Anthony T. Campagnoni - US grants

This is a proposal to engage in a collaborative research effort designed to carry out basic investigations directed toward an increase in information about mental retardation. The primary aim is to expand our knowledge rather than the development of practical applications, although we will not be blind to useful pragmatism. Specifically, we intend to: (1) explore in a close collaborative and cooperative fashion, areas of developmental biology which are relevant to mental retardation; (2) conduct further basic studies into etiology, pathogenesis, and prevention of mental retardation through the utilization of biomedical technology; (3) make available an attractive milieu for the training of young scientists with similar interests and increasing competence. A group of scientists and teachers primarily interested in biological and biomedical approaches to mental retardation has previously been assembled to form this program project. The direction of the investigation is via a time scale which includes the prenatal, perinatal, and postnatal periods; continues into adolescence and adulthood. The latter period is especially significant in both the disciplines of genetics and human gestational biology. Different levels of study to be undertaken and/or continued include genetics, biochemistry, ultrastructural, biomathematical, pharmacologic, behavioral and clinical. Together, the operatives in this cooperative program will direct their efforts toward a major public health problem.

The regulation of expression of the genes coding for myelin proteins is an important problem about which relatively little is known. The overall goal of this project is to examine the structural organization of these genes, beginning with those coding for the myelin basic proteins. In mice, four structurally-related myelin basic proteins exist and, although it is clear that four separate mRNAs code for these proteins, it is not yet known whether or not these proteins are specified by separate genes. With the availability of a cDNA probe to a part of the mouse myelin basic protein common to all four species, it is now possible to isolate MBP genes from a mouse genomic library and study their structure and organization. Two MBP clones have already been obtained which appear to be different from each other, suggesting that the mouse MBPs are coded for by more than one gene. These clones and the original cDNA clone will be used to determine the number of MBP genes in the mouse genome. Each gene will then be isolated from a genomic library and characterized by restriction enzyme analysis and sequencing of selected regions. The sequence information will be used to classify the MBP genes according to the type of MBP protein each codes for. The sequences will also be examined to identify potential regulatory sequences and splice sites and correlated with R-looping results and sequence data from cloned hybrid selected MBP mRNAs to identify introns. In the process of isolating and sequencing these genes, fragments of the genes will be subcloned and these fragments may prove to contain unique sequences which might be used as probes to discriminate the various MBP genes. The MBP cDNA probe will also permit the examination of the structure of the MBP genes in the dysmyelinating mutant mice, jimpy and shiverer. These two dysmyelinating mutants are defective in their ability to synthesize normal levels of the myelin basic proteins. Southern blots will be used to determine whether gross alterations exist in the structures of the MBP genes in these mutants. In addition, genomic libraries of the mutants will be prepared and the MBP genes will be isolated from these libraries and studied for sequence differences which may account for the mutant phenotype. In this way, structural alterations in the MBP genes or their controlling elements will be identified.

In view of the importance of the myelin sheath in the maintenance of a functionally normal brain, studies into the mechanism by which the membrane is formed are particularly important. Yet the mechanism of myelin assembly and the regulation of this process is an important neurobiological problem which is poorly understood. The overall objective of the proposed work is to examine, in detail, the expression of myelin basic protein (MBP) genes in normal mice and in dysmyelinating mutants which we have shown to have altered MBP metabolism. We propose to isolate and sequence cDNAs which correspond to the four mouse MBPs in order to establish the structural relationship among the 4 MBP mRNAs as well as the proteins themselves. Isolation and subcloning of the 4 MBP cDNAs will also provide probes with which to study MBP mRNA metabolism in the nucleus following transcription of the genes in normal and mutant mice. Several lines of evidence suggest that the MBP mRNAs are translated with different efficiencies and experiments are proposed to examine this in greater detail using inhibitors of specific steps of protein synthesis. Any differences in translational efficiencies will be related to structural differences noted in the primary sequences of the 4 MBP cDNAs. Since so little is known about MBP mRNA metabolism, experiments are also proposed to measure the levels of MBP mRNA precursors in the nuclei of normal and mutant mice, and to determine whether there occurs a build-up of precursor molecules in these mutants so that MPB mRNA does not reach cytoplasmic ribosomes. Finally using immunocytochemical and in situ hybridization techniques the mechanisms by which MBPs are transported from their sites of synthesis to their sites of assembly into myelin will be examined in normal mice and in quaking mice to determine where MPB assembly is blocked.

