1. TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia 
Felix L. Yeh, Yuanyuan Wang, Irene Tom, Lino C. Gonzalez, Morgan Sheng 
Neuron 
Volume 91, Issue 2, Pages (July 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1 Unbiased PM Screen for TREM2-Binding Partners
A PM containing 1,559 genes/proteins (randomly separated into two sublibraries 1 and 2 and spotted on separate microarray slides due to capacity limitations) was screened for binding to the extracellular domain of hTREM2 fused to IgG Fc.
(A and B) Representative images of PM slides (protein library 1; only a portion shown) screened with (A) TREM2-Fc or (B) TREM1-Fc. Slides were scanned for BSA-Cy3 (green) and TREM2-Fc binding through Cy5 (red; see the Experimental Procedures). BSA-Cy3 is spotted in pairs between each individual library protein, which is also spotted in duplicate, to help localize sample positions on the microarray. White boxes denote areas around three prominent hits for TREM2-Fc in library 1 of the microarray.
(C–E) Enlargements of the microarray are shown for the area around each hit and the identity of each protein is overlaid over the spots. The interactions between TREM2-Fc and (C) APOA1, (D) MDA-LDL, and (E) LDL are clearly visible as red dots. The data analysis and scoring system for the microarray are described in the Experimental Procedures.
(F–I) Each scatterplot represents two replicate microarray datasets (arrays 1 and 2), with scores for each protein in the library represented as a dot. An empirically set cutoff of 5 (inner square) was used to define hits. (F) LDL, MDA-LDL, APOA1, and GH were identified as hits for TREM2-Fc in library 1, and (G) SCGB2A2, UNC5B, and NRN1L were identified as hits for TREM2-Fc in library 2. (H and I) The PM library also was screened for binding to TREM1-Fc, which showed no positive hits.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2 TREM2 Binds to Apo/Lipoproteins Including CLU and APOE
Measurement of hTREM2 binding to lipoproteins and apolipoproteins by bio-layer interferometry (BLI). BLI measures analyte binding via an increase in optical thickness at the bait probe, which results in a wavelength shift measured in nanometers.
(A) TREM2-Fc bound to LDL, VLDL, Lp(a), HDL, HDL-2, and HDL-3 (25 μg/ml).
(B) TREM2-Fc also bound to purified APOB, lipidated (lipid) APOA1, and lipidated APOA2. TREM2-Fc showed no appreciable binding to purified APOA1, APOA2, lipidated APOB, or lipidation agent DMPC alone (lipid).
(C) TREM2-Fc bound to lipidated CLU, but no appreciable signal was seen with unlipidated CLU or lipid alone.
(D) TREM2-Fc also bound to lipidated APOE, but not unlipidated APOE. Protein concentrations were 10 μg/ml and lipid concentrations were 10 μg/ml for the BLI experiments, except where noted. Black arrows indicate when probe was switched into dissociation buffer (PBS).
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4. Figure 3
Disease Variants of TREM2 Reduce Binding to LDL, CLU, and APOE
(A) A schematic of TREM2 protein with alignment of the human and mouse sequences shows the variants in TREM2 (NHD variants Y38C and T66M; AD variants R47H, R62H, and D87N; and designer loss-of-function mutant K48M).
(B, D, and F) The average BLI binding responses from three technical replicates to (B) LDL, (D) lipidated CLU, and (F) lipidated APOE for WT as well as mutant forms of TREM2-Fc are shown by the solid lines, while the SEM is indicated by the corresponding lighter shading.
(C, E, and G) Quantitation of maximum binding responses for (C) LDL, (E) CLU, and (G) APOE to WT and different variants of TREM2-Fc. The NHD variants showed no binding while AD variants showed significant reduction (n = 3, one-way ANOVA with Bonferroni multiple comparison test). Error bars represent SEM (∗∗p < 0.01 and ∗∗∗p < 0.001). Black arrows indicate when probe was switched into dissociation buffer (PBS). All protein concentrations were 30 μg/ml and lipid concentrations were 10 μg/ml for lipidated material.
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5. Figure 4 TREM2 Is an Uptake Receptor for LDL, CLU, and APOE
(A) hTREM2-Dox cells were incubated with 25 μg/ml DiI-AcLDL for 4 hr and uptake of AcLDL was measured by DiI fluorescence (red). Cells were marked with CellTracker Green. Extracellular fluorescence was quenched with trypan blue. Representative confocal images show an increase in DiI-AcLDL uptake in induced (hTREM2-expressing) cells compared to uninduced cells. Scale bar, 20 μm.
(B) Time course shows DiI-AcLDL uptake, relative to cell area (marked by CellTracker Green), between induced and uninduced hTREM2-Dox stable 293 cells (n = 9, two-way ANOVA).
(C and D) Dox induction of hTREM2 also stimulated uptake of (C) lipidated CLU and (D) APOE, as measured by western blotting of cell lysates after 2-hr incubation with lipidated CLU or APOE (protein concentrations: 3 μg/ml; lipid concentrations: 10 μg/ml).
(E) Quantification of CLU and APOE band intensity demonstrated significantly increased CLU and APOE uptake in induced versus uninduced hTREM2-Dox cells (n = 6, one-way ANOVA with Bonferroni multiple comparison test).
