Triggering Receptor Expressed On Myeloid Cells 2 Mutations Research Articles

DAP12 interacts with RER1 and is retained in the secretory pathway before assembly with TREM2

DNAX-activating protein of 12 kDa (DAP12) is a transmembrane adapter protein expressed in lymphoid and myeloid lineage cells. It interacts with several immunoreceptors forming functional complexes that trigger intracellular signaling pathways. One of the DAP12 associated receptors is the triggering receptor expressed on myeloid cells 2 (TREM2). Mutations in both DAP12 and TREM2 have been linked to neurodegenerative diseases. However, mechanisms involved in the regulation of subcellular trafficking and turnover of these proteins are not well understood. Here, we demonstrate that proteasomal degradation of DAP12 is increased in the absence of TREM2. Interestingly, unassembled DAP12 is also retained in early secretory compartments, including the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC), thereby preventing its transport to the plasma membrane. We also show that unassembled DAP12 interacts with the retention in ER sorting receptor 1 (RER1). The deletion of endogenous RER1 decreases expression of functional TREM2-DAP12 complexes and membrane proximal signaling, and resulted in almost complete inhibition of phagocytic activity in THP-1 differentiated macrophage-like cells. These results indicate that RER1 acts as an important regulator of DAP12 containing immunoreceptor complexes and immune cell function.Graphical

TREM2 AD risk variants impact pTau and microglia and astrocyte related biomarkers in cognitively unimpaired subjects

AbstractBackgroundIn the central nervous system, Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is expressed primarily in microglia. TREM2 receptors are important for microglial functions including phagocytosis, lipid metabolism and inflammatory responses. TREM2 coding variants (e.g. R47H rs75932628 and R62H rs143332484) increase the risk of late onset Alzheimer’s Diseases (AD). However, the impact of TREM2 risk variants on microglial cell functions in humans remains to be elucidated.MethodCSF and plasma samples were collected from AD patients (n = 120) and cognitively unimpaired control subjects (n = 53) with subjective complaints who do not meet biomarker‐based criteria for AD diagnosis. TREM2 AD risk variant carriers (n = 52) were screened from over 6000 samples of the Amsterdam dementia cohort using a combination of genotype array and exome sequencing. We performed extensive biomarker analysis of CSF and plasma samples with multidisciplinary approaches. We compared the effect of TREM2 mutations on AD disease and microglial and astrocytic biomarkers in AD patients and in cognitively unimpaired control subjects, respectively. A linear model was used with age and gender adjusted for the data analysis.ResultWe found higher levels of pTau181 and pTau231 in CSF and plasma in cognitively unimpaired TREM2 mutation carriers compared to non‐carriers, while no difference was observed between TREM2 genotype among the AD patients. Microglia and astrocyte biomarkers including sTREM2, FABP3, OLR1, YKL40 and GFAP were higher in the CSF from cognitively unimpaired TREM2 mutation carriers compared to non‐carriers, but no difference was detected between TREM2 genotype among the AD patients. We also found different levels of lipid metabolites in CSF and plasma samples from the TREM2 mutation carriers compared to non‐carriers in both AD patients and cognitively unimpaired control subjects.ConclusionTREM2 AD risk variants in cognitively unimpaired subjects increase pTau biomarkers and microglial and astrocytic related biomarkers. In AD patients, the biomarker differences between TREM2 genotype are diminished likely due to the extensive microglial responses to AD pathology. Moreover, our data indicates TREM2 AD risk variants may impact lipid‐metabolic processes in microglial cells. These data provide the biomarker insights on how TREM2 mutations may increase the risk of AD.

The role of TREM2 in Alzheimer's disease: from the perspective of Tau.

