Signaling mechanisms in oligodendrocytes

描述(申请人提供)：这项建议的广泛、长期的目标是了解控制少突胶质细胞的生长、存活和分化的机制，少突胶质细胞是中枢神经系统的髓鞘产生细胞。因此，该项目与多发性硬化症等脱髓鞘疾病直接相关。建议的实验重点是PI3激酶的调节亚基P85在MBP基因表达中的作用。P85显著提高了MBP启动子的活性。该功能只需要SH2结构域，并且不依赖于PI3激酶活性。MBP基因只在成熟的少突胶质细胞中高水平表达。它的表达对来自多个信号通路的输入很敏感，这些信号通路调节从生长的祖细胞到终末分化的髓鞘产生细胞的进程。尽管控制这一过程的许多生长因子和激素已经被表征，但人们对它们产生的细胞内信号、这些信号的整合方式以及它们的分子靶标知之甚少。拟议中的实验应该会对其中一些过程提供重要的洞察。在目标1中，我们将寻求鉴定和表征与P85相互作用的蛋白质(S)。P85调节MBP表达的机制将通过测定少突胶质细胞发育不同阶段的P85分子相互作用来探索。这将通过GST下拉和免疫共沉淀实验来实现。目的2通过定义MBP启动子中的靶序列，并通过鉴定与这些元件相互作用的蛋白质，来确定P85激活MBP基因的分子基础。我们将使用突变来定义MBP启动子中对P85的反应所需的元件(S)。这将作为识别和表征参与这一过程的转录调控因子的基础。目的3是研究P85如何与其他信号通路整合，导致MBP表达增加。化学抑制剂以及已知参与MBP表达的信号通路组件的干扰表达构建将被用来确定P85是否与这些通路整合导致MBP启动子活性增加。所描述的研究的完成将为P85在调节少突胶质细胞分化中的作用提供重要的见解。

DESCRIPTION (provided by applicant): 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.