John P. Svaren - US grants

DESCRIPTION (provided by applicant): Formation of the myelin sheath is critical for rapid conduction of nerve signals in motor and sensory nerves. The EGR2/Krox20 transcription factor is a master regulator of the myelination program in peripheral nerve, and human genetics studies have identified several independent EGR2 mutations associated with myelination disorders such as Charcot-Marie-Tooth (CMT) disease and Dejerine-Sottas Syndrome, which constitute one of the most common types of human genetic diseases. Although EGR2 expression correlates with induction of many myelin-associated genes, very few direct targets of EGR2 activity have been identified, and interactions with other myelin-specific transcription factors remain relatively obscure. Novel techniques outlined in this proposal will analyze interaction of EGR2 with other transcription factors during myelination in vivo to address the following specific aims: To determine if EGR2 interaction with a conserved intron-associated element regulates Myelin Protein Zero expression. Myelin Protein Zero (MPZ) is one of the most highly expressed myelin genes, and is commonly mutated in peripheral neuropathies. This proposal will probe the mechanism by which EGR2 activates a newly discovered control element in the MPZ gene, and will elucidate the mechanism by which dominant EGR2 mutants cause disease. To test the role of EGR2/SREBP synergy in regulating myelination. This aim will explore the scope and mechanism by which EGR2 collaborates with Sterol Response Element Binding Proteins (SREBPs) to activate genes required for the myelination program. . To test the role of CHD4 in regulation of EGR2 target genes by NAB proteins during peripheral nerve myelination. This aim will provide the first mechanistic analysis of NAB repression of EGR2 target genes by investigating the role of the NuRD chromatin remodeling complex. Deficiencies in formation of the myelin sheath around nerve fibers are a central cause of several important medical disorders, including many types of muscular dystrophy, diabetic neuropathy, multiple sclerosis, and leprosy. Formation and maintenance of lipid-rich myelin is a complex process, and this proposal is directed towards understanding genetic control of the myelination process in the peripheral nervous system. These experiments will provide novel insights that will be vital for development, evaluation, and implementation of novel therapies for these diseases. .

The Cellular and Molecular Neuroscience Core is designed to facilitate the research objectives of the IDDRC and the scientific progress of individual IDDRC projects by providing state-of-the-art equipment, training, and expertise for IDDRC investigators employing, or wishing to employ, cellular and molecular biology in their research programs. In-house equipment includes advanced microscopy (laser scanning confocal and fluorescence microscopy with Stereology), genomic and proteomic analysis both in situ (flow cytometry) and in cell lysates (phosphonmaging and quantitative PCR), and informatics. During the current project period, the Waisman Center committed $500,000 of gift funds to upgrade the Core's existing proteomics and confocal microscopy capabilities. In addition, the CMN Core has well-established partnerships with nearby UW-Madison core facilities (UW-Biotechnology Center and UW-Cancer Center) to offer cost effective, state-of-the-art genomic and proteomic technology, such as 2-dimensional gel analysis, mass spectrometry, DNA sequencing and synthesis, DNA array and SNP analysis, NextGen sequencing, tiled array analysis of chromatin immunoprecipitation (ChIP) assays, and small molecule drug screening. In response to the growing interest for human-derived IDD models, in 2009 we created a new service of the CMN Core (with ARRA support) to generate induced pluripotent stem cells (iPSCs) from individuals with IDD conditions for IDDRC investigators. The IPSC service leverages the IDDRC's long-standing expertise in stem cell biology, access to well characterized patients with IDD (via the Research Participation Core), and clinical biomanufacturing (Waisman Biomanufacturing) for the production of IPSC viral vectors. Using these complementary resources, the iPSC service has successfully created 23 cell lines from patients with IDD including Rett syndrome, Alexander disease, FXS, DS, SMA, retinitis pigmentosa. Best disease, and others, and provided guidance and expertise in differentiating the cell lines into neuronal sub-types and astrocytes.

DESCRIPTION (provided by applicant): Hereditary peripheral neuropathies (also known as hereditary motor sensory neuropathies, HMSN) are among the most common genetic diseases affecting the nervous system. The mildest form of human peripheral neuropathy, Charcot-Marie- Tooth (CMT) disease, causes progressive deterioration of both motor and sensory nerves, muscular atrophy, and chronic pain/fatigue in affected individuals. A majority of inherited peripheral myelinopathies are caused by duplication of a critical myelin gene, Peripheral Myelin Protein 22 (PMP22), which is classified as CMT1A. Therefore, one of the most straightforward avenues for treating this inherited myelinopathy is to achieve a relatively subtle (<2-fold) change in gene regulation. Recent proof-of-principle studies using candidate compounds to reduce PMP22 expression levels have shown beneficial effects in rodent models of CMT1A. However, these agents have not yet been shown to be effective in clinical trials, and therefore it is criticl to develop effective screening tools that can be used to identify compounds that can achieve a therapeutic reduction in PMP22 levels. Our recent studies of PMP22 regulation have elucidated novel regulatory elements in this gene, and the goal of this proposal is to design and functionally validate novel assays to identify small molecules that reduce PMP22 expression. We propose to utilize a novel technology involving custom zinc finger nucleases, which will allow us to embed a series of orthologous reporters in the native Pmp22 locus. The assays will create a series of complementary assays for use in both primary and secondary screening assays. A series of validation assays are proposed to determine if the reporter assays are appropriately regulated, and such assays will be adapted for high throughput screening at the NIH Chemical Genomics Center.

