RESUMEN
Gestational alcohol exposure causes fetal alcohol spectrum disorder (FASD) and is a prominent cause of neurodevelopmental disability. Whole transcriptome sequencing (RNA-Seq) offer insights into mechanisms underlying FASD, but gene-level analysis provides limited information regarding complex transcriptional processes such as alternative splicing and non-coding RNAs. Moreover, traditional analytical approaches that use multiple hypothesis testing with a false discovery rate adjustment prioritize genes based on an adjusted p-value, which is not always biologically relevant. We address these limitations with a novel approach and implemented an unsupervised machine learning model, which we applied to an exon-level analysis to reduce data complexity to the most likely functionally relevant exons, without loss of novel information. This was performed on an RNA-Seq paired-end dataset derived from alcohol-exposed neural fold-stage chick crania, wherein alcohol causes facial deficits recapitulating those of FASD. A principal component analysis along with k-means clustering was utilized to extract exons that deviated from baseline expression. This identified 6857 differentially expressed exons representing 1251 geneIDs; 391 of these genes were identified in a prior gene-level analysis of this dataset. It also identified exons encoding 23 microRNAs (miRNAs) having significantly differential expression profiles in response to alcohol. We developed an RDAVID pipeline to identify KEGG pathways represented by these exons, and separately identified predicted KEGG pathways targeted by these miRNAs. Several of these (ribosome biogenesis, oxidative phosphorylation) were identified in our prior gene-level analysis. Other pathways are crucial to facial morphogenesis and represent both novel (focal adhesion, FoxO signaling, insulin signaling) and known (Wnt signaling) alcohol targets. Importantly, there was substantial overlap between the exomes themselves and the predicted miRNA targets, suggesting these miRNAs contribute to the gene-level expression changes. Our novel application of unsupervised machine learning in conjunction with statistical analyses facilitated the discovery of signaling pathways and miRNAs that inform mechanisms underlying FASD.
Asunto(s)
Exones/genética , Trastornos del Espectro Alcohólico Fetal/genética , MicroARNs/genética , Aprendizaje Automático no Supervisado , Animales , Macrodatos , Embrión de Pollo , Análisis por Conglomerados , Biología Computacional , Bases de Datos de Ácidos Nucleicos/estadística & datos numéricos , Modelos Animales de Enfermedad , Etanol/toxicidad , Femenino , Perfilación de la Expresión Génica/estadística & datos numéricos , Humanos , Embarazo , Análisis de Componente Principal , Aprendizaje Automático no Supervisado/estadística & datos numéricosRESUMEN
BACKGROUND: Prenatal alcohol exposure (PAE) causes long-term growth and neurodevelopmental deficits that are worsened by maternal iron deficiency (ID). In our preclinical rat model, PAE causes fetal anemia, brain ID, and elevated hepatic iron via increased maternal and fetal hepcidin synthesis. These changes are normalized by a prenatal iron-fortified (IF) diet. Here, we hypothesize that iron status and PAE dysregulate the major upstream pathways that govern hepcidin production-EPO/BMP6/SMAD and IL-6/JAK2/STAT3. METHODS: Pregnant, Long Evans rat dams consumed ID (2 to 6 ppm iron), iron-sufficient (IS, 100 ppm iron), or IF (500 ppm iron) diets and received alcohol (5 g/kg) or isocaloric maltodextrin daily from gestational days (GD) 13.5 to 19.5. Protein and gene expression were quantified in the 6 experimental groups at GD 20.5. RESULTS: PAE did not affect Epo or Bmp6 expression, but reduced p-SMAD1/5/8/SMAD1/5/8 protein ratios in both IS and ID maternal and fetal liver (all p's < 0.01). In contrast, PAE stimulated maternal hepatic expression of Il-6 (p = 0.03) and elevated p-STAT3/STAT3 protein ratios in both IS and ID maternal and fetal liver (all p's < 0.02). PAE modestly elevated maternal Il-1ß, Tnf-α, and Ifn-γ. Fetal cytokine responses to PAE were muted compared with dams, and PAE did not affect hepatic Il-6 (p = 0.78) in IS and ID fetuses. Dietary iron fortification sharply attenuated Il-6 expression in response to PAE, with IF driving a 150-fold decrease (p < 0.001) in maternal liver and a 10-fold decrease (p < 0.01) in fetal liver. The IF diet also normalized p-STAT3/STAT3 ratios in both maternal and fetal liver. CONCLUSIONS: These findings suggest that alcohol-driven stimulation of the IL-6/JAK2/STAT3 pathway mediates the elevated hepcidin observed in the PAE dam and fetus. Normalization of these signals by IF suggests that dysregulated hepcidin is driven by alcohol's disruption of the IL-6/JAK2/STAT3 pathway. Prenatal dietary IF represents a potential therapeutic approach for PAE that warrants further investigation.
