RESUMO
Zika virus (ZIKV) infection during human pregnancy may lead to severe fetal pathology and debilitating impairments in offspring. However, the majority of infections are subclinical and not associated with evident birth defects. Potentially detrimental life-long health outcomes in asymptomatic offspring evoke high concerns. Thus, animal models addressing sequelae in offspring may provide valuable information. To induce subclinical infection, we inoculated selected porcine fetuses at the mid-stage of development. Inoculation resulted in trans-fetal virus spread and persistent infection in the placenta and fetal membranes for two months. Offspring did not show congenital Zika syndrome (e.g., microcephaly, brain calcifications, congenital clubfoot, arthrogryposis, seizures) or other visible birth defects. However, a month after birth, a portion of offspring exhibited excessive interferon alpha (IFN-α) levels in blood plasma in a regular environment. Most affected offspring also showed dramatic IFN-α shutdown during social stress providing the first evidence for the cumulative impact of prenatal ZIKV exposure and postnatal environmental insult. Other eleven cytokines tested before and after stress were not altered suggesting the specific IFN-α pathology. While brains from offspring did not have histopathology, lesions, and ZIKV, the whole genome expression analysis of the prefrontal cortex revealed profound sex-specific transcriptional changes that most probably was the result of subclinical in utero infection. RNA-seq analysis in the placenta persistently infected with ZIKV provided independent support for the sex-specific pattern of in utero-acquired transcriptional responses. Collectively, our results provide strong evidence that two hallmarks of fetal ZIKV infection, altered type I IFN response and molecular brain pathology can persist after birth in offspring in the absence of congenital Zika syndrome.
Assuntos
Encéfalo/patologia , Doenças Fetais/epidemiologia , Feto/virologia , Interferon-alfa/metabolismo , Complicações Infecciosas na Gravidez/epidemiologia , Útero/virologia , Infecção por Zika virus/virologia , Animais , Antivirais/metabolismo , Encéfalo/metabolismo , Encéfalo/virologia , Doenças Transmissíveis/transmissão , Doenças Transmissíveis/virologia , Feminino , Doenças Fetais/metabolismo , Doenças Fetais/virologia , Feto/metabolismo , Feto/patologia , Masculino , Gravidez , Complicações Infecciosas na Gravidez/metabolismo , Complicações Infecciosas na Gravidez/virologia , Fatores Sexuais , Suínos , Útero/metabolismo , Útero/patologia , Zika virus/patogenicidade , Infecção por Zika virus/patologia , Infecção por Zika virus/transmissão , Infecção por Zika virus/veterináriaRESUMO
The CCCTC-binding factor (CTCF) is a central regulator of chromatin topology recently linked to neurodevelopmental disorders such as intellectual disability, autism, and schizophrenia. The aim of this study was to identify novel roles of CTCF in the developing mouse brain. We provide evidence that CTCF is required for the expression of the LIM homeodomain factor LHX6 involved in fate determination of cortical interneurons (CINs) that originate in the medial ganglionic eminence (MGE). Conditional Ctcf ablation in the MGE of mice of either sex leads to delayed tangential migration, abnormal distribution of CIN in the neocortex, a marked reduction of CINs expressing parvalbumin and somatostatin (Sst), and an increased number of MGE-derived cells expressing Lhx8 and other markers of basal forebrain projection neurons. Likewise, Ctcf-null MGE cells transplanted into the cortex of wild-type hosts generate fewer Sst-expressing CINs and exhibit lamination defects that are efficiently rescued upon reexpression of LHX6. Collectively, these data indicate that CTCF regulates the dichotomy between Lhx6 and Lhx8 to achieve correct specification and migration of MGE-derived CINs.SIGNIFICANCE STATEMENT This work provides evidence that CCCTC-binding factor (CTCF) controls an early fate decision point in the generation of cortical interneurons mediated at least in part by Lhx6. Importantly, the abnormalities described could reflect early molecular and cellular events that contribute to human neurological disorders previously linked to CTCF, including schizophrenia, autism, and intellectual disability.
