ABSTRACT
TREM2 is an innate immune receptor expressed by microglia in the adult brain. Genetic variation in the TREM2 gene has been implicated in risk for Alzheimer's disease and frontotemporal dementia, while homozygous TREM2 mutations cause a rare leukodystrophy, Nasu-Hakola disease (NHD). Despite extensive investigation, the role of TREM2 in NHD pathogenesis remains poorly understood. Here, we investigate the mechanisms by which a homozygous stop-gain TREM2 mutation (p.Q33X) contributes to NHD. Induced pluripotent stem cell (iPSC)-derived microglia (iMGLs) were generated from two NHD families: three homozygous TREM2 p.Q33X mutation carriers (termed NHD), two heterozygous mutation carriers, one related non-carrier, and two unrelated non-carriers. Transcriptomic and biochemical analyses revealed that iMGLs from NHD patients exhibited lysosomal dysfunction, downregulation of cholesterol genes, and reduced lipid droplets compared to controls. Also, NHD iMGLs displayed defective activation and HLA antigen presentation. This defective activation and lipid droplet content were restored by enhancing lysosomal biogenesis through mTOR-dependent and independent pathways. Alteration in lysosomal gene expression, such as decreased expression of genes implicated in lysosomal acidification (ATP6AP2) and chaperone mediated autophagy (LAMP2), together with reduction in lipid droplets were also observed in post-mortem brain tissues from NHD patients, thus closely recapitulating in vivo the phenotype observed in iMGLs in vitro. Our study provides the first cellular and molecular evidence that the TREM2 p.Q33X mutation in microglia leads to defects in lysosomal function and that compounds targeting lysosomal biogenesis restore a number of NHD microglial defects. A better understanding of how microglial lipid metabolism and lysosomal machinery are altered in NHD and how these defects impact microglia activation may provide new insights into mechanisms underlying NHD and other neurodegenerative diseases.
Subject(s)
Alzheimer Disease , Microglia , Adult , Humans , Microglia/metabolism , Lipid Metabolism/genetics , Loss of Function Mutation , Mutation/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Lysosomes/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Prorenin ReceptorABSTRACT
To develop drugs to treat Alzheimer's disease (AD) on the basis of the amyloid cascade hypothesis, the amyloid-ß (Aß) aggregation inhibitory activities of 110 extracts from mushrooms were evaluated by thioflavin T (Th-T) assays. The MeOH extract of Albatrellus yasudae inhibited Aß aggregation, and the bioactivity-guided fractionation of the extract afforded four novel meroterpenoids, named scutigeric acid (1), albatrelactone methyl ester (2), albatrelactone (3), and 10',11'-dihydroxygrifolic acid (4), together with two known compounds, grifolin (5) and grifolic acid (6). The structures of 1-4 were elucidated using NMR, MS, UV, IR, and induced ECD spectral data. The structure of 1 was determined as a methyl ester (1a) by 2D NMR spectroscopy. Th-T assays showed that compounds 1-4 and 1a possessed inhibitory activities against Aß aggregation, with IC50 values of 6.6, 40.7, 51.4, 53.3, and 50.3 µM, respectively. Notably, 1 possessed an inhibitory activity against Aß aggregation comparable to that of myricetin as a positive control. Moreover, 1-6 exhibited inhibitory activities against BACE1, with IC50 values of 1.6, 10.9, 10.5, 34.4, 6.1, and 1.4 µM, respectively.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Basidiomycota/chemistry , Terpenes/pharmacology , Agaricales/chemistry , Alzheimer Disease/drug therapy , Humans , Japan , Molecular Structure , Terpenes/isolation & purificationABSTRACT
BACE1 inhibitory activity-guided fractionation of an extract of the fruiting body of Boletinus asiaticus yielded five novel meroterpenoids (1-5) and one known compound (6; asiaticusin A). The structures of these compounds were determined by interpretation of NMR, MS, and IR spectral data. The five new compounds contain 4-hydroxybenzoic acid and geranylgeranoic acid units. Compounds 4-6 possessed BACE1 inhibitory activity (IC50 values: 14.7, 11.4, and 2.0 µM, respectively).
