAP (1:1000, Thermofisher), Rabbit-GFAP (1:1000, Dako), Goat-IBA1 (1:1000, Abcam), Rabbit-IBA1 (1:1000, Wako), Rabbit-LPL (1:50, Abcam), Rabbit-PLIN2 (1:500, Progen), Goat-SOX9 (1:200, R&D system), Mouse-SMI32 (1:3000, Biolegend), Sheep-TREM2 (1:250, R&D systems), Rabbit-YAP1 (1:200, Protintech).

Techniques: Comparison, Modification

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAP visualization of mouse hippocampal cell clusters classified by cell type based on DEG identified by Seurat v4. Violin plots represent the log-normalized expression of Itm2b and Trem2 across cell populations in mouse hippocampal cell clusters. ( B ) Human DFC cell clusters, classified by cell type as in ( A ). Violin plots represent the log-normalized expression of ITM2B and TREM2 across cell populations in human DFC cell clusters. ( C ) List of cell-type- specific marker genes used to annotate major brain populations. ( D ) Itm2b and Trem2 mRNA expression in mouse microglia and non-microglia cells analyzed by quantitative RT-PCR. Data information: The data sets analyzed are publicly available and are described in the two following papers: mouse scRNAseq data set (Zeisel et al, ), hippocampus n = 5 females, n = 5 males; human snRNAseq data set (Li et al, ), n = 10 (sex not specified). More information about these datasets can be found in the cited paper. Statistical comparisons between the groups shown in ( D ) was conducted using two-tailed unpaired t test **** P < 0.0001. The data are derived from are from 15-month-old w/w control animal, male n = 3, females n = 3; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing, Marker, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Control

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of objects Data 1 and Data 2 before filtering. ( B ) UMAPs of objects Data 1 and Data 2 after filtering. ( C ) UMAP of Object 1 after integration, filtering, and re-clustering, shows 16 microglia clusters. Data information: The data presented in this analysis are the result of two experiments, namely Data 1 and Data 2. To combine specific sample datasets from both Data 1 and Data 2, we employed the integration feature within the Seurat package. By utilizing the first 20 principal components, we integrated these datasets into a single entity referred to as “Object1,” which encapsulates information from a total of 297,215 cells. These cells derive from: Trem2-KO , 1 male and 1 female; Itm2b-KO , 2 males and 2 females; WT controls, 1 male and 2 females; Itm2b/Trem2-dKO , 1 male and 1 female. The scRNAseq data are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques:

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) UMAPs of microglia grouped by genotype. ( B ) Average scaled expression levels of selected signature genes per cluster and cluster’s annotation based on expression of signature genes. ( C ) Volcano plots showing differentially expressed genes in clusters I/T-D1, 2, 3 and 4. ( D ) Proportional contribution of each genotype and proportional contribution of individual samples of each genotype to cluster 3. ( E ) KEGG pathway enrichment analysis of pathways upregulated in cluster 3. Data information: The data presented in this analysis are the result of two experiments, namely Data 1 and Data 2. To combine specific sample datasets from both Data 1 and Data 2, we employed the integration feature within the Seurat package. By utilizing the first 20 principal components, we integrated these datasets into a single entity referred to as “Object1,” which encapsulates information from a total of 297,215 cells. Volcano plots in ( C ) were obtained using Fast Wilcoxon rank sum test and auROC. These cells derive from: Trem2-KO , n = 1 male and n = 1 female; Itm2b-KO , n = 2 males and n = 2 females; WT controls, n = 1 male and n = 2 females; Itm2b/Trem2-dKO , n = 1 male and n = 1 female. The scRNAseq data are deposited at https://www.ncbi.nlm.nih.gov/geo/info/seq.html , GSE233601 to allow public access once the data are published. All data are expressed as means +/− SEM.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) N2A or HEK293 cells were transfected with F-BRI2 (B) and Trem2 (T), either alone (V=empty pcDNA3.1vector) or in combination and analyzed by Western blot with anti-FLAG (M2) and anti-Trem2 (NT1) on total lysates (T.L.) and M2 immunoprecipitants (IP-M2). Immunoprecipitants bound to M2-Agarose beads were specifically eluted using the 3xFLAG peptide. For each cell line, two independent transfections were performed (Exp. 1 and Exp. 2). ( B ) Schematic representation of Trem2 and the two products of α-secretase cleavage, sTrem2, and Trem2-CTF. TM indicates the transmembrane region of Trem2. Red bars point to the antigenic regions used to produce the anti-Trem2 antibodies CT, NT1 and NT2. The cytosolic and intralumenal/extracellular regions of Trem2 are indicated. ( C ) Western blot analysis with anti-FLAG, anti-Trem2 NT1, and anti-Trem2 CT antibodies of T.L. and IP-M2 from HEK293 cells transfected with F-BRI2 and Trem2, either alone or in combination, with or without deglycosylation. *Indicates Trem2 species of unclear primary structure. Trem2 (f.l.) indicates full length Trem2. ( D ) Western blot analysis with anti-FLAG and anti-Trem2 CT antibodies of immunoprecipitants obtained with CT, NT1, and NT2 antibodies from HEK293 cells expressing either F-BRI2 alone or F-BRI2 plus Trem2. The nature of the bands migrating above 100 kDa in the NT2 IP samples is unknow. ( E ) Schematic representation of the F-BRI2 constructs used in ( F ). The Bri23 region, transmembrane region (TM), Brichos domain, APP-binding domain (APP BD), FLAG tag (F), cytosolic and intralumenal/extracellular regions are indicated. ( F ) WB analysis with anti-FLAG and anti-Trem2 antibodies of lysates and immunoprecipitants from HEK293 cells expressing F-BRI2 deletion mutants plus Trem2 or Trem2 alone (V). The * indicates Trem2 species of unclear primary structure. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Transfection, Western Blot, Expressing, Construct, Binding Assay, FLAG-tag

