Nora Perrone-Bizzozero - US grants

The operation of the nervous system depends on nerve cells communicating with each other through a fine network of cellular extensions known as axons and dendrites. Axons are usually longer extensions and their initial growth constitutes one of the crucial steps in determining the organization of the nervous system. A growth associated protein, known as GAP-43, enriches axons of nerve cells and is associated with axonal growth. While the relationship of this protein to growth and the biochemistry of this protein have been well established, little is known about the mechanisms that regulate the levels of GAP-43 in nerve cells. Previous work has indicated that the amounts of this protein are determined by the levels of messenger RNA, the intermediary molecule that links the activity of the gene with the synthesis of the protein. The investigator plans to study how the expression of GAP-43 is regulated by focusing on messenger RNA regulation. The levels of mRNA can be regulated at the level of synthesis or at the level of degradation. Preliminary work from the investigator's laboratory indicates that the key control point in the expression of GAP-43 may not depend on the level of synthesis but on the regulation of the stability of messenger RNA. The study will determine to what extent synthesis and degradation mechanisms participate in the control of GAP-43 mRNA levels during neuronal differentiation, in vivo and in cultures of isolated cells. These studies will provide fundamental information on the mechanisms that govern GAP-43 levels which will set the basis for future studies on the specific molecular aspects of these regulatory events. The elucidation of the mechanisms that regulate the expression of GAP-43 and other growth-related proteins will not only bring new insights into the question of gene expression control during neuronal differentiation, but will contribute to the understanding of the molecular events that underlie the development of the nervous system.

The growth of axons and the formation of synaptic connections are associated with the expression of the neuronal membrane phosphoprotein, GAP-43. Yet while the relationship of this protein to growth and many of its biochemical properties are well-established, very little is known about how its expression is controlled in the neuron. Our preliminary data suggest that GAP-43 expression is regulated primarily by post-transcriptional mechanisms, that operate on the rate of degradation of the mRNA. Here, we propose to study the nature of the physiological signals and of the molecular mechanisms that regulate GAP43 mRNA levels during neuronal differentiation. Our specific aims are: First, to investigate the nature of the extracellular signals that control GAP-43 mRNA levels during neuronal differentiation. To this end, we will determine the levels of this mRNA upon treatment of neuronal cell cultures and PCI2 cells with a variety of agents that either promote or inhibit neurite outgrowth. Second, to test the hypothesis of post-transcriptional regulation under several experimental conditions. These studies will examine the contribution of the rates of synthesis and degradation of GAP-43 mRNA on the levels of this mRNA by Northern blot analysis, run-on transcription assays, and pulse-chase labeling to determine the half-life of the mRNA . Finally, to study the molecular mechanisms that control the degradation of GAP-43 mRNA. Towards this goal, we will examine the specific RNA sequences and cytoplasmic factors that contribute to the NGF-mediated stabilization of GAP-43 mRNA in PCI2 cells. These studies will be performed using transfection experiments with constructs containing various amounts of coding and non-coding sequences of GAP-43 mRNA, and RNA gel retardation assays. The studies proposed here will provide fundamental information on the extracellular signals and intracellular mechanisms that control the degradation of GAP-43 mRNA during neurite outgrowth. Given that most of the research on the control of expression of neural genes has been focused almost exclusively on the transcriptional aspects of the regulation, our studies on the post-transcriptional regulation of GAP-43 will provide a first and unique opportunity to examine an area of research that has been so far poorly explored. The identification of specific sequences and factors that control the degradation of GAP-43 mRNA, will clearly contribute to establish the role of post-transcriptional mechanisms in controlling gene expression during neuronal differentiation.

