Review

Mitochondria as a central sensor for axonal degenerative stimuli

Felipe A. Court1,2 and Michael P. Coleman3
1 2

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

Axonal degeneration is a major contributor to neuronal dysfunction in many neurological conditions and has additional roles in development. It can be triggered by divergent stimuli including mechanical, metabolic, infectious, toxic, hereditary and inammatory stresses. Axonal mitochondria are an important convergence point as regulators of bioenergetic metabolism, reactive oxygen species (ROS), Ca2+ homeostasis and protease activation. The challenges likely to render axonal mitochondria more vulnerable than their cellular counterparts are reviewed, including axonal transport, replenishing nuclear-encoded proteins and maintenance of quality control, fusion and ssion in locations remote from the cell body. The potential for mitochondria to act as a decision node in axon loss is considered, highlighting the need to understand the biology of axonal mitochondria and their contributions to degenerative mechanisms for novel therapeutic strategies. Introduction Neuronal soma and axons die by very different mechanisms. Axon degeneration is in many cases an autonomous process that is distinct from classical apoptosis but can be genetically regulated. Conversely, a protein that protects axons, the slow Wallerian degeneration protein (WldS), has no known effect on survival of the soma [1,2]. As molecular mechanisms underlying axon survival and degeneration become better understood, it is important to ask what makes them specic for axons. Many mitochondrial dysfunctions lead to disorders in which axon degeneration is the predominant feature, or a prominent early step (Table 1). Thus, axons may face a greater challenge than neuronal soma or dendrites in maintaining sufcient functioning of mitochondria for survival. Mitochondria, long known to have multiple roles in cell death, differ strikingly between axons and soma. First, their elongated shape, at least in peripheral nerve axons, is specialized for life in a long narrow cylinder [3]. Maintenance of this shape is critical for efcient delivery by axonal transport so that even moderate swelling will block both their own transport and that of many other cargoes (Figure 1). Second, axonal mitochondria are discrete structures, in contrast to the syncytia more typically seen in
Corresponding authors: Court, F.A. (fcourt@bio.puc.cl); Coleman, M.P. (michael.coleman@babraham.ac.uk). Keywords: axonal degeneration; mitochondria; axonal transport; mitochondrial permeability transition pore; reactive oxygen species.

many cell types. Because of this discontinuity, the delivery of material and exchange with other mitochondria may be more dependent on mechanisms such as fusion and ssion. Third, their transport has to be carefully regulated to ensure that mitochondria are focused in the correct regions of the huge axonal compartment [4], which can be several hundred times larger than neuronal soma. In this way, high requirements for adenosine triphosphate (ATP) synthesis and Ca2+ buffering can be met. This review considers the extent to which unique features of axonal mitochondria underlie axon-specic degeneration mechanisms, making them a nodal point for decisions on axon survival. In some cases, axons degenerate through failure to maintain a healthy mitochondrial population at literally arms length from the cell body, whereas in others mitochondria may play a more active role in axon degeneration. The latter process may be much faster, whereas a steady decline in mitochondria quality in axons may take many years and only occur when axonal transport further declines with age. Challenges for axonal mitochondria Mitochondrial defects are surprisingly prevalent in axon degeneration disorders (Table 1). It is interesting to consider whether this reects properties of mitochondria that are important within axons in particular and how such properties may t together. Mitochondrial fusion and ssion in neurodegenerative conditions Mitochondrial fusion and ssion are particularly prominent among the functions of the proteins in Table 1. Proteins controlling these processes are mutated in subtypes of peripheral neuropathy, optic atrophy and hereditary spastic paraplegia (HSP). Mitofusin 2, an outer mitochondrial membrane protein mediating fusion is mutated in the pure axonal disorder Charcot-Marie-Tooth Type 2A [5], whereas optic atrophy 1 (OPA1), which has a related role in fusing the inner mitochondrial membrane, is defective in autosomal dominant optic atrophy [6]. Paraplegin (encoded by the SPG7 gene), which is mutated in some types of HSP, regulates control of mitochondrial size by OPA1 [7] and its absence results in giant mitochondria [8]. Ganglioside-induced differentiation-associated protein-1 (GDAP1), a protein that enhances mitochondria fragmentation, is mutated in the mixed axon/myelin disorder Charcot-Marie-Tooth Disease type 4A (CMT4A) [9].

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0166-2236/$ see front matter 2012 Elsevier Ltd. All rights reserved. http://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 Charcot-Marie-Tooth Protein Mitofusin 2 Disease type CMT2A Possible protein function Outer mitochondrial membrane protein ER protein Axonal mitochondrial transport ER-mitochondrial tethering Molecular chaperone Neurolament organization Microtubule binding Molecular chaperone Associated with autophagy Outer mitochondrial membrane protein Mitochondrial ssion Mitochondrial matrix iron chaperone Redox regulation Mitochondrial ATPase Mitochondrial chaperone Resistance to oxidative stress (?) Several protein partners, localization and functions Required for mitochondrial calcium uptake capacity Binds spastin Associates with microtubules ER-shaping and mitochondrial dynamics (?) Inner mitochondrial membrane protein Mitochondrial fusion Complex I subunit of mitochondria Decrease ATP production Cytosolic E3 ubiquitin ligase Mitochondrial quality control Microtubule dynamics Mitochondrial quality control Atypical peroxidase Regulation of oxidant defenses Partial mitochondrial localization Mitochondrial intermembrane space Pro-apoptotic Inner mitochondrial membrane Mitochondrial DNA synthesis, replication and repair Mitochondrial localization Anti apoptotic Bcl-2 family member Refs [5,32,103]

HSPB1 (HSP27)

CMT2F

[104,105]

HSPB8 (HSP22) Gdap1 Friedrichs ataxia Hereditary spastic paraplegia Frataxin Paraplegin Hsp60 Spartin Reep1

CMT2L CMT4A Friedrichs ataxia SPG7 SPG13 SPG20 SPG38

[106] [9] [22] [8] [107] [81] [108]

Optic atrophy/neuropathy

OPA1 Complex 1 subunits ND1, 4, 6 Parkin

Optic atrophy Lebers hereditary optic neuropathy PARK2

[6,109] [110] [18]

Parkinsons disease

PINK1 DJ-1 LRRK2 HTRA2 POLG1 Sensory neuropathy

a

PARK6 PARK7 PARK8 PARK13 Parkinsons disease, Alpers syndrome Small ber sensory neuropathy

[17] [111,112] [113] [114] [115] [67]

Bcl-w

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

The GTPase dynamin-related protein 1 (DRP1), in addition to being associated with mitochondrial ssion, is also required for neurite development or maintenance in primary culture [10]. In humans, a mutation in DRP1 has been associated with severe and lethal defects in the nervous system [11]. Fusion and ssion of axonal mitochondria may also be a primary target in toxic models, as chronic exposure to low levels of rotenone causes loss of dopaminergic neuronal processes without cell death in a process that may require mitochondrial ssion [12]. Mitochondria quality control and neuronal degeneration Quality control of mitochondria is another emerging theme, especially in Parkinsons disease (PD), where a growing body of evidence points to a dying back process of early axon and synapse loss [13]. Experiments in non-neuronal cell culture suggest that accumulation of phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) on the surface of dysfunctional mitochondria acts as a signal for their removal by mitophagy [14,15]. By contrast, regular voltage-dependent proteolysis of PINK1 on healthy mitochondria maintains low levels of this protein [14]. This

degradation stops if mitochondrial membrane potential falls, causing rapid accumulation of PINK1 and recruitment of Parkin leading to mitophagy. Recent data conrm that Parkin recruitment occurs in neuronal soma [16]. Genetic evidence supports a similar mechanism in vivo as loss-offunction mutations in PINK1 or Parkin cause PD [17,18] and Drosophila studies indicate that Parkin lies downstream of PINK1 [19]. Quality control becomes particularly intriguing in the context of the huge axonal arbors of dopaminergic neurons in mammalian brain [20]. Degeneration of these nerve terminals clearly precedes death of the soma [13] so any failure of quality control may occur here rst. In distal axons of sensory neurons, mitochondria fragments are engulfed by microtubule-associated protein 1A/1B-light chain 3 (LC3)-positive vesicles and the resulting autophagosomes fuse with lysosomes as they move retrogradely [21]. The importance of retrograde transport is consistent with the accumulation of depolarized mitochondria in distal axons in a Drosophila model of Friedreich ataxia, associated with a failure of retrograde axonal transport [22]. Whether these rst stages of mitophagy in axons use
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(a)

