Jeremy J. Day - US grants

The ability to form and maintain associations between environmental cues, actions, and rewarding stimuli is an elementary yet fundamental aspect of learned behavior that is necessary for survival. Multiple lines of research have identified that such reward-related learning is mediated by a distributed network of brain nuclei centered upon the nucleus accumbens and its innervation from dopamine neurons located in the midbrain. Both dopamine and nucleus accumbens neurons encode stimulus-reward relationships, and impaired processing in either area inhibits reward learning. Within the nucleus accumbens, dopamine operates through several well- defined intracellular signaling cascades to direct synaptic plasticity and alter neuronal output. Recent studies indicate that dopamine transmission within the nucleus accumbens induces epigenetic remodeling that is associated with both brief and prolonged changes in gene expression. However, the role that epigenetic changes play in reward learning is unclear. This proposal will examine whether two different types of epigenetic alteration (histone modification and DNA methylation) are induced by classical stimulus-reward conditioning. These changes will be investigated using a variety of cutting-edge techniques, including chromatin immunoprecipitation, direct bisulfite sequencing of DNA, and quantitative RT-PCR. These assays will allow us to determine not only whether reward learning is associated with epigenetic modification in the nucleus accumbens, but will also reveal which genes such changes are affecting. Furthermore, the ability of epigenetic changes to functionally modulate learning will be examined by blocking specific histone modifications or DNA methylation in the nucleus accumbens during conditioning. The results will provide novel insight into the epigenetic control of reward learning and enhance our understanding of the molecular pathways that regulate motivated behavior.

Drug addiction is a chronic, relapsing disorder in which drug-related associations (e.g., discrete drug cues, locations in which drugs were consumed, and drug paraphernalia) are capable of exerting tremendous control over behavior long after drug taking has ceased. A hallmark feature of drugs of abuse is that they result in persistent, long-lasting functional and structural alterations in brain reward circuits such as the nucleus accumbens. Recent discoveries have revealed that epigenetic modifications, such as methylation of cytosine nucleotides in DNA, are key regulators of long-term synaptic plasticity, learning, and long-term or even transgenerational behavioral change. Moreover, novel findings indicate that drugs of abuse such as cocaine induce epigenetic changes in the nucleus accumbens, and that these changes control cocaine-related neuroadaptations. However, very little is known about how experience with cocaine alters DNA methylation within the nucleus accumbens, and whether this modification subserves cocaine-induced neuronal and behavioral plasticity. This proposal will examine whether changes in DNA methylation (and a recently discovered brain-enriched intermediate modification, DNA hydroxymethylation) are induced by cocaine experience, and whether these changes are meaningfully related to cocaine-related behaviors. Changes in DNA methylation and hydroxymethylation will be investigated using a variety of cutting-edge techniques, including methylated DNA immunoprecipitation, direct bisulfite sequencing of DNA, and whole-genome next generation sequencing. Additional studies will examine for the first time whether these changes occur within individual cell populations in the nucleus accumbens. These assays will allow us to determine not only whether cocaine experience is associated with changes in DNA methylation in the nucleus accumbens, but will also reveal which genes and which neuronal subtypes such changes are affecting. Furthermore, the ability of DNA methylation changes to functionally modulate cocaine-induced behavioral plasticity will be examined by blocking or overexpressing critical DNA methylation and demethylation machinery during cocaine exposure. The results will provide groundbreaking insight into the epigenetic control of cocaine-related neuroadaptations and enhance our understanding of the molecular pathways that regulate motivated behavior.

? DESCRIPTION (provided by applicant): Drug addiction is a chronic, relapsing disorder in which drug-related associations are capable of exerting tremendous control over behavior long after drug taking has ceased. Epigenetic modifications in the central nervous system are critical for long-term behavioral and neuronal plasticity, and have been implicated in numerous features of motivated behavior, drug-related learning, and addiction. However, our ability to harness the therapeutic potential of epigenetic manipulations in the context of addiction has been limited by the lack of detailed insight into epigenetic dynamics following drug experiences, and the inability to target specific epigenetic alterations in real time to interrogate their molecular and behaviora function. This proposal seeks to utilize single-cell whole-epigenome sequencing approaches to define epigenetic signatures of drug experience in specific neuronal populations. In addition to revealing new therapeutic candidates, this information will be directly applied to targeted epigenetic editing strategies based on CRISPR technology, which will allow manipulation of epigenetic states at specific drug-regulated genes. This novel approach will demonstrate the necessity of unique epigenetic modifications at specific genes for drug-associated behaviors, and also enable the first investigation of whether an epigenetic modification is sufficient to alte reward function. Finally, we will integrate these tools with ontogenetic approaches to enable light-driven manipulation of epigenetic states in freely moving animals. Thus, in addition to revealing the exact nature and scope of epigenetic states following drug experience, this proposal will be the first to investigate how these modifications contribute to the function of reward circuits and ultimately to reward seeking behavior in general.