Asunto(s)
Anemia Ferropénica/complicaciones , Etanol/efectos adversos , Feto/efectos de los fármacos , Interleucina-6/sangre , Efectos Tardíos de la Exposición Prenatal/sangre , Factor de Transcripción STAT3/sangre , Animales , Modelos Animales de Enfermedad , Femenino , Feto/metabolismo , Interleucina-6/metabolismo , Hierro de la Dieta , Embarazo , Ratas , Ratas Long-Evans , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Prenatal alcohol exposure (PAE) remains a leading preventable cause of structural birth defects and permanent neurodevelopmental disability. The chicken (Gallus gallus domesticus) is a powerful embryological research model, and was possibly the first in which the teratogenicity of alcohol was demonstrated. Pharmacologically relevant exposure to alcohol in the range of 20-70 mmol/L (20-80 mg/egg) disrupt the growth of chicken embryos, morphogenesis, and behavior, and the resulting phenotypes strongly parallel those of mammalian models. The avian embryo's direct accessibility has enabled novel insights into the teratogenic mechanisms of alcohol. These include the contribution of IGF1 signaling to growth suppression, the altered flow dynamics that reshape valvuloseptal morphogenesis and mediate its cardiac teratogenicity, and the suppression of Wnt and Shh signals thereby disrupting the migration, expansion, and survival of the neural crest, and underlie its characteristic craniofacial deficits. The genetic diversity within commercial avian strains has enabled the identification of unique loci, such as ribosome biogenesis, that modify vulnerability to alcohol. This venerable research model is equally relevant for the future, as the application of technological advances including CRISPR, optogenetics, and biophotonics to the embryo's ready accessibility creates a unique model in which investigators can manipulate and monitor the embryo in real-time to investigate the effect of alcohol on cell fate.
Asunto(s)
Pollos , Modelos Animales de Enfermedad , Desarrollo Embrionario , Trastornos del Espectro Alcohólico Fetal/metabolismo , Organogénesis , Transducción de Señal , Teratogénesis , Animales , Embrión de Pollo , Trastornos del Espectro Alcohólico Fetal/patología , HumanosRESUMEN
Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Some affected individuals possess distinctive craniofacial deficits, but many more lack overt facial changes. An understanding of the mechanisms underlying these deficits would inform their diagnostic utility. Our understanding of these mechanisms is challenged because ethanol lacks a single receptor when redirecting cellular activity. This review summarizes our current understanding of how ethanol alters neural crest development. Ample evidence shows that ethanol causes the "classic" fetal alcohol syndrome (FAS) face (short palpebral fissures, elongated upper lip, deficient philtrum) because it suppresses prechordal plate outgrowth, thereby reducing neuroectoderm and neural crest induction and causing holoprosencephaly. Prenatal alcohol exposure (PAE) at premigratory stages elicits a different facial appearance, indicating FASD may represent a spectrum of facial outcomes. PAE at this premigratory period initiates a calcium transient that activates CaMKII and destabilizes transcriptionally active ß-catenin, thereby initiating apoptosis within neural crest populations. Contributing to neural crest vulnerability are their low antioxidant responses. Ethanol-treated neural crest produce reactive oxygen species and free radical scavengers attenuate their production and prevent apoptosis. Ethanol also significantly impairs neural crest migration, causing cytoskeletal rearrangements that destabilize focal adhesion formation; their directional migratory capacity is also lost. Genetic factors further modify vulnerability to ethanol-induced craniofacial dysmorphology and include genes important for neural crest development, including shh signaling, PDFGA, vangl2, and ribosomal biogenesis. Because facial and brain development are mechanistically and functionally linked, research into ethanol's effects on neural crest also informs our understanding of ethanol's CNS pathologies.
Asunto(s)
Trastornos del Espectro Alcohólico Fetal/patología , Cresta Neural/patología , Consumo de Bebidas Alcohólicas/efectos adversos , Animales , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Etanol/administración & dosificación , Etanol/efectos adversos , Femenino , Trastornos del Espectro Alcohólico Fetal/etiología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Cresta Neural/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/etiología , Efectos Tardíos de la Exposición Prenatal/patología , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Prenatal ethanol exposure causes persistent neurodevelopmental deficits by inducing apoptosis within neuronal progenitors including the neural crest. The cellular signaling events underlying this apoptosis are unclear. Using an established chick embryo model, we previously identified ethanol's activation of calmodulin-dependent protein kinase II (CaMKII) as a crucial early step in this pathway. Here, we report that CaMKII is pro-apoptotic because it mediates the loss of transcriptionally active ß-catenin, which normally provides trophic support to these cells. ß-catenin over-expression normalized cell survival in ethanol's presence. CaMKII inhibition similarly restored ß-catenin content and transcriptional activity within ethanol-treated cells and prevented their cell death. In contrast, inhibition of alternative effectors known to destabilize ß-catenin, including glycogen synthase kinase-3ß, Protein Kinase C, JNK, and calpain, failed to normalize cell survival and ß-catenin activity in ethanol's presence. Importantly, we found that purified CaMKII can directly phosphorylate ß-catenin. Using targeted mutagenesis we identified CaMKII phosphorylation sites within human ß-catenin at T332, T472, and S552. This is the first demonstration that ß-catenin is a phosphorylation target of CaMKII and represents a novel mechanism by which calcium signals could regulate ß-catenin-dependent transcription. These results inform ethanol's neurotoxicity and offer unexpected insights into other neurodevelopmental and neurodegenerative disorders having dysregulated calcium or ß-catenin signaling.