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Abstract

Axonal transport, an ATP demanding process, plays a critical role in maintaining axonal and neuronal health. ATP in neurons is synthesized by glycolysis and mitochondrial respiration, both driven by proper NAD+/NADH redox potentials. NMNAT2 is the major neuronal NAD+ synthesizing enzyme and is a key axonal maintenance factor. Here, we show that NMNAT2 co-migrates with fast vesicular cargos in axons and is required for fast axonal transport in the distal axons of cortical neurons. Using SoNar sensor imaging to detect axonal NAD+ and NADH, we show that NMNAT2 is critical in maintaining NAD+/NADH potentials in distal axons. With Syn-ATP sensor imaging to detect synaptic vesicle ATP levels (sv-ATP), we demonstrate that glycolysis is the major provider of sv-ATP and NMNAT2 deletion significantly reduces sv-ATP levels. NAD+ supplementation to NMNAT2 KO neurons restores sv-ATP levels and fast axonal transports in a glycolysis-dependent manner. Together, these data show that NMNAT2 maintains the local NAD+/NADH redox potential and sustains on-board glycolysis to meet the bioenergetic demands of fast vesicular transport in distal axons. Intriguingly, mitochondrial respiration contributes significantly to sv-ATP levels in NMNAT2 KO axons. This finding suggests that NMNAT2 deletion induces metabolic plasticity by engaging mitochondria. Surprisingly, supplying NMN, the substrate for NMNAT2 in NAD+ synthesis, restores sv-ATP and axonal transport in NMNAT2 KO axons with an efficacy similar to NAD+. The restoration of NMNAT2 KO distal axonal sv-ATP levels and vesicle transport by NMN requires mitochondrial respiration, while the rescue by NAD+ is independent of mitochondrial respiration. Based on these findings, we hypothesize that NMN rescues glycolysis to restore axonal transport in NMNAT2 KO axons by taking advantage of the compensatory ATP-generating metabolic pathways triggered by NMNAT2 loss.