Elsevier

Alcohol

Volume 60, May 2017, Pages 135-147
Alcohol

DNA Methylation program in normal and alcohol-induced thinning cortex

https://doi.org/10.1016/j.alcohol.2017.01.006Get rights and content

Highlights

  • The DNA methylation program progresses through cortical layer formation.

  • Fetal alcohol exposure during the first two trimesters leads to cortical thinning.

  • Cortical thinning occurs through reduced proliferation, migration, and maturity.

  • The DNA methylation program is dysregulated in parallel with abnormal phenotypes.

  • A hindered DNA methylation program may underlie alcohol-induced cortical deficits.

Abstract

While cerebral underdevelopment is a hallmark of fetal alcohol spectrum disorders (FASD), the mechanism(s) guiding the broad cortical neurodevelopmental deficits are not clear. DNA methylation is known to regulate early development and tissue specification through gene regulation. Here, we examined DNA methylation in the onset of alcohol-induced cortical thinning in a mouse model of FASD. C57BL/6 (B6) mice were administered a 4% alcohol (v/v) liquid diet from embryonic (E) days 7–16, and their embryos were harvested at E17, along with isocaloric liquid diet and lab chow controls. Cortical neuroanatomy, neural phenotypes, and epigenetic markers of methylation were assessed using immunohistochemistry, Western blot, and methyl-DNA assays. We report that cortical thickness, neuroepithelial proliferation, and neuronal migration and maturity were found to be deterred by alcohol at E17. Simultaneously, DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxcylmethylcytosine (5hmC), which progresses as an intrinsic program guiding normal embryonic cortical development, was severely affected by in utero alcohol exposure. The intricate relationship between cortical thinning and this DNA methylation program disruption is detailed and illustrated. DNA methylation, dynamic across the multiple cortical layers during the late embryonic stage, is highly disrupted by fetal alcohol exposure; this disruption occurs in tandem with characteristic developmental abnormalities, ranging from structural to molecular. Finally, our findings point to a significant question for future exploration: whether epigenetics guides neurodevelopment or whether developmental conditions dictate epigenetic dynamics in the context of alcohol-induced cortical teratogenesis.

Introduction

Children with Fetal Alcohol Spectrum Disorders (FASD) have been reported to suffer cognitive and neurological deficits, including learning disabilities, intellectual disabilities, and impairments of expressive and receptive language (Green, 2007, Jacobson et al., 2011, Jones et al., 2010, Lebel et al., 2012). Some of the underlying brain abnormalities of FASD include reduced brain volume (microcephaly), reduced grey matter (Nardelli, Lebel, Rasmussen, Andrew, & Beaulieu, 2011), and reduced corpus callosum (Yang, Phillips, et al., 2012). Cross-sectional neuroimaging studies have also recently revealed that children and adolescents suffering from FASD exhibit abnormalities in the thickness of different regions of the cerebral cortex, as compared to healthy controls (Robertson et al., 2016, Sowell et al., 2008, Yang et al., 2012b). While several observations have been made regarding the fundamental hindrance of alcohol on cortical development, e.g., apoptosis (Lebedeva et al., 2015), deficiency of neurotrophic factors, prevention of cell migration (Aronne et al., 2011, Chikhladze et al., 2011, Riar et al., 2016), and abnormal somatic morphologies of cortical neurons (Chikhladze et al., 2011, Lawrence et al., 2012), the mechanism underlying the structural abnormality systemically occurring in relationship to alcohol exposure is not clear.

