Albee Messing - US grants

Two groups of transgenic mice harboring the oncogene (T antigen) from SV40 virus have been produced which display distinct patterns of neuropathology. One group develops tumors in the choroid plexus; the second group develops a demyelinating/hypomyelinating peripheral neuropathy. The proposed study will characterize the pathology which develops in these transgenic mice in terms of the expression of the oncogene which was introduced. The developmental time courses of the choroid plexus tumors and the peripheral neuropathy will be studied morphologically and correlated with the temporal pattern of T antigen gene expression in various tissues. The peripheral neuropathy will be characterized by quantitative morphology. T antigen gene expression will be studied by in situ hybridization using cDNA probes to detect T antigen mRNA, and immunohistochemical localization of T antigen protein using polyclonal and monoclonal antibodies. Whether axons and/or Schwann cells are the primary defect in the peripheral neuropathy will be studied in vivo by transplantation of nerve grafts, and in vitro by recombinations of purified populations of neurons and Schwann cells.

Two groups of transgenic mice harboring the oncogene (T antigen) from SV40 virus have been produced which display distinct patterns of neuropathology. One group develops tumors in the choroid plexus; the second group develops a demyelinating/hypomyelinating peripheral neuropathy. The proposed study will characterize the pathology which develops in these transgenic mice in terms of the expression of the oncogene which was introduced. The developmental time courses of the choroid plexus tumors and the peripheral neuropathy will be studied morphologically and correlated with the temporal pattern of T antigen gene expression in various tissues. The peripheral neuropathy will be characterized by quantitative morphology. T antigen gene expression will be studied by in situ hybridization using cDNA probes to detect T antigen mRNA, and immunohistochemical localization of T antigen protein using polyclonal and monoclonal antibodies. Whether axons and/or Schwann cells are the primary defect in the peripheral neuropathy will be studied in vivo by transplantation of nerve grafts, and in vitro by recombinations of purified populations of neurons and Schwann cells.

DESCRIPTION (Adapted from applicant's abstract): Glial fibrillary acidic protein (GFAP) is one of the major structural proteins in astrocytes, and its expression is markedly up-regulated following injury. To explore the role of GFAP in astrocytes, during the previous funding period the investigator generated knockout and transgenic mice that are either completely deficient in or express elevated levels of, GFAP. The GFAP-null mice display only subtle alterations of astrocyte morphology, and are relatively normal unless stressed by disease or trauma. In contrast, the GFAP over-expressing mice develop astrocytes that are enlarged and contain complex inclusion bodies identical to the Rosenthal fibers seen in Alexander's disease. Mice in high expressing lines died before weaning. The phenotype of GFAP over-expressing mice raises important questions about how Rosenthal fibers form and how they affect astrocyte function. In addition, these results lead to a specific prediction regarding a genetic basis for Alexander's disease. During the next funding period , the investigator proposes to further explore the significance of GFAP over-expression by the following set of experiments. In specific aim 1, he will identify the molecular requirements for Rosenthal fiber formation by crossing the GFAP transgene into mice that are unable to express the endogenous mouse GFAP, vimentin, or alphaB-crystallin (three of the major components of Rosenthal fibers). In addition, he will determine whether it is over-expression per se, or the expression of two distinct GFAP polypeptides (mouse and human), that is responsible for Rosenthal fibers. In specific aim 2, he will test the hypothesis that Alexander's disease in humans results from a primary abnormality in the GFAP gene, by analyzing genomic DNA from patients and controls for mutations and gene duplications. In specific aim 3, he will test the hypothesis that formation of Rosenthal fibers in astrocytes is associated with fundamental physiological changes in astrocytes and in neuron-glial interactions.

