FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2950947069> ?p ?o ?g. }

• W2950947069 abstract "Research Article19 October 2018Open Access Source DataTransparent process High-affinity interactions and signal transduction between Aβ oligomers and TREM2 Christian B Lessard Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Samuel L Malnik Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Yingyue Zhou Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Thomas B Ladd Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Pedro E Cruz Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Yong Ran orcid.org/0000-0002-9748-4392 Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Thomas E Mahan Department of Neurology, Hope Center for Neurological Disorders, Knight ADRC, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Paramita Chakrabaty Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA McKnight Brain Institute, University of Florida, Gainesville, FL, USA Search for more papers by this author David M Holtzman orcid.org/0000-0002-3400-0856 Department of Neurology, Hope Center for Neurological Disorders, Knight ADRC, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Jason D Ulrich Department of Neurology, Hope Center for Neurological Disorders, Knight ADRC, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Marco Colonna Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Todd E Golde Corresponding Author [email protected] orcid.org/0000-0003-1867-7071 Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA McKnight Brain Institute, University of Florida, Gainesville, FL, USA Search for more papers by this author Christian B Lessard Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Samuel L Malnik Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Yingyue Zhou Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Thomas B Ladd Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Pedro E Cruz Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Yong Ran orcid.org/0000-0002-9748-4392 Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA Search for more papers by this author Thomas E Mahan Department of Neurology, Hope Center for Neurological Disorders, Knight ADRC, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Paramita Chakrabaty Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA McKnight Brain Institute, University of Florida, Gainesville, FL, USA Search for more papers by this author David M Holtzman orcid.org/0000-0002-3400-0856 Department of Neurology, Hope Center for Neurological Disorders, Knight ADRC, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Jason D Ulrich Department of Neurology, Hope Center for Neurological Disorders, Knight ADRC, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Marco Colonna Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Todd E Golde Corresponding Author [email protected] orcid.org/0000-0003-1867-7071 Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA McKnight Brain Institute, University of Florida, Gainesville, FL, USA Search for more papers by this author Author Information Christian B Lessard1, Samuel L Malnik1, Yingyue Zhou2, Thomas B Ladd1, Pedro E Cruz1, Yong Ran1, Thomas E Mahan3, Paramita Chakrabaty1,4, David M Holtzman3, Jason D Ulrich3, Marco Colonna2 and Todd E Golde *,1,4 1Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, Gainesville, FL, USA 2Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA 3Department of Neurology, Hope Center for Neurological Disorders, Knight ADRC, Washington University School of Medicine, St. Louis, MO, USA 4McKnight Brain Institute, University of Florida, Gainesville, FL, USA *Corresponding author. Tel: +1 352 273 9458; Fax +1 352 294 5060; E-mail: [email protected] EMBO Mol Med (2018)10:e9027https://doi.org/10.15252/emmm.201809027 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Rare coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with increased risk for Alzheimer's disease (AD), but how they confer this risk remains uncertain. We assessed binding of TREM2, AD-associated TREM2 variants to various forms of Aβ and APOE in multiple assays. TREM2 interacts directly with various forms of Aβ, with highest affinity interactions observed between TREM2 and soluble Aβ42 oligomers. High-affinity binding of TREM2 to Aβ oligomers is characterized by very slow dissociation. Pre-incubation with Aβ is shown to block the interaction of APOE. In cellular assays, AD-associated variants of TREM2 reduced the amount of Aβ42 internalized, and in NFAT assay, the R47H and R62H variants decreased NFAT signaling activity in