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• W3149228057 abstract "TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aβ and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aβ oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aβ oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aβ oligomerization and disaggregated preformed Aβ oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aβ fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aβ-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal–glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aβ but rather promotes Aβ protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aβ-induced release of sTREM2, which blocks Aβ aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aβ aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk. TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aβ and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aβ oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aβ oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aβ oligomerization and disaggregated preformed Aβ oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aβ fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aβ-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal–glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aβ but rather promotes Aβ protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aβ-induced release of sTREM2, which blocks Aβ aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aβ aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk. A prominent neuropathological feature of Alzheimer's disease (AD) is the presence of extracellular deposits of the amyloid β-peptide in amyloid plaques, surrounded by activated microglia (1Wong C.W. Quaranta V. Glenner G.G. Neuritic plaques and cerebrovascular amyloid in Alzheimer disease are antigenically related.Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8729-8732Crossref PubMed Scopus (307) Google Scholar, 2Edwards F.A. A unifying hypothesis for Alzheimer's disease: From plaques to neurodegeneration.Trends Neurosci. 2019; 42: 310-322Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 3Yuan P. Condello C. Keene C.D. Wang Y. Bird T.D. Paul S.M. Luo W. Colonna M. Baddeley D. Grutzendler J. TREM2 haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy.Neuron. 2016; 90: 724-739Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). The importance of this microglial response to the pathogenesis of AD has been highlighted by the recent discovery of sequence variants in multiple genes expressed in microglia that alter risk for AD. Prominent among these microglial AD-risk genes is the “triggering receptor expressed on myeloid cells 2” (TREM2) (4Guerreiro R. Wojtas A. Bras J. Carrasquillo M. Rogaeva E. Majounie E. Cruchaga C. Sassi C. Kauwe J.S. Younkin S. Hazrati L. Collinge J. Pocock J. Lashley T. Williams J. et al.TREM2 variants in Alzheimer's disease.N. Engl. J. Med. 2013; 368: 117-127Crossref PubMed Scopus (1543) Google Scholar, 5Jonsson T. Stefansson H. Steinberg S. Jonsdottir I. Jonsson P.V. Snaedal J. Bjornsson S. Huttenlocher J. Levey A.I. Lah J.J. Rujescu D. Hampel H. Giegling I. Andreassen O.A. Engedal K. et al.Variant of TREM2 associated with the risk of Alzheimer's disease.N. Engl. J. Med. 2013; 368: 107-116Crossref PubMed Scopus (1350) Google Scholar, 6Sims R. van der Lee S.J. Naj A.C. Bellenguez C. Badarinarayan N. Jakobsdottir J. Kunkle B.W. Boland A. Raybould R. Bis J.C. Martin E.R. Grenier-Boley B. Heilmann-Heimbach S. Chouraki V. Kuzma A.B. et al.Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease.Nat. Genet. 2017; 49: 1373-1384Crossref PubMed Scopus (358) Google Scholar), of which there are several missense mutations in the ectodomain, including R47H, associated with increased risk for AD (4Guerreiro R. Wojtas A. Bras J. Carrasquillo M. Rogaeva E. Majounie E. Cruchaga C. Sassi C. Kauwe J.S. Younkin S. Hazrati L. Collinge J. Pocock J. Lashley T. Williams J. et al.TREM2 variants in Alzheimer's disease.N. Engl. J. Med. 2013; 368: 117-127Crossref PubMed Scopus (1543) Google Scholar, 5Jonsson T. Stefansson H. Steinberg S. Jonsdottir I. Jonsson P.V. Snaedal J. Bjornsson S. Huttenlocher J. Levey A.I. Lah J.J. Rujescu D. Hampel H. Giegling I. Andreassen O.A. Engedal K. et al.Variant of TREM2 associated with the risk of Alzheimer's disease.N. Engl. J. Med. 2013; 368: 107-116Crossref PubMed Scopus (1350) Google Scholar, 6Sims R. van der Lee S.J. Naj A.C. Bellenguez C. Badarinarayan N. Jakobsdottir J. Kunkle B.W. Boland A. Raybould R. Bis J.C. Martin E.R. Grenier-Boley B. Heilmann-Heimbach S. Chouraki V. Kuzma A.B. et al.Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease.Nat. Genet. 2017; 49: 1373-1384Crossref PubMed Scopus (358) Google Scholar). The biological mechanisms underlying this association remain unclear. Full-length TREM2 is expressed on the plasma membrane of microglia, where it can be cleaved by one or more metalloproteases to produce (i) a membrane-bound C-terminal fragment (CTF); and (ii) an N-terminal fragment consisting of the soluble ectodomain of TREM2 (sTREM2), which is released into the extracellular space (7Wunderlich P. Glebov K. Kemmerling N. Tien N.T. Neumann H. Walter J. Sequential proteolytic processing of the triggering receptor expressed on myeloid cells-2 (TREM2) protein by ectodomain shedding and γ-secretase-dependent intramembranous cleavage.J. Biol. Chem. 2013; 288: 33027-33036Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 8Thornton P. Sevalle J. Deery M.J. Fraser G. Zhou Y. Ståhl S. Franssen E.H. Dodd R.B. Qamar S. Gomez Perez-Nievas B. Nicol L.S. Eketjäll S. Revell J. Jones C. Billinton A. et al.TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer's disease-associated H157Y variant.EMBO Mol. Med. 2017; 9: 1366-1378Crossref PubMed Scopus (50) Google Scholar, 9Schlepckow K. Kleinberger G. Fukumori A. Feederle R. Lichtenthaler S.F. Steiner H. Haass C. An Alzheimer-associated TREM2 variant occurs at the ADAM cleavage site and affects shedding and phagocytic function.EMBO Mol. Med. 2017; 9: 1356-1365Crossref PubMed Scopus (73) Google Scholar). sTREM2 has been thought of as a nonfunctional, degradation product of TREM2 and used as a biomarker of microglial activation (10Suárez-Calvet M. Morenas-Rodríguez E. Kleinberger G. Schlepckow K. Araque Caballero M.Á. Franzmeier N. Capell A. Fellerer K. Nuscher B. Eren E. Levin J. Deming Y. Piccio L. Karch C.M. Cruchaga C. et al.Early increase of CSF sTREM2 in Alzheimer's disease is associated with tau related-neurodegeneration but not with amyloid-β pathology.Mol. Neurodegener. 2019; 14: 1Crossref PubMed Scopus (54) Google Scholar, 11Suarez-Calvet M. Araque Caballero M.Á. Kleinberger G. Bateman R.J. Fagan A.M. Morris J.C. Levin J. Danek A. Ewers M. Haass C. et al.Early changes in CSF sTREM2 in dominantly inherited Alzheimer's disease occur after amyloid deposition and neuronal injury.Sci. Transl. Med. 2016; 8 (369ra178)Crossref PubMed Scopus (119) Google Scholar, 12Suárez-Calvet M. Kleinberger G. Araque Caballero M.