Special Issue: Circuit Development and Remodeling

Preserve and protect: maintainingaxons within functional circuitsSarah E. Pease1,2,3 and Rosalind A. Segal1,2,3

1 Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA2 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA3 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA

During development, neural circuits are initially gener-ated by exuberant innervation and are rapidly refined byselective preservation and elimination of axons. Theestablishment and maintenance of functional circuitstherefore requires coordination of axon survival anddegeneration pathways. Both developing and maturecircuits rely on interdependent mitochondrial and cyto-skeletal components to maintain axonal health and ho-meostasis; injury or diseases that impinge on thesecomponents frequently cause pathologic axon loss.Here, we review recent findings that identify mecha-nisms of axonal preservation in the contexts of develop-ment, injury, and disease.

Establishing and maintaining circuitsFunctional neural circuits depend on proper interconnec-tions formed by long-range axonal projections. As the fun-damental connective unit of neural circuits, axons must beprotected and maintained in the face of multiple potentialthreats. Axonal maintenance is particularly important be-cause most neurons cannot be replaced and must thereforebe preserved throughout the life of the organism. Axondegeneration is a broad term applied to various modes ofaxon death, with distinct instigators but similar final mor-phology of axon fragmentation. Although much progress hasbeen made toward elucidating the mechanisms underlyingaxonal degeneration, a complete understanding requiresstudy of the mechanisms opposing degeneration. Axon re-generation, the recovery and regrowth of axons followingacute or chronic trauma, is not the opposite of axon degen-eration, but rather a response to it. In reality the opposite ofaxon degeneration is the process of axonal survival.

It was classically thought that axons degenerate as aresult of cell body death, due to a lack of support from the cellbody. This theory was first challenged by the discovery of theWallerian degeneration slow (WldS) mutant mouse (seeGlossary), where neuronal expression of the WldS fusiongene delays degeneration of severed axons for weeks. Morerecent studies have provided direct evidence for active

Feature Review

Glossary

Apaf-1 (apoptotic protease activating factor 1): a key constituent of the

apoptotic machinery that binds cytochrome c and subsequently activates

caspase-9.

Bcl-w (Bcl-2-like protein 2): a pro-survival Bcl-2 family member that binds pro-

apoptotic Bcl-2 family members to prevent initiation of the axonal apoptotic

caspase cascade [4].

Calpains: a family of calcium-activated cysteine proteases that degrade

cytoskeletal components and are activated in both developmental and

pathological axon degeneration [30].

Calpastatin: an endogenous calpain inhibitor, which is degraded during

developmental and pathological axon degeneration [30].

Caspases (cysteine-aspartic proteases): a family of cysteine proteases essential

for cell body apoptosis and developmental axon degeneration. Caspases are

first synthesized as inactive pro-caspases, which are activated upon cleavage.

DLK (dual leucine zipper kinase): a mitogen-activated protein kinase kinase

kinase (MAPKKK) involved in axon degeneration and regeneration [92–97]. Its

Drosophila ortholog is Wallenda.

DR6 (death receptor 6): a tumor necrosis factor (TNF) receptor whose

activation induces apoptosis and axon degeneration [27,45].

IMPA1 (myo-inositol monophosphatase-1): an enzyme involved in synthesis of

myo-inositol and therefore essential for phosphophatidylinositol signaling

pathways such as neurotrophin signaling.

Lamin B2: a component of the nuclear skeleton that also localizes to axons and

is essential for axon maintenance [75].

MEC-17 (mechanosensory abnormality 17): an enzyme that catalyzes tubulin

acetylation, a posttranslational modification, and which also stabilizes micro-

tubules and preserves axons independent of its acetyltransferase activity [83].

Neurotrophins: a family of secreted growth factors that promote axonal and

neuronal survival by binding to transmembrane tropomyosin receptor kinase

(Trk) receptors.

NMNAT (nicotinamide mononucleotide adenylyltransferase): a family of NAD+

biosynthetic enzymes involved in axon maintenance, and a component of the

WldS fusion gene.

NAD+ (nicotinamide adenine dinucleotide): an essential coenzyme for cellular

metabolism and signaling, which functions in ATP metabolism and as a

substrate for several protein-modifying enzymes [48].

mPTP (mitochondrial permeability transition pore): a protein pore formed in

the inner membrane of the mitochondria under pathological conditions,

allowing mitochondrial release of calcium stores and reactive oxygen

species.

SARM1 (sterile a-motif-containing and armadillo-motif containing protein): a

Toll-like receptor adaptor essential for axon degeneration by an unknown

mechanism [1,2]. Its Drosophila ortholog is dSARM.

SCG10 (superior cervical ganglion 10): a microtubule destabilizing protein that

promotes axonal health. It is degraded in a c-Jun N-terminal kinase (JNK)-

dependent manner following axon injury [88].

UPS (ubiquitin proteasome system): the major non-lysosomal mechanism by

which the cell degrades proteins. Proteins are targeted for degradation by the

small protein tag ubiquitin via ubiquitin ligase enzymes, and tagged proteins

are subsequently degraded by the proteasome complex.

Wallerian degeneration: a form of axon degeneration resulting from focal nerve

transection wherein the axon distal to the injury site swells and fragments.

WldS (Wallerian degeneration slow): a chimeric fusion gene of the NAD+

biosynthetic enzyme NMNAT1 and the ubiquitination factor E4B (UBE4B),

Corresponding author: Segal, R.A. (rosalind_segal@dfci.harvard.edu).Keywords: axon injury; axon maintenance; axon survival; neurodegeneration;neurotrophins; Wallerian degeneration.

whose expression delays various forms of pathological axon degeneration.

XIAP (X-linked inhibitor of apoptosis protein): an inhibitor of apoptosis protein

that binds to and inhibits caspases to prevent initiation of cell body apoptosis

and the axonal apoptotic cascade [25,26].

0166-2236/

� 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tins.2014.07.007

572 Trends in Neurosciences, October 2014, Vol. 37, No. 10

Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

axonal death mechanisms, such as the pro-degenerationmolecule dSARM/SARM1 [1,2], as well as for pro-survivalmechanisms, such as the Bcl-2 family member Bcl-w (Bcl-2-like protein 2; Bcl2l2) [3–5]. Thus, it is now apparent that theaxonal compartment relies on distinctive pathways for sur-vival and degeneration, and these exhibit similarities to anddifferences from cell body survival and death mechanisms[5–13]. In this review, we first examine mechanisms ofdevelopmental axon survival and pruning. We then discusspathways promoting lifelong axonal maintenance andhealth, and the opposing degenerative processes triggeredby injury and disease. Recent reviews have addressed axonregeneration [14,15] and dendritic degeneration [11].

Developmental axon preservationA common theme in neural development is overproductionfollowed by elimination and refinement. This mechanismallows for great flexibility in potential circuit configuration[7]. In both the central and peripheral nervous systems,neurons initially extend excess axonal connections, andrefinement into a mature circuit requires coordinatedpruning of inappropriate connections and preservation ofappropriate connections. Pruning must therefore be in-duced in a selective subset of axons while the remainingaxons are protected and maintained. Further, the scale ofaxonal elimination must be closely regulated. Pruning canremove segments as small as axon terminals or as large aswhole axons, and can even include subsequent apoptosis ofthe cell body.

