RESUMO
Early studies in mouse neurodevelopment led to the discovery of the RE1 Silencing Transcription Factor (REST) and its role as a master repressor of neuronal gene expression. Recently, REST was reported to also repress neuronal genes in the human adult brain. These genes were found to be involved in pro-apoptotic pathways; and their repression, associated with increased REST levels during aging, were found to be neuroprotective and conserved across species. However, direct genome-wide REST binding profiles for REST in adult brain have not been identified for any species. Here, we apply this approach to mouse and human hippocampus. We find an expansion of REST binding sites in the human hippocampus that are lacking in both mouse hippocampus and other human non-neuronal cell types. The unique human REST binding sites are associated with genes involved in innate immunity processes and inflammation signaling which, on the basis of histology and recent public transcriptomic analyses, suggest that these new target genes are repressed in glia. We propose that the increases in REST expression in mid-adulthood presage the beginning of brain aging, and that human REST function has evolved to protect the longevity and function of both neurons and glia in human brain.SIGNIFICANCE STATEMENT The RE1 Silencing Transcription Factor (REST) repressor has served historically as a model for gene regulation during mouse neurogenesis. Recent studies of REST have also suggested a conserved role for REST repressor function across lower species during aging. However, direct genome-wide studies for REST have been lacking for human brain. Here, we perform the first genome-wide analysis of REST binding in both human and mouse hippocampus. The majority of REST-occupied genes in human hippocampus are distinct from those in mouse. Further, the REST-associated genes unique to human hippocampus represent a new set related to innate immunity and inflammation, where their gene dysregulation has been implicated in aging-related neuropathology, such as Alzheimer's disease.
Assuntos
Envelhecimento/metabolismo , Hipocampo/metabolismo , Neuroglia/metabolismo , Proteínas Repressoras/metabolismo , Idoso , Envelhecimento/imunologia , Animais , Feminino , Estudo de Associação Genômica Ampla , Hipocampo/imunologia , Humanos , Imunidade Inata/fisiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neuroglia/imunologia , Neurônios/metabolismo , Proteínas Repressoras/imunologiaRESUMO
The idea that astrocytes provide support for neurons has a long history, but whether neurons play an instructive role in these processes is poorly understood. To address this question, we co-culture astrocytes with genetically labeled neurons, permitting their separation by flow cytometry, and test whether the presence of neurons influences the astrocyte transcriptome. We find that numerous pathways are regulated in the co-cultured astrocytes, in a time-dependent matter coincident with synaptic maturation. In particular, the induction of glutathione metabolic genes is prominent, resulting in increased glutathione production. We show that the induction of the glutathione pathway is mediated by astrocytic metabotropic glutamate receptors. Using a candidate approach, we identify direct binding of the nuclear factor E2-related factor, NRF2, to several of the induced genes. Blocking nuclear accumulation of astrocytic NRF2 abolishes neuron-induced glutathione gene induction and glutathione production. Our results suggest that astrocyte transcriptional and metabolic profiles are tightly coupled to the activity of neurons, consistent with the model that astrocytes dynamically support healthy brain function.
Assuntos
Astrócitos/metabolismo , Regulação da Expressão Gênica/fisiologia , Glutationa/metabolismo , Neurônios/fisiologia , Animais , Astrócitos/citologia , Células Cultivadas , Técnicas de Cocultura , Hipocampo/citologia , Hipocampo/fisiologia , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/citologia , Receptores de Glutamato Metabotrópico/metabolismo , TranscriptomaRESUMO
AIM: The aim of this study was to determine the barriers and enablers influencing the uptake of two recommendations from a tertiary paediatric hospital's clinical practice guidelines by maternal and child health nurses (MCHNs) and emergency department (ED) doctors: (i) explaining normal crying; and (ii) avoiding attributing crying to gastro-oesophageal reflux (GOR) and limiting anti-reflux medication use. METHODS: The study was designed as 1-h focus group discussions, guided by the Theoretical Domains Framework, and a short questionnaire, with a purposive sample of MCHNs and ED doctors in Victoria, Australia in (March to September) 2015. Analyses were conducted by inductive content analysis to identify key barriers and enablers. RESULTS: A total of 53 MCHNs and 25 ED doctors participated in 11 discussions. For explaining normal crying, key enablers were: adequate experience/competency, perceiving it was their role to explain and belief it prevented over-medicalisation. The main barriers were time restriction and beliefs about parents' perceptions. For MCHNs, key barriers to avoid attributing crying to GOR were: lack of knowledge and confusion around their role in diagnosing GOR. For ED doctors, key barriers to limiting anti-reflux medication were: parents requesting medication, concern about disrupting the parent-primary-care practitioner relationship and belief it was not their role to cease anti-reflux medication. CONCLUSIONS: Overall, MCHN and ED doctors were proficient in describing normal crying. However, several barriers to best practice were identified, including time constraints and belief about consequences of intervening. These results will be used to develop effective interventions to address the identified barriers and enablers to optimise the management of infant colic.
