Advances in the development of novel antioxidant therapies as an approach for fetal alcohol syndrome prevention

Ethanol is the most common human teratogen, and its consumption during pregnancy can produce a wide range of abnormalities in infants known as fetal alcohol spectrum disorder (FASD). The major characteristics of FASD can be divided into: (i) growth retardation, (ii) craniofacial abnormalities, and (iii) central nervous system (CNS) dysfunction. FASD is the most common cause of nongenetic mental retardation in Western countries. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, the induction of oxidative stress is believed to be one central process linked to the development of the disease. Currently, there is no known effective strategy for prevention (other than alcohol avoidance) or treatment. In the present review we will provide the state of art in the evidence for the use of antioxidants as a potential therapeutic strategy for the treatment using whole‐embryo and culture cells models of FASD. We conclude that the imbalance of the intracellular redox state contributes to the pathogenesis observed in FASD models, and we suggest that antioxidant therapy can be considered a new efficient strategy to mitigate the effects of prenatal ethanol exposure. Birth Defects Research (Part A) 103:163–177, 2015. © 2014 Wiley Periodicals, Inc.     

Taurine protects cerebellar neurons of the external granular layer against ethanol-induced apoptosis in 7-day-old mice

Acute alcohol administration is harmful especially for the developing nervous system, where it induces massive apoptotic neurodegeneration leading to alcohol-related disorders of newborn infants. Neuroprotection against ethanol-induced apoptosis may save neurons and reduce the consequences of maternal alcohol consumption. Previously we have shown that taurine protects immature cerebellar neurons in the internal granular layer of cerebellum from ethanol-induced apoptosis. Now we describe a similar protective action for taurine in the external layer of cerebellum of 7-day-old mice. The mice were divided into three groups: ethanol-treated, ethanol + taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 0 h and 2.5 g/kg at 2 h) to the ethanol and ethanol + taurine groups. The ethanol + taurine group also received subcutaneously two injections of taurine (1 g/kg each, 1 h before the first dose of ethanol and 1 h after the second dose of ethanol). To verify apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I–X of the cerebellar vermis at 8 h after the first ethanol injection. Ethanol induced apoptosis in the cerebellar external granular layer. Taurine treatment significantly reduced the number of activated caspase-3-immunoreactive and TUNEL-positive cells. Taurine has thus a neuroprotective antiapoptotic action in the external granular layer of the cerebellum, preserving a number of neurons from ethanol-induced apoptosis.     

Face off against ROS: Tcof1/Treacle safeguards neuroepithelial cells and progenitor neural crest cells from oxidative stress during craniofacial development

One‐third of all congenital birth defects affect the head and face, and most craniofacial anomalies are considered to arise through defects in the development of cranial neural crest cells. Cranial neural crest cells give rise to the majority of craniofacial bones, cartilages and connective tissues. Therefore, understanding the events that control normal cranial neural crest and subsequent craniofacial development is important for elucidating the pathogenetic mechanisms of craniofacial anomalies and for the exploring potential therapeutic avenues for their prevention. Treacher Collins syndrome (TCS) is a congenital disorder characterized by severe craniofacial anomalies. An animal model of TCS, generated through mutation of Tcof1, the mouse (Mus musculus) homologue of the gene primarily mutated in association with TCS in humans, has recently revealed significant insights into the pathogenesis of TCS. Apoptotic elimination of neuroepithelial cells including neural crest cells is the primary cause of craniofacial defects in Tcof1 mutant embryos. However, our understanding of the mechanisms that induce tissue‐specific apoptosis remains incomplete. In this review, we describe recent advances in our understanding of the pathogenesis TCS. Furthermore, we discuss the role of Tcof1 in normal embryonic development, the correlation between genetic and environmental factors on the severity of craniofacial abnormalities, and the prospect for prenatal prevention of craniofacial anomalies.     

