Neuronal Epigenome Mapping in Autism and Schizophrenia - Schahram Akbarian
University of Massachusetts Medical School

Major psychiatric disease, including depression and schizophrenia, are likely to result from a complex interaction of heritable and environmental factors. Straightforward genetic causes are still lacking for a large majority of affected individuals. Here, I will focus on ‘epigenetic’ disease models that attribute brain phenotypes or gene expression alterations to mechanisms other than changes in genomic DNA sequence. The work presented here will include some of the first genome-wide mappings of nucleosome core histone modifications in neuronal chromatin from postmortem brain of subjects on the psychosis and autism spectrum. Recent technologies advances in chromatin extraction and sequencing technology make it possible to gain an unprecedented view of the developmental trajectory of cell -specific epigenomes from the human brain, and explore epigenetic differences between individuals, including potential alterations in disease.

Acknowledgement: We thank Dr. Ron Zielke and staff from Brain and Tissue Bank of the University of Maryland, Dr. Francine M. Benes and staff from the Harvard Brain Tissue Resource Center and the Autism Tissue Program (Director; Dr Jane Pickett), and Dr. William E. Bunney Jr. and Dr. Edward G. Jones at the University of California, Irvine and Davis, and Dr. Andree Lessard at the University of Maryland/Maryland Psychiatric Research Center for supplying some of the postmortem brain tissue used in this study, and Dr. Ellen Kittler and Dr. Maria Zapp from the UMMS Deep Sequencing Core and Dr. Richard Konz and staff from the UMMS Flow Cytometry core. The work was supported by Autism Speaks, the International Mental Health Research Organization, the National Alliance for Research on Schizophrenia and Depression, and the National Institute of Mental Health.

CIRCUITRY-BASED AND DIAGNOSIS SPECIFIC DISTURBANCES OF GENOMIC INTEGRITY EXPRESSION OF GAD67 REGULATORY GENES IN THE HIPPOCAMPUS OF SCHIZOPHRENIC AND BIPOLAR SUBJECTS - Francine M. Benes and Guoqing Sheng
Program in Structural and Molecular Neuroscience, McLean Hospital, Belmont, MA; Department of Psychiatry, Harvard Medical School

The maintenance of genomic integrity in the adult brain is believed to be involved in the pathogenesis of central nervous diseases. In psychotic disorders, a dysregulation of the 67 kDalton isoform of glutamate decarboxylase (GAD67) involves a network in GABA cells of the adult hippocampus that includes genes associated with kainate receptor subunits, TGFβ and Wnt signaling and transcription factors. Notably, this network also includes genes involved in the regulation of cell cycle and DNA repair. The association of genomic integrity with transcript expression in the healthy and diseased brain is not well understood. Laser microdissection was used to obtain samples of a GABA cell-rich layer in hippocampal sectors CA3/2 and CA1 of SZs and BDs where the expression changes were previously noted. In sectors CA3/2 and CA1, the results demonstrated that robust changes in CNVs were observed in both groups, however, in CA3/2 only, they correlated significantly with mRNA expression for the same target genes. The patterns noted in SZs and BDs were similar to those previously reported for expression changes and showed fundamental differences for the subjects in the respective groups. Overall, the data suggest that the association of CNVs with transcripts for specific genes involved in GABA cell regulation occurs in a circuitry-based and diagnosis-specific manner. Supported by MH42261, MH077175, MH31862, and the Test Endowment.

Roles of Histone Modifications in Neurodegeneration and Neuroprotection - De-Maw Chuang
National Institute of Mental Health, National Institutes of Health, Bethesda, MD

