Project Summary Metabolic abnormalities may play a role in neurodegeneration in the setting of mitochondrial complex I (CI) disease. CI deficiency decreases oxidative metabolism of glucose in the brain at a precise developmental window. However, neither the developmental changes leading to altered nutrient handling, nor the implications of decreased glucose flux have been described. This proposal will characterize the impact of CI deficiency (in the Ndufs4(KO) mouse) on glutamine flux pathways, a compensatory nutrient source, in the brain. Glutamine is unique from glucose as it supplies CI via pathways theoretically more effective than those of glucose in the presence of CI disease. However, they may also be pathogenic. The applicant will assess glutamine metabolic flux using stable isotope tracing assays in wildtype and CI defective Ndufs4(KO) mice. The goals of this proposal are to determine 1) the impact of CI defects on glutamine metabolic handling in brain tissue, 2) posttranslational changes to enzymes that facilitate glucose handling during development, and 3) whether mTOR inhibition, which rescues neurodegeneration in the Ndufs4(KO) mouse, impacts CNS glucose flux. Based on substantial preliminary data, CI dysfunction is hypothesized to increase glutamine flux, in response to decreased oxidative glucose flux. mTOR inhibition is predicted to rescue disease at least partly through alleviation of defective glucose flux, decreasing glutamine flux by extension. The metabolic impact of CI dysfunction will be characterized by 1) 13C glutamine flux experiments performed in cerebellar tissue from control and Ndufs4(KO) mice as a function of age and disease onset; 2) proteomic analysis of enzymes involved in glucose metabolism as a function of developmental age, and 3) similar 13C glucose flux experiments in the setting of mTOR inhibition. Relevance Genetic mitochondrial diseases include an array of symptoms, may affect one organ or present as a multisystem disorder, and are remarkably heterogeneous in severity. There are no validated effective treatment options for mitochondrial disease of any etiology. This work will help define the cellular and molecular mechanisms of mitochondrial diseases and dysfunction with the goal of leading to treatment. Environment and Training Plan Seattle Children’s Research Institute and the University of Washington provide an ideal environment for doctoral training, through outstanding faculty mentors, state-of-the-art facilities, and myriad opportunities for career development. The trainee will master biochemical techniques and mouse genetics. She will attend seminars and workshops to advance her conceptual knowledge of biochemistry, physiology, and neuroscience. Finally, the trainee will advance her science communication skills by presenting her work at conferences, and preparing at least two manuscripts for publication per year.