InteractiveFly: GeneBrief

Nicotinamide mononucleotide adenylyltransferase: Biological Overview | References

BIOLOGICAL OVERVIEW

Active zones are specialized presynaptic structures critical for neurotransmission. 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. It is proposed that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction (Zang, 2013).

Active zones are highly specialized presynaptic sites for synaptic vesicle docking and fusion, and fast, efficient and precise neurotransmission relies on their structural integrity. The protein composition of active zones has been identified, but the mechanism for maintaining their structural integrity is largely unknown. Chaperones have been implicated in synaptic function; for example, cysteine-string protein (CSP), a main synaptic vesicle and secretory granule protein, is known to act as a chaperone maintaining synaptic integrity. It is likely that molecular chaperones, the main mediators in maintenance of protein homeostasis, facilitate the localization of synaptic proteins and maintain synaptic structural integrity during neuronal activity. This study examined the role of a newly identified chaperone, nicotinamide mononucleotide adenylyltransferase (NMNAT), in active zone maintenance. Previous work has shown that that NMNAT is a protective factor required for maintaining neuronal integrity (Zhai, 2006; Zhai, 2008). Importantly, the neuroprotective ability of NMNAT was attributed partly to its chaperone function, where NMNAT was able to interact with misfolded protein oligomers and prevent the formation of large aggregates and also reduce the cellular load of aggregates, partly through the ubiquitin-proteasome pathway. In Drosophila, NMNAT is ubiquitously expressed and is localized at the synapse as well as in the cell body. This study directly examined the specific role of NMNAT at the synapse and reports a new mechanism of active zone maintenance by NMNAT, in which it stabilizes the primary active zone structure protein Bruchpilot (BRP). BRP was recently identified as an integral component of T-bar, the dense projection of the active zone, and was found to be essential for structural and functional integrity of the active zone. This study found that loss of NMNAT was in parallel with loss of synaptic BRP levels, leading to ubiquitination and redistribution of BRP from the synapse to the cell body, resulting in subsequent active zone degeneration. Moreover, this study shows that NMNAT interacts with BRP in an activity- under normal conditions, NMNAT functions to maintain synaptic BRP protein levels by preventi-e dinucleotide synthase and -mediated protein degradation, thereby maintaining active zone structural integrity during neuronal activity. This work describes NMNAT as an essential active zone maintenance factor and provides a new mechanism by which it sustains the proper structural integrity of active zones (Zang, 2013).

To investigate the specific role of NMNAT in synapses, the changes were examined in synaptic proteins in NMNAT-deficient neurons. By means of mosaic analysis with a repressible cell marker (MARCM) with eyFLP, mosaic nmat-null and heterozygous synapses in the lamina and observed that synaptic proteins, including synaptobrevin, synaptotagmin and BRP, were reduced in nmat mutant patches. Central brain synapses where NMNAT expression was reduced were examined using an RNA interference (RNAi)-mediated knockdown approach with a pan-neural c155-GAL4 driver, which achieved 76% knockdown of the NMNAT protein level. Consistently, reduced staining was observed of synaptic proteins in central brain synapses, including DLAR, synaptotagmin, CSP and BRP, and reduced protein levels as quantified by western analysis. These results indicate a loss of synapses induced by loss of NMNAT. Interestingly, BRP displayed a unique mislocalization phenotype. In contrast to the exclusively synaptic localization in wild-type synapses, in NMNAT RNAi-knockdown synapses BRP was found clustered in cell bodies, colocalizing with remnant NMNAT protein. The mislocalization was specific to BRP, as the distribution of other synaptic proteins including DLAR, synaptotagmin and CSP was not altered in NMNAT-knockdown brains, despite similar reduction in protein levels. These results indicate that NMNAT is required to maintain the level of synaptic proteins, and also specifically the synaptic localization of BRP. The specificity of NMNAT RNAi-mediated knockdown was confirmed by a rescue experiment in which overexpressing wild-type human NMNAT3 not targeted by Drosophila NMNAT RNAi suppressed the pupal lethality phenotype induced by NMNAT knockdown in motor neurons, and rescued the eclosion rate. Furthermore, the maintenance role of NMNAT on BRP is likely unidirectional, as knockdown of NMNAT reduced the BRP level; however, knockdown of BRP in the brains did not change the protein level of NMNAT (Zang, 2013).

The clustering and mislocalization of BRP is intriguing and cannot be explained by loss of synapses; therefore, this study investigated the underlying mechanism. Previous work indicated the role of the ubiquitin-proteasome pathway in regulating synaptic protein levels, such as dUNC-13 and Liprin α. To determine the biochemical nature of the mislocalized BRP protein clusters, BRP protein was immunoprecipitated with nc82 antibody from whole brain lysates of 2 DAE (days after eclosion) NMNAT RNAi flies (genotype: elav-GAL4C155>UAS-Dicer,UAS-NMNAT-RNAi), and probed for ubiquitin and HSP70, a molecular chaperone serving as a protein marker for aggregation. BRP protein in NMNAT RNAi flies was significantly ubiquitinated with several bands of different molecular weight, indicating the presence of poly-ubiquitinated BRP, while no ubiquitination of BRP was observed in control flies. Interestingly, HSP70 was present in the NMNAT RNAi brains but not in the control brains and, more importantly, interacted with BRP, indicating the expression of stress response protein HSP70 and the formation of BRP aggregates. As expected, the level of NMNAT pulled down by BRP antibody was reduced as a result of NMNAT RNAi knockdown and reduced BRP level. Consistent with the results described above, the level of BRP in NMNAT RNAi brains was reduced, but a higher molecular weight modification of BRP was detected, likely ubiquitinated BRP. In contrast, ubiquitination of other synaptic proteins including DLAR, synaptotagmin and CSP was not observed in NMNAT-knockdown neurons, suggesting a specific effect on BRP (Zang, 2013).

To further dissect the mechanism underlying the reduction and ubiquitination of BRP, the transcription of BRP was examined utilizing real-time PCR analysis, and it was observed that BRP transcripts were slightly increased on NMNAT knockdown. Therefore, the reduction of BRP protein is not owing to reduced transcription. Next, whether the ubiquitin proteasome pathway was involved in regulating BRP protein degradation was investigated. Proteasome function was examined by feeding flies a specific inhibitor MG-132, and examined the level of BRP ubiquitination was examined in wild-type or NMNAT overexpression flies. To specifically analyse the ubiquitinated pool of BRP protein, BRP was immunoprecipitated, probed with both anti-BRP and anti-ubiquitin antibody, and the percentage of ubiquitinated BRP was determined using multiplex western analysis, where only the protein bands detected by both BRP and ubiquitin antibodies were considered as ubiquitinated BRP. In wild-type flies, the percentage of ubiquitinated BRP increased when proteasome function was inhibited with MG132, indicating an accumulation of ubiquitinated BRP on proteasome inhibition. In NMNAT-overexpressing flies with three copies of the nmat gene, the percentage of ubiquitinated BRP was lower than that in wild-type with either dimethyl sulphoxide or MG132 treatment. This suggests that the ubiquitin-proteasome pathway is involved in regulating BRP protein degradation and that a higher level of NMNAT reduces the ubiquitination of BRP. Therefore, loss of NMNAT causes specifically the ubiquitination and aggregation of BRP and subsequent reduction in synaptic BRP protein level, likely through the proteasome pathway (Zang, 2013).

Loss-of-NMNAT studies indicate that under normal conditions, NMNAT functions to maintain synaptic BRP protein levels by preventing ubiquitination and aggregation of BRP. The dual function of NMNAT, as an nicotinamide adenine dinucleotide synthase and a chaperone, suggests two possible mechanisms: an indirect mechanism expressed through NMNAT-mediated synthesis of small molecules including nicotinamide adenine dinucleotide and other adaptor proteins; and a direct mechanism through protein-protein interactions, consistent with its chaperone function. To distinguish these, the localization of NMNAT was studied. BRP and NMNAT localize in photoreceptor and central brain synapses; however, the compact size of these synapses precludes a high-resolution analysis. Inasmuch as close proximity is a prerequisite for a 'direct' mechanism, the Drosophila larval neuromuscular junction (NMJ) is ideal for analysing synaptic localization, given its suitable spatial resolution. By confocal microscopy, it was observed that NMNAT is present at the NMJ and colocalizes strongly with BRP. With 3D-SIM Super-Resolution imaging (resolution 120 nm on XY axis), NMNAT localization was observed adjacent to synaptic membranes labelled with horseradish peroxidase (HRP) staining and some NMNAT puncta colocalized strongly with BRP puncta, with 48±11% of BRP colocalizing with NMNAT and 52±12% of NMNAT colocalizing with BRP, suggesting that NMNAT is localized within close proximity to the active zone and BRP. It was next observed that NMNAT co-immunoprecipitated specifically and reciprocally with BRP, but not with DLAR, synaptotagmin, CSP, syntaxin or DLG. These results indicate that NMNAT is localized to synaptic active zones and specifically interacts with the active zone protein BRP (Zang, 2013).

Recent studies have revealed the dynamic changes of active zones and active zone proteins responding to neuronal activity. To examine the functional relevance of NMNAT-BRP interaction during activity, the Drosophila visual system was used, as synaptic activity can be easily manipulated by altering light exposure. The synaptic and active zone structure was examined of wild-type and nmat-null synapses under normal (12 h dark/light condition, DL) or reduced activity (12 h dark/dark, DD). In nmat-null neurons the photoreceptor terminal and the active zone structure were compromised in 1 DAE flies under normal conditions, consistent with a previous report. However, when photoreceptor activity was diminished in complete darkness, active zone structure, as measured by the width of the T-bar, was maintained in nmat-null synaptic terminals for as long as 10 days. To further strengthen this finding, an alternative approach was taken to block neuronal activity utilizing a genetic mutation in NorpA (no receptor potential A), which attenuates the phototransduction cascade and synaptic transmission in photoreceptors. The NorpA mutation was introduced in the background of nmat null and the synaptic structures were examined NorpA and nmat double-mutant photoreceptor terminals. In nmat-null terminals, the active zone (T-bar) size declined with age under the normal (dark/light) condition, indicating age- and activity-dependent synaptic degeneration. Interestingly, in NorpA/nmat double-mutant photoreceptor terminals, larger T-bars were observed compared with those in nmat-null terminals at the same age, indicating reduced active zone degeneration when synaptic activity was attenuated. These results also indicate that NMNAT is not required for the assembly of T-bar, as normal active zone structure is observed in nmat-null neurons in dark-reared flies and in pharate adults, suggesting that during development the transport of BRP and synapse assembly was intact without NMNAT. Collectively, these results indicate that NMNAT protein is required for the maintenance of active zone only when synaptic activity is present (Zang, 2013).

To examine whether the interaction between NMNAT and BRP is also affected by neuronal activity, functional haemagglutinin (HA)-tagged NMNAT was expressed in the photoreceptors (using GMR-GAL4), and photoreceptor activity level was manipulated by light exposure. HA antibody was used to isolate the pool of NMNAT protein from the neurons (photoreceptors) subjected to activity alteration. At 10 DAE, whole brain lysates were immunoprecipitated with an HA antibody and probed for BRP. In brains where HA-NMNAT is not expressed (GMR-GAL4 control flies), no BRP was immunoprecipitated by HA antibody. When HA-NMNAT was expressed in the photoreceptors, HA antibody immunoprecipitated significantly more BRP from flies raised under normal activity conditions (DL) than from flies reared in darkness (DD). This difference is specific for HA-NMNAT expressed in the photoreceptors, as a similar level of BRP was immunoprecipitated by the anti-NMNAT antibody, indicating that the interaction between BRP and endogenous NMNAT in the entire brain was not significantly affected by the light exposure. It is also noted that the reduction of BRP immunoprecipitated by HA antibody was not owing to reduced HA-NMNAT expression in flies reared in the dark, as a similar amount of expressed HA-NMNAT and similar immunoprecipitation efficiency was observed between normal (DL) and dark (DD) conditions. These results indicate that the NMNAT-BRP interaction is affected by neuronal activity, and that increased activity leads to increased NMNAT-BRP interaction (Zang, 2013).

The findings of ubiquitinated, clustered and mislocalized BRP in loss-of-NMNAT neurons, and that increased activity leads to increased NMNAT-BRP interaction, together with the observation that active zone structure is maintained in nmat-null neurons when neuronal activity is reduced, suggest the following model of the activity-dependent role of NMNAT in active zone maintenance. Under normal activity conditions, NMNAT is required to maintain active zone structure by interacting with BRP and to prevent the ubiquitination of BRP, inasmuch as loss of NMNAT results in BRP ubiquitination, mislocalization, aggregation and reduced active zone size. When neuronal activity is minimized (for example, by blocking light stimulation (dark rearing), or by blocking phototransduction (NorpA)), the demand on maintenance by NMNAT is reduced. These studies have revealed a specific role of NMNAT in regulating homeostasis of the active zone protein BRP. The role of NMNAT in protein-protein interaction is consistent with the chaperone function of NMNAT. Chaperones, such as CSP, have been implicated in maintaining synaptic integrity. Moreover, recent studies have shown that an elevated activity level poses stress to synaptic proteins by highlighting the effect of CSP in maintaining synaptic function. It is expected that increased neuronal and/or synaptic activity will lead to increased protein misfolding and turnover, and therefore to an increase in the load/demand of maintenance of synaptic protein homeostasis. This notion is supported by a study showing that the level of ubiquitin conjugation of synaptic proteins is altered by the level of synaptic activity. These studies describe NMNAT as a synapse maintenance factor under normal activity conditions post assembly, when most of the BRP protein is present at the active zone and NMNAT protein is localized to the active zone area to carry out its maintenance function. The interesting observation of clustered BRP protein in the cell body away from the synapse in loss-of-NMNAT neurons indicates a possible defect in the transport of BRP. Two possibilities might explain this phenotype. One, NMNAT facilitates the anterograde transport of BRP during activity. Reduced NMNAT level leads to inefficient transport and subsequent clustering of BRP in the cell body. Two, these BRP clusters are retrogradely transported from the active zone en route to degradation in the cell body. Further work will be required to determine the direction of transport. In summary, this work has identified NMNAT as a chaperone for maintaining active zones, and for facilitating their maintenance during neuronal activity by binding to active zone structural protein BRP, adding NMNAT to the list of synaptic chaperones that are required to maintain functional and structural integrity in neurons (Zang, 2013).

Mitochondria and caspases tune Nmnat-mediated stabilization to promote axon regeneration

Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, this study investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury, it was found that mitochondrial fission in dendrites is upregulated, and reducing fission increases stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. Caspases were identified to be negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, it was found that Nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, requires Nmnat. Indeed Nmnat overexpression is sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos are also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, it was tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduce regeneration. In addition, overexpression of fos or JNK extend the timecourse of NP and dampen regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration (Chen, 2016).

The results lead to a model in which axon injury triggers opposing responses downstream of the initial DLK/JNK/fos signaling cascade. One early output of this conserved injury response pathway is NP, a global stabilization of the parts of the neuron still connected to the cell body. The central mediator of NP is Nmnat. One Nmnat effector is the dramatic increase in microtubule dynamics observed after axon injury. As axon damage is likely to be accompanied by disturbances in the surrounding tissue, making the cell more resistant to degeneration by turning on NP may help the neuron survive the initial trauma (Chen, 2016).

