Behavioural And Molecular Consequences Of Postnatal Stress In A Mouse Model Of Fetal Alcohol Spectrum Disorder

Fetal alcohol spectrum disorders (FASD) are caused by prenatal alcohol exposure (PAE) and affect 1‑5% of the North American population. Children born with FASD often face maternal separation throughout childhood. How this early life stress (ELS) affects the severity of FASD-related deficits is poorly understood. Using a mouse model, this dissertation establishes that behavioural deficits accumulate following prenatal alcohol exposure and early life stress, assessed using tests for activity, anxiety-like behaviour as well as learning and memory. Hippocampal gene expression was evaluated using RNA-seq followed by clustering of expression profiles through weighted gene co-expression network analysis (WGCNA). A set of transcripts are associated with anxiety-like behaviour (r = 0.79, p = 0.002) and treatment (r = 0.68, p = 0.015). Genes in this module are overrepresented by transcriptional regulation and neurodevelopment genes. One member of this module, Polr2a, is downregulated by the combination of treatments. Hippocampal promoter DNA methylation was assessed using methylated DNA immunoprecipitation sequencing (MeDIP-Seq). Methylation at different genes is affected by each treatment independently, and a unique set of genes are affected by the combination of treatments. PAE leads to altered promoter DNA methylation at genes important for transcriptional regulation and ELS leads to changes at genes important for histone methylation and immune response. The combination of treatments results in DNA methylation changes at genes important for neuronal migration and immune response. The results from the same samples show that genes with altered expression and promoter methylation are critical in brain development and function. Also, there is minimal complementarity between changes in promoter DNA methylation and gene expression. Mechanisms beyond promoter DNA methylation are likely involved in lasting gene expression changes leading to behavioural deficits seen in FASD. Although further research is required to elucidate the mechanism, the results included may be valuable towards early and reliable diagnosis, together with the development of novel strategies for the amelioration of FASD-related deficits

Exploring the Complexity of Intellectual Disability in Fetal Alcohol Spectrum Disorders

Brain development in mammals is long lasting. It begins early during embryonic growth and is finalized in early adulthood. This progression represents a delicate choreography of molecular, cellular, and physiological processes initiated and directed by the fetal genotype in close interaction with environment. Not surprisingly, most aberrations in brain functioning including intellectual disability (ID) are attributed to either gene(s), or environment or the interaction of the two. The ensuing complexity has made the assessment of this choreography, ever challenging. A model to assess this complexity has used a mouse model (C57BL/6J or B6) that is subjected to prenatal alcohol exposure. The resulting pups show learning and memory deficits similar to patients with fetal alcohol spectrum disorder (FASD), which is associated with life-long changes in gene expression. Interestingly, this change in gene expression underlies epigenetic processes including DNA methylation and miRNAs. This paradigm is applicable to ethanol exposure at different developmental times (binge at trimesters 1, 2, and 3 as well as continuous preference drinking (70%) of 10% alcohol by B6 females during pregnancy). The exposure leads to life-long changes in neural epigenetic marks, gene expression, and a variety of defects in neurodevelopment and CNS function. We argue that this cascade may be reversed postnatally via drugs, chemicals, and environment including maternal care. Such conclusions are supported by two sets of results. First, antipsychotic drugs that are used to treat ID including psychosis function via changes in DNA methylation, a major epigenetic mark. Second, post-natal environment may improve (with enriched environments) or worsen (with negative and maternal separation stress) the cognitive ability of pups that were prenatally exposed to ethanol as well as their matched controls. In this review, we will discuss operational epigenetic mechanisms involved in the development of intellectual ability/disability in response to alcohol during prenatal or post-natal development. In doing so, we will explore the potential of epigenetic manipulation in the treatment of FASD and related disorders implicated in ID

Long-Term Genomic and Epigenomic Dysregulation as a Consequence of Prenatal Alcohol Exposure: A Model for Fetal Alcohol Spectrum Disorders

There is abundant evidence that prenatal alcohol exposure leads to a range of behavioral and cognitive impairments, categorized under the term fetal alcohol spectrum disorders (FASDs). These disorders are pervasive in Western cultures and represent the most common preventable source of neurodevelopmental disabilities. The genetic and epigenetic etiology of these phenotypes, including those factors that may maintain these phenotypes throughout the lifetime of an affected individual, has become a recent topic of investigation. This review integrates recent data that has progressed our understanding FASD as a continuum of molecular events, beginning with cellular stress response and ending with a long-term footprint of epigenetic dysregulation across the genome. It reports on data from multiple ethanol-treatment paradigms in mouse models that identify changes in gene expression that occur with respect to neurodevelopmental timing of exposure and ethanol dose. These studies have identified patterns of genomic alteration that are dependent on the biological processes occurring at the time of ethanol exposure. This review also adds to evidence that epigenetic processes such as DNA methylation, histone modifications, and non-coding RNA regulation may underlie long-term changes to gene expression patterns. These may be initiated by ethanol-induced alterations to DNA and histone methylation, particularly in imprinted regions of the genome, affecting transcription which is further fine-tuned by altered microRNA expression. These processes are likely complex, genome-wide, and interrelated. The proposed model suggests a potential for intervention, given that epigenetic changes are malleable and may be altered by postnatal environment. This review accentuates the value of mouse models in deciphering the molecular etiology of FASD, including those processes that may provide a target for the ammelioration of this common yet entirely preventable disorder

