mitochondria as a central sensor for axonal degenerative stimuli

Mitochondria as a central sensor foraxonal degenerative stimuliFelipe A. Court1,2 and Michael P. Coleman3

1 Millennium Nucleus for Regenerative Biology, Faculty of Biology, Catholic University of Chile, Santiago 8331150, Chile2 Neurounion Biomedical Foundation, Santiago, Chile3 Signalling Programme, The Babraham Institute, Babraham Research Campus, Babraham, Cambridge CB22 3AT, UK

Review

Axonal degeneration is a major contributor to neuronaldysfunction in many neurological conditions and hasadditional roles in development. It can be triggered bydivergent stimuli including mechanical, metabolic, infec-tious, toxic, hereditary and inflammatory stresses. Axo-nal mitochondria are an important convergence point asregulators of bioenergetic metabolism, reactive oxygenspecies (ROS), Ca2+ homeostasis and protease activa-tion. The challenges likely to render axonal mitochondriamore vulnerable than their cellular counterparts arereviewed, including axonal transport, replenishingnuclear-encoded proteins and maintenance of qualitycontrol, fusion and fission in locations remote fromthe cell body. The potential for mitochondria to act asa decision node in axon loss is considered, highlightingthe need to understand the biology of axonal mitochon-dria and their contributions to degenerative mecha-nisms for novel therapeutic strategies.

IntroductionNeuronal soma and axons die by very different mecha-nisms. Axon degeneration is in many cases an autonomousprocess that is distinct from classical apoptosis but can begenetically regulated. Conversely, a protein that protectsaxons, the slow Wallerian degeneration protein (WldS), hasno known effect on survival of the soma [1,2]. As molecularmechanisms underlying axon survival and degenerationbecome better understood, it is important to ask whatmakes them specific for axons.

Many mitochondrial dysfunctions lead to disorders inwhich axon degeneration is the predominant feature, or aprominent early step (Table 1). Thus, axons may face agreater challenge than neuronal soma or dendrites inmaintaining sufficient functioning of mitochondria for sur-vival. Mitochondria, long known to have multiple roles incell death, differ strikingly between axons and soma. First,their elongated shape, at least in peripheral nerve axons, isspecialized for life in a long narrow cylinder [3]. Mainte-nance of this shape is critical for efficient delivery by axonaltransport so that even moderate swelling will block boththeir own transport and that of many other cargoes(Figure 1). Second, axonal mitochondria are discrete struc-tures, in contrast to the syncytia more typically seen in

Corresponding authors: Court, F.A. ([email protected]); Coleman, M.P.([email protected]).Keywords: axonal degeneration; mitochondria; axonal transport; mitochondrialpermeability transition pore; reactive oxygen species.

364 0166-2236/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http:/

many cell types. Because of this discontinuity, the deliveryof material and exchange with other mitochondria may bemore dependent on mechanisms such as fusion and fission.Third, their transport has to be carefully regulated toensure that mitochondria are focused in the correct regionsof the huge axonal compartment [4], which can be severalhundred times larger than neuronal soma. In this way,high requirements for adenosine triphosphate (ATP) syn-thesis and Ca2+ buffering can be met.

This review considers the extent to which unique fea-tures of axonal mitochondria underlie axon-specific degen-eration mechanisms, making them a nodal point fordecisions on axon survival. In some cases, axons degener-ate through failure to maintain a healthy mitochondrialpopulation at literally ‘arm’s length’ from the cell body,whereas in others mitochondria may play a more activerole in axon degeneration. The latter process may be muchfaster, whereas a steady decline in mitochondria quality inaxons may take many years and only occur when axonaltransport further declines with age.

Challenges for axonal mitochondriaMitochondrial defects are surprisingly prevalent in axondegeneration disorders (Table 1). It is interesting to con-sider whether this reflects properties of mitochondria thatare important within axons in particular and how suchproperties may fit together.