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Healthy axon

(b)

Healthy axon

(c)

Injured axon

(d)

Pathological (HSP)

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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 claried. Both proteins are present in axons, where they downregulate axonal transport of mitochondria [16,23] and Parkin recruitment can occur in neurites when mitochondria transport is blocked [16]. However, clear evidence for axonal Parkin recruitment in more physiological conditions has not yet been reported. Whatever their identity, the proteins targeting mitochondria for mitophagy in distal axons must be delivered by anterograde transport, so this transport is important for maintaining a pool of healthy axonal mitochondria. Mitochondrial transport in large neuronal compartments In addition to retrograde trafcking of autophagosomes, and anterograde transport of proteins for mitophagy, axonal transport plays a third critical role in maintaining axonal mitochondria. Unlike their somatic counterparts, 1030% of axonal mitochondria are trafcked over huge distances [3]. While the soma is the site where some axonal mitochondria are generated [24], mitochondrial biogenesis also occurs within isolated axons [25]. The need to trafc mitochondria from the soma when axons can generate their own probably reects the fact that mitochondrial DNA encodes only 13 out of hundreds of mitochondrial proteins [26]. Nuclear-encoded proteins need to be delivered to replace natural turnover. One attractive model is that motile mitochondria, which are usually smaller [3], deliver these nuclear-encoded proteins into axons, where they fuse with large, stationary mitochondria to replenish them (Figure 2), although local protein synthesis [27] and local mitochondrial import of
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nuclear-encoded proteins may also contribute to this delivery. According to this model, disruption of this anterograde trafcking should result in gradual deterioration of axonal mitochondria. It is interesting that such disruption has been observed to occur in animal models of familial amyotrophic lateral sclerosis (ALS) [28], tauopathy [29,30], HSP [31], CMT2A [32,33], Alzheimers disease (AD) [34], Huntingtons disease (HD) [35] and toxicity by A beta or 1-methyl-4-phenylpyridinium ion (MPP+) [3638]. Some studies report that axonal transport of mitochondria can be separated from pathogenesis in mouse models of ALS [39,40] but a contributory role to some axonal disorders does seem likely [41]. This could be particularly true in agerelated disorders, as transport decline during normal ageing [30] could render older axons less tolerant of any additional transport impairment. Anterograde transport, fusion, ssion and quality control are likely to act together to maintain the functionality of axonal mitochondria and long-term axon survival (Figure 2). In this model, stationary mitochondria are regularly serviced by delivery and fusion of small mobile mitochondria carrying newly-synthesized nuclear-encoded proteins. Consistent with this, mitochondrial ux increases when nuclear-encoded mitochondrial DNA polymerase Pol gamma is deleted, as though the axon were trying to compensate for the shortage [42]. Both fusion and ssion of mitochondria occur in axons [12] and asymmetric ssion has been shown in other cell types, producing daughter mitochondria with differing membrane potentials [43]. This should allow less competent mitochondria to be removed by quality control, potentially involving PINK1/Parkin. Defects in the anterograde transport of

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Proximal
Soma

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Distal Axon

(a) Moving, new Stationary, old

(b)

