Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2)
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TREM2 is an innate immune receptor specifically expressed in microglia. Coding variations in TREM2 have been reported to increase the risk for Alzheimer’s disease (AD) and other neurodegenerative diseases. While multiple studies support a role for TREM2 in microglial recruitment to amyloid plaques, the chemoattractant factor modulating TREM2-dependent microglial responses has not been defined.
Potential binding of oligomeric amyloid-β 1–42 (oAβ1–42) to TREM2 was tested by complementary approaches including solid phase binding, surface plasmon resonance and immunoprecipitation assays. The ability of oAβ1–42 to activate TREM2 signaling pathways was examined by analyzing the phosphorylation of Syk and Akt in primary microglia as well as TREM2-mediated signaling in a reporter cell system. Lastly, the functional outcome of oAβ1–42-TREM2 interaction was tested by examining impacts on microglial migration in vitro and clustering around oAβ1–42-bearing brain areas in vivo.
We found that oAβ1–42 bound to TREM2 with high affinity and activated TREM2-dependent signaling pathway. Neither monomeric nor scrambled Aβ bound to TREM2 supporting a specific interaction between oAβ and TREM2. The disease-associated mutations of TREM2 reduced its binding affinity to oAβ1–42. Furthermore, we identified several positively charged amino acids within residues 31–91 of TREM2 that were crucial for its interaction with oAβ1–42. Importantly, oAβ1–42 promoted microglial migration in vitro and clustering in vivo in a TREM2-dependent manner.
Our data establish a critical link between oAβ1–42, a major pathological component of AD, and TREM2, a strong genetic risk factor for AD expressed in microglia, and suggest that such interaction contributes to the pathogenic events in AD by modulating microglial responses.
Keywordsβ-amyloid microglia migration TREM2 Alzheimer’s disease
Central Nervous System
DNAX-activating Protein of 12 kDa
Granulocyte-Macrophage Colony-stimulating Factor
Oligomeric Amyloid-β 1–42
Receptor for Advanced Glycosylation Endproducts
Surface Plasmon Resonance
Triggering Receptor expressed on Myeloid Cells 1
Triggering Receptor expressed on Myeloid Cells 2
Alzheimer’s disease (AD) is the most common form of dementia with histopathological hallmarks of amyloid-β (Aβ) plaques and tau neurofibrillary tangles in the brain [1, 2]. While the mechanism by which neurodegeneration occurs in AD is not entirely clear, disturbed Aβ clearance and plaque removal by microglia likely contribute to Aβ accumulation and neuritic dystrophy [3, 4]. As the brain resident innate immune cells, microglia constantly survey the microenvironment and become activated in response to pathogenic components that disrupt the homeostasis of the nervous system . Intriguingly, microglia have been identified to constitute a protective barrier that prevents the outward extension of amyloid fibrils [6, 7]. The involvement of microglia in AD is further supported by recent genome-wide association and genome sequencing studies showing that rare coding variants in genes highly expressed in microglia as risk factors for late-onset AD [8, 9, 10, 11, 12, 13, 14, 15]. Single-cell RNA-sequencing in AD mouse model has also identified a novel microglia type that is associated with AD and restricts its development . Therefore, mounting evidence supports a causal role of microglial dysfunction in AD.
TREM2 is a cell surface receptor of the Ig superfamily that is specifically expressed by microglia within the central nervous system (CNS) . Interest in TREM2 stems from its genetic mutations associated with several neurodegenerative disorders . Homozygous missense mutations of TREM2 including Y38C or T66 M have been identified to be associated with Nasu-Hakola disease (NHD) . The R47H mutation in TREM2 constitutes one of the strongest single allele genetic risk factors for AD, with an odds ratio similar to that of carrying an apolipoprotein E (APOE) ε4 allele [8, 9, 19]. The R62H, D87N and T96 K mutations in TREM2 were also linked to AD after extensive analyses of TREM2 polymorphisms [8, 10, 20]. However, the precise effects of TREM2 mutations on AD pathogenesis remain elusive.