Mitochondrial fusion and fission in neurodegenerative

conditions

Mitochondrial fusion and fission are particularly promi-nent among the functions of the proteins in Table 1.Proteins controlling these processes are mutated insubtypes of peripheral neuropathy, optic atrophy and he-reditary spastic paraplegia (HSP). Mitofusin 2, an outermitochondrial membrane protein mediating fusion is mu-tated in the pure axonal disorder Charcot-Marie-ToothType 2A [5], whereas optic atrophy 1 (OPA1), which hasa related role in fusing the inner mitochondrial membrane,is defective in autosomal dominant optic atrophy [6]. Para-plegin (encoded by the SPG7 gene), which is mutated insome types of HSP, regulates control of mitochondrialsize by OPA1 [7] and its absence results in giant mitochon-dria [8]. Ganglioside-induced differentiation-associatedprotein-1 (GDAP1), a protein that enhances mitochondriafragmentation, is mutated in the mixed axon/myelin dis-order Charcot-Marie-Tooth Disease type 4A (CMT4A) [9].

/dx.doi.org/10.1016/j.tins.2012.04.001 Trends in Neurosciences, June 2012, Vol. 35, No. 6

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Table 1. Mitochondrial proteins mutated in disorders with prominent axonal degenerationa

Disease Protein Disease type Possible protein function Refs

Charcot-Marie-Tooth Mitofusin 2 CMT2A Outer mitochondrial membrane protein

ER protein

Axonal mitochondrial transport

ER-mitochondrial tethering

[5,32,103]

HSPB1 (HSP27) CMT2F Molecular chaperone

Neurofilament organization

Microtubule binding

[104,105]

HSPB8 (HSP22) CMT2L Molecular chaperone

Associated with autophagy

[106]

Gdap1 CMT4A Outer mitochondrial membrane protein

Mitochondrial fission

[9]

Friedrich’s ataxia Frataxin Friedrich’s ataxia Mitochondrial matrix iron chaperone

Redox regulation

[22]

Hereditary spastic

paraplegia

Paraplegin SPG7 Mitochondrial ATPase [8]

Hsp60 SPG13 Mitochondrial chaperone

Resistance to oxidative stress (?)

[107]

Spartin SPG20 Several protein partners, localization and functions

Required for mitochondrial calcium uptake capacity

[81]

Reep1 SPG38 Binds spastin

Associates with microtubules

ER-shaping and mitochondrial dynamics (?)

[108]

Optic atrophy/neuropathy OPA1 Optic atrophy Inner mitochondrial membrane protein

Mitochondrial fusion

[6,109]

Complex 1 subunits

ND1, 4, 6

Leber’s hereditary

optic neuropathy

Complex I subunit of mitochondria

Decrease ATP production

[110]

Parkinson’s disease Parkin PARK2 Cytosolic E3 ubiquitin ligase

Mitochondrial quality control

Microtubule dynamics

[18]

PINK1 PARK6 Mitochondrial quality control [17]

DJ-1 PARK7 Atypical peroxidase

Regulation of oxidant defenses

[111,112]

LRRK2 PARK8 Partial mitochondrial localization [113]

HTRA2 PARK13 Mitochondrial intermembrane space

Pro-apoptotic

[114]

POLG1 Parkinson’s disease,

Alper’s syndrome

Inner mitochondrial membrane

Mitochondrial DNA synthesis, replication and repair

[115]

Sensory neuropathy Bcl-w Small fiber sensory

neuropathy

Mitochondrial localization

Anti apoptotic Bcl-2 family member

[67]

aAbbreviations: HTRA2, high temperature requirement protein 2; LRRK2, leucine-rich repeat kinase 2; POLG1, mitochondrial DNA polymerase gamma 1; Reep1, receptor

expression enhancing protein 1.

Review Trends in Neurosciences June 2012, Vol. 35, No. 6

The GTPase dynamin-related protein 1 (DRP1), in additionto being associated with mitochondrial fission, is alsorequired for neurite development or maintenance in pri-mary culture [10]. In humans, a mutation in DRP1 hasbeen associated with severe and lethal defects in thenervous system [11]. Fusion and fission of axonal mito-chondria may also be a primary target in toxic models, aschronic exposure to low levels of rotenone causes loss ofdopaminergic neuronal processes without cell death in aprocess that may require mitochondrial fission [12].

