Richard H. Goodman - US grants

biomedical equipment purchase;

health science research support;

DESCRIPTION (provided by applicant): The overall goal of this training grant is to develop future leaders in the field of neuroendocrinology. There are two components to this effort. The first is to provide training to students enrolled in the Neuroscience Graduate Program, a degree-granting program administered through the Vollum Institute. Half of the 22 mentors listed in this proposal are members of the Vollum faculty;the others are members of the clinical and basic science departments at OHSU, including Pediatrics, Medicine, Biochemistry and Molecular Biology, Physiology and Pharmacology, and the Oregon National Primate Research Center. Support is requested for six predoctoral students per year. The second component is to support the training of four postdoctoral fellows each year, most of whom will be PhD's, although research- oriented MD's will also be considered. Graduate students will receive one or two years of support and are expected to complete the requirements for obtaining a PhD degree within five to six years. Most graduate students go on to further training by doing a postdoctoral fellowship in an academic institution. Postdoctoral fellows are similarly supported for one to two years and most also pursue academic careers. In addition to an extremely high concentration of neuroscience researchers, students and postdoctoral fellows are exposed to a wide range of neuroendocrine topics through journal clubs, seminars, trainee research presentations, and other didactic sessions. Research areas of the mentors include studies of central mechanisms of feeding behavior and weight regulation, cellular signaling, hormone action, neurotransmitter receptors and transporters, neuroendocrine aspects of diabetes, hypothalamic development, and reproduction. PUBLIC HEALTH RELEVANCE: The Multidisciplinary Training Program in Neuroendocrinology is designed to support the academic training of graduate students and postdoctoral fellows in neuroendocrinology research. Advances in this field are critical for new approaches to such diseases as obesity and diabetes and will require a new generation of investigators well-trained in basic neurobiology, neuroanatomy, neurophysiology, and cell signaling. This training grant has a long record of producing such investigators.

The regulation of gene expression by neurotransmitters and neuropeptides begins with the binding of ligands to specific membrane receptors. The overall goal of this Program is to understand the pathways used by receptor-activated second messenger systems to produce changes in gene expression. The first specific aim is to identify and characterize pathways that mediate signal transduction events important for cell growth and differentiation. These studies examine pathways involved in Drosophila embryogenesis and thyroid follicular cell growth. The second goal addresses how the seemingly ubiquitous CAMP second messenger pathway produces distinct patterns of biological effects. These studies focus on the mechanisms involved in the intracellular targeting of protein kinase A and the multifunctonal character of the CAMP-regulated enhancer. The third goal addresses how changes in intracellular calcium modulates expression of specific target genes. Core facilities provide support instrumentation, and reagents that insure the efficient accomplishment of each of these aims. These shared facilities include tissue culture, peptide synthesis and sequencing, antibody production, and administrative cores.

The longterm goals are to determine what nerve fibers provide functional vasomotor innervation to small resistance vessels (arterioles and muscular venules) in the gastrointestinal (GI) and intracerebral microcirculation and to determine under what circumstances nonfunctional innervation can become functional. A new optical method for on-line tracking of outside diameter from in vitro preparations of microvessels whose outside diameters range from 10-100 (mu)m will be used in combination with pharmacological studies, immunohistochemistry, selective denervations and intracellular filling with dyes of identified vasomotor neurons in order to fulfil these goals. These techniques will be used to: (1) determine the immunohistochemical and functional innervation to submucosal arterioles and muscular venules in the normal guinea-pig ileum, colon and in the human colon; (2) identify individual vasomotor neurons and the course of their projections in the GI microcirculation of the guinea-pig; (3) determine changes in the immunohistochemical and functional innervation to GI submucosal microvessels in the guinea-pig after experimental disruptions to their sympathetic and/or sensory inputs and to their intrinsic (enteric) nerve supply; (4) provide quantitative pharmacological characterization of presynaptic and postsynaptic receptors mediating neurotransmitter release, vasoconstriction and vasodilation in arterioles and muscular venules of the GI microcirculation as well as in rat intracerebral arterioles. Results from these studies will provide hitherto unavailable pharmacological and physiological information about the microcirculation along the GI tract in the guinea-pig and human and the nerves that control contractility in this vascular network. Such information is essential in order to design drug, genetic or environmental manipulations that may lead to more precise control of blood pressure.

