Jean de Vellis - US grants

The broad objective of the proposed research is to determine the natural history of lysosomal enzymes in normal cells from patients with genetic diseases. This research is designed to deduce the mechanisms by which cells selectively internalize hydrolytic enzymes and package them into lysosomes. The cell surface receptors for lysosomal hydrolases will be purified and characterized. Anti-receptor antibodies will be raised and used to follow the endocytosis of receptor and define the mechanisms of receptor recycling. Lysosomal organelles, residual bodies and GERL, will be isolated from cultured human fibroblasts. Lysosomal membrane will be analyzed for lipid and protein content and compared to membranes of cells from patients with lysosomal abnormalities. Lysosome fusion will be examined in vitro to detect membrane and enzyme exchange. Model systems will be developed with purified lysosomes and artificial vesicles called liposomes, to establish the chemical requirements that control organelle fusion.

In recent years, glial precursor cells surviving in cultures from neonatal rat optic nerve and forebrain have been partially characterized by cell morphology and immunocytochemical markers as they progress along their developmental trajectories. As a result, glial progenitor cultures have become a valuable model of cellular development and differentiation. A number of growth factors have been identified that regulate glial development. These extracellular signals activate a variety of intracellular pathways that can lead to proteins or protein complexes binding to target genes. Thus, extracellular signals triggering intracellular cascades can lead to a selected pattern of transcriptional events and gene expression during cellular differentiation. Recently, an entire family of genes, many encoding for nuclear proteins, has been identified which are induced with very rapid kinetics. Since these immediate-early or early response genes (ERGs) are regulated by the same growth factors which control the proliferation and differentiation of glial cells, we suggest they may be important components of the molecular mechanism that coordinates the regulation of specific genes necessary for normal glial growth and differentiation. We propose utilizing our laboratory's expertise in the molecular and biochemical analysis of glial development to study the pattern of early gene expression in cultures of glial progenitor cells that are progressing along a specific developmental lineage. This research will provide important data concerning the responsiveness of both developing and regenerating glial cells to extracellular signals. As such, our understanding of neural development and repair will be enhanced and potentially lead to new therapeutic approaches to neurodegenerative diseases.

The rapid advances in molecular biology and cell culture techniques have expanded our understanding of the mechanisms regulating cell growth and differentiation. At the same time, our ability to apply molecular biology techniques and reagents developed in vitro to the in vivo, cellular environment have lagged behind. Most studies have relied upon either vehicle-mediated transfer or facilitator-aided transport. These standard procedures work well for many cell lines, but provide little control over dose, require large numbers of cells and tend to perturb cell physiology. The advent of semi-automatic microinjection of cells in culture represents a technological bridge linking the most advanced techniques of molecular biology and cell culture model systems. In microinjection, the computer controlled movements of the glass micropipette penetrate the cell membrane to directly deliver to the cytoplasmic or nuclear compartment a wide variety of reagents by controlled volume displacement. Many of these reagents would not be able to normally enter the cell and reach their target molecules or the transfer method would require a large number of cells and in many cases, affect cell metabolism and phenotypic response. Thus, microinjection allows rapid and easy transfer of specific quantities of antibodies, proteins, RNA or DNA molecules. The automatic, computer controlled micromanipulation procedure permits the injection of up to 1-2000 cells per hour making possible reliable statistical evaluation of cell responses to injected reagents. This instrumentation will permit functional analysis of critical molecular processes that govern cell proliferation and differentiation. By directly manipulating levels of cellular macromolecules, it will be possible to unequivocally demonstrate causal relationships. We are proposing to set up at UCLA a Cell Microinjection System Core Facility that will serve the needs of an initial group of 9 cellular and molecular biology research groups. The enhancement of research through the application of this shared instrument is described in nine research projects.

