Xinyu Zhao - US grants

DESCRIPTION (provided by applicant): "Stem cell research offers enormous potential for treating a host of congenital, developmental, psychiatric or degenerative diseases for which there are no cures (NIH PA-04-101)." However, a thorough understanding of the molecular mechanisms that regulate adult neural stem cell (NSC) proliferation and differentiation is a pre-requisite for their therapeutic applications. Advances in the stem cell field have expanded our knowledge of transcriptional regulations in adult NSC functions. Recently, microRNA (miRNA) pathway has been shown to play important roles in the proliferation and differentiation of stem cells, indicating that translational regulation of adult NSCs, though less understood, is equally, if not more important in adult NSC function. The long-term goal of this project is to understand the role of translational regulation in adult neurogenesis. Fragile X mental retardation protein (FMRP), the protein that is lost in Fragile X syndrome, is a selective RNA-binding protein that forms a messenger ribonucleoprotein (mRNP) complex associating with polyribosomes. FMRP regulates protein translation and the loss of FMRP leads to abnormal translation of selective mRNAs, delayed maturation of dendritic spines, and abnormal microtubule stability in neurons. We found that FMRP is also highly expressed in neural stem/progenitor cells (NSPCs) derived from adult brains. Our recent studies and those of other groups have demonstrated the biochemical and genetic interactions between FMRP and the components of the miRNA pathway, including Dicer and Argonaute proteins, suggesting that translational regulation of specific mRNAs in adult NSCs could be achieved by collaborative actions between miRNA pathway and FMRP. We have isolated NSPCs from both wild type (wildtype) and Fmr1 knockout (KO) adult mice, and found that the loss of FMRP affects both proliferation and fate- specification of adult NSPCs in vitro. We hypothesize that the proliferation and differentiation of adult NSCs is regulated at the translational level by miRNA pathway and RNA binding protein, FMRP. Therefore, we propose: first to determine whether the translation of NSPC-specific mRNAs is regulated by miRNAs (Aim 1);then to determine whether translational regulation of adult NSCs is critical for NSC function in vivo (Aim 2);and finally to determine whether FMRP and miRNA pathway collaboratively regulate the proliferation and differentiation of adult NSCs (Aim 3). Our proposed work will be carried out by combining the efforts of the PI, Dr. Zhao, who has expertise in adult neurogenesis, and the Co-Pi, Dr. Jin, who has extensive experience in translational regulation and miRNA. The outcome of this work would add a new dimension to our knowledge of NSC regulation in the adult brain. It is our premise that a better understanding of these regulatory mechanisms is a pre-requisite for the therapeutic application of adult NSCs for human diseases.

DESCRIPTION (provided by applicant): Small noncoding RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), repeat-associated small interfering RNAs and piwi-associated RNAs, 21 to 30 nucleotides in length, could shape diverse cellular pathways. MiRNAs are sequence-specific regulators of post-transcriptional gene expression, and are believed to regulate the expression of thousands of target mRNAs, with each mRNA targeted by multiple miRNAs. Although it has been estimated that miRNAs could regulate as many as one-third of human genes, high-throughput sequencing data indicates that only a portion of small RNAs in the genome have been identified, and the identification of the remaining small RNAs is critical for understanding the small-RNA- mediated gene regulation. Therefore investigating the role of small RNA in depression is likely to be a fruitful, albeit currently understudied, area of research. Methyl-CpG binding protein 1 (MBD1) is a central player of epigenetic mechanism that is critical for gene expression regulation, chromatin structure establishment, and genomic stability maintenance. We have found that Mbd1-/- mice exhibit reduced adult neurogenesis, impaired learning, increased brain serotonin activity, increased anxiety and depression, and deficits in hypothalamic-pituitary-adrenal (HPA) axis. Given the important roles of