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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 (PAE), like other pregnancy complications, can result in placental insufficiency and fetal growth restriction, although the linking causal mechanisms are unclear. We previously identified 11 gestationally elevated maternal circulating miRNAs (HEamiRNAs) that predicted infant growth deficits following PAE. Here, we investigated whether these HEamiRNAs contribute to the pathology of PAE, by inhibiting trophoblast epithelial-mesenchymal transition (EMT), a pathway critical for placental development. We now report for the first time that PAE inhibits expression of placental pro-EMT pathway members in both rodents and primates, and that HEamiRNAs collectively, but not individually, mediate placental EMT inhibition. HEamiRNAs collectively, but not individually, also inhibited cell proliferation and the EMT pathway in cultured trophoblasts, while inducing cell stress, and following trophoblast syncytialization, aberrant endocrine maturation. Moreover, a single intravascular administration of the pooled murine-expressed HEamiRNAs, to pregnant mice, decreased placental and fetal growth and inhibited the expression of pro-EMT transcripts in the placenta. Our data suggest that HEamiRNAs collectively interfere with placental development, contributing to the pathology of PAE, and perhaps also, to other causes of fetal growth restriction.

Background: 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. Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Motivation Transcriptomics is a common approach to identify changes in gene expression induced by a disease state. Standard transcriptomic analyses consider differentially expressed genes (DEGs) as indicative of disease states so only a few genes would be treated as signals when the effect size is small, such as in brain tissue. For tissue with small effect sizes, if the DEGs do not belong to a pathway known to be involved in the disease, there would be little left in the transcriptome for researchers to follow up with. Results We developed RNA Solutions: Synthesizing Information to Support Transcriptomics (RNASSIST), a new approach to identify hidden signals in transcriptomic data by linking differential expression and co-expression networks using machine learning. We applied our approach to RNA-seq data of post-mortem brains that compared the Alcohol Use Disorder (AUD) group with the control group. Many of the candidate genes are not differentially expressed so would likely be ignored by standard transcriptomic analysis pipelines. Through multiple validation strategies, we concluded that these RNASSIST-identified genes likely play a significant role in AUD. Availability and implementation The RNASSIST algorithm is available at https://github.com/netrias/rnassist and both the software and the data used in RNASSIST are available at https://figshare.com/articles/software/RNAssist%5fSoftware%5fand%5fData/16617250. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Apoptotic endoplasmic reticulum (ER) stress is a major mechanism for dopaminergic (DA) loss in Parkinson's disease (PD). We assessed if low doses of the partial α4β2 nicotinic acetylcholine receptor agonist, cytisine attenuates apoptotic ER stress and exerts neuroprotection in substantia nigra pars compacta (SNc) DA neurons. Alternate day intraperitoneal injections of 0.2 mg/kg cytisine were administered to female and male mice with 6‐hydroxydopamine (6‐OHDA) lesions in the dorsolateral striatum, which caused unilateral degeneration of SNc DA neurons. Cytisine attenuated 6‐OHDA‐induced PD‐related behaviors in female, but not in male mice. We also found significant reductions in tyrosine hydroxylase (TH) loss within the lesioned SNc of female, but not male mice. In contrast to female mice, DA neurons within the lesioned SNc of male mice showed a cytisine‐induced pathological increase in the nuclear translocation of the pro‐apoptotic ER stress protein, C/EBP homologous protein (CHOP). To assess the role of estrogen in cytisine neuroprotection in female mice, we exposed primary mouse DA cultures to either 10 nM 17‐β‐estradiol and 200 nM cytisine or 10 nM 17‐β‐estradiol alone. 17‐β‐estradiol reduced expression of CHOP, whereas cytisine exposure reduced 6‐OHDA‐mediated nuclear translocation of two other ER stress proteins, activating transcription factor 6 and x‐box‐binding protein 1, but not CHOP. Taken together, these data show that cytisine and 17‐β‐estradiol work in combination to inhibit all three arms (activating transcription factor 6, x‐box‐binding protein 1, and CHOP) of apoptotic ER stress signaling in DA neurons, which can explain the neuroprotective effect of low‐dose cytisine in female mice. [ABSTRACT FROM AUTHOR]

Chronic alcohol consumption alters the levels of microRNAs and mRNAs in the brain, but the specific microRNAs and processes that target mRNAs to affect cellular function and behavior are not known. We examined the in vivo manipulation of previously identified alcohol-responsive microRNAs as potential targets to reduce alcohol consumption. Silencing of miR-411 by infusing antagomiR-411 into the prefrontal cortex of female C57BL/6J mice reduced alcohol consumption and preference, without altering total fluid consumption, saccharin consumption, or anxiety-related behaviors. AntagomiR-411 reduced alcohol consumption when given to mice exposed to a chronic alcohol drinking paradigm but did not affect the acquisition of consumption in mice without a history of alcohol exposure, suggesting that antagomiR-411 has a neuroadaptive, alcohol-dependent effect. AntagomiR-411 decreased the levels of miR-411, as well as the association of immunoprecipitated miR-411 with Argonaute2; and, it increased levels of Faah and Ppard mRNAs. Moreover, antagomiR-411 increased the neuronal expression of glutamate receptor AMPA-2 protein, a known alcohol target and a predicted target of miR-411. These results suggest that alcohol and miR-411 function in a homeostatic manner to regulate synaptic mRNA and protein, thus reversing alcohol-related neuroadaptations and reducing chronic alcohol consumption. [ABSTRACT FROM AUTHOR]

