Barbara Ranscht - US grants

Neurons require cell surface components to link the force- generating cytoskeleton with the extracellular environment to promote neurite outgrowth, fasciculation and the formation and stabilization of synapses. I am studying a specific component of the neuronal cell surface, GP 130, the only known membrane protein that is isolated specifically in association with the neuronal cytoskeleton. The aims of this research are a) to verify the structure of GP 130 as a transmembrane protein from the sequence of cDNA clones, b) to establish its functional interaction with the neuronal microenvironment and c) to localize GP 130 in the developing and adult nervous system, particularly in synapses. The structure of GP 130 will be derived from the sequence of full- length cDNA clones (Ranscht, in preparation). This will predict the GP 130 insertion into the plasma membrane, glycosylation sites and structural homologies with similar molecules in other cells. The major goal of this proposal is to establish the functional interaction of GP 130 with components of the neuronal microenvironment. To do this, I will develop direct binding assays of GP 130 with extracellular matrix and cellular substrates and attempt to purify the putative ligand by affinity chromatography. My preliminary work suggests that in the embryo, the expression of GP 130 is correlated with neurons reaching the target tissue and, in the adult, GP 130 is accumulated in synapses. To clarify the subcellular localization of GP 130 at synapses, I will conduct ultrastructural immunohistochemical studies of GP 130 in tissue sections of the chicken nervous system. The research I propose will contribute to our understanding of the role of the membrane cytoskeletal linkage in neuronal development and function.

The central theme of this program project is the study of interactions of cell surface proteins in the development of the vertebrate embryo. Two of these proteins are involved in the differentiation of the nervous system and two others will be tested for their role in the morphogenesis of skin and the development of the bony skeleton. These surface proteins are being investigated at three levels of biological organization; the cell, the tissue and the organism. The NILE glycoprotein will be studied for its role in mediating interactions between nerve cells, and a new surface glycoprotein, GP90, will be investigated for its function in the communication of nerve cells with non-neuronal tissues such as muscle. A cell surface-associated heparan sulfate proteoglycan will be studied for its role in the morphogenesis of the embryonic skin, and the membrane glycoprotein alkaline phosphatase will be considered for a possible structural role in the morphogenesis of the skin and the bony skeleton. All projects will use the most up-to-date methods in recombinant DNA technology, immunology, protein chemistry and cell biology to investigate in detail the structure and function of these surface proteins that are highly relevant to vertebrate development. The structure of the proteins will be deduced from the nucleotide sequence of cDNAs that encode them. Their functions will be investigated by identifying the functional domains on the molecules themselves and of the ligands with which they interact. An animal model system for the human inherited disorder hypophosphatasia will be developed as part of one project by introducing mutated forms of the normal allele into the germ line of mice. This transgenic system will serve as a paradigm for later studies on the role of the other cell surface molecules in the development of vertebrate embryos. Two core facilities will be established for this program project. One will support the molecular biological studies and the second one will be established for the quantitative analysis of the behavior of cells as a function of the surface molecules that are being studied. With this facility we will be able to record, store and process images of cells behaving under defined conditions and evaluate changes in their shape and motility as a response to other cells or their extracellular matrix.

Cadherins are a family of cell surface glycoproteins that mediate Ca2+- dependent, homophilic cell adhesion and thereby control tissue morphogenesis. All cadherins, reported thus far, contain a highly conserved cytoplasmic region that is crucially important for their function in cell adhesion by providing a linkage with the actin-based cytoskeleton. A member of the cadherin family, T-cadherin (T=truncated) was discovered in the developing chicken nervous system in this laboratory. T-cadherin lacks the cytoplasmic region and is anchored to the cellular plasma membrane through a glycosyl phosphatidyl inositol (GPI). Despite its different membrane anchor, T-cadherin is able to mediate Ca2+-dependent, homophilic cell adhesion in cellular aggregation assays. Thus, the function of T- cadherin must depend on a mechanism notably different from that of other cadherins. The aim of this proposal is to dissect the mechanism of T- cadherin mediated cell adhesion. The first step is to determine whether T- cadherin is localized to adherens type junctions like other cadherins or to different membrane subdomains. To do this, the localization of T-cadherin expressed in heterologous fibroblasts by DNA transfection will be determined. T-cadherin will also be localized in two cell populations of the chicken eye that express endogenous T-cadherin: Lens cells that form specialized epithelial type junctions and neural retina cells which form synaptic specializations. Second, to determine which domains of T-cadherin are necessary for its function in cell adhesion and subcellular distribution, chimeric molecules will be generated in which selected regions of T-cadherin are interchanged with the corresponding domains of N- cadherin. A). The domain for homophilic T-cadherin binding will be mapped by generating chimeric molecules that have a T-cadherin binding specificity and the membrane attachment of N-cadherin. This analysis is important since T-cadherin lacks a peptide, HisAlaVal, that contributes to the homophilic binding function of other cadherins. B). The region in T- cadherin that functionally substitutes for the cytoplasmic domain of other cadherins will be defined by generating chimeric molecules with a N- cadherin binding specificity and the GPI-membrane anchor of T-cadherin. This region may be as short as the sequences required for the attachment of the GPI-anchor or extend to sequences anywhere between EC2 and the carboxy terminus of T-cadherin. Lastly, membrane molecules putatively associated with and required for the function of T-cadherin will be identified by immunoprecipitation and affinity chromatography with T-cadherin antibodies. These molecules will be characterized and their interaction with T-cadherin will be determined. This work will contribute to understanding how cell adhesion molecules control the generation of specific tissues in developing embryos and sustain their function in the adult. In the long term, these studies may be applied to prevent birth defects and control malignant neoplasms.