Acetylation and deacetylation of histone proteins play a key role in regulating chromatin conformation and transcriptional activity. Inhibition of histone deacetylases (HDACs) results in enhanced histone acetylation, causing chromatin relaxation and gene expression. Valproic acid (VPA), long used to treat bipolar disorder and seizures, is an inhibitor of class I and IIa HDACs and exhibits neuroprotective, neurotrophic and anti-inflammatory properties. In cultured neurons, VPA and related HDAC inhibitors induced transcriptional activation of BDNF and HSP70, which can contribute to neuroprotection against glutamate excitotoxicity. VPA enhanced the synthesis and release of GDNF in astrocytes, triggering neurite outgrowth and protection against neuroinflammatory and apoptotic insults. We investigated the effects of HDAC inhibition in two experimental models of neurodegenerative disorders, cerebral ischemia and Huntington’s disease (HD), in which pathophysiology is associated with histone hypoacetylation. Using a rat middle cerebral artery occlusion (MCAO) model, post-insult VPA treatment suppressed brain infarction, caspase-3 activity, microglial activation, and neurological impairments. Histone acetylation, as well as HSP70 expression, was robustly increased by VPA in the ischemic brain. HDAC inhibition also alleviated MCAO-induced BBB disruption and promoted subsequent increase in neurogenesis and angiogenesis; regulation of MMP-9, BDNF and VEGF likely played important roles in these processes. In two transgenic mouse models of HD, N171-82Q and YAC128, dietary VPA treatment showed multiple benefits. VPA normalized behavioral performance in rotarod and open-field tests, showed anti-depressant and ant-anxiety effects, and prolonged survival time. Interestingly, these beneficial effects were potentiated by co-treatment with lithium, another mood stabilizer. Growing evidence supports the notion that chromatin remodeling by HDAC inhibition is a promising avenue for therapeutic intervention in a large number of neurodegenerative and neuropsychiatric conditions.

DNA methylation in the CNS - Guoping Fan
Department of Human Genetics, Brain Research Institute David Geffen School of Medicine, UCLA, Los Angeles, CA 90095 USA

DNA methylation is a major epigenetic factor involved in developmental gene regulation, genomic imprinting, X-inactivation and genome stability. Aberrant methylation patterns or mutations in methyl-CpG binding protein 2 (MeCP2) have been associated with several mental retardation disorders including Fragile-X, Rett, and ICF (Immunodeficiency, Centromere instability, and Facial anomaly) Syndrome, suggesting that DNA methylation is important for brain development and function. Intriguingly, a substantial level of DNA methyltransferases (Dnmts) is expressed in the developing and mature brain, particularly in postmitotic neurons that are already exit cell cycle. In order to gain insights into the role of Dnmts and DNA methylation in the central nervous system (CNS), we have generated a series of conditional mutant mice that are deficient of either single or multiple DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) in the CNS at distinct developmental stages. Our analysis showed that these mutant mice exhibit multiple defects in neuronal survival, neural cell differentiation, synaptic plasticity, and learning & memory behavior. Our results indicate that epigenetic alterations (e.g. DNA hypomethylation) can lead to abnormal brain development and function, supporting the notion that a number of human neuropsychiatric disorders may be mediated by aberrant epigenetic changes occurred in development or in adulthood.

GENOME-WIDE STUDY OF COCAINE INDUCED TRANSCRIPTION AND EPIGENOME CHANGE IN NUCLEUS ACCUMBENS - Jian Feng
Mount Sinai School of Medicine, New York, NY 10029

Repeated cocaine exposure induces alterations in genome-wide transcriptional regulatory networks, chromatin remodeling activity and gene expression profiles in the brain's reward circuitry. By using massive parallel DNA sequencing (mRNA seq and ChIP-seq), we try to identify cocaine induced gene transcription change as well as histone modification alterations in the nucleus accumbens (NAc), a key brain reward region. Taking the heterochromatin mark histone 3 lysine 9 trimethylation (H3K9me3) as an example, cocaine decreases H3K9me3 enrichment at specific genomic repeats regions [e.g., long interspersed nuclear element (LINE)-1 repeats] that accompanies the increased expression of LINE-1 retrotransposon-associated repetitive elements in NAc . This likely reflects global patterns of genomic destabilization in this brain region after repeated cocaine administration and indicates a putative role for heterochromatic regulation in the long-term actions of cocaine.

THE ROLE OF HISTONE ACETYLATION IN AGE-ASSOCIATED MEMORY IMPAIRMENT AND ALZHEIMERʼS DISEASE - André Fischer
Laboratory for Aging and Cognitive Diseases, European Neuroscience Institute, 37077 Goettingen, Germany

Changes in gene expression in the brain are essential for memory consolidation and proper genome-environment interaction is a prerequisite for cognitive function. Deregulation of this system may underlie cognitive deficits inherent to both normal aging and neurodegenerative disease. However, the mechanisms underlying pathological alterations in the brain transcriptome are little understood.