The regulation of expression of the genes coding for myelin proteins is an important problem about which relatively little is known. The overall goal of this project is to examine the structural organization of these genes, beginning with those coding for the myelin basic proteins. In mice, four structurally-related myelin basic proteins exist and, although it is clear that four separate mRNAs code for these proteins, it is not yet known whether or not these proteins are specified by separate genes. With the availability of a cDNA probe to a part of the mouse myelin basic protein common to all four species, it is now possible to isolate MBP genes from a mouse genomic library and study their structure and organization. Two MBP clones have already been obtained which appear to be different from each other, suggesting that the mouse MBPs are coded for by more than one gene. These clones and the original cDNA clone will be used to determine the number of MBP genes in the mouse genome. Each gene will then be isolated from a genomic library and characterized by restriction enzyme analysis and sequencing of selected regions. The sequence information will be used to classify the MBP genes according to the type of MBP protein each codes for. The sequences will also be examined to identify potential regulatory sequences and splice sites and correlated with R-looping results and sequence data from cloned hybrid selected MBP mRNAs to identify introns. In the process of isolating and sequencing these genes, fragments of the genes will be subcloned and these fragments may prove to contain unique sequences which might be used as probes to discriminate the various MBP genes. The MBP cDNA probe will also permit the examination of the structure of the MBP genes in the dysmyelinating mutant mice, jimpy and shiverer. These two dysmyelinating mutants are defective in their ability to synthesize normal levels of the myelin basic proteins. Southern blots will be used to determine whether gross alterations exist in the structures of the MBP genes in these mutants. In addition, genomic libraries of the mutants will be prepared and the MBP genes will be isolated from these libraries and studied for sequence differences which may account for the mutant phenotype. In this way, structural alterations in the MBP genes or their controlling elements will be identified.

In this application the hypothesis is being tested that the myelin basic protein (MBP) gene is more complex than originally thought, and that parts of the gene are expressed in mRNAs that do not encode MBPs. We have evidence for the expression of an mRNA in oligodendrocytes which is encoded by a gene that either overlaps or forms a part of the MBP gene. This MBP gene-Cl related product is expressed in oligodendrocytes (or their precursors) and in the spleen. In the oligodendrocyte, these mRNAs are expressed according to a different developmental pattern than that of the majority of MBP mRNAs. The overall goal of this project is to examine the structure and organization of this gene, to determine its expression in oligodendrocytes, and to deduce the role it plays during oligodendrocyte differentiation through an examination of its expression in both oligodendrocytes and cells of the immune system. We propose to complete the isolation, characterization and analysis of these mRNAs for which we currently have isolated only 40% of the entire message. The structure of the gene will be determined with respect to its exon/intron structure and its structural relationship to the MBP gene. Preliminary evidence indicates that the gene is expressed in either immature oligodendrocytes or their precursor cells. Accordingly, the cell type and the developmental stage at which oligodendrocytes express the gene will be determined. Experiments also will be performed to disrupt expression of the gene with antisense oligodeoxynucleotides and examine the subsequent differentiation of the oligodendrocyte. The ontogeny of expression of the gene will be examined in vivo and in vitro in several immunologically relevant tissues and cell lines by in situ hybridization to determine the stage of B cell and T cell development at which expression occurs. The hypothesis being examined in these experiments and those dealing with oligodendrocyte differentiation is that the gene plays some role in the differentiation of the oligodendrocyte. We believe that it may be possible to elucidate the role of this gene in oligodendrocytes through an examination of its expression in the immune system, where B cell and T cell differentiation is well understood.