TREM2 consists of an ectodomain, a transmembrane region and a short cytoplasmic tail. By coupling with DNAX-activating protein of 12 kDa (DAP12), TREM2 regulates a wide array of functions in microglia including cell survival, proliferation, phagocytosis, and inflammation [21, 22, 23, 24]. Recent studies in Trem2-deficient AD mouse models have showed contradictory results regarding the amounts of Aβ plaques accumulation [21, 25, 26]. A later study indicated that Trem2 deficiency has opposing effects on amyloid pathology at early and late stages of AD progression . Nevertheless, it has been consistently reported that Trem2 deficiency leads to reduced number of microglia around amyloid plaques, allowing greater amount of plaque pathology and related toxicity [26, 28]. However, it remains unknown how TREM2 affects the recruitment of microglia to the vicinity of plaque.
A diverse set of potential TREM2 ligands that are anionic or lipidic in nature have been proposed . Bacteria and poly-anionic molecules were the first ligands proposed for TREM2 [30, 31]. TREM2 was also found to bind macrophages , astrocytes  and neurons . Recently, TREM2 was shown to bind phospholipids , apolipoproteins (including apoE, apoJ and apoA) and lipoprotein particles (including low-density lipoprotein or “LDL”) [35, 36, 37]. The binding of apolipoproteins to TREM2 was shown to facilitate either the phagocytosis of apoptotic neurons or the uptake of Aβ by microglia [35, 37]. Interestingly, the AD risk-associated variants, including R47H, R62H and D87N, have reduced affinity to these ligands. While multiple TREM2 ligands have been proposed, the identification of bona fide endogenous TREM2 ligands and the exploration of their physiological and pathological roles remain elusive. In this study, we found that oAβ1–42, but not monomeric or scrambled Aβ, bound to TREM2 with high affinity. The disease-associated mutations of TREM2 reduced its binding affinity to oAβ1–42. We further identified the specific region of TREM2 encompassing the arginine 47 and 62 residues that is critical for binding to oAβ1–42. Functional analyses revealed that oAβ1–42 activated TREM2 signaling pathway, promoted microglial migration and clustering in oAβ1–42-bearing brain regions via a TREM2-dependent manner. Our data provide mechanistic insight by which microglia recognize Aβ-related pathologies in AD.
Reagents and antibodies
Granulocyte-macrophage colony-stimulating factor (GM-CSF, 415-ML-010), RAGE-Fc recombinant protein (1145-RG-050) and anti-Fc antibody (MAB110) were purchased from R&D systems. Anti-Phospho-Syk (Tyr525/526) (2711 s), anti-total-Syk (13,198 s), anti-Phospho-Akt (Ser473) (4060 s), anti-total-Akt (4685 s) and anti-β-actin antibody (4970 s) were purchased from Cell Signaling Technology. Anti-Aβ antibody (MOAB-2, ab126649) was from Abcam. Unlabeled Aβ1–42 (AS-20276), FAM-labeled Aβ1–42 (AS-23525-05) and scrambled Aβ42 (AS-25382) were from AnaSpec. The mAβ1–42 and oAβ1–42 were prepared as previously described with minor modification [38, 39]. Dissolved peptides in DMSO (5 mM) were diluted to 100 μM with cold phenol-free F-12 cell culture media (Gibco) and sonicated for 10 min to make mAβ1–42 which was used freshly. Part of the peptides were then incubated at 22 °C for 16 h followed by 24 h incubation at 4 °C, centrifuged at 16,000×g for 15 min, and the collected supernatant was used as oAβ1–42.
Cells were lysed with NP-40 lysis buffer (1% NP-40, 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride containing protease inhibitor mixture from Roche Applied Science) 24 h post transfection. Protein concentrations were determined using the BCA protein assay kit (Thermo Fisher Scientific, 23,225). Equal amounts of samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto PVDF membranes and probed with specific antibodies. Detection was performed using corresponding secondary antibodies and immunoreactive bands were quantified using ImageJ.
Two micrograms of oAβ1–42 were added into 400 μL of 1% solution of Tween-20 in TBS (1% TBST) with protease inhibitor cocktail. Two micrograms of sTREM2-Fc, sTREM1-Fc or Fc protein was pre-bound to 25 μL of protein A beads (Thermo Fisher Scientific, 20,334). The beads were then incubated with oAβ1–42 in 1% TBST at 4 °C overnight. Beads were washed 5 times with 1% TBST for 3 min, resuspended in 20 μL 2 × SDS loading buffer and subjected to electrophoresis on 4–12% Bis-Tris NuPAGE precast gels (Thermo Fisher Scientific, NP0322PK2).