Islet cell transplantation offers many theoretical advantages for the treatment of insulin-dependent diabetes compared to current modes of therapy. However, one of the main limitations to widespread use of islet cell transplantation is the difficulty of separating islets from contaminating cell populations, and the harshness of the methods sued for isolation which consequently compromise islet viability and yield. In the course of studies on inducible cell ablation in the central nervous system of transgenic mice, we stumbled upon an unexpected property of our transgene (GFAP-TK) which produces nearly complete ablation of pancreatic acinar cells when the mice are treated with ganciclovir. The resulting acinar-depleted pancreas consists largely of islets floating in loose adipose connective tissue. If this treatment can be optimized to completely eliminate acinar cells from the pancreas without harming islet cells, and it allows simpler and milder isolation procedures, it would justify continued efforts to generate transgenic pigs for use as uniform donors. As a pilot project to demonstrate feasibility of this approach, we propose two specific aims. The first is to further optimize ablation protocols in the existing transgenic mice, and evaluate the functional state of islets during this ablation process by immunohistochemical and clinical laboratory studies. We will also determine the mechanism by which acinar cells are killed, and generate newer transgenic mice using the acinar-specific elastase promoter that should have even better properties than the GFAP-TK mice. In the second aim we will harvest islets from the acinar-depleted pancreas and demonstrate their functional capacity in vivo by transplanting into streptozotocin-treated recipients to correct diabetes. If these transgenic mice offer the expected advantages for islet isolation, it suggests a novel approach for using transgenic animals in xenotransplantation.

DESCRIPTION: The objectives of the Pathology, Histology, and Stereology Core are to provide 1) pathological evaluation including morphological assessment and HPLC as well as other biochemical measurements, 2) histological services such as in situ RNA analysis and laser capture microdissection of tissues, and 3) stereological analysis to quantitatively assess cell numbers and of tissue specimens for the Center.

Alexander disease is a rare, fatal degenerative disease, classified among the leukodystrophies because of the severe hypomyelination seen in young infants or the demyelination seen in older children. The pathological signature of the disorder is the Rosenthal fiber, an accumulation of intermediate filaments and small heat shock proteins in astrocytes throughout the CNS. Sequencing the GFAP gene revealed single base changes in the coding region, predicting, non-conservative amino acid substitutions, in 12 of 13 patients examined to date. All mutations are heterozygous, suggesting a dominant, gain-of-function mechanism. Alexander disease therefore represents the first example of a primary genetic disorder of astrocytes, one of the major cell types in the vertebrate central nervous system. The goals of this Program Project are to investigate the means by which GFAP mutations lead to inclusion bodies, disruption of the astrocyte cytoskeleton, astrocyte, dysfunction, and severe consequences for oligodendrocytes in the central nervous system. We will continue genetic studies of Alexander disease patients with unusual clinical presentations to clarify the range of disorders associated with GFAP mutations; develop animal models carrying the same mutations as those identified in humans; and explore potential approaches for interfering with the effects of the mutant protein. Our studies span molecular, biochemical, cellular, and morphological approaches to these questions. The Program will link four laboratories; three of these already have a proven record of productive interactions, and a fourth group will bring unique expertise in studying filament assembly. The Program will promote an expanded effort on the role of glial filament dysfunction in disease, by fostering sharing of reagents, animals, and results between the four labs, cross-fertilization of ideas, and regular communication and meetings among laboratory members. These studies promise novel insights into the role of glial filaments in the cell biology of astrocytes, and the role of astrocytes in brain function and disease.