Triggering receptor expressed on myeloid cells 2 (TREM2), a pattern recognition receptor abundantly expressed on microglia, has been identified as one of the risk factors for Alzheimer's disease (AD). Several studies have already demonstrated the relationship between TREM2 and Tau. TREM2 mutations and altered expression play an important role in Tau phosphorylation. Furthermore, the level of Tau phosphorylation is correlated with soluble TREM2 (sTREM2). However, in different stages of AD, TREM2 seems to have varying effects on Tau pathology. The explicit interaction between TREM2 and Tau, as well as how they affect AD pathology, remains unclear, and there is much evidence to the contrary that requires rational interpretation. Reviewing the dual roles of TREM2 in AD will help identify a more appropriate development strategy for targeting TREM2 to treat AD. Therefore, this review focuses on the interplay between Tau and TREM2 in relation to AD.

Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells and macrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases, such as Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knockout (TREM2-KO) and wild-type (WT) mice. The TREM2-sol mouse is a new model with selective elimination of plasma membrane TREM2 and a reduced expression of soluble TREM2. In the acute cuprizone model demyelination and remyelination events were reflected by a T2-weighted signal intensity change in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). In contrast to WT and TREM2-IPD, TREM2-sol and TREM2-KO showed an additional increase in MRI signal during the recovery phase. Histological analyses of TREM2-IPD animals revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 and displayed enhanced cytokine/chemokine levels in the brain. TREM2-sol and, to a much lesser extent, TREM2-KO, however, despite presenting reduced levels of some cytokines/chemokines, showed persistent microgliosis and astrocytosis during recovery, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia without plasma membrane TREM2 and only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function, as observed in bone marrow-derived macrophages, leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication.

TREM2 differentially modulates parenchymal and vascular pathology in a mouse model of cerebral amyloid angiopathy

AbstractBackgroundCerebral amyloid angiopathy (CAA) features cerebral vascular deposition of pathological amyloid‐beta protein (Aβ) and plays an important role in the multimorbidity of aging brains including Alzheimer’s disease (AD), but it is less understood compared to parenchymal Aβ plaques. Triggering receptor expressed on myeloid cells 2 (TREM2), a key player in innate immune response, is exclusively expressed in microglia in the brain and mediates Aβ clearance and subsequently alleviates neuroinflammation. Loss‐of‐function mutations in TREM2 increase the risk of AD. Recent studies on TREM2 function using TREM2 knockout (KO) in transgenic (Tg) AD mice have shown that TREM2 modulates parenchymal Aβ deposition in the brain, although the direction of the effect depends on the specific Tg background and the stage of the disease development. However, the impact of TREM2 on CAA had not been investigated.MethodsTREM2 KO mice were crossed with SwDI mice (C57BL/6‐Tg(Thy1‐APPSwDutIowa)BWevn/Mmjax), a model of CAA and AD, to generate SwDI/TREM2WT, SwDI/TREM2Het, and SwDI/TREM2KO mice. At the age of 16 months, the mice were euthanized for tissue collection. ELISA was utilized for the assessment of Aβ40 and Aβ42 levels in brain tissue homogenates, and histochemical and immunohistochemical or immunofluorescent assays were used to evaluate amyloid deposition, astrogliosis, and microgliosis. All results were analyzed using GraphPad Prism 9.ResultsTREM2 deficiency markedly increased overall Aβ load in SwDI/TREM2KO mice compared to SwDI/TREM2WT or SwDI/TREM2Het in the cortex, hippocampus, and thalamus. Intriguingly, TREM2 deletion led to a dramatic decrease in CAA in vasculature‐enriched regions, such as thalamus, along with reduced microglial association with CAA. Consistent with previous reports, Aβ plaque‐associated microglia were significantly reduced in SwDI/TREM2KO mice. Additional experiments are underway to explore the molecular mechanisms underlying the distinct impact of TREM2 deficiency on Aβ plaques and CAA.ConclusionDeletion of TREM2 increases the overall brain Aβ load whereas decreases CAA, suggesting the differential role of TREM2 in the regulation of parenchymal versus vascular deposition of Aβ in the brain.