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Basidiomycota/chemistry , Fruiting Bodies, Fungal/chemistry , Terpenes/chemistry , Carbon-13 Magnetic Resonance Spectroscopy , Inhibitory Concentration 50 , Proton Magnetic Resonance Spectroscopy , Spectrometry, Mass, Electrospray Ionization , Terpenes/pharmacologyABSTRACT
AIM: Combination therapy with Daclatasvir (DCV) plus Asunaprevir (ASV) has been proven effective in patients with chronic hepatitis C virus (HCV) infection. However, little is known as to the effect of this therapy in patients with reduced renal function. Focusing on CKD patients whose renal function has declined, the present trial addresses the efficacy and safety of this combination therapy in CKD patients with reduced estimated glomerular filtration rate (eGFR). MATERIALS AND METHODS: The study design is a single-center, retrospective longitudinal observational study enrolling 106 patients with (n = 29) or without (n = 77) CKD. After the treatment with combined DCV with ASV for chronic HCV genotype 1b, patients were followed for a total of 48 weeks and the comparison was made in clinical parameters between the two groups. RESULTS: (1) The majority of patients in both groups achieved sustained virological response at 24 weeks (90.8 % in the non-CKD group, and 93.1 % in the CKD). (2)The reduction rate in HCV-RNA levels 2 weeks after commencing the treatment was faster in the CKD group than that in the non-CKD group (81.8 vs. 79.2 %, p < 0.01). (3) Three patients in the CKD group and 6 patients in the non-CKD group withdrew from the treatment because of the adverse events. CONCLUSION: Combination therapy with DCV plus ASV for chronic HCV genotype 1b infection is useful and tolerable, not only in patients with normal eGFR, but also in those with CKD with declined eGFR. Viral eradication at an early phase of the treatment appears to be faster in CKD patients.
Subject(s)
Antiviral Agents/therapeutic use , Glomerular Filtration Rate , Hepacivirus/drug effects , Hepatitis C, Chronic/drug therapy , Imidazoles/therapeutic use , Isoquinolines/therapeutic use , Kidney/physiopathology , Renal Insufficiency, Chronic/complications , Sulfonamides/therapeutic use , Aged , Aged, 80 and over , Antiviral Agents/adverse effects , Carbamates , Drug Therapy, Combination , Female , Genotype , Hepacivirus/genetics , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/diagnosis , Hepatitis C, Chronic/virology , Humans , Imidazoles/adverse effects , Isoquinolines/adverse effects , Japan , Longitudinal Studies , Male , Pyrrolidines , RNA, Viral/blood , RNA, Viral/genetics , Renal Insufficiency, Chronic/diagnosis , Renal Insufficiency, Chronic/physiopathology , Retrospective Studies , Sulfonamides/adverse effects , Sustained Virologic Response , Time Factors , Treatment Outcome , Valine/analogs & derivatives , Viral LoadABSTRACT
A fixed-dose formula that combines Ombitasvir (OBV), Paritaprevir (PTV) and Ritonavir (RTV) has been launched into the field of anti-HCV therapy in Japan for patients infected with HCV genotypes 1 and 2 in 2015. However, little is yet known as to the efficacy and safety of this novel therapy in patients on maintenance haemodialysis (HD). The present report describes a preliminary experience in 10 patients (five males and five females) who underwent maintenance HD. All of them had HCV genotype 1b, without having the resistance-associated variants at Y93 or L31 in the nonstructural proteins 5A (NS5A) region. After the treatment, eight patients successfully achieved virus eradication and sustained a virological response at 12 weeks (SVR12). In addition, mac-2 binding protein glycosylation isomer (M2BPGi), a biomarker for liver fibrosis, was reduced after the therapy. Two patients withdrew from the therapy due to the development of erythema multiforme and a strong drowsiness, respectively. These results suggest that triple therapy combining OBV, PTV and RTV is effective in achieving SVR12 in most of the HCV-infected patients on HD. In addition, this combination therapy contributed to retard the progression of liver fibrosis. However, we suggest that further trial will be required to establish its clinical efficacy and safety.