DESCRIPTION: (Adapted from the Investigator's Abstract) Learning disabilities are among the most subtle yet most pervasive deficits related to prenatal alcohol exposure in children. These learning deficits, which may not become apparent until a child is school-aged, can occur in the absence of other physical evidence of alcohol-related birth defects. Offspring of rats exposed to moderate levels of alcohol during gestation also show a significant impairment in learning when tested as young adults, validating the use of this animal model in studies of the effect of fetal alcohol exposure (FAE). Initial studies by our integrative research program suggested that these deficits are related to specific alterations in synaptic plasticity mechanisms which correlated with a failure in the establishment of long-term potentiation (LTP). The long-term objectives of this Integrated Research Program Grant (IRPG) are two-fold: 1)to delineate more clearly the molecular and neurochemical alterations of synaptic plasticity mechanisms caused by prenatal alcohol exposure and 2) to explore new treatment strategies to overcome these deficits. The overall hypothesis for the IRPG is that prenatal exposure to moderate levels of ethanol produces multiple defects in the mechanisms underlying glutamate levels of ethanol produces multiple defects in the mechanisms underlying glutamate receptor-dependent LTP in the hippocampus and medial frontal cortex. The specific goal of Project 3 in this IRPG is to define the impact of FAE on the levels and function of proteins involved in synaptic plasticity mechanisms. Based upon preliminary studies, our hypothesis is that protein kinase C (PKC) activity and the levels and phosphorylation of GAP-43 and other important plasticity-associated proteins is altered in specific brain regions of FAE rats. To test this idea, we propose the following Specific Aims: 1)to study PKC activity and the phosphorylation of GAP-43 and other PKC substrate proteins in the hippocampus and medial frontal cortex of FAE rats, both under basal conditions and after electrical stimulation 2)to examine the impact of FAE on activity-dependent changes in GAP-43 phosphorylation and gene expression during behavioral conditioning, 3) to characterize the causes for the deficit in PKC activity in FAE rats and to evaluate its significance in synaptic plasticity mechanisms and 4) to investigate the effects of different pharmacological treatments on PKC activity and GAP-43 phosphorylation in control and FAE rats and 5) to relate these to the behavioral and electrophysicological properties of the animals. The identification of the neurochemical basis for the alterations in synaptic plasticity in the hippocampus and medial frontal cortices of FAE rats will improve our understanding of the effects of prenatal alcohol exposure in the function of these important brain structures. Ultimately, this information will help design better therapeutic strategies to overcome behavioral and cognitive deficits in children affected with Alcohol Related Neurodevelopmental Disorders (ARND).

DESCRIPTION (provided by applicant): Basic biomedical research on alcohol has undergone remarkable growth at the University of New Mexico (UNM) in the past ten years. Currently, five Department of Neurosciences faculty are principal investigators on one or more extramural grants for alcohol research. Three other department faculty have NIAAA research grants pending. Having reached a critical mass of funded investigators with a strong record of multidisciplinary research collaboration, the Alcohol Research Group is striving to take the next step by establishing mechanisms to support an Alcohol Research Training in Neurosciences (ARTN) program. If this NIAAA-sponsored training grant application is funded, the ARTN Program would use these funds to provide three years of support for four additional graduate students recruited into UNM's Biomedical Sciences Graduate Program. First year ARTN program trainees would complete one year of required and selected coursework and conduct three rotations in laboratories of ARTN program core or affiliated faculty. Second-year trainees would take upper level neuroscience graduate courses and prepare a dissertation proposal. Third-year trainees would begin their dissertation research with a core faculty member and would apply for an F-31 award to support the remainder of their predoctoral training experience. Drs. Perrone-Bizzozero (as PI), Allan, Caldwell, Valenzuela and Savage, all Department of Neurosciences faculty members, will comprise the ARTN program core faculty steering committee. Eleven other faculty at UNM will serve as affiliated faculty. The Department of Neurosciences has a long history of effective teaching and research interactions by a group of faculty with diverse backgrounds in research training. During the past four years, the department has hired three new junior faculty, renovated most of its laboratory space, established a two-year cycle of upper level neuroscience graduate courses, and doubled its research revenues. In support of this training grant application, the department will commit resources to cost-share faculty salary support, student tuition, fees and health insurance, stipend supplements, recruiting expenses and expenses associated with an annual retreat. In addition, the department and the School of Medicine will provide ARTN program trainees access to its core facilities, which include state-of-the-art equipment for use in the study of molecular neurobiology, functional genomics and proteomics, neuroimaging, neuroanatomy, electrophysiology, and behavioral neuroscience. The ARTN program will encourage its trainees to take full advantage of the diversity in the neuroscience community at UNM to undertake multidisciplinary research projects that will increase their potential to become successful alcohol research investigators in the future.