Assuntos
Fator de Ligação a CCCTC/fisiologia , Córtex Cerebral/fisiologia , Interneurônios/fisiologia , Eminência Mediana/fisiologia , Animais , Fator de Ligação a CCCTC/genética , Contagem de Células , Movimento Celular/genética , Movimento Celular/fisiologia , Córtex Cerebral/citologia , Feminino , Proteínas com Homeodomínio LIM/biossíntese , Proteínas com Homeodomínio LIM/genética , Masculino , Eminência Mediana/citologia , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/citologia , Neocórtex/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Parvalbuminas/metabolismo , Somatostatina/metabolismo , Telencéfalo/citologia , Telencéfalo/crescimento & desenvolvimento , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Ácido gama-Aminobutírico/fisiologiaRESUMO
α-Thalassemia X-linked intellectual disability (ATR-X) syndrome is a neurodevelopmental disorder caused by mutations in the ATRX gene that encodes a SNF2-type chromatin-remodeling protein. The ATRX protein regulates chromatin structure and gene expression in the developing mouse brain and early inactivation leads to DNA replication stress, extensive cell death, and microcephaly. However, the outcome of Atrx loss of function postnatally in neurons is less well understood. We recently reported that conditional inactivation of Atrx in postnatal forebrain excitatory neurons (ATRX-cKO) causes deficits in long-term hippocampus-dependent spatial memory. Thus, we hypothesized that ATRX-cKO mice will display impaired hippocampal synaptic transmission and plasticity. In the present study, evoked field potentials and current source density analysis were recorded from a multichannel electrode in male, urethane-anesthetized mice. Three major excitatory synapses, the Schaffer collaterals to basal dendrites and proximal apical dendrites, and the temporoammonic path to distal apical dendrites on hippocampal CA1 pyramidal cells were assessed by their baseline synaptic transmission, including paired-pulse facilitation (PPF) at 50-ms interpulse interval, and by their long-term potentiation (LTP) induced by theta-frequency burst stimulation. Baseline single-pulse excitatory response at each synapse did not differ between ATRX-cKO and control mice, but baseline PPF was reduced at the CA1 basal dendritic synapse in ATRX-cKO mice. While basal dendritic LTP of the first-pulse excitatory response was not affected in ATRX-cKO mice, proximal and distal apical dendritic LTP were marginally and significantly reduced, respectively. These results suggest that ATRX is required in excitatory neurons of the forebrain to achieve normal hippocampal LTP and PPF at the CA1 apical and basal dendritic synapses, respectively. Such alterations in hippocampal synaptic transmission and plasticity could explain the long-term spatial memory deficits in ATRX-cKO mice and provide insight into the physiological mechanisms underlying intellectual disability in ATR-X syndrome patients.
Assuntos
Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Prosencéfalo/metabolismo , Sinapses/metabolismo , Proteína Nuclear Ligada ao X/deficiência , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/citologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Prosencéfalo/citologia , Proteína Nuclear Ligada ao X/genéticaRESUMO
Intellectual disability (ID) is a prevailing neurodevelopmental condition associated with impaired cognitive and adaptive behaviors. Many chromatin-modifying enzymes and other epigenetic regulators have been genetically associated with ID disorders (IDDs). Here we review how alterations in the function of histone modifiers, chromatin remodelers, and methyl-DNA binding proteins contribute to neurodevelopmental defects and altered brain plasticity. We also discuss how progress in human genetics has led to the generation of mouse models that unveil the molecular etiology of ID, and outline the direction in which this field is moving to identify therapeutic strategies for IDDs. Importantly, because the chromatin regulators linked to IDDs often target common downstream genes and cellular processes, the impact of research in individual syndromes goes well beyond each syndrome and can also contribute to the understanding and therapy of other IDDs. Furthermore, the investigation of these disorders helps us to understand the role of chromatin regulators in brain development, plasticity, and gene expression, thereby answering fundamental questions in neurobiology.
Assuntos
Epigênese Genética/genética , Deficiência Intelectual/etiologia , Deficiência Intelectual/genética , Epigenômica , HumanosRESUMO
Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes.
