Igor Ponomarev, PhD - US grants

DESCRIPTION (provided by applicant): The goal of the current application is to provide Dr. Igor Ponomarev with career development opportunities under the mentorship of Dr. R. Adron Harris. The excellent research environment, faculty and facilities at the University of Texas at Austin will allow him to sharpen his skills as a young investigator and to learn cellular neuroscience techniques such as brain microinjections and laser capture microdissection. These techniques will allow Dr. Ponomarev to identify and dissect individual neuronal populations for gene expression analysis and also to apply interference RNA and pharmacological agents to test functionality of differentially regulated candidate genes. Training under this award will better prepare Dr. Ponomarev for a career in academic science and compliment his expertise in behavioral neurogenetics and functional genomics. Because excessive alcohol consumption is a prerequisite for the development of alcohol dependence, it is important to identify molecular targets associated with high alcohol intake in order to understand the mechanisms of alcoholism progression, which mediate the switch from controlled alcohol consumption to alcohol abuse and alcohol dependence. The overall goal of this proposal is to determine the effects of genetic factors and drinking high amounts of alcohol on gene expression in specific brain regions believed to be involved in regulation of alcohol consumption and different populations of dopamine neurons projecting to these brain regions. We will use brain microinjections, laser capture microdisections and cDNA microarrays to study patterns of gene expression in brain regions and neurons of genetically selected mice with predisposition to high alcohol consumption. We hypothesize that different brain regions and neuronal populations of alcohol-related neurocircuitry can be distinguished by their individual patterns of gene expression and by their distinct transcriptional responses to genetic selection and alcohol drinking. The long-term goals of these studies are to determine roles of individual neuronal populations in alcohol actions and to identify cell type - specific alcohol-sensitive genes and gene products as potential therapeutic targets for alcoholism.

OVERVIEW Persistent changes in gene expression may mediate many effects of alcohol including reward learning, tolerance and dependence, suggesting that agents effective in changing alcohol-induced gene expression could be considered as therapeutic agents. Drugs targeting gene expression through inhibition of enzymes that regulate chromatin structure (epigenetic drugs) have been widely used in cancer research and recently emerged as potential therapeutics for neurodegenerative disorders and drug addiction. The main goals of this project are: 1) to identify epigenetic drugs that affect alcohol reward through testing their effects on alcohol consumption and conditioned place preference (CPP) and 2) to investigate the effects of selected epigenetic drugs on gene expression and cellular physiology in the reward pathway including the ventral tegmental area (VTA) and the nucleus accumbens (NA). The overall hypothesis is that some epigenetic drugs will reduce the rewarding properties of alcohol through changes in gene expression and cellular physiology in the reward pathway. Integration of electrophysiological and gene expression data will elucidate the drug's mechanisms of action and identify novel targets for drug development. This research will provide initial mechanistic evidence for the therapeutic potential of epigenetic drugs in treating alcohol addiction.

DESCRIPTION (provided by applicant): Chronic alcohol causes widespread changes in brain gene expression in humans and animal models, some of which contribute to alcohol addiction and alcohol dependence. Recent studies point to a central role of chromatin modifications, often referred to as epigenetic changes, in controlling alcohol- induced changes in gene expression and behavior. To understand how chromatin modifications mediate changes in gene expression in alcoholic brain, an integration of chromatin and transcriptional data at the genome level is required. Here we will test the hypothesis that chronic alcohol abuse changes gene expression via long-lasting changes in chromatin states. We will first measure genomic differences at two chromatin marks in postmortem brains of chronic alcoholics and control cases using chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq). We will then explore the genome-wide relationships between chromatin modifications and gene expression measured in the same samples by RNA-Seq using a novel systems approach. This approach will allow us to partition genomic variance into biologically meaningful patterns and identify robust correlations between ChIP- Seq and RNA-Seq data sets, which we will use to propose mechanistic links between chromatin marks and gene expression. This approach will also prioritize individual genes based on their importance in gene networks and correlations with chromatin marks and we will use this strategy to select several hub genes for validation. We will validate chromatin marks of several hub genes with ChIP followed by qRT- PCR and their expression levels with qRT-PCR. Overall, this proposal aims to develop a systems approach that will detect robust co-variation between ChIP-Seq and RNA-Seq data sets. We will identify epigenetic components critical for regulation of gene expression by chronic alcohol abuse and provide new targets for medication development for human alcoholism. In addition, this approach may serve as a prototype for analysis of the wealth of existing and emerging genomic data.

? DESCRIPTION (provided by applicant): Excessive alcohol consumption is a prerequisite for the development of alcohol dependence. In order to understand the mechanisms of alcoholism progression, which mediate the switch from controlled alcohol consumption to alcohol abuse and alcohol dependence, it is important to identify molecular targets affected by high alcohol intake in individual neuronal populations of the reward circuit. The main goal of this proposal is two-fold: 1) to determine the effects of high alcohol consumption on gene expression and cellular physiology in the nucleus accumbens (NA) neurons projecting to ventral tegmental area (VTA) and 2) to establish a methodology for labeling and profiling multiple neuronal populations in the reward circuit. We will use alcohol- preferring C57BL6 x FVB F1 hybrid mice that will drink intoxicating amounts of alcohol in a binge fashion for several days. Two populations of accumbal GABA-ergic medium spiny neurons, one projecting to VTA and the other projecting to the ventral pallidum will be labeled by injecting a retrograde tracer, cholera toxin subunit B conjugated to an Alexa Fluor, into the respective projection target areas. Part of the labeled NA neurons will be collected using laser capture microdissection and gene expression will be measured using microarrays and RNA sequencing. The other part will be used for electrophysiological recordings. A gene network approach will be used to identify individual genes and molecular pathways correlated with neuronal physiology and alcohol intake. This study will identify cell type - specific alcohol- sensitive molecular targets that may eventually b used to reduce alcohol intake via circuit- specific manipulation of neuronal activity. The long-term goal of this research is to determine molecular mechanisms of cellular adaptation to alcohol in individual neuronal populations of the neurocircuits that mediate alcohol actions. This knowledge may be used to develop new drugs for the prevention and treatment of alcoholism.

