Defining and targeting the compartmentalization of redox metabolism in aging using novel genetically encoded tools

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Abstract Multiple lines of evidence designate mitochondrial dysfunction and related cellular reduction-oxidation (redox) imbalance as one of the hallmarks of aging. The redox cofactor nicotinamide adenine dinucleotide (NAD+) plays a central role in cellular energy metabolism, and it is an essential cofactor for supporting mitochondrial oxidative phosphorylation (OXPHOS). Numerous studies have implicated lowering cellular NAD+ levels in aging- associated metabolic changes, but its precise role at present remains contentious. This is mostly because NAD+ and its phosphorylated form NADP+ are substrates in hundreds of redox reactions which are often times performed by paralogous enzymes found in different cellular compartments. Compartmentalization of cellular metabolism is one of the most fundamental properties of complex eukaryotic life and in order to support healthy cellular functions many metabolic pathways are spatially and temporally compartmentalized. To our knowledge, there have not been any comprehensive studies of the compartment-specific redox metabolism of the aging process, and the NAD+ cofactor is viewed only as a substrate for “NAD+-consuming” or signaling enzymes which are involved in epigenetic modifications (sirtuins) and DNA repair (poly(ADP-ribose) polymerase), widely ignoring its role in redox reactions. We recently developed genetically encoded tools which can be used to increase the NAD+-to-NADH or NADP+-to-NADPH ratios in the cytosol or mitochondria in mammalian cells. In this application we propose to study the role of redox compartmentalization in aging by expressing our tools in different cellular compartments (nucleus, cytosol, mitochondria, endoplasmic reticulum and peroxisomes) of both human primary fibroblasts and the multicellular nematode C. elegans. In both model systems we will explore how an increase in the NAD+-to-NADH or NADP+-to-NADPH ratios in different compartments tracks with cellular senescence, stress resistance and lifespan. Our current approach, for the first time, will allow us to identify both NAD- and NADP-coupled redox pathways or mechanisms which play key roles in the regulation of aging.