RESUMO
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.
Assuntos
Doença de Alzheimer , Microglia , Adulto , Humanos , Microglia/metabolismo , Metabolismo dos Lipídeos/genética , Mutação com Perda de Função , Mutação/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptor de Pró-ReninaRESUMO
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.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Basidiomycota/química , Terpenos/farmacologia , Agaricales/química , Doença de Alzheimer/tratamento farmacológico , Humanos , Japão , Estrutura Molecular , Terpenos/isolamento & purificaçãoRESUMO
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).
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Basidiomycota/química , Carpóforos/química , Terpenos/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Concentração Inibidora 50 , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Terpenos/farmacologiaRESUMO
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.
Assuntos
Antivirais/uso terapêutico , Taxa de Filtração Glomerular , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Imidazóis/uso terapêutico , Isoquinolinas/uso terapêutico , Rim/fisiopatologia , Insuficiência Renal Crônica/complicações , Sulfonamidas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antivirais/efeitos adversos , Carbamatos , Quimioterapia Combinada , Feminino , Genótipo , Hepacivirus/genética , Hepatite C Crônica/complicações , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/virologia , Humanos , Imidazóis/efeitos adversos , Isoquinolinas/efeitos adversos , Japão , Estudos Longitudinais , Masculino , Pirrolidinas , RNA Viral/sangue , RNA Viral/genética , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/fisiopatologia , Estudos Retrospectivos , Sulfonamidas/efeitos adversos , Resposta Viral Sustentada , Fatores de Tempo , Resultado do Tratamento , Valina/análogos & derivados , Carga ViralRESUMO
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.
Assuntos
Anilidas/uso terapêutico , Antivirais/uso terapêutico , Carbamatos/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Compostos Macrocíclicos/uso terapêutico , Diálise Renal , Insuficiência Renal Crônica/terapia , Ritonavir/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Anilidas/efeitos adversos , Antivirais/efeitos adversos , Carbamatos/efeitos adversos , Ciclopropanos , Progressão da Doença , Combinação de Medicamentos , Composição de Medicamentos , Feminino , Genótipo , Hepacivirus/genética , Hepatite C/complicações , Hepatite C/diagnóstico , Hepatite C/virologia , Humanos , Japão , Lactamas Macrocíclicas , Cirrose Hepática/diagnóstico , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/virologia , Compostos Macrocíclicos/efeitos adversos , Masculino , Pessoa de Meia-Idade , Prolina/análogos & derivados , RNA Viral/genética , Diálise Renal/efeitos adversos , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/diagnóstico , Ritonavir/efeitos adversos , Sulfonamidas , Resposta Viral Sustentada , Fatores de Tempo , Resultado do Tratamento , Valina , Carga ViralRESUMO
A new compound in cucumber, Cucumis sativus, nutrient solution that appears under iron-deficient conditions, but not under ordinary culture conditions, has been revealed by HPLC analysis. The chemical structure of this compound was identified using LC-MS and NMR techniques as that of 4'-ketoriboflavin. This is the first report to show that 4'-ketoriboflavin can be found in metabolites from organisms.
Assuntos
Cucumis sativus/metabolismo , Deficiências de Ferro , Raízes de Plantas/metabolismo , Riboflavina/metabolismo , Transporte Biológico , Cucumis sativus/efeitos dos fármacos , Meios de Cultura/química , Hidroponia , Ferro/farmacologia , Espectroscopia de Ressonância Magnética , Raízes de Plantas/efeitos dos fármacos , Riboflavina/análogos & derivados , Riboflavina/biossíntese , Estresse FisiológicoRESUMO
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.
Assuntos
Encéfalo/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microglia/metabolismo , Microglia/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Química Encefálica/genética , Proteínas de Ligação ao Cálcio , Linhagem Celular , Sequência Conservada , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Proteínas dos Microfilamentos , Doenças Neurodegenerativas/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Lipodistrofia/metabolismo , Lipodistrofia/patologia , Microglia/metabolismo , Microglia/patologia , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/biossíntese , Panencefalite Esclerosante Subaguda/metabolismo , Panencefalite Esclerosante Subaguda/patologia , Western Blotting , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/análiseRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Bombyx/virologia , Vetores Genéticos/genética , Proteínas de Membrana/biossíntese , Proteínas Mutantes/biossíntese , Nucleopoliedrovírus/genética , Engenharia de Proteínas/métodos , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Nucleopoliedrovírus/fisiologiaRESUMO
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.
