Sean P. Farris

Epigenetic modulation of brain gene networks for cocaine and alcohol abuse

Cocaine and alcohol are two substances of abuse that prominently affect the central nervous system (CNS). Repeated exposure to cocaine and alcohol leads to longstanding changes in gene expression, and subsequent functional CNS plasticity, throughout multiple brain regions. Epigenetic modifications of histones are one proposed mechanism guiding these enduring changes to the transcriptome. Characterizing the large number of available biological relationships as network models can reveal unexpected biochemical relationships. Clustering analysis of variation from whole-genome sequencing of gene expression (RNA-Seq) and histone H3 lysine 4 trimethylation (H3K4me3) events (ChIP-Seq) revealed the underlying structure of the transcriptional and epigenomic landscape within hippocampal postmortem brain tissue of drug abusers and control cases. Distinct sets of interrelated networks for cocaine and alcohol abuse were determined for each abusive substance. The network approach identified subsets of functionally related genes that are regulated in agreement with H3K4me3 changes, suggesting cause and effect relationships between this epigenetic mark and gene expression. Gene expression networks consisted of recognized substrates for addiction, such as the dopamine- and cAMP-regulated neuronal phosphoprotein PPP1R1B/DARPP-32 and the vesicular glutamate transporter SLC17A7/VGLUT1 as well as potentially novel molecular targets for substance abuse. Through a systems biology based approach our results illustrate the utility of integrating epigenetic and transcript expression to establish relevant biological networks in the human brain for addiction. Future work with laboratory models may clarify the functional relevance of these gene networks for cocaine and alcohol, and provide a framework for the development of medications for the treatment of addiction.

RNA-Seq Reveals Novel Transcriptional Reorganization in Human Alcoholic Brain

DNA microarrays have been used for over a decade to profile gene expression on a genomic scale. While this technology has advanced our understanding of complex cellular function, the reliance of microarrays on hybridization kinetics results in several technical limitations. For example, knowledge of the sequences being probed is required, distinguishing similar sequences is difficult because of cross-hybridization, and the relatively narrow dynamic range of the signal limits sensitivity. Recently, new technologies have been introduced that are based on novel sequencing methodologies. These next-generation sequencing methods do not have the limitations inherent to microarrays. Next-generation sequencing is unique since it allows the detection of all known and novel RNAs present in biological samples without bias toward known transcripts. In addition, the expression of coding and noncoding RNAs, alternative splicing events, and expressed single nucleotide polymorphisms (SNPs) can be identified in a single experiment. Furthermore, this technology allows for remarkably higher throughput while lowering sequencing costs. This significant shift in throughput and pricing makes low-cost access to whole genomes possible and more importantly expands sequencing applications far beyond traditional uses (Morozova & Marra, 2008) to include sequencing the transcriptome (RNA-Seq), providing detail on gene structure, alternative splicing events, expressed SNPs, and transcript size (Mane et al., 2009; Tang et al., 2009; Walter et al., 2009), in a single experiment, while also quantifying the absolute abundance of genes, all with greater sensitivity and dynamic range than the competing cDNA microarray technology (Mortazavi, Williams, McCue, Schaeffer, & Wold, 2008).

A molecular mechanism for choosing alcohol over an alternative reward

Finding the vulnerable minority “Only” about 10 to 15% of people exposed to alcohol develop alcohol-related problems. The behavioral repertoire of people confronted with opportunities to consume alcohol involves numerous choices between this drug reward and healthy alternatives. Augier et al. established a choice procedure that begins to address alcohol addiction in rats (see the Perspective by Spanagel). They found that a minority of outbred rats continued to self-administer alcohol even when a high-value alternative (such as sugar) was available. That minority displayed a remarkable constellation of behavioral traits resembling the human clinical condition, including a high motivation to obtain alcohol and continued use despite adverse consequences. The cause was impaired GABA (γ-aminobutyric acid) clearance in the central amygdala. Postmortem tissue analysis supported the possibility of a similar pathology in human alcoholism. Science, this issue p. 1321; see also p. 1298 Impaired GABA clearance within the central amygdala provides a molecular mechanism behind preferentially choosing alcohol. Alcohol addiction leads to increased choice of alcohol over healthy rewards. We established an exclusive choice procedure in which ~15% of outbred rats chose alcohol over a high-value reward. These animals displayed addiction-like traits, including high motivation to obtain alcohol and pursuit of this drug despite adverse consequences. Expression of the γ-aminobutyric acid (GABA) transporter GAT-3 was selectively decreased within the amygdala of alcohol-choosing rats, whereas a knockdown of this transcript reversed choice preference of rats that originally chose a sweet solution over alcohol. GAT-3 expression was selectively decreased in the central amygdala of alcohol-dependent people compared to those who died of unrelated causes. Impaired GABA clearance within the amygdala contributes to alcohol addiction, appears to translate between species, and may offer targets for new pharmacotherapies for treating this disorder.

