Greg E. Lemke - US grants

The vertebrate nervous system develops and functions through a set of cell-cell interactions. Among the most striking of these interactions is that which occurs between neurons and glia, and results in the elaboration of the myelin sheath, the electrical insulation which surrounds rapidly conducting axons. For the glial cells which elaborate myeline, production of the sheath entails a drastic reorganization of both morphology and metabolism, including the induction and high-level expression of a set of genes and proteins unique to myelin-forming cells. This proposal is concerned with an identification of the cis and trans-acting elements which mediate activation and expression of the major myelin genes, and with an analysis of the function of the proteins these genes encode. Specific questions addressed by this proposal include: by what mechanisms are the major myelin genes induced? What role do axons play in this process? What are the cis-acting regulatory regions of these genes which control their cell-specific transcription? When and where are the major myelin genes expressed during neural development? And what can we learn of the function of the proteins that these genes encode? To address these and related questions requires both probes for the relevant genes and proteins, as well as the ability to manipulate the cellular environment of glial cells. The experiments described below therefore employ the techniques of eukaryotic molecular genetics, and the in vitro culture of purified glial cells and cell lines, in conjunction with the application of recombinant DNA and antibody probes for the major myelin genes and proteins. The ultimate goal of this work is the delineation of the molecular pathway of myelin formation during normal development. Additionally, it may provide an understanding of glial cell gene expression and protein metabolism under circumstances in which glia are deprived of the influence of neurons, as occurs in central and peripheral neuropathies, and during episodes of demyelination and remyelination, as occur in multiple sclerosis.

The vertebrate nervous system develops and functions through a set of cellular interactions. Among the most striking of these interactions is one that occurs between neurons and glia, and results in the elaboration of the myelin sheath, the electrical insulation that surrounds all rapidly conducting axons. For the glial cells that elaborate myelin, production of the sheath involves a drastic reorganization of both morphology and metabolism, including the induction and high-level expression of a set of genes and proteins unique to myelin-forming cells. This proposal is concerned with the identification and functional description of the genes that mediate glial cell differentiation and myelination. Specific questions addressed include: By what mechanisms are the major and minor myelin genes induced? What role do neurons play in this process? What are the cis-acting regulatory elements of these genes that control their cell-specific transcription? When and where are the major and minor myelin genes expressed during neural development? And what can we learn of the function of the proteins these genes encode? To address these and related questions requires both probes for the relevant genes and proteins, as well as the ability to manipulate the cellular environment of glial cells. The experiments described below therefore employ the techniques of eukaryotic molecular genetics and the in vitro culture of purified glial cells and cell lines, in conjunction with the application of recombinant DNA and antibody probes for glial-specific genes and proteins. The ultimate goal of this work is the delineation of the molecular pathway of myelin formation during normal development. Additionally, it may provide an understanding of glial gene expression and protein metabolism under circumstances in which glia are deprived of the influence of neurons, as occurs in central and peripheral neuropathies, and during episodes of demyelination and remyelination, as occur in multiple sclerosis.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The differentiation of the nervous system depends on a sequence of related developmental decisions. These decisions - mediated by cell-cell interactions - include (1) the choice between neural versus non-neural cellular lineages, (2) the subsequent choice among the wide variety of specific neural cell fates, and (3) the choice of appropriate target cells with which to synapse. The long-term goal of this project is to understand the molecular basis of each of these important developmental decisions. In Project 1, led by Chris Kintner, we will study the role of a pair of interacting receptor-ligand proteins (Notch and Delta) in neural specification in the developing Xenopus embryo. In functional studies, we will test the hypothesis that, as in Drosophila, these two molecules play a determinative role in the initial choice of neural versus non- neural cell fate. In Project 2, led by Greg Lemke, we will study the structure and function of four new neural receptor tyrosines kinases (Tyrol 2, 3, and 6). Proteins related to the Tyros are known to function as receptors for neurotrophic regulators such as Nerve Growth Factor, and to directly control the choice between specific neural cell fates. In project 3, led by John Thomas, we have capitalized on the genetics of Drosophila to identify a gene (bendless) that controls the final choice of synaptic targets in a well-defined neuronal circuit. We will identify the bendless gene product and study its role in the target recognition process. These experiments will provide us with fundamental insights into the cellular interactions that mediate neural development, and a detailed understanding of their molecular basis.

