Triggering Receptor Expressed In Myeloid Cells 2 Research Articles

MTOR signaling regulates microglia metabolism and survival via 4E‐BP‐controlled mRNA translation

AbstractBackgroundRare loss‐of‐function variants in TREM2 (Triggering Receptor Expressed in Myeloid cells 2) are associated with an increased risk of AD. There are ongoing efforts to therapeutically boost microglial neuroprotective functions by targeting TREM2. However, the intracellular mechanisms underlying these functions in AD are not fully understood. TREM2 signaling maintains metabolic fitness through activation of the mechanistic target of rapamycin (mTOR) allowing microglia to sustain their metabolism during amyloidosis (Ulland et al., 2017). Detrimental decreases in microglial mTOR signalling during amyloidosis independent of TREM2 loss‐of‐function has also been reported (Baik et al., 2019).The mTOR pathway acts upstream of the eukaryotic mRNA translation by suppressing the translation repressor, eukaryotic translation initiation factor 4E (eIF4E)‐binding proteins (4E‐BPs). mTOR inhibits the 4E‐BPs via phosphorylation, thereby releasing eIF4E and allowing mRNA translation to initiate (Hay and Sonenberg, 2004). Work from our group demonstrated that mTOR boosts mitochondrial metabolism through 4E‐BP‐controlled mRNA translation (Morita et al., 2013). Therefore, we aimed to understand the dysregulated mechanisms at the intersection of mTOR‐controlled mRNA translation and metabolism in microglia response to Amyloid‐β (Aß).MethodWe used immunoblotting to investigate the effect on 4E‐BP phosphorylation following chronic exposure to Aß aggregates and TREM2 signaling inhibition in a microglia cell line. We manipulated the mTOR pathway by knocking out the downstream effectors, 4E‐BPs, to promote the translation of 4E‐BP‐controlled mRNAs in microglia in vitro and in vivo. We crossed the microglia‐specific 4E‐BPs knock out mouse with a RiboTag mouse to pull‐down ribosome‐bound mRNAs, providing a genome‐wide pool of actively translating mRNAs (the translatome) in the absence of 4E‐BPs in microglia.ResultWe showed that 4E‐BP‐controlled mRNA translation is inhibited upon chronic exposure to Aß or TREM2 signaling inhibition. The deletion of 4E‐BPs in microglia in‐vitro leads to a decrease in expression of pro‐inflammatory mediators and microglia cell death, and an increase in reliance on oxidative phosphorylation upon exposure to Aß. We also observed that 4E‐BP deletion changes the microglia translatome and decreases expression of inflammatory mediators in vivo after an intracranial Aß infusion.ConclusionWe conclude that mTOR signaling exerts key effects on microglia function and metabolism through 4E‐BP‐controlled mRNA translation.

EVOO Polyphenols Exert Anti-Inflammatory Effects on the Microglia Cell through TREM2 Signaling Pathway.

In Alzheimer's disease (AD), microglia, brain resident immune cells, become chronically inflammatory and neurotoxic. In recent years, neuroinflammation has attracted particular interest in the scientific community. The genetic variants of molecules associated with ''microgliopathies'', including the triggering receptor expressed in myeloid cells-2 (TREM2), result in increased risk of developing AD and cognitive decline. We performed a set of in vitro assays using human neuronal (SH-SY5Y) and microglial (BV2 and C13NJ) cell models. Cells were differentially treated with extra virgin olive oil (EVOO) polyphenols, oleuropein aglycone (OleA) and hydroxytyrosol (HT) before adding LPS. We evaluated the protective effects of these EVOO products by a set of biochemical and cell biology assays, including ELISA, MTT, ROS detection, Western blotting and immunofluorescence. Our results provide an integrated understanding of the neuroprotection exerted by polyphenols in terms of: (i) reduction of pro-inflammatory cytokines release (IL-6, IL-8, IP-10 and RANTES); (ii) activation of the TREM2-dependent anti-inflammatory pathway; (iii) enhancement of protective microglial activity favoring the M2 polarization phenotype. Such findings provide new and important insights into the mechanisms by which the dietary olive polyphenols exert beneficial properties against neuroinflammation and neuronal impairment.

