James L. Salzer - US grants

Long term objectives of this project are to characterize early events of myelinogenesis and to gain an understanding of the process by which the expression of genes for specific myelin proteins is regulated. To this end, the genes for the major peripheral myelin glycoproteins, Po and the myelin associated glycoprotein (MAG), will be studied. Po is an integral membrane protein and is the major peripheral myelin protein; it is likely to play and important role in the structural integrity of the peripheral myelin sheath. MAG has a localization within the myelin sheath that suggests a role in mediating glial-neuronal interactions; it is also preferentially lost in acute plaques of multiple sclerosis and it is the antigenic target of paraprotein induced peripheral neuropathies further underscoring its potential importance. cDNA clones for MAG and Po will be isolated by a common strategy. Antibodies against purified MAG and Po will be used to identify, in vitro translation experiments, mRNA fractions extracted from mouse peripheral nerves, which are enriched in sequences coding for MAG and Po. These fractions will be used as probes to isolate the respective cDNAs from a mouse peripheral nerve library (to be prepared). The primary structure of the proteins will be derived from the nucleotide sequence of the MAG and Po cDNAs which will also be used to isolate the genes for Po and MAG from a genomic library. The elucidation of the structure of the genes and an evaluation of intra-exon relationships may be useful in identifying possible functional domains of the proteins. Electron microscopic heteroduplex analysis of Po and PLP may reveal common sequences reflecting a common evolutionary origin. The defect in the peripheral dysmyelinating mouse mutatn Trembler, will be studied by determining mRNA levels of MAG, Po and the MBPs. Long range goals include studying the regulation of myelin protein gene expression in a peripheral myelin tissue culture system.

Long term objectives of this project are to characterize early events of myelinogenesis and to gain an understanding of the process by which the expression of genes for specific myelin proteins is regulated. To this end, the genes for the major peripheral myelin glycoproteins, Po and the myelin associated glycoprotein (MAG), will be studied. Po is an integral membrane protein and is the major peripheral myelin protein; it is likely to play and important role in the structural integrity of the peripheral myelin sheath. MAG has a localization within the myelin sheath that suggests a role in mediating glial-neuronal interactions; it is also preferentially lost in acute plaques of multiple sclerosis and it is the antigenic target of paraprotein induced peripheral neuropathies further underscoring its potential importance. cDNA clones for MAG and Po will be isolated by a common strategy. Antibodies against purified MAG and Po will be used to identify, in vitro translation experiments, mRNA fractions extracted from mouse peripheral nerves, which are enriched in sequences coding for MAG and Po. These fractions will be used as probes to isolate the respective cDNAs from a mouse peripheral nerve library (to be prepared). The primary structure of the proteins will be derived from the nucleotide sequence of the MAG and Po cDNAs which will also be used to isolate the genes for Po and MAG from a genomic library. The elucidation of the structure of the genes and an evaluation of intra-exon relationships may be useful in identifying possible functional domains of the proteins. Electron microscopic heteroduplex analysis of Po and PLP may reveal common sequences reflecting a common evolutionary origin. The defect in the peripheral dysmyelinating mouse mutatn Trembler, will be studied by determining mRNA levels of MAG, Po and the MBPs. Long range goals include studying the regulation of myelin protein gene expression in a peripheral myelin tissue culture system.

The long term goal of this project is to identify and characterize the membrane components that mediate the critical axonal-glial interactions of myelination, with the view that these constituents may be implicated in the pathogenesis of demyelinating diseases. The myelin-associated glycoproteins (the MAG proteins) are integral membrane proteins of myelinating glial cells that have been proposed to maintain the apposition of the glial membrane to the axon. Their precise role during myelinogenesis however, is not known. We will use the recently isolated and myelinogenesis however, is not known. We will use the recently isolated and characterized cDNA clones for these proteins in studies intended to elucidate their physiologic role in myelination, to define their functional domains, and eventually to identify the axonal constituents with which they interact. Specifically we will: i) verify the predicted disposition of these proteins within the phospholipid bilayer as a prelude to further investigations of their structure-function relationships. We will establish the orientation of specific domains of the proteins with monospecific antibodies, as well as determining the size of protease sensitive segments of the MAG proteins which have been cotranslationally inserted into microsome membranes or are being expressed in transfected heterologous cells. ii) We will determine the role of the MAG proteins in cell adhesion by studying the interaction of permanently transfected L-cells, which express these proteins at their cell surface, in co-cultures with rat sensory neurons at both the LM and EM levels. If a specific interaction is documented, then further studies of the role of the arg-gly-asp (RGD) peptide sequence of the MAG proteins in this interaction will be undertaken. We also propose to isolate a functionally active soluble form of the MAG proteins by using non-ionic detergents or by genetically engineering a secreted form of these proteins. This will facilitate functional studies of these proteins and, in the future, may aid in the identification of their axonal receptor by cross-linking techniques. iii) We will disrupt the function of the MAG proteins in myelinated peripheral nerve cultures. We will assess the effects in these cultures of monospecific and polyclonal antibodies, as well as of a synthetic peptide corresponding to the RGD sequence of the MAG proteins on the formation and integrity of the myelin sheath and periaxonal space.

The long term goal of this project is to identify and characterize the membrane components of axons and glial cells that mediate the cell-cell interactions of myelination in order to understand the normal development and the acquired disorders of myelinated nerve fibers. A major portion of our studies, has focused on the role of the myelin-associated glycoproteins (MAG). These proteins are integral membrane proteins of myelinating glial cells that are uniquely positioned to mediate the cell interactions of myelination - binding to specific axons via their ectodomain and, potentially, transducing intracellular signals through distinct cytoplasmic segments. To clarify MAG's physiologic role in myelination, we will characterize the structural elements that mediate and may regulate its adhesive function and identify the cytoplasmic constituents with which it interacts. In parallel studies, we have been characterizing surface components of those axons that are destined to become myelinated, with the view that proteins unique to these nerve fibers will provide important insights into their ensheathment fate by the glial cell and, in particular, the adhesion mediated by MAG. We have recently identified an apparently novel axonal protein that has a restricted fiber type distribution that we propose to characterize further. Specifically we will study wild type and recombinant MAG proteins (generated by site directed mutagenesis) in adhesion assays and in coprecipitation studies to: 1) identify the binding sequences of the ectodomain of MAG that promote adhesion, 2) determine whether this adhesion is regulated by the cytoplasmic segment, 3) determine if the two isoforms of MAG, that differ in their cytoplasmic sequences, are targeted to different membrane domains in cells, 4) determine whether these two isoforms differentially associate with cytoplasmic constituents, including kinases, 5) investigate the functional significance of the differential phosphorylation of the MAG isoforms and 6) investigate the axonal regulation of this phosphorylation. We will also purify and isolate cDNA clones for the novel PI linked protein of sensory nerve fibers that we have identified. Purified protein will be microsequenced and corresponding degenerate oligonucleotides will be synthesized and used to screen a cDNA library. cDNA clones will be isolated, sequenced, and used in functional assays. In parallel studies, the purified protein will be used in adhesion assays to determine its role in cell adhesion.

The long term goal of this project is to identify and characterize the membrane components of axons and glial cells that mediate the cell-cell interactions of myelination in order to understand the normal development and the acquired disorders of myelinated nerve fibers. A major portion of our studies, has focused on the role of the myelin-associated glycoproteins (MAG). These proteins are integral membrane proteins of myelinating glial cells that are uniquely positioned to mediate the cell interactions of myelination - binding to specific axons via their ectodomain and, potentially, transducing intracellular signals through distinct cytoplasmic segments. To clarify MAG's physiologic role in myelination, we will characterize the structural elements that mediate and may regulate its adhesive function and identify the cytoplasmic constituents with which it interacts. In parallel studies, we have been characterizing surface components of those axons that are destined to become myelinated, with the view that proteins unique to these nerve fibers will provide important insights into their ensheathment fate by the glial cell and, in particular, the adhesion mediated by MAG. We have recently identified an apparently novel axonal protein that has a restricted fiber type distribution that we propose to characterize further. Specifically we will study wild type and recombinant MAG proteins (generated by site directed mutagenesis) in adhesion assays and in coprecipitation studies to: 1) identify the binding sequences of the ectodomain of MAG that promote adhesion, 2) determine whether this adhesion is regulated by the cytoplasmic segment, 3) determine if the two isoforms of MAG, that differ in their cytoplasmic sequences, are targeted to different membrane domains in cells, 4) determine whether these two isoforms differentially associate with cytoplasmic constituents, including kinases, 5) investigate the functional significance of the differential phosphorylation of the MAG isoforms and 6) investigate the axonal regulation of this phosphorylation. We will also purify and isolate cDNA clones for the novel PI linked protein of sensory nerve fibers that we have identified. Purified protein will be microsequenced and corresponding degenerate oligonucleotides will be synthesized and used to screen a cDNA library. cDNA clones will be isolated, sequenced, and used in functional assays. In parallel studies, the purified protein will be used in adhesion assays to determine its role in cell adhesion.