The central aim of this proposed Program Project is to bring together individuals from a variety of disciplines to examine the expression of genes of neurobiological interest in order to understand their mode of action, regulation, and expression. It is clear that gene action can be regulated at a number of levels, which include transcription, RNA splicing, translation, and the post-translational processing/modification of proteins. It is also clear that agents such as hormones and growth/trophic factors, produced outside the nervous system (e.g. in the immune and endocrine systems) can regulate gene expression within the nervous system in important ways. Some of the consequences of this complex regulation by agents internal and external to the nervous system are: (a) the differentiation of precursors into cells with nervous system-specific functions, (b) proliferation and/or programmed death of groups of cells to give rise to specific anatomical and functional structures within the brain, and (c) the modulation of the production of important nervous system-specific proteins that result in modulation of cell function. A major goal of this proposal is to determine the mechanisms by which gene expression in neural cells is regulated in order to understand how function in the nervous system is regulated. Each project in this proposal is approaching the question of neural gene expression in the nervous system at a different level of investigation. The major objective of Project 1 will be to identify genes of importance in the functioning of the neostriatum, including putative genes that might involve Huntington's Disease. In Project 2, genomic elements that regulate the expression of two major myelin protein genes, myelin basic protein (MBP) and proteolipid protein, will be identified. In Project 3, elucidation of the multiple promoters of the MBP gene will be determined and the mechanism by which steroids influence the translation of the transcripts produced from those promoters will be determined. The focus of Project 4 will be to purify and clone a lymphocyte factor that induces the proliferation and differentiation of oligodendrocytes. In Project 5, the mechanisms by which steroids change the hypothalamic structure in the developing rat will be examined through the isolation and utilization of cDNAs specific for a sexually dimorphic nucleus in the brain. Collectively, these projects, which involve the cooperation and collaboration of the component research groups, seek to understand gene action at the molecular, cellular, and organismal levels.

Recent studies in our laboratory have shown that the 32 kb myelin basic protein (MBP) transcription unit is contained within an overlapping transcription unit, termed the Golli-mbp gene, which is approximately 105 kb in the mouse. The Golli-mbp transcription unit consists of eleven exons of which parts or all of the last seven exons form the MBP transcription unit. Expression of the MBP transcription unit begins at a promoter upstream of exon 5b of the Golli-mbp gene, and expression of the Golli-mbp transcription unit begins at exon 1, producing a number of tissue, cell and developmentally regulated splice products at least three of which contain one or more exons found within the MBP transcription unit. The overall objective of this proposal is to determine the developmental and tissue-specific regulatory element of the Golli-mbp transcription unit in the mouse using in vitro transfection of cells and transgenic mice. We will analyze the role of these elements in modulating the expression of the MBP transcription with respect to levels of transcription as well as the tissue, cell and developmental expression of the gene. Initially, we will analyze the region upstream of Golli-mbp exon 1 for elements involved in the tissue, cell and developmental regulation of this transcription unit. We w ill also examine the possibility that one or more of these regulatory elements are found elsewhere in the gene, particularly the first intron. We will prepare constructs of putative regulatory regions plus a reporter gene and test them by transfection analysis in cell lines that do and do not express the Golli-mbp gene. We will then test the most promising constructs in transgenic animals to provide proof that the putative regulatory regions are operative in vivo. In other experiments we will examine the influence of the Golli-mbp promoter and any other regulatory elements we find on the levels and nature of expression of the MBP transcription unit. The deletion in the shiverer mouse genome affects at least two products of the Golli-mbp transcription unit in addition to MBPs. In view of the pleiotropic nature of the shiverer deletion on oligodendrocyte function we will introduce a Golli-mbp minigene into shiverer mice and carefully examine all the effects known to be associated with the shiverer mutation (beyond MBP expression and the "shivering" phenotype). These studies could help us define the consequences of the expression of the Golli-mbp gene products. They would also help us determine if the produces themselves had an effect on the expression of the MBP transcription unit during oligodendrocyte development.

