John D. Sweatt - US grants

Long-term potentiation of synaptic transmission in the hippocampus (LTP) is a well-characterized, tractable model system for the study of mammalian learning and memory. Not only does a wide variety of evidence support a role for LTP in memory acquisition, the discrete, compact structure of the hippocampus allows for convenient monitoring of electrophysiological responses, and relative ease of in vitro manipulation. The development of the hippocampal slice preparation as a means to study LTP has provided a system in which to study the molecular and biophysical basis for this effect. The studies described in this application focus on the molecular mechanisms underlying the maintenance of LTP in the CA1 region of rat hippocampus. Using a variety of biochemical and pharmacological methods and approaches, we will investigate the involvement of protein kinase C in the maintenance of LTP. Specifically, the studies will address the following questions: By what mechanism is protein kinase C persistently activated in the maintenance phase of NMDA receptor-dependent LTP? How is the persistent activation of PKC reversed by depotentiating stimulation? These studies will serve as the foundation for future studies exploring in greater detail the biochemical mechanisms contributing to this form of synaptic plasticity, and should provide insight into mechanisms involved in long- lasting changes in neuronal function in the mammalian CNS. This in turn should increase our understanding of mechanisms for the development and prolongation of a variety of neuropsychiatric disorders.

DESCRIPTION: The objectives of the Mouse Behavior and Synaptic Plasticity Core are to establish, originally through interactions with Dr. Crnic at the University of Colorado MRRC and now by the newly recruited by Dr. Paylor, a facility that will provide Baylor MRRC PIs with access to behavioral assays in order to assess mutant mice in studies of MRDD, and to provide the ability to determine whether mutant mice exhibit derangements in hippocampal synaptic transmission and synaptic plasticity. Seventeen investigators (21 total projects) will access this core.

neuroregulation; potassium channel; phosphorylation; biological signal transduction; pyramidal cells; hippocampus; mitogen activated protein kinase; protein structure function; protein kinase C; norepinephrine; neural plasticity; neural transmission; second messengers; acetylcholine; electrophysiology; alpha methyldopamine; calmodulin dependent protein kinase; protein kinase A; voltage gated channel; cell line; western blottings; site directed mutagenesis; laboratory rat; electrical measurement; protein sequence;

In an effort to decipher the cellular dysfunction underlying Alzheimer's Disease (AD), transgenic (Tg) animal technology has been used to generate mouse strains that model aspects of the human disease. These models target the known autosomal dominant familial AD risk factors in order to model the amyloidogenesis- associated abnormalities observed in AD brain. This proposal utilizes a Tg mouse model for AD in which the transgene is the human 695 splice-variant of APP that contains the double mutation K670M, N671L driven by a hamster prion protein gene promoter. (This APP mutation was identified in a Swedish family with inherited AD). This is our AD mouse model of choice as this Tg mouse strain (Tg2576) approximates many of the pathological correlates of AD and, as importantly, is well characterized in several respects. We will use this model to characterize effects of overexpression of mutant APP in the mouse brain, especially as related to the signal transduction mechanisms subserving hippocampal synaptic plasticity and learning. Specific Aim number 1 - To test the hypothesis that hippocampal MAPK activation is aberrant in Tg2576 mice. It is our prediction that the chronic exposure of neurons to elevated levels of extracellular Abeta that occurs in Tg2576 mice affects p42 MAPK regulation. Specific Aim number 2 - To test the hypothesis that aberrant p42 MAPK activity underlies LTP deficits in Tg2576 mice. p42 MAPK is activated in LTP and this is necessary for LTP. We expect to find evidence for an interruption in p42 MAPK signaling following induction of CA1 LTP since hippocampal slices from aged Tg2576 mice fail to express this form of potentiation. Specific Aim number 3 - To test the hypothesis that the hippocampus- dependent associative learning impairment exhibited by Tg2576 is accompanied by deficits in hippocampal signaling mechanisms. Hippocampal MAPK is activated with fear conditioning. We will evaluate if this activation is altered in Tg2576 mice. Overall, these studies will yield new insights into the sequelae of mutant APP overproduction in the CNS.