DESCRIPTION (provided by applicant): This application is for continued support of the administrative and research cores of the UCLA Mental Retardation Research Center (MRRC). This MRRC was first established in 1969 and housed with the Department of Child Psychiatry in a four-floor addition to the Neuropsychiatric Institute. It will move next March to a new five-story neuroscience research building. This MRRC provides a comprehensive multidisciplinary research program in the field of mental retardation and developmental disabilities (MRDD). The investigations carried out by the Center investigators address 26 of the 31 MRRD research priorities listed in the current RFA issued by NICHD for MRDD Research Centers. The long-term goals of the Center are to discover ways to prevent mental retardation, and to improve the quality of life for mentally retarded and other developmentally disabled individuals. To accomplish this, four multidisciplinary research groups have been organized: Clinical Research, Cellular and Molecular Neuroscience and Neurogenetics, Systems Neuroscience, and Socio-Behavioral Research. To foster research innovation through interdisciplinary interactions among investigators, five cores have been organized: (A) Administration and Communications, (B) Neuroscience and Imaging, (C) Animal Models, (D) Functional Neurogenomics and Bioinformatics, and (E) Fieldwork Training and Qualitative Data. In addition, this application proposes support for a New Program Development project. Each of the cores is designed to offer investigators an integrated approach to support their multidisciplinary research projects from experimental design to data management and publication. This is achieved by the direct involvement in each core of several faculty and staff with expertise in the relevant scientific disciplines and techniques, working as a team rather than as a single provider of a technique. The mission of the Center is to provide an environment promoting the highest level of research in MRDD by fostering interaction among investigators and providing open access to cutting edge and efficient core services. The Center assigns high priority to the support of talented young investigators and the training of pre- and post-doctoral fellows in a variety of disciplines related to its goals. The Center's mission is facilitated by strong tie with faculty in more than ten departments and institutes at UCLA, excelling in neuroscience, genetics, imaging, molecular biology, psychology, psychiatry, neurology, neurosurgery, pharmacology, pediatrics, education, anthropology, and others. While the focus needs to be still on the molecular, cellular, and behavioral mechanisms of MRDD, the aim is to collaborate with MRDD scientists worldwide to translate this fundamental knowledge into novel diagnostic, preventive, and therapeutic approaches which can be ultimately applied to clinical care.

tissue /cell culture; mental retardation; biomedical facility; microinjections; organ culture; neurons; Schwann cells; oligodendroglia; glioma; microglia; PC12 cells; clone cells; cell line;

It is well known that growth factors, neurotrophins, cytokines and hormones regulate glial cell function. Alterations in the expression of these factors results in serious changes in cell morphology and molecular expression during CNS injury and disease. The number of factors to be considered is overwhelming. We have chosen to focus on those that we consider to be imperative for glia development and function. Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the etiology of many CNS disorders. It is thought to be involved in both beneficial and cytotoxic actions within an organism: however, little is known with respect to receptor expression. Our proposed studies, therefore, will phenotype the cell population(s) that express TNF receptor in the CNS. Reactive astrocytes and activated microglia are hallmarks of many CNS disorders. The mechanisms that regulate their activation are poorly understood. however, studies in our laboratory have highlighted two cytokines, ciliary neurotrophic factor (CNTF) and TNF-alpha, as playing a pivotal role. We propose therefore, to further examine the involvement of these cytokines in the initial stages of the inflammatory disease process. Platelet-derived growth factor-AA (PDGF- AA) is known to support oligodendrocyte (OL) survival and proliferation. In vivo data suggests OL compete for a limited amount of trophic factor and that PDGF may be a rate limiting factor in determining the number of OL that survive. Recent studies in our laboratory have shown that only cells early within the OL lineage express the PDGF-alpha(r). These findings are extremely important because they reveal that the main cell population in the CNS that is capable of responding to PDGF-AA is the OL: therefore, suggesting a possible therapeutic role for PDGF-AA in demyelinating disorders. We propose to test this hypothesis by providing experimental allergic encephalomyelitis (EAE) mice. mice inflicted with a CNS inflammatory and demyelinating disorder. with PDGF-AA growth factor treatment. Another growth factor that is known to enhance OL survival is neurotrophin-3 (NT-3). Recent studies in our laboratory support the presence of a biologically functional TrkC receptor in cells of the OL lineage. We plan to utilize normal, NT-3 and TrkC knockout mice to determine the potential roles and molecular mechanisms of NT-3 action on glia survival, proliferation and apoptosis in vivo and in vitro. It is well known that CNS disorders resulting from a variety of insults often demonstrate common injury responses. This overlap is observed in many adult and pediatric disorders associated with mental retardation, AIDS neuropathy, ischemia/anoxia, MS, leukodystrophies, head trauma, epilepsy and encephalitis. Our goals will attempt to understand the complex interactions of these factors during CNS development, disease and the injury response. It is our belief that some of these factors are essential during gliogenesis and glial recovery; while others play a detrimental role when expressed at high concentrations during CNS disease.