(F) Representative confocal images show DiI-AcLDL uptake in Dox-induced cells expressing WT and mutant forms of hTREM2. Scale bar, 10 μm.
(G) Quantification of DiI-AcLDL uptake by hTREM2 variants (normalized to WT hTREM2) showed a significant reduction of DiI-AcLDL internalization for all mutant forms of hTREM2 (n = 51, one-way ANOVA with Tukey’s multiple comparison test).
(H) Dox-induced cell lines expressing WT and mutant hTREM2 were incubated with lipidated CLU for 2 hr, and cell lysates were analyzed by western blot for CLU uptake (protein concentrations: 3 μg/ml; lipid concentrations: 10 μg/ml).
(I) Quantification of CLU uptake (normalized to WT hTREM2) showed significant reduction in hTREM2-Dox lines expressing Y38C, T66M, and R47H versus WT (n = 3, one-way ANOVA with Tukey’s multiple comparison test). Error bars represent SEM (∗∗p < 0.01 and ∗∗∗p < 0.001). All concentrations refer to protein concentrations.
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6. Figure 5 Loss of TREM2 Reduces Microglia Uptake of LDL as Well as CLU
(A) Representative confocal images of WT and Trem2 KO microglia that were incubated with 25 μg/ml (protein concentration) DiI-LDL for 4 hr. LDL uptake was measured by DiI fluorescence and normalized to cell number as marked by Hoechst dye. Extracellular fluorescence was quenched with trypan blue. Scale bar, 20 μm.
(B) Quantification of LDL uptake, normalized to WT, revealed an ∼40% reduction in DiI-LDL internalization by Trem2 KO (Trem2−/−) compared to WT microglia (n = 15, Student’s t test).
(C) Time course shows uptake of DiI-LDL by WT versus Trem2 KO microglia, as measured by flow cytometry.
(D and F) WT and Trem2 KO microglia were incubated with astrocyte-conditioned medium (ACM) for the indicated time, and uptake of (D) CLU and (F) APOE were measured by immunoblotting of the cell lysate.
(E and G) Quantification of time course of (E) CLU and (G) APOE uptake, normalized to t = 0 (n = 6, two-way ANOVA). Error bars represent SEM (∗p < 0.05 and ∗∗∗p < 0.001).
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7. Figure 6
Uptake and Degradation of Aβ Complexed with TREM2 Ligands Is Reduced in Trem2 KO Microglia
(A) Isolation of FITC-Aβ complexed with DiI-LDL by step-gradient centrifugation. Aβ that was bound to LDL (Aβ-LDL) floated to the interface between 0% and 24% Optiprep because of the lower density of LDL, and it was collected for use in microglial uptake experiments.
(B) FITC-Aβ-DiI-LDL complex isolated as shown in (A) or free FITC-Aβ (both at 0.1 μM concentration of Aβ) was added to microglia for 15 min, 30 min, 1 hr, 2 hr, 3 hr, or 4 hr. The uptake of FITC-Aβ or DiI-LDL was measured by flow cytometry based on the mean fluorescence intensity of FITC or DiI, respectively. The association with LDL greatly boosted the uptake of Aβ versus free Aβ (measured by FITC fluorescence intensity). Trem2 KO microglia were strongly impaired and Trem2 HET microglia were moderately impaired in uptake of the complex.
(C) DiI-LDL uptake, from FITC-Aβ-DiI-LDL complexes, was also TREM2 dependent, with a similar trend to Aβ (n = 8 for WT and Trem2 KO; n = 5 for Trem2 HET; two-way ANOVA).
(D) FITC-Aβ-APOJ complex was isolated by step gradient as in (A). Similar to LDL, Aβ-CLU complex was taken up more efficiently by microglia compared to free Aβ (both at 0.1 μM concentration of Aβ). Uptake of Aβ-CLU complex was TREM2 dependent (n = 5, two-way ANOVA). All points were normalized to WT microglia uptake of Aβ complexes at 4 hr.
(E and F) Time course of degradation of Aβ after uptake by WT versus Trem2 KO microglia. Microglia were incubated with either (E) free Aβ or (F) Aβ-LDL complexes for 2 hr. After washing, the amount of Aβ remaining at each time point was measured by Meso Scale Diagnostics Aβ immunoassay and normalized to t = 0 hr (immediately after Aβ-containing media were removed and cells were washed) (n = 4, two-way ANOVA). Error bars represent SEM (∗∗∗p < 0.001).
Neuron  , DOI: ( /j.neuron )
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8. Figure 7
Uptake of Aβ Complexed with LDL Is Reduced in Human MDMs from TREM2 R62H Carriers
(A and B) The individual (A) and averaged (B) traces for time course of uptake of FITC-Aβ-LDL complexes (Aβ-LDL, 0.02 μM Aβ) or FITC-Aβ (Aβ, 0.02 μM) by human MDMs from R62H carriers (n = 6) versus gender-/age-/ethnicity-matched controls (n = 10), as measured by flow cytometry. FITC-Aβ signal was normalized to the average intensity of matched control subjects at 4 hr. Association with LDL greatly boosted the uptake of Aβ by human MDMs. TREM2 R62H carriers had reduced uptake of Aβ-LDL. Error bars represent SEM (∗∗∗p < 0.001, two-way ANOVA).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions