RESUMO
Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon's (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits.
Assuntos
Alcoolismo/genética , Estudo de Associação Genômica Ampla , Seleção Genética , Alcoolismo/fisiopatologia , Álcoois/toxicidade , Animais , Modelos Animais de Doenças , Éxons/genética , Frequência do Gene , Genômica , Haplótipos , Humanos , Íntrons/genética , Herança Multifatorial/genética , Neurônios/efeitos dos fármacos , Fenótipo , RatosRESUMO
BACKGROUND: Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high- and low-alcohol drinking (HAD and LAD) rat lines. Here, we report allele-specific expression (ASE) differences, between the HAD2 and LAD2 rat lines. METHODS: The HAD2 and LAD2 rats, which have been sequenced, were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2, and for the other 5 litters, the sire was a LAD2. From these 10 litters, 2 males and 2 females were picked from each F1 litter (N = 40 total). The F1 pups were divided, balancing for sex and direction of cross, into an alcohol (15%) versus a water control group. Alcohol drinking started in the middle of adolescence (~postnatal day 35) and lasted 9 weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA-sequencing and ASE analyses. RESULTS: Analyses revealed that adolescent ethanol (EtOH) drinking, individual EtOH drinking levels, parentage, and sex-of-animal affected ASEs of about 300 genes. The identified genes included those associated with EtOH metabolism (e.g., Aldh2); neuromodulatory function (e.g., Cckbr, Slc6a7, and Slc1a1); ion channel activity (e.g., Kcnc3); and other synaptic and epigenetic functions. CONCLUSIONS: These data indicate that EtOH drinking differentially amplified paternal versus maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to EtOH-induced changes in cis-regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene-by-environment interactions mediating a genetic predisposition for, and/or the active development of, AUDs.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Alcoolismo/genética , Alelos , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Fatores Sexuais , Consumo de Bebidas Alcoólicas/fisiopatologia , Alcoolismo/fisiopatologia , Aldeído-Desidrogenase Mitocondrial/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Cruzamento/métodos , Cruzamentos Genéticos , Etanol/metabolismo , Transportador 3 de Aminoácido Excitatório/genética , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Interação Gene-Ambiente , Masculino , Ratos , Canais de Potássio Shaw/genéticaRESUMO
BACKGROUND: Down syndrome (DS) and Fetal Alcohol Syndrome (FAS) are two leading causes of birth defects with phenotypes ranging from craniofacial abnormalities to cognitive impairment. Despite different origins, we report that in addition to sharing many phenotypes, DS and FAS may have common underlying mechanisms of development. METHODS: Literature was surveyed for DS and FAS as well as mouse models. Gene expression and apoptosis were compared in embryonic mouse models of DS and FAS by qPCR, immunohistochemical and immunoflurorescence analyses. The craniometry was examined using MicroCT at postnatal day 21. RESULTS: A literature survey revealed over 20 comparable craniofacial and structural deficits in both humans with DS and FAS and corresponding mouse models. Similar phenotypes were experimentally found in pre- and postnatal craniofacial and neurological tissues of DS and FAS mice. Dysregulation of two genes, Dyrk1a and Rcan1, key to craniofacial and neurological precursors of DS, was shared in craniofacial precursors of DS and FAS embryos. Increased cleaved caspase 3 expression was also discovered in comparable regions of the craniofacial and brain precursors of DS and FAS embryos. Further mechanistic studies suggested overexpression of trisomic Ttc3 in DS embyros may influence nuclear pAkt localization and cell survival. CONCLUSIONS: This first and initial study indicates that DS and FAS share common dysmorphologies in humans and animal models. This work also suggests common mechanisms at cellular and molecular levels that are disrupted by trisomy or alcohol consumption during pregnancy and lead to craniofacial and neurological phenotypes associated with DS or FAS.