Extracellular cues

Extracellular cues from other neurons within a circuit orfrom nearby glial or target cells often determine whichaxons will initiate intracellular axon pro-survival path-ways and which will be removed. Critical cues that havebeen identified include network activation and secretion ofgrowth factors. During early postnatal development of theneuromuscular junction (NMJ), muscle cells are initiallyinnervated by multiple motor neuron terminal arbors.These overlapping inputs compete for survival in an activ-ity-dependent manner. Inputs delivering stronger andmore correlated activity are strengthened, and the remain-ing inputs are eliminated, such that each muscle cell isultimately innervated by a single motor neuron [7]. A simi-lar activity-dependent mechanism is used in the developingcerebellum to select for survival of a single climbing fiberinput onto a single Purkinje cell [16]. Activity-regulatedmechanisms, including changes in transcription as wellas cytoskeletal and morphological adaptation, enable main-tenance of axons connected within a functional circuit.

Neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins3 and 4 (NT3 and NT4), constitute the most well recog-nized growth factor family that promotes axonal andneuronal survival. In the peripheral nervous system,survival of sympathetic and sensory neurons dependson successful competition for a limited supply of tar-get-derived neurotrophins. Furthermore, local stimula-tion with neurotrophins regulates axonal growth,branching, and terminal arborization [8,17–20]. Neuro-trophins secreted by target cells bind to tropomyosin

receptor kinase (Trk) receptors located on innervatingaxon terminals and initiate both local and retrogradesignaling events in the axon. This paradigm has beenstudied in vitro through the use of various compartmen-ted culture platforms that spatially and fluidically isolatecell bodies and distal axons, and so replicate the separa-tion between axon terminals and cell bodies that occurswithin normal neuronal circuits. In these compartmen-ted culture platforms, cell bodies and axons can be inde-pendently deprived of or stimulated with neurotrophins,and changes within cell bodies and axons can be assayedseparately. In pioneering studies using sympathetic neu-rons grown in compartmented cultures, Campenot firstdemonstrated that local axonal neurotrophin stimula-tion, a correlate of in vivo target-derived neurotrophinstimulation, is required to promote axonal survival,whereas cell body survival is supported by either somaticor axonal neurotrophin stimulation [21].

Inhibitors of axonal apoptosis

Until recently, the involvement of the apoptotic cascade indevelopmental axon degeneration was largely discounted[22]. Seminal work from several groups has since describedan apoptotic caspase cascade within axons that is inducedby neurotrophin withdrawal, and identified anti-apoptoticproteins that promote developmental axon survival byinhibiting this specialized cascade (Figure 1).

Anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL (alsoknown as Bcl2l1), and Bcl-w (Bcl2l2) avert somatic apo-ptosis by binding and sequestering pro-apoptotic Bcl-2family members, thus preventing mitochondrial releaseof cytochrome c and subsequent activation of caspases.Of these closely related family members, only Bcl-w mRNAand protein are enriched in axons [4,5]. Bcl-w expressioncan be detected in late embryonic and early postnataldevelopment as axon terminals reach their peripheraltargets, and expression continues throughout adulthood[4]. Moreover, target-derived neurotrophins selectivelystimulate a pathway that relies on extracellular signal-regulated kinase 5 (ERK5) to induce transcription of Bcl-wand a set of retrograde response genes [3,23]. Bcl-w bindsand inhibits the anti-apoptotic Bcl-2 family member Bax,thus preventing changes in the mitochondrial membranepotential, cytochrome c release, and subsequent initiationof the axonal caspase cascade [5]. Genetic studies indicateBcl-w functions within the axon to promote axon survivaland prevent degeneration [3,5].

In contrast to Bcl-2 proteins, the inhibitor of apoptosis(IAP) proteins prevent initiation of the apoptotic cascadeby direct inhibition of caspases because they bind toactivated, cleaved caspase-3 [24,25]. Thus, X-linked inhib-itor of apoptosis protein (XIAP) provides a second mode ofcontrol that restrains axonal caspase activity and subse-quent axonal pruning. Cultured sensory neurons lackingXIAP degenerate more rapidly when deprived of neuro-trophin, and embryonic XIAP�/� mice show decreasedepidermal sensory innervation without a concomitant lossof cell bodies [25,26]. Furthermore, XIAP�/� neuronsgrown in compartmented cultures exhibit increased so-matic levels of active caspase-3 when neurotrophins arewithdrawn only from axons, suggesting a role for XIAP in

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Caspase-9

Caspase-3

Calpain

XIAP

Calpasta�n KIF2A

BaxCytochrome c

Bcl-w

Caspase-6

Cytoskeletal degrada�on

TrkATrkB

NGF/BDNF

DR6

SARM1

? ?

TRENDS in Neurosciences

Figure 1. Developmental axon survival and degeneration pathways. Following trophic withdrawal, parallel pro-degenerative cascades converge on a common pathway of

cytoskeletal degradation to induce axon degeneration. Pro-survival molecules (blue) actively inhibit pro-degenerative molecules (green). The neurotrophins NGF and BDNF

stimulate TrkA and TrkB receptors on the growing axon and induce axonal expression of the anti-apoptotic Bcl-2 family member Bcl-w. Bcl-w inhibits the pro-apoptotic Bcl-

2 family member Bax, preventing activation of the axonal apoptotic cascade [3,5]. The endogenous inhibitors XIAP and calpastatin also inhibit the degenerative proteases

caspase-3 and calpain, respectively, preventing downstream cytoskeletal degradation [25,26,30]. In the absence of neurotrophins, Bax elicits mitochondrial release of

cytochrome c and activation of the protease caspase-9 by an unknown mechanism [26,27]. Caspase-9 cleaves and activates caspase-3, which itself activates caspase-6 and

the calcium-sensitive protease calpain [13,25,27–30]. In addition, the receptor DR6 can initiate activation of caspase-6 [27,45]. The proteins KIF2A [46] and SARM1 [2] also

induce cytoskeletal degradation in the absence of trophic support, although it is unknown how they are regulated. Abbreviations: Bcl-w, Bcl-2-like protein 2; BDNF, brain-

derived neurotrophic factor; DR6, death receptor 6; KIF2A, kinesin superfamily protein 2A; NGF, nerve growth factor; TrkA, tropomyosin receptor kinase A receptor; TrkB,

tropomyosin receptor kinase B receptor; SARM1, sterile a-motif-containing and armadillo-motif containing protein; XIAP, X-linked inhibitor of apoptosis protein.

Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

spatially restricting apoptotic cascade activation to theaxon [26].

The caspase cascade activated in axonal degenerationinvolves an initial and essential catalytic function forcaspase-3, followed by activation of caspase-6 [13,25,27–30]. The roles of these two caspases in axonal degenerationhave been demonstrated by analysis of genetic models. Thecalcium-activated calpain family constitutes a second set ofproteases implicated in both developmental and patholog-ical axon degeneration. In neurotrophin-supported axons,calpastatin inhibits calpain activation. Upon neurotrophindeprivation, calpastatin is degraded by activated caspase-3, allowing downstream activation of calpain and subse-quent cleavage of calpain targets such as neurofilaments[30].

Although primarily regarded as mediating pathologi-cal axon degeneration, there is also some evidence that an

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NAD+-sensitive pathway operates in parallel with thecaspase cascade during axon pruning [13,31]. Combinedtreatment with NAD+ and caspase inhibitors completelyprotects wild type axons from neurotrophin withdrawal,whereas the individual inhibitors alone only exert incom-plete protective effects [13]. These results suggest thatNAD+ can promote survival of neurotrophin-deprivedaxons, but it is not yet known whether an NAD+-sensitivepathway is endogenously activated during axon pruning,and how it might interact with the axonal apoptoticcascade.