Apoptosis of premigratory neural crest cells in rhombomeres 3 and 5: consequences for patterning of the branchial region

In the avian hindbrain, premigratory neural crest cells undergo programmed cell death (apoptosis) in rhombomeres 3 and 5 (r3, r5). Here, we have attempted to analyze the significance of the loss of neural crest cells from these odd-numbered rhombomeres. When apoptosis is prevented in r3 and r5, r3 crest migrate into the first arch and r5 into the third arch. Interestingly, these extra neural crest cells contributed to the formation of ectopic muscle attachment sites that are also found in those species in which r3 and r5 neural crest cells do not undergo apoptosis. Thus, apoptosis in the odd-numbered rhombomeres appears to be an evolutionarily derived mechanism that is required to eliminate r3 and r5 crest migration into first and third arches and thereby remove these muscle attachment sites.     

Autophagy and ethanol neurotoxicity

Excessive ethanol exposure is detrimental to the brain. The developing brain is particularly vulnerable to ethanol such that prenatal ethanol exposure causes fetal alcohol spectrum disorders (FASD). Neuronal loss in the brain is the most devastating consequence and is associated with mental retardation and other behavioral deficits observed in FASD. Since alcohol consumption during pregnancy has not declined, it is imperative to elucidate the underlying mechanisms and develop effective therapeutic strategies. One cellular mechanism that acts as a protective response for the central nervous system (CNS) is autophagy. Autophagy regulates lysosomal turnover of organelles and proteins within cells, and is involved in cell differentiation, survival, metabolism, and immunity. We have recently shown that ethanol activates autophagy in the developing brain. The autophagic preconditioning alleviates ethanol-induced neuron apoptosis, whereas inhibition of autophagy potentiates ethanol-stimulated reactive oxygen species (ROS) and exacerbates ethanol-induced neuroapoptosis. The expression of genes encoding proteins required for autophagy in the CNS is developmentally regulated; their levels are much lower during an ethanol-sensitive period than during an ethanol-resistant period. Ethanol may stimulate autophagy through multiple mechanisms; these include induction of oxidative stress and endoplasmic reticulum stress, modulation of MTOR and AMPK signaling, alterations in BCL2 family proteins, and disruption of intracellular calcium (Ca2+) homeostasis. This review discusses the most recent evidence regarding the involvement of autophagy in ethanol-mediated neurotoxicity as well as the potential therapeutic approach of targeting autophagic pathways.     

Specification of neural crest cell formation and migration in mouse embryos

Of all the model organisms used to study human development, rodents such as mice most accurately reflect human craniofacial development. Collective advances in mouse embryology and mouse genetics continue to shape our understanding of neural crest cell development and by extrapolation the etiology of human congenital head and facial birth defects. The aim of this review is to highlight the considerable progress being made in our understanding of cranial neural crest cell patterning in mouse embryos.     

Analysis of Drosophila nervous system development following an early,brief exposure to ethanol

The effects of ethanol on neural function and development have been studied extensively, motivated in part by the addictive properties of alcohol and the neurodevelopmental deficits that arise in children with fetal alcohol spectrum disorder (FASD). Absent from this research area is a genetically tractable system to study the effects of early ethanol exposure on later neurodevelopmental and behavioral phenotypes. Here, we used embryos of the fruit fly, Drosophila melanogaster, as a model system to investigate the neuronal defects that arise after an early exposure to ethanol. We found several disruptions of neural development and morphology following a brief ethanol exposure during embryogenesis and subsequent changes in larval behavior. Altogether, this study establishes a new system to examine the effects of alcohol exposure in embryos and the potential to conduct large‐scale genetics screens to uncover novel factors that sensitize or protect neurons to the effects of alcohol.     

GSK3β in Ethanol Neurotoxicity

Alcohol consumption during pregnancy is a significant public health problem and may result in a wide range of adverse outcomes for the child. The developing central nervous system (CNS) is particularly susceptible to ethanol toxicity. Children with fetal alcohol spectrum disorders (FASD) have a variety of cognitive, behavioral, and neurological impairments. FASD currently represents the leading cause of mental retardation in North America ahead of Down syndrome and cerebral palsy. Ethanol exposure during development causes multiple abnormalities in the brain such as permanent loss of neurons, ectopic neurons, and alterations in synaptogenesis and myelinogenesis. These alcohol-induced structural alterations in the developing brain underlie many of the behavioral deficits observed in FASD. The cellular and molecular mechanisms of ethanol neurotoxicity, however, remain unclear. Ethanol elicits cellular stresses, including oxidative stress and endoplasmic reticulum stress. Glycogen synthase kinase 3β (GSK3β), a multifunctional serine/threonine kinase, responds to various cellular stresses. GSK3β is particularly abundant in the developing CNS, and regulates diverse developmental events in the immature brain, such as neurogenesis and neuronal differentiation, migration, and survival. Available evidence indicates that the activity of GSK3β in the CNS is affected by ethanol. GSK3β inhibition provides protection against ethanol neurotoxicity, whereas high GSK3β activity/expression sensitizes neuronal cells to ethanol-induced damages. It appears that GSK3β is a converging signaling point that mediates some of ethanol’s neurotoxic effects.     

