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We previously showed that ethanol did not kill fetal neural stem cells (NSCs), but that their numbers nevertheless are decreased due to aberrant maturation and loss of self-renewal. To identify mechanisms that mediate this loss of NSCs, we focused on a family of Gag-like proteins (GLPs), derived from retroviral gene remnants within mammalian genomes. GLPs are important for fetal development, though their role in brain development is virtually unexplored. Moreover, GLPs may be transferred between cells in extracellular vesicles (EVs) and thereby transfer environmental adaptations between cells. We hypothesized that GLPs may mediate some effects of ethanol in NSCs.Sex-segregated male and female fetal murine cortical NSCs, cultured ex vivo as nonadherent neurospheres, were exposed to a dose range of ethanol and to mitogen-withdrawal-induced differentiation. We used siRNAs to assess the effects of NSC-expressed GLP knockdown on growth, survival, and maturation and in silico GLP knockout, in an in vivo single-cell RNA-sequencing dataset, to identify GLP-mediated developmental pathways that were also ethanol-sensitive.PEG10 isoform-1, isoform-2, and PNMA2 were identified as dominant GLP species in both NSCs and their EVs. Ethanol-exposed NSCs exhibited significantly elevated PEG10 isoform-2 and PNMA2 protein during differentiation. Both PEG10 and PNMA2 were mediated apoptosis resistance and additionally, PEG10 promoted neuronal and astrocyte lineage maturation. Neither GLP influenced metabolism nor cell cycle in NSCs. Virtual PEG10 and PNMA2 knockout identified gene transcription regulation and ubiquitin-ligation processes as candidate mediators of GLP-linked prenatal alcohol effects.Collectively, GLPs present in NSCs and their EVs may confer apoptosis resistance within the NSC niche and contribute to the abnormal maturation induced by ethanol.

Summary: Prenatal alcohol exposure (PAE) results in cerebral cortical dysgenesis. Single-cell RNA sequencing was performed on murine fetal cerebral cortical cells from six timed pregnancies, to decipher persistent cell- and sex-specific effects of an episode of PAE during early neurogenesis. We found, in an analysis of 38 distinct neural subpopulations across 8 lineage subtypes, that PAE altered neural maturation and cell cycle and disrupted gene co-expression networks. Whereas most differentially regulated genes were inhibited, particularly in females, PAE also induced sex-independent neural expression of fetal hemoglobin, a presumptive epigenetic stress adaptation. PAE inhibited Bcl11a, Htt, Ctnnb1, and other upstream regulators of differentially expressed genes and inhibited several autism-linked genes, suggesting that neurodevelopmental disorders share underlying mechanisms. PAE females exhibited neural loss of X-inactivation, with correlated activation of autosomal genes and evidence for spliceosome dysfunction. Thus, episodic PAE persistently alters the developing neural transcriptome, contributing to sex- and cell-type-specific teratology.

Prenatal alcohol exposure (PAE) can result in cognitive and behavioral disabilities and growth deficits. Because alcohol-related neurobehavioral deficits may occur in the absence of overt dysmorphic features or growth deficits, there is a need to identify biomarkers of PAE that can predict neurobehavioral impairment. In this study, we assessed infant plasma extracellular, circulating miRNAs (exmiRNAs) obtained from a heavily exposed Cape Town cohort to determine whether these can be used to predict PAE-related growth restriction and cognitive impairment. PAE, controlling for smoking as a covariate, altered 27% of expressed exmiRNAs with clinically-relevant effect sizes (Cohen’s d ≥ 0.4). Moreover, at 2 weeks, PAE increased correlated expression of exmiRNAs across chromosomes, suggesting potential co-regulation. In confirmatory factor analysis, the variance in expression for PAE-altered exmiRNAs at 2 weeks and 6.5 months was best described by three-factor models. Pathway analysis found that factors at 2 weeks were associated with (F1) cell maturation, cell cycle inhibition, and somatic growth, (F2) cell survival, apoptosis, cardiac development, and metabolism, and (F3) cell proliferation, skeletal development, hematopoiesis, and inflammation, and at 6.5 months with (F1) neurodevelopment, neural crest/mesoderm-derivative development and growth, (F2) immune system and inflammation, and (F3) somatic growth and cardiovascular development. Factors F3 at 2 weeks and F2 at 6.5 months partially mediated PAE-induced growth deficits, and factor F3 at 2 weeks partially mediated effects of PAE on infant recognition memory at 6.5 months. These findings indicate that infant exmiRNAs can help identify infants who will exhibit PAE-related deficits in growth and cognition.

