Illuminating NAD⁺ Metabolism in Live Cells and In Vivo Using a Genetically Encoded Fluorescent Sensor

Understanding of NAD⁺ metabolism provides many critical insights into health and diseases, yet highly sensitive and specific detection of NAD⁺ metabolism in live cells and in vivo remains difficult. Here, we present ratiometric, highly responsive genetically encoded fluorescent indicators, FiNad, for monitoring NAD⁺ dynamics in living cells and animals. FiNad sensors cover physiologically relevant NAD⁺ concentrations and sensitively respond to increases and decreases in NAD⁺. Utilizing FiNad, we performed a head-to-head comparison study of common NAD⁺ precursors in various organisms and mapped their biochemical roles in enhancing NAD⁺ levels. Moreover, we showed that increased NAD⁺ synthesis controls morphofunctional changes of activated macrophages, and directly imaged NAD⁺ declines during aging in situ. The broad utility of the FiNad sensors will expand our mechanistic understanding of numerous NAD⁺-associated physiological and pathological processes and facilitate screening for drug or gene candidates that affect uptake, efflux, and metabolism of this important cofactor.