Myelinated fibers are organized into anatomically and physiologically distinct longitudinal domains - the internode, the paranodes and the node of Ranvier. This longitudinal organization results from complex interactions between the axon and myelinating glial cells that are essential for the rapid and efficient conduction of impulses via saltatory conduction. Voltage gated Na+ and delayed rectifier K+ channels are concentrated in the nodal and juxtaparanodal regions respectively, with specialized septate-like junctions interposed in-between. The precise function of these junctions is not known. They are likely to provide a site of adhesion between these cells and a partial barrier to ion flow; they may also regulate the distribution of voltage gated channels nd mediate reciprocal signaling between axons and glia. We have recently demonstrated that Caspr is a major axonal constituent of these junctions, the first such component to be identified. Critical unanswered questions include what regulates the formation of these junctions, what are their functions, and what other molecules are associated with Caspr in the paranodes? To address these question we will: i) Determine the sequence of events during formation of the node and the paranodes in a myelinating co- culture system and determine whether node formation is a prerequisite of Caspr localization, ii) Analyze the function of Caspr as an adhesion molecule and its role in the formation of the paranodal junctions. To this end, we will generate and purify a Caspr-Fc chimeric protein for binding studies with Schwann cells and oligodendrocytes and for perturbation studies of paranodal junction formation in vitro and iii) isolate novel proteins that are associated with Caspr in the paranodal junctions and characterize its interactions with several known proteins by isolating non-ionic detergent complexes enriched in Caspr and associated proteins. We will also use the Caspr-Fc to characterize its presumptive glial receptor by cross-linking and affinity purification techniques and continue to analyze proteins that may interact with the cytoplasmic domain of Caspr including PI 3-kinase.

DESCRIPTION (provided by applicant): Axons regulate the proliferation, survival and differentiation of Schwann cells. Neuregulin- I (NRG- 1), a family of neuronal growth factors, has an established and crucial role in the early events of Schwann cell development, promoting Schwann cell maturation, proliferation and survival. Neuregulin receptors, members of the erbB family of receptor tyrosine kinases, are expressed by Schwann cells. Following ligand binding, they undergo autophosphorylation and activate intracellular signaling pathways, notably investigator 3-kinase which we have shown is required for these early events. In this proposal, we will investigate whether NRG- 1 and the erbB receptors, exclusively mediate the initial interactions of axons and Schwann cells or whether they also mediate later interactions, including myelin and basal lamina (BL) and node of Ranvier formation. To investigate the role of NRG- 1 in these later events of development, we will coculture mature Schwann cells with neurons isolated from mice with either a conditional, neuron-specific knockout of NRG-1 or a knockout of the CRD-NRG-l isoform. We are particular interested in whether nrg- 1-I- neurons can still induce Schwann cell proliferation and survival and promote normal myelin and BL formation. We have recently found that erbB2 and several candidate interacting proteins, are enormously enriched in the Schwann cell microvilli which contact the nodal axolemma. To elucidate the signifance of this finding for local signaling and node organization, we will characterize the time course of its accumulation and its precise localization in the microvilli, determine whether erbB2 is complexed to and/or activates several candidate proteins in the microvilli and examine whether erbB2 regulates microvilli formation and nodal organization. Finally, we have found that MAP kinase and P1 3-kinase activation are activated not only during development, but also shortly after axotomy and NRG-induced demyelination. To investigate the role of MAP kinase and investigator 3-kinase activation during Wallerian degeneration and demyelination, we will examine whether activation of these pathways is a feature of demyelination in vivo, examine the effects of blocking their activation using pharmacologic inhibitors during Wallerian degeneration in vitro and determine whether the sequence of events during Wallenan degeneration is altered in cultures from NRG- 1 knockouts or in cultures treated with erbB2/e3rbB3-Fc, which inhibits NRG- 1 signaling.