The central theme of this program project is the study of interactions of cell surface proteins in the development of the vertebrate embryo. Two of these proteins are involved in the differentiation of the nervous system and two others will be tested for their role in the morphogenesis of skin and the development of the bony skeleton. These surface proteins are being investigated at three levels of biological organization; the cell, the tissue and the organism. The NILE glycoprotein will be studied for its role in mediating interactions between nerve cells, and a new surface glycoprotein, GP90, will be investigated for its function in the communication of nerve cells with non-neuronal tissues such as muscle. A cell surface-associated heparan sulfate proteoglycan will be studied for its role in the morphogenesis of the embryonic skin, and the membrane glycoprotein alkaline phosphatase will be considered for a possible structural role in the morphogenesis of the skin and the bony skeleton. All projects will use the most up-to-date methods in recombinant DNA technology, immunology, protein chemistry and cell biology to investigate in detail the structure and function of these surface proteins that are highly relevant to vertebrate development. The structure of the proteins will be deduced from the nucleotide sequence of cDNAs that encode them. Their functions will be investigated by identifying the functional domains on the molecules themselves and of the ligands with which they interact. An animal model system for the human inherited disorder hypophosphatasia will be developed as part of one project by introducing mutated forms of the normal allele into the germ line of mice. This transgenic system will serve as a paradigm for later studies on the role of the other cell surface molecules in the development of vertebrate embryos. Two core facilities will be established for this program project. One will support the molecular biological studies and the second one will be established for the quantitative analysis of the behavior of cells as a function of the surface molecules that are being studied. With this facility we will be able to record, store and process images of cells behaving under defined conditions and evaluate changes in their shape and motility as a response to other cells or their extracellular matrix.

Cadherins are a family of cell surface glycoproteins that mediate Ca2+- dependent, homophilic cell adhesion and thereby control tissue morphogenesis. All cadherins, reported thus far, contain a highly conserved cytoplasmic region that is crucially important for their function in cell adhesion by providing a linkage with the actin-based cytoskeleton. A member of the cadherin family, T-cadherin (T=truncated) was discovered in the developing chicken nervous system in this laboratory. T-cadherin lacks the cytoplasmic region and is anchored to the cellular plasma membrane through a glycosyl phosphatidyl inositol (GPI). Despite its different membrane anchor, T-cadherin is able to mediate Ca2+-dependent, homophilic cell adhesion in cellular aggregation assays. Thus, the function of T- cadherin must depend on a mechanism notably different from that of other cadherins. The aim of this proposal is to dissect the mechanism of T- cadherin mediated cell adhesion. The first step is to determine whether T- cadherin is localized to adherens type junctions like other cadherins or to different membrane subdomains. To do this, the localization of T-cadherin expressed in heterologous fibroblasts by DNA transfection will be determined. T-cadherin will also be localized in two cell populations of the chicken eye that express endogenous T-cadherin: Lens cells that form specialized epithelial type junctions and neural retina cells which form synaptic specializations. Second, to determine which domains of T-cadherin are necessary for its function in cell adhesion and subcellular distribution, chimeric molecules will be generated in which selected regions of T-cadherin are interchanged with the corresponding domains of N- cadherin. A). The domain for homophilic T-cadherin binding will be mapped by generating chimeric molecules that have a T-cadherin binding specificity and the membrane attachment of N-cadherin. This analysis is important since T-cadherin lacks a peptide, HisAlaVal, that contributes to the homophilic binding function of other cadherins. B). The region in T- cadherin that functionally substitutes for the cytoplasmic domain of other cadherins will be defined by generating chimeric molecules with a N- cadherin binding specificity and the GPI-membrane anchor of T-cadherin. This region may be as short as the sequences required for the attachment of the GPI-anchor or extend to sequences anywhere between EC2 and the carboxy terminus of T-cadherin. Lastly, membrane molecules putatively associated with and required for the function of T-cadherin will be identified by immunoprecipitation and affinity chromatography with T-cadherin antibodies. These molecules will be characterized and their interaction with T-cadherin will be determined. This work will contribute to understanding how cell adhesion molecules control the generation of specific tissues in developing embryos and sustain their function in the adult. In the long term, these studies may be applied to prevent birth defects and control malignant neoplasms.

The complex pattern of neuronal circuitry is established during embryonic development as neurons extend axons that navigate with temporal an spatial precision to their synaptic targets. The selection of specific axon pathways and innervation of synaptic target sites is controlled by molecules located in the environment of the tips of the growing axons. The recognition of these guidance cues is thought to be mediated by receptors on the growth cone surface which, upon activation, rearrange the neuronal cytoskeleton and control the extension and orientation of growing axons. The purpose of this program project is to elucidate cellular and molecular mechanisms that lead to the establishment of functional axon connections in the developing vertebrate nervous system. The participating laboratories each study cell surface or extracellular matrix molecules that are either known or promising candidates for the involvement in axon recognition and guidance. The specific molecules that are the subject of this program project are the immunoglobulin-like cell adhesion molecule NILE\L1 (project I), the GPI-linked cadherin cell adhesion molecule T-cadherin (project II), the Eph-related receptor-type tyrosine kinase Cek8 (Project III) and the secreted chrondroitin sulfate proteoglycan Brevican (Project IV). All of these molecules are expressed in the developing nervous system. Because they contain structural cell adhesion motifs, such as immunoglobulin-like domains (NILE/L1, Cek8, Brevican), fibronectin-type III repeats (NILE/L1, Cek8), a lectin-like domain closely related to selectins (Brevican) or cadherin structural repeats (T-cadherin), these molecules are all postulated to be involved in cell recognition events during neural development. The goal of this program is to determine the cellular and molecular interactions in developing neural tissues in which these molecules play a role. The functions of NILE/L1, T-cadherin, Cek8 and Brevican in neurite growth will be explored in vitro. The Program Project Core consists of a facility that will assist all participating laboratories with 1) a standardized service to establish primary neural cultures for the analysis of neurite growth and growth cone behavior and 2) computerized data collection and analysis of such cultures. This core component will be an extension of the Cell Behavior Analysis Core B of the current Program Project. The Program Project will provide an intellectually stimulating and interactive environment in which the molecular nature underlying the establishment of the complex neuronal circuitrary is the common and uniting theme.

