Drosophila NMNAT Maintains Neural Integrity Independent of Its NAD Synthesis Activity

Wallerian degeneration refers to a loss of the distal part of an axon after nerve injury. Wallerian degeneration slow (Wld^s) mice overexpress a chimeric protein containing the NAD synthase NMNAT (nicotinamide mononucleotide adenylyltransferase 1) and exhibit a delay in axonal degeneration. Currently, conflicting evidence raises questions as to whether NMNAT is the protecting factor and whether its enzymatic activity is required for such a possible function. Importantly, the link between nmnat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. Here we use the visual system of Drosophila as a model system to address these issues. We have isolated the first nmnat mutations in a multicellular organism in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmnat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmnat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmnat. Our data indicate an NAD-independent requirement of NMNAT for maintaining neuronal integrity that can be exploited to protect neurons from neuronal activity-induced degeneration by overexpression of the protein.     

MEK Inhibitor U0126 Reverses Protection of Axons from Wallerian Degeneration Independently of MEK–ERK Signaling

Wallerian degeneration is delayed when sufficient levels of proteins with NMNAT activity are maintained within axons after injury. This has been proposed to form the basis of ''slow Wallerian degeneration'' (Wld ^S), a neuroprotective phenotype conferred by an aberrant fusion protein, Wld^S. Proteasome inhibition also delays Wallerian degeneration, although much less robustly, with stabilization of NMNAT2 likely to play a key role in this mechanism. The pan-MEK inhibitor U0126 has previously been shown to reverse the axon-protective effects of proteasome inhibition, suggesting that MEK-ERK signaling plays a role in delayed Wallerian degeneration, in addition to its established role in promoting neuronal survival. Here we show that whilst U0126 can also reverse Wld^S-mediated axon protection, more specific inhibitors of MEK1/2 and MEK5, PD184352 and BIX02189, have no significant effect on the delay to Wallerian degeneration in either situation, whether used alone or in combination. This suggests that an off-target effect of U0126 is responsible for reversion of the axon protective effects of Wld^S expression or proteasome inhibition, rather than inhibition of MEK1/2-ERK1/2 or MEK5-ERK5 signaling. Importantly, this off-target effect does not appear to result in alterations in the stabilities of either Wld^S or NMNAT2.     

Reducing expression of NAD+ synthesizing enzyme NMNAT1 does not affect the rate of Wallerian degeneration

NAD(+) synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein Wld(S), which delays axon degeneration by 10-fold. NMNAT1 activity is necessary but not sufficient for Wld(S) neuroprotection in mice and 70 amino acids at the N-terminus of Wld(S), derived from polyubiquitination factor Ube4b, enhance axon protection by NMNAT1. NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed in vitro and partially in Drosophila. However, the role of endogenous NMNAT1 in normal axon maintenance and in Wallerian degeneration has not been elucidated yet. To address this question we disrupted the Nmnat1 locus by gene targeting. Homozygous Nmnat1 knockout mice do not survive to birth, indicating that extranuclear NMNAT isoforms cannot compensate for its loss. Heterozygous Nmnat1 knockout mice develop normally and do not show spontaneous neurodegeneration or axon pathology. Wallerian degeneration after sciatic nerve lesion is neither accelerated nor delayed in these mice, consistent with the proposal that other endogenous NMNAT isoforms play a principal role in Wallerian degeneration.     

Simultaneous Single-Sample Determination of NMNAT Isozyme Activities in Mouse Tissues

A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (Wld^S) mouse. Wld^S is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as Wld^S.     