Recently, alcohol has emerged as a key chemical player, which can reach nuclear chromatin and alter the core functions of DNA (for review see Resendiz, Lo, Badin, Chiu, & Zhou, 2016). We and other investigators (Perkins, Lehmann, Lawrence, & Kelly, 2013) have recently found that DNA methylation, an important regulator of gene expression, progresses in the developing nervous system as a program (Zhou, 2012), and is disturbed by alcohol in many aspects across neural tube (Zhou, Chen, & Love, 2011) and hippocampal development (Chen et al., 2013, Otero et al., 2012). Given the known intricacies of epigenetic mechanisms such as DNA methylation in gene regulation and cellular specification, we sought to characterize the epigenetic and phenotypic changes of chronic, moderate prenatal alcohol exposure in utero in the developing stages of the cortex. In this study, we report that beyond neural tube formation, the formation of the cortices adopt a systemic DNA methylation program (DMP) (including DNA methylation and its binding proteins), by which neuroepithelial cells (NEs) differentiate through the formation of cortical layers in a precise spatiotemporal manner. Aside from confirming cortical phenotypes of FASD, we also demonstrated novel deficiencies. These processes, together with global and cellular epigenetic mechanisms, may drive the consequential dysmorphology of the developing cortex. In this study, we demonstrate how alcohol interferes with the DMP, in parallel with cortical thinning and other abnormalities. Understanding the molecular drivers of alcohol-induced alteration of the highly ordered developmental cortical program is paramount toward uncovering how fetal environmental insults are established, maintained, and manifested into lasting cognitive and behavioral deficits.

Section snippets

Overview of experimental prenatal alcohol exposure

In this study, alcohol was administered via liquid diet according to the paradigm illustrated in Fig. 1A. The time course and types of analysis are summarized in Fig. 1B. Mice were conditioned to receive the liquid diet prior to mating. After conception, the liquid diet was re-introduced and alcohol was administered from E7–E16 (corresponding to brain development in the late first and second human trimester equivalent). The 4% alcohol liquid diet (v/v) administered in this paradigm has been

Results

First, we report the parallel development of the phenotypes and cellular features of normal corticogenesis alongside DNA methylation markers and their binding proteins layer-by-layer, revealing the cortical DNA methylation program of differentiating neuroepithelial cells into mature neurons. Subsequently, alcohol-induced aberrations of phenotypic and epigenetic features are demonstrably associated. Finally, a global analysis of the average cortical DNA methylation and MeCP2 protein are

Discussion

Fetal alcohol exposure has been associated with lasting cortical deficits through various molecular constructs and functional outcomes in human and rodent models of FASD (Abbott et al., 2016, El Shawa et al., 2013, Robertson et al., 2016, Zhou et al., 2011b). To date, however, the underpinnings of cortical thinning reported in human and animal models of FASD have not been clearly defined. We have previously demonstrated that an orderly progression of DNA methylation marks occurs parallel to the

Conclusion

Here we report that a dynamic DNA methylation program can be demonstrated throughout the distinct cortical laminae during development. 5mC and 5hmC, the two key methylation marks in the brain, demonstrated a differential distribution consistent with neural maturity. In the presence of fetal alcohol exposure, the DMP in the normal cortical condition was altered, globally, across individual cortical layers, and at a cellular level. More importantly, alcohol-induced alterations of the DMP

Funding sources

This work was supported by the National Institutes of Health AA024216, AA016698, and P50AA07611 to FCZ. Additional support was provided by the Indiana CTSI Cohort 11 core pilot grant to FCZ and BAP-SBEA 2011-5DR (Mersin University) to HÖ. NCÖ is supported in part by the Fulbright Visiting Researcher fellowship. MR is supported by National Institutes of Health training grant T32 AA007462.

References (55)

  • M. Resendiz et al.

    Alcohol metabolism and epigenetic methylation and acetylation

  • F.C. Zhou et al.

    Production and characterization of an anti-serotonin 1A receptor antibody which detects functional 5-HT1A binding sites

    Brain Research. Molecular Brain Research

    (1999)
  • F.C. Zhou et al.

    Moderate alcohol exposure compromises neural tube midline development in prenatal brain

    Brain Research. Developmental Brain Research

    (2003)
  • D. Zhou et al.

    Developmental cortical thinning in fetal alcohol spectrum disorders

    NeuroImage

    (2011)
  • C.W. Abbott et al.

    The impact of prenatal ethanol exposure on neuroanatomical and behavioral development in mice

    Alcoholism: Clinical and Experimental Research

    (2016)
  • J.B. Angevine et al.

    Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse

    Nature

    (1961)
  • B. Anthony et al.

    Alcohol exposure alters cell cycle and apoptotic events during early neurulation

    Alcohol and Alcoholism

    (2008)
  • M.P. Aronne et al.

