Triggering Receptor Expressed on Myeloid Cell 2 R47H Exacerbates Immune Response in Alzheimer's Disease Brain.

The R47H variant in the microglial triggering receptor expressed on myeloid cell 2 (TREM2) receptor is a strong risk factor for Alzheimer's disease (AD). To characterize processes affected by R47H, we performed an integrative network analysis of genes expressed in brains of AD patients with R47H, sporadic AD without the variant, and patients with polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), systemic disease with early-onset dementia caused by loss-of-function mutations in TREM2 or its adaptor TYRO protein tyrosine kinase-binding protein (TYROBP). Although sporadic AD had few perturbed microglial and immune genes, TREM2 R47H AD demonstrated upregulation of interferon type I response and pro-inflammatory cytokines accompanied by induction of NKG2D stress ligands. In contrast, PLOSL had distinct sets of highly perturbed immune and microglial genes that included inflammatory mediators, immune signaling, cell adhesion, and phagocytosis. TREM2 knockout (KO) in THP1, a human myeloid cell line that constitutively expresses the TREM2- TYROBP receptor, inhibited response to the viral RNA mimetic poly(I:C) and phagocytosis of amyloid-beta oligomers; overexpression of ectopic TREM2 restored these functions. Compared with wild-type protein, R47H TREM2 had a higher stimulatory effect on the interferon type I response signature. Our findings point to a role of the TREM2 receptor in the control of the interferon type I response in myeloid cells and provide insight regarding the contribution of R47H TREM2 to AD pathology.

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 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.

Adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) and Nasu-Hakola disease: lesion staging and dynamic changes of axons and microglial subsets.

The brains of 10 Japanese patients with adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) encompassing hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) and pigmentary orthochromatic leukodystrophy (POLD) and eight Japanese patients with Nasu-Hakola disease (N-HD) and five age-matched Japanese controls were examined neuropathologically with special reference to lesion staging and dynamic changes of microglial subsets. In both diseases, the pathognomonic neuropathological features included spherically swollen axons (spheroids and globules), axon loss and changes of microglia in the white matter. In ALSP, four lesion stages based on the degree of axon loss were discernible: Stage I, patchy axon loss in the cerebral white matter without atrophy; Stage II, large patchy areas of axon loss with slight atrophy of the cerebral white matter and slight dilatation of the lateral ventricles; Stage III, extensive axon loss in the cerebral white matter and dilatation of the lateral and third ventricles without remarkable axon loss in the brainstem and cerebellum; Stage IV, devastated cerebral white matter with marked dilatation of the ventricles and axon loss in the brainstem and/or cerebellum. Internal capsule and pontine base were relatively well preserved in the N-HD, even at Stage IV, and the swollen axons were larger with a higher density in the ALSP. Microglial cells immunopositive for CD68, CD163 or CD204 were far more obvious in ALSP, than in N-HD, and the shape and density of the cells changed in each stage. With progression of the stage, clinical symptoms became worse to apathetic state, and epilepsy was frequently observed in patients at Stages III and IV in both diseases. From these findings, it is concluded that (i) shape, density and subsets of microglia change dynamically along the passage of stages and (ii) increase of IBA-1-, CD68-, CD163- and CD204-immunopositive cells precedes loss of axons in ALSP.

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.

[Molecular Pathogenesis of Nasu-Hakola Disease Brain Lesions].

Nasu-Hakola disease (NHD) is a rare intractable autosomal recessive disorder, characterized by pathological bone fractures and progressive dementia owing to multifocal bone cysts and leukoencephalopathy, caused by various genetic mutations of either DAP12 or TREM2. Loss-of-function of TREM2-DAP12, constituting a signaling complex on osteoclasts and microglia, plays a central role in the pathogenesis of NHD. Recently, NHD has been recognized as the disease entity designated "microgliopathy". However, at present, TREM2-specific ligands in microglia and the precise molecular mechanism underlying leukoencephalopathy remain to be investigated in order to establish an effective molecular targeted therapy for NHD.

TMEM119 marks a subset of microglia in the human brain.