[unreadable] DESCRIPTION (provided by applicant): Alexander disease (AxD) patients carry heterozygous mutations within the coding region of GFAP. To facilitate mechanistic studies on the pathogenesis of AxD, and provide animal models suitable for testing potential therapies, our long-term goal has been to generate mouse models for this disorder. We have now generated knock-in lines of mice carrying several of the most common GFAP mu-tations found in human AxD (equivalent to R79H, R239H, R239C, and R416W), and found that expression of the mutant GFAPs induces Rosenthal fibers (the hallmark pathologic feature), but the mice are viable. Moreover, expressing the mutant GFAPs in the context of appropriate genetic modifiers (such as elevating wild type GFAP) results in a lethal phenotype. In addition, altering GFAP expression either by simple over-expression or production of mutant GFAPs leads to induction of multiple stress pathways (aB-crystallin, Nrf2) that suggest specific hypotheses about pathogenesis and, ultimately, strategies for therapy. With the goals of understanding more precisely the consequences of abnormal GFAP expression in astrocytes, and of generating mouse models that reflect key phenotypic features of AxD in humans, we propose the following specific aims: In Aim 1 we will test the consequences of expressing mutant GFAP protein and/or GFAP over-expression, for comparison with known features of the Alexander phenotype in humans. These studies will include both in vitro models using primary cultures of mouse astrocytes, as well as the in vivo knock-in models carrying point mutations in the endogenous mouse GFAP gene. A range of properties will be examined, including biochemical, functional, and behavioral. Reversibility will also be assessed. In Aims 2 and 3 we will test the roles of the small stress protein aB-crystallin, and the transcription factor Nrf2, respectively, as modifiers, by crossing the GFAP mice with knockouts or with newly generated transgenics that over-express the two genes. Specific mechanisms by which excess aB-crystallin or Nrf2 might achieve rescue will be evaluated. These studies promise novel information on the pathological significance of mutant intermediate filament expression in astrocytes, will suggest mechanisms by which primary astrocyte dysfunction leads to generalized CMS disease, and will identify critical stress pathways that could ultimately serve as the basis for therapeutic interventions to mitigate the devastating effects of this disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alexander disease is a rare and generally fatal disorder of the CNS that primarily affects young children. Recent genetic studies have identified heterozygous missense mutations in the astrocyte intermediate filament glial fibrillary acidic protein (GFAP) as the major cause of this disease. To begin a search for potential treatments of this devastating disease, the investigator proposes to screen a library of drugs that are already FDA-approved to identify compounds that suppress expression of GFAP. He will evaluate changes in GFAP expression at the level of both protein and gene transcription, in glioma cell lines (Specific Aim 1), primary cultures of astrocytes (Specific Aim 2), and organotypic explant cultures of spinal cord (Specific Aim 3). In future years using mice that express the same mutant GFAPs found in human patients (generated as part of another project) can then be tested candidate drugs identified as part of this screen. By restricting the focus to the FDA-approved list, successful identification of GFAP-suppressing drugs could offer immediate therapeutic benefit to Alexander disease patients.

[unreadable] DESCRIPTION (provided by applicant): The Waisman Center at the University of Wisconsin-Madison is one of thirteen NIH-funded centers focused on developmental disabilities and neurodegenerative diseases, with a broad mission that encompasses research, training, and clinical activities. Since its inception in 1973 the Center has included an animal facility as a key element of its infrastructure and one of its scientific core services, yet little has been done to upgrade the facility to allow it to keep pace with the needs of current science. During the past six years both internal and external reviews identified critical shortcomings in our animal facility that needed to be addressed. Eighteen months ago both the main and backup cage-washers failed and could not be repaired, forcing evacuation of the entire facility. We have formulated a comprehensive plan for renovation, the overall cost of which is approximately $2.7 million (the majority paid for by university and gift funds). The goal of this renovation is to support Waisman Center investigators and affiliates by 1) increasing capacity in what is now a rodent-only facility, 2) improving barrier functions through increased use of microisolation, purchase and installation of biohazard hoods for selected areas, purchase and installation of an on-site autoclave for support of immunodeficient animals, and creation of dedicated quarantine space, 3) upgrading infrastructure by replacing outdated equipment for cage washing, installing, a disposable pouch system for supplying drinking water, installing ceilings to improve environmental control, and repairing flooring throughout, 4) improving security by installation of video surveillance systems and card-access controls, and 5) creating a suite of rooms optimized for long-term behavioral studies that is separate from the barrier portion of the facility. [unreadable] [unreadable] [unreadable]

