Farah Lubin - US grants

DESCRIPTION (provided by applicant): The remodeling of neuronal circuits that develop in many types of epilepsy is thought to involve altered transcriptional activity and subsequent gene expression during epileptogenesis. Gene expression profiles are altered in epilepsy but the transcriptional regulatory mechanisms underlying these changes in epilepsy are not defined. The specific transcriptional regulator, nuclear factor-kappa B (NF-KappaB) has been identified as a potential key regulator of gene responses in epilepsy. We have pilot data suggesting that NF-KappaB activation correlates with acute seizures and the development of chronic epilepsy in the kainate model. Therefore, I propose to characterize NF-KB activation and transcriptional regulation of NF-KB responsive genes in the kainate epilepsy model and determine if NF-KappaB contributes to the epileptic phenotype. We anticipate that the proposed studies will help to further characterize the cellular and molecular mechanisms by which NF-KappaB is activated in epilepsy and to determine whether NF-KappaB contributes to altered gene expression in epilepsy. Investigations into the mechanisms involved in NF-KappaB activation in hippocampus will provide insights into the role of this transcription factor in epileptogenesis and possibly the maintenance of the epileptic circuitry. These studies may also identify novel targets for therapeutics in epilepsy.

[unreadable] DESCRIPTION (provided by applicant): A newly acquired memory becomes stable for long-term storage through a process known as memory consolidation, which requires de novo gene expression. Disruption of gene transcription during this process specifically blocks long-term memory formation. The brain-derived neurotrophic factor (BDNF) has been shown to play an essential role in the consolidation or storage of long-term memory. However, little is known about the regulation of exon-specific BDNF transcripts in the brain and how this level of BDNF gene regulation functions in the process of memory formation. The major scientific goal of this application is to identify epigenetic-regulating mechanisms for BDNF gene expression changes that serve to stabilize long-term memory. The underlying hypothesis of this grant is that aberrant epigenetic markings such as post- translational modification of histones, DNA methylation and transcription factor activation plays a role in exon-specific BDNF gene regulation in memory formation. The mentored phase of this application will dissect epigenetic mechanisms of exon-specific BDNF gene regulation during memory consolidation using a combination of approaches including measuring DNA methylation associated with exon-specific BDNF transcripts, using chromatin immunoprecipition (ChIP) technology to analyze levels of post-translational modification of histones, methyl CpG binding protein 2, and histone deacetylases at BDNF promoter regions, and investigating whether DNMT inhibition alters BDNF exon-specific mRNA expression during memory consolidation. During the first independent phase aim of the project, the role for NMDA receptor (NMDA-R) activation in the epigenetic regulation of the BDNF gene will be analyzed using a combination of novel technologies to assess cell-type specific chromatin remodeling of BDNF transcripts in hippocampus. The second independent phase aim will evaluate the functional impact of NMDA -R-mediated recruitment of the transcription factor nuclear factor kappa B (NF-KB) to BDNF regulatory elements within DNA and to identify the role of the NF-KB DNA-binding complex in the regulation of chromatin remodeling of the BDNF gene. This application explores the role of epigenetic mechanisms of BDNF gene regulation in long-term memory formation with a focus on identifying molecular mechanisms that may lead to drug discovery and development to intervene in the clinical features of mental disorders. Indeed, epigenetic mechanisms have been implicated in the etiology of mental illnesses, such as schizophrenia, depression, and bipolar disorder. Through the understanding of epigenetic-regulating mechanisms involved in BDNF gene expression during memory formation and potentially in mental disorders, other genes involved in this process may fall into a common biochemical pathway where disease intervention is possible. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal seeks to understand mechanisms of chromatin biology in memory. Histone methylation- dependent epigenetic mechanisms serve to regulate gene transcription in mature neurons of the adult nervous system. Here, we focus on the SET-domain/PHD-domain-containing protein lysine methyltransferase, G9a/GLP, that catalyses the histone H3 lysine 9 dimethylation (H3K9me2) mark and functions as a molecular linker between histone methylation, chromatin remodeling, and transcription regulation. However, very little is known about the role of G9a/GLP-H3K9me2 interactions in the nervous system or in the context of memory. AIM 1. Using two of the most innovative approaches in the field of neuroscience as it pertains to epigenetics, we will first selectively sort neuronal chromatin followed by massively parallel sequencing of