Nasu–Hakola Disease (Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy—PLOSL): A Dementia Associated with Bone Cystic Lesions. From Clinical to Genetic and Molecular Aspects

The authors review the clinical, radiological, electrophysiological, pathological, and molecular aspects of Nasu-Hakola disease (polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy or PLOSL). Nasu-Hakola disease is a unique disease characterized by multiple bone cysts associated with a peculiar form of neurodegeneration that leads to dementia and precocious death usually during the fifth decade of life. The diagnosis can be established on the basis of clinical and radiological findings. Recently, molecular analysis of affected families revealed mutations in the DAP12 (TYROBP) or TREM2 genes, providing an interesting example how mutations in two different subunits of a multi-subunit receptor complex result in an identical human disease phenotype. The association of PLOSL with mutations in the DAP12 or TREM2 genes has led to improved diagnosis of affected individuals. Also, the possible roles of the DAP12/TREM2 signaling pathway in microglia and osteoclasts in humans are just beginning to be elucidated. Some aspects of this peculiar signaling pathway are discussed here.     

TREM2 and β-catenin regulate bone homeostasis by controlling the rate of osteoclastogenesis

TREM2 is an immunoreceptor expressed on osteoclasts (OC) and microglia that transmits intracellular signals through the adaptor DAP12. Individuals with genetic mutations inactivating TREM2 or DAP12 develop the Nasu-Hakola disease (NHD) with cystic-like lesions of the bone and brain demyelination that lead to fractures and presenile dementia. The mechanisms of this disease are poorly understood. In this study, we report that TREM2-deficient mice have an osteopenic phenotype reminiscent of NHD. In vitro, lack of TREM2 impairs proliferation and β-catenin activation in osteoclast precursors (OcP) in response to M-CSF. This defect results in accelerated differentiation of OcP into mature OC. Corroborating the importance of a balanced proliferation and differentiation of OcP for bone homeostasis, we show that conditional deletion of β-catenin in OcP also results in reduced OcP proliferation and accelerated osteoclastogenesis in vitro as well as osteopenia in vivo. These results reveal that TREM2 regulates the rate of osteoclastogenesis and provide a mechanism for the bone pathology in NHD.     

Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as "Nasu-Hakola disease," is a globally distributed recessively inherited disease leading to death during the 5th decade of life and is characterized by early-onset progressive dementia and bone cysts. Elsewhere, we have identified PLOSL mutations in TYROBP (DAP12), which codes for a membrane receptor component in natural-killer and myeloid cells, and also have identified genetic heterogeneity in PLOSL, with some patients carrying no mutations in TYROBP. Here we complete the molecular pathology of PLOSL by identifying TREM2 as the second PLOSL gene. TREM2 forms a receptor signaling complex with TYROBP and triggers activation of the immune responses in macrophages and dendritic cells. Patients with PLOSL have no defects in cell-mediated immunity, suggesting a remarkable capacity of the human immune system to compensate for the inactive TYROBP-mediated activation pathway. Our data imply that the TYROBP-mediated signaling pathway plays a significant role in human brain and bone tissue and provide an interesting example of how mutations in two different subunits of a multisubunit receptor complex result in an identical human disease phenotype.     

TREMs in the immune system and beyond

Triggering receptors expressed by myeloid cells (TREMs) belong to a rapidly expanding family of receptors that include activating and inhibitory isoforms encoded by a gene cluster linked to the MHC. TREM1 and TREM2 activate myeloid cells by signalling through the adaptor protein DAP12. TREM1 triggers phagocyte secretion of pro-inflammatory chemokines and cytokines, amplifying the inflammation that is induced by bacteria and fungi. TREM2 activates monocyte-derived dendritic cells and regulates osteoclast development. Remarkably, TREM2 deficiency leads to a severe disease that is characterized by bone cysts and demyelination of the central nervous system, which results in dementia, implying that the function of TREM2 extends beyond the immune system.     

