RESUMO
INTRODUCTION: Despite improved management of traumatic brain injury (TBI), it still leads to lifelong sequelae and disability, particularly in children. Chronic neuroinflammation (the so-called tertiary phase), in particular, microglia/macrophage and astrocyte reactivity, is among the main mechanisms suspected of playing a role in the generation of lesions associated with TBI. The role of acute neuroinflammation is now well understood, but its persistent effect and impact on the brain, particularly during development, are not. Here, we investigated the long-term effects of pediatric TBI on the brain in a mouse model. METHODS: Pediatric TBI was induced in mice on postnatal day (P) 7 by weight-drop trauma. The time course of neuroinflammation and myelination was examined in the TBI mice. They were also assessed by magnetic resonance, functional ultrasound, and behavioral tests at P45. RESULTS: TBI induced robust neuroinflammation, characterized by acute microglia/macrophage and astrocyte reactivity. The long-term consequences of pediatric TBI studied on P45 involved localized scarring astrogliosis, persistent microgliosis associated with a specific transcriptomic signature, and a long-lasting myelination defect consisting of the loss of myelinated axons, a decreased level of myelin binding protein, and severe thinning of the corpus callosum. These results were confirmed by reduced fractional anisotropy, measured by diffusion tensor imaging, and altered inter- and intra-hemispheric connectivity, measured by functional ultrasound imaging. In addition, adolescent mice with pediatric TBI showed persistent social interaction deficits and signs of anxiety and depressive behaviors. CONCLUSIONS: We show that pediatric TBI induces tertiary neuroinflammatory processes associated with white matter lesions and altered behavior. These results support our model as a model for preclinical studies for tertiary lesions following TBI.
Assuntos
Lesões Encefálicas Traumáticas , Encéfalo , Modelos Animais de Doenças , Doenças Neuroinflamatórias , Animais , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/metabolismo , Camundongos , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/etiologia , Masculino , Encéfalo/metabolismo , Encéfalo/patologia , Astrócitos/metabolismo , Microglia/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Feminino , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Corpo Caloso/diagnóstico por imagem , Inflamação/metabolismo , Imagem de Tensor de Difusão/métodosRESUMO
During transcription, proteins assemble sequentially with nascent RNA to generate a messenger ribonucleoprotein particle (mRNP). The THO complex and its associated Sub2p helicase are functionally implicated in both transcription and mRNP biogenesis but their precise function remains elusive. We show here that THO/Sub2p mutation leads to the accumulation of a stalled intermediate in mRNP biogenesis that contains nuclear pore components and polyadenylation factors in association with chromatin. Microarray analyses of genomic loci that are aberrantly docked to the nuclear pore in mutants allowed the identification of approximately 400 novel validated target genes that require THO /Sub2p for efficient expression. Our data strongly suggests that the THO complex/Sub2p function is required to coordinate events leading to the acquisition of export competence at a step that follows commitment to 3'-processing.
Assuntos
Adenosina Trifosfatases/metabolismo , Poro Nuclear/metabolismo , Processamento de Terminações 3' de RNA , Transporte de RNA , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Adenosina Trifosfatases/genética , Cromatina/metabolismo , Proteínas de Choque Térmico/genética , Mutação , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Preterm infants often show pathologies of the cerebellum, which are associated with impaired motor performance, lower IQ and poor language skills at school ages. Using a mouse model of inflammation-induced encephalopathy of prematurity driven by systemic administration of pro-inflammatory IL-1ß, we sought to uncover causes of cerebellar damage. In this model, IL-1ß is administered between postnatal day (P) 1 to day 5, a timing equivalent to the last trimester for brain development in humans. Structural MRI analysis revealed that systemic IL-1ß treatment induced specific reductions in gray and white matter volumes of the mouse cerebellar lobules I and II (5% false discovery rate [FDR]) from P15 onwards. Preceding these MRI-detectable cerebellar volume changes, we observed damage to oligodendroglia, with reduced proliferation of OLIG2+ cells at P10 and reduced levels of the myelin proteins myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) at P10 and P15. Increased density of IBA1+ cerebellar microglia were observed both at P5 and P45, with evidence for increased microglial proliferation at P5 and P10. Comparison of the transcriptome of microglia isolated from P5 cerebellums and cerebrums revealed significant enrichment of pro-inflammatory markers in microglia from both regions, but cerebellar microglia displayed a unique type I interferon signaling dysregulation. Collectively, these data suggest that perinatal inflammation driven by systemic IL-1ß leads to specific cerebellar volume deficits, which likely reflect oligodendrocyte pathology downstream of microglial activation. Further studies are now required to confirm the potential of protective strategies aimed at preventing sustained type I interferon signaling driven by cerebellar microglia as an important therapeutic target.