Microglia are resident myeloid cells of the central nervous system (CNS), activated in the brains of various neurological diseases. Microglia are ontogenetically and functionally distinct from monocyte-derived macrophages that infiltrate the CNS under pathological conditions. However, a lack of specific markers that distinguish resident microglia from circulating blood-derived macrophages in human brain tissues hampers accurate evaluation of microglial contributions to the human brain pathology. By comparative analysis of five comprehensive microglial transcriptome datasets, we identified an evolutionarily conserved protein TMEM119 as the most promising candidate for human microglial markers. TMEM119 was expressed on immortalized human microglia, in which the expression levels were not elevated by exposure to lipopolysaccharide, IFNγ, IL-4, IL-13 or TGFß1. Notably, TMEM119 immunoreactivity was expressed exclusively on a subset of Iba1(+) CD68(+) microglia with ramified and amoeboid morphologies in the brains of neurodegenerative diseases, such as Alzheimer's disease (AD), whereas Iba1(+) CD68(+) infiltrating macrophages do not express TMEM119 in demyelinating lesions of multiple sclerosis and necrotic lesions of cerebral infarction. TMEM119 mRNA levels were elevated in AD brains, although the protein levels were not significantly different between AD and non-AD cases by western blot and morphometric analyses. TMEM119-positive microglia did not consistently express polarized markers for M1 (CD80) or M2 (CD163, CD209) in AD brains. These results suggest that TMEM119 serves as a reliable microglial marker that discriminates resident microglia from blood-derived macrophages in the human brain.

Immunohistochemical characterization of CD33 expression on microglia in Nasu-Hakola disease brains.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by formation of multifocal bone cysts and development of leukoencephalopathy, caused by genetic mutations of either DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). Although increasing evidence suggests a defect in microglial TREM2/DAP12 function in NHD, the molecular mechanism underlying leukoencephalopathy with relevance to microglial dysfunction remains unknown. TREM2, by transmitting signals via the immunoreceptor tyrosine-based activation motif (ITAM) of DAP12, stimulates phagocytic activity of microglia, and ITAM signaling is counterbalanced by sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs)-mediated immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling. To investigate a role of CD33, a member of the Siglecs family acting as a negative regulator of microglia activation, in the pathology of NHD, we studied CD33 expression patterns in five NHD brains and 11 controls by immunohistochemistry. In NHD brains, CD33 was identified exclusively on ramified and amoeboid microglia accumulated in demyelinated white matter lesions but not expressed in astrocytes, oligodendrocytes, or neurons. However, the number of CD33-immunoreactive microglia showed great variability from case to case and from lesion to lesion without significant differences between NHD and control brains. These results do not support the view that CD33-expressing microglia play a central role in the development of leukoencephalopathy in NHD brains.

LC3, an autophagosome marker, is expressed on oligodendrocytes in Nasu-Hakola disease brains.

BACKGROUND: 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 DAP12 or TREM2. TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Neuropathologically, NHD exhibits profound loss of myelin and accumulation of axonal spheroids, accompanied by intense gliosis accentuated in the white matter of the frontal and temporal lobes. At present, the molecular mechanism responsible for development of leukoencephalopathy in NHD brains remains totally unknown. METHODS: By immunohistochemistry, we studied the expression of microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, in 5 NHD and 12 control brains. RESULTS: In all NHD brains, Nogo-A-positive, CNPase-positive oligodendrocytes surviving in the non-demyelinated white matter intensely expressed LC3. They also expressed ubiquitin, ubiquilin-1, and histone deacetylase 6 (HDAC6) but did not express Beclin 1 or sequestosome 1 (p62). Substantial numbers of axonal spheroids were also labeled with LC3 in NHD brains. In contrast, none of oligodendrocytes expressed LC3 in control brains. Furthermore, surviving oligodendrocytes located at the demyelinated lesion edge of multiple sclerosis (MS) did not express LC3, whereas infiltrating Iba1-positive macrophages and microglia intensely expressed LC3 in MS lesions. CONCLUSIONS: These results propose a novel hypothesis that aberrant regulation of autophagy might induce oligodendrogliopathy causative of leukoencephalopathy in NHD brains.

Human astrocytes: secretome profiles of cytokines and chemokines.

Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1), IL-6, IL-8 (CXCL8), MCP-1 (CCL2), MIF and Serpin E1. Following stimulation with IL-1ß and TNF-α, activated astrocytes newly produced IL-1ß, IL-1ra, TNF-α, IP-10 (CXCL10), MIP-1α (CCL3) and RANTES (CCL5), in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.

Molecular network of ChIP-Seq-based NF-κB p65 target genes involves diverse immune functions relevant to the immunopathogenesis of multiple sclerosis.

