Project Summary Neonatal hypoxia/ischemia can result in severe damage to the infant brain. Reestablishing blood flow and oxygen delivery (reperfusion) is crucial for survival. However, reperfusion induces an accumulation of reactive oxygen species (ROS) produced by the mitochondria, culminating in oxidative stress and irreversible tissue damage. Current studies suggest that cardiolipin (CL) and its remodeling via oxidative injury to monolysocardiolipin (MLCL) directly participate in activation of mitophagy and lipidic pore formation to regulate cytochrome c release and facilitate induction of programmed cell death. The objectives of this research proposal are to investigate of the roles of CL and CL modification as molecular mechanisms in hypoxic brain injury. Aim 1 will focus on establishing the clinical relevance of CL modification and investigate the role of CL modification in brain injury following HIE. Utilizing the cutting-edge technology of cyclic ion mobility spectrometry mass spectrometry (cIMS-MS) CL and its isoforms will be quantitatively analyzed following hypoxia/ischemia in a neonatal piglet model of HIE. Unlike traditional mass spectrometry, cIMS-MS separates molecules based on structure as well as mass to charge ratio (m/z) allowing detailed analysis of CL species and to directly investigate its remodeling in I/R injury. Several novel transgenic mouse-lines will also be used to manipulate CL biosynthesis and remodeling in vivo. Mouse pups will be exposed to hypoxia/ischemia, then brains will be analyzed for injury to evaluate the contribution of CL modification on outcomes following HIE. Aim 2 will further explore the mechanisms regulated by CL modification following hypoxia/ischemia, including disruption to the mitochondrial network through mitophagy and mitochondrial dynamics. Mitophagic flux will be characterized in primary culture from mice possessing the mitochondrial quality control (mitoQC) reporter and disruptions to mitochondrial morphology will be assessed with our cutting-edge machine learning based quantification methodology. Finally, the mitochondrial specific antioxidant mitoTEMPO and phospholipase inhibitors will be used in combination with our genetic mouse models to define the contribution of oxidative modification and lipolysis to CL modification following hypoxia/ischemia. Together, these experiments will establish the mechanism of post-hypoxic CL modification, interrogated the casual role of CL and MLCL in the progression of brain injury , and define potential therapeutic targets. With exceptional scientific mentorship and rigorous academic study, this research will develop a deeper understanding of the role lipidomics play in HIE. This project will allow extensive training and allow me to gain expertise in a diverse range of animal models and molecular analysis of mechanisms of brain damage. The proposed project creates a unique environment to foster the development and critical thinking of a young scientist.