Assuntos
Doenças Cerebelares , Doenças do Prematuro , Inflamação , Interferon Tipo I , Interleucina-1beta , Microglia , Animais , Encefalopatias/induzido quimicamente , Encefalopatias/imunologia , Encefalopatias/patologia , Doenças Cerebelares/induzido quimicamente , Doenças Cerebelares/imunologia , Doenças Cerebelares/patologia , Cerebelo/efeitos dos fármacos , Cerebelo/imunologia , Cerebelo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/induzido quimicamente , Doenças do Prematuro/imunologia , Doenças do Prematuro/patologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Interferon Tipo I/imunologia , Interleucina-1beta/efeitos adversos , Interleucina-1beta/farmacologia , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/patologia , GravidezRESUMO
OBJECTIVES: Altered enteroendocrine cell (EEC) function in obesity and type 2 diabetes is not fully understood. Understanding the transcriptional program that controls EEC differentiation is important because some EEC types harbor significant therapeutic potential for type 2 diabetes. METHODS: EEC isolation from jejunum of obese individuals with (ObD) or without (Ob) type 2 diabetes was obtained with a new method of cell sorting. EEC transcriptional profiles were established by RNA-sequencing in a first group of 14 Ob and 13 ObD individuals. EEC lineage and densities were studied in the jejunum of a second independent group of 37 Ob, 21 ObD and 22 non obese (NOb) individuals. RESULTS: The RNA seq analysis revealed a distinctive transcriptomic signature and a decreased differentiation program in isolated EEC from ObD compared to Ob individuals. In the second independent group of ObD, Ob and NOb individuals a decreased GLP-1 cell lineage and GLP-1 maturation from proglucagon, were observed in ObD compared to Ob individuals. Furthermore, jejunal density of GLP-1-positive cells was significantly reduced in ObD compared to Ob individuals. CONCLUSIONS: These results highlight that the transcriptomic signature of EEC discriminate obese subjects according to their diabetic status. Furthermore, type 2 diabetes is associated with reduced GLP-1 cell differentiation and proglucagon maturation leading to low GLP-1-cell density in human obesity. These mechanisms could account for the decrease plasma GLP-1 observed in metabolic diseases.
Assuntos
Diabetes Mellitus Tipo 2 , Células Enteroendócrinas/metabolismo , Jejuno/citologia , Obesidade , Adulto , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/metabolismo , Células Enteroendócrinas/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/epidemiologia , Obesidade/metabolismoRESUMO
Ecological speciation entails divergent selection on specific traits and ultimately on the developmental pathways responsible for these traits. Selection can act on gene sequences but also on regulatory regions responsible for gene expression. Mimetic butterflies are a relevant system for speciation studies because wing colour pattern (WCP) often diverges between closely related taxa and is thought to drive speciation through assortative mating and increased predation on hybrids. Here, we generate the first transcriptomic resources for a mimetic butterfly of the tribe Ithomiini, Melinaea marsaeus, to examine patterns of differential expression between two subspecies and between tissues that express traits that likely drive reproductive isolation; WCP and chemosensory genes. We sequenced whole transcriptomes of three life stages to cover a large catalogue of transcripts, and we investigated differential expression between subspecies in pupal wing discs and antennae. Eighteen known WCP genes were expressed in wing discs and 115 chemosensory genes were expressed in antennae, with a remarkable diversity of chemosensory protein genes. Many transcripts were differentially expressed between subspecies, including two WCP genes and one odorant receptor. Our results suggest that in M. marsaeus the same genes as in other mimetic butterflies are involved in traits causing reproductive isolation, and point at possible candidates for the differences in those traits between subspecies. Differential expression analyses of other developmental stages and body organs and functional studies are needed to confirm and expand these results. Our work provides key resources for comparative genomics in mimetic butterflies, and more generally in Lepidoptera.