Assuntos
Anormalidades Craniofaciais/genética , Síndrome de Down/genética , Transtornos do Espectro Alcoólico Fetal/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Caspase 3/genética , Caspase 3/metabolismo , Anormalidades Craniofaciais/patologia , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Feminino , Transtornos do Espectro Alcoólico Fetal/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas Musculares/genética , Fenótipo , Gravidez , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Reação em Cadeia da Polimerase em Tempo Real , Quinases DyrkRESUMO
BACKGROUND: The basis for familial alcohol use disorder (AUD) remains an enigma due to various biological and societal confounds. The present study used three of the most adopted and documented rat models, combining the alcohol-preferring/non-alcohol-preferring (P/NP) lines and high alcohol-drinking/low alcohol-drinking (HAD/LAD) replicated lines, of AUD as examined through the lens of whole genomic analyses. METHODS: We used complete genome sequencing of the P/NP lines and previously published sequences of the HAD/LAD replicates to enhance the discovery of variants associated with AUD and to remove confounding with genetic background and random genetic drift. Specifically, we used high-order statistical methods to search for genetic variants whose frequency changes in whole sets of gene ontologies corresponded with phenotypic changes in the direction of selection, that is, ethanol-drinking preference. RESULTS: Our first finding was that in addition to variants causing translational changes, the principal genetic changes associated with drinking predisposition were silent mutations and mutations in the 3' untranslated regions (3'UTR) of genes. Neither of these types of mutations alters the amino acid sequence of the translated protein but they influence both the rate and conformation of gene transcription, including its stability and posttranslational events that alter gene efficacy. This finding argues for refocusing human genomic studies on changes in gene efficacy. Among the key ontologies identified were the central genes associated with the Na+ voltage-gated channels of neurons and glia (including the Scn1a, Scn2a, Scn2b, Scn3a, Scn7a, and Scn9a subtypes) and excitatory glutamatergic secretion (including Grm2 and Myo6), both of which are essential in neuroplasticity. In addition, we identified "Nociception or Sensory Perception of Pain," which contained variants in nociception (Arrb1, Ccl3, Ephb1) and enlist sodium (Scn1a, Scn2a, Scn2b, Scn3a, Scn7a), pain activation (Scn9a), and potassium channel (Kcna1) genes. CONCLUSION: The multi-model analyses used herein reduced the confounding effects of random drift and the "founders" genetic background. The most differentiated bidirectionally selected genes across all three animal models were Scn9a, Scn1a, and Kcna, all of which are annotated in the nociception ontology. The complexity of neuroplasticity and nociception adds strength to the hypothesis that neuroplasticity and pain (physical or psychological) are prominent phenotypes genetically linked to the development of AUD.
RESUMO
The serotonergic system is implicated in disordered emotional behavior. Autism is characterized by impaired processing of emotional information. The serotonergic (5-HT) system is also critically involved in brain development, and abnormal brain synthesis of serotonin is observed in autism. Furthermore, whole blood and platelet serotonin have been reported to be elevated in autism. The authors examined the CNS serotonin system in autism in vivo. 5-HT2 receptors were visualized by PET imaging of [18F]setoperone-binding in this pilot study of 6 high-functioning autistic adults and 10 matched-control participants. Autism subjects had less thalamic [18F]setoperone binding than controls, when covaried for age, but no difference reached significance in other areas. A negative relationship between thalamic binding and history of language impairment was also observed. Further studies will be needed to gain a clearer picture of the role of the 5-HT system in autism.
Assuntos
Transtorno Autístico/metabolismo , Radioisótopos de Flúor , Neuroimagem Funcional/psicologia , Pirimidinonas , Receptores 5-HT2 de Serotonina/metabolismo , Tálamo/metabolismo , Adulto , Transtorno Autístico/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Estudos de Casos e Controles , Feminino , Neuroimagem Funcional/métodos , Humanos , Transtornos da Linguagem/complicações , Transtornos da Linguagem/diagnóstico por imagem , Transtornos da Linguagem/metabolismo , Masculino , Projetos Piloto , Tomografia por Emissão de Pósitrons/métodos , Tomografia por Emissão de Pósitrons/psicologia , Ensaio Radioligante/métodos , Ensaio Radioligante/psicologia , Compostos Radiofarmacêuticos , Tálamo/diagnóstico por imagemRESUMO
BACKGROUND: We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. RESULT: Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. CONCLUSION: This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.
