Disturbed mitochondrial dynamics and neurodegenerative disorders.

REVIEWS Disturbed mitochondrial dynamics and neurodegenerative disorders Florence Burté, Valerio Carelli, Patrick F. Chinnery and Patrick Yu-Wai-Man Abstract | Mitochondria form a highly interconnected tubular network throughout the cell via a dynamic process, with mitochondrial segments fusing and breaking apart continuously. Strong evidence has emerged to implicate disturbed mitochondrial fusion and fission as central pathological components underpinning a number of childhood and adult-onset neurodegenerative disorders. Several proteins that regulate the morphology of the mitochondrial network have been identified, the most widely studied of which are optic atrophy 1 and mitofusin 2. Pathogenic mutations that disrupt these two pro-fusion proteins cause autosomal dominant optic atrophy and axonal Charcot–Marie–Tooth disease type 2A, respectively. These disorders predominantly affect specialized neurons that require precise shuttling of mitochondria over long axonal distances. Considerable insight has also been gained by carefully dissecting the deleterious consequences of imbalances in mitochondrial fusion and fission on respiratory chain function, mitochondrial quality control (mitophagy), and programmed cell death. Interestingly, these cellular processes are also implicated in morecommon complex neurodegenerative disorders, such as Alzheimer disease and Parkinson disease, indicating a common pathological thread and a close relationship with mitochondrial structure, function and localization. Understanding how these fundamental processes become disrupted will prove crucial to the development of therapies for the growing number of neurodegenerative disorders linked to disturbed mitochondrial dynamics. Burté, F. et al. Nat. Rev. Neurol. advance online publication 9 December 2014; doi:10.1038/nrneurol.2014.228

Introduction

Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK (F.B., P.F.C., P.Y.-W.-M.). IRCCS Institute of Neurological Sciences of Bologna, Bellaria Hospital, Via Altura 3, 40139 Bologna, Italy (V.C.). Correspondence to: P.Y.-W.-M. patrick.yu-wai-man@ ncl.ac.uk

Mitochondria do not exist as isolated organelles fixed at predetermined positions within the cell’s structure. Instead, they display a high degree of interconnectivity and plasticity, which are largely dictated by the cell’s metabolic demands under prevailing physiological conditions.1 The morphology of the mitochondrial network is in a constant state of flux, influenced by the delicate balance between opposing fusional and fissional forces. The main players in this intricate and tightly coordinated process were first identified in seminal experiments using yeast models. These mediators of mitochondrial dynamics have been highly conserved throughout evolution, which is in keeping with the critical regulatory roles of these proteins in both simple and complex organisms. Helped by the genomics revolution of the past decade, perturbation of mitochondrial dynamics has emerged as the central pathophysiological mechanism underpinning a broad range of human disorders, often with strikingly overlapping clinical features (Table 1). Unsurprisingly, pathogenic mutations have been identified in several pro-fusion and pro-fission nuclear genes, with disease phenotypes ranging from severe, early-onset and invariably lethal encephalomyopathies, through isolated optic atrophy and peripheral neuropathy, to more-complex late-onset multisystemic neuromuscular disorders. Competing interests The authors declare no competing interests.

Mutations in the pro-fusion genes optic atrophy 1 (OPA1) and mitofusin 2 (MFN2) were initially reported in families with autosomal dominant optic atrophy (DOA; OMIM #605290) and axonal Charcot–Marie–Tooth disease type 2A (CMT2A; OMIM #609260), respectively.2–4 By focusing on these two diseases, this Review will critically appraise the fundamental mechanisms linking disturbed mitochondrial dynamics with the development and progression of neurological deficits in both monogenic and complex neurodegenerative disorders.

Mitochondrial network dynamics Balancing mitochondrial fusion and fission OPA1 is an inner mitochondrial membrane protein, whereas MFN1 and MFN2 are embedded within the outer mitochondrial membrane.5,6 The two mitofusins are structurally and functionally complementary to OPA1 and, together, they choreograph a precise chain of mechanical events that sequentially trigger fusion of the outer, then the inner mitochondrial membranes (Figure 1).7,8 All three proteins belong to a large family of dynaminrelated mechanoenzymes characterized by a highly conserved dynamin GTPase domain, which is central to their multifaceted cellular roles and interactions.6 OPA1 is firmly anchored, via its transmembrane domain, within the narrow junctional regions that insulate the mitochondrial cristae in a zipper-like fashion from the rest of the intermembrane space.9,10 This specific