The cAMP-responsive enhancer (CRE), originally characterized in our lab, serves as the target for the transcription factor CREB. Although CRE sequences have been identified in hundreds of genes, the factors that determine the specificity of CREB binding are still unknown. We have obtained the first insights into this issue by solving the structure of the CREB : CRE complex by x-ray crystallography. These data have allowed us to refine our concept of the essential nature of CREB and the CRE. CREB is activated by phosphorylation at Ser 133, which allows the association of the co-activator CREB binding protein (CBP). Divergent CRE sequences appear to recruit CBP through distinct mechanisms. We have studied one such element, located in the human T cell leukemia virus (HTLV)-1 promoter, and have determined that CREB and CBP binding to this site involves a virally encoded accessory protein called Tax, which overcomes the requirement for CREB phosphorylation. Our specific aims include: Elucidating the mechanisms underlying the specificity of CREB dimerization and binding, defining the essential features of the CRE, and determining what features define membership in the CREB/CREM/ATF-1 gene family. The CREB: CRE complex contains a cavity that may be filled by a hydrated Mg2+ ion, which may explain our earlier observation that Mg2+ was required for CREB binding to DNA. We will quantitate the Mg2+ dependence of the CREB : CRE interaction using fluorescence polarization assays and test the efficacy of other divalent cations. The crystal structure also predicts that a novel Tyr - Glu interaction might contribute importantly to dimerization. We will test this hypothesis by mutational analysis, measuring the ability of the mutated CREB proteins to dimerize and bind to DNA by using fluorescence resonance energy transfer, crosslinking, and fluorescence anisotropy determinations. We will also determine how the virally encoded Tax protein allows CREB to recognize the atypical CREs in the HTLV-1 promoter by characterizing the structure of the Tax: CREB : HTLV-1 DNA complex. Finally, we will characterize the ability of the "CREB binding domain" of CBP to interact with other transcription factors activated or inhibited by Ser/Thr phosphorylation.

The cyclic AMP: CREB pathway has become a paradigm for second messenger-regulated transcriptional signaling in neurons. Phosphorylation of CREB permits binding to the co-activator CBP, which contacts proteins in the transcriptional initiation complex. Our main goal is to take advantage of a genetically-tractable system to resolve the role of CBP in transcriptional signal integration. To this end, we have cloned the Drosophila CBP homologue (dCBP) and have identified two transcription factors, dCREB-2a and cubitus interruptus (ci), that we believe utilize dCBP for gene activation. Already available mutations in the genes encoding dCBP, ci, and components of the PKA system will allow us to utilize a combination of genetic and biochemical approaches to address CBP function in the context of a living organism. We will additionally test the hypothesis that dCBP regulates gene expression not only by binding to general transcription factors, but also by recruiting proteins involved in chromatin remodeling. Our specific aims are to: (1) Define the dCBP mutant phenotype. DCBP mutants have been generated by excising a P- element insertion located in the 5 prime untranslated region of the dCBP gene. To confirm that the lethality of these deletions is due to mutations in the dCBP gene itself, we will attempt to rescue the mutant phenotype by using a sytem that allows us to express dCBP in specific cells under the control of a yeast transactivator. We will also use this system to assess the functions of the various dCBP protein interaction domains. (2) Test whether the PKA pathway in Drosophila activates transcription through dCBP. We will test whether phosphorylated dCREB-2a interacts with dCBP by using in vitro and in vivo binding assays and determine whether dCBP activity is a component of the PKA pathway in Drosophila imaginal disc development. (3) Test whether the transcription factor cubitus interruptus (ci) utilizes dCBP for transcriptional activation. DCBP augments ci-mediated transcription in cell culture and a dCBP mutation partially suppresses a dominant ci mutation in intact flies. We will determine whether dCBP mutations alter the phenotypes of hypomorphic and dominant ci gain-of-function mutations. We will also determine whether dCBP controls the expression patterns of wingless and decapentaplegic, which are believed to be downstream from ci. (4) Determine the role of 'chromatin organizers' in dCBP-mediated transcription. Recent evidence indicates that CBP interacts with a GCN5-like histone acetylase designated PAF. A Drosophila homologue of SIR2, which mediates silencing in yeast, binds to the dCBP 'CREB-binding domain'. We will test whether these putative 'chromatin organizers' are recruited to a promoter through dCBP and determine the functional consequences of these interactions.

CtBP (carboxyl-terminal binding protein) was initially identified through its ability to interact with a motif in the carboxyl-terminus of the adenoviral transforming protein, E1A. In many ways, the functions of CtBP exactly oppose those of the better-characterized E1A binding transcriptional co-activators, CBP and p300. While CBP and p300 have been extensively studied, the functions of CtBP have been relatively unexplored. This proposal describes experiments that should increase the appreciation of CtBP as a transcriptional co-regulator and adapter protein. The first aim is to determine the functional importance of interactions between CtBP and transcriptional regulators identified in a yeast two-hybrid screen. These studies will address, in particular, Teashirt, MyT1, Prox-1, RING3, and RIZ, proteins believed to e important for transcriptional repression and cell cycle regulation during development. Additional components of CTBP complexes will be identified in mass spectrometry. The second aim will be to test the hypothesis that nicotinamide adenine dinucleotide-induced alternations in CtBP structure allow the activities of selected transcriptional repressors to sense cellular redox state and hence, to monitor cellular energy levels. These studies are based on the finding that NAD and NADH dramatically stimulate the interaction of CtBP with its prototypical binding protein, E1A. If this mechanism can be generalized, it would imply that certain developmentally important transcriptional pathways could be regulated by nutrition and other factors that influence cellular metabolism. The third aim is to develop how an amino- terminally extended form of CtBP, termed Ribeye, contributes the unique functional properties of the ribbon synapse. These studies will use capacitance recordings and evanescent field microscopy to ask whether agents that modulate CtBP binding properties affect specific aspects of secretion in bipolar neurons.