Most persons with fetal alcohol spectrum disorders (FASDs) remain undiagnosed or are diagnosed in later life. To address the need for earlier diagnosis, we previously assessed miRNAs in the blood plasma of pregnant women who were classified as unexposed to alcohol (UE), heavily exposed with affected infants (HEa), or heavily exposed with apparently unaffected infants (HEua). We reported that maternal miRNAs predicted FASD-related growth and psychomotor deficits in infants. Here, we assessed whether fetal sex influenced alterations in maternal circulating miRNAs following prenatal alcohol exposure (PAE). To overcome the loss of statistical power due to disaggregating maternal samples by fetal sex, we adapted a strategy of iterative bootstrap resampling with replacement to assess the stability of statistical parameter estimates. Bootstrap estimates of parametric and effect size tests identified male and female fetal sex-associated maternal miRNA responses to PAE that were not observed in the aggregated sample. Additionally, we observed, in HEa mothers of female, but not male fetuses, a network of co-secreted miRNAs whose expression was linked to miRNAs encoded on the X-chromosome. Interestingly, the number of significant miRNA correlations for the HEua group mothers with female fetuses was intermediate between HEa and UE mothers at mid-pregnancy, but more similar to UE mothers by the end of pregnancy. Collectively, these data show that fetal sex predicts maternal circulating miRNA adaptations, a critical consideration when adopting maternal miRNAs as diagnostic biomarkers. Moreover, a maternal co-secretion network, predominantly in pregnancies with female fetuses, emerged as an index of risk for adverse birth outcomes due to PAE.

Prenatal alcohol exposure can have serious lifelong consequences on the individual. Despite that, at least 10% of women in the United States drink during pregnancy and the incidence of fetal alcohol spectrum disorders have been estimated to be 2%–5%. Decades of work describe effects in the central nervous system and show that basic biological mechanisms are affected. Now there is more attention paid to systemic effects including vulnerability to disease later in life. Outcomes of prenatal alcohol exposure are unpredictable as other factors contribute to vulnerability and resilience; these include genetic and epigenetic factors as well as extrinsic factors such as nutrition and environment. Such complexities contribute to difficulties with diagnosis and therapeutic intervention. Newly emerging fields such as the role of the gut–brain axis and investigation of novel therapeutic approaches will lend additional insight into these disorders.

Prenatal alcohol exposure can result in growth, cognitive, and behavioral deficits due to the toxicant and teratogenic effects of alcohol. Alcohol is an unusual toxicant, because, unlike other toxicants, it is consumed and has biological effects in the millimolar range. Cerebral cortical development is particularly vulnerable to both alcohol's acute and long-term reprogramming effects. Recent evidence suggests that neuroinflammation may be a persistent result of prenatal alcohol exposure and that modes of cellular communication capable of carrying miRNAs, such as extracellular vesicles, may be an integral part of long-term changes to cellular communication and inflammation following in utero alcohol exposure.

