Axon Degeneration And

CB28CH22-Bonini ARI 5 September 2012 15:57

Axon Degeneration andRegeneration: Insights fromDrosophila Models of NerveInjuryYanshan Fang and Nancy M. BoniniHoward Hughes Medical Institute and Department of Biology, University of Pennsylvania,Philadelphia, Pennsylvania, 19104; email: [email protected],[email protected]

Annu. Rev. Cell Dev. Biol. 2012. 28:57597

First published online as a Review in Advance onJuly 23, 2012

The Annual Review of Cell and DevelopmentalBiology is online at cellbio.annualreviews.org

This articles doi:10.1146/annurev-cellbio-101011-155836

Copyright c 2012 by Annual Reviews.All rights reserved

1081-0706/12/1110-0575$20.00

Keywords

axotomy, Wallerian degeneration, WldS, neuroprotection, axonregrowth

Abstract

Axon degeneration is the pivotal pathological event of acute traumaticneural injury as well as many chronic neurodegenerative diseases. It isan active cellular program and yet molecularly distinct from cell death.Much effort is devoted toward understanding the nature of axon degen-eration and promoting axon regeneration. However, the fundamentalmechanisms of self-destruction of damaged axons remain unclear, andthere are still few treatments for traumatic brain injury (TBI) or spinalcord injury (SCI). Genetically approachable model organisms such asDrosophila melanogaster, the fruit y, have proven exceptionally success-ful in modeling human neurodegenerative diseases. More recently, thissuccess has been extended into the eld of acute axon injury and re-generation. In this review, we discuss recent ndings, focusing on howthese models hold promise for accelerating mechanistic insight intoaxon injury and identifying potential therapeutic targets for TBI andSCI.

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Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 576WHY MODEL AND STUDY

NERVE INJURY USINGDROSOPHILA? . . . . . . . . . . . . . . . . . . 577

CURRENT FLY MODELS OFNEURAL INJURY . . . . . . . . . . . . . . 578The Head Model . . . . . . . . . . . . . . . . . 578The Olfactory System Model . . . . . 578The Whole-Brain Explant

Model . . . . . . . . . . . . . . . . . . . . . . . . 580The Fly Larval Models . . . . . . . . . . . 580The Wing Model . . . . . . . . . . . . . . . . 581

WLDS, NMNAT, AND AXONDEGENERATION. . . . . . . . . . . . . . 581Is the Protective Function of WldS

Owing to the Nmnat1Activity? . . . . . . . . . . . . . . . . . . . . . . 582

Where and How DoesWldS/Nmnat Act to ProtectInjured Axons? . . . . . . . . . . . . . . . . 582

Is Nmnats Axon ProtectionMediated by NAD+? . . . . . . . . . . 584

THE UBIQUITIN PROTEASOMESYSTEM AND NEURALINTEGRITY . . . . . . . . . . . . . . . . . . . . 584

The Ubiquitin ProteasomeSystem and WldS . . . . . . . . . . . . . . 584

Developmental Pruning VersusTraumatic AxonDegeneration . . . . . . . . . . . . . . . . . 585

The Ubiquitin Proteasome SystemIs a Double-Edged Sword. . . . . . 585

MOLECULAR SIGNALING INTHE NEURAL INJURYRESPONSE IN DROSOPHILA . . 585Amyloid Precursor

Protein/APP-Like . . . . . . . . . . . . . 585Cyclic AMP and Protein

Kinase A . . . . . . . . . . . . . . . . . . . . . . 586Jun N-Terminal Kinase . . . . . . . . . . . 586Dual Leucine Kinase/Wallenda

and Highwire . . . . . . . . . . . . . . . . . 588OTHER COMPONENTS

OF AXON DEGENERATIONAND REGENERATION . . . . . . . . 588Cytoskeleton and Microtubule

Dynamics . . . . . . . . . . . . . . . . . . . . . 589Axonal Transport. . . . . . . . . . . . . . . . . 589The Role of Glia . . . . . . . . . . . . . . . . . 589

PERSPECTIVES ANDCONCLUSIONS. . . . . . . . . . . . . . . . 590

Walleriandegeneration:degeneration of theaxon segment distal tothe site of injury

Pruning:programmeddisassembly of axonsand dendrites duringdevelopment

WldS: Walleriandegeneration slow

INTRODUCTIONAxons damaged by acute injury, toxic insults, orneurodegenerative diseases undergo Wallerianor Wallerian-like degeneration, in which theaxons become fragmented and are then clearedaway (Hilliard 2009, Vargas & Barres 2007).Herewe focus on axon degeneration (Walleriandegeneration) induced by an acute injury. Re-lated topics, such as axonal loss during normaldevelopment (pruning) or chronic degenera-tive disease (Wallerian-like degeneration), havebeen discussed in other reviews (Coleman 2005,Low & Cheng 2006, Luo & OLeary 2005,Saxena & Caroni 2007).

In a nerve injury, the axon segments distalto the injury site are often separated completelyor partially from the neuronal cell bodies. Forthis reason, Wallerian degeneration was long

thought to be a passive process resulting from alack of nutrients and support from the cell body.However, the discovery of the Wallerian degen-eration slow (WldS)mutantmouse, inwhich axondegeneration is dramatically delayed to almosttenfold the time the process takes in wild-typeanimals, demonstrated that axon degeneration,similar to programmed cell death, is an activeprocess of self-destruction (Lunn et al. 1989).Whereas the distal axons undergo Walleriandegeneration, the proximal segments eitheralso degenerate (die back), as in the centralnervous system (CNS) of humans and othermammals, or regenerate/regrow, as in the CNSof invertebrates including nematode, leech,and craysh and of some vertebrates such aszebrash as well as in the peripheral nervous

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SCI: spinal cordinjury

Axotomy: severing ofan axon

system (PNS) of mammals. Here we point outthat, although the two terms are often used in-terchangeably, regrowth is more accurate andappropriate than regeneration in most cases, assuccessful axon regeneration requires not onlysprouting and extension of the proximal axonend but also rewiring of the regrowing axonto the correct distal target(s) and, by morestringent denition, functional recovery.

In the past several decades, studies basedon vertebrate animal models as well as in vitrocultured mammalian neurons have providedvaluable insight into axon injury. However,the exact mechanisms underlying the intrinsicneuronal properties and the extrinsic factors,such as the role of glia and myelin in axondegeneration and regeneration, remain enig-matic. This is at least partly owing to thelimitations of classical experimental models inrodents, which, although important for neuralinjury research, are costly, time consuming, andchallenging for large-scale genetic screeningto identify unknown components.

