A Multisensor Framework Reveals Redox Constraints on Glycolysis in vivo

Genetically encoded biosensors have transformed the study of metabolism, yet measurements of individual metabolites often provide an incomplete view of pathway regulation. Here, we develop a multisensor framework in Caenorhabditis elegans neurons to interpret glycolytic dynamics and redox state in vivo. We combine biosensors for NADH/NAD, fructose-1,6-bisphosphate, lactate, and pyruvate to resolve metabolic responses during hypoxia and redox perturbation. To causally test how redox state modulates glycolysis in vivo, we cell-specifically expressed the NADH-producing enzyme EcSTH and the NADH oxidase LbNOX to bidirectionally tune neuronal NADH/NAD balance. These perturbations revealed that redox modulation is sufficient to constrain or relieve lower glycolytic activity. Elevation of NADH/NAD promoted accumulation of upper glycolytic intermediates while suppressing lower glycolytic responses during energetic stress, consistent with inhibition at the NAD-dependent GAPDH step. Conversely, oxidation of NADH relieved this constraint and shifted metabolite pools consistent with enhanced lower glycolytic activity. Elevated NADH/NAD ratios also impaired synaptic vesicle organization, linking redox-mediated glycolytic inhibition to neuronal function. As a case study for how integrated biosensor approaches can provide semi-quantitative insight into pathway-level metabolic regulation, we genetically perturbed endogenous NADH recycling pathways. These experiments revealed a hierarchical organization of neuronal redox buffering, with lactate dehydrogenase (LDH-1) serving as the dominant route for NAD regeneration during hypoxia and glycerol-3-phosphate dehydrogenase (GPDH-2) providing a secondary compensatory pathway. Graded impairment of NADH recycling resulted in corresponding increases in fructose-1,6-bisphosphate accumulation and synaptic defects, consistent with progressive inhibition of lower glycolysis. Together, these results establish a tractable in vivo system to probe causal relationships between redox state, glycolytic dynamics, and cellular physiology.