Regulating protein levels

Regulation of axonal degenerative cascades requiresprecisely controlled localization and quantities of protec-tive factors. New axonal proteins are supplied by antero-grade microtubule-dependent transport as well as by local

Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

translation. Inhibition of axonal protein synthesis withlocal cycloheximide treatment abolishes the axon survivaleffect of neurotrophins, suggesting that neurotrophins relyin part on locally synthesized factors to mediate axonsurvival [5]. Two such factors have been recently identified:Bcl-w and myo-inositol monophosphatase-1 (IMPA1). Asindicated previously, Bcl-w plays a role in local inhibitionof the caspase cascade, and also regulates mitochondrialmorphology and function [5]. IMPA1 is a key enzyme of theinositol cycle and therefore necessary for proper inductionof phosphoinositide pathways triggered by neurotrophinsignaling [32]. Given the thousands of mRNAs identified inembryonic axons, it is likely that additional protectivefactors are locally translated to stabilize axons connectedwithin a circuit [33].

Protein turnover is frequently regulated by the ubiqui-tin proteasome system (UPS). Global inhibition of theproteasome with pharmacological agents or genetic muta-tions have yielded conflicting results, with some studiesfinding it prevents axon pruning [30,34] and others findingit accelerates pruning [26]. One mechanism by whichproteasome inhibition may be protective is by preventingdegradation of pro-survival molecules. Axonal XIAPand calpastatin are both degraded upon neurotrophin

Degenera�on

Retrac�on

Axosome sh eddin g

(A)

(B)

(C)

Developmental

Bcl-wXIAP

Calpasta�n

CaspasesCalpainKIF2A

SARM1

Ephrin/EphSema3F/Plexin3A

Figure 2. Modes and mechanisms of axon elimination. Schematics of neurons during

axon degeneration, as occurs with trophic deprivation, results in cytoskeletal degrada

calpastatin and driven by caspases, calpain, KIF2A, and SARM1 [2–5,13,25–30]. (B) Retra

terminal branches. It can be driven by Ephrin/Eph [40,41] or Sema3F/plexin3A [39] sign

swelling of the axon tip and shedding of membrane-bound axon remnants called axos

degeneration is a type of pathological axon elimination induced by axon severing (indic

site undergoes swelling, cytoskeletal breakdown, and fragmentation. Various NMNAT

SARM1, DLK, and calpain drive degeneration [1,2,30,93]. (E) In many neurodegenerative

and fragmentation beginning distally and propagating in a proximal direction. Various

Abbreviations: Bcl-w, Bcl-2-like protein 2; DLK, dual leucine zipper kinase; KIF

adenylyltransferase; Sema3F, semaphorin 3F; SARM1, sterile a-motif-containing and

XIAP, X-linked inhibitor of apoptosis protein.

withdrawal, resulting in loss of axon viability [25,30]. XIAPis degraded by the UPS, thus releasing caspase-3 andallowing induction of the axonal apoptotic cascade[13,25] and activation of calpain [30]. Conversely, theUPS may also promote axonal survival by degradingpro-apoptotic proteins.

Developmental axon pruningAxons or axonal segments that are not selected to survivecan be eliminated by three distinct mechanisms(Figure 2A–C). The most well studied is axon degeneration,which culminates in cytoskeletal degradation, axon frag-mentation, and removal of debris by glia and possiblyepidermal cells (Figure 2A) [6–8,11,12,35]. By contrast,both axon retraction (Figure 2B) and axosome shedding(Figure 2C) have only been observed during small-scalepruning of synapses or terminal branches. Axon retractionis the pulling back and absorption of small segments ofaxon [7,8], and axosome shedding involves axon tip swell-ing and shedding of membrane-bound axonal remnantscalled axosomes [6,7,36]. Axon remnants are absorbed bythe neuron itself during retraction, whereas axosomes areengulfed by adjacent cells, such as the surroundingSchwann cells [36].

Wallerian degenera�on

Dying-back

(D)

(E)

Pathologica l

CalpainSARM1

DLK

Wlds/NMNA TBcl-w

Wlds/NMNA TCalpasta�n

TRENDS in Neurosciences

developmental pruning (A-C) and pathological axon loss (D-E). (A) Developmental

tion and axon fragmentation. Degeneration is prevented by Bcl-w, XIAP, and/or

ction involves pulling back and absorption of small axon segments, such as axon

aling pathways. (C) Axosome shedding also removes small axon segments, with

omes. The underlying molecular mechanisms are still unknown [36]. (D) Wallerian

ated by a dashed red line). Following a latency phase, the axon distal to the injury

isoforms, the WldS fusion protein, and calpastatin prevent degeneration, whereas

diseases, axons are eliminated by a dying-back process that involves axon swelling

NMNAT isoforms, the WldS fusion protein, and Bcl-w can oppose this process [4].

2A, kinesin superfamily protein 2A; NMNAT, nicotinamide mononucleotide

armadillo-motif containing protein; WldS, Wallerian degeneration slow protein;

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Extracellular signaling

Some cells in a circuit promote axon survival, whereasothers actively induce axon pruning. In sympathetic neu-rons, competition results in the more active axons eliminat-ing their competitors. NGF-TrkA and activity-dependentsignaling cascades maintain the winning axons. Meanwhile,the winning axons release proBDNF, which binds to p75neurotrophin receptor (p75NTR) in losing axons andinduces axon degeneration [37]. A similar mechanism ofp75NTR- mediated axon degeneration and Trk-mediatedaxon survival is critical for correct circuit connectivity inadult septal cholinergic neurons [38]. Several guidancemolecules secreted in target regions are also known toinduce axon pruning. These include semaphorin 3F andits receptor plexin-A3, which is involved in pruning ofhippocampal mossy fiber collaterals [39], and ephrins andtheir receptor Ephs, which mediate retinal ganglion cell(RGC) topographical mapping [7] and axon retraction[40,41].

Luo and colleagues demonstrated that ecdysone hor-mone signaling instigates axon pruning of mushroom body(MB) g neurons during Drosophila metamorphosis [7,42].Ecdysone hormone stimulates ecdysone receptor B1 (EcR-B1) expressed selectively on g neurons and induces axonpruning [42]. Glial cells control neuronal expression ofEcR-B1 by secreting the transforming growth factor beta(TGF-b) ligand myoglianin, activating TGF-b signalingpathways in g neurons [43,44].

Neurotrophin deprivation engages additional pathwaysto mediate axonal destruction. In particular, tumor necro-sis factor (TNF) family receptors such as death receptor 6(DR6) contribute to axonal degeneration via downstreamactivation of caspase-6 [27,45]. This cascade may be par-ticularly relevant in Alzheimer’s disease.

Intracellular signaling

Several intracellular signaling mechanisms play a role inaxon pruning. As discussed, the apoptotic cascade mediatesdevelopmental axon degeneration and is controlled by bothnegative (XIAP, Bcl-w) and positive (TNF receptor DR6)regulators. Neurotrophin withdrawal pathways converge onthe pro-apoptotic Bcl-2 family member Bax, which causescytochrome c release from mitochondria [26,27]. Interest-ingly, while cytochrome c binds apoptotic protease activat-ing factor 1 (Apaf-1) to activate caspase-9 in cell somaapoptosis, apparently Apaf-1 is not required for axon degen-eration, and so the mechanism for activation of caspase-9 inaxons is not yet clear [26]. Following activation of caspase-9,caspase-3 is cleaved and activated, and caspase-3 directlyactivates caspase-6 and indirectly activates calpain viacalpastatin cleavage [29,30]. Together these results suggestthat there are both similarities and distinctions between theaxonal and somatic apoptotic cascades that will be impor-tant to decipher. Furthermore, although the apoptotic ma-chinery is involved in some types of developmental axondegeneration (neurotrophin deprivation-induced degenera-tion, retinocollicular axon pruning) [13,25,27–30], it doesnot appear to be involved in others (MB g neuron pruning)[34]. These findings suggest there may be multiple mecha-nisms of developmental axon degeneration that converge ona common pathway of cytoskeletal breakdown.