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REVIEWS Key points ■■ Mitochondria do not exist as isolated organelles; instead, they form a highly interconnected tubular network throughout the cell ■■ The state of this dynamic mitochondrial network under both physiological and disease conditions is dictated by the balance between mitochondrial pro-fusion and pro-fission forces ■■ Optic atrophy protein 1 (OPA1) and mitofusin 2 (MFN2) are two important protein mediators of mitochondrial fusion ■■ Pathogenic OPA1 and MFN2 mutations cause autosomal dominant optic atrophy and axonal Charcot–Marie–Tooth disease type 2A, respectively ■■ Mounting evidence implicates disturbed mitochondrial dynamics in the pathogenesis of complex neurodegenerative disorders such as Alzheimer disease, Parkinson disease and Huntington disease ■■ Although treatment options for disorders of mitochondrial dynamics are currently limited, the future looks promising for the development of novel neuroprotective strategies and innovative gene therapy approaches

Table 1 | Human disorders associated with disturbed mitochondrial dynamics Gene

Locus

OMIM #

Phenotypes

1p36.22

609260

Charcot–Marie–Tooth disease axonal form type 2A (CMT2A)108 Hereditary motor and sensory neuropathy type 6 (HMSN6)111,132

Autosomal dominant Mitofusin 2 (MFN2)

601152 Optic atrophy 1 (OPA1)

3q28–q29

165500

Isolated optic atrophy and syndromic forms of dominant optic atrophy90,91,103

Ganglioside-induced differentiation associated protein 1 (GDAP1)

8q13.1–12.3

607831

Charcot–Marie–Tooth disease axonal form type 2K (CMT2K)199

Dynamin-related protein 1 (DRP1)

12p11.21

603850

Severe infantile neurodegenerative disease200,201

Optic atrophy 3 (OPA3)

19q13.2–q13.3

165300

Optic atrophy and premature cataracts202

GDAP1

8q13.1–12.3

214400

Charcot–Marie–Tooth disease demyelinating form type 4A (CMT4A)203

OPA3

19q13.2–q13.3

258501

3-methylglutaconic aciduria type III (Costeff syndrome)204

Autosomal recessive

Permission obtained from Springer © Yu-Wai-Man, P. et al. in Mitochondrial Disorders Caused by Nuclear Genes (ed. Wong, L.‑J. C.) 141–161 (2013).5

transmembrane protein domain is followed by a series of coiled-coil segments that allow OPA1 to homopolymerize into cylindrical tubular structures, which in turn facilitate the tethering of opposing membranes. A pathological reduction in OPA1 levels results in fragmentation of the mitochondrial network due to unopposed mitochondrial fission (Figure 2).11,12 Although much attention has focused on these main pro-fusion and pro-fission proteins (Table 2), a host of equally critical intermediaries are starting to emerge. These novel mitochondrial dynamic proteins include molecules that promote fission, such as endophilin‑B1, mitochondrial fission factor (MFF), mitochondrial dynamics proteins MID49 and MID51, mitochondrial protein 18 kDa (MTP18; also known as mitochondrial fission process protein 1), and mitochondrial fission regulator 1

(MTFR1).13–16 Endophilin‑B1 has also been linked with the regulation of autophagy.17

Proteolytic control Mitochondrial fusion is modulated by the proteolytic processing of two major mediators, OPA1 and MFN2. The degradation of MFN2 through ubiquitination is dependent on PTEN-induced putative kinase 1 (PINK1, also called mitochondrial serine/threonine-protein kinase PINK1), and this process is tightly linked to the loss of mitochondrial membrane potential and the autophagic process. OPA1 exists as eight isoforms that arise from alternative splicing of exons 4, 4b and 5b.18,19 Following its import into the mitochondrial inter membrane space, OPA1 is first processed by the matrix metalloproteases, which remove the mitochondrial targeting signal, and then by proteases of the mitochondrial AAA+ (ATPases associated with diverse cellular activities) superfamily, which leads to the formation of long and short forms of OPA1.20,21 The nature of the proteases involved in the maturation steps of OPA1 remains an area of intense investigation. The mitochondrial AAA+ proteases AFG3-like protein 2 (AFG3L2) and para plegin cleave OPA1 at site S1 within exon 5,20,22 whereas the ATP-dependent zinc metalloprotease YME1L constitutively cleaves OPA1 at S2 within exon 5b.23,24 Other important mitochondrial membrane proteases involved in OPA1 cleavage include the presenilins- associated rhomboid-like (PARL) protease and the metalloendopeptidase OMA1.25–28 Mitochondrial morphology and bioenergetics The state of the mitochondrial network is a reflection of the cell’s metabolic needs under both physiological and stress conditions. Although the precise mechanisms still need to be elucidated, both OPA1 and MFN2 are involved in the regulation of mitochondrial respiratory chain coupling and oxidative phosphorylation.5,29 MFN2 is also thought to exert a direct influence on mitochondrial biogenesis by regulating the expression of nuclear-encoded respiratory chain subunits.30 Fibroblasts from patients harbouring pathogenic OPA1 or MFN2 mutations exhibit a mitochondrial coupling defect with reduced mitochondrial membrane potential and impaired ATP synthesis.11,12,30–32 There is now direct experimental evidence that OPA1 regulates the assembly and stability of the respiratory chain supercomplexes through its influence on remodelling of mitochondrial cristae.33 Recent data also suggest that proteolytic activation of OPA1 is sufficient to stimulate mitochondrial inner membrane fusion in a process that is sensitive to the oxidative phosphorylation output from the mitochondrial respiratory chain.29 These elegant tuning mechanisms ensure a close match between mitochondrial morphology and the energetic demands of the cell.29,33 Impaired oxidative phosphorylation has been found consistently with the use of in vivo phosphorus magnetic resonance spectroscopy in the calf muscle of patients harbouring a broad range of OPA1 mutations.34–36 Similarly, a serious biochemical defect in the visual cortex