Á. Brendel M. Rominger A. Alcolea D. Fortea J. Lleó A. Blesa R. Gispert J.D. Sánchez-Valle R. Antonell A. Rami L. Molinuevo J.L. Brosseron F. et al.sTREM2 cerebrospinal fluid levels are a potential biomarker for microglia activity in early-stage Alzheimer's disease and associate with neuronal injury markers.EMBO Mol. Med. 2016; 8: 466-476Crossref PubMed Scopus (202) Google Scholar). However, several recent observations suggest the possibility that sTREM2 per se may play a role protecting against AD by interacting with Aβ. First, oligomeric Aβ binds to TREM2 and to sTREM2-Fc fusion protein (13Zhao Y. Wu X. Li X. Jiang L.L. Gui X. Liu Y. Sun Y. Zhu B. Piña-Crespo J.C. Zhang M. Zhang N. Chen X. Bu G. An Z. Huang T.Y. TREM2 is a receptor for beta-amyloid that mediates microglial function.Neuron. 2018; 97: 1023-1031Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 14Zhong L. Wang Z. Wang D. Wang Z. Martens Y.A. Wu L. Xu Y. Wang K. Li J. Huang R. Can D. Xu H. Bu G. Chen X.F. Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2).Mol. Neurodegener. 2018; 13: 15Crossref PubMed Scopus (51) Google Scholar, 15Lessard C.B. Malnik S.L. Zhou Y. Ladd T.B. Cruz P.E. Ran Y. Mahan T.E. Chakrabaty P. Holtzman D.M. Ulrich J.D. Colonna M. Golde T.E. High-affinity interactions and signal transduction between Aβ oligomers and TREM2.EMBO Mol. Med. 2018; 10e9027Crossref PubMed Scopus (26) Google Scholar), suggesting that sTREM2 might bind Aβ and potentially affect its aggregation state. Second, injection or expression of sTREM2 in the hippocampus of 5xFAD mice reduces both amyloid plaque load and memory deficits (16Zhong L. Xu Y. Zhuo R. Wang T. Wang K. Huang R. Wang D. Gao Y. Zhu Y. Sheng X. Chen K. Wang N. Zhu L. Can D. Marten Y. et al.Soluble TREM2 ameliorates pathological phenotypes by modulating microglial functions in an Alzheimer's disease model.Nat. Commun. 2019; 10: 1365Crossref PubMed Scopus (69) Google Scholar), indicating that sTREM2 inhibits amyloid pathology somehow. Third, in transgenic mouse models overproducing Aβ, the knockout of TREM2 expression accelerates amyloid plaque seeding (17Parhizkar S. Arzberger T. Brendel M. Kleinberger G. Deussing M. Focke C. Nuscher B. Xiong M. Ghasemigharagoz A. Katzmarski N. Krasemann S. Lichtenthaler S.F. Müller S.A. Colombo A. Monasor L.S. Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE.Nat. Neurosci. 2019; 22: 191-204Crossref PubMed Scopus (131) Google Scholar) and the plaques have increased protofibrillar halos and hotspots (3Yuan P. Condello C. Keene C.D. Wang Y. Bird T.D. Paul S.M. Luo W. Colonna M. Baddeley D. Grutzendler J. TREM2 haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy.Neuron. 2016; 90: 724-739Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Zhong L. Xu Y. Zhuo R. Wang T. Wang K. Huang R. Wang D. Gao Y. Zhu Y. Sheng X. Chen K. Wang N. Zhu L. Can D. Marten Y. et al.Soluble TREM2 ameliorates pathological phenotypes by modulating microglial functions in an Alzheimer's disease model.Nat. Commun. 