Chaperoning the synapse—NMNAT protects Bruchpilot from crashing

Maintaining active zone structure is crucial for synaptic function. In this issue of EMBO reports, NMNAT is shown to act as a chaperone that protects the active zone structural protein Bruchpilot from degradation.EMBO reports (2013) 14 1, 87–94 doi:10.1038/embor.2012.181Synapses perform several tasks independently from the cell body of the neuron, including synaptic vesicle recycling through endocytosis or local protein maturation and degradation. Failure to regulate protein function locally is detrimental to the nervous system as evidenced by neuronal dysfunctions that arise as a consequence of synaptic ageing. This relative synaptic autonomy comes with a need for mechanisms that ensure correct protein (re)folding, and there is accumulating evidence that key chap-erones have a central role in the regulation and maintenance of synaptic structural integrity and function [1]. Work by Grace Zhai''s group, published in this issue of EMBO reports, demonstrates a key role of the Drosophila nicotinamide mononucleotide adenylyltransferase (NMNAT) chaperone in the protection of active zone components against activity-induced degeneration (Fig 1; [2]).Open in a separate windowFigure 1Results reported by Zang and colleagues [2] reveal a specific role of nicotinamide mononucleotide adenylyltransferase (NMNAT) in preserving active zone structure against use-dependent decline. This protection is exerted by direct interaction with BRP and protection of this key structural protein against ubiquitination and subsequent degradation. BRP, Bruchpilot; Ub, ubiquitin.Active zones, the specialized sites for neurotransmitter release at presynaptic terminals, are characterized by a dense protein network called the cytomatrix at the active zone (CAZ). The protein machinery of the CAZ is responsible for efficient synaptic vesicle tethering, docking and fusion with the presynaptic membrane and, thus, for reliable signal transmission from the neuron to the postsynaptic cell. Clearly, proteins in the CAZ are tightly regulated, especially in response to external cues such as synaptic activity [3,4]. Yet, this particularly crowded protein environment might be favourable for the formation of non-functional—and sometimes toxic—protein aggregates. Chaperones that act at the synapse reduce the probability of crucial protein aggregation by preventing and reverting these inappropriate interactions, which happen as a result of environmental stress.One of these chaperones, the Drosophila neuroprotective NMNAT, was identified in a genetic screen for factors involved in synapse function [5]. Its chaperone activity was later confirmed by using in vitro and in vivo protein folding assays [6]. NMNAT null mutants show severe and early onset neurodegeneration, whereas neurodevelopment does not seem to be strongly affected. Interestingly, degeneration of photoreceptors lacking NMNAT can be significantly attenuated by limiting synaptic activity, either by rearing flies in the dark or by introducing the no receptor potential A (norpA) mutation that blocks phototransduction [5]. These results indicate that NMNAT protects adult neurons from activity-induced degeneration.In this issue of EMBO reports, Zang and colleagues report a role for NMNAT at the synapse. They observed that loss or reduced levels of NMNAT leads to a concomitant loss of several synaptic markers including cysteine-string protein (CSP), synaptotagmin and the active zone structural protein Bruchpilot (BRP). Remarkably, BRP was the only one of these proteins found to co-immunoprecipitate with NMNAT from brain lysates. Both proteins show approximately 50% co-localization at the neuromuscular junction when imaged by 3D-SIM^™ super-resolution microscopy, suggesting that NMNAT might act directly as a chaperone for maintaining a functional BRP conformation.Consistent with a protective role of NMNAT against BRP degradation, RNA interference-mediated NMNAT knockdown leads to BRP ubiquitination, whereas this modification was not detected in control brain lysates. Given the involvement of the ubiquitin proteasome pathway in regulating synaptic development and function [1], the authors tested the effect of the proteasome inhibitor MG-132 on BRP ubiquitination. They observed an increased level of BRP ubiquitination in wild-type flies fed with this drug, suggesting a role for the proteasome in the clearance of ubiquitinated BRP. By contrast, overexpression of NMNAT reduces the level of BRP ubiquitination both in the absence and the presence of MG-132, providing further evidence for the protective role of this chaperone against ubiquitination of BRP (Fig 1).a key role of the […] nicotinamide mononucleotide adenylyltransferase (NMNAT) chaperone in the protection of active zone components against activity-induced degenerationBRP is a cytoskeletal-like protein that is an integral component of T-bars—electron-dense structures that project from the presynaptic membrane and around which synaptic vesicles cluster. In agreement with a protective role of NMNAT against BRP ubiquitination, reduced levels of this chaperone give rise to a marked decrease in T-bar size in an age-dependent manner (Fig 1). Active zones are known to show dynamic changes in response to synaptic activity, and NMNAT was previously reported to protect photoreceptors against activity-induced degeneration [5]. The authors thus tested the effect of minimizing photoreceptor activity on active zone structure by keeping flies in the dark or inhibiting phototransduction by means of the norpA mutation. Both manipulations largely reversed the effect of NMNAT knockdown on T-bar size. Absence of light exposure also significantly reduced the amount of BRP that co-immunoprecipitates with NMNAT, indicating that neuronal activity regulates NMNAT–BRP interaction. Further experiments are needed to examine whether there is a positive correlation between synaptic activity and BRP ubiquitination levels, and whether NMNAT can indeed keep T-bar structure intact by protecting BRP against this modification under conditions of high synaptic activity.Finally, the study shows that reduced NMNAT levels not only caused a loss of BRP from the synapse but also a specific mislocalization of this protein to the cell body, where it accumulates in clusters together with the remaining NMNAT protein. Under these conditions BRP co-immunoprecipitated with the stress-induced Hsp70, a chaperone classically used as a marker for protein aggregation. It is still unclear whether these BRP clusters form as a result of defective anterograde trafficking and/or of enhanced retrograde transport of BRP. In the absence of light stimulation T-bars are properly assembled in nmnat null photoreceptors, but at this stage a role of NMNAT in regulating the axonal transport of BRP under conditions of normal synaptic activity cannot be excluded. Noticeably, two independent recent reports show involvement of NMNAT in mitochondrial mobility [7,8].As BRP and NMNAT co-localize and interact with one another, the simplest model that accounts for all the observations by Zang et al is that NMNAT directly prevents activity-induced ubiquitination of BRP and subsequent degradation. Yet, as its name indicates, this chaperone is an essential enzyme in NAD synthesis. It was previously shown by the Bellen lab that mutant versions of NMNAT, impaired for NAD production, rescue photoreceptor degeneration caused by loss of NMNAT [5]. This strongly suggests that NAD production is not required for stabilization of BRP but this might need further scrutiny [9].…reduced levels of this chaperone [NMNAT] give rise to a marked decrease in T-bar sizeWhile providing further insights into the role of NMNAT at the active zone in Drosophila, the paper by Zang et al might also have important implications for neurodegeneration in mammals. When ectopically expressed in mice, Nmnat has a protective role against Wallerian degeneration, that is, synapse and axon degeneration that rapidly occurs distal from an axonal wound in wild-type animals. This process is significantly delayed in mice overexpressing a chimaeric protein consisting of the amino-terminal 70 residues of the ubiquitination factor E4B (Ube4b) fused through a linker to Nmnat1, known as the Wallerian degeneration slow (Wld^s) protein. Conversely, mutations in the human NMNAT1 gene were characterized in several families with Leber congenital amaurosis—a severe, early-onset neurodegenerative disease of the retina [10,11,12,13]. As Wld^s or Nmnat1 overexpression protects axons from degeneration in various disease models [9], Nmnat1 emerges as a promising candidate for developing protective strategies against axonal degeneration in peripheral neuropathies such as amyotrophic lateral sclerosis but also in glaucoma, AIDS and other diseases [9].     