Recent advances in the understanding ofthe molecular and genetic basis of axon de-generation and regeneration have come frommodeling nerve injury in invertebrate animalssuch as Caenorhabditis elegans (nematode) (forrecent reviews, see Chen & Chisholm 2011,Chiu et al. 2011), Hirudo medicinalis (leech) (fora review, see Mladinic et al. 2009), and recentlyDrosophila melanogaster (fruit y). In thisreview, we rst discuss the reasons and need todevelop Drosophila models of axon injury. Wethen summarize current paradigms of nerveinjury in ies, highlighting the advantages andlimitations of each model. Next, we review thekey genes and molecular signaling pathwaysinvolved in regulating axon degeneration andregeneration, emphasizing more recent contri-butions from studies of the Drosophila models.Finally, we conclude with future perspectiveson nerve injury using the y as a model.

WHY MODEL AND STUDY NERVEINJURY USING DROSOPHILA?

As noted, many studies of axon injury havethe ultimate goal of providing the foundation

for treatments for spinal cord injury (SCI)in human patients (for a recent review, seeTuszynski & Steward 2012). For this reason,the concept of studying such a topic in the y,an animal that does not even have a spine, maynot sound rational to many neurologists andneurobiologists studying human conditions.But in fact, the fundamental mechanisms inaxon injury and regeneration are evolutionarilyconserved through species including ies andhumans, and the armamentaria of moleculargenetic tools in such model organisms to ad-dress and dene gene function are staggering(see Related Resources). On the other hand,precise axotomy can be achieved readily bytargeted laser ablation in C. elegans, a modelorganism that is also genetically tractable butwith an even simpler nervous system thanDrosophila. Recently, systematic screens andfocused investigations have been performedusing C. elegans models to identify genes thatmodulate axon regrowth (Bejjani & Hummar-lund 2012, Chen et al. 2011, Hammarlundet al. 2009). So, why would there be a need foradditional models of axon injury in Drosophila?

The nervous system of C. elegans is indeedvery simplean adult nematode has only 302neurons, and the genealogy and morphologyof each neuron has been well established. Thesimplicity of C. elegans facilitates the study ofaxon injury and regeneration greatly, althoughit also presents limitations. For example, al-though C. elegans nerves have the advantage ofsingle axon morphology, these differ from thenerve tracts of higher systems that are typicallycomprised of many axons and wrapped by gliaand myelin. The glial scar and myelin-derivedfactors are known to form an inhibitoryenvironment to axon regeneration in the mam-malian CNS (Yiu & He 2006). This differencemay be one reason why severed C. elegans axonsundergo robust spontaneous regrowth, whichoften occurs before the distal axon segmentdegenerates, therefore allowing reconnec-tion (Chen & Chisholm 2011, Chiu et al.2011).

The fruit y, although seemingly smalland simple, in fact has an extensive brain and

www.annualreviews.org Drosophila Models of Axon Injury 577

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APP: amyloidprecursor protein

TBI: traumatic braininjury

nervous system, and it has proven instrumentalin dening genes and signal transductionpathways that are strikingly conserved to hu-mans (Bier 2005, Lessing & Bonini 2009). Ex-amples of conserved mechanisms between iesand mammals range from human neurodegen-erative diseases (Ambegaokar et al. 2010,Marsh& Thompson 2006, Shulman et al. 2003), can-cer (Potter et al. 2000, Vidal & Morin 2007),ethanol intoxication (Guarnieri & Heberlein2003, Rodan &Rothenuh 2010), and learningand memory (Fiala 2007, Keene & Waddell2007) to circadian rhythms and sleep (Crocker& Sehgal 2010, Sehgal et al. 2007). The highsimilarity and conservation also applies to theresponse and the molecular mechanisms ofnerve injury. For example, injured Drosophilanerves indeed undergo axonal fragmentation,the morphology and progressive timing ofwhich exhibit striking similarities to Walleriandegeneration in human patients and rodentmodels (Ayaz et al. 2008, Fang et al. 2012,MacDonald et al. 2006). And, as in vertebrates,Drosophila nerve tracts are typically comprisedof many axons, which, although lackingmyelin,are ensheathed by glial cells (Banerjee et al.2006, Bellen et al. 1998). Moreover, expressionof mouse WldS can potently suppress injury-induced axonal loss in ies (Avery et al. 2009,Fang et al. 2012,Hoopfer et al. 2006,MacDon-ald et al. 2006, Wen et al. 2011), which furtherargues for mechanistic and functional conser-vation between ies and mammals. Given theextensive biological and gene-pathway overlap,the vast molecular genetic tools to manip-ulate gene function in Drosophila almost atwillwith upregulation, downregulation, andtissue-selective gene manipulation (see RelatedResources)are a prime advantage to test,dene, and discover new genes whose activitiesimpact neural injury and regrowth in a centralway.

CURRENT FLY MODELS OFNEURAL INJURY

The Head ModelA y head injury model was developed in astudy that focused initially on the role of human

amyloid precursor protein (APP) andDrosophilaAPP-Like (APPL) in postdevelopmental axonalarborization. To determine whether DrosophilaAPPL was upregulated in response to brain in-jury, Leyssen et al. (2005) took a very straight-forward approachstabbing the y head withneedles (Figure 1a). Despite the brutal injury,some ies survived, and a few of them even livedup to two weeks after trauma. This is one ofthe earliest attempts to model traumatic braininjury (TBI) in ies. The main limitations ofmanually stabbing the y head with needles in-clude a lack of precise control of the injury anda variable extent of damage. These issues makeit difcult to perform the type of quantiableanalysis that is increasingly demanded in scien-tic research.

The Olfactory System Model

One year later, two groups reported indepen-dently the use of theDrosophila olfactory systemto model and study axon injury (Hoopfer et al.2006, MacDonald et al. 2006). To sever theolfactory nerve, the third antennal segments, ormaxillary palps, which house the olfactory re-ceptor neurons, were physically removed fromthe head (Figure 1b). The axotomy-inducedresponse was then examined in the antennal ormaxillary nerve that projected into the antennallobe of the brain. Because the axon injury inthis model is achieved by nonlethal surgicalablation, organismal survival is improved sig-nicantly. Also, the axon injury induced by thismethod is precise and consistent. Moreover,because the Drosophila olfactory system hasbeen well established (Brochtrup & Hummel2010, Imai et al. 2010), many useful reagents,such as subset-specic odorant receptor genepromoter-Gal4 driver lines, are available toselectively label olfactory receptor neurons andto manipulate genes in this system. Further-more, this model has proven useful in the studyof the glial response and axon-glia interactionsin nerve injury (MacDonald et al. 2006,Ziegenfuss et al. 2008). This is a robust modelfor the study of axon injury and degeneration.However, the axotomy procedure removes the

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a Stabbed headb Antennaremoval

c Brain explant

e Wing cut

d Crushing or laserablation of larva

Figure 1Approaches to induce traumatic neural injury in ies. The current Drosophila paradigms of acute neuralinjury include: (a) stabbing a needle through the y head, (b) removing the antenna to sever the olfactorynerve, (c) transecting central nervous system (CNS) axons in y brain explants, (d ) crushing or laser ablatinglarval axons, and (e) cutting the wing to sever the L1 nerve along the wing margin. Further details of eachinjury paradigm are summarized in Table 1.