light microscopy; biomedical facility; digital imaging; image processing; histology; DVD /CD ROM;

charge coupled device camera; tissue /cell preparation; image processing; neurogenesis; biomedical facility;

ABSTRACT IBN-9723934 RANSCHT Brain function critically depends on the wiring of nerve fibers into a complex neuronal network. This network is established during embryonic development as neurons extend processes, called axons, that carry on their distal tip a mobile structure, the growth cone. The growth cones of each neuron type sense cues in their cellular environment to navigate to, recognize and form functional contacts with specific target cells, such as other neurons or effector organs such as muscle, glands or skin. In order to recognize the guidance cues in their environment and seek out the proper cells to contact, growth cones explore their surroundings with an array of molecular sensors. Contactin, a cell adhesion/recognition molecule discovered by Dr. Ranscht, is one of the molecular sensors exposed on the growth cone surface and is therefore a strong candidate to mediate growth cone recognition. To test the function of contactin in the establishment of axon connections in developing embryos, Dr. Ranscht's laboratory has recently generated contactin-deficient mice by manipulating the mouse genome. In support of the hypothesis that contactin is required for the formation of neuronal circuitry, the mutant mice show neurological defects: Their movements are severely uncoordinated and characteristic of those of patients diagnosed with ataxia. The mutant animals die shortly after birth. As the phenotype of contactin mutant mice is indicative of defects in neuronal circuits controlling motor behavior, Dr. Ranscht will produce a detail map of contactin distribution in brain areas controlling and modulating motor functions. Specifically, she will identify the neuron populations that use contactin for axonal pathfinding and clarify which of the identified contactin-binding proteins are distributed in the surroundings of contactin-positive neurons. To understand which molecular interactions are disrupted in the contactin mutant mice, the distribution and expression levels of contactin-binding proteins will also be examined in these mutants. Results from this project will contribute to understanding the role of contactin and its molecular interactions in neuronal circuits controlling motor functions.

DESCRIPTION (Adapted from the applicant's abstract): The goal of this application is to understand the molecular signals that underlie the formation of cytoarchitecture in cerebellar development. Over the past few years, a number of studies have addressed cell adhesion/recognition molecules of the immunoglobulin gene superfamily (IgSF) in the nervous system. However, studies on the function of these molecules in neural development in vivo are sparse, and the understanding of their roles in establishing neuronal architecture remains largely elusive. The current application will address the role of the IgSF cell adhesion/recognition molecule contactin in cerebellar development using a combination of in vivo and in vitro approaches. Several lines of evidence indicate that contactin plays a pivotal role in regulating cellular interactions that control cerebellar development. This investigator has generated mice with disrupted contactin gene function which develop a severely ataxic phenotype with an onset of postnatal day 9. The mutation is lethal by postnatal day 18. Preliminary analyses of the mutant cerebellum have identified defects in granule neuron development. This neuron population, which, in the wild type, expresses contactin throughout postnatal cerebellar development, provides an opportune model to study the role of contactin in neural development. This project will focus on two major functions of contactin that are indicated from preliminary analyses of the knockout mice. First, using the knock-out mice, the investigator has obtained evidence for a role of contactin in regulating the number of granule cell precursors in the external germinal layer (EGL). The project will test several hypotheses, which will address if contactin-mediated cellular interactions are required for the proliferation, survival and/or maintenance of the undifferentiated state of granule neuron precursors in the EGL. Second, additional preliminary data have revealed a later role of contactin in the fasciculation of parallel fiber axons in the cerebellar molecular layer. The investigator proposes to investigate the mechanism by which contactin regulates this function, and will determine in vitro the ligands with which contactin interacts in controlling axon-axon interactions. These studies will move forward the understanding of the molecular basis of cerebellar development, and, in particular, provide an opportunity to dissect the function of an IgSF cell adhesion/recognition molecule in a biological setting. Thus, this work may set an example for future studies on a host of similar proteins.

Description: This core consists of a facility that assists all participating laboratories with tissue culture experiments, and provides assistance with histological experiments. Tissue culture will be used in all four components of this program to analyze the role and mechanism of cell adhesion molecules (Ranscht), receptor protein tyrosine kinases (Pasquale) and cell surface proteoglycans (Stallcup and Yamaguchi) by cell transfection assays and/or assays for neurite growth, synaptogenesis, and spine formation. The Tissue Culture will assist investigations of this Program Project with the transfection of cDNA constructs in cell lines and primary neural cultures, and setup primary cultures from the hippocampus to assay neuronal differentiation and synaptogenesis. One major advantage of this core is that the hippocampal culture system is standardized, so that results obtained for one of the studied molecules can be related to those for others. This will broaden the understanding of the molecular nature of neuronal differentiation and synaptogenesis, and foster additional interactions and joint experiments between the program project laboratories. The Core will also provide assistance with histological experiments by cutting frozen and paraffin sections, and perform immunostaining with various antibodies.