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REVIEWS Endoplasmic reticulum APP PS1 Secretase complex PS2

PS

PC Phospholipid transport

2

VDAC

Fission

3

IP3R

Grp75

DRP1

MAM

Ca2+

DRP1

MFN2 MFN1/2 MICU1

Fusion 1 MFN1/2

PE FIS1 DRP1 FIS1 OPA3 DRP1 FIS1 DRP1 FIS1 GDAP1

MCU MAM signalling

OPA1 OPA1

Mitochondrion

Figure 1 | Mitochondrial dynamics and interactions. OPA1, MFN1 and MFN2 are essential mediators of the sequential fusion of the outer and inner membranes of Nature Reviews | Neurology adjacent mitochondria (1). During fission, DRP1 is recruited from the cytosol to the outer mitochondrial membrane, where it assembles with FIS1 to constrict the mitochondrial tubule (2). Interaction between the endoplasmic reticulum and mitochondria at MAMs is essential for many processes, including Ca2+ signalling, γ-secretase activity, and phospholipid, cholesterol ester and fatty acid metabolism (3). MFN2 at MAM interfaces is involved in endoplasmic-reticulum–mitochondria tethering via interactions with both MFN1 and MFN2 on the outer mitochondrial membrane. Abbreviations: APP, amyloid precursor protein; DRP1, dynamin-related protein 1; FIS1, mitochondrial fission 1 protein; GDAP1, Ganglioside-induced differentiation-associated protein 1; Grp75, 75 kDa glucose-regulated protein; IP3R, inositol 1,4,5-trisphosphate receptor; MAM, mitochondria-associated membrane; MCU, mitochondrial calcium uniporter protein; MFN, mitofusin; MICU1, mitochondrial calcium uptake protein 1; OPA, optic atrophy protein; PC, phosphatidylcholine; PE, phosphatidylethalonamine; PS, phosphatidylserine; PS1, presenilin 1; PS2, presenilin 2; VDAC, voltage-dependent anion channel.

was observed in MFN2 mutation carriers with impaired vision secondary to optic atrophy.37

Endoplasmic reticulum interactions The crosstalk between the endoplasmic reticulum and mitochondrial compartments is currently a hot topic of research.38,39 Some fascinating insights have been uncovered over the past 5 years, with a number of seminal experiments gradually dissecting the intricate physical interactions and interorganellar signalling pathways operating at these interfaces (Figure 1). These socalled mitochondria-associated membranes (MAMs) create unique microenvironments that influence lipid biosynthesis and calcium homeostasis.40 Calcium regulates a broad range of functions that are directly related to cell survival, including intracellular signalling, the response to excitotoxic glutaminergic stimuli, and the initiation and propagation of programmed cell death.41,42 Intracellular calcium is predominantly sequestered within the endoplasmic reticulum and the mitochondrial reticulum with the bidirectional flux between these two compartments being strictly controlled. However, spatial coupling alone cannot explain the highly