BACKGROUND: The transcription factor nuclear factor-kappa B (NF-κB) acts as a central regulator of immune response, stress response, cell proliferation, and apoptosis. Aberrant regulation of NF-κB function triggers development of cancers, metabolic diseases, and autoimmune diseases. We attempted to characterize a global picture of the NF-κB target gene network relevant to the immunopathogenesis of multiple sclerosis (MS). METHODS: We identified the comprehensive set of 918 NF-κB p65 binding sites on protein-coding genes from chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) dataset of TNFα-stimulated human B lymphoblastoid cells. The molecular network was studied by a battery of pathway analysis tools of bioinformatics. RESULTS: The GenomeJack genome viewer showed that NF-κB p65 binding sites were accumulated in promoter (35.5%) and intronic (54.9%) regions with an existence of the NF-κB consensus sequence motif. A set of 52 genes (5.7%) corresponded to known NF-κB targets by database search. KEGG, PANTHER, and Ingenuity Pathways Analysis (IPA) revealed that the NF-κB p65 target gene network is linked to regulation of immune functions and oncogenesis, including B cell receptor signaling, T cell activation pathway, Toll-like receptor signaling, and apoptosis signaling, and molecular mechanisms of cancers. KeyMolnet indicated an involvement of the complex crosstalk among core transcription factors in the NF-κB p65 target gene network. Furthermore, the set of NF-κB p65 target genes included 10 genes among 98 MS risk alleles and 49 molecules among 709 MS brain lesion-specific proteins. CONCLUSIONS: These results suggest that aberrant regulation of NF-κB-mediated gene expression, by inducing dysfunction of diverse immune functions, is closely associated with development and progression of MS.

Pathway Analysis of ChIP-Seq-Based NRF1 Target Genes Suggests a Logical Hypothesis of their Involvement in the Pathogenesis of Neurodegenerative Diseases.

Nuclear respiratory factor 1 (NRF1) serves as a transcription factor that activates the expression of a wide range of nuclear genes essential for mitochondrial biogenesis and function, including mitochondrial respiratory complex subunits, heme biosynthetic enzymes, and regulatory factors involved in the replication and transcription of mitochondrial DNA. Increasing evidence indicates that mitochondrial function is severely compromised in the brains of aging-related neurodegenerative diseases. To identify the comprehensive set of human NRF1 target genes potentially relevant to the pathogenesis of neurodegenerative diseases, we analyzed the NRF1 chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) dataset retrieved from the Encyclopedia of DNA Elements (ENCODE) project. Overall, we identified 2,470 highly stringent ChIP-Seq peaks on protein-coding genes in SK-N-SH human neuroblastoma cells. They were accumulated in the proximal promoter regions with an existence of the NRF1-binding consensus sequence. The set of ChIP-Seq-based NRF1 target genes included known NRF1 targets such as EIF2S1, EIF2S2, CYCS, FMR1, FXR2, E2F6, CD47, and TOMM34. By pathway analysis, the molecules located in the core pathways related to mitochondrial respiratory function were determined to be highly enriched in NRF1 target genes. Furthermore, we found that NRF1 target genes play a pivotal role in regulation of extra-mitochondrial biological processes, including RNA metabolism, splicing, cell cycle, DNA damage repair, protein translation initiation, and ubiquitin-mediated protein degradation. We identified a panel of neurodegenerative disease-related genes, such as PARK2 (Parkin), PARK6 (Pink1), PARK7 (DJ-1), and PAELR (GPR37) for Parkinson's disease, as well as PSENEN (Pen2) and MAPT (tau) for Alzheimer's disease, as previously unrecognized NRF1 targets. These results suggest a logical hypothesis that aberrant regulation of NRF1 and its targets might contribute to the pathogenesis of human neurodegenerative diseases via perturbation of diverse mitochondrial and extra-mitochondrial functions.

A Comprehensive Profile of ChIP-Seq-Based STAT1 Target Genes Suggests the Complexity of STAT1-Mediated Gene Regulatory Mechanisms.

Interferon-gamma (IFNγ) plays a key role in macrophage activation, T helper and regulatory cell differentiation, defense against intracellular pathogens, tissue remodeling, and tumor surveillance. The diverse biological functions of IFNγ are mediated by direct activation of signal transducer and activator of transcription 1 (STAT1) as well as numerous downstream effector genes. Because a perturbation in STAT1 target gene networks is closely associated with development of autoimmune diseases and cancers, it is important to characterize the global picture of these networks. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) provides a highly efficient method for genome-wide profiling of DNA-binding proteins. We analyzed the STAT1 ChIP-Seq dataset of IFNγ-stimulated HeLa S3 cells derived from the ENCODE project, along with transcriptome analysis on microarray. We identified 1,441 stringent ChIP-Seq peaks of protein-coding genes. They were located in the promoter (21.5%) and more often in intronic regions (72.2%) with an existence of IFNγ-activated site (GAS) elements. Among the 1,441 STAT1 target genes, 212 genes are known IFN-regulated genes (IRGs) and 194 genes (13.5%) are actually upregulated in response to IFNγ by transcriptome analysis. The panel of upregulated genes constituted IFN-signaling molecular networks pivotal for host defense against infections, where interferon-regulatory factor (IRF) and STAT transcription factors serve as a hub on which biologically important molecular connections concentrate. The genes with the peak location in intronic regions showed significantly lower expression levels in response to IFNγ. These results indicate that the binding of STAT1 to GAS is not sufficient to fully activate target genes, suggesting the high complexity of STAT1-mediated gene regulatory mechanisms.