    Prenatal ethanol exposure reduces the expression of the transcriptional factor Pax6 in the developing rat brain

    Annals of the New York Academy of Sciences

    (2008)
  • C. Camarillo et al.

    Ethanol exposure during neurogenesis induces persistent effects on neural maturation: Evidence from an ex vivo model of fetal cerebral cortical neuroepithelial progenitor maturation

    Gene Expression

    (2008)
  • M. Chahrour et al.

    MeCP2, a key contributor to neurological disease, activates and represses transcription

    Science

    (2008)
  • Y. Chen et al.

    Diversity of two forms of DNA methylation in the brain

    Frontiers in Genetics

    (2014)
  • Y. Chen et al.

    DNA methylation program in developing hippocampus and its alteration by alcohol

    PLoS One

    (2013)
  • R.T. Chikhladze et al.

    The spectrum of hemispheral cortex lesions in intrauterine alcoholic intoxication

    Georgian Medical News

    (2011)
  • N. Diotel et al.

    5-hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system

    The Journal of Comparative Neurology

    (2016)
  • H. El Shawa et al.

    Prenatal ethanol exposure disrupts intraneocortical circuitry, cortical gene expression, and behavior in a mouse model of FASD

    The Journal of Neuroscience

    (2013)
  • A.J. Garro et al.

    Ethanol consumption inhibits fetal DNA methylation in mice: Implications for the fetal alcohol syndrome

    Alcoholism: Clinical and Experimental Research

    (1991)
  • J.H. Green

    Fetal alcohol spectrum disorders: Understanding the effects of prenatal alcohol exposure and supporting students

    The Journal of School Health

    (2007)
  • Cited by (23)

    • Conditioned media of mouse macrophages modulates neuronal dynamics in mouse hippocampal cells

      2023, International Immunopharmacology
      Citation Excerpt :

      When we evaluated the staining pattern of CR immune reactivity on the positive control tissue (rat brain sections) and HT-22 cells prior to the main ICC experiments, we observed differentially stained cells rather than clearly countable cells (like negative or positive manner). Therefore, we performed an immunocytochemical scoring analysis in which we measured the intensity of specific fluorescent content in each cell as a proxy of immunocytochemical expression of CR protein [25–27]. The presence of positive staining was judged based on the camera's capture of a distinguishable image above background (black) level and by overlay of DAPI staining.

    • Rescue of ethanol-induced FASD-like phenotypes via prenatal co-administration of choline

      2020, Neuropharmacology
      Citation Excerpt :

      Choline influences neurodevelopment in a multitude of ways including acting as a methyl-group donor in one-carbon metabolism (OCM), the cellular pathway that generates methyl groups used for epigenetic modifications to DNA and histones (reviewed in Zeisel, 2006). Here, we report global DNA hypomethylation in rostral and caudal cortices of newborn EtOH mice, similar to previous reports (Öztürk et al., 2017). However, this effect was mitigated by choline supplementation, suggesting choline supplementation may prevent epigenetic dysregulation due to PrEE as previously reported (Otero et al., 2012; Bekdash et al., 2013).

    • Fetal toxicology

      2019, Handbook of Clinical Neurology
      Citation Excerpt :

      Later in gestation, exposure to ethanol significantly activates glycogen synthase kinase 3β in cortical neuroblasts and therefore the upregulation of programmed cell death protein (Pdcd4) (Riar et al., 2014), which leads to reduced protein synthesis in the developing cerebral cortex and reduced neuronal differentiation (Joya et al., 2014). Öztürk et al. (2017) demonstrated in a mouse model of FASD a significant reduction in cortical thickness, neuroepithelial proliferation, neuronal migration, and neuronal maturity, which were accompanied by a global reduction in DNA methylation. Also in a mouse model, Chen et al. (2013) demonstrated that prenatal alcohol exposure hindered hippocampal neuronal differentiation and maturation spatiotemporally, together with reduced acquisition and progression of methylation marks and altered chromatin translocation of these marks, which was correlated with developmental retardation.

    • DNA hydroxymethylation: Implications for toxicology and epigenetic epidemiology

      2018, Toxicoepigenetics: Core Principles and Applications
    View all citing articles on Scopus
    View full text