Qiang Chang - US grants

DESCRIPTION (provided by applicant): MeCP2 (methyl-CpG binding protein 2) functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Several lines of evidence exist to support the possibility that differential phosphorylation of MeCP2 in response to neuronal activity may serve as a molecular switch in dynamically modulating neuronal gene expression, which underlies the activity-dependent phase of mammalian brain development. First, MeCP2 expression is dramatically up-regulated in mature neurons during the period of synaptic refinement. Second, Rett Syndrome (RTT, an autism spectrum developmental disorder caused by mutations in the MECP2 gene) patients are born normal (suggesting MeCP2 is not required for activity-independent embryonic brain development), but become symptomatic during the period of synaptic refinement (suggesting MeCP2 is required for activity-dependent postnatal brain development). Third, two in vitro studies showed that neuronal activity-induced phosphorylation at serine 421 (S421) precedes the release of MeCP2 from the neuronal specific promoter of the brain-derived neurotrophic factor (BDNF) gene and the subsequent expression of BDNF. Finally, comprehensive biochemical analysis has identified 8 phosphorylation sites on the MeCP2 protein. Among these, serine 80 (S80) is phosphorylated in resting neurons but dephosphorylated in active neurons, whereas S421 is dephosphorylated in resting neurons but phosphorylated in active neurons. To determine how neuronal activity induced differential phosphorylation of MeCP2 fine-tunes the promoter occupancy of MeCP2 across the genome and induces corresponding changes in chromatin marks, we have generated several novel Mecp2 knock-in alleles carrying point mutations that either abolish or mimic phosphorylation at S80 and S421 on the MeCP2 protein, as well as a FLAG tag at the carboxyl terminal of the MeCP2 protein. As a part of our long-term goal to understand the dynamic role of MeCP2 in DNA methylation-dependent epigenetic regulation of mammalian brain development and functions, we propose to: 1) perform ChIP-chip (chromatin immunoprecipitation followed by hybridization onto a DNA oligo array) experiments to reveal how changes in the phosphorylation status of MeCP2 cause changes in its ability to bind to gene promoters across the entire genome;2) perform ChIP-chip experiments to reveal how changes in the phosphorylation status of MeCP2 induce corresponding changes in chromatin marks at its target gene promoters across the entire genome. PUBLIC HEALTH RELEVANCE: Mutations in the X-linked human MECP2 gene (methyl-CpG binding protein 2) cause Rett syndrome (RTT), an autism spectrum developmental disorder that predominantly affects females. To understand the molecular mechanism of RTT, it is important to study how MeCP2 dynamically regulates gene transcription. Results from this study will advance our understanding of the molecular mechanism of Rett syndrome (RTT). Furthermore, because of the considerable overlap in clinical features between RTT and autistic spectrum disorders, the lessons learned studying RTT might also benefit the general understanding of autism.

DESCRIPTION (provided by applicant): MeCP2 (methyl-CpG binding protein 2) functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Mutations in the X-linked human MECP2 gene cause of Rett syndrome (RTT), an autism spectrum developmental disorder that predominantly affects females. To understand the molecular mechanism of RTT, it is important to study how MeCP2 dynamically regulates gene transcription, and to reveal the physiological significance of such regulation. Recent biochemical analysis has identified 8 phosphorylation sites on the MeCP2 protein. Among these, serine 80 (S80) is phosphorylated in resting neurons but dephosphorylated in active neurons, whereas serine 421 (S421) is dephosphorylated in resting neurons but phosphorylated in active neurons. Two in vitro studies have shown that neuronal activity- induced phosphorylation at S421 precedes the release of MeCP2 from the neuronal specific promoter of the brain-derived neurotrophic factor (BDNF) gene and the subsequent expression of BDNF. Collectively, those studies raise the possibility that differential phosphorylation of MeCP2 in response to neuronal activity may serve as a molecular switch in dynamically modulating neuronal gene expression, leading to important consequences in development and function of the adult brain. To test this hypothesis in vivo, we have generated several novel Mecp2 knockin alleles carrying point mutations that either abolish or mimic phosphorylation at S80 and S421 on the MeCP2 protein. As a part of our long-term goal to understand the dynamic role of MeCP2 in DNA methylation-dependent epigenetic regulation of mammalian brain development and functions, we propose to: 1) study the effects of manipulating MeCP2 phosphorylation on animal behavior; 2) study the effects of manipulating MeCP2 phosphorylation on adult neurogenesis; 3) study how MeCP2 phosphorylation regulates its binding to the Bdnf promoter, remodels chromatin and subsequently alters BDNF expression and neuronal activity. Together, the experiments proposed in these three specific aims will provide insights into the central role of neuronal activity induced differential phosphorylation of MeCP2 in regulating neuronal gene expression, and its functional significance in neuronal development and animal behavior. These insights will not only bring us closer to understand the molecular mechanism of RTT and find potential treatments for RTT, but also benefit the general understanding of autism. PUBLIC HEALTH RELEVANCE: Results from this study will not only help elucidate the central role of MeCP2 in DNA methylation-dependent epigenetic regulation of brain function, but also advance our understanding of the molecular mechanism of Rett syndrome (RTT). Furthermore, because the considerable overlap in clinical features between RTT and autistic spectrum disorders, the lessons learned studying RTT might also benefit the general understanding of autism.