576

Developmental axon degeneration can be initiated bythe mammalian Toll receptor adaptor sterile a-motif-containing and armadillo-motif containing protein(SARM1) [2]. SARM1 is activated in parallel with thecaspase cascade during neurotrophin deprivation andappears to function primarily at an early stage of degen-eration [2]. The mechanism by which SARM1 mediatesaxon degeneration is unknown, but its Toll interleukin-1receptor and sterile a motif domains are necessary for itsdestructive function [2]. Like the caspase cascade, theDrosophila ortholog dSARM does not appear to play a rolein MB g neuron axon pruning [1], but both SARM1 anddSARM promote injury-induced axon degeneration [1,2].

Cytoskeletal breakdown is a common, late feature of axondegeneration and does not seem to be involved in the morerestricted processes of axosome shedding or axon retraction.Recent advances have provided some insight into the mech-anisms of cytoskeletal breakdown during degeneration. Thekinesin superfamily protein 2A (KIF2A), a microtubuledepolymerizing protein, is a key executor of microtubulebreakdown and axonal degeneration during neurotrophinwithdrawal-induced axon pruning, and so mice lackingKIF2A exhibit delayed degeneration of sensory neuronsinnervating the skin [46]. In the future, it will be importantto ascertain how KIF2A is regulated to selectively depoly-merize the microtubule cytoskeleton in degenerating axons.

Lifelong axon maintenanceNumerous mechanisms control the health and homeosta-sis of axons throughout life and oppose stressors such asexcitation and aging. Injury and disease induce axon de-generation both by compromising maintenance mecha-nisms and promoting active self-destruction pathways.Expression of the Wallerian degeneration slow (WldS)mutant protein, a chimeric fusion of the NAD+ biosyntheticenzyme nicotinamide mononucleotide adenylyltransferase1 (NMNAT1) and a fragment of the ubiquitination factorE4B (UBE4B), delays axonal degeneration induced bynumerous pathological insults (see Conforti et al. for anextensive summary of the effects of WldS/NMNAT onvarious axon pathologies [47]) [48–50]. Study of the WldS

protein and its constituents has provided great insight intomechanisms of axonal viability that rely on the interrelat-ed processes of metabolic homeostasis, calcium buffering,axonal transport, and protein synthesis.

Metabolic maintenance

Proper metabolism is integral to axon viability and func-tionality. Axons require large amounts of energy andmetabolites to support membrane depolarization and syn-aptic transmission [51], and thus the NAD+ biosyntheticNMNAT enzymes are essential in axon maintenance [48–50]. Mammalian NMNAT isoforms include the nuclearNMNAT1, the golgi-associated NMNAT2, and the mito-chondrial NMNAT3 [50]. WldS is a chimeric version ofNMNAT1, and its ability to delay axon degenerationrequires NAD+ synthetic activity in the cytoplasm, andpossibly in the axon [52–54]. Overexpression of eitherNMNAT2 [55–57] or NMNAT3 [54] delays degenerationof injured axons, whereas only endogenous NMNAT2 isrequired for maintenance of healthy axons [55,58,59]. WldS

Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

may confer its protection by directly substituting for themore labile NMNAT2, because NMNAT2 is rapidly de-graded following injury whereas WldS is degraded moreslowly [55,59]. Furthermore, mutant forms of NMNAT2with prolonged half-life exhibit a level of axon protectioncomparable to WldS [59].

Although NMNAT enzymatic activity is necessary foraxon viability, it is less clear whether this effect requiresthe enzymatic product NAD+ [52–54]. Injured axonsexhibit a dramatic decrease in NAD+ prior to morpholog-ical degradation, and degeneration is mitigated in vitroby exogenous NAD+ [60,61]. However, protection by ex-ogenous NAD+ requires supra-physiological levels[52,60,62,63], increasing cellular NAD+ levels by inhibit-ing NAD+-consuming enzymes does not protect injuredaxons [64], nor does the inhibition of NAD+ biosynthesisabolish WldS-mediated protection [52]. Furthermore,WldS does not detectably increase overall cellular levelsof NAD+ [64,65]. A possible explanation that reconcilesthese disparate results is that WldS and NMNATs inducea local increase in NAD+, perhaps at mitochondria,rather than increasing total cytoplasmic content ofNAD+ [12,54]. This hypothesis is supported by evidencethat cytoplasmic WldS copurifies with mitochondria[53,54,66].

Metabolism

NMNAT/Wlds

Fusion/fission

Mitofusin 2

OPA1

DRP1

Traffick

NAD+ ATP

Mitofusin 2

Figure 3. Mitochondria are central to axon health and homeostasis. The interdepende

health and are impaired by injury and disease. WldS, a chimeric fusion of the enzyme

NMNAT/WldS produces NAD+, which is used by mitochondria to produce ATP. NMNAT

ATP [60]. Innate mitochondrial calcium buffering maintains calcium homeostasis and i

axoplasmic calcium levels [12,69,70,72]. NMNAT/WldS increases the calcium buffering

Several processes are necessary for mitochondrial function and localization, including

health by the proteins Lamin B2 [75] and Bcl-w [4]. Mutations in the a tubulin acetyltrans

implicated in axon degenerative diseases [78–80]. Abbreviations: ATP, adenosine trip

mechanosensory abnormality 17; mPTP, mitochondrial permeability transition pore; N

adenylyltransferase; OPA1, optic atrophy 1; UBE4B, ubiquitination factor E4B; WldS, W

If NAD+ is a pro-survival factor, how does it act toprotect axons? Axonal energy is primarily supplied bymitochondria, which use NAD+ to produce ATP. Axonalinjury decreases both NAD+ and ATP levels even beforemorphological degeneration [60]. Exogenous application ofNAD+ or expression of WldS sustains both NAD+ and ATPlevels and delays subsequent degeneration [60]. In addi-tion, WldS mitochondria exhibit enhanced ability to gener-ate ATP, suggesting that NMNAT/WldS may thereforeprotect axons by maintaining mitochondrial bioenergetics(Figure 3) [54].

Calcium homeostasis

Calcium, an important regulator of synaptic transmission,mitochondrial transport and function, and of diverse sig-naling cascades [51,67], also activates pro-degenerativeaxonal components [30,68]. Traumatic injury elevatesintra-axonal calcium by impairing both extracellular cal-cium efflux and mitochondrial calcium sequestration, andthereby promotes axon degeneration [12,69,70]. Mito-chondria normally control calcium levels by cytosolic cal-cium uptake (Figure 3) [71]. However, excess intracellularcalcium levels can cause mitochondrial overloading, for-mation of the mitochondrial permeability transitionpore (mPTP), and subsequent release of calcium from

mPTP

Health

Bcl-w

Lamin B2

ing

Ca2+

buffering

Ca2+

MEC-17

TRENDS in Neurosciences

nt mitochondrial processes of metabolism and calcium buffering maintain axonal

NMNAT1 and UBE4B, delays axonal degeneration induced by injury and disease.

/WldS may promote axonal survival by preventing injury-induced loss of NAD+ and

s overwhelmed by axon injury, resulting in formation of the mPTP and increased

capacity of axonal mitochondria [66], possibly by increasing ATP synthesis [60].

fusion and fission, mitochondrial trafficking, and maintenance of mitochondrial

ferase MEC-17 and the fusion and fission proteins OPA1, mitofusin2, and DRP1 are

hosphate; Ca2+, calcium ion; DRP1, GTPase dynamin-related protein 1; MEC-17,

AD+, nicotinamide adenine dinucleotide; NMNAT, nicotinamide mononucleotide

allerian degeneration slow protein.