immunoprecipitates (ChIP-seq) to obtain insight into the H3K9me2 landscape in mature neurons from the hippocampus, entorhinal cortex, and amygdala. We will then define the epigenetic readers recruited to the H3K9me2 methylation marks in a gene promoter-specific manner in mature neurons. The effect of genetically manipulating G9a/GLP activity will also be determined at gene promoter regions and behavioral outcomes will be assessed. This genetic data, together with information gathered on the H3K9me2 landscape, will strongly implicate G9a/GLP as a major regulator of gene transcription in the adult brain during memory consolidation. AIM 2. Currently, nothing is known about the signaling mechanisms coupled to these molecular processes for any cell-type. Thus, we will determine the signaling pathways coupled to G9a/GLP-H3K9me2 interactions in neuronal cell types recruited by NMDA receptor activation during memory consolidation using pharmacological approaches and laser-capture microdissection technology. AIM 3. NF-¿B (p65) is a non-histone target of protein lysine methyltransferases, and once methylated NF¿B can associate with proteins such as G9a/GLP. Here, we will determine how this transcription factor serves as an epigenetic initiator of the G9a/GLP- H3K9me2 interaction in mature neurons during memory consolidation. Through genetic knockdown of p65, blocking peptides, and lysine demethylase inhibitors, we will manipulate the p65-G9a/GLP interaction and assess behavioral outcomes. Together, the research studies proposed will provide the first glimpse into the epigenetic initiators and writers of the H3K9me2 methylation mark in the adult brain. Interestingly, subtelomeric deletion of the human chromosome 9 (9q34), which includes G9a/GLP, is associated with human mental retardation or intellectual disability disorders characterized by major defects in learning and cognition. Thus, this basic scientific study will clearly impact cognitive dysfunction by helping to develop new therapeutic approaches based on manipulating the epigenome to improve learning and memory deficits associated with aging, schizophrenia, depression, and post-traumatic stress disorder.

? DESCRIPTION (provided by applicant): This is a new R25 application requesting support for the Neuroscience Roadmap Scholars Program at the University of Alabama at Birmingham (UAB). Although the number of U.S. citizens from underrepresented minority (URM) groups earning doctoral degrees in science has increased over the past decade, URMs continue to represent a small proportion of the scientists in the United States. UAB is uniquely positioned to support the training of diverse group of trainees. Evidence of this readiness is the significant (36%) undergraduate enrollment of URM students, predominantly African American, given our location in the Southeastern US. In addition, UAB is the home for several students with disabilities at the undergraduate and graduate levels (approximately 400). Importantly, there are several programs already in place, such as the Office of Equity and Diversity, the Comprehensive Neuroscience Center, and the focus on Neuroscience in the School of Medicine Strategic Plan serve as a firm footing for the development of a program targeting diversity in Neuroscience. The Comprehensive Neuroscience Center functions as the epicenter for the Neurosciences on UAB campus and will be the home of the Neuroscience Roadmap Scholars Program. The CNC has a membership of approximately 365 members, with faculty from 11 basic science and clinical departments from 5 Schools. Since 2010, 89 new faculty have been recruited across several neuroscience-related departments and the number of students from diverse backgrounds applying to the Neuroscience Theme graduate program represents 27% of the domestic applicant pool in 2014. Our proposed Neuroscience Roadmap Scholars Program will target the obstacles impeding success with the goal of attracting an increased number of diversity trainees to neuroscience research and providing them with the necessary tools and skills early in their PhD careers which are essential to making this a life-long career choice. The specific value added components of the Neuroscience Roadmap Scholars Program include the annual southeast regional NEURAL (National Enhancement of Under-Represented Academic Leaders) summer conference, interactions with Career Coaches (separate from the scholar's Research mentor), Peer-to peer mentoring and undergraduate mentees, and distinct extracurricular activities. As the UAB SOM and office of Diversity and Equity have focused on diversity in the neurosciences as an area for major investment with $414,000 in stipend support and $20 million towards strategic planning over the next five years, the future of the Neuroscience Roadmap Scholars Program is secure and it will benefit substantially from this investment with the addition of talented and qualified academic leaders. The result should be an increase in the number and quality of both applicants and matriculating students from a diverse background at UAB in neuroscience at the graduate level, with a positive impact at the undergraduate, postdoctoral and faculty levels as well.