Fusion replenishes

(c) Mitophagy PINK1? Fission

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Figure 2. Proposed model for quality control of axonal mitochondria. (a) A small anterogradely moving mitochondrion [3] (blue) is proposed to contain new nuclearencoded 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 mitophagy, and defects in fusion or ssion or mitophagy would all disrupt this mechanism. Consistent with this, there are indications that mitochondrial fusion and ssion are disrupted in HD [35,44] and AD [34,45]. In addition, short fragmented mitochondria accumulate in dystrophic axons close to amyloid plaques [46], whereas a-synuclein also stimulates mitochondrial ssion [47] and defects in autophagy lead to severe swelling in Purkinje cell axons [48,49]. Finally, complex IV negative mitochondria accumulate in axons of multiple sclerosis (MS) patients as though quality control has failed at some level [50]. Participation of mitochondria in Wallerian degeneration Recent data suggest that mitochondria have important roles in one specic axon degeneration mechanism known as Wallerian degeneration (WD). WD is triggered by mechanical disconnection of axons from cell bodies and serves as a model for axon degeneration when axonal transport is disrupted [51]. The supporting evidence is that an aberrant protein generated by a spontaneous mutation, WldS, is able to delay axon degeneration both after injury and in some axonal transport disorders, genetically linking these mechanisms [52]. It has been demonstrated that several parallels exists between axonal degeneration triggered by mechanical injury (i.e. WD) and the axonal degenerative events activated by some toxic, inammatory or metabolic stressors in diverse neurodegenerative conditions [53]. Therefore, WD represents an experimental model extensively used to dene the mechanisms involved in axonal degeneration. After mechanical nerve injury, axons exhibit a latent phase in which the structures remain apparently normal

