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The past 20 years of research on axon degeneration has revealed fine details on how NAD biology controls axonal survival. Extensive data demonstrate that the NAD precursor NMN binds to and activates the pro-degenerative enzyme SARM1, so a failure to convert sufficient NMN into NAD leads to toxic NMN accumulation and axon degeneration. This involvement of NMN brings the axon degeneration field to an unexpected overlap with research into ageing and extending healthy lifespan. A decline in NAD levels throughout life, at least in some tissues, is believed to contribute to age-related functional decay and boosting NAD production with supplementation of NMN or other NAD precursors has gained attention as a potential anti-ageing therapy. Recent years have witnessed an influx of NMN-based products and related molecules on the market, sold as food supplements, with many people taking these supplements daily. While several clinical trials are ongoing to check the safety profiles and efficacy of NAD precursors, sufficient data to back their therapeutic use are still lacking. Here, we discuss NMN supplementation, SARM1 and anti-ageing strategies, with an important question in mind: considering that NMN accumulation can lead to axon degeneration, how is this compatible with its beneficial effect in ageing and are there circumstances in which NMN supplementation could become harmful?, Competing Interests: Declaration of interests M.P.C. holds funding jointly provided by AstraZeneca for academic research and consults for Nura Bio, neither of which is directly relevant to this review., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

SARM1 is an inducible TIR-domain NAD + hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD + , which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD + hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD + -related metabolites might activate SARM1. Here, we show the nicotinamide analog 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN, which activates SARM1 and induces SARM1-dependent NAD + depletion, axon degeneration, and neuronal death. In mice, systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to the peripheral nerve induces SARM1-dependent axon degeneration. We identify 2-aminopyridine as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity and suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy., Competing Interests: Declaration of interests A.J.B. and Y.S. have consulted for Disarm Therapeutics. A.D. and J.M. are co-founders, scientific advisory board members, and shareholders of Disarm Therapeutics, a wholly owned subsidiary of Eli Lilly & Company., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The programmed axon degeneration pathway has emerged as an important process contributing to the pathogenesis of several neurological diseases. The most crucial events in this pathway include activation of the central executioner SARM1 and NAD + depletion, which leads to an energetic failure and ultimately axon destruction. Given the prevalence of this pathway, it is not surprising that inhibitory therapies are currently being developed in order to treat multiple neurological diseases with the same therapy. Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of neurological diseases that may also benefit from this therapeutic approach. To evaluate the appropriateness of this strategy, the contribution of the programmed axon degeneration pathway to the pathogenesis of different CMT subtypes is being actively investigated. The subtypes CMT1A, CMT1B and CMT2D are the first to have been examined. Based on the results from these studies and advances in developing therapies to block the programmed axon degeneration pathway, promising therapeutics for CMT are now on the horizon., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD + ) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration., Competing Interests: KK, NT, CP, MA, SP, MZ, NK, SC, ZW, AC, YJ No competing interests declared, (© 2018, Kim et al.)

Axons require the axonal NAD-synthesizing enzyme NMNAT2 to survive. Injury or genetically induced depletion of NMNAT2 triggers axonal degeneration or defective axon growth. We have previously proposed that axonal NMNAT2 primarily promotes axon survival by maintaining low levels of its substrate NMN rather than generating NAD; however, this is still debated. NMN deamidase, a bacterial enzyme, shares NMN-consuming activity with NMNAT2, but not NAD-synthesizing activity, and it delays axon degeneration in primary neuronal cultures. Here we show that NMN deamidase can also delay axon degeneration in zebrafish larvae and in transgenic mice. Like overexpressed NMNATs, NMN deamidase reduces NMN accumulation in injured mouse sciatic nerves and preserves some axons for up to three weeks, even when expressed at a low level. Remarkably, NMN deamidase also rescues axonal outgrowth and perinatal lethality in a dose-dependent manner in mice lacking NMNAT2. These data further support a pro-degenerative effect of accumulating NMN in axons in vivo. The NMN deamidase mouse will be an important tool to further probe the mechanisms underlying Wallerian degeneration and its prevention., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Overexpression of the NAD + biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD + or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD + metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD + synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD + consumption that is central to axon degeneration., Competing Interests: YS: May derive benefit from a licensing agreement with ChromaDex, which did not provide any support for this work. JM: May derive benefit from a licensing agreement with ChromaDex, which did not provide any support for this work. The other authors declare that no competing interests exist.