adult neurogenesis, serotonin, and HPA axis in depression, MBD1 could play important roles in the pathogenesis of depression. Using our established technologies for small RNA analyses, including miRNA array, quantitative RT-PCR of miRNAs and high-throughput sequencing of small RNA libraries, we have found that the loss of MBD1 alters the expression of specific miRNAs in both adult neural stem/progenitor cells (aNSCs) and medial frontal cortex (MFC). In addition, we have identified several known miRNAs critical for the proliferation and differentiation in aNSCs as well as more than two thousand previously unidentified small RNAs in wildtype aNSCs. Functional crosstalk between epigenetic modulation and small RNA pathway has been clearly demonstrated in other species, and our preliminary data prompts us to investigate whether such crosstalk is the key regulatory mechanism for complex functions and related disorders in mammalian brains. These two linked- R01 applications will test the hypothesis that the expression of small RNAs modulated by DNA methylation- mediated epigenetic mechanism plays critical roles in the etiology of depression disorder. We propose a multidisciplinary approach that will apply our combined expertise to first identify the small RNAs and their mRNA targets in aNSCs that are critical for adult neurogenesis (Aim 1);then test the hypothesis that MBD1 regulates the expression of small RNAs that modulate the expression of the key factors of serotonin and HPA axis (Aim 2);and finally determine whether small RNA-mediated gene regulation is involved in the effect of antidepressants (Aim 3). The success of the proposed work should advance our understanding of the role of small RNAs in the etiology of depression, and provide new targets for further research and therapeutic development.

An important mechanism for the control of gene expression in neurons is DNA methylation-mediated epigenetic regulation. The protein MeCP2 binds to methylated DNA and regulates the transcription of genes that are important for normal neurodevelopment such as BDNF. Alterations in the expression and/or function of MeCP2 cause Rett syndrome, a genetic mental retardation syndrome. However, little is known about the role of this protein in mental retardation syndromes caused by environmental insults. Given that developmental exposure to alcohol is a leading environmental cause of mental retardation, it is important to study the role of MeCP2 in fetal alcohol spectrum disorder. We recently reported that acute ethanol exposure potently stimulates network activity in the CAS region of the rat neonatal hippocampus (J Neurochem. 94:1500-11, 2005) and the literature indicates that this effect could modulate MeCP2 levels and/or activity. We therefore performed preliminary in vitro experiments with hippocampal slices and found that acute exposure to ethanol rapidly increases MeCP2 expression levels and decreases the phospho-MeCP2/total MeCP2 ratio. Based on these in vitro preliminary data, we hypothesize that acute EtOH exposure during the third trimester equivalent will produce the same effects in vivo, increasing MeCP2 binding to its DNA targets. In aim #1, we will assess whether ethanol affects total MeCP2 or phospho-MeCP2 levels in vivo. Rat dams and their respective neonatal offspring will be exposed to ethanol in inhalation chambers. We will then investigate the effect of ethanol on total MeCP2 and phospho-MeCP2 expression levels using Western immunoblotting and immunohistochemical techniques. In aim #2, we will investigate whether in vivo ethanol exposure affects MeCP2 binding to its DNA targets. We will initially assess whether ethanol affects MeCP2 binding to the BDNF promoter using a chromatin immunoprecipitation assay (ChIP). We will also examine whether EtOH affects MeCP2 binding to other target DNAs using a state-of-art ChlP-microarray (ChlP-onchip) assay. The results of these pilot studies will form the basis for future detailed mechanistic studies of ethanol's action on MeCP2 expression and/or function. Lay Description: This pilot project will investigate the role of a protein that has been linked to genetic mental retardation syndromes in the mechanism of action of alcohol during development;developmental alcohol exposure is the leading environmental cause of mental retardation in the U.S.A..