2019; 10: 1365Crossref PubMed Scopus (69) Google Scholar, 18Wang Y. Ulland T.K. Ulrich J.D. Song W. Tzaferis J.A. Hole J.T. Yuan P. Mahan T.E. Shi Y. Gilfillan S. Cella M. Grutzendler J. DeMattos R.B. Cirrito J.R. Holtzman D.M. et al.TREM2-mediated early microglial response limits diffusion and toxicity of amyloid plaques.J. Exp. Med. 2016; 213: 667-675Crossref PubMed Scopus (291) Google Scholar), indicating that either TREM2 or sTREM2 inhibits plaque formation. Fourth, in the earliest presymptomatic stages of AD, at the time of Aβ deposition, sTREM2 levels in cerebrospinal fluid (CSF) are lower than in healthy controls (10Suárez-Calvet M. Morenas-Rodríguez E. Kleinberger G. Schlepckow K. Araque Caballero M.Á. Franzmeier N. Capell A. Fellerer K. Nuscher B. Eren E. Levin J. Deming Y. Piccio L. Karch C.M. Cruchaga C. et al.Early increase of CSF sTREM2 in Alzheimer's disease is associated with tau related-neurodegeneration but not with amyloid-β pathology.Mol. Neurodegener. 2019; 14: 1Crossref PubMed Scopus (54) Google Scholar, 19Ma L.Z. Tan L. Bi Y.L. Shen X.N. Xu W. Ma Y.H. Li H.Q. Dong Q. Yu J.T. Dynamic changes of CSF sTREM2 in preclinical Alzheimer's disease: The CABLE study.Mol. Neurodegener. 2020; 15: 25Crossref PubMed Scopus (13) Google Scholar), consistent with sTREM2 being an endogenous inhibitor of Aβ deposition. However, the CSF Aβ levels rise in the early symptomatic stages of AD and then decline again at later stages of AD (11Suarez-Calvet M. Araque Caballero M.Á. Kleinberger G. Bateman R.J. Fagan A.M. Morris J.C. Levin J. Danek A. Ewers M. Haass C. et al.Early changes in CSF sTREM2 in dominantly inherited Alzheimer's disease occur after amyloid deposition and neuronal injury.Sci. Transl. Med. 2016; 8 (369ra178)Crossref PubMed Scopus (119) Google Scholar, 12Suárez-Calvet M. Kleinberger G. Araque Caballero M.Á. Brendel M. Rominger A. Alcolea D. Fortea J. Lleó A. Blesa R. Gispert J.D. Sánchez-Valle R. Antonell A. Rami L. Molinuevo J.L. Brosseron F. et al.sTREM2 cerebrospinal fluid levels are a potential biomarker for microglia activity in early-stage Alzheimer's disease and associate with neuronal injury markers.EMBO Mol. Med. 2016; 8: 466-476Crossref PubMed Scopus (202) Google Scholar). Fifth, mild cognitive impairment (MCI) and AD patients with higher sTREM2 levels in CSF have slower brain atrophy, cognitive decline, and clinical decline (20Ewers M. Franzmeier N. Suárez-Calvet M. Morenas-Rodriguez E. Caballero M.A.A. Kleinberger G. Piccio L. Cruchaga C. Deming Y. Dichgans M. Trojanowski J.Q. Shaw L.M. Weiner M.W. Haass C. Increased soluble TREM2 in cerebrospinal fluid is associated with reduced cognitive and clinical decline in Alzheimer's disease.Sci. Transl. Med. 2019; 11: 507Crossref Scopus (57) Google Scholar, 21Franzmeier N. Suárez-Calvet M. Frontzkowski L. Moore A. Hohman T.J. Morenas-Rodriguez E. Nuscher B. Shaw L. Trojanowski J.Q. Dichgans M. Kleinberger G. Haass C. Ewers M. Alzheimer's Disease Neuroimaging Initiative (ADNI), Higher CSF sTREM2 attenuates ApoE4-related risk for cognitive decline and neurodegeneration.Mol. Neurodegener. 2020; 15: 57Crossref PubMed Scopus (5) Google Scholar), consistent with sTREM2 inhibiting AD progression. Sixth, healthy controls and MCI patients with higher sTREM2 levels in CSF have slower progression of amyloid and tau deposition (22Ewers M. Biechele G. Suárez-Calvet M. Sacher C. Blume T. Morenas-Rodriguez E. Deming Y. Piccio L. Cruchaga C. Kleinberger G. Shaw L. Trojanowski J.Q. Herms J. Dichgans M. Alzheimer's Disease Neuroimaging Initiative (ADNI) et al.Higher CSF sTREM2 and microglia activation are associated with slower rates of beta-amyloid accumulation.EMBO Mol. Med. 2020; 12: e12308Crossref PubMed Scopus (13) Google Scholar), consistent with sTREM2 inhibiting Aβ aggregation and subsequent tau pathology. All