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of the bicistronic expression plasmids used for HEK293 cell transfections in Panel ( B ): TREM2-3xFLAG + Myc-BRI2, 3xFLAG + Myc-BRI2 1-131 , 3xFLAG + Myc-BRI2 1-80 , and 3xFLAG + Myc-BRI2δ 80-131 . The TREM2-3xFLAG is expressed by the 5’ cistron, while BRI2 proteins are expressed by the 3’ cistron. ( B ) Western blot analysis using anti-FLAG, anti-Myc, and anti-human BRI2 antibodies of Total Lysate and Immunoprecipitation (M2-IP) samples from transfected HEK293 cells. “Glyc.” indicates glycosylated TREM2. The anti-human BRI2 antibody exhibits reactivity toward Myc-BRI2 and Myc-BRI2 1-131 suggesting recognition of an epitope located within the amino acids 80-131 region of BRI2. ( C ) Schematic representation of the bicistronic expression plasmids employed for HEK293 cell transfections in Panel ( D ): 3xFLAG-BRI2 + TREM2-Myc, 3xFLAG-BRI2 + TREM2-δIg-like-Myc, 3xFLAG-BRI2 + TREM2-CTF-Myc, 3xFLAG-BRI2 + TREM2-W198Ter-Myc, and 3xFLAG-BRI2 + TREM2-δ/α-site-Myc. The 3xFLAG-BRI2 is expressed by the 5’ cistron, while TREM2 proteins are expressed by the 3’ cistron. ( D ) Western blot analysis with anti-FLAG, anti-human TREM2-NT, and anti-human TREM2-CT antibodies of Total Lysate (T.L.) and Immunoprecipitation (IP) samples from transfected HEK293 cells. “Glyc.” indicates glycosylated TREM2. * Indicates protein signals of unclear nature. ** and *** indicate TREM2W198Ter signals of unclear primary structure. A longer exposure (Long Exposure) of the Anti-hTREM2-CT Western blot for the 3xFLAG-BRI2 + TREM2-Myc transfection revealed traces of TREM2-CTF precipitating with BRI2. Data information: This figure represents one of three independent experiments conducted. Data from the other two experiments are presented in Fig. . We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Western blot analysis with anti-FLAG, anti-Myc antibodies, and anti-human BRI2 antibodies of total lysates and immunoprecipitated samples (IP-M2) from transfected HEK293 cells. These experiments are biological replicates of the experiment shown in Fig. . ( B ) Western blot analysis with anti-FLAG, anti-human TREM2-NT, and anti-human TREM2-CT antibodies of total lysates (T.L.) and immunoprecipitated samples (IP-M2) from transfected HEK293 cells. These experiments are biological replicates of the experiment shown in Fig. . ( C ) Co-immunoprecipitation of endogenous Bri2 and Trem2 from mouse primary macrophages. Samples were deglycosylated before Western blot. Data Information: Panels ( A ) and ( B ) represent two independent experiments conducted similarly to those in Figs. B and , respectively. Panel ( C ) shows the only co-immunoprecipitation of endogenous Bri2 and Trem2 performed to date. The complete membrane images used for Western blot analyses are included without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Western Blot, Immunoprecipitation, Transfection, Membrane