nucleic acid sequence; nucleic acid chemical synthesis; biomedical facility; synthetic nucleotide; oligonucleotides;

The vertebrate nervous system develops and functions through a set of cellular interactions. Among the most striking of these interactions is one that occurs between neurons and glia, and results in the elaboration of the myelin sheath, the electrical insulation that surrounds all rapidly conducting axons. For the glial cells that elaborate myelin, production of the sheath involves a drastic reorganization of both morphology and metabolism, including the induction and high-level expression of a set of genes and proteins unique to myelin-forming cells. This proposal is concerned with the identification and functional characterization of the genes that mediate glial cell differentiation and myelination. Specific questions addressed include: By what mechanisms are myelination-specific genes induced and regulated? What role do neurons play in these processes? What are the cis-acting regulatory elements of these genes that control their cell-specific transcription and what are the trans-acting proteins that bind these elements? When and where are myelination genes expressed during neural development? And what can we learn of the function of the proteins these genes encode? To address these and related questions requires both probes for the relevant genes and proteins, as well as the ability to manipulate the cellular environment and development of myelinating glia. The experiments described below therefore employ the techniques of eukaryotic molecular genetics and the in vitro culture of purified glial cells and cell lines, in conjunction with the application of recombinant DNA and antibody probes for glial- specific genes and proteins. The ultimate goal of this work is the delineation of the molecular pathway of myelin formation during normal development. Additionally, it may provide an understanding of glial gene expression and protein metabolism under circumstances in which glia are deprived of the influence of neurons, as occurs in central and peripheral neuropathies, and during episodes of demyelination and remyelination, as occur in Multiple Sclerosis.

Neural development requires the generation of a remarkable diversity of neural cell types and the stereotyped and often highly ordered specification of synaptic connections between these cells. A large number of studies have demonstrated that receptor-configured protein-tyrosine kinases (PTKs) play central roles in both of these events. Recent work from our laboratory and others has provided strong circumstantial evidence that several members of what is the largest sub-family of receptor PTKs in vertebrates-the Eph receptors-function to pattern topographic axonal projects that map ordered synaptic activity in one set of neurons onto another. Perhaps the best studied of such maps connects the retinal ganglion cells of the eye to their targets in the superior colliculus (SC) of the midbrain. In this map, a gradient of EphA receptors in the retina is hypothesized to interact with a complementary gradient of axon-repellant ephrin-A ligands in the SC. We propose to experimentally manipulate and analyze a batter of recently generated mouse lines that carry both loss-of-function and gain-of-functions mutations in the genes encoding many of these receptors and ligands, and to thereby assess in vivo the validity of previously advanced but largely untested hypotheses as to the mechanism of action of the EphA signaling system. We will also use these same mouse mutants to experimentally assess the functional relevance of this signaling system to the specificity of synaptic innervation from motor neurons in the spinal cord to their muscle targets in the limbs and the body wall.

Neural development requires the generation of an enormous diversity of distinct cell types, and the early assumption of individual cell fates in large part controls the subsequent development of the nervous system. A large body of evidence, from work in Drosophila, C. elegans, and mammalian systems, has suggested that transcription factors of the POU domain family play a central role in the initial commitment to specific cellular lineages, in the transactivation and repression of neural- specific genes, and in the terminal differentiation and function of neural cells. We wish to understand the role of one particular POU domain protein, specifically with regard to the determination of neural cell fate and the timing of neural cell differentiation. This transcription factor - designated SCIP - is transiently expressed by differentiating Schwann cells, oligodendrocytes, and a subset of central nervous system neurons. It is hypothesized that initial activation of the SCIP gene is required for lineage commitment in these cells, and that a subsequent deactivation of this gene is necessary in order for these cells to terminally differentiate. To dissect SCIP function in the developing nervous system, we propose to generate and analyze conditional knock-outs of the mouse SCIP gene in neurons and glia, and to identify, clone, and analyze transcriptional co-factors essential for SCIP action. We also propose to use antibodies to SCIP to define developmental stages of the mammalian Schwann cell lineage. We hope through these experiments to elucidate fundamental mechanisms underlying POU domain regulation of the development of neurons and glia.

