Glycerol-3-phosphate Biosynthesis Antagonizes NADH Reductive Stress And Alleviates Mitochondrial Disease

The cellular NADH/NAD+ redox balance is fundamental to metabolism.The oxidized form NAD+functions as an electron acceptor in diverse metabolic pathways such as glycolysis,fatty acid oxidation,amino acid degradation,citric acid cycle,serine synthesis,and one carbon metabolism.Electron transport chain(ETC)oxidizes NADH to regenerate NAD+,and transfers electrons to oxygen.ETC inhibition,resulting from either hypoxia or genetic/pharmacological disruption of ETC function,impairs NADH oxidation and elevates the NADH/NAD+ ratio,a condition termed NADH reductive stress.NADH reductive stress causes metabolic derangements and has important physiological and pathophysiological roles.Mitochondrial diseases are characterized by ETC dysfunction and often show metabolic signatures of NADH reductive stress;NAD+regeneration is a therapeutic target for mitochondrial diseases.NADH reductive stress also limits tumor growth under hypoxia.But how cells manage NADH reductive stress remains elusive.To identify ETC-independent endogenous NAD+regeneration pathways,we analyzed NAD+-dependent and NAD(P)+dual-specific dehydrogenases/reductases in human and yeast genomes.After experimental validation in yeast,C.elegans,cancer cells and mice,we reveal glycerol-3-phosphate(Gro3P)biosynthesis from glucose as a conserved NAD+-regeneration pathway under both ETC dysfunction and hypoxia.We find that ETC inhibition or hypoxia rewires glucose metabolism towards Gro3P biosynthesis with concurrent transfer of electrons from NADH to Gro3P.Abolishing Gro3P synthesis impairs proliferation of yeast and shortens lifespan of C.elegans under ETC dysfunction,and sensitizes cancer cells to ETC inhibition and hypoxia both in culture and in xenograft model.Genetic inactivation of Gro3P synthesis in mouse liver exacerbates hepatic NADH reductive stress under complex I inhibition.We find that the Gro3P shuttle,one of the three downstream pathways of Gro3P synthesis,selectively regenerates NAD+under Complex-I inhibition.Abolishing Gro3P shuttle increased cellular sensitivity to complex I inhibition.Surprisingly,enhancing Gro3P synthesis promotes the shuttle activity to alleviate NADH reductive stress and to restore proliferation of complex-I-impaired cells.We futher find that Gro3P synthesis enzymes are expressed at much lower levels in mouse brain and human brain.Augmenting Gro3P synthesis enzymes in cultured neurons promotes Gro3P shuttle activity,alleviates NADH reductive stress and rescues ATP reduction under complex-I inhibition.More importantly,AAV mediated expression of Gro3P synthesis enzyme in mouse brain suppresses neuroinflammation,rescues neurological symptoms and extends lifespan of the Ndufs4-/-mice,a complex I deficiency model.Collectively,our findings reveal the central and conserved function of Gro3P metabolism in coordinating NADH/NAD+redox balance,and implicate its role in cancer biology and mitochondrial disease pathogenesis.Beyond this,we present a novel therapeutic target for mitochondrial diseases.