of these in vivo findings are compatible with the hypothesis that sTREM2 might protect against AD, potentially by impacting Aβ aggregation. To explore this hypothesis and the mechanisms involved, we investigated the interaction of Aβ with sTREM2 in vitro. We report that soluble Aβ oligomers bind TREM2 receptor on microglia and induce shedding of sTREM2. Next, we show that sTREM2 can bind and disaggregate Aβ oligomers, block Aβ fibrillization, and reduce Aβ neurotoxicity. These activities are attenuated in the R47H TREM2 holoprotein and R47H sTREM2. Moreover, the R47H sTREM2 promotes formation of morphologically distinct Aβ protofibrils. These data indicate additional mechanisms by which TREM2 protects against Alzheimer's disease and a previously unrecognized mechanism by which the R47H mutant increases risk. To confirm that TREM2 can act as a cell surface receptor for Aβ oligomers, we treated mouse primary microglia or TREM2-transfected HeLa cells with Aβ42 oligomers that have been characterized both by electron microscopy and by oligomer/fibril specific antibodies (Fig. S1; note only Aβ42 was used in this work and will be referred to as Aβ henceforth). We then immunoprecipitated TREM2 from lysates of these cells and found that Aβ co-immunoprecipitated with endogenous TREM2 on primary microglia from wild-type mice, but not on microglia from TREM2−/− knockout mice (Fig. S2i). The Aβ binding was prevented by TREM2-blocking antibody (Fig. S2ii). Oligomeric Aβ bound to TREM2 but not TREM1 (Fig. S2iii). Monomeric Aβ co-immunoprecipitated with TREM2 less efficiently than oligomeric Aβ (Fig. S2, iv and v, p = 0.03). The TREM2: Aβ oligomer interaction is at least partially specific because neither of two other neurodegeneration-associated oligomeric proteins (oligomeric α-synuclein or oligomeric Tau) bound to TREM2 even in HeLa cells overexpressing TREM2 and DAP12 (Fig. S3). These results confirm and extend the work of other groups showing that oligomeric Aβ binds TREM2 (13Zhao Y. Wu X. Li X. Jiang L.L. Gui X. Liu Y. Sun Y. Zhu B. Piña-Crespo J.C. Zhang M. Zhang N. Chen X. Bu G. An Z. Huang T.Y. TREM2 is a receptor for beta-amyloid that mediates microglial function.Neuron. 2018; 97: 1023-1031Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 14Zhong L. Wang Z. Wang D. Wang Z. Martens Y.A. Wu L. Xu Y. Wang K. Li J. Huang R. Can D. Xu H. Bu G. Chen X.F. Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2).Mol. Neurodegener. 2018; 13: 15Crossref PubMed Scopus (51) Google Scholar, 15Lessard C.B. Malnik S.L. Zhou Y. Ladd T.B. Cruz P.E. Ran Y. Mahan T.E. Chakrabaty P. Holtzman D.M. Ulrich J.D. Colonna M. Golde T.E. High-affinity interactions and signal transduction between Aβ oligomers and TREM2.EMBO Mol. Med. 2018; 10e9027Crossref PubMed Scopus (26) Google Scholar). We next tested the consequences of Aβ oligomers binding to TREM2 on primary microglia. Aβ oligomers induced release of sTREM2 into the medium of primary microglia from wild-type mice, but not microglia from mice engineered to express R47H TREM2 (Fig. S4i, p < 0.05). To study this effect in more detail, we stably expressed human TREM2 (together with human DAP12) in HEK293 cells. Treatment of these cells with Aβ oligomers resulted in dose-dependent release of sTREM2 into the medium and the accumulation of TREM2-CTF in cell lysates (Fig. 1, A and B & Fig. S4ii). In contrast, treatment with Aβ monomers or fibrils resulted in no modulation of sTREM2's release (Figs. 