DESCRIPTION (provided by applicant): Myelination of axons in the nervous system is critical for not only conduction of action potentials, but also for providing tropic support to ensure long term survival of neurons in both the central and peripheral nervous systems. Myelin disorders are a major cause of neurological disease, and can be caused by genetic disorders, infectious disease, and inflammation. Therefore, understanding the pathways that control gene expression patterns in myelinating cells is a critical step in not only elucidating developmental pathways, but also to provide insight into means by which remyelination after nerve injury can be accelerated. The genetic control of myelination has been a major focus of research, and critical transcription factors and their target gene networks have begun to be elucidated. Interestingly, recent studies have demonstrated that formation of myelin-and it longs term maintenance-depends upon not only gene activation, but also downregulation of genes that inhibit myelin formation. Although substantial progress has been made to identify gene expression changes that coordinate myelination, there have been relatively few studies examining the chromatin modifications required for myelination. For example, myelin maintenance depends upon a program of gene repression, but practically nothing is known regarding the role of histone/DNA methylation in this vital aspect of myelination. The long term objective of our laboratory is to elucidate an integrated mechanism of myelination based on critical genetic and epigenetic switches required for myelin formation and maintenance. Specifically, this proposal focuses on testing the involvement of the polycomb epigenetic pathway in peripheral nerve myelination. Chromatin immunoprecipitation analyses will be used to determine the developmental regulation of epigenetic markers in response to injury and aging. The analysis will focus on epigenetic changes that occur in gene loci that are repressed during the myelination process, and test for the first time the involvement of the polycomb pathway in formation and long term maintenance of myelin. Finally, this proposal also takes advantage of several unique aspects of peripheral nerve, which facilitate the epigenetic analysis that we have proposed here.

DESCRIPTION (provided by applicant): Hereditary peripheral neuropathies (also known as hereditary motor sensory neuropathies, HMSN) are among the most common genetic diseases affecting the nervous system. The mildest form of human peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, causes progressive deterioration of both motor and sensory nerves, muscular atrophy, and chronic pain/fatigue in affected individuals. A majority of inherited peripheral myelinopathies are caused by duplication of a critical myelin gene, Peripheral Myelin Protein 22 (PMP22), which is classified as CMT1A. Since this disorder results from gene dosage effects, achieving a slight (<2-fold) reduction in PMP22 expression would effectively treat this inherited myelinopathy. Recent proof-of-principle studies using candidate compounds to reduce PMP22 expression levels have shown beneficial effects in rodent models of CMT1A. Before such candidate compounds enter clinical trials, it will be critical to achieve a comprehensive understanding of their molecular targets and how they impact PMP22 regulation. Our recent studies of PMP22 regulation have elucidated novel regulatory elements controlled by two major regulators of Schwann cell development-Egr2/Krox20 and Sox10-and the goal of this proposal is to test the function of these elements by using genome editing to delete them in the endogenous PMP22 locus. In addition, we will use zebrafish analysis to elucidate the developmental control of these enhancers. This proposal will also explore the function of cooperating transcription factors that amplify PMP22 expression to the high levels found in myelinating Schwann cells. Finally, using the mechanistic analysis that we have developed, we propose to identify the molecular targets of proteasome inhibitors, which are a candidate treatment for CMT1A as they have been recently identified in a drug screen to lower the expression level of PMP22.