DESCRIPTION (provided by applicant): Neurogenesis is defined as generation and maturation of new neurons. Postnatal neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. Deciphering these regulatory mechanisms represents a step towards understanding the development and plasticity of postnatal mammalian brains, and realizing the therapeutic potential of neural stem/progenitor cells (NSCs). Epigenetic mechanisms, including DNA methylation and histone modification, are known to play significant roles in the modulation of stem cell proliferation and differentiation. Methyl-CpG binding proteins, including MBD1 and MeCP2, are central players in epigenetic regulation, and can translate DNA methylation into gene expression changes. MBD1 deficiency has been reported in sporadic human cancers, consistent with its role in cellular growth control. Despite its ubiquitous expression pattern, we found that MBD1 deficiency in mice results largely in brain-associated phenotypes during the postnatal period, including impaired adult neurogenesis and related behavioral deficits such as defective hippocampus-dependent learning and susceptibility to anxiety and depression. Recently, MBD1 mutations were found in a subset of autistic patients and were correlated with more severe phenotypes. However, the precise role of MBD1 in postnatal neuronal development and molecular pathway mediating its effect is not fully clear. During the past three-year funding period, we have discovered that MBD1 regulates the expression of a number of miRNAs and some of these miRNAs exhibit an important regulatory role in neurogenesis. For example, miR-184 promotes proliferation and represses differentiation of adult NSCs by repressing the expression of Numblike (Nbl), a regulator of Notch signaling. The complete picture of this regulatory network is still lacking. In addition to its role in NSC proliferation and neuronal differentiation, we discovered that MBD1 also had important roles in maturation of new neurons. Indeed, some of MBD1-regulated miRNAs have been implicated in neuronal maturation. Taken together, these breakthrough discoveries serve as the basis of this proposal which is aimed towards a better understanding of the epigenetic mechanisms controlling multiple stages of postnatal neurogenesis. We will test the hypothesis that MBD1 regulation of miRNAs and their subsequent downstream targets is critical for postnatal neurogenesis. Therefore we propose to determine how MBD1-regulated miRNAs govern the proliferation and differentiation of aNSCs (Aim 1), to determine whether and how MBD1-regulated miRNAs modulate the maturation of new neurons (Aim 2), and to explore the mechanism underlying MBD1 regulation of small RNAs (Aim 3). The results will provide novel insights into the epigenetic mechanisms governing postnatal neurogenesis.

DESCRIPTION (provided by applicant): Neural stem/progenitor cells (NSCs) in postnatal and adult brains may play a major role in both normal brain functions, such as learning and memory, as well as the brain's response to injury and disease. Understanding NSCs and adult neurogenesis holds the key to therapeutic applications of not only NSCs but also many other types of stem cells. In addition, NSCs make an excellent model system for studying neurodevelopment and related disorders with postnatal etiology, such as autism spectrum disorders. Our ultimate goal is to reveal mechanisms regulating NSCs and uncover new therapeutic targets for treating mental disorders. Neurogenesis is defined as generation and maturation of new neurons. Although the specific purpose of adult neurogenesis is not entirely clear, work from ours and others' have provided evidence supporting its important roles in adult neuroplasticity and hippocampus-dependent learning. Both adult hippocampal neurogenesis and learning are altered in a number of pathological conditions. However how they contribute to human intellectual disability, a deficiency in learning and memory, is still unclear. Fragile X mental retardation protein (FMRP) is a neuron- enriched selective RNA-binding protein associated with polyribosomes and it is known to regulate protein translation. Functional loss of FMRP leads to Fragile X syndrome, most common monogenetic form of inherited intellectual disability and autism, with learning disability. Despite extensive effort, the mechanisms underlying the learning deficits in Fragile X syndrome remain unclear. During the current funding period, we have found that FMRP is highly expressed in adult NSCs and regulates the translational of several proteins involved in NSC fate specification. Using null and conditional inducible mouse genetics, we have demonstrated that FMRP deficiency impairs both hippocampal neurogenesis and hippocampus-dependent learning (PloS Genet 2010; Nat Med 2011). In addition, manipulation of FMRP-regulated pathways, such as treatment by a Gsk3¿ inhibitor, rescues both neurogenesis and learning deficits of FMRP null mice (Hum Mol Genet 2011). These data provide direct evidence for the role of FMRP in postnatal neurogenesis and learning and present us a unique opportunity for understanding the specific roles and functional impact of RNA binding protein- mediated translational regulation in postnatal/adult neurogenesis and learning disabilities. Built upon these exciting data and our strength, the current proposal aims to test the hypothesis that FMRP regulates multiple stages of adult neurogenesis and its deficiency disrupts the development and impairs the function of new neurons. To test this hypothesis, we will define the roles of FMRP in stem and progenitor cells in the adult DG (Aim 1); determine specific function of FMRP in new DG neurons (Aim 2); and determine the mechanism underlying FMRP regulation of adult neurogenesis (Aim 3). These data will provide critical information regarding not only the function of FMRP in neurogenesis but also the function of stage-specific neurogenesis in learning and memory. PUBLIC HEALTH RELEVANCE: Characterizing the role of FMRP family of RNA binding proteins in regulating postnatal neurogenesis and learning will provide insights into the mechanisms underlying mammalian neuronal development and learning, which could have significant implication in understanding and treating mental disorders including not only Fragile X syndrome but also autism, depression, and other learning deficits.