The success of neurobiological research depends upon the availability of suitable simple systems for the study of complex problems. Injury response in the CNS clearly involves a complex and interactive chain of events which impact upon multiple cell types. In the CNS, oligodendrocytes perform two major functions critical for proper development and viability of the organism throughout life. These two functions are myelination and iron homeostasis. Our previous work showed that oligodendrocytes are highly sensitive to environmental changes that make them extremely vulnerable to various forms of injury or diseases. Oligodendrocyte's plasticity, discovered largely in vitro as well as in vivo studies during the last 15 years, allows us to consider yet unexplored general aspects of the modulation of the injury caused by the disruption of iron homeostasis. Oligodendrocytes synthesize and secrete transferrin, an iron transport glycoprotein, that acts as a trophic and survival factor for the various cell types in the CNS, and as an autocrine differentiation actor that may affect the myelination/remyelination process. Here, we propose to further investigate the regulatory region of the rat Tf gene by means of in vitro and transgenic studies. Having demonstrated the translocation of Tf into the nucleus of maturing oligodendrocytes (but not of other neural cells), we propose to elucidate its putative role as a transcription factor. We will examine the mechanisms involved in this phenomenon, and identify putative target genes. The elucidation of these issues will provide new insights on the modulation of injury response. We propose to study CNS repair particularly the process of remyelination by using progenitor cell grafting. This system allows grafted cells to migrate, integrate and myelinate within the host parenchyma. We propose to establish and characterize human oligodendrocytes and use our transplantation system to study their behavior upon grafting. We feel that these multi-disciplinary studies will contribute to a better understanding of glia, CNS injury and repair. More than sixty inherited white matter disorders have been identified and many are associated with mental retardation and inflammation. Furthermore, iron deficiency is a major problem in the world that affects myelination and results in developmental and cognitive dysfunction. These studies will provide fundamental knowledge that can be applied to develop new modalities to simulate CNS regeneration.

CELLULAR AND MOLECULAR NEUROSCIENCE AND NEUROGENETICS Jean de Vellis and Anthony Campagnoni, Group Coordinators During the last decade, the research groups headed by Jean de Vellis and Anthony Campagnoni have increasingly taken similar approaches to investigate the underlying mechanisms directing the normal development of the nervous system as well as the pathogenesis causing developmental dysfunctions, i.e. mental retardation and developmental disabilities (MRDD). They use genetics, molecular biology, and cellular and molecular neuroscience to analyze mechanisms in genetically-engineered animal models of human developmental disorders. The majority of MRRC researchers throughout this group are interested in the development of the nervous system. Another reason to present the Campagnoni and de Vellis groups together is that they will move to the third floor of the Neuroscience Research/MRRC Building in early spring. They have set up shared governance to utilize large shared laboratories, procedure rooms, vivarium, and a large linear equipment room. Therefore they expect to generate more collaborations in their efforts to elucidate the neurobiology and genetics of developmental disorders. Some of the geneticists are already located in the nearby Department of Human Genetics. One of the common research interests is the study of stem cells, an area relevant to the recent California initiative that will hopefully supplement funding. Harley Kornblum, the director of the budding Neural Stem Cell Center has been recruited as an MRRC core faculty and Psychiatry and Biobehavioral Sciences will become his primary department. Kornblum joins Sun, Fan, Waschek, de Vellis and Cederbaum who have major interest in stem cells for their role in development and MRDD. The research progress for these two groups is listed alphabetically by investigator.

DESCRIPTION (provided by applicant): This application is designed to provide for didactic and research training for four predoctoral and six postdoctoral trainees annually in the problems and methods of interdisciplinary research in mental retardation and developmental disabilities (MRDD). Training will primarily take place in laboratories and field settings of the six major research groups at the UCLA Mental Retardation Research Center (MRRC): Molecular and Developmental Neuroscience, Neurobiochemistry and Molecular Genetics, Systems Neuroscience, Plasticity and Behavior, Sociobehavior, and Developmental Disabilities Clinical Research. MRRC faculty members approach problems in MRDD from a number of different disciplinary viewpoints supported by nine scientific and service core facilities funded by an NICHD Center Core Grant as well as University support. This problem-oriented and interdisciplinary approach will be emphasized in the formal didactic training program. The MRRC faculty has undergone an extensive renewal during the last several years, a number of them retired or left, and thirteen new faculty members were recruited to the MRRC from outside or from campus departments. The three highest priority areas for development at UCLA School of Medicine are: neuroscience, human genetics, and aging. A Department of Human Genetics has been formed, and a new Neuroscience and Human Genetics Research Building has opened. These will provide for enriching the MRRC research and training relevant to MRDD. A designed program of classes and seminars will serve 1) to introduce the research trainees to the nature and range of problems in the area of mental retardation and developmental disabilities; 2) to facilitate trainees with access to the ideas and methods of disciplines other than their own; 3) to provide forums for initiating cross-disciplinary discussion, and 4) to enable trainees to extend their knowledge in their own fields of specialization. Postdoctoral candidates must have Ph.D.s in relevant discipline or have M.D.s and intend to devote their careers to biomedical research. Predoctoral candidates will have been accepted as graduate students in Ph.D. programs in which members of the MRRC are affiliated. Predoctoral candidates will have completed one or more years of graduate education. It is anticipated that trainees will pursue research careers that focus on mental retardation or related developmental problems. They will assume positions in medical schools, in research institutes and hospitals, and in traditional academic departments.