Assuntos
Embrião de Mamíferos/efeitos dos fármacos , Etanol/efeitos adversos , Perfilação da Expressão Gênica , Neurulação , Animais , Análise por Conglomerados , Técnicas de Cultura Embrionária , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , TeratogênicosRESUMO
BACKGROUND: Prenatal alcohol exposure can result in varying degrees of neurodevelopmental deficits, growth retardation, and facial dysmorphology. Variation in these adverse outcomes not only depends on the dose and pattern of alcohol exposure but also on less well understood interactions among environmental, genetic, and maternal factors. The current study tested the hypothesis that fetal genotype is an important determinant of ethanol teratogenesis by evaluating effects of ethanol exposure via embryo culture in 3 inbred strains of mice known to differ in the vulnerability of prenatal alcohol exposure in vivo. METHODS: Three strains of mice, C57BL/6N (B6), DBA/2 (D2), and 129S6/SvEvTac (129S6) were assessed in a whole embryo culture beginning on embryonic day 8.25, with or without alcohol administration at 88 mM for 6 hours followed by 42 hours culture in ethanol-free media. RESULTS: Contrasting strain differences in susceptibility were observed for the brain, the face, and other organ systems using the Maele-Fabry and Picard scoring system. The forebrain, midbrain, hindbrain, heart, optic vesicle, caudal neural tube, and hindlimbs of the B6 mice were severely delayed in growth, whereas compared to the respective controls, only the forebrain and optic vesicle were delayed in the D2 mice, and no effects were found in the 129S6 mice. A large number of cleaved (c)-caspase 3 positive (+) cells were found in regions of the brain, optic vesicles, cranial nerve nuclei V, VII, VIII, and IX as well as the craniofacial primordial; only a few were found in corresponding regions of the B6 controls. In contrast, only a small number of c-caspase 3 immunostaining cells were found in either the alcohol treated or the controls of the D2 embryos and in 129S6 embryos. The independent apoptotic markers TUNEL and Nile blue staining further confirmed the strain differences in apoptotic responses in both the neural tube and craniofacial primordia. CONCLUSIONS: Under embryo culture conditions, in which alcohol exposure factors and fetal developmental staging were controlled, and maternal and intrauterine factors were eliminated, the degree of growth retardation and the extent and type of neurodevelopmental teratogenesis varied significantly across strains. Notably, the 129S6 strain was remarkably resistant to alcohol-induced growth deficits, confirming a previous in vivo study, and the D2 strain was also significantly less affected than the B6 strain. These findings demonstrate that fetal genotype is an important factor that can contribute to the variation in fetal alcohol spectrum disorder.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Etanol/toxicidade , Animais , Desenvolvimento Embrionário/genética , Feminino , Predisposição Genética para Doença , Genótipo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Técnicas de Cultura de Órgãos , Gravidez , Especificidade da EspécieRESUMO
BACKGROUND: Potential epigenetic mechanisms underlying fetal alcohol syndrome (FAS) include alcohol-induced alterations of methyl metabolism, resulting in aberrant patterns of DNA methylation and gene expression during development. Having previously demonstrated an essential role for epigenetics in neural stem cell (NSC) development and that inhibiting DNA methylation prevents NSC differentiation, here we investigated the effect of alcohol exposure on genome-wide DNA methylation patterns and NSC differentiation. METHODS: Neural stem cells in culture were treated with or without a 6-hour 88 mM ("binge-like") alcohol exposure and examined at 48 hours, for migration, growth, and genome-wide DNA methylation. The DNA methylation was examined using DNA-methylation immunoprecipitation followed by microarray analysis. Further validation was performed using Independent Sequenom analysis. RESULTS: Neural stem cell differentiated in 24 to 48 hours with migration, neuronal expression, and morphological transformation. Alcohol exposure retarded the migration, neuronal formation, and growth processes of NSC, similar to treatment with the methylation inhibitor 5-aza-cytidine. When NSC departed from the quiescent state, a genome-wide diversification of DNA methylation was observed-that is, many moderately methylated genes altered methylation levels and became hyper- and hypomethylated. Alcohol prevented many genes from such diversification, including genes related to neural development, neuronal receptors, and olfaction, while retarding differentiation. Validation of specific genes by Sequenom analysis demonstrated that alcohol exposure prevented methylation of specific genes associated with neural development [cut-like 