Role of Extracellular Matrix in Hypoxic-Ischemic Perinatal White Matter Injury

项目摘要 治疗早产儿白质损伤(WMI)的进展受到以下方面的根本差距的阻碍 再髓鞘形成失败的分子机制。我们寻求通过破坏透明质酸来促进髓鞘再生 酸(HA)介导的信号转导，阻止白质修复和功能可塑性。HA由CNS处理 透明质酸酶为大小不等的HA片段(HAF)。我们发现~210 kDa的HAF抑制少突胶质细胞 祖细胞(OPC)在体外成熟，在体内阻止髓鞘形成。210HAf促进OPC利基市场 利用AKT-FOXO_3信号通路抑制OPC分化的髓鞘形成的代价。它 我们的统一假设是210HAf通过三种互补的机制阻止髓鞘形成 刺激OPC增殖，阻断Preol成熟，偏向小胶质细胞释放抗炎因子， 限制OPC差异化。我们将使用新生儿将遗传、细胞和生化方法结合在一起 大鼠缺氧缺血模型、转基因小鼠、原代OPC和前脑切片培养。在目标1中，我们将 确定肿瘤抑制因子Merlin通过210HAf调节OPC增殖的新的中枢神经系统作用。我们 将在Merlin下游定义一条途径，通过激活表皮生长促进OPC的增殖 因子受体。在目标2中，我们发现210HAf能刺激小胶质细胞释放与“抗肿瘤抗体”相关的因子。 炎症性“状态。我们假设210HAf促进小胶质细胞释放这些因子以破坏白质 通过抑制OPC分化进行物质修复。我们将测定小胶质细胞因子的表达。 受210HAf和HA受体及信号通路的刺激，可能促进OPC生态位的形成。 在目标2结束时，我们将进行体内研究，以测试一种广谱透明质酸酶抑制剂， 我们已经证明的VCPAL在成人WMI后促进髓鞘形成。我们将确定VCPAL是否会改变平衡 从促进OPC走向促使OPC成熟为少突胶质细胞的因素。在目标3中，我们 将定义AKT调节的转录因子FOX03的下游靶点，该转录因子由AKT长期激活 210HAf抑制OPC成熟。我们将定义FOX03通过哪些新机制与 我们最近发现的染色质重塑因子BRG1通过以下方式调节OPC的规格和分化 控制与早期少突胶质细胞分化有关的基因的表达，如寡核苷酸2。我们还将定义 FOXO_3作为人类髓鞘形成失败的分子标志物的作用 促进OPC成熟的干预措施。我们的长期目标是通过 哪个210HAf信号调节WM炎症和OPC存活、增殖和 差异化。对这些密切相关的过程的详细的分子理解将提供关键的 需要有洞察力来开发新的策略来促进髓鞘形成。

Project Summary Progress to treat white matter injury (WMI) in preterm neonates has been hampered by fundamental gaps in the molecular mechanisms of remyelination failure. We seek to promote myelin regeneration by disrupting hyaluronic acid (HA)-mediated signaling that prevents white matter repair and functional plasticity. HA is processed by CNS hyaluronidases to HA fragments (HAf) of varying size. We found that a HAf of ~210 kDa inhibits oligodendrocyte progenitor cell (OPC) maturation in vitro and blocks myelination in vivo. The 210HAf promotes an OPC niche at the expense of myelination by utilizing an AKT-FoxO3 signaling pathway that constrains OPC differentiation. It is our unifying hypothesis that 210HAf blocks myelination through three complementary mechanisms that stimulate OPC proliferation, block preOL maturation and bias microglia to release “anti-inflammatory” factors that constrain OPC differentiation. We will integrate genetic, cellular, and biochemical approaches using a neonatal rat model of hypoxia-ischemia, transgenic mice, primary OPCs and forebrain slice cultures. In aim 1, we will determine a novel CNS role for the tumor suppressor Merlin that regulates OPC proliferation via 210HAf. We will define a pathway downstream of Merlin that promotes OPC proliferation via activation of epidermal growth factor receptor. In aim 2, we found that 210HAf stimulates microglia to release factors associated with “anti- inflammatory” states. We hypothesize that 210HAf promotes microglial release of these factors to disrupt white matter repair by constraining OPC differentiation. We will determine the expression of microglial cytokines stimulated by 210HAf and the HA receptors and signaling pathways involved that may promote an OPC niche. At the conclusion of aim 2, we will undertake in vivo studies to test a broad-spectrum hyaluronidase inhibitor, VCPAL that we have shown promotes myelination after adult WMI. We will determine if VCPAL shifts the balance from factors that promote OPCs toward a state that drives OPC maturation to oligodendrocytes. In aim 3, we will define down-stream targets of the AKT-regulated transcription factor FoxO3 that is chronically activated by 210HAf to constrain OPC maturation. We will define novel mechanisms through which FoxO3 interacts with the chromatin remodeling factor Brg1, which we recently showed regulates OPC specification and differentiation by controlling expression of genes involved in early oligodendrocyte differentiation, like Olig2. We will also define the role of FoxO3 as a molecular marker of human myelination failure to define the window and response to interventions to promote OPC maturation. Our long-term objective is to define molecular mechanisms through which 210HAf signals to regulate WM inflammation and the balance between OPC survival, proliferation and differentiation. A detailed molecular understanding of these closely related processes will provide critically needed insights to develop new strategies to promote myelination.