DESCRIPTION (provided by applicant): Abstract Project Summary: The objective of our research is to elucidate the role of myelin gene mutation on oligodendrocyte (OL) cell maturation and ensheathment of axons, failure of which results in hypomyelination that leads to leukodystrophy. Canavan disease (CD) is an example of such leukodystrophy which is caused by a metabolic mutation in aspartoacylase (ASPA) gene. Now recognized as a marker for OL cells, ASPA metabolizes N-acetyl aspartate (NAA) to release acetate and aspartic acid. The pathophysiology of this childhood disorder includes a spongy white matter morphology, mental retardation, megalencephaly, NAA aciduria, motor deficit, and early death. Because young children affected by Canavan disease display hypomyelination, the question arises whether this early event is indicative of OL dysfunction. An ASPA knockout (KO) mouse strain lacks expression of ASPA protein and enzyme activity, and closely resembles symptoms of human CD. Our preliminary work has shown that the ASPA knockout OLs exhibit lack of mature myelin basic proteins and lack of immunostaining of myelin proteins in cell processes in the CNS, even though presence of transcripts for myelin genes were identified. This suggests that expression of ASPA is critical during early stages of development for the maturation of OLs. In addition, a massive apoptotic cell death occurs in the CNS of ASPA knockout mice. Gene therapy was approved to another group of researchers, who administered an adenoviral-associated viral (AAV)-mediated ASPA gene therapy in a cohort of CD children, and in rodent models of CD after the peak period of myelination. No improvement in spongy white matter morphology was detected. During early development, OL progenitors arise embryonically, and yet another OL lineage is generated post- birth to populate the forebrain. Therefore, we propose to target these two early populations of OLs for delivery of ASPA gene to induce OL maturation and function in the KO brain. The findings will help understand the role of ASPA in prevention of leukodystrophy and will be useful for future development of therapies. Abstract Narrative: Objective of this proposal is to restore aspartoacylase activity during the embryonic and post-natal period of mouse model of Canavan Disease (CD). We will demonstrate that activity of this gene is critically necessary for the normal development of the myelin forming cells and the ensheathment of axons that is essential for normal CNS function. If successful, information from this study could be used to develop appropriate therapeutic interventions for the treatment of children affected by CD, the most devastating disease that causes mental retardation and death.

Organizational Chart of Core A: Administration and Communication The Core has three components providing different services. This section describes the Administration and Translation/Education Components and the next section describes the Communication component.

Overall Objective and Rationale: The existing Neuroscience and Imaging Core has been reorganized and is now entitled Cell Biology and Cellular Imaging. The aim is to provide expertise, services and equipment to broadly support cellular and molecular neuroscience research which, in our Center, is focused on elucidating the mechanisms of pathophysiology associated with the genetic and environmentally-induced developmental diseases affecting CNS development and function. We upgraded tissue processing for histology and cellular imaging with stateof-the art instruments like the Zeiss Laser Scanning Confocal Microscope LSM 510 META and the Leica Laser Cell Capture Microdissection system. We also equipped 3 new cell culture rooms to support our expanding needs for neural stem/progenitor cell culture methodologies to study glial and neuronal cell biology and for transplantation studies. The histology component has now been eliminated, as similar services are now available from the BRI histology core. Although a new and separate Stem Cell Core is proposed to focus on human embryonic stem cells (Core C), the Cell Biology and Cellular Imaging Core will continue to support cell culture of rodent and human brain cell subpopulations, and will expand the cellular imaging component. The demand for the latter has grown dramatically, becoming particular heavily used by faculty working with animal models of developmental diseases. We will also initiate a new subcomponent to provide expertise in the area of cellular immunology. This is done with the growing recognition that immune function and/or inflammatory responses impact a great number of IDD, and is a critical of component of investigation in human studies as well as our many animal models. This new function is included in this Core because it utilizes cell culture and cellular imaging as principle tools, although the methodologies in many cases are highly specialized, for example, the use of FACS to examine and investigate the cellular and molecular character of the immune response.