coordinated nature of calcium transfer between these two compartments. This riddle was clarified with the identification of the various proteins that constitute the mitochondrial calcium uniporter, in particular the mitochondrial calcium uniporter protein (MCU) and the mitochondrial calcium uptake protein 1 (MICU1).43 MCU is a transmembrane protein located within the mitochondrial inner membrane and it assembles to form the conducting pore of this highly selective ion channel.44 MICU1 is the calcium sensor that activates the mitochondrial calcium uniporter, and loss-of-function mutations that disrupt this critical accessory protein result in a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder.45–47 Predictably, pathogenic OPA1 and MFN2 mutations cause significant disruption of all the homeostatic mechanisms that operate at MAM interfaces, clearly highlighting the important role of mitochondrial dynamics in the b uffering of intracellular calcium concentrations.48–50 MFN2 is an outer mitochondrial membrane protein but, unlike MFN1, it is also enriched at the interface between the endoplasmic reticulum and the mitochondria.38,39 Ablation or silencing of MFN2 disrupts endoplasmic reticulum morphology and loosens the interactions with mitochondria, thereby reducing the efficiency of mitochondrial calcium uptake in response to stimuli that generate inositol‑1,4,5-trisphosphate.51 The current genetic and biochemical evidence supports a model in which MFN2 on the endoplasmic reticulum acts as a structural bridge by engaging in homotypic and heterotypic complexes with MFN1 or MFN2 located on the outer mitochondrial surface. The close physical interaction between the endoplasmic reticulum and the mitochondria is likely to be even more complex than is currently understood, and identi fication of the other mediators involved in the formation of MAMs is actively being pursued. Intriguingly, the areas of contact between these two organelles seem to mark future sites of mitochondrial division before the recruitment of dynamin-related protein 1 (DRP1, also called dynamin-1-like protein) to these sites.52 In this proposed model, which seems to be evolutionarily conserved between yeast and mammals, the endoplasmic reticulum tubules wrap around a mitochondrial segment, constricting this area and facilitating the recruitment of DRP1 to complete the fission process. An increasing number of neurodegenerative diseases, including Alzheimer disease (AD) and Parkinson disease (PD), are being linked to disturbed interactions between the endoplasmic reticulum and mitochondria, and this list is set to grow even further as the molecular bases for as yet undefined neuropathological entities are uncovered.53,54

Mitochondrial cell death pathways Programmed cell death involves several players operating along overlapping pathways (Figure 3). The actual sequence of events is still the subject of much debate but, regardless of the triggers and mediators involved, mitochondria remain central to the irreversible process



REVIEWS a

fusion,10,19,26,62,63 there is also evidence, albeit to a lesser extent, to support a contribution by MFN2.64,65

b

c

d

Figure 2 | Mitochondrial network fragmentation in OPA1-mutant fibroblasts. Nature Reviews | Neurology Staining was performed with MitoTrackerTM (Invitrogen, UK), a mitochondrionspecific fluorescent dye (green), and Hoechst 33342 (Invitrogen, UK) for the nucleus (blue). Images were captured as a series of Z‑stacks with 16 slices, each 0.2 μm apart, using an inverted epifluorescence microscope (Leica, UK). HuygensTM image processing software (Scientific Volume Imaging, Netherlands) generates a data file that includes information on the total number of mitochondrial fragments per cell, their individual lengths and their volumes. A false colour scheme aids visualization of network morphology. A control fibroblast, a | before and b | after image deconvolution and reconstruction, shows a highly interconnected mitochondrial network. c,d | Similarly processed images in an OPA1-mutant fibroblast reveal a fragmented mitochondrial network secondary to haploinsufficiency. Abbreviation: OPA1, optic atrophy 1. Permission obtained from Springer © Yu-Wai-Man, P. et al. in Mitochondrial Disorders Caused by Nuclear Genes (ed. Wong, L.‑J. C.) 141–161 (2013).5

that will ultimately result in the cell’s elimination.55,56 Proapoptotic and antiapoptotic signals are detected and balanced by a complex network of Bcl‑2 family proteins that includes both proapoptotic members— for example, apoptosis regulators BAK and BAX, and BAD (Bcl-2-associated agonist of cell death)—and anti apoptotic members such as Bcl‑2 (also known as Bcl-2like protein 1) and its isoform Bcl‑X(L).55–57 Once the point of no return is reached, BAX and BAD result in permeabilization of the mitochondrial outer membrane, and the release of a potent cocktail of proapoptotic molecules, including cytochrome c, from the inner membrane space.58,59 The nature of this ‘death squad’ remains controversial, and a number of the suggested mediators, such as mitochondrial apoptosis-inducing factor 1 (AIFM‑1) and apoptotic protease-activating factor 1 (APAF‑1), could eventually prove to be innocent bystanders that are simply released into the cytosol as the mitochondrial content implodes.60 The central role of cytochrome c in apoptosis remains undisputed and, because of its potent nature, it remains carefully sequestered within the mitochondrial cristae. OPA1 ensures the tightness of these cristae junctions and, crucially, it controls the structural remodelling that occurs before cytochrome c is released during apoptosis.10,61 In addition to the anti apoptotic properties of OPA1-mediated mitochondrial