[unreadable] DESCRIPTION (provided by applicant): Besides transcription, post-transcriptional mechanisms such as RNA processing, mRNA stability and local translation are also important for controlling the expression of many nervous system-specific genes. For a large number of neuronal genes, expression levels are controlled by changes in mRNA stability. These processes are regulated by specific interactions between RNA-binding proteins and instability-conferring sequences in the mRNAs. One of the best characterized post-transcriptionally regulated genes in neurons is that for GAP-43. Work done under our previous grants demonstrated that GAP-43 gene expression is regulated by selective changes in the stability of its mRNA and that this process depends on the interaction of a highly conserved regulatory element in the 3'untranslated region (3'UTR) of the mRNA with the neuronal-specific RNA-binding protein HuD. Not only is HuD capable of stabilizing GAP-43 mRNA in developing neurons in culture, but also overexpression of this protein in transgenic mice increases GAP-43 gene expression in the hippocampus and neocortex. We have recently found that the pro-destabilizing RNA-binding protein KSRP also binds to the GAP-43 mRNA, suggesting that this protein may be responsible for the fast degradation of the GAP-43 mRNA observed in mature dentate granule cells. Based upon our preliminary studies, we propose that the stability of GAP-43 and other post- transcriptionally-regulated neuronal genes is controlled by the interplay of pro-stabilization factors such as HuD and pro-degradation factors such as KSRP. To test this hypothesis, we plan to perform the studies under the following two specific aims: Aim 1. To explore the mechanism by which HuD and KSRP control the stability of neuronal mRNAs. Aim 2. To define the function of HuD and KSRP in the post-transcriptional control of neuronal gene expression in vivo during developmental and adult plasticity. Although the aims are focused on GAP-43, our studies will include other targets of HuD such as neuroserpin and tau. These mRNAs were chosen because they are axonally-localized, developmentally- regulated, up-regulated in response to injury and thus, likely to be controlled by similar mechanisms. The proposed studies will characterize the mechanisms of control of GAP-43 and other post- transcriptionally-regulated neuronal mRNAs. Given the role of these proteins in nervous system development, synaptic plasticity, and nerve regeneration, the elucidation of regulatory mechanisms controlling their mRNAs has a broad range of potential applications, from the treatment of neurodevelopmental disorders to the recovery from brain trauma and spinal cord injury. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in the establishment of addictive behaviors has received very little attention. Amongst these, mRNA stability is estimated to control about 10% of all human genes. One of the most studied cis-acting elements is the AU-rich element (ARE) present in the 3'untranslated region (3'UTR) of several unstable mRNAs. These sequences are targets of RNA-binding proteins such as the neuronal-specific and plasticity-associated RNA-binding protein HuD. We have recently identified three new binding motifs for HuD;two of which are U-rich and thus termed HuD-AREs. Using bioinformatics analyses, we found that there is an overrepresentation of HuD-ARE motifs in the 3'UTRs of mRNAs that are associated with mechanisms of addiction, including BDNF and GAP-43. ARE sequences are not only targets of RNA-binding proteins but also they were recently shown to be targets of specific microRNAs. In the case of BDNF and GAP-43, the same ARE motifs that are recognized by HuD also contain target sequences for miR-495. Given that the overlap between sequences recognized by RNA-binding proteins and microRNAs and the overrepresentation of these sites in the 3'UTRs of addiction-related genes (ARGs), we propose that RNA-binding proteins such as HuD could compete with microRNAs such as miR-495 for the post-transcriptional control of genes associated with substance abuse. To test this hypothesis we propose: 1) To use bioinformatics tools to mine data from public domain databases and from our own microarray studies to investigate the prevalence and co-localization of HuD-AREs and microRNA sites in the 3'UTR of genes associated with drug addiction and 2) To experimentally test the functional competition between HuD and miR-495 on the stability of the BDNF and GAP-43 mRNAs in vitro and to assess the significance of these interactions in vivo in an animal model of cocaine-self-administration. The experiments described in this R03 grant are the first steps to address the possible interactions of microRNAs and RNA-binding proteins in the control of addiction-related gene expression, a novel and important regulatory process. PUBLIC HEALTH RELEVANCE: Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in the establishment of addictive behaviors has received very little attention. Our preliminary results indicate that 1) genes associated with drug addiction have an unusual high number of post-transcriptional regulatory elements, 2) these elements are recognized by two key post-transcriptional regulators, microRNAs and RNA-binding proteins and 3) these molecules could compete to control gene expression. The experiments described in this R03 grant are the first steps to address the possible interactions of microRNAs and RNA-binding proteins in the control of addiction-related gene expression, a novel and important regulatory process.