Prenatal ethanol exposure can result in loss of neural stem cells (NSCs) and decreased brain growth. Here, we assessed whether a noncoding RNA (ncRNA) related to the NSC self-renewal factor Oct4/Pou5f1, and transcribed from a processed pseudogene locus on mouse chromosome 9 (mOct4pg9), contributed to the loss of NSCs due to ethanol. Mouse fetal cortical-derived NSCs, cultured ex vivo to mimic the early neurogenic environment of the fetal telencephalon, expressed mOct4pg9 ncRNA at significantly higher levels than the parent Oct4/Pou5f1 mRNA. Ethanol exposure increased expression of mOct4pg9 ncRNA, but decreased expression of OCT4/POU5F1. Gain- and loss-of-function analyses indicated that mOct4pg9 overexpression generally mimicked effects of ethanol exposure, resulting in increased proliferation and expression of transcripts associated with neural maturation. Moreover, mOct4pg9 associated with Ago2 and with miRNAs, including the anti-proliferative miR-328-3p, whose levels were reduced following mOct4pg9 overexpression. Finally, mOct4pg9 inhibited Oct4/Pou5f1-3’UTR-dependent protein translation. Consistent with these observations, data from single-cell transcriptome analysis showed that mOct4pg9-expressing progenitors lack Oct4/Pou5f1, but instead overexpress transcripts for increased mitosis, suggesting initiation of transit amplification. Collectively, these data suggest that the inhibitory effects of ethanol on brain development are explained, in part, by a novel ncRNA which promotes loss of NSC identity and maturation.

Stroke is the fifth leading cause of death and acquired disability in aged populations. Women are disproportionally affected by stroke, having a higher incidence and worse outcomes than men. Numerous preclinical studies have discovered novel therapies for the treatment of stroke, but almost all of these have been shown to be unsuccessful in clinical trials. Despite known sex differences in occurrence and severity of stroke, few preclinical or clinical therapeutics take into account possible sex differences in treatment. Reanalysis of data from studies of tissue plasminogen activator (tPA), the only currently FDA-approved stroke therapy, has shown that tPA improves stroke outcomes for both sexes and also shows sexual dimorphism by more robust improvement in stroke outcome in females. Experimental evidence supports the inclusion of sex as a variable in the study of a number of novel stroke drugs and therapies, including preclinical studies of anti-inflammatory drugs (minocycline), stimulators of cell survival (insulin-like growth factor-1), and inhibitors of cell death pathways (pharmacological inhibition of poly[ADP-ribose] polymerase-1, nitric oxide production, and caspase activation) as well as in current clinical trials of stem cell therapy and cortical stimulation. Overall, study design and analysis in clinical trials as well as in preclinical studies must include both sexes equally, consider possible sex differences in the analyses, and report the differences/similarities in more systematic/structured ways to allow promising therapies for both sexes and increase stroke recovery. © 2016 Wiley Periodicals, Inc.

Early developmental exposure to ethanol, a known teratogen, can result in a range of neurodevelopmental disorders, collectively referred to as Fetal Alcohol Spectrum Disorders (FASDs). Changes in the environment, including exposure to teratogens, can result in long term alterations to the epigenetic landscape of a cell, thereby altering gene expression. Noncoding RNAs (ncRNAs) can affect transcription and translation of networks of genes. ncRNAs are dynamically expressed during development and have been identified as a target of alcohol. ncRNAs therefore make for attractive targets for novel therapeutics to address the developmental deficits associated with FASDs.

Early developmental exposure to ethanol, a known teratogen, can result in a range of neurodevelopmental disorders, collectively referred to as Fetal Alcohol Spectrum Disorders (FASDs). Changes in the environment, including exposure to teratogens, can result in long term alterations to the epigenetic landscape of a cell, thereby altering gene expression. Noncoding RNAs (ncRNAs) can affect transcription and translation of networks of genes. ncRNAs are dynamically expressed during development and have been identified as a target of alcohol. ncRNAs therefore make for attractive targets for novel therapeutics to address the developmental deficits associated with FASDs.

• Epigenetic modifications change global gene expression in response to environmental factors.