Diapause Formation and Downregulation of Insulin-Like Signaling via DAF-16/FOXO Delays Axonal Degeneration and Neuronal Loss

Axonal degeneration is a key event in the pathogenesis of neurodegenerative conditions. We show here that mec-4d triggered axonal degeneration of Caenorhabditis elegans neurons and mammalian axons share mechanistical similarities, as both are rescued by inhibition of calcium increase, mitochondrial dysfunction, and NMNAT overexpression. We then explore whether reactive oxygen species (ROS) participate in axonal degeneration and neuronal demise. C. elegans dauers have enhanced anti-ROS systems, and dauer mec-4d worms are completely protected from axonal degeneration and neuronal loss. Mechanistically, downregulation of the Insulin/IGF-1-like signaling (IIS) pathway protects neurons from degenerating in a DAF-16/FOXO–dependent manner and is related to superoxide dismutase and catalase-increased expression. Caloric restriction and systemic antioxidant treatment, which decrease oxidative damage, protect C. elegans axons from mec-4d-mediated degeneration and delay Wallerian degeneration in mice. In summary, we show that the IIS pathway is essential in maintaining neuronal homeostasis under pro-degenerative stimuli and identify ROS as a key intermediate of neuronal degeneration in vivo. Since axonal degeneration represents an early pathological event in neurodegeneration, our work identifies potential targets for therapeutic intervention in several conditions characterized by axonal loss and functional impairment.     

The Wallerian degeneration slow (Wld(s)) gene does not attenuate disease in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by loss of spinal α-motor neurons, resulting in the paralysis of skeletal muscle. SMA is caused by deficiency of survival motor neuron (SMN) protein levels. Recent evidence has highlighted an axon-specific role for SMN protein, raising the possibility that axon degeneration may be an early event in SMA pathogenesis. The Wallerian degeneration slow (Wld^s) gene is a spontaneous dominant mutation in mice that delays axon degeneration by approximately 2-3 weeks. We set out to examine the effect of Wld^s on the phenotype of a mouse model of SMA. We found that Wld^s does not alter the SMA phenotype, indicating that Wallerian degeneration does not directly contribute to the pathogenesis of SMA development.     