DESCRIPTION (provided by applicant): The striking concentration of Na+ channels at the node results from complex interactions between axons and myelinating glial cells, i.e. Schwann cells in the PNS and oligodendrocytes in the CNS. The nature of these interactions are poorly understood as are the mechanisms by which Na+ channels are targeted to and assemble into a larger complex with other proteins at the node. Associated proteins include the 186 kD isoform of neurofascin, which may bind to receptors on overlying glial processes, and ankyrin G which links cell adhesion molecules to Na+ channels. The node is flanked by paranodal junctions, which are dispensible for initial node formation, but may regulate the maturation and channel density at the node. We propose to address important features of this model by characterizing the targeting of proteins to the node, the role of glial processes that overlie and flank the node and the potential role of neurofascin in directing assembly of the node. Specifically, we will i) determine whether proteins targeted to the node redistribute from existing pools and/or are newly synthesized and transported to this site by labeling live cocultures of neurons with anti-NrCAM Fab fragments as they undergo myelination, analyzing whether nodes form after transecting axons of the Wlds/Ola mouse, and characterizing axonal transport of Na+ channels and other proteins to the node; ii) investigate the role of the ERM+ Schwann cell processes in PNS node formation by blocking their formation by dominant negative strategies or the use of Rho kinase inhibitors; iii) characterize the role of the flanking paranodal processes and junctions in the development and maturation of the node in Caspr deficient mice which lack paranodal junctions focusing on node width, channel density and Na+ channel subtypes and iv) determine the role of the L1 family of cell adhesion molecules, in particular neurofascin, in node formation by generating mice with a conditional, neuron-specific knockout of neurofascin and determine whether they exhibit aberrant initial segment, node or paranode organization and, if minimally affected, by crossing these mice to an existing line of NrCAM knockout mice for analysis of the double knockouts. These studies should provide important new insights into the mechanisms responsible for the assembly of the node, the nature of the glial signals that direct its assembly and may clarify the functional deficits that accompany demyelinating disorders and interrupt normal saltatory conduction.