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Table 1 Summary of Drosophila paradigms of neural injury

Model Injury methodNeuronal

processes damaged Degeneration timingRegenerative

regrowth Major referencesHead Stab with needles Various brain

regionsNot determined Not determined Leyssen et al. 2005

Olfactorysystem

Remove thesensory organ

Axonal projectionsof ORNs

As early as 1 day,complete by 5 days

Not determined MacDonald et al. 2006,Hoopfer et al. 2006

Brainexplant

Cut with amicrodissector

Brain projection ofsLN-v

45 days Minimalspontaneousregeneration

Ayaz et al. 2008

Larvae Crush withforceps

Segmental axons ofmotor neurons

Within 24 hours Regenerativesprouting

Xiong et al. 2010

Laser axotomy Axons of da neurons Within 24 hours A dendrite isconverted to aregrowing axon

Stone et al. 2010

Wing Cut with scissors Axons of sensoryneurons along thewing margin

57 days in the wing,2448 hours in thethorax

Not determined Fang et al. 2012

neuronal cell bodies, which prohibits the utilityof this system for study of the regenerativecapacity of Drosophila neurons.

The Whole-Brain Explant Model

This model was developed by the same groupthat developed the TBI model (Leyssen et al.(2005). In order to generate a more precise andreproducible CNS axon injury, in this system,the researchers dissected out the y brain fromthe head cuticle and then cultured the y brainin vitro (Figure 1c). They then used amicrodis-section technique to transect the axons of aspecic subset of uorescently labeled neurons(Ayaz et al. 2008). The neurons selected are thesmall ventral lateral neurons (s-LNv), the cir-cadian oscillator cells that form a stereotypicprojection pattern in the y brain (Nitabach &Taghert 2008). Aswith the olfactorymodel, thisparadigm also takes advantage of the moleculargenetic tools developed fromprevious studies ofthe Drosophila circadian system. Furthermore,because the cell bodies and the proximal ax-ons are preserved, axon regeneration can be as-sessed in this model; these explant experimentsshowed that, as in mammals, the CNS axonsin the wild-type y exhibited limited regrowth

after axotomy (Ayaz et al. 2008). Although thismodel presents the opportunity to assay bothdegeneration and regeneration of injured yaxons, the extensive technical manipulation in-volved in dissecting and culturing the whole-brain explantmakes it no longer a simplemodel.

The Fly Larval Models

Like C. elegans, Drosophila larvae have relativelysimpleneuroanatomywith a translucent cuticle,making the animal amenable to in vivo imagingat this stage. For this reason, y geneticists havesought to model nerve injury in larvae. Usingeither a UV laser to completely sever the axonsof dendritic arborization (da) neurons (Stoneet al. 2010) or forceps to crush the segmentalnerve (Xiong et al. 2010), precise axon injurycan be achieved in y larvae (Figure 1d). As inadult ies, the injured larval axons also degener-ate (Stone et al. 2010, Tao &Rolls 2011, Xionget al. 2010). Because of the simplicity of visualiz-ing axons in living Drosophila larvae, Ghannad-Rezaie et al. (2012) recently developed a poten-tially high throughput method, a microuidiclarva chip, for in vivo imaging of cellular re-sponses to nerve injury and axon regrowth, suchas calcium uxes and actin dynamics. Although

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DRG: dorsal rootganglion

Nmnat:Nicotinamidemononucleotideadenylyltransferase, acritical enzymeinvolved in NAD+biosynthesis

larvae are a powerful tool, a limitation to lar-val models is the rapid Drosophila developmen-tal cycle, as the time window to study injury-induced axon degeneration and regeneration inlarvae is on the order of hours to a few days. An-other important consideration is that y larvaeare still undergoing development. The molec-ular mechanisms regulating axon degenerationduring developmental stages can be distinctfrom those in the adult, as discussed below (alsosee Hoopfer et al. 2006, Tao & Rolls 2011).

The Wing Model

Most recently, Fang et al. (2012) developeda model using the adult Drosophila wingnerve that combines the advantages of in vivoimaging, precise axotomy, and accessibility ofgenetic manipulation. Furthermore, the adultstage negates the short time frame in larvaeand the confounding effects of development.The y wing is semitransparent, and the wingaxon bundle is easily visible when labeled withuorescent protein markers, which allowsprecise and reproducible axotomy by a simplescissor cut (Figure 1e). Along the wing mar-gin, approximately 250 mechanosensory andchemosensory neurons send axons to form thewing nerve and project into the thoracic gan-glion of the CNS (Garca-Alonso 1999). Thesignicant length of the wing nerve (comparethe wing with the head in Figure 1) providesa much larger window in which to resolvethe rapid process of axon degeneration. Forexample, injury-induced axon fragmentationexhibits a retrograde directionality in the wing(Fang et al. 2012), which is evident in rodentmodels and human patients but is not presentedin previous y models. Also, because the wingis dispensable for y survival, injury-inducedresponses can be followed on live animalsthroughout their entire life span. Furthermore,unlike the previous adult y paradigms, notime-consuming CNS dissection is required.This model is therefore especially suitable forunbiased, large-scale genetic screens in whicha fast and easy readout of axon degenerationis desired (Fang et al. 2012). In contrast to

the olfactory model, the wing paradigm offersthe potential to study axon regeneration ifequipped with a laser ablation system as usedin C. elegans and y larvae. However, similarto C. elegans, a technical limitation of the wingmodel comes from the cuticle barrier: histo-chemical approaches such as immunostainingcan be challenging. Thus, the wing modelrelies on in vivo labeled probes to visualizeinjury-induced responses and cellular changes.

The goal of using model genetic systems isto identify the molecules that control axon de-generation and regeneration. Before discussingthese genetic and molecular mechanisms, itshould be pointed out that varying or even op-posite results about a genes function sometimescan be observed in different models and distinctsettings. Therefore, to conclude that a gene isa positive or negative modier of axon degen-eration and/or regeneration is often an over-simplication. Instead, as reviewed below, it isnecessary to consider carefully the precise sys-tem, and what exactly is being measured, wheninterpreting ndings.

WLDS, NMNAT, AND AXONDEGENERATION

Ever since the discovery of the spontaneousWldS mutant mouse (Glass et al. 1993, Ludwin& Bisby 1992, Lunn et al. 1989), research onWldS and its constituent proteins have pro-vided valuable insight into the understandingof axon injury and degeneration. In addition toslower degeneration in response to mechanicaldamage, other responses are delayed in WldS

compared with wild-type nerves, includingneurolament degradation upon additionof the calcium-activated protease m-calpain(Bernier et al. 1999) and degeneration ofdorsal root ganglia (DRG) neurites exposed totoxins such as vincristine (Wang et al. 2001) ortaxol (Wang et al. 2002). The protective WldS

gene was later identied to encode a chimericprotein formed by fusion of the N terminus ofUbe4b, an E4 ubiquitin ligase, to the completesequence of nicotinamide mononucleotideadenylyltransferase 1 (Nmnat1) (Conforti et al.