? DESCRIPTION (provided by applicant): Methyl-CpG binding proteins (MBPs) are central players of epigenetic regulation by translating DNA methylation into phenotypic changes. Mutations in MeCP2 result in Rett Syndrome and certain autism- spectrum disorders. Alterations in MBD1 are found in a subset of autistic patients and intellectual disabilities. We have demonstrated that MBD1 null mice display learning impairment and autism-like behavioral deficits. Thus, both MeCP2 and MBD1 are autism-associated genes (SFARI). We have shown that both MeCP2 and MBD1 are expressed in immature stem cells and new neurons in the postnatal brains and regulate neurogenesis and neuronal maturation. We found that such regulations are at least in part through controlling the expression of noncoding microRNAs. Although many studies have focused on MeCP2 target identification, MeCP2 targets in specific neural cell types remain largely unknown. In addition, little has been done to identify the targets of MBD1 in the brain. Literatures have suggested that MBD1 and MeCP2 may have different sets of targets, however both protein exhibit similar heterochromatin binding and share at least several noncoding gene targets. Functional relationship between these two proteins during neuronal development has not been explored. We observed that mice lacking both MBD1 and MeCP2 die much earlier than single mutant mice, at postnatal day 21, which coincides with the postnatal synaptogenesis and neuronal maturation period as well as the dynamic changes in DNA methylation in neurons. Therefore a lack of both MBD1 and MeCP2 during this period is extremely detrimental but the reason is unknown. In addition, despite the apparent functional compensations between these two proteins, questions remained why MBD1 cannot fully compensate for MeCP2 deficiency and which genomic binding targets might be responsible for this lack of compensation. A direct comparison of genome-wide MBD1 and MeCP2 targets will yield valuable information for understanding MBD regulation of neurodevelopment and disorders. The PI and collaborators are uniquely suited to fill these knowledge gaps. In this exploratory R21 proposal, we aim to take advantage of a MeCP2-FLAG line created by collaborator Dr Chang and our newly created MBD1-FLAG mouse line to directly compare the targets between MBD1 and MeCP2 in developing brain. We aim to test the hypothesis that MBD1 and MeCP2 have both common and unique targets which underlie their specific functions during postnatal development. We will first identify genomic binding profiles of these proteins. We will then determine whether the genomic binding specificities have impact on gene expression and neuronal maturation. The proposed study is built upon the complimentary expertise of the PI and collaborators. The results of this study will provide much needed understanding in biological functions of MBP and its significance in gene regulation. Since both MBPs are important for autism and neurodevelopmental disorders, the study will unveil the novel genes and pathways involved in developmental disorders.