response to Aβ42. These studies demonstrate i) a high-affinity interaction between TREM2 and Aβ oligomers that can block interaction with another TREM2 ligand and ii) that AD-associated TREM2 variants bind Aβ with equivalent affinity but show loss of function in terms of signaling and Aβ internalization. Synopsis Rare coding variants of TREM2 (R47H, R62H) are associated with increased risk for Alzheimer's disease (AD), but how they confer this risk remains uncertain. Using BioLayer Interferometry and other biochemical methods, TREM2 and AD-associated variants binding to Aβ and APOE is examined. High-affinity binding of TREM2 to Aβ oligomers is characterized by very slow dissociation which is almost “irreversible”. Pre-incubation of TREM2 with Aβ oligomers is shown to block its interaction with APOE. AD-associated TREM2 variants bound Aβ with equivalent affinity. AD-associated TREM2 variants reduced Aβ42 internalization and NFAT signaling activity. AD-associated TREM2 variants showed a partial loss of function. Introduction The most prevalent form of dementia, Alzheimer's disease (AD), is hypothesized to be triggered by accumulation of aggregated amyloid-β (Aβ) followed by a “cascade-like” chain of events that includes induction of tauopathy, neurodegeneration, and alterations in innate immune signaling (Hardy & Selkoe, 2002; Musiek & Holtzman, 2015). The later feature is reflected by the presence of a reactive astrocytosis, microgliosis, and increased level and accumulation of a variety of immune molecules. A more central role of the innate immune system and microglial cells in particular has emerged from genetic studies. These studies show that numerous loci encoding immune genes are associated with altered risk for AD. Even more compelling are data showing that coding variants in three different genes (TREM2, PLCG2, and ABI3), whose transcripts are expressed primarily in microglial cells in the brain, alter risk for AD (Golde et al, 2013; Jin et al, 2014; Sims et al, 2017). Despite these associations between the immune system and AD, there is little consensus as to how alterations in the immune system mechanistically alter AD risk. Modeling studies reveal a complex relationship between immune activation states, the proteinopathies found in AD, and neurodegeneration—a phenomenon we refer to as immunoproteostasis (Chakrabarty et al, 2015). Further, definitive insight into how genetic variations within immune loci that alter AD risk alter immune function has remained enigmatic. Further understanding of how genetic risk factors impact the immune function will be important to guide therapeutic development, that to date has largely focused on anti-inflammatory strategies (Chakrabarty et al, 2015). The rare protein coding variants in TREM2, R47H, and R62H, which are reproducibly associated with increased risk for developing AD, are being intensively studied as these variants were the first coding variants in an innate immune gene that have been reproducibly shown to alter risk for AD (Cruchaga et al, 2013; Jin et al, 2015; Lill et al, 2015). Prior to association with AD, homozygous or compound heterozygous mutations in TREM2 had been identified in a disease called polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL) or Nasu-Hakola disease (Bianchin et al, 2006). PLOSL is characterized by fractures, frontal lobe syndrome, and progressive presenile dementia beginning in the fourth decade. Numerous studies of the PLOSL-associated TREM2 variants suggest that these variants are loss of function altering maturation and cell-surface expression of TREM2 (Paloneva et al, 2001, 2002). Both structural and cell biology studies of the AD TREM2 variants show they lie on the surface of the TREM2 ectodomain, possibly framing a binding pocket, whereas the PLOSL variants are within the core structure (Kleinberger et al, 2014; Kober et al, 2016). Maturation studies demonstrate folding and maturation deficits induced by PLOSL-associated TREM2 variants, whereas the AD variants do not consistently show these same functional impairments. Notably, a DNA polymorphism within a DNase hypersensitive site 5′ of TREM2, rs9357347-C, also associates with reduced AD risk and increased TREML1 and TREM2 levels (Carrasquillo et al, 2017). This variant is associated with decreased risk for AD, which would be consistent with the hypothesis that the coding variants in TREM2 associated with increased AD risk are partial loss of function. TREM2 is a type I transmembrane glycoprotein that interacts with the transmembrane region of DAP12 (TYROBP) to mediate