Mitochondria quality control and neuronal degeneration

Quality control of mitochondria is another emerging theme,especially in Parkinson’s disease (PD), where a growingbody of evidence points to a ‘dying back’ process of earlyaxon and synapse loss [13]. Experiments in non-neuronalcell culture suggest that accumulation of phosphatase andtensin homolog (PTEN)-induced putative kinase 1 (PINK1)on the surface of dysfunctional mitochondria acts as a signalfor their removal by mitophagy [14,15]. By contrast, regularvoltage-dependent proteolysis of PINK1 on healthy mito-chondria maintains low levels of this protein [14]. This

degradation stops if mitochondrial membrane potentialfalls, causing rapid accumulation of PINK1 and recruitmentof Parkin leading to mitophagy. Recent data confirm thatParkin recruitment occurs in neuronal soma [16]. Geneticevidence supports a similar mechanism in vivo as loss-of-function mutations in PINK1 or Parkin cause PD [17,18]and Drosophila studies indicate that Parkin lies down-stream of PINK1 [19].

Quality control becomes particularly intriguing in thecontext of the huge axonal arbors of dopaminergic neuronsin mammalian brain [20]. Degeneration of these nerveterminals clearly precedes death of the soma [13] so anyfailure of quality control may occur here first. In distalaxons of sensory neurons, mitochondria fragments areengulfed by microtubule-associated protein 1A/1B-lightchain 3 (LC3)-positive vesicles and the resulting autopha-gosomes fuse with lysosomes as they move retrogradely[21]. The importance of retrograde transport is consistentwith the accumulation of depolarized mitochondria indistal axons in a Drosophila model of Friedreich ataxia,associated with a failure of retrograde axonal transport[22]. Whether these first stages of mitophagy in axons use

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(a) Healthy axon

(c) (d)

**

(b) Healthy axon Injured axon Pathological (HSP)

TRENDS in Neurosciences

Figure 1. Mitochondrial morphology in healthy and degenerating axons. (a) Healthy axonal mitochondria (depicted by the arrow) are often elongated discrete structures

that are oriented along the axon shaft, as shown in this electron micrograph of mouse optic nerve. Scale bar, 2 mm. Reproduced, with permission, from [102]. (b) Healthy

mitochondria (arrows) in axons from mouse optic nerves occupy a small fraction of axonal diameter but after nerve injury (c) mitochondria (*) swell to dimensions likely to

perturb axonal transport of both themselves and other cargoes. Scale bars in (b) and (c), 300 nm. Reprinted, with permission, from [60]. (d) Similar morphologies occur in

axonal pathology, such as in the spinal cord of SPG7-deficient mice, which model hereditary spastic paraplegia (HSP). Scale bar, 800 nm. Reproduced, with permission,

from [8].


the PINK1/Parkin mechanism remains to be clarified.Both proteins are present in axons, where they downreg-ulate axonal transport of mitochondria [16,23] and Parkinrecruitment can occur in neurites when mitochondriatransport is blocked [16]. However, clear evidence for axo-nal Parkin recruitment in more physiological conditionshas not yet been reported. Whatever their identity, theproteins targeting mitochondria for mitophagy in distalaxons must be delivered by anterograde transport, so thistransport is important for maintaining a pool of healthyaxonal mitochondria.

Mitochondrial transport in large neuronal

compartments

In addition to retrograde trafficking of autophagosomes,and anterograde transport of proteins for mitophagy, axo-nal transport plays a third critical role in maintainingaxonal mitochondria. Unlike their somatic counterparts,10–30% of axonal mitochondria are trafficked over hugedistances [3]. While the soma is the site where some axonalmitochondria are generated [24], mitochondrial biogenesisalso occurs within isolated axons [25]. The need to trafficmitochondria from the soma when axons can generatetheir own probably reflects the fact that mitochondrialDNA encodes only 13 out of hundreds of mitochondrialproteins [26]. Nuclear-encoded proteins need to be deliv-ered to replace natural turnover.