Description All of the proposed projects require conventional and fluorescence microscopy, image analysis, and time lapse cinematography. Thus, to efficiently accomplish the microscopic analysis of our data, we request a microscopy Core Facility. This Facility will be staffed with an experienced senior research technician (to be recruited when the program is funded), and will provide assistance with sample preparation, execution of the experiments, image collection, data analysis and editing of confocal and other microscopic images. The Program PI's concurred that this facility is essential to efficiently achieve the goals of the proposed program. In our current set-up, individual postdocs are trained in the use of the Metamorph Image Analysis system and the confocal microscope by the Manager of the Cell Analysis Facility, Dr. Edward Monosov. This training provides members of the program with skills to operate the systems to address a specific question. By nature, however, this training provides neither continuity within the program, nor depth of knowledge to an individual to independently sept-up and operate the Confocal Microscope and Image Analysis Systems for numerous applications. In order to overcome these problems, the Program PI's want to place these operations in the hands of an experienced microscopist/computer analyst. This person will be trained by and interface with the manager of the Institute's Cell Analysis Facility in order to conduct confocal and image analyses for the Program. Operation The core will be house in a 300 square foot room on the first floor on the Burnham Institute's Fitch Building (Building 4), adjacent to three of the Program Laboratories. This room currently houses the Program's 405M Axiovert and the attached image analysis system, and the Burnham Institute's second Confocal microscope, a Zeiss LSM 410. Plans are to accommodate the staff of Program Project Core B in this room. A core staff person with extensive experience in microscopy and computing will be recruited when this grant is funded. The staff will report to Dr. Ranscht, who will serve as the core director (6% effort). Training of the staff will be provided by Dr. Edward Monosov, the manager of the Institute's Cell Analysis Shared Facility The Burnham Institute charges for training time $40/hour, and for unsupervised time on the confocal microscope $20 per hour to offset costs for service contracts and maintenance of the MRC1024-MP confocal instrument. Equipment 1) Zeiss 405M inverted Microscope and Metamorph Image Analysis System. The Program Project extensively uses an inverted Zeiss 405M Microscope that was acquired for the Core of the existing Program Grant in year 1 (1989). This is an excellent microscope that is equipped with optics for phase contrast, differential interference contrast, darkfield, bright field and fluorescence in the red, green and ultra violet spectra. The instrument has been updated over the years with new filter sets and objectives. Attached peripherals included a heated stage for the cultures and an Eppendorff microinjection system suited for the injection of cultured cell. We have extended the capability of the system to do lapse time cinematography using an Optronics ZVS-47E intensified CCD video camera, a Sony Trinitron Color Video Monitor (analog) and a time lapse laser videodisk recorder (Sony LVR 3000N, analog).

DESCRIPTION (Adapted from applicant's description): This program investigates the cellular and molecular mechanisms by which developing neural cells recognize, translate and respond to extracellular signals. Each of the participating Program laboratories examines the function and molecular mechanisms underlying distinct interactions of neural cells with their environment, including axonal recognition, synapse formation, plasticity, and oligodendrocyte precursor migration. Specifically, the functions and mechanisms of operation of cadherin, receptor-type, tyrosine kinases of the Eph-family and their ephrin-ligands, and membrane-bound proteoglycans will be investigated. Dr. Ranscht's project examines the role of T-cadherin in axonal pathway recognition and synapse formation in the hippocampus using mice with disrupted T- cadherin function. Dr. Yamaguchi's project addresses the function of proteoglycan syndecan-2 in dendritic spine maturation of hippocampal neurons Dr. Pasquale's project investigates the bi-directional signaling mechanisms of Ephbeta2 and its ephrin-B1 ligand, a receptor ligand pair that is expressed in hippocampal synapses. Dr. Stallcup's project determines the mechanism by which proteoglycan NG2 signals oligodendrocyte precursor cells to migrate.

Cadherins are calcium-dependent cell adhesion that play pivotal roles in the specification of embryonic cells and their segregation into functionally distinct groups. They operate by connecting cells expressing the same cadherin type through a homophilic binding mechanism, and require interactions on both sides of the membrane for this function. In the nervous system, cadherins are expressed by e distinct axon populations, and are implicated to regulate neuronal specificity. Cadherin molecules have recently been localized in synaptic junctions, and are suggested to play a role in synapse specification and plasticity. This project will investigate in the hippocampal formation the function and mechanism of operation of T-cadherin that is anchored to the membrane through a glycosylphosphatidyl anchor. T-cadherin is discretely localized in hippocampal laminae where axons and dendrites interact to form synaptic contacts. This suggests that T-cadherin plays a distinct role in axonal interactional interactions either during axon guidance and target selection, and/or at synapses. Using mice deficient for T-cadherin gene function, this project will investigate the function of T-cadherin in the establishment of hippocampal circuitry. First, we will determine if T- cadherin is a component of synaptic junctions, and how its expression relates to that of the classical cadherins that are expressed in synapses in areas of T-cadherin expression. Second, we will address the role of T- cadherin in axonal interactions of mossy fiber axons during pathfinding and synapse formation in the stratum lucidum in T-cadherin-deficient mouse mutants. Third, we will dissect T-cadherin's role in neuronal differentiation and synapse formation in an in vitro model. Lastly, we will begin to analyze the mechanism of T-cadherin's function by characterizing proteins associated with T-cadherin. This work will contribute to the molecular understanding of the mechanisms underlying the formation of the intriguing neuronal circuitry involved in cognitive functions.