Fos injury signaling also triggers Drp1-mediated mitochondrial fission in the first few hours after axon injury, and this leads to dampening of NP by caspases. Positive and negative regulation of NP are envisioned as balancing one another in different ways through time after injury. Eventually the negative pathway must outweigh the positive or regeneration is dampened by persistent NP. It is possible that the timing of this balance shift is controlled by additional signals that report whether the environment is conducive for regeneration (Chen, 2016).

This model suggests that rather than DLK/JNK/fos directly regulating regeneration, this signaling pathway kicks off a multi-step response to axon injury that includes regeneration as a relatively late event. Indeed, although this pathway is known as the conserved axon regeneration pathway, this study found that it first turns on a response that inhibits regeneration. Although this idea is surprising, this model does make sense in the overall picture of neuronal injury responses and stabilization. For example, in mammals and flies the AP-1 transcription factor fos is activated soon after axon injury, but its role in regeneration is not as clear as that of some other transcription factors like jun. The data suggests that this early activation could be because fos orchestrates the injury response that precedes regeneration (Chen, 2016).

The results also touch on the role of caspases in axon regeneration. A study in C. elegans demonstrated that caspases are positive regulators of axon regeneration, which is surprising considering their involvement in self-destruct programs like apoptosis and dendrite pruning. This study confirmed that in Drosophila caspases are pro-regenerative. In addition, the data suggests that this effect is not through a direct role in regeneration, but because caspases down-regulate NP, which inhibits regeneration (Chen, 2016).

A negative role for mitochondria in NP is also intriguing. Mitochondria seem to promote axonal stability, and there are studies in several systems that suggest that mitochondria are required for the neuroprotective effects of Nmnat or Wld_s, an extraordinary protein discovered in mice that is formed by fusing a Ube4b sequence to Nmnat1. However, mitochondria can play prodegenerative roles in other contexts. More specifically mitochondrial fission can promote degeneration. This study demonstrates that mitochondria, Drp1 and caspases all counteract NP, suggesting that caspase activation may regulate NP downstream of mitochondrial fission. This does not mean that mitochondria are not also positive regulators of this type of NP. Indeed the data in this study combined with others suggests that mitochondria are critical nodes for control of neuronal stability and both positive and negative regulation likely converge on them (Chen, 2016).

Like mitochondria, the role of Nmnat in injury responses has been difficult to classify simply as either positive or negative. Its ability to prevent injury-induced Wallerian degeneration, as well as to act as an endogenous neuroprotective factor has led to the idea that it has a purely positive influence on neuronal health. However, the myriad ways in which it can be regulated suggest that it is useful only in exactly the right dose. Indeed this study showed that when its regulation is disrupted, Nmnat inhibits a different type of neuronal resilience: axon regeneration. Thus upregulation of Nmnat as a potential therapeutic strategy to counteract neurodegeneration could have negative outcomes due to dampened regeneration (Chen, 2016).

While the experiments support the idea that endogenous Nmnat is a central regulator of neuronal stability, the way it exerts this effect remains unclear. Nmnat is an enzyme that uses ATP and NMN (nicotinamide mononucleotide) to make NAD+. Protective effects of endogenous or overexpressed Nmnat have been proposed to be due to maintenance of high NAD levels, keeping levels of the precursor NMN low, acting as a chaperone, and through maintaining mitochondrial integrity or function. This study shows that Nmnat also acts upstream of increased microtubule dynamics after axon injury. This increased microtubule dynamics in response to axon injury is also seen in mammalian neurons, and so this part of the NP response is likely to be conserved. Although increased microtubule dynamics plays a role in NP, and now show that Nmnat overexpression is sufficient to increase microtubule dynamics, it is possible that Nmnat has other effectors that can mediate NP (Chen, 2016).

In conclusion, a model is proposed in which DLK signaling initiates key injury responses before axon regeneration begins. These responses include upregulation of Nmnat-mediated NP, microtubule dynamics and mitochondrial fission. Mitochondrial fission likely counteracts NP through caspase activation, although it is possible that mitochondria and caspases regulate NP independently. Although this early response is downstream of the core axon regeneration kinase cascade, it actually inhibits regeneration if unchecked. This multi-step model of injury responses downstream of DLK helps explain the function of caspases in promoting regeneration. It is anticipated that understanding the transition between early injury responses and regeneration itself will suggest strategies for promoting axon regeneration without overactivating NP, which would, in turn, dampen regeneration. A more complete understanding of the relationship between NP and regeneration is essential to designing any therapeutic approach to either stabilize neurons or to enhance regeneration (Chen, 2016).

Axon death pathways converge on Axundead to promote functional and structural axon disassembly

Axon degeneration is a hallmark of neurodegenerative disease and neural injury. Axotomy activates an intrinsic pro-degenerative axon death signaling cascade involving loss of the NAD+ biosynthetic enzyme Nmnat/Nmnat2 in axons, activation of dSarm/Sarm1, and subsequent Sarm-dependent depletion of NAD+. This study has identified Axundead (Axed) as a mediator of axon death. axed mutants suppress axon death in several types of axons for the lifespan of the fly and block the pro-degenerative effects of activated dSarm in vivo. Neurodegeneration induced by loss of the sole fly Nmnat ortholog is also fully blocked by axed, but not dsarm, mutants. Thus, pro-degenerative pathways activated by dSarm signaling or Nmnat elimination ultimately converge on Axed. Remarkably, severed axons morphologically preserved by axon death pathway mutations remain integrated in circuits and able to elicit complex behaviors after stimulation, indicating that blockade of axon death signaling results in long-term functional preservation of axons (Neukomm, 2017).

Maintenance of the morphological integrity of neurons is essential for sustained nervous system function throughout an animal's lifespan. Nervous system injury or neurological disease leads to axonal and synaptic degeneration and, in turn, loss of neural circuit connectivity and function. Molecular pathways driving axonal degeneration remain poorly defined in any context; however, recent work on Wallerian degeneration (WD) has revealed that axon injury activates an intrinsic, conserved, pro-degenerative (axon death) signaling pathway. Previously identified dSarm/Sarm1 (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) as a key mediator of axon death signaling. Loss of dSarm in Drosophila, or Sarm1 in mouse, resulted in severed distal axons remaining morphologically preserved for weeks after injury (Gerdts, 2013, Osterloh, 2012), indicating that dSarm/Sarm1 pro-degenerative signaling is an ancient mechanism used by axons to drive self-destruction. How dSarm/Sarm1 signals to execute axon death remains unclear, but dSarm/Sarm1 has recently been linked to the NAD+ metabolic pathway, which based on extensive evidence appears to be a central mediator of axonal integrity (Neukomm, 2017).

The first evidence supporting a role for NAD+ in axon maintenance came from the identification and characterization of the slow Wallerian degeneration (WldS) mouse, where severed axons fibers survived for weeks when detached from their cell bodies. This remarkable neuroprotective phenotype effect was due to a chromosomal rearrangement that led to the generation of the novel Wld^S molecule, a fusion protein consisting of the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) and a short fragment of Ube4b. Consistent with a positive role for NAD+ in sustaining axons, NAD+ levels were found to plummet in axons immediately prior to granular fragmentation. Axon degeneration could be rescued by exogenous NAD+ or its precursors, and axotomy-induced NAD+ depletion was blocked by Wld^S. Numerous studies have demonstrated neuroprotective roles for NAD+-related metabolites. The current proposed mechanism for activation of axon degeneration after injury is the depletion of Nmnat2, a labile form of Nmnat found in mammalian axons that is normally transported down axons from the soma. Nmnat2 is seen as a critical regulator of axon survival: its half-life approximates the latent phase prior to explosive axon fragmentation, depletion of Nmnat2 from axons induces spontaneous degeneration, and stabilization of Nmnat2 can phenocopy the effects of Wld^S (Neukomm, 2017).

The second line of evidence supporting a role for NAD+ in axonal protection came from drug screens for molecules that promoted neurogenesis (or neuroprotection) in vivo. P7C3 was identified as an activity-enhancing compound of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in the NAD+ salvage pathway, which likely leads to increased levels of NAD+ in injured axons to help sustain integrity. Derivatives of the P7C3 series have since been shown to be neuroprotective in many models of neurodegenerative disease, and in some models of neural injury . However, whether P7C3 harbors the ability to attenuate axon death remains to be determined (Neukomm, 2017).

Finally, recent work has demonstrated that NAD+ depletion after axotomy is blocked in Sarm1-/- mutants, and dimerization of the Sarm1 TIR domain can drive rapid depletion of NAD+ from cells and axons (Gerdts, 2015). Surprisingly, it appears that the Sarm1 TIR domain harbors endogenous NAD+ hydrolase activity (Essuman, 2017). Thus an emerging stepwise model of WD is that axotomy leads to depletion of labile pools of Nmnat2 in axons detached from their cell bodies; this in turn results in an initial depletion of NAD+ levels due to lack of new NAD+ synthesis and/or salvage, dSarm/Sarm1 signaling is then activated followed by rapid depletion of NAD+ levels below a threshold needed to maintain axonal integrity, and finally explosive degeneration of the axon ensues. NAD+ depletion from axons is a compelling model for WD, although alternative mechanisms cannot be excluded, due to limited understanding of the genetics of axon death signaling. Besides dSarm/Sarm1, only one other molecule, the E3 ubiquitin ligase Highwire/Phr1, has been shown to be required for axon death in vivo: loss-of-function mutations in highwire/Phr1 potently suppress axon death in fly nerve injury models and mammalian sciatic nerve lesion experiments. Highwire/Phr1 likely acts upstream of dSarm/Sarm1, and is required for the normal turnover of Nmnat/Nmnat2. Loss of Highwire/Phr1 is therefore proposed to stabilize Nmnat/Nmnat2 and maintain axonal pools of NAD+. Are there other signaling molecules acting downstream of dSarm/Sarm1 to execute axonal death, or is NAD+ depletion the final step? Sarm1 has also been reported to drive axon death through a downstream MAPK signaling cascade (Yang, 2015), similar to the signaling mechanism used by C. elegans TIR-1 during regulation of odorant receptor expression; however, this remains controversial. A recent contradictory study argues that MAPKs exert their effect by fine-tuning levels of Nmnat2 upstream of Sarm1 signaling (Neukomm, 2017).

This paper reports the identification and characterization of Axundead (Axed), an axonal BTB and BACK domain protein that signals downstream of dSarm. Axed is essential for injury-induced axon death signaling, and axed mutations can fully suppress degeneration induced by activated dSarm, or complete elimination of Nmnat activity from axons. Thus, Axed is a novel pro-degenerative signaling molecule, and the neuroprotective effects of loss of Axed exceed those of dsarm null mutants (Neukomm, 2017).

This study presents the identification and initial characterization of the BTB and BACK domain molecule Axed. Loss of Axed function was sufficient to block axon death for the lifespan of the fly, and axed mutants were neuroprotective in all neurons tested. Axed, like dSarm, appears to function selectively in axon death during WD, as axed mutants blocked neither cell death nor developmental pruning of axons or dendrites, supporting the notion that the Nmnat-depletion/dSarm/Axed signaling pathway is engaged specifically in response to axonal injury. However, it remains possible that while Axed or dSarm elimination is not sufficient to block cell death, redundant genetic pathways might work together with dSarm and Axed in the context of apoptotic cell death (Neukomm, 2017).

Axed encodes a previously uncharacterized BTB and BACK domain protein. The best-characterized role for BTB-containing proteins is the recruitment of substrates to Cullin Ring Ubiquitin Ligase (CRL) complexes for ubiquitin tagging and proteasome degradation, where BTB domains function in homo- or hetero-dimerization while the BACK domain (often with the BTB domain) interacts directly with Cullins. Typically, the C-terminal regions in BTB domains molecules function within CRL complexes to bind target substrates designated either for ubiquitination by the CRL and subsequent degradation by the proteasome or for signaling. The BTB and BACK domains of Axed are required for optimal Axed function in vivo based on their partial abilities to rescue axon death phenotypes in axed mutant backgrounds. In contrast, the C terminus appears to be absolutely essential for Axed function as Axed^δCterm completely fails to rescue axon death. This is not likely due to destabilization of the molecule lacking the C terminus, since Axed^δCterm can be stably expressed in Drosophila S2 cells. Despite extensive attempts to implicate the wide array of CRLs in Drosophila in axon death and Axed function, no evidence was found supporting a role for CRLs in axon degeneration. Given that there are five Cullin-like molecules in the Drosophila genome, it seems possible that genetic redundancy between Cullins might explain the lack of phenotype. Alternatively, Axed could be functioning to promote axon death in a completely novel Cullin-independent manner. Future studies aimed at identifying direct binding partners for Axed will be essential to resolve these issues. If Axed functions as a bridging molecule for CRL ubiquitination activity, of particular interest will be identifying molecules that bind to the Axed C-terminal putative substrate binding domain (Neukomm, 2017).

Sarm1 functions in axons after injury (Gerdts, 2015). Given that Axed functions genetically downstream of Sarm1, it is proposed that Axed also functions in axons. Using a functional, endogenously enhanced GFP-tagged version of Axed (Axed^eGFP), it was found that Axed protein is enriched in the neuropil in both the larva and the adult nervous system. The Drosophila larval neuropil is highly enriched for dendrites and axons and is the site of all CNS synapse formation. Axed^eGFP signals overlapped extensively with axons, dendrites, and synapses, further supporting a cell-autonomous role for Axed in neurons during axon death. Axed^eGFP is also present in the adult neuropil, and Axed^eGFP signals transiently increased at 4 and 6 hr in the antennal lobe after injury but returned to baseline levels by 24 hr, a time point at which most severed axons have fragmented. The return to baseline levels likely represents Axed^eGFP staining in local and projection interneurons in the antennal lobe that were not severed by antennal ablation. Why Axed^eGFP signals might transiently increase remains unclear. Perhaps Axed^eGFP is relocalized in a way that increases GFP exposure, or new Axed^eGFP protein may be locally synthesized. Regardless of the precise mechanisms, the observations indicate that Axed^eGFP dynamics in vivo are sensitive to axonal injury (Neukomm, 2017).

axed mutant phenotypes are indistinguishable from dsarm mutants with respect to axon preservation after injury. In addition, axed mutants completely blocked the pro-degenerative activity of the gain-of-function dSarm molecule dSarm^Δdsarm in vivo. These genetic data strongly support a model whereby Axed functions genetically downstream of dSarm, and argue that dSarm and Axed drive axon death through the same genetic pathway (Neukomm, 2017).

dSarm^Δdsarm appears to induce a Wallerian-like program in vivo based on several observations. First, the explosive fragmentation observed is morphologically similar to WD. Second, dSarm^Δdsarm-induced degeneration can be suppressed partially by Wld^S, whose activity is highly selective to WD. Third, dSarm^Δdsarm pro-degenerative activity can be fully suppressed in axed mutants. Finally, all structural and biochemical studies to date support the notion that elimination of the N-terminal ARM domain of dSarm (Drosophila), TIR-1(C. elegans), or Sarm1 (mammals) leads to the production of a gain-of-function molecule that activates signaling. That dSarm^Δdsarm signaling in the absence of injury is potently suppressed by axed mutants provides a compelling argument for Axed to act downstream of dSarm. The nature of the genetic program that drives death of neuronal cell bodies after dSarm/Sarm1 activation remains mysterious. It could not be blocked with the broad caspase inhibitor P35. Related studies in mouse found a lack of evidence for apoptotic signaling molecules, necroptosis, and parthanatos in death induced by activated Sarm1. The observation that it was possible to induce robust degeneration of axons, dendrites, and cell bodies by adult-specific induction of dSarm^Δdsarm in adult PDF+ neurons, even in the absence of injury, suggests that dSarm^Δdsarm could provide a useful tool for conditional removal of selected neurons from adult circuits for functional studies (Neukomm, 2017).