[unreadable] DESCRIPTION (provided by applicant): The long term goal of this research program is to identify the signals on axons that determine whether they are ensheathed or myelinated by Schwann cells and to elucidate the downstream pathways they activate in Schwann cell that regulate these distinct phenotypes. We have been characterizing the neuronal growth factor, neuregulin-1 (NRG1) in these events. NRG1 has three major isoforms (types I, II, and III), which differ in their amino terminal sequences and modes of signaling. Types I and II are paracrine signals that are shed from the axon surface by metalloproteinase cleavage whereas type III is a juxtacrine signal retained at the membrane. We have recently found that type III NRG1 levels provide the long sought instructive signal that determines the ensheathment fate of axons and does so by activating PI-kinase in Schwann cells, a key signaling pathway we have found to be essential for ensheathment and myelination. To elucidate how type NRG1, and PI-kinase, drives Schwann cell myelination and to examine its potential role in myelin maintenance, we are characterizing its function in a myelinating coculture system that faithfully replicates key events in the development of myelinating fibers in the PNS in vivo. In particular, we propose to: i) determine whether the type III NRG1 isoform is specific in promoting myelination or whether other NRG1 isoforms can substitute for its activity, whether its ability to trigger myelination requires that it be tethered to the axon, and the significance of its cleavage by metalloproteinases, ii) characterize which of the downstream effectors of PI-kinase are critical to its ability to promote Schwann cell ensheathment and myelination, and iii) determine whether NRG1 signals are required, not only during development, but also to maintain the integrity of the axon-myelinating Schwann cell unit in the adult. These studies should provide major insights into how the axon drives formation of the myelin sheath, which is essential for normal function of the nervous system. They are likely to have important implications for an understanding of the neurologic disability and pathogenesis of peripheral neuropathies, and of other disorders of myelinated fibers, and may lead to the development of new strategies to promote repair and remyelination of such disorders. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Action potentials are generated at the axon initial segment (AIS) and are propagated via saltatory conduction at nodes of Ranvier. These domains are highly enriched in voltage gated Na+ channels, which form a multimeric complex with beta subunits, neuronal cell adhesion molecules, notably neurofascin (NF) 186, and the cytoskeletal proteins ankyrin G and piv spectrin. We have recently demonstrated that targeting of NF186 to PNS nodes is mediated via its extracellular interactions, that it has an essential role in node formation by recruiting ankyrin G via its cytoplasmic domain, and that ankyrin G in turn, is required for the accumulation of sodium channels. In contrast, ankyrin accumulation at initial segments, which is also critical for domain organization is intrinsically specified, independent of NF186. These studies raise a number of key questions. What is the source of proteins targeted to the node and how do they traffic to this site? Are nodal components, once assembled, stably expressed at the node or do they continuously turnover and, if so, is this turnover enhanced by demyelination? Finally, do CNS nodes assemble like PNS nodes (i.e. directed by extracellular signals) or more akin to initial segments (i.e. directed from the inside-out via interactions with ankyrin G)? In this proposal, we address these questions and further characterize mechanisms of node assembly by: i) determining how proteins traffic to PNS nodes, including whether they are recruited from cell surface pools or via directed vesicular transport, ii) live image nodes to examine dynamic changes that occur during development and with demyelination, and iii) examine the dependence of CNS nodes on NF186- dependent signals in cocultures and targeting signals in transgenic mice. These studies should provide important new insights into the axo-glial interactions that regulate the assembly and maintenance of nodes of Ranvier. They will also be an important foundation for elucidating pathogenetic changes at nodes that result from demyelination. [unreadable] [unreadable]

The nodes of Ranvier of myelinated axons are critical for action potential propagation by saltatory conduction and are a striking example of the exquisite domain organization characteristic of neurons. Nodes are comprised of a multimeric complex that includes voltage gated sodium and potassium channels, accessory beta subunits, cell adhesion molecules (CAMs), and a cytoskeletal complex that includes ankyrin G to which channels and CAMs bind. Our recent studies indicate that the node assembles from distinct sources. CAMs, notably NF186, accumulate at forming nodes by redistribution from existing pools on the axon surface via diffusion trapping from interactions with the Schwann cell. In contrast, channels and cytoskeletal proteins are delivered to the node primarily via axonal transport. Surprisingly, there also appears to be a pool of sodium channels that traffics and accumulates at the node independent of transport and of ankyrin G. Finally, our studies suggest there is an active program to clear nodal proteins from extranodal sites that further reinforces their selective enrichment at the node. To extend these findings and further elucidate the mechanisms of node assembly, we will investigate trafficking and assembly of components of the node, and how they are cleared from extranodal sites, by live imaging strategies in myelinating cocultures. In particular, we will: i) determine whether vesicles that transport components to the nodes segregate into those specific for CAMs and others for ion channels, and exhibit any domain specificity, ii) characterize transport-dependent and -independent trafficking of sodium channel components to the node, including the roles of ankyrin G and NF186, respectively, and determine whether the sodium channel complex assembles locally, and i) investigate how nodal components are cleared from extranodal sites, focusing on NF186 to examine further the role of its cytoplasmic segment and that of endocytosis and proteolysis in clearing the internodal, surface pool of NF186. Relevance: These studies will elucidate the mechanisms that regulate the assembly of the node of Ranvier, which is critical to the ability of nerve fibers to conduct electrical impulses appropriately. Findings in this study may therefore have important implications for our understanding of the pathogenesis of disorders of myelinated fibers, including neuropathies that result in aberrant nerve conduction, and may thereby lead to new therapeutic strategies for these neurological disorders.