Circulating Soluble TREM2 and Cardiovascular Outcome in Cohort Study of Coronary Atherosclerosis Patients.

Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) is a membrane receptor in myeloid cells that mediates cellular phagocytosis and inflammation. TREM2 and its soluble extracellular domain are clearly implicated in neuroinflammation and neurodegeneration. sTREM2 is also expressed in atherosclerotic macrophages. We hypothesized that sTREM2 would predict cardiovascular mortality in patients with established coronary atherosclerosis (CAD). Consecutive patients undergoing coronary angiography with the establishment of the diagnosis of CAD (n = 230) and without CAD (n = 53) were tested for their baseline serum sTREM2 levels. All patients were followed up for 84 months or until death occurred. sTREM2 correlated with age; however, no association was found between sTREM2 and the number of atherosclerotic vessels involved (p = 0.642). After 84 months of follow-up, 68 out of the 230 CAD patients had died. After adjusting for age and other risk factors, the adjusted hazard ratio for the highest quartile of sTREM2 was 2.37 (95% confidence interval 1.17-4.83) for death. In patients with established CAD, serum sTREM2 appears to predict cardiovascular death as a potential surrogate for plaque rupture. TREM2 and its soluble extracellular form might be implicated in the fate of the atherosclerotic plaque, but corroboration within larger studies is needed.

Long-term running exercise improves cognitive function and promotes microglial glucose metabolism and morphological plasticity in the hippocampus of APP/PS1 mice

BackgroundThe role of physical exercise in the prevention of Alzheimer’s disease (AD) has been widely studied. Microglia play an important role in AD. Triggering receptor expressed in myeloid cells 2 (TREM2) is expressed on microglia and is known to mediate microglial metabolic activity and brain glucose metabolism. However, the relationship between brain glucose metabolism and microglial metabolic activity during running exercise in APP/PS1 mice remains unclear.MethodsTen-month-old male APP/PS1 mice and wild-type mice were randomly divided into sedentary groups or running groups (AD_Sed, WT_Sed, AD_Run and WT_Run, n = 20/group). Running mice had free access to a running wheel for 3 months. Behavioral tests, [18]F-FDG-PET and hippocampal RNA-Seq were performed. The expression levels of microglial glucose transporter (GLUT5), TREM2, soluble TREM2 (sTREM2), TYRO protein tyrosine kinase binding protein (TYROBP), secreted phosphoprotein 1 (SPP1), and phosphorylated spleen tyrosine kinase (p-SYK) were estimated by western blot or ELISA. Immunohistochemistry, stereological methods and immunofluorescence were used to investigate the morphology, proliferation and activity of microglia.ResultsLong-term voluntary running significantly improved cognitive function in APP/PS1 mice. Although there were few differentially expressed genes (DEGs), gene set enrichment analysis (GSEA) showed enriched glycometabolic pathways in APP/PS1 running mice. Running exercise increased FDG uptake in the hippocampus of APP/PS1 mice, as well as the protein expression of GLUT5, TREM2, SPP1 and p-SYK. The level of sTREM2 decreased in the plasma of APP/PS1 running mice. The number of microglia, the length and endpoints of microglial processes, and the ratio of GLUT5+/IBA1+ microglia were increased in the dentate gyrus (DG) of APP/PS1 running mice. Running exercise did not alter the number of 5-bromo-2′-deoxyuridine (BrdU)+/IBA1+ microglia but reduced the immunoactivity of CD68 in the hippocampus of APP/PS1 mice.ConclusionsRunning exercise inhibited TREM2 shedding and maintained TREM2 protein levels, which were accompanied by the promotion of brain glucose metabolism, microglial glucose metabolism and morphological plasticity in the hippocampus of AD mice. Microglia might be a structural target responsible for the benefits of running exercise in AD. Promoting microglial glucose metabolism and morphological plasticity modulated by TREM2 might be a novel strategy for AD treatment.

Does Soluble TREM2 Protect Against Alzheimer's Disease?

Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) is a pattern recognition receptor on myeloid cells, and is upregulated on microglia surrounding amyloid plaques in Alzheimer's disease (AD). Rare, heterozygous mutations in TREM2 (e.g., R47H) increase AD risk several fold. TREM2 can be cleaved at the plasma membrane by metalloproteases to release the ectodomain as soluble TREM2 (sTREM2). Wild-type sTREM2 binds oligomeric amyloid beta (Aβ) and acts as an extracellular chaperone, blocking and reversing Aβ oligomerization and fibrillization, and preventing Aβ-induced neuronal loss in vitro. Whereas, R47H sTREM2 increases Aβ fibrillization and neurotoxicity. AD brains expressing R47H TREM2 have more fibrous plaques with more neuritic pathology around these plaques, consistent with R47H sTREM2 promoting Aβ fibrillization relative to WT sTREM2. Brain expression or injection of wild-type sTREM2 reduces pathology in amyloid models of AD in mice, indicating that wild-type sTREM2 is protective against amyloid pathology. Levels of sTREM2 in cerebrospinal fluid (CSF) fall prior to AD, rise in early AD, and fall again in late AD. People with higher sTREM2 levels in CSF progress more slowly into and through AD than do people with lower sTREM2 levels, suggesting that sTREM2 protects against AD. However, some of these experiments can be interpreted as full-length TREM2 protecting rather than sTREM2, and to distinguish between these two possibilities, we need more experiments testing whether sTREM2 itself protects in AD and AD models, and at what stage of disease. If sTREM2 is protective, then treatments could be designed to elevate sTREM2 in AD.

Maprotiline ameliorates isoflurane-induced microglial activation via regulating triggering receptor expressed in myeloid cells 2 (TREM2)

ABSTRACT Isoflurane-induced neurotoxicity has attracted much interest. Recent studies suggest that isoflurane causes microglial activation, resulting in an inflammatory response and microglial insult. Maprotiline is a novel drug that has been licensed as an antidepressant with considerable anti-inflammatory activity. However, it is still unknown whether maprotiline possesses a protective effect against isoflurane-induced microglial insult. Here, we found that maprotiline ameliorated isoflurane-caused reduction in BV2 microglial cell viability and lactate dehydrogenase (LDH) release. Maprotiline mitigated isoflurane-induced oxidative stress by inhibiting reactive oxygen species (ROS) production and increasing superoxide dismutase (SOD) activity. Isoflurane-induced expression and production of inflammatory markers including tumor necrosis factor (TNF-α), interleukin (IL)-1β, cyclooxygenase‐2 (COX-2), and prostaglandin E2 (PGE2) were decreased in maprotiline-treated cells. Maprotiline inhibited the mRNA and protein levels of Iba1, a marker of microglial activation, in isoflurane-induced BV2 cells. Maprotiline treatment restored isoflurane-induced reduction of TREM2 in BV2 microglial cells. In addition, the knockdown of TREM2 abolished the beneficial effects of maprotiline against isoflurane. Collectively, maprotiline exerted protective effects against isoflurane-caused oxidative stress, inflammatory response, and cell injury via regulating TREM2. These findings show that maprotiline prevented the isoflurane-induced microglial activation, indicating that maprotiline might be used as an optimal therapeutic agent for preventing the isoflurane-caused neurotoxicity.

Mitochondrial Dysfunction and Alzheimer's Disease: Role of Microglia.

In 1907, Alois Alzheimer observed, as he quoted, development of “numerous fibers” and “adipose saccules” in the brain of his diseased patient Auguste Deter. The neurodegenerative disease became known as Alzheimer’s disease (AD) and is the most common cause of dementia worldwide. AD normally develops with aging and is mostly initiated because of the imbalance between the formation and clearance of amyloid-β (Aβ). Formation of neurofibrillary tangles (NFTs) of hyperphosphorylated tau is another hallmark of AD. Neuroinflammation plays a significant role in the development and pathology of AD. This chapter explores the role of mitochondrial dysfunction in microglia in case of AD. Mitochondrial oxidative stress in microglia has been linked to the development of AD. Elevated generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential through various mechanisms have been observed in AD. Aβ interacts with microglial receptors, such as triggering receptor expressed in myeloid cells 2 (TREM2), activating downstream pathways causing mitochondrial damage and aggravating inflammation and cytotoxicity. Fibrillar Aβ activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in microglia leading to elevated induction of mitochondrial ROS which further causes neurotoxicity. Elevated ROS in microglia causes activation of inflammatory and cell death pathways. Production of ATP, regulation of mitochondrial health, autophagy, and mitophagy in microglia play significant roles in the AD pathology. Understanding microglial physiology and mitochondrial dysfunction will enable better therapeutic interventions.