DESCRIPTION: (Adapted from the Investigator's Abstract) Signal transduction via protein phosphorylation plays a critical role in learning and memory. In a prominent candidate mechanism for mammalian learning and memory, hippocampal long-term potentiation (LTP), a diverse set of protein kinases plays an important role. Thus, the cAMP-dependent protein kinase (PKA), calcium/phospholipid-dependent kinase (PKC), calcium/calmodulin-dependent protein kinase (CaMKII), and mitogen-activated protein kinase (MAP kinase) all are necessary for the induction of various phases of LTP. While great progress has been made in identifying the kinases involved in LTP, there are many gaps in our knowledge of how the protein kinases are regulated by NMDA receptor-dependent and independent processes during LTP. In addition, little is understood concerning the downstream targets of these kinases. To address this question, three specific aims are proposed: Specific Aim 1, to investigate the regulation of phosphorylation of the Shal-type K+ channel Kv4.2 in LTP, Specific Aim 2, to investigate the biochemical mechanisms for the activation of MAP kinase during LTP and Specific Aim 3, to investigate the role of protein kinases in regulating CREB phosphorylation during LTP. These studies will provide insight into mechanisms involved in long-lasting changes in neuronal function in the mammalian CNS and should increase our understanding of the molecular basis of neuropsychiatric disorders.

DESCRIPTION (provided by applicant): We have isolated clones encoding the members of the nicotinic acetylcholine receptor (nAChR) gene family, sequenced these clones, determined the loci in the rat brain of the cells that express the genes encoding these sequences, expressed these clones in the Xenopus oocyte to form functional neuronal nAChRs, and have discovered several interesting and unanticipated properties of the expressed receptors and of their synthesis. We also have used these clones, other new reagents we have created, and our knowledge of the properties of these receptors to address issues of their composition, assembly, disposition on the cell surface, and role in synapse formation. In the studies we are now proposing we will use the reductionist approach that has characterized our ongoing 30-year research endeavor, and for this phase of our studies focus intently on one specific nAChR subtype: the alpha7 nAChR. In our studies of the alpha7 nAChR we will focus on its physiologic roles, mechanisms of action, and its potential contribution to neurological disorders. In our studies we will ask the following questions: What are the hippocampal signal transduction mechanisms to which alpha7 nAChRs couple? What is the role of alpha7 nAChRs in the regulation of gene expression in the hippocampus? What is the role of the alpha7 nAChRs system in amyloid-beta peptide toxicity and Alzheimer's disease? These experiments are motivated by our new appreciation of the diversity of the ligand-gated ion channels and are designed to move our study of the neuronal nAChRs from the clones that encode them to the properties of the expressed proteins and their roles in synaptic function and modification in the central nervous system.

