Metabolic Landscape and Mitochondrial ROS Balance in Brain Ischemia/Reperfusion

Project Summary Ischemia-reperfusion (IR) injury after perinatal asphyxia causes neurological disability, morbidity, and even death in infants. To develop therapeutic strategies, we need a better mechanistic understanding of the metabolic changes in cerebral tissue during IR and IR-driven oxidative stress. IR-associated disruptions in glycolysis, the TCA cycle, amino acid and nucleotide metabolism, oxidative phosphorylation, and reactive oxygen species (ROS) balance lead to oxidative stress and contribute to cerebral tissue injury. The initial acute injury occurs via ROS elevation whose origin is not yet completely known. Recently, we found that oxygen deprivation in the brain results in so-called reverse electron transfer in mitochondrial Complex I leading to high ROS production and dissociation of its natural cofactor, flavin (FMN). The oxygen deprivation also degrades amino acids and purine nucleotides, which causes accumulation of ammonia. Ammonia increases ROS generation by dihydrolipoamide dehydrogenase (DLD), a component of ketoacids dehydrogenases of Krebs cycle. This contributes to a cell-type specific increase of ROS during reoxygenation and redox imbalance. Our preliminary results prompt further study of this metabolic pathway in the brain IR. Our overarching objective is to determine the main contributors to redox balance of ROS during brain IR. We hypothesize that ROS imbalance in brain IR is tissue-specific and involves at least two metabolic mechanisms: 1) a ROS decrease due to mitochondrial FMN release, and 2) a ROS increase due to ammonia-dependent activation of TCA cycle DLD. We will use advanced mass spectrometry imaging to determine the spatial distribution of small molecules in brain sections obtained after IR for simultaneous visualization of various metabolites (Aim 1). In Aim 2 we will establish the role of succinate and glycerol 3-phosphate-induced RET as modulators of ROS production in brain using transgenic mouse model expressing alternative oxidase. Mitochondria from these mice do not catalyze RET and do not produce ROS at the CxI level. In Aim 3 we will evaluate the relative contribution of neurons and astrocytes to ammonia accumulation and ROS balance. We will use freshly isolated, primary cultures of neurons and astrocytes to analyze their ammonia-generating capabilities in response to lack of oxygen These new, unrecognized, and unexplored mechanisms for the regulation of ROS production in IR explain published observations showing the transient peak of ROS during brain IR. Knowledge of the molecular details and regulation of mitochondrial ROS production is vital to clarify the fundamental principles behind brain IR injury.