Oligodendrocyte Lineage Gene Function in the CNS

描述（由申请人提供）：本文提出的研究的广泛目标是确定调节bHLH转录因子Olig 2对神经祖细胞增殖和分化的相反功能的分子机制。在初步研究中，我们已经鉴定了Olig 2的氨基末端中的三个丝氨酸残基（“三S基序”）的簇，其在循环神经祖细胞中被磷酸化，但在分化的后代中不被磷酸化。突变分析表明Olig 2 Triple-S基序的磷酸化是神经干细胞自我更新所需的，但不需要Olig 2依赖性的未成熟少突胶质细胞的规范。本文所建议的工作以这些初步意见为基础。我们有五个具体目标：目的一是利用小分子激酶抑制剂和全激酶组的ShRNAi文库筛选Olig 2磷酸化的蛋白激酶。目的二是使用“TAP-标记”的野生型、磷酸化无效和磷酸化模拟Olig 2蛋白来定义由磷酸化调节的Olig 2蛋白-蛋白相互作用。目的三是阐明Olig 2磷酸化在神经前体细胞自我更新或退出细胞周期和分化中的转录功能。我们将使用ChIP/Seq方案来鉴定磷酸化Olig 2的直接遗传靶标。目的四是利用一种新的、双功能Olig 2-tva-cre转基因小鼠神经管外植体系统，确定Olig 2 Triple-S磷酸化在胚胎脊髓和运动神经元发育中的可能需要。目的五是确定体内少突胶质细胞成熟和CNS肿瘤发生过程中Olig 2磷酸化的可能需求。原位移植研究将确定Olig 2磷酸化是否是少突胶质细胞髓鞘形成所必需的，或者是恶性胶质瘤小鼠模型中的肿瘤发生。 公共卫生相关性：这项工作可能有助于阐明在恶性胶质瘤、脊髓损伤和脱髓鞘疾病中调节神经祖细胞的分子机制。

DESCRIPTION (provided by applicant): The broad objective of the research proposed here is to define the molecular mechanisms that regulate oppositional functions of the bHLH transcription factor Olig2 on proliferation and differentiation of neural progenitor cells. In preliminary studies, we have identified a cluster of three serine residues ("Triple-S motif") in the amino terminus of Olig2 that are phosphorylated in cycling neural progenitors, but not in differentiated progeny. Mutational analysis indicates that phosphorylation of the Olig2 Triple-S motif is required for self-renewal of neural stem cells but is not required for Olig2-dependent specification of immature oligodendrocytes. The work proposed here builds upon these preliminary observations. We have five Specific Aims: Aim One is to identify the protein kinase(s) that phosphorylate Olig2 using small molecule kinase inhibitors and a kinome-wide ShRNAi library. Aim Two is to define Olig2 protein-protein interactions that are regulated by phosphorylation using "TAP- Tagged" wild type, phospho-null and phosphomimetic Olig2 proteins. Aim Three is to define the transcriptional functions of Olig2 phosphorylation to dictate the decision of neural progenitors to self renew or exit the cell cycle and differentiate. We will use ChIP/Seq protocols to identify direct genetic targets of phosphorylated Olig2. Aim Four is to determine a possible requirement for Olig2 Triple-S phosphorylation in embryonic patterning of spinal cord and in motor neuron development using a novel, bifunctional Olig2-tva-cre transgenic mouse neural tube explant system. Aim Five is to determine a possible requirement for Olig2 phosphorylation during oligodendrocyte maturation and CNS tumorgenesis in vivo. Orthotopic grafting studies will determine if Olig2 phosphorylation is essential for oligodendrocyte myelination, or alternatively, tumorgenesis in a mouse model of malignant glioma. PUBLIC HEALTH RELEVANCE: The proposed work may shed light on molecular mechanisms that regulate neural progenitors in malignant gliomas, spinal cord injury and demyelinating diseases.