1C and S5). We have previously shown that this shedding event occurs rapidly, within 1 h of Aβ oligomer binding, but that FL-TREM2 levels remain constant due to ongoing new synthesis of FL-TREM2 (8Thornton P. Sevalle J. Deery M.J. Fraser G. Zhou Y. Ståhl S. Franssen E.H. Dodd R.B. Qamar S. Gomez Perez-Nievas B. Nicol L.S. Eketjäll S. Revell J. Jones C. Billinton A. et al.TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer's disease-associated H157Y variant.EMBO Mol. Med. 2017; 9: 1366-1378Crossref PubMed Scopus (50) Google Scholar). The release of sTREM2 induced by oligomeric Aβ was attenuated in HEK293 cells expressing R47H TREM2 (Fig. 1B & Figs. S4ii and S5). Thus, oligomeric Aβ stimulates release of sTREM2 (potentially by endoproteolysis) from cells expressing wild-type TREM2, but less so from cells expressing R47H TREM2. Because Aβ oligomers bind to TREM2 and induce shedding of sTREM2, it is of interest whether sTREM2 itself binds Aβ oligomers. Three groups reported that wild-type sTREM2 fused to the Fc domain of IgG binds oligomeric Aβ (13Zhao Y. Wu X. Li X. Jiang L.L. Gui X. Liu Y. Sun Y. Zhu B. Piña-Crespo J.C. Zhang M. Zhang N. Chen X. Bu G. An Z. Huang T.Y. TREM2 is a receptor for beta-amyloid that mediates microglial function.Neuron. 2018; 97: 1023-1031Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 14Zhong L. Wang Z. Wang D. Wang Z. Martens Y.A. Wu L. Xu Y. Wang K. Li J. Huang R. Can D. Xu H. Bu G. Chen X.F. Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2).Mol. Neurodegener. 2018; 13: 15Crossref PubMed Scopus (51) Google Scholar, 15Lessard C.B. Malnik S.L. Zhou Y. Ladd T.B. Cruz P.E. Ran Y. Mahan T.E. Chakrabaty P. Holtzman D.M. Ulrich J.D. Colonna M. Golde T.E. High-affinity interactions and signal transduction between Aβ oligomers and TREM2.EMBO Mol. Med. 2018; 10e9027Crossref PubMed Scopus (26) Google Scholar). However, Zhao et al. (13Zhao Y. Wu X. Li X. Jiang L.L. Gui X. Liu Y. Sun Y. Zhu B. Piña-Crespo J.C. Zhang M. Zhang N. Chen X. Bu G. An Z. Huang T.Y. TREM2 is a receptor for beta-amyloid that mediates microglial function.Neuron. 2018; 97: 1023-1031Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and Zhong et al. (14Zhong L. Wang Z. Wang D. Wang Z. Martens Y.A. Wu L. Xu Y. Wang K. Li J. Huang R. Can D. Xu H. Bu G. Chen X.F. Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2).Mol. Neurodegener. 2018; 13: 15Crossref PubMed Scopus (51) Google Scholar) found that R47H sTREM2-Fc bound less than wild-type to Aβ, while Lessard et al. (15Lessard C.B. Malnik S.L. Zhou Y. Ladd T.B. Cruz P.E. Ran Y. Mahan T.E. Chakrabaty P. Holtzman D.M. Ulrich J.D. Colonna M. Golde T.E. High-affinity interactions and signal transduction between Aβ oligomers and TREM2.EMBO Mol. Med. 2018; 10e9027Crossref PubMed Scopus (26) Google Scholar) found that they bound equally. We applied three orthogonal assays using an approach that did not require fusion of sTREM2 to the Fc domain of IgG, to reassess this question. Firstly, we examined this interaction at the molecular level using single-molecule total internal reflection fluorescence (TIRF) microscopy-based two-color coincidence detection (23Horrocks M.H. Lee S.F. Gandhi S. Magdalinou N.K. Chen S.W. Devine M.J. Tosatto L. Kjaergaard M. Beckwith J.S. Zetterberg H. Iljina M. Cremades N. Dobson C.M. Wood N.W. Klenerman D. Single-molecule imaging of individual amyloid protein aggregates in human biofluids.ACS Chem. Neurosci. 