Day 2: Saturday, October 30, 2010

Session III: Development

Alterations of DNA Methylation Associated With Growth Restriction And Infant Neurobehavior

Carmen J. Marsit PhD^1,2, Amanda Filiberto¹, Cailey Bromer¹, Carolyn Banister, PhD¹, Devin Koestler², E. Andres Houseman ScD², James F. Padbury, MD³ and Barry Lester, PhD³

¹Department of Pathology and Laboratory Medicine
²Department of Community Health
³Department of Pediatrics, Brown Center for the Study of Children at Risk, Warren Alpert Medical School, Brown University, Women and Infants Hospital, Providence, RI

The period of intrauterine development constitutes one of the most critical periods during which modifications to the control of the genome through alterations to the epigenome can influence not only fetal development but also lead to programming of health outcomes including neurodevelopment throughout life. In a large, population-based study of growth restricted infants and matched controls, we are examining, using candidate gene and genomic approaches, the associations between gene-specific DNA methylation profiles in term placental tissue and infant growth and neurobehavior assessed using validated phenotypic assessments of newborn neurobehavior. We have identified that the extent of methylation of the glucocorticoid receptor exon 1F in the placenta is significantly associated with infant birth weight. Examining data from the Illumina Infinium Methylation BeadArray on a sample of over 200 placentas, we have also identified and validated a panel of 30 loci whose pattern of DNA methylation is highly predictive of infant birth weight. Two profiles of methylation in particular, could predict infant weights significantly reduced by 270 and 395 grams in models controlled for known confounders. Continuing analyses are examining how these gene-specific and genomic profiles are associated with measures of neurodevelopment as well as in identifying the functional consequences of the methylation patterns identified in these gene promoters. Taken together, these results indicate that the intrauterine environment mediates its phenotypic effects on infant outcomes through epigenetic alterations and that further examination of the genes and pathways targeted for alteration can provide critical insight into the developmental basis of mental health.

Epigenetic Mechanisms Underlying Persistent Alterations In Medial Prefrontal Cortical Function In Mice Exposed To Cocaine In Utero

Barry Kosofsky MD, PhD^1,2, Zeeba Daruwalla^1,2, Stephen Ra², Anjali Rajadhyaksha, PhD^1,2

¹Department of Neuroscience, Weill Cornell Graduate School of Biomedical Sciences, New York, NY
²Division of Pediatric Neurology, Department of Pediatrics, Weill Medical College of Cornell University, New York, NY

Prenatal exposure to cocaine has been shown to disrupt cognitive function in some children, which has also been seen in a number of animal models, including one we have developed in mice. It is likely that some of these cognitive deficits are a result of prenatal cocaine-induced persistent changes in the structure and function of the brain possibly due to epigenetic modifications of the genome. Our work has focused on the medial prefrontal cortex, a key brain region involved in cognitive functions including learning. We have been studying 2 molecules: one called brain derived neurotrophic factor (BDNF) and another called early growth response protein 1 (egr-1), two proteins that are induced by synaptic activation. We have identified changes in the expression of these genes in the medial prefrontal cortex of prenatally cocaine-exposed mice: they are increased in juvenile mice, but decreased in adults. We have also identified epigenetic modifications in the promoter regions of the BDNF and egr-1 genes in the medial prefrontal cortex of prenatally cocaine-exposed mice that may mediate the changes in expression that we observe. We have found decreased binding of the transcription factor methyl cytosine binding protein 2 (MeCP2) to the BDNF and egr-1 promoters in adult mice, and increased acetylation of one of the core histone proteins, namely histone 3, to the BDNF promoter in juvenile mice following prenatal cocaine exposure. Additional behavioral testing revealed that the cocaine-exposed offspring, when tested in adulthood, were able to process fearful and emotional stimuli but had a deficit in their ability to subsequently learn to ignore a fearful stimulus that was no longer threatening. We hypothesize that the observed molecular changes in juvenile and adult prenatally cocaine-exposed mice are the basis for such learning deficits, and that such molecular and behavioral changes evident in mice may also be evident in humans, which would have significant clinical implications (supported by K02DA00354 to BK).