Fragile X mental retardation protein (FMRP), encoded by the FMR1 gene on the X chromosome, is an RNA-binding protein that regulates the expression of hundreds of genes and plays a key role in brain development and function. The 5' untranslated region of the human FMR1 gene contains various numbers of CGG trinucleotide repeats. The modal number of repeats in the population is 30, whereas expansion of these CGG repeats above 200 leads to fragile X syndrome (FXS). CGG repeats between 55 and 200 are classified as ?premutation?, since these individuals are at increased risk for expansion into the FXS range. There is considerable polymorphism in the number of CGG repeats below 55, and controversy over the true definition of ?normal?. Indeed, the significance of either ?Gray Zone? (variously defined as 45-54 or 41-54) or ?Low Zone? (?23) CGG repeats, although much more prevalent than FXS and the premutation in the general population, are largely unexplored apart from the clinical literature. We have preliminary evidence to suggest that older adults with Low Zone repeats may be at increased risk of difficulties in cognitive and motor functioning as well as for having a child with a disability. We also found, in unpublished preliminary analyses, that Gray Zone expansions are associated with increased psychiatric and other health problems. Here we propose to validate and further explore these intriguing findings in a population-based cohort, a unique opportunity afforded by the Personalized Medicine Research Program (PMRP) of the Marshfield Clinic. In addition, we have established human embryonic stem cell (ESC) lines with defined numbers of CGG repeats. These lines, together with induced pluripotent stem cells (iPSC) derived from PMRP individuals with Low and Gray Zone CGGs, as well as controls, will provide an opportunity to assess whether having CGG repeats at either end of the distribution that currently is considered normal impairs expression, localization, and functions of FMRP. Building on our novel observations and unique collaborative opportunities, our two specific aims are to test the hypotheses that Low or Gray Zone CGG repeats in FMR1 are associated at the organismal level with compromised health and an increased risk of having a child with a disability; and at the cellular level with alterations in FMRP function. In Aim 1 we will determine, in a population-based sample, whether individuals with Low or Gray Zone CGG repeats are at increased risk of neurocognitive, motor, and physical and mental health problems, and having a child with a disability. We will use a combination of questionnaires and in-person assessments, as well as supervised machine learning algorithms on the comprehensive database of electronic health records available through the PMRP. In Aim 2 we will test the hypothesis that Low Zone or Gray Zone CGGs affect FMRP function and neural differentiation/maturation. We will use a combination of human ES cell lines engineered to have various CGG repeat lengths, and a series of induced pluripotent stem cell lines derived from patients with defined genotypes.

Abstract Although most neurons in the mammalian brain are born before birth, neuronal development continues postnatally both in the form of neuronal maturation and in the mode of postnatal neurogenesis. Dysregulation of postnatal neuronal development is linked to a number of neurodevelopmental and neuropsychiatric disorders. Therefore understanding the regulatory mechanism of postnatal neuronal development has significant impact on human health. Epigenetic mechanisms, including DNA methylation and histone modification, play significant roles in brain development and plasticity, as well as in mediating environmental impact on brain functions. The precise mechanism remains unclear. Methyl-CpG binding proteins, including MBD1 and MeCP2 can translate DNA methylation into gene expression changes. MBD1 gene mutations and polymorphism are found in a subset of autistic patients and chromosome 18q21 deletion syndrome. We have shown that MBD1 deficiency in mice results in impaired postnatal neurogenesis, neuronal maturation and behavioral deficits including learning defects, impaired social interaction, anxiety, reduced prepulse inhibition Using genome-wide approaches, we found that although MBD1 is recognized as a transcriptional repressor, majority of MBD1 bound genes showed decreased expression in KO neurons. We