DESCRIPTION (Adapted from the applicant's abstract): The goal of this application is to understand the molecular signals that underlie the formation of cytoarchitecture in cerebellar development. Over the past few years, a number of studies have addressed cell adhesion/recognition molecules of the immunoglobulin gene superfamily (IgSF) in the nervous system. However, studies on the function of these molecules in neural development in vivo are sparse, and the understanding of their roles in establishing neuronal architecture remains largely elusive. The current application will address the role of the IgSF cell adhesion/recognition molecule contactin in cerebellar development using a combination of in vivo and in vitro approaches. Several lines of evidence indicate that contactin plays a pivotal role in regulating cellular interactions that control cerebellar development. This investigator has generated mice with disrupted contactin gene function which develop a severely ataxic phenotype with an onset of postnatal day 9. The mutation is lethal by postnatal day 18. Preliminary analyses of the mutant cerebellum have identified defects in granule neuron development. This neuron population, which, in the wild type, expresses contactin throughout postnatal cerebellar development, provides an opportune model to study the role of contactin in neural development. This project will focus on two major functions of contactin that are indicated from preliminary analyses of the knockout mice. First, using the knock-out mice, the investigator has obtained evidence for a role of contactin in regulating the number of granule cell precursors in the external germinal layer (EGL). The project will test several hypotheses, which will address if contactin-mediated cellular interactions are required for the proliferation, survival and/or maintenance of the undifferentiated state of granule neuron precursors in the EGL. Second, additional preliminary data have revealed a later role of contactin in the fasciculation of parallel fiber axons in the cerebellar molecular layer. The investigator proposes to investigate the mechanism by which contactin regulates this function, and will determine in vitro the ligands with which contactin interacts in controlling axon-axon interactions. These studies will move forward the understanding of the molecular basis of cerebellar development, and, in particular, provide an opportunity to dissect the function of an IgSF cell adhesion/recognition molecule in a biological setting. Thus, this work may set an example for future studies on a host of similar proteins.

DESCRIPTION (provided by applicant): The Cell Imaging and Histology Shared Resource provides the equipment and expertise for three types of services: 1) High resolution microscopic techniques to obtain information about the morphology of cells, and sophisticated imaging techniques to analyze the distribution of molecules and their interactions within their cellular environment; 2) Histological and immunohistological techniques to study the morphology of normal and pathological specimens and the distribution of molecules in tissues; and 3) electron and immunoelectron microscopy to analyze the ultrastructure of of cells and the association of molecules with subcellular organelles. The Resource operates four light micsroscopes for confocal and standard imaging and an electron microscope for ultrastructural analysis. Equipment and services are provided for tissue embedding, sectioning, laser capture dissection, staining, and microscopic analysis of specimens. The goals for the next five years include expansion of existing services and introduction of new services. An increased effort will be placed on assisting users with the design and set-up of experiments, including sample collection and processing. New advanced optical imaging strategies for visualizing molecular events in living cells will be implemented, such as: 1) Optical imaging based on the principle of fluorescence resonance energy transfer (FRET) and fluorescent lifetime imaging (FLIM); 2) Total internal reflection fluorescence microscopy (TIRF) to register real-time interactions and trafficking of fluorescent molecules, and to study the dynamics of protein-protein interactions and enzymatic reactions in live cells; and 3) Fluorescence recovery after photobleaching (FRAP) to examine the lateral diffusion and mobility of fluorophore-labeled molecules into an area that has been photobleached. Overall the Cell Imaging and Histology Shared Resource provides essential microscopic and histological services and will accommodate the increased use and requirement for new services in response to new research initiatives at the Cancer Center.

DESCRIPTION (provided by applicant): The rapid propagation of nerve impulses in myelinated nerve depends on its segregation into specialized membrane domains. Myelin segments along the nerve are interspersed by the unmyelinated nodes of Ranvier that contain clusters of sodium channels and serve to regenerate the action potential. On both sides of the node, septate-like junctions tightly attach the terminal myelin loops to the axon membrane. These paranodal junctions physically segregate the sodium channel clusters at the node from potassium channel clusters at the juxtaparanode just underneath the myelin. The molecular interactions that control the formation and maintenance of specialized membrane domains in myelin remain poorly understood. We have established an important function for Contactin in the formation of the septate-like paranodal junctions in both the CNS and PNS (Boyle et al., 2001; and in preparation). Contactin associates with Caspr (Contactin-associated Protein) and this interaction is necessary for Caspr trafficking to the axon membrane where the complex engages in junctional adhesion. However, little is known about how the expression of the Contactin-Caspr complex is regulated at central and peripheral paranodes. We have identified a novel Caspr-interacting protein that may regulate the availability and stability of the Caspr-Contactin complex on neuronal cell surfaces. Nothing is currently known about the distribution and function of this novel protein within cells and in the intact nervous system. The goal of this proposal is to reveal the functional association of this novel protein with Caspr and determine how this interaction regulates myelin assembly and function. This work will shed new light on the regulation of a protein complex that is vital for the development and function of myelin. Understanding the molecular signals that control the assembly, disassembly and compartmentalized localization of protein complexes in myelin will help in the design of strategies aimed at preventing and restoring proper functions in individuals affected by demyelinating diseases.