DESCRIPTION (provided by applicant): Controlling motivation for cocaine is the goal of long-term treatment success of cocaine addiction, which may require reversal of drug-induced changes in gene expression. Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in addictive behaviors has received little attention. RNA binding proteins and microRNAs serve as master switches controlling gene expression, with mRNA stability estimated to control about 20% of brain-expressed genes. Our research suggests that the RNA binding protein HuD and the microRNA miR-495 play opposite roles in the control of addiction-related gene expression and behavior: 1) They are predicted to bind the same GU-rich sequence in mRNAs; 2) Their binding sites are overrepresented in transcripts from an addiction-related gene (ARG) database; 3) They show differential regulation by cocaine in addiction-related brain regions, with miR-495 being downregulated and HuD upregulated; 3) In vitro manipulations of these molecules result in opposite effects on the expression of two of their target genes, BDNF and arc; 5) Most importantly, in vivo manipulations of these molecules show contrasting effects on motivation for cocaine. Based upon these results, we hypothesize that HuD and miR- 495 play a role in drug abuse by post-transcriptionally competing for binding to the same sequences and controlling the expression of ARGs in opposing directions. To test this hypothesis, we will: 1) test the functional competition between HuD and miR-495 for a) specific mRNA binding sites and the control of ARG gene expression, and b) cocaine conditioned place preference in mice overexpressing HuD in forebrain neurons; 2) determine the effects of viral-mediated gene transfer of miR- 495 and 3) HuD to the nucleus accumbens shell of rats using the following 3 models of motivation for cocaine: i) break point on a progressive ratio schedule of cocaine reinforcement, ii) extinction of cocaine-seeking behavior, and iii) reinstatement of extinguished cocaine-seeking behavior; 4) examine changes in the levels of miR-495 and HuD and selected target genes, including BDNF and arc in rats which have been manipulated to express varying degrees of motivation for cocaine. The proposed work synergistically combines the expertise of Dr. Perrone-Bizzozero in mRNA stability, HuD function, and target analyses and Dr. Neisewander in animal models of addiction and the neurocircuitry involved. The outcome of this work will provide new knowledge about the post-transcriptional mechanisms regulating addiction-related gene expression, an exciting new area of neuroscience research. A better understanding of these regulatory mechanisms is a pre-requisite for the application of these new tools in addiction research and ultimately in the treatment of this disorder. PUBLIC HEALTH RELEVANCE: Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in the establishment of addictive behaviors has received very little attention. Therefore, characterizing the competing roles of mR-495 and HuD in the control of addiction-related genes will unveil a new mechanism underlying the maladaptive changes in synaptic plasticity during drug addiction and provide potential new targets for intervention.

DESCRIPTION (provided by applicant): Controlling motivation for cocaine is critical for the successful long-term treatment of cocaine addiction, which may require reversal of drug-induced changes in gene expression. Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in drug abuse has received little attention. RNA binding proteins and microRNAs serve as master switches controlling gene expression, with mRNA stability estimated to control about 20% of brain-expressed genes. Our research suggests that the RNA-binding protein HuD and the microRNA miR-495 play opposite roles in the control of addiction-related gene expression and behavior: 1) they are predicted to bind the same GU-rich sequence in mRNAs; 2) their binding sites are overrepresented in transcripts from an addiction-related gene (ARG) database; 3) they show differential regulation by cocaine, with miR-495 downregulated and HuD upregulated in the nucleus accumbens; 4) in vitro manipulations of these molecules result in opposite effects on the expression of two of their target genes, BDNF and arc; and 5) most importantly, in vivo manipulations of these molecules show contrasting effects on the motivation for cocaine. Based upon these results, we hypothesize that HuD and miR-495 compete for binding to the same sequences to control the expression of ARGs and motivation for drug in opposing directions. To test this hypothesis, in the CEBRA application we propose the following two specific aims: Aim 1: To identify mRNA targets of miR-495 and HuD and to map their specific binding sites using a novel UV- crosslinking and RNA immunoprecipitation and high throughput sequencing (CLIP-seq) protocol on the new Ion Torrent PGMTM semiconductor sequencers. These studies will use in vitro and in vivo validation methods as well as unique bioinformatics tools and novel competition studies to define how HuD and miR-495 are able to regulate the same targets in an opposite manner. Aim 2: To test the functional consequences of the competitive interactions of HuD vs. miR495 with their targets in vivo both on the motivation of animals for cocaine (Aim 2a) and the post-transcriptional control of shared target mRNAs (Aim 2b). These studies will use HuD overexpressor mice and stereotaxic injections of lentiviral constructs to increase or decrease miR-495 levels in the nucleus accumbens shell. We consider our application to be appropriate for the CEBRA initiative as the cutting-edge sequencing method proposed in Aim 1 to simultaneously identify the common targets and binding sites of HuD and miR-495 is highly innovative and potentially of high risk. Our application is also markedly significant and potentialy of high-impact as these studies will uncover novel molecular targets and unexplored molecular mechanisms involving the competition between RNA-binding proteins and microRNAs in drug addiction. A better understanding of these regulatory mechanisms is a pre-requisite for the application of these new tools in addiction research and ultimately in the treatment of this disorder.