Assuntos
Cromatina/metabolismo , Mitose/genética , Neoplasias/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Cromatina/genética , Instabilidade Cromossômica/genética , Segregação de Cromossomos , Cultura , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Mutação/genética , Fenótipo , Proteína do Retinoblastoma/genética , Análise de Sobrevida , Proteínas Supressoras de Tumor/genéticaRESUMO
ATRX is a chromatin remodeling protein involved in deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. It also influences the expression level of specific genes; however, deposition of H3.3 at transcribed genes is currently thought to occur independently of ATRX. We focused on a set of genes, including the autism susceptibility gene Neuroligin 4 (Nlgn4), that exhibit decreased expression in ATRX-null cells to investigate the mechanisms used by ATRX to promote gene transcription. Overall TERRA levels, as well as DNA methylation and histone modifications at ATRX target genes are not altered and thus cannot explain transcriptional dysregulation. We found that ATRX does not associate with the promoter of these genes, but rather binds within regions of the gene body corresponding to high H3.3 occupancy. These intragenic regions consist of guanine-rich DNA sequences predicted to form non-B DNA structures called G-quadruplexes during transcriptional elongation. We demonstrate that ATRX deficiency corresponds to reduced H3.3 incorporation and stalling of RNA polymerase II at these G-rich intragenic sites. These findings suggest that ATRX promotes the incorporation of histone H3.3 at particular transcribed genes and facilitates transcriptional elongation through G-rich sequences. The inability to transcribe genes such as Nlgn4 could cause deficits in neuronal connectivity and cognition associated with ATRX mutations in humans.
Assuntos
DNA Helicases/metabolismo , Regulação da Expressão Gênica , Guanina/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Proteínas Nucleares/metabolismo , Transcrição Gênica , Talassemia alfa/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , DNA Helicases/genética , Metilação de DNA , Quadruplex G , Histonas/metabolismo , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/embriologia , Deficiência Intelectual Ligada ao Cromossomo X/genética , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/genética , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Proteína Nuclear Ligada ao X , Talassemia alfa/embriologia , Talassemia alfa/genéticaRESUMO
BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus infecting pigs of all ages with high morbidity and mortality among newborn piglets. Currently, there is no effective vaccine available to protect the pigs from PEDV. The N-terminal subunit of spike protein (S1) is responsible for virus binding to the cellular receptor and contains a number of neutralizing antibody epitopes. Thus, we expressed and produced recombinant S1 protein to protect newborn piglets by immunization of sows. METHODS: Affinity tagged PEDV S1 protein was expressed in a secretory form in yeast, insect and mammalian cells to identify the most suitable production system. Purified recombinant protein was analysed by SDS-PAGE, Western blot and deglycosylation assay. A pregnant sow was intramuscularly immunized three times with adjuvanted recombinant protein prior to farrowing. PEDV-specific immune responses in sera and colostrum of the sow and piglets were assayed by ELISA and virus neutralization assays. Piglets were challenged orally with PEDV, and clinical parameters were monitored for 6 days post-challenge. RESULTS AND CONCLUSION: Of three eukaryotic expression systems tested (yeast, insect-cell, and mammalian), expression by HEK-293 T cells gave the highest yield of protein that was N-glycosylated and was the most appropriate candidate for vaccination. Administration of the subunit vaccine in a sow resulted in induction of S1-specific IgG and IgA that were passively transferred to the suckling piglets. Also, high virus neutralization titres were observed in the serum of the vaccinated sow and its piglets. After PEDV challenge, piglets born to the vaccinated sow exhibited less severe signs of disease and significantly lower mortality compared to the piglets of a control sow. However, there were no significant differences in diarrhea, body weight and virus shedding. Thus, vaccination with S1 subunit vaccine failed to provide complete protection to suckling piglets after challenge exposure, and further improvements are needed for the development of a subunit vaccine that fully protects against PEDV infection.
Assuntos
Antígenos Virais/imunologia , Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Antígenos Virais/genética , Colostro/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Feminino , Injeções Intramusculares , Testes de Neutralização , Vírus da Diarreia Epidêmica Suína/genética , Gravidez , Soro/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved. Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation. We show that ATRX binds many imprinted domains individually but that transient co-localization between imprinted domains in the nuclei of neurons does not require ATRX. We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy. These findings indicate that MeCP2 and ATRX regulate gene expression at a subset of imprinted domains by maintaining a nucleosome configuration conducive to CTCF binding and to the maintenance of higher order chromatin structure.