Microglia-Related Gene Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) Is Upregulated in the Substantia Nigra of Progressive Supranuclear Palsy.

The role of the microglia-related gene triggering receptor expressed in myeloid cells 2 (TREM2) in primary tauopathies, such as progressive supranuclear palsy (PSP), still remains unclear. The objective of this study was to profile overall and transcript-specific TREM2 expression levels in the substantia nigra (SN) of PSP patients and controls. SN samples from neuropathologically confirmed PSP cases (n = 24) and controls (n = 14) were used to measure TREM2 and TREM2-modulating gene Membrane-spanning 4-domains subfamily A member 4A (MS4A4A) mRNA levels by real-time quantitative polymerase chain reaction. Correlation with hyperphosphorylated tau protein burden was assessed. Overall TREM2 and each of the 3 TREM2 transcripts mRNA levels were significantly increased in the SN of PSP cases versus controls. TREM2 mRNA levels positively correlated with hyperphosphorylated tau burden in SN, specifically in neurons. The MS4A4A gene was also upregulated in PSP patients versus controls. These results add evidence to the involvement of microglia in the disease process of PSP. These findings support the idea that different tauopathies may share common patterns of deregulation in innate immune molecular pathways. © 2020 International Parkinson and Movement Disorder Society.

Caldecrin inhibits lipopolysaccharide-induced pro-inflammatory cytokines and M1 macrophage polarization through the immunoreceptor triggering receptor expressed in myeloid cells-2

Caldecrin was previously isolated as a serum calcium-decreasing factor from the pancreas and is known to suppress receptor activator of nuclear factor-κB ligand (RANKL)-induced calcium oscillation pathways in osteoclasts. Here, we explored the effects of caldecrin on lipopolysaccharide (LPS)–Toll-like receptor-4 (TLR-4) signaling pathways in macrophages. Caldecrin inhibited the LPS-induced gene expression of pro-inflammatory cytokines and M1 macrophage polarization in mouse bone marrow macrophages and the RAW264.7 mouse macrophage cell line. Next, we focused on triggering receptor expressed in myeloid cells-2 (TREM-2) as a co-receptor common to RANKL receptor and TLR-4, and established Trem2-KO RAW264.7 cells, in which Trem2 gene was deleted using the CRISPR/Cas9 system. Caldecrin-mediated alterations in pro-inflammatory cytokine expression and M1 macrophage polarization were not observed in Trem2-KO RAW264.7 cells. These results suggest that caldecrin is not only an inhibitor of osteoclast activation but also a negative regulator of LPS-induced inflammatory responses, functioning via TREM-2.

Microglial Drug Targets in AD: Opportunities and Challenges in Drug Discovery and Development.

Alzheimer’s disease (AD) is a large and increasing unmet medical need with no disease-modifying treatment currently available. Genetic evidence from genome-wide association studies (GWASs) and gene network analysis has clearly revealed a key role of the innate immune system in the brain, of which microglia are the most important element. Single-nucleotide polymorphisms (SNPs) in genes predominantly expressed in microglia have been associated with altered risk of developing AD. Furthermore, microglia-specific pathways are affected on the messenger RNA (mRNA) expression level in post-mortem AD tissue and in mouse models of AD. Together these findings have increased the interest in microglia biology, and numerous scientific reports have proposed microglial molecules and pathways as drug targets for AD. Target identification and validation are generally the first steps in drug discovery. Both target validation and drug lead identification for central nervous system (CNS) targets and diseases entail additional significant obstacles compared to peripheral targets and diseases. This makes CNS drug discovery, even with well-validated targets, challenging. In this article, we will illustrate the special challenges of AD drug discovery by discussing the viability/practicality of possible microglia drug targets including cluster of differentiation 33 (CD33), KCa3.1, kynurenines, ionotropic P2 receptor 7 (P2X7), programmed death-1 (PD-1), Toll-like receptors (TLRs), and triggering receptor expressed in myeloid cells 2 (TREM2).

