Susan M. Smith

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.

Background: Prenatal alcohol exposure (PAE) causes neurodevelopmental disabilities, and gestational iron deficiency (ID) selectively worsens learning

Background: Gestational alcohol exposure causes lifelong physical and neurocognitive deficits collectively referred to as fetal alcohol spectrum disorders (FASDs). Micronutrient deficiencies are common in pregnancies of alcohol‐abusing women. Here we show the most common micronutrient deficiency of pregnancy—iron deficiency without anemia—significantly worsens neurocognitive outcomes following perinatal alcohol exposure.Methods: Pregnant rats were fed iron‐deficient (ID) or iron‐sufficient diets from gestational day 13 to postnatal day (P) 7. Pups received alcohol (0, 3.5, 5.0 g/kg) from P 4 to P 9, targeting the brain growth spurt. At P 32, learning was assessed using delay or trace eyeblink classical conditioning (ECC). Cerebellar interpositus nucleus (IPN) and hippocampal CA1 cellularity was quantified using unbiased stereology.Results: Global analysis of variance revealed that ID and alcohol separately and significantly reduced ECC learning with respect to amplitude (ps ≤ 0.001) and conditioned response [CR] percentage (ps ≤ 0.001). Iron and alcohol interacted to reduce CR percentage in the trace ECC task (p = 0.013). Both ID and alcohol significantly reduced IPN (ps < 0.001) and CA1 cellularity (ps < 0.005). CR amplitude correlated with IPN cellularity (delay: r = 0.871, trace: r = 0.703, ps < 0.001) and CA1 cellularity (delay: r = 0.792, trace: r = 0.846, ps < 0.001) across both tasks. The learning impairments persisted even though the offsprings' iron status had normalized.Conclusions: Supporting our previous work, gestational ID exacerbates the associative learning deficits in this rat model of FASD. This is strongly associated with cellular reductions within the ECC neurocircuitry. Significant learning impairments in FASD could be the consequence, in part, of pregnancies in which the mother was also iron inadequate.

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 β‐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. Birth Defects Research (Part C) 102:210–220, 2014.

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 β-catenin/calcium, sonic hedgehog, and mTOR signaling. Genomewide SNP analysis links PDGF receptor genes 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.

Background Fetal alcohol spectrum disorders (FASD) is a leading cause of neurodevelopmental disability. Genetic factors can modify vulnerability to FASD, but these elements are poorly characterized. Methods We performed high‐throughput transcriptional profiling to identify gene candidates that could potentially modify vulnerability to ethanol's (EtOH's) neurotoxicity. We interrogated a unique genetic resource, neuroprogenitor cells from 2 closely related Gallus gallus lines having well‐characterized robust or attenuated EtOH responses with respect to intracellular calcium mobilization and CaMKII/β‐catenin‐dependent apoptosis. Samples were not exposed to EtOH prior to analysis. Results We identified 363 differentially expressed genes in neuroprogenitors from these 2 lines. Kyoto Encyclopedia of Genes and Genomes analysis revealed several gene clusters having significantly differential enrichment in gene expression. The largest and most significant cluster comprised ribosomal proteins (38 genes, p = 1.85 × 10⁻⁴⁷). Other significantly enriched gene clusters included metabolism (25 genes, p = 0.0098), oxidative phosphorylation (18 genes, p = 1.10 × 10⁻¹¹), spliceosome (13 genes, p = 7.02 × 10⁻⁸), and protein processing in the endoplasmic reticulum (9 genes, p = 0.0011). Inspection of gene ontogeny (GO) terms identified 24 genes involved in the calcium/β‐catenin signals that mediate EtOH's neurotoxicity in this model, including β‐catenin itself and both calmodulin isoforms. Conclusions Four of the identified pathways with altered transcript abundance mediate the flow of cellular information from RNA to protein. Importantly, ribosome biogenesis also senses nucleolar stress and regulates p53‐mediated apoptosis in neural crest. Human ribosomopathies produce craniofacial malformations and 11 known ribosomopathy genes were differentially expressed in this model of neural crest apoptosis. Rapid changes in ribosome expression are consistently observed in EtOH‐treated mouse embryo neural folds, a model that is developmentally similar to ours. The recurring identification of ribosome biogenesis suggests it is a candidate modifier of EtOH vulnerability. These results highlight this approach's efficacy to formulate new, mechanistic hypotheses regarding EtOH's developmental damage.

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 EtOH_int, 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 EtOH_ext levels across the range from 50 to 300 mM EtOH_ext, 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 EtOH_ext 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.

Background: Prenatal alcohol exposure (PAE) is a leading cause of significant neurobehavioral and neurocognitive deficits. Its potential consequences for eating behaviors, nutritional status, and other nutritional issues in childhood have received little attention.Methods: Nineteen children (11 boys, 8 girls) of mean age 9.6 years, referred for fetal alcohol spectrum disorder (FASD) screening and assessment, were analyzed with physical exams and caregiver questionnaires to identify possible abnormalities in food and eating behaviors. Fourteen children contributed 24‐hour diet recalls and were assessed for nutritional status.Results: Seventy‐nine percent of participants were diagnosed with FASD and 63.2% had confirmed PAE. Fifty percent of girls were overweight or obese, whereas 37% of boys had reduced stature, weight, or body mass index for their age. Recurring feeding problems included constant snacking (36.8%), lack of satiety (26.3%), and picky eating/poor appetite (31.6%). None had oral feeding problems. Constipation was common (26.3%). Macronutrient intakes were largely normal, but sugar consumption was excessive (140 to 190% of recommendations) in 57% of subjects. Vitamin A intake exceeded the upper limit for 64% of participants, whereas ≥50% had intakes <80% of recommended daily allowances (RDAs) for choline, vitamin E, potassium, β‐carotene, and essential fatty acids; 100% had vitamin D intakes <80% of the RDA.Conclusions: PAE may be associated with altered acquisition and distribution of body mass with increasing age. Disordered eating was common. The increased feeding behaviors surrounding lack of satiety suggest that self‐regulation may be altered. Constipation could reflect low dietary fiber or altered gastrointestinal function. These exploratory data suggest that children with PAE may be at risk for nutritional deficiencies, which are influenced by inappropriate food preferences, disordered eating patterns, medication use, and the stressful dynamics surrounding food preparation and mealtime.

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 β‐catenin, which normally provides trophic support to these cells. β‐catenin over‐expression normalized cell survival in ethanol's presence. CaMKII inhibition similarly restored β‐catenin content and transcriptional activity within ethanol‐treated cells and prevented their cell death. In contrast, inhibition of alternative effectors known to destabilize β‐catenin, including glycogen synthase kinase‐3β, Protein Kinase C, JNK, and calpain, failed to normalize cell survival and β‐catenin activity in ethanol's presence. Importantly, we found that purified CaMKII can directly phosphorylate β‐catenin. Using targeted mutagenesis we identified CaMKII phosphorylation sites within human β‐catenin at T332, T472, and S552. This is the first demonstration that β‐catenin is a phosphorylation target of CaMKII and represents a novel mechanism by which calcium signals could regulate β‐catenin‐dependent transcription. These results inform ethanol's neurotoxicity and offer unexpected insights into other neurodevelopmental and neurodegenerative disorders having dysregulated calcium or β‐catenin signaling. image We proposed that ethanol initiates apoptosis in early neuroprogenitors through an intracellular calcium transient that activates CaMKII, which in turn destabilizes transcriptionally‐active beta‐catenin, perhaps through direct phosphorylation at Thr332, Thr472 and Ser552. This represents a novel mechanism of calcium‐modulation of Wnt signaling and of ethanol's neurotoxicity.