This proposal is to investigate the molecular basis for activity-dependent and neurotransmitter-dependent modulation of the biophysical and biochemical properties of potassium channels. We are testing the hypothesis that short-term and long-term regulation of the Kv4.2 channel is mediated by phosphorylation. Specifically we hypothesize that protein kinase-dependent regulation of Kv4.2 is mediated by direct phosphorylation of the pore-forming alpha subunit. In our studies over the last few years we have identified 9 different phosphorylation sites on Kv4.2, mediated by 4 different second-messenger-regulated kinases. Moreover, we have found that PKC phosphorylation of the Kv4.2 C-terminal cytoplasmic domain can regulate the capacity of ERK MAP Kinase to phosphorylate this same channel subregion. Addressing the mechanism for this PKC/ERK interaction will be the first Specific Aim of this project: To determine the protein structure/function relationships underlying PKC modulation of ERK phosphorylation of the C-terminal cytoplasmic domain of Kv4.2. Another example of the complexity of phosphorylationdependent regulation of Kv4.2 is illustrated by considering PKA regulation of Kv4.2. In recent studies we found that PKA regulation of Kv4.2-encoded currents required the presence of a KChlP ancillary subunit. Despite the ability of PKA to phosphorylate the Kv4.2 alpha subunit in the absence of KChlP3, PKA was unable to alter channel biophysical properties by this mechanism alone. Thus, we propose Specific Aim 2 of the project: To determine the structure/function relationships for KChlP modulation of phosphoregulation of Kv4.2. Our final Specific Aim will focus on Calcium/calmodulin-dependent Protein Kinase II (CaMKII) regulation of Kv4.2. We have identified two sites of CaMKII phosphorylation on Kv4.2 and determined that phosphorylation at one of these sites (T438) is necessary and sufficient for CaMKII stabilization and enhanced surface expression of Kv4.2 in COS cells. Additional preliminary results indicate that CaMKII regulation of Kv4.2 surface expression occurs in hippocampal pyramidal neurons as well. We will test the hypothesis that phosphorylation-dependent protein-protein interactions mediate this effect in the final Specific Aim: To investigate the mechanism of CaMKII regulation of Kv4.2 expression and protein stabilization. These studies should allow for the first time the definition of specific phosphorylation events, of known physiologic consequence, as mechanisms for Kv4.2 A-type potassium channel regulation.

DESCRIPTION (provided by applicant): This project focuses on the role of signal transduction mechanisms in Long-term Potentiation (LTP) and memory formation. Lately we have been exploring the role of the Mitogen-Activated Protein Kinase (MAPK) family of signal transduction cascades in hippocampal synaptic plasticity and learning. We initiated our studies in this area about 10 years ago by determining that the Extracellular-Signal Regulated Kinase (ERK) isoforms of MAPK are activated with LTP-inducing stimulation in hippocampal slices, and that ERK activation is necessary for NMDA receptor-dependent LTP in area CA1. We then transitioned to studies in the behaving animal and discovered that ERK is activated in the hippocampus with contextual associative conditioning, and that ERK activation is necessary for fear conditioning and for spatial learning in the Morris water maze. Studies from a wide variety of laboratories have now shown that MAPK signaling is involved in many forms of synaptic plasticity and learning, in essentially every species that has so far been examined. Given the clear importance of understanding the roles and regulation of ERK in synaptic plasticity and learning, for the next project period we propose to continue our investigations into the upstream regulators and downstream targets of ERK in the hippocampus. We will pursue the following three Specific Aims: 1: To test the hypothesis of a role for the scaffolding protein Kinase Suppressor of Ras (KSR) in hippocampal ERK activation, LTP, and hippocampus-dependent memory. 2: To test the hypothesis that Histone Acetyl Transferases (HATs) are a target of ERK regulation in the hippocampus. 3: To test the hypothesis that the dual-specificity MAPK phosphatase MKP-3 is a negative feedback regulator of ERK. These studies will give us insights into key functional loci in the hippocampal ERK MAP Kinase cascade, a new signal transduction pathway involved in transcriptional regulation, synaptic plasticity, and memory formation.