CORE A - ADMINISTRATIVE CORE - ABSTRACT The goal of the Administrative Core is to coordinate regular communication among all the Project Investigators, plan the periodic meetings of the investigators and the External Advisory Committee, prepare progress reports and grant renewals, and maintain financial accounting between the home institution (Wisconsin), the other participating institutions (Rochester and Brigham and Women's Hospital), and the NIH. An administrative assistant manages day-to-day Program business. This person will be responsible for grant accounting at Wisconsin, coordination with the consortium sites on grant accounting and regulatory compliance issues, collection of updated materials for progress reports and grant renewals, and coordination of biannual meetings of Project Investigators and the annual meetings with the External Advisory Committee. The administrative assistant also acts as liaison between the Program Director, Project Investigators and NIH grant management personnel. An Internal Advisory Committee (composed of senior administrative officials from each of the participating institutions) will advise on resolution of potential conflicts.

[unreadable] DESCRIPTION (provided by applicant): Alexander disease is a rare and typically fatal neurodegenerative disease that results from heterozygous mutations in the gene encoding the type III intermediate filament protein GFAP. The pathological signature of the disorder is the Rosenthal fiber, a cytoplasmic inclusion containing intermediate filaments and small heat shock proteins that accumulates in astrocytes throughout the CMS. Prior investigations by our groups have let to the acceptance of GFAP mutations as the cause for nearly all cases of Alexander disease, and the rapid translation of this information to clinical practice as the standard for diagnosis. However, the mechanisms by which GFAP mutations cause astrocyte dysfunction and disease remain unclear. Based on results obtained during the previous grant period we have developed a hypothesis to guide experiments that emphasizes expression of elevated levels of GFAP in concert with induction of a cellular stress response and formation of inclusions as key elements of pathogenesis. The goals of this Program Project are to investigate molecular mechanisms and functional consequences of the stress response, to explore biochemical composition and downstream effects of inclusion body formation, and to identify and characterize genetic modifiers of disease phenotype. Our studies span genetic, biochemical, cellular, physiological, and morphological approaches to these questions. The Program will link five laboratories; three of these continue from the previous grant period, and two new groups join that bring novel approaches and techniques (physiology and Drosophila). The Program will promote a focussed effort on the role of glial filament dysfunction in disease, by fostering sharing of reagents, animals, and results among the five labs, through cross-fertilization of ideas, and by regular communication and meetings among laboratory members. These studies promise novel insights into the role of glial filaments in the cell biology of astrocytes, and the role of astrocytes in brain function and disease. [unreadable] [unreadable] PROJECT 1 [unreadable] [unreadable] Principal Investigator: Michael Brenner [unreadable] [unreadable] Title: Biochemistry and Genetics of Alexander Disease [unreadable] [unreadable] Description (provided by applicant): Glial fibrillary acidic protein (GFAP) is a structural protein found almost exclusively in astrocytes. Our laboratory recently found that mutations in the coding region of the GFAP gene cause Alexander disease (AxD), a rare but usually fatal disorder of the central nervous system. This disease is characterized by the presence of protein aggregates which have GFAP as a primary constituent. The purpose of this proposal is to develop additional information about the mechanism by which the GFAP mutations cause AxD. In Aim 1 we will determine the composition of the RFs to obtain clues for the disease mechanism, an approach that has proved fruitful for other protein aggregate disorders. The aggregates will be partially purified, and their protein components identified by mass spectrometry. In Aim 2 we will determine if the GFAP is abnormally deiminated or phosphorylated, two modifications of GFAP known to affect its polymerization and to be present in other neurodegenerative disorders. In Aim 3 we will also determine whether the mutant form specifically accumulates in the aggregates. This will test the hypothesis that mutant GFAP is not toxic per se, but produces disease by causing GFAP accumulation. [unreadable] [unreadable] [unreadable]