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/farmacología , Depresores del Sistema Nervioso Central/toxicidad , Etanol/toxicidad , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/metabolismo , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismo , Animales , Western Blotting , Muerte Celular/efectos de los fármacos , Embrión de Pollo , Electroporación , Inmunohistoquímica , Cresta Neural/enzimología , Cresta Neural/metabolismo , Fosforilación , Factor 1 de Transcripción de Linfocitos T/genética , Transcripción Genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. Nonhuman animal models offer novel insights into its underlying mechanisms. Although the developing zebrafish has great promise for FASD research, a significant challenge to its wider adoption is the paucity of clear, mechanistic parallels between its ethanol (EtOH) responses and those of nonpiscine, established models. Inconsistencies in the published pharmacodynamics for EtOH-exposed zebrafish, alongside the use of comparatively high EtOH doses, challenge the interpretation of this model's clinical relevance. METHODS: To address these limitations, we developed a binge, single-exposure model of EtOH exposure in the early zebrafish embryo. RESULTS: Brief (3-hour) EtOH exposure is sufficient to cause significant neural crest losses and craniofacial alterations, with peak vulnerability during neurogenesis and early somitogenesis. These losses are apoptotic, documented using TUNEL assay and secA5-YFP-reporter fish. Apoptosis is dose dependent with an EC50 = 56.2 ± 14.3 mM EtOHint , a clinically relevant value within the range producing apoptosis in chick and mouse neural crest. This apoptosis requires the calcium-dependent activation of CaMKII and recapitulates the well-described EtOH signaling mechanism in avian neural crest. Importantly, we resolve the existing confusion regarding zebrafish EtOH kinetics. We show that steady-state EtOH concentrations within both chorion-intact and dechorionated embryos are maintained at 35.7 ± 2.8% of EtOHext levels across the range from 50 to 300 mM EtOHext , a value consistent with several published reports. Equilibrium is rapid and complete within 5 minutes of EtOH addition. CONCLUSIONS: The calcium/CaMKII mechanism of EtOH's neurotoxicity is shared between an amniote (chick) and teleost fish, indicating that this mechanism is evolutionarily conserved. Our data suggest that EtOHext concentrations >2% (v/v) for chorion-intact embryos and 1.5% (v/v) for dechorionated embryos have limited clinical relevance. The strong parallels with established models endorse the zebrafish's relevance for mechanistic studies of EtOH's developmental neurotoxicity.
Asunto(s)
Trastornos del Espectro Alcohólico Fetal/etiología , Síndromes de Neurotoxicidad/embriología , Animales , Apoptosis/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Etanol/farmacología , Etiquetado Corte-Fin in Situ , Cresta Neural/efectos de los fármacos , Cresta Neural/embriología , Neurogénesis/efectos de los fármacos , Síndromes de Neurotoxicidad/etiología , Pez CebraRESUMEN
BACKGROUND: Prenatal alcohol exposure is a leading cause of permanent neurodevelopmental disability. Diagnosis is often initiated by a distinctive craniofacial appearance that originates, in part, from the apoptotic deletion of craniofacial progenitors, a stem cell lineage called the neural crest (NC). We recently demonstrated that alcohol causes nucleolar stress in NC through its suppression of Ribosome Biogenesis (RBG) and this suppression is causative in their p53/MDM2-mediated apoptosis. Here, we show that this nucleolar stress originates from alcohol's activation of AMPK, which suppresses TORC1 and the p70/S6K-mediated stimulation of RBG. METHODS: Alcohol-exposed cells of the pluripotent, primary cranial NC line O9-1 were evaluated with respect to their p70/S6K, TORC1, and AMPK activity. The functional impact of these signals with respect to RBG, p53, and apoptosis were assessed using gain-of-function constructs and small molecule mediators. RESULTS: Alcohol rapidly (<2hr) increased pAMPK, p-Raptor, p-mTOR(S2446), and reduced both total and p-p70/S6K in NC cells. These changes persisted for at least 12hr to 18hr following alcohol exposure. Attenuation of these signals via gain- or loss-of-function approaches prevented alcohol's suppression of rRNA synthesis and the induction of p53-stimulated apoptosis. CONCLUSIONS: We conclude that alcohol induces ribosome dysbiogenesis and activates their p53/MDM2-mediated apoptosis via its activation of pAMPK, which in turn activates Raptor to suppress the TORC1/S6K-mediated promotion of ribosome biogenesis. This represents a novel mechanism underlying alcohol's neurotoxicity and is consistent with findings that TORC1/S6K networks are critical for cranial NC survival.