Mitochondrial quality control In addition to its link with monogenic diseases, mitochondrial dysfunction has been associated with the normal ageing process and with cancer.66 During evolution, eukaryotic cells have acquired a number of defensive mechanisms to identify and eliminate damaged dysfunctional mitochondria that would otherwise be detrimental to their survival.67,68 Multiple proteins and pathways are implicated in mitochondrial quality control, known as mitophagy, and the emerging associ ation between this process and human diseases provides a tantalizing link to possible future treatment strategies.69 Proteins that regulate mitochondrial dynamics are closely involved in the autophagic process, especially the initiation and formation of autophagosomes.70 Two important protein mediators in this process are PINK1 and the E3 ubiquitin-protein ligase parkin (Figure 3).71 PINK1 selectively accumulates on the outer membrane of depolarized mitochondria, whereas parkin is a cytosolic component that ubiquitinates proteins targeted for degradation.72–74 The mechanism by which PINK1 recruits parkin from the cytosol to the outer mitochondrial membrane is still being hotly debated, but an intriguing role for MFN2 has recently emerged.75 When a mitochondrial segment loses its membrane potential, PINK1 accumulates on the outer mitochondrial membrane, leading to the phosphorylation of MFN2. The phosphorylated form of MFN2 is thought to be a highly effective receptor for parkin, which then undergoes a conformational change to its active form, in addition to being drawn into close juxtaposition with PINK1 on the outer mitochondrial membrane.76 This recruitment pathway is completed when PINK1 phosphorylates parkin, thereby activating its ubiquitin ligase activity and initiating the ubiquitination of several downstream proteins, including DRP1, voltage-dependent anion-selective channel protein (VDAC1), Bcl‑2, parkin-interacting substrate (PARIS), NF‑κB essential modulator (NEMO), and mitochondrial Rho GTPase protein 1 (MIRO1).77–79 The proteosomal degradation of MIRO1 causes kinesin to detach from the mitochondrial surface, which represents a sophisticated way of putting damaged mitochondria into quarantine by severing their connection to the microtubule network before their eventual destruction.80,81 Parkin can also reduce cytochrome c release from mitochondria in response to proapoptotic stimuli; this stress-protective activity is mediated by OPA1 operating downstream of NEMO. The latter protein is essential for canonical NF‑κB signalling, and its ubiquitination by parkin results in activation of the NF‑κB pathway, followed by transcriptional upregulation of OPA1.79 Despite the considerable amount of in vitro evidence that links parkin to the regulation of mitochondrial dynamics and mitophagy, the situation in vivo might prove rather more complex. In one study, dopaminergic neurons were isolated from a ‘MitoPark’ mouse model that mirrors the key features of PD, namely, a slowly



REVIEWS Table 2 | Major mitochondrial pro-fusion and pro-fission mediators Protein

Location

Functions

OPA1

Inner mitochondrial membrane

Fusion of the inner mitochondrial membrane7,188 Maintenance of cristae junctions and regulation of cytochrome c release10,19,62 Assembly and stability of the mitochondrial respiratory chain complexes12 Mitochondrial DNA maintenance with a postulated role in anchoring nucleoids to the inner mitochondrial membrane136

MFN1/MFN2*

Outer mitochondrial membrane and endoplasmic reticulum

Fusion of the outer mitochondrial membrane7,8 Interactions between endoplasmic reticulum and mitochondria, and Ca2+ homeostasis (MFN2)51 Axonal transport of mitochondria via interaction with the MIRO1–Milton complex189,190 Regulation of mitochondrial respiratory chain coupling and oxidative phosphorylation30

DRP1

Cytoplasm

Permeabilization of the outer mitochondrial membrane mediated by the apoptosis regulator BAX191,192

FIS1

Outer mitochondrial membrane

Interacts with the endoplasmic reticulum to regulate apoptosis193

OPA3


Control of lipid metabolism and thermogenesis194–196

GDAP1


Regulation of oxidative phosphorylation and apoptosis197,198

Pro-fusion

Pro-fission

*MFN2 is located on both the outer mitochondrial membrane and the endoplasmic reticulum. Abbreviations: DRP1, dynamin-related protein 1; FIS1, mitochondrial fission 1 protein; GDAP1, ganglioside-induced differentiation associated protein 1; MFN, mitofusin; MIRO1, mitochondrial Rho GTPase protein 1; OPA, optic atrophy protein.

progressive degeneration of substantia nigra neurons, and impaired spontaneous locomotion in the adult animals.82 Although these dopaminergic neurons showed marked respiratory chain deficiency with mitochondrial network fragmentation, rather surprisingly, parkin was not recruited to dysfunctional mitochondria, and the loss of parkin did not inhibit mitophagy or modify the neurodegenerative process in the MitoPark mice.