Background: Neural stem cells (NSCs) generate most of the neurons of the adult brain in humans, during the mid‐first through second‐trimester period. This critical neurogenic window is particularly vulnerable to prenatal alcohol exposure, which can result in diminished brain growth. Previous studies showed that ethanol (EtOH) exposure does not kill NSCs, but, rather, results in their depletion by influencing cell cycle kinetics and promoting aberrant maturation, in part, by altering NSC expression of key neurogenic miRNAs. NSCs reside in a complex microenvironment rich in extracellular vesicles, shown to traffic miRNA cargo between cells. Methods: We profiled the miRNA content of extracellular vesicles from control and EtOH‐exposed ex vivo neurosphere cultures of fetal NSCs. We subsequently examined the effects of one EtOH‐sensitive miRNA, miR‐140‐3p, on NSC growth, survival, and maturation. Results: EtOH exposure significantly elevates levels of a subset of miRNAs in secreted extracellular vesicles. Overexpression of one of these elevated miRNAs, miR‐140‐3p, and its passenger strand relative, miR‐140‐5p, significantly increased the proportion of S‐phase cells while decreasing the proportion of G0/G1 cells compared to controls. In contrast, while miR‐140‐3p knockdown had minimal effects on the proportion of cells in each phase of the cell cycle, knockdown of miR‐140‐5p significantly decreased the proportion of cells in G2/M phase. Furthermore, miR‐140‐3p overexpression, during mitogen‐withdrawal–induced NSC differentiation, favors astroglial maturation at the expense of neural and oligodendrocyte differentiation. Conclusions: Collectively, the dysregulated miRNA content of extracellular vesicles following EtOH exposure may result in aberrant neural progenitor cell growth and maturation, explaining brain growth deficits associated with prenatal alcohol exposure. [ABSTRACT FROM AUTHOR]

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 m Oct4pg9 ncRNA at significantly higher levels than the parent Oct4/Pou5f1 mRNA. Ethanol exposure increased expression of m Oct4pg9 ncRNA, but decreased expression of Oct4/Pou5f1. Gain- and loss-of-function analyses indicated that m Oct4pg9 overexpression generally mimicked effects of ethanol exposure, resulting in increased proliferation and expression of transcripts associated with neural maturation. Moreover, m Oct4pg9 associated with Ago2 and with miRNAs, including the anti-proliferative miR-328-3p, whose levels were reduced following m Oct4pg9 overexpression. Finally, m Oct4pg9 inhibited Oct4/Pou5f1–3'UTR-dependent protein translation. Consistent with these observations, data from single-cell transcriptome analysis showed that m Oct4pg9-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. • Oct4pg9 is a novel mouse pseudogene-encoded noncoding RNA (ncRNA), related to the gene encoding the transcription factor Oct4/Pou5f1, is expressed in neural stem cells (NSCs), and is induced by alcohol (ethanol) exposure • Oct4pg9 ncRNA and Oct4/Pou5f1 mRNA are expressed in non-overlapping sub-populations of neural stem cells. • Oct4pg9 associates with miRNA-chaperone Ago2, and with the antiproliferative miRNA, miR-328-3p. • Oct4pg9 expression results in inhibition of Oct4/Pou5f1 3'UTR-dependent translation. • Gain- and loss-of-function studies indicate that Oct4pg9 upregulation explains pro-maturation and teratogenic effects of ethanol on NSCs. [ABSTRACT FROM AUTHOR]

Alcohol use disorder (AUD) is a prevalent neuropsychiatric disorder that is a major global health concern, affecting millions of people worldwide. Past molecular studies of AUD used underpowered single cell analysis or bulk homogenates of postmortem brain tissue, which obscures gene expression changes in specific cell types. Here we performed single nuclei RNA-sequencing analysis of 73 post-mortem samples from individuals with AUD (N=36, N nuclei = 248,873) and neurotypical controls (N=37, N nuclei = 210,573) in both sexes across two institutional sites. We identified 32 clusters and found widespread cell type-specific transcriptomic changes across the cortex in AUD, particularly affecting glia. We found the greatest dysregulation in novel microglial and astrocytic subtypes that accounted for the majority of differential gene expression and co-expression modules linked to AUD. Analysis for cell type-specific enrichment of aggregate genetic risk for AUD identified subtypes of microglia and astrocytes as potential key players not only affected by but causally linked to the progression of AUD. These results highlight the importance of cell-type specific molecular changes in AUD and offer opportunities to identify novel targets for treatment.

In congenital stationary night blindness type 2 (CSNB2)-a disorder involving the Ca v 1.4 (L-type) Ca 2+ channel-visual impairment is mild considering that Ca v 1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Ca v 1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Ca v 1.4. Surprisingly, Ca v 3 (T-type) Ca 2+ currents were detected in cones of G369i KI mice and Ca v 1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Ca v 1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Ca v 1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Ca v 1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Ca v 3 channels maintain cone synaptic output provided that the nonconducting role of Ca v 1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

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., (Copyright © 2017. Published by Elsevier Inc.)