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Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

intra-mitochondrial stores [67]. Therefore, inhibitingmPTP activation or decreasing intracellular calcium pre-vents degeneration of injured axons [72].

NMNAT/WldS functions upstream of increased intra-axonal calcium, because inhibition of mPTP does not fur-ther delay degeneration of WldS axons [72] and exogenouscalcium treatment abolishes WldS-mediated axon protec-tion [73]. A recent study demonstrated that WldS sup-presses injury-induced axonal calcium elevation, andthat WldS mitochondria have increased calcium bufferingcapacity [66]. Given that mitochondrial ATP production,membrane potential, and calcium buffering are highlyinterrelated [67], it is possible that NMNAT/WldS

increases mitochondrial calcium buffering as a result ofincreasing ATP synthesis [66]. However, detailed temporaland spatial examinations of mitochondrial ATP and calci-um levels are needed to test this theory.

Mitochondrial quality control and localization

Given the importance of mitochondria in metabolism andcalcium buffering, maintaining a population of healthymitochondria is essential for axon viability. Numerousmechanisms promote mitochondrial health and removedysfunctional mitochondria; impairment of these processesis central to many neurodegenerative diseases (Figure 3)[74].

Several proteins contribute to the health of axonalmitochondria. Bcl-w is necessary for axonal maintenancein mature animals, in addition to its function in develop-ment [4]. Mice lacking Bcl-w exhibit elongated axonalmitochondria together with adult-onset degeneration ofperipheral sensory axons [4]. Axonal Lamin B2, a nuclearskeleton protein, is essential for axon viability in XenopusRGCs [75]. In the absence of Lamin B2, axonal mitochon-dria are elongated and exhibit impaired membrane poten-tial [75]. As previously discussed, NMNAT/WldS promotesmitochondrial function, enhancing mitochondrial ATP syn-thesis [54], calcium buffering capacity [66], and motility[66,76]. Mitochondrial fusion and fission are opposingprocesses whose balance maintains mitochondrial mor-phology and function [77]. Mutations in components con-trolling fusion and fission are responsible for several axondegenerative diseases. Notably, mutations affecting themitochondrial fusion protein optic atrophy 1 (OPA1) orthe mitochondrial fission protein GTPase dynamin-relatedprotein 1 (DRP1) result in atrophy of optic nerve axons[78,79]. In addition, mutation of the mitochondrial fusionprotein mitofusin 2 suppresses both mitochondrial fusionand transport and causes Charcot–Marie Tooth Disease[80].

Axonal transport both delivers new mitochondria andremoves dysfunctional mitochondria, and so disruptionsin mitochondrial transport contribute to multiple neuro-degenerative diseases [74,81]. Mitochondria are activelytransported to regions with high energetic demands, suchas synapses, in order to maintain proper circuit connec-tivity [74,81]. Mitophagy, or mitochondrial autophagy,which removes damaged mitochondria from axons, alsorelies on axonal transport [74,82]. Although axon injurycauses cessation of mitochondrial movement, WldS pre-vents mitochondrial stalling after injury and increases

578

basal mitochondrial motility in uninjured axons [66].The ability of WldS to enhance mitochondrial motilitycorrelates with improved calcium buffering capacity bymitochondria [66]. Similarly, cytoplasmically-targetedNMNAT1 partially protects against chemotoxic injury-induced deficits in mitochondrial transport [76]. Mito-chondrial motility is essential for WldS or NMNAT1 toprotect axons after injury, highlighting the essential roleof mitochondrial transport in axon viability [66,76]. Mito-chondrial transport is closely tied to cytoskeletal balance,as demonstrated by a recent study in Caenorhabditiselegans showing that loss of the a-tubulin acetyltransfer-ase mechanosensory abnormality 17 (MEC-17) causesmicrotubule destabilization, reduced axonal mitochondrianumbers, and spontaneous axon degeneration [83].

Cytoskeletal stability

The axonal cytoskeleton consists of microtubules, actin, andneurofilaments. Microtubules, the main highway for long-range retrograde and anterograde transport, are in a con-stant state of dynamic instability, with continuous depo-lymerization and repolymerization [84]. Pharmacologicalhyperstabilization [85] or destabilization [86] of axonalmicrotubules causes degeneration. Furthermore, injured[87], MEC-17 mutant [83], or neurotrophin-deprived [46]axons, which typically undergo microtubule destabilization,can be protected by the microtubule stabilizing agent pacli-taxel. Thus, paclitaxel, which is toxic to healthy axons [85],may act to restore cytoskeletal balance in degeneratingaxons. These results suggest that carefully balanced micro-tubule stability is critical for axon health (Figure 4).

Cytoskeletal stability is regulated by several protectiveor degenerative molecules. For example, superior cervicalganglion 10 (SCG10) is a microtubule destabilizing proteinthat promotes axonal health. SCG10 is rapidly degradedfollowing axon injury, and maintaining SCG10 levels pre-serves mitochondrial motility and delays axon degenera-tion [88]. Although SCG10 knockdown does not causespontaneous axon degeneration, it does accelerate inju-ry-induced degeneration [88]. Conversely, inhibition ofmicrotubule destabilizing proteins can protect axons withpathological microtubule instability. Progressive motorneuronopathy ( pmn) mice have a mutation in the tubulinchaperone Tbce gene that impairs microtubule polymeri-zation and causes motor axon degeneration [89]. The tran-scription factor signal transducer and activator oftranscription 3 (STAT3) locally inhibits axonal stathmin,a microtubule destabilizing protein in the same family asSCG10, and rescues axonal pathology in cultured pmnmotor neurons [90]. Microtubule stabilizing proteins havealso been shown to be necessary for axon health. Thecollapsin response mediator protein 2 (CRMP2) promotesmicrotubule stability. Axon injury causes CRMP2 degra-dation, and maintaining CRMP2 levels delays degenera-tion [91].

Accumulating evidence suggests that balanced cytoskel-etal stability regulates the pro-degeneration molecule dualleucine zipper kinase (DLK) and its Drosophila orthologWallenda. Genetic loss of the cytoskeletal stabilizing pro-tein spectraplakin short stop (Shot) or the tubulin chaper-one T-complex protein 1 (TCP1) leads to cytoskeletal

Axondegenera�on

Axondegenera�on

e.g.,Paclitaxelloss of SCG10

e.g.,Vincris�ne

loss of MEC-17loss of stabilizer s

(CRMP2, TBCE, TCP, Shot)

e.g.,PaclitaxelStathmin inhibi�on

Excessstability

Balance ddynamics

Excessinstability

TRENDS in Neurosciences

Figure 4. Balanced cytoskeletal dynamics are essential for axon maintenance. Microtubules are in a constant state of depolymerization and polymerization. Genetic and

pharmacological insults that either stabilize microtubules (such as the chemotherapeutic paclitaxel or loss of the microtubule destabilizing protein SCG10) or destabilize

microtubules (such as the chemotherapeutic vincristine, loss of MEC-17, or loss of microtubule stabilizing proteins CRMP2, TBCE, TCP, or Shot) cause axon degeneration

[85,86,88,89,91,92]. Axons with pathologically destabilized microtubules can be protected by treatment with the microtubule stabilizing agent paclitaxel [46,83,87] or

inhibition of the microtubule destabilizing protein stathmin [90]. Abbreviations: CRMP2, collapsin response mediator protein 2; MEC-17, mechanosensory abnormality 17;

SCG10, superior cervical ganglion 10; TBCE, tubulin-specific chaperone E; TCP, T-complex protein; Shot, short stop.

Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

instability and DLK activation [92]. DLK/Wallenda med-iates a degenerative response by activating the c-Jun N-terminal kinase (JNK) [93]. Notably, DLK function iscontext dependent and can promote both axon regenera-tion [94–97] and presynaptic bouton development [98].

Maintaining protein levels

As in developmental axon preservation, axonal mainte-nance requires supply, localization, and turnover of protein.Axonal transport and local translation provide maintenancefactors to the axon. Conversely, UPS-mediated protein deg-radation promotes axon degeneration by reducing levels ofseveral axonal maintenance factors [99–102]. Although lo-cal translation is required for mitochondrial function andaxon viability [103], only the protective factor Lamin B2 isknown to be translated locally in mature axons [75]. Bycontrast, the maintenance factors NMNAT2 and SCG10 arerapidly degraded in axons by the UPS and must be constant-ly renewed by anterograde transport from the cell body[55,59].

Pathological axon degenerationAxons damaged by injury or disease must be activelyeliminated. In some cases, removal of damaged axonsenables axon regrowth and maintains neural circuitry,such as following lesions of peripheral axons. However,when axon trauma is more widespread or axons are notcapable of regenerating, pathological axon degenerationcompromises neural circuit functionality.

Following nerve transection, axons undergo Walleriandegeneration (Figure 2D). During an initial latency stage,the injured axon remains intact and electrically functional;this is followed by a rapid degenerative phase involvingswelling, cytoskeletal degradation, and axon fragmenta-tion. Because the precise timing and location of injury canbe controlled, nerve transection is commonly used as asimple model of axon degenerative diseases. In Walleriandegeneration, transport is interrupted by the physicalsevering of the axon, resulting in a loss of labile pro-survival factors such as NMNAT2 [59,55,101,102] andSCG10 [88]. Axonal ATP levels rapidly decline [60], axonalmitochondria stall [66], and the innate calcium bufferingcapabilities of the axon fail [12,69,70]. Future studies willbe needed to define the exact temporal and causal relation-ships among these events. Many neurodegenerative dis-eases also exhibit axon loss, with a distal to proximalgradient of swelling and fragmenting that resembles Wal-lerian degeneration, described as a dying-back axonopathy(Figure 2E). Impaired axonal mitochondrial integrity,function, and transport are common pathologies of neuro-degenerative diseases, including Alzheimer’s disease,Huntington’s disease, Parkinson’s disease, amyotrophiclateral sclerosis, and others [74].

Concluding remarks and future directionsThe establishment and functionality of neural networksrequires precise control of axon survival and elimination indevelopment and throughout life. Recent studies describe

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Box 1. Outstanding questions

� What are the similarities and differences between developmental

and pathological axon survival and death pathways?

� Are multiple axon survival or death factors locally synthesized in

the axon?

� How is axonal caspase-9 activated in the absence of Apaf-1?

� Is the axonal apoptotic cascade involved in pathological axon

degeneration?

� What is the significance of the similarities and differences

between cell soma survival/death and axon survival/death? Do

these differences restrict degeneration to the axon?

� What is the role of electrical activity in maintaining mature axons

in a functional circuit?

Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

core mechanisms that preserve connections between cellsin a circuit and eliminate surplus or damaged connections.The mechanisms that govern axon survival and elimina-tion have similarities to and differences from cell somaviability and death. In particular, interdependent mito-chondrial and cytoskeletal processes are central to axonsurvival, and impairment of these processes by injury ordisease leads to pathological axon degeneration.

The field of axonal survival and death has been veryactive recently, including studies across multiple organ-isms and multiple types of neurons. These studies havebenefited from improved in vivo methodologies as well asimproved spatially compartmented culture systems thatwill now enable future studies to address the criticalquestions that remain (Box 1). A major question is thedegree of overlap between developmental and matureaxon survival pathways as well as their opposing devel-opmental and pathological axon destruction mechanisms.In identifying key players in these processes, recentstudies have already defined some commonalities anddifferences. Another major question is whether pro-de-generation factors or other pro-survival factors are trans-lated locally in the axon. Despite the abundance ofmRNAs in both developing and mature axons [34], fewpro-survival factors are known to be locally synthesized.Investigations into these questions and others will eluci-date mechanisms that maintain healthy axons withinneural networks.

AcknowledgmentsOur research is supported by the National Institutes of Health grantR01NS050674, the Barr Weaver Award, and the Harvard/MassachusettsInstitute of Technology (MIT) Joint Research Grant in Basic Neu-roscience to R.A.S., and by the Edward R. and Anne G. Lefler CenterPredoctoral Fellowship to S.E.P. We thank Dr Katharina Cosker, SaraFenstermacher, and Maria Pazyra-Murphy for critical reading of themanuscript.

References1 Osterloh, J. et al. (2012) dSarm/Sarm1 is required for activation of an

injury-induced axon death pathway. Science 337, 481–4842 Gerdts, J. et al. (2013) Sarm1-mediated axon degeneration requires

both SAM and TIR interactions. J. Neurosci. 33, 13569–135803 Pazyra-Murphy, M. et al. (2009) A retrograde neuronal survival

response: target-derived neurotrophins regulate MEF2D and bcl-w.J. Neurosci. 29, 6700–6709

4 Courchesne, S. et al. (2011) Sensory neuropathy attributable to loss ofBcl-w. J. Neurosci. 31, 1624–1634

5 Cosker, K. et al. (2013) Target-derived neurotrophins coordinatetranscription and transport of bclw to prevent axonal degeneration.J. Neurosci. 33, 5195–5207

580

6 Low, L. and Cheng, H. (2005) A little nip and tuck: axon refinementduring development and axonal injury. Curr. Opin. Neurobiol. 15,549–556

7 Luo, L. and O’Leary, D. (2005) Axon retraction and degeneration indevelopment and disease. Annu. Rev. Neurosci. 28, 127–156

8 Saxena, S. and Caroni, P. (2007) Mechanisms of axon degeneration:from development to disease. Prog. Neurobiol. 83, 174–191

9 Coleman, M. (2005) Axon degeneration mechanisms: commonalityamid diversity. Nat. Rev. Neurosci. 6, 889–898

10 Yan, T. et al. (2010) Axon degeneration: mechanisms and implicationsof a distinct program from cell death. Neurochem. Int. 56, 529–534

11 Maor-Nof, M. and Yaron, A. (2013) Neurite pruning and neuronal celldeath: spatial regulation of shared destruction programs. Curr. Opin.Neurobiol. 23, 990–996

12 Wang, J. et al. (2012) Axon degeneration: molecular mechanisms of aself-destruction pathway. J. Cell Biol. 196, 7–18

13 Schoenmann, Z. et al. (2010) Axonal degeneration is regulated by theapoptotic machinery or a NAD+-sensitive pathway in insects andmammals. J. Neurosci. 30, 6375–6386

14 Chen, Z. et al. (2007) Peripheral regeneration. Annu. Rev. Neurosci.30, 209–233

15 Hilliard, M.A. (2009) Axonal degeneration and regeneration: amechanistic tug-of-war. J. Neurochem. 108, 23–32

16 Hashimoto, K. and Kano, M. (2003) Functional differentiation ofmultiple climbing fiber inputs during synapse elimination in thedeveloping cerebellum. Neuron 38, 785–796

17 LeMaster, A. et al. (1999) Overexpression of brain-derivedneurotrophic factor enhances sensory innervation and selectivelyincreases neuron number. J. Neurosci. 19, 5919–5931

