Ferroptosis in knock-in sepiapterin reductase mutation rabbits

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.