2 (cutl2), insulin-like growth factor 1 (Igf1), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (Efemp1), and SRY-box-containing gene 7 (Sox 7)]; eye development, lens intrinsic membrane protein 2 (Lim 2); the epigenetic mark Smarca2 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2); and developmental disorder [DiGeorge syndrome critical region gene 2 (Dgcr2)]. Specific sites altered by DNA methylation also correlated with transcription factor binding sites known to be critical for regulating neural development. CONCLUSION: The data indicate that alcohol prevents normal DNA methylation programming of key neural stem cell genes and retards NSC differentiation. Thus, the role of DNA methylation in FAS warrants further investigation.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Etanol/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Depressores do Sistema Nervoso Central/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Análise em Microsséries , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacosRESUMO
BACKGROUND: Epigenetic changes are believed to be among the earliest key regulators for cell fate and embryonic development. To support this premise, it is important to understand whether or not systemic epigenetic changes coordinate with the progression of development. We have demonstrated that DNA methylation is programmed when neural stem cells differentiate (Zhou et al.,2011). Here, we analyzed the DNA methylation events that occur during early neural tube development. METHODS AND RESULTS: Using immunocytochemistry, we demonstrated that the DNA methylation marks - 5-methylcytosine (5-MeC), DNA methylation binding domain 1 (MBD1), and DNA methytransferases 1 (DNMT1) were highly coordinated in temporal and spatial patterns that paralleled the progress of embryonic development. The above ontogenic program of DNA methylation was, however, subjected to environmental modification. Alcohol exposure during fetal development, which is known to cause fetal alcohol spectrum disorder, altered the density and distribution of the DNA methylation marks. The alcohol exposure (88 mM) over 6 or 44 hours at gestation day 8 (GD-8) to GD-10 altered timely DNA methylation and retarded embryonic growth. We further demonstrated that the direct inhibiting of DNA methylation with 5-aza-cytidine (5-AZA) resulted in similar growth retardation. CONCLUSIONS: We identified a temporal and spatial cellular DNA methylation program after initial erasure, which parallels embryonic maturation. Alcohol delayed the cellular DNA methylation program and also retarded embryonic growth. Since direct inhibiting of DNA methylation resulted in similar retardation, alcohol thus can affect embryonic development through a epigenetic pathway.
Assuntos
Azacitidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Etanol/farmacologia , Neurulação/efeitos dos fármacos , 5-Metilcitosina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Tubo Neural/efeitos dos fármacos , Tubo Neural/embriologia , Neurulação/genética , Neurulação/fisiologia , Gravidez , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Preservation of neural stem cells (NSCs) in the adult peripheral nervous system (PNS) has recently been confirmed. However, it is not clear whether peripheral NSCs possess predestined, bona fide phenotypes or a response to innate developmental cues. In this study, we first demonstrated the longevity, multipotency, and high fidelity of sensory features of postmigrating adult dorsal root ganglia (aDRG) stem cells. Derived from aDRG and after 4-5 years in culture without dissociating, the aDRG NSCs were found capable of proliferation, expressing neuroepithelial, neuronal, and glial markers. Remarkably, these aDRG NSCs expressed sensory neuronal markers vesicular glutamate transporter 2 (VGluT2--glutamate terminals), transient receptor potential vanilloid 1 (TrpV1--capsaicin sensitive), phosphorylated 200 kDa neurofilaments (pNF200--capsaicin insensitive, myelinated), and the serotonin transporter (5-HTT), which normally is transiently expressed in developing DRG. Furthermore, in response to neurotrophins, the aDRG NSCs enhanced TrpV1 expression upon exposure to nerve growth factor (NGF), but not to brain-derived neurotrophic factor (BDNF). On the contrary, BDNF increased the expression of NeuN. Third, the characterization of aDRG NSCs was demonstrated by transplantation of red fluorescent-expressing aDRG NSCs into injured spinal cord. These cells expressed nestin, Hu, and beta-III-tubulin (immature neuronal markers), GFAP (astrocyte marker) as well as sensory neural marker TrpV1 (capsaicin sensitive) and pNF200 (mature, capsaicin insensitive, myelinated). Our results demonstrated that the postmigrating neural crest adult DRG stem cells not only preserved their multipotency but also were retentive in sensory potency despite the age and long-term ex vivo status.