RESUMEN
Prenatal alcohol exposure (PAE) causes cognitive impairment and a distinctive craniofacial dysmorphology, due in part to apoptotic losses of the pluripotent cranial neural crest cells (CNCs) that form facial bones and cartilage. We previously reported that PAE rapidly represses expression of >70 ribosomal proteins (padj = 10-E47). Ribosome dysbiogenesis causes nucleolar stress and activates p53-MDM2-mediated apoptosis. Using primary avian CNCs and the murine CNC line O9-1, we tested whether nucleolar stress and p53-MDM2 signaling mediates this apoptosis. We further tested whether haploinsufficiency in genes that govern ribosome biogenesis, using a blocking morpholino approach, synergizes with alcohol to worsen craniofacial outcomes in a zebrafish model. In both avian and murine CNCs, pharmacologically relevant alcohol exposure (20mM, 2hr) causes the dissolution of nucleolar structures and the loss of rRNA synthesis; this nucleolar stress persisted for 18-24hr. This was followed by reduced proliferation, stabilization of nuclear p53, and apoptosis that was prevented by overexpression of MDM2 or dominant-negative p53. In zebrafish embryos, low-dose alcohol or morpholinos directed against ribosomal proteins Rpl5a, Rpl11, and Rps3a, the Tcof homolog Nolc1, or mdm2 separately caused modest craniofacial malformations, whereas these blocking morpholinos synergized with low-dose alcohol to reduce and even eliminate facial elements. Similar results were obtained using a small molecule inhibitor of RNA Polymerase 1, CX5461, whereas p53-blocking morpholinos normalized craniofacial outcomes under high-dose alcohol. Transcriptome analysis affirmed that alcohol suppressed the expression of >150 genes essential for ribosome biogenesis. We conclude that alcohol causes the apoptosis of CNCs, at least in part, by suppressing ribosome biogenesis and invoking a nucleolar stress that initiates their p53-MDM2 mediated apoptosis. We further note that the facial deficits that typify PAE and some ribosomopathies share features including reduced philtrum, upper lip, and epicanthal distance, suggesting the facial deficits of PAE represent, in part, a ribosomopathy.
Asunto(s)
Apoptosis , Etanol , Cresta Neural , Ribosomas , Proteína p53 Supresora de Tumor , Pez Cebra , Animales , Cresta Neural/metabolismo , Cresta Neural/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/efectos de los fármacos , Etanol/toxicidad , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Ratones , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/efectos de los fármacos , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Cráneo/patología , Cráneo/metabolismo , Cráneo/efectos de los fármacos , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Prenatal alcohol exposure (PAE) is a leading cause of neurodevelopmental disability through its induction of neuronal growth dysfunction through incompletely understood mechanisms. Ribosome biogenesis regulates cell cycle progression through p53 and the nucleolar cell stress response. Whether those processes are targeted by alcohol is unknown. Pregnant C57BL/6J mice received 3 g alcohol/kg daily at E8.5-E17.5. Transcriptome sequencing was performed on the E17.5 fetal cortex. Additionally, primary neural stem cells (NSCs) were isolated from the E14.5 cerebral cortex and exposed to alcohol to evaluate nucleolar stress and p53/MDM2 signaling. Alcohol suppressed KEGG pathways involving ribosome biogenesis (rRNA synthesis/processing and ribosomal proteins) and genes that are mechanistic in ribosomopathies (Polr1d, Rpl11; Rpl35; Nhp2); this was accompanied by nucleolar dissolution and p53 stabilization. In primary NSCs, alcohol reduced rRNA synthesis, caused nucleolar loss, suppressed proliferation, stabilized nuclear p53, and caused apoptosis that was prevented by dominant-negative p53 and MDM2 overexpression. Alcohol's actions were dose-dependent and rapid, and rRNA synthesis was suppressed between 30 and 60 min following alcohol exposure. The alcohol-mediated deficits in ribosomal protein expression were correlated with fetal brain weight reductions. This is the first report describing that pharmacologically relevant alcohol levels suppress ribosome biogenesis, induce nucleolar stress in neuronal populations, and involve the ribosomal/MDM2/p53 pathway to cause growth arrest and apoptosis. This represents a novel mechanism of alcohol-mediated neuronal damage.
Asunto(s)
Células-Madre Neurales , Efectos Tardíos de la Exposición Prenatal , Embarazo , Humanos , Femenino , Animales , Ratones , Proteína p53 Supresora de Tumor/metabolismo , Ratones Endogámicos C57BL , Apoptosis , Etanol , Células-Madre Neurales/metabolismo , Encéfalo/metabolismoRESUMEN
Genetic selection for rapid growth in broilers has inadvertently resulted in increased susceptibility to heat stress, particularly in male birds. Increased oxidative stress associated with hyperthermia may be reduced by avian uncoupling protein (avUCP), which has been proposed to modulate free radical production. However, the relationship between avUCP expression and current heat stress management strategies is unclear. Embryonic acclimation or thermal manipulation (TM) and dietary fat source are 2 heat stress interventions that may alter avUCP expression and oxidative stress, but the literature is inconclusive. The objective of this trial was to investigate the effect of TM and dietary fat source on avUCP gene expression and oxidative damage in the breast meat of market age broilers before and after acute heat challenge. The influence of bird sex was also evaluated as broilers exhibit a high degree of sexual dimorphism in growth and stress susceptibility. Concentration of thiobarbituric acid reactive substances (TBARS) was measured as a marker of oxidative damage. Embryonic TM occurred from incubation d 7 to 16 for 12 h daily at 39.5°C. Dietary treatments were applied during the finisher period using either poultry fat, soya oil, or olive oil supplemented at 4.5% in the diet. Acute heat stress (AHS) occurred on d 43 at 32°C for 4 h. Bird performance was decreased by TM, but no significant differences were noted between dietary fat source treatments. Neither avUCP nor TBARS concentrations were significantly influenced by TM or dietary fat source. Downregulation of avUCP was observed following AHS, concurrent with an increase in TBARS concentration. Male birds exhibited higher levels of both avUCP expression and TBARS compared to females and a significant interaction was noted for heat stress by sex, with avUCP expression being greatest in males prior to AHS. The increase in avUCP expression and TBARS concentrations in male birds may be associated with an increased susceptibility to stress arising from the increased growth rate noted for male broilers.