One attractive model is that motile mitochondria, whichare usually smaller [3], deliver these nuclear-encodedproteins into axons, where they fuse with large, stationarymitochondria to replenish them (Figure 2), although localprotein synthesis [27] and local mitochondrial import of

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nuclear-encoded proteins may also contribute to this deliv-ery. According to this model, disruption of this anterogradetrafficking should result in gradual deterioration of axonalmitochondria. It is interesting that such disruption hasbeen observed to occur in animal models of familial amyo-trophic lateral sclerosis (ALS) [28], tauopathy [29,30], HSP[31], CMT2A [32,33], Alzheimer’s disease (AD) [34],Huntington’s disease (HD) [35] and toxicity by A beta or1-methyl-4-phenylpyridinium ion (MPP+) [36–38]. Somestudies report that axonal transport of mitochondria canbe separated from pathogenesis in mouse models of ALS[39,40] but a contributory role to some axonal disordersdoes seem likely [41]. This could be particularly true in age-related disorders, as transport decline during normal age-ing [30] could render older axons less tolerant of anyadditional transport impairment.

Anterograde transport, fusion, fission and quality con-trol are likely to act together to maintain the functionalityof axonal mitochondria and long-term axon survival(Figure 2). In this model, stationary mitochondria areregularly serviced by delivery and fusion of small mobilemitochondria carrying newly-synthesized nuclear-encodedproteins. Consistent with this, mitochondrial fluxincreases when nuclear-encoded mitochondrial DNA poly-merase Pol gamma is deleted, as though the axon weretrying to compensate for the shortage [42]. Both fusion andfission of mitochondria occur in axons [12] and asymmetricfission has been shown in other cell types, producingdaughter mitochondria with differing membrane poten-tials [43]. This should allow less competent mitochondriato be removed by quality control, potentially involvingPINK1/Parkin. Defects in the anterograde transport of

(a)

(b)

(c)Mitophagy

PINK1?

Moving, new Stationary, old

Fusion replenishes

Fission

Proximal Distal

AxonSoma


Figure 2. Proposed model for quality control of axonal mitochondria. (a) A small anterogradely moving mitochondrion [3] (blue) is proposed to contain new nuclear-

encoded proteins consistent with biogenesis of some mitochondria within the cell body [24]. By contrast, the larger, stationary mitochondrion (red) is proposed to contain

older proteins that have resided in the axon for some time. (b) Fusion of moving mitochondrion to stationary mitochondrion [12] mixes their contents (purple). (c) A

stationary mitochondrion is depicted as undergoing asymmetric fission [43]. The smaller daughter mitochondrion (red) is targeted for mitophagy within the axon [21],

potentially involving axonally localized PINK1 [23], and retrogradely trafficked for lysosomal degradation [16,21]. The large stationary mitochondrion is replenished by the

delivery of new proteins and disposal of less functional material. In this model, axonal transport, mitochondrial fusion and fission, and signaling proteins important for

mitophagy are all required to maintain functionality of the axonal mitochondria population.


mitochondria themselves, or of proteins needed for mito-phagy, and defects in fusion or fission or mitophagy wouldall disrupt this mechanism. Consistent with this, there areindications that mitochondrial fusion and fission are dis-rupted in HD [35,44] and AD [34,45]. In addition, shortfragmented mitochondria accumulate in dystrophic axonsclose to amyloid plaques [46], whereas a-synuclein alsostimulates mitochondrial fission [47] and defects in autop-hagy lead to severe swelling in Purkinje cell axons [48,49].Finally, complex IV negative mitochondria accumulate inaxons of multiple sclerosis (MS) patients as though qualitycontrol has failed at some level [50].

Participation of mitochondria in Wallerian degenerationRecent data suggest that mitochondria have important rolesin one specific axon degeneration mechanism known asWallerian degeneration (WD). WD is triggered by mechani-cal disconnection of axons from cell bodies and serves as amodel for axon degeneration when axonal transport is dis-rupted [51]. The supporting evidence is that an aberrantprotein generated by a spontaneous mutation, WldS, is ableto delay axon degeneration both after injury and in someaxonal transport disorders, genetically linking these mech-anisms [52].