In the larger context of axon death signaling, the finding that mutations in hiw do not suppress dSarm^Δdsarm in vivo agrees with the proposed model that Hiw/Phr1 acts genetically upstream of dSarm/Sarm1 and Nmnat degradation. The position of the MAPK signaling cascade relative to dSarm/Sarm1 is still debated in the field and remains to be determined. The fact that the Drosophila genome houses only a single JNK family member, and the observation that null alleles had no axon death phenotype, argues strongly against a central role for JNK signaling in axon death (Neukomm, 2017).

With respect to their roles in the functional disassembly of axons, this study has demonstrated that blocking axon death with mutations in dsarm, axed, or highwire is sufficient to preserve axons in a functional state in neural circuits where they can elicit complex grooming behaviors for weeks after axotomy. Therefore, axon death mutants, like the Wld^S molecule, exert their neuroprotective effects very early in the axon death genetic program, and their therapeutic blockade is likely to lead to the preservation of functional axons in the context of neurological disease (Neukomm, 2017).

A number of recent studies have led to the following model for axon death signaling: (1) axotomy results in the degradation of pools of labile Nmnat/Nmnat2 in distal severed axons; (2) depletion of Nmnat/Nmnat2 in turn leads to decreases in axonal NAD+; (3) loss of Nmnat/Nmnat2 or decreased NAD+ somehow activates dSarm/Sarm1 signaling, and (4) Sarm1 NAD+ hydrolase activity then drives pathological depletion of axonal NAD+ pools, causing catastrophic energy failure that ultimately drives axon degeneration. A number of observations were made that are difficult to rectify with a simple Nmnat2/Sarm1-dependent NAD+ depletion model. The first is that expression of dSarm^Δdsarm, which based on previous reports should rapidly degrade axonal NAD+ and normally leads to rapid axonal degeneration (Essuman, 2017), can be completely suppressed by loss of Axed. If the terminal step in axonal death were Nmnat2/Sarm1-dependent depletion of NAD+ pools, it is hard to imagine how the NAD+ hydrolase activity of the Sarm1 TIR domain, once unleashed, could be suppressed by the loss of a BTB and BACK domain protein (i.e., Axed). The alternative hypothesis is favored that Sarm1 signaling, perhaps through its TIR domain NAD+ hydrolase activity, activates an Axed-dependent downstream signaling pathway essential for axon death. ADP-ribosylation of targets is one mechanism that allows certain E3 ubiquitin ligases to bind substrates. There is certainly a tight association between NAD+ metabolism and axon death signaling, and the NAD+ hydrolase activity associated with the Sarm1 TIR domain can lead to the production of ADPr. Perhaps BTB and BACK domain proteins like Axed also require ADP-ribosylation of selected targets during axon death. In this model, the degradation of NAD+ would be a byproduct of the reaction, not a driving force (Neukomm, 2017).

Nmnat2-/- mutant mice die perinatally with neurons containing short axons, but these embryos can be rescued to adulthood by Sarm1-/- null mutations. It is likely that Nmnat2-/- animals and axons can survive in Sarm1-/- backgrounds because Nmnat1 and Nmnat3, the additional isoforms of Nmnat found in mice, could partially compensate. An nmnat^RNAi experiment may therefore more closely resemble conditions in the Nmnat2-/-, Sarm1-/- mouse, with respect to the partial, but not complete elimination of Nmnat function. dsarm null alleles could only partially protect axons from Nmnat depletion with nmnat^RNAi, as far fewer axons and cell bodies were observed compared to controls. Consistent with a requirement for Nmnat activity in cell survival, this study found null clones of the sole fly Nmnat molecule die within 1 day after eclosion. Remarkably, this phenotype can be completely overcome by loss of Axed, since axed, nmnat double mutant clones survive for weeks. However, it was surprising to find that loss of dSarm fails to block this degeneration. Whether Sarm1-/- animals could suppress the degeneration of mammalian neurons completely lacking Nmnat function would require the simultaneous elimination of all Nmnat isoforms (i.e., Nmnat1,2,3 triple mutants), which has not been explored. In the absence of Nmnat activity, invertebrate cells should not be able to autonomously synthesize NAD+. How these cells survive remains a mystery, but a number of possibilities might explain this phenotype. Perhaps yet-to-be-identified NAD+ biosynthetic pathways exist that can function without Nmnat. Alternatively, axons in vivo could be supplemented with NAD+ or key intermediates by surrounding glia and thereby sustain their integrity in axed, nmnat double mutant axons. Finally, it is possible that in the absence of Axed, Nmnat protein may not be turned over appropriately in axons and that perdurance of small amounts of Nmnat may generate sufficient levels of NAD+ for axon survival. Direct, in vivo measurement of NAD+ levels in axons in Drosophila will be essential to answer this question, but current NAD+ sensors have not yet provided sufficient sensitivity for this analysis (Neukomm, 2017).

Axed appears to sit genetically at a convergence point in axon death signaling. There are three ways to activate axon death: axotomy, Nmnat/Nmnat2 depletion, or expression of gain-of-function dSarm/Sarm1. Axed mutants, but not dsarm, can suppress each of these treatments, and, amazingly, axed mutants can even survive the combination of all three insults at once. The neuroprotective effects of axed therefore exceed those of dsarm mutants. It remains to be determined whether any of the four putative mammalian paralogs (BTBD1, BTBD2, BTBD3, and BTBD6) play a role in axon death, but this seems likely based on the strong conservation of dSarm/Sarm1 function in axon degeneration. If so, these would represent important new therapeutic targets for blocking axon death in neurological diseases such as traumatic brain injury, peripheral neuropathy, or nerve injury, where dSarm/Sarm1 signaling is known to drive axon loss (Neukomm, 2017).

Axonal self-destruction and neurodegeneration

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. This study reports a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. This study uncovered that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons (Izadifar, 2021).

A hallmark in the generation of neuronal cell type diversity is the acquisition of diverse morphologies, which requires the formation of axonal and dendritic compartments ranging from simple to highly complex, depending on the degree of neurite branching. Specifying the degree and pattern of neurite branching is crucial in brain development, as it directly impacts the total number and spatial distribution of synaptic contacts of each circuit element. However, the identity of the molecular effectors determining how diverse, cell-type-specific patterns of axon arborization are established and how they are stabilized as well as maintained throughout the life of an organism remains a major challenge (Izadifar, 2021).

A reverse genetic screen was performed to identify novel regulators of axon branching by utilizing an experimental system in Drosophila that combines efficient single-neuron labeling and simultaneous knockdown of candidate genes. Clear orthologs of selective candidates were then further examined in vertebrates. Using this approach, it was found that loss of the Drosophila dWnk kinase specifically disrupts axon growth and branch patterning of mechanosensory neurons. Surprisingly, unlike other essential regulators of axon branching, this study discovered that dWnk is also continuously required in mature neurons for axon maintenance. Moreover, comparative studies in mouse cortical pyramidal neurons (PNs) provide strong evidence that both of these functions of Wnk kinases are conserved and required in PNs, i.e., long-range projecting mammalian neurons of the central nervous system (CNS) (Izadifar, 2021).

Wnk kinases are present in most multicellular organisms, including plants and some unicellular organisms, but not in yeast. Mammals have four Wnk kinases (WNK1-4) and Drosophila one (dWnk). The WNK ('with no K(lysine)') kinases are catalytically active but are referred to as 'atypical kinases' because a catalytically important lysine residue is swapped from subdomain II to subdomain I. WNK proteins are involved in a broad spectrum of diseases (e.g., hypertension, sensory and autonomic neuropathy, osteoporosis, and many different cancers) (Izadifar, 2021).

The majority of studies on human WNK kinases have been conducted in the context of blood pressure regulation, due to the identification of mutations in human patients with hereditary hypertension (familial hyperkalemia and hypertension [FHHt] or Gordon's syndrome. For this reason, a major focus in dissecting WNK function has been on studying renal regulation of ion transport. However, regulation of ion homeostasis is only one of multiple functions of WNK kinases, and they are broadly expressed, including in the developing as well as mature brain. In rare cases, WNK function has been linked to a severe form of peripheral sensory neuropathy (hereditary sensory and autonomic neuropathy type 2, HSNA2); however, their developmental, cellular, and molecular mechanisms are poorly understood in neurons. The fact, however, that most identified mutations cluster in a neuron-specific alternatively spliced exon (HSN2) of human Wnk1 supports the notion that Wnk1 kinase plays an important role in sensory neurons (Izadifar, 2021).

In the process of studying the role of dWnk kinase in fly sensory neurons, this study identified novel interactors of Wnk kinases. Specifically, it was found that nicotinamide mononucleotide adenylyltransferase (Nmnat), Sarm, and Axundead (Axed) are molecular interactors of dWnk. While Nmnat is broadly required for axon maintenance, Sarm and Axed are primarily studied for their roles as effectors in active axon degeneration (e.g., in Wallerian degeneration) in response to axon injury. This study provides evidence that dWnk and Nmnat have synergistic functions in axon growth and branching but are also required in post-developmental processes to continuously support axon maintenance. The function of dWnk is evolutionarily conserved, as their mouse orthologs WNK1 and WNK2 are both required in cortical PNs during axon morphogenesis as well as maintenance. Genetic epistasis analysis demonstrates that both dWnk and Nmnat functions during axon development and axon maintenance are mediated by antagonizing the axon destruction function of Sarm and Axed. Depletion of axon-protective factors (e.g., dWnk/WNK1/2 and Nmnat) during development leads to axon branching defects, whereas their depletion in mature neurons eliminates their safeguard function and initiates spontaneous axon degeneration in the absence of axon injury (Izadifar, 2021).

This study reports that the function of the Wnk-family kinases (Drosophila dWnk and mammalian WNK1/2) are required in developmental axonal branch patterning as well as axon maintenance. Phenotypic defects observed in mechanosensory axons of dWnk mutant neurons are indistinguishable from defects observed in Nmnat mutant neurons. Both dWnk and Nmnat mutant mechanosensory axons can grow from the periphery to the VNC but require dWnk as well as Nmnat function during axonal branch growth and patterning. Moreover, a post-developmental knockdown of dWnk triggered progressive degeneration in mature and fully developed mechanosensory axons. Such an adult-specific function is also analogous to the well-documented role of Nmnat in axon maintenance. Although Nmnat has been studied most extensively for its role in axon maintenance in injury models or neurodegenerative disease models, it has been reported previously that LOF mutants are lethal and show axonal defects. Furthermore, this study shows that the dual developmental and maintenance functions of dWnk are evolutionary conserved, as knockdown of WNK1 and WNK2 results in remarkably similar axon morphogenesis defects and trigger axon degeneration in mouse cortical neurons. It is concluded that, in neurons, Wnk kinases exert novel functions that are analogous and synergistic to conserved Nmnat functions. The discovery of Wnk kinases having important neuroprotective roles analogous to Nmnat offers new tools and insights to further dissect the complex regulatory network underlying active axon degeneration (Izadifar, 2021).

Formally, the adult maintenance defects that were observed upon Wnk depletion could be a consequence of developmental defects. However, several reasons argue strongly against this possibility: first, the in vivo knockdown of WNK1 and WNK2 after P30 in mouse cortical layer 2/3 PNs triggered degeneration of axons of mature neurons. Second, knockdown of dWnk at post-developmental (late pupal) stages does not alter the axonal branching or targeting of mechanosensory neurons but triggered spontaneous degeneration of adult mechanosensory axons after 3 days post-eclosion. Third, a single-copy RNAi knockdown of dWnk resulted in strong axon branching defects. Fourth, in previous studies, several mutants were identified and characterized that lead to severe axon branching defects of mechanosensory axons. For example, in Dscam1-null mutant clones, the axon branching defects are even more severe than in dWnk mutant clones, yet no sign of axon degeneration was found even in 1 week or older flies. Fifth, even in cases of very short axon branches in dWnk or other mutants with a complete absence of contralateral projecting axon collaterals, the distal part of the mutant axons still reaches the corresponding ipsilateral target area. Given that putative trophic signals would have to support axons on both sides of the VNC, it seems highly unlikely that target-derived trophic signals would only support contralateral projecting axon arbors. In summary, the data provide strong evidence that Wnk kinases have dual roles: first, during developmental axon morphogenesis and, second and independently, during continuous axon maintenance in mature neurons (Izadifar, 2021).

A key question raised by these results is: how similar are the molecular processes in developmental axon growth and branching and adult maintenance? A related question has been discussed in a hallmark review (Raff, 2002). This perspective article discussed the discovery that neurons can activate a self-destructive program independent of general apoptosis. The authors noted that neurons 'apparently have a second, molecularly distinct self-destruct program in their axon.' And the authors raise the incisive question: 'Do neurons also use this second program to prune their axonal tree during development and to conserve resources in response to chronic insults?' (Izadifar, 2021).

It is assumed that 'pruning of axonal branches during development' could-as a cellular mechanism-also be involved in axonal branch patterning as analyzed in this study. Specifically, the developmental axon branching of mechanosensory axons is viewed as a process where continuous competitive interactions among nascent branches select for stabilization or retraction (timescale minutes or few hours). In contrast, the well-characterized pruning of axons during metamorphosis (e.g., mushroom body remodeling) is initiated when axon branches and connectivity have been already established. This type of pruning (remodeling) requires primarily a destabilization of a fully established axon projection and is likely different from axonal branch selection as investigated in this study. Consistent with this idea is the finding that Nmnat is not required in axon pruning of mushroom body neurons (Izadifar, 2021).

The results now provide genetic and molecular data that support the notion that components of a 'distinct self-destruct program' are involved in axon branch stabilization and destabilization. It is further suggested that molecular control mechanisms of axon branching and axon destruction (or preventing axon destruction, i.e., axon maintenance) are mechanistically related. Specifically, the dynamics of axon branching requires the selection of an exuberant number of nascent axon branches by either stabilizing or destabilizing nascent branches. During axon morphogenesis, the majority of filopodia and nascent branches are retracted to maintain just a few that are consolidated into axon collaterals. It seems plausible, therefore, that axon branch retraction in developing neurons might involve molecular effectors that are also required for a distinct type of axon branch pruning. Based on in vitro studies, it is speculated that regulation of de novo protein synthesis could be the molecular process that is targeted in both axon branching and axon maintenance (Izadifar, 2021).