Asunto(s)
Pollos , Trastornos de Estrés por Calor , Animales , Pollos/fisiología , Dieta/veterinaria , Suplementos Dietéticos , Trastornos de Estrés por Calor/veterinaria , Respuesta al Choque Térmico , Masculino , Proteínas Desacopladoras Mitocondriales/metabolismo , Aceite de Oliva/metabolismo , Estrés Oxidativo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Prenatal ethanol exposure causes significant neurodevelopmental deficits through its induction of apoptosis in neuronal progenitors including the neural crest. Using an established chick embryo model, we previously showed that clinically relevant ethanol concentrations cause neural crest apoptosis through mobilization of an intracellular calcium transient. How the calcium transient initiates this cell death is unknown. In this study, we identify CaMKII as the calcium target responsible for ethanol-induced apoptosis. Immunostaining revealed selective enrichment of activated phosphoCaMKII(Thr286) within ethanol-treated neural crest. CaMKII activation in response to ethanol was rapid (< 60 s) and robust, and CaMKII activity was increased 300% over control levels. Treatment with CaMKII-selective inhibitors but not those directed against CaMKIV or PKC completely prevented the cell death. Forced expression of dominant-negative CaMKII prevented ethanol's activation of CaMKII and prevented the ethanol-induced death, whereas constitutively active CaMKII in ethanol's absence significantly increased cell death to levels caused by ethanol treatment. In summary, CaMKII is the key signal that converts the ethanol-induced, short-lived Ca(i) (2+) transient into a long-lived cellular effector. This is the first identification of CaMKII as a critical mediator of ethanol-induced cell death. Because neural crest differentiates into several neuronal lineages, our findings offer novel insights into how ethanol disrupts early neurogenesis.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Depresores del Sistema Nervioso Central/toxicidad , Activación Enzimática/efectos de los fármacos , Etanol/toxicidad , Cresta Neural/efectos de los fármacos , Cresta Neural/patología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/patología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Calcio/metabolismo , Embrión de Pollo , ADN Complementario/biosíntesis , ADN Complementario/genética , Inmunohistoquímica , Proteína Quinasa C/metabolismo , Estimulación QuímicaRESUMEN
Fetal alcohol syndrome (FAS) is a common birth defect in many societies. Affected individuals have neurodevelopmental disabilities and a distinctive craniofacial dysmorphology. These latter deficits originate during early development from the ethanol-mediated apoptotic depletion of cranial facial progenitors, a population known as the neural crest. We showed previously that this apoptosis is caused because acute ethanol exposure activates G-protein-dependent intracellular calcium within cranial neural crest progenitors, and this calcium transient initiates the cell death. The dysregulated signals that reside downstream of ethanol's calcium transient and effect neural crest death are unknown. Here we show that ethanol's repression of the transcriptional effector ß-catenin causes the neural crest losses. Clinically relevant ethanol concentrations (22-78 mM) rapidly deplete nuclear ß-catenin from neural crest progenitors, with accompanying losses of ß-catenin transcriptional activity and downstream genes that govern neural crest induction, expansion, and survival. Using forced expression studies, we show that ß-catenin loss of function (via dominant-negative T cell transcription factor [TCF]) recapitulates ethanol's effects on neural crest apoptosis, whereas ß-catenin gain-of-function in ethanol's presence preserves neural crest survival. Blockade of ethanol's calcium transient using Bapta-AM normalizes ß-catenin activity and prevents the neural crest losses, whereas ionomycin treatment is sufficient to destabilize ß-catenin. We propose that ethanol's repression of ß-catenin causes the neural crest losses in this model of FAS. ß-Catenin is a novel target for ethanol's teratogenicity. ß-Catenin/Wnt signals participate in many developmental events and its rapid and persistent dysregulation by ethanol may explain why the latter is such a potent teratogen.
