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

Department of Neuroscience and Experimental Therapeutics, Texas A&M Health Science Center, College of Medicine, Bryan, Texas.
Alcoholism Clinical and Experimental Research (Impact Factor: 3.21). 06/2013; 37(10). DOI: 10.1111/acer.12139
Source: PubMed


Fetal alcohol exposure produces multiorgan defects, making it difficult to identify underlying etiological mechanisms. However, recent evidence for ethanol (EtOH) sensitivity of the miRNA miR-9 suggests one mechanism, whereby EtOH broadly influences development. We hypothesized that loss of miR-9 function recapitulates aspects of EtOH teratology.
Zebrafish embryos were exposed to EtOH 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 postfertilization (hpf). We also assessed the expression of developmentally important, and known miR-9 targets, FGFR-1, FOXP2, and the nontargeted 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.
EtOH treatment and miR-9 knockdown resulted in similar cranial defects including microcephaly. Additionally, EtOH transiently suppressed miR-9, as well as FGFR-1 and FOXP2, and alterations in miR-9 expression were correlated with severity of EtOH-induced teratology. In mNSCs, EtOH increased CpG dinucleotide methylation at the miR-9-2 locus and accumulation of pre-miR-9-3.
EtOH 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 EtOH exposure. EtOH also disrupts the relationship between miR-9 and target gene expression, suggesting a nuanced relationship between EtOH 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.