INTRODUCTION TO REVISED APPLICATION The reviewers of the A1 application considered the Core essential for the successful accomplishment of all the projects in the Program. Discrepancies between the budget and the grant text have been corrected in the current revision. The letter from Ms. Karin Eastham, Vice President and Chief Operating Officer of the Burnham Institute, is enclosed at the end of section II and also in the Appendix. This letter states that the Burnham Institute will provide, as part of its commitment to this Program, two rigs for electrophysiological recording from slices and cell cultures. It is also now clearly stated that both Research Associates, Dr. Hadieh Badie-Mahdavi and Dr. Barbara Fredette, will contribute their expertise to the work in Core A starting in year 1. Their effort has been reduced to 50%, as recommended by the reviewers. This also reflects decreased needs due to elimination of the previous Project 1. Dr. Ranscht's effort, on the other hand, has been increased to 15% due to her ability to dedicate increased effort to the preparation of mixed neuron-glia cultures and also due to the need for her consultation to evaluate the effects of heparan sulfate proteoglycans and NG2 at the nodes of Ranvier, as recommended by the reviewers. The revised portions of the application are indicated with a line on the left margin. The overall editorial modifications introduced to unify the style of the application are not marked. Objectives The goal of this Program is to analyze the molecular signals exchanged between neurons and glia at synapses and in myelinated axons. The program has identified cell surface components implicated in neuron-glia communication and now intends to study the function of these molecules in vivo and in tissue culture models that closely mimic the in vivo interactions. Core A of this Program Project will provide the infrastructure and the expertise to accomplish this goal. The Core will offer support for two aspects of the work, analysis of neuronal function by electrophysiology and modeling neuron-glia interactions in suitable culture systems. The Electrophysiology component adds a new research dimension that will provide the Program with the tools and know-how for functional and activity-dependent studies designed to acquire knowledge of the electrophysiological changes occurring in neurons in response to glial cells or glial-derived ligands. It is now apparent that neuron-glia interactions contribute to the functional properties not only of myelin but also of synapses. Addition of the electrophysiology component to the Program will overcome previous limitations and enable the Program to functionally analyze functional defects resulting from the genetic disruption of neural cell surface proteins and their associated signal transduction pathways. The electrophysiological approach is geared towards the analysis of functional neuron-glia interactions at the level of nerve impulse conduction along axons, as well as synaptic function and plasticity. Electrophysiological recordings will assess the modifications in neuronal membrane properties resulting from changes in the expression or function of glial proteoglycans. The Program will employ transgenic and knockout mice that are already in hand and have a sufficiently long survival time for conducting the electrophysiological analyses. Electrophysiological recordings will be conducted on brain slices, single cells, and isolated nerves. Slice recordings will focus on determining the role of the glial protein ephrin-A3 in synaptic efficacy changes during LTP and LTD. Single-cell recording will assess the contribution of ephrin- A3 to neuronal excitability and plasticity through its neuronal receptor, EphA4. Whole nerve recording will detect conduction velocity changes in the compound action potential caused by malfunction of the myelin sheath. Recordings at these different levels are necessary for gaining insights into the molecular interactions that underlie neuron-glia crosstalk as outlined in the individual projects. The second critical aspect of the proposed work is to probe neuron-glia interactions at the cellular level using material from genetically manipulated mice. This is effectively accomplished using suitable systems of primary neural cultures that mimic specific in vivo interactions. Specifically, Core A will provide the co-cultures of neurons and myelin-forming Schwann cells or oligodendrocytes for studies of myelinogenesis. The use of hippocampal cultures for studies on the influence of glial ephrin-A3 in regulating synaptic function and plasticity will be a continuation of the current Core.

The reviewers of the A1 application considered the Core essential for the successful accomplishment of all the projects in the Program. Discrepancies between the budget and the grant text have been corrected in the current revision. The letter from Ms. Karin Eastham, Vice President and Chief Operating Officer of the Burnham Institute, is enclosed at the end of section II and also in the Appendix. This letter states that the Burnham Institute will provide, as part of its commitment to this Program, two rigs for electrophysiological recording from slices and cell cultures. It is also now clearly stated that both Research Associates, Dr. Hadieh Badie-Mahdavi and Dr. Barbara Fredette, will contribute their expertise to the work in Core A starting in year 1. Their effort has been reduced to 50%, as recommended by the reviewers. This also reflects decreased needs due to elimination of the previous Project 1. Dr. Ranscht's effort, on the other hand, has been increased to 15% due to her ability to dedicate increased effort to the preparation of mixed neuron-glia cultures and also due to the need for her consultation to evaluate the effects of heparan sulfate proteoglycans and NG2 at the nodes of Ranvier, as recommended by the reviewers. The revised portions of the application are indicated with a line on the left margin. The overall editorial modifications introduced to unify the style of the application are not marked

Our study using a conditional knockout of EXT1, te gene encoding an enzyme essential for heparan sulfate biosynthesis, has demonstrated the crucial ole of heparan sulfate proteoglycans (HSPGs) in multiple CMS morphogenetic evets (Inatani et al., Science 302:1144-1146, 2003). Yet the role of HSPGs in the nervous system is not limited to the CNS. There is evidece that HSPGs also play critical roles in the development and physiology of the peripheral nervous systm (PNS) by mediating molecular communications between axons and gia cells. This Component will focus on the role of heparan sulfate and HSPGs in neuron-glia interactions using Schwann cell-axon interactio as a model system. Aim 1. Role of heparan sulfate in Schwann cell development: We have found that in ur EXT1 conditional knockout mice, the number of Schwann cells in peripheral nerves is greatly reduced. This suggests that HSPGs play a crucial role in the proliferation and/or migration of Schwann cells, the developmental events in which axon-derived factors and adhesive interactions with axons play significan roles. We will employ the EXT1 conditional knockout mice, cultures of Schwan cells, and neuregulin-1 knockout mice to investigate the role of HSPGs in Schwann cell development and their involvement in neuregulin-ErB signaling. Aim 2. Role of heparan sulfate in myelin formation and function: Molecules that bind SPGs, such as laminins and neuregulins, are known to play criticalroles in myelination, and our preliminary studies have demonstrated aberrant myelination in the EXT1 conditional knockout mice. There are also clinicl reports suggesting that HSPGs are involved in the development of neuropathies. In this aim, by enetic, cell biological, and physiological experiments, we will investigate the role of HSPGs in developmental and physiological aspects of PNS myelin ensheathment. Overall, this Component will generatenew insight into the function of HSPGs in various types of neuron-glia interactions. Furthermore, through a series of collaborative studies, we will explore new lines of research at the interface between Components, obtaining answers to important questions beyond the immediate reaches of each Component.

