David R. Colman - US grants

The goal is to understand how plasma membranes of myelinating cells in the central and peripheral nervous systems (CNS, PNS) become organized into mature myelin sheaths. Two major integral proteins unique to myelin are thought to be mediators of myelin compaction in the PNS (protein zero, P(o)) and CNS (the myelin proteolipid protein, PLP). These proteins are unrelated to each other biochemically, but in their respective nervous system divisions they are each believed to be responsible for the formation and maintenance of the intraperiod line (IPL), formed by the close apposition of the extracellular leaflets of the myelin bilayer. In non-glial cells transfected with a P(o) cDNA, P(o) expression at the plasma membranes of adjacent cells is accompanied by the formation of intercellular adhesive contacts, readily detectable by immunofluorescence, which ultrastructurally resemble an IPL. The formation of these contacts is mediated by a 'functional domain' in the extracellular segment of P(o) that will be identified (Aim I). By using site-directed mutagenesis methods, segments of the cDNA encoding the P(o) molecule will be mutated and expressed in non-glial cells. The capacity of each construction to induce adhesive zones at cell-cell borders will be assessed immunocytochemically by confocal microscopy, and by routine and immunoelectron microscopy of stable transfectants.Stable transfectants will be used in an assay for adhesion that measures the ability of single cells to form aggregates in suspension. Poly- and monospecific antibodies directed against peptide segments, and truncated P(o) molecules secreted by stable transfectants will be used to inhibit cell-cell adhesion, mediated by the intact P(o) molecule. Several models for the topology of PLP have been proposed. An accurate topological map (Aim II) is essential for functional studies on this molecule. intramembranous, cytoplasmic and extracellular domains of the PLP molecule will be defined using proteases, and poly- and monospecific antibodies as probes. The role of specific segments of the polypeptide in establishing the disposition of PLP in the bilayer will be assessed by site-directed mutagenesis studies. Mutagenized cDNAs will be expressed in a coupled transcription-translation system containing membranes derived from rough endoplasmic reticulum, that are the acceptor membranes for the nascent polypeptide in vivo. Cultured oligodendrocytes and non-glial transfectants expressing high levels of PLP will be used in conjunction with PLP antibodies of defined specificity in immunocytochemical experiments that will localize the extracellular and intracellular domains.

Our long range goal is to understand the molecular mechanisms that underlie the formation of the mature myelin sheath, in the realization that this may suggest ways by which the repair process can be encouraged after myelin destruction. An essential step towards achievement of this goal is to precisely define the functional roles played by the myelin basic proteins (MBPs) over the course of the myelinogenesis program that is implemented by the oligodendrocyte. For many of the studies outlined below, we will rely on the cDNA transfection systems we have developed and refined over the past two years, that employ specific MBP antisera and confocal immunomicroscopy to map intracellular distributions of the expressed proteins. Our specific aims are: I.To identify the functional domains within the 14K and 18.5K MBPs that mediate association of these isoforms with the plasma membrane. Using site-directed mutagenesis, we will (1) delete internal segments or (2) truncate the N or C terminus of the 14K and 18.5K MBP cDNAs, and express the mutated constructs in non-glial cells, and in shiverer oligodendrocytes. the intracellular distribution of each mutated polypeptide will be mapped immunocytochemically to determine minimum polypeptide lengths and sequences required for membrane association. In parallel, (3) the mutated polypeptides will be expressed and tested in biochemical and ultrastructural experiments for the capacity to bind to and aggregate oriented vesicles derived from shiverer brain plasma membranes. II.To begin to explore the mechanisms by which the exon II-containing 21.5K and 17K MBPs are translocated from their site of synthesis in the cell cytoplasm across the nuclear pore complex into the nuclear matrix. In non-glial cells, and in shiverer mouse oligodendrocytes transfected with the 21.5K or 17K MBP cDNAs, the expressed proteins are found within the cytoplasm and nucleoplasm. Significantly, in the normal developing mouse brain, oligodendrocytes are readily detected whose nuclei contain high concentrations of MBPs. In many cell types, proteins that are translocated into the nucleus contain elements within their primary amino acid sequences that act as nuclear localization signals (NLSs) which enable selective entry through the nuclear pore complex into the nuclear compartment where the translocated proteins can exert regulatory functions. We have identified a region in the exon II-containing MBP isoforms that bears strong homology to known NLSs. We will test the capacity of this, and other sequences in these MBPs to function as authentic NLSs in transient transfectants. Three criteria that are used to rigorously define NLSs in other systems will be applied: (1) NLS-mediated entry of MBP into the nucleus is likely to be an energy-requiring process, and therefore should be sensitive to disruption by (a) chilling, or (b) ATP depletion; (2) deletion or mutation of MBP NLS sequence(s) should cause cytoplasmic accumulation of the mutated protein; and (3) when the putative MBP NLS sequence is engineered into an unrelated non-nuclear protein, such as bovine serum albumin, the chimeric protein should be translocated into the nucleus.

