Timothy Bredy - US grants

DESCRIPTION (provided by applicant): Many neuropsychiatric diseases involve associative learning. The most obvious of these are the addictions (in which neutral cues in the environment come to evoke strong craving for drugs of abuse) and anxiety disorders (e.g., post-traumatic stress disorder, PTSD, in which neutral cues in the environment of the traumatic event come to evoke strong fear reactions). Epigenetics refers to the process by which cellular traits are established and inherited without a change in DNA sequence. These mechanisms of cellular memory also orchestrate the activity of genes in the adult brain, and recent findings suggest a role for epigenetic factors in the etiology of psychiatric disease. Thus, given the prevalence and co-morbidity of cocaine addiction and fear-related anxiety, and the increasing appreciation that environmental factors play a major role in determining mental health ("DNA is not destiny"), a deeper understanding of common epigenetic mechanisms associated with learning in coordinated models of addiction and fear is both timely and relevant. Aim 1 will employ a genome-wide approach using state-of-the-art technology to investigate epigenetic mechanisms regulating gene expression during the acquisition and extinction of cocaine-seeking behavior in a self-administration model of cocaine addiction, and of fear-related learning in a parallel model of auditory conditioned fear. Aim 2 will validate candidate genes by using ChIP and bisulphite mapping to examine histone modifications and DNA methylation around, and within, individual gene promoters, and correlate them with gene expression in brain regions supporting both forms of learning. These effects will be examined at different time points post-training in an effort to elucidate their role in spontaneous incubation and relapse. Aim 3 will study whether manipulations that induce local histone hyperacetylation, histone demethylation or inhibition of DNA methylation, during cocaine-self administration and fear training will prejudice animals toward incubation, while the same manipulation during extinction will prevent relapse of cocaine-seeking and of conditioned fear. These studies aim to elucidate common epigenetic factors contributing to psychiatric disease, and to explore the epigenome as a therapeutic point of intervention for cocaine addiction, PTSD and phobia. PUBLIC HEALTH RELEVANCE: Using analagous procedures, we will study the importance of common epigenetic mechanisms in animal models of cocaine addiction and fear-related anxiety disorders. These studies may yield improved therapeutic intervention strategies based on facilitating extinction learning through the use of specialized pharmacological adjuncts during psychotherapy for co-morbid psychiatric disorders.

DESCRIPTION (provided by applicant): Although epigenetic regulation of gene expression has been shown to be associated with experience- dependent neural plasticity and memory formation, due to technical and conceptual limitations, the full repertoire of DNA base modifications and their role in neuropsychiatric disease remains equivocal. In this project, new technology will be developed to more precisely identify activity-dependent epigenetic mechanisms in neuronal populations selectively activated by learning and challenge current concepts related to the full repertoire of DNA base modifications and their role in regulating gene expression. Specifically, we will determine whether there is a causal relationship between various DNA base modifications and rapid behavioral adaptation, and we will advance the state of knowledge regarding the molecular mechanisms underlying the memory in a preclinical model of fear-related anxiety disorder.

DESCRIPTION (provided by applicant): This proposal is a resubmission of application R21MH103812-01, Long non-coding RNAs, learning and memory, which was reviewed in November 2013 by the MNG study section. Its focus is to establish a functional role for activity-dependent long non-coding RNAs in cortex. We are particularly interested in how non-coding RNAs may mediate epigenetic regulation of the gene expression underlying behavior and cognition. Eventually, we hope to examine how these enigmatic transcripts are involved in the fundamental molecular transactions underlying memory, and how they contribute to the development of fear- related psychiatric disorders. We have made an effort to address every issue that has been raised, and the grant has been substantially revised both to clarify the experimental approach and to include additional data. As a young investigator, I sincerely appreciate the thought and constructive criticism that went into the initial review, and I believe that the revised application is significantly stronger than the initial submission. Reviewer 1 indicated that, time should have been spent discussing what results would be expected from the ChIRP experiments and how they would be interpreted, and to propose to study how interaction of the lncRNA with the promoter of the protein coding genes leads to transcription regulation. We have now addressed this issue by including experiments utilizing ChIP assay for interrogation of the chromatin landscape surrounding TSS of protein coding gene targets, RIP for candidate lncRNA-chromatin modifying complex interactions and ChIRP for lncRNA-target protein coding gene promoter interactions. Moreover, we now describe how they will shed light on the role of lncRNAs in regulating gene expression associated with learning. Reviewer 1 also stated that, it would be instructive to know if and how the lncRNA acts as a decoy, a scaffold or a guide for regulating expression of the protein coding genes. We believe this is a great question and, although it is beyond the scope of the current exploratory R21 grant, it will definitely be subject of a future R01 application within the context of cell-type specific gene regulation supporting behavioral adaptation. Finally, it was unclear to reviewer 1 with whom the PI would collaborate and/or what services they will provide that will facilitate success of this project. Dr. Sha Sun in the Department of Developmental and Cell Biology at UCI is an expert in both lncRNAs and epigenetic programming (Sun et al., 2013) and has offered to serve as a collaborator on this project (letter of support attached) by providing strategic advice on the project as it progresses, and practical support in sharing expertise in the application of experimental validation of the novel findings to assist in the efficient and successful prosecution of this project. Both Reviewer 2 and 3 expressed concern that, the ASO technology may not be efficient at knocking down lncRNA, and that no other alternative approach was mentioned. It is important to note that we already have successfully manipulated Gomafu in primary cortical neurons in culture (Barry et al., 2013) and preliminary data, Aim 2). In the event that we can't reduce Gomafu or other lncRNAs in vivo using ASO, we propose to use CRISPR/Cas9 technology to knockdown these lncRNA. However, we also acknowledge that this approach carries some degree of risk, due to the permanent nature of the knockout, which may lead to compensatory effects on the expression of genes that were under inhibitory control by the target lncRNA. Finally, we have a Gomafu lncRNA overexpression construct that will also be tested in Aim 3. Reviewer 3 indicated that, it would also be important to determine whether there is indeed a functional relationship between lncRNA and its protein-coding targets. E.g. can the effect of manipulating lncRNA be counter-acted by the manipulation of protein-coding target? We completely agree with this reviewer; however, due to time limitations for the R21 project, these experiments will be included in a future R01 designed to explore in greater detail the functional relationship between lncRNA-mediated epigenetic regulation of gene expression, once a role for candidate lncRNAs in regulating learning and memory have been firmly established.

? DESCRIPTION (provided by applicant): RNA modification, and N6 methyladenosine (m6A) in particular, is a newly discovered epigenetic mechanism in the adult brain that is has recently been shown to be highly dynamic and, as indicated by our preliminary evidence, appears to be involved in fear-related learning and memory. The overarching goal of this research program is to establish, for the first time, a causal relationship between the epitranscriptomic regulation of gene expression and the formation and maintenance of memory in a preclinical model of fear-related anxiety disorder. We can then capitalize on this information to design better treatments for neuropsychiatric disorders characterized by impairments in cognitive function. Successful completion of these experiments also has the potential to dramatically change the way we think about mechanisms of adaptive plasticity by shedding new light on how the qualitative nature of RNA, rather than its overall abundance, is involved in a key learning process with implications for our understanding of neuropsychiatric disorders characterized by abnormally intense memories. This will be achieved through a potent combination of advance high-throughput sequencing approaches, robust behavioral paradigms and viral-mediated manipulation of gene activity in the adult brain.