(12 days in laboratory animals), followed by a catastrophic phase in which all axonal structures collapse rapidly, in a well-ordered sequence [51]. With a few exceptions, collapse of severed axons occurs in animals of widely separated phyla [51]. Agents that interfere with microtubular function also trigger degeneration, again pointing to a role for disruption of microtubule-assisted transport in axonal degeneration [51]. Interestingly, several links have emerged between WD and mitochondria. Overexpression of the mitochondrial NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) delays axonal degeneration [54]. Furthermore, the WD-protective WldS protein, which is a chimera between nuclear NMNAT1 and a non-catalytic sequence from a ubiquitin ligase, is also partially localized to axonal mitochondria [54,55]. The enzymatic activity of NMNAT3 or WldS is required for their protective action. Although this was originally thought to implicate NAD+ in axonal degeneration, it is possible that another metabolite handled by these enzymes has a critical role [56]. Thus, localization of WldS and NMNAT3 in axonal mitochondria could be an important requirement for the protective effect, but because neither is conned to mitochondria this still requires conrmation. Although the protective mechanism of overexpressed NMNATs has not yet been elucidated, the involvement of NAD+-dependent pathways in mitochondrial energy metabolism and redox capacity in the cytoplasm or mitochondria [57] suggest a mechanism linking loss of mitochondrial function to axonal degeneration [58]. For example, this could occur when one or more NMNATs are not properly trafcked to axons [59].
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Recent data indicate a direct association between WD and activation of the mitochondrial permeability transition pore (mPTP). Genetic or pharmacologic inhibition of mPTP activation delays axonal degeneration downstream WldS protection [60]. The mPTP has been functionally associated to several neurodegenerative conditions harboring axonal degeneration, including MS, AD, ALS and cerebral ischemia [6164]. Interestingly, two of the main activators of the mPTP in excitable cells, Ca2+ and ROS [65], are pathways experimentally dened as participants in the axonal degeneration program. Moreover, a recent report shows that WldS enhances basal mitochondrial motility and increases Ca2+ buffering capability of axonal mitochondria [66], consistent with the involvement of mPTP in axonal degeneration [60]. The mPTP is molecularly distinct from mitochondrialdependent apoptosis. This is important because classical apoptosis involving B cell lymphoma 2 (Bcl-2)-associated X protein (Bax), BCL2-homologous antagonist/killer (Bak), or Bcl-2, is not required for WD [51]. Therefore, activation of the mPTP could be part of an axonal-specic program of degeneration that reects specic axonal characteristics such as distance from the nucleus and unique requirements of axonal mitochondria, in contrast to classic apoptosis taking place in the neuronal soma. Interestingly, a recent report suggests an axon-specic role also for the mitochondrial anti apoptotic protein Bcl-w (also known as Bcl-2L2), at least in a subset of sensory axons [67]. Thus, there may be several pathways in which mitochondria contribute to the specic degeneration of axons. Mitochondrial changes after degenerative stimuli Axonal degeneration after heterogeneous induction stimuli is considered to proceed through a Wallerian-like mechanism if it is genetically delayed by WldS [51]. Thus, diverse activators converge onto a common degeneration pathway, suggesting one or more integration points. Axonal mitochondria appear to be one such integration point, where pro-degenerative stimuli combine to produce the appropriate survival or degeneration response. Features of mitochondrial dysfunction occur in many disorders, including Ca2+ dyshomeostasis, ATP depletion, ROS generation and mitochondrial swelling, suggesting mitochondria act as a central sensor for degenerative stimuli. According to this model, degradative processes should be activated when concerted stimuli surpass the mitochondrial homeostatic capacity. Mitochondria control dynamic changes in the cytoplasmic levels of Ca2+, ATP and ROS, and these cellular pathways are connected by several feedback loops [68]. Stimuli affecting one or more of these components are integrated by axonal mitochondria and if a new homeostatic state is not reached, ATP levels decline, intracellular Ca2+ levels increase and ROS is produced, which culminate in the activation of positive feedback loops that target axons for destruction. Importantly, the sensitivity of mitochondria to diverse stimuli will depend on genetic and environmental factors, including the cell- and tissuespecic context. For example, mutations that change mitochondrial parameters such as trafcking, respiratory activity, Ca2+ buffering or ROS generation constitute
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genetic risk factors that increase axonal vulnerability to sublethal stimuli (Table 1). Several mitochondrial properties, including ATP production, Ca2+ uptake, buffering and release, exhibit regional, cellular and subcellular variations in the nervous system [69]. Mitochondria in axons with unique morphology and metabolic requirements, such as the huge axonal arbors of dopaminergic neurons or meter long peripheral nerve axons, are likely to differ in susceptibility to stressors compared to somatic ones (see above). Environmental factors including caloric intake and toxins such as heavy metals modify the strength of feedback loops between Ca2+, ATP and ROS [68]. Finally, aging, a well-established risk factor for neurodegenerative conditions [70], profoundly affects the homeostasis of the Ca2+/ATP/ROS triad, especially in excitable cells, with a decrease in axonal transport in aging axons as one contributing factor [30]. Energetic metabolism and ionic homeostasis in axons Excitability comes at a high energetic price. Maintaining ionic gradients and action potential propagation use most cellular energy, exceeding the demands of associated glia by fourfold [71]. Mitochondrial ATP production provides most axonal energy, and ATP consumption is central to mitochondrial homeostatic capacities regulating Ca2+ and ROS [68]. Energy metabolism, therefore, is a key parameter in axons. Mutations in mitochondrial DNA (mtDNA) affecting the electron transport chain, such as Lebers Hereditary Optic Neuropathy (LHON), reduce ATP production and increase ROS generation leading to optic nerve degeneration ([72] and Table 1) and decreased ATP production is also associated with reduced mitochondrial transport and dying-back neuropathy in Friedreich ataxia [22]. Neuronal activity consumes ATP and high frequency stimulation leads to axonal degeneration [73] and neuronal hyperexcitability or ion channel mutations leading to persistent currents are associated to axonopathies and neuronal cell death [7476]. When the energy supply fails in axons, Na+-K+ ATPase in membranes stop working, causing intra-axonal Na+ accumulation, reversal of the Na+-Ca2+ exchanger and increase in Ca2+ [77]. Normally, mitochondria face regular increases in Ca2+ concentration, which is integrated in a homeostatic mechanism regulating mitochondrial transport and ATP production [4,78] but reduced Ca2+ handling underlies neuronal degeneration in several diseases [79]. Mutant huntingtin-expressing neurons show enhanced Ca2+ sensitivity and reduced mitochondrial Ca2+ uptake [80], and in a form of HSP caused by a mutation in the spartin (SPG20) gene, mitochondria show a decreased membrane potential and reduced Ca2+ buffering [81]. Oxidative stress as a degenerative intermediate in axons On the dark side of the mitochondrial triad is ROS, historically considered a damaging by product of respiration, but recently shown to have physiological signaling functions [82]. Excess ROS becomes deleterious and axons seem particularly sensitive [38]. Consequently, several neurodegenerative conditions are associated with increased ROS [83]. Increase in axonal ROS and axonal degeneration triggered by the mitochondrial electron transport inhibitor