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DESCRIPTION (provided by applicant): Molecular biological and biochemical studies of the members of the nicotinic acetylcholine receptor (nAChR) Family, including those studies supported in the past by this Project, have allowed the discovery of several interesting and unanticipated properties of these prototypical ligand-gated ion channels. Nowhere is this more apparent than in studies of one specific nAChR subtype: the alpha7 nAChR. For example, we now know that the expression of the mRNA encoding a single alpha7 subunit is capable of allowing expression of a fully functional acetylcholine-gated channel, which surprisingly fluxes calcium with a permeability on par with the NMDA receptor. Moreover, we know that the alpha7 receptor is expressed in the hippocampus, a brain region important for memory formation, and that the alpha7 receptor modulates neuronal function in this brain region. Finally, in the last Project Period, we made two discoveries concerning alpha7 receptors that also were surprising. We discovered that hippocampal alpha7 receptors activate the Mitogen-Activated Protein Kinase (MAPK) cascade, the prototypical signal transduction cascade for regulating gene expression and triggering long-term cellular change in the hippocampus. We, along with several other groups, also discovered that the likely causative agent for Alzheimer's Disease, the amyloid beta peptide, is a high-affinity ligand for alpha7 receptors. These latter two discoveries provide the basis for the studies we are proposing for the next Project Period. In the next phase of our studies we will ask the following three questions: 1. Does the alpha7/MAPK pathway regulate the BDNF gene as a target in hippocampal neurons, and is regulation of chromatin structure involved in this process? 2. What are the long-term physiologic consequences of the A-beta peptide interaction with the alpha7 nAChR at hippocampal Schaffer/collateral synapses? 3. What are the behavioral consequences of the A-beta/alpha7 nAChR interaction, studied in vivo using genetically engineered mouse models? These experiments are motivated by our desire to understand the physiologic roles and mechanisms of action of the alpha7 nAChR in the CNS, and to understand its potential contribution to neurological disorders.

DESCRIPTION (provided by applicant): We are requesting partial support for the next three Molecular and Cellular Cognition (MCC) Meetings. The Conference will be held in the San Diego Convention Center on November 1st and 2nd, 2007, right before the annual meeting of the Society for Neuroscience. The MCC meeting brings together junior and senior scientists that combine molecular (pharmacology, genetics, transgenics, viral approaches, etc) and physiological (electrophysiology, optical physiology) and other cellular approaches to study behavior, including learning and memory. The general goal of these studies is to derive explanations of cognitive processes that integrate molecular, cellular, and behavioral mechanisms, as well as to use this information and related animal models in the search for treatments for cognitive, psychiatric and neurological disorders in children, adults and the elderly. These meetings have been organized under the sponsorship and leadership of the Molecular and Cellular Cognition Society (MCCS) (www.molcellcog.org), a relatively new group whose main function is to organize meetings and promote interaction and collaborations among laboratories working in this general area. Although there are a few learning and memory meetings in the USA and abroad, the Molecular and Cellular Cognition meeting is unique because it brings together individuals that integrate molecular, physiological and behavioral approaches in studies of memory and learning related disorders. Although the molecular and cellular cognition field is relatively new, it has already had a profound impact on neuroscience research. Currently, this is the only periodic meeting in the field, an invaluable opportunity to exchange information, and develop this young field. The 2005 and 2006 meetings were highly successful, attracting each year a diverse group of approximately 400 participants from North America, Europe, and Asia, and we have every reason to believe that the 2007-2009 meetings will be equally successful. (Summaries at http://www.molcellcog.org/meetings.htm). The Molecular and Cellular Cognition Society meeting brings together junior and senior scientists that combine molecular (pharmacology, genetics, transgenics, viral approaches, etc) and physiological (electrophysiology, optical physiology) and other cellular approaches to study behavior, including learning and memory. The general goal of these studies is to derive explanations of cognitive processes that integrate molecular, cellular, and behavioral mechanisms, as well as to use this information and related animal models in the search for treatments for cognitive, psychiatric and neurological disorders in children, adults and the elderly.