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity‐induced ubiquitin–proteasome‐mediated degradation. NMNAT localizes to the peri‐active zone and interacts biochemically with BRP in an activity‐dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein–protein interaction.     

NAD and axon degeneration: From the Wlds gene to neurochemistry

Neurodegenerative diseases have become a global issue due to the aging population. These disorders affect a vast patient population and represent a huge area of unmet therapeutic need. Axon degeneration is a common pathological character of those neurodegenerative diseases. It results in the loss of communication between neurons. Two decades ago, the Wallerian degeneration slow (Wlds) mouse strain was identified, in which the degeneration of transected axons is delayed. The phenotype is attributed to the overexpression of a chimeric protein Wlds which contains a short fragment of the ubiquitin assembly protein UFD2 and the full-length nicotinamide adenine dinucleotide (NAD) synthetic enzyme Nicotinamide mononucleotide adenylyl-transferase-1 (Nmnat-1). However, the underlying molecular mechanism remains largely unknown. Recently, it''s reported by independent researchers that the full length coding sequence of mouse Nmnat-1 could mimic the axonal protective effect of the Wlds gene when overexpressed in primary neural cultures. Together with a significant number of subsequential reports, this finding highlighted the substantial role of nicotinamide adenine dinucleotide (NAD) in the process of axon degeneration. Here we reviewed the history of axon degeneration research from a neurochemical standpoint and discuss the potential involvement of NAD synthesis, NAD consumption and NAD-dependent proteins and small molecules in axon degeneration.Key words: axon degeneration, Wallerian degeneration, Wlds, NAD, UPS, neurodegenerative diseases     

A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration

NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld^S prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld^S protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.Axon degeneration in disease shares features with the progressive breakdown of the distal segment of severed axons as described by Augustus Waller in 1850 and named Wallerian degeneration.¹ The serendipitous discovery of Wallerian degeneration slow (Wld^S) mice, where transected axons survive 10 times longer than in wild types (WTs),² suggested that axon degeneration is a regulated process, akin to apoptosis of the cell bodies but distinct in molecular terms.^3,4 This process appears conserved in rats, flies, zebrafish and humans.^{5, 6, 7, 8} Wld^S blocks axon degeneration in some disease models, indicating a mechanistic similarity.³ Therefore understanding the pathway it influences is an excellent route towards novel therapeutic strategies.Wld^S is a modified nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) enzyme, whose N-terminal extension partially relocates NMNAT1 from nuclei to axons, conferring gain of function.^9,10 In mammals, three NMNAT isoforms, nuclear NMNAT1, cytoplasmic NMNAT2 and mitochondrial NMNAT3, catalyse nicotinamide adenine dinucleotide (NAD) synthesis from nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP; Figure 1a).^11,12 Several reports indicate Wld^S protects injured axons by maintaining axonal NMNAT activity.^{13, 14, 15} In WT injured axons, without Wld^S, NMNAT activity falls when the labile, endogenous axonal isoform, NMNAT2, is no longer transported from cell bodies.¹⁶ NMNAT2 is required for axon maintenance¹⁶ and for axon growth in vivo and in vitro,^17,18 and modulation of its stability by palmitoylation¹⁹ or ubiquitin-dependent processes both in mice or when ectopically expressed in Drosophila^{19, 20, 21} has a corresponding effect on axon survival.Open in a separate windowFigure 1FK866 acts within axons to delay degeneration after injury. (a) The salvage pathway of NAD biosynthesis from nicotinamide (Nam) and nicotinic acid (Na). Only NAD biosynthesis from Nam is sensitive to FK866, which potently inhibits NAMPT while having no effect on nicotinic acid phosphoribosyltransferase (NaPRT).²⁹ The reaction catalysed by bacterial NMN deamidase is also shown. (b) SCG explants were treated with 100 nM FK866 for the indicated times, and then the whole explants (top panel) or the cell bodies (bottom left panel) and neurite fractions (bottom right panel) were separately collected. NAD was determined with an HPLC-based method (see Materials and Methods; n=3, mean and S.D. shown). (c) SCG neurites untreated (top panels) or treated with 100 nM FK866 the day before transection (bottom panels) and imaged after transection at the indicated time points. (d) SCG explants were treated with 100 nM FK866 1 day before or at the indicated times after cutting their neurites. Degeneration index was calculated from three fields in 2–4 independent experiments. The effect of treatment is highly significant when the drug is preincubated or added at 0–4 h after cut (mean ±S.E.M., n=6–12, one-way ANOVA followed by Bonferroni''s post-hoc test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with untreated)Wld^S partially colocalizes with mitochondria^14,22 and was shown to increase mitochondria motility and Ca²⁺-buffering capacity.²³ Inhibiting mitochondrial permeability transition pore protects degenerating axons.²⁴ However, Wld^S is protective in axons devoid of mitochondria,⁸ and targeting a cytosolic variant of NMNAT2 to mitochondria abolished its protective effect,¹⁹ suggesting a late mitochondrial involvement in Wallerian degeneration.Despite the importance of NMNAT activity in axon survival and degeneration, the molecular players remain elusive. Although NMNAT activity is required for protection,¹³ the hypothesis that increased NAD levels are responsible^25,26 does not fit some data.^27,28While further investigating the role of NAD, we found that blocking nicotinamide phosphoribosyltransferase (NAMPT, the enzyme preceding NMNAT, Figure 1a), was surprisingly axon-protective despite lowering NAD. NAMPT catalyses the synthesis of NMNAT-substrate NMN, the rate-limiting step in the NAD salvage pathway from nicotinamide (Nam) (Figure 1a). Here, we show that NMN accumulates after axon injury, and we provide genetic and pharmacological evidence supporting a role for this NMN increase in axon degeneration when NMNAT2 is depleted. We reveal an unexpected new direction for research into the degenerative mechanism, a novel class of protective proteins and new players in an axon-degeneration pathway sensitive to drugs under development for cancer.     

Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants

Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD⁺ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD⁺ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome.     

The chemical biology of NAD+ regulation in axon degeneration

During axon degeneration, NAD⁺ levels are largely controlled by two enzymes: nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha and toll interleukin motif containing protein 1 (SARM1). NMNAT2, which catalyzes the formation of NAD⁺ from NMN and ATP, is actively degraded leading to decreased NAD⁺ levels. SARM1 activity further decreases the concentration of NAD⁺ by catalyzing its hydrolysis to form nicotinamide and a mixture of ADPR and cADPR. Notably, SARM1 knockout mice show decreased neurodegeneration in animal models of axon degeneration, highlighting the therapeutic potential of targeting this novel NAD⁺ hydrolase. This review discusses recent advances in the SARM1 field, including SARM1 structure, regulation, and catalysis as well as the identification of the first SARM1 inhibitors.     

Identification of Palmitoyltransferase and Thioesterase Enzymes That Control the Subcellular Localization of Axon Survival Factor Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2)

The NAD-synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is a critical survival factor for axons and its constant supply from neuronal cell bodies into axons is required for axon survival in primary culture neurites and axon extension in vivo. Recently, we showed that palmitoylation is necessary to target NMNAT2 to post-Golgi vesicles, thereby influencing its protein turnover and axon protective capacity. Here we find that NMNAT2 is a substrate for cytosolic thioesterases APT1 and APT2 and that palmitoylation/depalmitoylation dynamics are on a time scale similar to its short half-life. Interestingly, however, depalmitoylation does not release NMNAT2 from membranes. The mechanism of palmitoylation-independent membrane attachment appears to be mediated by the same minimal domain required for palmitoylation itself. Furthermore, we identify several zDHHC palmitoyltransferases that influence NMNAT2 palmitoylation and subcellular localization, among which a role for zDHHC17 (HIP14) in neuronal NMNAT2 palmitoylation is best supported by our data. These findings shed light on the enzymatic regulation of NMNAT2 palmitoylation and highlight individual thioesterases and palmitoyltransferases as potential targets to modulate NMNAT2-dependent axon survival.     

Endogenous Nmnat2 Is an Essential Survival Factor for Maintenance of Healthy Axons

The molecular triggers for axon degeneration remain unknown. We identify endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnat1 and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured Wld ^S neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that Wld^S does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. We conclude that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and propose that, when present, the more long-lived, functionally related Wld^S protein substitutes for Nmnat2 loss after axon injury. Endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders.     