Observations of molecular changes in patient tissues or caused by genetic or experimental manipulations in mice and cancer cells in culture critically depend on histological and immunohistological analyses in cells and tissues through advanced imaging techniques. The mission of the Cell Imaging and Histopathology Shared Resource is to provide state-of-the-art imaging and histopathology tools, training, and services to Cancer Center researchers. The Shared Resource is divided into two adjacent Facilities: 1) The Cell /mag/ng facility houses four confocal microscopes and five additional fluorescence microscopes. All the microscopes are equipped with advanced digital cameras and image analysis software. Available systems include laser scanning confocal microscopes with single and multi-photon lasers, as well as a spinning disk confocal microscope for extended live cell imaging. Time-lapse imaging systems are available to study cell motility, proliferation, and differentiation, with FRET and TIRF systems to image intracellular dynamic interactions. A full range of wide-field imaging approaches are also supported. The facility transmission electron microscope service provides analysis of cell and tissue ultrastructure. Facility staff maintains the imaging equipment and provides investigator training and support for imaging and image analysis, as well as imaging service, and active development of new imaging tools. 2) The Histopathology facility offers the preparation, sectioning, staining and analysis of tissue samples for histological and histopathological analyses and probe-specific detection of cellular and molecular changes in tissue sections. For analysis of identified cells or cell groups within tissue sections, the facility offers Laser Capture Microdissection services. An automated slide scanning system affords efficient histological data acquisition, image archiving, analysis and quantitation. A staff pathologist with expertise in both human and mouse specimens provides consultation, as well as assistance with tissue microarrays. The Facility staff provides consultation, user training, development and dissemination of imaging-, histology- and immunohistochemical protocols, and assistance with instrumentation for efficient histology data documentation, analysis and storage. The Shared Resource supported the studies of 35 Cancer Center Members in the past year, and is essential to advance the understanding of molecular and cellular events leading to cancer development and spreading. Overall, $185,234 in CCSG support is requested in the first year, representing 18.4% of the total projected annual operating budget.

DESCRIPTION (provided by applicant): Obesity-related metabolic syndrome is major risk factor for cardiovascular diseases. Adiponectin (APN), a circulating fat-secreted cytokine, is implicated in protecting against obesity-related metabolic and cardiovascular dysfunctions. Levels of APN decrease with the expansion of adipose tissue and low levels are associated with coronary artery disease, hypertension, heart infarct and other cardiovascular dysfunctions. In animal models of cardiac hypertrophy and ischemic heart disease, administration of APN improves pathological myocardial remodeling. While the beneficial functions of APN are well described, little is known about membrane receptors that enable APN's physiological functions in the cardiovascular system. T-cadherin, an APN binding protein implicated by genetic linkage analysis in cardiovascular functions, is a candidate cell surface glycoprotein to mediate APN functions. This R21 application will explore the functions of T-cadherin in a mouse model of heart disease and relate a potential role to the functions of adiponectin. PUBLIC HEALTH RELEVANCE: Obesity contributes to metabolic and cardiovascular disorders by altering the levels of adipocyte-secreted pro- and anti-inflammatory cytokines in the circulation. Adiponectin is a fat-secreted anti-inflammatory cytokine with beneficial actions in regulating metabolic and cardiovascular functions. Adiponectin associates with cardiomyocyte and endothelial cell surfaces to exert beneficial functions. The receptors that enable the engagement of adiponectin with cell surfaces and lead to activation of downstream signaling cascades remain poorly understood. Research proposed in this application will explore the contributions of T- cadherin, a novel adiponectin-binding cell surface glycoprotein, in functions of the heart and vasculature. Specifically, the proposed experiments will test if T-cadherin is essential for the cardioprotective actions of adiponectin. This work will contribute to understanding adiponectin's cardiovascular-protective functions and - if successful - form a new basis for exploring the T-cadherin-adiponectin interaction as a possible drug target for recovery from cardiovascular injury.

DESCRIPTION (provided by applicant): A growing body of evidence indicates the importance of cell adhesion molecules in regulating synapse formation, stability and function in the central nervous system (CNS). T-cadherin encoded by the Cdh13 gene, is a unique GPI-linked cadherin type adhesion protein discovered and extensively studied in our laboratory. Our work in the cardiovascular system established T- cadherin as a physiological key receptor/co-receptor for APN functions (Hebbard et al. 2008; Denzel et al 2010). Mutations in the human CDH13 gene have been associated with hypoadiponectemia and cardiovascular dysfunctions. In the nervous system, human case- control studies have linked clusters of nearby CDH13 SNPs to attention deficit hyperactivity and comorbid neuropsychiatric disorders. Elucidating functions for T-cadherin in the nervous system in Cdh13-deficient mice, we discovered defects in hippocampal synaptic function and plasticity manifesting in dendritic spine changes of Cdh13-deficient principal neurons. T-cadherin is expressed by GABAergic interneurons synapsing on dendrites of the principal pyramidal neurons that do not express T-cadherin. A major discovery that could bear on this issue is the fact that Adiponectin (APN), genetically requiring T-cadherin for regulating functions in the periphery, is expressed by hippocampal pyramidal cell receiving inputs from T-cadherin- expressing interneurons. In combination, these findings raise the intriguing hypothesis that APN is part of the T-cadherin signaling pathway that modulates circuits in the hippocampus, with profound affects on associative memory behaviors as Cdh13-KO mice show significant impairments in fear- and reward-based conditioning tests. In this exploratory proposal, we will genetically test the role of APN in hippocampal synaptic functions in relation to T-cadherin.