Human embryonic kidney 293 T cells (HEK 293 T) were transfected with pmCherry-N1, pmCherry-TREM1 or pmCherry-TREM2 plasmid. Twenty-four hours after transfection, cells were washed 3 times with DMEM and incubated with 1.0 μM FAM-labeled oAβ1–42 for 2 h at 4 °C. After 3 washes with PBS, cells were fixed with 4% paraformaldehyde for 15 min. Cells were then stained with DAPI for 3 min and then washed twice with PBS for 15 min. Coverslips were mounted on the glass slide using antifade reagent (Thermo Fisher Scientific, P36935) and observed using NIKON A1R Plus confocal microscope.
Expression and purification of sTREM2-Fc fusion proteins
The human TREM2 extracellular domain tagged with Fc that carries different fragments and mutations were cloned and purified from the conditioned medium of HEK 293 T cells as previously described . Purified proteins were quantified with the BCA protein assay kit and the purity was determined on silver-stained SDS-PAGE gels.
Solid phase binding assay
A 96-well plate (Corning Incorporated) was coated with 100 nM oAβ1–42 in PBS overnight at 4 °C. Wells were washed with 0.05% PBST and blocked with 4% BSA for 1 h at 37 °C. Purified Fc or sTREM2-Fc proteins diluted in PBST containing 0.5% BSA were added and incubated for 30 min at 37 °C. After washing, the bound proteins were detected with biotinylated-anti-Fc antibody for 1 h at 37 °C. Plates were washed and then incubated with avidin-HRP for 30 min at 37 °C, washed again, and developed with TMB substrate solution (Sigma Aldrich, T5569), and read at 620 nm.
Mouse primary microglial culture
All animal experiments were conducted in compliance with the protocols of the Institutional Animal Care and Use Committee at Xiamen University. Trem2-knockout mice (KO, on C57BL/6 N background) and wild-type (WT) C57BL/6 N mice were obtained from the UC Davis Knockout Mouse Project (KOMP) repository as described previously . Primary microglial cultures were prepared as previously described [35, 40]. WT or Trem2-KO mice at postnatal day 1–3 were used to prepare mixed glial cultures. Cells were plated onto poly-lysine coated flasks and grown in fresh DMEM (Gibco) supplemented with 10% FBS (Gibco). Medium was changed after 3 days to that contains 25 ng/mL GM-CSF and 10% FBS. Primary microglial cells were harvested after 10–12 days in culture and once every 3 days thereafter (up to two harvests).
Cell migration assay was performed with Transwell cell culture inserts comprised of two chambers separated by an 8.0 μm polycarbonate membrane (Costar, 3422). Primary microglial cells (5 × 104) suspended in serum-free DMEM were added to the upper chamber of the inserts with serum-free DMEM in the bottom chamber. After 30 min incubation at 37 °C, the bottom medium was replaced with DMEM containing vehicle, 0.5 μM oAβ1–42 or 10% FBS. After 20 h, cells remained on the upper surface of the membrane were removed by scraping with a cotton swab. Cells that migrated through the membrane were fixed with 4% paraformaldehyde, stained with hematoxylin and eosin, and counted under Nikon inverted microscope.
TREM2 reporter assay
BWZ thymoma reporter cells (WT and TD4) were a generous gift from Dr. William E. Seaman (UCSF). BWZ cells express lacZ under control of the promoter for nuclear factor of activated T cells (NFAT) . The TD4 cells are stably transfected to co-express TREM2 and DAP12 . TD4 or WT cells were seeded in 96-well plates at 105 cells/well in RPMI media supplemented with 10 ng/mL phorbol 12-myristate 13-acetate (PMA). Ionomycin was added to a final concentration of 1 μM to serve as a positive control. Reporter cells were stimulated with different concentrations of oAβ1–42, mAβ1–42 or scAβ42 for 16 h at 37 °C, washed once with PBS, and lysed in a buffer containing 100 mM 2-mercaptoethanol, 9 mM MgCl2, 0.125% NP-40, and 0.3 mM chlorophenol red-β-D-galactopyranoside. After 4 h at 37 °C, absorbance was measured at 595 nm, with correction for background absorbance at 630 nm.
Surface Plasmon Resonance assay
Surface Plasmon Resonance (SPR) analysis was performed at 25 °C using the Biacore T200 (GE Healthcare). Purified sTREM2-Fc and Fc protein were immobilized onto Biacore CM5 (GE Healthcare, BR-1005-30) chip using amine coupling kit (GE Healthcare, BR-1000-50). oAβ1–42, mAβ1–42 or scAβ42 was tested with a gradient concentration of 1.25 μM, 2.5 μM, 5.0 μM and 10.0 μM respectively.