Based on new findings, it is suggested that Wnk as well as Nmnat are necessary components of axon branching as well as axon maintenance. Support for this model comes from the results of our genetic epistasis analysis. The loss of dWnk during axon branching is only a problem if Sarm or Axed are present: in the absence of Axed (the most downstream effector of the destruction program in Drosophila known so far), loss of dWnk does not cause defects in axonal branching or maintenance. Implicit to this model is that dWnk is unlikely instructing axon branching but rather provides a safeguard function curbing destructive effectors such that retraction, pruning, or branch destruction can be restricted and constrained in a spatially restricted manner. A further implication of this model is the notion that loss of dWnk effectively represents a gain of function (on switch) of an axonal destruction program in developing axons (Izadifar, 2021).

In this context, it is also interesting to note that expression of Nmnat can compensate (i.e., rescue) for loss of dWnk in both axon branching and maintenance. It is well established that local depletion of Nmnat in adult neurons does directly lead to axon degeneration, i.e., has a role in axon maintenance independent of its developmental role. This is consistent with the new finding that post-developmental inactivation of dWnk or mammalian Wnk1/2 triggers axon degeneration (Izadifar, 2021).

Nmnat has been previously shown to protect from axon degeneration following axotomy by counter-acting Sarm-induced NAD+ depletion and Axed activity. This study shows that, even in the absence of axon injury, loss of Nmnat as well as loss of dWnk or overexpression of dSarm and Axed leads to progressive degeneration of adult mechanosensory axons without injury. This is consistent with previous reports showing that Nmnat loss in sensory neurons of the wing leads to spontaneous axon degeneration in adult flies or that loss of NMNAT2 leads to truncation of peripheral nerve and CNS axon tracts in mice. A role of Wnk1 in axon maintenance is also consistent with the finding that WNK function has been linked to a severe form of peripheral neuropathy (Izadifar, 2021).

The results, therefore, support the notion that both axon morphogenesis and maintenance require a constitutive involvement of Wnk and Nmnat (Izadifar, 2021).

A link between dWnk, Nmnat, and axon destructive factors is further corroborated by biochemical experiments co-expressing these factors and using co-immunoprecipitations to analyze protein-protein interactions of dWnk or mammalian WNK1/2. First, the results suggest the possibility that dWnk, Nmnat, Sarm1, and Axed are able to form mixed complexes. Moreover, mammalian WNK1 can interact with WNK2 and Nmnat2 as well as SARM1. Although previous work has not identified Nmnat proteins as potential Wnk kinase substrates, the current results suggest that this possibility is worthwhile to examine in future experiments. Second, whereas a vertebrate ortholog of Axed has not been described, it was found that mammalian SARM1 overexpression strongly downregulates levels of WNK1, WNK2, NMNAT2, and NMNAT1. However, future experiments will have to confirm that this downregulation of proteins by Sarm1 is also occurring in neurons in vivo (Izadifar, 2021).

It has been reported that inhibition of axon degeneration can be accomplished by increasing or stabilizing levels of Nmnat protein. Moreover, Highwire/Phr1, which is an additional conserved factor functioning in Wallerian degeneration, directly promotes the downregulation of Nmnat, and axon degeneration is strongly inhibited in Highwire/Phr1 mutants. This previously described regulation of Nmnat levels is mediated via mitogen-activated protein kinase (MAPK) signaling and ubiquitin-dependent proteolysis. The findings of this study suggest that Sarm1 may rather inhibit de novo protein synthesis in order to deplete Nmnat and other axon protective factors. Future studies will have to investigate in detail how the SARM1-dependent depletion of NAD+ also leads to a Wnk/Nmnat protein depletion as described in this study. Particularly interesting will be to determine how the destructive activity of Sarm protein can be limited during development to selective axon branch compartments in order to enable local axon branch pruning but prevent progressive axon degeneration (Izadifar, 2021).

Finally, Nmnat function has not only been involved in neuroprotection as a response to injury, such as axotomy, but also in a diverse range of neurodegenerative diseases, such as spinocerebellar ataxia, fronto-temporal dementia (FTD) and Parkinsonism, or glaucomatous optic neuropathy, or following growth factor deprivation. Future studies will need to consider the possibility that the newly identified dWnk and WNK1 and WNK2 kinases may also play similar neuroprotective roles in diverse types of neurodegenerative conditions (Izadifar, 2021).

Phagocytosis and self-destruction break down dendrites of Drosophila sensory neurons at distinct steps of Wallerian degeneration

After injury, severed dendrites and axons expose the 'eat-me' signal phosphatidylserine (PS) on their surface while they break down. The degeneration of injured axons is controlled by a conserved Wallerian degeneration (WD) pathway, which is thought to activate neurite self-destruction through Sarm-mediated nicotinamide adenine dinucleotide (NAD(+)) depletion. While neurite PS exposure is known to be affected by genetic manipulations of NAD(+), how the WD pathway coordinates both neurite PS exposure and self-destruction and whether PS-induced phagocytosis contributes to neurite breakdown in vivo remain unknown. This study shows that in Drosophila sensory dendrites, PS exposure and self-destruction are two sequential steps of WD resulting from Sarm activation. Surprisingly, phagocytosis is the main driver of dendrite degeneration induced by both genetic NAD(+) disruptions and injury. However, unlike neuronal Nmnat loss, which triggers PS exposure only and results in phagocytosis-dependent dendrite degeneration, injury activates both PS exposure and self-destruction as two redundant means of dendrite degeneration. Furthermore, the axon-death factor Axed is only partially required for self-destruction of injured dendrites, acting in parallel with PS-induced phagocytosis. Lastly, injured dendrites exhibit a unique rhythmic calcium-flashing that correlates with WD. Therefore, both NAD(+)-related general mechanisms and dendrite-specific programs govern PS exposure and self-destruction in injury-induced dendrite degeneration in vivo (Ji, 2022).

Physical insults to the nervous system often disrupt neuronal connectivity and function by damaging dendritic or axonal processes of neurons. Injured axons break down through a series of stereotypical events collectively called Wallerian degeneration (WD). Dendrites undergo a similar program of degeneration after injury (AI). Before neurons can regenerate their processes and restore connections, the debris from damaged neurites has to be promptly cleared by phagocytes, which are cells that engulf dead cells or cell debris. Inefficient clearance can lead to neuroinflammation and further exacerbate the damage to the surrounding tissues. Although WD is mainly considered to be a neurite-intrinsic, self-destructive process, whether phagocytosis actively contributes to degeneration of injured neurites in vivo, rather than merely passively removing neuronal debris, remains unclear (Ji, 2022).

WD is governed by an evolutionarily conserved pathway, which is also called 'axon-death' pathway, because it was discovered in studies focused primarily on axon degeneration in Drosophila and rodents. In this pathway, injury induces activation of the E3 ubiquitin ligase Highwire/Phr1 in severed axons, which in turn causes degradation of nicotinamide mononucleotide adenyltransferase (Nmnat), an enzyme required for the synthesis of nicotinamide adenine dinucleotide (NAD+). The decrease of NAD+ resulting from Nmnat degradation, together with an accumulation of the NAD+ precursor nicotinamide mononucleotide (NMN), activates Sarm/SARM1, a sterile alpha/Armadillo/Toll-Interleukin receptor homology domain protein. Due to its NADase activity, Sarm/SARM1 is thought to subsequently cause local NAD+ depletion in injured axons. In Drosophila, axundead (axed) is required downstream of Sarm for axon degeneration of olfactory receptor neurons and wing sensory neurons. The loss of axed blocks axon degeneration even when Sarm is dominantly activated, suggesting that Axed activation is a key switch of WD. In addition, pebbled (peb) encodes a Drosophila transcription factor required for axon degeneration of glutamatergic but not cholinergic sensory neurons in the wing. Although how exactly Axed and/or NAD+ depletion lead to axon breakdown is still mysterious, it is generally believed that Sarm activity initiates a neurite-intrinsic self-destruction program that ultimately is responsible for WD of axons. While the WD pathway is primarily characterized in axons, evidence suggests that NAD+ reduction is also an essential step in injury-induced dendrite degeneration. However, which components of the WD pathway are conserved in dendrites remains unknown (Ji, 2022).

Neuronal debris is recognized by resident phagocytes of the nervous system through specific 'eat-me' signals exposed on the neuronal surface. A highly conserved eat-me signal is phosphatidylserine (PS), a negatively charged phospholipid normally found in the inner leaflet of the plasma membrane of healthy cells. During apoptosis, PS is externalized to the outer leaflet of the plasma membrane to mark the cell for engulfment (Sapar, 2018). Genetic analyses of certain PS-binding bridging molecules and cell membrane receptors in mice and zebrafish suggest that PS recognition contributes to the phagocytosis of neurons. Similarly, clearance of injured axons and dendrites in Drosophila requires Draper (Drpr), an engulfment receptor that binds to PS. PS exposure has also been directly observed on injured axons of mouse neurons in vitro and on injured dendrites of Drosophila sensory neurons in vivo. Although axonal PS exposure can be induced independently of axon degeneration in vitro, ectopically induced PS exposure resulted in engulfment-dependent neurite reduction of otherwise healthy neurons in both the central nervous system and the peripheral nervous system (PNS) of Drosophila, pointing to a dominant effect of PS exposure in inducing phagocytosis. Recent studies revealed a link between neuronal PS exposure and the WD pathway. Overexpression (OE) of WldS, a fusion protein containing the full-length murine Nmnat1, in Drosophila sensory neurons suppresses PS exposure of injured dendrites. In addition, Sarm1 ablation and NAD+ supplementation in neuronal culture reduce PS exposure on injured axons. These observations raise two important questions: How does the WD pathway regulate and coordinate both neurite PS exposure and self-destruction? What are the relative contributions of PS-mediated phagocytosis and neurite self-destruction in WD in vivo (Ji, 2022)?

To address these questions, Drosophila class IV dendritic arborization (C4da) neurons on the larval body wall, an established in vivo model of injury-induced dendrite degeneration, were used. In this system, degenerating dendrites of C4da neurons are phagocytosed by epidermal cells through the engulfment receptor Drpr. This study shows that PS exposure and dendrite self-destruction are two distinct steps downstream of Sarm activation and that in vivo, phagocytosis is the main driving force of dendrite degeneration induced by injury and Nmnat loss-of-function (LOF). Furthermore, unlike in axons, Axed is not required for dendrite degeneration: it contributes to injury-induced dendrite self-destruction but is not involved in dendrite PS exposure. Lastly, dendrite degenerations induced by injury and Nmnat LOF differ in phagocytosis dependency, membrane disruption, and dendrite calcium dynamics. Injured dendrites exhibit a unique rhythmic calcium-flashing, which is disrupted by WldS OE and axed loss, indicating a potential role of calcium-flashing in dendrite self-destruction (Ji, 2022).

This study investigated mechanisms of dendrite degenerations caused by injury and genetic activation of the WD pathway in the Drosophila PNS. Although neurite phagocytosis has been observed after neuronal injury both in vivo and in vitro, WD is generally considered as a result of neurite self-destruction triggered by NAD+ depletion. However, the current results using engulfment-deficient drpr LOF strongly suggest that PS-mediated phagocytosis is the main driving force for WD-related dendrite degeneration in vivo. It is solely responsible for dendrite degeneration of Nmnat KO neurons and greatly accelerates the degeneration of SarmGOF OE neurons. In injury, phagocytosis is responsible for at least half of the dendrite fragmentation by 10 h AI and may contribute more to dendrite breakdown than self-destruction at earlier stages. Supporting this idea, ectopic PS exposure on injured dendrites is sufficient to revert the blockage of dendrite fragmentation by WldS OE or Sarm KO, even though ectopically induced PS exposure is much lower than natural PS exposure on injured dendrites. Time-lapse analyses of PS exposure, the final calcium surge, and dendrite rupture also support the possibility that PS-mediated phagocytosis breaks down injured dendrites at the time of dendrite fragmentation. Therefore, at least in the context of dendrite injury, phagocytosis is the major factor driving WD, while self-destruction acts as a secondary mechanism to ensure complete fragmentation (Ji, 2022).

NAD+ reduction is thought to be responsible for neuronal PS exposure and neurite self-destruction during WD. How may NAD+ reductions coordinate the two different events? The results of this study support a hypothesis that NAD+ disruption controls PS exposure and neurite self-destruction in two separate steps of WD. In the existing model, Sarm activation is believed to cause catastrophic NAD+ depletion that is sufficient to initiate neurite self-destruction. However, this study found that downstream of Sarm activation and before the initiation of self-destruction, neurites first expose PS to engage in phagocytosis-mediated nonautonomous degeneration. Therefore, in a revised model, dendrites respond to at least three distinct, increasingly severe levels of NAD+ reduction by eliciting different molecular events. Between the NAD+ level required for Sarm activation (SA level) and the level that initiates self-destruction (the self-destruction level), Sarm activity lowers NAD+ to a level that causes neurons to expose PS on their surface (which is called the PSE level). This PS exposure is sufficient to cause phagocytosis-mediated dendrite degeneration, which can be completely prevented by blocking engulfment activity of phagocytes. However, below the self-destruction level, neurites spontaneously fragment even in the absence of phagocytosis (Ji, 2022).

This model is consistent with an apparent correlation between the expected kinetics of NAD+ reduction and the severity of neurite degeneration. Nmnat KO is expected to cause slow NAD+ reduction, due to gene perdurance and the time required for natural NAD+ turnover, and correspondingly causes engulfment-dependent dendrite degeneration only in late third instar larvae. In contrast, SarmGOF OE should lead to a more-rapid NAD+ depletion and in fact causes engulfment-dependent dendrite degeneration as early as the first instar and dendrite self-destruction by the third instar. Injury is known to cause even more rapid NAD+ reduction in axons (20) and is correlated with the fastest dendrite degeneration-initiation at around 4 h AI and completion usually by 10 h AI. Directly validating this model will likely require a sensitive NAD+ indicator that can measure NAD+ levels in dendrites in vivo (Ji, 2022).

How may NAD+ reduction cause PS exposure? A direct consequence of NAD+ loss is the decline of neurite ATP levels due to the requirement of NAD+ in glycolysis and oxidative phosphorylation. Consistent with ATP reduction playing a role in inducing PS exposure, suppressing mitochondria ATP synthesis in dorsal root ganglia (DRG) culture caused gradual axonal PS exposure. However, how ATP reduction may induce PS exposure remains elusive. Although the maintenance of membrane PS asymmetry by flippases requires ATP, flippase KO in C4da neurons causes a much-milder PS exposure than injury, suggesting that mechanisms other than flippase inhibition must be contributing to the rapid PS exposure seen AI. Identifying the PS transporters responsible for PS exposure on injured neurites will be a key step for revealing the mechanisms of NAD+ regulation of PS exposure (Ji, 2022).

Genetic analyses in Drosophila identified Axed as a key switch of WD, whose activity is absolutely required for axon degeneration caused by injury and Sarm GOF. How does Axed regulate neurite degeneration? The data suggest that Axed is not required for PS-mediated phagocytosis but contributes to the self-destruction of injured dendrites, placing its activation below the self-destruction level of NAD+ in the model. Surprisingly, Axed seems to play a minor role in dendrite degeneration, as its LOF only slowed down but did not block self-destruction, indicating the existence of other factors that promote self-destruction of injured dendrites (Ji, 2022).