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of TREM2-ECD, sTREM2, BRI2-ECD and BRI2-BRICHOS recombinant proteins. TREM2-ECD encompasses the entire extracellular domain of TREM2, and BRI2-ECD encompasses the entire extracellular domain of BRI2, including the second putative TREM2-interacting domain. BRI2-BRICHOS and BRI2-ECD were fused with a 3xFLAG tag at their N-terminus, enabling immunoprecipitation using anti-FLAG M2-Agarose beads for the purification of protein complexes in a cell-free system via elution with a 3xFLAG peptide. The diagram highlights the signal peptides (SP), 7-Histidine tag (7-His, employed for protein purification), the 3XFLAG tag (3xF, utilized for complex purification), Ig-like domain (of TREM2), and BRICHOS domain (of BRI2). ( B ) BRI2-BRICHOS + sTREM2, BRI2-BRICHOS + TREM2-ECD, BRI2-ECD + sTREM2, BRI2-ECD + TREM2-ECD, sTREM2 alone, and TREM2-ECD alone were incubated overnight at 4 degrees Celsius with M2-Agarose beads at a concentration of 2 μM for each protein. Following extensive washing, complexes bound to M2-Agarose beads were specifically eluted using the 3xFLAG peptide. Unbound proteins and eluates (3xFLAG elu.) were analyzed by Western blot using either the anti-FLAG antibody M2 or an anti-human TREM2 N-terminal antibody (TREM2-NT). sTREM2 and TREM2-ECD were not recovered in the eluates when BRI2 recombinant proteins were absent. The * indicates residual BRI2-BRICHOS and BRI2-ECD dimers and oligomers. ( C ) BRI2-BRICHOS + sTREM2, BRI2-BRICHOS + TREM2-ECD, BRI2-ECD + sTREM2, BRI2-ECD + TREM2-ECD, 3xFLAG + sTREM2, 3xFLAG + TREM2-ECD, sTREM2 alone, and TREM2-ECD alone were incubated as in B. Proteins eluted with the 3xFLAG peptide were analyzed by Western blot using either M2 or TREM2-NT. sTREM2 and TREM2-ECD were not recovered in the eluates when BRI2 recombinant proteins were absent. The * indicates residual BRI2-BRICHOS and BRI2-ECD dimers and oligomers. ( D ) Western blot analysis using M2 and TREM2-NT antibodies of a new experiment mirroring the setup in ( B ). Eluates were separated under reducing and non-reducing conditions. BRI2-BRICHOS and BRI2-ECD monomers, dimers, trimers, and tetramers are indicated by the numbers 1, 2, 3, and 4, respectively. Higher multimolecular complexes are present but not labeled. The sTREM2 and TREM2-ECD bound to BRI2-BRICHOS and BRI2-ECD analyzed under non-reducing conditions show no significant increase in molecular weight compared to those analyzed under reducing conditions. ( E ) Decreasing concentrations (4, 2, 1, 0.5, 0.25, and 0 μM) of BRI2-BRICHOS and BRI2-ECD were incubated with 2 μM of either sTREM2 or TREM2-ECD and analyzed as described in ( C ). The bottom panel displays a Western blot of deglycosylated eluates using the TREM2-NT antibody. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Recombinant, Immunoprecipitation, Purification, Protein Purification, Incubation, Concentration Assay, Western Blot, Labeling, Molecular Weight

