Marie T. Filbin, Ph.D. - US grants

The formation of myelin by Schwann cells requires the precise timing of expression and the exact positioning of a number of specific proteins. How these proteins are delivered to the correct location and what specific functions they have is unknown. The major peripheral myelin protein, Po, is thought to interact with itself (hemophilic binding) possibly via its carbohydrate residues and thus play a role in compaction of myelin. Moreover, changes in the glycosylation of Po have been associated with a changes in its expression, although evidence for a direct correlation is lacking. The overall goals of this proposal are to determine whether the oligosaccharide chain of Po is involved in hemophilic interaction and whether changes in glycosylation constitute the key mechanism for regulation Po expression and sorting in the Schwann cell. As an abundance of Po is required for these studies, the immediate goals are to over-express the glycoprotein by gene amplification using the dihydrofolate reductase (DHFR) strategy in cells transfected with the cDNA for Po. For the adhesion studies, cells that have no endogenous myelin proteins, Chinese hamster ovary (CHO) cells, will be used which will assist in the accurate interpretation of the data. Through specific manipulation of the sugar structure, assessment of its contribution of adhesion will be made. Studies on the expression/sorting of Po will be carried out in Schwann cells. However, since in Schwann cells the transfected Po must be distinguished from the endogenous glycoprotein, a "reporter" group of 7-10 amino acids will be attached to the C-terminal portion of the transfected glycoprotein. To ensure that Po with and without the reporter peptide is expressed/sorted in an identifical manner, both will be characterized in CHO cells, prior to the studies with Schwann cells. Eventually, the expression of Po-reporter will be monitored in Schwann cells in relation to the addition of axolemma and/or extracellular matrix to the culture, before and after treatment with specific inhibitors of glycosylation. Finally the long term goals of this study are to apply the expertise gained in this study ot other specific myelin proteins and hence to gain an understanding of myelin-ation in the peripheral nervous system. Such information is critical in understanding the basic mechanism and pathology of any peripheral neuropathy which involves demyelination and remyelination e.g., Charcot-Marie-Tooth disease.

The formation of myelin by Schwann cells requires the precise timing of expression and the exact positioning of a number of specific proteins. How these proteins are delivered to the correct location and what specific functions they have is unknown. The major peripheral myelin protein, Po, is thought to interact with itself (hemophilic binding) possibly via its carbohydrate residues and thus play a role in compaction of myelin. Moreover, changes in the glycosylation of Po have been associated with a changes in its expression, although evidence for a direct correlation is lacking. The overall goals of this proposal are to determine whether the oligosaccharide chain of Po is involved in hemophilic interaction and whether changes in glycosylation constitute the key mechanism for regulation Po expression and sorting in the Schwann cell. As an abundance of Po is required for these studies, the immediate goals are to over-express the glycoprotein by gene amplification using the dihydrofolate reductase (DHFR) strategy in cells transfected with the cDNA for Po. For the adhesion studies, cells that have no endogenous myelin proteins, Chinese hamster ovary (CHO) cells, will be used which will assist in the accurate interpretation of the data. Through specific manipulation of the sugar structure, assessment of its contribution of adhesion will be made. Studies on the expression/sorting of Po will be carried out in Schwann cells. However, since in Schwann cells the transfected Po must be distinguished from the endogenous glycoprotein, a "reporter" group of 7-10 amino acids will be attached to the C-terminal portion of the transfected glycoprotein. To ensure that Po with and without the reporter peptide is expressed/sorted in an identifical manner, both will be characterized in CHO cells, prior to the studies with Schwann cells. Eventually, the expression of Po-reporter will be monitored in Schwann cells in relation to the addition of axolemma and/or extracellular matrix to the culture, before and after treatment with specific inhibitors of glycosylation. Finally the long term goals of this study are to apply the expertise gained in this study ot other specific myelin proteins and hence to gain an understanding of myelin-ation in the peripheral nervous system. Such information is critical in understanding the basic mechanism and pathology of any peripheral neuropathy which involves demyelination and remyelination e.g., Charcot-Marie-Tooth disease.