Epigenetic processes like DNA methylation and histone acetylation are central regulators of genome-environment interactions. In line with this, it has been shown that the epigenetic machinery is essential for cognitive function. In turn, there is now accumulating evidence that altered chromatin plasticity and histone acetylation is also involved in cognitive aging, neurodegeneration, and neuropsychiatric diseases. Targeting the epigenome is therefore currently discussed as a promising therapeutic strategy. For example, histone deacetylase (HDAC) inhibitors exhibit neuroprotective and neuroregenerative properties in animal models of various brain diseases.

My presentation will specifically address the role of histone acetylation in age-associated memory impairment and Alzheimer’s disease and ask why targeting the epigenome could be a suitable strategy for neuroprotection and neuroregeneration.

Epigenetic regulation of the Reelin and GAD67 Promoters - Dennis R. Grayson
University of Illinois at Chicago

γ-amino acid decarboxylase 67 (GAD₆₇) corresponds to one of two enzymes that decarboxylates glutamate to produce γ-aminobutyric acid, the main inhibitory neurotransmitter in the mammalian central nervous system, hence defining the phenotype of a diverse set of inhibitory interneurons of the brain. In contrast, reelin (Reln) is an extracellular matrix protein expressed in GABAergic neurons that functions in the elaboration of dendritic spines in adult neurons.  Reduced cortical GAD67 and Reln mRNA levels have consistently been reported in schizophrenia and bipolar disorder with psychosis suggesting an inhibitory hypofunction as contributing to the core symptoms associated with this disease. We have analyzed the human GAD₆₇ and Reln promoters using transient transfection analysis of upstream and downstream sequences. Interestingly, results from these studies show that cis-acting regulatory elements are located downstream of the RNA start site and are in the region corresponding to the first exon. Trans-acting factors bind to sequences active in promoting downstream reporter expression that show higher levels of DNA methylation. The Class I histone deacetylase inhibitor MS-275 potently activates GAD₆₇ and Reln mRNA expression in NT2 cells suggesting the possibility that the promoter is sensitive to drugs that induce chromatin remodeling. Using methyl DNA immunoprecipitation of MS-275-treated NT2 cells, we provide data showing that Class I HDAC inhibition facilitates increases in GAD₆₇ and Reln mRNA expression and this is accompanied by decreased promoter methylation. Moreover, the reduced levels of DNA methylation are highest in those regions proximal to the location of the in vitro defined cis-acting regulatory elements. Our data suggest that changes in promoter methylation associated with gene regulation are not random but overlap the locations of proximal cis-acting elements. The search to find clear cut differences in DNA methylation in postmortem brain tissue of schizophrenia subjects at specific gene promoters has caused some confusion as to whether DNA methylation plays a key role in the pathogenesis of the disease. However, studying human genomic DNA for subtle differences in methylation is more about knowing where to look for these changes.

Acknowledgement.  I acknowledge the many contributions of Ying Chen, M.D. to results presented.

Dr. Costa’s Psychiatric Research Legacy - Alessandro Guidotti
University of Illinois at Chicago

In 1996, Dr. Costa was invited by Prof. Boris Astrachan, then Chairman of the Department of Psychiatry at the University of Illinois at Chicago, to direct the research of the Psychiatric Institute, Department of Psychiatry, School of Medicine, at the University of Illinois at Chicago. He was asked to develop a seminal research program on psychiatric disorders. Viewed in retrospect, Dr. Costa met and surpassed the challenge, as was usual for him.

To elucidate the molecular mechanisms whereby nurture (epigenetic factors) and nature (genetic factors) interact to cause major psychiatric disorders was at the center of Dr. Costa’s mission for the last 15 years of his research at the Psychiatric Institute.

The challenge for Dr. Costa and his colleagues (Drs. Auta, Caruncho, Davis, Grayson, Guidotti, Impagnatiello, Kiedrowski, Larson, Manev, Pappas, Pesold, Pinna, Sharma, Smalheiser, Sugaya, Tueting, Veldic) had always been to find new ways to prevent and treat psychiatric disorders with pharmacological agents that failed to have major unwanted side effects.

From the start, Dr. Costa was aware of the importance of GABAergic transmission in maintaining an efficient synchronization of cortical activity and also that the reciprocal interaction between GABAergic interneurons and glutamatergic pyramidal neurons was disrupted in the cortex and hippocampus of schizophrenia patients.