Mitochondrial dynamics and disease Autosomal dominant optic atrophy DOA is the most common inherited optic nerve disorder seen in clinical practice, and a population-based epidemiological study in northern England estimated the minimum prevalence at 1 in 25,000.83,84 The pathological hallmark of this disorder is preferential loss of the retinal ganglion cell (RGC) layer within the inner retina, which leads to optic nerve degeneration and subsequent visual failure.85 DOA has an insidious onset, and it typically presents in early childhood with bilateral, symmetrical central visual loss and dyschromatopsia.86,87 Visual loss is invariably progressive, and almost all affected indi viduals will eventually fulfil the legal requirement for blind registration.88,89 The majority (50–65%) of families with DOA harbour pathogenic mutations within the OPA1 gene, which consists of 30 coding exons spanning over 100 kb of genomic DNA.83,90–92 OPA1 codes for a 960-amino-acid, dynamin-related GTPase that localizes to the inner mitochondrial membrane. The gene is highly expressed within the RGC layer, although the protein is ubiquitous, and abundant levels have also been identified in photoreceptors and other nonocular tissues such as the inner ear and the brain.93–96 Over 200 disease-causing

variants have been reported so far in this highly polymorphic gene, with mutational hot spots in the catalytic GTPase domain (exons 8–15) and the dynamin central domain (exons 16–23).83,92 The majority of OPA1 mutations result in premature termination codons, and the resultant truncated mRNA species are highly unstable, being rapidly degraded by protective surveillance mech anisms operating via nonsense-mediated mRNA decay. Haploinsufficiency, therefore, is a major disease mech anism in DOA, and the pathological consequences of a dramatic reduction in OPA1 protein levels is highlighted by those rare families who are heterozygous for microdeletions spanning the entire OPA1 coding region.97 Progressive visual failure remains the defining feature of DOA but, with greater availability of genetic testing, a specific OPA1 mutation in exon 14 (c.1334G>A, p.Arg445His) has been found to have a particular predilection for causing sensorineural deafness.11,98,99 The phenotypes associated with OPA1-linked disease have expanded even further to encompass a wide range of prominent neuromuscular features such as ataxia, myo pathy, peripheral neuropathy, and classical chronic progressive external ophthalmoplegia (CPEO).100–102 These so-called DOA+ variants are mechanistically relevant, as they highlight the deleterious consequences of OPA1 mutations not only for RGCs, but also for other CNS populations, peripheral nerves, and skeletal muscle. Although DOA+ was only recently recognized as a distinct clinical entity, up to 20% of OPA1 mutation carriers are now thought to be at risk of developing DOA+ features (Figure 4), which has major implications for patient counselling.103 Furthermore, OPA1 screening is increasingly performed as part of diagnostic panels for patients with unexplained neurodegenerative disorders, and



REVIEWS

Mitochondrion

BAX Bcl-2

VDAC BAX

OPA1 OPA1OPA1 OPA1 OPA1

2 YME1L

OMA1 AFG3L2

OPA1

Respiratory chain complexes

Cristae remodelling

3 NEMO PARIS

Mitophagy

OPA1

Apoptosis

Cytochrome c

PINK1

Depolarized membrane

mtDNA

SPG7

MFN2 P

1

Bcl-2

MIRO1

Parkin Ubiquitin

Axonal transport

Kinesin/dynein/dynectin Destabilize from microtubule Microtubule

Figure 3 | Major pathways implicated in neurodegenerative disorders. Nature ReviewsUnder | Neurology stress conditions, AFG3L2 triggers OPA1 cleavage via an OMA1-dependent pathway. The resulting OPA1 isoform loses its fusion and antiapoptotic properties, resulting in a fragmented mitochondrial network, release of cytochrome c into the cytosol, and cell death (1). Cleavage of OPA1 by YME1L and SPG7 regulates several mitochondrial parameters, including activity of the respiratory chain complexes, mitochondrial cristae structure and mtDNA stability (2). Accumulation of PINK1 leads to phosphorylation of MFN2 (red circle) and recruitment of cytosolic parkin to the outer mitochondrial membrane. Activated parkin ubiquitinates a range of membrane proteins that inhibit axonal transport, leading to mitophagy (3). Abbreviations: AFG3L2, AFG3-like protein 2; MFN2, mitofusin 2; MIRO1, mitochondrial Rho GTPase protein 1; mtDNA, mitochondrial DNA; NEMO, NFκB essential modulator; PARIS, parkin-interacting substrate; PINK1, PTENinduced putative kinase 1; OMA1, metalloendopeptidase; OPA1, optic atrophy protein 1; SPG7, paraplegin; VDAC, voltage-dependent anion channel; YME1L, ATP-dependent zinc metalloprotease YME1L.