Compaction of the myelin membranes is essential for it to function as insulator of axons. As the Po glycoprotein is the most abundant protein of peripheral nervous system (PNS) myelin and is a member of the immunoglobulin (Ig) gene superfamily, this molecule is believed to hold these membrane lamellae together via interactions of both its extracellular and its cytoplasmic domains. The long term goals of this study are to identify in molecular terms the role of Po in the formation and in the maintenance of compact myelin, in the hope of contributing to our understanding of the mechanisms of demyelinating diseases such as Guillain-Barre Syndrome (GBS). While it has been shown that the extracellular domains of Po interact homophilically it is not known what precise regions of the molecule are responsible for these interactions. Even less is known about the role of Po's cytoplasmic domain in myelination. Thus, the overall goals of this proposal are to determine what roles various regions of Po play in the synthesis and compaction of myelin. The adhesion assay we have developed will allow us to pursue the following aims. First, to determine by point-mutation, whether a 5-amino acid sequence, SDNGT, in Po's Ig domain, is crucial to adhesion. As Po is believed to be the closest relative to the ancestral gene for the whole Ig superfamily, and because of the conservation of this 5-amino acid sequence in a sub-set of Ig domains, SDNGT may be an important motif to all Ig adhesion molecules in which it is found. Also this sequence is also present in an influenza virus which elicitated Guillain-Barre syndrome in hundreds of people when administered as a vaccine. The connection between the SDNGT sequence and GBS will be addressed by screening sera from these particular patients for SDNGT antibodies and by determining if SDNGT peptide antibodies recognize only the strain of virus used in this vaccine and not others. If this amino acid sequence is found to be involved in demyelination then it is imperative that future vaccines are screened for its presence. Second, to establish if the cytoplasmic domain of Po is necessary for its extracellular adhesion, the adhesion of truncated Po will be assessed. In addition, by detergent extraction and affinity chromatography, the putative interactions of Po's cytoplasmic domain with cytoskeletal components will be identified and characterized; an interaction that may be crucial in the initial stages of compaction. Third, to elucidate the interactions of Po with myelin associated glycoprotein (MAG) , an interaction that may also be essential in the early stages of myelination, mixed adhesion assays will be conducted with Po-expressing and MAG-expressing cells. In addition, as MAG and Po are expressed in the same and opposing membranes simultaneously, adhesion as says with cells expressing both Po and MAG will be carried out. Accomplishment of these aims should substantially advance the long term understanding of the mechanism of myelin compaction, organization and the role of adhesion molecules in demyelinating diseases.

DESCRIPTION: (Adapted from Applicant's Abstract): Axons are insulated and are allowed to function efficiently by the multi-lamellar membrane, myelin. Peripheral nervous system myelin is held compact by P0, a protein that contain a single immunoglobulin (Ig)-domain. P0 holds myelin together at both the extracellular (via homophilic interactions) and at the cytoplasmic (putatively via interactions with lipids) membrane leaflets. Furthermore, by measuring the adhesion of P0 in transfected cells, a dynamic relationship between the interaction of the extracellular and cytoplasmic domain of P0 was demonstrated; the cytoplasmic domain must be intact, initially, interacting with the cytoskeleton, for adhesion of the extracellular sequences to take place. Similarly, P0 mutated in either the extracellular or the cytoplasmic domain of P0 can exert a dominant negative effect on adhesion, implying a cis interaction, within the membrane. Loss of any of these interactions of P0 could result in a loss of function of the molecule. Recently, mutations and deletions of P0 have been associated with the peripheral neuropathy. Charcot-Marie-Tooth (CMT),1B. The longterm objectives of this application are to characterize the interactions of P0 as a prototype for all Ig-members and to determine how mutations in P0 bring about CMT 1B. In this proposal, the cytoskeletal interactions will be investigated by identifying and characterizing P0-associated proteins, which may be a link to the cytoskleleton. Also, using a transfection/adhesion strategy, how post-translational modifications of P0 s cytoplasmic domain affect adhesion and cytoskeletal interactions will be addressed. The ability of P0 to cluster, an indication cis interactions, will be determined by cross-linking surface P0. In addition, monoclonal antibodies to a soluble, extracellular domain of P0, in its native form, will be raised and used to distinguish mutations that disrupt conformation from those that affect binding directly. Finally, with this information, how the mutations in P0 associated with CMT1B, affect its function will be addressed both in transfected cells and in transgenic mice. The various CMT1B-mutated P0 protein will be expressed in cells by transfection. Surface expression, clustering, antibody binding and adhesion will be assessed. Then the mutated proteins will be co-expressed with wild type P0 to determine if they exert a dominant negative effect. To complement these studies, transgenic mice, initially, expressing a tagged P0 will be examined both morphologically and biochemically. Eventually, mice co-expressing wildtype and mutant P0 will be produced and examined for a dysmyelination. In this way, how the various mutations in P0 bring about the disease phenotype in CMT1B can be addressed directly.

