Mechanisms of Neonatal Erythropoietin Neuroprotection

描述(申请人提供)：早产儿容易出现脑瘫、癫痫、认知迟缓和行为问题，但目前的干预措施未能减少神经系统的发病率。通常，全身性缺氧缺血(HI)、感染，或者更常见的是两者的组合，会在出生前导致产前中枢神经系统(CNS)损伤，出生后神经元和少突胶质细胞长期丢失，最终导致神经回路形成障碍。神经细胞的丢失主要是由于新一波祖细胞无法存活而导致的细胞凋亡。促红细胞生成素(EPO)及其同源受体EPOR是胎儿脑发育所必需的，特别是在妊娠的后半期，EPO信号局部调节神经细胞的存活，以修剪神经细胞的过度生产。未结合的EPOR促使神经细胞凋亡，而与配体结合的EPOR激活生存信号通路。促红细胞生成素还具有神经保护特性，可以穿越血脑屏障。我们使用建立的子宫动脉阻断的产前一过性全身性缺氧模型，以及一过性全身性缺氧加宫颈内脂多糖(LPS)来模拟人类全身性产前缺氧和缺血/炎性联合损伤。我们发现，新生儿促红细胞生成素治疗逆转了成年大鼠出生前HI损伤后的组织学和功能缺陷。我们的初步数据揭示了EPO信号的一种新的分子机制，有助于解释产前侮辱后发生的过度凋亡，并建议在新生儿期使用外源性EPO进行新的药物干预。我们发现，系统性产前缺氧似乎上调了最脆弱的神经细胞、神经元和少突胶质细胞上的EPOR，外源性新生儿EPO似乎促进了产前缺氧后神经细胞的存活和突起形成。我们假设，胎儿期的侮辱上调了神经细胞上的EPOR，如果没有足够的EPO存在，这些细胞就会发生凋亡。我们认为，在产前损伤新生儿外源性EPO后，可以挽救神经细胞，提高其存活和分化能力，最终改善神经回路的形成，从而导致功能恢复。我们将使用我们的产前HI模型，在有和没有宫颈内脂多糖(LPS)的情况下，产生产前侮辱来检验我们的假设。在目标1中，我们将确定EPO、EPOR的表达模式，并鉴定产前缺氧和脂多糖加缺氧后受损发育神经细胞中活跃的上下游信号分子。在目标2中，我们将使用剂量-反应曲线和特定的分子抑制剂来测试EPO信号是否在体外调节神经细胞的存活和分化。最后，在目标3中，我们将操纵EPOR在受损发育神经细胞中的表达，以阐明EPOR的过度表达或沉默是否支持我们的预测。总之，这些研究将阐明EPO诱导的产前损伤后神经细胞恢复的机制。他们将提供急需的临床前基础，潜在地将这种使用新生儿促红细胞生成素的有希望的治疗选择转化为早产婴儿，并优化这些儿童无缺陷发育的机会。早产儿患有脑瘫、癫痫、认知迟缓和行为问题，给这些儿童、他们的家庭和社会带来了巨大的负担。目前的干预措施未能改善他们的结果。为了治疗早产儿，我们提出了一种新的药物干预，使用促红细胞生成素，一种目前用于治疗贫血的药物，因为促红细胞生成素既提供神经保护，又促进最容易受到早产侮辱损害的神经细胞的发育。

DESCRIPTION (provided by applicant): Children who are born very preterm are prone to cerebral palsy, epilepsy, cognitive delay and behavioral problems, but current interventions have failed to reduce the neurologic morbidity. Typically, systemic hypoxia- ischemia (HI), infection, or more commonly a combination of both, cause prenatal central nervous system (CNS) injury prior to birth with prolonged postnatal loss of neurons and oligodendrocytes, culminating in impaired circuit formation. Neural cell loss occurs primarily by apoptosis with failure of new waves of progenitors to survive. Erythropoietin (EPO) and its cognate receptor EPOR are required for prenatal brain development, especially in the second half of gestation, where EPO signaling locally regulates neural cell survival to prune neural cell overproduction. Unbound EPOR drives neural cells to apoptosis, while ligand- bound EPOR activates survival signaling pathways. EPO also has neuroprotective properties and crosses the blood-brain barrier. We used an established model of prenatal transient systemic HI from uterine artery occlusion, and transient systemic HI plus intracervical lipopolysaccharide (LPS) to mimic human systemic prenatal HI insults and combined ischemic/inflammatory insults. We found neonatal EPO treatment reverses the histological and functional deficits of adult rats after prenatal HI injury. Our pilot data reveal a novel molecular mechanism of EPO signaling that helps to explain the excess apoptosis that occurs after prenatal insults, and suggests a novel drug intervention using exogenous EPO in the neonatal period. We found that systemic prenatal HI appears to upregulate neural cell EPOR on the most vulnerable neural cells, neurons and oligodendrocytes, and that exogenous neonatal EPO appears to enhance survival and process formation by neural cells after prenatal HI. We hypothesize that prenatal insults upregulate EPOR on neural cells and that without adequate EPO present these cells undergo apoptosis. We propose that after prenatal insults neonatal exogenous EPO rescues neural cells, enhances their survival and differentiation, ultimately improving circuit formation, and leading to functional recovery. We will use our model of prenatal HI with and without intracervical lipopolysaccharide (LPS) to produce prenatal insults to test our hypothesis. In Aim 1 we will define the expression pattern of EPO, EPOR and identify the up and downstream signaling molecules active in damaged developing neural cells after prenatal HI, and LPS plus HI. In Aim 2 we will test the whether EPO signaling regulates neural cell survival and differentiation in vitro using dose-response curves and specific molecular inhibitors. Lastly, in Aim 3 we will manipulate the expression of EPOR in damaged developing neural cells to clarify whether over-expression or silencing of EPOR supports our predictions. Together, these studies will clarify the mechanism of EPO-induced neural cell recovery after prenatal injury. They will provide the much needed preclinical foundation for potentially translating this promising therapeutic option using neonatal EPO to infants born prematurely, and optimize the chance these children develop without deficits. Children who are born very preterm suffer from cerebral palsy, epilepsy, cognitive delay and behavioral problems, placing a tremendous burden on these children, their families, and society. Current interventions have failed to improve their outcome. To treat infants born preterm, we propose a novel drug intervention with erythropoietin, a drug currently used to treat anemia, as EPO both provides neuroprotection and enhances development of the neural cells most vulnerable to damage from preterm insults.