DAP12 Stabilizes the C-terminal Fragment of the Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) and Protects against LPS-induced Pro-inflammatory Response

Triggering receptor expressed on myeloid cells 2 (TREM2) is a DAP12-associated receptor expressed in microglia, macrophages, and other myeloid-derived cells. Previous studies have suggested that TREM2/DAP12 signaling pathway reduces inflammatory responses and promotes phagocytosis of apoptotic neurons. Recently, TREM2 has been identified as a risk gene for Alzheimer disease (AD). Here, we show that DAP12 stabilizes the C-terminal fragment of TREM2 (TREM2-CTF), a substrate for γ-secretase. Co-expression of DAP12 with TREM2 selectively increased the level of TREM2-CTF with little effects on that of full-length TREM2. The interaction between DAP12 and TREM2 is essential for TREM2-CTF stabilization as a mutant form of DAP12 with disrupted interaction with TREM2 failed to exhibit such an effect. Silencing of either Trem2 or Dap12 gene significantly exacerbated pro-inflammatory responses induced by lipopolysaccharides (LPS). Importantly, overexpression of either full-length TREM2 or TREM2-CTF reduced LPS-induced inflammatory responses. Taken together, our results support a role of DAP12 in stabilizing TREM2-CTF, thereby protecting against excessive pro-inflammatory responses.     

The Triggering Receptor Expressed on Myeloid Cells 2: A Molecular Link of Neuroinflammation and Neurodegenerative Diseases

The triggering receptor expressed on myeloid cells (TREM) 2 is a member of the immunoglobulin superfamily of receptors and mediates signaling in immune cells via engagement of its co-receptor DNAX-activating protein of 12 kDa (DAP12). Homozygous mutations in TREM2 or DAP12 cause Nasu-Hakola disease, which is characterized by bone abnormalities and dementia. Recently, a variant of TREM2 has also been associated with an increased risk for Alzheimer disease. The selective expression of TREM2 on immune cells and its association with different forms of dementia indicate a contribution of this receptor in common pathways of neurodegeneration.     

Disease‐Associated Mutations of TREM2 Alter the Processing of N‐Linked Oligosaccharides in the Golgi Apparatus

The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune‐modulatory receptor involved in phagocytosis and inflammation. Mutations of Q33X, Y38C and T66M cause Nasu‐Hakola disease (NHD) which is characterized by early onset of dementia and bone cysts. A recent, genome‐wide association study also revealed that single nucleotide polymorphism of TREM2, such as R47H, increased the risk of Alzheimer's disease (AD) similar to ApoE4. However, how these mutations affect the trafficking of TREM2, which may affect the normal functions of TREM2, was not known. In this study, we show that TREM2 with NHD mutations are impaired in the glycosylation with complex oligosaccharides in the Golgi apparatus, in the trafficking to plasma membrane and further processing by γ‐secretase. Although R47H mutation in AD affected the glycosylation and normal trafficking of TREM2 less, the detailed pattern of glycosylated TREM2 differs from that of the wild type, thus suggesting that precise regulation of TREM2 glycosylation is impaired when arginine at 47 is mutated to histidine. Our results suggest that the impaired glycosylation and trafficking of TREM2 from endoplasmic reticulum/Golgi to plasma membrane by mutations may inhibit its normal functions in the plasma membrane, which may contribute to the disease.      