2016; 7: 399-406Crossref PubMed Scopus (65) Google Scholar). HiLyte 488-dye labeled Aβ was oligomerized and TAMRA-dye labeled sTREM2 added and imaged. Monomeric Aβ bleaches rapidly, so colocalization of Aβ and sTREM2 before and after bleaching enabled us to distinguish between monomers and oligomers. These experiments revealed that wild-type sTREM2 bound oligomeric Aβ much more than monomeric Aβ (Fig. 1, D and E) and that R47H sTREM2 bound less well to oligomeric Aβ than wild-type sTREM2 did (Fig. 1F). Second, this result was confirmed by a semiquantitative dot blot assay, which showed that wild-type sTREM2 preferentially bound oligomeric Aβ over monomeric Aβ and that R47H sTREM2 bound less of both forms of Aβ (Fig. S6, i and ii). Third, we used Bio-Layer Interferometry (BLI) (24Concepcion J. Witte K. Wartchow C. Choo S. Yao D. Persson H. Wei J. Li P. Heidecker B. Ma W. Varma R. Zhao L.S. Perillat D. Carricato G. Recknor M. et al.Label-free detection of biomolecular interactions using Bio-Layer Interferometry for kinetic characterization.Comb. Chem. High Throughput Screen. 2009; 12: 791-800Crossref PubMed Scopus (189) Google Scholar) to quantitatively assess the Aβ oligomer: sTREM2 interaction by immobilizing biotin-Aβ oligomers on Streptavidin biosensors and then exposing them to sTREM2. These studies suggested that sTREM2 associated with Aβ oligomers with two different rates and dissociated from Aβ oligomers with two different rates (Fig. S6, iii and iv), consistent with sTREM2 binding to different Aβ oligomer sizes with different affinities. Best fit association and dissociation curves for both wild-type and R47H sTREM2 were with a 2:1 heterogeneous ligand model. In agreement with the semiquantitative experiments described above, these BLI studies confirmed that R47H sTREM2 bound Aβ oligomers less well than wild-type sTREM2 (Table S1: TREM2 WT KD1 = 2.00 ± 0.15 μM, KD2 = 0.29 ± 0.08 μM; TREM2 R47H mutant KD1 = 11.70 ± 5.97 μM, KD2 = 1.22 ± 0.30 μM; where KD1 is the dissociation constant of the predominant interaction within the heterogeneous population of Aβ species). Because sTREM2 bound to Aβ oligomers, we next investigated whether sTREM2 affected Aβ oligomerization. We incubated 22 μM monomeric Aβ ± 0.22 or 0.44 μM sTREM2 (wild-type or R47H) for 3 h at 37 °C, i.e., conditions known to generate Aβ oligomers. The formation of Aβ oligomers was then assessed by transmission electron microscopy (TEM) (Figs. 2 and S7); dot blotting with A11 antioligomer antibody; and by high-performance liquid chromatography size-exclusion chromatography (HPLC-SEC) experiments (Fig. S8). These experiments revealed that preincubation of Aβ monomers with wild-type sTREM2 (at molar ratios of 1:50 and 1:100 sTREM2: Aβ) inhibited Aβ oligomerization (Fig. 2A) quantified by area (Fig. 2B) or number (Fig. S7i). SEC and antibody dot blots confirmed that sTREM2 inhibited formation of Aβ oligomers (Fig. S8i). Analysis of the size distribution of Aβ oligomers by TEM revealed that wild-type sTREM2 reduced the abundance of small oligomers (oligomer size <60 nm2) and eliminated larger oligomers (oligomer size >70 nm2) (Fig. S9i). In contrast, R47H sTREM2 did not block Aβ oligomerization, but instead promoted the formation of morphologically-distinct Aβ protofibrils (Fig. 2, C and D; Figs. S7ii and S9ii). Thus, the presence of wild-type sTREM2 reduces the formation of Aβ oligomers, particularly larger oligomers, and in stark contrast, R47H sTREM2 induces Aβ monomers to form Aβ protofibrils. To