5.2 Abstract - CELL IMAGING AND HISTOLOGY (Core Group A) The Cell Imaging & Histology Shared Resource (Core Group A) provides Cancer Center researchers with modern imaging tools and histology services, facilitates microscope-acquired data documentation and analysis, and enables small and large volume electronic data sharing and storage. The Core supports studies of pathological and molecular changes in human cancers as well as in experimental animal and cell culture models that require histological examinations and probing of molecular changes in tissues and cells through advanced imaging techniques. The Cell Imaging Facility provides state-of-the-art microscopy resources. Data acquired using three confocal and six additional microscopes, supported 130 publications during the past grant cycle. In the past year, the imaging capabilities of the Facility were utilized by 26 Cancer Center laboratories. Expanded imaging capabilities, added through acquisition of a Zeiss LSM710 NLO multi-photon Laser Scanning Microscope in 2011, permits deep laser scanning confocal microscopy, and extended live cell imaging. High-resolution imaging and confocal microscopy, in combination with other imaging techniques, including FRET, TIRF and time-lapse imaging, permit a spectrum of applications, including studies of cancer cells motility, proliferation, and differentiation, protein localization and dynamic molecular interactions in cells, time-lapse visualization of three-dimensional cancer cell accumulation, and studies of cellular responses to drugs. Facility personnel maintain the microscopes, train investigators in optimal microscopy and imaging techniques, and in advanced image analysis. Full-service imaging is also available. The Histology Core supports histological and immunohistochemical analysis of cancer tissues, performs tissue preparation, fixation and processing, paraffin- and cryosectioning, and histological and immunological stains to monitor morphological and molecular alterations in cancer tissues. Last year, the Facility produced over 20,000 slides for 25 Cancer Center laboratories, and processed over 5,000 for histology or immunological stains. The Facility is taking a central role in archiving and sharing microscope-acquired data by scanning slides into the Aperio XT- and -FL scanners, and supports automated data analysis and quantitation. The availability of easily shared high-resolution scans of histological samples aids in utilizing an extended network of specialized consulting pathologists who assist the Cancer Center. The core's web site provides contact information for a number of pathologists, and the Facility Manager assists in matching a project with specific expertise. The Histology Facility, which supported over 125 publications in the previous granting period, is currently launching a service to assist investigators in obtaining human tissues. Addition of a Tissue Procurement Specialist will help investigators locate sources for tumor tissues, facilitate inter-institutional agreements and IRB approvals, coordinating and managing the entire procurement process. Centralized and expert assistance with tissue procurement will greatly enhance the efficiency of connecting basic research with early translational studies.

PROJECT SUMMARY The goal of the proposed study is to develop a platform for peptide-targeted delivery of therapeutics into myelin lesions in Multiple Sclerosis (MS) patients. MS is a chronic inflammatory disease that progressively destroys the protective myelin sheaths that encircle the axons in the central nervous system. Loss of myelin and oligodendrocytes, the myelin-forming cells in the CNS, impairs neuronal functions and leads to neurodegeneration. Current therapies target the immune response and can reduce frequency and severity of autoimmune attacks. However, there are no clinical therapies for repairing damaged myelin and stopping progressive neurodegenerative decline and patient disability. As research efforts identify novel strategies for myelin maintenance and repair, a recurring problem is how to deliver drugs or treatment agents to sites of myelin injury to achieve disease improvement. For example, inhibition of LINGO-1 (Leucine-Rich Repeat And Immunoglobulin Domain-Containing Protein 1) in vitro and in mouse MS animals improves maturation of oligodendrocyte progenitor cells and myelination, and treatments with humanized LINGO-1 antibodies are in clinical trials for optic neuritis. Systemic application to target lesions in brain, however, has limits as antibodies are diluted in the circulation, may have off-target effects elsewhere, and may not reach effective concentrations at lesion sites. We here propose to test a novel targeting approach for MS treatment in which we will use a unique tetrapeptide that selectively recognizes an epitope in injured but not uninjured myelin. The targeting peptide was identified in an in vivo phage display peptide library screen for penetrating brain injuries in the mouse, and targets to an extracellular matrix epitope selectively upregulated in injured myelin. We will test targeting specificity of the peptide to myelin lesions in mouse models of inflammatory demyelinating disease (EAE), and attempt to induce myelin repair and improve disease symptoms by selective targeting of Lingo-1 siRNA to myelin lesions through packaging the siRNA into peptide-coated nanoparticles for administration into the circulation. The peptide coat on the nanoparticle surface presents an address code to deliver the siRNA cargo through the open BBB specifically to the binding epitope at the lesion site where the siRNAi's are released from the non- coated particle core for target gene silencing during particle degradation. The development of peptide targeting technology for MS offers a new approach to beneficially modify disease through specific high local concentrations of disease modifying activity with reduced systemic effects. If successful, the proposed work will set the stage for developing peptide-targeting applications for a range of agents to MS lesions.