Assuntos
Borboletas , Animais , Borboletas/genética , Perfilação da Expressão Gênica , Isolamento Reprodutivo , Transcriptoma , Asas de AnimaisRESUMO
The choroid plexus is an important blood barrier that secretes cerebrospinal fluid, which essential for embryonic brain development and adult brain homeostasis. The OTX2 homeoprotein is a transcription factor that is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative functional roles for OTX2 in adult choroid plexus function, including cell signaling and adhesion, and show that OTX2 regulates the expression of factors that are secreted into the cerebrospinal fluid, notably transthyretin. We also show that Otx2 expression impacts choroid plexus immune and stress responses, and affects splicing, leading to changes in the mRNA isoforms of proteins that are implicated in the oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell-autonomous target regions, such as the visual cortex and subventricular zone, we identify putative targets that are involved in cell adhesion, chromatin structure, and RNA processing. Thus, OTX2 retains important roles for regulating choroid plexus function and brain homeostasis throughout life.
Assuntos
Encéfalo/fisiologia , Plexo Corióideo/metabolismo , Regulação da Expressão Gênica , Homeostase , Ventrículos Laterais/metabolismo , Fatores de Transcrição Otx/fisiologia , Córtex Visual/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , TranscriptomaRESUMO
The tentacular system of Clytia hemisphaerica medusa (Cnidaria, Hydrozoa) has recently emerged as a promising experimental model to tackle the developmental mechanisms that regulate cell lineage progression in an early-diverging animal phylum. From a population of proximal stem cells, the successive steps of tentacle stinging cell (nematocyte) elaboration, are spatially ordered along a "cellular conveyor belt". Furthermore, the C. hemisphaerica tentacular system exhibits bilateral organisation, with two perpendicular polarity axes (proximo-distal and oral-aboral). We aimed to improve our knowledge of this cellular system by combining RNAseq-based differential gene expression analyses and expression studies of Wnt signalling genes. RNAseq comparisons of gene expression levels were performed (i) between the tentacular system and a control medusa deprived of all tentacles, nematogenic sites and gonads, and (ii) between three samples staggered along the cellular conveyor belt. The behaviour in these differential expression analyses of two reference gene sets (stem cell genes; nematocyte genes), as well as the relative representations of selected gene ontology categories, support the validity of the cellular conveyor belt model. Expression patterns obtained by in situ hybridisation for selected highly differentially expressed genes and for Wnt signalling genes are largely consistent with the results from RNAseq. Wnt signalling genes exhibit complex spatial deployment along both polarity axes of the tentacular system, with the Wnt/ß-catenin pathway probably acting along the oral-aboral axis rather than the proximo-distal axis. These findings reinforce the idea that, despite overall radial symmetry, cnidarians have a full potential for elaboration of bilateral structures based on finely orchestrated deployment of an ancient developmental gene toolkit.
Assuntos
Padronização Corporal/genética , Hidrozoários/genética , Via de Sinalização Wnt/genética , Animais , Biologia do Desenvolvimento/métodos , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hidrozoários/metabolismoRESUMO
BACKGROUND: The degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei. Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30. RESULTS: Experimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to i) a positive correlation between lactose concentration and cellulase production, ii) a particular dependence of the lactose onto the ß-glucosidase regulation and iii) a negative regulation of the development process and growth. CONCLUSIONS: This work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1, clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the ß-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to better cellulase-producing strains in industry-like conditions.
Assuntos
Celulase , Trichoderma , Celulase/genética , Redes Reguladoras de Genes , Glucose , Hypocreales , Lactose , Trichoderma/genéticaRESUMO
BACKGROUND: Alternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells. RESULTS: ONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build "splice trees", a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation. CONCLUSION: ONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells.
