Abstract One of the most important organizing principles in all life forms is the uninterrupted flow of electrons through reactions that involve reduction-oxidation (redox) changes. Not surprisingly, an imbalance in this fundamental cellular process, i.e. redox homeostasis, has been attributed to numerous diseases, including mitochondrial disorders, cancer, diabetes, neurodegeneration and the aging process itself. The redox cofactors, NADH and NADPH, and their oxidized forms are key contributors to the cellular redox environment, but it is unclear whether perturbations in their metabolism contribute directly to disease etiology or is simply a reflection of ongoing pathology. For most of these conditions, it is not known whether the observed redox imbalance is linked to altered bioenergetic efficiency or to a cellular process that is neither linked to ATP production nor to maintenance of the mitochondrial membrane potential. Another major challenge is that some of these redox reactions are redundant, i.e. have overlapping substrate dependency (towards NAD(P)H) or are found in more than one cellular compartment. To systematically address these pressing questions, methodology to modulate the steady-state concentrations of the NADH and NADPH cofactors is needed. Recently, we have developed genetically encoded tools to selectively decrease the NADH/NAD+ and NADPH/NADP+ ratios in live cells that are based on the heterologous expression of native or engineered versions of bacterial H 2O- forming NAD(P)H oxidases. In this proposal, we plan to expand our toolkit by developing a genetically encoded tool for the direct modulation of NADH reductive stress (i.e. increased NADH/NAD+ ratio) (Project 1). Preliminary screening of several bacterial enzymes has furnished promising candidates for driving NADH overproduction in different cellular compartments. The development of compartment-specific tools will enable studies to elucidate how overproduction of reducing equivalents in one cellular compartment is communicated to another and how NADH reductive stress remodels cellular metabolism (Project 2a). Multiple lines of evidence indicate that NAD(P)H-consuming redox cycling agents at low concentrations mildly exhaust antioxidant systems and that the resulting pro-oxidative shift promotes stress resistance and improves heathspan in several model organisms. We are using Drosophila as a model organism, to directly test whether redox modulation in either the oxidative or reductive direction are correlated with stress resistance, healthspan and lifespan (Project 2b). A third goal is to develop variants of our genetically encoded tools that are controlled by small molecules or by light to afford greater spatiotemporal control (Project 3). The latter is especially important as many redox processes crucial for redox signaling or energy metabolism and dysregulated in pathologies, occur rapidly (on an acute time scale). The successful completion of our studies will lead to ...