Increasing evidence indicates that the majority of children adversely affected by prenatal alcohol exposure (PAE) do not present with the physical features characteristic of Fetal Alcohol Syndrome (FAS). In the absence of these features, a confirmation of maternal drinking is required both for diagnosing Fetal Alcohol Spectrum Disorder (FASD) and initiating interventions. However, maternal self-report of drinking can be unreliable and conventional ethanol biomarkers are not sensitive enough for a diagnosis of drinking in many pregnant women, especially those who are moderate drinkers. The advent of high-throughput screening technologies for assessing the expression of genes, proteins and other metabolites in biological fluids has created new opportunities for developing more sensitive and specific biomarkers. MicroRNAs (miRNAs) are particularly appealing in this respect as they are very stable in plasma and serum and have shown great promise as biomarkers of a variety of disease conditions from cancer to myocardial infarction. Thus far, the utility of miRNAs as biomarkers for diagnosis of FASD has not been explored in detail. Our preliminary data suggest that alterations in the levels of at least two serum miRNAs could be used to reliably detect alcohol consumption during pregnancy. Furthermore, given that one of these miRNAs is expressed in the maternal reproductive system and the other in the fetal brain, these miRNAs could also serve as biomarkers of alcohol-induced tissue damage. The establishment of a panel of miRNAs as a clinically useful diagnostic tool will require replication of these findings in a larger clinical sample as well as a systematic evaluation of various factors that could affect the levels of circulating miRNAs during pregnancy. Thus, the goals of this translational research proposal are twofold. Aim 1: To identify a panel of serum miRNAs that predicts maternal alcohol consumption in pregnant women with higher specificity and sensitivity than conventional biomarkers, even in the presence of co-exposures with other drugs such as nicotine and opioids. The tissue origin and targets of these miRNAs will also be evaluated to assess potential mechanisms of alcohol-induced teratogenicity. In Aim 2. we will use a rodent model of prenatal alcohol exposure to further examine the specificity of these novel biomarkers in relationship to co-exposure with either methadone (Aim 2A) or nicotine (Aim 2B), the window of detectability after the last drinking episode (Aim 2C) and the impact of ethanol dose (Aim 2D). It is expected that this highly translational project will advance the field of FASD by providing novel biomarkers for an earlier and more accurate diagnosis of affected children.

? DESCRIPTION (provided by applicant): Extension and maturation of axons and dendrites are essential developmental steps that allow the nervous system to function. This development requires precise regulation of gene expression, with coordinated activation and inactivation of gene expression programs associated with growth, maturation, and function of neurons. Growth of neuronal processes or `neurites' must be precisely timed and regulated to generate functional neural circuits. Regulation of gene expression extends beyond transcribing DNA into mRNAs, and it has become increasingly clear that much regulation occurs post-transcriptionally in neurons. Regulatory steps include splicing, subcellular localization, and translational control of mRNAs. Stability of mRNAs plays a critical role in gene expression by modifying the amount of an individual mRNA available as a template for generating new protein over time. Stabilization and destabilization of mRNAs within growing neurites also impacts where new proteins are produced. Despite increased recognition of importance of this mechanism, we have little understanding of how neuronal mRNA stability is regulated. Recent work from the PI's and Co- PI's labs have uncovered a mechanism for modulation of mRNA stability in neurons. The RNA binding proteins KSRP and HuD compete for binding to GAP-43 mRNA. Both these RNA binding proteins are known to have multiple functions, and our data suggest that KSRP and HuD have antagonistic functions. For GAP-43 mRNA, KSRP binding destabilizes the transcript while HuD binding stabilizes the transcript. By initial CLIP analyses, KSRP and HuD can bind to overlapping cohorts of mRNAs and cytoplasmic KSRP appears to provide a governor to limit neurite length by destabilizing mRNAs. These data have led us to hypothesize that competitive interactions of HuD and KSRP with specific cohorts of ARE-containing mRNAs control the temporal and spatial pattern of neuronal protein expression during the initiation and termination of neurite outgrowth through changes in mRNA stability. We will test this hypothesis with three specific aims: 1) Does KSRP destabilize neuronal mRNA cohorts? 2) Do KSRP or HuD interactions alter stability of localized mRNAs? 3) Do KSRP and HuD compete for binding to a shared cohort of mRNAs with antagonistic functions? Completion of these aims will fill a gap in knowledge on mechanisms of neuronal mRNA stability and its contributions to brain development.