DESCRIPTION (provided by applicant): Studying the Glial Contribution to RTT Pathogenesis Using Patient-Specific iPSCs Abstract Rett syndrome (RTT) is an autism spectrum disorder (ASD) that predominantly affects females. The identification of mutations in the X-linked MECP2 (methyl-CpG binding protein 2) gene as the cause of RTT has led to the creation of mouse models for studying disease mechanism. However, mouse models have limitations in mimicking human RTT mutations and in drug screening. Induced pluripotent stem cells (iPSCs) have been generated from RTT patients as an in vitro human model to validate and extend knowledge obtained from mouse studies. Currently, all studies involving RTT iPSCs have focused on studying neuronal pathologies in the absence of astrocytes. As the other major cell type in the brain, astrocytes also express MeCP2. There is strong evidence, from work in RTT mouse models, that astrocytes play a critical role in disease progression. Thus, we hypothesize that astrocytes differentiated from mutant RTT-iPSC lines will significantly impair neuronal growth and maturation when cultured together with neurons. In the current proposal, we will focus our attention on revealing the glial contribution to RTT pathology in a neuron/astrocyte co-culture system. To minimize phenotypic variation caused by different genetic backgrounds across iPSC lines generated from different individuals, we have established several pairs of isogenic iPSC lines from the same female RTT patients (carrying either common or rare RTT mutations) skin cells that clonally express either the wild type copy or the mutant copy (but not both) of the MECP2 gene. Using these unique tools, we plan to test 1) whether mutant iPSC-derived astrocytes may impair the growth and maturation of wild type iPSC-derived neurons in the neuron/astrocyte co-culture; 2) whether wild type iPSC-derived astrocytes may rescue the growth and maturation defects of mutant type iPSC-derived neurons in the neuron/astrocyte co-culture; and 3) whether the potential astrocyte influnce is mediated by cell-cell contact or secreted molecules. Better understanding of the glial contribution to RTT pathology will not only provide insight into disease mechanisms, but also lay the groundwork for future drug screens using RTT iPSC-derived neurons and/or astrocytes. Furthermore, in light of the recent evidence that MECP2 may be altered at both the genomic level and the expression level in many autism patients, the lessons learned and the experimental approaches used in studying RTT might also benefit the general understanding of autism. PUBLIC HEALTH RELEVANCE: Studying the Glial Contribution to RTT Pathologenesis Using Patient-Specific iPSCs Project Narrative Mutations in the X-linked human MECP2 gene (methyl-CpG binding protein 2) cause Rett syndrome (RTT), an autism spectrum developmental disorder that predominantly affects females. To fully understand the molecular mechanism of RTT, it is important to study the role of astrocytes in disease progression in human cells. The successful completion of this project will significantly advance the understanding of RTT disease mechanism and facilitate future development of therapies to treat RTT. First, the proposal aims at studying RTT pathogenesis in human RTT neurons and astrocytes, the two major cell types affected in RTT patients. This is necessary because mice are not humans. Any knowledge obtained in mouse studies or any treatment showing efficacy in mouse models should be validated in human systems. Second, the proposal aims at revealing the glial contribution to RTT pathology, which will open up a research area that has not been explored in human cells. This is very important for treating RTT, because strategies aimed at correcting neuronal defects may not work efficiently if glial defects are left untreated. Third, the proposal will establish RT induced pluripotent stem cell (iPSC)-derived neurons and astrocytes as in vitro platforms for future drug screens, which is the next step to translate discoveries at the bench side to treatments at the bed side. Finally, because of the considerable overlap in clinical features between RTT and autism spectrum disorders and the recent reports of MECP2 alterations in autism patients, the lessons learned studying RTT might also benefit the general understanding of autism.