DESCRIPTION (provided by applicant): Long term changes in gene expression are believed to contribute importantly to the mechanisms underlying drug addiction. Considerable attention has been devoted to the cAMP second messenger pathway because it is upregulated by opiates and other drugs of abuse. Both tolerance and dependence have been attributed to changes in function of the transcription factor CREB, which mediates cAMP-dependent gene expression. It is currently believed that CREB binds constitutively to a promoter element termed the CRE. This proposal challenges this model and tests a new hypothesis-that chronic exposure to morphine induces CREB binding to some genes but not others. To address this hypothesis the lab has developed a novel approach termed SACO for examining CREB binding to target genes in vivo. This method combines chromatin immunoprecipitation with a modification of Long SAGE (an approach designed for analysis of mixtures of RNA). SACO will be used to identify the entire complement of CREB targets and measure how the selection of these targets is affected by agents, such as morphine, that upregulate the cAMP pathway. Additional studies will address the mechanisms underlying the morphological changes in dendritic processes induced by CREB that occur after treatment with other drugs of abuse, namely cocaine and amphetamine. Specific goals of this project are to identify the entire set of CREB targets in human neuroblastoma cells and determine whether this set is altered by acute exposure to cAMP or chronic exposure to morphine. Previous studies have indicated that CREB regulates the expression of both protein-coding and noncoding transcripts. Many protein-coding transcripts and most noncoding transcripts are missing from conventional microarrays, however. Studies in this proposal will examine both classes of RNAs by developing a custom microarray representing the CREB transcriptome. One specific microRNA, designated miR132, was found to be induced by CREB in PC12 cells and to stimulate changes in dendritic morphology in neurons that are highly reminiscent of those caused by CREB activators. A candidate target for this miRNA has been identified and experiments are designed to elucidate the mechanism of miR132 action. The concept that CREB signaling induces mediators of translational arrest has profound implications for the understanding of drug action.

DESCRIPTION (provided by applicant): Long term changes in gene expression are believed to contribute importantly to the mechanisms underlying drug addiction. Considerable attention has been devoted to the cAMP second messenger pathway because it is upregulated by opiates and other drugs of abuse. Both tolerance and dependence have been attributed to changes in function of the transcription factor CREB, which mediates cAMP-dependent gene expression. It is currently believed that CREB binds constitutively to a promoter element termed the CRE. This proposal challenges this model and tests a new hypothesis-that chronic exposure to morphine induces CREB binding to some genes but not others. To address this hypothesis the lab has developed a novel approach termed SACO for examining CREB binding to target genes in vivo. This method combines chromatin immunoprecipitation with a modification of Long SAGE (an approach designed for analysis of mixtures of RNA). SACO will be used to identify the entire complement of CREB targets and measure how the selection of these targets is affected by agents, such as morphine, that upregulate the cAMP pathway. Additional studies will address the mechanisms underlying the morphological changes in dendritic processes induced by CREB that occur after treatment with other drugs of abuse, namely cocaine and amphetamine. Specific goals of this project are to identify the entire set of CREB targets in human neuroblastoma cells and determine whether this set is altered by acute exposure to cAMP or chronic exposure to morphine. Previous studies have indicated that CREB regulates the expression of both protein-coding and noncoding transcripts. Many protein-coding transcripts and most noncoding transcripts are missing from conventional microarrays, however. Studies in this proposal will examine both classes of RNAs by developing a custom microarray representing the CREB transcriptome. One specific microRNA, designated miR132, was found to be induced by CREB in PC12 cells and to stimulate changes in dendritic morphology in neurons that are highly reminiscent of those caused by CREB activators. A candidate target for this miRNA has been identified and experiments are designed to elucidate the mechanism of miR132 action. The concept that CREB signaling induces mediators of translational arrest has profound implications for the understanding of drug action.