The Wallenda (Wnd)/dual leucine zipper kinase (DLK)-Jnk pathway is an evolutionarily conserved MAPK signaling pathway that functions during neuronal development and following axonal injury. Improper pathway activation causes defects in axonal guidance and synaptic growth, whereas loss-of-function mutations in pathway components impairs axonal regeneration and degeneration after injury. Regulation of this pathway is in part through the E3 ubiquitin ligase Highwire (Hiw), which targets Wnd/DLK for degradation to limit MAPK signaling. To explore mechanisms controlling Wnd/DLK signaling, we performed a large-scale genetic screen in Drosophila to identify negative regulators of the pathway. Here we describe the identification and characterization of SkpA, a core component of SCF E3 ubiquitin ligases. Mutants in SkpA display synaptic overgrowth and an increase in Jnk signaling, similar to hiw mutants. The combination of hypomorphic alleles of SkpA and hiw leads to enhanced synaptic growth. Mutants in the Wnd-Jnk pathway suppress the overgrowth of SkpA mutants demonstrating that the synaptic overgrowth is due to increased Jnk signaling. These findings support the model that SkpA and the E3 ligase Hiw function as part of an SCF-like complex that attenuates Wnd/DLK signaling. In addition, SkpA, like Hiw, is required for synaptic and axonal responses to injury. Synapses in SkpA mutants are more stable following genetic or traumatic axonal injury, and axon loss is delayed in SkpA mutants after nerve crush. As in highwire mutants, this axonal protection requires Nmnat. Hence, SkpA is a novel negative regulator of the Wnd-Jnk pathway that functions with Hiw to regulate both synaptic development and axonal maintenance., (Copyright © 2014 the authors 0270-6474/14/348398-13$15.00/0.)

Depending upon the stimulus, neuronal cell death can either be triggered from the cell body (soma) or the axon. We investigated the origin of the degeneration signal in amyloid β (Aβ) induced neuronal cell death in cultured in vitro hippocampal neurons. We discovered that Aβ1-42 toxicity-induced axon degeneration precedes cell death in hippocampal neurons. Overexpression of Bcl-xl inhibited both axonal and cell body degeneration in the Aβ-42 treated neurons. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) blocks axon degeneration in a variety of paradigms, but it cannot block neuronal cell body death. Therefore, if the neuronal death signals in Aβ1-42 toxicity originate from degenerating axons, we should be able to block neuronal death by inhibiting axon degeneration. To explore this possibility we over-expressed Nmnat1 in hippocampal neurons. We found that inhibition of axon degeneration in Aβ1-42 treated neurons prevented neuronal cell death. Thus, we conclude that axon degeneration is the key component of Aβ1-42 induced neuronal degeneration, and therapies targeting axonal protection can be important in finding a treatment for Alzheimer's disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.

Abstract: Peripheral axons can re-extend robustly after nerve injury. Soon after a nerve crush regenerating axons grow through the nerve segment distal to the lesion in close proximity to distal axons that are still morphologically and molecularly preserved. Hence, following the progress of regenerating axons necessitates markers that can distinguish between regenerating and degenerating axons. Here, we show that axonal levels of superior cervical ganglion 10 (SCG10) are dynamically regulated after axonal injury and provide an efficient method to label regenerating axons. In contrast to the rapid loss of SCG10 in distal axons (Shin et al., 2012b), we report that SCG10 accumulates in the proximal axons within an hour after injury, leading to a rapid identification of the lesion site. The increase in SCG10 levels is maintained during axon regeneration after nerve crush or nerve repair and allows for more selective labeling of regenerating axons than the commonly used markers growth-associated protein 43 (GAP43) and YFP. SCG10 is preferentially expressed in regenerating sensory axons rather than motor axons in the sciatic nerve. In a mouse model of slow Wallerian degeneration, SCG10 labeling remains selective for regenerating axons and allows for a quantitative analysis of delayed regeneration in this mutant. Taken together, these data demonstrate the utility of SCG10 as an efficient and selective marker of sensory axon regeneration. [Copyright &y& Elsevier]

Abstract: WldS mutation protects axons from degeneration in diverse experimental models of neurological disorders, suggesting that the mutation might act on a key step shared by different axon degeneration pathways. Here we test the hypothesis that WldS protects axons by preventing energy deficiency commonly encountered in many diseases. We subjected compartmentally cultured, mouse cortical axons to energy deprivation with 6mM azide and zero glucose. In wild-type (WT) culture, the treatment, which reduced axon ATP level ([ATP]axon) by 65%, caused immediate axon depolarization followed by gradual free calcium accumulation and subsequent irreversible axon damage. The calcium accumulation resulted from calcium influx partially via L-type voltage-gated calcium channel (L-VGCC). Blocking L-VGCC with nimodipine reduced calcium accumulation and protected axons. Without altering baseline [ATP]axon, the presence of WldS mutation significantly reduced the axon ATP loss and depolarization, restrained the subsequent calcium accumulation, and protected axons against energy deprivation. WldS neurons possessed higher than normal nicotinamide mononucleotide adenylyltransferase (NMNAT) activity. The intrinsic WldS NMNAT activity was required for the WldS -mediated energy preservation and axon protection during but not prior to energy deprivation. NMNAT catalyzes the reversible reaction that produces nicotinamide adenine dinucleotide (NAD) from nicotinamide mononucleotide (NMN). Interestingly, preventing the production of NAD from NMN with FK866 increased [ATP]axon and protected axons from energy deprivation. These results indicate that the WldS mutation depends on its intrinsic WldS NMNAT activity and the subsequent increase in axon ATP but not NAD to protect axons, implicating a novel role of WldS NMNAT in axon bioenergetics and protection. [Copyright &y& Elsevier]