Microglial-specific transcriptome changes following chronic alcohol consumption.

ABSTRACT Microglia are fundamentally important immune cells within the central nervous system (CNS) that respond to environmental challenges to maintain normal physiological processes. Alterations in steady‐state cellular function and over‐activation of microglia can facilitate the initiation and progression of neuropathological conditions such as Alzheimers disease, Multiple Sclerosis, and Major Depressive Disorder. Alcohol consumption disrupts signaling pathways including both innate and adaptive immune responses that are necessary for CNS homeostasis. Coordinate expression of these genes is not ascertained from an admixture of CNS cell‐types, underscoring the importance of examining isolated cellular populations to reveal systematic gene expression changes arising from mature microglia. Unbiased RNA‐Seq profiling was used to identify gene expression changes in isolated prefrontal cortical microglia in response to recurring bouts of voluntary alcohol drinking behavior. The voluntary ethanol paradigm utilizes long‐term consumption ethanol that results in escalated alcohol intake and altered cortical plasticity that is seen in humans. Gene coexpression analysis identified a coordinately regulated group of genes, unique to microglia, that collectively are associated with alcohol consumption. Genes within this group are involved in toll‐like receptor signaling and transforming growth factor beta signaling. Network connectivity of this group identified Siglech as a putative hub gene and highlighted the potential importance of proteases in the microglial response to chronic ethanol. In conclusion, we identified a distinctive microglial gene expression signature for neuroimmune responses related to alcohol consumption that provides valuable insight into microglia‐specific changes underlying the development of substance abuse, and possibly other CNS disorders. HIGHLIGHTSMost microglial expression changes are not identified in a cellular admixture.Cortical microglia have distinct ethanol‐responsive gene networks.TLR and TGF‐&bgr; signaling genes are altered in microglia in response to ethanol.

FMRP regulates an ethanol-dependent shift in GABABR function and expression with rapid antidepressant properties.

Alcohol promotes lasting neuroadaptive changes that may provide relief from depressive symptoms, often referred to as the self-medication hypothesis. However, the molecular/synaptic pathways that are shared by alcohol and antidepressants are unknown. In the current study, acute exposure to ethanol produced lasting antidepressant and anxiolytic behaviours. To understand the functional basis of these behaviours, we examined a molecular pathway that is activated by rapid antidepressants. Ethanol, like rapid antidepressants, alters γ-aminobutyric acid type B receptor (GABABR) expression and signalling, to increase dendritic calcium. Furthermore, new GABABRs are synthesized in response to ethanol treatment, requiring fragile-X mental retardation protein (FMRP). Ethanol-dependent changes in GABABR expression, dendritic signalling, and antidepressant efficacy are absent in Fmr1-knockout (KO) mice. These findings indicate that FMRP is an important regulator of protein synthesis following alcohol exposure, providing a molecular basis for the antidepressant efficacy of acute ethanol exposure.

Astrocyte-specific transcriptome responses to chronic ethanol consumption

Astrocytes play critical roles in central nervous system (CNS) homeostasis and are implicated in the pathogenesis of neurological and psychiatric conditions, including drug dependence. Little is known about the effects of chronic ethanol consumption on astrocyte gene expression. To address this gap in knowledge, we performed transcriptome-wide RNA sequencing of astrocytes isolated from the prefrontal cortex (PFC) of mice following chronic ethanol consumption. Differential expression analysis revealed ethanol-induced changes unique to astrocytes that were not identified in total homogenate preparations. Astrocyte-specific gene expression revealed calcium-related signaling and regulation of extracellular matrix genes as responses to chronic ethanol use. These findings emphasize the importance of investigating expression changes in specific cellular populations to define molecular consequences of chronic ethanol consumption in mammalian brain.