Hippocampal transcriptome analysis following maternal separation implicates altered RNA processing in a mouse model of fetal alcohol spectrum disorder

Background: Fetal alcohol spectrum disorders (FASD) are common, seen in 1-5% of the population in the USA and Canada. Children diagnosed with FASD are not likely to remain with their biological parents, facing early maternal separation and foster placements throughout childhood. Methods: We model FASD in mice via prenatal alcohol exposure and further induce early life stress through maternal separation. We use RNA-seq followed by clustering of expression profiles through weighted gene co-expression network analysis (WGCNA) to analyze transcriptomic changes that result from the treatments. We use reverse transcription qPCR to validate these changes in the mouse hippocampus. Results: We report an association between adult hippocampal gene expression and prenatal ethanol exposure followed by postnatal separation stress that is related to behavioral changes. Expression profile clustering using WGCNA identifies a set of transcripts, module 19, associated with anxiety-like behavior (r = 0.79, p = 0.002) as well as treatment group (r = 0.68, p = 0.015). Genes in this module are overrepresented by genes involved in transcriptional regulation and other pathways related to neurodevelopment. Interestingly, one member of this module, Polr2a, polymerase (RNA) II (DNA directed) polypeptide A, is downregulated by the combination of prenatal ethanol and postnatal stress in an RNA-Seq experiment and qPCR validation (q = 2e-12, p = 0.004, respectively). Conclusions: Together, transcriptional control in the hippocampus is implicated as a potential underlying mechanism leading to anxiety-like behavior via environmental insults. Further research is required to elucidate the mechanism involved and use this insight towards early diagnosis and amelioration strategies involving children born with FASD

Coordinated Tcf7l2 regulation in a mouse model implicates Wnt signaling in Fetal Alcohol Spectrum Disorders

Mouse models of fetal alcohol spectrum disorders (FASD) have repeatedly identified genes with long-term changes in expression, DNA methylation, noncoding RNA and histone modifications in response to neurodevelopmental alcohol exposure. Articulation of FASD is achieved via alcoholâ s effect on gene expression, likely involving epigenetic regulation. The list of genes affected is large and heterogeneous, depending on experimental protocol. We present reanalysis and synthesis of results highlighting Wnt transcription factor 7 like 2 (Tcf7l2) gene as uniquely compatible with hippocampal DNA methylation, histone modifications, and gene expression changes in a coordinated response to neurodevelopmental alcohol exposure. We data-mined literature for Tcf7l2 alterations in response to prenatal alcohol exposure. Four studies identified changes in brain Tcf7l2 expression in different FASD models. Further, we performed an in silico TCF7L2 binding site analysis for FASD mouse model datasets. Seven of these published gene lists were significantly enriched for TCF7L2 binding, indicating potential functional relationships. Finally, TCF7L2 is involved in regulation of hundreds of genes, with a role in brain development, myelination, and neuronal function. Tcf7l2 may be involved in neurological defects associated with alcohol exposure via dysregulation of many genes through Wnt signaling. Further functional work is warranted to validate this model for FASD.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Alteration of gene expression, DNA methylation, and histone methylation in free radical scavenging networks in adult mouse hippocampus following fetal alcohol exposure

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse\u27s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as Free radical scavenging . We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylationaffected genes was Peroxisome biogenesis ; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD

Pathways significantly enriched with differentially expressed genes.

<p>Pathways significantly enriched with differentially expressed genes.</p

Location of differentially methylated CpG position in of <i>Acaa1</i> gene.

<p>Bars denote <i>Acaa1</i> exons, lines denote introns, grey bars denote untranslated regions, and black bars denote coding sequence. Yellow bar shows location of DMR from microarray. Red line shows location of 3.2% decrease in methylation at cytosine in CpG site in ethanol-exposed mice (Student’s t-test). Not pictured an additional DMR 3.7 kb upstream, 1.2 kb in size.</p

MicroRNAs predicted to target mRNAs with reciprocal expression changes.

<p>MicroRNAs predicted to target mRNAs with reciprocal expression changes.</p

Global changes in DNA methylation, histone methylation, miRNA expression, and gene expression in adult mice in response to neonatal ethanol exposure.

<p>Tracks show alterations in: (A) DNA methylation as measured by absolute methylation score (AMS) <i>p</i><0.001; (B) H3k27me3 and (C) H3k4me3 measured by model-based analysis of tiling arrays (MAT) score, <i>p</i><0.001; (D) miRNA expression and (E) gene expression <i>p</i><0.05, fold-change>1.2.</p