Assuntos
Atitude do Pessoal de Saúde , Cólica/terapia , Choro , Padrões de Prática Médica , Adulto , Feminino , Grupos Focais , Refluxo Gastroesofágico , Pesquisas sobre Atenção à Saúde , Hospitais Pediátricos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , VitóriaRESUMO
De novo mutations in the X-linked gene encoding the transcription factor methyl-CpG binding protein 2 (MECP2) are the most frequent cause of the neurological disorder Rett syndrome (RTT). Hemizygous males usually die of neonatal encephalopathy. Heterozygous females survive into adulthood but exhibit severe symptoms including microcephaly, loss of purposeful hand motions and speech, and motor abnormalities, which appear after a period of apparently normal development. Most studies have focused on male mouse models because of the shorter latency to and severity in symptoms, yet how well these mice mimic the disease in affected females is not clear. Very few therapeutic treatments have been proposed for females, the more gender-appropriate model. Here, we show that self-complementary AAV9, bearing MeCP2 cDNA under control of a fragment of its own promoter (scAAV9/MeCP2), is capable of significantly stabilizing or reversing symptoms when administered systemically into female RTT mice. To our knowledge, this is the first potential gene therapy for females afflicted with RTT.
Assuntos
Comportamento Animal/efeitos dos fármacos , Proteína 2 de Ligação a Metil-CpG/administração & dosagem , Síndrome de Rett/fisiopatologia , Síndrome de Rett/terapia , Animais , Comportamento Animal/fisiologia , Contagem de Células , Dependovirus/fisiologia , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Proteína 2 de Ligação a Metil-CpG/biossíntese , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Mutação/genética , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Fosfopiruvato Hidratase/metabolismo , Pletismografia , Equilíbrio Postural/genética , Equilíbrio Postural/fisiologia , Reconhecimento Psicológico/fisiologia , Respiração , Síndrome de Rett/genética , Síndrome de Rett/patologia , Teste de Desempenho do Rota-RodRESUMO
The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation.
Assuntos
Encéfalo/embriologia , Diferenciação Celular , Neurogênese , Neurônios/fisiologia , Proteínas Repressoras/metabolismo , Células-Tronco/fisiologia , Animais , Ciclo Celular , Técnicas de Silenciamento de Genes , CamundongosRESUMO
The bivalent hypothesis posits that genes encoding developmental regulators required for early lineage decisions are poised in stem/progenitor cells by the balance between a repressor histone modification (H3K27me3), mediated by the Polycomb Repressor Complex 2 (PRC2), and an activator modification (H3K4me3). In this study, we test whether this mechanism applies equally to genes that are not required until terminal differentiation. We focus on the RE1 Silencing Transcription Factor (REST) because it is expressed highly in stem cells and is an established global repressor of terminal neuronal genes. Elucidation of the REST complex, and comparison of chromatin marks and gene expression levels in control and REST-deficient stem cells, shows that REST target genes are poised by a mechanism independent of Polycomb, even at promoters which bear the H3K27me3 mark. Specifically, genes under REST control are actively repressed in stem cells by a balance of the H3K4me3 mark and a repressor complex that relies on histone deacetylase activity. Thus, chromatin distinctions between pro-neural and terminal neuronal genes are established at the embryonic stem cell stage by two parallel, but distinct, repressor pathways.
Assuntos
Diferenciação Celular , Histona Desacetilases/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Complexo Repressor Polycomb 2/metabolismo , Proteínas Repressoras/metabolismo , Animais , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Lisina/metabolismo , Metilação , Camundongos , Fenótipo , Regiões Promotoras GenéticasRESUMO
Receptor-interacting protein kinase 4 (RIPK4) is required for epidermal differentiation and is mutated in Bartsocas-Papas syndrome. RIPK4 binds to protein kinase C, but its signaling mechanisms are largely unknown. Ectopic RIPK4, but not catalytically inactive or Bartsocas-Papas RIPK4 mutants, induced accumulation of cytosolic ß-catenin and a transcriptional program similar to that caused by Wnt3a. In Xenopus embryos, Ripk4 synergized with coexpressed Xwnt8, whereas Ripk4 morpholinos or catalytic inactive Ripk4 antagonized Wnt signaling. RIPK4 interacted constitutively with the adaptor protein DVL2 and, after Wnt3a stimulation, with the co-receptor LRP6. Phosphorylation of DVL2 by RIPK4 favored canonical Wnt signaling. Wnt-dependent growth of xenografted human tumor cells was suppressed by RIPK4 knockdown, suggesting that RIPK4 overexpression may contribute to the growth of certain tumor types.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Proteínas Desgrenhadas , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Transplante de Neoplasias , Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transplante Heterólogo , Proteína Wnt3A/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMO
mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eµ-myc Burkitt's lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk. DOI:http://dx.doi.org/10.7554/eLife.00822.001.
Assuntos
Apoptose/fisiologia , MicroRNAs/fisiologia , Oncogenes , Animais , Apoptose/genética , Células Cultivadas , CamundongosRESUMO
As astrocytes are becoming recognized as important mediators of normal brain function, studies into their roles in neurological disease have gained significance. Across mouse models for neurodevelopmental and neurodegenerative diseases, astrocytes are considered key regulators of disease progression. In Rett syndrome and Parkinson's disease, astrocytes can even initiate certain disease phenotypes. Numerous potential mechanisms have been offered to explain these results, but research into the functions of astrocytes in disease is just beginning. Crucially, in vivo verification of in vitro data is still necessary, as well as a deeper understanding of the complex and relatively unexplored interactions between astrocytes, oligodendrocytes, microglia, and neurons.