By exploring the different mechanisms employed in Nmnat KO- and injury-induced dendrite degenerations, dynamic calcium activities were discovered that are only present in injured dendrites, including a calcium-flashing pattern prior to any obvious degenerative event and a final calcium surge that coincides with dendrite fragmentation. Calcium surge at the time of neurite fragmentation is a shared feature between injured axons of zebrafish and injured dendrites of Drosophila da neurons. Although calcium influx is required for WD and may activate calcium-dependent lipid scramblases, time-lapse analyses suggest that the final calcium surge is more likely a result of phagocytosis-induced membrane rupture rather than the cause of fragmentation. In comparison, the calcium-flashing soon after the injury is unique to dendrites and may play an active role in dendrite degeneration in ways similar to the compartmentalized calcium-flashing that occurs during developmental pruning of C4da neurons. Consistent with this possibility, the calcium-flashing is suppressed by WldS OE and axed KO. Interestingly, these two manipulations block the calcium-flashing in opposite ways, with WldS OE dampening the calcium level and axed KO keeping the calcium level high. This distinction may be a useful clue for understanding the regulation of PS exposure and self-destruction. For example, it is possible that elevated calcium levels prepare dendrites for PS exposure and drastic changes of calcium levels promote dendrite self-destruction (Ji, 2022).

Previous studies have shown that axons and dendrites share common features in neurite degeneration: they both undergo PS exposure and WD AI, they are both subject to PS exposure-induced degeneration, and injury-induced PS exposure and degeneration of both can be blocked by manipulations to increase NAD+ levels. These analyses on Nmnat and Sarm additionally show that dendrites are similar to axons in SARM-dependent spontaneous degeneration associated with NMNAT deficiency. Unexpectedly, the results also reveal important differences between dendrites and axons. First, while Axed is absolutely required for axon degeneration in injury and in Sarm GOF, it is only partially involved in dendrite degeneration AI. Second, calcium-flashing is found in dendrites that are injured or undergoing developmental pruning but not in injured axons. These results suggest that dendrites and axons utilize both shared and neurite type-specific programs in degeneration. Lastly, as impairment of NAD+ metabolism is a general feature of neurodegenerative disorders including Leber congenital amaurosis, Alzheimer's disease, Parkinson's disease, and retinal degenerations, phagocytosis may play important roles in the pathogenesis of these diseases through dysregulated neuronal PS exposure (Ji, 2022).

Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress

Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a conserved enzyme in the NAD synthetic pathway. It has also been identified as an effective and versatile neuroprotective factor. However, it remains unclear how healthy neurons regulate the dual functions of NMNAT and achieve self-protection under stress. This study shows that Drosophila Nmnat (DmNmnat) is alternatively spliced into two mRNA variants, RA and RB, which translate to protein isoforms with divergent neuroprotective capacities against spinocerebellar ataxia 1-induced neurodegeneration. Isoform PA/PC translated from RA is nuclear-localized with minimal neuroprotective ability, and isoform PB/PD translated from RB is cytoplasmic and has robust neuroprotective capacity. Under stress, RB is preferably spliced in neurons to produce the neuroprotective PB/PD isoforms. These results indicate that alternative splicing functions as a switch that regulates the expression of functionally distinct DmNmnat variants. Neurons respond to stress by driving the splicing switch to produce the neuroprotective variant and therefore achieve self-protection (Ruan, 2015).

This study identified a critical role of alternative splicing in regulating the maintenance capacity of the nervous system. The results demonstrated that, through alternative splicing, two sets of protein products are generated from the DmNmnat gene. PC translated from RA is nuclear-localized, has strong 'holdase' activity, minimum refolding activity and no neuroprotective activity against hAtx-1[82Q]-induced neurodegeneration. In contrast, PD translated from RB is localized to the cytoplasm, has strong refolding activity and robust protective activity against hAtx-1[82Q]-induced neurodegeneration. Since RA and RB are mutually exclusive, alternative splicing functions as a switch in DmNmnat expression between RA and RB: RA to produce nuclear protein isoforms, and RB to produce cytoplasmic neuroprotective factors. Importantly, this switch is regulated by stress in the nervous system. Under normal conditions, both RA and RB are transcribed, with slightly more RA (RA:RB=1.46:1); therefore, both PC and PD proteins are expressed to sustain basic physiological needs. When neurons are under stress from either external (heat or hypoxia) or internal (proteotoxic) origin, the transcription of the DmNmnat gene is upregulated and the splicing of pre-mRNA is shifted to RB, therefore allowing the production of PD with robust neuroprotective activity. Such regulation at the level of pre-mRNA splicing allows neurons to quickly respond to stress and achieve self-protection (Ruan, 2015).

Stress response is a key process for all cells to maintain homeostasis. Central to the stress response is the increased synthesis of molecular chaperone (HSPs) that function to prevent protein misfolding and aggregation to maintain protein homeostasis. On the onset of stress, the transcription of HSPs is quickly initiated by stress transcription factors such as HSFs or hypoxia-inducible factors. The mRNA transcripts of inducible HSPs are often labile to facilitate rapid downregulation of the expression of HSP proteins after stress. Indeed, the half-life of Hsp70 or Hsp26 mRNA is less than 1 h, much shorter than those of some of the housekeeping genes; for instance, over 8 h for rp49 mRNA60. Previously, it was shown that DmNmnat is a stress-response factor and its transcription is upregulated under stress by the stress transcription factor HSF, similar to HSPs35. Interestingly, this study found that only RB is increased under stress, and DmNmnat variants have very different half-lives: RA has a long half-life of 16.1 h, and RB has short half-life of 2.97 h, which is further reduced to 2.04 h under stress. Therefore, the transcription and regulation patterns of RA and RB fit the profiles of housekeeping genes and stress-response factors, respectively. Collectively, these findings present a model in which DmNmnat assumes two identities through alternative splicing: a housekeeping variant that is constitutively expressed with a long half-life and a stress-response variant that exhibits increased expression of unstable transcripts under stress. This mode of post-transcriptional regulation increases cellular tolerance to stress without adding new genes (Ruan, 2015).

Mammals have three Nmnat genes, Nmnat1, -2 and -3, with distinct tissue expression and subcellular localizations. It is possible that mammals achieve functional diversity of NMNAT through gene duplication rather than alternative splicing. For example, Human NMNAT1 and -2 are highly expressed in the nervous system, and NMNAT1 protein is nuclear-localized while NMNAT2 is cytoplasmic. Both NMNAT1 and -2 have NAD synthetic activity and have been shown to be neuroprotective when expressed in the cytoplasm. Nuclear-localized NMNAT1 plays a role in preventing cell death after DNA damage, as its NAD synthetic activity is essential for the function of DNA repair enzymes such as PARP-1 . It is likely that the PC isoform is required in the nucleus for NAD metabolism, a hypothesis that requires experimental confirmation (Ruan, 2015).

It is worth noting that the mechanism of transcriptional regulation by alternative splicing may be conserved between Drosophila and mammalian Nmnat homologues, as all three human NMNAT genes are multiexonic and predicted to be alternatively spliced. In a recent study, two splice variants of human NMNAT3 (v1 and V3-FKSG76) have been experimentally identified. It will be intriguing to investigate the alternative splicing and transcriptional regulation of human NMNAT1 and -2 in the nervous system. Exploring the role of alternative splicing in regulating neuronal maintenance and protection will prove both interesting and promising for the discovery of new therapeutic strategies for neuroprotection (Ruan, 2015).

The regulation of alternative splicing occurs in multiple ways, including developmental stage-specific, sex-specific or tissue-specific manners and even in response to environmental or cellular (ER) stress. The current findings also indicate that differential splicing events of DmNmnat could occur in a cell-type-specific manner. It has been reported that cell-specific alternative splicing not only participates in neuronal development, but is also involved in human diseases such as cancer and neurodegeneration. Recent studies have revealed that cell-specific alternative splicing in vertebrates involves repression in the inappropriate cell type as well as activation in the appropriate cell type. Future work is required to identify the molecular components in the splicing machinery that sense and respond to stress in a cell-specific manner (Ruan, 2015).

Maintaining neuronal homeostasis is a prerequisite for proper neurological activity. Healthy neurons are able to maintain their integrity throughout the life of an animal, suggesting the existence of a maintenance mechanism that allows neurons to sustain, mitigate or even repair internal or external damage. The identification of alternative splicing functioning as a switch to enhance the neuroprotective role of DmNmnat indicates that the neuronal maintenance capacity is regulated endogenously and enhanced under stress for neurons to confer self-protection and higher resistance to adverse conditions (Ruan, 2015).

SkpA restrains synaptic terminal growth during development and promotes axonal degeneration following injury

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, a large-scale genetic screen was performed in Drosophila to identify negative regulators of the pathway. This study describes 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 (Brace, 2014).

Molecular chaperones protect against JNK- and Nmnat-regulated axon degeneration in Drosophila

Axon degeneration is observed at the early stages of many neurodegenerative conditions and this often leads to subsequent neuronal loss. Previous work has shown that inactivating the c-Jun N-terminal kinase (JNK) pathway leads to axon degeneration in Drosophila mushroom body (MB) neurons. To understand this process, candidate suppressor genes were screened, and it was found that the Wallerian degeneration slow (Wld_S) protein blocked JNK axonal degeneration. Although the nicotinamide mononucleotide adenylyltransferase (Nmnat1) portion of Wld_S is required, it was found that its nicotinamide adenine dinucleotide (NAD(+)) enzyme activity and the Wld_S N-terminus (N70) are dispensable, unlike axotomy models of neurodegeneration. It is suggest that Wld_S-Nmnat protects against axonal degeneration through chaperone activity. Furthermore, ectopically expressed heat shock proteins (Hsp26 and Hsp70) also protected against JNK and Nmnat degeneration phenotypes. These results suggest that molecular chaperones are key in JNK- and Nmnat-regulated axonal protective functions (Rallis, 2013).

Wld^S was discovered from the molecular cloning of spontaneously generated slow Wallerian degeneration (Wld^S) mutant mice that showed a strong capacity to promote axonal survival following acute physical lesion. The Wld^S protein has neuroprotective effects across different species and in different neurodegeneration models. The Wld^S gene product results from the fusion of first 70 residues of the UBE4B gene (N70), that is involved in polyubiquitination, with the entire nicotinamide mononucleotide adenylyltransferase protein sequence (Nmnat1) that is involved in nicotinamide adenine dinucleotide (NAD⁺) biosynthesis. Different portions of Wld^S can confer neuroprotective function (Coleman, 2010). However, Wld^S function remains unclear. For example, despite its predominant nuclear localisation, it is axonal localisation that appears to be key to neuroprotection, even though Wld^S and different Nmnat isoforms have subtle and distinct subcellular locations. Also, while in many neurodegenerative paradigms the Nmnat enzyme activity is essential, it is unclear how the NAD⁺ pathway contributes to axonal protection. Furthermore, some studies suggest Nmnat neuroprotective functions are enzyme-independent. To date, the relationship between Wld^S function(s) and axon-neuronal damage and repair also remains unclear, although recent data suggest Wld^S-Nmnat regulation of mitochondrial motility and calcium buffering functions may underlie key neuroprotective responses to physical injury in Drosophila and mouse axons. A further report suggests Drosophila Nmnat (dNmnat or nmnat) also controls axonal mitochondria levels and their availability is key to neuroprotection following acute injury (Fang, 2012). Previous data suggest Wld^S-Nmnat localisation within mitochondria may also be the underlying basis of axonal neuroprotection (Rallis, 2013).

When tested ectopically, many Nmnat isoforms and homologs show axonal-protective effects even though some appear to be weaker, possibly due to labile effects. However, apart from Drosophila Nmnat, currently only mouse Nmnat2 has an endogenous role in promoting axonal stability. It is important to note, beyond their neuronal roles, Nmnats also have obligate roles in NAD⁺ metabolism and multiple cellular processes across species. Very recent reports show Nmnat1 mutations cause Leber congenital amaurosis (LCA), highlighting its importance in retinal degenerative diseases in humans (Rallis, 2013).

This study shows that the Wld^S protein protects against axon degeneration triggered by JNK inactivation. Contrary to previous models, while the Nmnat1 region is sufficient, this study found that its enzyme activity is dispensable for Wld^S neuroprotection. The results suggest that Nmnat and JNK axonal-protective functions occur through molecular chaperones (Rallis, 2013).

One previous report showed that Drosophila Nmnat has a non-enzyme function that involves molecular chaperone activity (Zhai, 2008). Drosophila Nmnat was recruited together with the molecular chaperone, Heat shock protein (Hsp) Hsp70 to polyglutamine expanded spinocerebellar ataxin-1 (SCA-1) containing aggregates. Non-enzyme Nmnat functions were involved in regulating protein folding and blocking SCA-1 neurotoxicity. Very recent results show non-enzyme Nmnat also functions to clear tau oligomers in vivo. This study tested the effect of Heat shock proteins (Hsps) on the bsk phenotypes in two ways. In bsk-null neuroblast clones, it was found that, like Wld^S and Nmnats1 and 3, ectopic Hsp70 or Hsp26 also blocked the bsk axon degeneration (Rallis, 2013).

Compared to wild-type axons, bsk axons showed more abnormal protrusions and swellings along the axons and terminals. When Hsp70, Wld^S, Nmnat and Nmnat enzyme-inactive forms were expressed in these clones, these were reduced suggesting that this phenotype is also linked to Hsps and non-enzyme Nmnat activities (Rallis, 2013).

To further test the neuroprotective activity of Hsps, Nmnat RNAi assays was used. When Nmnat RNAi was expressed in MB neurons, this resulted in a β-axon loss phenotype similar to nmnat¹ loss-of-function clones above. Some neuronal loss was visible in newly eclosed adults (1-day-old adults). However, almost all neurons were lost in 7-day-old adults, suggesting that Nmnat is an obligate maintenance factor, consistent with previous reports. The Nmnat RNAi axon and neuronal cell loss was rescued by enzyme-inactive forms of mNmnat1 (H24A) and Wld^S-dead. Furthermore, Hsp26 and Hsp70 expression also partially suppressed the Nmnat RNAi phenotype. Together, these results suggest non-enzyme Nmnat and chaperone activities are linked to JNK axonal functions (Rallis, 2013).

Using the GAL80ts system to control JNK temporal expression, it has been shown that JNK activity is required throughout development, even though the axon degeneration phenotype occurs mainly at adult stages. To determine Nmnat's temporal requirements, Nmnat RNAi was coupled to GAL80ts control, and the loss-of-function phenotype was induced at various stages of development. It was found that RNAi throughout the development and adult phase caused the strongest neuronal loss phenotype. RNAi induction at pupal or adult stages also caused neuronal loss, albeit at a weaker levels. These results suggest Nmnat is required throughout development as well as adult stages. Even though the Nmnat RNAi phenotype is more severe in adults, as in bsk mutants, unlike bsk, Nmnat's genetic requirements extend beyond the developmental stages and are essential at adult stages. This suggests Drosophila Nmnat may have additional roles at adult stages that may be independent of JNK activity (Rallis, 2013).