SARM1 signaling mechanisms in the injured nervous system

Axon degeneration is a prominent feature of the injured nervous system, occurs across neurological diseases, and drives functional loss in neural circuits. We have seen a paradigm shift in the last decade with the realization that injured axons are capable of actively driving their own destruction through the sterile-alpha and TIR motif containing 1 (SARM1) protein. Early studies of Wallerian degeneration highlighted a central role for NAD⁺ metabolites in axon survival, and this association has grown even stronger in recent years with a deeper understanding of SARM1 biology. Here, we review our current knowledge of SARM1 function in vivo and our evolving understanding of its complex architecture and regulation by injury-dependent changes in the local metabolic environment. The field is converging on a model whereby SARM1 acts as a sensor for metabolic changes that occur after injury and then drives catastrophic NAD⁺ loss to promote degeneration. However, a number of observations suggest that SARM1 biology is more complicated, and there remains much to learn about how SARM1 governs nervous system responses to injury or disease.     

Impact of Genetic Reduction of NMNAT2 on Chemotherapy-Induced Losses in Cell Viability In Vitro and Peripheral Neuropathy In Vivo

Nicotinamide mononucleotide adenylyl transferases (NMNATs) are essential neuronal maintenance factors postulated to preserve neuronal function and protect against axonal degeneration in various neurodegenerative disease states. We used in vitro and in vivo approaches to assess the impact of NMNAT2 reduction on cellular and physiological functions induced by treatment with a vinca alkaloid (vincristine) and a taxane-based (paclitaxel) chemotherapeutic agent. NMNAT2 null (NMNAT2-/-) mutant mice die at birth and cannot be used to probe functions of NMNAT2 in adult animals. Nonetheless, primary cortical cultures derived from NMNAT2-/- embryos showed reduced cell viability in response to either vincristine or paclitaxel treatment whereas those derived from NMNAT2 heterozygous (NMNAT2+/-) mice were preferentially sensitive to vincristine-induced degeneration. Adult NMNAT2+/- mice, which survive to adulthood, exhibited a 50% reduction of NMNAT2 protein levels in dorsal root ganglia relative to wildtype (WT) mice with no change in levels of other NMNAT isoforms (NMNAT1 or NMNAT3), NMNAT enzyme activity (i.e. NAD/NADH levels) or microtubule associated protein-2 (MAP2) or neurofilament protein levels. We therefore compared the impact of NMNAT2 knockdown on the development and maintenance of chemotherapy-induced peripheral neuropathy induced by vincristine and paclitaxel treatment using NMNAT2+/- and WT mice. NMNAT2+/- did not differ from WT mice in either the development or maintenance of either mechanical or cold allodynia induced by either vincristine or paclitaxel treatment. Intradermal injection of capsaicin, the pungent ingredient in hot chili peppers, produced equivalent hypersensitivity in NMNAT2+/- and WT mice receiving vehicle in lieu of paclitaxel. Capsaicin-evoked hypersensitivity was enhanced by prior paclitaxel treatment but did not differ in either NMNAT2+/- or WT mice. Thus, capsaicin failed to unmask differences in nociceptive behaviors in either paclitaxel-treated or paclitaxel-untreated NMNAT2+/- and WT mice. Moreover, no differences in motor behavior were detected between genotypes in the rotarod test. Our studies do not preclude the possibility that complete knockout of NMNAT2 in a conditional knockout animal could unmask a role for NMNAT2 in protection against detrimental effects of chemotherapeutic treatment.     