The long term goal of this research program is to elucidate how axons drive Schwann cell ensheathment and myelination. We are investigating the role of neuregulin-1 (NRG1), a neuronal growth factor which regulates the entire Schwann cell lineage, including the binary choice between ensheathment and myelination in the PNS. The mechanisms by which type III NRG1 drives Schwann cell ensheathment and myelination remain poorly understood. NRG1 binds to erbB receptors on Schwann cells, activating the PI 3-kinase, PLC-3, and MAP kinase signaling pathways. Our general model is that Schwann cell survival, ensheathment and myelin wrapping all require NRG1-dependent activation of PI 3-kinase whereas other signaling pathways may drive the sequential expression of myelin-specific transcription factors (TFs). Using a recently generated transgenic mouse that expresses an activated, membrane targeted form of Akt1, we will investigate further how NRG1 drives axon ensheathment, myelination, and survival. Specifically, we will: i) investigate whether Akt is a primary effector of PI 3-kinase- dependent survival and ensheathment by analyzing mice deficient in Akt isoforms and by expressing activated Akt in Schwann cells interacting with NRG1 deficient neurons, ii) characterize the role(s) of the TORC1 and TORC2 pathways in ensheathment and myelination, and iii) analyze whether Akt cooperates with other signals, including NFAT, during myelination, by crossing transgenic mice expressing activated Akt and NFAT transgenes and examine potential cooperative effects on myelination. These studies should provide important insights into how type III NRG1 on the axon drives survival, ensheathment and myelination by Schwann cells. Relevance: These studies seek to identify signals that promote myelin sheath formation, the cellular insulation that surrounds nerve fibers and is critical for their proper function and long term integrity. Findings in this study may therefore have important implications for the pathogenesis of disorders of myelinated fibers, including neuropathies, and lead to new therapeutic strategies to promote their repair.

Project Summary Both oligodendrocyte progenitors (OPCs) and neural stem cells (NSCs) in the subventricular zone (SVZ) are known sources of remyelinating oligodendrocytes. Their precise contribution to remyelination and what limits their effectiveness in repair are active areas of research with important therapeutic implications. We have found that Sonic hedgehog (Shh)-responsive adult NSCs are a significant source of remyelinating cells. These NSCs, which are enriched in the ventral SVZ, normally give rise to parenchymal astrocytes and interneurons. They only enter white matter tracts upon demyelination, including to the the demyelinated corpus callosum (CC), where they are robustly recruited and differentiate preferentially into oligodendroglia. Unexpectedly, their recruitment to such lesions and their differentiation into remyelinating oligodendrocytes is significantly enhanced by genetic ablation or pharmacological inhibition of the Shh-dependent transcription factor Gli1. Further, pharmacological inhibition of Gli1 enhances remyelination in the adult and improves functional recovery from inflammatory demyelination. Important questions include whether their contribution to remyelination is impacted by competition with parenchymal OPCs during repair, what recruits these cells to demyelinated lesions, and how Gli1 limits NSC repair. In Aim 1, we examine whether NSCs expand their contribution to repair if OPC remyelination is blocked to assess whether NSC and OPCs compete to remyelinate the same lesion sites. In Aim 2, we assess the role of microglia (MG) and astrocytes, which are both activated in lesion sites, in the expansion and recruitment of NSCs. Our preliminary studies suggest MG are essential for NSC expansion and recruitment but do not indicate if this is a direct effect or is secondary to activation of astroglia. In Aim 3, we will investigate a novel NSC phenotype revealed by RNAseq that is only upregulated with demyelination and is Gli1- dependent. This phenotype includes a number of inflammation-related mediators and the C1q complex. We will characterize this altered NSC phenotype further, examine its potential role in regulating the number and phenotype of MG in the SVZ, and assess its potential impact on NSC clearance and remyelination. These studies will provide important, new insights into the signals (cells and molecules) that regulate the contribution of stem cells to repair and may thereby guide therapeutic efforts to promote remyelination.