Assuntos
Esclerose Múltipla/genética , Receptores de Calcitriol/genética , Vitamina D/genética , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Calcitriol/imunologia , Receptores de Calcitriol/metabolismo , Transdução de Sinais/fisiologia , Vitamina D/imunologia , Vitamina D/metabolismo , Elemento de Resposta à Vitamina D/genética , Elemento de Resposta à Vitamina D/imunologiaRESUMO
A chemical investigation of Digitalis purpurea seeds led to the isolation of three new cardenolide glycosides (1, 8 and 11), together with 12 known cardenolide glycosides (2-7, 9, 10 and 12-15). The structures of 1, 8 and 11 were determined by 1D and 2D NMR spectroscopic analyses and the results of an acid or enzymatic hydrolysis. The cytotoxic activity of the isolated compounds (1-15) against HL-60 leukemia cells was examined. Compounds 2, 9, 11 and 12 showed potent cytotoxicity against HL-60 cells with respective 50% inhibition concentration (IC50) values of 0.060, 0.069, 0.038, and 0.034 µM. Compounds 2, 9 and 11 also exhibited potent cytotoxic activity against HepG2 human liver cancer cells with respective IC50 values of 0.38, 0.79, and 0.71 µM. An investigation of the structure-activity relationship showed that the cytotoxic activity was reduced by the introduction of a hydroxy group at C-16 of the digitoxigenin aglycone, methylation of the C-3' hydroxy group at the fucopyranosyl moiety, and acetylation of the C-3' hydroxy group at the digitoxopyranoyl moiety.
Assuntos
Cardenolídeos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Glicosídeos Digitálicos/farmacologia , Extratos Vegetais/farmacologia , Cardenolídeos/química , Glicosídeos Digitálicos/química , Humanos , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Sementes/químicaRESUMO
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.
RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Lipodistrofia/genética , Lipodistrofia/patologia , Proteínas de Membrana/genética , Monócitos/metabolismo , Monócitos/patologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/patologia , Transcriptoma/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Linhagem Celular Tumoral , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes/métodos , Redes Reguladoras de Genes/genética , Humanos , Lipodistrofia/diagnóstico , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Células Mieloides/metabolismo , Células Mieloides/patologia , Osteocondrodisplasias/diagnóstico , RNA Interferente Pequeno/genética , Panencefalite Esclerosante Subaguda/diagnósticoRESUMO
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.
Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Rim Policístico Autossômico Dominante/complicações , Tuberculose dos Genitais Masculinos/microbiologia , Tuberculose Miliar/microbiologia , Tuberculose Pulmonar/microbiologia , Tuberculose Renal/microbiologia , Adulto , Antituberculosos/uso terapêutico , Técnicas Bacteriológicas , Diagnóstico Tardio , Humanos , Masculino , Nefrectomia , Orquiectomia , Fatores de Tempo , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Tuberculose dos Genitais Masculinos/diagnóstico , Tuberculose dos Genitais Masculinos/terapia , Tuberculose Miliar/diagnóstico , Tuberculose Miliar/terapia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/terapia , Tuberculose Renal/diagnóstico , Tuberculose Renal/terapiaRESUMO
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.
Assuntos
Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipodistrofia/enzimologia , Osteocondrodisplasias/enzimologia , Proteínas Tirosina Quinases/metabolismo , Panencefalite Esclerosante Subaguda/enzimologia , Regulação para Cima/fisiologia , Adulto , Idoso , Encéfalo/metabolismo , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Feminino , Hipocampo/enzimologia , Hipocampo/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Lipodistrofia/patologia , Masculino , Pessoa de Meia-Idade , Osteocondrodisplasias/patologia , Fosforilação/fisiologia , Proteínas Tirosina Quinases/biossíntese , Panencefalite Esclerosante Subaguda/patologia , Quinase SykRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Imunossupressores/farmacologia , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/fisiologia , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Inibidores de Caspase , Linhagem Celular , Cloridrato de Fingolimode , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Imunossupressores/uso terapêutico , Análise em Microsséries , Microglia/citologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Toxina Pertussis/farmacologia , Fosforilação , Propilenoglicóis/uso terapêutico , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/farmacologia , Esfingosina/uso terapêutico , Proteína de Ligação a Elemento Regulador de Esterol 2/genéticaRESUMO
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.
Assuntos
Encéfalo/metabolismo , Lipodistrofia/metabolismo , Microglia/metabolismo , Osteocondrodisplasias/metabolismo , Panencefalite Esclerosante Subaguda/metabolismo , Adulto , Idoso , Proteínas de Ligação ao Cálcio , Biologia Computacional , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Lectinas/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Receptores Imunológicos/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Adulto , Idoso , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Terciária de ProteínaRESUMO
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.