The broad, long term goal of this proposal is to understand the mechanisms that control the growth, survival, and differentiation of oligodendrocytes, the myelin producing cells of the central nervous system. The project is therefore directly relevant to demyelinating diseases such as multiple sclerosis. The proposed experiments focus on the rote of the regulatory subunit of PI3 kinase, p85, in expression of the MBP gene. p85 greatly enhances MBP promoter activity. This function requires only the SH2 domains and is independent of PI3 kinase activity. The MBP gene is only expressed at high levels in mature oligodendrocytes. Its expression is sensitive to inputs from multiple signaling pathways that regulate the progression from growing progenitors to terminally differentiated myelin-producing cells. Although many of the growth factors and hormones that control this process have been characterized, much less is known about the intracellular signals that they generate, the way these signals are integrated, and their molecular targets. The proposed experiments should provide significant insight into some of these processes.
In aim 1 we will seek to identify and characterize the protein(s) with which p85 interacts. The mechanism by which p85 modulates MBP expression will be explored by determining p85 molecular interactions at various stages of oligodendrocyte development. This will be pursued using GST pull downs and co-immunoprecipitation experiments.
Aim 2 seeks to determine the molecular basis through which p85 activates the MBP gene by defining the target sequences in the MBP promoter, and by characterizing the proteins that interact with these elements. We will use mutagenesis to define the element(s) in the MBP promoter that is required for the response to p85. This will serve as the basis for identifying and characterizing the transcription regulatory factors involved in this process.
Aim 3 is to investigate how p85 integrates with other signaling pathways in leading to increased MBP expression. Chemical inhibitors as well as interfering expression constructs for components of signaling pathways known to be involved in MBP expression will be used to determine whether p85 integrates with these pathways in leading to increased MBP promoter activity. Completion of the studies described will provide significant insight into the role of p85 in regulating oligodendrocyte differentiation. ? ?