Molecular network analysis of human microRNA targetome: from cancers to Alzheimer's disease.

MicroRNAs (miRNAs), a class of endogenous small noncoding RNAs, mediate posttranscriptional regulation of protein-coding genes by binding chiefly to the 3' untranslated region of target mRNAs, leading to translational inhibition, mRNA destabilization or degradation. A single miRNA concurrently downregulates hundreds of target mRNAs designated "targetome", and thereby fine-tunes gene expression involved in diverse cellular functions, such as development, differentiation, proliferation, apoptosis and metabolism. Recently, we characterized the molecular network of the whole human miRNA targetome by using bioinformatics tools for analyzing molecular interactions on the comprehensive knowledgebase. We found that the miRNA targetome regulated by an individual miRNA generally constitutes the biological network of functionally-associated molecules in human cells, closely linked to pathological events involved in cancers and neurodegenerative diseases. We also identified a collaborative regulation of gene expression by transcription factors and miRNAs in cancer-associated miRNA targetome networks. This review focuses on the workflow of molecular network analysis of miRNA targetome in silico. We applied the workflow to two representative datasets, composed of miRNA expression profiling of adult T cell leukemia (ATL) and Alzheimer's disease (AD), retrieved from Gene Expression Omnibus (GEO) repository. The results supported the view that miRNAs act as a central regulator of both oncogenesis and neurodegeneration.

Molecular network profiling of U373MG human glioblastoma cells following induction of apoptosis by novel marine-derived anti-cancer 1,2,3,4-tetrahydroisoquinoline alkaloids.

BACKGROUND: Glioblastoma is the most aggressive form of brain tumors showing resistance to treatment with various chemotherapeutic agents. The most effective way to eradicate glioblastoma requires the concurrent inhibition of multiple signaling pathways and target molecules involved in the progression of glioblastoma. Recently, we obtained a series of 1,2,3,4-tetrahydroisoquinoline alkaloids with potent anti-cancer activities, including ecteinascidin-770 (ET-770; the compound 1a) and renieramycin M (RM; the compound 2a) from Thai marine invertebrates, together with a 2'-N-4"-pyridinecarbonyl derivative of ET-770 (the compound 3). We attempted to characterize the molecular pathways responsible for cytotoxic effects of these compounds on a human glioblastoma cell line U373MG. METHODS: We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. RESULTS: All of these compounds induced apoptosis of U373MG cells at nanomolar concentrations. The compound 3 reduced the expression of 417 genes and elevated the levels of 84 genes, while ET-770 downregulated 426 genes and upregulated 45 genes. RM decreased the expression of 274 genes and increased the expression of 9 genes. The set of 196 downregulated genes and 6 upregulated genes showed an overlap among all the compounds, suggesting an existence of the common pathways involved in induction of apoptosis. We identified the ErbB (EGFR) signaling pathway as one of the common pathways enriched in the set of downregulated genes, composed of PTK2, AKT3, and GSK3B serving as key molecules that regulate cell movement and the nervous system development. Furthermore, a GSK3B-specific inhibitor induced apoptosis of U373MG cells, supporting an anti-apoptotic role of GSK3B. CONCLUSION: Molecular network analysis is a useful approach not only to characterize the glioma-relevant pathways but also to identify the network-based effective drug targets.

Disseminated kidney tuberculosis complicating autosomal dominant polycystic kidney disease: a case report.

Mycobacterium tuberculosis infection in patients with autosomal dominant polycystic kidney disease (ADPKD) is rare, and its diagnosis and treatment are difficult because numerous cysts are exposed to infection and antibiotics do not easily penetrate infected cysts. Here, we report the case of a 43-year-old Japanese man with disseminated urogenital tuberculosis (TB) and ADPKD without human immunodeficiency virus (HIV) infection. Delayed diagnosis and ineffective anti-TB chemotherapy worsened his condition. Finally, he underwent bilateral nephrectomy but experienced postoperative complications. In conclusion, kidney TB should be recognized as a cause of renal infection in ADPKD, and surgical treatment should be instituted without delay. The importance of early diagnosis and treatment cannot be overemphasized to prevent kidney TB deterioration.