The Triggering Receptor Expressed on Myeloid Cells 2 Binds Apolipoprotein E

Abstract

The triggering receptor expressed on myeloid cells 2 (TREM2) is an Ig-like V-type receptor expressed by populations of myeloid cells in the central nervous system and periphery. Loss-of-function mutations in TREM2 cause a progressive, fatal neurodegenerative disorder called Nasu-Hakola disease. In addition, a TREM2 R47H coding variant was recently identified as a risk factor for late-onset Alzheimer disease. TREM2 binds various polyanionic molecules but no specific protein ligands have been identified. Here we show that TREM2 specifically binds apolipoprotein E, a well established participant in Alzheimer disease. TREM2-Ig fusions efficiently precipitate ApoE from cerebrospinal fluid and serum. TREM2 also binds recombinant ApoE in solution and immobilized ApoE as detected by ELISA. Furthermore, the Alzheimer disease-associated R47H mutation, and other artificial mutations introduced in the same location, markedly reduced the affinity of TREM2 for ApoE. These findings reveal a link between two Alzheimer disease risk factors and may provide important clues to the pathogenesis of Nasu-Hakola disease and other neurodegenerative disorders.

Keywords: Alzheimer disease; Nasu-Hakola disease; PLOSL; TREM2; apolipoprotein E (ApoE); genetic polymorphism; high-density lipoprotein (HDL); myeloid cell; neurodegenerative disease.

FIGURE 1.

TREM2-Ig recognizes PS and other lipids on lipid arrays, but not on the surface of apoptotic cells. A, the TREM2-Ig fusion was used to probe an array spotted with various lipids as indicated in the legend on the right. Dark spots show lipids recognized by TREM2-Ig as detected by chemiluminescence. As shown by the left membrane, TREM2-Ig bound strongly to spots containing phosphatidic acid (PA), PS, and cardiolipin (CL). A human IgG1 isotype control exhibited no appreciable lipid binding. These results were reproducible with at least two different lots of arrays. PA and PS binding were also observed on sphingolipid strips (that do not include CL, not shown). B, Jurkat cells were treated for 12 h with actinomycin D1 (actD1) to induce apoptosis and expose PS on the cell surface. Cells were then stained with PS-binding reagents ANXAV and TIM1-Ig, with TREM2-Ig, or with a human IgG1 isotype control antibody. The membrane-impermeant vital stain propidium iodide (PI) was used to discriminate between dead cells (PI⁺) and apoptotic or viable cells (PI⁻). The vertical axis of each plot shows the relative intensity of ANXAV/Ig fusion staining and the horizontal axis shows the intensity of PI staining. Known PS-binding proteins ANXAV and TIM-1 labeled apoptotic cells (ANXAV⁺, PI⁻) as indicated in the bottom left plot. TREM2-Ig, however, failed to label apoptotic cells, indicating that it does not bind PS in the context of intact cellular membranes.

FIGURE 2.

TREM2-Ig precipitates ApoE from cerebrospinal fluid. A, TREM2-Ig, a human IgG1 isotype control antibody (hIgG), or unbound protein G beads were used as bait for immunoprecipitation of cynomolgus macaque CSF. Input (CSF), the TREM2-Ig regent by itself, and the precipitated products were separated by reducing SDS-PAGE and visualized by silver staining. TREM2-Ig, but neither hIgG nor beads alone, precipitated a doublet band of ∼36 kDa (white arrowhead), and a single band of ∼22 kDa (black arrowhead). Dashed lines indicate bands corresponding to TREM2-Ig, the 50-kDa IgG heavy chain (HC), and the 25-kDa IgG light chain (LC). B, immunoprecipitation (IP) of macaque CSF was repeated and analyzed by Western blot. The 36-kDa doublet was confirmed as ApoE and the 22-kDa single band as ApoA-I. ApoA-II, although not visible with silver staining, was detected by Western blot in both the CSF and TREM2-Ig precipitate lanes. The band indicated by the white arrowhead is the result of secondary antibody cross-reaction with the hIgG1 light chain. IB, immunoblot.

FIGURE 3.

TREM2-Ig precipitates ApoE from serum. A, immunoprecipitation (IP) of cynomolgus macaque serum was performed in an experiment analogous to that shown in Fig. 1. The precipitated proteins are shown next to input (serum) following reducing SDS-PAGE and silver staining. ApoE was not visible on the silver-stained gel but ApoA-I was visible beneath the IgG light chain in both serum and the TREM2-Ig IP lane. B, Western blot confirmed the presence of ApoE and ApoA-I in the TREM2-Ig precipitate. The white arrowhead indicates the hIgG1 light chain band. IB, immunoblot.

FIGURE 4.