Assuntos
Encéfalo/metabolismo , Cromatina/química , DNA Helicases/fisiologia , Impressão Genômica , Proteína 2 de Ligação a Metil-CpG/fisiologia , Proteínas Nucleares/fisiologia , Nucleossomos/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Encéfalo/crescimento & desenvolvimento , Fator de Ligação a CCCTC , Proteínas de Ligação ao Cálcio , Núcleo Celular/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Deleção de Genes , Fator de Crescimento Insulin-Like II/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/genética , Proteína Nuclear Ligada ao XRESUMO
An increasing number of proteins involved in genome organization have been implicated in neurodevelopmental disorders, highlighting the importance of chromatin architecture in the developing CNS. The CCCTC-binding factor (CTCF) is a zinc finger DNA binding protein involved in higher-order chromatin organization, and mutations in the human CTCF gene cause an intellectual disability syndrome associated with microcephaly. However, information on CTCF function in vivo in the developing brain is lacking. To address this gap, we conditionally inactivated the Ctcf gene at early stages of mouse brain development. Cre-mediated Ctcf deletion in the telencephalon and anterior retina at embryonic day 8.5 triggered upregulation of the p53 effector PUMA (p53 upregulated modulator of apoptosis), resulting in massive apoptosis and profound ablation of telencephalic structures. Inactivation of Ctcf several days later at E11 also resulted in PUMA upregulation and increased apoptotic cell death, and the Ctcf-null forebrain was hypocellular and disorganized at birth. Although deletion of both Ctcf and Puma in the embryonic brain efficiently rescued Ctcf-null progenitor cell apoptosis, it failed to improve neonatal hypocellularity due to decreased proliferative capacity of rescued apical and outer radial glia progenitor cells. This was exacerbated by an independent effect of CTCF loss that resulted in depletion of the progenitor pool due to premature neurogenesis earlier in development. Our findings demonstrate that CTCF activities are required for two distinct events in early cortex formation: first, to correctly regulate the balance between neuroprogenitor cell proliferation and differentiation, and second, for the survival of neuroprogenitor cells, providing new clues regarding the contributions of CTCF in microcephaly/intellectual disability syndrome pathologies.
Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células-Tronco Neurais/fisiologia , Proteínas Repressoras/fisiologia , Animais , Antimetabólitos , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Encéfalo/anormalidades , Bromodesoxiuridina , Fator de Ligação a CCCTC , Morte Celular/fisiologia , Imunoprecipitação da Cromatina , Éxons/genética , Feminino , Imunofluorescência , Genes p53/genética , Genes p53/fisiologia , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Knockout , Nestina/genética , Nestina/fisiologia , Gravidez , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , Retina/citologia , Retina/fisiologia , Telencéfalo/citologia , Telencéfalo/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
ATR-X syndrome is a rare genetic disorder caused by mutations in the ATRX gene. Affected individuals are cognitively impaired and display a variety of developmental abnormalities, including skeletal deformities. To investigate the function of ATRX during skeletal development, we selectively deleted the gene in the developing forelimb mesenchyme of mice. The absence of ATRX in the limb mesenchyme resulted in shorter digits, or brachydactyly, a defect also observed in a subset of ATR-X patients. This phenotype persisted until adulthood, causing reduced grip strength and altered gait in mutant mice. Examination of the embryonic ATRX-null forelimbs revealed a significant increase in apoptotic cell death, which could explain the reduced digit length. In addition, staining for the DNA damage markers γ-histone 2A family member X (γ-H2AX) and 53BP1 demonstrated a significant increase in the number of cells with DNA damage in the embryonic ATRX-null forepaw. Strikingly, only one large bright DNA damage event was observed per nucleus in proliferating cells. These large γ-H2AX foci were located in close proximity to the nuclear lamina and remained largely unresolved after cell differentiation. In addition, ATRX-depleted forelimb mesenchymal cells did not exhibit hypersensitivity to DNA fork-stalling compounds, suggesting that the nature as well as the response to DNA damage incurred by loss of ATRX in the developing limb fundamentally differs from other tissues. Our data suggest that DNA damage-induced apoptosis is a novel cellular mechanism underlying brachydactyly that might be relevant to additional skeletal syndromes.