DESCRIPTION (provided by applicant): Rett syndrome is a neurodevelopmental disorder typically due to mutations in the methyl CpG binding protein, MeCP2. An aspect of MeCP2 biology that has received relatively little attention is its regulation at the level of translation. The long isoform of the MeCP2 transcript (the predominant transcript found in brain) contains blocks of sequence that are highly conserved evolutionary, including some regions that have not undergone a single nucleotide change over tens of millions of years. We propose that these sequences are binding sites for a family of microRNAs whose expression is driven by CREB, a critical signal-dependent transcriptional activator. Our overall hypothesis is that CREB directs the expression of a family of microRNAs that regulate MeCP2 translation. The ability of CREB to stimulate microRNA expression is, in turn, under the control of REST, a well-known represser of neural genes. This model provides a mechanism for CREB (as well as the pathways that activate CREB function) to regulate MeCP2 protein levels over the long term. This mechanism is proposed to prevent the deleterious effects of increased MeCP2 protein levels that may occur after certain types of neuronal activity. Our specific aims are to 1) Determine whether five CREB-regulated microRNAs, namely miR132, miR191, miR320, miR219-1, and miR328 inhibit MeCP2 protein expression. Specific 2'-O-methyl oligonucleotides will be used singly and in combination to determine whether they block the microRNA-mediated changes in MeCP2 levels. The effects of the microRNAs on the binding of MeCP2 to the BDNF promoter will also be determined;-2) Characterize the developmental and signaling pathways that regulate expression of the five microRNAs;3) Determine whether the developmental regulation of MeCP2 by miR132 is controlled by REST. There is virutally nothing known about the transcriptional regulation of microRNAs or the factors that control MeCP2 protein expression. The studies described in this proposal address a completely novel mechanism of gene regulation that has implications for the understanding of MeCP2 biology and neural development in general.

DESCRIPTION (provided by applicant): Long term changes in gene expression are believed to contribute importantly to the mechanisms underlying drug addiction. Considerable attention has been devoted to the cAMP second messenger pathway because it is upregulated by opiates and other drugs of abuse. Both tolerance and dependence have been attributed to changes in function of the transcription factor CREB, which mediates cAMP-dependent gene expression. It is currently believed that CREB binds constitutively to a promoter element termed the CRE. This proposal challenges this model and tests a new hypothesis-that chronic exposure to morphine induces CREB binding to some genes but not others. To address this hypothesis the lab has developed a novel approach termed SACO for examining CREB binding to target genes in vivo. This method combines chromatin immunoprecipitation with a modification of Long SAGE (an approach designed for analysis of mixtures of RNA). SACO will be used to identify the entire complement of CREB targets and measure how the selection of these targets is affected by agents, such as morphine, that upregulate the cAMP pathway. Additional studies will address the mechanisms underlying the morphological changes in dendritic processes induced by CREB that occur after treatment with other drugs of abuse, namely cocaine and amphetamine. Specific goals of this project are to identify the entire set of CREB targets in human neuroblastoma cells and determine whether this set is altered by acute exposure to cAMP or chronic exposure to morphine. Previous studies have indicated that CREB regulates the expression of both protein-coding and noncoding transcripts. Many protein-coding transcripts and most noncoding transcripts are missing from conventional microarrays, however. Studies in this proposal will examine both classes of RNAs by developing a custom microarray representing the CREB transcriptome. One specific microRNA, designated miR132, was found to be induced by CREB in PC12 cells and to stimulate changes in dendritic morphology in neurons that are highly reminiscent of those caused by CREB activators. A candidate target for this miRNA has been identified and experiments are designed to elucidate the mechanism of miR132 action. The concept that CREB signaling induces mediators of translational arrest has profound implications for the understanding of drug action.

DESCRIPTION (provided by applicant): This proposal seeks support for hiring a young husband and ife team from the HHMI Janelia Farm Research Campus who will add great strength to the Vollum Institute. Drs. Haining Zhong and Tianyi Mao utilize newly developed optical approaches to solve problems involving neural circuits and signaling, an area complementary to the Vollum's existing strengths. Dr. Zhong recently completed his postdoctoral fellowship with Karel Svoboda and Eric Betzig, co-inventor of photoactivated localization microscopy (PALM). Dr. Mao also did her postdoctoral fellowship in the Svoboda lab. Our intent is to use these PSO funds to help offset the costs of their sophisticated microscopic needs so that the available start up funds can be used for longer term commitment. These two investigators will have extensive intersections with existing Vollum faculty, and there is widespread excitement at the prospect of hiring them. PSO funds are critical for insuring their successful transition to independence. Traditional electrophysiological and biochemical approaches, although powerful in addressing many questions, lack the necessary spatiotemporai resolution for addressing key questions. Dr. Zhong utilizes newly developed imaging methodologies that extend beyond the diffraction limit of conventional light microscopy to explore signaling pathways at the synapse. He will develop methods to combine PALM with electron microscopy to analyze the regulation of PKA localization in synaptic spines and use two-photon fluorescence lifetime imaging (FLIM) to measure direct interaction of PKA with associated proteins.Dr. Mao also uses advanced imaging techniques, together with molecular and physiology tools such as laser scanning photostimulation (LSPS), channelrhodopsin-assisted circuitry mapping (CRACM), and genetically encoded calcium sensors to map local circuits underlying reward-related behaviors in the basal ganglia. She initially will focus on comparing how the inputs from various areas of the cortex and the thalamus differentially influence striatal neuronal activities within and between different compartments. A longer-term goal will be to characterize circuit changes through different stages of behavior {e.g., goal-directed vs. habitual) as animal models of addiction. This set of recruitments would allow the Vollum to further its preeminence in synaptic modulation and signaling, the central focus of its program, and extend into the area of neuronal circuitry. PUBLIC HEALTH RELEVANCE: Synaptic function and plasticity are controlled by complex signal ransduction networks. Although many components of these networks have been identified, the challenge remains to understand how they contribute to synaptic signaling mechanisms. Resolving the authentic synaptic signaling events requires greater spatial and temporal resolution than previously available. Drs. Zhong and Mao utilize advanced imaging approaches to address key questions in synaptic regulation.

