Sridevi Balaraman

MICRORNA DYSREGULATION FOLLOWING SPINAL CORD CONTUSION: IMPLICATIONS FOR NEURAL PLASTICITY AND REPAIR

Spinal cord injury (SCI) is medically and socioeconomically debilitating. Currently, there is a paucity of effective therapies that promote regeneration at the injury site, and limited understanding of mechanisms that can be utilized to therapeutically manipulate spinal cord plasticity. MicroRNAs (miRNAs) constitute novel targets for therapeutic intervention to promote repair and regeneration. Microarray comparisons of the injury sites of contused and sham rat spinal cords, harvested 4 and 14 days following SCI, showed that 32 miRNAs, including miR124, miR129, and miR1, were significantly down-regulated, whereas SNORD2, a translation-initiation factor, was induced. Additionally, three miRNAs including miR21 were significantly induced, indicating adaptive induction of an anti-apoptotic response in the injured cord. Validation of miRNA expression by qRT-PCR and in situ hybridization assays revealed that the influence of SCI on miRNA expression persists up to 14 days and expands both anteriorly and caudally beyond the lesion site. Specifically, changes in miR129-2 and miR146a expression significantly explained the variability in initial injury severity, suggesting that these specific miRNAs may serve as biomarkers and therapeutic targets for SCI. Moreover, the pattern of miRNA changes coincided spatially and temporally with the appearance of SOX2, nestin, and REST immunoreactivity, suggesting that aberrant expression of these miRNAs may not only reflect the emergence of stem cell niches, but also the reemergence in surviving neurons of a pre-neuronal phenotype. Finally, bioinformatics analysis of validated miRNA-targeted genes indicates that miRNA dysregulation may explain apoptosis susceptibility and aberrant cell cycle associated with a loss of neuronal identity, which underlies the pathogenesis of secondary SCI.

Maternal and Neonatal Plasma MicroRNA Biomarkers for Fetal Alcohol Exposure in an Ovine Model

BACKGROUND Plasma or circulating miRNAs (cir miRNAs) have potential diagnostic value as biomarkers for a range of diseases. Based on observations that ethanol (EtOH) altered intracellular miRNAs during development, we tested the hypothesis that plasma miRNAs were biomarkers for maternal alcohol exposure, and for past in utero exposure, in the neonate. METHODS Pregnant sheep were exposed to a binge model of EtOH consumption resulting in an average peak blood alcohol content of 243 mg/dl, for a third-trimester-equivalent period from gestational day 4 (GD4) to GD132. MiRNA profiles were assessed by quantitative PCR analysis in plasma, erythrocyte, and leukocytes obtained from nonpregnant ewes, and plasma from pregnant ewes 24 hours following the last binge EtOH episode, and from newborn lambs, at birth on ~GD147. RESULTS Pregnant ewe and newborn lamb cir miRNA profiles were similar to each other and different from nonpregnant female plasma, erythrocyte, or leukocyte miRNAs. Significant changes in cir miRNA profiles were observed in the EtOH-exposed ewe and, at birth, in the in utero, EtOH-exposed lamb. Cir miRNAs including miR-9, -15b, -19b, and -20a were sensitive and specific measures of EtOH exposure in both pregnant ewe and newborn lamb. Additionally, EtOH exposure altered guide-to-passenger strand cir miRNA ratios in the pregnant ewe, but not in the lamb. CONCLUSIONS Shared profiles between pregnant dam and neonate suggest possible maternal-fetal miRNA transfer. Cir miRNAs are biomarkers for alcohol exposure during pregnancy, in both mother and neonate, and may constitute an important shared endocrine biomarker that is vulnerable to the maternal environment.

MiR-153 targets the nuclear factor-1 family and protects against teratogenic effects of ethanol exposure in fetal neural stem cells

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.