DESCRIPTION: This is a request for support for a meeting entitled "Frontiers of Myelin Biology and Demyelinating Diseases" that will be held as a satellite meeting to the ISN/ASN in Mystic, Connecticut (7/26/97 to 7/29/97). The meeting will be chaired by Drs. Marie Filbin and Steve Pfeiffer and will run for three days with nine scientific sessions and one plenary lecture. The overall theme of the meeting is to bring together scientists engaged in the basic research of oligodendrocytes and myelin with physicians/scientists involved in clinical management of neurological disorders affecting oligodendrocytes and myelin, such as multiple sclerosis and Charcot-Marie-Tooth disease. Each scientific session will address specific topics of myelin biology and will include two invited speakers and three to four additional speakers who will be selected on the basis of abstracts received. Facilities for poster presentations are also available and it is anticipated that posters will be on view throughout the entire meeting, allowing ample time for presentation and discussion of the data. The meeting location can provide accommodation for 260 participants, with additional rooms available within walking distance if necessary. The proceedings will be published as a supplement to the Journal of Developmental Neuroscience. Funds are requested to cover registration for 19 speakers (at $400 per person) plus travel from Boston (19 at $40), with an additional $500 per person for 4 speakers not attending the ISN meeting in Boston. Funds are also requested to cover registration and travel from Boston for 6 students/postdoctoral fellows and student travel stipends for 6 at $200, as well as expenditures for audiovisual requirements and advertising.

DESCRIPTION (Investigator's Abstract): The mammalian brain and spinal cord do not regenerate after injury. It is believed that a predominance of inhibitory molecules, particularly in myelin, is responsible for this effect and that blocking these molecules would allow regeneration. Until recently a precise molecule(s) in myelin that could account for this inhibition had not been identified. Myelin associated glycoprotein (MAG), a glycoprotein specific to both CNS and PNS myelin, has been shown by the applicants to potently inhibit axonal outgrowth from all post-natal cerebellar and adult dorsal root ganglion (DRG) neurons but to promote outgrowth from newborn DRG neurons. In addition, MAG can account for a majority of the inhibitory properties of CNS myelin in vitro and has been shown to inhibit regeneration in vivo. Furthermore, a soluble form of MAG, MAG-Fc (the extracellular domain of MAG fused to the Fc portion of IgG) binds specifically to both cerebellar and DRG neurons via a sialo-glycoprotein and can inhibit axonal regeneration. Importantly, desialyation of neurons reverses inhibition, but because MAG mutated at its sialic acid recognition site, Arg118, still inhibits outgrowth when expressed by cells, implies there are two inter-dependent yet distinct epitopes on MAG; a sialic acid binding epitope and neurite inhibition epitope. The overall goals of this proposal are to characterize the interaction of MAG with its receptor and, using this information, test agents likely to block/disrupt the interaction and so prevent MAG's inhibition of spinal cord regeneration. First, using mutated forms of MAG and a panel of MAG monoclonal antibodies, the inhibition site will be precisely mapped. Second, MAG mutants and antibodies, identified in this way, will be tested for their ability to reverse the inhibition of wildtype MAG first in culture (MAG expressed by CHO cells) and then in vivo (by addition of the antibodies/mutated MAG to injured spinal cord). Also, in transgenic mice the effect of mis-expressing MAG in the PNS during normal regeneration will be assessed. Third, using MAG-Fc, MAG-binding proteins from neurons that are inhibited and that are promoted will be identified by molecular weight and compared. Antibodies will be raised to these proteins and tested for their ability to reverse the inhibition/promotion of neurite outgrowth. Accomplishment of these specific aims will advance our understanding of, not only inhibition of neurite outgrowth with a view to preventing such an inhibition after injury, but also of how the same molecule can, depending on the age and type of neuron, have opposite effects.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this proposal is to develop a strong Neuroscience Research Program at[unreadable] Hunter College by bringing together a strong core of Neuroscientists with similar research interests.[unreadable] It is envisaged that the theme of this Neuroscience Program will be "Repairing the Damaged[unreadable] Nervous System". A core of molecular/cellular Neurobiologists will be established, with similar,[unreadable] complementary interests in topics such as neuronal death and survival, axonal regeneration and[unreadable] guidance during development, and myelination, both during development and after disease/injury.[unreadable] By developing this theme in Neuroscience the strengths already at Hunter College will be built upon[unreadable] and expanded. The specific aims of this proposal are: Aim #1: To strengthen the general research[unreadable] theme of "Repairing the Damaged Nervous System" through 3 related projects, which in year 3 of[unreadable] the proposal will be submitted as R01s. It is of note that aspects of these projects are translational[unreadable] and related to health disparities Aim #2: To strengthen and expand technology and its sharing[unreadable] and to promote collaborations via a viral vector core, an electrophysiology core and shared models[unreadable] of regeneration. Aim #3: To develop a sub-theme of Neuroscience within the Biology Department[unreadable] of Hunter College. Funding of this proposal will also allow more outside speakers to be invited to[unreadable] give seminars on Neuroscience. A strong Neuroscience Research Program will also (a) expose[unreadable] undergraduates to Neuroscience at a very early stage of their career and so stimulate an interest in;[unreadable] neuroscience, (b) provide graduate students with more Neuroscience labs to choose from for their![unreadable] PhD research. New ideas would be stimulated and they would be exposed to a variety of state-of-j[unreadable] the-art techniques and (c) attract strong post-doctoral fellows.[unreadable]

