Project Summary Neonatal hypoxic ischemic encephalopathy (HIE) is characterized by a protracted series of pathophysiological events that, without intervention, are devastating. HIE can result in neurodevelopmental delays, epilepsy, cognitive and motor issues, or death. The current standard of clinical care for infants born at term with severe hypoxia-ischemia (HI) is hypothermia. Therapeutic hypothermia reduces the likelihood of death and lessens deficits in some behavioral outcome measures. However, neuroprotection is far from complete. The major goal of this proposal is to use the well-established Rice-Vannucci HI rodent model to develop a translational strategy for improving neuroprotection by targeting mitochondrial Complex I. Although Complex I function is necessary for the recovery of brain energy metabolism after acute energy failure, reactive oxygen species (ROS) produced by Complex I during the recovery phase contribute to secondary injury. Apoptotic cell death and neuroinflammation also contribute to hypoxic-ischemic brain injury. It is unknown whether these mechanisms depend on Complex I alterations in vivo. Our published findings demonstrate that the preclinical drug mdivi-1 inhibits Complex I-dependent ROS production while only mildly and reversibly inhibiting mitochondrial respiration. Our new data suggest a direct interaction of mdivi-1 with a subunit of Complex I. We find that mdivi- 1 significantly reduces the occurrence of severe rat brain tissue loss measured 3 days after HI and decreases 3-nitrotyrosine labeling, a marker of oxidative stress, in the hippocampus,. Mechanistically, we find that mdivi-1 interacts with Complex I in microglia, the innate immune cells mediating the persistent neuroinflammatory response after neonatal HI. Mdivi-1 shows inhibition of microglial pro-inflammatory responses in vitro and in vivo. The central hypothesis of this study is that mdivi-1 will additively enhance hypothermic neuroprotection in neonatal hypoxic-ischemic brain injury models by reducing Complex I-dependent oxidative stress. We predict benefit in both male and female animals, though through potentially sexually dimorphic mechanisms. Males and females differ in dominant cell death pathways, and males exhibit greater vulnerability to chronic microglial activation after HI. A novel mouse model of partial Complex I deficiency showing normal neurodevelopment will be used to establish whether neuroprotection by mdivi-1 or hypothermia is occluded by a moderate reduction in the level of assembled Complex I. This genetic method will also reveal whether Complex I dysfunction contributes to HI-induced apoptosis or neuroinflammation in the immature brain of either sex. Positive outcomes will support the development of reversible Complex I inhibitors for clinical use, with the hope that this class of drugs may ultimately help millions by reducing the devastating consequences of neonatal hypoxic ischemic encephalopathy.