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Mammalian Nmnats

Mammals, including humans, have three Nmnat genes: Nmnat1,Nmnat2, andNmnat3. The protein encoded byNmnat1, themaincomponent in the aberrant WldS protein, is located predomi-nantly in the nucleus, and its overexpression is sufcient to delayWallerian degeneration. The Nmnat2 protein is found mostly inthe Golgi complex, and downregulation of the gene in culturedneurites induces spontaneous axon degeneration. The Nmnat3protein is localized to themitochondrial matrix, and it is the mostpotent among the three Nmnats in protecting injured axons (Lauet al. 2009, Jayaram et al. 2011).

Dying-back: aretrograde manner ofaxon degeneration

2000, Mack et al. 2001). Nmnat is a critical en-zyme in the NAD+ biosynthesis pathway and isessential for many cellular processes ( Jayaramet al. 2011, Lau et al. 2009). For a detailed lookat WldS, Nmnat, and axon degeneration, somerecent reviews are recommended (Coleman &Freeman 2010, Feng et al. 2010). Below, wefocus on the current view of its mechanisms,with an emphasis on the new knowledge thatrecent Drosophila studies have contributed.

Is the Protective Function of WldSOwing to the Nmnat1 Activity?

A central question regarding the protectiveactivity of WldS is the extent to which theactivity in neural protection can be attributedto the Nmnat1 activity of the chimeric protein.Although in one study Nmnat1-overexpressingtransgenic mice failed to show a delay inWallerian degeneration in severed sciaticnerves (Conforti et al. 2007), a larger body ofinvestigations based on both mammalian andy models demonstrates that overexpression ofNmnat1 alone is sufcient to prevent axon de-generation (Araki et al. 2004; Avery et al. 2009;Babetto et al. 2010; Conforti et al. 2009; Press& Milbrandt 2008; Sasaki et al. 2006, 2009a;Sasaki & Milbrandt 2010; Vohra et al. 2010;Yahata et al. 2009). Furthermore, Nmnat islikely also essential for normal axon mainte-nance. This idea emerged from an in vitromodel of cultured mouse superior cervical

ganglia neurons (Gilley & Coleman 2010), inwhich RNA interference of Nmnat2 (see side-bar, Mammalian Nmnats) induced Wallerian-like degeneration in cultured superior cervicalganglia neurites. However, a follow-up studycould not conrm this effect in vivo: Het-erozygous Nmnat1 knockout mice showedno spontaneous axon degeneration, and thelesioned sciatic nerve degenerated with normaltiming, whereas homozygous Nmnat1 knock-out mice were not viable (Conforti et al. 2011).

In ies, loss of the Drosophila Nmnat(dNmnat) function causes neurodegenerationin photoreceptor cells (Zhai et al. 2006) andfragmentation of developing dendrites andaxons in y larvae (Wen et al. 2011). How-ever, whether endogenous Nmnat activity isrequired for maintaining healthy axons in theadult was unknown until recently, when the ywing model was developed. Taking advantageof tissue-specic genetic manipulation, thissystem reveals that downregulation of dNmnatin the wing nerve leads to robust dying-backfragmentation that markedly resembles Walle-rian degeneration (Fang et al. 2012). Together,upregulation of the Nmnat activity protects,whereas downregulation induces, axon degen-eration, indicating that Nmnat plays a centralregulatory role in controlling axon integrity.

Where and How Does WldS/NmnatAct to Protect Injured Axons?

Because WldS and Nmnat1 are found predom-inantly in the nucleus, their axon-protectivefunction initially was thought to involve a nu-clear mechanism (Mack et al. 2001). Lend-ing some support to this hypothesis, mam-malianNmnat1-mediated neuroprotection wasshown to require an NAD+-dependent nucleardeacetylase, Sirt1 (Araki et al. 2004). A yearlater, however, a conicting paper reported thatSirt1 was not required for NAD+-dependentaxon protection (Wang et al. 2005). In agree-ment with this assessment, a recent study basedon ablation of the y olfactory nerve indicatedthat genetic knockdown of Sir2, the Drosophilahomolog of mammalian Sirt1, did not affect the

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WldS-mediated protection (Avery et al. 2009).Similarly, overexpression of WldS or Nmnatcould still dramatically delay Wallerian degen-eration of the severed y wing nerve when Sir2or other members of the sirtuin protein familywere downregulated (Y. Fang & N.M. Bonini,unpublished observations).

Other studies support a nonnuclear func-tion for the WldS/Nmnatmediated axonprotection. In transgenic mice, deletion of thenuclear localization signal of WldS, which re-distributes the WldS protein from the nucleusto the cytoplasm, appeared to provide evenstronger axon protection than the native WldS

protein (Beirowski et al. 2009). Moreover,expression of a mutant form of the Nmnat1protein localized to the cytoplasm was suf-cient to delay axon degeneration in culturedneurons (Sasaki et al. 2006). And although theinitial Nmnat1-overexpressing transgenic miceshowed no effect to delay Wallerian degenera-tion (Conforti et al. 2007), the transgenic miceoverexpressing cytNmnat1 (an engineeredmutant version of Nmnat1 that was targeted tothe cytoplasm) or Nmnat1 fused with an axonaltargeting peptide derived fromAPPmanifestedrobust axon protection (Babetto et al. 2010,Sasaki et al. 2009a). Furthermore, deletion of a16amino acid N-terminal sequence of WldS,which binds to valosin-containing protein(VCP)/TER97 in the cytoplasm, signicantlyreduced the effectiveness of the protein in axonprotection in both transgenic mice (Confortiet al. 2009) and Drosophila (Avery et al. 2009).Also, local transduction of the Nmnat proteininto severed axons potently blocked Walleriandegeneration (Sasaki & Milbrandt 2010),and interruption of Nmnat1 axonal deliveryabolished its axon protection (Babetto et al.2010). Taken together, these data indicate animportant role for cytoplasmic/axoplasmic lo-calization ofWldS/Nmnat rather than a nuclearfunction.