Review
rotenone or by exogenous oxidants is prevented by NMNAT3 overexpression; and vincristine, which causes axonal degeneration, leads to increase in ROS, which is also prevented by NMNAT3 overexpression [84]. Cellular antioxidative mechanisms including superoxide dismutases (SODs) and glutathione generating enzymes normally keep ROS at a low level [85] and genetic deletion of the antioxidant enzyme SOD-1, which is localized to axonal mitochondria in addition to other neuronal domains, triggers axonal degeneration in vitro [86]. Taken together, these ndings point to an important role of oxidative stress in an as yet undened step of the axonal degeneration program. One possibility is that overexpressed NMNATs, either within or associated with mitochondria, could exert their protective function by blocking or reducing ROS produced by very diverse pro-degenerative stimuli. A recent report demonstrates that WldS also protects from diabetesinduced peripheral neuropathies and retinal ganglion cell degeneration, an effect that might be related to reduction in oxidative stress and energy depletion [87]. ROS generation occurs in virtually all conditions involving microglia and astrocyte activation or macrophage inltration, common features of most neurodegenerative diseases [88]. Nitric oxide (NO) induces axonal degeneration, especially in electrically active axons [89], an example of the synergistic induction of axonal degeneration. In the experimental autoimmune encephalomyelitis (EAE) model of MS, characterized by axonal degeneration, inammatory episodes lead to elevation of NO by T-cell-activated macrophages, inhibiting mitochondrial respiration and ATP production, and inducing free radical production [88]. Loss of mitochondrial SOD-2 in damaged motor axons accelerates axonal degeneration [90], suggesting that after injury antioxidant enzymes control excess ROS. Integration by mitochondria: the Ca2+/ATP/ROS triad out of control Once intra-axonal homeostatic capability is exceeded, mitochondria are confronted by high Ca2+ and ROS levels,

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ideal conditions to activate mitochondrial cell death processes (Figure 3). Poor quality control of mitochondria (above) is likely to reduce this capability. In these conditions, mitochondrial dysfunction and mPTP activation would produce a catastrophic resolution of pro-degenerative stimuli, most likely executed by Ca2+-dependent calpain activation [91]. Importantly, the threshold for mPTP activation decreases with age [92,93], a likely underlying factor in age-related axonal degeneration found in several neurodegenerative conditions, such as AD [70]. mPTP sensitivity is also tissue dependent [94] and neuronal mitochondria are more sensitive to Ca2+ overload and opening of the mPTP than astrocytic mitochondria [95,96]. Whether diverse neuronal populations also differ in sensitivity to mPTP induction, and hence, in their susceptibility to genetic and toxic insults, remains to be investigated. Axonal endoplasmic reticulum Finally, other organelles are likely to participate in a concerted program that ultimately leads to axonal demise. In axons, the endoplasmic reticulum (ER) forms an extended network in close association with the plasmalemma and mitochondria [97]. ROS and ATP also modulate extramitochondrial Ca2+ pools; ROS targets the ryanodine receptor in the ER, enhancing Ca2+ release [98]; Ca2+ uptake into the ER by the sarcoplasmic-endoplasmic reticulum Ca2-ATPase (SERCA) pump is ATP-dependent. Therefore, intra-axonal Ca2+ homeostasis is probably jointly regulated by ER and mitochondria [99], and the close apposition of the ER to the axonal plasma membrane might provide a structural support to achieve Ca2+ control [77]. Interestingly, mitofusin-2, which is mutated in the pure axonal disorder CMT2A with mitochondrial transport defects [33], mediates ER-mitochondrial tethering [100], which may be important for both Ca2+ handling and mitochondrial transport. It is also worth noting that a recent study has suggested that ER tubules play an important role in dening the position of mitochondrial division sites
Glia

