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.