Waisman Center investigators study Intellectual and Developmental Disabilities (IDD) through elucidation of molecular pathways that are altered in genetic and epigenetic conditions affecting nervous system function. Compared with other genetically tractable model organisms, rodents afford the highest similarity to humans in anatomy, physiology, and genetics. Thus, study of IDD has relied on development of rodent models of various disorders. In recent years, complementary development of human stem cell-based models to study similar processes has greatly expanded the scope and relevance of studies of IDD. Investigators of the Waisman Center have spearheaded research in a number of IDD conditions, and many investigators rely on complementary mouse and human stem cell models to study different facets of these disorders. Use of these models provides the means to evaluate candidate therapeutics, as well as develop novel screens to identify promising compounds. The resources of the IDD Models Core, almost all housed within the Waisman Center itself, provide the tools for development of mouse and human stem-cell derived models of IDD. Therefore, we propose two specific aims for the next project period. Aim 1 is to create and analyze rodent models of IDD. The objectives of this aim are to: 1) provide advanced technical services in the generation, cryopreservation, and maintenance of mutant or genetically engineered strains of mice and rats; 2) provide advanced behavioral testing services and facilities for the phenotypic characterization of novel strains of rodents; 3) provide training and consultation to investigators and laboratory personnel; and 4) facilitate maintenance and exchange of unique rodent models between institutions. Aim 2 is to to develop human stem cell models of IDD and facilitate phenotyping of neurodevelopmental defects. The objectives of this aim are to: (1) support the generation of induced pluripotent stem cell (iPSC) lines directly from individuals diagnosed with IDD; (2) support development of IDD-specific PSC lines through genome editing; (3) provide high quality, cost effective, cellular and molecular neuroscience technology and expertise to IDDRC investigators to facilitate phenotyping of neurodevelopmental defects in human PSC derived cells and (4) provide training and technical support in human stem cell culture, phenotyping and gene editing to promote usage of new approaches by our investigators. These initiatives are further served by state-of-the-art imaging and molecular biology analysis tools housed within the Waisman Center. With support from the Administrative Core, linkages are provided to complementary capabilities elsewhere on campus, such as those offered by the UW Biotechnology Center, UW Comprehensive Cancer Center, and other partnerships. These resources and partnerships are leveraged to facilitate usage of a large repertoire of techniques to enhance multidimensional analysis of IDD models, and to promote the development and testing of potential therapeutics.

Abstract Myelination of axons in the nervous system is critical for not only conduction of action potentials, but also for providing tropic support to ensure long term survival of neurons in both the central and peripheral nervous systems. Myelin disorders are a major cause of neurological disease, and can be caused by genetic disorders, infectious disease, and inflammation. Therefore, understanding the pathways that control gene expression patterns in myelinating cells is a critical step in not only elucidating developmental pathways, but also to provide insight into means by which remyelination after nerve injury can be accelerated. The peripheral nervous system has substantial plasticity in being able to regenerate after nerve injury, and critical transcription factors and their target gene networks have begun to be elucidated. Interestingly, recent studies have demonstrated that Schwann cell reprogramming to a new differentiated state is a critical and rate limiting factor in peripheral nerve regeneration. Although substantial progress has been made to identify gene expression changes that occur after injury, there have been relatively few studies examining the chromatin modifications required for Schwann cell reprogramming to the injured state. The long term objective of our laboratory is to elucidate an integrated mechanism of Schwann cell reprogramming after nerve injury based on critical microRNA and epigenomic switches that we have identified. Specifically, this proposal focuses on testing how reversal of the polycomb pathway is required for Schwann cell responses to peripheral nerve injury. Chromatin immunoprecipitation analyses will focus on epigenetic changes that occur during nerve injury, and test for the first time the involvement of histone demethylases in nerve injury responses. Finally, this proposal also takes advantage of several unique aspects of peripheral nerve, which facilitate the epigenomic analysis that we have proposed here.

The IDD Models Core is designed to foster development and analysis of human stem cell and rodent models of IDD conditions. The core supports basic science studies of molecular pathways and cellular interactions that are altered in genetic and epigenetic disorders affecting nervous system function. In addition, the core enables drug and biomarker studies, as well as preclinical studies that are aimed at developing novel translational approaches to IDD. A historic strength of the IDD Models core is the use of human pluripotent stem cell (hPSC)-based models, which include both patient-specific induced pluripotent stem cells (iPSCs) and their modification by genome editing, to study diverse types of IDD conditions. The core has incorporated novel approaches to meet the increased demands for rigor and reproducibility by meeting international/peer institution standards in the creation and characterization of patient-specific iPSCs and hESCs. The refined gene editing capabilities develop innovative cellular tools for sophisticated analysis of altered nervous system development and function. These models also provide the means to evaluate candidate therapeutics in disorder-relevant cells, as well as develop novel screens to identify promising compounds in small- and large-scale library screening. The core also empowers analysis of rodent models of IDD conditions, not only by providing support for their creation, but also by providing a comprehensive and cutting edge behavioral testing service. The service incorporates a battery of cognitive/nervous system analyses under the supervision of a skilled core manager to ensure the rigor and reproducibility of these IDD model studies. Our specific aims for the next project period are: (1) to create and analyze human stem cell models of IDD; and (2) to create and analyze rodent models of IDD. Recognizing that sophisticated analysis of these models is required to elucidate pathogenesis and novel therapeutic approaches, the IDD Models Core uses extensive on-site research resources and leverages an extended network of complementary research cores on the UW- Madison campus to provide the infrastructure and skilled personnel that support integrated analysis at molecular, genomic, epigenomic, cellular, electrophysiological, and behavioral levels. Assembly of these diverse resources is designed to broaden the scope and depth of IDD model studies and to facilitate progress of IDD research from basic science to translational studies.