also discovered that MBD1 interacts with several proteins in the BAF ( Brg/Brahma-associated factors or SWI/SNF) ATP-dependent chromatin remodeling complex. BAF is critical for neuronal development and mutations of its core components are strongly associated with intellectual disabilities, including schizophrenia and autism. How BAF targets to specific genes remain unclear. An interaction between a known transcriptional activation complex and a canonical transcriptional repressor has not been established. Thus the functional significance of such regulation in neurodevelopment is unclear. Our preliminary data shows that the MBD1 and BAF170 depend on the presence of each other to effectively bind and activate gene expression in developing neurons. Based on these observations, we hypothesize that MBD1 interacts with BAF complex to activate a subset of genes important for neuronal maturation. We will determine whether and how MBD1 interacts with BAF proteins and potential roles of this interaction (Aim 1); whether BAF is important for MBD1 regulation of neurodevelopmental genes (Aim 2) and whether MBD1 and BAF deletion during postnatal development lead to similar social and cognitive behavioral deficits. Our results will unveil novel epigenetic mechanisms regulating neuronal development. (Synomymous: BRM/Smarca2/SNF2L2; BAF47/Smarcb1/SNF5; BAF53b/ACTL6B; BAF170/Smarcc2)

The dentate gyrus generates new neurons throughout life, long after the neurogenesis of early development has ended. Adult neurogenesis makes important contributions to neuroplasticity and learning, and its impairment has been linked to neurodegeneration, learning disability, and epilepsy. New adult-born neurons integrate into existing mature neural circuitry, but retain the physiological attributes of immature neurons. Compared to mature neurons, new adult-born neurons form synapses more readily, are more excitable, and are more plastic with respect to induction of long-term potentiation of synaptic transmission. The study of adult neurogenesis is a very active field, and dramatic advances are being made in understanding the molecular control mechanisms and cellular properties of new adult-born neurons. Although the circuitry and network properties of new adult-born neurons ultimately play a critical role in their functions, the lack of suitable experimental tools and methods has impeded progress in this direction. This proposal will investigate network activity of new adult-born neurons by using a genetically targetable fluorescent probe in the hybrid voltage sensor (hVOS) family. hVOS employs a fluorescent protein that can be targeted for expression in defined populations of cells. hVOS imaging can record action potentials and subthreshold synaptic potentials from single neurons in hippocampal slices in a single trial (without averaging). We propose to express hVOS probe in newborn neurons and image their electrical activity. We will express probe in newborn neurons with retrovirus, as well as with a new hVOS Cre reporter mouse that can target probe expression to genetically defined populations of cells by crossing with Cre drivers. We will use an inducible Tbr2-CreERT2 driver to target probe to new adult-born neurons with high temporal resolution. The two expression approaches will be compared to check for consistency. hVOS imaging in hippocampal slices will then allow us to monitor the electrical activity of multiple new adult-born neurons simultaneously. We will stimulate the different granule cell inputs, perforant path axons and mossy cells, and use hVOS to record subthreshold synaptic potentials and action potentials. Experiments will test hypotheses about the organization of newborn neuron circuits by evaluating the properties, correlations, and synchronization of evoked electrical responses recorded in many new adult-born neurons simultaneously. We will directly test the hypothesis that mature granule cells synapse with immature granule cells. We will use this imaging approach to determine whether new neurons integrate into existing neural circuitry as functional clusters with shared synaptic inputs, evaluate clones of newborn neurons descended from a common progenitor cell, and explore the evolution of circuits as neurons mature. This work will reveal the circuit relations of new adult-born neurons, and will introduce a novel technique for the study of neural networks to the field of adult neurogenesis.