Soluble TREM2 inhibits secondary nucleation of Aβ fibrillization and enhances cellular uptake of fibrillar Aβ

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single-pass transmembrane receptor of the immunoglobulin superfamily that is secreted in a soluble (sTREM2) form. Mutations in TREM2 have been linked to increased risk of Alzheimer's disease (AD). A prominent neuropathological component of AD is deposition of the amyloid-β (Aβ) into plaques, particularly Aβ40 and Aβ42. While the membrane-bound form of TREM2 is known to facilitate uptake of Aβ fibrils and the polarization of microglial processes toward amyloid plaques, the role of its soluble ectodomain, particularly in interactions with monomeric or fibrillar Aβ, has been less clear. Our results demonstrate that sTREM2 does not bind to monomeric Aβ40 and Aβ42, even at a high micromolar concentration, while it does bind to fibrillar Aβ42 and Aβ40 with equal affinities (2.6 ± 0.3 µM and 2.3 ± 0.4 µM). Kinetic analysis shows that sTREM2 inhibits the secondary nucleation step in the fibrillization of Aβ, while having little effect on the primary nucleation pathway. Furthermore, binding of sTREM2 to fibrils markedly enhanced uptake of fibrils into human microglial and neuroglioma derived cell lines. The disease-associated sTREM2 mutant, R47H, displayed little to no effect on fibril nucleation and binding, but it decreased uptake and functional responses markedly. We also probed the structure of the WT sTREM2-Aβ fibril complex using integrative molecular modeling based primarily on the cross-linking mass spectrometry data. The model shows that sTREM2 binds fibrils along one face of the structure, leaving a second, mutation-sensitive site free to mediate cellular binding and uptake.

TREM2 Mediates Microglial Anti-Inflammatory Activations in Alzheimer's Disease: Lessons Learned from Transcriptomics.

Alzheimer’s disease (AD) is a lethal neurodegenerative disorder primarily affecting the aged population. The etiopathogenesis of AD, especially that of the sporadic type, remains elusive. The triggering receptor expressed on myeloid cells 2 (TREM2), a member of TREM immunoglobulin superfamily, plays a critical role in microglial physiology. Missense mutations in human TREM2 are determined as genetic risk factors associated with the development of sporadic AD. However, the roles of TREM2 in the pathogenesis of AD are still to be established. In this review, we outlined the influence of Trem2 on balance of pro- and anti-inflammatory microglial activations from a perspective of AD mouse model transcriptomics. On this basis, we further speculated the roles of TREM2 in different stages of AD, which may shed light to the development of TREM2-targeted strategy for the prevention and treatment of this neurodegenerative disorder.

Differential regulation of TREM2 and CSF1R in CNS macrophages in an SIV/macaque model of HIV CNS disease.

HIV-associated neuroinflammation is primarily driven by CNS macrophages including microglia. Regulation of these immune responses, however, remains to be characterized in detail. Using the SIV/macaque model of HIV, we evaluated CNS expression of triggering receptor expressed on myeloid cells 2 (TREM2) which is constitutively expressed by microglia and contributes to cell survival, proliferation, and differentiation. Loss-of-function mutations in TREM2 are recognized risk factors for neurodegenerative diseases including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Nasu-Hakola disease (NHD); recent reports have also indicated a role for TREM2 in HIV-associated neuroinflammation. Using in situ hybridization (ISH) and qRT-PCR, TREM2 mRNA levels were found to be significantly elevated in frontal cortex of macaques with SIV encephalitis compared with uninfected controls (P = 0.02). TREM2 protein levels were also elevated as measured by ELISA of frontal cortex tissue homogenates in these animals. Previously, we characterized the expression of CSF1R (colony-stimulating factor 1 receptor) in this model; the TREM2 and CSF1R promoters both contain a PU.1 binding site. While TREM2 and CSF1R mRNA levels in the frontal cortex were highly correlated (Spearman R = 0.79, P < 0.001), protein levels were not well correlated. In SIV-infected macaques released from ART to study viral rebound, neither TREM2 nor CSF1R mRNA increased with rebound viremia. However, CSF1R protein levels remained significantly elevated unlike TREM2 (P = 0.02). This differential expression suggests that TREM2 and CSF1R play unique, distinct roles in the pathogenesis of HIV CNS disease.