Subject(s)
Anilides/therapeutic use , Antiviral Agents/therapeutic use , Carbamates/therapeutic use , Hepacivirus/drug effects , Hepatitis C/drug therapy , Macrocyclic Compounds/therapeutic use , Renal Dialysis , Renal Insufficiency, Chronic/therapy , Ritonavir/therapeutic use , Aged , Aged, 80 and over , Anilides/adverse effects , Antiviral Agents/adverse effects , Carbamates/adverse effects , Cyclopropanes , Disease Progression , Drug Combinations , Drug Compounding , Female , Genotype , Hepacivirus/genetics , Hepatitis C/complications , Hepatitis C/diagnosis , Hepatitis C/virology , Humans , Japan , Lactams, Macrocyclic , Liver Cirrhosis/diagnosis , Liver Cirrhosis/drug therapy , Liver Cirrhosis/virology , Macrocyclic Compounds/adverse effects , Male , Middle Aged , Proline/analogs & derivatives , RNA, Viral/genetics , Renal Dialysis/adverse effects , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/diagnosis , Ritonavir/adverse effects , Sulfonamides , Sustained Virologic Response , Time Factors , Treatment Outcome , Valine , Viral LoadABSTRACT
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.
Subject(s)
Brain/pathology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microglia/metabolism , Microglia/pathology , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Brain Chemistry/genetics , Calcium-Binding Proteins , Cell Line , Conserved Sequence , Cytokines/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Humans , Macrophages/metabolism , Male , Microfilament Proteins , Neurodegenerative Diseases/pathology , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
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.
Subject(s)
Lipodystrophy/metabolism , Lipodystrophy/pathology , Microglia/metabolism , Microglia/pathology , Osteochondrodysplasias/metabolism , Osteochondrodysplasias/pathology , Sialic Acid Binding Ig-like Lectin 3/biosynthesis , Subacute Sclerosing Panencephalitis/metabolism , Subacute Sclerosing Panencephalitis/pathology , Blotting, Western , Female , Humans , Immunohistochemistry , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Sialic Acid Binding Ig-like Lectin 3/analysisABSTRACT
A Japanese patient with Nasu-Hakola disease was found to have a serine-to-asparagine (S39N) substitution in human DNAX-activation protein 12 (DAP12). To elucidate the functional abnormalities of mutant-type DAP12, we expressed wild-type and mutant-type recombinant DAP12 protein with Bombyx mori nucleopolyhedrovirus (BmNPV) vector, and successfully purified the respective proteins from the hemolymph of recombinant BmNPV infected B. mori larvae.
Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , Bombyx/virology , Genetic Vectors/genetics , Membrane Proteins/biosynthesis , Mutant Proteins/biosynthesis , Nucleopolyhedroviruses/genetics , Protein Engineering/methods , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Nucleopolyhedroviruses/physiologyABSTRACT
BACKGROUND: Vitamin D is a liposoluble vitamin essential for calcium metabolism. The ligand-bound vitamin D receptor (VDR), heterodimerized with retinoid X receptor, interacts with vitamin D response elements (VDREs) to regulate gene expression. Vitamin D deficiency due to insufficient sunlight exposure confers an increased risk for multiple sclerosis (MS). OBJECTIVE: To study a protective role of vitamin D in multiple sclerosis (MS), it is important to characterize the global molecular network of VDR target genes (VDRTGs) in immune cells. METHODS: We identified genome-wide VDRTGs collectively from two distinct chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) datasets of VDR-binding sites derived from calcitriol-treated human cells of B cell and monocyte origins. We mapped short reads of next generation sequencing (NGS) data on hg19 with Bowtie, detected the peaks with Model-based Analysis of ChIP-Seq (MACS), and identified genomic locations by GenomeJack, a novel genome viewer for NGS platforms. RESULTS: We found 2997 stringent peaks distributed on protein-coding genes, chiefly located in the promoter and the intron on VDRE DR3 sequences. However, the corresponding transcriptome data verified calcitriol-induced upregulation of only a small set of VDRTGs. The molecular network of 1541 calcitriol-responsive VDRTGs showed a significant relationship with leukocyte transendothelial migration, Fcγ receptor-mediated phagocytosis, and transcriptional regulation by VDR, suggesting a pivotal role of genome-wide VDRTGs in immune regulation. CONCLUSION: These results suggest the working hypothesis that persistent deficiency of vitamin D might perturb the complex network of VDRTGs in immune cells, being responsible for induction of an autoimmune response causative for MS.
