The goal of this research is to decipher the signals and identify the molecular players that drive oligodendrocytes (OLGs) to assemble myelin. Such knowledge would pave the way to an understanding of demyelinating diseases such as multiple sclerosis and others. The NOVOcans are the products of a single gene that is developmentally regulated. The predicted amino acid sequence contains domains that have been conserved from Drosophila to mammals. NOVOcan has been localized to OLGs and ependymal cells. The hypothesis is that NOVOcan is a receptor that transduces extracellular signals; and, as such, that it likely partakes in driving OLGs toward myelinogenesis. It is further postulated that proteoglycans are the physiological ligands for NOVOcan, and that their binding alters the state of NOVOcan phosphorylation which, in turn, initiates a signaling cascade that drives a change in gene expression. The proposed research is geared to test these hypotheses and, thereby, shed light on the role of NOVOcan in myelination. The proposal has two specific aims. The objectives of Specific Aim 1) are to assess the contribution of the BTB/POZ domains, glycosaminoglycan-attachment, N-glycosylation and phosphorylation motifs to the function of NOVOcan. Site-directed mutations of these domains will be engineered into the novocan cDNA. Plasmids containing the cDNA, either the native or mutated sequence, will then be transfected into cells, such as OLGs, and expressed in large quantities in order to assess functional effects of the mutations. The goals of Specific Aim 2 are to generate transgenic mouse models to study when and where the novocan gene is expressed during development, as well as to determine the consequences of eliminating NOVOcan. For these studies it is proposed to use a """"""""knock-in"""""""" mouse model, which is created when a reporter gene is inserted into the gene of interest and is transcribed in essentially the same manner as the interrupted (targeted) gene. This research should yield insight on the function of NOVOcan and foster the understanding of the biology of myelination.