? DESCRIPTION (provided by applicant): A consensus is building that several epilepsy disorders, including Temporal lobe epilepsy (TLE), a partial adult onset form of human epilepsy, are often associated with significant long-term cognitive impairments. Currently, no effective treatment options exist to prevent or reverse epilepsy-related memory loss, and the underlying molecular mechanisms remain elusive. Recent work from our lab and others has implicated abnormal epigenetic DNA methylation (DNAme) regulation in epilepsy. Thus, we propose that DNAme may be a major contributor of aberrant gene transcription in the epileptic hippocampus. Because, previous studies have demonstrated that brain derived neurotrophic factor (Bdnf) gene expression is required for memory formation and is significantly dysregulated in the hippocampi of both epileptic patients and spontaneously seizing rats, we will focus our studies by investigating epigenetic regulation of Bdnf at excitatory synapses in the hippocampus during memory formation with TLE. Intriguingly, the role of epigenetics in mediating behaviorally- induced Bdnf gene expression changes in the epileptic hippocampus is largely uncharacterized. We will manipulate DNAme with DNMT inhibitors and Methionine in our experimental model of TLE, to gain insights into potential therapeutic targets for the treatment of epilepsy-related memory loss. Since these inhibitors are already in use clinically or being aggressively studied and developed in oncology, there is the potential to rapidly translate to treatments for epilepsy-related memory impairments and other comorbidities such as depression and anxiety in humans. The central hypothesis of this proposal is that aberrant DNAme mediated transcriptional regulation of memory permissive genes, like Bdnf, in the epileptic hippocampus contributes to epilepsy-related memory impairments. Using novel methodical and technical approaches, the aims of this proposal are as follows: 1) to test whether Bdnf gene regulation by DNAme is altered in CA1 pyramidal neurons during memory formation in an experimental rodent model of TLE, and 2) to test whether treatment with methionine reverses two-epileptic phenotypes, hippocampal network hyperactivity and hippocampus-dependent memory formation in TLE. Understanding the pathophysiological mechanisms of memory deficits associated with TLE at the cellular and molecular levels and evaluation of potential therapeutic strategies undoubtedly take priority in the path of research on cognitive impairments associated with this neurological disorder.