[unreadable] DESCRIPTION (provided by applicant): An explosion of interest in cognitive neurobiology over the last 15 years has produced a corresponding demand for highly trained cognitive neurobiologists, and in addition a high demand for students trained in basic science with an interest and expertise in translating basic science advances into patient treatments and cures. In response to this demand, we propose to develop a four-year Training Program in the Neurobiology of Cognition and Cognitive Disorders at the University of Alabama at Birmingham (UAB). UAB is ideally poised to provide this novel type of Training Program. In the past decade UAB has made major financial, infrastructural and scholarly commitments to neuroscience research and training, including the recruitment of new outstanding chairs and faculty in neuroscience and the establishment of the McKnight Brain Institute, and new UAB Centers in Comprehensive Neuroscience, Glial Biology in Medicine, Neurodegeneration and Experimental Therapeutics, and Functional Neuroimaging. The proposed Training Program will have its administrative base in the Department of Neurobiology and include 42 training faculty from six basic science and six clinical departments in the UAB Schools of Medicine and Optometry. The goal of the Program is to train the next generation of leaders and thinkers in neurobiology research who have a solid foundation in molecular, cellular, and cognitive neuroscience, have exposure to current concepts in clinical research, value the importance of translating basic research into advancements in the treatment of cognitive disorders, and are prepared to use multidisciplinary and innovative research approaches to understand cognition and cognitive disorders. To achieve this goal, we propose to bring together a group of outstanding UAB faculty members working in cognition and cognitive disorders and allow them to teach a specialized curriculum including cqursework in molecular, cellular, developmental, integrative, medical, and cognitive neuroscience and diseases of the nervous system. In addition to this unique coursework, the specific "value added" components of the Training Program are a special translational/clinical training experience in patient-oriented research, dual basic science/clinical science mentorship and distinct extracurricular activities. [unreadable]

DESCRIPTION (provided by applicant): This is a competitive renewal application for the Training Program in Neurobiology of Cognition and Cognitive Disorders at the University of Alabama at Birmingham (C&CD Program). Since the C&CD Program was implemented in 2007 under the leadership of Dr. David Sweatt, UAB has made an impressive investment in neuroscience with the areas of cognition and cognitive disorders now being identified as a focal point for strategic development across the entire institution. The C&CD Program is housed in the Department of Neurobiology, and includes 47 faculty from 11 basic science and clinical departments in the School of Medicine (SoM), School of Optometry, and College of Arts and Sciences. The goal of the C&CD Program is to develop the next generation of leaders and scholars in neuroscience research who: have a foundation in molecular, cellular, and cognitive neuroscience theories and research findings; value the importance of using findings from basic science to understanding the basis of cognitive disorders; are prepared to use innovative research approaches to understand cognition and brain-behavior relationships; and will be able to translate this knowledge to treatments and cures of cognitive disorders in the future. The success of the C&CD Program in its first funding period is evidenced by its robust participation (35 total students-18 trainees funded), graduation of a talented cadre of doctoral students (ten total graduates-six funded), with an average of more than four publications each, and all transitioning to competitive postdoctoral positions or completing clinical training. This renewal builds on the successful components of the Program Plan by providing: (1) A curriculum with courses in cellular, molecular, cognitive and translational neuroscience; (2) Clinical shadowing in the Clinical Evaluation of Cognitive Disorders course; (3) An outstanding seminar series; (4) Dissertation research with both a basic science and clinical mentor, using experimental approaches that address questions with direct relevance to cognition and cognitive disorders; and (5) Opportunities to hone presentation skills at retreats and national meetings, and instruction in ethical conduct of research, and career development. The specific value added components of the C&CD Program include the special clinical/translational training experience in patient-oriented research, interactions with both basic science and clinician scientist mentors, and distinct extracurricular activities. As the UAB SoM Strategic Plan highlighted cognition and cognitive diseases as an area for major investment with 15 new faculty and $20 million over the next five years, the future of the C&CD Program is secure and it will benefit substantially from this investment with the addition of dynamic new faculty mentors and research facilities.