Stereotactic injection of oAβ1–42
WT or Trem2-KO male mice at 6 months of age were anesthetized and placed in a stereotaxic frame. Investigators were blind to the condition of each animal during the experimental procedure and the quantification and analysis stages. A skin incision was made and holes were drilled at defined x (± 2.0 mm from bregma) and y (− 2.2 mm from bregma) positions. The left and the right hemisphere were injected with FAM-oAβ1–42 (a total of 1.5 μg in 3.0 μL) or vehicle at 0.30 μL/min with z-depths of 2.0 mm, respectively. Before being withdrawn slowly, the syringe was left in place for 10 min after each injection. Four or sixteen hours after injection, mice were anesthetized and perfused with ice-cold PBS. Brains were fixed in 4% PFA overnight at 4 °C, transferred to 30% sucrose for 48 h before embedded for cryostat sectioning.
Immunohistochemistry and microscopy
Twelve-μm thick cryosections were washed in PBS for 15 min, permeabilized in 5% normal donkey serum and 0.2% Triton X-100 for 1 h, followed by 48 h’ incubation with Iba1 antibodies at 4 °C. The cryosections were washed with PBS for 30 min and treated with the Alexa-fluorophore-conjugated secondary antibodies for 2 h at RT. Sections were stained with DAPI (5 μg/mL), washed and mounted with anti-fade reagent. For confocal microscopy, 6 μm z-stacks (consisting of 13 optical slices of 0.5 μm thickness) were acquired using a NIKON A1R Plus confocal microscope.
Data were obtained from at least three independent experiments, and presented as mean ± standard deviation (SD). Statistical analyses were performed using Graphpad Prism 5.0 software or SPSS software. Statistical significance was determined by one-way ANOVA or two-way ANOVA when more than two groups were compared and unpaired Student’s t-test for single comparisons. p < 0.05 was considered significant.
Oligomeric Aβ1–42 binds to TREM2 and activates TREM2 signaling pathway
To determine if oAβ1–42 can functionally engage TREM2 and initiate intracellular signaling, we utilized TREM2 reporter cells (TD4) which express both TREM2 and DAP12 and produce β-galactosidase upon TREM2 engagement . As a positive control, ionomycin stimulated the expression of reporter β-galactosidase in both wild-type (WT) and TREM2 reporter cells (Additional file 1: Figure S2A and Additional file 1: Figure S2B). Consistent with oAβ1–42 as a specific ligand for TREM2, it selectively activated TREM2 reporter cells but not WT cells in a dose-dependent manner, suggesting the requirement of TREM2 engagement. Consistent with the binding data, neither mAβ1–42 nor scAβ42 stimulated the reporter activity in TD4 cells. To gain more direct evidence that oAβ1–42 stimulates TREM2 signaling, we compared the levels of phosphorylated Syk (p-Syk) and Akt (p-Akt) known to represent TREM2 activation in microglia . The stimulation with oAβ1–42 induced significantly higher level of p-Syk in WT but not Trem2-KO microglia (Fig. 1k and l). Furthermore, oAβ1–42 increased to a greater extent of p-Akt in WT than Trem2-KO microglia at both the 30- and 60-min time points (Fig. 1m and n). Taken together, these results indicate that oAβ1–42 binds to TREM2 and activates TREM2-dependent signaling pathway in microglia.
The disease-associated mutations reduce TREM2 binding to oAβ1–42
Residues 31–91 of TREM2, in particular the positively charged amino acids within this region, are crucial for its interaction with oAβ1–42
Oligomeric Aβ1–42 modulates microglial responses dependent on TREM2 receptor
Human genetic association studies increasingly highlight microglial genes, such as TREM2, CD33, and CR1 playing important roles in the pathogenesis of AD . Although mechanistic understanding of TREM2 action remains elusive, TREM2 presumably carries out its function in AD by binding to endogenous ligand(s). The many diverse ligands reported for TREM2 suggest that TREM2 is a highly promiscuous receptor. In this study, we found that oAβ specifically bound to TREM2 with high affinity. The interaction is functionally relevant to the pathogenesis of AD as evidenced by the observation that oAβ triggers microglial migration in vitro and microglial clustering around oAβ-bearing regions in vivo in a TREM2-dependent manner. Therefore, different ligands of TREM2 likely trigger ligand-specific events to accomplish the multi-functions reported for TREM2, in the case of oAβ microglial migration.