INTRODUCTION The Rodent Models Core provides technical support for the generation and characterization of transgenic and knockout strains of mice, access to expensive behavioral equipment for mice and rats that can be shared among several investigators, and expertise in the behavioral phenotyping of mouse and rat models of MRDD and related disorders of the CNS. In addition, the Core is responsible for the care, feeding, and recordkeeping related to the use of mice and rats by MRDDRC projects and the maintenance of specific rodent models of development, developmental disabilities, and neurodegenerative diseases. This Core has been renamed during the present project period, reflecting a sharpening of its focus and considerable new expertise and services added during the past five years in order to be both responsive to the growing and changing needs of MRDDRC investigators and to contribute to the evolving scientific agenda of the MRDDRC. The space allocated to this Core is in the midst of a major renovation that, as of January 2005, resulted in the temporary relocation of all animals to other facilities on campus, with reassignment of the caretaker staff to those facilities. While renovation is taking place, Core services are continuing uninterrupted at those alternate locations. This description of the Rodent Models Core includes an explanation of how this Core will operate when the animals return in 2007, with newly trained staff who will operate this exceptional and fully modernized facility serving the diverse scientific needs of MRDDRC investigators using rodent models. In the description below, the present tense is used to portray resources and services that continue uninterrupted in temporary locations during the renovation, past tense is used to refer to improvements made prior to the renovation, and future tense is used to refer to new Core services that will be provided after the renovation is completed in 2007. Therefore, the description of the Rodent Models Core reflects the strengths of past accomplishments as well as the promise of future achievements.

DESCRIPTION: Alexander disease is a rare and typically fatal neurodegenerative disease of childhood that results from heterozygous mutations in the gene encoding the type III intermediate filament protein GFAP. The pathological signature of the disorder is the Rosenthal fiber, a cytoplasmic inclusion containing intermediate filaments and small heat shock proteins that accumulates in astrocytes throughout the CNS. Prior investigations by our group have let to the acceptance of GFAP mutations as the cause for nearly all cases of Alexander disease, and the rapid translation of this information to clinical practice as the standard for diagnosis. However, the mechanisms by which GFAP mutations cause astrocyte dysfunction and disease remain unclear. The goals of this Program Project are to develop novel model systems of human astrocytes, investigate the impact of mutant GFAP and GFAP excess in astrocytes on neuronal development, viability, and function, and study the pathways by which mis-folded proteins are cleared from the brain. In addition, we will identify and characterize genetic modifiers of disease phenotype and test potential strategies for treatment. Our studies span genetic, biochemical, cellular, physiological, and morphological approaches to these questions, and include model systems ranging from invertebrate through human. The Program will link four laboratories, one using human induced pluripotent stem cells, one using Drosophila, and two using mouse models. An administrative core will coordinate financial reporting, monitor IACUC approvals, and support communication between the groups as well as with the internal and external advisory committees. The Program will promote a focused effort on the role of GFAP in disease, by fostering sharing of reagents, animals, and results among the four labs, through cross-fertilization of ideas, and by regular communication and meetings among laboratory members. These studies promise novel insights into the consequences of GFAP toxicity and the role of astrocytes in brain function and disease. Our hope is that such studies will ultimately lea to strategies for mitigating the devastating effects of astrocyte dysfunction.

