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Research Component 3/8: Adolescent Alcohol, Epigenetics and Behavioral Changes in Adulthood.

 

Principal Investigator: Subhash Pandey, Ph.D., University of Illinois, Chicago

 Abstract

Several clinical and preclinical studies suggest that adolescent binge drinking is one of the major risk factors for the development of psychiatric disorders, including anxiety and alcohol use disorder (AUD) later in life.

Adolescence represents an important stage of brain development, and epigenetic mechanisms are known to orchestrate developmental maturation of the brain from adolescence to adulthood. This research component of

Neurobiology of Adolescent Drinking in Adulthood (NADIA) consortium will examine how epigenetic modifications induced by adolescent intermittent ethanol (AIE) at the genome-wide level lead to an aberrant gene network pathway regulating various synaptic mechanisms in the amygdala that are responsible for anxiety-like and alcohol drinking behavior in adulthood. This component will identify closed and open domains of the epigenome after AIE in adult amygdala using Assay for Transposase-Accessible Chromatin-sequencing (ATACseq), and the corresponding regulation of the transcriptome by merging this dataset with existing RNA-seq data.

 

The overarching hypothesis of this proposal is that AIE will produce differential gene expression due to altered status of the epigenome in adult amygdala. The dynamic changes in epigenetic targets (HATs/EZH2/G9a/LSD1/DNMT3b) will induce changes in the transcriptome that regulate synaptic mechanisms in the amygdala after AIE in adulthood, ultimately regulating anxiety-like and alcohol-drinking behaviors. Specific Aim 1 will examine a) the status of chromatin accessibility and loci of genomic epigenetic marks using ATAC-seq in the adult amygdala of rats (male & female) after AIE. The emerging data set will be merged with existing RNA-seq data to identify epigenetically regulated transcriptomic changes, and b) To validate expression and epigenetic enrichments of new genes in the amygdala after AIE and use them for functional studies. Specific Aim 2 will examine the effects of neuronal epigenomic editing in the CeA using CRISPR/dCas9-p300, CRISPR/dCas9-KRAB, CRISPR/dCas9-Lsd1 (histone acetylation/methylation), or CRISPR/dCAS9-Tet1 (DNA methylation mechanism) on AIE-induced changes in gene expression and on anxiety-like and alcohol drinking behaviors in adult male and female rats. Specific Aim 3 will examine whether treatment with G9a inhibitor (UNC0642) will normalize epigenetic, gene expression, and dendritic spine changes in the amygdala and attenuate AIE-induced anxiety-like and alcohol drinking behaviors in adult male and female rats. Finally, Specific Aim 4 will translate epigenetic dynamics and expression of novel genes that will be identified from RNA-seq and ATAC-seq in the AIE rat amygdala to post-mortem amygdala of human alcoholics with early age onset and correlate them to drinking data (daily or weekly ethanol intake). The proposed studies will provide new information about epigenetic regulation of the whole transcriptome in the amygdala after AIE, leading to identification of epigenetic targets for drug development for the treatment of adult psychopathology.

 

 Specific Aims

Adolescence represents an important stage of brain development, and epigenetic mechanisms such as histone acetylation and DNA methylation are known to orchestrate developmental maturation of the brain from adolescence to adulthood (1-4). Gene expression is regulated by histone or DNA chemical modifications, and histone deacetylases (HDACs), histone acetyltransferases (HATs), and DNA methyltransferases (DNMTs) are the key enzymes implicated in these processes (4-8). It is well known that adolescent binge drinking is one of the crucial factors for the development of alcohol use disorder (AUD) and mental illnesses, including anxiety, later in life (9-12). This research component of NADIA will examine how epigenetic modifications induced by adolescent intermittent ethanol (AIE) at the genome-wide level lead to an aberrant gene network pathway regulating various synaptic mechanisms in the amygdala that are responsible for anxiety-like and alcohol drinking behavior in adulthood.