molecular biology; mental retardation; biomedical facility; recombinant proteins; nucleic acid sequence; molecular cloning; genetic library; nucleic acid probes; complementary DNA; genetic manipulation;

DESCRIPTION: The Golli-mbp gene encodes two families of proteins, the Golli proteins and the myelin basic proteins. The overall goal of this application is to identify the genetic regulatory elements that govern specificity in the regulation of transcription start sites for this gene in oligodendrocytes and neurons in the nervous system and in macrophages and other cells in the immune system. A part of this application is to use a promoter element, as well as transgenic mice expressing a reporter gene under its control to investigate the developmental biology of these cortical pioneer neurons and the fate of the subplate that they form.

The overall goal of this proposal is to include within the Microscopic Imaging Core of the UCLA Brain Research Institute a two-photon laser scanning microscope (TPLSM) equipped for investigation of physiological processes in living cells. The intention is to complement other imaging technologies, such as confocal microscopy and electron microscopy, which are already available within the Core. Many important new experimental avenues will be made possible by utilizing two-photon excitation in a cellular imaging system equipped for complex cellular manipulation using electrophysiology and microinjection. The TPLSM allows measurement of fluorescence within a diffraction limited volume, without the need for a confocal pinhole and with minimal photobleaching and photodamage outside the focal plane. This makes two- photon excitation ideal for measurements in living cells. In addition, the use of longer wavelengths for illumination allows imaging deep within tissue. The projects in this proposal come from a wide spectrum of neuroscience faculty with different research interests. Several of the major users propose to measure ion concentrations (Ca2+ or Zn2+) in cells within intact tissue. These include slices from various regions of the brain (Istvan Mody, Chris Colwell, Michael Levine), retina (Nicholas Brecha), neuroendocrine organs (Nancy Wayne, Jonathan Monck), and skeletal muscle fibers (Julio Vergara). Another user will study inspiratory-modulated synaptic inputs to various neuron types with fluorophores (Jack Feldman). Several users are interested in the organization of the nervous system during development and propose to use fluorescent labels (green fluorescent protein (GFP), carboxycyanines) to trace neural projections or synaptic connections (Anthony Campagnoni, Susana Cohen-Cory, Ellen Carpenter). Two users are tracing GFP targeted proteins expressed in specific neuronal populations in transgenic mice and C. elegans (Anthony Campagnoni, Alex van der Bliek). Another user proposes to use GFP to study receptor internalization in neurons within intact gastrointestinal tissue (Catia Sternini). The availability of the TPLSM will significantly enhance and expand the research directions of these investigators, most of whom have ongoing NIH-funded research programs.

Description ( from the applicant's abstract): Recently, our conception of the two principal myelin protein genes, i.e. the myelin basic protein (MBP) and proteolipid protein (PLP) genes, has begun to change in view of new information about their structure, expression patterns and possible non-myelin related functions of their products. There is a considerable body of evidence to suggest that the PLP/DM20 gene plays roles other than that encoding the major myelin structural proteins in non-myelinating cells. This could occur either through multiple activities of the "classic" PLP and DM20, or through the existence of other products of the gene that have not yet been identified. We have isolated two new srPLP/DM20 gene products and identified a new exon of the gene. These products are expressed in neurons as well as oligodendrocytes, and they are localized in the somata of oligodendrocytes and neurons and are absent from the myelin sheath. On the basis of these and other preliminary data, we propose the existence of other products of the PLP/DM20 gene in addition to the "classic" and sr-PLP/DM20 variants. The overall objective of this application is to isolate and identify these other variants of the PLP/DM20 gene and to examine possible non-myelin functions of the "classic" PLP and DM20 as well as the variant products of the gene. These include: (1) identifying and characterizing novel products of the PLP/DM20 gene and examining their cellular and regional localization in the developing nervous system; (2)[re-] evaluating the hypothesis that cell death in the jimpy mutant is due solely to toxic effects of the mutated protein by prepare cell lines stably transfected with normal and variant/mutant PLP/DM20 cDNAs and comparing levels of expression in conditionally-immortalized OL lines on cell survival; and (3) examining aspects of the non-myelin roles of PLP/DM20 gene products.