More recently, the cellular site of Nmnat-mediated axon protection has been linked tomitochondria. Despite its predominant nuclearlocalization, the WldS protein is also detected

in cytoplasmic fractions that include mito-chondria (Beirowski et al. 2009). Yahata et al.(2009) showed that the axon protective func-tion of Nmnat is correlated with its local-ization to the mitochondrial matrix, and Ba-betto et al. (2010) showed that axon-targetedNmnat1 is transported coordinately with mi-tochondrial movement in transfected neuritecultures. Furthermore, expression of the mi-tochondrial isoform of the mammalian Nm-nat, Nmnat3 (Nikiforov et al. 2011), protectsinjured axons as potently as WldS in culturedDRG neurons (Sasaki et al. 2006), transgenicmice (Yahata et al. 2009), and Drosophila (Av-ery et al. 2009). In addition to axotomy, Nmnatalso delays axon degeneration induced by mito-chondrial or oxidative stress (Press&Milbrandt2008). In contrast, however, Antenor-Dorsey&OMalley (2012) reported that, in a Parkinsonsdisease (PD) model induced by N-methyl-4-phenylpyridinium (MPP+; thought to impairmitochondrial function), WldS but not Nm-nat1, Nmnat3, or cytoplasm-targeted Nmnat1protected dopamine axons.Most recently, stud-ies using the Drosophila wing model demon-strate that WldS/Nmnat prevents injury-induced mitochondrial loss and subsequentaxon degeneration in vivo (Fang et al. 2012).This function may involve Nmnats essentialrole in NAD+ biosynthesis and energy pro-duction, as mitochondria puried from Nmnattransgenic mice appeared to have increased ca-pacity for ATP production (Yahata et al. 2009).Furthermore, when mitochondria are removedfrom the axons of the ywing by genetic knock-down of the mitochondrial transporter Milton(Gorska-Andrzejak et al. 2003, Stowers et al.2002), overexpression of WldS or Nmnat canno longer delay axon degeneration (Fang et al.2012). Similarly, reduction of another mito-chondrial adaptor, Miro (Wang & Schwarz2009), also suppresses the neuroprotective ef-fect of WldS in the injured olfactory nerve(Avery et al. 2012). Together, ndings fromthese studies argue for a mitochondrial siteof action and mechanism for Nmnat-mediatedaxon protection.


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UPS: ubiquitinproteasome system

Is Nmnats Axon Protection Mediatedby NAD+?

Several in vitro studies indicate that exogenousapplication of NAD+ or its precursors cansuppress axon degeneration in mammalianneurite cultures (Araki et al. 2004, Sasakiet al. 2006, Wang et al. 2005), although noprotection is observed in severed axons fromprimary neuronal cultures suppliedwithNAD+

(Conforti et al. 2007). In addition, NAD+

content is not increased in tissues from WldS

mice (Mack et al. 2001) or in cultured neuronsoverexpressing WldS or Nmnat1 (Araki et al.2004, Sasaki et al. 2009b). A recent studysuggested that the NAD+-mediated protectionmay function differentially in acute traumaticinjury compared with chronic secondary axonalinjury. Application of nicotinamide, the Nm-nat1 precursor, transiently delayed Walleriandegeneration in an in vivo model of peripheralnerve injury, but it did not delay axonal loss inthe rat model of chronic Charcot-Marie-Toothdisease (Meyer zu Horste et al. 2011).

Fly geneticists have approached this ques-tion from a different angleby mutating theactive sites in the Nmnat protein (enzyme-dead) and then assessing the requirement forNmnats enzymatic activity in neuroprotection.First, in vivo expression of the enzyme-deadNmnat retained strong neuroprotection in yphotoreceptor cells, which suggests an NAD+-independentmechanism (Zhai et al. 2006). Thesame group later revealed that Nmnat mayprovide protection against neurodegenerationby acting like a chaperone (Zhai et al. 2008)and that Nmnat is a stress response proteinthat is transcriptionally regulated during sev-eral different stress conditions (Ali et al. 2011).In axons, however,Nmnat-mediated neuropro-tection appears to engage a different mecha-nism. For example, WldS dramatically atten-uated axonal loss and disease progression in aprogressive motor neuronopathy (pmn) model(Ferri et al. 2003), but apoptosis of the mo-tor neurons in pmn mice was not prevented byWldS (Simonin et al. 2007). In addition, expres-sion of enzymatically inactive Nmnat1 orWldS

severely reduced the ability of these proteins toprotect axons in the y olfactory nerve model(Avery et al. 2009). Taken together with recentmammalian studies (Conforti et al. 2009, Sasakiet al. 2009b, Yan et al. 2010), the current viewof NAD+ and Nmnats enzymatic activity inthe eld is that the activity is required for Nm-nat to be fully functional in axon protection,but additional Nmnat substrates and down-stream effectors are likely involved (Coleman&Freeman 2010, Sorci et al. 2007).

THE UBIQUITIN PROTEASOMESYSTEM AND NEURALINTEGRITY

Ubiquitination and proteasome-mediatedprotein degradation play important roles inmaintaining protein homeostasis in neuronalcell bodies and synapses and are thought tocontribute to the pathophysiology of neu-rodegenerative diseases including Alzheimersdisease (AD) (Upadhya &Hegde 2007) and PD(Lim&Tan 2007). Recent studies also indicatea crucial role for the ubiquitin proteasome sys-tem (UPS) in axon and dendrite maintenanceand neuropathology (for reviews, see Korho-nen & Lindholm 2004, Lehman 2009). Here,we focus on comparing the role of the UPSand WldS/Nmnat in traumatic axon injury anddevelopmental pruning, as well as the dualfunction of the UPS in axon maintenance.

The Ubiquitin ProteasomeSystem and WldS

Because the chimeric WldS protein containsthe Ube4b substrate-binding domain but hasno ubiquitylation enzymatic activity, it wasspeculated that WldS might have a dominant-negative effect on theUPS pathway. Consistentwith this hypothesis, pharmacologic or geneticdisruption of UPS activity profoundly delaysWallerian degeneration (Hoopfer et al. 2006,MacInnis & Campenot 2005, Wakatsukiet al. 2011, Zhai et al. 2003) and impairsdevelopmental axon pruning (Hoopfer et al.2006, 2008; Watts et al. 2003), which suggestsa mechanistic overlap between these two

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NGF: nerve growthfactor

processes. Similar to Nmnats effect on axons,proteasome inhibitors such asMG132 preserveaxonal mitochondrial function in transected ax-ons of rat sympathetic neurons and delay axondegeneration induced by withdrawal of nervegrowth factor (NGF) (MacInnis & Campenot2005). However, Simonin et al. (2007) foundthat, contrary to the hypothesis that WldS maynegatively affect the UPS, WldS partially res-cued the proteasome impairment in the motorneuron cell bodies and axons of pmn mice. Inaddition, Nmnat was found to suppress Tau-associated toxicity in Drosophila by interactingwith phosphorylated Tau and promoting itsubiquitination and degradation (Ali et al. 2012).Thus, the precise effects of WldS/Nmnat andthe effect of the UPS vary depending uponthe situation, whether in an acute injury,developmental pruning, or disease context.

