Project description:Prenatal alcohol exposure (PAE) alters the development of neurobiological systems, leading to lasting neuroendocrine, neuroimmune, and neurobehavioral deficits. Although the etiology of this reprogramming remains unknown, emerging evidence suggests DNA methylation as a potential mediator and biomarker for the effects of PAE due to its responsiveness to environmental cues and relative stability over time. Here, we utilized a rat model of PAE to examine the DNA methylation profiles of rat hypothalami and leukocytes at four time points during early development to assess the genome-wide impact of PAE on the epigenome and identify potential biomarkers of PAE. Our model of PAE approximates to 1-2x the legal limit of drunk driving in most jurisdictions (blood alcohol 80-150 mg/dl) throughout the equivalent of the first two trimesters of human pregnancy. Hypothalami were analyzed on postnatal (P) days 1, 8, 15, 22 and leukocytes at P22 to compare central and peripheral markers. Genome-wide DNA methylation analysis was performed by methylated DNA immunoprecipitation followed by next-generation sequencing. PAE resulted in lasting changes to DNA methylation profiles across all four ages, with 118 differentially methylated regions (DMRs) displaying persistent alterations across the developmental profile at a false-discovery rate (FDR) <0.05. By contrast, 299 DMRs showed the same direction of change in the hypothalamus and leukocytes of P22 pups at an FDR<0.05, with some genes overlapping with the developmental profile findings. The majority of these DMRs were located in intergenic regions, which contained several computationally-predicted transcription factor binding sites. Differentially methylated genes were generally involved in immune function, epigenetic remodeling, metabolism, and hormonal signaling, as determined by gene ontology analyses. Persistent DNA methylation changes in the hypothalamus may be associated with the long-term deficits observed in PAE. Furthermore, correlations between epigenetic alterations in peripheral tissues and those in the brain will provide a foundation for the development of biomarkers of fetal alcohol spectrum disorder (FASD). Finally, findings from studies of PAE provide important insight into the etiology neurodevelopmental and mental health disorders, as they tend to share numerous symptoms and comorbidities.

Project description:Background: Prenatal alcohol exposure (PAE) is associated with alterations in numerous physiological systems, including the stress and immune systems. We have previously shown that PAE increases the course and severity of arthritis in an adjuvant-induced arthritis (AA) model. While the molecular mechanisms underlying these effects are not fully known, changes in neural gene expression are emerging as important factors in the etiology of PAE effects. As the prefrontal cortex (PFC) and hippocampus (HPC) play key roles in neuroimmune function, PAE-induced alterations to their transcriptome may underlie abnormal steady-state functions and responses to immune challenge. The current study examined brains from adult PAE and control females from our recent AA study to determine whether PAE causes long-term alterations in gene expression and whether these mediate the altered severity and course of arthritis in PAE females Methods: Adult females from PAE, pair-fed [PF], and ad libitum-fed control [C]) groups were injected with either saline or complete FreundM-bM-^@M-^Ys adjuvant. Animals were terminated at the peak of inflammation or during resolution (days 16 and 39 post-injection, respectively); cohorts of saline-injected PAE, PF and C females were terminated in parallel. Gene expression was analyzed in the PFC and HPC using whole genome mRNA expression microarrays. Results: Significant changes in gene expression in both the PFC and HPC were found in PAE compared to controls in response to ethanol exposure alone (saline-injected females), including genes involved in neurodevelopment, apoptosis, and energy metabolism. Moreover, in response to inflammation (adjuvant-injected females), PAE animals showed unique expression patterns, while failing to exhibit the activation of genes and regulators involved in the immune response observed in control and pair-fed animals. Conclusions: These results support the hypothesis that PAE affects neuroimmune function at the level of gene expression, demonstrating long-term effects of PAE on the CNS response under steady-state conditions and following an inflammatory insult. Key words: prenatal alcohol exposure (PAE), ethanol, inflammation, arthritis, gene expression, rat. 192 samples, including 20 hybridization replicates