Beyond genome-wide significance: integrative approaches to the interpretation and extension of GWAS findings for alcohol use disorder: Beyond GWAS

Alcohol use disorder (AUD) is a heritable complex behavior. Due to the highly polygenic nature of AUD, identifying genetic variants that comprise this heritable variation has proved to be challenging. With the exception of functional variants in alcohol metabolizing genes (e.g. ADH1B and ALDH2), few other candidate loci have been confidently linked to AUD. Genome‐wide association studies (GWAS) of AUD and other alcohol‐related phenotypes have either produced few hits with genome‐wide significance or have failed to replicate on further study. These issues reinforce the complex nature of the genetic underpinnings for AUD and suggest that both GWAS studies with larger samples and additional analysis approaches that better harness the nominally significant loci in existing GWAS are needed. Here, we review approaches of interest in the post‐GWAS era, including in silico functional analyses; functional partitioning of single nucleotide polymorphism heritability; aggregation of signal into genes and gene networks; and validation of identified loci, genes and gene networks in postmortem brain tissue and across species. These integrative approaches hold promise to illuminate our understanding of the biological basis of AUD; however, we recognize that the main challenge continues to be the extremely polygenic nature of AUD, which necessitates large samples to identify multiple loci associated with AUD liability.

Bioinformatic and biological avenues for understanding alcohol use disorder.

Alcohol Use Disorder (AUD) is a multifarious psychiatric condition resulting from complex relationships between genetics, gene expression, neuroadaptations, and environmental influences. Understanding these complex relationships is essential to uncovering the mechanisms involved in the development and progression of AUD, with the ultimate goal of devising effective behavioral and therapeutic interventions. Technical advances in the fields of omics-based research and bioinformatics have yielded insights into gene interactions, biological networks, and cellular responses across humans and animal models. This review highlights several of the newly developed sequencing methodologies and resultant discoveries in neuroscience, as well as the importance of a multi-faceted and integrative approach for determining causal factors in AUD.

Moving Toward Understanding the Proteome Involved in Substance Abuse

Addiction to alcohol and other drugs of abuse is a persistent mental health disorder adversely affecting the individuals with the disorder as well as society at large. Initial substance use and subsequent chronic abuse affect multiple brain systems and biological pathways. The transition to a substance use disorder entails interacting molecular components within the central nervous system (CNS), forming a dense network of proteins capable of being usurped by abused substances. Deciphering the affected components throughout the life cycle of addiction is essential for understanding the neurobiological complexity of this mental health disorder and for rational design of pharmacotherapies. Since the end of the 20th century, there has been a steady increase in the number of publications using proteomicsbased approaches (Figure 1). Proteomics seeks to conduct an unbiased, simultaneous assessment of all the proteins altered by an experimental condition. Using two-dimensional differential in-gel electrophoresis followed by matrix assisted laser desorption ionization tandem time-of-flight, Salling et al. (1) examined expression changes for a subset of proteins within the amygdala following a period of ad libitum alcohol consumption. Paired with proprietary bioinformatics software, which assists in the curation of the existing biomedical literature (Ingenuity Pathway Analysis; QIAGEN, Redwood City, California), the authors identified two main literatureassociated networks for proteins altered within the amygdala of adult male C57BL/6J mice voluntarily consuming alcohol. Following the proteomics findings, Salling et al. (1) focused their efforts on calcium/calmodulin-dependent protein kinase II alpha (CAMKIIα) and through a series of elegant experiments demonstrated the physiologic relevance of CAMKIIα within the central amygdala on alcohol drinking behavior. The GENCODE consortium currently estimates that between 19,881 and 21,936 protein-coding genes exist in the mammalian genome. The two-dimensional differential ingel electrophoresis experiments conducted by Salling et al. (1) identified 29 proteins extracted from the amygdala, leaving .99% of the amygdala proteome open to future scrutiny. Only a small portion of all proteomics studies published to date have focused on either the amygdala or alcohol-related research (Figure 1). Technical limitations surrounding current proteomics methodologies and the complexity of the CNS and addiction research significantly hinder the widespread adoption of functional proteomics experimental designs. Any individual brain region contains a heterogeneous mixture of numerous CNS cell types, wherein specific proteins may show distinctive expression patterns. Characterizing the abundance and distribution of the complete proteome among discrete neuronal and glial cells is a daunting task, yet one that must be