signaling events through DAP12's immunoreceptor tyrosine-based activation motif (ITAM) domain (Peng et al, 2010). The extracellular, immunoglobulin-like domain V type, domain of TREM2 is glycosylated and shows cell-surface localization. TREM2 is highly expressed in microglia and peripheral macrophages (Colonna & Wang, 2016). TREM2 is one member of a family of receptors that have diverged between mouse and humans. In both mouse and human, the family members are clustered within a single chromosomal region on chromosome 6. In the context of neurodegenerative disease, the other family members have not been intensively studied as they are typically expressed at much lower levels in the brain. Like many type I membrane receptors, TREM2 can undergo ectodomain shedding and subsequent intramembrane cleavage by γ-secretase (Wunderlich et al, 2013). The physiologic implications of these proteolytic cleavages remain unclear, though increased soluble TREM2 has been reported in the CSF of early-stage Alzheimer's disease (Suarez-Calvet et al, 2016), and study of a variant p.H157Y reported to be associated with AD in the Han Chinese population has been shown to increase ectodomain shedding (Jiang et al, 2016a; Schlepckow et al, 2017; Thornton et al, 2017). Enhanced shedding activity from the mutation p.H157Y located in the ectodomain cleavage site leads to a reduced cell-surface TREM2 and phagocytic activity (Schlepckow et al, 2017; Thornton et al, 2017). TREM2 has been implicated in multiple functions including migration, survival and proliferation, cytokine release, and phagocytosis (Kleinberger et al, 2014; Ulrich & Holtzman, 2016; Yuan et al, 2016). TREM2 appears to be a promiscuous receptor and has been shown to interact with cellular debris, bacteria, anionic phospholipids, nucleic acids, and apolipoprotein E (APOE; Atagi et al, 2015; Bailey et al, 2015; Yeh et al, 2016; Kober & Brett, 2017; Song et al, 2017). Alterations in signaling and binding of TREM2 AD variants compared to wild-type (WT) TREM2 have been reported, though the pathogenic relevance of these alterations is not yet completely clear (Kleinberger et al, 2014; Kober et al, 2016). Mouse modeling studies show increased TREM2 expression in multiple models of neurodegenerative diseases, and TREM2-positive microglial cells appear to surround plaques in APP models (Jiang et al, 2014; Ulrich et al, 2014; Yuan et al, 2016; Jay et al, 2017). TREM2 also shows an altered distribution in microglia surrounding the amyloid plaque, with much of the TREM2 immunoreactivity localized within the plasma membrane of the microglial cells adjacent to the plaque. In TREM2 knockout mice crossed with APP, there are fewer microglial cells surrounding plaques and an increase in plaques with “specular” amyloid morphology as well as more neuritic dystrophy (Wang et al, 2015, 2016). Such data have led to the hypothesis that TREM2 may regulate the interaction of microglia with plaques and protect other brain cells from toxicity. In this study, we investigated whether TREM2 and its AD variants interact directly with Aβ. We found no significant changes in the binding affinity between Aβ42 with TREM2 or the AD variants or with their mouse counterpart Trem2, Trem1, and Treml1. Despite this lack of difference in the binding affinity, we observed an attenuated activation of NFAT signaling and a reduced amount of Aβ42 internalized by cells expressing the TREM2 AD variants. We show that TREM2, TREM1 and TREML1 bind APOE and Aβ variably. Our study reveals that the TREM2 and other family members can bind various forms of Aβ and that Aβ oligomers can induce singling events. Further, our data are consistent with the hypothesis that AD risk variants in TREM2 induce a partial loss of function (Song et al, 2018), but do not directly alter binding affinity for either Aβ or APOE. Results Soluble TREM2-Fc and soluble TREML1-Fc interact with Aβ42 fibrils The ectodomain of TREM2 interacts with various ligands including bacterial liposaccharides and phospholipids. We explored whether TREM2 might interact with Aβ and whether this interaction might be affected by the AD variants. We also analyzed mouse Trem2 in order to compare its relative binding ability to human TREM2. Initial studies focused on whether TREM2 could interact with fibrillar Aβ42 (fAβ), using an Aβ42 pull-down assay we have previously validated (Chakrabarty et al, 2015). Conditioned media from transiently transfected HEK 293 cells expressing the soluble ectodomains of (i) human TREM2 fused to the Fc domain of human IgG4(sTREM2-Fc), (ii) AD-associated