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

项目摘要 治疗早产儿白色损伤（白质损伤）的进展一直受到以下因素的阻碍： 髓鞘再生失败的分子机制。我们试图通过破坏透明质酸， 酸（HA）介导的信号传导，阻止白色物质修复和功能可塑性。HA由CNS处理 透明质酸酶转化为不同大小的HA片段（HAf）。我们发现，约210 kDa的HAf抑制少突胶质细胞， 在体外抑制前体细胞（OPC）成熟并在体内阻断髓鞘形成。210 HAf促进OPC利基市场， 通过利用限制OPC分化的AKT-FoxO 3信号传导途径来消耗髓鞘形成。它 是我们的统一假设，即210 HAf通过三种互补机制阻断髓鞘形成， 刺激OPC增殖、阻断前OL成熟并偏向小胶质细胞释放“抗炎”因子， 限制OPC差异化。我们将整合遗传，细胞和生化方法，使用新生儿 缺氧缺血大鼠模型、转基因小鼠、原代OPCs和前脑切片培养。在目标1中， 确定肿瘤抑制因子Merlin通过210 HAf调节OPC增殖的新CNS作用。我们 将定义Merlin下游的途径，其通过激活表皮生长促进OPC增殖 因子受体在目的2中，我们发现210 HAf刺激小胶质细胞释放与“抗- “煽动性”国家。我们假设210 HAf促进小胶质细胞释放这些因子，以破坏白色。 通过限制OPC分化进行物质修复。我们将确定小胶质细胞因子的表达 受210 HAf和HA受体刺激，以及可能促进OPC小生境的信号通路。 在目标2结束时，我们将进行体内研究以测试广谱透明质酸酶抑制剂， 我们已经证明的VCPAL促进了成年脊髓损伤后的髓鞘形成。我们将确定VCPAL是否会改变平衡 从促进OPC向驱动OPC成熟为少突胶质细胞的状态的因子。在目标3中，我们 将定义AKT调节的转录因子FoxO 3的下游靶点，FoxO 3被 210 HAf限制OPC成熟。我们将定义新的机制，通过它FoxO 3相互作用与 染色质重塑因子Brg 1，我们最近发现，调节OPC规范和分化， 控制参与早期少突胶质细胞分化的基因表达，如Olig 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。我们的长期目标是确定分子机制， 其中210 HAf信号调节WM炎症和OPC存活、增殖和增殖之间的平衡， 分化对这些密切相关的过程的详细分子理解将提供关键的 需要深入了解来开发促进髓鞘形成的新策略。

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.