Probing Role of Tetrahydrobiopterin in Cerebral Palsy by Using Transgenic Rabbits

项目摘要/摘要 患有运动障碍的儿童是社会的一大负担。疾病的负担非常重，因为 对患者、照顾者和社会机构的终生后果。目前没有治愈的方法或 脑性瘫痪(CP)的预防性治疗，因为疾病的机制仍不清楚。人类 关键酶途径的突变构成了儿童运动障碍的遗传原因。与 转基因兔模型的出现，为研究转基因兔的致病机制提供了千载难逢的机会。 大脑导致运动障碍，因为兔子更有可能出现模仿的运动障碍 就像人类一样。兔子和人类一样，也是围产期大脑的发育者。人血清白蛋白的酶基因突变 四氢生物蝶呤途径导致运动障碍。四氢生物蝶呤是一种酶辅助因子，其 补充先天缺乏症可改善运动障碍。因此，可能会有一个 四氢生物蝶呤在运动障碍的发展中的关键作用，如CP。我们开发了一种 在一种四氢生物蝶呤合成酶中引入特定突变的基因敲除兔， 七叶蝶呤还原酶。胎儿缺氧缺血后，新生兔出现高张和困难 保持平衡。四氢生物蝶呤在不同脑区发育不良的胎兔有一个 更容易发展成高张力症。磁共振成像(MRI)使我们能够预测 胎儿在出生后会出现高眼压。这一进展使得能够识别早期的关键途径 导致高张力。我们的目标是阐明围产期脑损伤的分子机制。 通过使用异源基因降低四氢生物蝶呤水平而导致儿童运动障碍的突变 兔纯合子基因敲除转基因方法。这项提案中提出的主要问题是 选择性脑区四氢生物蝶呤可导致严重的运动障碍 由额外的产前侮辱确定，如缺氧、缺血或炎症。利用基因敲除技术 我们可以进一步降低脑内四氢生物蝶呤的水平，并研究 由此导致的运动缺陷会增加，或者我们需要更少的侮辱程度才能达到同样的运动缺陷。 第一个目标确定增加的胎儿侮辱、缺氧缺血或炎症是否来自 脂多糖增强海燕蛋白het/纯合子还原酶基因敲除中的运动障碍 兔子。第二个目标将确定神经元或少突胶质细胞损伤是否解释了 基因敲除兔的运动障碍。我们使用创新的出生前和出生后磁共振生物标记物 组织流式细胞术和高效液相电化学法测定高渗血症 侦测。通过进行依赖时间的、特定器官的和特定细胞的致病研究，我们将获得 全面了解这种辅因子在运动障碍的围产期发病机制中的作用。

PROJECT SUMMARY/ABSTRACT Children with movement disorders are a big burden to society. The burden of disease is very high because of the life-long consequences to the patient, caretakers, and social institutions. Currently there are no cures or preventative treatments for cerebral palsy (CP), as the mechanisms of disease remain poorly defined. Human mutations in key enzymatic pathways constitute genetic causes of childhood movement disorders. With the advent of transgenic rabbit models, a golden opportunity has arisen to study the pathogenetic mechanisms in brain leading to movement disorders, as rabbits are more likely to present with movement disorders mimicking that of humans. Rabbits are perinatal brain developers like humans. Mutations in enzymes of tetrahydrobiopterin pathway result in movement disorders. Tetrahydrobiopterin is an enzyme co-factor and its supplementation in congenital deficiency disorders ameliorates the movement disorder. Thus, there may be a critical role of tetrahydrobiopterin in the development of movement disorders, such as CP. We developed a knockout rabbit that introducing a specific mutation in one of the tetrahydrobiopterin synthesis enzymes, sepiapterin reductase. Following fetal hypoxia-ischemia, newborn rabbits present with hypertonia and difficulty with balance. Fetal rabbits showing low developmental tetrahydrobiopterin in discrete brain regions have a greater disposition to develop hypertonia. Magnetic resonance imaging (MRI) allows us to predict which fetuses will develop postnatal hypertonia. This advance allows the identification of early critical pathways causing hypertonia. Our objective is to elucidate molecular mechanisms of perinatal brain injury in human mutations causing childhood movement disorders, by decreasing tetrahydrobiopterin levels using a hetero- and homozygous knockout transgenic approach in the rabbit. The main question asked in this proposal is whether tetrahydrobiopterin in selective brain regions contributes to the development of motor disorders with a severity determined by an added prenatal insult such as hypoxia-ischemia or inflammation. Using genetic knockout of sepiapterin reductase, we can further lower the tetrahydrobiopterin levels in brain and investigate whether the resulting motor deficits are increased or that we need less degree of insult to achieve the same motor deficits. The first Aim determines whether an added fetal insult, hypoxia-ischemia or inflammation from lipopolysaccharide, enhances movement disorders in the sepiapterin het/homozygous reductase knockout rabbit. The second Aim will determine if neuronal or oligodendroglial injury explains the development of movement disorders in the knockout rabbit. We use innovative pre- and postnatal MRI biomarkers of hypertonia with tissue flow cytometry and high-performance liquid chromatography with electrochemical detection. By conducting a time-dependent, organ-specific and cell-specific pathogenetic study, we will obtain a comprehensive picture of the role of this cofactor in perinatal pathogenesis of movement disorders.