DESCRIPTION (Abstract verbatim): The experiments of this application are designed to assess the function of the three cell surface receptor tyrosine kinases of the Tyro 3 family. These proteins, designated Tyro 3, Ax1, and Mer, are expressed in a variety of differentiated cell types in the central and peripheral nervous systems, in the immune and hematopoietic systems, and in the endocrine and reproductive systems, where they are thought to receive extracellular signals that regulate trophic interactions and cellular homeostasis. These signals are conveyed by Gas6 and protein S, two polypeptide ligands that bind to, and thereby activate Tyro 3 family receptors. The studies proposed rely on a unique set of genetic reagents -- mutant mice that lack the function of each of the receptors, both singly and in combination. These mice, particularly those that carry mutations in more than one of the three receptor genes, display devastating major organ defects, neurological abnormalities, and physiological deficits as adults. They are infertile, blind, and prone to epileptiform seizures. Their immune systems are seriously compromised, their vasculature appears to be weakened, and their brains, livers, and reproductive organs undergo widespread cellular degeneration. Most remarkably, these phenomena appear to be truly degenerative as opposed to developmental: each of the affected tissues undergoes superficially normal embryonic and early postnatal development, and only then is the degeneration of cells and tissues observed. We will use a combination of genetic, biochemical, and immunohistochemical methods to understand the cellular basis of the degeneration observed in each of the affected tissues. We will determine what cells are degenerating and how they are degenerating, when degeneration begins and ends, what cells in the affected tissues express which and how many of the receptor genes that have been inactivated, whether the receptor-expressing cells are also those that degenerate, and what cells in the affected tissues express which ligands. Together, these studies will reveal the basics of cell-to-cell signaling through Tyro 3, Axl, and Mer -- three receptors of genuine biological importance.

DESCRIPTION (From the Applicant's Abstract): Reciprocal signaling interactions between differentiating glia and neurons shape many of the salient features of the developing nervous system. These include the consolidation of its dorsal-ventral, anterior-posterior, and medial-lateral axes, the regulation of axon growth and guidance, the control of cell proliferation and survival in functionally-related networks, and the myelination of axons. Among the most dramatic examples of glial-neuron interaction are those related to axon guidance. In this proposal, we address two nuclear proteins that function as key regulators of this process. These proteins - Vax1 and Vax2 - are novel homeodomain transcription factors unique to vertebrate nervous systems. Previous genetic studies in our laboratory have suggested that Vax 1 controls the expression of essential axon guidance cues, including the chemoattractant netrin-1 and the receptor tyrosine kinase EphB3, in a set of specialized glial cells at the ventral anterior midline of the developing forebrain and in the optic nerve. Additional preliminary work is consistent with a similar role for Vax2 in a set of specialized guidance glia in the peripheral nervous system. In the experiments detailed in the five specific aims of this proposal, we will exploit a battery of existing mouse mutants, and generate new mutants, to genetically dissect Vax1 and Vax2 function in the developing CNS and PNS. We will identify the targets of Vax1 action in the astrocyte precursors of optic nerve, and assess the genetic interaction of the Vax1, netrin-1, EphB3 and slit-1 genes in the developing forebrain. We will similarly identify targets of Vax2 action in the developing eye field, and assess the role that this transcription factor plays in establishing the dorsal-ventral axis of the retina. We will also study its function in a subset of peripheral glia (Schwann cells) in developing peripheral nerves. Finally, we will investigate the strong genetic interaction that we have recently observed between the Vax genes in forebrain development. Together, these studies will yield fundamental new insights into the transcriptional regulation of glial-neuron interactions in the developing central and peripheral nervous systems.