Asunto(s)
Apoptosis/genética , Calcio/metabolismo , Etanol/toxicidad , Trastornos del Espectro Alcohólico Fetal/metabolismo , Cresta Neural/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , beta Catenina/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Embrión de Pollo , Pollos , Modelos Animales de Enfermedad , Femenino , Trastornos del Espectro Alcohólico Fetal/genética , Humanos , Neuronas/metabolismo , Embarazo , Transducción de Señal , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Prenatal alcohol exposure (PAE) causes developmental abnormalities known as fetal alcohol spectrum disorder (FASD). Maternal iron status modulates the severity of these defects in the offspring. Because the placenta is central in supporting fetal development, we investigated whether maternal iron status similarly modulates alcohol's effects in the placenta. We hypothesized that PAE causes placental insufficiency by decreasing placental weight and efficiency, and we hypothesized that these are worsened by maternal iron deficiency (ID) and alleviated by dietary iron fortification (IF). We also determined whether altered placental iron flux and inflammatory balance contribute to placental insufficiency. Pregnant Long-Evans rats consumed an iron-deficient (ID; 2-6 ppm), iron-sufficient (IS; 100 ppm), or iron-fortified (IF; 500 ppm) diet. Alcohol (5 g/kg body weight) or isocaloric maltodextrin (MD) was gavaged daily from gestational day (GD) 13.5-19.5. Placental outcomes were evaluated on GD20.5. PAE reduced fetal weight (p < 0.0001), placental weight (p = 0.0324), and placental efficiency (p = 0.0043). PAE downregulated placental transferrin receptor (p = 0.0032); it also altered placental Il1b and Tnf expression and the Il6:Il10 ratio (p = 0.0337, 0.0300, and 0.0034, respectively) to generate a response favoring inflammation. ID-PAE further reduced fetal growth and placental efficiency and induced a heightened pro-inflammatory placental profile. IF did not rescue the alcohol-reduced fetal weight, but it normalized placental efficiency and decreased placental inflammation. These placental cytokines correlated with fetal and placental growth, and explained 45% of the variability in fetal weight and 20% of the variability in placental efficiency. In summary, alcohol induces placental insufficiency and is associated with a pro-inflammatory cytokine profile exacerbated by maternal ID and mitigated by maternal IF. Because the placenta is closely linked to intrauterine growth, the placental insufficiency reported here may correlate with the lower birth weights in a subgroup of individuals who experienced PAE.
Asunto(s)
Citocinas/metabolismo , Etanol/administración & dosificación , Trastornos del Espectro Alcohólico Fetal , Deficiencias de Hierro , Hierro de la Dieta/administración & dosificación , Fenómenos Fisiologicos Nutricionales Maternos , Placentación/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Femenino , Inflamación , Embarazo , Ratas , Ratas Long-EvansRESUMEN
BACKGROUND: Prenatal alcohol exposure causes distinctive craniofacial anomalies that arise, in part, from the apoptotic elimination of neural crest (NC) progenitors that form the face. This vulnerability of NC to alcohol is puzzling as they normally express the transcriptional repressor Snail1/2 (in chick Snai2), which suppresses apoptosis and promotes their migration. Here, we investigate alcohol's impact upon Snai2 function. METHODS: Chick cranial NC cells were treated with acute alcohol (52 mM, 2 hr). We evaluated NC migration, gene expression, proliferation, and apoptosis thereafter. RESULTS: Transient alcohol exposure induced Snai2 (191% ± 23%; p = .003) and stimulated NC migration (p = .0092). An alcohol-induced calcium transient mediated this Snai2 induction, and BAPTA-AM blocked whereas ionomycin mimicked these pro-migratory effects. Alcohol suppressed CyclinD1 protein content (59.1 ± 12%, p = .007) and NC proliferation (19.7 ± 5.8%, p < .001), but these Snai2-enriched cells still apoptosed in response to alcohol. This was explained because alcohol induced p53 (198 ± 29%, p = .023), and the p53 antagonist pifithrin-α prevented their apoptosis. Moreover, alcohol counteracted Snai2's pro-survival signals, and Bcl2 was repressed (68.5 ± 6.0% of controls, p = .016) and PUMA was not induced, while ATM (1.32-fold, p = .01) and PTEN (1.30-fold, p = .028) were elevated. CONCLUSIONS: Alcohol's calcium transient uncouples the Snai2/p53 regulatory loop that normally prevents apoptosis during EMT. This represents a novel pathway in alcohol's neurotoxicity, and complements demonstrations that alcohol suppresses PUMA in mouse NC. We propose that the NCs migratory behavior, and their requirement for Snai2/p53 co-expression, makes them vulnerable to stressors that dysregulate Snai2/p53 interactions, such as alcohol.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Aviares/metabolismo , Señalización del Calcio/efectos de los fármacos , Etanol/efectos adversos , Cresta Neural/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Embrión de Pollo , Etanol/farmacología , Cresta Neural/patología , Células-Madre Neurales/patologíaRESUMEN
Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Individuals with FASD may exhibit a characteristic facial appearance that has diagnostic utility. The mechanism by which alcohol disrupts craniofacial development is incompletely understood, as are the genetic factors that can modify individual alcohol vulnerability. Using an established avian model, we characterized the cranial transcriptome in response to alcohol to inform the mechanism underlying these cells' vulnerability. Gallus gallus embryos having 3-6 somites were exposed to 52 mM alcohol and the cranial transcriptomes were sequenced thereafter. A total of 3422 genes had significantly differential expression. The KEGG pathways with the greatest enrichment of differentially expressed gene clusters were Ribosome (P = 1.2 x 10-17, 67 genes), Oxidative Phosphorylation (P = 4.8 x 10-12, 60 genes), RNA Polymerase (P = 2.2 x 10-3, 15 genes) and Spliceosome (P = 2.6 x 10-2, 39 genes). The preponderance of transcripts in these pathways were repressed in response to alcohol. These same gene clusters also had the greatest altered representation in our previous comparison of neural crest populations having differential vulnerability to alcohol-induced apoptosis. Comparison of differentially expressed genes in alcohol-exposed (3422) and untreated, alcohol-vulnerable (1201) transcriptomes identified 525 overlapping genes of which 257 have the same direction of transcriptional change. These included 36 ribosomal, 25 oxidative phosphorylation and 7 spliceosome genes. Using a functional approach in zebrafish, partial knockdown of ribosomal proteins zrpl11, zrpl5a, and zrps3a individually heightened vulnerability to alcohol-induced craniofacial deficits and increased apoptosis. In humans, haploinsufficiency of several of the identified ribosomal proteins are causative in craniofacial dysmorphologies such as Treacher Collins Syndrome and Diamond-Blackfan Anemia. This work suggests ribosome biogenesis may be a novel target mediating alcohol's damage to developing neural crest. Our findings are consistent with observations that gene-environment interactions contribute to vulnerability in FASD.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Etanol/toxicidad , Perfilación de la Expresión Génica , Biogénesis de Organelos , Ribosomas/metabolismo , Teratogénesis/efectos de los fármacos , Animales , Embrión de Pollo , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Cresta Neural/efectos de los fármacos , Cresta Neural/metabolismo , Reproducibilidad de los Resultados , Teratogénesis/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Precise regulation of retinoid levels is critical for normal heart development. Retinol-binding protein (RBP), an extracellular retinol transporter, is strongly secreted by cardiogenic endoderm. This study addresses whether RBP gene ablation affects heart development. Despite exhibiting an >85% decrease in serum retinol, adult RBP-null mice are viable, breed, and have normal vision when maintained on a vitamin A-sufficient diet. Comparison of RBP-null with wild-type (WT) hearts from embryos at day 9.0 (E9.0) through E12.5 revealed an RBP-null phenotype similar to that of other retinoid-deficient models. At an early stage, RBP-null hearts display retarded trabecular development, which recovers by E9.5. This is accompanied at E9.5 and E10.5 by precocious differentiation of subepicardial cardiac myocytes. Most remarkably, RBP-null hearts display augmented deposition of fibronectin protein in the cardiac jelly at E9.0 through E10.5 and in the outflow tract at E12.5. This phenomenon, which was detected by immunohistochemistry and Western blotting without increased fibronectin transcript levels, is accompanied by increased numbers of mesenchymal cells in the outflow tract but not in the atrioventricular canal. RBP-null cardiac myocytes, especially in the subepicardial layer, display increased cell proliferation. This phenotype may present a model of subclinical retinoid insufficiency characterized by alteration of an extracellular matrix component and altered cellular differentiation and proliferation, changes that may have functional consequences for adult cardiac function. This murine model may have relevance to fetal development in human populations with inadequate retinoid intake.
Asunto(s)
Fibronectinas/metabolismo , Corazón/embriología , Miocardio/metabolismo , Proteínas de Unión al Retinol/fisiología , Animales , Northern Blotting , Western Blotting , División Celular/fisiología , Embrión de Mamíferos/metabolismo , Femenino , Fibronectinas/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Miocardio/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al Retinol/genética , Factores de TiempoRESUMEN
Prenatal alcohol exposure (PAE) causes distinctive facial characteristics in some pregnancies and not others; genetic factors may contribute to this differential vulnerability. Ethanol disrupts multiple events of neural crest development, including induction, survival, migration, and differentiation. Animal models and genomic approaches have substantially advanced our understanding of the mechanisms underlying these facial changes. PAE during gastrulation produces craniofacial changes corresponding with human fetal alcohol syndrome. These result because PAE reduces prechordal plate extension and suppresses sonic hedgehog, leading to holoprosencephaly and malpositioned facial primordia. Haploinsufficiency in sonic hedgehog signaling increases vulnerability to facial deficits and may influence some PAE pregnancies. In contrast, PAE during early neurogenesis produces facial hypoplasia, preceded by neural crest reductions due to significant apoptosis. Factors mediating this apoptosis include intracellular calcium mobilization, elevated reactive oxygen species, and loss of trophic support from ß-catenin/calcium, sonic hedgehog, and mTOR signaling. Genome-wide SNP analysis links PDGFRA with facial outcomes in human PAE. Multiple genomic-level comparisons of ethanol-sensitive and - resistant early embryos, in both mouse and chick, independently identify common candidate genes that may potentially modify craniofacial vulnerability, including ribosomal proteins, proteosome, RNA splicing, and focal adhesion. In summary, research using animal models with genome-level differences in ethanol vulnerability, as well as targeted loss-and gain-of-function mutants, has clarified the mechanisms mediating craniofacial change in PAE. The findings additionally suggest that craniofacial deficits may represent a gene-ethanol interaction for some affected individuals. Genetic-level changes may prime individuals toward greater sensitivity or resistance to ethanol's neurotoxicity.