Subject(s)
Multiple Sclerosis/genetics , Receptors, Calcitriol/genetics , Vitamin D/genetics , Chromatin Immunoprecipitation , High-Throughput Nucleotide Sequencing , Humans , Multiple Sclerosis/immunology , Multiple Sclerosis/metabolism , Oligonucleotide Array Sequence Analysis , Receptors, Calcitriol/immunology , Receptors, Calcitriol/metabolism , Signal Transduction/physiology , Vitamin D/immunology , Vitamin D/metabolism , Vitamin D Response Element/genetics , Vitamin D Response Element/immunologyABSTRACT
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.
ABSTRACT
Nasu-Hakola disease (NHD), also designated polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, is a rare autosomal recessive disorder characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss-of-function mutation of DAP12 or TREM2. TREM2 and DAP12 constitute a receptor/adaptor complex expressed on osteoclasts, dendritic cells, macrophages, monocytes, and microglia. At present, the precise molecular mechanisms underlying development of leukoencephalopathy and bone cysts in NHD remain largely unknown. We established THP-1 human monocyte clones that stably express small interfering RNA targeting DAP12 for serving as a cellular model of NHD. Genome-wide transcriptome analysis identified a set of 22 genes consistently downregulated in DAP12 knockdown cells. They constituted the molecular network closely related to the network defined by cell-to-cell signaling and interaction, hematological system development and function, and inflammatory response, where NF-κB acts as a central regulator. These results suggest that a molecular defect of DAP12 in human monocytes deregulates the gene network pivotal for maintenance of myeloid cell function in NHD.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Lipodystrophy/genetics , Lipodystrophy/pathology , Membrane Proteins/genetics , Monocytes/metabolism , Monocytes/pathology , Osteochondrodysplasias/genetics , Osteochondrodysplasias/pathology , Subacute Sclerosing Panencephalitis/genetics , Subacute Sclerosing Panencephalitis/pathology , Transcriptome/genetics , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/deficiency , Cell Line, Tumor , Down-Regulation/genetics , Gene Knockdown Techniques/methods , Gene Regulatory Networks/genetics , Humans , Lipodystrophy/diagnosis , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/deficiency , Myeloid Cells/metabolism , Myeloid Cells/pathology , Osteochondrodysplasias/diagnosis , RNA, Small Interfering/genetics , Subacute Sclerosing Panencephalitis/diagnosisABSTRACT
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.
Subject(s)
Mycobacterium tuberculosis/isolation & purification , Polycystic Kidney, Autosomal Dominant/complications , Tuberculosis, Male Genital/microbiology , Tuberculosis, Miliary/microbiology , Tuberculosis, Pulmonary/microbiology , Tuberculosis, Renal/microbiology , Adult , Antitubercular Agents/therapeutic use , Bacteriological Techniques , Delayed Diagnosis , Humans , Male , Nephrectomy , Orchiectomy , Time Factors , Tomography, X-Ray Computed , Treatment Outcome , Tuberculosis, Male Genital/diagnosis , Tuberculosis, Male Genital/therapy , Tuberculosis, Miliary/diagnosis , Tuberculosis, Miliary/therapy , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/therapy , Tuberculosis, Renal/diagnosis , Tuberculosis, Renal/therapyABSTRACT
Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss-of-function mutation of DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adaptor complex on myeloid cells. The post-receptor signals are transmitted via rapid phosphorylation of the immunoreceptor tyrosine-based activating motif (ITAM) of DAP12, mediated by Src protein tyrosine kinases, followed by binding of phosphorylated ITAM to Src homology 2 (SH2) domains of spleen tyrosine kinase (Syk), resulting in autophosphorylation of the activation loop of Syk. To elucidate the molecular mechanism underlying the pathogenesis of NHD, we investigated Syk expression and activation in the frontal cortex and the hippocampus of three NHD and eight control brains by immunohistochemistry. In NHD brains, the majority of neurons expressed intense immunoreactivities for Syk and Y525/Y526-phosphorylated Syk (pSyk) chiefly located in the cytoplasm, while more limited populations of neurons expressed Src. The levels of pSyk expression were elevated significantly in NHD brains compared with control brains. In both NHD and control brains, substantial populations of microglia and macrophages expressed pSyk, while the great majority of reactive astrocytes and myelinating oligodendrocytes did not express pSyk, Syk or Src. These observations indicate that neuronal expression of pSyk was greatly enhanced in the cerebral cortex and the hippocampus of NHD brains, possibly via non-TREM2/DAP12 signaling pathways involved in Syk activation.