Assuntos
Células-Tronco Adultas/citologia , Gânglios Espinais/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco Adultas/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Gânglios Espinais/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Multipotentes/metabolismo , Fator de Crescimento Neural/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismoRESUMO
This is a first report, using our MotifModeler informatics program, to simultaneously identify transcription factor (TF) and microRNA (miRNA) binding sites from gene expression microarray data. Based on the assumption that gene expression is controlled by combinatorial effects of transcription factors binding in the 5'-upstream regulatory region and miRNAs binding in the 3'-untranslated region (3'-UTR), we developed a model for (1) predicting the most influential cis-acting elements under a given biological condition, and (2) estimating the effects of those elements on gene expression levels. The regulatory regions, TF and miRNA, which mediate the differential genes expression in fetal alcohol syndrome were unknown; microarray data from alcohol exposure paradigm was used. The model predicted strong inhibitory effects of 5' cis-acting elements and stimulatory effects of 3'-UTR under alcohol treatment. Current predictive model derived a key hypothesis for the first time a novel role of miRNAs in gene expression changes associated with abnormal mouse embryo development after alcohol exposure. This suggests that disturbance of miRNA functions may contribute to the alcohol-induced developmental deficiencies.
Assuntos
Transtornos do Espectro Alcoólico Fetal/metabolismo , Regulação da Expressão Gênica/genética , MicroRNAs/metabolismo , Modelos Genéticos , Fatores de Transcrição/metabolismo , Sítios de Ligação/genética , Biologia Computacional/métodos , Feminino , Transtornos do Espectro Alcoólico Fetal/genética , Humanos , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Fatores de Transcrição/genéticaRESUMO
INTRODUCTION: Prenatal alcohol exposure via maternal liquid diet consumption by C57BL/6 (B6) mice causes conspicuous midline neural tube deficit (dysraphia) and disruption of genesis and development of serotonin (5-HT) neurons in the raphe nuclei, together with brain growth retardation. The current study tested the hypothesis that concurrent treatment with either an activity-dependent neurotrophic factor (ADNF) agonist peptide [SALLRSIPA, (SAL)] or an activity-dependent neurotrophic protein (ADNP) agonist peptide [NAPVSIPQ, (NAP)] would protect against these alcohol-induced deficits in brain development. METHODS: Timed-pregnant B6 dams consumed alcohol from embryonic day 7 (E7, before the onset of neurulation) until E15. Fetuses were obtained on E15 and brain sections processed for 5-HT immunocytochemistry, for evaluation of morphologic development of the brainstem raphe and its 5-HT neurons. Additional groups were treated either with SAL or NAP daily from E7 to E15 to assess the potential protective effects of these peptides. Measures of incomplete occlusion of the ventral canal and the frequency and extent of the openings in the rhombencephalon were obtained to assess fetal dysraphia. Counts of 5-HT-immunostained neurons were also obtained in the rostral and caudal raphe. RESULTS: Prenatal alcohol exposure resulted in abnormal openings along the midline and delayed closure of ventral canal in the brainstem. This dysraphia was associated with reductions in the number of 5-HT neurons both in the rostral raphe nuclei (that gives rise to ascending 5-HT projections) and in the caudal raphe (that gives rise to the descending 5-HT projections). Concurrent treatment of the alcohol-consuming dams with SAL prevented dysraphia and protected against the alcohol-induced reductions in 5-HT neurons in both the rostral and caudal raphe. NAP was less effective in protecting against dysraphia and did not protect against 5-HT loss in the rostral raphe, but did protect against loss in the caudal raphe. CONCLUSIONS: These findings further support the potential usefulness of these peptides for therapeutic interventions in pregnancies at risk for alcohol-induced developmental deficits. Notably, the ascending 5-HT projections of the rostral raphe have profound effects in regulating forebrain development and function, and the descending 5-HT projections of the caudal raphe are critical for regulating respiration. Protection of the rostral 5-HT-system may help prevent structural and functional deficits linked to abnormal forebrain development, and protection of the caudal systems may also reduce the increased risk for sudden infant death syndrome associated with prenatal alcohol exposure.
