Oligodendrocytes are the CNS cells that synthesize myelin, which is essential for normal nervous system function. The overall goal of our research program is to elucidate how oligodendrocyte development and CNS myelination are regulated, and to apply this information toward the development of strategies to promote remyelination in multiple sclerosis and other myelin disorders. In the first funding period we developed immortalized oligodendrocyte lines as experimental material for these investigations. Oligodendrocytes and oligodendrocyte progenitors were stably transformed with a construct containing a copy of the SV40 T antigen gene under the control of a fragment of the mouse metallothionein I gene promoter, which is inducible by Zn++. The cell proliferate with a mean generation time of approximately 48 hours in the presence of 100 muM Zn++; whereas, when Zn++ is removed, T antigen mRNA becomes undetectable, the doubling time of the cells increases to greater than 10 days, and expression of myelin components including 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), myelin proteolipid protein (PLP), DM20 protein and myelin-associated glycoprotein (MAG) increases. We now propose to use these immortalized oligodendrocytes for two new projects. In the first project we will investigate myelination by immortalized oligodendrocytes. The cells will be cocultured with neurons and with developing rat CNS cells in aggregate cultures to test parameters that affect myelin synthesis by immortalized oligodendrocytes; and immortalized oligodendrocytes will be transplanted into spinal cord of myelin deficient (md) rats to investigate myelination by immortalized oligodendrocytes as a means of therapy of dysmyelinated or demyelinated CNS lesions in vivo. In the second project we will use our immortalized clones to investigate the function of CNP, a major myelin protein whose function remains unknown. CNP gene expression will be blocked by addition of antisense oligonucleotides to the culture medium or by stable transfection of immortalized oligodendrocytes with CNP antisense constructs, and CNP-null mutants will be produced by homologous recombination (""""""""knockout""""""""). The ability of the cells to differentiate and to myelinate in vitro and in vivo will be investigated. Thirdly, we propose to generate a new series of immortalized oligodendrocyte clones by combining a temperature-sensitive (ts) SV40 T antigen gene sequence with an inducible promoter so that pre-existing T antigen can be inactivated by a brief exposure to elevated temperature at the same time that T antigen synthesis is terminated by removal of Zn++. These new clones will be characterized and used in studies as described above. The proposed studies will give new insight into the role of the major oligodendroglial and myelin protein CNP in oligodendrocyte development and myelin synthesis, and will explore a potential approach to therapy of demyelinated lesions.
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