? DESCRIPTION (provided by applicant) Rett syndrome (RTT) is a debilitating neurodevelopmental disorder primarily affecting females. Although it is clear that mutations in a single gene (MECP2) cause the disease, the precise mechanism underlying the pathologies is not well understood. Natural history studies of RTT patients and research using mouse models of RTT and RTT patient iPSC models have all contributed to our current understanding of the disease. Because each experimental platform has its unique strength and limitations, before insights gained on a particular platform become useful for therapy development, it is critical to validate the findings on multiple platforms acros disciplines. Recently, a suppressor screen in a RTT mouse model has identified altered cholesterol metabolism as a likely influence on disease progression, opening the door to potential intervention, since many key players in the pathway are drug targets in other unrelated diseases. However, the mouse study did not identify the cellular origin of the altered cholesterol metabolism in the brain. Two subsequent studies further demonstrated some impairment in cholesterol metabolism in the plasma and fibroblasts from RTT patients. Yet, the significance of these studies is limited because plasma cholesterol levels are not correlated with brain cholesterol homeostasis and fibroblasts are notoriously poor models of brain cells, hence leaving unanswered the question of a potential contribution of abnormal cholesterol metabolism to the pathogenesis of Rett syndrome, a widely recognized neurobiological condition. Also unanswered with these two studies is the question of a correlation between the observed cholesterol abnormalities and the RTT clinical phenotype or disease progression. To begin addressing these unanswered questions, we propose to 1) determine whether sterol metabolism is altered in astrocytes differentiated from mutant RTT induced pluripotent stem cells (iPSCs) and whether manipulation of the cholesterol synthesis pathway may rescue the cellular phenotypes in RTT astrocytes; and 2) perform a refined exploration of sterol metabolism in RTT patients facilitating future correlations between sterol biomarkers and clinical phenotypes. Results from our proposed study will help to determine whether sterol metabolism is altered in RTT patients or brain cells derived from these patients (as suggested by findings in RTT mouse models), and reveal the significance of such alteration in the context of other clinical symptoms. If successful, our study will not only advance our understanding of RTT disease mechanisms, but also lead to potential treatments for RTT patients.

Astrocyte Dysfunction in Rett Syndrome Abstract Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. The disease is complex, as all key cell types (neurons, astrocytes, microglia, and oligodendrocytes) in the brain have been shown to contribute to the disease etiology. To fully understand the disease mechanism and develop effective treatments, it is essential to define the key phenotypes, link the phenotypes with loss of MeCP2 function, and reveal the consequences of the phenotypes in each cell type; and to study how these cells interact. While earlier research mostly focused on neuronal dysfunction in RTT, more recent studies have clearly demonstrated that astrocytes express MeCP2, loss of MeCP2 in astrocytes causes neuronal defects, and restoring MeCP2 expression to normal in astrocytes alleviates disease symptoms. Although a few studies have reported gene expression changes and phenotypes in Mecp2 mutant mouse astrocytes, it is not clear how these alterations contribute to RTT pathogenesis either by directly changing astrocyte functions or by indirectly changing neuronal functions. Therefore, it is necessary to systematically investigate the astrocyte cell autonomous phenotypes, the underlying mechanism of the phenotypes, and the functional consequence of the phenotypes. We have discovered significant changes in cytosolic calcium homeostasis in RTT astrocytes in the absence and presence of neurons, revealed potential molecular and cellular mechanisms underlying the abnormal calcium homeostasis, and identified major functional consequence of this astrocyte cell autonomous phenotype on neighboring neurons and the neural network. Here, we propose to 1) dissect the cell type specific contribution to the phenotypes of increase TRPC4 expression and abnormal calcium activity in astrocytes, the phenotype of excessive activation of extrasynaptic NMDA receptor (eNMDAR) in neighboring neurons, and the phenotype of increased network excitability in RTT mouse models; 2) reveal the functional significance of abnormal calcium activity in RTT disease progression; and 3) investigate the contribution of TRPC4 to the phenotype of abnormal calcium homeostasis in RTT astrocytes, and reveal the functional significance of ectopic TRPC4 expression in RTT disease progression. Our study employs various RTT mouse models (germline knockout mice, cell type specific conditional knockout mice, and cell type specific conditional reactivation mice), the RTT patient- specific induced pluripotent stem cell (iPSC) model, and genetically engineered human embryonic stem cell (hESC) model. By combining the strengths from these complementary models, we can cross-validate our findings between species and between in vitro and in vivo, therefore generating more valid insights. The successful completion of this project will significantly advance the understanding of RTT disease mechanism and facilitate future development of therapies to treat RTT.