[unreadable] DESCRIPTION (provided by applicant): Project Summary: The vertebrate eye is among the most exquisitely polarized of sensory tissues, and its axial patterning during development is essential to both its orderly connection to the brain and its mature function. Although the retina appears superficially uniform and featureless, it is in fact a highly ordered epithelium that displays gradients of signaling proteins and their receptors, enzymatic activities, and transcription factors. We wish to understand how these gradients are established during eye development. Our goal in this project is the functional analysis of genes that control axial patterning. The experiments of the proposal focus on Vax1 and Vax2, which encode two closely-related transcription factors that play key roles in the specification of the eye's dorsal-ventral axis. We will exploit a set of existing mouse mutants, and will also generate new mutants, in which the structure, expression, and activity of the Vax genes is systematically altered. We will use these genetic reagents, together with biochemical and cell biological assays, to perform a detailed dissection of Vax function during two distinct phases of eye development. We will identify and analyze the target genes whose expression is regulated by Vax 1 and Vax2 during both of these phases. We will study the mechanisms by which Vax2 activity within cells is regulated by the subcellular trafficking and localization of the protein. And we will identify the'protein partners that interact with, and modulate the activity of, Vax1 and Vax2. Together, these experiments will reveal the molecular mechanisms through which transcription factor gradients specify the Cartesian coordinates of the eye. Relevance: Our ability to interpret the light that falls onto the surface of the retina - to see the world around us - requires an ordered set of connections between the eye and the brain. Order within these connections is first established by the coordinate axes of the eye. The experiments of this proposal are designed to provide fundamental insights into the genetic and biochemical mechanisms through which these axes are organized - from top to bottom and from side to side - during development. [unreadable] [unreadable]

by e-mail and telephone. As is also described in the Introductory Overview, review of scientific progress toward the objectives of the program will be provided, as it has over the past eight years, by the Salk Institute's non-resident fellows, who meet once per year at the Institute at the time of the annual faculty meeting (early April). Ms. Becky Hensley (50%), Administrative Assistant III,will continue to devote a substantial portion of her daily efforts to the coordination of lab meetings, purchase orders, seminar scheduling, service requirements, and budget management directly related to the component projects of the program. $5,837 is requested for computer and office supplies (computer software and discs, computer maintenance, notebooks, lab books, general office supplies, xerox supplies, etc.)to support administration of the program project. $2,500 is requested for service charges directly related to the administration of the program project. 246 Lemke, Greg E. CoreB ANALYTICAL CORE Dennis D.M. O'Leary, PI 247

DESCRIPTION (provided by applicant): The experiments of this application are designed to elucidate the roles that the TAM receptor tyrosine kinases - Tyro 3, Axl, and Mer - play in the homeostatic regulation of innate immunity. We have discovered that these receptors, together with their agonists Gas 6 and Protein S, are essential intrinsic inhibitors of the inflammatory response to infection. Tyro 3, Axl, and Mer are expressed by macrophages and dendritic cells (DCs). These cells respond to invading bacteria and viruses by producing a panoply of pro-inflammatory and inflammatory cytokines, which, while essential to the defeat of pathogens, are also injurious to normal cells and tissues. If unchecked at the end of the innate immune response, production of cytokines such as TNF-1 leads to chronic inflammatory disease and autoimmunity. The proposed studies exploit a powerful set of genetic reagents - mutant mice that lack the function of each of the receptors and of the activating ligands. As adults, these mice display devastating immune deficits, including lymphoproliferation marked by severe splenomegalogy and lymphadenopathy, systemic hyperactivation of both antigen-presenting cells and of B and T lymphocytes, defects in Natural Killer cell function, and broad-spectrum autoimmune disease. We will investigate the cellular and molecular basis of these phenomena. We will determine the relative contributions of Gas6 and Protein S to TAM receptor activation, and investigate the biochemical mechanisms that underlie suppression of innate immune responses by TAM signaling. We have found that a central mechanism of TAM-mediated suppression involves activation of the transcription factor STAT1 - which is also initially required to activate cytokine production. This sets up a classic negative feedback loop for the regulation of inflammation. We will also elucidate the role that TAM receptors play in triggering the phagocytosis and clearance of apoptotic cells, and dissect the extracellular and intracellular components of the pathway through which the receptors receive and transduce signals required for this process. Together, these studies will provide basic insights into a new signaling system of critical importance to the regulation of immune responses and the development of autoimmunity. Relevance: Autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis exact a major toll on human health. Our studies have revealed that the TAM signaling system is a key regulator of the physiology of the cells that become chronically activated in these diseases, that deficient TAM signaling contributes to the development of autoimmunity, and that hyper-activation of TAM signaling may play a role in sepsis. This receptor system is therefore a novel and attractive target for therapeutic modulation and intervention in disease treatment.