Subject(s)
Brain/enzymology , Gene Expression Regulation, Enzymologic , Intracellular Signaling Peptides and Proteins/metabolism , Lipodystrophy/enzymology , Osteochondrodysplasias/enzymology , Protein-Tyrosine Kinases/metabolism , Subacute Sclerosing Panencephalitis/enzymology , Up-Regulation/physiology , Adult , Aged , Brain/metabolism , Cerebral Cortex/enzymology , Cerebral Cortex/pathology , Female , Hippocampus/enzymology , Hippocampus/pathology , Humans , Intracellular Signaling Peptides and Proteins/biosynthesis , Lipodystrophy/pathology , Male , Middle Aged , Osteochondrodysplasias/pathology , Phosphorylation/physiology , Protein-Tyrosine Kinases/biosynthesis , Subacute Sclerosing Panencephalitis/pathology , Syk KinaseABSTRACT
A synthetic analog of sphingosine named FTY720 (Fingolimod), phosphorylated by sphingosine kinase-2, interacts with sphingosine-1-phosphate (S1P) receptors expressed on various cells. FTY720 suppresses the disease activity of multiple sclerosis (MS) chiefly by inhibiting S1P-dependent egress of autoreactive T lymphocytes from secondary lymphoid organs, and possibly by exerting anti-inflammatory and neuroprotective effects directly on brain cells. However, at present, biological effects of FTY720 on human microglia are largely unknown. We studied FTY720-mediated apoptosis of a human microglia cell line HMO6. The exposure of HMO6 cells to non-phosphorylated FTY720 (FTY720-non-P) induced apoptosis in a dose-dependent manner with IC50 of 10.6 ± 2.0 µM, accompanied by the cleavage of caspase-7 and caspase-3 but not of caspase-9. The apoptosis was inhibited by Z-DQMD-FMK, a caspase-3 inhibitor, but not by Pertussis toxin, a Gi protein inhibitor, suramin, a S1P3/S1P5 inhibitor, or W123, a S1P1 competitive antagonist, although HMO6 expressed S1P1, S1P2, and S1P3. Furthermore, both phosphorylated FTY720 (FTY720-P) and SEW2871, S1P1 selective agonists, did not induce apoptosis of HMO6. Genome-wide gene expression profiling and molecular network analysis indicated activation of transcriptional regulation by sterol regulatory element-binding protein (SREBP) in FTY720-non-P-treated HMO6 cells. Western blot verified activation of SREBP2 in these cells, and apoptosis was enhanced by pretreatment with simvastatin, an activator of SREBP2, and by overexpression of the N-terminal fragment of SREBP2. These observations suggest that FTY720-non-P-induced apoptosis of HMO6 human microglia is independent of S1P receptor binding, and positively regulated by the SREBP2-dependent proapoptotic signaling pathway.