DESCRIPTION (provided by applicant): Although we are at the end of the Congressionally-declared Decade for Pain Control and Research, chronic pain continues to be a leading public health epidemic, affecting more than 50 million Americans and costing more than $165 billion per year in treatment expenses and lost work productivity. Of particular note are those patients with chronic diseases, such as cancer and HIV, who suffer from treatment-related pain that cannot be controlled with conventional analgesics. Treatment-related pain affects approximately 50% of these patients, depending on drug and dosing regimen, which translates into nearly 700,000 Americans per year. The predominant symptom is excruciating unremitting pain that is resistant to traditional pharmacological treatments. As therapeutics become increasingly aggressive and diseases become chronic, this becomes a risk/benefit issue; patients must choose between increased life expectancies and constant suffering with decreased quality of life. This decision is untenable, and thus, the crucial question that must be answered is how to prevent or reverse persistent disease treatment-related pain. Recent mechanistic studies have found that the development and persistence of neuropathic pain stems in part from central sensitization in the spinal dorsal horn. Much like memory formation in the hippocampus, central sensitization in the spinal cord produces pain hypersensitivity that is uncoupled from noxious input. More simply put, innocuous inputs produce long lasting pain hypersensitivity. Recently, we made the preliminary discovery that antiretroviral treatment produces up-regulation of Brain-derived Neurotrophic Factor (BDNF) in the mouse spinal dorsal horn, causing central sensitization (hyperexcitability of spinal dorsal horn neurons) and the development and persistence of nocifensive (pain) behavior. Reducing levels of BDNF expression in vivo reversed this phenotype. These data implicating BDNF in treatment-related pain suggest that BDNF may represent a new target for drug development. However, further studies to determine precisely how chemotherapeutic drugs alter BDNF expression and that of the BDNF receptor, tyrosine kinase receptor B (trkB) are crucial first steps. One likely possibility is via epigenetic regulation of BDNF and trkB gene expression. In support of this idea, we have shown that alterations in DNA methylation and histone modifications in promoter regions produce changes in BDNF gene expression that regulate the capacity for long-term memory formation. Thus, we hypothesize that complex epigenetic regulation of BDNF and trkB may produce gene dysregulation that is mechanistically linked to long-term chemotherapy-induced neuropathic pain. In addition, we posit that other genes, not previously linked with nociception, may undergo epigenetic regulation and we will examine this question using chromatin immunoprecipitation (ChIP) followed by high-throughput next generation sequencing (ChIP-seq). Results from these studies will have broad impact beyond treatment-related pain to improve our understanding of chronic pain persistence and the role for epigenetic modifications.

DESCRIPTION (provided by applicant): The biochemical signaling mechanisms underlying the sustenance and perpetuation of long-lasting, experience-dependent functional change in the CNS remain mysterious. Although several appealing potential mechanisms in this context have been identified, including CaMKII autophosphorylation, PKM-zeta production, and AMPA receptor auto-regulation, transcription-regulating mechanisms have received little attention to date. This Project will investigate the hypothesis that a potent transcription-regulating mechanism, altered DNA methylation, might serve as a lasting signal in the hippocampus and cortex to subserve persisting alterations in gene expression, cellular properties, and circuit function. We will test this idea by executing two Specific Aims that test important predictions of the concept. In Specific Aim 1 we will test the hypothesis that DNA methylation controls arc gene expression and hippocampal place field stability in vivo. Prior results have demonstrated that application of a variety of DNMT inhibitors, and conditional deletion of the DNMT 1 and 3A genes, leads to deficits in hippocampal LTP and deficits in hippocampus-dependent long-term contextual learning. However, it is not known how the deficits in hippocampal plasticity lead to memory deficits in the behaving animal. In this Aim we will investigate the role of DNA methylation at the cellular and systems level by investigating experience-driven long-term and short-term changes in DNA methylation in specific hippocampal neuronal subtypes using both immunohistochemistry and laser-capture dissection. In additional studies we will investigate the capacity of DNA methylation to regulate hippocampal arc gene expression, and by investigating the capacity of DNA methylation to control the formation and stabilization of hippocampal place cell firing patterns. In Specific Aim 2 we will test the hypothesis that DNA methylation controls the storage of remote memory in the anterior cingulate cortex. Recent work from several laboratories has demonstrated that remote, i.e. very long-lasting, contextual memories are consolidated and stored in the anterior cingulate cortex. It is intriguing to consider that lasting changes in DNA methylation might contribute to stabilization of remote memory in the cortex, and in this Aim we will test whether remote memory formation is associated with altered DNA methylation in the anterior cingulate cortex, and whether disrupting cortical DNA methylation leads to remote memory destabilization. PUBLIC HEALTH RELEVANCE: The study of the signaling mechanisms that contribute to long-lasting behavioral change will lead to the identification of novel neuropharmacological targets for drug development, and will contribute to a better general understanding of regulation of neurobehavioral function. This Project will specifically be relevant to the idea that drugs affecting gene expression may potentially be useful therapies for memory dysfunction and for facilitating long-term behavioral modification.