study reversal of oligomerization, preassembled Aβ oligomers were diluted to 2 μM and mixed with wild-type or R47H sTREM2 (at molar ratios of sTREM2: total Aβ of: 1:5 and 1:1) and incubated for 30 min at 37 °C. The Aβ assemblies were then assessed using the same methods as above. This experiment revealed that wild-type sTREM2 disaggregated preformed Aβ oligomers (TEM in Fig. 2, C and D and Fig. S7ii; anti-A11 dot blot and HPLC-SEC assays in Fig. S8ii). Wild-type sTREM2 strongly reduced the abundance of all but the very smallest oligomers (Fig. 2C & Fig. S9ii). In contrast, R47H sTREM2 increased the abundance of Aβ oligomers and induced the formation of morphologically-distinct Aβ protofibrils (Fig. 2C & Figs. S7ii and S9ii). Aβ protofibrils are large, linear Aβ oligomers, formed from Aβ monomers in a variety of conditions (25Harper J.D. Wong S.S. Lieber C.M. Lansbury Jr., P.T. Assembly of A beta amyloid protofibrils: An in vitro model for a possible early event in Alzheimer's disease.Biochemistry. 1999; 38: 8972-8980Crossref PubMed Scopus (443) Google Scholar, 26Nichols M.R. Moss M.A. Reed D.K. Amyloid-beta protofibrils differ from amyloid-beta aggregates induced in dilute hexafluoroisopropanol in stability and morphology.J. Biol. Chem. 2005; 280: 2471-2480Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and are observed in CSF of MCI and AD patients but not healthy controls (27De S. Whiten D.R. Ruggeri F.S. Hughes C. Rodrigues M. Sideris D.I. Taylor C.G. Aprile F.A. Muyldermans S. Knowles T.P.J. Vendruscolo M. Bryant C. Blennow K. Skoog I. Kern S. Soluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer's disease progression.Acta Neuropathol. Commun. 2019; 7: 120Crossref PubMed Scopus (20) Google Scholar). We repeated these experiments at lower concentrations, using preformed Aβ oligomers at 100 nM Aβ (monomer equivalent) incubated with 20 or 100 nM sTREM2. Wild-type sTREM2 (at either concentration) induced rapid dissolution of the Aβ oligomers (Figs. S10 and S11). In contrast, treatment with R47H sTREM2 induced aggregation of Aβ oligomers into much larger Aβ assemblies (Figs. S10 and S11). Because wild-type sTREM2 inhibited Aβ oligomerization and disaggregated Aβ oligomers, we decided to test whether sTREM2 affected Aβ fibrillization. In the absence of sTREM2, Aβ (10 μM) fibrillized with standard lag-phase kinetics as measured with Thioflavin T fluorescence. However, 1 μM of wild-type sTREM2 almost completely prevented Aβ fibrillization (Fig. 3, A–C). In total, 1 μM of R47H sTREM2 did not prevent Aβ fibrillization, but the Thioflavin T fluorescence was lower (Fig. 3A), suggesting fibrils with less β sheet structure. Repeating these experiments, at lower concentrations and higher temperature, gave similar results. Thus, fibrillization of 2 μM Aβ was substantially delayed by 20 nM sTREM2 (i.e., a 100:1 ratio) and prevented by 1 μM sTREM2 (Fig. 3D). In contrast, at the same low dose (20 nM), R47H sTREM2 had minimal effect on Aβ fibrillization (Fig. 3E). To test whether sTREM2 could disaggregate fibrils of Aβ, small fibrils were preassembled and di" @default.
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• W3149228057 title "Wild-type sTREM2 blocks Aβ aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aβ aggregation" @default.
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• W3149228057 doi "https://doi.org/10.1016/j.jbc.2021.100631" @default.
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