Assuntos
Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Proteínas de Homeodomínio/agonistas , Proteínas do Tecido Nervoso/agonistas , Defeitos do Tubo Neural/prevenção & controle , Fragmentos de Peptídeos/uso terapêutico , Animais , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Modelos Animais de Doenças , Etanol/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/induzido quimicamente , Degeneração Neural/prevenção & controle , Defeitos do Tubo Neural/induzido quimicamente , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeos , Oligopeptídeos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Lesões Pré-Natais/induzido quimicamente , Lesões Pré-Natais/prevenção & controle , Respiração/efeitos dos fármacos , Serotonina/metabolismo , Disrafismo Espinal/induzido quimicamente , Disrafismo Espinal/prevenção & controleRESUMO
BACKGROUND: Fetal alcohol exposure causes growth deficits, microencephaly, and neurological abnormalities. Although the effects of alcohol on developmental delay and growth-related deficits have been hypothesized, little is understood about how alcohol alters, in particular, the cyclin pathway within the cell cycle, which is critical to proliferation and apoptotic control. In this study, we examined cell cycle proteins pertinent to the G1-S phase transition and apoptosis, to determine if cell cycle misregulation can be attributed to apoptotic induction and growth defects. METHODS: We examined cell cycle regulation during G1 and S-phase, and DNA fragmentation damage, using E14 dorsal root ganglia neural stem cells (DRG-NC), and cultured mouse embryos exposed to 200 and 400 mg/dl ethanol. RESULTS: Alcohol-exposed DRG-NC demonstrated a dose-dependent increase in cells expressing increased cyclin D1 protein, and increased DNA fragmentation. Western blot analysis, using embryos, demonstrated an overexpression of cyclin D1, D2, and E2F1, key G1 to S-phase cell cycle regulatory components, and increases in p53, linking the cell cycle and apoptotic pathways. Bromodeoxyuridine incorporation indicated reduced DNA synthesis and growth in several embryonic regions. Propidium iodide staining demonstrated decreases in DNA content and increases in DNA fragmentation in several embryonic tissues. CONCLUSIONS: This study indicated that retarded growth of DRG-NC and embryos, induced by alcohol, is associated with altered expression of cell cycle and apoptotic proteins and concurrent inhibition of proliferation and increased DNA fragmentation. We suggest that alcohol induces an increase in cyclin D1 expression, premature S-phase entry, and disjointed DNA synthesis with increased apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Etanol/administração & dosagem , Neurulação/efeitos dos fármacos , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/patologia , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/biossíntese , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Etanol/toxicidade , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurulação/fisiologia , Gravidez , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Alcohol is known to affect glutamate transmission. However, how chronic alcohol affects the synaptic structure mediating glutamate transmission is unknown. Repeated alcohol exposure in a subject with familial alcoholic history often leads to alcohol addiction. The current study adopts alcohol-preferring rats, which are known to develop high drinking. Two-photon microscopy analysis indicates that chronic alcohol of 14 weeks either, under continuous alcohol (C-Alc) or with repeated deprivation (RD-Alc), causes dysmorphology--thickened, beaded, and disoriented dendrites that are reminiscent of reactive astrocytes--in a subpopulation of medium spiny neurons. The density of dendritic spines was found differentially lower in the nucleus accumbens of RD-Alc and C-Alc groups as compared with those of Water groups. Large-sized spines and multiple-headed spines were increased in the RD-Alc group. The NMDA receptor subunit NR1 proteins, as analyzed with Western blot, were upregulated in C-Alc, but not in RD-Alc. The upregulated NMDA receptor subunits of NR1 however, are predominantly a splice variant isoform with truncated exon 21, which is required for membrane-bound trafficking or anchoring into a spine synaptic site. These maladaptations may contribute to the transformation of spines. The changes, in density and head-size of spines and the corresponding NMDA receptors, demonstrated an alteration of microcircuitry for glutamate reception. The current study demonstrates for the first time that chronic alcohol exposure causes structural alteration of dendrites and their spines in the key reward brain region in animals that have a genetic background leading to alcohol addiction.