Assuntos
Processamento Alternativo/genética , Infecções por HIV/virologia , HIV-1/genética , RNA Viral/genética , Linfócitos T CD4-Positivos/virologia , Regulação Viral da Expressão Gênica , Humanos , Sequenciamento por Nanoporos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sítios de Splice de RNA , RNA Viral/metabolismo , Transcriptoma , Vírion/genéticaRESUMO
In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.
Assuntos
Epigênese Genética/genética , Genoma/genética , Padrões de Herança/genética , Paramecium tetraurellia/genética , RNA Interferente Pequeno/genética , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica , Genes/genética , Dados de Sequência Molecular , Paramecium tetraurellia/fisiologia , Regiões Promotoras Genéticas/genética , Reprodução/genética , Reprodução/fisiologia , Deleção de Sequência/genéticaRESUMO
The non-cell autonomous transfer of OTX2 homeoprotein transcription factor into juvenile mouse cerebral cortex regulates parvalbumin interneuron maturation and critical period timing. By analyzing gene expression in primary visual cortex of wild-type and Otx2+/GFP mice at plastic and nonplastic ages, we identified several putative genes implicated in Otx2-dependent visual cortex plasticity for ocular dominance. Cortical OTX2 infusion in juvenile mice induced Gadd45b/g expression through direct regulation of transcription. Intriguingly, a reverse effect was found in the adult, where reducing cortical OTX2 resulted in Gadd45b/g upregulation. Viral expression of Gadd45b in adult visual cortex directly induced ocular dominance plasticity with concomitant changes in MeCP2 foci within parvalbumin interneurons and in methylation states of several plasticity gene promoters, suggesting epigenetic regulation. This interaction provides a molecular mechanism for OTX2 to trigger critical period plasticity yet suppress adult plasticity.
Assuntos
Antígenos de Diferenciação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Plasticidade Neuronal/fisiologia , Fatores de Transcrição Otx/metabolismo , Córtex Visual/fisiologia , Animais , Dominância Ocular/fisiologia , Epigênese Genética , Regulação da Expressão Gênica , Interneurônios/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Parvalbuminas/metabolismoRESUMO
Lipid homeostasis is dysregulated in some neurodegenerative diseases and after brain injuries due to excess glutamate or lack of oxygen. However the kinetics and cell specificity of dysregulation in different groups of lipids during excitotoxic neuronal death are not clear. Here we examined the changes during excitotoxic neuronal death induced by injecting kainic acid (KA) into the CA1 region of mouse hippocampus. We compared neuronal loss and glial cell proliferation with changes in lipid-related transcripts and markers for different lipid groups, over 12 days after KA-treatment. As neurons showed initial signs of damage, transcripts and proteins linked to fatty acid oxidation were up-regulated. Cholesterol biosynthesis induced by transcripts controlled by the transcription factor Srebp2 seems to be responsible for a transient increase in neuronal free cholesterol at 1 to 2 days. In microglia, but not in neurons, Perilipin-2 associated lipid droplets were induced and properties of Nile red emissions suggest lipid contents change over time. After microglial expression of phagocytotic markers at 2 days, some neutral lipid deposits co-localized with lysosome markers of microglia and were detected within putative phagocytotic cups. These data delineate distinct lipid signals in neurons and glial cells during excitotoxic processes from initial neuronal damage to engagement of the lysosome-phagosome system.