TREM2 variants (sTREM2-R47HFc,sTREM2-R62H), (iii) mouse TREM2 (sTrem2-FC) or (iv) Fc control was incubated with fAβ. These were spun at 18,000 g to pellet fAβ associated proteins. sTREM2, sTREM2-R47H and sTREM2-R62H were enriched in the fAβ pellet (Figs 1A and EV1A), whereas the Fc control was not detected in the pellet. Mouse sTrem2 also showed binding to Aβ fibrils. Similar pull-down assay between fAβ and full-length TREM2 was conducted using the cleared RIPA lysate from TREM2, TREM2-R47H, and TREM2-R62H transiently transfected into HEK293T cells. Although less efficient, presumably because of detergent present in the lysate, full-length TREM2 and the AD variants were pulled down by fAβ (Figs 1B and EV1B). Figure 1. Soluble TREM2 and soluble TREML1-Fc bind Aβ42 fibrils A. Media from soluble TREM2-Fc transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original media samples. Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10. B. RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original RIPA lysate samples, and top right panel shows the presence of TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10. C. Media from soluble TREM2-Fc and APOE3 co-transfected HEK293T cells incubated with agarose beads anti-human IgG Fc. Left panels show original media samples. Right panels show the purified soluble TREM2-Fc (bottom) and the presence of APOE3 with the purified soluble TREM2-Fc (top). D. Media from soluble TREM-Fc family member transfected HEK293T cells incubated with Aβ42 fibrils. Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet. Bottom panel shows original media and Aβ42 pellet probed with 6E10. E. Media from soluble TREM-Fc family members and APOE3 co-transfected HEK293T cells incubated with agarose beads anti-human IgG Fc. Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3. Bottom panel shows original media and purified soluble TREM-Fc family members probed with anti-V5. Data information: All experiments were replicated 3 independent times. Western blots in panels (A, B and C) have been cropped. The originals showing the supernatant fractions are available as Fig EV1. Source data are available online for this figure. Source Data for Figure 1 [emmm201809027-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Original uncropped images of the Western blots in Fig 1 showing the supernatant fractions A. Original Western blot from Fig 1A. B. Original Western blot from Fig 1B. C. Original Western blot from Fig 1C. Download figure Download PowerPoint Multiple studies show TREM2 ectodomain interacts with APOE (Atagi et al, 2015; Bailey et al, 2015; Yeh et al, 2016). To confirm that the sTREM2-Fc proteins are functional and bind APOE3, we co-transfected TREM2-Fc constructs with APOE3 and pulled down proteins that bound sTREM2-Fc with anti-IgG Fc agarose beads. These data show that the TREM2 ectodomain binds APOE and that there is no major difference in binding between the AD variants and WT TREM2 (Figs 1C and EV1C). In this experiment, a minor interaction between the human sIgG4-Fc domain and APOE3 is observed. TREM2 is a member of a multiprotein family that is divergent between human and mice. We explored whether select members of the TREM2 family bind Aβ42 fibrils and APOE. We repeated the fAβ pull-down experiments in Fig 1A with two other soluble family members, which share low sequence homology with each and other: TREM1-Fc and TREML1-Fc. In Fig 1D, we confirm presence of soluble TREM2-Fc and TREML1-Fc in the pelleted Aβ42 fibrils. Soluble TREM1-Fc was not detected in the pelleted fAβ. Soluble TREM1-Fc like soluble TREM2-Fc interacts with APOE3, but there is lack of detectable interaction between soluble TREML1-Fc and APOE3 (Fig 1E). Soluble TREM2-Fc and AD variants interact with Aβ monomers or APOE with a similar affinity, and soluble TREM-Fc family members interact with Aβ42 oligomers with a higher affinity We used BioLayer Interferometry (BLI) to further explore the interaction between Aβ and TREM2 and other TREM family members. By immobilizing the TREM proteins on the BLI sensor, ligand interaction can be monitored in real time, enabling precise determination of the association and dissociation constants. As poorly soluble fAβ does not produce detectable binding in this assay even with high-affinity control anti-Aβ antibodies, we were only able to assess binding with Aβ42 oligomers and Aβ40 and Aβ42 monomers. We initially explored interactions between Aβ42 oligomers and sTREM2-Fc in the BLI assay. These data revealed that sTREM2 bound Aβ42 oligomers with high affinity largely attributable