Ferroptosis in knock-in sepiapterin reductase mutation rabbits

项目总结/摘要 本项目旨在研究一种罕见的神经代谢紊乱，Sepiapterin还原酶的交叉 (SPR)缺乏症，以及一种相对罕见的儿童疾病，脑瘫（CP）。都有运动障碍 有时候临床表现也是相似的SPR基因的突变导致 四氢生物蝶呤（BH 4）。BH 4不仅是大脑中五种重要酶的辅因子， 在细胞死亡的途径，特别是涉及氧化剂。在非凋亡性细胞死亡的形式中， 是由大量氧化剂沿着及BH 4引起的。鉴于我们过去对氧化剂效应的兴趣 和抗氧化剂减少运动缺陷，我们建议在我们的CP兔模型中研究铁凋亡。的 BH 4的确切作用将通过比较在BH 4基因敲入和BH 4基因敲入之间的铁凋亡途径的变化来研究。 在兔子和野生型兔子中的人类突变。生成代表患者的精确敲入家兔 突变直到最近才成为可能，这是由我们的多个主要研究者团队实现的。敲入 的人R150 G突变是通过成簇间隔短回文重复序列完成的 （CRISPR）基因编辑平台。采用与人急性缺氧缺血性脑病（H-I）相似的缺氧缺血性脑病（hypoxia-ischemia，H-I）建立兔CP模型 早产时的胎盘功能不全，基于人类胎盘发育。这个胎儿H-I模型是 第一个可靠地导致CP，使我们不仅能够严格测试机制途径， 运动缺陷的治疗，目前还没有。随着代孕母亲的发展 磁共振成像（MRI）的标志物，我们可以预测哪些胎儿将发展产后运动 赤字这一进展使得早期识别引起张力亢进的关键途径， 由于胎儿损伤后早期发生铁性下垂，因此可以研究铁性下垂。我们的假设是细胞死亡 通过与BH 4相关的氧化应激的特定途径决定了后期CP运动缺陷的发展 第一个目标是确定在兔子中敲入人类突变R150 G是否会增加易感性 运动缺陷第二个目标是确定与BH 4有关的特定氧化剂是否确定关键的 导致运动障碍的铁性下垂基本的生化机制将利用 确定胎儿注定要得到出生后高张力和研究整个大脑，脑区和细胞 停职提出的创新是MRI作为替代标记物与血流的系统整合 流式细胞术技术，荧光和电子显微镜，自动蛋白质印迹，电子顺磁 光谱、高效液相色谱和质谱分析到独特的动物模型中 探索铁下垂的生化基础，并测试未来可能的治疗方法。这些研究将 阐明导致运动缺陷的临界细胞死亡周围的早期事件。临床重要性在于， 拟议的研究为系统开发急需的 用于预防先天性BH 4缺乏和CP引起的运动缺陷的疗法。

PROJECT SUMMARY/ABSTRACT This project seeks to investigate the intersection of a rare neurometabolic disorder, sepiapterin reductase (SPR) deficiency, and a relatively rare childhood disorder, cerebral palsy (CP). Both present with motor deficits and sometimes the clinical presentation can be similar. Mutations in the SPR gene result in deficiency of tetrahydrobiopterin (BH4). BH4 is not only a cofactor of five important enzymes in the brain, but is also involved in the pathways of cell death, especially involving oxidants. Of the forms of non-apoptotic cell death, ferroptosis is caused by a slew of oxidants along with the involvement of BH4. Given our past interest in oxidant effects and that antioxidants reduce motor deficits, we propose to study ferroptosis in our rabbit model of CP. The exact role of BH4 will be investigated by comparing the changes in ferroptosis pathways between a knock-in of a human mutation in the rabbit and wild-type rabbits. Generating precise knock-in rabbits representing patient mutations has not been possible until recently, achieved by our multi-principal-investigator team. The knock-in of the human R150G mutation was done through the clustered-regularly-interspaced-short-palindromic-repeats (CRISPR) gene editing platform. The rabbit model of CP utilizes hypoxia-ischemia (H-I) akin to human acute placental insufficiency at preterm gestation, based on the human abruptio placentae. This fetal H-I model is the first to reliably lead to CP, allowing us to rigorously test not only mechanistic pathways but also possible therapies for motor deficits, for which there is none currently available. With the development of a surrogate marker of magnetic resonance imaging (MRI), we can predict which fetuses will develop postnatal motor deficits. This advance allows the early identification of critical pathways causing hypertonia, making an in-depth study of ferroptosis possible, as ferroptosis occurs early after the fetal insult. Our hypothesis is that cell death by specific pathways of oxidant stress related to BH4 determines the development of later CP motor deficits The first Aim will determine whether knock-in of the human mutation R150G in rabbits increases susceptibility to motor deficits. The second Aim will determine whether specific oxidants related to BH4 determine critical ferroptosis that leads to motor deficits. The underlying biochemical mechanisms will be studied utilizing the identification of fetuses destined to get postnatal hypertonia and studying entire brain, brain regions and cell suspensions. Innovations proposed are the systemic integration of MRI as a surrogate marker with flow cytometry techniques, fluorescent and electron microscopy, automated western blot, electron paramagnetic spectroscopy, high performance liquid chromatography, and mass spectrometry into the unique animal model to probe the biochemical basis of ferroptosis, with testing of possible future therapies. These studies will elucidate the early events around critical cell death that cause motor deficits. The clinical importance is that the proposed studies provide the mechanistic understanding for the systematic development of much-needed therapies for prevention of motor deficits from congenital BH4 deficiency and CP.