Developmental Pruning VersusTraumatic Axon Degeneration

It seems that both WldS/Nmnat and the UPSfunction in regulating nerve degeneration.But do they always work together? Evidencesuggests that they can be parsed into distinctpathways. Expression of either WldS or theubiquitin proteaseUBP2 blocks injury-inducedaxon degeneration. In contrast, the WldS pro-tein has no effect on normal developmentalpruning in the mouse retinal ganglion cells orthe Drosophila mushroom body axons (Hoopferet al. 2006). However, Schoenmann et al.(2010) showed that WldS suppresses dendriticpruning in the y larval da neurons. Thesame study also showed that inhibition of theeffector caspase-3 or caspase-6 is not sufcientto confer axon protection in cultured mouseDRG explants (Schoenmann et al. 2010).This is contrary to an earlier report in whichdownregulation of the caspase-6 activity blocksNGF deprivationinduced axon degenerationboth in culture and in vivo in mice (Nikolaevet al. 2009). More recently, Tao & Rolls (2011)demonstrated with the larval y laser ablationsystem that expression of the WldS proteindelays developmental pruning of dendrites,but the protective effect in this developmental

context is not as potent as protection seenby WldS upon dendritic injury. Furthermore,they found that overexpression of effectorcaspase inhibitors such as P35 and DIAP1(Drosophila inhibitor of apoptosis), or geneticdownregulation of the initiator caspase Dronc,all potently delayed dendritic pruning. Suchinhibition or knockdown, however, could notprotect the dendrites from injury-induced de-generation (Tao & Rolls 2011). Thus, not onlymay WldS/Nmnat and the UPS act separately,but traumatic injuryinduced degenerationand programmed developmental pruning alsoappear to use distinct molecular mechanisms.

The Ubiquitin Proteasome SystemIs a Double-Edged Sword

The UPS alone appears to play a complicatedrole in axon maintenance. First, although im-pairing the UPS in injured axons delays de-generation (Hoopfer et al. 2006, MacInnis &Campenot 2005, Wakatsuki et al. 2011, Zhaiet al. 2003) or can even induce neurite out-growth (Inoue et al. 2004, Song et al. 2009),there are contrasting situations in which pro-teasome inhibition leads to axon degeneration(Kane et al. 2003, Laser et al. 2003). Further-more, deleting one copy of Ube4b causes ax-onal dystrophy and degeneration rather thanaxonprotection (Kaneko-Oshikawa et al. 2005).Moreover, a study of an axon stretch injurymodel suggests that activation of UPS activitymight actually have a protective role in TBI.In this model, the transient stretch injury didnot induce primary axotomy, but a large por-tion of the stretched axons developed secondaryaxonal loss, and inhibition of UPS activity ac-celerated progression of the nerve to secondaryinjury stages (Staal et al. 2009).

MOLECULAR SIGNALING INTHE NEURAL INJURY RESPONSEIN DROSOPHILA

Amyloid Precursor Protein/APP-Like

APP is the precursor of amyloid (A), whichis found in the extracellular senile plaques ofAD patients (Glenner & Wong 1984, Kang


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JNK: Jun N-terminalkinase

et al. 1987). The Drosophila homolog APPLis expressed in neurons but does not con-tain the A sequence (Martin-Morris & White1990), although fragments are generated thatmay have toxicity analogous to mammalian A(Carmine-Simmen et al. 2009). APP/APPL isinvolved in synaptic action and brain develop-ment as well as in response to traumatic injury.[For reviews of the physiological function ofAPP, see Kim&Tsai (2009), Panegyres (2001),and Zhou et al. (2011).] TBI can cause AD-likebrain pathology (Ikonomovic et al. 2004), andAPP levels increase after TBI in humans andin mammalian models (Murakami et al. 1998,Van den Heuvel et al. 1999). Similarly, the yAPPL is upregulated in brain regions damagedin needle stabbing experiments (Leyssen et al.2005). Furthermore, themortality rate after thebrain damage is increased in appl mutant iescompared with the wild type, suggesting thatinjury-induced upregulation of APP may be animportant antitrauma mechanism.

What are the downstream effectors ofAPP/APPL? APPL interacts with the Abelson(Abl) tyrosine kinase and actin-binding pro-tein Prolin (Lanier & Gertler 2000, Zandy& Pendergast 2008), which could activateAbl-dependent actin remodeling and thusregulate axonal arborization (Leyssen et al.2005). APP is also linked to caspases via thedeath receptor 6 (DR6) in mammals, as APP isreleased as a DR6 ligand in response to NGFdeprivation (Nikolaev et al. 2009). Specically,the extracellular N terminus of APP becomesbound to DR6 in axons and activates caspase6 (but not caspase 3, which is required for cellbody apoptosis) to trigger axon fragmentation.Interestingly, Park & Strittmatter (2007)found that APP and A also interact with theNogo-66 receptor, which reduces pathologicalchanges in a mouse AD model. The Nogo-66receptor is thought to mediate glial inhibitionof axon regeneration in the mammalian CNS(Vargas & Barres 2007, Zhang et al. 2008).

Cyclic AMP and Protein Kinase A

As mentioned earlier, injured Drosophila CNSaxons, like their mammalian counterparts,

essentially fail to regenerate in the y brainexplant model (Ayaz et al. 2008). Cyclic AMP(cAMP) signaling has long been known topromote regeneration of mammalian CNSaxons after SCI (for a recent review, seeHannila & Filbin 2008), and protein kinaseA (PKA) inhibitors can block this effect (Qiuet al. 2002). Consistent with these ndings,when the activity of PKA is upregulated in yneurons, the regenerative capacity of injureds-LNv axons in the y brain explant model isimproved signicantly (Ayaz et al. 2008). Thisagain conrms that mechanisms regulatingaxon regeneration in mammals and ies shareevolutionary conservation.

Jun N-Terminal Kinase

Another key molecular signaling pathway thatpromotes axon regeneration is the Jun N-terminal kinase ( JNK) cascade (Ayaz et al.2008). JNK signaling is upregulated uponinjury in the y brain needle-injury model(Leyssen et al. 2005) and the crushed larval mo-tor neuron axon model (Xiong et al. 2010). Inthe explanted y brain, expression of a consti-tutively active form of the Drosophila JNK ki-nase Hemipterous (Adachi-Yamada et al. 1999)induces robust regrowth of the severed axons,one-third of which even extend to reenter thetarget area (Ayaz et al. 2008). In a separate studyin which a Drosophila larval dendrite was con-verted to a growing axon in response to axo-tomy, JNK activation was also found to initiatemicrotubule rearrangements that preceded theanatomical conversion (Stone et al. 2010).

JNK activity not only is necessary and suf-cient for axon extension (Leyssen et al. 2005,Srahna et al. 2006) but also appears to be acore pathway that interacts with other molec-ular signals to orchestrate the complex cellu-lar response to neural injury (Figure 2). Forexample, although injury-induced activation ofJNK signaling is independent of the APP-Ablpathway (Leyssen et al. 2005), the function ofthese pathways in regulating axonal outgrowthseems linked: expression of Prolin and othercytoskeleton-interacting proteins can be up-regulated by the JNK pathway ( Jasper et al.