DESCRIPTION (provided by applicant): This application addresses Challenge Area 06, Enabling technologies;Specific challenge topic 06-MH-103, "New technologies for neuroscience research" MicroRNAs are believed to contribute to schizophrenia, autism, Tourette's syndrome, mood disorders, and fragile X syndrome. MicroRNA networks in brain are quite complex, however, largely because each microRNA can have hundreds of mRNA targets and each target can, potentially, be regulated by dozens of microRNAs. Research in this area has hit a roadblock because of two major gaps in the understanding of microRNA function. First, what determines which microRNAs bind to a specific mRNA target is very poorly understood. This problem has not been approachable because there has not been an empirical way to determine which particular microRNA interactions occur under specific conditions. Second, the populations of mRNA targets regulated by individual microRNAs are poorly defined. Linking the dysregulation of a microRNA pathway to a particular mRNA target requires this information, which cannot be obtained through bioinformatics alone. We have developed a general method to solve the first problem and will apply it to identify microRNAs that interact with the 3'UTR sequences of mRNAs encoding p250GAP and LimKI, two proteins that have been linked to dendritic spine formation and synaptic plasticity. We have also optimized a complementary method for identifying additional targets of the two microRNAs, miR132 and miR134, that have been proposed to regulate p250GAP and LimKI expression at the synapse. The novel methods outlined in this proposal will have a major impact on the understanding of how microRNAs contribute to synaptic plasticity that could lead to new therapeutic approaches. The approach that we have developed utilizes a fusion gene containing a 3'UTR probe linked to the sequence, MS2, which is recognized by the bacteriophage MS2 binding protein. The MS2-tagged 3'UTR probes will be introduced into primary cortical cells and transgenic mice, and complexes containing the associated microRNAs will be purified by affinity chromatography and identified by multiplex PCR. We have already established the efficacy of this approach and will now characterize the populations of microRNAs interacting with the p250GAP and LimKI transcripts and determine whether these associations are influenced by growth factors and neuronal activity using cell culture and transgenic mouse models. We will then identify new mRNA targets of miR132 and miR134 by immunopurifying RNA-induced silencing complexes (RISC) containing an epitope-tagged version of Ago2. A few targets of these microRNAs have been identified using various prediction algorithms, but most remain to be discovered. These studies will allow us to characterize the full complement of miR132 and miR134 targets and determine whether miR132 and miR134 regulate distinct, overlapping, or functionally related sets of mRNA targets. We predict that the majority of miR132 and miR134 targets will be distinct, but that these targets will converge on pathways involved in mediating synaptic plasticity. PUBLIC HEALTH RELEVANCE: This project describes a novel method for determining which microRNAs bind to the 3'UTR sequences of p250GAP and LimKI, two proteins that have been proposed to regulate the formation of synaptic spines in response to growth factors and neuronal activity. This method is critical for understanding the contribution of microRNA pathways to neurological and psychiatric disease.

DESCRIPTION (provided by applicant): Long term changes in gene expression are believed to contribute importantly to the mechanisms underlying drug addiction. Considerable attention has been devoted to the cAMP second messenger pathway because it is upregulated by opiates and other drugs of abuse. Both tolerance and dependence have been attributed to changes in function of the transcription factor CREB, which mediates cAMP-dependent gene expression. It is currently believed that CREB binds constitutively to a promoter element termed the CRE. This proposal challenges this model and tests a new hypothesis-that chronic exposure to morphine induces CREB binding to some genes but not others. To address this hypothesis the lab has developed a novel approach termed SACO for examining CREB binding to target genes in vivo. This method combines chromatin immunoprecipitation with a modification of Long SAGE (an approach designed for analysis of mixtures of RNA). SACO will be used to identify the entire complement of CREB targets and measure how the selection of these targets is affected by agents, such as morphine, that upregulate the cAMP pathway. Additional studies will address the mechanisms underlying the morphological changes in dendritic processes induced by CREB that occur after treatment with other drugs of abuse, namely cocaine and amphetamine. Specific goals of this project are to identify the entire set of CREB targets in human neuroblastoma cells and determine whether this set is altered by acute exposure to cAMP or chronic exposure to morphine. Previous studies have indicated that CREB regulates the expression of both protein-coding and noncoding transcripts. Many protein-coding transcripts and most noncoding transcripts are missing from conventional microarrays, however. Studies in this proposal will examine both classes of RNAs by developing a custom microarray representing the CREB transcriptome. One specific microRNA, designated miR132, was found to be induced by CREB in PC12 cells and to stimulate changes in dendritic morphology in neurons that are highly reminiscent of those caused by CREB activators. A candidate target for this miRNA has been identified and experiments are designed to elucidate the mechanism of miR132 action. The concept that CREB signaling induces mediators of translational arrest has profound implications for the understanding of drug action.