other hitherto unreported pathological manifestations are bound to emerge.104–107 Interestingly, two large Italian families with an extreme DOA+ phenotype have been described, with clinical features dominated by CPEO and neurodegenerative features—namely, parkinsonism and dementia—but with a distinct lack of marked optic atrophy among affected patients (V. Carelli, unpublished work). These two families were found to harbour closely located missense OPA1 mutations within the catalytic GTPase domain (p.Gly488Arg and p.Ala495Val), further reinforcing the close pathophysiological links between mitochondrial dynamics, mitophagy and neuronal loss.

Charcot–Marie–Tooth disease CMT encompasses an important group of inherited peripheral neuropathies with an estimated prevalence of approximately 1 in 2,500 in the general population.3,4 CMT is characterized by progressive degeneration of the peripheral nerves, and the clinical classification depends on whether the underlying pathological process is predominantly axonal or demyelinative. Affected patients

develop distal muscle weakness and sensory loss but, depending on the actual causative gene, there can be quite marked variations in age of onset and rate of clinical progression.3,4 MFN2 mutations were initially identified in families with CMT2A, an autosomal dominant form of CMT.108 The MFN2 gene consists of 19 exons and codes for a 757-amino-acid, dynamin-related GTPase protein, which is anchored within the outer mitochondrial membrane. The majority of patients with CMT2A develop a severe peripheral neuropathy that may be primarily motor or accompanied by prominent proprioceptive loss.109,110 However, there is a degree of phenotypic variability, and in some affected MFN2 mutation carriers, the peripheral neuropathy can be complicated by subacute visual failure and optic atrophy.111 This specific CMT subtype is also known as hereditary sensory motor neuropathy type 6 (HSMN6; OMIM #601152) and, although rare, it is mechanistically highly relevant because of the fascinating link it draws between disturbed MFN2 function, RGC axonal loss and optic nerve degeneration. Visual acuity is usually reduced to 20/200 or worse in patients with HSMN6, but despite the severity of the initial visual loss, some patients can experience significant visual recovery in later life.110 Recently, compound heterozygous MFN2 mutations have also been identified in patients with early-onset peripheral neuropathy and, overall, a more severe neurological prognosis. 112 The carrier parents were asymptomatic, which is consistent with an autosomal recessive mode of inheritance for some MFN2 mutations.

Pathological mechanisms of disease Insights from animal models Research into DOA and CMT2A has been severely limited by a lack of access to diseased human tissues. To circumvent these practical difficulties, several animal models have been used to investigate the developmental impact of OPA1 (Box 1) and MFN2 (Box 2) knockdown in tissues that are normally affected in human disease. An elegant Cre–loxP recombinase strategy was used to create a conditional Mfn2 knockout mouse model that allowed embryonic development to proceed to term by sparing placental tissues.113 These Mfn2loxP mutant mice exhibited striking histopathological changes, with marked Purkinje cell neurodegeneration in the cere bellum associated with increased levels of apoptosis throughout the cerebellar layers. A marked respiratory chain defect was detected in the surviving Purkinje cells, and the mitochondrial network was fragmented, with aberrant mitochondrial distribution and ultrastructure visible on electron microscopy. An even more fundamental observation in this conditional Mfn2 knockout mouse model was the direct correlation between a lack of mitochondrial fusion and loss of mitochondrial DNA (mtDNA) nucleoids within affected cells, resulting in severe mtDNA depletion.114 Therefore, the mitofusins are central to mtDNA stability and fidelity, as is OPA1 (see below). Although chronic inhibition of mitochondrial fusion clearly results in