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NgR

Profilin

Ca2+

JNK

APP

DR6

cAMP

PKA

Hiw Hiw

Caspase 6

Abl

Profilin JIPs

Cytoskeletal integrity/microtubule dynamics/axonal transport

Glia

Axon

Nucleus

JNK

Axot

omy

Shark

Src42A

P

Draper

DLK/Wnd

DLK/Wnd

Figure 2Molecular signaling in Drosophila axon injury and regeneration. Axon injury in Drosophila activates multiple, cross-linked signalingpathways including amyloid precursor protein (APP), the DLK/Wnd-JNK cascade, and the cAMP-PKA pathway. These pathwaysregulate cytoskeletal integrity, microtubule (MT) dynamics, and axonal transport, and they thus mediate axon degeneration andregeneration. Specically, axotomy increases APP levels, which activates Abelson (Abl)-dependent actin remodeling via Prolin; APP isalso released as the death receptor 6 (DR6) ligand to activate caspase 6mediated axon degeneration; and APP interacts with the glialreceptor Nogo-66 receptor (NgR), which inhibits axon regeneration. The E3 ligase Hiw, which negatively regulates DLK/Wnd levels,is downregulated upon axotomy. This activates the DLK/Wnd-JNK cascade, which not only directly regulates MT dynamics andaffects axonal transport (e.g., via JIPs) but also can be transported retrogradely into the nucleus to reprogram gene expression in theinjured neuron. Similarly, axotomy elevates Ca2+ levels, which activates the cAMP-PKA pathway. These pathways are not independentbut rather interact with each other often. For example, JNK is associated with APP via JIPs, and the JNK cascade regulates transcriptionof cytoskeleton-interacting proteins such as Prolin. Furthermore, massive cross talk occurs between the cAMP-PKA and theMAPK-JNK pathways (for a recent review, see Gerits et al. 2008). In addition, axon injury activates the glial surface receptor Draperthrough Shark-Src42Adependent phosphorylation, which recruits glia to the degenerating axons and triggers phagocytosis to clean upthe axon debris. The orange owchart arrows indicate direction of signal transduction and the black arrows represent biological eventssuch as degradation and phosphorylation (solid), potential protein-protein interaction (dotted), and nuclear translocation (long broken).Abbreviations: DLK, dual leucine kinase; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.


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MAPK: mitogen-activated proteinkinase

2001), and JNK-interacting protein-1b ( JIP-1b) may scaffold APP/APPL with JNK in hu-mans and ies (Matsuda et al. 2001, Taru et al.2002). In addition, as reviewed below, JNKalso sits in the center of the dual leucine ki-nase (DLK)mitogen-activated protein kinase(MAPK) pathway in regulating axon degenera-tion and regeneration (Chen&Chisholm 2011,Wang & Jin 2011).

Dual Leucine Kinase/Wallendaand Highwire

DLK is a MAPK kinase kinase that is upstreamof JNK in the MAPK cascade (Gallo & John-son 2002). In Drosophila, the DLK orthologWallenda (Wnd) was identied initially in a ge-netic screen for suppressors of mutations in thehighwire (hiw) gene. Hiw is an evolutionarilyconserved E3 ubiquitin ligase that negativelyregulates synaptic growth in the Drosophilaneuromuscular junction (NMJ) (Wan et al.2000), and hiwmutants exhibit dramatic synap-tic overgrowth inDrosophila larvae (Collins et al.2006). Massaro et al. (2009) reported that en-hanced MAPK-JNK-Fos signaling and loss ofhiw [which results in increased levels of Wndprotein (Collins et al. 2006)] suppressed NMJretraction in spectrin mutants, which suggeststhat upregulation ofWnd prevents cytoskeletalderangements. Consistent with this idea, over-expression of Wnd is sufcient to protect ax-ons from degeneration in the larval nerve crushmodel (Xiong & Collins 2012). Thus, thesendings highlight a protective role for upregu-lated Wnd. In contrast, however, Miller et al.(2009) reported that severed olfactory axons inadult ies in a strongWnd loss-of-functionmu-tant background were signicantly preserved.The discrepancy between the two y mod-els may represent different regulatory mecha-nisms employed by the developing PNS of ylarvae and the mature CNS of adult ies; inthe larval NMJ model, the projections are inthe PNS, whereas in the adult olfactory nervemodel, the axonal trajectories are within thebrain. It should be pointed out that the Wndmutant is, to date, the only known loss-of-

function mutant that has displayed robust sup-pression of axon degeneration following injury(Miller et al. 2009). The degenerative effect byDLK/Wnd is supported by studies in mouseDRGcultures inwhichDLK-decient neuronsshow substantially delayed axon degenerationinduced by multiple methods including physi-cal injury, chemical insults such as vincristin [achemotherapeutic that is known to induce neu-ropathy (Wang et al. 2000)], and deprivation ofNGF (Ghosh et al. 2011, Miller et al. 2009).

Although Wnd seems to be essential forWallerian degeneration in the distal axonstumps, several other studies indicate a re-generative function of the Wnd protein inthe proximal axons following injury. Xionget al. (2010) show that axonal sprouting of thecrushed larval nerves is inhibited strongly in theloss-of-function mutant ofWnd and dominant-negativemutants of JNKandFos, but it is accel-erated in the hiw mutant, which indicates thatWnd/JNK/Fos signaling is required for effec-tive axonal regrowth (Xiong et al. 2010). Moreinterestingly, injury induced a rapid increase ofthe Wnd protein concomitant with a decreaseof the Hiw protein. The decrease of Hiw levelsnot only appears to slow down Wnd turnoverbut also may enhance retrograde transport ofthe Wnd protein (Xiong et al. 2010). As Wndcan be transported back into the nucleus, it mayserve as an axon injury signal to activate the nu-clear downstream targets of the JNK pathway,thus reprogramming the axon to regrow.A con-served regulatorymechanismby theMAPKsig-naling pathway is also seen in the axon injury re-sponse and regeneration inmammals (Itoh et al.2009, Lewcock et al. 2007, Nix et al. 2011) andC. elegans (Chen et al. 2011, Ghosh-Roy et al.2010,Hammarlund et al. 2009, Yan et al. 2009).

OTHER COMPONENTSOF AXON DEGENERATIONAND REGENERATION

The components discussed below are allessential factors that contribute signicantly tomaintaining healthy axons and allowing a quickresponse to nerve injury. They are summarizedtogether here in this review because the focus is

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on new insight from recent studies ofDrosophilamodels of axon injury, and the mechanisms ofthe following subjects, unlike those discussedabove, have not yet been addressed extensivelyusing y models.