DESCRIPTION (provided by applicant): The fact that individual cardiac cells can express hundreds of microRNAs, each with potentially hundreds of targets, raises the question of how such a complex mode of regulation can possibly achieve specificity. We hypothesize that this occurs through the ability of individual 3'UTRs to interact with multiple microRNAs simultaneously, such that concurrent binding of two (or more) microRNAs is required for biologically meaningful regulation. Using a method that we developed to identify microRNA-mRNA interactions biochemically, we showed that miR-1 and miR-133a associate simultaneously with the 3'UTR of the cardiac transcription factor, Hand2. This is probably the best example to date in any system of concurrent binding by two microRNAs to a single target. We hypothesize that binding of miR-1 and miR-133a to the Hand2 3'UTR is mutually interdependent, such that both microRNAs must associate with their recognition elements (MREs) to achieve efficient repression. Such a mechanism would increase signaling complexity, generating specificity of targeting and constraining the number of effective mRNA targets for an individual microRNA. This scenario may be characteristic of other microRNA targets, and we will use a new assay developed in our lab to identify additional cardiac mRNAs that are subject to a similar dual mode of regulation. These studies could answer a seminal question in microRNA signaling-how the multitude of microRNAs and predicted targets can achieve specificity. To accomplish our goals, we will determine how miR-1 and miR-133a cooperate to regulate Hand2 expression. Our data indicates that binding of miR-1 and miR-133a is mutually interdependent, which has never before been described. We will elucidate the functional implications of this finding by utilizing a set of novel bidirectional ratiometric sensors. To elucidate the mechanism underlying the interdependency of miR-1 and miR-133a binding, we will target Ago2, a core component of the RNA induced silencing complex (RISC), to the mutated Hand2 miR-1 or miR-133a MREs to determine whether recruitment of Ago2 and associated proteins can rescue effects of the MRE mutations. We will also determine whether the coordinate regulation by miR-1 and miR-133a is shared by other cardiac mRNAs. Bioinformatic algorithms designed to predict microRNA targets are notoriously imprecise in their ability to identify authentic interactions, missing many interactions and falsely predicting others. We developed a novel approach for identifying these targets that employs a dominant negative RISC component to trap microRNA-mRNA intermediates prior to degradation. We will use this approach to identify other cardiac mRNAs that associate with both miR-1 and miR-133a and test whether microRNA binding and function are similarly interdependent. Understanding how combinations of microRNAs affect expression of targets is essential for developing effective microRNA-based therapies. PUBLIC HEALTH RELEVANCE: This project uses several novel approaches to examine the role of micro RNA pathways in regulating cardiac transcription factors with implications for human congenital and acquired heart disease. Characterization of these pathways may identify new therapies for treating cardiovascular disease.

DESCRIPTION (provided by applicant): MicroRNAs contribute to critical brain functions and participate in multiple neurological and psychiatric conditions. Exactly how specific microRNAs relate to these processes remains unclear, however, for several reasons. First, genetic knockouts of brain-specific microRNAs have been difficult to generate. Second, predicting microRNA targets remains extraordinarily challenging. Third, methods for measuring microRNA levels in individual cells and, more importantly, linking these levels to the regulation of particuar targets are lacking. The overall objective of this application is to determine how the CREB-regulated microRNA, miR-132, directs dendritic growth and plasticity. Our specific aims are to: 1) Characterize the role of miR-132 in brain using a conditional knockout. We have already generated a mouse strain containing a floxed miR-132 allele and have shown, using a Cre-expressing retroviral vector, that newborn hippocampal neurons lacking miR-132 display a dramatic decrease in dendritic growth and branching. This was the first example of a neural-specific microRNA knockout, and we will use these mice to examine the role of miR-132 in adult neurogenesis, dendritic growth, and behaviors associated with activity-induced morphological changes, such as spatial learning and fear conditioning. 2) Determine whether heparin-binding (Hb)-EGF and the CDK inhibitor, p21, two miR-132 targets identified in Ago2-immunopurification assays, contribute to miR-132 effects on neuronal survival, morphology, and function. 3) Determine how changes in microRNA expression in individual neurons correlates with expression of specific microRNA targets. Assays of microRNA function typically average responses of large populations of cells. Using a new ratiometric microRNA sensor developed in our laboratory, we observed that individual neurons display large differences in the expression of miR-132. We will now ask whether these differences in miR-132 levels are associated with differences in target mRNA expression using, initially, cultured neurons and, subsequently, in vivo models.