Abstract: Dendrites can be maintained for extended periods of time after they initially establish coverage of their receptive field. The long-term maintenance of dendrites underlies synaptic connectivity, but how neurons establish and then maintain their dendritic arborization patterns throughout development is not well understood. Here, we show that the NAD synthase Nicotinamide mononucleotide adenylyltransferase (Nmnat) is cell-autonomously required for maintaining type-specific dendritic coverage of Drosophila dendritic arborization (da) sensory neurons. In nmnat heterozygous mutants, dendritic arborization patterns of class IV da neurons are properly established before increased retraction and decreased growth of terminal branches lead to progressive defects in dendritic coverage during later stages of development. Although sensory axons are largely intact in nmnat heterozygotes, complete loss of nmnat function causes severe axonal degeneration, demonstrating differential requirements for nmnat dosage in the maintenance of dendritic arborization patterns and axonal integrity. Overexpression of Nmnat suppresses dendrite maintenance defects associated with loss of the tumor suppressor kinase Warts (Wts), providing evidence that Nmnat, in addition to its neuroprotective role in axons, can function as a protective factor against progressive dendritic loss. Moreover, motor neurons deficient for nmnat show progressive defects in both dendrites and axons. Our studies reveal an essential role for endogenous Nmnat function in the maintenance of both axonal and dendritic integrity and present evidence of a broad neuroprotective role for Nmnat in the central nervous system. [Copyright &y& Elsevier]

Abstract: Axon degeneration has been proposed to be a new therapeutic target for neurodegenerative diseases, because it usually occurs earlier than neuronal cell body death with a distinct active program from apoptosis and necrosis. Overexpression of WldS or Nmnats (nicotinamide mononucleotide adenylytransferase, EC2.7.7.1) has been demonstrated to delay axon degeneration initiated by various insults. NAD synthesis activity of WldS and Nmnats was shown to be responsible for their axon-protective function. The mitochondrial Nmnat3 and cytoplasm-localized mutants of WldS and Nmnat1 have similar or even stronger effect than WldS to delay axon degeneration, which suggest that increased mitochondrial or local NAD synthesis might contribute to the protective function of WldS and Nmnats. Further studies show NAD synthesis pathway and ubiquitin proteasome system play important roles in delaying axon degeneration. WldS mice are resistant to a variety of neurodegenerative diseases, but the role of Nmnats in neurodegenerative diseases are largely unknown. NAD plays key roles in energy metabolism, mitochondrial functions and aging, and is suggested to be involved in neuron degenerative diseases. Future studies to identify the upstream factors inducing NAD depletion and the downstream NAD effectors responsible for the axon-protective function will provide more meaningful insights into the molecular mechanisms of axon degeneration in neurodegenerative diseases. [Copyright &y& Elsevier]

Axonal degeneration is a prominent feature of many neurological disorders that are associated with mitochondrial dysfunction, including Parkinson's disease, motor neuron disease, and inherited peripheral neuropathies. Studies of the Wlds mutant mouse, which undergoes delayed Wallerian degeneration in response to axonal injury, suggest that axonal degeneration is an active process. Wlds mice also have slower axonal degeneration and disease progression in numerous models of neurodegenerative disease. The Wlds mutation results in the production of a chimeric protein that contains the full-length coding sequence of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), which alone is sufficient for axonal protection in vitro. To test the effects of increased Nmnat expression on axonal degeneration induced by mitochondrial dysfunction, we examined dorsal root ganglion (DRG) neurons treated with rotenone. Rotenone induced profound axonal degeneration in DRG neurons; however, this degeneration was delayed by expression of Nmnat. Nmnat-mediated protection resulted in decreased axonal accumulation and sensitivity to reactive oxygen species (ROS) but did not affect the change in the rate of rotenone-induced loss in neuronal ATP. Nmnat also prevented axonal degeneration caused by exposure to exogenous oxidants and reduced the level of axonal ROS after treatment with vincristine, further supporting the idea that Nmnat promotes axonal protection by mitigating the effects of ROS. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor that mediates numerous biological processes in all living cells. Multiple NAD+ biosynthetic enzymes and NAD+-consuming enzymes are involved in neuroprotection and axon regeneration. The nematode Caenorhabditis elegans has served as a model to study the neuronal role of NAD+ because many molecular components regulating NAD+ are highly conserved. This review focuses on recent findings using C. elegans models of neuronal damage pertaining to the neuronal functions of NAD+ and its precursors, including a neuroprotective role against excitotoxicity and axon degeneration as well as an inhibitory role in axon regeneration. The regulation of NAD+ levels could be a promising therapeutic strategy to counter many neurodegenerative diseases, as well as neurotoxin-induced and traumatic neuronal damage. [ABSTRACT FROM AUTHOR]