 

NADIA discoveries have shown that adolescent intermittent ethanol (AIE) exposure upregulates HDAC2 and DNMT3b, downregulates lysine demethylase 6B (KDM6B), CREB binding protein (CBP)/p300, and lysine demethylase 1 (LSD1), causes deficits in histone acetylation (H3K9Ac, H3K27Ac) and increases in histone methylation (H3K27me3, H3K9me2), and DNA methylation at regulatory sites of brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton-associated protein (Arc). This decreases dendritic spines and synapses in the amygdala, produces anxiety-like behaviors, and increases alcohol intake during adulthood in rats. Blocking HDAC activity with the pan-HDAC inhibitor, trichostatin A (TSA), and DNMT activity with 5-Azacytidine, in adulthood attenuates AIE-induced anxiety and alcohol intake, restores deficits in histone acetylation and inhibits DNA and histone methylation of synaptic genes, such as BDNF, Arc and neuropeptide Y (NPY) in the amygdala (4,13-20). Together, these data suggest the possibility that AIE may lead to altered chromatin structure at the genome-wide level in the adult amygdala.

Our pilot data using RNA-sequencing identified axon-guidance associated genes (Ephb6, Sema3e, Slit2, and Limk1) that are decreased in the adult amygdala by AIE. However, the epigenomic determinants of transcriptomic (axon guidance and synaptic genes) changes produced by AIE at the whole genome level in the amygdala and their role in AIE-induced anxiety and alcohol intake in adulthood are currently unknown. It is possible that specific epigenetic targets identified during the current funding period may be altering the epigenome and regulating synaptic events in the amygdala after AIE in adulthood, thereby regulating anxiety and alcohol intake. The proposed research will test this possibility using next-generation sequencing and bioinformatic approaches that include assay for transposase-accessible chromatin using sequencing (ATACseq), and by merging this dataset with our existing RNA-seq data. This will identify closed and open domains of the epigenome and the corresponding regulation of the transcriptome in adult amygdala after AIE. We hypothesize that AIE will produce differential gene expression due to altered status of the epigenome in adult amygdala. The dynamic changes in epigenetic targets (HATs/EZH2/G9a/LSD1/DNMT3b) will induce changes in the transcriptome that regulate synaptic mechanisms in the amygdala after AIE in adulthood, ultimately regulating anxiety-like and alcohol drinking behaviors. The following Specific Aims will test this hypothesis:

 

Aim 1: a) To examine the status of chromatin accessibility and loci of genomic epigenetic marks using ATACseq in the adult amygdala of rats (male & female) after AIE. The emerging data set will be merged with existing RNA-seq data to identify epigenetically regulated transcriptomic changes, b) To validate expression and epigenetic enrichments of new genes in the amygdala after AIE and use them for functional studies.

 

Aim 2: To examine the effects of neuronal epigenomic editing in CeA using CRISPR/dCas9-p300,

CRISPR/dCas9-KRAB, CRISPR/dCas9-Lsd1 (Histone acetylation/methylation), or CRISPR/dCAS9-Tet1 (DNA methylation mechanism) on AIE-induced changes in gene expression (identified from RNA-seq) and on anxiety-like and alcohol drinking behaviors in adult male and female rats.

 

Aim 3: To examine whether treatment with G9a inhibitor (UNC0642) will normalize epigenetic, gene expression, and dendritic spine changes in the amygdala and attenuate AIE-induced anxiety-like and alcohol drinking behaviors in adult male and female rats.

 

Aim 4: To translate epigenetic dynamics and expression of novel genes that will be identified from RNA-seq and ATAC-seq in the AIE rat amygdala to post-mortem amygdala of human alcoholics with early age onset and correlate them to drinking data (daily or weekly ethanol intake).

 

The proposed preclinical and translational studies will provide new information on the epigenetic regulation of the whole transcriptome in the amygdala after AIE. These studies may also pave the way to identify novel epigenetic targets for the potential development of drugs for the treatment of AIE-induced psychopathologies that lead to AUD and associated psychiatric disorders, including anxiety, during adulthood.

 

References Cited:

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