Function of stimulus-induced MeCP2 phosphorylation Abstract MeCP2 is a key player in recognizing methylated DNA and interpreting the epigenetic information encoded in different DNA methylation patterns. Alterations in sequence or copy number of the X-linked human MECP2 gene cause either Rett syndrome (RTT) or MECP2 duplication syndrome. In addition to RTT-causing mutations, many missense mutations with unknown functional significance have been identified in the MECP2 gene in humans. Two of those mutations are at or close to serine 80 or serine 421, whose phosphorylation status is important for regulating MeCP2 function. To fully understand the significant role of MECP2 in regulating the development and function of the nervous system, it is important to study all aspects of MeCP2 function. We have previously demonstrated that phosphorylation at serine 421 (S421) can be induced by spatial learning, and that S421 phosphorylation plays critical roles in regulating MeCP2 binding to gene promoters, neuronal gene transcription, excitatory synaptogenesis, two types of synaptic plasticity (long-term potentiation and synaptic scaling), locomotion, learning and memory. Most recently, we discovered that S421 is also phosphorylated in adult neural progenitor cells (aNPC) isolated from the hippocampus. Interestingly, the stimulus, the regulation and the function of S421 phosphorylation in aNPCs are completely different from those in post-mitotic neurons. In aNPCs, MeCP2 S421 phosphorylation is induced by growth factors, linked to cell cycle, directly regulated by aurora kinase B, and plays critical roles in regulating the proliferation and differentiation of aNPCs through the Notch signaling pathway. These new findings further generalize MeCP2 phosphorylation as a common regulatory module in cellular functions. More interestingly, phosphorylation at serine 80 (S80) appears to be regulated differentially from phosphorylation at S421 in post-mitotic neurons, and plays opposing functional roles against S421 phosphorylation in both post-mitotic neurons and aNPCs. Collectively, these studies raise the possibility that dynamic phosphorylation states at multiple sites on MeCP2 may form a combinatorial code, presenting another epigenetic regulatory module in addition to DNA methylation and histone codes. To test this hypothesis, we propose to generate several novel Mecp2 knockin alleles carrying combinations of point mutations that either abolish or mimic phosphorylation at S80 and S421, and study the functional output of each combinatorial code in the well-established paradigm of neurogenesis in both mouse and human models. Our specific aims are: 1) To study the effects of double mutations at S80 and S421 on aNPC proliferation and differentiation in mouse models, 2) To study the effects of single and double mutations at S80 and S421 on NPC proliferation and differentiation in human stem cell models, and 3) To define the molecular mechanism linking MeCP2 phosphorylation with its functional output. Studying posttranslational modification of MeCP2 provides a critical angle of understanding the basic function of MeCP2, which will expand our knowledge of MeCP2 beyond RTT and MECP2 duplication syndrome.