Microglial inflammation and phagocytosis in Alzheimer's disease: Potential therapeutic targets.

One of the largest unmet medical needs is a disease‐modifying treatment for Alzheimer's disease (AD). Recently, the role of microglia in disease, particularly AD, has gained great interest, following the identification of several disease risk‐associated genes that are highly expressed in microglia. Microglia play a critical homeostatic role in the brain, with neuroinflammatory and phagocytic mechanisms being of particular importance. Here, we review the role of NLRP3, the complement system, and the triggering receptor expressed in myeloid cells 2 (TREM2) in modulating microglial functions. We have reviewed the targets, their molecular pathways and the therapeutic interventions aimed at modulating these targets, in the hope of discovering a novel therapeutic approach for the treatment of AD.Linked ArticlesThis article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc

Effective Knockdown of Gene Expression in Primary Microglia With siRNA and Magnetic Nanoparticles Without Cell Death or Inflammation.

Microglia, the resident immune cells of the brain, have multiple functions in physiological and pathological conditions, including Alzheimer’s disease (AD). The use of primary microglial cell cultures has proved to be a valuable tool to study microglial biology under various conditions. However, more advanced transfection methodologies for primary cultured microglia are still needed, as current methodologies provide low transfection efficiency and induce cell death and/or inflammatory activation of the microglia. Here, we describe an easy, and effective method based on the Glial-Mag method (OZ Biosciences) using magnetic nanoparticles and a magnet to successfully transfect primary microglia cells with different small interfering RNAs (siRNAs). This method does not require specialist facilities or specific training and does not induce cell toxicity or inflammatory activation. We demonstrate that this protocol successfully decreases the expression of two key genes associated with AD, the triggering receptor expressed in myeloid cells 2 (TREM2) and CD33, in primary microglia cell cultures.

Loss of Trem2 in microglia leads to widespread disruption of cell coexpression networks in mouse brain.

Rare heterozygous coding variants in the triggering receptor expressed in myeloid cells 2 (TREM2) gene, conferring increased risk of developing late-onset Alzheimer's disease, have been identified. We examined the transcriptional consequences of the loss of Trem2 in mouse brain to better understand its role in disease using differential expression and coexpression network analysis of Trem2 knockout and wild-type mice. We generated RNA-Seq data from cortex and hippocampus sampled at 4 and 8 months. Using brain cell-type markers and ontology enrichment, we found subnetworks with cell type and/or functional identity. We primarily discovered changes in an endothelial gene-enriched subnetwork at 4 months, including a shift toward a more central role for the amyloid precursor protein gene, coupled with widespread disruption of other cell-type subnetworks, including a subnetwork with neuronal identity. We reveal an unexpected potential role of Trem2 in the homeostasis of endothelial cells that goes beyond its known functions as a microglial receptor and signaling hub, suggesting an underlying link between immune response and vascular disease in dementia.

Erythromyeloid-Derived TREM2: A Major Determinant of Alzheimer's Disease Pathology in Down Syndrome.

Background:Down syndrome (DS; trisomy 21) individuals have a spectrum of hematopoietic and neuronal dysfunctions and by the time they reach the age of 40 years, almost all develop Alzheimer’s disease (AD) neuropathology which includes senile plaques and neurofibrillary tangles. Inflammation and innate immunity are key players in AD and DS. Triggering receptor expressed in myeloid cells-2 (TREM2) variants have been identified as risk factors for AD and other neurodegenerative diseases.Objective:To investigate the effects of TREM2 and the AD-associated R47H mutation on brain pathology and hematopoietic state in AD and DS.Methods:We analyzed peripheral blood, bone marrow, and brain tissue from DS, AD, and age-matched control subjects by immunohistochemistry and western blotting. TREM2-related phagocytosis was investigated using a human myeloid cell line.Results:TREM2 protein levels in brain and sera declined with age and disease progression in DS. We observed soluble TREM2 in brain parenchyma that may be carried by a subset of microglia, macrophages, or exosomes. Two DS cases had the AD-associated TREM2-R47H mutation, which manifested a morphologically extreme phenotype of megakaryocytes and erythrocytes in addition to impaired trafficking of TREM2 to the erythroid membrane. TREM2 was shown to be involved in phagocytosis of red blood cells. TREM2 was seen in early and late endosomes. Silencing TREM2 using siRNA in THP1 cells resulted in significant cell death.Conclusion:We provide evidence that peripheral TREM2 originating from erythromyeloid cells significantly determines AD neuropathology in DS subjects. Understanding the molecular signaling pathways mediated by TREM2 may reveal novel therapeutic targets.