Subject(s)
Apoptosis/drug effects , Immunosuppressive Agents/pharmacology , Microglia/drug effects , Microglia/pathology , Microglia/physiology , Propylene Glycols/pharmacology , Sphingosine/analogs & derivatives , Sterol Regulatory Element Binding Protein 2/metabolism , Animals , Caspase 3/metabolism , Caspase 7/metabolism , Caspase Inhibitors , Cell Line , Fingolimod Hydrochloride , Gene Expression Profiling , Gene Regulatory Networks , Humans , Immunosuppressive Agents/therapeutic use , Microarray Analysis , Microglia/cytology , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Pertussis Toxin/pharmacology , Phosphorylation , Propylene Glycols/therapeutic use , Receptors, Lysosphingolipid/antagonists & inhibitors , Receptors, Lysosphingolipid/genetics , Receptors, Lysosphingolipid/metabolism , Sphingosine/pharmacology , Sphingosine/therapeutic use , Sterol Regulatory Element Binding Protein 2/geneticsABSTRACT
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 DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adapter signaling complex expressed on osteoclasts, dendritic cells (DC), macrophages and microglia. Previous studies using knockout mice and mouse brain cell cultures suggest that a loss-of-function of DAP12/TREM2 in microglia plays a central role in the neuropathological manifestation of NHD. However, there exist no immunohistochemical studies that focus attention on microglia in NHD brains. To elucidate a role of microglia in the pathogenesis of NHD, we searched NHD-specific biomarkers and characterized their expression on microglia in NHD brains. Here, we identified allograft inflammatory factor 1 (AIF1, Iba1) and sialic acid binding Ig-like lectin 1 (SIGLEC1) as putative NHD-specific biomarkers by bioinformatics analysis of microarray data of NHD DC. We studied three NHD and eight control brains by immunohistochemistry with a panel of 16 antibodies, including those against Iba1 and SIGLEC1. We verified the absence of DAP12 expression in NHD brains and the expression of DAP12 immunoreactivity on ramified microglia in control brains. Unexpectedly, TREM2 was not expressed on microglia but expressed on a small subset of intravascular monocytes/macrophages in control and NHD brains. In the cortex of NHD brains, we identified accumulation of numerous Iba1-positive microglia to an extent similar to control brains, while SIGLEC1 was undetectable on microglia in all the brains examined. These observations indicate that human microglia in brain tissues do not express TREM2 and DAP12-deficient microglia are preserved in NHD brains, suggesting that the loss of DAP2/TREM2 function in microglia might not be primarily responsible for the neuropathological phenotype of NHD.
Subject(s)
Brain/metabolism , Lipodystrophy/metabolism , Microglia/metabolism , Osteochondrodysplasias/metabolism , Subacute Sclerosing Panencephalitis/metabolism , Adult , Aged , Calcium-Binding Proteins , Computational Biology , DNA-Binding Proteins/metabolism , Female , Humans , Immunohistochemistry , Lectins/metabolism , Male , Membrane Glycoproteins/metabolism , Microfilament Proteins , Middle Aged , Receptors, Immunologic/metabolism , Sialic Acid Binding Immunoglobulin-like LectinsABSTRACT
TAR DNA-binding protein-43 (TDP-43) is a 43-kDa nuclear protein involved in regulation of gene expression. Abnormally, phosphorylated, ubiquitinated, and aggregated TDP-43 constitute a principal component of neuronal and glial cytoplasmic and nuclear inclusions in the brains of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS), although the molecular mechanism that triggers aggregate formation remains unknown. By Western blot analysis using anti-TDP-43 antibodies, we identified a band with an apparent molecular mass of 86-kDa in HEK293, HeLa, and SK-N-SH cells in culture. It was labeled with both N-terminal-specific and C-terminal-specific TDP-43 antibodies, enriched in the cytosolic fraction, and the expression levels were reduced by TDP-43 siRNA but unaltered by treatment with MG-132 or by expression of ubiqulin-1 or casein kinase-1. By immunoprecipitation analysis, we found the interaction between the endogenous full-length TDP-43 and the exogenous Flag-tagged TDP-43, and identified the N-terminal half of TDP-43 spanning amino acid residues 3-183 as an intermolecular interaction domain. When the tagged 86-kDa tandemly connected dimer of TDP-43 was overexpressed in HEK293, it was sequestered in the cytoplasm and promoted an accumulation of high-molecular-mass TDP-43-immunoreactive proteins. Furthermore, the 86-kDa band was identified in the immunoblot of human brain tissues, including those of ALS. These results suggest that the 86-kDa band represents dimerized TDP-43 expressed constitutively in normal cells under physiological conditions.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Protein Multimerization , Protein Structure, Quaternary , Adult , Aged , Animals , Brain/cytology , Brain/metabolism , Cell Line , DNA-Binding Proteins/genetics , Female , Humans , Male , Middle Aged , Protein Structure, TertiaryABSTRACT