DESCRIPTION (provided by applicant): Considerable progress has been made understanding the effects of microRNAs on neuronal development, but microRNAs are also expressed in mature neurons where they have been proposed to control synaptic plasticity. This is an appealing concept because local regulation of protein translation is essential for this process and microRNAs can regulate dendritic growth and spine formation, properties that underlie plasticity. The idea that microRNAs contribute to axonal growth has received less attention, in part because the existence of polyribosomes in this compartment has been controversial. We argue in this proposal that microRNA regulation of axonal growth does not necessarily require translation to occur within the axon and that palmitoylation enzymes, regulated by microRNAs elsewhere in the neuron, can direct trafficking of key signaling molecules to axonal membranes. This proposal focuses on miR-134, a microRNA initially characterized by virtue of its activity-dependence and ability to regulate dendritic spine size. Using a set of ratiometric microRNA sensors, we found, unexpectedly, that miR-134 activity in mature cortical neurons was limited to inhibitory somatostatin (SST)-producing interneurons, contradicting a widely held view of miR-134 function. The mechanisms responsible for restricting miR-134 expression to SST-interneurons are unknown, and we propose that this is accomplished via cell-specific processing of the miR-134 precursor. We will establish whether the ability of neurons to generate mature, functional miR-134 is due to transcriptional or post-transcriptional mechanisms, identify RNA-binding proteins that interact with the precursor, and test whether these factors affect processing of the miR-134 precursor in a cell- specific manner using miR-Glo, a novel fluorescence assay. Using a new method termed RISC-trap designed to capture microRNA-mRNA interactions prior to mRNA degradation, we discovered that miR-134 targets the palmitoylation enzyme, DHHC9, which controls Ras trafficking to the cell membrane. We will test whether the miR-134 regulation of DHHC9 in inhibitory SST interneurons, and the consequent palmitoylation of Ras, controls Ras trafficking to axonal growth cones and, consequently, axon morphology in SST interneurons. We hypothesize that activity-regulation of miR- 134 negatively influences axon growth and is related to the unique axonal branching pattern characteristic of these cells. The ability of microRNAs such as miR-134 to regulate palmitoylation enzymes, and thereby membrane trafficking of signaling molecules like Ras, could be an important component of synaptic plasticity, particularly in relation to axonal growth.

DESCRIPTION (provided by applicant): Newborn neurons are produced in the hippocampus throughout life and may contribute to specific types of learning and memory. Presumably, this requires their functional integration into pre-existing circuits. Adult hippocampal neurogenesis decreases with age, but this decline can be lessened by physical exercise, growth factors, and certain types of environmental stimuli. We hypothesize that each stage of adult newborn hippocampal neuron maturation is associated with a characteristic transcriptional profile, that newborn neurons are uniquely responsive transcriptionally, and that integration of these cells into functional circuits fundamentally alters their patterns of gene expression. Addressing these hypotheses requires new tools to determine the transcriptional profiles of a small population of cells within the intact brain. We will use retroviruses expressing Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) to label adult newborn hippocampal neurons population specifically at their birth. Only these cells will be able to incorporate the nucleotide analogue, -thiouracil (4-TU), into nascent RNA transcripts. TU-tagged RNAs will be biotinylated, purified, and analyzed by RNASeq. Studies will characterize the transcriptional profiles of adult newborn hippocampal neurons in mice at various stages of maturation and after exposure to exercise, treatment with the phosphodiesterase inhibitor, Rollipam, and activation of TrkB signaling, comparing these profiles to those of pre-existing neurons. Adult newborn neuron transcripts will be analyzed to determine whether treatments that augment neurogenesis and maturation stimulate or repress particular signaling pathways that could underlie their characteristic plasticity. Retrovirally-expressed shRNAs will be utilized to test the contributions of selected gene products to morphological and functional features of newborn neuron maturation. TU-tagging performed using wild type and NMDA receptor- deficient adult newborn hippocampal neurons will allow us to determine how the excitatory synaptic activity that occurs upon functional integration changes their transcriptional program. Changes in specific transcripts will be correlated with morphological maturation, assessed using confocal microscopy, and functional properties, determined electrophysiologically. Selected transcripts and protein products will be monitored by single-cell PCR and immunohistochemistry at defined times after retroviral marking to determine their precise onset of expression. Retrovirally-expressed shRNAs directed against selected transcripts will be utilized to test whether they perturb functional properties of the integrated neurons. This project takes advantage of the combined expertise of the Goodman and Westbrook labs in genomics, transcription, and hippocampal circuitry.