It has been demonstrated that several parallels existsbetween axonal degeneration triggered by mechanicalinjury (i.e. WD) and the axonal degenerative events acti-vated by some toxic, inflammatory or metabolic stressorsin diverse neurodegenerative conditions [53]. Therefore,WD represents an experimental model extensively used todefine the mechanisms involved in axonal degeneration.After mechanical nerve injury, axons exhibit a latentphase in which the structures remain apparently normal

(1–2 days in laboratory animals), followed by a cata-strophic phase in which all axonal structures collapserapidly, in a well-ordered sequence [51]. With a few excep-tions, collapse of severed axons occurs in animals of widelyseparated phyla [51]. Agents that interfere with microtu-bular function also trigger degeneration, again pointing toa role for disruption of microtubule-assisted transport inaxonal degeneration [51].

Interestingly, several links have emerged between WDand mitochondria. Overexpression of the mitochondrialNAD+ synthesizing enzyme nicotinamide mononucleo-tide adenylyltransferase 3 (NMNAT3) delays axonaldegeneration [54]. Furthermore, the WD-protective WldS

protein, which is a chimera between nuclear NMNAT1and a non-catalytic sequence from a ubiquitin ligase, isalso partially localized to axonal mitochondria [54,55].The enzymatic activity of NMNAT3 or WldS is requiredfor their protective action. Although this was originallythought to implicate NAD+ in axonal degeneration, it ispossible that another metabolite handled by theseenzymes has a critical role [56]. Thus, localization ofWldS and NMNAT3 in axonal mitochondria could bean important requirement for the protective effect, butbecause neither is confined to mitochondria this stillrequires confirmation.

Although the protective mechanism of overexpressedNMNATs has not yet been elucidated, the involvementof NAD+-dependent pathways in mitochondrial energymetabolism and redox capacity in the cytoplasm or mito-chondria [57] suggest a mechanism linking loss of mito-chondrial function to axonal degeneration [58]. Forexample, this could occur when one or more NMNATsare not properly trafficked to axons [59].

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Recent data indicate a direct association between WDand activation of the mitochondrial permeability transitionpore (mPTP). Genetic or pharmacologic inhibition of mPTPactivation delays axonal degeneration downstream WldS

protection [60]. The mPTP has been functionally associat-ed to several neurodegenerative conditions harboring axo-nal degeneration, including MS, AD, ALS and cerebralischemia [61–64]. Interestingly, two of the main activatorsof the mPTP in excitable cells, Ca2+ and ROS [65], arepathways experimentally defined as participants in theaxonal degeneration program. Moreover, a recent reportshows that WldS enhances basal mitochondrial motilityand increases Ca2+ buffering capability of axonal mito-chondria [66], consistent with the involvement of mPTPin axonal degeneration [60].

The mPTP is molecularly distinct from mitochondrial-dependent apoptosis. This is important because classicalapoptosis involving B cell lymphoma 2 (Bcl-2)-associated Xprotein (Bax), BCL2-homologous antagonist/killer (Bak),or Bcl-2, is not required for WD [51]. Therefore, activationof the mPTP could be part of an axonal-specific program ofdegeneration that reflects specific axonal characteristicssuch as distance from the nucleus and unique require-ments of axonal mitochondria, in contrast to classic apo-ptosis taking place in the neuronal soma. Interestingly, arecent report suggests an axon-specific role also for themitochondrial anti apoptotic protein Bcl-w (also known asBcl-2L2), at least in a subset of sensory axons [67]. Thus,there may be several pathways in which mitochondriacontribute to the specific degeneration of axons.

Mitochondrial changes after degenerative stimuliAxonal degeneration after heterogeneous inductionstimuli is considered to proceed through a Wallerian-likemechanism if it is genetically delayed by WldS [51]. Thus,diverse activators converge onto a common degenerationpathway, suggesting one or more integration points. Axo-nal mitochondria appear to be one such integration point,where pro-degenerative stimuli combine to produce theappropriate survival or degeneration response. Featuresof mitochondrial dysfunction occur in many disorders,including Ca2+ dyshomeostasis, ATP depletion, ROS gen-eration and mitochondrial swelling, suggesting mitochon-dria act as a central sensor for degenerative stimuli.According to this model, degradative processes should beactivated when concerted stimuli surpass the mitochon-drial homeostatic capacity.

