Soren Impey, PhD - US grants

DESCRIPTION (provided by applicant): Activity-regulated gene expression plays a fundamental role in both the remodeling of synaptic circuitry and in neuronal survival. Activity-dependent increases in intracellular Ca2+ trigger the expression of hundreds or perhaps thousands of genes. The cAMP response element (CRE) was identified as a major Ca2+ responsive element via analysis of immediate early gene (IEGs) promoters and many if not most Ca2+ responsive promoters contain CREs. The basic leucine zipper transcription factor, CREB, binds to the CRE as a dimer and can be activated by Ca2+, cAMP/PKA, and growth factor signaling pathways. The transcription factor CREB is a major target of activity-induced Ca2+ influx and is a key regulator of both neuronal survival and adaptive synaptic plasticity. Nevertheless, the mechanisms by which Ca2+ activates CREB have not been clearly elucidated. It is well established that PKA phosphorylates Ser 133 of CREB and creates a phospho-serine docking motif that recruits its coactivator CBP (or the homologue, p300). Recruitment of CBP is thought to enhance transcriptional activation either via its association with the general transcriptional machinery or via its intrinsic histone acetyltransferase activity. Although activity-induced Ca2+ influx is widely believed to regulate CREB function in an analogous manner, the signaling cascades that promote CBP recruitment and transcriptional activation have not been clearly defined. A major theme of this proposal is to determine whether the PKA-CREB-CBP paradigm also pertains to Ca2+-activated transcription. Thus, an initial goal of this study will be to determine whether neuronal activity induces the recruitment of CBP to CREB and whether CBP recruitment is required for transcriptional activation. Four specific aims are proposed. (1) Determine whether synaptic activity induces the recruitment of CBP to CREB in neurons and whether recruitment of CBP is sufficient for full transcriptional activation. (2) Decipher the activity-regulated signaling cascades that promote the phosphorylation and activation of CREB and CBP. (3) Determine how synaptic activity and Ca2+ influx regulate CBP function. (4) Analyze the regulation of CBP and CREB function in mice deficient for CaM kinase IV. This study seeks to dissect the biochemical events result in synaptic activity-mediated phosphorylation of CREB, recruitment of CBP, and "activation" of CBP-dependent transcription. Gaining insight into the mechanisms of activity-mediated transcription has relevance in the treatment of neurodegenerative pathologies that may result from excitotoxic cell death or apoptosis. Through a mentored career development plan, the Principal Investigator will gain an enhanced capacity to be productive in the field of molecular neurobiology with a focus on gene regulation. This will be accomplished by conducting the above described research, attending and presenting at local and national seminars and meetings, and participating in courses teaching molecular biology and neurophysiology techniques.

[unreadable] DESCRIPTION (provided by applicant): Embryonic stem (ES) cells isolated from the inner cell mass of murine blostocysts are pluripotent, capable of indefinite symmetric cell division, and can generate chimeric animals. The recent isolation of human ES cells holds great promise for the treatment of a variety of degenerative disorders including, but not limited to, Parkinson's disease and diabetes. The homeodomain transcription factors, Oct3/4 and Nanog, are believed to play critical roles in sustaining ES cell pluripotency. Surprisingly, targets of Nanog are completely unknown and few Oct3/4 targets have been proposed. Thus, the molecular mechanisms that repress differentiation of ES cells and promote self-renewal are poorly defined. We have developed a novel approach for identifying complex metazoan regulons called SACO (Serial Analysis of Chromatin Occupancy), which combines chromatin immunoprecipitation with a modification of Long SAGE. We will use SACO to identify the entire complement of Nanog and Oct3/4 genomic targets in mouse ES cells. Such knowledge would not only aid efforts to characterize mechanisms that govern cell-fate commitment of stem cells, but could also enable the indefinite propagation of existing human stem cell lines. Insight into these transcriptional networks could also lead to the generation of pluripotent stem cells from adult tissues or cells. Our studies are designed to provide a complete definition of Nanog and Oct3/4 targets in pluripotent stem cells. By characterizing the regulation of the corresponding transcripts, we expect to characterize the molecular pathways that control self-renewal and pluripotency of ES cells. The microarrays representing novel Nanog and Oct3/4 driven transcripts and the catalog of genomic binding sites will be provided to the stem cell research community. The creation of microarrays representing novel Nanog and Oct3/4 targets will facilitate high-throughput analysis of their expression during early embryogenesis and their regulation by pathways that regulate differentiation and pluripotency. This set of studies will represent the most comprehensive analysis to date of the regulation of transcription factor binding in any metazoan system. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): MicroRNA is a recently characterized class of small, non-coding, RNA that repress mRNA translation. Work over the past several years has revealed important roles for microRNA in a vast array of developmental and disease-related processes. Within the developing mammalian central nervous system, results from dicer null mice support a role for microRNAs in neuronal morphogenesis and neuronal survival. However, relatively little is known about how neuronal activity regulates microRNA expression patterns in the mature nervous system and, importantly, whether microRNA regulate neuronal plasticity and cell viability. Based on recent work by a number of investigators, and on the preliminary data reported here, we propose that microRNA plays a key role in activity-dependent structural plasticity in the mature nervous system. To test this hypothesis we have assembled a novel set of genetically modified mouse models, and an array of genetic and functional screening assays. In Aim 1, we propose to utilize the Solexa deep sequence method to examine activity-dependent expression of non-coding RNA in the hippocampus. We will also examine the contribution of transcriptional networks that underlie activity-dependent neuronal plasticity and perform a series of experiments to identify functionally relevant microRNA targets. In Aim 2 we propose to determine the contribution of microRNA to adult neuronal structural plasticity and neuroprotection. To this end, we will employ an inducible form of Cre-recombinase to disrupt Dicer expression. The effects on both physiological and pathophysiological levels of neuronal activity will be examined. In Aim 3, we propose to determine the role of the microRNA-132 locus in activity-induced structural remodeling in vivo. A combination of knockout and tet-inducible microRNA mouse strains will be used to test this question. The data generated here should provide a wealth of new insights regarding how neuronal activity sculpts microRNA expression patterns, and, in turn, how these changes affect key aspects of neuronal plasticity and pathology. PUBLIC HEALTH RELEVANCE: microRNAs are small molecules that act as potent silencers of protein expression. With respect to brain health, dysregulation of microRNAs expression has been suggested to contribute to a number of neurological disorders, including Down syndrome, Rett syndrome and schizophrenia. In this proposal, our goal is to provide the first comprehensive examination of how neuronal activity regulates microRNA expression in the adult mammalian central nervous system and, in turn, how microRNA regulates key aspects of neuronal plasticity and pathology. These data should provide a framework to begin to develop therapeutic approaches designed to regulate microRNA expression.