Genetic modulation of NAD(P)H homeostasis reveals distinct redox-coupled metabolic vulnerabilities [dataset 1]

ABSTRACT: 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 aging remains poorly understood. Here, we show that mitochondrial electron transport chain (ETC) inhibition in primary human fibroblasts induces NADH-reductive stress that coexists with profound NADPH depletion. Using the E. coli soluble transhydrogenase (EcSTH) to model this redox coupling in cells with intact mitochondria, we demonstrate that simultaneous perturbation of both NAD(H) and NADP(H) redox pairs elicits coordinated metabolomic and transcriptomic reprogramming, culminating in proliferation arrest with senescence features. Notably, normalizing the NADH/NAD⁺ ratio alone, while NADPH depletion persisted, failed to rescue proliferation in EcSTH-expressing primary human cells, in contrast to transformed HeLa cells. Stable-isotope tracing revealed that primary fibroblasts rely more on NADPH-driven reductive carboxylation than HeLa cells, explaining why normalization of the NADH/NAD⁺ ratio alone is insufficient to rescue proliferation of primary cells. To directly test this NADPH dependency, we developed SaNADK, a constitutively active Staphylococcus aureus NAD kinase that expands the mammalian NADP(H) pool. In fibroblasts, SaNADK restored reductive carboxylation, rescued de novo pyrimidine biosynthesis, reversed proliferation arrest, delayed replicative senescence, and enhanced oxidative stress resistance. In aged male mice, hepatic SaNADK expression partially alleviated transcriptional profiles 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 developing interventions that counteract aging-associated metabolic decline.

ORGANISM(S): Homo sapiens

PROVIDER: GSE343499 | GEO | 2026/09/15

REPOSITORIES: GEO