Between 1998 (Impagnatiello et al.) and 2000 (Guidotti et al.), Dr. Costa’s laboratory reported that a GABAergic neuropathology is evident in the hippocampus and cortex in postmortem brains of SZ and BP disorder patients. This GABAergic neuropathology is characterized by a decrease in the expression of GAD67 and reelin mRNAs and protein.

The next question was “what is the cause of GABAergic neurotransmitter downregulation in prefrontal cortex of SZ and BP patients?” This question was addressed by Dr. Costa and his colleagues in a seminal paper entitled “Reelin and schizophrenia: a disease at the interface of the genome and the epigenome.” The hypothesis was that SZ or BP disorder psychopathologies are the consequence of synergistic interactions of multiple susceptibility genes with neuroepigenetic factors.

The findings reviewed in this presentation are an outcome of the pioneering work of Dr. Costa and his collaborators (1996-2009) and suggest that an epigenetic downregulation of telencephalic GABAergic genes may be a contributing factor to the behavioral and cognitive impairments experienced by SZ and BP disorder patients.

The pioneering research on psychosis and the breakthroughs in the study of aberrant epigenetic molecular mechanisms operative in SZ and BP disorder represent yet another example of Dr. Costa’s prodigious output of important discoveries during the 60 years of his productive career. Dr. Costa pursued science with relentless intensity and was enormously productive. He was never interested in the safe, predictable regions of the scientific arena; he was always more comfortable at the cutting edge and he was always provocative. It is striking how consistently innovative he was!

Dr. Costa left us not only with the rich legacy of his work in Neuroscience but also with the many talented students and postdocs he trained.

The Sixth DNA Base in Brain Development - Peng Jin
Department of Human Genetics, Emory University School of Medicine, Atlanta, GA

5-Methylcytosine (5-mC) is an important DNA modification found in eukaryotes that impacts gene regulation and disease pathogenesis. Recently, 5-hydroxymethylcytosine (5-hmC), another form of DNA modification, has been identified. It has been shown that 5-hmC is significantly enriched in the central nervous system relative to many other tissues and cell types. To elucidate the biology of 5-hmC, we have recently developed a selective chemical labeling method for 5-hmC by utilizing T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC, which in turn can chemically incorporate a biotin group for detection, affinity enrichment, and sequencing. Using this technology, we have generated the detailed genome-wide maps of 5-hmC in mouse cerebellum and hippocampus during development and aging, as well as in proliferating and differentiated neural stem cells. Our analyses suggest a dynamic regulation of 5-hmC during neuronal differentiation, neurodevelopment and aging.

Genetics and Epigenetics of Human Brain Development and Schizophrenia - Joel E. Kleinman, M.D., Ph.D.
Chief, Section on Neuropathology, CBDB, GCAP, IRP, NIMH, NIH

Introduction. Advances in methodology have made possible genome wide studies of genetic variation, mRNA transcript expression and epigenetic methylation. These can be applied to postmortem human brain in order to study development and brain diseases/syndromes including schizophrenia.

Methods. Using microarray technology from illumina we have analyzed prefrontal cortex and to a lesser degree hippocampus in large cohorts of patients with schizophrenia (n=113) and normal controls (n= 287 from week 14 in the fetus to 80 years of age) to examine associations of genetic variation with expression and methylation (the latter involved only a subset of the normals, n= 109). A number of these findings have been confirmed with qRT-PCR. Moreover, a number of novel transcripts were detected in human brain using exon by exon amplification as well as 5’ and 3” RACE

Results. Novel transcripts have been found in a number of genes including GRM3, KCNH2, DISC1, NRG1 and 3. Schizophrenia associated risk alleles are associated with alternative transcripts in these genes that are preferentially expressed in postmortem human brain. A GWAs study of genetic variation and expression has found thousands of associations between SNPs and expression with significance between 10^-8 and 10^-79. Lastly, there are a number of significant associations between methylation and expression and stages of brain development.

Conclusions. Genome wide studies have made possible a number of findings that may contribute to improved understanding of schizophrenia.

References. Kleinman JE, Law AJ, Lipska BK, Hyde TM, Ellis JK, Harrison PJ and Weinberger DR. Genetic neuropathology of schizophrenia: new approaches to an old question and new uses for postmortem human brains.Biological Psychiatry 69: 140-145, 2011.

