Mechanism of hypoxia mediated failure of oligodendrocyte generation

项目摘要 在美国，每10个孩子中就有1个早产，尽管在这方面取得了进展， 围产期护理导致早产儿的存活率增加，其中许多儿童继续表现出 神经发育缺陷导致显著的认知和运动功能障碍。最常见的一 早产后的神经损伤是弥漫性白色物质损伤（DWMI），其被认为是由 肺发育和脑发育的不成熟状态对发育中的脑造成缺氧性损伤 没有治愈的方法白色物质是中枢神经系统内通讯所必需的， 主要由髓磷脂组成，髓磷脂是一种脂肪鞘，包围着神经元轴突，使其能够有效地发挥作用 潜在传播髓鞘是由成熟的少突胶质细胞产生的，其通过分化产生， 少突胶质祖细胞（OPCs）。在DWMI的情况下，缺氧导致细胞凋亡， 少突胶质细胞谱系随后增殖和随后的少突胶质细胞再生失败， 残余OPC。缺氧后OPCs产生少突胶质细胞的缺陷可以通过以下方法消除： 敲除缺氧诱导因子（HIF），HIF是DNA结合转录因子， 在OPCs中缺氧并在常氧下迅速降解。然而，HIFs如何阻断 少突胶质细胞的生成仍然是难以捉摸的。利用我们实验室的能力， 在OPC中，我对HIF 1a和H3 K27 Ac（活性染色质的标志物）进行了ChIP-seq，以确定 OPC基因组中的假定HIF靶点。基于HIF 1a结合的最佳候选靶点以及 H3 K27 Ac的富集表明了一种转录因子，其已被证明对干细胞的生长和分化是重要的。 在中枢神经系统外的组织中维持作为OPCs中HIF的靶点。我证明， 该转录因子的过表达足以抑制OPC向少突胶质细胞的分化， 在DWMI小鼠模型中缺氧损伤后上调。这项建议旨在进一步调查 这些发现通过1）利用细胞，分子和遗传技术的组合来确定 该转录因子抑制OPC分化的机制2）确定下调是否 这种转录因子的表达将促进体外缺氧损伤后少突胶质细胞形成的恢复， 3)使用一种新的方法来表征这种转录因子在体内上调的时空动力学， DWMI小鼠模型。本提案中概述的实验将增加我们对机制的理解 在缺氧条件下阻止OPCs生成少突胶质细胞， 用于治疗这种高度流行且使人衰弱的神经发育状况。

PROJECT SUMMARY Nearly 1 out of every 10 children are born prematurely in the United States and although advancements in perinatal care have resulted in the increased survival of preterm infants, many of these children go on to exhibit neurodevelopmental deficits leading to significant cognitive and motor dysfunction. One of the most common neurologic insults following preterm birth is diffuse white matter injury (DWMI), which is thought to arise from hypoxic injury to the developing brain caused by the immature state of lung development and cerebral vasculature and has no cure. White matter is required for communication within the central nervous system and is primarily composed of myelin, which is a fatty sheath that surrounds neuronal axons to allow efficient action potential propagation. Myelin is generated by mature oligodendrocytes, which arise via differentiation of oligodendrocyte progenitor cells (OPCs). In the context of DWMI, hypoxia leads to apoptosis of cells of the oligodendrocyte lineage followed by proliferation and failure of subsequent oligodendrocyte regeneration from residual OPCs. This deficit in oligodendrocyte generation from OPCs following hypoxia can be abrogated by knocking out hypoxia inducible factors (HIFs), which are DNA-binding transcription factors that accumulate under hypoxia and are rapidly degraded in normoxia, in OPCs. However, the mechanism of how HIFs block oligodendrocyte generation remains elusive. Leveraging our lab’s ability to generate large and pure populations of OPCs, I performed ChIP-seq for HIF1a and H3K27Ac, a marker of active chromatin, in order to determine putative HIF targets across the OPC genome. The top candidate target based on HIF1a binding as well as enrichment of H3K27Ac suggested a transcription factor that has been shown to be important for stem cell maintenance in tissues outside the central nervous system as a target of HIF in OPCs. I demonstrate that overexpression of this transcription factor is sufficient to inhibit differentiation of OPCs to oligodendrocytes and is upregulated following hypoxic injury in a mouse model of DWMI. This proposal seeks to further investigate these findings by 1) utilizing a combination of cellular, molecular and genetic techniques to determine the mechanism by which this transcription factor inhibits OPC differentiation 2) determining whether downregulation of this transcription factor will facilitate recovery of oligodendrocyte formation following hypoxic injury in vitro and 3) characterizing the spatiotemporal dynamics of the upregulation of this transcription factor in vivo using a mouse model of DWMI. The experiments outlined in this proposal will increase our understanding of mechanisms that impede oligodendrocyte generation from OPCs under hypoxic conditions, and will uncover novel avenues for therapeutic intervention for this highly prevalent and debilitating neurodevelopmental condition.