The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Using a Drosophila injury model this study has identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw, the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury (Xiong, 2012).

Since the discovery of the dramatic inhibition of degeneration by the WldS mutation, many studies have focused upon the action of the NAD+ biosynthetic enzyme isoforms, Nmnat1, Nmnat2, and Nmnat3, which in some circumstances can confer protection against axonal degeneration (reviewed in Coleman, 2010). Most of these studies involve gain-of-function overexpression experiments; it has been difficult to address the role of endogenous Nmnat enzymes in this process. Recent observations indicate that endogenous Nmnat activity plays an essential role in neuronal survival, and its depletion leads to neurodegeneration. In addition, recent studies in vertebrate neurons suggest that the cytoplasmic isoform, Nmnat2, has a short half-life in neurons. An attractive model proposes that Nmnat2 is rapidly turned over in axons, and that its loss in the distal stump of an axon, which has become disconnected from its cell body, leads to the initiation of Wallerian degeneration (Xiong, 2012).

Some aspects of this model are supported by current in vivo characterization in Drosophila. This study identfies Hiw, a highly conserved protein with features of an E3 ubiquitin ligase, as an important regulator of Wallerian degeneration. Hiw's role in this process involves the Nmnat protein, whose levels in axons and synapses are regulated post-transcriptionally by Hiw function. In hiw mutants, Wallerian degeneration is strongly inhibited, and the increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration (Xiong, 2012).

While the localization of endogenous Hiw in Drosophila is not known, homologues in mice and Caenorhabditis elegans have been detected in axons and at synapses, so it is in the appropriate location to target the destruction of Nmnat in distal axons. However, it remains to be determined whether the down-regulation of Nmnat in the distal stump per se is the trigger for Wallerian degeneration. When HA-Nmnat was overexpressed, axons were protected from degeneration long after the rapid disappearance of detectable protein in the distal stump. It is possible that even very low levels of Nmnat protein are sufficient to protect from degeneration. It is also formally possible that the basal levels of Nmnat before injury, rather than the disappearance of Nmnat after injury, is an important determinant of degeneration. It is also acknowledged that axonal degeneration likely involves additional steps downstream or in parallel to the regulation of Nmnat by Hiw. While overexpression of Hiw can induce a reduction in HA-Nmnat levels, it was not possible to observe an enhanced rate of degeneration when Hiw was overexpressed (Xiong, 2012).

Studies almost a decade ago suggested a role for the ubiquitin protease system (UPS) in the initiation of Wallerian degeneration (Zhai, 2003). It is tempting to propose that this role is manifested by the regulation of Nmnat by Hiw. However the current observations caution against a simple interpretation that Hiw regulates Nmnat via the UPS, since Hiw can promote disappearance of Nmnat protein in cells in a manner unaffected by proteasome inhibitors. Moreover, in vivo, inhibition of the proteasome had only a minor effect upon Nmnat levels in a wild-type background. However in hiw mutants, Nmnat levels were very sensitive to the function of the proteasome. It is interpreted that additional ubiquitin ligases and the UPS may regulate Nmnat independently of Hiw (Xiong, 2012).

Regardless of the role of the proteasome, the current observations suggest that ubiquitin plays an important role in Nmnat regulation. Overexpression of the yeast de-ubiquitinating protease UBP2 leads to increased levels of Nmnat protein and inhibition of Wallerian degeneration, in a manner that requires endogenous Nmnat. Future studies of the mechanism by which Hiw regulates Nmnat will therefore consider potential proteasome-independent roles of ubiquitination. Of note, in yeast UBP2 has been shown to preferentially disassemble polyubiquitin chains linked at Lys63, which have been found to perform non-proteolytic functions in DNA repair pathways, kinase activation, and receptor endocytosis. The possibility should also be considered that Hiw regulates Nmnat indirectly: since thus far it has not been possible to detect any ubiquitinated Nmnat species, it is possible that an intermediate, yet unknown, regulator of Nmnat may be the actual substrate of ubiquitination. Nevertheless, co-immunoprecipitation studies from S2R+ cells indicate that Hiw and Nmnat have the capacity to interact (Xiong, 2012).

The mechanism and cellular location of Nmnat's protective action is a highly debated subject. Observations in the literature point to both NAD+-dependent and NAD+-independent models for the strong protection by the WldS mutation (Coleman, 2010). The location of its protective action may be the mitochondria, since mitochondrially localized Nmnat can protect axons from degeneration. However golgi/endosomal localized Nmnat2 can also be protective. The findings suggest that mutation of hiw leads to an increase in the pool of endogenous Nmnat that functionally impacts degeneration (Xiong, 2012).

While the site of endogenous Nmnat function during axonal degeneration remains to be identified, this study found that the levels of ectopically expressed mouse Nmnat2 were specifically increased in the hiw mutant background. In contrast, the levels of nuclearly localized mNmnat1 or mitochondrially localized mNmnat3 were unaffected by Hiw. Since Nmnat2 has a short half-life in vertebrate neurons, it is intriguing to propose that it is regulated by Hiw orthologs via an analogous mechanism (Xiong, 2012).

Since Nmnat2 does not appear to localize to mitochondria, does this favor a non-mitochondrial activity, such as function as a chaperone, for the protective action? It remains challenging to determine the exact location of protection, since the most apparent changes in Nmnat protein may not necessarily be the functionally relevant changes (Xiong, 2012).

A previously characterized target of Hiw regulation is the Wallenda (Wnd) MAP kinase kinase kinase. This axonal kinase is also capable of inhibiting Wallerian degeneration in motoneurons. The protective action of Wnd requires a downstream signaling cascade and changes in gene expression mediated by the Fos transcription factor. Loss of nmat does not affect this signaling cascade nor does it affect the protective action of Wnd. Conversely, loss of wnd does not affect the protection caused by overexpressing nmat. Importantly, the regulation of Nmnat by Hiw does not appear to require Wnd function, and Wnd and Nmnat can protect axons independently of each other. These findings favor the model that Wnd and Nmnat are both regulated by Hiw and influence axonal degeneration through independent mechanisms (Xiong, 2012).

The Wnd kinase plays additional roles in neurons, which can be genetically separated from Nmnat function. These include regulation of synaptic growth: a dramatic synaptic overgrowth phenotype in hiw mutants is fully suppressed by mutation of wnd, but is not at all affected by knockdown of nmat. Wnd/DLK also promotes axonal sprouting in response to axonal injury, which is also unaffected by nmat knockdown. It is therefore clear that by regulating both Wnd and Nmnat, Hiw regulates multiple independent pathways in neurons (Xiong, 2012).

It is intriguing that the actions of both Wnd and Nmnat promote cellular responses to axonal injury. Axonal regeneration requires an initiation of a growth program within the axon, which depends upon the function of Wnd and its homologues. Equally important is a clearance of the distal stump to make room for the regenerating axon. Since both Wnd and Nmnat are transported in axons a model is proposed in which Hiw function in the distal axon terminal could simultaneously promote destruction of Nmnat in the distal stump, and accumulation of Wnd in the proximal stump. The latter is observed after injur, and is required to promote new axonal growth. The actual location in which Hiw regulates Nmnat remains to be determined. As an upstream regulator of both sprouting in the proximal stump and degeneration of the distal stump, Hiw may play a central role in regulating the ability of a neuron to regenerate its connection after injury (Xiong, 2012).

Importantly, the protective action of Nmnat may not be limited to Wallerian degeneration. The WldS mutation can protect neurons from degeneration in a wide variety of paradigms, from models of neurodegenerative disease, diabetic neuropathy, excitotoxity, and loss of myelination. These findings suggest that action and regulation of Nmnat function is broadly important for neuronal function and maintenance. As a critical regulator of Nmnat, the Hiw ubiquitin ligase and its vertebrate homologues deserve further scrutiny for potential roles in human health and disease (Xiong, 2012).

The role of autophagy in Nmnat-mediated protection against hypoxia-induced dendrite degeneration

The selective degeneration of dendrites precedes neuronal cell death in hypoxia-ischemia (HI) and is a neuropathological hallmark of stroke. While it is clear that a number of different molecular pathways likely contribute to neuronal cell death in HI, the mechanisms that govern HI-induced dendrite degeneration are largely unknown. This study show sthat the NAD synthase nicotinamide mononucleotide adenylyltransferase (Nmnat) functions endogenously to protect Drosophila class IV dendritic arborization (da) sensory neurons against hypoxia-induced dendritic damage. Whereas dendrites of wild-type class IV neurons are largely resistant to morphological changes during prolonged periods of hypoxia (<1.0% O₂, class IV neurons of nmat heterozygous mutants exhibit significant dendrite loss and extensive fragmentation of the dendritic arbor under the same hypoxic conditions. Although basal levels of autophagy are required for neuronal survival, this study demonstrates that autophagy is dispensable for maintaining the dendritic integrity of class IV neurons. However, it was found that genetically blocking autophagy can suppress hypoxia-induced dendrite degeneration of nmat heterozygous mutants in a cell-autonomous manner, suggestive of a self-destructive role for autophagy in this context. It was further shown that inducing autophagy by overexpression of the autophagy-specific kinase Atg1 is sufficient to cause dendrite degeneration of class IV neurons under hypoxia and that overexpression of Nmnat fails to protect class IV dendrites from the effects of Atg1 overexpression. These studies reveal an essential neuroprotective role for endogenous Nmnat in hypoxia and demonstrate that Nmnat functions upstream of autophagy to mitigate the damage incurred by dendrites in neurons under hypoxic stress (Wen, 2013).

A novel Drosophila model of nerve injury reveals an essential role of Nmnat in maintaining axonal integrity

Axons damaged by acute injury, toxic insults, or during neurodegenerative diseases undergo Wallerian or Wallerian-like degeneration, which is an active and orderly cellular process, but the underlying mechanisms are poorly understood. Drosophila has been proven to be a successful system for modeling human neurodegenerative diseases. This study established a novel in vivo model of axon injury using the adult fly wing. The wing nerve highlighted by fluorescent protein markers can be directly visualized in living animals and be precisely severed by a simple wing cut, making it highly suitable for large-scale screening. Using this model, an axonal protective function of Wld^S and nicotinamide mononucleotide adenylyltransferase (Nmnat) was confirmed. It was further revealed that knockdown of endogenous Nmnat triggered spontaneous, dying-back axon degeneration in vivo. Intriguingly, axonal mitochondria were rapidly depleted upon axotomy or downregulation of Nmnat. The injury-induced mitochondrial loss was dramatically suppressed by upregulation of Nmnat, which also protected severed axons from degeneration. However, when mitochondria were genetically eliminated from axons, upregulation of Nmnat was no longer effective to suppress axon degeneration. Together, these findings demonstrate an essential role of endogenous Nmnat in maintaining axonal integrity that may rely on and function by stabilizing mitochondria (Fang, 2012).

This study presents a novel in vivo model for axon injury and degeneration based on the adult fly wing. Using this model, the following was uncovered: (1) endogenous dNmnat is required for axonal integrity, (2) axonal mitochondria are depleted rapidly upon axotomy or downregulation of dNmnat, (3) upregulation of dNmnat preserves mitochondria in injured axons and delays Wallerian degeneration, and (4) removal of mitochondria from axons abolishes the protective effect of Wld^S and Nmnat. The levels of mNmnat2 rapidly decline in mammalian neurite culture upon injury. Drosophila has only one gene encoding Nmnat, and axon degeneration was observed as early as 18 hr after axotomy, suggesting a rapid turnover of dNmnat. Hence, reduction of Nmnat levels, either by rapid turnover of Nmnat upon axotomy or genetic knockdown of dNmnat, may render mitochondria unstable and/or dysfunctional, thus triggering axon degeneration. The self-destructive mechanisms of axon degeneration in injury and loss of endogenous Nmnat appear to converge at axonal mitochondria. This may underlie the morphological similarity between Wallerian degeneration and spontaneous axon degeneration in dying-back diseases. As such, endogenous Nmnat and axonal mitochondria may be key to identifying additional downstream events and therefore providing exciting new targets for therapeutic interventions of both acute neural injury and chronic axonal disorders (Fang, 2012).

NMNAT suppresses tau-induced neurodegeneration by promoting clearance of hyperphosphorylated tau oligomers in a Drosophila model of tauopathy

Tauopathies, including Alzheimer's disease, are a group of neurodegenerative diseases characterized by abnormal tau hyperphosphorylation that leads to formation of neurofibrillary tangles. Drosophila models of tauopathy display prominent features of the human disease including compromised lifespan, impairments of learning, memory and locomotor functions and age-dependent neurodegeneration visible as vacuolization. This study used a Drosophila model of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), in order to study the neuroprotective capacity of a recently identified neuronal maintenance factor, nicotinamide mononucleotide (NAD) adenylyl transferase (NMNAT), a protein that has both NAD synthase and chaperone function. NMNAT is essential for maintaining neuronal integrity under normal conditions and has been shown to protect against several neurodegenerative conditions. However, its protective role in tauopathy has not been examined. This study shows that overexpression of NMNAT significantly suppresses both behavioral and morphological deficits associated with tauopathy by means of reducing the levels of hyperphosphorylated tau oligomers. Importantly, the protective activity of NMNAT protein is independent of its NAD synthesis activity, indicating a role for direct protein-protein interaction. Next, it was shown that NMNAT interacts with phosphorylated tau in vivo and promotes the ubiquitination and clearance of toxic tau species. Consequently, apoptosis activation was significantly reduced in brains overexpressing NMNAT, and neurodegeneration was suppressed. This report on the molecular basis of NMNAT-mediated neuroprotection in tauopathies opens future investigation of this factor in other protein foldopathies (Ali, 2012).

Nmnat exerts neuroprotective effects in dendrites and axons

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. This study shows 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 nmat 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 nmat heterozygotes, complete loss of nmat function causes severe axonal degeneration, demonstrating differential requirements for nmat 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 nmat show progressive defects in both dendrites and axons. These 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 (Wen, 2011).

Nicotinamide mononucleotide adenylyltransferase is a stress response protein regulated by the heat shock factor/hypoxia-inducible factor 1α pathway

Stress responses are cellular processes essential for maintenance of cellular integrity and defense against environmental and intracellular insults. Neurodegenerative conditions are linked with inadequate stress responses. Several stress-responsive genes encoding neuroprotective proteins have been identified, and among them, the heat shock proteins comprise an important group of molecular chaperones that have neuroprotective functions. However, evidence for other critical stress-responsive genes is lacking. Recent studies on the NAD synthesis enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) have uncovered a novel neuronal maintenance and protective function against activity-, injury-, or misfolded protein-induced degeneration in Drosophila and in mammalian neurons. This study shows that NMNAT is also a novel stress response protein required for thermotolerance and mitigation of oxidative stress-induced shortened lifespan. NMNAT is transcriptionally regulated during various stress conditions including heat shock and hypoxia through heat shock factor (HSF) and hypoxia-inducible factor 1α in vivo. HSF binds to nmat promoter and induces NMNAT expression under heat shock. In contrast, under hypoxia, HIF1α up-regulates NMNAT indirectly through the induction of HSF. These studies provide an in vivo mechanism for transcriptional regulation of NMNAT under stress and establish an essential role for this neuroprotective factor in cellular stress response (Ali, 2011).

NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration. This study shows that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. These results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. These studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions (Zhai, 2008).

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 nmat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. This study used the visual system of Drosophila as a model system to address these issues. The first nmat mutations in a multicellular organism were isolated in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmat. These 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 (Zhai, 2006).

The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons

Neuron-glia communication is central to all nervous system responses to trauma, yet neural injury signaling pathways remain poorly understood. This study explores cellular and molecular aspects of neural injury signaling in Drosophila. Transected Drosophila axons undergo injury-induced degeneration that is morphologically similar to Wallerian degeneration in mammals and can be suppressed by the neuroprotective mouse Wallerian degeneration slow (Wld^s) protein. Axonal injury elicits potent morphological and molecular responses from Drosophila glia: glia upregulate expression of the engulfment receptor Draper, undergo dramatic changes in morphology, and rapidly recruit cellular processes toward severed axons. In draper mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the CNS. Thus Draper appears to act as a glial receptor for severed axon-derived molecular cues that drive recruitment of glial processes to injured axons for engulfment (MacDonald, 2006).

Whether severed Drosophila axons undergo Wallerian degeneration and whether Drosophila glia respond to axon injury were tested. This study used the adult olfactory system to study neuron-glia interactions following nerve transection. This tissue is well-defined histologically and a number of useful reagents are available to label and manipulate olfactory receptor neurons (ORNs) and glia (MacDonald, 2006).

ORN cell bodies are housed in the third antennal segments or maxillary palps of adult Drosophila with axons projecting to the antennal lobe of the brain via the antennal or maxillary nerve, respectively. Axon projections from ORNs expressing the same odorant receptor (OR) gene converge on common, spatially discrete glomerular targets in the antennal lobe. Conveniently, these subsets of ORNs and their axons can be labeled and genetically manipulated using a number of available OR gene promoter-Gal4 driver lines, which drive UAS-regulated expression in highly reproducible subsets of ORNs. Glial cells in the antennal lobe can be identified based on their expression of the reversed polarity (repo) gene and labeled or manipulated using the repo-Gal4 driver. Glial cell nuclei were found at the edge of the antennal lobe neuropil; glial membranes delineate the boundaries of the antennal lobe and extend into the neuropil where they ensheath individual glomeruli (MacDonald, 2006).

Axon injury was induced by nonlethal surgical ablation of third antennal segments or maxillary palps. This ablation completely removed ORN cell bodies and fully transected the antennal or maxillary nerve, respectively. Degeneration of severed ORN axons and glial responses to axonal injury were monitored in vivo for several weeks after injury. For the majority of the experiments the OR22a-Gal4 driver was used to label subsets of antennal ORNs, or OR85e-Gal4 was used to label subsets of maxillary palp ORNs; however, similar results were obtained using additional OR-Gal4 driver lines (MacDonald, 2006).

Attempts were made to determine the time course and morphology of ORN axon degeneration after nerve transection. Three markers were used to visualize axons: UAS-mCD8::GFP (axonal membranes); UAS-α-tubulin::GFP (axonal microtubule cytoskeleton); and UAS-n-Synaptobrevin::YFP::YFP::YFP (UAS-n-Syb::3XYFP) (axon terminals). In control uninjured animals, OR22a⁺ axons (labeled with mCD8::GFP) had a smooth morphology as they projected across the antennal lobe, and GFP intensity in glomeruli was very strong. However, 1 day after ablation of third antennal segments, axon fibers outside glomeruli appeared highly fragmented and were undetectable at 3 or 5 days after injury. GFP signals in the OR22a-innervated glomeruli (termed DM2) remained near control levels 1 day after injury and were reduced to ~30% of control levels 3 days after injury, and nearly all GFP⁺ material was cleared from the CNS within 5 days. A similar profile of axon removal was observed with α-tubulin::GFP-labeled axons. It was found that axonal fibers were largely fragmented 1 day after injury and were absent by 3 days after injury. Interestingly, 1 day after injury, GFP signals within DM2 glomeruli were already reduced to ~50% of control levels. This observation suggests that the microtubule cytoskeleton may degenerate more rapidly than axonal membranes. GFP signals within DM2 glomeruli were near 15% of control levels; by 5 days after injury nearly all α-tub::GFP was cleared from the CNS. Finally, when OR22a⁺ axon terminals were marked with n-Syb::3XYFP, it was found that clearance of YFP⁺ axonal material occurred within 9 days after injury. Thus, axonal membranes and the microtubule cytoskeleton rapidly degenerate when Drosophila ORN axons are severed, clearance of axonal material within the DM2 glomerulus (i.e., membrane and cytoskeletal elements) was slightly delayed relative to axons outside glomeruli, and nearly all degenerating GFP⁺/YFP⁺-labeled axonal material was cleared from the antennal lobe within 5–9 days after axons were severed. A similar time course and morphology of axon fragmentation was observed when ORN axons were marked with the OR85e-Gal4 driver and ablated maxillary palps (MacDonald, 2006).

Axon degeneration appears to occur simultaneously in all portions of the severed axon rather than in a linear fashion along the length of the axon. For example, maxillary palp ORN axons in the maxillary nerve are closest to the site of injury (i.e., the ablated maxillary palp), while midline-crossing ORN axons within the antennal lobe commissure are the most distal to the site of injury. OR85e⁺ maxillary palp ORNs were labeled with mCD8::GFP, ablated maxillary palps, and it was found that bright GFP⁺ puncta (indicative of fragmentation) first appeared ~4 hr after injury. This occurred coincidently in the maxillary nerve and in midline crossing axons, suggesting that axon fragmentation can be initiated at any point along the axon. It was also found that axon destruction was specific to severed ORN axons. For example, if OR85e⁺ maxillary palp ORNs were labeled with mCD8::GFP and then ablated third antennal segments, OR85e⁺ ORN axons showed normal morphology even 9 days after injury. At this time point all antennal ORN axons were removed from the antennal lobe. Thus the cellular mechanisms mediating clearance of severed axons can discriminate between healthy and injured neurons, which suggests that severed axons are autonomously tagged for clearance from the CNS (MacDonald, 2006).

The above data show severed Drosophila axons undergo injury-induced fragmentation that is morphologically similar to mammalian Wallerian degeneration. How similar are these events at the molecular level? Wallerian degeneration had long been thought to represent a simple wasting away of the severed axons due to a lack of nutrients supplied by the cell body. However, when nerves are transected in the spontaneous mutant mouse line C57BL/Wld^s (for Wallerian degeneration slow) severed axons survive in a functionally competent state for weeks after injury (Glass, 1993; Lunn, 1989). This observation suggests that severed axons, rather than simply wasting away, may use an active autodestruction program to drive degeneration. Axon-sparing activity in the C57Bl/Wld^s line has recently been found to map to a novel chimeric protein termed Wld^s, which is generated from the fusion of two genes: Ube4b, an E4 ubiquitin ligase, and Nmnat1, a nicotinamide mononucleotide adenylyltransferase (see Drosophila Nmnat), a NAD⁺ biosynthetic and salvaging enzyme (Conforti, 2000; Mack, 2001). To determine whether Wld^s-modulated mechanisms regulate axon degeneration in Drosophila, the ability of mouse Wld^s to protect severed Drosophila axons from injury-induced degeneration was tested (MacDonald, 2006).

UAS-Wld^s and UAS-mCD8::GFP were coexpressed in antennal ORNs using OR22a-Gal4, ablated third antennal segments, and subsequently scored axon morphology. Control animals (OR22a-Gal4 driving only UAS-mCD8::GFP) exhibited a normal rate of axon degeneration with all mCD8::GFP being absent by 5 days after injury. In striking contrast, Wld^s+ axons did not undergo Wallerian degeneration: animals bearing a single copy of the UAS-Wld^s transgene exhibited normal mCD8::GFP fluorescence in severed ORN axons 5 days after injury, and Wld^s+ axons maintained normal morphology. Neuroprotective effects of Wld^s was further explored by labeling axon terminals (with n-Syb::3XYFP) and the axonal microtubule cytoskeleton (with α-tubulin::GFP) and inducing injury. Impressively, in severed Wld^s+ axons, n-Syb::3XYFP signals appeared morphologically normal and YFP intensity remained at control levels, even 9 days after injury. Similarly, in Wld^s+ axons α-tubulin::GFP labeling appeared morphologically normal 5 days after injury, and GFP intensities in glomeruli remained at levels comparable to uninjured animals. Similar levels of protection of axons and their terminals were found with an independent UAS-Wld^s insertion line (MacDonald, 2006).

How long can Wld^s protect severed Drosophila axons? Severed axon morphology was examined in Wld^s+ ORNs at 20, 30, and 50 days after injury. Remarkably, even 20 or 30 days after antennal ablation, Wld^s+ ORN axons maintained largely normal morphology, and GFP intensity within glomeruli remained close to control levels. By 50 days after injury, the axons of Wld^s+ neurons had degenerated significantly; only ~20% of antennal lobes contained detectable GFP⁺ axonal fibers, but mCD8::GFP signals within glomeruli remained at ~35% of control levels. These observations indicate that mouse Wld^s can protect severed Drosophila axons from Wallerian degeneration for weeks, but severed Drosophila axons ultimately degenerate between 30–50 days after injury. It was also noted that these data show that mCD8::GFP is stable in axons for up to 50 days in the absence of transcription (MacDonald, 2006).

In vitro studies with mammalian DRG neurons suggest that (Nicotinamide mononucleotide adenylyltransferase 1) Nmnat1 provides the activity essential for Wld^s-mediated neuroprotection (Araki, 2004; Wang, 2005). To determine whether Drosophila Nmnat (dNmnat) could protect severed axons from Wallerian degeneration in vivo, a UAS-dNmnat transgenic line was generated, dNmnat was coexpressed in OR22a⁺ ORNs with mCD8::GFP, ablated third antennal segments, and axon morphology was assayed 5 days after injury. dNmnat was found to protect severed axons from Wallerian degeneration 5 days after injury. The efficacy of protection may be reduced compared to Wld^s, since 5 days after injury axon degeneration occurs at some level in dNmnat⁺, but not Wld^s+, axons. Nevertheless, these data argue that Wld^s-mediated axon protection in vivo occurs through dNmnat-dependent mechanisms. Together these data show that severed Drosophila axons undergo Wallerian degeneration, and the observation that Wld^s potently suppresses this process in Drosophila argues strongly that Wld^s-modulated mechanisms of severed axon autodestruction are conserved in Drosophila and mammals (MacDonald, 2006).

Glial cells are responsible for mediating postinjury events in the mammalian nervous system, but glial responses to injury have not been explored in Drosophila. This study explored morphological and molecular responses of Drosophila antennal lobe glia to axon injury. In control, uninjured animals, glial membranes were found to delineate the borders of the antennal lobe and were intimately associated with antennal lobe glomeruli. However, 1 day after antennal ablation, glia exhibited dramatic changes in morphology and appeared to significantly increase their membrane surface area. This expansion of glial membranes appeared to be a local response since antennal lobe glia, but not glia in surrounding brain regions, responded to ablation of third antennal segments in this way. During this injury response glial nuclei consistently remained at the periphery of the antennal lobe, indicating that glia responded to ORN injury by extending membranes toward severed axons rather than by migrating as an entire cell into the antennal lobe (MacDonald, 2006).

Mammalian microglia and Schwann cells, but not astrocytes, normally proliferate in response to neuronal injury; therefore Drosophila glia were assayed for injury-induced proliferative events. Third antennal segments were ablated, adult brains were costained with anti-Repo antibodies and the mitotic marker anti-phosphohistone H3 (PHH3), and Repo⁺/PHH3⁺ cells were assayed at 1, 3, 5, 7, and 9 days after ORN injury. No examples of mitotic glia were found at any of these time points after ORN injury, no gross increase in glial numbers surrounding the antennal lobe, nor evidence for proliferation in any other cell types in the brain after injury at these time points. Peripheral glial responses along the antennal nerve were assayed. In response to antennal ablation these peripheral glia do not proliferate, and no gross changes were observe in glial numbers along the antennal nerve (MacDonald, 2006).

To gain insight into how Drosophila glia might be responding to severed axons at the molecular level, a collection of embryonically expressed glial genes were assayed for those specifically enriched in antennal lobe glia 1 day after antennal ablation. Among these candidates draper, the Drosophila ortholog of the C. elegans cell corpse engulfment gene ced-1 was detected; draper has been shown to be required for glial engulfment of apoptotic neuronal cell corpses during embryonic development (Freeman, 2003). Such an engulfment receptor is an excellent candidate for driving glial removal of severed axons, though it would be somewhat surprising in light of the fact that CED-1/Draper is thought to play a role in the recognition of cell corpses, and severed axons degenerate via mechanisms that are molecularly distinct from apoptosis (MacDonald, 2006).

Using an antibody specific to Draper it was found that Draper is expressed in all Repo⁺ adult brain glia, including antennal lobe glia. Interestingly, 1 day after ablation of third antennal segments a dramatic increase was observed in Draper immunoreactivity in antennal lobe glia. Similar to the expansion of antennal lobe glial membranes after antennal ablation, increased glial Draper was found to be a local response to injury as only antennal lobe glia exhibited increased Draper after antennal ORN injury. All observed staining represents glial expression of Draper: anti-Draper immunoreactivity was absent in drpr^Δ5 null mutants, and glial specific knockdown of draper mRNA by dsRNAi removes all detectable Draper immunoreactivity in adult brains even after antennal ablation. Together these data indicate that Drosophila glia rapidly respond to ORN axon injury with changes in morphology and Draper protein levels. How glial membranes specifically interact with severed axons has been further explored, and the Draper receptor is shown to be essential for all glia responses to ORN axon injury (MacDonald, 2006).

The third antennal segments each house ~600 ORNs, and their ablation severs axons that project to ~44/50 antennal lobe glomeruli. Such ablations resulted in a robust upregulation of Draper and a dramatic expansion of antennal lobe glial membranes, but precisely how antennal lobe glia were interacting with severed axons under this conditions was unclear. For example, were glial membranes invading the antennal lobe and extending specifically toward severed axons? To explore glial membrane dynamics after ORN axon injury with higher resolution a second ORN injury assay was used: maxillary palp ablation. The maxillary palp houses only ~60 ORNs which collectively innervate ~6 glomeruli, all positioned in the ventro-medial region of the antennal lobe. Ablation of maxillary palps should therefore result in the injury of only this small subset of ORNs. If glial processes are recruited to severed ORNs, it is predicted that glial Draper and membranes would be specifically targeted to this subset of maxillary palp-innervated antennal lobe glomeruli after maxillary palp ablation (MacDonald, 2006).