Epigenetic Programming by Maternal Care

Michael Meaney PhD
McGill University, Montreal, Canada

Maternal care alters adaptive behavioral and endocrine responses to stress, as well as reproductive phenotypes in the rat. The mechanisms for these Œmaternal effects¹ involve stable changes in gene expression. Thus, the adult offspring of mothers that exhibit increased pup licking/grooming (LG) show increased hippocampal glucocorticoid receptor (GR) mRNA expression.
Effects on reproductive phenotypes in females involve altered estrogen receptor alpha expression in regions such as the medial preoptic area. The data presented here will review the evidence addressing two hypothesis: first, that sustained effects on gene expression are mediated by specific epigenetic states and 2) that intracellular signals derived from the tactile stimulation associated with specific forms of maternal care directly induce the relevant epigenetic states. Such processes reveal experience-dependent plasticity in the chemistry of the DNA and chromatin structure.

Session IV: Neuropsychiatry

Histone Methylation-Dependent Transcriptional Regulation Of Cocaine-Induced Behavioral And Structural Plasticity

Ian Maze PhD¹, Herbert E. Covington, III, PhD², David M. Dietz, PhD², Quincey LaPlant, PhD², HaoSheng Sun, BS², Alexander Tarakhovsky, MD, PhD³, and Eric J. Nestler, MD, PhD²

¹Rockefeller University, Laboratory of Chromatin Biology and Epigenetics, New York, NY
²Mount Sinai School of Medicine, Department of Neuroscience, New York, NY
³Rockefeller University, Laboratory of Lymphocyte Signaling, New York, NY

Cocaine-induced alterations in gene expression cause changes in neuronal morphology and behavior that may underlie cocaine addiction. We have identified an essential role for histone 3 lysine 9 dimethylation (H3K9me2) and the lysine dimethyltransferase G9a in cocaine-induced structural and behavioral plasticity. Repeated cocaine administration reduces global levels of H3K9me2 in nucleus accumbens (NAc). This reduction is mediated through repression of G9a expression in this brain region, which is regulated by the cocaine-induced transcription factor ∆FosB. Using conditional mutagenesis and viral-mediated gene transfer, we found that G9a downregulation increases dendritic spine plasticity of NAc neurons and enhances preference for cocaine. Due to high incidences of co-morbidity between substance abuse and stress-related illnesses, administration of drugs, such as cocaine, causes neural adaptations that may promote vulnerability to later stress experiences. We have identified histone methylation in NAc as a candidate mechanism linking drug exposure to enhancement of stress vulnerability. Repeated cocaine administration, prior to sub-chronic social defeat stress, potentiates depressive behaviors in mice through decreased H3K9me2 expression in NAc. Cre-mediated reduction of G9a promotes increased susceptibility to social stress, similar to that observed with repeated cocaine, whereas overexpression of G9a after repeated cocaine protects mice from the ensuing consequences of social stress. This form of resiliency is mediated, in part, through repression of Ras, a member of the BDNF-TrkB signaling cascade, which is induced after both repeated cocaine and chronic stress. Identifying such common regulatory mechanisms may aid in the development of therapeutics aimed at alleviating severe cases of addiction and depression.

