InteractiveFly: GeneBrief

Nicotinamide mononucleotide adenylyltransferase: Biological Overview | References


Gene name - Nicotinamide mononucleotide adenylyltransferase

Synonyms -

Cytological map position - 96B11-96B11

Function - enzyme

Keywords - NAD salvage pathway - catalyzing the last step of NAD synthesis - stress response protein - required for thermotolerance and mitigation of oxidative stress-induced shortened lifespan - protects against axonal degeneration through chaperone activity - protects against neurodegeneration through a proteasome-mediated pathway - required for maintaining active zone structural integrity by interacting Bruchpilot - regulated post-transcriptionally by Highwire function

Symbol - Nmnat

FlyBase ID: FBgn0039254

Genetic map position - chr3R:24,945,237-24,947,400

NCBI classification - Nicotinamide/nicotinate mononucleotide adenylyltransferase, Eukaryotic

Cellular location - cytoplasmic



NCBI links: EntrezGene, Nucleotide, Protein
Recent literature
Russo, A., Goel, P., Brace, E. J., Buser, C., Dickman, D. and DiAntonio, A. (2019). The E3 ligase Highwire promotes synaptic transmission by targeting the NAD-synthesizing enzyme dNmnat. EMBO Rep. PubMed ID: 30692130
Summary:
The ubiquitin ligase Highwire restrains synaptic growth and promotes evoked neurotransmission at NMJ synapses in Drosophila. Highwire regulates synaptic morphology by downregulating the MAP3K Wallenda, but excess Wallenda signaling does not account for the decreased presynaptic release observed in highwire mutants. Hence, Highwire likely has a second substrate that inhibits neurotransmission. Highwire targets the NAD(+) biosynthetic and axoprotective enzyme dNmnat to regulate axonal injury responses. dNmnat localizes to synapses and interacts with the active zone protein Bruchpilot, leading to a hypothesis that Highwire promotes evoked release by downregulating dNmnat. This study shows that excess dNmnat is necessary in highwire mutants and sufficient in wild-type larvae to reduce quantal content, likely via disruption of active zone ultrastructure. Catalytically active dNmnat is required to drive defects in evoked release, and depletion of a second NAD(+) synthesizing enzyme is sufficient to suppress these defects in highwire mutants, suggesting that excess NAD(+) biosynthesis is the mechanism inhibiting neurotransmission. Thus, Highwire downregulates dNmnat to promote evoked synaptic release, suggesting that Highwire balances the axoprotective and synapse-inhibitory functions of dNmnat.
Russo, A., Goel, P., Brace, E. J., Buser, C., Dickman, D. and DiAntonio, A. (2019). The E3 ligase Highwire promotes synaptic transmission by targeting the NAD-synthesizing enzyme dNmnat. EMBO Rep 20(3). PubMed ID: 30692130
Summary:
The ubiquitin ligase Highwire restrains synaptic growth and promotes evoked neurotransmission at NMJ synapses in Drosophila. Highwire regulates synaptic morphology by downregulating the MAP3K Wallenda, but excess Wallenda signaling does not account for the decreased presynaptic release observed in highwire mutants. Hence, Highwire likely has a second substrate that inhibits neurotransmission. Highwire targets the NAD(+) biosynthetic and axoprotective enzyme dNmnat to regulate axonal injury responses. dNmnat localizes to synapses and interacts with the active zone protein Bruchpilot, leading to a hypothesis that Highwire promotes evoked release by downregulating dNmnat. This study also shows that excess dNmnat is necessary in highwire mutants and sufficient in wild-type larvae to reduce quantal content, likely via disruption of active zone ultrastructure. Catalytically active dNmnat is required to drive defects in evoked release, and depletion of a second NAD(+) synthesizing enzyme is sufficient to suppress these defects in highwire mutants, suggesting that excess NAD(+) biosynthesis is the mechanism inhibiting neurotransmission. Thus, Highwire downregulates dNmnat to promote evoked synaptic release, suggesting that Highwire balances the axoprotective and synapse-inhibitory functions of dNmnat.
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 Wlds, 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).

