Project summary We are working on new in vivo methods to study the intermediary metabolite nicotinamide adenine dinucleotide (NAD+). NAD+ is critical for cellular metabolism and health, and its decreased steady-state levels have been linked to human pathologies such as neurodegeneration, cardiovascular disease, metabolic syndrome, and cancer. NAD+ concentrations are highly compartmentalized by cell type, subcellular localization, and its protein- bound or free fractions. A lack of methods that can monitor free NAD+ in cells with spatial and temporal information has hindered our learning about the relevant pools, threshold concentrations, and fluctuations that NAD+ may undergo leading to disease onset. This precludes our ability to identify treatments or approaches to intervene before NAD+ levels are misregulated. In particular, there are no genetically-encoded methods for in vivo mitochondrial NAD+ measurements. Free mitochondrial NAD+ is required for cellular respiration, and diminished levels due to genetics, age, or infection can promote reductive metabolism. Thus, to address this gap we will improve the brightness (Aim 1) of a variant of the current NAD+ sensor, which is able to monitor free mitochondrial NAD+ in cells. We will also generate a series of constructs that will be used to test a new approach for improving the dynamic range of fluorescent readout (Aim 2). Together, data from these the aims will identify a feasible method to robustly obtain free mitochondrial NAD+ measurements in vivo.