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) HEK293 cells were transfected with Trem2 and either empty vector (V) or F-BRI2 (B). NT are non-transfected cells. Western blot of cell lysates with either the anti-FLAG antibody M2 or the anti-Trem2 antibody CT. Western blot of deglycosylated culture supernatants with the anti-Trem2 antibody NT1. *Indicates Trem2 species of unclear primary structure. ( B ) Quantification of Trem2, Trem2-CTF and sTrem2 levels detected by Western blot in ( A ). ( C ) HEK293 cells were transfected with Trem2 and either empty vector (V), F-BRI2 or deletion mutant F-BRI2 1-80 . Western blot of cell lysates with either the anti-FLAG antibody M2 or the anti-Trem2 antibody CT. Western blot of deglycosylated culture supernatants with the anti-Trem2 antibody NT1 (lower panel). The * indicates Trem2 species of unclear primary structure. ( D ) Quantification of Trem2, Trem2-CTF and sTrem2 levels detected by Western blot in ( C ). ( E ) HEK293 cells were transfected with either Trem2 (T) or empty vector (V). Following transfection, lysates and media underwent deglycosylation and were subsequently analyzed by Western blot using anti-Trem2 antibodies CT and NT1. In the CT Western blot, the asterisk (*) indicates a Trem2-derived polypeptide that retains the CT epitope and is likely to lack part of the N-terminal Trem2 sequence, causing a reduction in size. In the NT1 Western blot, the double asterisk (**) highlights a Trem2-derived polypeptide that retains the NT1 epitope and is likely to lack part of the C-terminal sequence. The presence of this band primarily in cell lysates suggests potential retention of the transmembrane region and/or localization within intracellular compartments. Its low-level detection in the media further suggests intracellular origin. The band marked as sTrem2 is marked as sTrem2 because: (1) is of the expected size for deglycosilated sTrem2; (2) it is notably enriched in the media, consistent with the preferential localization of sTrem2 in extracellular fluids. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using two-tailed unpaired t test ( B ) and one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences ( C , D ). * P < 0.05, ** P < 0.01, *** P < 0.001. The data presented are derived from are from: Trem2+Vector transfectant n = 5, Trem2+F-BRI2 transfectant n = 5 ( A , B ); Trem2+Vector transfectant n = 3, Trem2+F-BRI2 transfectant n = 3, Trem2+F-BRI2 1-80 n = 3 ( C , D ); the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Derivative Assay, Sequencing, Two Tailed Test

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Schematic representation of ELISA 1 and ELISA 2. Both ELISAs use the same Biotinylated-αTrem2 capture antibody (in black). ELISA 1 uses αTrem2-CT (red) + Sulfo-αRabbit (blue) detection antibodies. ELISA 2 uses αTrem2-NT (orange) + Sulfo-αRat (green) detection antibodies. Trem2 can be detected by both ELISAs, sTrem2 can be detected only by ELISA 2: neither ELISA can detect Trem2-CTF. ( B ) Quantification of Trem2 and sTrem2 in the P100 and S100 brain fractions of ~245 days old w/w control, Itm2b-KO and Trem2-KO mice. ( C ) Western blot analysis of P100 fractions from a representative w/w, Trem2-KO and Itm2b-KO P100 sample with αTrem2-CT and an αBri2 antibody. *Indicates a non-specific band. ( D ) Detection and quantification of Trem2-CTF in the P100 fraction by Western blot analysis and with Image Lab software; GAPDH was used as a loading control. ( E ) ELISA measurements of endogenous Aβ40 and Aβ42 in brain homogenates of w/w, Trem2-KO and Itm2b-KO animals. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. The membrane in ( C ) was cut at the 20 and 15 kDa molecular weight marker (MWM). The upper section was probed with the anti-Bri2 antibody, while the lower section was probed with the Trem2-CT antibody. Similarly, in ( D ), the two membranes were divided at the 20 and 15 kDa MWM. The upper portion was probed with the anti-Gapdh antibody, while the lower portion was probed with the Trem2-CT antibody. Statistical comparisons among the groups were conducted using one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test when ANOVA showed significant differences ( B , E ); two–way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences ( D ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from are from w/w control, females n = 7, males n = 12; Itm2b-KO females n = 6, males n = 7; Trem2-KO , females n = 6, males n = 7; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Software, Membrane, Molecular Weight, Marker, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) CD11b and CD45 staining, and FACS analysis of brain cells isolated from Cx3cr1 CreER/wt and Cx3cr1 wt/wt animals. ( B ) FACS analysis of sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from Itm2b f/f :Cx3cr1 CreER/wt animals. ( C ) Schematic representation of the PCR test used to identify the Itm2b f and Itm2b KO alleles. ( D ) PCR analysis of genomic DNA isolated from EYFP + and EYFP - cells sorted from Itm2b f/f :Cx3cr1 CreER/wt brains. ( E ) Analysis of Itm2b and Trem2 mRNA expression in sorted EYFP + (microglia) and EYFP - (non-microglia) brain cell populations from ~14 months-old Itm2b f/f :Cx3cr1 CreER/wt and Itm2b w/w :Cx3cr1 CreER/wt animals. ( F ) ELISA 2 was used to measure sTrem2 levels in Itm2b f/f :Cx3cr1 CreER/wt and Itm2b f/f :Cx3cr1 wt/wt littermates. Data information: Statistical comparisons among the groups were conducted two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA showed significant differences ( E ); two-tailed unpaired t test ( F ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data presented are derived from: (E) Itm2b f/f :Cx3cr1 CreER/wt , females n = 5, males n = 7; Itm2b w/w :Cx3cr1 CreER/wt , females n = 3, males n = 4; ( F ) Itm2b f/f :Cx3cr1 CreER/wt , females n = 15, males n = 12; Itm2b f/f :Cx3cr1 wt/wt , females n = 10, males n = 11; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Staining, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of sTrem2 in the S100 brain fractions of ~245 days old control and FDD-KI mice. Data were analyzed by unpaired T -test. All data are shown as means +/− SEM: **** P < 0.0001. ( B ) Quantification of Trem2-CTF in the P100 fraction by Western blot analysis and with Image Lab software. ( C ) Red Ponceau staining (upper panel) was used to normalize the Trem2-CTF signal (lover panel) obtained by Western blot. Data information: Statistical comparisons among the groups were conducted using unpaired T -test. **** P < 0.0001. The data presented are derived from: ( A ) w/w mice (females, n = 7; males, n = 12) and FDD-KI mice (females, n = 8; males, n = 9) mice; ( B ) w/w mice (females, n = 3; males, n = 3) and FDD-KI mice (females, n = 6; males, n = 7) mice; the letter “n” indicates biological replicates. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Control, Western Blot, Software, Staining, Derivative Assay