18 Causing, C. et al. (1997) Synaptic innervation density is regulated byneuron-derived BDNF. Neuron 18, 257–267

19 Albers, K. et al. (1994) Overexpression of nerve growth factor inepidermis of transgenic mice causes hypertrophy of the peripheralnervous system. J. Neurosci. 14, 1422–1432

20 Zhou, F. et al. (2004) NGF-induced axon growth is mediated bylocalized inactivation of GSK-3beta and functions of themicrotubule plus end binding protein APC. Neuron 42, 897–912

21 Campenot, R. (1982) Development of sympathetic neurons incompartmentalized cultures. II. Local control of neurite survival bynerve growth factor. Dev. Biol. 93, 13–21

22 Finn, J. et al. (2000) Evidence that Wallerian degeneration andlocalized axon degeneration induced by local neurotrophindeprivation do not involve caspases. J. Neurosci. 20, 1333–1341

23 Watson, F. et al. (2001) Neurotrophins use the Erk5 pathwayto mediate a retrograde survival response. Nat. Neurosci. 4,981–988

24 Salvesen, G. and Duckett, C. (2002) IAP proteins: blocking the road todeath’s door. Nat. Rev. Mol. Cell Biol. 3, 401–410

25 Unsain, N. et al. (2013) XIAP regulates caspase activity indegenerating axons. Cell Rep. 4, 751–763

26 Cusack, C. et al. (2013) Distinct pathways mediate axondegeneration during apoptosis and axon-specific pruning. Nat.Commun. 4, 1876

27 Nikolaev, A. et al. (2009) APP binds DR6 to trigger axon pruning andneuron death via distinct caspases. Nature 457, 981–989

28 Vohra, B. et al. (2010) Amyloid precursor protein cleavage-dependentand -independent axonal degeneration programs share a commonnicotinamide mononucleotide adenylyltransferase 1-sensitivepathway. J. Neurosci. 30, 13729–13738

29 Simon, D. et al. (2012) A caspase cascade regulating developmentalaxon degeneration. J. Neurosci. 32, 17540–17553

30 Yang, J. et al. (2013) Regulation of axon degeneration after injury andin development by the endogenous calpain inhibitor calpastatin.Neuron 80, 1175–1189

31 Deckwerth, T. and Johnson, E. (1994) Neurites can remain viableafter destruction of the neuronal soma by programmed cell death(apoptosis). Dev. Biol. 165, 63–72

32 Andreassi, C. et al. (2010) An NGF-responsive element targets myo-inositol monophosphatase-1 mRNA to sympathetic neuron axons.Nat. Neurosci. 13, 291–301

33 Gumy, L.F. et al. (2011) Transcriptome analysis of embryonic andadult sensory axons reveals changes in mRNA repertoire localization.RNA 17, 85–98

Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

34 Watts, R. et al. (2003) Axon pruning during Drosophilametamorphosis: evidence for local degeneration and requirement ofthe ubiquitin-proteasome system. Neuron 38, 871–885

35 Han, C. et al. (2014) Epidermal cells are the primary phagocytes in thefragmentation and clearance of degenerating dendrites in Drosophila.Neuron 81, 544–560

36 Bishop, D. et al. (2004) Axon branch removal at developing synapsesby axosome shedding. Neuron 44, 651–661

37 Singh, K. et al. (2008) Developmental axon pruning mediated byBDNF-p75NTR-dependent axon degeneration. Nat. Neurosci. 11,649–658

38 Park, K. et al. (2010) p75NTR-dependent, myelin-mediated axonaldegeneration regulates neural connectivity in the adult brain. Nat.Neurosci. 13, 559–566

39 Bagri, A. et al. (2003) Stereotyped pruning of long hippocampal axonbranches triggered by retraction inducers of the semaphorin family.Cell 113, 285–299

40 Xu, N. and Henkemeyer, M. (2009) Ephrin-B3 reverse signalingthrough Grb4 and cytoskeletal regulators mediates axon pruning.Nat. Neurosci. 12, 268–276

41 Petros, T.J. et al. (2010) Ephrin-B2 elicits differential growth conecollapse and axon retraction in retinal ganglion cells from distinctretinal regions. Dev. Neurobiol. 70, 781–794

42 Lee, T. et al. (2000) Cell-autonomous requirement of the USP/EcR-Becdysone receptor for mushroom body neuronal remodeling inDrosophila. Neuron 28, 807–818

43 Awasaki, T. et al. (2011) Glia instruct developmental neuronalremodeling through TGF-b signaling. Nat. Neurosci. 14, 821–823

44 Yu, X. et al. (2013) Plum, an immunoglobulin superfamily protein,regulates axon pruning by facilitating TGF-b signaling. Neuron 78,456–468

45 Olsen, O. et al. (2014) Genetic analysis reveals that amyloid precursorprotein and death receptor 6 function in the same pathway to controlaxonal pruning independent of beta-secretase. J. Neurosci. 34, 6438–6447

46 Maor-Nof, M. et al. (2013) Axonal pruning is actively regulated by themicrotubule-destabilizing protein kinesin superfamily protein 2A.Cell Rep. 3, 971–977

47 Conforti, L. et al. (2014) Wallerian degeneration: an emerging axondeath pathway linking injury and disease. Nat. Rev. Neurosci. 15,394–409

48 Wang, J. and He, Z. (2009) NAD and axon degeneration: from the Wldsgene to neurochemistry. Cell Adh. Migr. 3, 77–87

49 Coleman, M. and Freeman, M. (2010) Wallerian degeneration, wld(s),and nmnat. Annu. Rev. Neurosci. 33, 245–267

50 Ali, Y. et al. (2013) NMNATs, evolutionarily conserved neuronalmaintenance factors. Trends Neurosci. 36, 632–640

51 Sheng, Z. (2014) Mitochondrial trafficking and anchoring in neurons:new insight and implications. J. Cell Biol. 204, 1087–1098

52 Conforti, L. et al. (2009) Wld S protein requires Nmnat activity and ashort N-terminal sequence to protect axons in mice. J. Cell Biol. 184,491–500

53 Beirowski, B. et al. (2009) Non-nuclear Wld(S) determines itsneuroprotective efficacy for axons and synapses in vivo. J.Neurosci. 29, 653–668

54 Yahata, N. et al. (2009) Nicotinamide mononucleotideadenylyltransferase expression in mitochondrial matrix delaysWallerian degeneration. J. Neurosci. 29, 6276–6284

55 Gilley, J. and Coleman, M.P. (2010) Endogenous Nmnat2 is anessential survival factor for maintenance of healthy axons. PLoSBiol. http://dx.doi.org/10.1371/journal.pbio.1000300

56 Feng, Y. et al. (2010) Overexpression of Wld(S) or Nmnat2 inmauthner cells by single-cell electroporation delays axondegeneration in live zebrafish. J. Neurosci. Res. 88, 3319–3327

57 Yan, T. et al. (2010) Nmnat2 delays axon degeneration in superiorcervical ganglia dependent on its NAD synthesis activity. Neurochem.Int. 56, 101–106

58 Milde, S. et al. (2013) Deletions within its subcellular targetingdomain enhance the axon protective capacity of Nmnat2 in vivo.Sci. Rep. 3, 2567

59 Milde, S. et al. (2013) Subcellular localization determines the stabilityand axon protective capacity of axon survival factor Nmnat2. PLoSBiol. http://dx.doi.org/10.1371/journal.pbio.1001539

60 Wang, J. et al. (2005) A local mechanism mediates NAD-dependentprotection of axon degeneration. J. Cell Biol. 170, 349–355