Epigenetic Targets in Neurodegenerative and Psychiatric Disorders

Ted Abel PhD
University of Pennsylvania, Philadelphia, PA

Transcriptional activation is thought to be a key process in long-lasting forms of memory and synaptic plasticity. This activation is directed by transcription factors and their coactivators, which regulate gene expression via chromatin remodeling, histone modification and interactions with the basal transcription machinery. One type of histone modification associated with transcriptional activation is acetylation, which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that add or remove acetyl groups from histones, respectively. Recently, we have demonstrated that the transcriptional coactivator CREB-binding protein (CBP), a potent HAT, is involved in specific forms of long-term memory and synaptic plasticity. Mutant mice in which CBP activity in neurons is reduced either by the transgenic expression of an inhibitory form of cbp lacking the HAT domain or by knocking in a mutation of the CREB transcription factor-binding KIX domain of cbp exhibit deficits in spatial and contextual memory and in long-lasting forms of hippocampal synaptic plasticity. A complementary method to study the role of histone acetylation in synaptic plasticity and memory is to examine the effects of HDAC inhibitors, which increase the level of histone acetylation that correlates with transcriptional activation. We found that increasing histone acetylation using the HDAC inhibitor TSA enhances long-term contextual memory and facilitates synaptic plasticity via the transcription factor CREB. Using genetic approaches, we have found that conditional deletion of the transcriptional corepressor Sin3a results in enhanced contextual memory, consistent with the idea that HDACs are recruited to specific genes by Sin3a-containing complexes. We have identified a family of nuclear receptors that appears to be among the gene targets of HDAC inhibition critical for this cognitive enhancing activity. Histone acetylation may provide an epigenetic mechanism for establishing gene-specific modifications that result in the coordinate expression of genes required for long-term memory storage and HDAC inhibitors may provide a novel therapeutic approach to treat the cognitive deficits that accompany many psychiatric disorders.

Epigenetic Mechanisms Regulating Synapse Function And Behavior

Lisa M. Monteggia PhD
UT Southwestern Medical Center, Dallas, TX

Alterations in synaptic function contribute to the pathophysiology associated with several neuropsychiatric diseases. Modifications in synaptic vesicle trafficking can cause frequency-dependent changes in neurotransmission, alter information coding in neural circuits, and affect long-term plasticity. Rett syndrome, a neurodevelopmental disorder that arises from mutations in the methyl-CpG-binding protein-2 (MeCP2) gene, is a salient example for such a disease state in which synaptic transmission—in particular, spontaneous neurotransmission and short-term synaptic plasticity, have been altered. MeCP2 binds to methylated CpG islands in the promoter region of genes to silence expression and is part of a protein complex with histone deacetylases that also participates in repression of gene activation. We have therefore started to investigate the role of histone deacetylation and DNA methylation, two key epigenetic mechanisms, in the regulation of synaptic function. The regulation of histone deacetylation and DNA methylation by synaptic activity and how these epigenetic alterations affect neurotransmission will be critical to elucidate the mechanisms underlying Rett syndrome as well the roles these factors have in basic cellular processes. This work is essential in delineating key mechanisms that regulates properties of neurotransmission in the central nervous system that may underlie additional neuropsychiatric disorders.

Epigenetic Risk Factors In Social-Communication Disorders

David H. Skuse MD, PhD
Institute of Child Health, University College London, United Kingdom

Genomic imprinting involves the setting of epigenetic marks that result in differential gene expression from chromosomes that are of paternal or maternal origin. We proposed, over a decade ago, that imprinting of the X chromosome could result in sexually dimorphic characteristics. Our theory predicted that sexually dimorphic (male) vulnerability to neurodevelopmental disorders such as autism could occur, whether the silencing of alleles was confined to chromosomes that were either paternally- or maternally-derived. The X-chromosome is enriched for genes that are involved in brain function in both humans and mice. X-monosomy in humans (Turner syndrome) provides a model by which these putative mechanisms may be studied. In general, females with a single X chromosome of paternal origin have rather better social communication skills than those whose single X-chromosome is maternal in origin. They are also more empathic. These characteristics have been shown, in our longitudinal studies, to persist from childhood into adulthood.

Our human observations were followed up by a series of studies in X-monosomic mice. Replicated findings included preferential expression of alleles from the maternally-derived X-chromosome (specifically Xlr3b). This allele (invariably expressed in males) was associated with perseverative behavior. No evidence for preferential expression from the paternally-derived X-chromosome has been adduced yet, but recent work from Gregg et al (Science, July 2010) has provided new support for this controversial theory.