Our overall goal is to define the roles that cadherins play during myelination of model neural structures derived from the CNS (optic nerve) and PNS (sciatic nerve). To this end our specific aims are: 1. TO COMPLETE THE CHARACTERIZATION OF A NOVEL OPTIC NERVE-ENRICHED CADHERIN WE HAVE IDENTIFIED, O-CADHERIN (OCAD), AND TO IDENTIFY THE FULL COMPLEMENT OF CADHERINS EXPRESSED IN OPTIC AND SCIATIC NERVES; (1) We will complete the sequence of OCAD by isolating cDNAs from an optic nerve cDNA library that we have prepared from mouse tissue, using as probes partial OCAD cDNAs, and/or polyclonal antibodies we have raised against an OCAD fusion protein. The complete OCAD primary sequence will be compared with those of other cadherins, an interpreted in light of the anticipated solution of the crystal structure of the first extracellular domain (ECI) of NCAD that we are completing in related experiments. (2) Using specific and degenerate oligonucleotide primers representing segments of the homologous calcium binding domains and cytoplasmic segments common to the cadherin family, we will identify the full complement of cadherins from mouse optic and sciatic nerves, which are heavily myelinated neural structures. We expect that several cadherins are expressed in these nerves, and for each novel cadherin, we will generate cDNA probes, and prokaryotic fusion polypeptides from which specific antisera will be raised. We will identify the cell(s) of origin in optic or sciatic nerve that express each cadherin, in particular for OCAD, and for any other novel cadherin we identify we will provide tissue distributions and developmental profiles using protein blotting, in situ hybridization, and immunocytochemical methods. II. TO EXPLORE CADHERIN FUNCTION IN NERVE MYELINATION: The capacity of individual cadherins identified in optic and/or sciatic nerves to interact with each other will be assessed (1) by confocal immunomicroscopy to precisely localize each cadherin in situ. In particular, high resolution imaging with specific antisera will be useful in revealing putative co- localization patterns for cadherins, their associated protein (e.g., catenins and actin) and other adhesion molecules at cell; cell interfaces; (2) and in transfection studies. Full length cadherin cDNA clones (O-, N-, E-and/or R-cadherin, for example) will be used singly and in various combinations for expression in host cells. The capacity of the expressed cadherins to associate with each other within a single cell, and induce homophilic and/or heterophilic adhesive contacts between plasma membranes at apposed cell-cell borders will be assessed by confocal immunomicroscopy. (3) Stable cadherin transfectants and co-transfectants will be used in assays for adhesion that measures the ability of single cells to form aggregates. The sensitivity of observed adhesive interactions to inhibition by antibodies and soluble cadherin fragments will be tested collaboratively in tissue culture systems that his laboratory has established.

biomedical facility; genetically modified animals; animal colony; nervous system transplantation; microinjections; laboratory mouse;

Protein zero (P-0, mw = 28,500) is the major adhesive glycoprotein of the myelin sheath in the mammalian peripheral nervous system. Mutations in the P0 gene in humans underlie certain myelin diseases that affect the peripheral nervous system (Charcot-Marie-Tooth and Dejerine-Sottas Diseases). P0 is also the "simplest" transmembrane member of the immunoglobulin gene (Ig) superfamily, and so this molecule has attracted great interest as a prototypic representative of Ig superfamily interactions. Definitively establishing the structural basis for P0:P0 membrane adhesive interactions will have direct bearing on understanding the binding elements by which other Ig superfamily molecules exert their' actions. Our single Specific Aim is to experimentally evaluate a proposed model for P0:P0 molecular interactions which lead to strong membrane adhesion. In the first phase of these studies, we will engineer full length P0 cDNAs encoding polypeptides mutagenized by point mutation in the extracellular domain. Strategic mutations will be placed such that we may test the roles for amino acid residues at several specific contact points first revealed by analysis of the P0 extracellular domain. Each mutagenized cDNA will be used as template for mRNA synthesis, which in turn will program a coupled frog oocyte system we devised to test for intermembrane adhesion. In parallel studies, permanent expressing pure clonal cell lines of mutagenized P0 expressors will be used in cellular assays for adhesion and aggregation in normally non-adherent, non-aggregating cells such as HeLa or L-cells.