Paternal Genetic Contribution Influences Fetal Vulnerability to Maternal Alcohol Consumption in a Rat Model of Fetal Alcohol Spectrum Disorder

Background

Fetal alcohol exposure causes in the offspring a collection of permanent physiological and neuropsychological deficits collectively termed Fetal Alcohol Spectrum Disorder (FASD). The timing and amount of exposure cannot fully explain the substantial variability among affected individuals, pointing to genetic influences that mediate fetal vulnerability. However, the aspects of vulnerability that depend on the mother, the father, or both, are not known.

Methodology/Principal Findings

Using the outbred Sprague-Dawley (SD) and inbred Brown Norway (BN) rat strains as well as their reciprocal crosses, we administered ethanol (E), pair-fed (PF), or control (C) diets to the pregnant dams. The dams'' plasma levels of free thyroxine (fT4), triiodothyronine (T3), free T3 (fT3), and thyroid stimulating hormone (TSH) were measured to elucidate potential differences in maternal thyroid hormonal environment, which affects specific aspects of FASD. We then compared alcohol-exposed, pair fed, and control offspring of each fetal strain on gestational day 21 (G21) to identify maternal and paternal genetic effects on bodyweight and placental weight of male and female fetuses.

Conclusions

SD and BN dams exhibited different baseline hypothalamic-pituitary-thyroid function. Moreover, the thyroid function of SD dams was more severely affected by alcohol consumption while that of BN dams was relatively resistant. This novel finding suggests that genetic differences in maternal thyroid function are one source of maternal genetic effects on fetal vulnerability to FASD. The fetal vulnerability to decreased bodyweight after alcohol exposure depended on the genetic contribution of both parents, not only maternal contribution as previously thought. In contrast, the effect of maternal alcohol consumption on placental weight was consistent and not strain-dependent. Interestingly, placental weight in fetuses with different paternal genetic contributions exhibited opposite responses to caloric restriction (pair feeding). In summary, these novel findings demonstrate both maternal and paternal genetic contributions to in utero vulnerability to alcohol, refining our understanding of the genetically-based heterogeneity seen in human FASD.     

Common mechanisms in development and disease: BMP signaling in craniofacial development

BMP signaling is one of the key pathways regulating craniofacial development. It is involved in the early patterning of the head, the development of cranial neural crest cells, and facial patterning. It regulates development of its mineralized structures, such as cranial bones, maxilla, mandible, palate, and teeth. Targeted mutations in the mouse have been instrumental to delineate the functional involvement of this signaling network in different aspects of craniofacial development. Gene polymorphisms and mutations in BMP pathway genes have been associated with various non-syndromic and syndromic human craniofacial malformations. The identification of intricate cellular interactions and underlying molecular pathways illustrate the importance of local fine-regulation of Bmp signaling to control proliferation, apoptosis, epithelial-mesenchymal interactions, and stem/progenitor differentiation during craniofacial development. Thus, BMP signaling contributes both to shape and functionality of our facial features. BMP signaling also regulates postnatal craniofacial growth and is associated with dental structures life-long. A more detailed understanding of BMP function in growth, homeostasis, and repair of postnatal craniofacial tissues will contribute to our ability to rationally manipulate this signaling network in the context of tissue engineering.     

Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels

Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental malformations caused by alcohol consumption during pregnancy. Fetal Alcohol Syndrome (FAS), the strongest manifestation of FASD, results in short stature, microcephally and facial dysmorphogenesis including microphthalmia. Using Xenopus embryos as a model developmental system, we show that ethanol exposure recapitulates many aspects of FAS, including a shortened rostro-caudal axis, microcephally and microphthalmia. Temporal analysis revealed that Xenopus embryos are most sensitive to ethanol exposure between late blastula and early/mid gastrula stages. This window of sensitivity overlaps with the formation and early function of the embryonic organizer, Spemann's organizer. Molecular analysis revealed that ethanol exposure of embryos induces changes in the domains and levels of organizer-specific gene expression, identifying Spemann's organizer as an early target of ethanol. Ethanol also induces a defect in convergent extension movements that delays gastrulation movements and may affect the overall length. We show that mechanistically, ethanol is antagonistic to retinol (Vitamin A) and retinal conversion to retinoic acid, and that the organizer is active in retinoic acid signaling during early gastrulation. The model suggests that FASD is induced in part by an ethanol-dependent reduction in retinoic acid levels that are necessary for the normal function of Spemann's organizer.     

Neural crest cells: From developmental biology to clinical interventions

Neural crest cells are multipotent cells, which are specified in embryonic ectoderm in the border of neural plate and epiderm during early development by interconnection of extrinsic stimuli and intrinsic factors. Neural crest cells are capable of differentiating into various somatic cell types, including melanocytes, craniofacial cartilage and bone, smooth muscle, and peripheral nervous cells, which supports their promise for cell therapy. In this work, we provide a comprehensive review of wide aspects of neural crest cells from their developmental biology to applicability in medical research. We provide a simplified model of neural crest cell development and highlight the key external stimuli and intrinsic regulators that determine the neural crest cell fate. Defects of neural crest cell development leading to several human disorders are also mentioned, with the emphasis of using human induced pluripotent stem cells to model neurocristopathic syndromes. Birth Defects Research (Part C) 102:263–274, 2014. © 2014 Wiley Periodicals, Inc.     

Early expression of Tubulin Beta-III in avian cranial neural crest cells

Neural crest cells are a transient stem-like cell population that forms in the dorsal neural tube of vertebrate embryos and then migrates to various locations to differentiate into diverse derivatives such as craniofacial bone, cartilage, and the enteric and peripheral nervous systems. The current dogma of neural crest cell development suggests that there is a specific hierarchical gene regulatory network (GRN) that controls the induction, specification, and differentiation of these cells at specific developmental times. Our lab has identified that a marker of differentiated neurons, Tubulin Beta-III (TUBB3), is expressed in premigratory neural crest cells. TUBB3 has previously been identified as a major constituent of microtubules and is required for the proper guidance and maintenance of axons during development. Using the model organism, Gallus gallus, we have characterized the spatiotemporal localization of TUBB3 in early stages of development. Here we show TUBB3 is expressed in the developing neural plate, is upregulated in the pre-migratory cranial neural crest prior to cell delamination and migration, and it is maintained or upregulated in neurons in later developmental stages. We believe that TUBB3 likely has a role in early neural crest formation and migration separate from its role in neurogenesis.     

Temporal control of neural crest lineage generation by Wnt/β-catenin signaling

Wnt/β-catenin signaling controls multiple steps of neural crest development, ranging from neural crest induction, lineage decisions, to differentiation. In mice, conditional β-catenin inactivation in premigratory neural crest cells abolishes both sensory neuron and melanocyte formation. Intriguingly, the generation of melanocytes is also prevented by activation of β-catenin in the premigratory neural crest, which promotes sensory neurogenesis at the expense of other neural crest derivatives. This raises the question of how Wnt/β-catenin signaling regulates the formation of distinct lineages from the neural crest. Using various Cre lines to conditionally activate β-catenin in neural crest cells at different developmental stages, we show that neural crest cell fate decisions in vivo are subject to temporal control by Wnt/β-catenin. Unlike in premigratory neural crest, β-catenin activation in migratory neural crest cells promotes the formation of ectopic melanoblasts, while the production of most other lineages is suppressed. Ectopic melanoblasts emerge at sites of neural crest target structures and in many tissues usually devoid of neural crest-derived cells. β-catenin activation at later stages in glial progenitors or in melanoblasts does not lead to surplus melanoblasts, indicating a narrow time window of Wnt/β-catenin responsiveness during neural crest cell migration. Thus, neural crest cells appear to be multipotent in vivo both before and after emigration from the neural tube but adapt their response to extracellular signals in a temporally controlled manner.     

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.