Interestingly, the disease-associated mutations of TREM2 reduced its binding affinity to Aβ. Although coding variants of TREM2 are linked to the risk of neurodegenerative diseases, the underlying mechanisms remain unknown. Several recent studies have shed light on how TREM2 missense mutations affect TREM2 function. First, the disease-associated mutations of TREM2 have impaired cell surface expression as reported previously [48, 49] and in our current study. Second, TREM2 missense mutations have been reported to reduce its shedding by ADAM proteases and impair the phagocytic activity of TREM2-expressing cells . Furthermore, AD risk-associated mutations abrogate the function of soluble TREM2 in both suppressing apoptosis and triggering inflammatory responses . Importantly, TREM2 variants exhibited deficits in binding to a wide array of ligands, including phospholipids, apolipoproteins, lipoprotein particles [21, 35, 36, 37] and oAβ1–42 as reported in the current study. Taken together, the disease-associated mutations of TREM2 likely modify the disease progression via multiple pathways.
Although our current study emphasizes a critical role of membrane-bound TREM2 which associates with DAP12 for downstream signaling, we could not rule out a potential contribution by sTREM2, a shed product of membrane-bound TREM2, that binds to a yet identified microglial receptor . Hence, both full-length TREM2 and sTREM2 might be involved in microglial recruitment to the vicinity of plaques. Although the molecular mechanism underlying each pathway remains to be defined in future, our data suggest that the membrane-bound TREM2 signals through Syk and Akt, while sTREM2 might act on a separate microglial cell-surface receptor .
One striking feature of microglia in AD brain is their universal clustering around amyloid plaque. It was consistently reported that the number of microglia around amyloid plaques was reduced in Trem2-deficient amyloid mouse models, leading to an increase in less compact plaques [26, 28]. A critical function of microglia is thought to remove Aβ deposits via phagocytosis . Indeed, loss of Trem2 has been reported to impair the phagocytosis of Aβ by microglia in vitro . Therefore, the physical interaction between TREM2 and Aβ likely regulates microglial mobility, potentially contributing to both the barrier function of microglia to compact the plaques and the phagocytic activity of microglia in removing the plaques. Future studies elucidating the biological function of their interaction might uncover targetable pathways for AD therapy.
Our current study demonstrates that oAβ binds to TREM2 with high affinity and activates TREM2-dependent signaling pathways in microglia. The absence of TREM2 impairs the oAβ-induced microglial migration in vitro and microglial clustering around oAβ-bearing brain regions in vivo. The identification of Aβ as a novel TREM2 ligand links TREM2 to the most prominent pathological features of AD and provides a critical mechanism by which microglia recognize and react to Aβ pathology.
The authors thank Junjie Chen from the Analysis and Measurement Center, School of Pharmaceutical Sciences, Xiamen University for kindly performing the data analysis on surface plasmon resonance assay.
X.C., G.B., L.Z., Z.W. and H.X. designed research; L.Z., Z.W., D.W., Z.W., Y.M., L.W., K.W., Y.X., J.L., R.H. and D.C. performed experiments; L.Z., Z.W., D.W., Z.W., Y.M. and X.C. analyzed data; X.C., Z.W. L.Z. and G.B. wrote the manuscript. All authors read and approved the final manuscript.
Research by the authors was supported by a grant from the Natural Science Foundation of Guangdong Province 2016A030306005 (to X.C.), grants from the National Natural Science Foundation of China 81370459, 31400914 (to X.C.), U1505227 (to G.B.), U1405222, 81771163 (to H.X.), 81701079 (to L.Z.), a grant from the Fundamental Research Funds for the Central Universities (20720180055, to X.C.), NIH grants RF1AG056130 (to G.B. and H.X.), R01AG035355 (to G.B.), R37AG027924 (to G.B.), RF1AG056114 (to H.X.); grants from the Postdoctoral Science Foundation of China 2016 M600503 and 2017 T100469 (to L.Z.), and the Cure Alzheimer’s Fund (to G.B. and H.X.).
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All animal experiments were conducted in compliance with the protocols of the Institutional Animal Care and Use Committee at Xiamen University.
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