NADPH plays a central role in the protection from oxidative stress as it fuels the glutathione and thioredoxin antioxidant systems. Drops in NAD+ levels with aging likely lead to declines in (free) [NADPH] that drive the loss of oxidative stress resistance in aged individuals that frequently results in the development of aging-related diseases. This has been difficult to study in the past due to inadequate tools to measure [NADPH] levels in vivo. Recently a new targetable fluorescent protein indicator iNAP has been developed to bypass this bottleneck in the NADPH research field. In this study the kinetics of the losses of NAD+, NADPH, GSH/GSSG, and ATP with aging in whole worm and isolated mitochondrial extracts will be measured. In addition it will be determined the extent to which increasing or decreasing NADPH levels in the cytoplasm or mitochondria alters lifespan and the loss of NAD+, mitochondrial electron transport chain function, and ATP levels with aging. It will also be determined if supplementation of the lifespan-extending, potentially NADPH generating metabolic intermediates malate, isocitrate, or gluconate increase [NADPH] as a mechanism of lifespan extension. This research program is novel because it combines both genetic and metabolic strategies for increasing NADPH levels to delay aging- induced redox imbalance and extend lifespan. The researchers who make up the project team for this three year investigation consist of the PI, one second year Ph.D. student, one third year Ph.D. student, one master?s student and two undergraduate students. The project will provide an important component of the graduate training program for the graduate students. The findings will provide important insight into three important questions at the intersections of the fields of redox regulation, metabolism and aging. First, the kinetics of the changes in cytoplasmic and mitochondrial NADPH levels with aging will be measured and compared to the rates of change of other markers of aging. Second, the major enzymes regulating NADPH levels and lifespan will be identified through the use of gene knockdown and C. elegans transgenics. And third, the importance of tissue- specific and organelle-specific changes in NADPH levels on lifespan will be determined. The results from these experiments will provide a unique understanding as to how NADPH levels regulate lifespan and provide the rationale for the development of therapies to increase NADPH levels to prevent and treat human aging-related disorders.
Aging and aging-related disorders are characterized by increased oxidative stress that drive pathology. Decreases in the important cellular cofactors NAD+ and NADPH are hypothesized to be at least partially responsible for the increased oxidative stress that occurs with aging, but conclusive evidence implicating NADPH in the regulation of the aging process is lacking. Therefore, using the short-lived model worm C. elegans and novel methods for measuring subcellular compartment-specific NADPH levels in live animals, we will determine the extent that NADPH levels regulate lifespan to lay the groundwork for future therapies aimed at raising NADPH levels for the treatment of human aging-related disorders.
