Mechanisms of Neonatal Erythropoietin Neuroprotection

描述（由申请人提供）：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降低神经系统发病率。通常，全身性缺氧-缺血（HI），感染，或更常见的两者的结合，导致产前中枢神经系统（CNS）损伤，在出生前延长出生后神经元和少突胶质细胞的损失，最终损害电路的形成。神经细胞的损失主要是由于新一代祖细胞的凋亡而无法存活。促红细胞生成素（EPO）及其同源受体EPOR是产前大脑发育所必需的，特别是在妊娠后半期，EPO信号局部调节神经细胞的存活以减少神经细胞的过量生产。未结合的EPOR驱动神经细胞凋亡，而配体结合的EPOR激活存活信号通路。EPO还具有神经保护作用，能穿过血脑屏障。我们建立了一个由子宫动脉闭塞引起的产前一过性全身性HI模型，以及一过性全身性HI加宫颈内脂多糖（LPS）来模拟人类全身性产前HI损伤和缺血/炎症联合损伤。我们发现新生儿EPO治疗逆转了成年大鼠产前HI损伤后的组织学和功能缺陷。我们的实验数据揭示了EPO信号传导的一种新的分子机制，有助于解释产前损伤后发生的过度凋亡，并建议在新生儿期使用外源性EPO进行一种新的药物干预。我们发现，全身性产前HI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神经细胞EPOR，并且外源性新生儿EPO似乎增强了产前HI后神经细胞的存活和过程形成。我们假设产前损伤上调神经细胞的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，在产前损伤后，新生儿外源性EPO拯救了神经细胞，增强了它们的存活和分化，最终改善了回路的形成，并导致功能恢复。我们将使用我们的产前HI模型，有和没有宫颈内脂多糖（LPS）来产生产前损伤，以检验我们的假设。在Aim 1中，我们将定义EPO、EPOR的表达模式，并识别在产前HI和LPS加HI后受损发育中的神经细胞中活跃的上下游信号分子。在Aim 2中，我们将使用剂量-反应曲线和特异性分子抑制剂测试EPO信号是否调节体外神经细胞存活和分化。最后，在Aim 3中，我们将在受损的发育中的神经细胞中操纵EPOR的表达，以阐明EPOR的过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，以潜在地将新生儿促生成素这一有前途的治疗选择转化为早产儿，并优化这些儿童无缺陷发育的机会。非常早产的儿童患有脑瘫、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巨大负担。目前的干预措施未能改善其结果。为了治疗早产婴儿，我们提出了一种新的药物干预，即促红细胞生成素（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损伤的神经细胞的发育。

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.