Creating Zinc Monkey Wrenches – HDAC6 Selective Inhibitors for Use in Inflammation, Transplant Medicine, and Neurological Disorders. - Alan P, Kozikowski
Drug Discovery Program Department of Medicinal Chemistry University of Illinois at Chicago

CHROMATIN DYNAMICS OF PROXIMAL 15q IN AUTISM-SPECTRUM DISORDERS - Janine M. LaSalle

Dag H. Yasui¹, Haley A. Scoles¹, Karen N. Leung¹, Weston Powell¹, Shin-ichi Horike³, Makiko Meguro-Horike³, Keith W. Dunaway¹, Diane I. Schroeder¹ and Janine M. LaSalle^1,2

¹Department of Medical Microbiology and Immunology, Genome Center, University of California Davis School of Medicine, One Shields Avenue Davis, CA 95616. ²UC Davis MIND Institute, 2825 50^th Street, Sacramento, CA. ³Frontier Science Organization, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan.

Autism is an increasingly common disorder of complex etiology, affected by multiple genetic, epigenetic, and environmental influences. Methylation of CpG dinucleotides and methyl-specific binding proteins are part of an epigenetic pathway essential for parental imprinting and chromatin dynamics during normal brain development. Autism has several phenotypic features in common with the neurodevelopmental disorders with altered epigenetic pathways. Prader-Willi (PWS), Angelman (AS), and 15q duplication syndromes are imprinted disorders caused by paternal or maternal 15q11-13 deficiency or duplication, respectively. Copy number variants within 15q11-13 are also associated with a spectrum of neurodevelopmental disorders, including schizophrenia and epilepsy. Rett syndrome (RTT) is an X-linked pervasive developmental disorder caused by mutations in MECP2, which encodes methyl-CpG-binding protein 2 (MeCP2). Previous results establish that MeCP2 binds to the imprinting control region (PWS-IC) and is required for optimal expression of the 15q11.2-13.3 genes GABRB3 and UBE3A. In addition, the paternal allele expressing multiple imprinted small nucleolar RNAs undergoes extensive chromatin decondensation specifically in postnatal neurons. To identify long-range interactions of the PWS-IC with distant loci, chromosome capture conformation on chip (4C) analysis for a 13 Mb region of 15q11.2-13.3 was performed on human SH-SY5Y neuroblastoma cells. Differentiated SH-SY5Y neurons displayed 2.84 fold fewer 15q11.2-13.3 chromatin interactions with the PWS-IC than undifferentiated neuroblasts, revealing a developmental increase in chromatin decondensation. When total 15q11.2-13.3 PWS-IC interactions identified by 4C were compared with previously identified MeCP2 binding sites, five sites were found to significantly overlap. Remarkably, overlapping PWS-IC and MeCP2 bound sites mapped to 15q13.3 containing CHRNA7 encoding the cholinergic receptor, nicotinic, alpha 7. Subsequent quantitative transcriptional analyses of frontal cortex from Rett syndrome and autism patients revealed significantly reduced CHRNA7 expression compared to controls. PWS-IC interaction with CHRNA7 was independently confirmed by fluorescence in situ hybridization analysis of SH-SY5Y neuroblasts and neurons. Together, these results suggest that transcription of CHRNA7 is modulated by chromatin interactions with the PWS-IC. Therefore, loss of long-range chromatin interactions within 15q11.2-13.3 may contribute to shared mechanisms underlying multiple human neurodevelopmental disorders.

NEURONAL ACTIVITY-INDUCED GADD45B PROMOTES EPIGENETIC DNA DEMETHYLATION AND ADULT NEUROGENESIS - Dengke Ma
Massachusetts Institute of Technology, Boston, MA, USA

We initiated an expression screen for epigenetic regulators of adult neurogenesis and found the striking induction of Gadd45b by neuronal activity. Mice with Gadd45b deletion exhibit specific deficits in neural activity-induced proliferation of neural progenitors and dendritic growth of newborn neurons in the adult hippocampus. Mechanistically, Gadd45b is required for activity-induced DNA demethylation of specific promoters and expression of corresponding genes critical for adult neurogenesis, including brain-derived neurotrophic factor and fibroblast growth factor. Reversible neuronal DNA methylation in the mammalian nervous system has been previously implicated, but its molecular mechanisms and functional significance remain largely unclear. We suggest that Gadd45b translates transient neuronal activity stimuli to lasting epigenetic DNA modifications in neurons, with specific functional impact on activity-regulated aspects of adult neurogenesis. Our studies also support emerging lines of evidence that active DNA demethylation in mammals can occur through DNA excision repair-like mechanisms. Gadd45b may facilitate the second step of DNA demethylation by promoting completion of DNA excision repair following actions of 5-methylcytosine hydroxylasese and cytidine deaminase.