61 Sasaki, Y. et al. (2006) Stimulation of nicotinamide adeninedinucleotide biosynthetic pathways delays axonal degenerationafter axotomy. J. Neurosci. 26, 8484–8491

62 Araki, T. et al. (2004) Increased nuclear NAD biosynthesis andSIRT1 activation prevent axonal degeneration. Science 305, 1010–1013

63 Conforti, L. et al. (2007) NAD(+) and axon degeneration revisited:Nmnat1 cannot substitute for Wld(S) to delay Walleriandegeneration. Cell Death Differ. 14, 116–127

64 Sasaki, Y. et al. (2009) Nicotinamide mononucleotide adenylyltransferase-mediated axonal protection requires enzymatic activitybut not increased levels of neuronal nicotinamide adeninedinucleotide. J. Neurosci. 29, 5525–5535

65 Mack, T. et al. (2001) Wallerian degeneration of injured axons andsynapses is delayed by a Ube4b/Nmnat chimeric gene. Nat. Neurosci.4, 1199–1206

66 Avery, M. et al. (2012) WldS prevents axon degeneration throughincreased mitochondrial flux and enhanced mitochondrial Ca2+

buffering. Curr. Biol. 22, 596–60067 Brookes, P. et al. (2004) Calcium, ATP, and ROS: a mitochondrial love-

hate triangle. Am. J. Physiol. 287, 3368 George, E.B. et al. (1995) Axotomy-induced axonal degeneration is

mediated by calcium influx through ion-specific channels. J. Neurosci.15, 6445–6452

69 Stirling, D. and Stys, P. (2010) Mechanisms of axonal injury:internodal nanocomplexes and calcium deregulation. Trends Mol.Med. 16, 160–170

70 Marambaud, P. et al. (2009) Calcium signaling in neurodegeneration.Mol. Neurodegen. 4, 20

71 Rizzuto, R. et al. (2000) Mitochondria as all-round players of thecalcium game. J. Physiol. 529 (Pt 1), 37–47

72 Barrientos, S. et al. (2011) Axonal degeneration is mediated by themitochondrial permeability transition pore. J. Neurosci. 31, 966–978

73 Glass, J. et al. (1994) Calcium-mediated degeneration of the axonalcytoskeleton in the Ola mouse. J. Neurochem. 62, 2472–2475

74 Court, F. and Coleman, M. (2012) Mitochondria as a central sensor foraxonal degenerative stimuli. Trends Neurosci. 35, 364–372

75 Yoon, B. et al. (2012) Local translation of extranuclear lamin Bpromotes axon maintenance. Cell 148, 752–764

76 Fang, C. et al. (2014) Axonal transport plays a crucial role inmediating the axon-protective effects of NmNAT. Neurobiol. Dis.68, 78–90

77 Chan, D. (2006) Mitochondrial fusion and fission in mammals. Annu.Rev. Cell Dev. Biol. 22, 79–99

78 Waterham, H.R. et al. (2007) A lethal defect of mitochondrial andperoxisomal fission. N. Engl. J. Med. 356, 1736–1741

79 Delettre, C. et al. (2000) Nuclear gene OPA1, encoding amitochondrial dynamin-related protein, is mutated in dominantoptic atrophy. Nat. Genet. 26, 207–210

80 Misko, A.L. et al. (2012) Mitofusin2 mutations disrupt axonalmitochondrial positioning and promote axon degeneration. J.Neurosci. 32, 4145–4155

81 Schwarz, T.L. (2013) Mitochondrial trafficking in neurons. ColdSpring Harb. Perspect. Biol. http://dx.doi.org/10.1101/cshperspect.a011304

82 Maday, S. et al. (2012) Autophagosomes initiate distally and matureduring transport toward the cell soma in primary neurons. J. CellBiol. 196, 407–417

83 Neumann, B. and Hilliard, M.A. (2014) Loss of MEC-17 leads tomicrotubule instability and axonal degeneration. Cell Rep. 6, 93–103

84 Mitchison, T. and Kirschner, M. (1984) Dynamic instability ofmicrotubule growth. Nature 312, 237–242

85 Yang, I.H. et al. (2009) Compartmentalized microfluidic cultureplatform to study mechanism of paclitaxel-induced axonaldegeneration. Exp. Neurol. 218, 124–128

86 Silva, A. et al. (2006) Evidence for direct axonal toxicity in vincristineneuropathy. J. Peripher. Nerv. Syst. 11, 211–216

87 King, A.E. et al. (2013) Excitotoxin-induced caspase-3 activationand microtubule disintegration in axons is inhibited by taxol.Acta Neuropathol. Commun. 1, http://dx.doi.org/10.1186/2051-5960-1-59

581

Feature Review Trends in Neurosciences October 2014, Vol. 37, No. 10

88 Shin, J. et al. (2012) SCG10 is a JNK target in the axonal degenerationpathway. Proc. Natl. Acad. Sci. U.S.A. 109, 705

89 Schaefer, M.K. et al. (2007) Progressive motor neuronopathy: a criticalrole of the tubulin chaperone TBCE in axonal tubulin routing from theGolgi apparatus. J. Neurosci. 27, 8779–8789

90 Selvaraj, B. et al. (2012) Local axonal function of STAT3 rescues axondegeneration in the pmn model of motoneuron disease. J. Cell Biol.199, 437–451

91 Wakatsuki, S. et al. (2011) ZNRF1 promotes Wallerian degenerationby degrading AKT to induce GSK3B-dependent CRMP2phosphorylation. Nat. Cell Biol. 13, 1415–1423

92 Valakh, V. et al. (2013) Loss of the spectraplakin short stop activatesthe DLK injury response pathway in Drosophila. J. Neurosci. 33,17863–17873

93 Miller, B.R. et al. (2009) A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration. Nat.Neurosci. 12, 387–389

94 Xiong, X. and Collins, C. (2012) A conditioning lesion protects axonsfrom degeneration via the Wallenda/DLK MAP kinase signalingcascade. J. Neurosci. 32, 610–615

95 Xiong, X. et al. (2010) Protein turnover of the Wallenda/DLK kinaseregulates a retrograde response to axonal injury. J. Cell Biol. 191,211–223

582

96 Watkins, T.A. et al. (2013) DLK initiates a transcriptional programthat couples apoptotic and regenerative responses to axonal injury.Proc. Natl. Acad. Sci. U.S.A. 110, 4039–4044

97 Shin, J.E. et al. (2012) Dual leucine zipper kinase is required forretrograde injury signaling and axonal regeneration. Neuron 74,1015–1022

98 Klinedinst, S. et al. (2013) Independent pathways downstream of theWnd/DLK MAPKKK regulate synaptic structure, axonal transport,and injury signaling. J. Neurosci. 33, 12764–12778

99 Hoopfer, E. et al. (2006) Wlds protection distinguishes axondegeneration following injury from naturally occurringdevelopmental pruning. Neuron 50, 883–895

100 Zhai, Q. et al. (2003) Involvement of the ubiquitin-proteasomesystem in the early stages of wallerian degeneration. Neuron 39,217–225

101 Xiong, X. et al. (2012) The highwire ubiquitin ligase promotes axonaldegeneration by tuning levels of Nmnat protein. PLoS Biol. http://dx.doi.org/10.1371/journal.pbio.1001440

102 Babetto, E. et al. (2013) The Phr1 ubiquitin ligase promotes injury-induced axon self-destruction. Cell Rep. 3, 1422–1429

103 Hillefors, M. et al. (2007) Axon viability and mitochondrial functionare dependent on local protein synthesis in sympathetic neurons. Cell.Mol. Neurobiol. 27, 701–716