Distal enhancer elements in DNA enable higher-order chromatin interactions that facilitate gene expression programs and thus contribute to cellular phenotype and function. Enhancers regulate numerous aspects of cell and tissue-specific gene expression patterns in the developing and adult brain, and have been highly implicated in mental health and disease. In neuronal systems, enhancer elements are subject to widespread, bidirectional transcription that yields non-coding enhancer RNA (eRNA). However, the precise function of eRNAs is still unclear, with different models proposing unique regulatory functions. Emerging evidence suggests that eRNAs function to modify epigenetic states at gene regulatory elements, which are critical for long-term behavioral and neuronal plasticity. Specific Aim 1 of this proposal seeks to utilize whole-genome sequencing approaches to define transcriptional and epigenetic signatures of neuronal activity at enhancers. In addition to revealing new therapeutic candidates, this aim will employ novel targeted eRNA delivery strategies based on CRISPR technology to allow manipulation of eRNAs at specific enhancers to determine their function. Specific Aim 2 will examine interactions between eRNAs and epigenetic profiles at enhancers and promoters of linked genes, which will establish the hierarchical relationships between molecular interactions at enhancers. Specific Aim 3 will examine the contributions of eRNAs to neuronal function and memory formation in the adult brain using hippocampus-dependent contextual fear conditioning, a robust assay of learning and memory that requires activity-dependent gene transcription. This novel approach will demonstrate the necessity of unique eRNAs at specific genes for neuronal activity and behavior, and also enable the first investigation of whether modulation of eRNAs is sufficient to alter memory function. In addition to revealing the exact nature and scope of eRNAs in neuronal gene regulation, this proposal will pave the way for future experiments that will explore how manipulation of enhancers could be used to reprogram circuits that have become maladaptive in mental illness and cognitive disease states.

The ability to form and maintain associations between environmental cues, actions, and rewarding stimuli is an elementary yet fundamental aspect of learned behavior that is necessary for survival. Multiple lines of research have identified that such reward-related learning is mediated by dopamine neurons located in the midbrain. Dopamine neurons encode stimulus-reward relationships in a way that is dynamically modified during learning, and impaired dopamine neuron functioning inhibits reward learning. However, almost nothing is known about how the molecular mechanisms in dopamine neurons that are relevant for learning. While pioneering studies have identified that Egr1 (Early growth response 1; an activity-responsive transcription factor) is rapidly upregulated in dopamine neurons after learning and correlates with learning strength, the functional consequence of this upregulation for learning and mechanistic consequences of this upregulation has not been examined. Specific Aim 1 of this proposal will use bidirectional CRISPR-based manipulation of Egr1 to determine it?s role in reward learning. Specific Aim 2 will define downstream gene targets of Egr1 induction using innovative single-cell sequencing as well as chromatin immunoprecipitation. This novel approach will demonstrate the necessity of Egr1 for learning and will pave the way for future experiments to explore how experience-dependent gene expression programs contribute to motivated behavior.

Psychostimulant abuse is a public health crisis that affects millions of individuals in the United States and results in profound economic, social, and individual harm. However, despite rapid increases in overdose deaths linked to stimulant drugs like cocaine, there are still no approved therapeutic options for stimulant abuse disorders. Psychostimulant drugs act through well-defined signaling mechanisms to elevate dopaminergic neurotransmission in the nucleus accumbens (NAc), a key reward-linked brain structure that integrates information from diverse brain regions to directly influence motivated behavior. Further, cocaine causes epigenetic and transcriptional reorganization in medium spiny neurons (MSNs) in the NAc, promoting maladaptive shifts in cell signaling and synaptic function. Our preliminary data indicates that expression of Reln mRNA, which codes for the large secreted extracellular matrix protein Reelin, is enriched in a subpopulation of MSNs that are robustly activated by cocaine. Although Reelin knockout animals exhibit impaired response to psychostimulants and Reelin plays a critical role in synaptic plasticity and memory formation in other brain regions, the role of Reelin in cocaine-related cellular and behavioral adaptations has never been studied. In this proposal, we will test the overarching hypothesis that Reelin signaling is required for the maladaptive molecular, physiological, and behavioral effects of cocaine in the NAc. Specific Aim 1 of this proposal will combine bidirectional CRISPR-based manipulations and single-cell RNA sequencing to determine how Reelin signaling impacts transcriptional responses to cocaine and dopamine receptor activation. Specific Aim 2 will use in vitro and in vivo single unit recordings and ex vivo slice electrophysiology to test the hypothesis that Reelin regulates cocaine response by modulation of physiological and synaptic properties of MSNs. Finally, Specific Aim 3 will use cell-specific in vivo Reelin manipulations in combination with behavioral assays of cocaine and natural reward to test the hypothesis that Reelin enhances the behavioral effects of cocaine. Together, these experiments will identify Reelin target genes in the NAc, dissect molecular signaling pathways by which Reelin alters MSN function and physiology, and determine whether Reelin expression within the NAc modulates cocaine-related behavioral plasticity. These studies will reveal fundamental mechanisms by which Reelin contributes to psychostimulant response, and will pave the way for future experiments to explore how this unique Reln-expressing cell population contributes to motivated behavior.