Multi-PI: Xinyu Zhao, Meyer Jackson, University of Wisconsin-Madison. Title: Integration of Experience-Induced Gene Expression and Circuit Functions Understanding the complex relationships between cells, gene networks, neural circuits, and behavior requires techniques that can probe the molecular makeup of distinct types of neurons, evaluate their properties, and test their roles in higher level functions. Genes expressed within specific populations of neurons determine their electrical properties and these properties together with their synaptic connectivity collectively shape the electrical activity of neural circuits. This is especially well illustrated by a population of neurons defined by expression of the Ca2+ binding protein parvalbumin (PV). PV interneurons (PVIs) are sparsely distributed, fast-spiking cells that provide feedback and feedforward inhibition to principal neurons. One of the most well-defined network functions of PVIs is in the coordination of neuronal networks and their associated oscillations. PVIs entrain cortical networks to drive gamma oscillations (30-100 Hz) and control their frequency and strength. PVI-mediated gamma oscillations are known to have important roles in sensory processing, attention, working memory, and cognition. However, the gene networks that control PVI functions and their impact on gamma oscillations remain unclear. PVIs are readily modified by environmental conditions and experience. PV immunoreactivity increases after exploration of a novel environment, rearing under environmental enrichment (EE), and voluntary running (VR). These changes occur in brain regions associated with cognition, including hippocampus, prefrontal cortex, and amygdala. The molecular mechanisms underlying PVI changes during behavioral adaptation remain unknown. Although studies suggest that behavioral adaptions affect gamma oscillations, a role for PVIs in the link between behavioral adaption and gamma oscillations has not been established. This application takes a multidisciplinary approach to address the fundamental question of how PVIs contribute to behavioral adaptations. Our overarching hypothesis is that changes in gene expression that modify the cellular properties of PVIs will alter network oscillations, enabling PVIs to serve as a critical hub in behavioral adaptations. We will determine whether behavioral adaptation mobilizes networks of genes in PVIs, and assess the contributions of these networks to PVI physiology and gamma oscillations. This project combines the unique expertise of co-PIs Zhao (genetic regulation of neurodevelopment) and Jackson (neurophysiology and neural circuits) and co-Is Roy (system biology and machine learning) and Rosenberg (computational and system neuroscience). By integrating experimental data with gene network analysis and computational modeling of multicellular networks, this work will reveal how changes in molecular/cellular properties impact the emergent properties of neural circuits.

ABSTRACT Fragile X Mental Retardation Protein (FMRP) is an RNA binding protein that binds to specific mRNAs to control their location and protein translation, thus regulating the expression of important genes in neuronal development, neuronal function and synaptic plasticity. FMRP?s critical role is demonstrated by the neurodevelopmental disorder, Fragile X syndrome (FXS) that is caused by a lack of FMRP due to mutations in the FMR1 gene. Although the function of FMRP has been extensively explored in animal models, how FMRP functions in human neuronal development and how a lack of FMRP causes the devastating characteristics in FXS patients are largely unknown. The experiments in this discovery-based proposal seek to identify mRNA targets of FMRP in human neurons for the first time. Human pluripotent stem cells with the endogenous FMR1 gene FLAG tagged will be differentiated into forebrain excitatory and inhibitory neurons. FMRP targets will be identified by crosslinking immunoprecipitation (CLIP) with an antibody against the FLAG tag, followed by deep sequencing (RNA-seq). We will then investigate whether FMRP targets identified in mouse neurons are also regulated by FMRP in human neurons as well as validate the FMRP bound mRNAs identified in Aim 1 and perform initial functional analyses of these mRNAs. The data will unveil potential mechanisms for FMRP?s role in human neuronal development. The data will enable better understanding of how lack of FMRP in FXS causes intellectual disability and potentially autism.

