Apolipoprotein E Is a Ligand for Triggering Receptor Expressed on Myeloid Cells 2 (TREM2)

Abstract

Several heterozygous missense mutations in the triggering receptor expressed on myeloid cells 2 (TREM2) have recently been linked to risk for a number of neurological disorders including Alzheimer disease (AD), Parkinson disease, and frontotemporal dementia. These discoveries have re-ignited interest in the role of neuroinflammation in the pathogenesis of neurodegenerative diseases. TREM2 is highly expressed in microglia, the resident immune cells of the central nervous system. Along with its adaptor protein, DAP12, TREM2 regulates inflammatory cytokine release and phagocytosis of apoptotic neurons. Here, we report apolipoprotein E (apoE) as a novel ligand for TREM2. Using a biochemical assay, we demonstrated high-affinity binding of apoE to human TREM2. The functional significance of this binding was highlighted by increased phagocytosis of apoE-bound apoptotic N2a cells by primary microglia in a manner that depends on TREM2 expression. Moreover, when the AD-associated TREM2-R47H mutant was used in biochemical assays, apoE binding was vastly reduced. Our data demonstrate that apoE-TREM2 interaction in microglia plays critical roles in modulating phagocytosis of apoE-bound apoptotic neurons and establish a critical link between two proteins whose genes are strongly linked to the risk for AD.

Keywords: Alzheimer disease; TREM2; apolipoprotein; apolipoprotein E (apoE); microglia; neuroinflammation.

FIGURE 1.

ApoE specifically binds to TREM2 with high affinity. A, representative dot blot of apoE binding to TREM2. A nitrocellulose membrane was spotted with 200 ng each of recombinant human IgG Fc region (hIgG-Fc), recombinant human TREM1, or TREM2 extracellular region conjugated to Fc (TREM1-Fc and TREM2-Fc). Membrane strips were then incubated with 10 nm recombinant or immortalized astrocyte-derived apoE2, -E3, or -E4. Bound apoE was detected using biotinylated anti-apoE antibody. B, quantification of three independent dot blots. Data are plotted as mean ± S.E. (n = 3, one-way analysis of variance with Tukey post hoc analysis, *, p < 0.05). C, saturation binding curve and dissociation constant (K_d) of apoE3 binding to TREM2-Fc. Membrane strips spotted with hIgG-Fc, TREM1-Fc, or TREM2-Fc were incubated with increasing concentrations of recombinant apoE3, and bound apoE was detected using appropriate antibodies. The curve fit and K_d were derived using GraphPad Prism nonlinear fit for one site total binding. Data are plotted as mean ± S.E. (n = 3).

FIGURE 2.

ApoA1 and apoB also bind to TREM2. A, a representative dot blot of TREM2 binding to apoA1 and apoB. Membrane strips spotted with 200 ng each of normal mouse IgG (mIgG), apoA1, or apoB were incubated with 80 nm hIgG-Fc, TREM1-Fc, or TREM2-Fc. Bound Fc protein was detected with biotinylated anti-Fc antibody. B, a representative dot blot showing binding specificity of TREM2 to apoE and not to two other LRP1 ligands, tPA or RAP. Membrane strips spotted with 200 ng each of mIgG, apoE3, tPA, or RAP were incubated with 80 nm hIgG-Fc, TREM1-Fc, or TREM2-Fc, and bound Fc protein was detected with biotinylated anti-Fc antibody. C, apoA1 competes with apoE for binding to TREM2. Membrane strips spotted with hIgG-Fc, TREM1-Fc, or TREM2-Fc were incubated with 10 nm apoE3 along with increasing concentrations of apoA1, and bound apoE was detected and quantified using biotinylated anti-apoE antibody. Data are plotted as mean ± S.E. (n = 3).

FIGURE 3.

ApoE binds to apoptotic neurons and increases TREM2-mediated phagocytosis by microglia. A, Western blot analysis of neuronal N2a cells incubated with apoE3 or apoE4. Control or staurosporin-induced apoptotic N2a cells were incubated with 10 nm apoE for 1 h. After washing with PBS, cells were lysed and analyzed for cell-bound apoE. Actin was used as a loading control. B, phagocytic uptake of apoptotic N2a cells by WT mouse primary microglia. CM-DiI-labeled apoptotic N2a cells were added to mouse primary microglia with or without 10 nm apoE for 2 h. Microglia were labeled with CD11b-APC antibody, and analyzed by flow cytometry. Percent of CM-DiI⁺/CD11b⁺ cells were quantified using CFlow. Data are plotted as mean ± S.E. (n = 4, Student's t test, ***, p < 0.001). C, phagocytosis assay of apoptotic N2a cells by WT and Trem2-KO mouse primary microglia. WT or Trem2-KO microglia were incubated with 10 nm apoE3 and CM-DiI-labeled apoptotic N2a for 2 h. Percent of CM-DiI⁺/CD11b⁺ cells were quantified using CFlow. Data are plotted as mean ± S.E. (n = 3, Student's t test; *, p < 0.05).

FIGURE 4.

Expression and purification of soluble TREM2-Fc. A, schematic representations of human full-length TREM2, and TREM2-Fc and TREM2-R47H-Fc chimeric proteins. SP, signal peptide; TM, transmembrane domain. The cDNA encoding the extracellular domain of TREM2 or TREM2-R47H mutant (amino acids 1–174) was transfected into HEK293 cells and TREM2 protein in the conditioned medium was purified by protein A-agarose beads. The purified TREM2-Fc and TREM2-R47H-Fc fusion proteins were analyzed by silver-stained SDS-PAGE (B) and Western blotting using anti-TREM2 (C) and anti-Fc (D) antibodies.

FIGURE 5.

The AD-associated R47H mutation in TREM2 impairs apoE binding. A, a representative dot blot of apoE binding to TREM2. Membrane strips spotted with 200 ng each of hIgG-Fc, TREM2-Fc, or TREM2-R47H-Fc were incubated with 10 nm apoE, and bound apoE was detected using biotinylated anti-apoE antibody. Membrane strips spotted with the same proteins were also incubated with anti-Fc antibody to ensure equal protein loading. B–D, quantification of dot blot of TREM2 and mutant TREM2-R47H binding to apoE2 (B), apoE3 (C), and apoE4 (D). Data are plotted as mean ± S.E. (n = 3, Student's t test, **, p < 0.01; ***, p < 0.001). E-G, solid phase binding analysis of TREM2 and TREM2-R47H mutant to apoE2 (E), apoE3 (F), and apoE4 (G). A 96-well plate coated with apoE was incubated with 12.5 and 25 nm TREM2 or mutant TREM2-R47H-Fc. The bound TREM2-Fc proteins were detected using biotinylated anti-Fc antibody. Uncoated wells incubated with TREM2-Fc proteins were used as background controls. Data are plotted as mean ± S.E. (n = 3, two-way analysis of variance with Sidak post hoc correction, *, p < 0.05; **, p < 0.01; ***, p < 0.001).