Project Summary/Abstract Activation of the innate immune system results in the release of pro-inflammatory cytokines, which may lead to changes in neuroimmune signaling and behavioral abnormalities, such as high alcohol (ethanol) consumption, which may ultimately lead to the development of alcohol use disorder (AUD). We lack understanding of how changes in neuroimmune signaling are integrated into neuronal networks that mediate the transition from normal (social) drinking to excessive alcohol consumption. Here, we propose the neuroimmune model of excessive alcohol consumption, where repeated injections of the immune activator Poly(I:C) produce progressive escalation of alcohol intake over several weeks of drinking. We hypothesize that immune activation induces cell type-specific changes in gene expression, which are integrated to affect neuronal functions and drive excessive drinking. We will use a combination of molecular, cellular, behavioral and computational approaches to test this hypothesis. Gene expression will be measured in neurons, astrocytes and microglia to investigate cell type-specific molecular mechanisms of immune activation. We will then use molecular signatures from different cell types and computationally-driven drug-repurposing approaches to identify and test pharmacological compounds with the potential to reduce high alcohol drinking. We will investigate roles of specific neural networks in immune-induced escalation of drinking by measuring neuronal functions and manipulating excitability of critical projection neurons. Temporal profiling across three critical time points will identify dynamic changes in cell type-specific transcriptomes and neuronal functions. Results of the proposed experiments will advance our understanding of the role of neuroimmune signaling in the transition to AUD and will be widely applicable to brain disorders with pro- inflammatory phenotypes.

Project Summary/Abstract Activation of the innate immune system results in the release of pro-inflammatory cytokines, which may lead to changes in neuroimmune signaling and behavioral abnormalities, such as high alcohol (ethanol) consumption, which may ultimately lead to the development of alcohol use disorder (AUD). We lack understanding of how changes in neuroimmune signaling are integrated into neuronal networks that mediate the transition from normal (social) drinking to excessive alcohol consumption. Here, we propose the neuroimmune model of excessive alcohol consumption, where repeated injections of the immune activator Poly(I:C) produce progressive escalation of alcohol intake over several weeks of drinking. We hypothesize that immune activation induces cell type-specific changes in gene expression, which are integrated to affect neuronal functions and drive excessive drinking. We will use a combination of molecular, cellular, behavioral and computational approaches to test this hypothesis. Gene expression will be measured in neurons, astrocytes and microglia to investigate cell type-specific molecular mechanisms of immune activation. We will then use molecular signatures from different cell types and computationally-driven drug-repurposing approaches to identify and test pharmacological compounds with the potential to reduce high alcohol drinking. We will investigate roles of specific neural networks in immune-induced escalation of drinking by measuring neuronal functions and manipulating excitability of critical projection neurons. Temporal profiling across three critical time points will identify dynamic changes in cell type-specific transcriptomes and neuronal functions. Results of the proposed experiments will advance our understanding of the role of neuroimmune signaling in the transition to AUD and will be widely applicable to brain disorders with pro- inflammatory phenotypes.

Project Summary/Abstract Repeated alcohol (ethanol) exposure results in cellular adaptations that are thought to be critical for the transition from low/moderate drinking to excessive alcohol consumption, which is a prerequisite for the development of Alcohol Use Disorder (AUD). Local protein synthesis (translation) in dendrites and axons plays a central role in synaptic plasticity and neuroadaptations to environmental challenges including exposure to drugs. Acute and chronic alcohol changes RNA abundance in synaptic terminals, suggesting a change in local protein synthesis, but we do not know which specific mRNAs undergo active translation at the synapse (the synaptic translatome) in response to alcohol. Actively translated synaptic mRNAs are mechanistically linked to neuronal functions and provide primary mechanistic candidates for alcohol-induced neuroadaptations. Here, we propose to use a combination of synaptoneurosome preparations and polysome profiling followed by RNA sequencing (RNA-Seq) to determine changes in the synaptic translatome after chronic alcohol drinking in mice. Synaptoneurosomes have been widely used to study molecular functions at the synapse in health and disease and polysome profiling is the gold standard for studying cellular translatomes. Because the synaptoneurosome/polysome profiling combination has not been used in alcohol studies, our initial goal is to develop/optimize the existing methodology for alcohol research. Alcohol-preferring C57BL/6J mice will be exposed to alcohol for several weeks using a 2-bottle choice chronic intermittent alcohol drinking paradigm that is used as a model of escalating alcohol consumption. The prefrontal cortex, a critical brain region implicated in AUD, will be dissected and subjected to the synaptoneurosome/polysome profiling followed by RNA-Seq. We hypothesize that chronic alcohol intake will affect the synaptic translatome and change local protein synthesis in the prefrontal cortex, which may contribute to neuroadaptations underlying high alcohol drinking. These initial experiments will identify alcohol-sensitive actively translated synaptic mRNAs (genes) that may eventually be targeted to reduce alcohol intake via manipulation of synaptic functions. The long-term goal of this research is to determine the role of local protein synthesis in brain mechanisms underlying AUD traits. This knowledge may be used to develop new therapies for the prevention and treatment of AUD.