Engulfment of Axon Debris by Microglia Requires p38 MAPK Activity

The clearance of debris after injuries to the nervous system is a critical step for restoration of the injured neural network. Microglia are thought to be involved in elimination of degenerating neurons and axons in the central nervous system (CNS), presumably restoring a favorable environment after CNS injuries. However, the mechanism underlying debris clearance remains elusive. Here, we establish an in vitro assay system to estimate phagocytosis of axon debris. We employed a Wallerian degeneration model by cutting axons of the cortical explants. The cortical explants were co-cultured with primary microglia or the MG5 microglial cell line. The cortical neurites were then transected. MG5 cells efficiently phagocytosed the debris, whereas primary microglia showed phagocytic activity only when they were activated by lipopolysaccharide or interferon-β. When MG5 cells or primary microglia were co-cultured with degenerated axons, p38 mitogen-activated protein kinase (MAPK) was activated in these cells. Engulfment of axon debris was blocked by the p38 MAPK inhibitor SB203580, indicating that p38 MAPK is required for phagocytic activity. Receptors that recognize dying cells appeared not to be involved in the process of phagocytosis of the axon debris. In addition, the axons undergoing Wallerian degeneration did not release lactate dehydrogenase, suggesting that degeneration of the severed axons and apoptosis may represent two distinct self-destruction programs. We observed regrowth of the severed neurites after axon debris was removed. This finding suggests that axon debris, in addition to myelin debris, is an inhibitory factor for axon regeneration.Axon degeneration is an active, tightly controlled, and versatile process of axon segment self-destruction. The lesion-induced degeneration process was first described by Waller (1) and has since been known as Wallerian degeneration (2, 3). This degeneration involves rapid blebbing and fragmentation of an entire axonal stretch into short segments, which are then removed by locally activated phagocytic cells. Phagocytic removal of damaged axons and their myelin sheaths distal to the injury is important for creating a favorable environment for axonal regeneration in the nervous system. Although the debris of degenerated axons and myelin is cleared by phagocytes in the peripheral nervous system (PNS), the debris is removed very slowly in the central nervous system (CNS)³ (4, 5). This is considered to be one of the obstacles for regeneration of the injured axons in the CNS.Apoptotic neurons are also engulfed by activated phagocytic cells. Apoptosis is very well documented in the CNS where a significant proportion of neurons undergo programmed cell death (6). To prevent the diffusion of damaging degradation products into surrounding tissues, dying neurons are phagocytosed. In the brain, apoptotic cells are engulfed mainly by the resident population of phagocytes known as microglia. Microglia are generally considered to be immune cells of the CNS (7). They respond to any kind of pathology with a reaction termed “microglial activation.” After injuries to the CNS, microglia react within a few hours with a migratory response toward the lesion site.Although insight into the mechanism of phagocytosis of dying cells by microglia has improved, little is known about the mechanism of clearance of degenerated axons and myelin debris by microglia after axonal injury in the CNS. Interestingly, the axons undergoing Wallerian degeneration do not seem to possess detectable activation of the caspase family (8), suggesting that Wallerian degeneration and apoptosis may represent two distinct self-destruction programs. Thus, the mechanism of microglial phagocytosis of dying cells might be different from that of axon/myelin debris. We aimed to elucidate the mechanism of debris clearance by microglia after an axonal injury. We established an in vitro assay system to estimate phagocytosis of degenerated axon debris. We found that p38 mitogen-activated protein kinase (MAPK) was critical for the phagocytic activity of microglia. Treatment with lipopolysaccharide (LPS) or interferon-β (IFN-β) was necessary for the primary microglia to become phagocytic. In addition, clearance of degenerated axon debris allowed axonal growth from the severed neurites, suggesting that removal of the axon debris provides a favorable environment for axonal regeneration.     

The relationship of neuromuscular synapse elimination to synaptic degeneration and pathology: Insights from WldS and other mutant mice

Neuromuscular synapse elimination, Wallerian degeneration and peripheral neuropathies are not normally considered as related phenomena. However, recent studies of mutant and transgenic mice, particularly the Wld ^S mutant—in which orthograde degeneration is delayed following axotomy—suggest that re-evaluation of possible links between natural, traumatic and pathogenic regression of synapses may be warranted. During developmental synapse elimination from polyneuronally innervated junctions, some motor nerve terminals progressively and asynchronously vacate motor endplates. A form of asynchronous synapse withdrawal, strongly resembling synapse elimination, also occurs from mononeuronally-innervated motor endplates following axotomy in young adult Wld ^S mutant mice. A similar pattern is observed in skeletal muscles of several neuropathic mutants, including mouse models of dying-back neuropathies, motor neuron disease and—remarkably—models of neurodegenerative diseases such as Huntington's and Alzheimer's diseases. Taken together with recent analysis of synaptic remodelling at neuromuscular junctions in Drosophila, a strong candidate for a common regulatory mechanism in these diverse conditions is one based on protein ubiquitination/deubiquitination. Axotomised neuromuscular junctions in Wld ^S mutant mice offer favourable experimental opportunities for examining developmental mechanisms of synaptic regression, that may also benefit our understanding of how degeneration in the synaptic compartment of a neuron is initiated, and its role in progressive, whole-cell neuronal degeneration.