DESCRIPTION (provided by applicant): The receptor tyrosine kinases (RTKs) of the TAM family - Tyro3, Axl, and Mer - are exploited by flaviviruses, such as West Nile Virus and Dengue Virus, to infect their target cells. The experiments of this proposal are designed to elucidate how TAM potentiation of infection is achieved. TAM RTKs are expressed on the surface of many cells, including dendritic cells and macrophages of the immune system, and neurons of the central nervous system. The ligands that bind to and activate the TAMs - Gas6 and Protein S (ProS) - have the ability to attach both to the phospholipid phosphatidylserine, expressed on the surface of flavivirus particles, and also to their cognate TAM receptor. In this way, the TAM ligands serve as a 'bridge' that links flaviviruses to the surface of cells that they will infect. t the same time, TAM receptor activation by Gas6 or ProS results in the inhibition of type I interferon (IFN) signaling, to which flaviviruses and many other viruses are sensitive. In Aim 1, genetic, biochemical, and cell biological methods will be used to dissect the mechanisms through which TAM receptors facilitate flavivirus entry into human cells in vitro. In Aim 2, these same methods will be used in experiments in dendritic cells and macrophages prepared from mice that carry targeted mutations in the Axl and Mer genes, and in CNS neurons prepared Tyro3 mutant mice, to define the TAM receptors and ligands that promote infection in different cellular settings, and to determine the extent to which TAM facilitation of flavivirus infection is dependent on the inhibition of type I IFN signaling. In Aim 3, mice that carry mutations in the Tyro3, Axl, Mer, Gas6, and ProS genes will be used to assess the cellular and immunological consequences of specific deficits in TAM signaling for infection by West Nile Virus in vivo. Together, these experiments will delineate the molecular, cellular, and physiological features of a heretofore unknown pathway of flavivirus infection.

DESCRIPTION (provided by applicant): Deficiencies in the control of central nervous system (CNS) inflammation precipitate or exacerbate a plethora of debilitating human diseases, yet our understanding of the basic mechanisms that regulate neuroinflammation is incomplete. Microglia, the distinctive tissue macrophages of the brain and spinal cord, are key players in this process. These sentinel cells display two activities that are fundamental to the maintenance of neural homeostasis - (i) immune surveillance and (ii) the phagocytosis of apoptotic cells (ACs) and membranes. Studies of macrophages outside of the CNS have demonstrated that both of these activities are strictly controlled by signaling through TAM receptor tyrosine kinases. Although TAM receptors are also prominently expressed by microglia, the importance of TAM signaling to microglial activation and function in the CNS is - remarkably - unknown. The experiments of this proposal address this question. In Aim 1, genetic and cell biological methods that rely on TAM receptor and ligand mouse mutants will be used to assess the importance of TAM signaling in the homeostatic, non- inflammatory phagocytosis of ACs that occurs continuously in the healthy mammalian brain. In Aim 2, similar methods, coupled with confocal and two-photon imaging of microglia in vivo, will be used to determine the role of TAM signaling in the localized phagocytosis that underlies synaptic pruning and the remodeling of neuronal connections in postnatal neural development. In Aim 3, a series of pro- and anti- inflammatory challenges will be applied to TAM receptor- and ligand-deficient mice to determine if inhibition of the innate inflammatory response in microglia is under TAM control, as it is in cells of the immune system. Finally, in Aim 4, the knowledge gained from the earlier aims will be applied to investigate the role that TAM regulation plays in neurodegeneration, as assessed in both acute and progressive mouse models of Parkinson's disease. Together, these studies will delineate the basic molecular, cellular, and physiological features of a fundamentally new pathway of immune homeostasis in the CNS, and potentially identify TAM receptors and ligands as new targets for therapeutic intervention in neuroinflammatory and neurodegenerative disease.

