1. ABSTRACT. Mitochondrial complex V (CV) subunit gene mutations cause a variety of severe metabolic diseases that impair child health and development, with strokes, neuropathy, ataxia, vision loss, and cardiomyopathy. However, much remains to be learned about underlying mechanisms and potential therapies for CV diseases. We Hypothesize that (a) CV subunit mutations evoke assorted changes in its structure and function that may result in a variety of discrete biochemical defects, and (b) CV disease severity is directly influenced by mTORC1 activity and cellular nutrient status.
Specific Aims of this work are to [Aim 1] Identify the precise biochemical processes disrupted by CV deficiency; [Aim 2] Characterize the impact of cellular nutrients and nutrient-sensing signaling through the AMPK/mTOR pathway on CV regulation; and [Aim 3] Evaluate organ- level effects of CV deficiency and targeted signaling therapies in a zebrafish vertebrate model animal, given extensive evolutionary conservation of CV. Methods will include in vitro cellular assessment of mitochondrial CV structure and function (blue native gel), mitochondrial physiology (mitochondrial membrane potential, oxidative stress), and activities of central nodes in the integrated nutrient-sensing signaling network in human fibroblasts, using cells from healthy individuals, genetic-based CV diseases, and pharmacologic CV inhibition (oligomycin). Cellular analyses will be performed in response to modulation of cellular nutrients (glucose, leucine) and mTORC1 activity (rapamycin, probucol). We will also generate and characterize pharmacologic (oligomycin) and genetic (morpholino, CRISPR/Cas9) zebrafish model animals of CV disease in which to evaluate the organ-level sequelae of CV diseases as well as the potential therapeutic effects of cellular nutrients and mTORC1 activity regulators on CV functions. These studies will establish the foundation on which to future develop clinical diagnostic assays to confirm CV mutation pathogenicity and evaluate potential treatment responsiveness, and inform organ-specific effects of disease and potential therapies in a novel vertebrate model animal of CV disease.

Public Health Relevance

. Genetic diseases that cause primary mitochondrial respiratory chain deficiency result in severe disease, affecting nearly every organ system, including brain, heart, liver and eyes and are largely currently untreatable. Making a diagnosis of primary respiratory chain deficiency is difficult, especially in the subset of diseases that affect Complex V (ATP Synthase; CV), the primary source of energy in the cell. This work seeks to understand how CV is regulated by nutrients in health and disease states, providing a potential avenue for future diagnosis and therapies in these conditions.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Clinical Investigator Award (CIA) (K08)
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Kidney, Urologic and Hematologic Diseases D Subcommittee (DDK)
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Spain, Lisa M
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Children's Hospital of Philadelphia
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Barca, Emanuele; Ganetzky, Rebecca D; Potluri, Prasanth et al. (2018) USMG5 Ashkenazi Jewish founder mutation impairs mitochondrial complex V dimerization and ATP synthesis. Hum Mol Genet 27:3305-3312
Ahrens-Nicklas, Rebecca C; Ganetzky, Rebecca D; Rush, Peggy W et al. (2018) Characteristics and outcomes of patients with formiminoglutamic aciduria detected through newborn screening. J Inherit Metab Dis :
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Ganetzky, Rebecca D; Falk, Marni J (2018) 8-year retrospective analysis of intravenous arginine therapy for acute metabolic strokes in pediatric mitochondrial disease. Mol Genet Metab 123:301-308