Description (Provided by applicant): In the most contemporary view, the central nervous System synapse may be thought of as comprising 2 discrete subdomains which overlap structurally and functionally. The first domain is the synaptic "scaffold" which is observed by electron microscopy, consisting of apposed, rigorously parallel presynaptic and postsynaptic plasma membrane thickenings bound together by "crossbridges" that span the synaptic cleft. The scaffold is retained even after vigorous fractionation and detergent extraction of synaptosomes, and it seems clear that it is held together by adhesion molecules, whose identities remain unknown at present. The second subdomain is the neurotransmissional machinery through which the synapse mediates its primary physiological functions. This subdomain is superimposed upon the scaffold and interacts with it via molecular forces we don't understand as yet. Much effort has been focused on analyzing the physiological components of the synapse; however, the major constituents of the scaffold and how its intercellular adhesive components interact have remained elusive. This is because of inadequate fractionation techniques for the purification of intact CNS synaptic junctions, and the bewildering array of candidate proteins that may operate at different synapses. It is clear that efforts to recognize and evaluate changes in synaptic proteins which occur during degenerative processes must rely first on a complete catalog of the structural proteins involved in synaptic development and maintenance and how they interact with each other; and second, on an understanding of the intracellular binding partners for these scaffolding molecules which function in synaptic signaling phenomena, and in attachment to the underlying subsynaptic components. Long range, we want to understand exactly how molecular adhesive forces organize and stabilize the pre-to post-synaptic scaffold of the synaptic junctional complex in the CNS. We propose to: I) use a novel cell fractionation procedure we devised to purify synaptic junctional complexes in high yield, and then use newly developed methods in mass spectrometry to identify the component adhesion and adhesion associated molecules, and II) begin studies on the interactions between these molecules which lead to the assembly of the synaptic junctional complex in the CNS.

DESCRIPTION (provided by applicant) This is a collaborative proposal between the laboratories of David R. Colman at Mount Sinai, NY, and of Graciela L. Boccaccio at the Institute Leloir (formerly Institute for Biochemical Research Campomar Foundation), Buenos Aires, Argentina. This proposal extend our ongoing work on the Functional Organization of the Myelin Axoglial Junction (grant # 1R01 NS40560; 07/01/00 - 06/30/05) to the study of intracellular elements of the glial cell cytoplasm adjacent to the junction. Specifically, we are aimed to elucidate the cell mechanisms mediating the localized synthesis of proteins in the myelinating cells of the central nervous system, that has being for long time of major interest to both, the applicant's and the foreign collaborator's research group. We will investigate the participation of the double stranded RNA binding protein Staufen in the localization and translation of mRNAs in oligodendrocytes. This is likely relevant to two related processes: 1. synthesis of components required for membrane maintenance and remodeling, and 2, signal transduction at the oligodendrocyte side of the axoglial junction. We will 1) use mass spectrometry to unambiguously identify novel protein components of the mRNA localization apparatus and 2) begin to asses the role of Staufen and novel components in mRNA localization. An important aspect of this proposal is its complementary nature combining the scientific interests and the methodological strength of two laboratories. Dr. Boccaccio's group have developed primary culture of oligodendrocytes and biochemical analysis of brain subcellular components, as well as a functional assay for mRNA localization in cultured cells. A number of methods that are strictly required for the present project have been established in our laboratory in Mount Sinai, namely, protein analysis via mass spectrometry and analysis of protein-protein interactions by yeast two-hybrid and "pull-down" experiments. Thus, this project will allow the training of the foreign investigator and Ph.D. students on state-of-the-art techniques that are currently not established at their home institute, thus representing a valuable contribution