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 (WldS) protein blocked JNK axonal degeneration. Although the nicotinamide mononucleotide adenylyltransferase (Nmnat1) portion of WldS is required, it was found that its nicotinamide adenine dinucleotide (NAD(+)) enzyme activity and the WldS N-terminus (N70) are dispensable, unlike axotomy models of neurodegeneration. It is suggest that WldS-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).

WldS was discovered from the molecular cloning of spontaneously generated slow Wallerian degeneration (WldS) mutant mice that showed a strong capacity to promote axonal survival following acute physical lesion. The WldS protein has neuroprotective effects across different species and in different neurodegeneration models. The WldS 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 WldS can confer neuroprotective function (Coleman, 2010). However, WldS function remains unclear. For example, despite its predominant nuclear localisation, it is axonal localisation that appears to be key to neuroprotection, even though WldS 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 WldS function(s) and axon-neuronal damage and repair also remains unclear, although recent data suggest WldS-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 WldS-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 WldS 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 WldS 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 WldS 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, WldS, 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 WldS-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 WldS 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 WldS 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 (Wlds) 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 (Wlds) 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/Wlds (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/Wlds line has recently been found to map to a novel chimeric protein termed Wlds, 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 Wlds-modulated mechanisms regulate axon degeneration in Drosophila, the ability of mouse Wlds to protect severed Drosophila axons from injury-induced degeneration was tested (MacDonald, 2006).

UAS-Wlds 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, Wlds+ axons did not undergo Wallerian degeneration: animals bearing a single copy of the UAS-Wlds transgene exhibited normal mCD8::GFP fluorescence in severed ORN axons 5 days after injury, and Wlds+ axons maintained normal morphology. Neuroprotective effects of Wlds 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 Wlds+ axons, n-Syb::3XYFP signals appeared morphologically normal and YFP intensity remained at control levels, even 9 days after injury. Similarly, in Wlds+ 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-Wlds insertion line (MacDonald, 2006).

How long can Wlds protect severed Drosophila axons? Severed axon morphology was examined in Wlds+ ORNs at 20, 30, and 50 days after injury. Remarkably, even 20 or 30 days after antennal ablation, Wlds+ ORN axons maintained largely normal morphology, and GFP intensity within glomeruli remained close to control levels. By 50 days after injury, the axons of Wlds+ 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 Wlds 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 Wlds-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 Wlds, since 5 days after injury axon degeneration occurs at some level in dNmnat+, but not Wlds+, axons. Nevertheless, these data argue that Wlds-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 Wlds potently suppresses this process in Drosophila argues strongly that Wlds-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 drprΔ5 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 drprΔ5 mutants, antennal lobe glia showed no noticeable changes in morphology or GFP intensity. It is noted that antennal lobe morphology was grossly normal in drprΔ5 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 drprΔ5 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 drprΔ5 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 drprΔ5 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 drprΔ5 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 drprΔ5 mutants. Severed axons showed signs of fragmentation as early as 5 hr after injury in drprΔ5 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, Wlds 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 Wlds and dNmnat were used as tools to block ORN axon degeneration and glial responses were assayed to these severed, but nondegenerating, axons. UAS-Wlds 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, Wlds-expressing axons showed control levels of Draper immunoreactivity. Thus Wlds 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 Wlds 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 Wlds and are consistent with axon fragmentation being essential for robust glial responses to injury. In addition, the results indicate that Wlds acts in a cell-autonomous fashion in Drosophila, as the recruitment of glial processes to severed axons is suppressed in Wlds-expressing axons but not in adjacent wild-type injured axons (MacDonald, 2006).

The mechanism by which Wlds exerts its neuroprotective effects remains unclear. One model proposes that Wlds 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 Wlds 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 Wlds in protecting axons and suppressing glial responses to axon injury. The results therefore support the notion that Nmnat activity is an important component of Wlds neuroprotective function in vivo, but indicate that Wlds somehow provides more effective neuroprotection than dNmnat (MacDonald, 2006).


REFERENCES

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, wlds, 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

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

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

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


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date revised: 26 July 2018

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