Assuntos
Transtornos do Sistema Nervoso Induzidos por Álcool/patologia , Alcoolismo/patologia , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/patologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/patologia , Transtornos do Sistema Nervoso Induzidos por Álcool/fisiopatologia , Alcoolismo/fisiopatologia , Animais , Depressores do Sistema Nervoso Central/efeitos adversos , Doença Crônica , Etanol/efeitos adversos , Ácido Glutâmico/metabolismo , Citometria por Imagem , Imuno-Histoquímica , Masculino , Microscopia Confocal , Núcleo Accumbens/fisiopatologia , Ratos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Recompensa , Transtornos Relacionados ao Uso de Substâncias/patologia , Transtornos Relacionados ao Uso de Substâncias/fisiopatologia , Sinapses/efeitos dos fármacos , Sinapses/patologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Epigênese Genética , Biologia Molecular/métodos , Células-Tronco Neurais , Optogenética/métodos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Perfilação da Expressão Gênica , Luz , Camundongos , Regiões Promotoras GenéticasRESUMO
While cerebral underdevelopment is a hallmark of fetal alcohol spectrum disorders (FASD), the mechanism(s) guiding the broad cortical neurodevelopmental deficits are not clear. DNA methylation is known to regulate early development and tissue specification through gene regulation. Here, we examined DNA methylation in the onset of alcohol-induced cortical thinning in a mouse model of FASD. C57BL/6 (B6) mice were administered a 4% alcohol (v/v) liquid diet from embryonic (E) days 7-16, and their embryos were harvested at E17, along with isocaloric liquid diet and lab chow controls. Cortical neuroanatomy, neural phenotypes, and epigenetic markers of methylation were assessed using immunohistochemistry, Western blot, and methyl-DNA assays. We report that cortical thickness, neuroepithelial proliferation, and neuronal migration and maturity were found to be deterred by alcohol at E17. Simultaneously, DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxcylmethylcytosine (5hmC), which progresses as an intrinsic program guiding normal embryonic cortical development, was severely affected by in utero alcohol exposure. The intricate relationship between cortical thinning and this DNA methylation program disruption is detailed and illustrated. DNA methylation, dynamic across the multiple cortical layers during the late embryonic stage, is highly disrupted by fetal alcohol exposure; this disruption occurs in tandem with characteristic developmental abnormalities, ranging from structural to molecular. Finally, our findings point to a significant question for future exploration: whether epigenetics guides neurodevelopment or whether developmental conditions dictate epigenetic dynamics in the context of alcohol-induced cortical teratogenesis.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Etanol/toxicidade , Transtornos do Espectro Alcoólico Fetal/genética , Neocórtex/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/metabolismo , Consumo de Bebidas Alcoólicas/patologia , Animais , Feminino , Transtornos do Espectro Alcoólico Fetal/metabolismo , Transtornos do Espectro Alcoólico Fetal/patologia , Genótipo , Idade Gestacional , Exposição Materna , Camundongos Endogâmicos C57BL , Neocórtex/embriologia , Neocórtex/metabolismo , Fenótipo , GravidezRESUMO
Previous studies have indicated that the nigrostriatal dopaminergic (DA) pathway can be reconstructed in hemiparkinsonian rats with a bridge transplantation technique involving fetal ventral mesencephalic transplants and glial cell line-derived neurotrophic factor. In this study, we examined if the nigrostriatal pathway can be restored by combining peripheral nervous tissue with the fetal ventral mesencephalon transplants. Adult rats were injected with 6-hydroxydopamine into left median forebrain bundle. Those with marked rotational behavior, which has been previously shown to indicate complete DA dennervtion, were used for transplant treatments. One month after the lesion, fetal ventral mesencephalic cells were transplanted into the nigral region followed by nigral-striatal grafting of peripheral nerves as a bridge. The bridging nerves (sciatic or intercostals) were pretreated with basic fibrous growth factor (nerve+bFGF+) or Hank's saline (nerve+bFGF-). We found that (a) animals receiving transplants of VM and bFGF+ nerve had a reduction in rotational behavior; (b) animals receiving bFGF-- nerve bridge only had a partial improvement in rotation. Reinnervation of tyrosine hydroxylase (TH)-immunoreactive (ir) fibers into the striatum was found in both of the above groups with more innervation in the former than in the latter. No TH-ir fibers in lesioned striatum or reduction in rotational behavior were found in animals receiving VM only, or VM plus bFGF. Taken together, our data indicate that peripheral nerve, with the aid of bFGF, greatly facilitates the reconstitution of the TH pathway from nigra to striatum and improves motor function in hemiparkinsonian rats.