DESCRIPTION (provided by applicant): Abnormalities in microRNA signaling have been associated with multiple neurological and psychiatric diseases. Understanding these associations cannot proceed further, however, without better methods for determining microRNA targets. Because of the incomplete complementarity of microRNAs and their targets, predicting authentic microRNA:mRNA interactions can be difficult. Predictions typically rely on bioinformatics, but it is well known that the concurrence of different algorithms is distressingly low. Thus, empirical approaches for characterizing microRNA:mRNA interactions have received increasing attention. We have developed an approach that utilizes an epitope-tagged, dominant negative version of GW182, a component of the RNA-induced silencing complex (RISC), to purify microRNA:mRNA complexes prior to microRNA-mediated mRNA degradation. This method, which we term RISC-Trap, is considerably more robust than previously reported approaches and allows us to address fundamental problems in microRNA biology, such as, what proportion of mRNA targets undergo degradation versus translational arrest, which targets are direct or indirect, and whether some interactions occur specifically in neural versus non-neural cells. To begin to address these questions, we will use RNASeq to examine complexes containing miR-124, an abundant, neural-specific microRNA with a large data set of previously characterized targets. Our approach should be generally applicable to other systems, however, and should significantly increase understanding of the contributions of microRNAs to neurodevelopmental processes, plasticity, and certain neurological and psychiatric diseases. Our first specific aim is to use the RISC-trap approach to determine which miR-124 targets in HEK293 cells are regulated by mRNA degradation versus translational arrest. These studies will provide a comprehensive picture of the regulatory effects of an important brain microRNA and a straightforward and easily applicable method for identifying microRNA targets in general. Our second aim will be to identify miR-124 targets specific to neuronal cells and determine whether the mode of microRNA action differs. Although many important miR-124 targets are expressed in both neural and non-neural cells, specific neuronal targets are likely to exist as well. We will identify these neural-specific targets by infecting SH-SY5Y neuroblastomas and primary hippocampal neurons with a dnGW182 lentivirus and analyzing the targets as described above. Some targets will be detected only in neuronal cells because their expression is linked to this cell type. Neural-specific microRNA interactions involving mRNAs expressed in both cell types may indicate the involvement of essential RNA binding proteins. We believe that the RISC-trap assay may uncover new aspects of microRNA function.

Project Summary Nicotinamide adenine dinucleotide (NAD+) is the substrate for sirtuins and polyADP-ribose polymerases (PARPs), linking it to gene expression and genomic stability. These enzymatic activities also connect NAD+ to such aging-related conditions as diabetes and muscle weakness. NAD+ alterations also figure prominently in the relationship between calorie restriction (CR) and disease prevention, but the exact nature of this link remains unknown. One model is that CR elevates intracellular NAD+, which then controls activity of sirtuins that regulate fuel utilization and expression of nutrient-responsive genes. PARP activation can also influence cellular energy homeostasis by depleting NAD+, ultimately leading to cell death. It is important to remember that the contribution of NAD+ to sirtuins and PARPs depends entirely upon the free NAD+ concentration and not on redox, that is, the NAD+/NADH ratio. Although measuring the NAD+/NADH ratio is straightforward, monitoring NAD+ is not?our development of an NAD+ biosensor has provided the first glimpses into NAD+ regulation within subcellular compartments of intact cells. This is an important advance because previous studies could not distinguish free from bound NAD+ or monitor differences in NAD+ regulation across compartments. With this novel sensor in hand, we will determine how NAD+ levels in pancreatic beta cells and skeletal muscle are regulated during aging and whether age-related changes can be prevented by CR or augmentation of NAD+ production. Although we have gained significant insights into NAD+ biology using the current sensor, it would be of great value to extend our studies into intact animals. Thus, our first goal is to develop a conditional transgenic mouse line expressing the NAD+ biosensor. We have already made significant progress optimizing our sensor for in vivo dynamic measurements and describe strategies for increasing its sensitivity and dynamic range further. We will then test whether aging decreases, and CR or NAD+ precursor administration increases, NAD+ levels in pancreatic beta cells and skeletal muscle cells by generating tissue- specific sensor strains capable of monitoring NAD+ levels in the nucleus, cytoplasm, and mitochondria. NAD+ depletion is thought to mediate age-related decreases in insulin secretion. Similarly, age-dependent NAD+ decreases have been proposed to underlie muscle weakness and impairments in muscle regenerative capacity. Our biosensor provides an unprecedented opportunity to examine the effect of aging on NAD+ levels, the contribution of NAD+ to age-related disorders, and the efficacy of several proposed approaches to ameliorating these conditions.