Title: The Role of MDM2 in FMRP regulation of neuronal development Fragile X Mental Retardation Protein (FMRP) is an RNA binding protein that binds to specific mRNAs to control their stability, localization, and protein translation. Loss of FMRP leads to Fragile X syndrome (FXS), the most common heritable cause of intellectual disability, and is also the greatest single-gene contributor to autism. Despite extensive effort, the mechanisms underlying the learning deficits in FXS are not fully understood and an effective therapy for this devastating disorder is lacking. Disappointing results from recent clinical trials underscore the pressing need for innovation in both target selection and cell type consideration. The development of an appropriate neural network is a prerequisite for normal brain functions. Although most neurons in the mammalian brain are born during embryonic neurogenesis, a significant amount of neuronal development continues postnatally. Neuronal maturation, including dendritic and axonal morphogenesis, spine development, synaptogenesis/pruning, and circuit integration, is critical for proper brain function and human health. In addition, new glutamatergic neurons are continuously produced in the dentate gyrus (DG) of the hippocampus, one of a few brain regions, possibly the only region in humans, with lifelong neurogenesis. Postnatal neurogenesis is important for cognitive outcomes and its impairment is implicated in both neuropsychiatric disorders and neurodegenerative diseases. Our lab has pioneered the investigation of FMRP in postnatal neurogenesis and our work has provided a causal link between postnatal neurogenesis and cognitive function in FXS, a postnatal developmental disorder. Using postnatal neurogenesis as a model system, we recently discovered that FMRP controls the levels of active (phosphorylated- or P-) MDM2. We were able to use a low dosage of Nutlin-3, an MDM2 inhibitor in clinical trials as a cancer treatment, to rescue cognitive deficits of adult FXS mice. Elevated MDM2 activity is found in a number of disease conditions, mostly cancers, and has been a focus for drug targeting. However, how elevated MDM2 impacts neurodevelopment and neuropsychiatric disorders is unclear. Our preliminary data show that FMRP-deficiency neurons also have elevated MDM2 levels and MDM2 inhibition rescue neuronal dendritic deficits of these neurons. Our exciting results have presented us with a set of lingering questions that are central to our understanding of FMRP regulation of neuronal development and developing novel treatment for FXS. This proposal aims to test the hypothesis that MDM2 is a key mediator of FMRP regulation of neurodevelopment. We will determine whether genetic reduction of MDM2 during postnatal development rescue certain behavioral deficits of FMRP-deficient mice (Aim 1), determine whether MDM2 dysregulation contributes to developmental deficits of FMRP-deficient neurons (Aim 2), and identify proteins and pathways that mediate MDM2 inhibition rescue of FMRP deficiency (Aim 3). The outcome of this study will yield important new information leading to novel therapeutic applications for FXS and potentially other neurodevelopmental disorders as well.

Abstract Fragile X related protein 1 (FXR1P) is a brain-enriched RNA binding protein. Its loss of function is intolerant for both mice and humans. Large-scale genome wide association studies and recent gene network analyses have identified FXR1 as a high confidence risk gene for mental illness. FXR1P has unique protein domains and mechanisms of action that are distinct from its X-linked homolog fragile X mental retardation protein. Due to neonatal lethality of FXR1P-null mice the impact of FXR1P deficiency on brain development and postnatal brain function is largely unexplored. FXR1P is widely expressed in both excitatory and inhibitory neurons in the mammalian brain throughout postnatal development and in adults. Limited literature and our preliminary data suggest that FXR1P may have distinct functions in inhibitory versus excitatory neurons. The function of FXR1P in interneurons has not been studied. Complex neuronal information processing depends on precise spatial and temporal coordination of principal excitatory neurons, which requires intimate interactions between excitatory and inhibitory interneurons. Among inhibitory neurons, fast spiking, parvalbumin (PV)-expressing interneurons (PVIs) have emerged as critical players in many forms of circuit activities. PVIs provide both feedback and feedforward inhibition to excitatory neurons and entrain cortical networks to drive gamma oscillations and control their frequency and strength. Extensive studies have shown that gamma oscillations are important for sensory processing, attention, working memory, and cognition, which are impaired in a number of mental disorders, including autism and schizophrenia. However, the regulation of gene expression in PVIs has received limited attention. We found that FXR1P is expressed in a majority of PVIs of adult mouse cortex. Our preliminary data show that mice with PVI-specific deletion of FXR1P exhibited deficits in behaviors that require proper function of prefrontal cortex (PFC). Interestingly, these behavioral changes are not found in mice with FXR1P deletion only in forebrain excitatory neurons. We hypothesize that FXR1P regulates gene expression in PVIs in the PFC to control PVI excitability, synaptic plasticity, and circuit function and FXR1P deficiency in PVIs alters cortical circuit activities leading to behavioral deficits. We will determine whether FXR1P deficiency in PVIs in the PFC leads to deficits in PVI physiology and connectivity, impairs PFC- dependent behaviors, and changes in specific gene networks. This work brings state-of-art techniques together in a multidisciplinary approach to investigate how FXR1P deficiency impacts the function of an important type of interneuron. Our approach provides a potential framework for assessing other potentially important genes with unclear functions, in PVIs and other genetically defined populations of neurons.