Behavioral and transcriptomic analysis of Trem2-null mice: not all knockout mice are created equal.

It is clear that innate immune system status is altered in numerous neurodegenerative diseases. Human genetic studies have demonstrated that triggering receptor expressed in myeloid cells 2 (TREM2) coding variants have a strong association with Alzheimer’s disease (AD) and other neurodegenerative diseases. To more thoroughly understand the impact of TREM2 in vivo, we studied the behavioral and cognitive functions of wild-type (WT) and Trem2−/− (KO) mice during basal conditions and brain function in the context of innate immune stimulation with peripherally administered lipopolysaccharide (LPS). Early markers of neuroinflammation preceded Aif1 and Trem2 upregulation that occurred at later stages (24–48 h post-LPS). We performed a transcriptomic study of these cohorts and found numerous transcripts and pathways that were altered in Trem2−/− mice both at baseline and 48 h after LPS challenge. Importantly, our transcriptome analysis revealed that our Trem2−/− mouse line (Velocigene allele) results in exaggerated Treml1 upregulation. In contrast, aberrantly high Treml1 expression was absent in the Trem2 knockout line generated by the Colonna lab and the Jackson Labs CRISPR/Cas9 Trem2 knockout line. Notably, removal of the floxed neomycin selection cassette ameliorated aberrant Treml1 expression, validating the artifactual nature of Treml1 expression in the original Trem2−/− Velocigene line. Clearly further studies are needed to decipher whether the Treml1 transcriptional artifact is functionally meaningful, but our data indicate that caution is warranted when interpreting functional studies with this particular line. Additionally, our results indicate that other Velocigene alleles or targeting strategies with strong heterologous promoters need to carefully consider downstream genes.

Expression and processing analyses of wild type and p.R47H TREM2 variant in Alzheimer's disease brains.

BackgroundGenetic analyses showed that the triggering receptor expressed in myeloid cells 2 (TREM2) p.R47H variant increases the risk for Alzheimer’s disease (AD). The question of whether the p.R47H mutation affects expression or function of the receptor remains unanswered. To address this question we quantified mRNA and analyzed protein profiles of WT and p.R47H TREM2 in human brains.MethodsQuantitative real-time PCR (qPCR) was performed using 2 sets of primers one that detects all TREM2 mRNA isoforms and one specific for the alternative spliced isoform (TREM2alt) that encodes for the extracellular domain (soluble TREM2). Because in the brain TREM2 is expressed primarily in microglial cells, we also assessed the levels of IBA1 to control for microglial variability across samples. For TREM2 protein quantitation and N-glycosylation processing, RIPA brain extracts were analyzed by Western blot before and after EndoH and PNGaseF treatments.ResultsWe identified statistically significant increased levels of TREM2 transcripts in the temporal cortex of AD subjects when compared with controls; TREM2alt was likewise higher in AD cases, but was not significant after adjustment for covariates. Quantitative analysis of TREM2 protein confirmed qPCR results that showed higher levels in AD than in control brains. Among AD subjects, we observed a trend towards higher mRNA and protein TREM2 levels in carriers of the p.R47H risk allele. Analysis of individual TREM2 species found no difference in the relative amounts of mature and immature species, and carboxyl terminal fragments between non carriers and p.R47H samples. Furthermore, TREM2 species from either non carriers or p.R47H brains were equally susceptible to EndoH and PNGaseF treatments.ConclusionsOur results suggest that TREM2 expression is increased in AD. Furthermore, we provide evidence indicating that p.R47H mutation does not affect the levels of TREM2 either directly by altering expression or indirectly by affecting processing of the protein. Our data support previous findings that suggest that p.R47H variant affects TREM2 function by altering binding properties of the receptor rather than expression.

