Genetic modulation of NAD(P)H homeostasis reveals distinct redox-coupled metabolic vulnerabilities.

An elevated NADH/NAD+ ratio (NADH-reductive stress) is a hallmark of mitochondrial dysfunction, yet how it drives pathologies ranging from primary mitochondrial disorders to ageing remains poorly understood1,2. Here, we show that mitochondrial respiratory chain inhibition in primary human fibroblasts induces NADH-reductive stress that coexists with profound NADPH depletion. Using the Escherichia coli soluble transhydrogenase (EcSTH)3 to model this redox coupling in cells with intact mitochondria, we demonstrate that perturbation of both NAD(H) and NADP(H) redox pairs elicits metabolomic and transcriptomic reprogramming, culminating in proliferation arrest with senescence features. Notably, normalizing the NADH/NAD+ ratio alone while NADPH depletion persists fails to rescue proliferation in EcSTH-expressing primary human cells, unlike in transformed HeLa cells. Stable isotope tracing reveals that primary fibroblasts rely more on NADPH-driven reductive carboxylation than HeLa cells, explaining why NADH/NAD+ normalization alone is insufficient for rescue. To test this NADPH dependency, we develop a constitutively active Staphylococcus aureus NAD kinase (SaNADK) that expands the mammalian NADP(H) pool. In fibroblasts, SaNADK restores reductive carboxylation and de novo pyrimidine biosynthesis, and reverses proliferation arrest and senescence features. Furthermore, hepatic SaNADK expression in aged male mice partially alleviates transcriptional signatures of inflammation and metabolic dysregulation. Together, these findings establish EcSTH and SaNADK as xenotopic tools for dissecting NAD(P)H redox coupling and provide a framework for counteracting ageing-associated metabolic decline.