Biochemical and Physiological Phenotypes of CV Dysfunction In Human Cell Models - ABSTRACT. Complex V (CV, or ATP synthase) of the electron transport chain is the central enzyme of cellular energy capture. CV synthesizes ATP driven by the proton gradient generated by the electron transport chain. The advent of clinical gene sequencing has highlighted the devastating effects of deficiency of this critical enzyme. Pathogenic variants in CV subunits give rise to multi-system disease, including strokes, neuropathy, ataxia, retinopathy, and cardiomyopathy. Genetic variation in CV is frequent, and the inability to distinguish pathogenic mutations from the variants of unknown significance is a clinical challenge preventing understanding of prognosis and rational approach to management. However, no clinical test for CV function exists. This thwarts our ability to classify genetic variants. Our Goal is to develop a biochemical approach to evaluating CV function and ultimately predicting the clinical significance of CV variants. We previously demonstrated that basal ATP levels are normal with CV deficiency while the rate of ATP synthesis can be low, suggesting that clinical symptoms result from an inability of CV to accommodate an increased metabolic demand. Direct enzymatic testing of ATP synthesis by CV is impossible, as the substrate for CV is the proton motive force, which is dissipated when the enzyme is purified. We therefore propose to assay ATP flux in our human cell (fibroblast, transmitochondrial cybrid) models of diverse CV genetic variants, including novel candidate genes. We have observed that the biochemical effects of CV variants result in diverse biochemical sequelae; therefore, we will also assay oxygen consumption, mitochondrial membrane potential, matrix pH, CV assembly, and mitochondrial cristae structure and correlate results with clinical manifestations. We anticipate that this approach will furnish a biochemical and morphologic profile that informs the pathogenicity of the variant. We hypothesize that the observation that steady-state ATP levels are normal in CV deficient cell-lines despite low enzymatic flux implies that CV function is responsive to cellular metabolic state. We will introduce a series of provocative (stimulus-response) testing procedures that involve modulation of nutrient levels (glucose, αKG) and exposure to cell stress (galactose, lipopolysaccharide). By rigorously investigating the biochemical consequences of CV deficiency including in dynamic models of cellular stress, we will establish the foundation on which to develop clinical diagnostic assays to confirm CV mutation pathogenicity and treatment response. The Central Hypothesis of this proposal is: pathogenic variants in CV subunit genes evoke changes in CV bioenergic function resulting in diverse downstream biochemical defects that predict clinical presentation. Further, we propose that the clinical manifestations of Complex V deficiency severity are influenced by the biochemical and nutritional milieu in which the genetic deficiency finds itself. Experimental manipulation of this milieu may identify nutritional therapeutic approaches.