DESCRIPTION (provided by applicant): The basic helix-loop-helix transcription factor TCF4 (aka E2-2) has been implicated broadly in human CNS function. TCF4 was confirmed in several large GWAS studies as one of the rare highly replicated schizophrenia susceptibility genes, and when haplo-insufficient TCF4 is the causative factor for Pitt-Hopkins Syndrome (PTHS). PTHS has compelling attributes in terms of human cognitive function, being associated with pronounced memory deficits, autistic behaviors, and importantly an almost complete lack of language development. Thus, understanding the roles and function of TCF4 in the CNS is highly significant with respect to human language and auditory cognition, memory function, autism spectrum behavior, and schizophrenia susceptibility. Despite the clear importance of TCF4 function in the human CNS, the basic neurobiology of TCF4 has been only sparsely studied. Thus, this Project will use genetically engineered mouse models to assess the role of TCF4 in memory, social interactions and communication, hippocampal synaptic plasticity, synaptic anatomy, and epigenomic and transcriptional regulation in the CNS. The central hypothesis of the proposed studies is that TCF4 regulates the brain's ability to trigger long-term synaptic plasticity and memory formation by actively regulating transcriptional activity in response to behavioral experience. The approach will be to use both germline constitutive knockout and acute knockdown or deletion of TCF4 in the adult mouse hippocampus, coupled with extensive behavioral, electrophysiological, and transcriptomic/epigenomic characterization, to determine if TCF4 function is necessary in an ongoing fashion for normal memory and synaptic plasticity in the mature CNS. The studies will capitalize not only upon the available germline knockout line but also upon an already-available floxed TCF4 allele mouse line, and combine these mice with both virus- driven cre expression in the adult hippocampus and post-developmental CaMKII promoter-driven forebrain neuron-selective cre expression to achieve inducible post-developmental attenuation of CNS TCF4 function in vivo. Using these novel mouse models we will undertake three Specific Aims. Aim 1 will test the hypothesis that TCF4-deficient mice exhibit cognitive memory and social interaction deficits, and also test the hypothesis that TCF4-deficient mice exhibit altered synaptic plasticity. Aim 2 will comprise a significant translational component of the studies and will test the hypothesis that transcription-promoting Histone DeAcetylase Inhibitors (HDACi) will restore learning, memory, and synaptic plasticity in TCF4-deficient mice, as proof-of- concept that behavioral deficits and plasticity deficits in PTHS model mice can be reversed pharmacologically with HDACi. Aim 3 will test the hypothesis that loss of TCF4 function triggers secondary alterations in chromatin structure, DNA methylation, and gene transcription in the mature CNS. Toward this end, we will use the unbiased approach of utilizing MBD-seq, mRNA-seq, and small RNA-seq approaches for a comprehensive analysis of potential transcriptomic and epigenomic alterations in TCF4-deficient mice.