Project description:Although alcohol consumption during pregnancy is a major cause of behavioral and learning disabilities, most FASD infants are late- or even misdiagnosed due to clinician’s difficulties achieving early detection of alcohol-induced neurodevelopmental impairments. Neuroplacentology has emerged as a new field of research focusing on the role of the placenta in fetal brain development. Several studies have reported that prenatal alcohol exposure (PAE) dysregulates a functional placenta–cortex axis, which is involved in the control of angiogenesis and leads to neurovascular-related defects. However, these studies were focused on PlGF, a pro-angiogenic factor. The aim of the present study is to provide the first transcriptomic “placenta–cortex” signature of the effects of PAE on fetal angiogenesis. Whole mouse genome microarrays of paired placentas and cortices were performed to establish the transcriptomic inter-organ “placenta–cortex” signature in control and PAE groups at gestational day 20. Genespring comparison of the control and PAE signatures revealed that 895 and 1501 genes were only detected in one of two placenta–cortex expression profiles, respectively. Gene ontology analysis indicated that 107 of these genes were associated with vascular development, and String protein–protein interaction analysis showed that they were associated with three functional clusters. PANTHER functional classification analysis indicated that “intercellular communication” was a significantly enriched biological process, and 27 genes were encoded for neuroactive ligand/receptors interactors. Protein validation experiments involving Western blot for one ligand–receptor couple (Agt/AGTR1/2) confirmed the transcriptomic data, and Pearson statistical analysis of paired placentas and fetal cortices revealed a negative correlation between placental Atg and cortical AGTR1, which was significantly impacted by PAE. In humans, a comparison of a 38WG control placenta with a 36WG alcohol-exposed placenta revealed low Agt immunolabeling in the syncytiotrophoblast layer of the alcohol case. In conclusion, this study establishes the first transcriptomic placenta–cortex signature of a developing mouse. The data show that PAE markedly unbalances this inter-organ signature; in particular, several ligands and/or receptors involved in the control of angiogenesis. These data support that PAE modifies the existing communication between the two organs and opens new research avenues regarding the impact of placental dysfunction on the neurovascular development of fetuses. Such a signature would present a clinical value for early diagnosis of brain defects in FASD.

Project description:Glial cells are central players in the pathogenesis of neurodevelopmental disorders like Fetal Alcohol Spectrum Disorder (FASD). Ethanol can modulate glial differentiation and astrocyte function imposing dramatic changes to the developing brain. Since ethanol can affect a vast number of intracellular targets and signaling pathways and many effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to a long-lasting change in astrocyte secretome in vitro. Animals were treated every other day with ethanol (5g/kg) or saline between post-natal day (P)10-30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids (SILAC). 24h-conditioned media of cells obtained from ethanol- or saline-treated animals (ET-CM or SAL-CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET-CM to SAL-CM. Among 59 proteins found reduced in ET-CM we found components of the extracellular matrix like Laminin subunits α2, α4, β1, β2 and γ1 and the proteoglycans Biglycan, Heparin Sulfate Proteoglycan 2 and Lumican. Proteins with trophic function like Insulin-Like Growth Factor Binding Protein 4, Pigment Epithelium-Derived Factor and Clusterin as well as proteins involved on modulation of proteolysis like TIMP-1 and PAI-1 were also found reduced. On the other hand, pro-synaptogeneic proteins like Thrombospondin-1, Hevin as well as the modulator of extracelular matrix expression, Angiotensinogen, were found increased in ET-CM. The analysis of interactome maps through Ingenuity Pathway Analysis demonstrated that the Amyloid beta A4 protein precursor (APP), which was found reduced in ET-CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET-CM, rendering it a suitable target for a more accurate analysis about the effects of ethanol on its biology. Taken together our results strongly suggest that early exposure to teratogens such alcohol may lead to an enduring change in astrocyte secretome, affecting mainly the expression of trophic and matricellular proteins, which in turn may lead to permanent alterations in brain function.

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Prenatal alcohol exposure: profiling developmental DNA methylation patterns in central and peripheral tissues

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