Assuntos
Braquidactilia/genética , DNA Helicases/genética , Membro Anterior/anormalidades , Mesoderma/metabolismo , Proteínas Nucleares/genética , Animais , Braquidactilia/metabolismo , Morte Celular/genética , Condrócitos/metabolismo , DNA Helicases/deficiência , DNA Helicases/metabolismo , Modelos Animais de Doenças , Feminino , Membro Anterior/embriologia , Membro Anterior/fisiopatologia , Estudos de Associação Genética , Histonas/genética , Histonas/metabolismo , Hidroxiureia/farmacologia , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Masculino , Mesoderma/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Fenótipo , Proteína Nuclear Ligada ao XRESUMO
Limited protection of current vaccines and antiviral drugs against influenza A virus infection underscores the urgent need for development of novel anti-influenza virus interventions. While short interfering RNA (siRNA) has been shown to be able to inhibit influenza virus infection in a gene-specific manner, activation of the retinoic acid-inducible gene I protein (RIG-I) pathway has an antiviral effect in a non-gene-specific mode. In this study, we designed and tested the anti-influenza virus effect of a short double-stranded RNA, designated 3p-mNP1496-siRNA, that possesses dual functions: an siRNA-targeting influenza NP gene and an agonist for RIG-I activation. This double-stranded siRNA possesses a triphosphate group at the 5' end of the sense strand and is blunt ended. Our study showed that 3p-mNP1496-siRNA could potently inhibit influenza A virus infection both in cell culture and in mice. The strong inhibition effect was attributed to its siRNA function as well as its ability to activate the RIG-I pathway. To the best of our knowledge, this is the first report that the combination of siRNA and RIG-I pathway activation can synergistically inhibit influenza A virus infection. The development of such dual functional RNA molecules will greatly contribute to the arsenal of tools to combat not only influenza viruses but also other important viral pathogens.
Assuntos
RNA Helicases DEAD-box/metabolismo , Inativação Gênica , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Fosfatos/química , RNA Interferente Pequeno/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Galinhas , Chlorocebus aethiops , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Cães , Feminino , Células HEK293 , Humanos , Interferon beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Receptores Imunológicos , Células VeroRESUMO
BACKGROUND: ATRX is an ATP-dependent chromatin remodeling protein with essential roles in safeguarding genome integrity and modulating gene expression. Deficiencies in this protein cause ATR-X syndrome, a condition characterized by intellectual disability and an array of developmental abnormalities, including features of autism. Previous studies demonstrated that deleting ATRX in mouse forebrain excitatory neurons postnatally resulted in male-specific memory deficits, but no apparent autistic-like behaviours. METHODS: We generated mice with an earlier embryonic deletion of ATRX in forebrain excitatory neurons and characterized their behaviour using a series of memory and autistic-related paradigms. RESULTS: We found that mutant mice displayed a broader spectrum of impairments, including fear memory, decreased anxiety-like behaviour, hyperactivity, as well as self-injurious and repetitive grooming. Sex-specific alterations were also observed, including male-specific aggression, sensory gating impairments, and decreased social memory. CONCLUSIONS: Collectively, the findings indicate that early developmental abnormalities arising from ATRX deficiency in forebrain excitatory neurons contribute to the presentation of fear memory deficits as well as autistic-like behaviours.
Assuntos
Transtorno Autístico , Feminino , Camundongos , Masculino , Animais , Transtorno Autístico/complicações , Transtorno Autístico/genética , Neurônios/fisiologia , Transtornos da Memória/etiologia , CogniçãoRESUMO
Myelin, an extension of the oligodendrocyte plasma membrane, wraps around axons to facilitate nerve conduction. Myelination is compromised in ATR-X intellectual disability syndrome patients, but the causes are unknown. We show that loss of ATRX leads to myelination deficits in male mice that are partially rectified upon systemic thyroxine administration. Targeted ATRX inactivation in either neurons or oligodendrocyte progenitor cells (OPCs) reveals OPC-intrinsic effects on myelination. OPCs lacking ATRX fail to differentiate along the oligodendrocyte lineage and acquire a more plastic state that favors astrocytic differentiation in vitro and in vivo. ATRX chromatin occupancy in OPCs greatly overlaps with that of the chromatin remodelers CHD7 and CHD8 as well as H3K27Ac, a mark of active enhancers. Overall, our data indicate that ATRX regulates the onset of myelination systemically via thyroxine, and by promoting OPC differentiation and suppressing astrogliogenesis. These functions of ATRX identified in mice could explain white matter pathogenesis observed in ATR-X syndrome patients.