Mechanistic insights into the deleterious roles of Nasu-Hakola disease associated TREM2 variants

Recently, the critical roles played by genetic variants of TREM2 (Triggering Receptor Expressed on Myeloid cells 2) in Alzheimer’s disease have been aggressively highlighted. However, few studies have focused on the deleterious roles of Nasu-Hakola disease (NHD) associated TREM2 variants. In order to get insights into the contributions made by these variants to neurodegeneration, we investigated the influences of four NHD associated TREM2 mutations (Y38C, W50C, T66M, and V126G) on loss-of-function, and followed this with in silico prediction and conventional molecular dynamics simulation. NHD mutations were predicted to be highly deleterious by eight different in silico bioinformatics tools and found to induce conformational changes by molecular dynamics simulation. As compared with the wild-type, the four variants produced substantial differences in the collective motions of loop regions, which not only promoted structural remodeling in the CDR2 (complementarity-determining region 2) loop but also in the CDR1 loop, by changing inter- and intra-loop hydrogen bonding networks. In addition, structural studies in a free energy landscape analysis showed that Y38, T66, and V126 are crucial for maintaining the structural features of CDR1 and CDR2 loops, and that mutations in these positions produced steric clashes and loss of ligand binding. These results showed the presence of mutations in the TREM2 ectodomain induced flexibility and caused structural alterations. Dynamical scenarios, as provided by the present study, may be critical to our understanding of the roles of these TREM2 mutations in neurodegenerative diseases.

Distinct Signaling Pathways Regulate TREM2 Phagocytic and NFκB Antagonistic Activities.

Several genetic variants of the Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) have been shown to increase the risk of developing Alzheimer’s disease (AD) supporting a role of microglia and immune cells in the pathobiology of AD. We have employed an ectopic model of TREM2 and DAP12 expression in HEK293 cells to study selectively TREM2 dependent signaling and phagocytic functions and evaluated the effects of some of the TREM2 mutations associated with AD. We show that shedding of the TREM2 N-terminal domain does not affect the inhibition of NFκB activation induced by TREM2 while it completely blocks phagocytosis suggesting that TREM2 anti-inflammatory properties can be mediated by the TREM2 C-terminal fragment while the phagocytic activity requires the full-length receptor. In addition, we confirm in that model that apolipoprotein E (APOE) is a ligand for TREM2 and triggers TREM2 signaling. In particular, we show that APOE4 stimulates spleen tyrosine kinase (SYK) activation more potently than APOE2 in a TREM2 dependent manner. Interestingly, TREM2 appears to antagonize NFκB activation induced by phorbol ester but is unable to prevent TNFα induction of NFκB activation suggesting that TREM2 antagonizes inflammatory events triggered downstream of PKC. TREM2 mutations drastically impact TREM2 phagocytosis as well as its ability to antagonize NFκB activation and notably prevent the activation of the PI3K/AKT pathway observed with wild-type TREM2. Overall our data suggest that TREM2 dependent phagocytosis requires an activation of the SYK/PI3K/AKT/PLCγ pathways while the suppression of NFκB activation by TREM2 is independent of SYK, PI3K, and PLCγ activities. This model of ectopic TREM2-DAP12 co-expression appears suitable to study TREM2 signaling as several biological functions of TREM2 and TREM2 mutations that have been previously described in myeloid and microglial cells were also replicated in this model.

TREM2 Variants and Neurodegenerative Diseases: A Systematic Review and Meta-Analysis.