5-Hydroxymethylcytosine in Aging Brain - Hari Manev
Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago

In all higher organisms, development and aging are characterized by rather particular patterns of gene expression that are highly susceptible to epigenetic/environmental influences. Neuronal DNA appears to be sensitive to aging-associated alterations such as the oxidative stressinduced bases oxidation that is typically related to neurodegenerative disorders. In addition, significant progress has been made in research aimed at clarifying mechanisms of and a role for DNA methylation at the 5-position of cytosine (5-mC) in brain functioning. Recently, a different type of DNA cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been detected at high levels in the brain including cerebellar Purkinje neurons. A putative mechanism leading to 5- hmC formation includes a family of TET (ten-eleven-translocation) genes. Only rudimentary information is available on general mechanisms leading to the 5-hmC type of DNA modification in an aging brain. In this talk, I will use our findings obtained in a mouse model of aging to stress the importance of methodological characteristics, brain-region differences, and cell-type specificity of aging-related alterations of DNA 5-hmC modifications. Our data point to a new direction in 5-hmC DNA research.

Acknowledgement: I acknowledge the contribution by Svetlana Dzitoyeva, PhD and Hu Chen, PhD and support from the NIH - National Institute on Aging grant R01AG015347.

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.

Anxiety and Alcoholism: A Perspective from Epigenetic Studies - Subhash C. Pandey, PhD
Dept. of Psychiatry, University of Illinois at Chicago & Jesse Brown VA Medical Center, Chicago, IL 60612

Epigenetic mechanisms of gene expression are an emerging field that has been implicated in the pathophysiology of brain disorders. We investigated the role of amygdaloid chromatin remodeling in the co-morbidity of anxiety and alcoholism using well-established animal model of alcoholism. The selectively-bred alcohol preferring (P) rats display heightened anxiety and drink higher amounts of ethanol compared to alcohol non-preferring (NP) rats. We found that nuclear, but not cytosolic, HDAC activity was higher in the amygdala of P rats compared to NP rats. This is due to higher protein levels of HDAC-2 (class I HDAC), but not HDAC-4 (class II HDAC) in the central (CeA) and medial amygdala (MeA) of P compared with NP rats. We also found that acetylation of histones (H3-K9 and H4-K8) were lower and dimethylation of H3 (K-9) levels were higher in the CeA and MeA of P rats compared with NP rats. Treatment with trichostatin A (TSA), an HDAC inhibitor, inhibited HDAC activity and decreased HDAC-2, but not HDAC-4 protein levels, and corrected the deficits in histone acetylation and neuropeptide Y expression in the CeA and MeA of P rats. TSA treatment attenuated the anxiety-like behaviors, and also decreased the voluntary alcohol-drinking behaviors in P rats, but had no effects on these behaviors in NP rats. It has been shown that HDAC-2 regulates synaptic plasticity, we therefore examined changes in HDAC-2 and dendritic spine density (DSD) during ethanol exposure in P and NP rats. Acute ethanol exposure normalizes the innately higher nuclear HDAC activity, HDAC-2 protein levels and histone (H3) methylation, and also normalizes the lower histone (H3) acetylation and lower expression of synaptic plasticity related genes (brain-derived neurotrophic factor and activity-regulated cytoskeleton-associated protein) in the amygdaloid structures of P, but not of NP rats. Acute ethanol also corrected the deficits in DSD in the amygdaloid structures and produced anxiolytic effects in P, but not in NP rats. Interestingly, knockdown of HDAC2 in the CeA by siRNA, produced anxiolytic effects in P rats and corrected the deficits in histone acetylation and DSD in the CeA of P rats. Taken together, these results indicate that upregulation of HDAC-2 in the neurocircuitry of the amygdala may be responsible for abnormal chromatin architecture that may be involved in the co-morbidity of anxiety and alcoholism and HDAC inhibitors may serve as potential therapeutic agents for the treatment of alcoholism (Supported by NIH-NIAAA grants and VA Merit and Career Scientist Grants to SCP).