Alexander disease (AxD) is a homogeneous disorder from a genetic perspective, with over 95% of patients accounted by mutations in a single gene, GFAP, and no other genes currently associated with the disease. Despite this genetic homogeneity, patients exhibit a wide range of clinical phenotypes, with ages of onset ranging from fetal through the seventh decade of life, differing distributions of lesions, varying degrees of leukodystrophy, and mild to severe courses. Using cell culture and animal models we have found that a key factor in pathogenesis is the subsequent accumulation of GFAP protein above a toxic threshold. The accumulation of GFAP results from a combination of both increased synthesis and decreased degradation. By engineering mouse strains that are exact genetic mimics of the human disease, and placing the GFAP mutations on different genetic backgrounds, we have discovered marked differences between strains in the levels of GFAP accumulation, and corresponding measures of disease severity, that follow from expression of mutant GFAP. These data strongly suggest the presence of one or more genetic modifiers that influence the induction of GFAP in response to injury. In addition, results from our collaborators studying Drosophila models of AxD implicate nitric oxide as a key mediator of glial-neuronal toxicity, and one that is amenable to both genetic and pharmacological investigation. Recent experiments from our lab also demonstrate deficits in adult neurogenesis, a new phenotype that has not previously been studied in AxD. In Specific Aim 1 we will perform expression QTL analysis to identify genetic modifiers that regulate GFAP accumulation in AxD, with a focus on hippocampus and corpus callosum. In Specific Aim 2 we will test the hypothesis that one particular gene, encoding inducible nitric oxide synthase (iNOS), is a key modifier of the overall AxD phenotype in mice. In Specific Aim 3 we will determine whether the deficit in adult neurogenesis results from the expression of mutant GFAP in neural stem cells or mature astrocytes. These studies promise novel insights into the genetic influences on disease severity in AxD, will test the validity of a target that may be amenable to therapeutic interventions, and will identify the cellular origin of the newly identified deficit in adult neurogenesis.

DESCRIPTION (provided by applicant): We seek continued support for the Waisman Intellectual and Developmental Disabilities Research Center, a comprehensive interdisciplinary program on IDD spanning the biological, biobehavioral, and behavioral sciences. The Waisman IDDRC brings together 49 PIs from 25 academic departments from the UW-Madison Schools of Medicine and Public Health, Veterinary Medicine, Pharmacy, Agriculture and Life Sciences, Letters and Science, Education, 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; ad four innovative scientific core services: Research Participation (Core B), providing services, resources, and training in the recruitment of human participants, clinical assessment, and behavioral methods development; 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; Rodent Models (Core D), providing resources, expertise, and technical services in the generation, cryopreservation, and maintenance of mutant or genetically engineered strains of mice, and advanced behavioral testing facilities for phenotypic characterization; and Cellular and Molecular Neuroscience (Core E), providing cellular and molecular neuroscience technology, training, and expertise, and supporting the generation of iPS cell lines from humans with IDD conditions. We propose to provide core support to 95 research projects headed by 49 PIs organized into three research groups: Molecular and Genetic Sciences, Communicative and Cognitive Sciences, and Social and Affective Sciences. Collectively, the core services and research groups 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 serves as the liaison between the IDDRC and the UW-Madison, the NIH, and national and international organizations. The core functions to create a highly visible and comprehensive center that systematically integrates all aspects of research, training, and clinical services in the field of IDD at the University of Wisconsin-Madison. Messing and the previous Director, Marsha Mailick, 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 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 board, internal and external advisory groups, consultants, and visiting scientists. To obtain additional resources, the Administrative Core works closely with UW-Madison administration, 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, allocating support for special interest groups that involve IDDRC faculty from multiple disciplines who share a common area of focus (e.g., fragile X, epigenetics), provision of seed money for groups of investigators who seek to develop multi-component grant applications for interdisciplinary IDD research, funding for speakers in the Seminar Series, and creation of an environment of mutual respect for diverse disciplinary approaches to studying IDD conditions. Ultimately, the Administrative Core of the Waisman Center 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 cures or treatments for IDD conditions. Specific Aim 3 is to further connect research and clinical activities and strengthen community partnerships. The Administrative Core facilitates interactions between research and clinical activities within the Center, works to ensure access to individuals with IDD throughout the UW health care system and in community settings such as the public schools, and nurtures and maintains the relationships with key campus partners such as the CTSA, and with the Marshfield Clinic Research Foundation. The core maintains close connections with parent and patient groups as well as agencies and organizations active in the field of IDD.