Assuntos
Região CA1 Hipocampal/metabolismo , Perfilação da Expressão Gênica , Ácido Caínico/farmacologia , Gotículas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Microglia/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Animais , Biomarcadores/metabolismo , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colesterol/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microscopia Eletrônica , Microscopia de Fluorescência por Excitação Multifotônica , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Neurônios/efeitos dos fármacos , Regulação para CimaRESUMO
Microglia, the immune cells of the brain, are highly plastic and possess multiple functional phenotypes. Differences in phenotype in different regions and different states of epileptic human brain have been little studied. Here we use transcriptomics, anatomy, imaging of living cells and ELISA measurements of cytokine release to examine microglia from patients with temporal lobe epilepsies. Two distinct microglial phenotypes were explored. First we asked how microglial phenotype differs between regions of high and low neuronal loss in the same brain. Second, we asked how microglial phenotype is changed by a recent seizure. In sclerotic areas with few neurons, microglia have an amoeboid rather than ramified shape, express activation markers and respond faster to purinergic stimuli. The repairing interleukin, IL-10, regulates the basal phenotype of microglia in the CA1 and CA3 regions with neuronal loss and gliosis. To understand changes in phenotype induced by a seizure, we estimated the delay from the last seizure until tissue collection from changes in reads for immediate early gene transcripts. Pseudotime ordering of these data was validated by comparison with results from kainate-treated mice. It revealed a local and transient phenotype in which microglia secrete the human interleukin CXCL8, IL-1B and other cytokines. This secretory response is mediated in part via the NRLP3 inflammasome.
Assuntos
Encéfalo/imunologia , Encéfalo/patologia , Epilepsia do Lobo Temporal/imunologia , Epilepsia do Lobo Temporal/patologia , Microglia/patologia , Adulto , Idoso , Animais , Epilepsia do Lobo Temporal/metabolismo , Feminino , Humanos , Interleucina-10/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fenótipo , Transcriptoma , Adulto JovemRESUMO
In the normal brain, immune cell trafficking and immune responses are strictly controlled and limited. This unique homeostatic equilibrium, also called brain immune quiescence, is crucial to maintaining proper brain functions and is altered in various pathological processes, from chronic immunopathological disorders to cognitive and psychiatric impairments. To date, the precise nature of factors regulating the brain/immune system interrelationship is poorly understood. In the present study, we demonstrate that one of these regulating factors is Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the blood-brain barrier (BBB) interface. We show that, by setting the activated state of cerebral endothelium, astroglial Cx43 controls immune recruitment as well as antigen presentation mechanisms in the mouse brain. Consequently, in the absence of astroglial Cx43, recruited immune cells elaborate a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein of the brain. Altogether, our results demonstrate that Cx43 is a new astroglial factor promoting the immune quiescence of the brain.
Assuntos
Astrócitos/metabolismo , Encéfalo/citologia , Encéfalo/imunologia , Conexina 43/metabolismo , Citocinas/metabolismo , Imunidade Humoral/fisiologia , Leucócitos/fisiologia , Fatores Etários , Albuminas/metabolismo , Animais , Astrócitos/ultraestrutura , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/ultraestrutura , Complexo CD3/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Isótopos de Carbono/farmacocinética , Movimento Celular/genética , Células Cultivadas , Conexina 43/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Proteína Glial Fibrilar Ácida , Imunidade Humoral/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Sacarose/farmacocinéticaRESUMO
ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Regulação Fúngica da Expressão Gênica , Transativadores/metabolismoRESUMO
Thyroid hormones are involved in many biological processes such as neurogenesis, metabolism, and development. However, compounds called endocrine disruptors can alter thyroid hormone signaling and induce unwanted effects on human and ecosystems health. Regulatory tests have been developed to detect these compounds but need to be significantly improved by proposing novel endpoints and key events. The Xenopus Eleutheroembryonic Thyroid Assay (XETA, OECD test guideline no. 248) is one such test. It is based on Xenopus laevis tadpoles, a particularly sensitive model system for studying the physiology and disruption of thyroid hormone signaling: amphibian metamorphosis is a spectacular (thus easy to monitor) life cycle transition governed by thyroid hormones. With a long-term objective of providing novel molecular markers under XETA settings, we propose first to describe the differential effects of thyroid hormones on gene expression, which, surprisingly, are not known. After thyroid hormones exposure (T3 or T4), whole tadpole RNAs were subjected to transcriptomic analysis. By using standard approaches coupled to system biology, we found similar effects of the two thyroid hormones. They impact the cell cycle and promote the expression of genes involves in cell proliferation. At the level of the whole tadpole, the immune system is also a prime target of thyroid hormone action.
Assuntos
Ecossistema , Hormônios Tireóideos , Animais , Humanos , Xenopus laevis/metabolismo , Hormônios Tireóideos/metabolismo , Glândula Tireoide/metabolismo , Proliferação de CélulasRESUMO
Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.