Role of TREM1-DAP12 in Renal Inflammation during Obstructive Nephropathy

Tubulo-interstitial damage is a common finding in the chronically diseased kidney and is characterized by ongoing inflammation and fibrosis leading to renal dysfunction and end-stage renal disease. Upon kidney injury, endogenous ligands can be released which are recognized by innate immune sensors to alarm innate immune system. A new family of innate sensors is the family of TREM (triggering receptor expressed on myeloid cell). TREM1 is an activating receptor and requires association with transmembrane adapter molecule DAP12 (DNAX-associated protein 12) for cell signaling. TREM1-DAP12 pathway has a cross-talk with intracellular signaling pathways of several Toll-like receptors (TLRs) and is able to amplify TLR signaling and thereby contributes to the magnitude of inflammation. So far, several studies have shown that TLRs play a role in obstructive nephropathy but the contribution of TREM1-DAP12 herein is unknown. Therefore, we studied TREM1 expression in human and murine progressive renal diseases and further investigated the role for TREM1-DAP12 by subjecting wild-type (WT), TREM1/3 double KO and DAP12 KO mice to murine unilateral ureter obstruction (UUO) model. In patients with hydronephrosis, TREM1 positive cells were observed in renal tissue. We showed that in kidneys from WT mice, DAP12 mRNA and TREM1 mRNA and protein levels were elevated upon UUO. Compared to WT mice, DAP12 KO mice displayed less renal MCP-1, KC and TGF-β1 levels and less influx of macrophages during progression of UUO, whereas TREM1/3 double KO mice displayed less renal MCP-1 level. Renal fibrosis was comparable in WT, TREM1/3 double KO and DAP12 KO mice. We conclude that DAP12, partly through TREM1/3, is involved in renal inflammation during progression of UUO.     

Inactivation of DAP12 in PMN Inhibits TREM1-Mediated Activation in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by dysregulated and chronic systemic inflammatory responses that affect the synovium, bone, and cartilage causing damage to extra-articular tissue. Innate immunity is the first line of defense against invading pathogens and assists in the initiation of adaptive immune responses. Polymorphonuclear cells (PMNs), which include neutrophils, are the largest population of white blood cells in peripheral blood and functionally produce their inflammatory effect through phagocytosis, cytokine production and natural killer-like cytotoxic activity. TREM1 (triggering receptor expressed by myeloid cells) is an inflammatory receptor in PMNs that signals through the use of the intracellular activating adaptor DAP12 to induce downstream signaling. After TREM crosslinking, DAP12’s tyrosines in its ITAM motif get phosphorylated inducing the recruitment of Syk tyrosine kinases and eventual activation of PI3 kinases and ERK signaling pathways. While both TREM1 and DAP12 have been shown to be important activators of RA pathogenesis, their activity in PMNs or the importance of DAP12 as a possible therapeutic target have not been shown. Here we corroborate, using primary RA specimens, that isolated PMNs have an increased proportion of both TREM1 and DAP12 compared to normal healthy control isolated PMNs both at the protein and gene expression levels. This increased expression is highly functional with increased activation of ERK and MAPKs, secretion of IL-8 and RANTES and cytotoxicity of target cells. Importantly, based on our hypothesis of an imbalance of activating and inhibitory signaling in the pathogenesis of RA we demonstrate that inhibition of the DAP12 signaling pathway inactivates these important inflammatory cells.     

The surface-exposed chaperone, Hsp60, is an agonist of the microglial TREM2 receptor

Triggering receptor expressed in myeloid (TREM) cells 2, a receptor expressed by myeloid cells, osteoclasts and microglia, is known to play a protective role in bones and brain. Mutations of the receptor (or of its coupling protein, DAP12) sustain in fact a genetic disease affecting the two organs, the polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (PLOSL or Nasu-Hakola disease). So far, specific agonist(s) of TREM2 have not been identified and its (their) transduction mechanisms are largely unknown. Heat shock protein 60 (Hsp60) is a mitochondrial chaperone that can also be harboured at the cell surface. By using constructs including the extracellular domain of TREM2 and the Fc domain of IgGs we have identified Hsp60 as the only TREM2-binding protein exposed at the surface of neuroblastoma N2A cells and astrocytes, and lacking in U373 astrocytoma. Treatment with Hsp60 was found to stimulate the best known TREM2-dependent process, phagocytosis, however, only in the microglial N9 cells rich in the receptor. Upon TREM2 down-regulation, the Hsp60-induced stimulation of N9 phagocytosis was greatly attenuated. Hsp60 is also released by many cell types, segregated within exosomes or shedding vesicles which might then undergo dissolution. However, the affinity of its binding ( K _d = 3.8 μM) might be too low for the soluble chaperone released from the vesicles to the extracellular space to induce a significant activation of TREM2. It might in contrast be appropriate for the binding of TREM2 to Hsp60 exposed at the surface of cells closely interacting with microglia. The ensuing stimulation of phagocytosis could play protective effects on the brain.     

Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes

We have identified new activating receptors of the Ig superfamily expressed on human myeloid cells, called TREM (triggering receptor expressed on myeloid cells). TREM-1 is selectively expressed on blood neutrophils and a subset of monocytes and is up-regulated by bacterial LPS. Engagement of TREM-1 triggers secretion of IL-8, monocyte chemotactic protein-1, and TNF-alpha and induces neutrophil degranulation. Intracellularly, TREM-1 induces Ca2+ mobilization and tyrosine phosphorylation of extracellular signal-related kinase 1 (ERK1), ERK2 and phospholipase C-gamma. To mediate activation, TREM-1 associates with the transmembrane adapter molecule DAP12. Thus, TREM-1 mediates activation of neutrophil and monocytes, and may have a predominant role in inflammatory responses.     

Apolipoprotein E Is a Ligand for Triggering Receptor Expressed on Myeloid Cells 2 (TREM2)

Several heterozygous missense mutations in the triggering receptor expressed on myeloid cells 2 (TREM2) have recently been linked to risk for a number of neurological disorders including Alzheimer disease (AD), Parkinson disease, and frontotemporal dementia. These discoveries have re-ignited interest in the role of neuroinflammation in the pathogenesis of neurodegenerative diseases. TREM2 is highly expressed in microglia, the resident immune cells of the central nervous system. Along with its adaptor protein, DAP12, TREM2 regulates inflammatory cytokine release and phagocytosis of apoptotic neurons. Here, we report apolipoprotein E (apoE) as a novel ligand for TREM2. Using a biochemical assay, we demonstrated high-affinity binding of apoE to human TREM2. The functional significance of this binding was highlighted by increased phagocytosis of apoE-bound apoptotic N2a cells by primary microglia in a manner that depends on TREM2 expression. Moreover, when the AD-associated TREM2-R47H mutant was used in biochemical assays, apoE binding was vastly reduced. Our data demonstrate that apoE-TREM2 interaction in microglia plays critical roles in modulating phagocytosis of apoE-bound apoptotic neurons and establish a critical link between two proteins whose genes are strongly linked to the risk for AD.     

Cutting edge: inhibition of TLR and FcR responses in macrophages by triggering receptor expressed on myeloid cells (TREM)-2 and DAP12

DAP12 is an ITAM-containing adapter that associates with receptors in myeloid and NK cells. DAP12-associated receptors can give activation signals leading to cytokine production; however, in some situations, DAP12 inhibits cytokine production stimulated through TLRs and FcRs. Here we show that Triggering Receptor Expressed on Myeloid cells (TREM)-2 is responsible for the DAP12-mediated inhibition in mouse macrophages. A chimeric receptor composed of the extracellular domain of TREM-2 and the cytoplasmic domain of DAP12 inhibited the TLR- and FcR-induced TNF production of DAP12-deficient macrophages, whereas a TREM-1 chimera did not. In wild-type macrophages, TREM-2 knockdown increased TLR-induced TNF production. A TREM-2 Fc fusion protein bound to macrophages, indicating that macrophages express a TREM-2 ligand. Thus, the interaction of TREM-2 and its ligand results in an inhibitory signal that can reduce the inflammatory response.     

DAP12 Overexpression Induces Osteopenia and Impaired Early Hematopoiesis

ITAM-bearing transmembrane signaling adaptors such as DAP12 and FcRγ are important players in bone homeostasis, but their precise role and functions are still unknown. It has been shown that osteoclast differentiation results from the integration of the RANK and of the DAP12 and FcRγ signaling pathways. DAP12-deficient mice suffer from a mild osteopetrosis and culture of their bone marrow cells in the presence of M-CSF and RANKL, fails to give rise to multinucleated osteoclasts. Here, we report that mice overexpressing human DAP12 have an osteopenic bone phenotype due to an increased number of osteoclasts on the surface of trabecular and cortical bone. This enhanced number of osteoclasts is associated with an increased number of proliferating myeloid progenitors in Tg-hDAP12 mice. It is concomitant with an arrest of B cell development at the Pre-Pro B/Pre B stage in the bone marrow of Tg-hDAP12 mice and important decrease of follicular and marginal B cells in the spleen of these animals. Our data show that the overexpression of DAP12 results in both increased osteoclastogenesis and impaired hematopoiesis underlining the relationship between bone homeostasis and hematopoiesis.     

TREM2 mRNA Expression in Leukocytes Is Increased in Alzheimer’s Disease and Schizophrenia

TREM2 and TYROBP are causal genes for Nasu–Hakola disease (NHD), a rare autosomal recessive disease characterized by bone lesions and early-onset progressive dementia. TREM2 forms a receptor signaling complex with TYROBP, which triggers the activation of immune responses in macrophages and dendritic cells, and the functional polymorphism of TREM2 is reported to be associated with neurodegenerative disorders such as Alzheimer’s disease (AD). The objective of this study was to reveal the involvement of TYROBP and TREM2 in the pathophysiology of AD and schizophrenia. Methods: We investigated the mRNA expression level of the 2 genes in leukocytes of 26 patients with AD and 24 with schizophrenia in comparison with age-matched controls. Moreover, we performed gene association analysis between these 2 genes and schizophrenia. Results: No differences were found in TYROBP mRNA expression in patients with AD and schizophrenia; however, TREM2 mRNA expression was increased in patients with AD and schizophrenia compared with controls (P < 0.001). There were no genetic associations of either gene with schizophrenia in Japanese patients. Conclusion: TREM2 expression in leukocytes is elevated not only in AD but also in schizophrenia. Inflammatory processes involving TREM2 may occur in schizophrenia, as observed in neurocognitive disorders such as AD. TREM2 expression in leukocytes may be a novel biomarker for neurological and psychiatric disorders.     

Developmental Regulation of TREM2 and DAP12 Expression in the Murine CNS: Implications for Nasu-Hakola Disease

Trem2 is an orphan, DAP12 associated receptor constitutively expressed in vivo by subsets of microglia in the healthy adult murine CNS and in vitro by subsets of oligodendrocytes in neonatal mixed glial cultures. Loss of a functional Trem2 signaling pathway is the genetic cause of Nasu-Hakola disease. Whether the early onset cognitive dementia and myelin-pallor associated with this disorder are due to deficits in functional Trem2 signaling in microglia and/or oligodendrocytes is still being debated. Here, we find that Trem2/DAP12 expression is detected in embryonic day 14 CNS mRNA. Using dual immunohistochemistry/in situ hybridization, we find that both Trem2 and DAP12 expression always co-localized with markers of microglia/macrophages. However, Trem2/DAP12 positive microglia are found in very close apposition with CNP+ oligodendrocytes prior to myelination (post-natal day 1). In addition, CNS expression of TREM2 and DAP12 are not detected in PU.1KO which lack microglia and macrophages. Our data provide continuing support for Nasu-Hakola disease being identified as a cognitive disorder caused by a primary dysfunction of CNS microglia. Special issue article in honor of Dr. George DeVries.     

OH MYeloid! Immune cells wreaking havoc on brain homeostasis

Genetic mutations responsible for neurodegenerative Nasu‐Hakola disease have been localized to the gene TREM2 and its adaptor DAP12, but it remained unclear what causes the brain to deteriorate. In this issue of The EMBO Journal, Kleinberger et al (2017) provide intriguing evidence suggesting a TREM2 mutation alone can lead to striking microglial dysfunction and precipitate changes in cerebral blood flow and metabolism in mice.