? DESCRIPTION (provided by applicant): This project focuses on the role of signal transduction mechanisms in hippocampal neuronal plasticity and memory formation. In the most recent Project Period we have been exploring the role of the TET Dioxygenase family of signal transduction cascades in hippocampal neuronal plasticity and learning, focusing on the mechanisms through which this pathway controls memory-associated gene transcription. We initiated our studies in this area about 10 years ago by determining that epigenetic mechanisms including histone acetylation and DNA Methyltransferase activity are necessary for NMDA receptor-dependent LTP in area CA1. We then transitioned to studies in the behaving animal and discovered that epigenetic mechanisms are activated in the hippocampus with contextual associative conditioning, and that alterations in epigenetic mechanisms are necessary for fear conditioning and for spatial learning. Studies from a wide variety of laboratories have now shown that epigenetic signaling is involved in many forms of synaptic plasticity and learning, in essentially every species that has so far been examined including humans. Moreover, in the last Project Period, we as well as the Tsai lab discovered that TET1 Oxygenase is a controller of active DNA demethylation in the CNS, and that this pathway regulates memory capacity in the behaving animal. Given the clear importance of understanding the roles and regulation of TET Oxygenase-dependent epigenetic mechanisms in neural plasticity and learning, for the next Project Period we propose to continue our investigations into the genomic, epigenomic, and functional targets of TET Oxygenase regulation in the hippocampus. We will pursue the following three Specific Aims: 1: To test the hypothesis that TET1 drives active demethylation at memory-associated genes. 2: To test the prediction that site-specific gene demethylation is sufficient to alter gene expression and regulate memory formation. 3: To test the specific hypothesis that TET1 regulates memory capacity via controlling homeostatic synaptic plasticity and cell-wide excitability in hippocampal pyramidal neurons. By focusing on these important new targets for the TET Oxygenase pathway in hippocampus, we will continue to formulate a comprehensive model of epigenetic regulation of transcription-dependent molecular mechanisms in neuronal plasticity and memory.

? DESCRIPTION (provided by applicant): Histone subunit exchange represents an entire branch of epigenetics that is the subject of rigorous experimentation in many model systems, including yeast, plants, and cancer, but its role in the nervous system is virtually unknown. We recently conducted the first in vivo experimental investigation of activity-induced histone subunit exchange in the nervous system, focusing specifically on the histone variant H2A.Z in rodent hippocampus and cortex. In these studies we discovered that behavioral experience triggers histone subunit exchange and attendant alterations in gene transcription in the adult CNS. The characterization of experience- dependent histone subunit exchange in the brain represents a significant step forward in our knowledge of activity-regulated epigenetic mechanisms in the nervous system and provides crucial insights into the general function of this process for the field of epigenetics in general. These findings introduce a novel mechanism for regulating the three-dimensional structure of chromatin in neurons, triggering attendant alterations in gene readout, and driving experience-dependent changes in behavior. These discoveries also open up the possibility that targeting histone subunit exchange may be a novel target for therapeutic intervention in a broad range of CNS disorders, including drug addiction, cognitive disorders, and disorders of neural plasticity in general. Given this background of new information concerning a role for histone H2A.Z subunit exchange in the CNS, for this Project we propose to pursue the following four Specific Aims: Aim 1: To test the hypothesis that the SIRT1 histone/lysine de-acetylase signaling cascade regulates H2A.Z subunit exchange in neurons. Aim 2: To test the hypothesis that H2A.Z controls transcription and CpG methylation of plasticity- associated genes using a genome-wide approach. Aim 3: To enable the selective pharmacologic inhibition of H2A.Z by developing antisense oligonucleotide-based constructs that are sufficient to alter H2A.Z mRNA and protein expression and augment the acquisition of long-term behavioral change. Aim 4: To test the hypotheses that H2A.Z regulates neural plasticity via controlling both synaptic plasticity and homeostatic synaptic scaling in neurons. We anticipate that our results will be broadly applicable to understanding experience- and drug-induced neural plasticity involved in the induction and maintenance of lasting behavioral change.