Assuntos
Corpo Estriado/citologia , Feto/citologia , Fatores de Crescimento de Fibroblastos/farmacologia , Mesencéfalo/citologia , Mesencéfalo/transplante , Doença de Parkinson/terapia , Nervos Periféricos/efeitos dos fármacos , Anfetamina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Transplante de Células , Imuno-Histoquímica , Fibras Nervosas/imunologia , Fibras Nervosas/transplante , Oxidopamina/farmacologia , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/imunologiaRESUMO
Alcohol has been shown to affect glutamate (GLU) and dopamine (DA) release and their correlated receptors in the key reward center--extended amygdala--which includes the shell of nucleus accumbens (sNAc) and central nucleus of amygdala (cAmg). It is unclear to date whether there is an alteration in the number of presynaptic GLU/DA nerve terminals. In this study, we investigated the number of GLU and DA terminals in the extended amygdala of alcohol-preferring (P) rats that chronically drank ethanol. P rats have a propensity to drink ethanol to intoxication and develop an alcohol dependency. The P rats were divided into (1) Water group given ad libitum chow and water for 14 weeks; (2) Continuous alcohol group (C-Alc) given ad libitum chow and choice of 15 or 30% (v/v) ethanol or water for 14 weeks; and (3) Repeated deprivation (RD-Alc) group given the same choice of ethanol or water for 6 weeks, followed by a twice repeated cycle of 2 weeks without ethanol followed by 2 weeks with ethanol. Two subpopulations of GLU terminals were labeled by immunostaining for the vesicular GLU transporter 1 (vGLUT1) and vesicular GLU transporter 2 (vGLUT2). DA terminals were labeled by immunostaining for tyrosine hydroxylase (TH). The GLU and DA immunostained (im) varicosities were quantified and analyzed using stereological methods. We found that chronic alcohol did not alter the number of TH-im terminals in the extended amygdala in either the C-Alc or RD-Alc drinking paradigms. Thus, the increases in extracellular levels of DA previously reported following chronic alcohol are likely due to a change in the efficiency of DA release rather than a change in the number of DA terminals. The number of vGLUT1-im terminals was also unchanged in the extended amygdala; however, the number of vGLUT2-im terminals, which represent the greater population of GLU terminals, was increased in the sNAc of the RD-Alc group compared to the Water group. Chronic alcohol is known to affect GLU release, and our findings indicate that repeated alcohol deprivation may preferentially increase GLU terminals in the sNAc bearing the vGLUT2, which are primarily afferents from the thalamus. Our results further indicate that repeated deprivation of alcohol can change the ratio of GLU to DA innervation in the sNAc, a key region of the reward circuitry.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Tonsila do Cerebelo/fisiopatologia , Terminações Pré-Sinápticas/metabolismo , Receptores Dopaminérgicos/metabolismo , Receptores de Glutamato/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Masculino , Plasticidade Neuronal , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology.