Translational regulation and protein-coding capacity of the 5' untranslated region of human TREM2.

TREM2 is a transmembrane receptor expressed in microglia and macrophages. Elevated TREM2 levels in these cells are associated with age-related pathological conditions, including Alzheimer's disease. However, the regulatory mechanism underlying the protein expression of TREM2 remains unclear. In this study, we uncover the role of the 5' untranslated region (5'-UTR) of human TREM2 in translation. An upstream start codon (uAUG) in the 5'-UTR of TREM2 is specific to some primates, including humans. The expression of the conventional TREM2 protein, starting from the downstream AUG (dTREM2), is repressed by the 5'-UTR in a uAUG-mediated manner. We also detect a TREM2 protein isoform starting from uAUG (uTREM2) that is largely degraded by proteasomes. Finally, the 5'-UTR is essential for the downregulation of dTREM2 expression in response to amino acid starvation. Collectively, our study identifies a species-specific regulatory role of the 5'-UTR in TREM2 translation.

Alzheimer's Disease-Associated Alternative Splicing of CD33 Is Regulated by the HNRNPA Family Proteins.

Genetic variations of CD33 have been implicated as a susceptibility factor of Alzheimer's disease (AD). A polymorphism on exon 2 of CD33, rs12459419, affects the alternative splicing of this exon. The minor allele is associated with a reduced risk of AD and promotes the skipping of exon 2 to produce a shorter CD33 isoform lacking the extracellular ligand-binding domain, leading to decreased suppressive signaling on microglial activity. Therefore, factors that regulate the splicing of exon 2 may alter the disease-associated properties of CD33. Herein, we sought to identify the regulatory proteins of CD33 splicing. Using a panel of RNA-binding proteins and a human CD33 minigene, we found that exon 2 skipping of CD33 was promoted by HNRNPA1. Although the knockdown of HNRNPA1 alone did not reduce exon 2 skipping, simultaneous knockdown of HNRNPA1 together with that of HNRNPA2B1 and HNRNPA3 promoted exon 2 inclusion, suggesting functional redundancy among HNRNPA proteins. Similar redundant regulation by HNRNPA proteins was observed in endogenous CD33 of THP-1 and human microglia-like cells. Although mouse Cd33 showed a unique splicing pattern of exon 2, we confirmed that HNRNPA1 promoted the skipping of this exon. Collectively, our results revealed novel regulatory relationships between CD33 and HNRNPA proteins.

Reactive astrocytes express Aggregatin (FAM222A) in the brains of Alzheimer's disease and Nasu-Hakola disease.

By combining genomic data and brain imaging data, a recent study has identified a novel gene named FAM222A that participates in the formation of amyloid-ß (Aß) plaques and brain atrophy in Alzheimer's disease (AD). FAM222A encodes a 47-kDa protein designated Aggregatin that accumulates in the center of amyloid plaques and physically interacts with Aß to facilitate Aß aggregation. Aggregatin is expressed predominantly in the central nervous system (CNS) and its levels are increased in brains of the patients with AD and in mouse models of AD. However, at present, the precise cell types that express Aggregatin in the human CNS remain unknown. By immunohistochemistry, we studied Aggregatin expression in the frontal lobe of the patients with AD, Nasu-Hakola disease (NHD), and the subjects who died of non-neurological causes (NNC). We identified the clusters of Aggregatin-positive reactive astrocytes distributed widely in the cerebral cortex of most cases examined. In contrast, small numbers of cortical neurons showed variable immunoreactivities for Aggregatin, whereas microglia and oligodendrocytes did not express Aggregatin. Importantly, amyloid plaques were not clearly labelled with anti-Aggregatin antibody. These results suggest that Aggregatin plays a primarily role in generation of reactive astrocytes in the human CNS.

CELF2 regulates the species-specific alternative splicing of TREM2.

Genetic variations of TREM2 have been implicated as a risk factor of Alzheimer's disease (AD). Recent studies suggest that the loss of TREM2 function compromises microglial responses to the accumulation of amyloid beta. Previously, we found that exon 3 of TREM2 is an alternative exon whose skipping leads to a reduction in full-length TREM2 protein by inducing nonsense-mediated mRNA decay. Here, we aimed to identify factors regulating TREM2 splicing. Using a panel of RNA-binding proteins, we found that exon 3 skipping of TREM2 was promoted by two paralogous proteins, CELF1 and CELF2, which were both linked previously with risk loci of AD. Although the overexpression of both CELF1 and CELF2 enhanced exon 3 skipping, only CELF2 reduced the expression of full-length TREM2 protein. Notably, the TREM2 ortholog in the green monkey, but not in the mouse, showed alternative splicing of exon 3 like human TREM2. Similarly, splicing regulation of exon 3 by CELF1/2 was found to be common to humans and monkeys. Using chimeric minigenes of human and mouse TREM2, we mapped a CELF-responsive sequence within intron 3 of human TREM2. Collectively, our results revealed a novel regulatory factor of TREM2 expression and highlighted a species-dependent difference of its regulation.

Microglia express GPNMB in the brains of Alzheimer's disease and Nasu-Hakola disease.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein first identified in low-metastatic human melanoma cell lines as a regulator of tumor growth. GPNMB is widely expressed in various tissues, where it is involved in cell differentiation, migration, inflammation/anti-inflammation, tissue regeneration, and neuroprotection. GPNMB is identified in microglia of adult rat brains, neurons and astrocytes of GPNMB transgenic (Tg) mouse brains, and motor neurons of amyotrophic lateral sclerosis (ALS) patients. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of either TYROBP (DAP12) or TREM2. TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Pathologically, the brains of NHD patients exhibit leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and remarkable activation of microglia predominantly in the white matter of frontal and temporal lobes and the basal ganglia. At present, molecular mechanisms responsible for development of leukoencephaolpathy in NHD brains remain totally unknown. Recent evidence indicates that disease-associated microglia (DAM) that cluster around amyloid plaques express high levels of GPNMB in Alzheimer's disease (AD) brains. Because microglia act as a key regulator of leukoencephalopathy in NHD brains, it is proposed that GPNMB expressed on microglia might play a protective role in progression of leukoencephalopathy possibly via active phagocytosis of myelin debris. In the present study using immunohistochemistry, we have attempted to clarify the expression of GPNMB in NHD brains, compared with AD brains. We found that microglia accumulating in the white matter express an intense GPNMB immunoreactivity in both NHD and AD brains, suggesting that the accumulation of GPNMB-immunoreactive microglia is a general phenomenon in neurodegenerative brains.

Microglia express TMEM119 in the brains of Nasu-Hakola disease.

We previously identified an evolutionarily conserved protein named transmembrane protein 119 (TMEM119) as the most reliable maker for human microglia. Recent studies showed that under homeostatic conditions, microglia intensely express TMEM119, whereas the expression levels are greatly reduced in disease-associated microglia (DAM) activated at the site of neurodegeneration. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, pathologically characterized by leukoencephalopathy, astrogliosis, axonal spheroids, and accumulation of microglia. However, it remains unknown whether microglia are homeostatic or activated in NHD brains. In the present study, we identified TMEM119 on microglia in NHD brains by immunohistochemistry. TMEM119 was expressed on microglia in NHD brains as well as in the brains of non-neurological controls (NC) and Alzheimer's disease (AD) patients, although TMEM119-immunolabeled areas exhibited great variability from case to case without significant differences among the study population. These results suggest that TMEM119 expression on microglia might play a key role in steady-state brain maintenance in NHD, AD and controls.

Microglia express gamma-interferon-inducible lysosomal thiol reductase in the brains of Alzheimer's disease and Nasu-Hakola disease.

Gamma-interferon-inducible lysosomal thiol reductase (GILT), expressed in antigen-presenting cells (APCs), facilitates the reduction of disulfide bonds of endocytosed proteins in the endocytic pathway and they are further processed for presentation of immunogenic peptides loaded on major histocompatibility complex (MHC) class II. Although the constitutive and IFNγ-inducible expression of GILT was observed in various APCs, such as dendritic cells, monocytes/macrophages, and B cells, GILT-expressing cell types remain unknown in the human central nervous system (CNS). Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder characterized by sclerosing leukoencephalopathy and multifocal bone cysts, caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2, both of which are expressed on microglia. A rare heterozygous variant of the TREM2 gene encoding p.Arg47His causes a 3-fold increase in the risk for late-onset Alzheimer's disease (LOAD), suggesting that both NHD and AD are induced by dysfunction of the microglial TREM2 signaling pathway in the brains. We studied by immunohistochemistry GILT expression in NHD and AD brains. GILT was expressed on amoeboid microglia with the highest levels of expression in AD brains, compared with those in non-neurological control (NC) brains and in NHD brains. In AD brains, the clusters of amoeboid microglia surrounding amyloid-beta (Aꞵ) deposition strongly expressed GILT. Furthermore, a human microglial cell line expressed GILT in response to IFNγ. These results indicate that microglia, expressing constitutively high levels of GILT, act as a principal cell type of APCs in AD brains, in contrast to baseline levels of GILT expression in NHD brains.

Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation.

Loss-of-function mutations in TREM2 cause Nasu-Hakola disease (NHD), a rare genetic disease characterized by early-onset dementia with leukoencephalopathy and bone cysts. An NHD-associated mutation, c.482 + 2 T > C, disrupts the splice donor site of intron 3 and causes aberrant skipping of exon 3, resulting in the loss of full-length TREM2 protein. Here, we examined the efficacy of artificial U1 and U7 small nuclear RNAs (snRNAs) designed to enhance exon 3 inclusion. Using mutant TREM2 minigenes, we found that some modified U1, but not U7, snRNAs enhanced exon 3 inclusion and restored TREM2 protein expression. Unexpectedly, we found that exon 3 of wild-type TREM2 is an alternative exon, whose skipping leads to reduced expression of the full-length protein. Indeed, TREM2 protein levels were modulated by modified snRNAs that either promoted or repressed exon 3 inclusion. The splice donor site flanking exon 3 was predicted to be weak, which may explain both the alternative splicing of exon 3 under normal conditions and complete exon skipping when the c.482 + 2 T > C mutation was present. Collectively, our snRNA-based approaches provide a potential therapeutic strategy for NHD-associated mis-splicing and novel insights into the post-transcriptional regulation of TREM2.

Alzheimer's disease pathology in Nasu-Hakola disease brains.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of either triggering receptor expressed on myeloid cells 2 (TREM2) or TYRO protein tyrosine kinase binding protein (TYROBP), alternatively named DNAX-activation protein 12 (DAP12), both of which are expressed on microglia in the brain and form the receptor-adaptor complex that chiefly recognizes anionic lipids. TREM2 transmits the signals involved in microglial survival, proliferation, chemotaxis, and phagocytosis. A recent study indicated that a loss of TREM2 function causes greater amounts of amyloid-ß (Aß) deposition in the hippocampus of a mouse model of Alzheimer's disease (AD) owing to a dysfunctional response of microglia to amyloid plaques, suggesting that TREM2 facilitates Aß clearance by microglia. TREM2/DAP12-mediated microglial response limits diffusion and toxicity of amyloid plaques by forming a protective barrier. However, the levels of Aß deposition in postmortem brains of NHD, where the biological function of the TREM2/DAP12 signaling pathway is completely lost, remain to be investigated. By immunohistochemistry, we studied the expression of Aß and phosphorylated tau (p-tau) in the frontal cortex and the hippocampus of five NHD cases. Although we identified several small Aß-immunoreactive spheroids, amyloid plaques were almost undetectable in NHD brains. We found a small number of p-tau-immunoreactive neurofibrillary tangle (NFT)-bearing neurons in NHD brains. Because AD pathology is less evident in NHD than the full-brown AD, it does not play an active role in the development of NHD.

Microglia express ABI3 in the brains of Alzheimer's disease and Nasu-Hakola disease.

Nasu-Hakola disease (NHD) is a rare autosomal recessive leukoencephalopathy caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2 expressed in microglia. A rare variant of the TREM2 gene encoding p.Arg47His causes a 3-fold increase in the risk for late-onset Alzheimer's disease (LOAD). A recent study demonstrated that a rare coding variant p.Ser209Phe in the ABI family member 3 (ABI3) gene, a regulator of actin cytoskeleton organization, confers risk of developing of LOAD, although the pattern of ABI3 expression in AD and NHD brains with relevance to microglial pathology remains to be characterized. We investigated the cell type-specific expression of ABI3 in the brains derived from four non-neurological controls (NC), ten AD and five NHD cases by immunohistochemistry. We identified an intense ABI3 immunoreactivity chiefly on a subset of microglia with ramified or amoeboid morphology located in the grey matter and the white matter of the frontal cortex and the hippocampus of NC, AD, and NHD cases. The immunolabeled area of ABI3-positive microglia was not significantly different among NC, AD, and NHD cases due to great variability from case to case. The clusters of ABI3-immunoreactive microglia were found exclusively in AD brains and they were associated with amyloid plaques. Although these observations do not actively support the view that ABI3-immunoreactive microglia play a central role in the development of leukoencephalopathy in NHD brains and the neurodegeneration in AD brains, the intense expression of ABI3 on microglia might regulate their migration under conditions of health and disease in the central nervous system (CNS).

Expression of GPR17, a regulator of oligodendrocyte differentiation and maturation, in Nasu-Hakola disease brains.

The G protein-coupled receptor 17 (GPR17), a Gi-coupled GPCR, acts as an intrinsic timer of oligodendrocyte differentiation and myelination. The expression of GPR17 is upregulated during differentiation of oligodendrocyte precursor cells (OPCs) into premyelinating oligodendrocytes (preoligodendrocytes), whereas it is markedly downregulated during terminal maturation of myelinating oligodendrocytes. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2. Pathologically, the brains of NHD patients exhibit extensive demyelination designated leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and activation of microglia predominantly in the white matter of frontal and temporal lobes. Although GPR17 is a key regulator of oligodendrogenesis, a pathological role of GPR17 in NHD brains with relevance to development of leukoencephalopathy remains unknown. We studied the expression of GPR17 in five NHD brains and eight control brains by immunohistochemistry. We identified GPR17-immunoreactive preoligodendrocytes with a multipolar ramified morphology distributed in the white matter and the grey matter of all cases examined. However, we did not find statistically significant differences in the number of GPR17-expressing cells between NHD and control brains both in the white matter and the grey matter due to great variability from case to case. These observations do not support the view that GPR17-positive preoligodendrocytes play a central role in the development of leukoencephalopathy in NHD brains.

Targeted sequencing approach to identify genetic mutations in Nasu-Hakola disease.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder characterized by sclerosing leukoencephalopathy and multifocal bone cysts, caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2. TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Premortem molecular diagnosis of NHD requires genetic analysis of both TYROBP and TREM2, in which 20 distinct NHD-causing mutations have been reported. Due to genetic heterogeneity, it is often difficult to identify the exact mutation responsible for NHD. Recently, the revolution of the next-generation sequencing (NGS) technology has greatly advanced the field of genome research. A targeted sequencing approach allows us to investigate a selected set of disease-causing genes and mutations in a number of samples within several days. By targeted sequencing using the TruSight One Sequencing Panel, we resequenced genetic mutations of seven NHD cases with known molecular diagnosis and two control subjects. We identified homozygous variants of TYROBP or TREM2 in all NHD cases, composed of a frameshift mutation of c.141delG in exon 3 of TYROBP in four cases, a missense mutation of c.2T>C in exon 1 of TYROBP in two cases, or a splicing mutation of c.482+2T>C in intron 3 of TREM2 in one case. The results of targeted resequencing corresponded to those of Sanger sequencing. In contrast, causative variants were not detected in control subjects. These results indicate that targeted sequencing is a useful approach to precisely identify genetic mutations responsible for NHD in a comprehensive manner.

Expression of gp91phox and p22phox, catalytic subunits of NADPH oxidase, on microglia in Nasu-Hakola disease brains.

The superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex of phagocytes (phox) plays a key role in production of reactive oxygen species (ROS) by microglia. The catalytic subunits of the NADPH oxidase are composed of p22phox and gp91phox. Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder caused by a loss-of-function mutation of either TYROBP (DAP12) or TREM2. Pathologically, the brains of NHD patients exhibit extensive demyelination designated leukoencephalopathy, astrogliosis, accumulation of axonal spheroids, and remarkable activation of microglia predominantly in the white matter of frontal and temporal lobes. However, a pathological role of the gp91phox-p22phox complex in generation of leukoencephalopathy in NHD remains unknown. We clarified the expression of gp91phox and p22phox in the white matter of the frontal cortex derived from five NHD and eight control subjects. We identified the expression of p22phox and gp91phox immunoreactivity almost exclusively on microglia. Microglia overexpressed gp91phox in NHD brains and p22phox in myotonic dystrophy (MD) brains, when compared with non-neurological control (NC) brains. These results suggest that the enhanced expression of gp91phox by microglia might contribute to overproduction of ROS highly toxic to myelinating oligodendrocytes, resulting in oligodendrocyte cell death that induces leukoencephalopathy in NHD brains.