14 Reads
  • Source
    • "While uncontrollable nociceptive stimulation in SCI animals attenuated this decrease in miR1 observed following SCI alone, effectively resulting in increased miRNA expression, the expression of IGF-1 remained unchanged from the shock-only condition. These data suggest a dissociation between miRNA and target gene networks following SCI and intermittent noxious stimulation, as we have previously observed in other models of neural damage (Pappalardo-Carter et al., 2013). While such dissociation may be simply the result of miRNA and mRNAs being expressed in distinct and non-overlapping cell cohorts, this dissociation in ventral spinal cord may be a secondary consequence of activating the dorsal spinal cord. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Uncontrollable nociceptive stimulation adversely affects recovery in spinally contused rats. Spinal cord injury (SCI) results in altered microRNA (miRNA) expression both at, and distal to the lesion site. We hypothesized that uncontrollable nociception further influences SCI-sensitive miRNAs and associated gene targets, potentially explaining the progression of maladaptive plasticity. Our data validated previously described sensitivity of miRNAs to SCI alone. Moreover, following SCI, intermittent noxious stimulation decreased expression of miR124 in dorsal spinal cord 24 h after stimulation and increased expression of miR129-2 in dorsal, and miR1 in ventral spinal cord at 7 days. We also found that brain-derived neurotrophic factor (BDNF) mRNA expression was significantly down-regulated 1 day after SCI alone, and significantly more so, after SCI followed by tailshock. Insulin-like growth factor-1 (IGF-1) mRNA expression was significantly increased at both 1 and 7 days post-SCI, and significantly more so, 7 days post-SCI with shock. MiR1 expression was positively and significantly correlated with IGF-1, but not BDNF mRNA expression. Further, stepwise linear regression analysis indicated that a significant proportion of the changes in BDNF and IGF-1 mRNA expression were explained by variance in two groups of miRNAs, implying co-regulation. Collectively, these data show that uncontrollable nociception which activates sensorimotor circuits distal to the injury site, influences SCI-miRNAs and target mRNAs within the lesion site. SCI-sensitive miRNAs may well mediate adverse consequences of uncontrolled sensorimotor activation on functional recovery. However, their sensitivity to distal sensory input also implicates these miRNAs as candidate targets for the management of SCI and neuropathic pain.
    Frontiers in Neural Circuits 09/2014; 8:117. DOI:10.3389/fncir.2014.00117 · 3.60 Impact Factor
  • Source
    • "We identified miR-153 as one of a small cohort of miRNAs that were significantly decreased in fetal NSCs, following ethanol exposure (Sathyan et al., 2007). In that, and subsequent studies (Pappalardo-Carter et al., 2013), we also showed that the suppression of ethanol-sensitive miRNAs individually and collectively, explained some of the teratogenic effects of ethanol. Recently, developmental ethanol exposure was shown to also result in decreased miR-153 in a zebrafish model, and dysregulation of miR-153 in that model in turn resulted in neurobehavioral impairment (Tal et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ethanol exposure during pregnancy is an established cause of birth defects, including neurodevelopmental defects. Most adult neurons are produced during the second trimester-equivalent period. The fetal neural stem cells (NSCs) that generate these neurons are an important but poorly understood target for teratogenesis. A cohort of miRNAs, including miR-153, may serve as mediators of teratogenesis. We previously showed that ethanol decreased, while nicotine increased miR-153 expression in NSCs. To understand the role of miR-153 in the etiology of teratology, we first screened fetal cortical NSCs cultured ex vivo, by microarray and quantitative RT-PCR analyses, to identify cell-signaling mRNAs and gene networks as important miR-153 targets. Moreover, miR-153 over-expression prevented neuronal differentiation without altering neuroepithelial cell survival or proliferation. Analysis of 3'UTRs and in utero over-expression of pre-miR-153 in fetal mouse brain identified Nfia (nuclear factor-1A) and its paralog, Nfib, as direct targets of miR-153. In utero ethanol exposure resulted in a predicted expansion of Nfia and Nfib expression in the fetal telencephalon. In turn, miR-153 over-expression prevented, and partly reversed, the effects of ethanol exposure on miR-153 target transcripts. Varenicline, a partial nicotinic acetylcholine receptor agonist that, like nicotine, induces miR-153 expression, also prevented and reversed the effects of ethanol exposure. These data collectively provide evidence for a role for miR-153 in preventing premature NSC differentiation. Moreover, they provide the first evidence in a preclinical model that direct or pharmacological manipulation of miRNAs have the potential to prevent or even reverse effects of a teratogen like ethanol on fetal development.
    Biology Open 07/2014; 3(8). DOI:10.1242/bio.20147765 · 2.42 Impact Factor
  • Source
    • "In particular, microRNAs (miRs, ∼22 nucleotide single-stranded non-coding RNA) regulate the translation of proteins important for neuronal development during embryogenesis, postnatal neuronal maintenance and survival, and hippocampal neurogenesis throughout life [32], [33], [34], [35], [36], [37]. Moreover, disruption of mature miR expression and/or function has been linked to alcohol-induced neurological afflictions including addiction and fetal alcohol spectrum disorder (FASD) [38], [39]. In this study we used a Wistar rat model to identify EtOH-sensitive miRs that target genes involved in regulating hippocampal processes, such as memory and mood. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Adolescent binge alcohol abuse induces long-term changes in gene expression, which impacts the physiological stress response and memory formation, two functions mediated in part by the ventral (VH) and dorsal (DH) hippocampus. microRNAs (miRs) are small RNAs that play an important role in gene regulation and are potential mediators of long-term changes in gene expression. Two genes important for regulating hippocampal functions include brain-derived neurotrophic factor (BDNF) and sirtuin-1 (SIRT1), which we identified as putative gene targets of miR-10a-5p, miR-26a, miR-103, miR-495. The purpose of this study was to quantify miR-10a-5p, miR-26a, miR-103, miR-495 expression levels in the dorsal and ventral hippocampus of male Wistar rats during normal pubertal development and then assess the effects of repeated binge-EtOH exposure. In addition, we measured the effects of binge EtOH-exposure on hippocampal Drosha and Dicer mRNA levels, as well as the putative miR target genes, BDNF and SIRT1. Overall, mid/peri-pubertal binge EtOH exposure altered the normal expression patterns of all miRs tested in an age- and brain region-dependent manner and this effect persisted for up to 30 days post-EtOH exposure. Moreover, our data revealed that mid/peri-pubertal binge EtOH exposure significantly affected miR biosynthetic processing enzymes, Drosha and Dicer. Finally, EtOH-induced significant changes in the expression of a subset of miRs, which correlated with changes in the expression of their predicted target genes. Taken together, these data demonstrate that EtOH exposure during pubertal development has long-term effects on miRNA expression in the rat hippocampus.
    PLoS ONE 01/2014; 9(1):e83166. DOI:10.1371/journal.pone.0083166 · 3.23 Impact Factor
Show more