TREM2 (triggering receptor expressed on myeloid cells 2) gene variants were reported to increase the risk of Alzheimer's disease (AD) and even other neurodegenerative diseases (frontotemporal dementia (FTD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS)), but so far, no definite conclusion has been drawn. The aim of our systematic review and meta-analysis was to investigate the role of TREM2 variants in neurodegenerative diseases. A total of 39 papers (including 26 case-control studies and 13 case reports) were retrieved from PubMed, MEDLINE, EMBASE, and the Cochrane library in this study. A fixed effect model was used to pool results in the analysis. Three variants in TREM2 (rs75932628 (R47H), rs2234255 (H157Y), and rs143332484 (R62H)) were significantly associated with AD risk, but the similar associations between rs104894002 (Q33X), rs2234253 (T96K), rs142232675 (D87N), rs2234256 (L211P), and AD were not proven. Rs75932628 also increased risk of PD in North Americans and FTD, but not PD in Europeans or ALS. In the systematic review, 12 biallelic TREM2 mutations (e.g., rs104894002, rs201258663 (T66M), and rs386834144, etc.) have been described to cause Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy (PLOSL) in 14 families. And homozygous mutations also have been reported to cause FTD without typical bone phenotypes in 7 families. This study demonstrates that multiple variants in TREM2 have association with the onset of AD, FTD, and PD in North Americans and also play a key role in the phenotypes of the rare familial genetic disorder.

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Mutations: a Potential Common Cause of Alzheimer's Disease and Musculoskeletal Disorders

Alzheimer's disease (AD), a worldwide growing health issue, is a purely neurodegenerative form of age‐induced dementia. Interestingly, weight loss is frequently associated with disease severity and clinical progression and has been shown to begin at least ten years before the diagnosis of the disease. Bone frailty and sarcopenia have each also been linked to the onset of this disease, although very little is known about the pathogenesis of these conditions in subjects with AD. Indeed, although a common link between dementia and osteoporosis has been suggested, it has never been conclusively demonstrated. The prevalence of both conditions increase with aging, and some genetic conditions can lead to both AD and osteoporosis. Examples for the potential common cause of the 2 conditions are mutations of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) gene, primarily expressed in myeloid cells or microglia in brain and in osteoclasts in bone, which result in AD and dementia, as well as in bone fragility. In a recent Genome Wide Association Study, a single nucleotide polymorphism (SNP) encoding the R47H variant in TREM2 was linked with strong single allele genetic risk factors for AD, as well as with increased risk for Parkinson's disease, amyotrophic lateral sclerosis and frontotemporal dementia. In addition, recent studies showed that mice expressing the R47H TREM2 variant exhibit loss of TREM2 function and neuritic dystrophy. In a similar manner, another TREM2 mutation, the Y38C variant, was previously described in the pathogenesis of Nasu‐Hakola disease (also known as PLOSL) which is characterized by extensive white matter loss and frontotemporal‐like dementia and polycystic lipomembranous osteodysplasia. To investigate whether mutations in the TREM2 gene, previously associated with AD and frontotemporal dementia, also affect the skeleton, we analyzed the musculoskeletal phenotype of mice in which the Y38C and R47H variants of the TREM2 gene are ubiquitously expressed, generated by Drs. Lamb and Landreth at the Stark Neuroscience Research Institute, Indiana University School of Medicine. Our studies showed decreased bone mass in mice expressing the TREM2 mutants at 6–8 months of age. Interestingly, even though TREM2 expression was absent in skeletal muscle of wild type mice, mice expressing the R47H TREM2 variant exhibit decrease muscle mass. In summary, our results suggest a common cause of cognitive disorders and bone loss, and support an indirect role of TREM2 mutations on the loss of muscle mass, secondary to the altered bone remodeling.Support or Funding InformationNIH/NIAMS R01AR067210ICMH Collaborative Pilot Project AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 133: Triggering Receptor Expressed on Myeloid Cells-2 Improves Post-stroke Neurological Function and Regulates Microglia/Macrophage Phenotype in Aged Female Mice

