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.

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): 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.

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.