to a very slow dissociation. In these initial studies, we also found no interaction between the Fc alone and Aβ42 oligomers. Further, when a high-affinity anti-Aβ1-16 antibody (Ab5) was incubated following the dissociation step, we were able to demonstrate that the Aβ oligomers were indeed tightly complexed with sTREM2 on the sensor (Fig EV2A). After further optimization of the assay (see methods), we systematically evaluated the binding of sTREM-Fc proteins to various concentrations of Aβ42 oligomers, Aβ42 monomers Aβ40 monomers, as well as lipidated recombinant APOE3 and APOE4 (Figs 2A and B, and EV2B). Under these optimized conditions, no sFc binding is observed, and non-specific binding of APOE3 or APOE4 eliminated. These studies enabled precise determination of the Kon, Kdis (Koff), and the overall KD (Fig 2C) of various form of Aβ and APOE under identical conditions. These studies revealed very high-affinity binding between Aβ42 oligomers and sTREM2-Fc and the AD variants (<1.0 E-12 M). This strong binding is largely attributable to the apparent irreversibility of the binding. Indeed, the dissociation constants are the maximal measurable (Kdis = 1.0 E-07 1/s) on the Octet Red instrument. In contrast to the binding observed between sTREM2 and Aβ42 oligomers, binding to Aβ42 monomers was much weaker and reversible (KD from 1.4 to 2.9 E-07 M). The KDs of the WT TREM2 were not statistically different from the AD variants. Binding to Aβ40 monomers was similar to binding to Aβ42 monomers (KDs from 1.4 to 2.7 E-07 M), and again, the KDs of the WT TREM2 were not statistically different from the AD variants. We also established KDs in the nm range for binding of lipidated APOE3 and APOE4 with sTREM2. KDs for APOE3 binding sTREM2 or the AD variants were very similar and not statistically different. Binding of APOE4 revealed a slight increase in binding affinity of R47H variant (1.7 fold) and the R62H variant (1.56-fold) with APOE4 that was statistically significant. Given the irreversible nature of the Aβ42 oligomers binding to sTREM2, we explored whether the binding of Aβ42 oligomers could block interaction with APOE3. We evaluated whether pre-incubation with Aβ42 oligomers to sTREM2 could block subsequent interaction with APOE3. As shown in Fig 3, binding of Aβ42 oligomers blocked APOE3 binding. In contrast, addition of Aβ42 oligomers to sTREM2 previously bound to APOE3 did not block subsequent interaction with Aβ42 oligomers. We next examined the binding of select soluble mouse and human TREM family members with Aβ42 oligomers by BLI (Fig 2D). TREML1 showed a similar high-affinity binding of Aβ42 oligomers to TREM2 but the interaction between TREM1 and Aβ42 oligomers was much weaker (1.1E-7 M). In contrast, both mouse Trem1 and Treml1 bound Aβ42 oligomers with high affinity. Click here to expand this figure. Figure EV2. Complementary experiments for the BioLayer Interferometry results in Fig 2 A. Schematic representation an Aβ antibody association on soluble TREM2-Fc pre-incubated with Aβ42 oligomers. B. Western blot analysis of purified soluble TREM-Fc and soluble trem2-Fc family members and Aβ oligomers. Download figure Download PowerPoint Figure 2. Binding affinity of soluble TREM-Fc family members and AD variants with Aβ42 oligomers, Aβ42 monomers, Aβ40 monomers, and APOE3 and APOE4BioLayer Interferometry experiments were performed to measure ligand binding affinity with soluble TREM-Fc family members and mouse soluble TREM-Fc family members. A. Schematic representation of a full-length experiment. B. Schematic representation of Aβ42 oligomers (right panel) and APOE4 (left panel) binding with soluble TREM2-Fc at various concentrations. Curves were fit by setting buffer control (no ligand) to y = 0. C. Calculated Kon, Kdis, and KD values for Aβ42 oligomers or Aβ42 monomers, Aβ40 monomers, or APOE3 or APOE4 association/dissociation with soluble TREM2-Fc and AD variants. Kinetic constants were calculated using five different concentrations of ligand, and the entire experiment (including new ligand preparations and purifications of soluble TREM-Fc) was repeated twice (#P < 0.05, ANOVA, Tukey's multiple comparison test, see Appendix Table S1 for exact P-value). D. Calculated Kon, Kdis, and KD value for Aβ42 oligomers association/dissociation with soluble human vs. mouse TREM-Fc family members. Kinetic constants were calculated using five different concentrations of ligand, and the entire experiment (including new ligand preparations and purifications of soluble TREM-Fc) was repeated twice. Data information: Kon; constant of association, Kdis; constant of dissociation, KD; equilibrium constant of dissociation. Download figure Download PowerPoint Figure 3. Binding of Aβ42 oligomers on soluble TREM-Fc prevents binding of APOE3 in BioLayer InterferometryBioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2-Fc pre-incubated with Aβ42 oligomers. Schematic representations of two sequential associations of ligand on soluble TREM2-Fc are represented by I and II. Soluble TREM2-Fc loaded on a sensor is first incubated with APOE3 (association I) followed by a second incubation with Aβ42 oligomers (association II) or the reverse-incubated with Aβ42 oligomers (association I) followed by a second incubation with APOE3 (association II). All experiments were replicated 3 independent times. Download figure Download PowerPoint We further investigated the binding of fAβ with soluble TREM2-Fc and the AD variants by an ELISA binding assay due to their unsuitability with BioLayer Interferometry. We compared the binding of fAβ, Aβ42 monomers, and Aβ42 oligomers with soluble TREM2-Fc and the AD variants. In Fig EV3, Aβ42 fibrils incubated with soluble TREM2-Fc and the AD variants or soluble Trem2-Fc increased weakly the signal compared to the Fc control and showed no significant differences. The Aβ42 oligomers incubated with soluble TREM2-Fc or the AD variants or soluble Trem2-FC produced a signal significantly higher compared to Fc control and around two times stronger than the one from the Aβ42 monomers. In Fig 4A, we tested Aβ42 monomers at higher concentrations. A significant signal between Fc control and the soluble TREM2-Fc or the AD variants or soluble Trem2-Fc is obtained at 1,000 nM Aβ42 monomers and more. This result confirms the binding of Aβ42 monomers with soluble TREM2 and the AD variants and soluble Trem2-Fc without significant differences between all of them. We then repeated a similar ELISA assay with soluble TREM-Fc family members and Aβ42 oligomers. In Fig 4B, only soluble TREM2-Fc and soluble TREML1-Fc produced a significant signal compared to Fc control. This result confirms the binding of Aβ42 oligomers with soluble TREM2-Fc and soluble TREML1-Fc and lack of binding with soluble TREM1-Fc. Click here to expand this figure. Figure EV3. Aβ42 fibrils failed to bind soluble TREM2-Fc measured by ELISA A, B. ELISA plates coated with soluble TREM2-Fc and AD variants were incubated with various concentrations of Aβ42 aggregates or monomers or oligomers: (A) 100 nM and (B) 500 nM. Results were expressed as the OD450 averaged ± standard error (n = 2, **P < 0.01, ****P < 0.0001, two-way ANOVA, Bonferroni multiple comparisons, compared to Fc, see Appendix Table S4 for exact P-values). Download figure Download PowerPoint Figure 4. Binding of Aβ42 monomers and Aβ42 oligomers with soluble TREM2-Fc measured by ELISA assayELISA plates were coated with purified soluble TREM2-Fc and then incubated with various concentrations of Aβ42 monomers or 1 μM Aβ42 oligomers. A. Binding of Aβ42 monomers with soluble TREM2-Fc shows a dose-dependent signal. Results were expressed as the OD450 averaged ± standard error (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA, Bonferroni multiple comparisons, see Appendix Table S2 for exact P-values). B. Binding of 1 μM Aβ42 oligomers with soluble TREM-Fc family members. Results were expressed as the OD450 averaged ± standard error (n = 4, *P < 0.05, ****P < 0.0001, ANOVA, Bonferroni multiple comparisons, see Appendix Table S2 for exact P-values). Download figure Download PowerPoint Collectively, these biochemical experiments confirm a direct interaction between Aβ42 and TREM2 and the AD variants TRE" @default.
• W2950947069 created "2019-06-27" @default.
• W2950947069 creator A5001585929 @default.
• W2950947069 creator A5011714808 @default.
• W2950947069 creator A5016324624 @default.
• W2950947069 creator A5022819121 @default.
• W2950947069 creator A5028067127 @default.
• W2950947069 creator A5048918490 @default.
• W2950947069 creator A5053489094 @default.
• W2950947069 creator A5055691289 @default.
• W2950947069 creator A5058073147 @default.
• W2950947069 creator A5058688886 @default.
• W2950947069 creator A5062989062 @default.
• W2950947069 creator A5079657285 @default.
• W2950947069 date "2018-10-19" @default.
• W2950947069 modified "2023-10-15" @default.
• W2950947069 title "High‐affinity interactions and signal transduction between Aβ oligomers and <scp>TREM</scp> 2" @default.
• W2950947069 cites W1464665613 @default.
• W2950947069 cites W1770378670 @default.
• W2950947069 cites W1972133700 @default.