RESUMEN
The avian embryo is a long-standing model for developmental biology research. It also has proven utility for toxicology research both in ovo and in explant culture. Like mammals, avian embryos have an allantois and their developmental pathways are highly conserved with those of mammals, thus avian models have biomedical relevance. Fertile eggs are inexpensive and the embryo develops rapidly, allowing for high-throughput. The chick genome is sequenced and significant molecular resources are available for study, including the ability for genetic manipulation. The absence of a placenta permits the direct study of an agent's embryotoxic effects. Here, we present protocols for using avian embryos in toxicology research, including egg husbandry and hatch, toxicant delivery, and assessment of proliferation, apoptosis, and cardiac structure and function.
Asunto(s)
Embrión de Pollo/citología , Modelos Animales de Enfermedad , Crianza de Animales Domésticos , Animales , Apoptosis , Proliferación Celular , Embrión de Pollo/diagnóstico por imagen , Técnicas de Cultivo , Biología Evolutiva , Ecocardiografía/métodos , Teratología , Pruebas de Toxicidad/métodosRESUMEN
Fetal alcohol spectrum disorders (FASD) are the leading non-genetic cause of neurodevelopmental disability in children. Although alcohol is clearly teratogenic, environmental factors such as gravidity and socioeconomic status significantly modify individual FASD risk despite equivalent alcohol intake. An explanation for this variability could inform FASD prevention. Here we show that the most common nutritional deficiency of pregnancy, iron deficiency without anemia (ID), is a potent and synergistic modifier of FASD risk. Using an established rat model of third trimester-equivalent binge drinking, we show that ID significantly interacts with alcohol to impair postnatal somatic growth, associative learning, and white matter formation, as compared with either insult separately. For the associative learning and myelination deficits, the ID-alcohol interaction was synergistic and the deficits persisted even after the offsprings' iron status had normalized. Importantly, the observed deficits in the ID-alcohol animals comprise key diagnostic criteria of FASD. Other neurobehaviors were normal, showing the ID-alcohol interaction was selective and did not reflect a generalized malnutrition. Importantly ID worsened FASD outcome even though the mothers lacked overt anemia; thus diagnostics that emphasize hematological markers will not identify pregnancies at-risk. This is the first direct demonstration that, as suggested by clinical studies, maternal iron status has a unique influence upon FASD outcome. While alcohol is unquestionably teratogenic, this ID-alcohol interaction likely represents a significant portion of FASD diagnoses because ID is more common in alcohol-abusing pregnancies than generally appreciated. Iron status may also underlie the associations between FASD and parity or socioeconomic status. We propose that increased attention to normalizing maternal iron status will substantially improve FASD outcome, even if maternal alcohol abuse continues. These findings offer novel insights into how alcohol damages the developing brain.
Asunto(s)
Consumo Excesivo de Bebidas Alcohólicas/metabolismo , Etanol/farmacología , Trastornos del Espectro Alcohólico Fetal/metabolismo , Deficiencias de Hierro , Intercambio Materno-Fetal , Complicaciones del Embarazo/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Animales , Animales Recién Nacidos , Consumo Excesivo de Bebidas Alcohólicas/fisiopatología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Cognición/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Trastornos del Espectro Alcohólico Fetal/etiología , Trastornos del Espectro Alcohólico Fetal/patología , Trastornos del Espectro Alcohólico Fetal/fisiopatología , Feto/efectos de los fármacos , Número de Embarazos , Aprendizaje/efectos de los fármacos , Embarazo , Complicaciones del Embarazo/fisiopatología , Efectos Tardíos de la Exposición Prenatal/etiología , Efectos Tardíos de la Exposición Prenatal/patología , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Ratas , Factores de RiesgoRESUMEN
Prenatal alcohol exposure is a leading cause of childhood neurodevelopmental disability. The adverse behavioral effects of alcohol exposure during the second and third trimester are well documented; less clear is whether early first trimester-equivalent exposures also alter behavior. We investigated this question using an established chick model of alcohol exposure. In ovo embryos experienced a single, acute ethanol exposure that spanned gastrulation through neuroectoderm induction and early brain patterning (19-22h incubation). At 7 days posthatch, the chicks were evaluated for reflexive motor function (wingflap extension, righting reflex), fearfulness (tonic immobility [TI]), and fear/social reinstatement (open-field behavior). Chicks exposed to a peak ethanol level of 0.23-0.28% were compared against untreated and saline-treated controls. Birds receiving early ethanol exposure had a normal righting reflex and a significantly reduced wingflap extension in response to a sudden descent. The ethanol-treated chicks also displayed heightened fearfulness, reflected in increased frequency of TI, and they required significantly fewer trials for its induction. In an open-field test, ethanol treatment did not affect latency to move, steps taken, vocalizations, defecations, or escape attempts. The current findings demonstrate that early ethanol exposure can increase fearfulness and impair aspects of motor function. Importantly, the observed dysfunctions resulted from an acute ethanol exposure during the period when the major brain components are induced and patterned. The equivalent period in human development is 3-4 weeks postconception. The current findings emphasize that ethanol exposure during the early first trimester equivalent can produce neurodevelopmental disability in the offspring.