Mitochondria control dynamic changes in the cyto-plasmic levels of Ca2+, ATP and ROS, and these cellularpathways are connected by several feedback loops [68].Stimuli affecting one or more of these components areintegrated by axonal mitochondria and if a new homeo-static state is not reached, ATP levels decline, intracellularCa2+ levels increase and ROS is produced, which culminatein the activation of positive feedback loops that targetaxons for destruction. Importantly, the sensitivity of mito-chondria to diverse stimuli will depend on genetic andenvironmental factors, including the cell- and tissue-specific context. For example, mutations that changemitochondrial parameters such as trafficking, respiratoryactivity, Ca2+ buffering or ROS generation constitute

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genetic risk factors that increase axonal vulnerability tosublethal stimuli (Table 1). Several mitochondrial proper-ties, including ATP production, Ca2+ uptake, buffering andrelease, exhibit regional, cellular and subcellular varia-tions in the nervous system [69]. Mitochondria in axonswith unique morphology and metabolic requirements, suchas the huge axonal arbors of dopaminergic neurons ormeter long peripheral nerve axons, are likely to differ insusceptibility to stressors compared to somatic ones (seeabove). Environmental factors including caloric intake andtoxins such as heavy metals modify the strength of feed-back loops between Ca2+, ATP and ROS [68]. Finally,aging, a well-established risk factor for neurodegenerativeconditions [70], profoundly affects the homeostasis of theCa2+/ATP/ROS triad, especially in excitable cells, with adecrease in axonal transport in aging axons as one con-tributing factor [30].

Energetic metabolism and ionic homeostasis in axons

Excitability comes at a high energetic price. Maintainingionic gradients and action potential propagation use mostcellular energy, exceeding the demands of associated gliaby fourfold [71]. Mitochondrial ATP production providesmost axonal energy, and ATP consumption is central tomitochondrial homeostatic capacities regulating Ca2+ andROS [68]. Energy metabolism, therefore, is a key parame-ter in axons. Mutations in mitochondrial DNA (mtDNA)affecting the electron transport chain, such as Leber’sHereditary Optic Neuropathy (LHON), reduce ATP pro-duction and increase ROS generation leading to optic nervedegeneration ([72] and Table 1) and decreased ATP pro-duction is also associated with reduced mitochondrialtransport and dying-back neuropathy in Friedreich ataxia[22]. Neuronal activity consumes ATP and high frequencystimulation leads to axonal degeneration [73] and neuronalhyperexcitability or ion channel mutations leading to per-sistent currents are associated to axonopathies and neu-ronal cell death [74–76].

When the energy supply fails in axons, Na+-K+ ATPasein membranes stop working, causing intra-axonal Na+

accumulation, reversal of the Na+-Ca2+ exchanger andincrease in Ca2+ [77]. Normally, mitochondria face regularincreases in Ca2+ concentration, which is integrated in ahomeostatic mechanism regulating mitochondrial trans-port and ATP production [4,78] but reduced Ca2+ handlingunderlies neuronal degeneration in several diseases [79].Mutant huntingtin-expressing neurons show enhancedCa2+ sensitivity and reduced mitochondrial Ca2+ uptake[80], and in a form of HSP caused by a mutation in thespartin (SPG20) gene, mitochondria show a decreasedmembrane potential and reduced Ca2+ buffering [81].

Oxidative stress as a degenerative intermediate in axons

On the dark side of the mitochondrial triad is ROS, histori-cally considered a damaging by product of respiration, butrecently shown to have physiological signaling functions[82]. Excess ROS becomes deleterious and axons seemparticularly sensitive [38]. Consequently, several neurode-generative conditions are associated with increased ROS[83]. Increase in axonal ROS and axonal degeneration trig-gered by the mitochondrial electron transport inhibitor


rotenone or by exogenous oxidants is prevented byNMNAT3 overexpression; and vincristine, which causesaxonal degeneration, leads to increase in ROS, which is alsoprevented by NMNAT3 overexpression [84].

