Ferroptosis in knock-in sepiapterin reductase mutation rabbits

敲入墨蝶呤还原酶突变兔的铁死亡

基本信息

批准号：
10747716
负责人：
SIDHARTHA TAN
金额：
$ 60.55万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-21 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10747716
关键词：
Abbreviations Abruptio Placentae Acute Affect Anabolism Animal Model Antioxidants Apoptosis Biochemical Birth Brain Brain Hypoxia-Ischemia Brain Injuries Brain region Carotid Arteries Cause of Death Cell Death Cerebral Palsy Chemicals Child Childhood Clinical Clustered Regularly Interspaced Short Palindromic Repeats Critical Pathways DHFR gene Development Diffusion Dihydrofolate Reductase Discipline of obstetrics Disease Disorder of neurometabolic regulation Early identification Electron Microscopy Electron Spin Resonance Spectroscopy Electrons Endoplasmic Reticulum Enzymes Equation Ethidium Etiology Event Failure Family suidae Fetus Flow Cytometry Future GTP Cyclohydrolase I Genes Guide RNA Heterozygote High Pressure Liquid Chromatography Homozygote Human Injury Institution Iron Chelation Knock-in Knock-out Lead Ligation Link Lipid Peroxidation Lipid Peroxides Magnetic Resonance Imaging Mammals Mediating Modeling Motor Muscle Hypertonia Mutation Necrosis Neuroprotective Agents Nitric Oxide Synthase Type I Oryctolagus cuniculus Outcome Oxidants Oxidation-Reduction Oxidative Stress Oxidative Stress Pathway Pathogenesis Pathway interactions Patients Pattern Perinatal Phenotype Phospholipids Placental Insufficiency Predisposition Pregnancy Prevention Prevention therapy Principal Investigator Propidium Diiodide Quadriplegia Recycling Reperfusion Therapy Resistance Rhodamine 123 Rodent Role Scientific Inquiry Sepiapterin reductase Sheep Societies Spectrum Analysis Superoxides Surrogate Markers Suspensions Techniques Testing Tetrahydrobiopterin deficiency Western Blotting antenatal brain cell burden of illness cell type cofactor coping cost drug development fetal fetal brain injury fetus hypoxia human fetal brain hydroethidine inhibitor innovation insertion/deletion mutation insight interest liquid chromatography mass spectrometry magnetic resonance imaging biomarker motor deficit neuroimaging marker nonhuman primate oxidant stress oxidation peroxidation phenyl-N-tert-butylnitrone postnatal prevent sepiapterin reductase deficiency social spasticity targeted treatment tetrahydrobiopterin

项目摘要

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.

项目摘要/摘要该项目旨在研究罕见的神经代谢障碍，神经蛋白还原酶的交集。（SPR）缺乏症和相对罕见的儿童障碍，脑瘫（CP）。两者都出现运动不足有时临床表现可能相似。 SPR基因的突变导致缺乏四氢无菌蛋白酶（BH4）。 BH4不仅是大脑中五种重要酶的辅助因子，而且还涉及在细胞死亡的途径中，尤其是涉及氧化剂。非凋亡细胞死亡的形式由大量氧化剂以及BH4的参与引起。考虑到我们过去对氧化作用的兴趣抗氧化剂减少了运动缺陷，我们建议在我们的CP兔模型中研究铁铁作用。这 BH4的确切作用将通过比较在敲门之间的螺旋病途径的变化来研究兔子和野生型兔子中的人类突变。产生代表患者的精确敲打兔子直到最近，我们的多本文投资者团队才能实现突变。敲门人R150g突变是通过簇状的定期间隔 - 折线 - palindromic重复进行的（CRISPR）基因编辑平台。 CP的兔模型利用类似于人类急性的低氧 - 异化（H-I）基于人类突发胎盘的早产妊娠不足。该胎儿H-I模型是首先可靠地导致CP，使我们不仅可以严格测试机械途径，而且可以测试运动缺陷的疗法，目前没有任何可用的疗法。随着代理的发展磁共振成像（MRI）的标记，我们可以预测哪些胎儿将发展产后运动缺陷。这一进步允许早期识别引起高血压的关键途径，使得深入由于胎儿损伤后早期发生铁铁作用的研究。我们的假设是细胞死亡通过与BH4相关的氧化应激的特定途径决定了后来CP运动缺陷的发展第一个目标将确定兔子中人类突变R150g的敲击是否会增加易感性到运动不足。第二个目的将确定与BH4相关的特定氧化剂是否确定关键导致运动缺陷的铁铁作用。将研究基本的生化机制。鉴定注定要获得产后高血压并研究整个大脑，大脑和细胞的胎儿悬架。提出的创新是MRI作为流量的替代标记的系统整合细胞仪技术，荧光和电子显微镜，自动蛋白质印迹，电子顺磁性光谱，高性能液相色谱法和质谱法进入独特的动物模型探测甲状腺吞噬作用的生化基础，并测试可能的未来疗法。这些研究会阐明导致运动缺陷的关键细胞死亡周围的早期事件。临床重要性是拟议的研究为急需的系统发展提供了机械理解预防先天性BH4缺乏症和CP的运动缺陷的疗法。