TREM2-Ig binds ApoE specifically and irrespective of genotype. A, TREM2-Ig or control beads were used to immunoprecipitate (IP) solutions of the indicated purified apolipoproteins. Shown here are the results of SDS-PAGE and Western blots for each of the indicated apolipoproteins. The leftmost lane in each blot contains a sample of the input as a positive control. These results are representative of three independent experiments. B, ApoE co-immunoprecipitates (IP) with C terminally FLAG-tagged TREM2 when both constructs are co-transfected into HEK293T cells. Cells were co-transfected with three different plasmid mixtures as indicated at the top of the figure. The top two blots (Input) show immunostaining of the clarified lysates and the bottom two blots (IP FLAG) show staining of the corresponding immunoprecipitates. The left lane demonstrates the specificity of the ApoE antibody. The middle lane shows that FLAG-tagged TREM2 bound to ApoE, whereas the right lane includes an untagged, rather than FLAG-tagged, TREM2 as a negative control. The faint ApoE band in the rightmost lane of the IP FLAG blot shows the level of background binding of ApoE to the Sepharose support of the anti-FLAG beads. Recovery of ApoE from cell lysates was generally inefficient compared with other experiments so the (IP FLAG) ApoE blot was exposed 5 times longer than the corresponding (Input) ApoE blot. These results are representative of three independent experiments. C, ApoE2, E3, and E4 were expressed in HEK293T cells. Levels of ApoE in culture supernatants were approximately equivalent as shown by SDS-PAGE and Coomassie staining (left). A dilution series of these supernatants were used as immobilized ligands for ELISA. The left graph shows the binding of TREM2-Ig to each of the ApoE variants, whereas the right graph shows the binding of a polyclonal control serum. Error bars show 95% confidence intervals for triplicate wells. Similar results were obtained with a second preparation of ApoE (not shown).

FIGURE 5.

The TREM2 R47H polymorphism reduces the affinity of TREM2 for ApoE. A, immunoprecipitation (IP) of macaque CSF was performed as in the legend to Fig. 1A with the addition of a TREM2-Ig bait containing the R47H mutation. CSF input (leftmost lane) and precipitates were separated by SDS-PAGE and blotted for the indicated apolipoproteins. The R47H mutation decreases, but does not abolish, the precipitation of ApoE from CSF. This experiment is representative of two. B, wild-type TREM2-Ig, mutant TREM2-Ig constructs, and CD4-Ig were used as primary immunoreagents in ELISAs against recombinant ApoE and ApoA-I (E. coli produced), ApoA-II (from human plasma), and ApoJ (of NS-20 NS0 mouse cell origin). TREM2-Ig (wild-type) bound strongly to ApoE. The R47H mutation severely impaired binding of TREM2-Ig as did similar mutations. Error bars show 95% confidence intervals for triplicate wells. C, the effects R47H and related experimental mutations on TREM2/ApoE interaction were confirmed with ApoE purified from human plasma. In this, and following experiments, the concentration of ApoE used to coat the plate was varied rather than the concentration of Ig fusion. Error bars show 95% confidence intervals of triplicate wells of one of two experiments. D, the specificity of TREM2-Ig for ApoE was confirmed via ELISA with plates coated with apoproteins E, A-I, and A-II all purified from human plasma. The dashed gray line indicates the mean level of CD4-Ig binding to the highest concentration of ApoE on the plate (mean signal from 3 wells). Error bars show 95% confidence intervals for triplicate wells from a representative experiment. E, either ApoE antiserum (AS) or control goat serum (CS) were titered onto immobilized purified plasma-derived ApoE or a BSA control ligand. ApoE antiserum, but not the control serum, blocked TREM2-Ig binding in a concentration-dependent manner. Error bars show upper (AS) or lower (CS) 95% confidence intervals for the TREM2 binding signal. BSA error bars are omitted for clarity. The serum dilution series begins at a 1:50 dilution (corresponding to a relative dilution factor of 1 on the graph). The dotted gray line shows the TREM2/ApoE binding signal in the absence of serum (mean value from 6 wells).

FIGURE 6.

A model for TREM2/ApoE interaction in neurodegenerative disease. We speculate that, under normal conditions, TREM2 recognizes ApoE in at least two contexts: in association with Aβ and in association with lipid debris. TREM2 promotes clearance of these injurious substances by activating phagocytes and possibly modulating the inflammatory response. Hypomorphic mutations lead to mild impairment of ApoE recognition that may ultimately pre-dispose to Aβ accumulation and Alzheimer disease late in life. Complete loss of TREM2 function impairs phagocytic clearance of lipid debris (such as myelin) resulting in the early onset leukoencephalopathy and fatty, cystic degenerative changes in peripheral organs that constitute Nasu-Hakola disease.