DESCRIPTION (provided by applicant) Funds are requested to support the sixth Gordon Research Conference on Myelin to be held from 3-8th Feb. 2002 at the Clarion Ventura Beach Hotel, Ventura, California. It is hoped that speakers' registration and travel can be supported. In addition, travel and registration will be provided for at least 10 students or post-doctoral fellows. Women and under-represented minorities will be encouraged to apply for these stipends. The high attendance at previous Gordon conference meetings on Myelin is testament to their popularity, their importance and their good track record of outstanding and cutting edge science. The program plan can be grouped under two general themes 1) Interfacing molecular biology of myelin with diseases of myelin. The outcome of clinical trials for MS will be assessed against the backdrop of basic research. This has been successful in this respect in the past, particularly in stimulating collaborations and interaction between clinicians and scientists. 2) Broadening the program. In addition to covering the molecular aspects of myelin biology other aspects of what has perhaps been considered more peripheral aspects of glial/myelin biology will be addressed. In particular, the focus will be on the role of myelin on regeneration of the CNS. In addition, the role of glial cells at the synapse will be covered. In this way, we hope to stimulate new avenues of research and new collaborations for glial/myelin cell biologists. The meeting will be for four and one half days and in that time there will be nine scientific sessions. In addition posters will be displayed throughout the meeting. The program includes nationally and internationally renowned scientists and is complete. The sessions are as follows: Perspectives; Immune Cells in Regeneration and Re-myelination; Oligodendrocyte Determination; Schwann Cell Determination; Glial/Neuron Signaling; Stem Cells; Non-Compact Myelin; Signaling and Cell Cycle in Myelinating Cells. Each session has 3 or 4 speakers and a discussion leader.

DESCRIPTION (provided by applicant): One of the major obstacles to axonal regeneration in the adult CNS is inhibitors associated with myelin, such as MAG. However, axonal growth can be encouraged in an inhibitory environment both in culture and in vivo if the neuronal cAMP levels are elevated, either with analogues such as db cAMP or by priming neurons with neurotrophins. One situation where spontaneous CNS axon regeneration does occur is of dorsal root ganglion axons if the peripheral branch of the same neuron, the dorsal root ganglion (DRG) neuron, is lesioned one week before - the conditioning lesion (CL) effect, which is cAMP dependent. Both the cAMP and the CL effects are dependent on transcription and one gen that is up-regulated is for the enzyme Arginase I (Arg I), which is key in the synthesis of polyamines. The polyamine, spermidine, can overcome inhibition by MAG in culture and promotes optic nerve regeneration in vivo. Furthermore, spermidine promotes regeneration by activating the kinase CDK5 by hypusinating the eukaryotic initiation factor 5A (eIF5A), resulting in an increase in translation of the CDK5 activator p35. In Aim 1a the CDK5 substrates that are activated in response to polyamine will be identified and characterized for their role in overcoming inhibition and promoting regeneration in vivo. Aim 1b will address the cross-talk between the neurotrophin and MAG signaling pathway, focusing on the ability of MAG to block the activation of the small GTPase, Rap1, by neurotrophin. Strong preliminary data suggest that both the cAMP and CL effects require local translation in the axon to promote regeneration in an inhibitory environment. In Aim 2 mRNA and microRNAs that increase in the processes after both a CL and treatment with db cAMP will be identified. In Aim 3 those mRNAs and microRNAs that increase after both conditions will be characterized (over-expression, knock-down) for a possible role in promoting regeneration in the presence of MAG in culture and in promoting regeneration in vivo. Through the experiments described in this proposal not only will our understanding be advanced of the mechanism of action of agents known to promote regeneration in vivo but novel agents will be identified. This in turn will reveal novel targets for intervention and drug development to promote axonal regeneration in vivo. PUBLIC HEALTH RELEVANCE: The identification of novels agents/mechanisms that promote axonal regeneration will reveal novel targets for therapeutic intervention and drug development to promote axonal regeneration in humans.