PROJECT SUMMARY/ABSTRACT The receptor tyrosine kinases of the TAM family ? Tyro3, Axl, and Mer ? are essential regulators of infection by enveloped viruses, and Axl and Mer are especially important to arbovirus infection of cells of the central nervous system (CNS). Infection of the CNS by encephalitic arboviruses, including West Nile virus (WNV), often has devastating consequences, both acutely and after recovery, and deciphering the molecular mechanisms through which TAM receptors control virus entry, propagation, and clearance is therefore a key objective. Genetic, molecular biologic, cell biologic, and behavioral assays will be used to elucidate these mechanisms. In Aim 1, a set of new conditional mouse mutants and cell-specific Cre drivers will be used to investigate the CNS cell types through which TAM receptors control infection by WNV and two other neurotropic enveloped arboviruses - La Crosse encephalitis virus and Venezuelan equine encephalitis virus. These studies will elucidate the specific roles played by Axl and Mer in brain microvascular endothelial cells (BMECs), microglia, astrocytes, and pericytes in neuroinvasion and CNS pathogenesis by these viruses. In Aim 2, a new mouse model of learning impairment after recovery from CNS infection by WNV will be used to probe the role that Axl and Mer in microglia and astrocytes play in spatial learning and memory after infection. These experiments also will assess the role that cell-specific TAM signaling plays in synapse elimination and neurogenesis subsequent to WNV infection of the brain. In Aim 3, molecular genetics and cell-based signaling assays will be used to elucidate the molecular architecture of TAM receptor-Interferon receptor (IFNAR) interaction, which is crucial to the phenomena of Aims 1 and 2, in BMECs, microglia, macrophages, and dendritic cells. These studies will identify the signaling pathways activated by cooperative TAM receptor/INFAR signaling in these cells, and assess the ability of these interacting receptor systems to reorganize actin cytoskeletons. Together, the experiments of this proposal will guide the formulation of novel strategies for inhibiting virus entry into the CNS, attenuating virus infection of neural cells, and promoting the repair and recovery of infected neural tissues.

PROJECT SUMMARY/ABSTRACT Unrestrained inflammation of the central nervous system (CNS) precipitates and exacerbates a wide range of debilitating neurodegenerative diseases, including Alzheimer?s disease (AD), yet our understanding of the mechanisms that regulate neuroinflammation is incomplete. Microglia, the distinctive tissue macrophages of the CNS, display two coupled activities that are fundamental to this regulation ? the inhibition of neurotoxic inflammatory cytokines and chemokines, and the phagocytosis of apoptotic cells and membranes. Multiple lines of evidence demonstrate that these coupled microglial activities are controlled by signaling through TAM receptor tyrosine kinases. The experiments of this proposal address how the TAM receptors Axl and Mer (gene name Mertk), whose microglial expression is markedly elevated in AD and its animal models, act to restrain disease. In Aim 1, genetic and cell biological methods that exploit mouse models of AD will be used to quantify expression of TAM signaling components in the CNS with respect to amyloid burden, cytokine expression, and lifespan during the course of disease. In Aim 2, mice that carry Axl and Mertk mutations, or mutations in the genes encoding the TAM ligands Gas6 and Pros1, will be crossed to AD models to assess how global loss of TAM signaling affects the course of disease. Survival, cytokine elevation, amyloid burden, physiological dysfunction, and cognitive decline will be quantified in these compound mutants. Tests will also be performed to quantify the effects of drugs that inhibit TAM receptor activity, or alternatively, boost TAM receptor expression, on AD development and progression. Several TAM inhibitors are now being given to patients in clinical trials as cancer therapies, with no appreciation of their potential effect on AD. In Aim 3, cell-specific inactivation of the Axl and Mertk genes - only in microglia, or alternatively, only in TAM-expressing brain vascular endothelial cells - will be performed. Analyses of these mice will quantify the relative contribution of TAM action in each of these cell types to the control of AD. Together, these studies will delineate the molecular, cellular, and physiological features of a fundamental pathway of immune homeostasis in the CNS, and elucidate how TAM receptors and their ligands may be exploited as new targets for therapeutic intervention in AD and other neurodegenerative disorders.