Abstract It is now clear that epigenetic mechanisms control gene transcription in area CA1 of the hippocampus that are necessary for proper memory formation in young and mature adulthood. For example, we have discovered that epigenetic mechanisms thought to be static after development, remain active in postmitotic neurons in adulthood and very much responsive to environmental experiences. Specifically, histone lysine methylation (HKM) mechanisms have been identified as a crucial transcriptional mechanism in area CA1 subserving the process of associative memory formation. Surprisingly little is known about how environmental influences like aging and stress experiences contribute to memory decline with age. Our long-term goal is to identify HKM modifications induced by stress to impact memory formation with aging. This has tremendous implications with regard to long- lasting epigenetic effects, which is rarely observed in the field of cognitive neuroepigenetics. For this grant proposal, we will focus on the histone lysine methyltransferase, G9a that mediates histone H3-lysine 9 dimethylation (H3K9me2) marks in area CA1 neurons. With this in mind, our preliminary results demonstrate that severe stress increased G9a-H3K9me2 activity in area CA1 of young adult rats that was sustained with aging. Furthermore, H3K9me2 hypermethylation strongly correlated with memory decline in young adults and in aged adults. Preliminary results also demonstrate that inhibiting G9a activity in the hippocampus reverses stress- induced memory impairments. Based on these preliminary results, we plan to rigorously investigate the beneficial effects of manipulating G9a-H3K9me2 activity in CA1 following stress and determine effects on age-related memory decline. To gain further mechanistic insight into stress-related G9a-H3K9me2 mediated gene transcription, we will use state-of-the-art approaches such as siRNA and CRISPR-dCas9-VP64 technology to directly target G9a in our animal model system. Our central hypotheses are 1) stress abnormally increases G9a- H3K9me2- mediated silencing of hippocampal genes in young adult rats similar to what is observed in normal aging adults, 2) stress-mediated G9a-H3K9me2 hypermethylation will correlate with aged-impaired but not aged- unimpaired animals, and 3) stress experienced in young adults results in latent effects on H3K9me2 hypermethylation with aging that can be overcome by G9a inhibition. Our Specific Aims are as follows: Specific Aim 1: Stress interferes with memory formation by increasing G9a-H3K9me2 activity in the hippocampus of young adults; Specific Aim 2: Stress has differing effects on G9a-H3K9me2 in the hippocampus of aged- impaired versus aged-unimpaired animals; and Specific Aim 3: Stress experience increases G9a-H3K9me2 activity in the hippocampus of young adults contributing to memory decline with age. Collectively, this proposal will identify long-lasting epigenetic mechanisms involved in stress-related memory decline with age. Moreover, we hope to add to our understanding of stress-induced effects on memory decline as the brain ages.

Project Summary We propose to investigate the contribution of the long non-coding RNA (lncRNA) Neat1 in astrocytes and identify the epigenetic mechanisms in these glia cells involved in enhancing memory resiliency with age. Interventions to enhance memory resilience within the aging population are possible. However, research studies that inform resiliency in this area are still lacking. Within the aging hippocampus, it is now clear that abnormal epigenetic control of gene transcription contributes to memory deficits. Nearly all the suggested epigenetic mechanisms controlling memory formation have mostly been attributed to neuronal cells within the hippocampus, largely disregarding these mechanisms in astrocytes. Thus, little is known about how astrocytic epigenetic mechanisms influence memory resiliency with age. Our long-term goal is to study the role of lncRNAs in astrocytes and to identify how these powerful epigenetic regulators impact memory formation with aging. Our pilot data demonstrate that Neat1 expression is decreased in area CA1 of young adult mice and overexpressed in aged mice. Furthermore, we demonstrate that inhibiting Neat1 expression in area CA1 of the hippocampus of aged mice reverses memory impairments. Pilot studies also suggest a strong relationship between G9a mediated H3K9me2 hypermethylation with Neat1 overexpression in aged adults. Based on these preliminary results, we plan to rigorously investigate the effects of manipulating Neat1 in astrocytes and determine effects on age-related memory decline. To gain further mechanistic insight into Neat1 mediated gene transcription in astrocytes in the aging hippocampus, we will use state-of-the-art approaches such as CRISPR reprogramming and chemogenetics to elucidate the epigenetic mechanisms in astrocytes in our aging animal model system. Our overarching hypothesis is that Neat1 contributes to age-associated changes in hippocampal astrocyte diversity, astrocyte function and vulnerability to memory dysfunction. Our Specific Aims are as follows: Specific Aim 1: Test the hypothesis that Neat1 is associated with astrocyte diversity with aging; Specific Aim 2: To determine the mechanism by which Neat1 acts to influence chromatin restructuring in astrocytes from young versus aged animals; and Specific Aim 3: To determine the contribution of astrocytic Neat1 to memory resiliency with age. Collectively, these studies will identify epigenetic mechanisms in astrocytes involved in age-related memory decline, with broad implications for treatment options for age-related dementia and Alzheimer?s disease.