Prior to injury Draper immunoreactivity was only weakly detectable within antennal lobe glomeruli and along the length of the maxillary nerve. However, 1 day after maxillary palp ablation, Draper immunoreactivity was found at high levels in ~6 ventro-medially positioned antennal lobe glomeruli—one of these was positively identified as maxillary palp-innervated with the OR85e-Gal4 driver—and along the entire length of the maxillary nerve that was visible in preparations. Draper localization to severed axons was observed beginning as early as 4 hr after maxillary palp ablation, colocalizing coincidently with the appearance of GFP⁺ puncta from degenerating axons, and Draper levels on severed axons appeared to be strongest between 12 hr and 1 day after injury. Draper protein was maintained at elevated levels on all injured axonal elements while they were still detectable (by GFP signals) and returned to control levels after axons had been cleared from the CNS. Interestingly, maxillary palp ablation did not lead to a dramatic upregulation of Draper protein in all antennal lobe glia as was seen after ablation of the third antennal segment. Thus, injuring a smaller number of ORN axons through maxillary palp ablation leads to a qualitatively different response by glia in the antennal lobe (MacDonald, 2006).

Glial membranes showed a similar pattern of rapid and specific localization to severed maxillary palp ORN axons after injury. Prior to injury GFP-labeled glial membranes were detectable at low levels around (but not within) glomeruli and at low levels along the maxillary nerve. However, 1 day after ablation of maxillary palps, glial membranes were found to be enriched within ~6 ventro-medially positioned antennal lobe glomeruli and along the entire maxillary nerve. Glial membrane-decorated glomeruli perfectly overlapped with those showing high-level Draper immunoreactivity and presumably represent glomeruli housing severed maxillary palp ORN axons (MacDonald, 2006).

In summary, glial-expressed Draper and glial membranes are rapidly and specifically recruited to severed ORN axons; their localization is coincident with the initiation of severed axon fragmentation; and Draper protein remains associated with degenerating axons until they are cleared from the CNS. These data further demonstrate that severed Drosophila axons generate molecular cues that elicit potent responses from glia and that these neuron→glia injury signals are sufficient to drive the selective recruitment of glial processes to severed axons (MacDonald, 2006).

In C. elegans, CED-1 has been shown to be essential for the engulfment of cell corpses and is believed to act as a recognition receptor for molecular cues presented by corpses (Zhou, 2001). Additional cellular targets for CED-1 have not been identified. This study shows that Draper is required to drive injury-induced changes in glial morphology and for glial clearance of severed axons from the CNS (MacDonald, 2006).

The requirements for draper in glial morphological responses to antennal ORN injury were determined. In control animals 1 day after antennal ablation a widespread expansion of antennal lobe glial membranes was observed. In contrast, when third antennal segments were ablated in drpr^Δ5 mutants, antennal lobe glia showed no noticeable changes in morphology or GFP intensity. It is noted that antennal lobe morphology was grossly normal in drpr^Δ5 mutants: ORN axons projected to the correct glomeruli, glomeruli appeared well-defined morphologically, and glial membrane processes were found in their normal positions ensheathing antennal lobe glomeruli. Together these data indicate that Draper is not required for antennal lobe development, but that glial morphological responses to antennal ORN axon injury require Draper signaling (MacDonald, 2006).

Maxillary palps were ablated and glial recruitment to the severed maxillary palp ORN axons was assayed. In control animals glial membranes were found to be highly enriched in maxillary palp-innervated glomeruli and on the maxillary nerve 1 day after injury. However, these responses were blocked in drpr^Δ5 mutants: glial processes did not accumulate in glomeruli housing severed maxillary palp ORN axons, nor were they recruited at high levels to the maxillary nerve. The autonomy of Draper function was further assayed by glial-specific RNAi knockdown of Draper. It was found that blocking Draper function in glia also fully suppressed glial responses to antennal or maxillary palp ablation, consistent with a requirement for Draper in glia. Thus all morphological changes exhibited by glia after injury (i.e., membrane expansion and process extension toward severed axons) require Draper signaling. These observations suggest that Draper may act as a glial receptor for severed axon-derived cues that drive glial responses to axon injury (MacDonald, 2006).

CED-1/Draper encodes a receptor essential for engulfment of cell corpses in both C. elegans (Zhou et al., 2001) and Drosophila (Freeman, 2003). Is Draper required for glial clearance of degenerating axons from the CNS? To test this the OR22a⁺ subset of antennal ORN axons in the drpr^Δ5 null mutant background was marked with mCD8::GFP and removal of injured axons was assayed. In control animals severed ORN axons outside glomeruli were cleared from the CNS by 3 days after injury (20/20 antennal lobes), and GFP⁺ axonal material was cleared from the CNS within 5 days. In striking contrast, the majority of axonal debris lingered in the CNS of drpr^Δ5 mutants after ablation of the third antennal segments. For example, 3 days after injury there was an abundance of GFP-labeled OR22a⁺ axon fibers remaining in the antennal lobe (20/20 antennal lobes). Similarly, 5 days after injury in drpr^Δ5 mutants, ~65% of antennal lobes retained GFP⁺ axonal fibers and GFP levels in DM2 glomeruli remained at levels comparable to control uninjured animals. Severed axon fragmentation was not blocked in drpr^Δ5 mutants. Severed axons showed signs of fragmentation as early as 5 hr after injury in drpr^Δ5 mutants—this approximates the earliest time points at which axons fragmented in control animals—and axon fibers were highly fragmented in these animals 3 and 5 days after injury. These data indicate that Draper is essential for glial clearance of degenerating axons from the Drosophila CNS, and axons thus represent a new engulfment target for this receptor. These observations also indicate that severed axons actively communicate with glia (via Draper) to promote their clearance from the CNS.

It is proposed that Draper acts as a glial receptor for severed axon-derived molecular cues, and that a Draper ligand encodes a neuron→glia injury signal that drives glial responses to axon injury. The ligand on cell corpses recognized by CED-1 remains to be identified, but is thought to encode the “eat-me” signal that initiates engulfment. Do severed axons, developmentally pruned axons, and cell corpses present similar molecular cues for engulfment? This may indeed be the case since CED-1/Draper is required for the clearance of each of these engulfment targets. However, Wallerian degeneration, developmental axon pruning, and apoptotic cell death are clearly distinct at the molecular level. For example, Wld^s expression in neurons does not block apoptosis in neuronal cell bodies, nor the developmental pruning of Drosophila mushroom body γ neurons. Reciprocally, inhibition of canonical cell death pathways is not sufficient to block Wallerian degeneration. Nevertheless, it is possible that Wallerian degeneration, developmental axon pruning, and apoptotic cell death, though promoted by different molecular mechanisms, lead to the production of the same engulfment cue that acts as a ligand for CED-1/Draper (MacDonald, 2006).

Degenerating axons and cell corpses could also generate distinct engulfment cues that are each recognized by CED-1/Draper. One model would be that CED-1/Draper would not discriminate these targets, but only drive their nonspecific engulfment. Alternatively, distinct Draper receptor isoforms might recognize specific ligands presented by a cell corpse or a severed axon and potentially play an important role in discriminating these engulfment targets. Intriguingly, at least two isoforms of Draper have been identified in Drosophila that vary significantly in their extracellular domains; perhaps these bind distinct ligands on engulfment targets in vivo. Future studies aimed at defining the functional requirements of specific Draper receptor isoforms in the engulfment of cell corpses, developmentally pruned axons, and severed axons, as well as studies aimed at identifying the CED-1/Draper ligand(s) presented by these engulfment targets, should help resolve these issues (MacDonald, 2006).

Draper becomes localized to severed axons ~4 hr after injury, coincident with the initiation of axon fragmentation. Is axon degeneration essential to recruit glial processes in Drosophila? Mouse Wld^s and dNmnat were used as tools to block ORN axon degeneration and glial responses were assayed to these severed, but nondegenerating, axons. UAS-Wld^s or UAS-dNmnat were coexpressed with UAS-n-Syb::3XYFP in OR85e⁺ maxillary palp ORNs, ablated maxillary palps, and subsequently Draper localization to severed axons was assayed. Interestingly, while Draper was detected at high levels 1 day after maxillary palp ablation in 5/6 maxillary palp ORN-innervated glomeruli, Wld^s-expressing axons showed control levels of Draper immunoreactivity. Thus Wld^s expression is sufficient to potently suppress glial recruitment to severed axons. dNmnat was also found to suppress glial recruitment to severed axons, though, as was found in severed axon protection experiments, dNmnat was not as efficient at suppressing glial recruitment to severed axons. Nevertheless, this observation suggests that dNmnat activity is an important requirement for Wld^s to block glial responses to severed axons. Together these data indicate that production of the molecular cues in severed axons that elicit glial responses is genetically downstream of Wld^s and are consistent with axon fragmentation being essential for robust glial responses to injury. In addition, the results indicate that Wld^s acts in a cell-autonomous fashion in Drosophila, as the recruitment of glial processes to severed axons is suppressed in Wld^s-expressing axons but not in adjacent wild-type injured axons (MacDonald, 2006).

The mechanism by which Wld^s exerts its neuroprotective effects remains unclear. One model proposes that Wld^s acts prior to injury in the nucleus through the NAD binding histone deacetylase Sirt1 to effect neuroprotective changes in gene expression. In contrast, a second model proposes that Wld^s acts locally in axons after injury to maintain high NAD levels which ultimately block axonal degeneration. Although transfection of Nmnat1 into DRG explant cultures indeed suppressed Wallerian degeneration in vitro, it was not known whether Nmnat1 could protect severed axons in vivo. This study has shown that dNmnat can suppress severed axon autodestruction in Drosophila; however, dNmnat appears less efficient than Wld^s in protecting axons and suppressing glial responses to axon injury. The results therefore support the notion that Nmnat activity is an important component of Wld^s neuroprotective function in vivo, but indicate that Wld^s somehow provides more effective neuroprotection than dNmnat (MacDonald, 2006).

Search PubMed for articles about Drosophila Nmnat

Ali, Y. O., McCormack, R., Darr, A. and Zhai, R. G. (2011). Nicotinamide mononucleotide adenylyltransferase is a stress response protein regulated by the heat shock factor/hypoxia-inducible factor 1α pathway. J Biol Chem 286(21): 19089-19099. PubMed ID: 21478149

Ali, Y. O., Ruan, K. and Zhai, R. G. (2012). NMNAT suppresses tau-induced neurodegeneration by promoting clearance of hyperphosphorylated tau oligomers in a Drosophila model of tauopathy. Hum Mol Genet 21(2): 237-250. PubMed ID: 21965302

Brace, E. J., Wu, C., Valakh, V. and DiAntonio, A. (2014). SkpA restrains synaptic terminal growth during development and promotes axonal degeneration following injury. J Neurosci 34: 8398-8410. PubMed ID: 24948796

Chen, L., Nye, D.M., Stone, M.C., Weiner, A.T., Gheres, K.W., Xiong, X., Collins, C.A. and Rolls, M.M. (2016). Mitochondria and caspases tune Nmnat-mediated stabilization to promote axon regeneration. PLoS Genet 12: e1006503. PubMed ID: 27923046

Coleman, M. P. and Freeman, M. R. (2010). Wallerian degeneration, wld^s, and nmat. Annu Rev Neurosci 33: 245-267. PubMed ID: 20345246

Fang, Y., Soares, L., Teng, X., Geary, M. and Bonini, N. M. (2012). A novel Drosophila model of nerve injury reveals an essential role of Nmnat in maintaining axonal integrity. Curr Biol 22(7): 590-595. PubMed ID: 22425156

Izadifar, A., Courchet, J., Virga, D. M., Verreet, T., Hamilton, S., Ayaz, D., Misbaer, A., Vandenbogaerde, S., Monteiro, L., Petrovic, M., Sachse, S., Yan, B., Erfurth, M. L., Dascenco, D., Kise, Y., Yan, J., Edwards-Faret, G., Lewis, T., Polleux, F. and Schmucker, D. (2021). Axon morphogenesis and maintenance require an evolutionary conserved safeguard function of Wnk kinases antagonizing Sarm and Axed. Neuron. PubMed ID: 34384519

Ji, H., Sapar, M. L., Sarkar, A., Wang, B. and Han, C. (2022). Phagocytosis and self-destruction break down dendrites of Drosophila sensory neurons at distinct steps of Wallerian degeneration. Proc Natl Acad Sci U S A 119(4). PubMed ID: 35058357

MacDonald, J. M., Beach, M. G., Porpiglia, E., Sheehan, A. E., Watts, R. J. and Freeman, M. R. (2006). The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron 50(6): 869-881. PubMed ID: 16772169

Neukomm, L. J., Burdett, T. C., Seeds, A. M., Hampel, S., Coutinho-Budd, J. C., Farley, J. E., Wong, J., Karadeniz, Y. B., Osterloh, J. M., Sheehan, A. E. and Freeman, M. R. (2017). Axon death pathways converge on Axundead to promote functional and structural axon disassembly. Neuron 95(1): 78-91.e75. PubMed ID: 28683272

Raff, M. C., Whitmore, A. V. and Finn, J. T. (2002). Axonal self-destruction and neurodegeneration. Science 296(5569): 868-871. PubMed ID: 11988563

Rallis, A., Lu, B. and Ng, J. (2013). Molecular chaperones protect against JNK- and Nmnat-regulated axon degeneration in Drosophila. J Cell Sci 126(Pt 3): 838-849. PubMed ID: 23264732

Ruan, K., Zhu, Y., Li, C., Brazill, J. M. and Zhai, R. G. (2015). Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress. Nat Commun 6: 10057. PubMed ID: 26616331

Sapar, M. L., Ji, H., Wang, B., Poe, A. R., Dubey, K., Ren, X., Ni, J. Q. and Han, C. (2018). Phosphatidylserine Externalization Results from and Causes Neurite Degeneration in Drosophila. Cell Rep 24(9): 2273-2286. PubMed ID: 30157423

Wen, Y., Parrish, J. Z., He, R., Zhai, R. G. and Kim, M. D. (2011). Nmnat exerts neuroprotective effects in dendrites and axons. Mol Cell Neurosci 48(1): 1-8. PubMed ID: 21596138

Wen, Y., Zhai, R. G. and Kim, M. D. (2013). The role of autophagy in Nmnat-mediated protection against hypoxia-induced dendrite degeneration. Mol Cell Neurosci 52: 140-151. PubMed ID: 23159780

Xiong, X., Hao, Y., Sun, K., Li, J., Li, X., Mishra, B., Soppina, P., Wu, C., Hume, R. I. and Collins, C. A. (2012). The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein. PLoS Biol 10(12): e1001440. PubMed ID: 23226106

Zang, S., Ali, Y. O., Ruan, K. and Zhai, R. G. (2013). Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot. EMBO Rep 14: 87-94. PubMed ID: 23154466

Zhai, R. G., Cao, Y., Hiesinger, P. R., Zhou, Y., Mehta, S. Q., Schulze, K. L., Verstreken, P. and Bellen, H. J. (2006). Drosophila NMNAT maintains neural integrity independent of its NAD synthesis activity. PLoS Biol 4(12): e416. PubMed ID: 17132048

Zhai, R. G., Zhang, F., Hiesinger, P. R., Cao, Y., Haueter, C. M. and Bellen, H. J. (2008). NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration. Nature 452(7189): 887-891. PubMed ID: 18344983

date revised: 1 March 2024

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