Assuntos
Bainha de Mielina , Tiroxina , Proteína Nuclear Ligada ao X , Animais , Humanos , Masculino , Camundongos , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Bainha de Mielina/metabolismo , Neurogênese , Oligodendroglia/metabolismo , Tiroxina/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , NeurogliaRESUMO
Increasing the number of CpG dinucleotides in RNA viral genomes, while preserving the original amino acid composition, leads to impaired infection which does not cause disease. Beneficially, impaired infection evokes antiviral host immune responses providing a cutting-edge vaccine approach. For example, we previously showed that CpG-enriched Zika virus variants cause attenuated infection phenotypes and protect against lethal challenge in mice. While CpG recoding is an emerging and promising vaccine approach, little is known about infection phenotypes caused by recoded viruses in vivo, particularly in non-rodent species. Here, we used well-established mouse and porcine models to study infection phenotypes of the CpG-enriched neurotropic and congenital virus-Zika virus, directly in the target tissues-the brain and placenta. Specifically, we used the uttermost challenge and directly injected mice intracerebrally to compare infection phenotypes caused by wild-type and two CpG-recoded Zika variants and model the scenario where vaccine strains breach the blood-brain barrier. Also, we directly injected porcine fetuses to compare in utero infection phenotypes and model the scenario where recoded vaccine strains breach the placental barrier. While overall infection kinetics were comparable between wild-type and recoded virus variants, we found convergent phenotypical differences characterized by reduced pathology in the mouse brain and reduced replication of CpG-enriched variants in fetal lymph nodes. Next, using next-generation sequencing for the whole virus genome, we compared the stability of de novo introduced CpG dinucleotides during prolonged virus infection in the brain and placenta. Most de novo introduced CpG dinucleotides were preserved in sequences of recoded Zika viruses showing the stability of vaccine variants. Altogether, our study emphasized further directions to fine-tune the CpG recoding vaccine approach for better safety and can inform future immunization strategies.
Assuntos
Vírus , Infecção por Zika virus , Zika virus , Animais , Encéfalo , Feminino , Feto , Genoma Viral , Linfonodos , Camundongos , Fenótipo , Placenta , Gravidez , Suínos , Vírus/genéticaRESUMO
BACKGROUND: Isolation of cell types of interest from the brain for molecular applications presents several challenges, including cellular damage during tissue dissociation or enrichment procedures, and low cell number in the tissue in some cases. Techniques have been developed to enrich distinct cell populations using immunopanning or fluorescence activated cell/nuclei sorting. However, these techniques often involve fixation, immunolabeling and DNA staining steps, which could potentially influence downstream omics applications. NEW METHOD: Taking advantage of readily available genetically modified mice with fluorescent-tagged nuclei, we describe a technique for the purification of cell-type specific brain nuclei, optimized to decrease sample preparation time and to limit potential artefacts for downstream omics applications. We demonstrate the applicability of this approach for the purification of glial cell nuclei and show that the resulting cell-type specific nuclei obtained can be used effectively for omics applications, including ATAC-seq and RNA-seq. RESULTS: We demonstrate excellent enrichment of fluorescently-tagged glial nuclei, yielding high quality RNA and chromatin. We identify several critical steps during nuclei isolation that help limit nuclei rupture and clumping, including quick homogenization, dilution before filtration and loosening of the pellet before resuspension, thus improving yield. Sorting of fluorescent nuclei can be achieved without fixation, antibody labelling, or DAPI staining, reducing potential artifactual results in RNA-seq and ATAC-seq analyses. We show that reproducible glial cell type-specific profiles can be obtained in transcriptomic and chromatin accessibility assays using this rapid protocol. COMPARISON WITH EXISTING METHODS: Our method allows for rapid enrichment of glial nuclei populations from the mouse brain with minimal processing steps, while still providing high quality RNA and chromatin required for reliable omics analyses. CONCLUSIONS: We provide a reproducible method to obtain nucleic material from glial cells in the mouse brain with a quick and limited sample preparation.