ANALYSIS OF WILD TYPE AND R47H MUTANT TREM2 EXPRESSION IN ALZHEIMER'S DISEASE BRAINS

Genetic analyses showed that the triggering receptor expressed in myeloid cells 2 (TREM2) R47H variant increases the risk for AD. In the brain TREM2 is predominantly expressed in microglia. The question of whether the R47H mutation affects expression or function of the receptor remains unanswered. In this study we quantitated mRNA and analyzed protein profiles of WT and R47H TREM2 in human brains to determine if the mutation affects expression or processing of TREM2. There are at least three TREM2 transcripts that are expressed in human brain including one encoding for a soluble form of the receptor. qRT-PCR was performed using 2 sets of primers one that detects all TREM2 mRNA isoforms and one specific for the alternative spliced isoform (TREM2alt) that encodes for the extracellular domain. All analysis were carried out in R software using linear regression adjusting for Age, Sex, APOEε4dose, qRT-PCR plate, RNA integrity number, and expression levels of AIF1 (as a microglia marker). For TREM2 protein quantitation and N-glycosylation processing, RIPA brain extracts were analyzed by Western blot before and after Endo H and PNGAse F treatments. We identified significantly increased levels of TREM2 transcripts in the temporal cortex of AD subjects when compared with age matched controls (p=8.8E-03); TREM2alt was likewise higher in AD cases, but did not reach significance (p=6.9E-02). Among AD subjects (N=41), we observed suggestively higher TREM2 levels in carriers of the R47H risk allele (N=18, p=8.5E-02). Expression levels of TREM2 and TREM2alt were highly correlated in both sets of samples assessed (r2 > 0.72). At the protein level, SDS-gel profiles that included the carboxy-terminal fragment of WT and R47H samples were undistinguishable. Furthermore, TREM2 from either WT or R47H brains was equally susceptible to Endo H and PNGase F treatments. 1) The levels of all TREM2 transcripts are increased in AD. 2) In R47H carriers higher levels of TREM2 transcripts were found, but it failed to reach significance. 3) R47H mutation does not affect processing of TREM2 protein. Our data support previous findings that suggest that the R47H variant affects TREM2 function by altering binding properties of the receptor.

Alzheimer's disease (AD) as a disorder of the plasma membrane

GENERAL COMMENTARY article Front. Physiol., 15 February 2013Sec. Membrane Physiology and Membrane Biophysics https://doi.org/10.3389/fphys.2013.00024

Identification of LRRc17 as a Negative Regulator of Receptor Activator of NF-κB Ligand (RANKL)-induced Osteoclast Differentiation

Osteoblasts are the primary cells responsible for bone formation. They also support osteoclast formation from bone marrow precursors in response to osteotropic factors by inducing receptor activator of NF-kappaB ligand (RANKL) expression and down-regulating osteoprotegerin (OPG) production. In addition to the RANKL-RANK-OPG signaling axis, other factors produced by osteoblasts/stromal cells are involved in osteoclastogenesis. Here, we describe the identification and characterization of leucine-rich repeat-containing 17 (LRRc17), a member of the LRR superfamily that acts as a negative regulator of RANKL-induced osteoclast differentiation. Osteoblasts showed high levels of LRRc17 expression, which was down-regulated in response to the pro-osteoclastogenic factor 1,25-dihydroxyvitamin D(3). Recombinant LRRc17 protein inhibited RANKL-induced osteoclast differentiation from bone marrow precursors, whereas it did not affect the differentiation or activation of macrophages and dendritic cells. These results suggest that among the cell types derived from common myeloid precursors, LRRc17 specifically regulates osteoclasts. Further analysis revealed that LRRc17 attenuated RANKL-induced expression of NFATc1 by blocking phospholipase C-gamma signaling, which, in turn, inhibited RANKL-mediated osteoclast differentiation. Taken together, our results demonstrated a novel inhibitory activity of LRRc17 in RANKL-induced osteoclastogenesis.

TREM2, a DAP12-associated receptor, regulates osteoclast differentiation and function.

Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation. TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor gamma chain (FcRgamma) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption. We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed pre-osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts. Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF. Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.