We seek continued support of the Waisman Center Intellectual and Developmental Disabilities Research Center, a comprehensive interdisciplinary program focused on IDD spanning the biological, biobehavioral, and bio-behavioral sciences. The Waisman IDDRC brings together 46 PIs from 21 academic departments from the UW-Madison's Schools of Medicine and Public Health, Veterinary Medicine, Pharmacy, Agriculture and Life Sciences, Letters and Science, Education, Engineering, and Human Ecology. This application requests support for an Administrative Core (Core A), providing scientific leadership, program and faculty development, facilitation of interdisciplinary collaboration, and biostatistical and bioinformatics expertise; and three innovative scientific core services: Clinical Translational (Core B), providing services, resources, and training in the recruitment of human participants, clinical assessment, behavioral methods development, and cGMP biomanufacturing of therapeutics; Brain Imaging (Core C), providing access to state-of-the-art neuroimaging instrumentation (3T MRI, PET, and microPET scanners for human, non-human primate, and rodent scanning, and an EEG recording system), as well as expertise and tools for image acquisition and analysis; and IDD Models (Core D), providing resources, expertise, and technical services in the generation and characterization of mutant or genetically engineered strains of mice and rats, the generation of induced pluripotent stem cell lines from humans with IDD conditions, and facilities for phenotypic characterization of these models using molecular, cellular and behavioral technologies. In addition, we request support for a Research Project that is focused on the impact of variations in CGG repeat length in the FMR1 gene on health and function at the cellular through organismal level, using both cell culture models and human subjects. We propose to provide core support to 79 research projects headed by 46 PIs addressing three broad themes relevant to IDD: 1) nervous system development and pathogenesis, 2) IDD conditions, and 3) assessment, interventions, and therapeutics. Collectively the core services and the research project of the Waisman Center IDDRC will stimulate new interdisciplinary IDD research and enhance existing IDD investigations sharpening our focus on discovery, prevention, and treatment for IDD conditions, and improvement of the quality of life of individuals with IDD and their families.

We seek continued support of the Waisman Center Intellectual and Developmental Disabilities Research Center, a comprehensive interdisciplinary program focused on IDD spanning the biological, biobehavioral, and bio-behavioral sciences. The Waisman IDDRC brings together 46 PIs from 21 academic departments from the UW-Madison's Schools of Medicine and Public Health, Veterinary Medicine, Pharmacy, Agriculture and Life Sciences, Letters and Science, Education, Engineering, and Human Ecology. This application requests support for an Administrative Core (Core A), providing scientific leadership, program and faculty development, facilitation of interdisciplinary collaboration, and biostatistical and bioinformatics expertise; and three innovative scientific core services: Clinical Translational (Core B), providing services, resources, and training in the recruitment of human participants, clinical assessment, behavioral methods development, and cGMP biomanufacturing of therapeutics; Brain Imaging (Core C), providing access to state-of-the-art neuroimaging instrumentation (3T MRI, PET, and microPET scanners for human, non-human primate, and rodent scanning, and an EEG recording system), as well as expertise and tools for image acquisition and analysis; and IDD Models (Core D), providing resources, expertise, and technical services in the generation and characterization of mutant or genetically engineered strains of mice and rats, the generation of induced pluripotent stem cell lines from humans with IDD conditions, and facilities for phenotypic characterization of these models using molecular, cellular and behavioral technologies. In addition, we request support for a Research Project that is focused on the impact of variations in CGG repeat length in the FMR1 gene on health and function at the cellular through organismal level, using both cell culture models and human subjects. We propose to provide core support to 79 research projects headed by 46 PIs addressing three broad themes relevant to IDD: 1) nervous system development and pathogenesis, 2) IDD conditions, and 3) assessment, interventions, and therapeutics. Collectively the core services and the research project of the Waisman Center IDDRC will stimulate new interdisciplinary IDD research and enhance existing IDD investigations sharpening our focus on discovery, prevention, and treatment for IDD conditions, and improvement of the quality of life of individuals with IDD and their families.