Background and Purpose: We previously reported that triggering receptor expressed on myeloid cells-2 (TREM2) expression on microglia/macrophage activates its phagocytic function and promotes stroke recovery. Although its importance has been demonstrated, it is unclear whether there is a similar effect in females. Furthermore, TREM2 mutations have been linked to dementia. In the present study, we explored the influence of TREM2 deficiency in aged female mice subjected to middle cerebral artery occlusion (MCAO). We assessed histology, sensorimotor and cognitive functions along with post stroke immune responses. Methods: Age matched (≥8months) female TREM2 knockout (TREM2-KO) and Wild type (Wt) mice were subjected to permanent distal MCAO. Sensorimotor and cognitive (Y-maze test) functions and infarct size were assessed up to 14d after stroke (n=6/each). Microglial activation (isolectin B4, IB4 &amp; CD68), phagocytosis (oil red O stain) and M1 (NO/iNOS) and M2 (arginase, Arg) markers 1,3,7 and 14d post MCAO. Results: At baseline, aged female mice lacking TREM2 had similar cognitive &amp; sensorimotor function. However, post stroke, TREM2-KO females, compared to Wt, showed worsened cognitive (p&lt;0.01) and neurological function (p&lt;0.05). Female TREM2 knockout mice also had larger infarct volumes (p&lt;0.05) and reduced phagocytic activity (p&lt;0.01). Microglia/macrophage accumulation, which peaked by 7d, was significantly inhibited in TREM2-KO mice (p&lt;0.01). TREM2-KO mice also showed fewer CD68+ phagocytes; however, iNOS positive CD68 cells were significantly increased at days 3 &amp; 7 (p&lt;0.05), while, Arg positive CD68 cells was significantly decreased at days 7 &amp; 14 (p&lt;0.05) in the TREM2-KO group, compared to the Wt group and suggests a M2 bias for TREM2. Conclusions: Similar to male mice, TREM2 seems to have beneficial properties in female mice. TREM2 deficient female mice had larger infarct sizes and worsened neurological performance. Microglial responses also seemed to suggest that TREM2 promotes beneficial and/or recovery pathways.

A novel homozygous mutation in TREM2 found in a Chinese early-onset dementia family with mild bone involvement

Variants in triggering receptor expressed on myeloid cells 2 (TREM2) are associated with both behavioral variant frontotemporal lobar degeneration and Alzheimer's disease. TREM2 homozygous mutations cause Nasu-Hakola disease, an early-onset recessive form of dementia preceded by bone cysts and fractures. The same type of mutations has been shown to cause frontotemporal dementia without the presence of any bone phenotype. Herein, we report a Chinese Han consanguineous family carrying a novel TREM2 mutation, presenting with early-onset dementia similar to behavioral variant frontotemporal dementia with mild radiological bone involvement. Minigene reporter assay showed the variant disturbed splicing by preservation of intron 2 in transcription. In our investigation, the clinical and genetic spectra of Chinese early-onset dementia patients were expanded; TREM2 mutations should be screened in familial and Chinese early-onset dementia patients.

Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia

SummaryThe derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2), which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations.

TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function

Mutations in triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to increased Alzheimer's disease (AD) risk. Neurobiological functions of TREM2 and its pathophysiological ligands remain elusive. Here we found that TREM2 directly binds to β-amyloid (Aβ) oligomers with nanomolar affinity, whereas AD-associated TREM2 mutations reduce Aβ binding. TREM2 deficiency impairs Aβ degradation in primary microglial culture and mouse brain. Aβ-induced microglial depolarization, K+ inward current induction, cytokine expression and secretion, migration, proliferation, apoptosis, and morphological changes are dependent on TREM2. In addition, TREM2 interaction with its signaling adaptor DAP12 is enhanced by Aβ, regulating downstream phosphorylation of SYK and GSK3β. Our data demonstrate TREM2 as a microglial Aβ receptor transducing physiological and AD-related pathological effects associated with Aβ.

