NAD homeostasis maintained by NMNAT2 supports vesicular glycolysis and fuels fast axonal transport in distal axons of cortical glutamatergic neurons in mice

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.

How neurons maintain their axons long-term: an integrated view of axon biology and pathology

“…Further fundamental aspects of transport regulation remain little understood: (a) coordination of cargo loading/unloading at source/target sites ( Kelliher et al, 2019 ); (b) mechanisms of MT track choice and transport duration (shown to involve phosphorylation of motors; alterations of MTs through posttranslational modification or decoration with MT-binding proteins Karasmanis et al, 2018 ; Monroy et al, 2020 ; Banerjee and Gunawardena, 2023 ; Genova et al 2023 ; Li et al, 2023 ); (c) how transport can overcome physical barriers especially in narrow axons ( Yu et al, 2017 ; Wang et al, 2020 ; images in Bowen et al, 2007 ); (d) Vesicular transport was shown to be fuelled by ‘on-board’ glycolysis ( Figure 2 ; Zala et al, 2013 ; Hinckelmann et al, 2016 ; Yang et al, 2022a ), but how ATP supply is ensured for the transport of other cargoes is not known ( Chamberlain and Sheng, 2019 ).…”

“…Many links relate to failed organelle transport which will be discussed in the organelle section below. Another link relates to failed transport of the NAD + -producing enzyme NMNAT2 which leads to the accumulation of its substrate NMN which, in turn, triggers axonal degeneration mediated by the NAD + -catabolising enzyme SARM1 ( Gilley et al, 2017 ; Hopkins et al, 2021 ; Li et al, 2022 ; Yang et al, 2022a ; Loreto et al, 2023 ; Figure 2C ).…”