The Administrative Core is the nucleus of the IDDRC, providing scientific, administrative, and fiscal leadership, and high quality, cost-effective core services, in a strongly collaborative spirit. The core functions to create a highly visible and comprehensive center that systematically integrates all aspects of research, training, clinical services, and outreach in the field of IDD at the University of Wisconsin-Madison, and serves as the liaison to the NIH, and national and international organizations. Chang and the previous director, Messing, have raised considerable private funds for improvement of IDDRC physical facilities, purchase of scientific equipment for IDDRC cores, program development, and recruitment and retention of investigators. Specific Aim 1 is to develop new and coordinate existing scientific resources to strategically support the research of IDDRC investigators. IDDRC core resources and services have been designed to complement UW-Madison resources to enhance the scientific work involving all phases of the translational research cycle, from basic discovery to clinical application. The Administrative Core?s leadership team (Executive Committee) gathers information about research needs from surveys of investigators as well as from each core?s user advisory committee, internal and external advisory committees, consultants, and visiting scientists. To obtain additional resources, the Administrative Core works closely with UW-Madison administration including the Office of the Vice Chancellor for Research and Graduate Education, academic deans, department chairs, center directors, and directors of other relevant research units. Specific Aim 2 is to promote interdisciplinary and collaborative research in high priority areas of IDD. This includes provision of start-up funds to attract faculty to the Waisman Center, allocation of support for special interest groups that involve IDDRC faculty from multiple disciplines who share a common area of focus (e.g., Down syndrome, genome editing in human pluripotent stem cells, functional genomics), provision of seed money support for groups of investigators who seek to develop multi-component grant applications for interdisciplinary IDD research (e.g. our signature research project), funding for speakers in the John Wiley Seminar Series, and most importantly the creation of an environment of mutual respect for diverse disciplinary approaches to studying IDD conditions. Ultimately, the Administrative Core of the Waisman Center IDDRC seeks to create a nexus where multiple angles of vision are focused on a shared commitment to understanding the causes and consequences of, and discovering treatments for IDD conditions. Specific Aim 3 is to further connect research and clinical activities and strengthen community partnerships for efficient dissemination of knowledge and best practices to improve the lives of individuals and families affected by IDD. The Administrative Core not only facilitates interactions between research and clinical activities within the Center, but also works to engage individuals and families affected by IDD throughout the UW health care system and in community settings such as the public schools. The Core nurtures and maintains the relationships with key campus partners, with the Marshfield Clinic Research Foundation, with All of Us, and with state partners such as the Department of Health Services. The core develops and maintains close connections with parent and patient groups as wells as agencies and organization active in the field of IDD.

We seek continued support of the Waisman Center Intellectual and Developmental Disabilities Research Center, a comprehensive interdisciplinary program focused on IDD spanning the biological, behavioral, and biomedical sciences. The Waisman IDDRC brings together 60 PIs from 24 academic departments from the UW-Madison?s Schools of Medicine and Public Health, Veterinary Medicine, Agriculture and Life Sciences, Letters and Science, Education, Engineering, Social Work, and Human Ecology. This application requests support for an Administrative Core (Core A), providing scientific leadership, program and faculty development, facilitation of interdisciplinary collaboration, oversight of training programs, biostatistical and bioinformatics expertise; and dissemination of knowledges and best practices; and three innovative scientific core services: Clinical Translational (Core B), providing services, resources, and training in the recruitment of human participants, clinical research coordination/navigation, clinical assessment, behavioral methods development, and production of clinical grade biotherapeutics for use in clinical trials; Brain Imaging (Core C), providing access to state-of- the-art neuroimaging instrumentation for both human and animal studies (3T MRI, PET, and microPET scanners for human, non-human primate, and rodent scanning, an fNIRS, and an EEG recording system), as well as expertise and tools for image acquisition and analysis; and IDD Models (Core D), providing resources, expertise, and technical services in cellular and molecular neuroscience, the generation and manipulation of human pluripotent stem cell (hPSC) lines from humans with IDD conditions, as well as the generation and behavioral phenotyping of mutant or genetically engineered strains of mice and rats as models of IDD conditions. In addition, we request support for a Research Project that addresses a fundamental question on the emergence of ADHD symptoms in children with ASD, using a multidisciplinary approach that combines the power of neurobehavioral, brain imaging, statistical genomics, and machine learning analyses. We propose to provide core support to 69 research projects headed by 44 PIs addressing three broad themes relevant to IDD: 1) neurodevelopment and mechanisms, 2) disorders of the nervous system, and 3) assessments and interventions. Collectively the core services and the research project of the Waisman Center IDDRC will stimulate new interdisciplinary IDD research and enhance existing IDD investigations, with a sharp focus on discovery, prevention, and treatment for IDD conditions, and improvement of the quality of life of individuals with IDD and their families.