• W2950947069 cites W1996596731 @default.
• W2950947069 cites W1997816650 @default.
• W2950947069 cites W2006876731 @default.
• W2950947069 cites W2011041653 @default.
• W2950947069 cites W2014200126 @default.
• W2950947069 cites W2026279003 @default.
• W2950947069 cites W2055995786 @default.
• W2950947069 cites W2082429191 @default.
• W2950947069 cites W2089101207 @default.
• W2950947069 cites W2096401612 @default.
• W2950947069 cites W2096410114 @default.
• W2950947069 cites W2107590165 @default.
• W2950947069 cites W2111606353 @default.
• W2950947069 cites W2121590561 @default.
• W2950947069 cites W2123047934 @default.
• W2950947069 cites W2124341714 @default.
• W2950947069 cites W2124905160 @default.
• W2950947069 cites W2132154794 @default.
• W2950947069 cites W2133631618 @default.
• W2950947069 cites W2155344647 @default.
• W2950947069 cites W2167516157 @default.
• W2950947069 cites W2194170515 @default.
• W2950947069 cites W2243723775 @default.
• W2950947069 cites W2246389678 @default.
• W2950947069 cites W2256729275 @default.
• W2950947069 cites W2275354472 @default.
• W2950947069 cites W2292528698 @default.
• W2950947069 cites W2339534121 @default.
• W2950947069 cites W2408084759 @default.
• W2950947069 cites W2413770764 @default.
• W2950947069 cites W2482717739 @default.
• W2950947069 cites W2500839489 @default.
• W2950947069 cites W2534674603 @default.
• W2950947069 cites W2560062133 @default.
• W2950947069 cites W2562065042 @default.
• W2950947069 cites W2567168289 @default.
• W2950947069 cites W2592518050 @default.
• W2950947069 cites W2605569345 @default.
• W2950947069 cites W2606080622 @default.
• W2950947069 cites W2752344732 @default.
• W2950947069 cites W2753509052 @default.
• W2950947069 cites W2762857072 @default.
• W2950947069 cites W2784206224 @default.
• W2950947069 cites W2790983771 @default.
• W2950947069 cites W2793872352 @default.
• W2950947069 cites W2803198382 @default.
• W2950947069 cites W2807252917 @default.
• W2950947069 cites W4249119281 @default.
• W2950947069 doi "https://doi.org/10.15252/emmm.201809027" @default.
• W2950947069 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6220267" @default.
• W2950947069 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30341064" @default.
• W2950947069 hasPublicationYear "2018" @default.
• W2950947069 type Work @default.
• W2950947069 sameAs 2950947069 @default.
• W2950947069 citedByCount "79" @default.
• W2950947069 countsByYear W29509470692018 @default.
• W2950947069 countsByYear W29509470692019 @default.
• W2950947069 countsByYear W29509470692020 @default.
• W2950947069 countsByYear W29509470692021 @default.
• W2950947069 countsByYear W29509470692022 @default.
• W2950947069 countsByYear W29509470692023 @default.
• W2950947069 crossrefType "journal-article" @default.
• W2950947069 hasAuthorship W2950947069A5001585929 @default.
• W2950947069 hasAuthorship W2950947069A5011714808 @default.
• W2950947069 hasAuthorship W2950947069A5016324624 @default.
• W2950947069 hasAuthorship W2950947069A5022819121 @default.
• W2950947069 hasAuthorship W2950947069A5028067127 @default.
• W2950947069 hasAuthorship W2950947069A5048918490 @default.
• W2950947069 hasAuthorship W2950947069A5053489094 @default.
• W2950947069 hasAuthorship W2950947069A5055691289 @default.
• W2950947069 hasAuthorship W2950947069A5058073147 @default.
• W2950947069 hasAuthorship W2950947069A5058688886 @default.
• W2950947069 hasAuthorship W2950947069A5062989062 @default.
• W2950947069 hasAuthorship W2950947069A5079657285 @default.
• W2950947069 hasBestOaLocation W29509470691 @default.
• W2950947069 hasConcept C12554922 @default.
• W2950947069 hasConcept C185592680 @default.
• W2950947069 hasConcept C55493867 @default.
• W2950947069 hasConcept C62478195 @default.
• W2950947069 hasConcept C86803240 @default.

• next