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

描述（由申请人提供）：白质损伤（WMI）是早产幸存者终身神经功能障碍的主要原因。我们最近在一个大型人体解剖系列中证明（Buser et al., 2012） WMI的主要形式包括弥漫性病变，坏死是次要成分。弥漫性WMI中髓鞘形成失败的机制涉及晚期少突胶质细胞祖细胞（preOLs）成熟受阻，且无明显的轴突病变。预ol骤停与星形胶质细胞增生的大小显著相关，这支持了星形胶质细胞病变导致预ol骤停的假设。弥漫性人WMI富含星形胶质细胞相关透明质酸（HA），弥漫性积聚在细胞外基质中。我们发现，高分子量（MW）形式的透明质酸被透明质酸酶加工成低分子量形式的透明质酸，在体外抑制ol前成熟并延迟实验诱导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在中枢神经系统的表达，该酶定位于新生儿大脑的preol，并且在白质病变的preol和星形胶质细胞中表达升高。我们建议定义新的机制，包括将HA加工成具有生物活性的低分子量形式，从而在体外和体内阻止ol前成熟。我们将验证WMI中PH20表达的增强介导低分子量HA的产生，从而抑制ol前成熟到髓鞘化ol的整体假设。目的1解决了一系列相关的机制问题，这些问题将确定在新生大鼠慢性WMI的两种互补模型中介导预ol阻滞的HA分子的具体大小范围：一种是缺氧缺血的早产等效模型（H-I），另一种是显示强大的星形胶质细胞形成和预ol阻滞的片培养模型。目的2与目的1结合，确定正常对照和H-I演变为新生儿WMI的体内组织HA水平的调节。我们将量化HA合酶和透明质酸酶的发育表达，包括PH20。目的3将确定PH20的获得或丧失对ol前成熟和髓鞘形成的影响。我们将量化在体外和新生儿H-I后正常WM发育期间、慢性WMI中由慢病毒驱动的PH20过表达介导的preOL阻滞的程度。补充研究将量化PH20抑制剂递送至H-I引起的慢性白质病变所促进的ol前成熟和髓鞘形成的程度。目的4将定义慢性人类WMI中髓鞘形成失败的时间过程，以及它是否持续存在或显示部分消退。这些人类研究将与目标1-3中的啮齿动物研究相结合，以确定人类ol前阻滞与反应性星形胶质细胞形成的生物标志物（GFAP和CD44）、PH20表达和总HA水平的关系。我们期望对慢性WMI中引发正常髓鞘形成失败的机制获得新的见解，从而定位于制定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.