We propose to explore the functions of two putative cell adhesion molecules, myelin associated glycoprotein (MAG) and myelin/oligodendrocyte glycoprotein (MCG), in the processes of myelin assembly and maintenance. We will modulate the level of mRNAs encoding these proteins through novel molecular genetic means and produce transgenic mouse lines in which oligodendrocytes are either impoverished or enriched in their content of the MAG and MOG proteins. Using this set of mutant mice with graded abilities to express the MAG or MOG protein, we will examine the morphological results of their altered levels of expression. We will conduct systematic analyses of these mice using double label immunofluorescence and confocal microscopy, as well as biochemical analyses. The organization of the myelin internode and its environment will be accessed by electron microscopy. Because adequate visualization of subtle changes in oligodendrocyte or myelin may only be achieved early in development when myelinating oligodendrocytes are sparse and not obscured by large amounts of myelin, we will carry out these microscopic anlyses at early, as well as late, developmental times. To crete the these mice we will use a powerful oligodendrocyte specific expression vector developed by us to express MAG and MOG cDNAs in either the sense or antisense orientation. The simple antisense blockade of functional mRNAs are seldom 100% efficient but produce a """"""""phenocopy"""""""" of a """"""""leaky"""""""" mutation. To produce phenocopies of more severely restricted expression of the MAG and MOG genes we will take two additional experimental tacts. In the first, we will develop transgenic mice that carry and express genes encoding ribozymes that specifically cleave either MOG or MAG mRNA. The ribozymes we propose will be targeted to either the S or the L MAG mRNA separately and we anticipate that mice bearing these ribozymes as transgenes will be impoverished in only one or the other isoforms of MAG. In the second, we will produce mice carrying null mutations in either their endogenous MAG or MOG gene. One line of MAG null mutant mice already has been created and made them available to us for this study. Because of this windfall, we have discontinued our work on creating a MAG knock-out mouse until we evaluate these animals and determine whether additional lines would be advantageous. The initial characterization of these mice by Dr. Roder and his colleagues revealed a subtle pathology characterized by a collapse of the periaxonal cytoplasmic collar of the myelinated axon. The study proposed here will carry the analyses to early developmental times when the interactions between oligodendrocyte and neuron are more easily visualized.
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