Using a xenotopic approach to manipulate redox metabolism in a tissue- and compartment-specific manner to study aging

A steady decline in cellular and physiological function is a recognized aspect of the aging process. It is now widely accepted that in humans, aging is the major risk factor for multiple diseases, including cancer, cardiovascular diseases, and neurodegeneration. Thus, the research community has become interested in developing specific methods and strategies to modulate aging-associated metabolic changes — including those in cellular energy metabolism — to better understand the basic mechanisms of aging. If successful, insights from such approaches could lead to an improved healthspan in humans by potentially enhancing resistance to aging- associated diseases. Currently, it is not possible to modulate redox imbalance often associated with mitochondrial dysfunction, which further prevents our ability to assess how it promotes aging-associated metabolic changes. In particular, it is currently not feasible to modulate levels of redox cofactors nicotinamide adenine dinucleotide (NAD) and related nicotinamide adenine dinucleotide phosphate (NADP), key contributors to the cellular redox environment, with tissue- and compartment-specific precision. The main objective of this grant proposal is to develop and validate novel genetically encoded tools that directly modulate both NADH/NAD+ and NADPH/NADP+ ratios when expressed in different tissues and cellular compartments of Drosophila. We will use these tools to test our hypothesis that direct targeting of redox metabolism extends health- and lifespan in a tissue- and organelle specific manner. In Aim 1, we will determine the specific tissues responsible for the beneficial effects of targeting NAD metabolism with a water-forming NADH oxidase LbNOX on lifespan and sleep in Drosophila. We already demonstrated using two transgenic Drosophila lines expressing LbNOX that a sex- dependent pro-oxidative shift (a decrease) in the NADH/NAD+ ratio co-occurs with a pro-reductive shift (an increase) in the NADPH/NADP+ ratio in these flies. Most importantly, we established that cytosolic LbNOX expression can lead to multiple beneficial effects, such as lifespan extension, resistance to oxidative stress, improved neuromuscular function and sleep. In addition, we demonstrated that LbNOX was much more effective for rescuing paraquat-induced oxidative stress when localized to the muscle compared to whole-body or neuronal-specific expression. Building on our preliminary results, we will determine which Drosophila tissues mediate the beneficial effects of LbNOX expression on aging and sleep and whether improved sleep is required for the extension of lifespan. Because our preliminary experiments showed that modulation of NAD+ regeneration led to an increase in NADP(H) levels and NADPH/NADP+ ratio, Aim 2 will focus on addressing whether the anti- aging effects and aging-related signatures of targeting NAD metabolism with LbNOX are mediated by the conversion of NAD+ to NADP+. For this aim, we will explore if ubiquitous and/or tissue-specific beneficial effects of LbNOX in Drosophila depend on NADK (an enzyme which converts NAD+ into NADP+). In summary, the growing arsenal of enzyme-based genetically encoded tools we are developing for tissue- and compartment- specific modulation of redox metabolism will establish a transformative biochemical and genetic discovery pipeline that can be used to systematically identify redox pathways that contribute to the pathobiology of aging. Project Number: 1R21AG101323-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Valentin Cracan (+1 co-PI) | Institution: SCINTILLON INSTITUTE FOR PHOTOBIOLOGY, SAN DIEGO, CA | Award Amount: $501,160 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 CDB-H (90)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11357281