[unreadable] DESCRIPTION (provided by applicant): The golli proteins are one of two families of proteins produced from the MBP gene with more ubiquitous expression than the classic MBP family. In the last funding period we have obtained preliminary evidence that golli proteins are involved in process and sheet formation in oligodendrocytes and neurons, and that they serve as modulators of signal transduction, particularly of the PKC pathway. The overall objective of this application is to determine the functions of the golli proteins in oligodendrocytes (OLs) and the role they play in myelination. In this application we will test several hypotheses: (1) Golli proteins are involved in OL process extension in myelination, and possibly in OL migration. (2) Golli proteins are modulators of signal transduction pathways in OLs and other cells. (3) Golli proteins may be involved in one or more points of intracellular signaling events that cause cytoskeletal rearrangements leading to OL process extension during myelination and migration. We have also generated both golli loss-of-function and gain-of-function mouse mutants to help determine the function of the golli proteins. These mutants, however, have not been completely characterized and another aim of this proposal is to complete their phenotypic analysis. The specific aims of this proposal are: (1) To analyze the oligodendrocyte phenotype in the golli knock out and golli overexpressing mice in vivo and in vitro. (2) To test the hypothesis that golli associates with microfilaments in the growing tips of oligodendrocytes and neurons during process formation/elaboration. (3) To test the hypothesis that golli modulates signal transduction in oligodendrocytes and plays a role in signaling processes important for process formation and myelination. Successful completion of these aims will, we believe, establish that another function of the myelin basic protein gene, through its golli products, is to modulate signaling events important for process and neurite extension in oligodendrocytes and neurons. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A new mouse model with a myelinating disorder has been generated. It is severely hypomyelinated between birth and P50, and exhibits severe tremors during this period. After P50, myelin begins to accumulate in the brain, but it never completely achieves control levels. Hypomyelination occurs over the 'critical" period of brain development when axonal sprouting, neural networks, synaptic connections, and neuronal-glial relationships are being established. The overall objective of this proposal is to use this mouse as a unique model to study the cellular basis for the "delayed" myelination, to determine how the brain can "recover" from a major hypomyelinating event, to determine the cellular and functional consequences of retarded myelination during development and to determine how transient hypomyelination during a critical period of brain development influences the survival and structural integrity of neurons und neuronal connectivity in the brain. This application consists of three specific aims: (1) Examine myelination in the brains of the JOE (i.e. J37 Over-Expressing) mice. In this aim myelination will be examined in JOE and WT mice using an array of morphological and biochemical approaches; (2) determine the cellular mechanisms responsible for the delay in myelination in the JOE mice; (3) define the nature and extent of axon pathology, neuronal degeneration and neuronal abnormalities in the JOE mice. The proposed experiments will determine if the retarded myelination in JOE mice is normal or abnormal and if the mice form structurally normal myelin. The studies will assess the effect of golli J37 overexpression on oligodendrocyte (OL) development, survival and maturation; whether golli overexpression occurs in OL precursors, late progenitors, or immature OLs; how this affects the survival, differentiation or migration of these cells in JOE mice; and which cells are responsible for the later myelination in JOE mice. The effects of hypomyelination during the "critical" period of brain development on several aspects of neuronal biology will be determined. These include: (a) cerebellar and cerebral cortical organization; (b) susceptibility to hypomyelination and survival; (c) axonal integrity and (d) axonal and dendritic organization. These studies are relevant to demyelinating diseases, like MS and leukodystrophies, and will enhance our understanding of myelination, remyelination and the effects of hypomyelination on neural function.