Epigenomics and Complex Psychiatric Disease - Art Petronis
The Krembil Family Epigenetics Laboratory, Centre for Addiction and Mental Health, and University of Toronto, Toronto, Ontario, Canada

The traditional aetiological approach to understanding the causes of complex psychiatric disease has focussed on the interplay between genetic (DNA) and environmental factors. There are, however, numerous epidemiological, clinical, and molecular peculiarities associated with psychiatric diseases that are hard to explain using traditional DNA- and environment-based approaches. For example, discordance between monozygotic twins, relatively late age of onset, sexual dimorphism, parental-origin effects, and significant fluctuation of disease course suggest that epigenetic factors may also play a role in disease aetiology. In addition to DNA, epigenetic variation in the germline can be a secondary molecular substrate of heritability. Epigenetics may also allow contribute to the understanding of the molecular effects of environmental hazards. Finally, a non-environmental epigenetic metastability could be an important etiological player in psychiatric disease. All these epigenetic aspects will be illustrated by experimental findings from our ongoing DNA methylome studies of schizophrenia and bipolar disorder.

EPIGENETIC MECHANISMS IN MEMORY FORMATION - J. David Sweatt
UAB School of Medicine, Birmingham, Alabama

This presentation will address the idea that conservation of epigenetic mechanisms for information storage represents a unifying model in biology, with epigenetic mechanisms being utilized for cellular memory at levels from behavioral memory to development to cellular differentiation.

The area of epigenetics is unfamiliar to many neurobiologists: epigenetic mechanisms typically involve alterations in chromatin structure, which in turn regulate gene expression. “Epigenetics” is functionally equivalent to the mechanisms allowing stable maintenance of gene expression that involve physically “marking” DNA or its associated proteins through post-translational modification. Thus, regulation of chromatin structure and regulation of direct methylation of DNA are the principal mechanisms of epigenetic regulation.

Do epigenetic mechanisms operate in behavioral memory formation? We have generated several lines of evidence that support this idea. 1. Contextual fear conditioning triggers alterations in hippocampal histone acetylation, and contextual latent inhibition training triggers similar but distinct changes in histone acetylation. 2. The methyl-DNA binding protein MeCP2 (the Rett mental retardation syndrome gene product) alters chromatin structure and regulates hippocampal LTP and memory formation. 3. Inhibitors of DNA methylation block both hippocampal LTP and associative learning in vivo.

Conclusions - An emerging idea is that the regulation of chromatin structure, mechanistically via histone modification and DNA methylation, may mediate long-lasting behavioral change and learning and memory. We find this idea fascinating because similar mechanisms are used for triggering and storing long-term "memory" at the cellular level, for example when cells differentiate.

Epigenetic Programming of Behavior by the Early Life Social Environment - Moshe Szyf
McGill University Montreal Canada

One of the outstanding questions in behavioral disorders is untangling the complex relationship between nurture and nature. Although epidemiological data provides evidence that there is an interaction between genetics (nature) and the social and physical environments (nurture) in a spectrum of behavioral disorders; the main open question remains the mechanism. Emerging data supports the hypothesis that DNA methylation a covalent modification of the DNA molecule that is a component of its chemical structure serves as an interface between the dynamic environment and the fixed genome. We propose that modulation of DNA methylation in response to environmental cues early in life serves as a mechanism of life-long genome adaptation. Under certain contexts this adaptation can turn maladaptive resulting in behavioral disorders. We will discuss emerging evidence from rodent, primate and human studies.

Function of Long Noncoding RNAs in the Nervous System - Claes Wahlestedt MD PhD
University of Miami, Miami FL 33136, USA

Much of the mammalian genome is transcribed into long noncoding RNAs of different categories. This lecture will primarily be concerned with natural antisense transcripts (most of which are long noncoding RNAs) which regulate gene expression through several distinct mechanisms. Inhibition/perturbation of endogenous natural antisense transcripts by AntagoNATs, in vitro or in vivo, reveals concordant or discordant regulation and results in down- or up-regulation of conventional (protein-coding) gene expression, respectively. Examples of CNS prime drug targets that are under the regulation of long noncoding RNAs will be used to illustrate these phenomena.