Neural stem cells (NSC) with self-renewal and multipotent properties serve as an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. To efficiently induce neuronal lineage cells from NSC for neuron replacement therapy, we should clarify the intrinsic genetic programs involved in a time- and place-specific regulation of human NSC differentiation. Recently, we established an immortalized human NSC clone HB1.F3 to provide an unlimited NSC source applicable to genetic manipulation for cell-based therapy. To investigate a role of neurogenin 1 (Ngn1), a proneural basic helix-loop-helix (bHLH) transcription factor, in human NSC differentiation, we established a clone derived from F3 stably overexpressing Ngn1. Genome-wide gene expression profiling identified 250 upregulated genes and 338 downregulated genes in Ngn1-overexpressing F3 cells (F3-Ngn1) versus wild-type F3 cells (F3-WT). Notably, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a novel stem cell marker, showed an 167-fold increase in F3-Ngn1, although transient overexpression of Ngn1 did not induce upregulation of LGR5, suggesting that LGR5 is not a direct transcriptional target of Ngn1. KeyMolnet, a bioinformatics tool for analyzing molecular relations on a comprehensive knowledgebase, suggests that the molecular network of differentially expressed genes involves the complex interaction of networks regulated by multiple transcription factors. Gene ontology (GO) terms of development and morphogenesis are enriched in upregulated genes, while those of extracellular matrix and adhesion are enriched in downregulated genes. These results suggest that stable expression of a single gene Ngn1 in F3 cells induces not simply neurogenic but multifunctional changes that potentially affect the differentiation of human NSC via a reorganization of complex gene regulatory networks.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Cell Lineage/genetics , Nerve Tissue Proteins/genetics , Neurons/metabolism , Receptors, G-Protein-Coupled/genetics , Stem Cells/metabolism , Biomarkers/metabolism , Cell Line, Transformed , Central Nervous System/embryology , Central Nervous System/growth & development , Central Nervous System/metabolism , Computational Biology , Gene Expression Profiling , Gene Expression Regulation/genetics , Gene Expression Regulation, Developmental/genetics , Gene Regulatory Networks/genetics , Genome-Wide Association Study , Humans , Molecular Biology/methods , Morphogenesis/genetics , Neurons/cytology , Software , Stem Cells/cytology , Transcriptional Activation/genetics , Up-Regulation/geneticsABSTRACT
MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate translational repression of multiple target mRNAs. The miRNAs in a whole cell regulate greater than 30% of all protein-coding genes. The vast majority of presently identified miRNAs are expressed in the brain in a spatially and temporally controlled manner. They play a key role in neuronal development, differentiation, and synaptic plasticity. However, at present, the pathological implications of deregulated miRNA expression in neurodegenerative diseases remain largely unknown. This review will briefly summarize recent studies that focus attention on aberrant miRNA expression in Alzheimer's disease brains.
Subject(s)
Alzheimer Disease/metabolism , Brain/metabolism , Gene Expression Regulation/physiology , MicroRNAs/metabolism , Neurodegenerative Diseases/metabolism , Alzheimer Disease/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , MicroRNAs/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/genetics , Neurons/metabolism , Neurons/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Nogo-A, a neurite outgrowth inhibitor, is expressed exclusively on oligodendrocytes and neurons in the CNS. The central domain of Amino-Nogo spanning amino acids 567-748 in the human Nogo-A designated NIG, mediates persistent inhibition of axonal outgrowth and induces growth cone collapse by signaling through an as yet unidentified NIG receptor. We identified 82 NIG-interacting proteins by screening a high-density human protein microarray composed of 5000 proteins with a recombinant NIG protein as a probe. Following an intensive database search, we selected 12 neuron/oligodendrocyte-associated NIG interactors. Among them, we verified the molecular interaction of NIG with 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a cell type-specific marker of oligodendrocytes, by immunoprecipitation and cell imaging analysis. Although CNP located chiefly in the cytoplasm of oligodendrocytes might not serve as a cell-surface NIG receptor, it could act as a conformational stabilizer for the intrinsically unstructured large segment of Amino-Nogo.