Fibrinogen-mediated mechanisms of preterm infant brain injury

项目摘要/摘要 早产儿脑损伤常与血脑屏障(BBB)破坏和成熟改变有关 正常大脑发育和髓鞘形成所需的中枢神经系统(CNS)前体细胞。 损伤微环境中抑制中枢神经系统前体细胞成熟的分子信号还不完全 为人所知。因此，没有可用的治疗方案来预防与以下方面相关的发育障碍 早产。血脑屏障中断通过允许血液蛋白进入中枢神经系统而改变了中枢神经系统祖细胞的生态位。 纤维蛋白原是一种凝血蛋白，可以穿过泄漏的血脑屏障，是神经炎症的关键因素。 胶质瘢痕形成、神经变性和中枢神经系统修复受阻。我们假设纤维蛋白原是一种 早产儿脑损伤微环境中抑制中枢神经前体细胞的关键成分 成熟会损害大脑生长和髓鞘形成。我们的初步研究表明：(1)新生小鼠受到 慢性缺氧表现为显著的小脑病理，包括纤维蛋白原沉积，髓鞘形成 缺陷和小脑发育受损；(2)新生小鼠脑室注射纤维蛋白原中断 (3)纤维蛋白原激活骨形态发生蛋白受体激活素 A受体I型(ACVR1)在少突胶质前体细胞(OPC)中抑制OPC成熟和 髓鞘形成，(4)纤维蛋白原在体外抑制神经前体细胞的神经发生。我们的具体目标将是 测试我们的工作模型，在该模型中，血脑屏障中断后纤维蛋白原沉积诱导ACVR1介导的BMP 中枢神经系统前体细胞抑制神经发生和髓鞘形成的信号导致异常 神经发育。在目标1中，我们将在体内确定纤维蛋白原在早产儿脑损伤中的作用。 使用纤维蛋白原突变小鼠。在目标2中，我们将确定导致早产的细胞机制。 活体双光子显微镜观察纤维蛋白原沉积部位的婴幼儿脑损伤(下午2：00)。在《目标3》中，我们将 用体外结合和分子生物学方法确定纤维蛋白原诱导ACVR1活化的分子机制 细胞分析。这些研究将揭示血脑屏障破坏和中枢神经系统衰竭之间的分子联系 早产儿脑损伤中的祖细胞成熟。我的目标是成为一名独立的医生- 科学家和新生儿脑损伤领域的领导者。为了继续朝着这个目标前进，我将建立 凭借格莱斯顿学院和加州大学旧金山分校的专业知识，我在以下领域扩大了研究技能： (1)2 PM活体实验，研究大鼠脑内血脑屏障破坏和纤维蛋白原沉积的动态细胞反应 新生儿脑，(2)RNA测序和转录组分析以确定纤维蛋白原介导的机制 外源性抑制，以及(3)发现中枢神经系统新的纤维蛋白原受体的结合分析和抑制剂研究 祖细胞。从这项建议中获得的知识和经验将使我能够竞争R01 资金集中在针对早产儿脑损伤抑制微环境的治疗上。

PROJECT SUMMARY/ABSTRACT Preterm infant brain injury is often associated with blood-brain barrier (BBB) disruption and altered maturation of the central nervous system (CNS) progenitor cells required for normal brain development and myelination. The molecular signals in the injured microenvironment that inhibit CNS progenitor maturation are not fully known. Thus, no therapeutic options are available to prevent the developmental disabilities associated with preterm birth. BBB disruption alters the CNS progenitor niche by allowing blood proteins into the CNS. Fibrinogen, a blood coagulation protein, crosses a leaky BBB and is a key contributor to neuroinflammation, glial scar formation, neurodegeneration, and inhibition of CNS repair. We hypothesize that fibrinogen is a critical component of the microenvironment in preterm infant brain injury that inhibits CNS progenitor cell maturation to impair brain growth and myelination. Our preliminary studies show: (1) neonatal mice subjected to chronic hypoxia display prominent cerebellar pathology that includes fibrinogen deposition, myelination deficits, and impaired cerebellar growth; (2) Intraventricular injection of fibrinogen in neonatal mice disrupts cerebellar development in vivo; (3) Fibrinogen activates the bone morphogenetic protein (BMP) receptor activin A receptor type I (ACVR1) in oligodendrocyte progenitor cells (OPCs) to inhibit OPC maturation and myelination, (4) Fibrinogen inhibits neurogenesis from neuronal progenitor cells in vitro. Our specific aims will test our working model, whereby fibrinogen deposition after BBB disruption induces ACVR1-mediated BMP signaling in CNS progenitor cells to inhibit neurogenesis and myelination leading to abnormal neurodevelopment. In Aim 1, we will define the contribution of fibrinogen to preterm infant brain injury in vivo using fibrinogen mutant mice. In Aim 2, we will identify the cellular mechanisms that contribute to preterm infant brain injury at sites of fibrinogen deposition using in vivo two-photon microscopy (2PM). In Aim 3, we will determine the molecular mechanism of fibrinogen-induced activation of ACVR1 using in vitro binding and cellular assays. These studies will reveal the molecular link between BBB disruption and failure of CNS progenitor cell maturation in preterm infant brain injury. My goal is to become an independent physician- scientist and leader in the field of newborn brain injury. To continue my progress towards this goal, I will build upon the expertise of the Gladstone Institutes and UCSF to expand my research skills in the following areas: (1) in vivo 2PM to study the dynamic cellular responses to BBB disruption and fibrinogen deposition in the neonatal brain, (2) RNA-sequencing and transcriptome analysis to identify fibrinogen-mediated mechanisms of extrinsic inhibition, and (3) binding assays and inhibitor studies to discover novel fibrinogen receptors on CNS progenitor cells. The knowledge and experience gained from this proposal will allow me to compete for R01 funding focused on therapeutics that target the inhibitory microenvironment in preterm infant brain injury.