Assuntos
Antivirais , Decitabina , Infecções por Herpesviridae , Herpesvirus Equídeo 4 , Doenças dos Cavalos , Imunidade Inata , Animais , Antivirais/farmacologia , Linhagem Celular , Decitabina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/virologia , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/imunologia , Herpesvirus Equídeo 4/efeitos dos fármacos , Doenças dos Cavalos/virologia , Doenças dos Cavalos/tratamento farmacológico , Doenças dos Cavalos/imunologia , Cavalos , Imunidade Inata/efeitos dos fármacos , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: The blood-brain barrier (BBB) is pivotal for the maintenance of brain homeostasis and it strictly regulates the cerebral transport of a wide range of endogenous compounds and drugs. While fasting is increasingly recognized as a potential therapeutic intervention in neurology and psychiatry, its impact upon the BBB has not been studied. This study was designed to assess the global impact of fasting upon the repertoire of BBB transporters. METHODS: We used a combination of in vivo and in vitro experiments to assess the response of the brain endothelium in male rats that were fed ad libitum or fasted for one to three days. Brain endothelial cells were acutely purified and transcriptionaly profiled using RNA-Seq. Isolated brain microvessels were used to assess the protein expression of selected BBB transporters through western blot. The molecular mechanisms involved in the adaptation to fasting were investigated in primary cultured rat brain endothelial cells. MCT1 activity was probed by in situ brain perfusion. RESULTS: Fasting did not change the expression of the main drug efflux ATP-binding cassette transporters or P-glycoprotein activity at the BBB but modulated a restrictive set of solute carrier transporters. These included the ketone bodies transporter MCT1, which is pivotal for the brain adaptation to fasting. Our findings in vivo suggested that PPAR δ, a major lipid sensor, was selectively activated in brain endothelial cells in response to fasting. This was confirmed in vitro where pharmacological agonists and free fatty acids selectively activated PPAR δ, resulting in the upregulation of MCT1 expression. Moreover, dosing rats with a specific PPAR δ antagonist blocked the upregulation of MCT1 expression and activity induced by fasting. CONCLUSIONS: Altogether, our study shows that fasting affects a selected set of BBB transporters which does not include the main drug efflux transporters. Moreover, we describe a previously unknown selective adaptive response of the brain vasculature to fasting which involves PPAR δ and is responsible for the up-regulation of MCT1 expression and activity. Our study opens new perspectives for the metabolic manipulation of the BBB in the healthy or diseased brain.
Assuntos
Barreira Hematoencefálica , PPAR delta , Ratos , Masculino , Animais , Barreira Hematoencefálica/metabolismo , PPAR delta/metabolismo , Células Endoteliais/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Encéfalo/metabolismo , JejumRESUMO
Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Radiação Ionizante , Tardígrados , Transcriptoma , Tardígrados/genética , Tardígrados/metabolismo , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Dano ao DNA , Tolerância a Radiação/genéticaRESUMO
Importance: Cerebral small vessel diseases (CSVDs) account for one-fifth of stroke cases. Numerous familial cases remain unresolved after routine screening of known CSVD genes. Objective: To identify novel genes and mechanisms associated with familial CSVD. Design, Setting, and Participants: This 2-stage study involved linkage analysis and a case-control study; linkage analysis and whole exome and genome sequencing were used to identify candidate gene variants in 2 large families with CSVD (9 patients with CSVD). Then, a case-control analysis was conducted on 246 unrelated probands, including probands from these 2 families and 244 additional probands. All probands (clinical onset Assuntos
Regiões 3' não Traduzidas
, Doenças de Pequenos Vasos Cerebrais
, Colágeno Tipo IV
, Adulto
, Feminino
, Humanos
, Pessoa de Meia-Idade
, Regiões 3' não Traduzidas/genética
, Alelos
, Estudos de Casos e Controles
, Doenças de Pequenos Vasos Cerebrais/genética
, Colágeno Tipo IV/metabolismo
, Isoformas de Proteínas
, Mutagênese Insercional