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

描述(由申请人提供)：白质损伤(WMI)是早产幸存者终生神经残疾的主要原因。我们最近在一个大型的人类尸检系列中证明了WMI的主要形式包括弥漫性病变，坏死是次要成分。弥漫性WMI的髓鞘失效机制包括晚期少突胶质细胞前体细胞(PreOL)成熟受阻，并且没有明显的轴索病变。Preol停滞与星形胶质细胞增生症的程度显著相关，这支持了来自星形胶质细胞损伤的因素与Preol停滞有关的假说。弥漫性人类WMI富含星形胶质细胞相关的透明质酸(HA)，这种透明质酸弥漫地聚集在细胞外基质中。我们发现，高相对分子质量(MW)形式的HA被透明质酸酶加工成低相对分子质量形式，从而在体外抑制Preol的成熟，并延迟实验诱导的脱髓鞘后的再髓鞘形成。我们发现了一种新的GPI锚定的透明质酸酶PH20的CNS表达，该酶定位于新生儿脑中的前OL，并在白质病变中的前OL和星形胶质细胞中表达上调。我们建议定义新的机制，涉及将HA加工成具有生物活性的低分子量形式，以防止Preol在体外和体内成熟。我们将检验这一总体假设，即在WMI中PH20的增强表达介导了低分子量HA的产生，从而抑制了Preol成熟为髓鞘OL。目的1解决一系列相关的机制问题，以确定在两种互补的新生大鼠慢性WMI模型中介导Preol停滞的HA分子的特定大小范围：早产等效的缺氧-缺血(H-I)模型和表现出强大的星形胶质细胞增生和Preol停滞的切片培养模型。AIM 2与AIM 1整合以确定正常对照组体内HA组织水平的调节以及从H-I演变而来的新生儿WMI的调节。我们将量化HA合成酶和透明质酸酶的发育表达，包括PH20。目标3将确定PH20功能的获得或丧失对胎盘成熟和髓鞘形成的影响。我们将量化慢病毒驱动的PH20在正常的WM发育过程中以及在体外和新生儿H-I后的慢性WMI中过度表达所介导的Preol停滞的程度。补充性研究将量化PH20抑制剂对H-I引起的慢性白质损伤所促进的Preol成熟和髓鞘形成的程度。目标4将定义慢性人类WMI髓鞘失败的时间过程，以及它是持续的还是部分消失的。这些人类研究将与AIMS 1-3中的啮齿动物研究相结合，以确定人类Preol停滞与反应性星形胶质细胞增多症(GFAP和CD44)、PH20表达和总HA水平的生物标记物之间的关联。我们期望对触发慢性WMI患者正常髓鞘形成失败的机制获得新的见解，并因此能够开发新的策略来促进早产幸存者的正常髓鞘形成。

DESCRIPTION (provided by applicant): White matter injury (WMI) is the leading cause of life-long neurological disability in survivors of premature birth. We recently demonstrated in a large human autopsy series (Buser et al., 2012) that the major form of WMI comprises diffuse lesions and necrosis is a minor component. The mechanism of myelination failure in diffuse WMI involves arrested maturation of late oligodendrocyte progenitors (preOLs) and occurs without significant axonopathy. PreOL arrest was significantly associated with the magnitude of astrogliosis, which supports the hypothesis that factors derived from the astrogliotic lesion contribute to preOL arrest. Diffuse human WMI was enriched in astrocyte-associated hyaluronic acid (HA) that diffusely accumulated in the extracellular matrix. We found that higher molecular weight (MW) forms of HA are processed by hyaluronidases to lower MW forms that inhibit preOL maturation in vitro and delay re-myelination after experimentally-induced demyelination. We identified novel CNS expression of a GPI-anchored hyaluronidase, PH20, that localizes to preOLs in neonatal brain and which shows elevated expression within preOLs and astrocytes in white matter lesions. We propose to define novel mechanisms that involve processing of HA to bioactive lower MW forms that prevent preOL maturation in vitro and in vivo. We will test the overall hypothesis that the enhanced expression of PH20 in WMI mediates the generation of lower MW forms of HA that inhibit preOL maturation to myelinating OLs. Aim 1 addresses a series of related mechanistic questions that will define the specific size ranges of HA molecules that mediate preOL arrest in two complementary models of chronic WMI in the neonatal rat: a preterm- equivalent model of hypoxia-ischemia (H-I) and a slice culture model that displays robust astrogliosis and preOL arrest. Aim 2 integrates with aim 1 to define the regulation of tissue levels of HA in vivo in normal controls and in evolving neonatal WMI from H-I. We will quantify the developmental expression of both the HA synthases and hyaluronidases, including PH20. Aim 3 will define the effect of PH20 gain or loss of function on preOL maturation and myelination. We will quantify the magnitude of preOL arrest mediated by lentiviral-driven PH20 over-expression during normal WM development and in chronic WMI in vitro and after neonatal H-I. Complementary studies will quantify the magnitude of preOL maturation and myelination promoted by delivery of PH20 inhibitors to chronic white matter lesions arising from H-I. Aim 4 will define the temporal course of myelination failure in chronic human WMI and whether it persists or shows partial resolution. These human studies will integrate with the rodent studies in aims 1-3 to define the association of human preOL arrest with biomarkers of reactive astrogliosis (GFAP and CD44), PH20 expression, and total HA levels. We expect to gain new insights into mechanisms that trigger a failure of normal myelination in chronic WMI, and, thereby, be positioned to develop new strategies to promote normal myelination in survivors of premature birth.