Poverty, conflict, chemical contaminants, and substance abuse are examples of environmental conditions that are associated with malnutrition, stress, vulnerability to obesity, cancer, psychiatric and other diseases, and diminished life expectancy. We have come to expect that conditions such as these result in deleterious consequences for target populations. However, what if the effects of these conditions transcend generations? What if grandparents’ adverse life experiences influence health and disease susceptibility of their grandchildren or even great-grandchildren? What are the implications for the practice of medicine and social justice? Evidence from human and animal studies in the rapidly growing field of transgenerational epigenetics suggests that the inherited consequences of adverse ancestral life experiences are real. Biology can be programmed in one generation to produce long-lasting, multigenerational maladaptation. Periods of embryonic development and epigenetic programing, which modify the output of genes, are specifically vulnerable to environmental perturbations. This chapter discusses the somewhat checkered history of enquiry into the heritability of acquired traits, along with more recent experimental and epidemiological data that validates the phenomenon of transgenerational epigenetics. Last, we focus on specific examples of environmental perturbations, diet, exposure to toxic chemicals, and addiction that can be modeled in the laboratory, to show how transgenerational epigenetic mechanisms may underlie human susceptibility to disease.

Within neurons of several regions of the CNS, mature dendrite architecture is attained via extensive reorganization of arbor during the developmental period. Since dendrite morphology determines the firing patterns of the neuron, morphological refinement of dendritic arbor may have important implications for mature network activity. In the neocortex, a region of brain that is sensitive to activity-dependent structural rearrangement of dendritic arbor, the proportion of AMPA receptors increases over the developmental period. However, it is unclear whether changes in AMPA receptor expression contribute to maturation of dendritic architecture. To determine the effects of increasing AMPA receptor expression on dendrite morphology and connectivity within the neocortex, and to determine whether these effects are dependent on specific AMPA receptor subunits, we overexpressed the AMPA glutamate receptor subunit 1 (GluR1) and glutamate receptor subunit 2 (GluR2) in cultured rat neocortical neurons at the time that AMPA receptors would normally be incorporated into synapses. Following expression of GluR1 or GluR2 we observed increases in the length and complexity of dendritic arbor of cortical neurons, and a concurrent reduction in motility of spines. In addition, expression of either subunit was associated with an increased density of excitatory postsynaptic puncta. These results suggest that AMPA receptor expression is an important determinant of dendrite morphology and connectivity in neocortical neurons, and further, that contrary to other regions of the CNS, the effects of AMPA receptors on dendrite morphology are not subunit-specific.

The level and integrity of glutamate transmission during critical periods of postnatal development plays an important role in the refinement of pyramidal neuron dendritic arbor, synaptic plasticity, and cognition. Presently, it is not clear how excitatory transmission via the two predominant isoforms of the vesicular glutamate transporter (VGLUT1 and VGLUT2) participate in this process. To assess a neurodevelopmental role for VGLUT2 in pyramidal neuron maturation, we generated recombinant VGLUT2 knock-out mice and inactivated VGLUT2 throughout development using Emx1-Cre+/+knock-in mice. We show that VGLUT2 deficiency in corticolimbic circuits results in reduced evoked glutamate transmission, release probability, and LTD at hippocampal CA3-CA1 synapses during a formative developmental period (postnatal days 11–14). In adults, we find a marked reduction in the amount of dendritic arbor across the span of the dendritic tree of CA1 pyramidal neurons and reduced long-term potentiation and levels of synaptic markers spinophilin and VGLUT1. Loss of dendritic arbor is accompanied by corresponding reductions in the number of dendritic spines, suggesting widespread alterations in synaptic connectivity. Conditional VGLUT2 knock-out mice exhibit increased open-field exploratory activity yet impaired spatial learning and memory, endophenotypes similar to those of NMDA receptor knock-down mice. Remarkably, the impairment in learning can be partially restored by selectively increasing NMDA receptor-mediated glutamate transmission in adult mice by prolonged treatment withd-serine and ad-amino acid oxidase inhibitor. Our data indicate that VGLUT2 expression is pivotal to the proper development of mature pyramidal neuronal architecture and plasticity, and that such glutamatergic deficiency leads to cognitive malfunction as observed in several neurodevelopmental psychiatric disorders.