Suppression and Epigenetic Regulation of MiR‐9 Contributes to Ethanol Teratology: Evidence from Zebrafish and Murine Fetal Neural Stem Cell Models

Abstract

Background Fetal alcohol exposure produces multi-organ defects, making it difficult to identify underlying etiological mechanisms. However, recent evidence for ethanol sensitivity of the miRNA miR-9, suggests one mechanism whereby ethanol broadly influences development. We hypothesized that loss of miR-9 function recapitulates aspects of ethanol teratology. Methods Zebrafish embryos were exposed to ethanol during gastrulation, or injected with anti-miR-9 or nonsense control morpholinos during the 2-cell stage of development and collected between 24 and 72 hours post fertilization (hpf). We also assessed the expression of developmentally important, and known miR-9 targets, FGFR-1, FOXP2, and the non-targeted transcript, MECP2. Methylation at CpG islands of mammalian miR-9 genes was assessed in fetal murine neural stem cells (mNSCs) by methylation-specific PCR, and miRNA processing assessed by qRT-PCR for pre-miR-9 transcripts. Results Ethanol treatment and miR-9 knockdown resulted in similar cranial defects including microcephaly. Additionally, ethanol transiently suppressed miR-9, as well as FGFR-1 and FOXP2, and alterations in miR-9 expression were correlated with severity of ethanol-induced teratology. In mNSCs, ethanol increased CpG dinucleotide methylation at the miR-9-2 locus and accumulation of pre-miR-9-3. Conclusions Ethanol exerts regulatory control at multiple levels of miR-9 biogenesis. Moreover, early embryonic loss of miR-9 function recapitulated the severe range of teratology associated with developmental ethanol exposure. Ethanol also disrupts the relationship between miR-9 and target gene expression, suggesting a nuanced relationship between ethanol and miRNA regulatory networks in the developing embryo. The implications of these data for the expression and function of mature miR-9 warrant further investigation.

Ethanol deregulates Mecp2/MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements

“…Differentiating neural stem cells are frequently used as in vitro models to study the underlying molecular mechanisms of ethanol tera-togenesis and FASD pathogenesis (Pappalardo-Carter et al, 2013;Vangipuram and Lyman, 2012;Zhou et al, 2011a). Alcoholmediated neurological symptoms and microcephaly are usually associated with disrupted communications between neural cell types due to aberrant neurite outgrowth (Bingham et al, 2004) and glial cell growth or morphology (Guerri et al, 2001).…”

“…Control experiments were carried out without ethanol treatment. As established in previous similar studies (Pappalardo-Carter et al, 2013;Vangipuram and Lyman, 2012;Zhou et al, 2011a), cells were treated once with ethanol for 48 h to study the effect of single time or binge-like alcohol exposure. To study the effects of continuous/chronic ethanol exposure, differentiating cells were treated with ethanol for 8 days (until D8).…”

Developmental Consequences of Fetal Exposure to Drugs: What We Know and What We Still Must Learn

“…Transcriptional analyses of gene products regulated by fetal ethanol exposure in both mouse and human cells also highlight genes related to neural development, such as cell proliferation, neuronal migration, and differentiation (Hashimoto-Torii et al, 2011;Hicks et al, 2010). Emerging data also implicate microRNAs and epigenetic mechanisms Mantha et al, 2014;Pappalardo-Carter et al, 2013;Ungerer et al, 2013). For example, fetal alcohol results in alterations in DNA methylation and microRNA expression and function (Pappalardo-Carter et al, 2013).…”

“…Emerging data also implicate microRNAs and epigenetic mechanisms Mantha et al, 2014;Pappalardo-Carter et al, 2013;Ungerer et al, 2013). For example, fetal alcohol results in alterations in DNA methylation and microRNA expression and function (Pappalardo-Carter et al, 2013). An intriguing hypothesis suggests that fetal alcohol may induce a dual state of central nervous system insulin resistance and oxidative stress and proposes that adverse effects may be prevented or treated by treatment with peroxisome-proliferated activated receptor agonists (de la Monte and Wands, 2010).…”

MicroRNAs and Ethanol Toxicity

“…Ago-2 along with its bound miRNA serves as the minimal RISC (MacRae et al, 2008). Peri-pubertal alcohol exposure in rodent models has recently been shown to result in dynamic temporal changes in both Drosha and Dicer mRNA transcripts (Prins, Przybycien-Szymanska, Rao, & Pak, 2014), and alcohol exposure in neural stem cell (NSC) cultures has been shown to result in accumulation of pre-miR-9-3 transcripts (Pappalardo-Carter et al, 2013), suggesting that miRNA processing may be broadly influenced by alcohol exposure. On the other hand, miRNA expression analyses (Balaraman et al, 2014;Guo, Chen, Carreon, & Qiang, 2012;Lewohl et al, 2011;Sathyan, Golden, & Miranda, 2007;Steenwyk, Janeczek, & Lewohl, 2013;Wang, Zhang, et al, 2009) indicate that alcohol exposure does not result in broad changes in miRNA expression, suggesting that miRNA processing may not be a significant target.…”