Soma

Axon

Glia

Ca2+

VDAC+uniporter

Ca2+
mPTP

MITO

Ca2+

ATP ROS

Calpain activation

Axon

Axonal degeneration
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Figure 3. The contribution of axonal mitochondria in axonal degeneration. A simplified scheme of the factors directly or indirectly controlled by mitochondria that might contribute to axonal degeneration. As shown in the lower diagram, representing a segment of the axon, mitochondria (MITO) physiologically interact with interconnected pathways (black arrows) that regulate energetic metabolism, Ca2+ homeostasis and ROS generation. High Ca2+ in the mitochondria, channeled from the axonal cytoplasm by the voltage-dependent anion channel (VDAC) and the uniporter, together with elevated ROS levels, constitute ideal conditions to activate the mitochondrial permeability transition pore (mPTP). When the energy supply fails in axons, Ca2+ influx through neuronal Na+-Ca2+ exchangers, as well as by voltage dependent Ca2+ channels, will contribute to Ca2+ increase in the axonal cytoplasm (dashed red arrows). Oxidative damage to axonal proteins and lipids together with Ca2+-activated calpains probably constitute the point of no return for axonal destruction (red arrows).

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[101]. Although this study was not performed in neurons, it is interesting to speculate that mitochondrial ssion within axons may be inuenced by the ER in a similar manner. Concluding remarks and future perspectives Genetic as well as correlative data suggest that mitochondrial dysfunction is tightly associated with several neurodegenerative conditions characterized by axonal degeneration. The unique morphological and functional features of axons impose a challenge for mechanisms of mitochondrial transport and quality control, increasing susceptibility of axonal mitochondria to genetic and toxic insults compared to their somatic counterparts. Mitochondria regulate energetic metabolism, Ca2+ homeostasis and ROS generation, which are interconnected pathways that will respond to a variety of noxious stimuli resulting in homeostatic control or axonal destruction (Figure 3). A pathway involved in many cellular processes is a difcult therapeutic target, but its commonality to multiple neurodegenerative conditions makes it an attractive one nevertheless. Important questions remain about topics discussed above. It is important to clarify how nuclear-encoded proteins reach stationary axonal mitochondria, whether through axonal transport within moving mitochondria, transport as cytoplasmic proteins, or transport of mRNAs encoding them. The answer will help indicate the respective importance of mitochondrial fusion, local protein import and synthesis in replenishing the mitochondrial proteome, and may of course differ for different proteins. A better understanding is needed of the wider roles of mitochondrial axonal transport, fusion and ssion and how this relates to the many neurodegenerative disorders in which these processes are perturbed. For example, does the failure of increased mitochondrial transport to alleviate motor decits in a mouse model of familial ALS [40] mean that reduced mitochondrial transport in this disorder is an epiphenomenon or does it reect subtleties of mitochondrial transport that we do not yet understand? The regulation of mitochondrial quality control, now reasonably well understood in cell lines and neuronal soma, needs to be placed in an axonal context where most of the neuronal cytoplasm resides. In WD, there is no indication yet as to how NMNAT activity links to the mPTP activation step, or whether NMNAT exerts its axon protective effect within mitochondria or in an upstream cytoplasmic location. Fundamental points about mitochondrial NAD+ synthesis remain unclear, such as whether and how NAD+ or its precursor nicotinamide mononucleotide (NMN+) enters mitochondria. Little is known about how axonal mitochondria compare to somatic mitochondria in terms of Ca2+ handling, ATP production and ROS generation, or about whether moving and stationary mitochondria are equally functional in these regards. It will be important to determine whether intrinsic differences between WldS and wild type mitochondria underlie axon protection or whether events upstream of the mitochondria determine the differential response to axon injury. In conclusion, axon survival depends on mitochondria both in the short term, to integrate the survival and death response to a wide array of axonal stresses, and in the long
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term, where quality control requires effective transport, fusion, ssion and selective removal of mitochondria. Advances in molecular genetics and live imaging are set to drive further understanding of these critically important processes.
Acknowledgments
Our research is supported by Fondo Nacional de Desarrollo Cientico y Tecnologico (FONDECYT) no. 1110987, Millennium Nucleus no. P07-011F (to F.C.) and a Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant (to M.C.).

References
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