Assuntos
Núcleo Celular , Cromatina , Animais , Encéfalo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Camundongos , RNA-SeqRESUMO
An effective single-dose vaccine that protects the dam and her suckling offspring against infectious disease would be widely beneficial to livestock animals. We assessed whether a single-dose intramuscular (i.m.) porcine epidemic diarrhea virus (PEDV) vaccine administered to the gilt 30 days post-breeding could generate mucosal and systemic immunity and sufficient colostral and mature milk antibodies to protect suckling piglets against infectious challenge. The vaccine was comprised of polymeric poly-(lactide-co-glycolide) (PGLA)-nanoparticle (NP) encapsulating recombinant PEDV spike protein 1 (PEDVS1) associated with ARC4 and ARC7 adjuvants, a muramyl dipeptide analog and a monophosphoryl lipid A (MPLA) analog, respectively (NP-PEDVS1). To establish whether prior mucosal exposure could augment the i.m. immune response and/or contribute to mucosal tolerance, gilts were immunized with the NP-PEDVS1 vaccine via the intrauterine route at breeding, followed by the i.m. vaccine 30 days later. Archived colostrum from gilts that were challenged with low-dose PEDV plus alum was used as positive reference samples for neutralizing antibodies and passive protection. On day 100 of gestation (70 days post i.m. immunization), both vaccinated groups showed significant PEDVS1-specific IgG and IgA in the serum, as well as in uterine tissue collected on the day of euthanasia. Anti-PEDVS1 colostral IgG antibody titers collected at farrowing were significantly higher relative to the negative control gilts indicating that the NP vaccine was effective in contributing to the colostral antibodies. The PEDVS1-specific colostral IgA and anti-PEDVS1 IgG and IgA antibodies in the mature milk collected 6 days after farrowing were low for both vaccinated groups. No statistical differences between the vaccinated groups were observed, suggesting that the i.u. priming vaccine did not induce mucosal tolerance. Piglets born to either group of vaccinated gilts did not receive sufficient neutralizing antibodies to protect them against infectious PEDV at 3 days of age. In summary, a single i.m. NP vaccine administered 30 days after breeding and a joint i.u./i.m. vaccine administered at breeding and 30 days post-breeding induced significant anti-PEDVS1 immunity in systemic and mucosal sites but did not provide passive protection in suckling offspring.
RESUMO
ATRX is an SWI/SNF-like chromatin remodeling protein that is mutated in several X-linked mental retardation syndromes, including the ATR-X syndrome. In mice, Atrx expression is widespread and attempts to understand its function in brain development are hampered by the lethality associated with ubiquitous or forebrain-restricted ablation of this gene. One way to circumvent this problem is to study its function in a region of the brain that is dispensable for long-term survival of the organism. The retina is a well-characterized tractable model of CNS development and in our review of 202 ATR-X syndrome patients, we found ocular defects present in approximately 25% of the cases, suggesting that studying Atrx in this tissue will provide insight into function. We report that Atrx is expressed in the neuroprogenitor pool in embryonic retina and in all cell types of the mature retina with the exception of rod photoreceptors. Conditional inactivation of Atrx in the retina during embryogenesis ultimately results in a loss of only two types of neurons, amacrine and horizontal cells. We show that this defect does not arise from a failure to specify these cells but rather a defect in interneuron differentiation and survival post-natally. The timing of cell loss is concomitant with light-dependent changes in synaptic organization in the retina and with a change in Atrx subnuclear localization within these interneurons. Moreover, these interneuron defects are associated with functional deficits as demonstrated by reduced b-wave amplitudes upon electroretinogram analysis. These results implicate a role for Atrx in interneuron survival and differentiation.
Assuntos
DNA Helicases/metabolismo , Interneurônios/fisiologia , Deficiência Intelectual Ligada ao Cromossomo X/fisiopatologia , Proteínas Nucleares/metabolismo , Visão Ocular , Adulto , Células Amácrinas/fisiologia , Animais , Sobrevivência Celular , DNA Helicases/genética , Feminino , Expressão Gênica , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/embriologia , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteína Nuclear Ligada ao XRESUMO
The regulation of genome architecture is essential for a variety of fundamental cellular phenomena that underlie the complex orchestration of mammalian development. The ATP-dependent chromatin remodeling protein ATRX is emerging as a key regulatory component of nucleosomal dynamics and higher order chromatin conformation. Here we provide an overview of the role of ATRX at chromatin and during development, and discuss recent studies exposing a repertoire of ATRX functions at heterochromatin, in gene regulation, and during mitosis and meiosis. Exciting new progress on several fronts suggest that ATRX operates in histone variant deposition and in the modulation of higher order chromatin structure. Not surprisingly, dysfunction or absence of ATRX protein has devastating consequences on embryonic development and leads to human disease.
Assuntos
Cromatina/metabolismo , DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Doenças do Sistema Nervoso/genética , Proteínas Nucleares/metabolismo , Animais , Cromatina/química , DNA Helicases/genética , Feminino , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/genética , Camundongos , Proteínas Nucleares/genética , Síndrome de Rett/genética , Proteína Nuclear Ligada ao X , Talassemia alfa/genéticaRESUMO
Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410), AA79 (PR8-412), or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410, PR8-411, and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection, their growth potential is similar to that of the WT virus during a single cycle of infection, and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus, Tx91-445, which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.