Cytoskeleton and MicrotubuleDynamics

Axon injury triggers rapid alteration of thecytoskeleton, such as changes in actin lamentsand microtubule (MT) dynamics (Bradke et al.2012, Hur et al. 2011). In ies, such dynamicchanges are best represented by the amazingconversion of a dendrite into a regeneratingaxon in the experimentalmodel that axotomizeslarval da neurons (Stone et al. 2010) (seeTable 1). Because neurons are highly po-larized cells, their axons and dendrites havedistinct cytoskeletal arrangements for the pur-pose of transporting different cargoes (Conde& Caceres 2009, Hoogenraad & Bradke 2009,Witte & Bradke 2008). Drosophila neurons,similar to vertebrate neurons, have oppositepolarity in axons and dendrites: the axonalMTs are plus-end out, whereas more than90% of dendritic MTs are minus-end out(Stone et al. 2008). Upon axon injury, however,the number of growing MTs is ten timesupregulated, and the MT polarity is reversedin the dendrite. This injury response is specicto the axon, as a similar injury to a dendritedoes not elicit such a change of MT dynamicsin the remaining dendrites of the same neuron.Consistent with JNK regulating MT in axons(Gelderblom et al. 2004), the injury-inducedMT rearrangement in ies relies on activationof the JNK cascade (Stone et al. 2010).

Axonal Transport

Axonal transport of nutrients, proteins, RNAs,lipids, vesicles, and organelles is crucial tothe normal function and integrity of neurons;defective axonal transport is associated withneurodegenerative diseases such as AD, PD,Huntingtons disease, and amyotrophic lateralsclerosis (Perlson et al. 2010). However, it isunclear whether alteration of axonal transporthas a causative role in triggering these diseases

or if it is a secondary defect, although it stillmay contribute to the degenerative process. Inies, disruption of mitochondrial transport inthe adult y wing nerve causes spontaneous,progressive, retrograde axon degeneration(Fang et al. 2012), although larval axons appearto have a better tolerance for mitochondrialdepletion (Glater et al. 2006, Stowers et al.2002, Tao & Rolls 2011). In addition, it hasbecome clear that axonal transport plays animportant role in the injury response. Exam-ples in invertebrate models include retrogradeaxonal transport of injury signals, such as theWnd protein in ies (Xiong et al. 2010), andanterograde axonal transport and local trans-lation, such as the CCAAT/enhancer-bindingprotein-1 (cebp-1) mRNA in C. elegans (Yan et al.2009). In mammals, JNK and JNK-interactingprotein ( JIP) 3/Sunday Driver (Syd) aretransported retrogradely in response to injury(Cavalli et al. 2005), whereas JIPs bind directlyto kinesin, one of the major axonal transportmotor proteins (Koushika 2008, Verhey &Rapoport 2001). Therefore, once again, JNKsignaling appears to be key to sensing axoninjury and initiating axon regeneration.

The Role of Glia

As in vertebrates, theDrosophila nervous systemhas multiple types of glia. In fact, all four majortypes of glial cells in mammals have compara-ble counterparts in ies (Doherty et al. 2009,Freeman & Doherty 2006). This provides aunique opportunity to study the glial response,as well as axon-glial interactions in neuralinjury, in a manner that is not readily availablein lower model organisms. In 2006, two groupsdiscovered independently that the glial cellsurface receptor Draper is required for efcientclearance of degenerating axons during devel-opmental pruning and nerve injury (Hoopferet al. 2006, MacDonald et al. 2006). Draperis the homolog of the C. elegans gene ced-1,which is required for cell corpse engulfment(Freeman et al. 2003, Zhou et al. 2001).Severing the olfactory nerve by removing theantenna induced rapid and robust upregulationof Draper, and the glial membrane and Draper


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were recruited to surround the severed axons(MacDonald et al. 2006). In the Draper mutantor in ies with reduced Draper activity in glia,the severed axons still degenerate, but theaxonal debris lingers in the y brain (MacDon-ald et al. 2006). In addition, Ziegenfuss et al.(2008) revealed that Draper-dependent glialphagocytosis is mediated by a nonreceptortyrosine kinase, Shark, through Src and Sykfamily kinase signaling (Ziegenfuss et al. 2008).

Interestingly, axonal expression ofWldS notonly protects axons from degeneration but alsosuppresses the axotomy-induced glial response(MacDonald et al. 2006). This is strong evi-dence that the recruitment of glia and activationof Draper are not general injury responses butrather specic to degenerating/degenerated ax-ons. This suggests also that during the processof axon degeneration, some molecular signalslikely are released from the axons that are thenperceived by glia and activate the subsequentglial response.

PERSPECTIVES ANDCONCLUSIONS

Since Drosophila models entered the eldof neural injury, their study has added sig-nicantly to our understanding of the basiccellular and molecular mechanisms in axondegeneration and regeneration. Study of thesemodels is just beginning, and there are manyunsolved, interesting questions, such as howNmnat protects injured axons and maintainsaxonal integrity, how theMAPK-JNK pathwayis activated following injury, how cytoskeletaldynamics and axonal transport are regulatedin response to injury, how transcription in thenucleus is reprogrammed, and how glia arerecruited to the dying axons.

The discovery of the WldS mouse andnumerous subsequent studies have had asignicant impact in moving the eld of

axon degeneration forward. However, arethere other unknown components that canprovide axon protection like WldS/Nmnat?Similarly, the JNK cascade seems to be a corepathway that is activated to coordinate theinjury-induced molecular responses. But whatof other signaling pathways that also respondto axon injury? Intriguingly, Wakatsuki et al.(2011) recently revealed a parallel ZNRF1-AKT-GSK3B-CRMP2 pathway that regulatesaxon degeneration in cultured mammalianDRG neurons. Thus, little is known beyonda few genetic players that have been studiedextensively. Rather, the powerful approachesof simple model systems have entered the sceneonly recently, and insight from these systemsinto pathways and genes that impact acute axoninjury and trigger regeneration is still at earlystages. We maintain that Drosophila, as a modelorganism that is highly amenable to rapid,unbiased, large-scale genetic and genomicscreens, will show its full power in identifyingunsuspected and novel genes and pathways inthe years to come, especially with advancessuch as the recently streamlined larva chips(Ghannad-Rezaie et al. 2012) and the newlydeveloped adult y wing model (Fang et al.2012).

In conclusion, the simplicity of visualizingand severing Drosophila axons and the avail-ability of vast genetic toolsa fortune owingto the large, open, and generously collabora-tive y research communitycoupled with thehigh degree of conservation of pathways andmechanisms between ies and humans, makeDrosophila an exceptional model for the study offundamental mechanisms of nerve injury. Suchinsight has the promise to revolutionize our un-derstanding and will provide the foundation fornovel therapeutic targets for the enhancementof axon protection and the promotion of neuralregeneration in humans.

DISCLOSURE STATEMENT

The authors are not aware of any afliations, memberships, funding, or nancial holdings thatmight be perceived as affecting the objectivity of this review.

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ACKNOWLEDGMENTS

We thank M. Parisi, S.Y. Shieh, A. Berson, L. Soares and other members of the Bonini lab forcomments and critical reading of the manuscript. This work was supported by an NIH EUREKAaward (grant 1R01NS066312) toN.M.B.N.M.B. is an investigator of theHowardHughesMedicalInstitute.

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