Probing Role of Tetrahydrobiopterin in Cerebral Palsy by Using Transgenic Rabbits

项目概要/摘要 患有运动障碍的儿童是社会的一大负担。疾病的负担非常高，因为 对病人、看护人和社会机构的终身影响。目前还没有治愈或 预防性治疗脑性瘫痪（CP），因为疾病的机制仍然不清楚。人类 关键酶途径的突变构成儿童运动障碍的遗传原因。与 转基因兔模型的出现，为研究其发病机制提供了一个极好的机会。 大脑导致运动障碍，因为兔子更有可能出现模仿运动障碍 人类的。兔子和人类一样是围产期大脑发育者。酶的突变 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因子， 在先天性缺陷性疾病中补充可改善运动障碍。因此，可能有一个 四氢生物蝶呤在运动障碍如CP的发展中的关键作用。我们开发了一个 在四氢生物蝶呤合成酶之一中引入特定突变的敲除兔， 乌贼蝶呤还原酶胎兔缺氧缺血后，新生兔出现肌张力增高， 保持平衡胎兔在不连续的脑区显示出低发育的四氢生物蝶呤， 更容易出现肌张力亢进。磁共振成像（MRI）使我们能够预测 胎儿会在出生后出现张力亢进。这一进展使早期关键途径的识别 引起张力亢进。我们的目的是阐明人类围产期脑损伤的分子机制 突变引起的儿童运动障碍，通过使用异源和 纯合敲除转基因方法。本提案提出的主要问题是， 选择性脑区域中的四氢生物蝶呤有助于运动障碍的发展， 由附加的产前损伤如缺氧-缺血或炎症确定。使用基因敲除 通过sepiapterin还原酶，我们可以进一步降低大脑中的四氢生物蝶呤水平，并研究是否 导致的运动缺陷增加，或者我们需要更小程度的侮辱来实现相同的运动缺陷。 第一个目的是确定是否增加了胎儿损伤，缺氧缺血或炎症， 脂多糖，增强sepiapterin het/纯合还原酶敲除中的运动障碍 兔子第二个目标是确定神经元或少突胶质细胞损伤是否解释了 被击倒的兔子的运动障碍我们使用创新的产前和产后MRI生物标志物， 组织流式细胞术和高效液相色谱电化学检测 侦测通过进行时间依赖性、器官特异性和细胞特异性的发病机制研究，我们将获得 全面了解这种辅助因子在围产期运动障碍发病机制中的作用。

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.