Cellular antioxidative mechanisms including superoxidedismutases (SODs) and glutathione generating enzymesnormally keep ROS at a low level [85] and genetic deletionof the antioxidant enzyme SOD-1, which is localized toaxonal mitochondria in addition to other neuronal domains,triggers axonal degeneration in vitro [86]. Taken together,these findings point to an important role of oxidative stressin an as yet undefined step of the axonal degenerationprogram. One possibility is that overexpressed NMNATs,either within or associated with mitochondria, could exerttheir protective function by blocking or reducing ROS pro-duced by very diverse pro-degenerative stimuli. A recentreport demonstrates that WldS also protects from diabetes-induced peripheral neuropathies and retinal ganglion celldegeneration, an effect that might be related to reduction inoxidative stress and energy depletion [87].

ROS generation occurs in virtually all conditions involv-ing microglia and astrocyte activation or macrophage in-filtration, common features of most neurodegenerativediseases [88]. Nitric oxide (NO) induces axonal degenera-tion, especially in electrically active axons [89], an exampleof the synergistic induction of axonal degeneration. In theexperimental autoimmune encephalomyelitis (EAE) modelof MS, characterized by axonal degeneration, inflammato-ry episodes lead to elevation of NO by T-cell-activatedmacrophages, inhibiting mitochondrial respiration andATP production, and inducing free radical production[88]. Loss of mitochondrial SOD-2 in damaged motor axonsaccelerates axonal degeneration [90], suggesting that afterinjury antioxidant enzymes control excess ROS.

Integration by mitochondria: the Ca2+/ATP/ROS triad out

of control

Once intra-axonal homeostatic capability is exceeded, mi-tochondria are confronted by high Ca2+ and ROS levels,

Soma

Glia

VDAC+uniporter

MITO

Axon

?

Ca2+

mPTP

R

Figure 3. The contribution of axonal mitochondria in axonal degeneration. A simplified

contribute to axonal degeneration. As shown in the lower diagram, representing a segm

pathways (black arrows) that regulate energetic metabolism, Ca2+ homeostasis and ROS

by the voltage-dependent anion channel (VDAC) and the uniporter, together with elevate

transition pore (mPTP). When the energy supply fails in axons, Ca2+ influx through ne

contribute to Ca2+ increase in the axonal cytoplasm (dashed red arrows). Oxidative dam

constitute the point of no return for axonal destruction (red arrows).

ideal conditions to activate mitochondrial cell death pro-cesses (Figure 3). Poor quality control of mitochondria(above) is likely to reduce this capability. In these condi-tions, mitochondrial dysfunction and mPTP activationwould produce a catastrophic resolution of pro-degenera-tive stimuli, most likely executed by Ca2+-dependent cal-pain activation [91]. Importantly, the threshold for mPTPactivation decreases with age [92,93], a likely underlyingfactor in age-related axonal degeneration found in severalneurodegenerative conditions, such as AD [70]. mPTPsensitivity is also tissue dependent [94] and neuronalmitochondria are more sensitive to Ca2+ overload andopening of the mPTP than astrocytic mitochondria[95,96]. Whether diverse neuronal populations also differin sensitivity to mPTP induction, and hence, in theirsusceptibility to genetic and toxic insults, remains to beinvestigated.

Axonal endoplasmic reticulum

Finally, other organelles are likely to participate in aconcerted program that ultimately leads to axonal demise.In axons, the endoplasmic reticulum (ER) forms an extend-ed network in close association with the plasmalemma andmitochondria [97]. ROS and ATP also modulate extra-mitochondrial Ca2+ pools; ROS targets the ryanodine re-ceptor in the ER, enhancing Ca2+ release [98]; Ca2+ uptakeinto the ER by the sarcoplasmic-endoplasmic reticulumCa2-ATPase (SERCA) pump is ATP-dependent. Therefore,intra-axonal Ca2+ homeostasis is probably jointly regulat-ed by ER and mitochondria [99], and the close apposition ofthe ER to the axonal plasma membrane might provide astructural support to achieve Ca2+ control [77]. Interest-ingly, mitofusin-2, which is mutated in the pure axonaldisorder CMT2A with mitochondrial transport defects [33],mediates ER-mitochondrial tethering [100], which may beimportant for both Ca2+ handling and mitochondrial trans-port. It is also worth noting that a recent study hassuggested that ER tubules play an important role indefining the position of mitochondrial division sites

Glia

Axon

Ca2+

Ca2+

ATP Calpain activation

Axonal degenerationOS


scheme of the factors directly or indirectly controlled by mitochondria that might

ent of the axon, mitochondria (MITO) physiologically interact with interconnected

generation. High Ca2+ in the mitochondria, channeled from the axonal cytoplasm

d ROS levels, constitute ideal conditions to activate the mitochondrial permeability

uronal Na+-Ca2+ exchangers, as well as by voltage dependent Ca2+ channels, will

age to axonal proteins and lipids together with Ca2+-activated calpains probably

369


[101]. Although this study was not performed in neurons, itis interesting to speculate that mitochondrial fission with-in axons may be influenced by the ER in a similar manner.

Concluding remarks and future perspectivesGenetic as well as correlative data suggest that mitochon-drial dysfunction is tightly associated with severalneurodegenerative conditions characterized by axonaldegeneration. The unique morphological and functionalfeatures of axons impose a challenge for mechanisms ofmitochondrial transport and quality control, increasingsusceptibility of axonal mitochondria to genetic and toxicinsults compared to their somatic counterparts. Mitochon-dria regulate energetic metabolism, Ca2+ homeostasis andROS generation, which are interconnected pathways thatwill respond to a variety of noxious stimuli resulting inhomeostatic control or axonal destruction (Figure 3). Apathway involved in many cellular processes is a difficulttherapeutic target, but its commonality to multiple neuro-degenerative conditions makes it an attractive one never-theless.

Important questions remain about topics discussedabove. It is important to clarify how nuclear-encoded pro-teins reach stationary axonal mitochondria, whetherthrough axonal transport within moving mitochondria,transport as cytoplasmic proteins, or transport of mRNAsencoding them. The answer will help indicate the respec-tive importance of mitochondrial fusion, local protein im-port and synthesis in replenishing the mitochondrialproteome, and may of course differ for different proteins.A better understanding is needed of the wider roles ofmitochondrial axonal transport, fusion and fission andhow this relates to the many neurodegenerative disordersin which these processes are perturbed. For example, doesthe failure of increased mitochondrial transport to allevi-ate motor deficits in a mouse model of familial ALS [40]mean that reduced mitochondrial transport in this disor-der is an epiphenomenon or does it reflect subtleties ofmitochondrial transport that we do not yet understand?The regulation of mitochondrial quality control, now rea-sonably well understood in cell lines and neuronal soma,needs to be placed in an axonal context where most of theneuronal cytoplasm resides. In WD, there is no indicationyet as to how NMNAT activity links to the mPTP activationstep, or whether NMNAT exerts its axon protective effectwithin mitochondria or in an upstream cytoplasmic loca-tion. Fundamental points about mitochondrial NAD+ syn-thesis remain unclear, such as whether and how NAD+ orits precursor nicotinamide mononucleotide (NMN+) entersmitochondria. Little is known about how axonal mitochon-dria compare to somatic mitochondria in terms of Ca2+

handling, ATP production and ROS generation, or aboutwhether moving and stationary mitochondria are equallyfunctional in these regards. It will be important to deter-mine whether intrinsic differences between WldS and wildtype mitochondria underlie axon protection or whetherevents upstream of the mitochondria determine the differ-ential response to axon injury.

In conclusion, axon survival depends on mitochondriaboth in the short term, to integrate the survival and deathresponse to a wide array of axonal stresses, and in the long

370

term, where quality control requires effective transport,fusion, fission and selective removal of mitochondria.Advances in molecular genetics and live imaging are setto drive further understanding of these critically importantprocesses.

AcknowledgmentsOur research is supported by Fondo Nacional de Desarrollo Cientifico yTecnologico (FONDECYT) no. 1110987, Millennium Nucleus no. P07-011-F (to F.C.) and a Biotechnology and Biological Sciences Research Council(BBSRC) Institute Strategic Programme Grant (to M.C.).

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