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia using ELISA (left panel) and Western blot of deglycosylated conditioned media with Trem2 NT antibody (quantification of Western blot is shown in the second panel). Trem2 CT antibody does not show any signal. ( B ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia after 5 h of serum starvation by ELISA. ( C ) Quantification of sTrem2 in the conditioned media of WT and Itm2b-KO primary microglia after 1 and 2 h with either vehicle (Veh) or E. coli by ELISA. ( D ) Western blot of deglycosylated cell lysates from WT and Itm2b-KO primary microglia, 2 h after E. coli stimulation, with Trem2 CT antibody to visualize Trem2 f.l. and Trem2-CTF, along with quantification of the Trem2-CTF/Trem2 f.l. ratio. ( E ) Quantification of the Trem2-CTF/Trem2 f.l. ratio for only the two untreated (Veh) groups. ( F ) Analysis of Itm2b and Trem2 mRNA expression in WT and Itm2b-KO primary microglia using quantitative RT-PCR. ( G ) A second set of biological replicates was analyzed following the same procedure as in panel ( C ). ( H ) A second set of biological replicates was analyzed following the same procedure as in panel ( D ). ( I ) A second set of biological replicates was analyzed following the same procedure as in panel ( F ). Data information: Statistical comparisons among the groups were conducted using either a two-tailed unpaired t -test ( A , B , E , F , I ) or a two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA indicated significant differences ( C , D , G , H ). * P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001. The data presented are derived from WT primary microglia cultures ( n = 3 for ( A , C , D , E – I ), n = 6 for ( B )) and Itm2b-KO primary microglia ( n = 3 for Exp. and n = 3 for ( A , C , D , E – I ), n = 6 for ( B )); the letter “n” indicates biological replicates, except for ( B ), which includes 3 biological replicates with 2 technical replicates each. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Western blot analysis with Trem2 CT antibody of deglycosylated cell lysates from Itm2b-KO microglia treated with either vehicle (PBS) or a 2 μM concentration of BRI2-ECD. Quantification of the Trem2-CTF/Trem2 f.l. ratios from the Western blot shown in the right panel. ( B ) sTrem2 ELISA on conditioned media from these cell cultures (left panel). The right panel shows an ELISA performed using media from cells treated with vehicle and incubated before and during the ELISA with either vehicle (PBS) or 2 μM of BRI2-ECD. The evidence that incubation with BRI2-ECD does not change the ELISA quantification indicates that BRI2-ECD does not interfere with the quantification of sTrem2 by ELISA. ( C ) Western blot analysis with anti-Syk and anti-pSyk antibodies of cell lysates from Itm2b-KO microglia treated with either vehicle (PBS) or a 2 μM concentration of BRI2-ECD. Quantification of the pSyk/Syk ratios from the Western blot is shown in the right panel. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using a two-tailed unpaired t -test. * P < 0.05, ** P < 0.01, **** P < 0.0001. The data presented are derived from Itm2b-KO primary microglia ( n = 3 for each condition); the letter “n” indicates biological replicates. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: Reagents and tools.

Article Snippet: The plates were incubated with 0.25 μg/ml biotinylated sheep anti-mouse TREM2 antibody (R&D Systems, BAF1729) for 1 h at RT with shaking.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Gene Expression, Blocking Assay, Western Blot, Isolation, Biomarker Discovery, Software, Imaging, Real-time Polymerase Chain Reaction