DESCRIPTION: Provided by Applicant: This application is for continued support of the administrative and research cores of the UCLA Intellectual and Developmental Disabilities Research Center (IDDRC). This IDDRC provides a comprehensive multidisciplinary research program in the field of intellectual and developmental disabilities. Importantly, the research investigations carried out by the Center Investigators encompass 23 of the 31 IDD research priorities listed in the current P30 RFA. This Center maintains and sharpens its focus on discovering ways to prevent and treat IDD, and to improve the quality of life for intellectual and developmentally disabled individuals, by fostering research innovation into the pathophysiological mechanisms of developmental disorders in animal models and also by patient-based research through interdisciplinary interactions among investigators. Newly emerging areas of excellence at the IDDRC, which extensively combine studies of human IDD and animal models, include studies of fragile X syndrome, autism spectrum disorders, pediatric brain tumors, and pediatric epilepsy. To serve these and other areas of excellence, the cores have been reorganized to provide much greater support for translational research while expanding cutting edge technology for the basic sciences. The eight cores are: (A) Administration and Communication, (B1) Neurogenomics and Bioinformatics and (B2) Epigenetics, (C) Stem Cells, (D) Cell Biology and Cellular Imaging, (E) Animal Models, (F) Electrophysiological Assessment, and (G) Translational Core for Human Phenotyping and Imaging. These cores are strengthened by the expertise of the increased number of IDDRC faculty involved with the cores, and the increased synergy among them. The mission of the Center is to provide an environment promoting the highest level of research in IDD by providing investigators open access to cutting edge and efficient core services in the IDDRC. The Center also organizes seminars, mini-symposia, an annual retreat, and core-based workshops to foster cohesive scientific discourse and collaboration. The Center assigns high priority to the support of talented young investigators and the training of pre- and postdoctoral fellows in a variety of disciplines related to its goals. The Center's mission is facilitated by strong ties with faculty in more than ten departments and institutes at UCLA excelling in neuroscience, genetics, human and animal imaging, nanotechnology, molecular biology, psychology, psychiatry, neurology, neurosurgery, pathology, pharmacology, pediatrics, education, and anthropology. The Center has recently begun interactions with a new IDD program at the University of Southern California, and it has increased interactions with national and international IDD researchers to share resources and knowledge to accelerate research breakthroughs and their translation.

? DESCRIPTION (provided by applicant): This submission is a new application for the UCLA IDDRC, entitled UCLA Center for Translational Research in Neurodevelopment (UC-TRaN). The fundamental purpose of our Center is to provide an optimal environment for performing outstanding research into the causes, mechanisms, and treatments of intellectual and developmental disorders. The new leadership model in the center emphasizes collaborations among Center investigators, core personnel, and community partners, with support for research spanning basic research and clinical practice. Our aims are 1) to create cutting edge and cost effective infrastructure resources in facilities and personnel for IDD investigators; 2) to provide integrated services within cores that support research from multidisciplinary teams and multiple levels of analysis; 3) to promote interactions between cores and among research investigators to promote collaborative, multidisciplinary and translational research, and 4) to create educational and outreach opportunities within the center, in collaboration with a West Coast Consortium of IDDRCs and community partners. We propose five interacting cores: 1) Administration, Education and Outreach; 2) Genetics, Genomics and BioInformatics, which performs genetic analysis, sequencing, expression, and provides Big Data resources; 3) Cells, Circuits and Systems analysis, supporting electrophysiology, human iPSC and cerebral organoid culture development, and optogenetics; 4) Structural and Functional Visualization, which provides access to and analysis of imaging tools from miniaturized microscopes through animal and human MRI; and 5) Clinical Translation, supporting clinical trial development, recruitment, phenotyping across species, and collecting biosamples. The Center proposes an exciting model research project, Neurophysiological biomarkers of cognition in Dup15 syndrome: From mouse models to patients. Dup15q11.2-13.1 is associated with marked cognitive impairment and other symptoms of developmental delay. The study examines a novel EEG biomarker in a cohort of children with Dup15q and in two separate mouse models of Dup15q syndrome, one defined by an overexpression of a single gene, UBE3A, and the other from a full duplication of the region. In the animal model the project will test three mechanism-based pharmacologic treatments: d-serine, rapamycin, and a GABA-A ?5 receptor subunit inverse agonist, to determine whether these treatments change cell activation patterns and behavior. Together, the project and cores support our primary goal of optimizing outcomes of individuals with intellectual and developmental disabilities by promoting outstanding translational research.