Intracellular trafficking of TREM2 is regulated by presenilin 1

Genetic mutations in triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to a variety of neurodegenerative diseases including Alzheimer’s disease, amyotrophic lateral sclerosis, frontotemporal dementia and Parkinson’s disease. In the brain, TREM2 is highly expressed on the cell surface of microglia, where it can transduce signals to regulate microglial functions such as phagocytosis. To date, mechanisms underlying intracellular trafficking of TREM2 remain elusive. Mutations in the presenilin 1 (PS1) catalytic subunit of the γ-secretase complex have been associated with increased generation of the amyloidogenic Aβ (amyloid-β) 42 peptide through cleavage of the Aβ precursor amyloid precursor protein. Here we found that TREM2 interacts with PS1 in a manner independent of γ-secretase activity. Mutations in TREM2 alter its subcellular localization and affects its interaction with PS1. Upregulation of PS1 reduces, whereas downregulation of PS1 increases, steady-state levels of cell surface TREM2. Furthermore, PS1 overexpression results in attenuated phagocytic uptake of Aβ by microglia, which is reversed by TREM2 overexpression. Our data indicate a novel role for PS1 in regulating TREM2 intracellular trafficking and pathophysiological function.

Neurodegeneration-associated mutant TREM2 proteins abortively cycle between the ER and ER–Golgi intermediate compartment

Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane protein expressed on microglia within the brain. Several rare mutations in TREM2 cause an early-onset form of neurodegeneration when inherited homozygously. Here we investigate how these mutations affect the intracellular transport of TREM2. We find that most pathogenic TREM2 mutant proteins fail to undergo normal maturation in the Golgi complex and show markedly reduced cell-surface expression. Prior research has suggested that two such mutants are retained in the endoplasmic reticulum (ER), but we find, using a cell-free coat protein complex II (COPII) vesicle budding reaction, that mutant TREM2 is exported efficiently from the ER. In addition, mutant TREM2 becomes sensitive to cleavage by endoglycosidase D under conditions that inhibit recycling to the ER, indicating that it normally reaches a post-ER compartment. Maturation-defective TREM2 mutants are also efficiently bound by a lectin that recognizes O-glycans added in the ER-Golgi intermediate compartment (ERGIC) and cis-Golgi cisterna. Finally, mutant TREM2 accumulates in the ERGIC in cells depleted of COPI. These results indicate that efficient ER export is not sufficient to enable normal cell-surface expression of TREM2. Moreover, our findings suggest that the ERGIC may play an underappreciated role as a quality-control center for mutant and/or malformed membrane proteins.

A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer's disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.

Functional involvement of γ-secretase in signaling of the triggering receptor expressed on myeloid cells-2 (TREM2).

BackgroundTriggering receptor expressed on myeloid cells-2 (TREM2) exerts important functions in the regulation of monocytes, like dendritic cells, osteoclasts, tissue macrophages, and microglia. Mutations in TREM2 are associated with several diseases, including Nasu-Hakola disease, frontotemporal dementia, and Alzheimer’s disease (AD). TREM2 undergoes sequential proteolytic processing by ectodomain shedding and intramembrane proteolysis.FindingsWe show that inhibition of γ-secretase-dependent cleavage of the TREM2 C-terminal fragment in cellular membranes interferes with TREM2-dependent signaling and cellular function. Inhibition of γ-secretase decreases membrane-proximal signaling and intracellular Ca2+ response. Decreased signaling alters morphological changes and phagocytic activity of cells upon selective stimulation of TREM2.ConclusionsThe data demonstrate the importance of γ-secretase-dependent intramembrane processing in TREM2-mediated signaling and, thus, a functional relation of two AD-associated proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0479-9) contains supplementary material, which is available to authorized users.