Plasma miRNA Profiles in Pregnant Women Predict Infant Outcomes following Prenatal Alcohol Exposure

Fetal alcohol spectrum disorders (FASD) are difficult to diagnose since many heavily exposed infants, at risk for intellectual disability, do not exhibit craniofacial dysmorphology or growth deficits. Consequently, there is a need for biomarkers that predict disability. In both animal models and human studies, alcohol exposure during pregnancy resulted in significant alterations in circulating microRNAs (miRNAs) in maternal blood. In the current study, we asked if changes in plasma miRNAs in alcohol-exposed pregnant mothers, either alone or in conjunction with other clinical variables, could predict infant outcomes. Sixty-eight pregnant women at two perinatal care clinics in western Ukraine were recruited into the study. Detailed health and alcohol consumption histories, and 2nd and 3rd trimester blood samples were obtained. Birth cohort infants were assessed by a geneticist and classified as unexposed (UE), heavily prenatally exposed and affected (HEa) or heavily exposed but apparently unaffected (HEua). MiRNAs were assessed in plasma samples using qRT-PCR arrays. ANOVA models identified 11 miRNAs that were all significantly elevated in maternal plasma from the HEa group relative to HEua and UE groups. In a random forest analysis classification model, a combination of high variance miRNAs, smoking history and socioeconomic status classified membership in HEa and UE groups, with a misclassification rate of 13%. The RFA model also classified 17% of the HEua group as UE-like, whereas 83% were HEa-like, at least at one stage of pregnancy. Collectively our data indicate that maternal plasma miRNAs predict infant outcomes, and may be useful to classify difficult-to-diagnose FASD subpopulations.

The BAF (BRG1/BRM-Associated Factor) chromatin-remodeling complex exhibits ethanol sensitivity in fetal neural progenitor cells and regulates transcription at the miR-9-2 encoding gene locus

Fetal alcohol spectrum disorders are a leading cause of intellectual disability worldwide. Previous studies have shown that developmental ethanol exposure results in loss of microRNAs (miRNAs), including miR-9, and loss of these miRNAs, in turn, mediates some of ethanols teratogenic effects in the developing brain. We previously found that ethanol increased methylation at the miR-9-2 encoding gene locus in mouse fetal neural stem cells (NSC), advancing a mechanism for epigenetic silencing of this locus and consequently, miR-9 loss in NSCs. Therefore, we assessed the role of the BAF (BRG1/BRM-Associated Factor) complex, which disassembles nucleosomes to facilitate access to chromatin, as an epigenetic mediator of ethanols effects on miR-9. Chromatin immunoprecipitation and DNAse I-hypersensitivity analyses showed that the BAF complex was associated with both transcriptionally accessible and heterochromatic regions of the miR-9-2 locus, and that disintegration of the BAF complex by combined knockdown of BAF170 and BAF155 resulted in a significant decrease in miR-9. We hypothesized that ethanol exposure would result in loss of BAF-complex function at the miR-9-2 locus. However, ethanol exposure significantly increased mRNA transcripts for maturation-associated BAF-complex members BAF170, SS18, ARID2, BAF60a, BRM/BAF190b, and BAF53b. Ethanol also significantly increased BAF-complex binding within an intron containing a CpG island and in the terminal exon encoding precursor (pre)-miR-9-2. These data suggest that the BAF complex may adaptively respond to ethanol exposure to protect against a complete loss of miR-9-2 in fetal NSCs. Chromatin remodeling factors may adapt to the presence of a teratogen, to maintain transcription of critical miRNA regulatory pathways.

MicroRNAs in Alcohol Abuse and Toxicity

Alcohol, though widely consumed, can be addictive, toxic, tumorigenic, and teratogenic. The biology of alcoholism and its consequences is often viewed as insurmountably complex. However, recent data showing the involvement of microRNAs (miRNAs) in alcohol addiction and pathology provides evidence for a new layer of cellular regulation that may explain and integrate the complexity of alcohol’s effects. MiRNAs are small, non-protein-coding RNA molecules that control networks of protein-coding genes by translation repression and by other more recently discovered mechanisms, including epigenetics and endocrine signaling. In this chapter, we will focus mainly on the brain effects of ethanol and discuss the small but growing literature that shows that common miRNAs mediate aspects of alcoholism and the emergence of alcohol-associated pathology and teratology. We will also examine mechanisms that regulate miRNA biogenesis and function, which are potentially vulnerable to drugs of abuse such as alcohol.