REVIEWS a

b

c 9.9 kb

II:1

II:2 II:3 II:4 II:5 II:6 L

C

II:2

d III:1 III:2 III:3 III:4 III:5 III:6 III:7

IV:1 IV:2

TTAAGSCTTGC

IV:3

Nature Reviews | Neurology Figure 4 | DOA+ phenotype and mtDNA instability. a | A large British family with early-onset optic atrophy and progressive external ophthalmoplegia, and variable features including sensorineural deafness, ataxia and peripheral neuropathy. At their last follow-up, the affected children (IV:2 and IV:3) were A, p.Ser545Arg) segregates with disease status in this family. The arrow indicates the pathogenic variant on a sequence chromatogram. Abbreviations: C, control; DOA+, dominant optic atrophy plus; L, DNA ladder; mtDNA, mitochondrial DNA; OPA1, optic atrophy 1. © Hudson, G. et al. Mutation of OPA1 causes dominant optic atrophy with external ophthalmoplegia, ataxia, deafness and multiple mitochondrial DNA deletions: a novel disorder of mtDNA maintenance. Brain (2008), 131(2), 329–337, by permission of Oxford University Press.101

marked mtDNA depletion, recent experimental data suggest that the observed mitochondrial dysfunction is caused primarily by reduced stability of the respiratory chain supercomplexes.33 More-sophisticated transgenic models harbouring specific pathogenic OPA1 and MFN2 mutations have also been created by making use of available N‑ethyl‑Nnitrosourea (ENU) libraries or site-directed mutagenesis of embryonic stem cells. Mitochondrial genome instability Molecules of mtDNA are packaged within intricate replicative structures known as nucleoids.115–118 Several proteins with critical roles in mtDNA replication are also associated with mammalian nucleoids; these include mitochondrial polymerase‑γ (POLG, also called DNA polymerase subunit gamma-1), the Twinkle protein (which unwinds double-stranded mtDNA at the replication fork), the mitochondrial single-stranded binding protein, and the accessory topoisomerase and ligase proteins.119 Nuclear disorders of mtDNA maintenance represent an important group of human diseases.120,121 The common molecular hallmark is mitochondrial genome instability, which manifests itself either as a reduction in mtDNA copy number (depletion), or accumulation of high levels of somatic mtDNA mutations in affected tissues.122–124 These mutations are predominantly multiple mtDNA deletions, but mtDNA point mutations have also been reported in association with some specific nuclear genetic defects.125–127 There is mounting evidence that the accumulation of these secondary quantitative and qualitative mtDNA abnormalities disrupt oxidative

phosphorylation, triggering a biochemical defect at the cellular level, and eventually contributing to the onset of overt clinical disease.128–130 From a diagnostic perspective, the mitochondrial biochemical defect can be readily observed in skeletal muscle biopsies as cytochrome c oxidase (COX)-negative fibres (Figure 4).131 Crucially, both DOA and CMT2A have been linked with impaired mtDNA maintenance. 98,99,101,132 COXnegative fibres harbouring high levels of mtDNA deletions have been identified in skeletal muscle biopsies from patients harbouring pathogenic OPA1 or MFN2 mutations. This finding raises the distinct possibility that the accumulation of these somatic mtDNA defects is contributing, at least partly, to multisystemic cellular dysfunction.132–135 In support of this hypothesis, the level of COX-negative muscle fibres was found to be over four times higher in patients with DOA+ phenotypes than in those who only developed pure optic atrophy.101,128 Different mechanisms have been postulated to account for the mtDNA deletions, including an imbalance of the intramitochondrial nucleotide pool, and impaired ability of the N‑terminal domain of OPA1, encoded by exon 4b, to physically anchor nucleoids to the inner mitochondrial membrane. 133,134,136 Circumstantial evid ence obtained with ultra-deep next-generation sequencing suggests that OPA1 mutations lead to an increased rate of clonal expansion of somatic mtDNA mutations, triggering the emergence of COX-negative muscle fibres once a critical mutational threshold has been exceeded.137 Marked mitochondrial proliferation is frequently observed in tissues from patients with DOA or CMT2A, presumably as a cellular compensatory mechanism in the face of impaired oxidative phosphorylation.138–140 Depletion of mtDNA has, so far, not been reported in the context of OPA1 mutations. However, a patient was recently described with optic atrophy and an earlyonset progressive multisystemic disorder secondary to a de novo missense mutation involving the MFN2 GTPase domain (c.628G>T, p.Asp210Tyr).141 A skeletal muscle biopsy revealed COX-negative fibres, lipid accumulation, dysmorphic mitochondria, and severe mtDNA depletion at 28% residual level. This rather unexpected finding is in sharp contrast with another MFN2 missense mutation altering the same amino acid (c.629A>T, p.Asp210Val), which had previously been identified in a large Tunisian family with a complicated neuro-ophthalmological phenotype.132 COX-negative fibres were present in skeletal muscle biopsies from several affected individuals in this family, and multiple mtDNA deletions were detected on long-range PCR analysis of DNA extracted from skeletal muscle homogenates. However, the p.Asp210Val amino acid substitution, in contrast to p.Asp210Tyr, did not lead to mtDNA depletion. The divergent molecular consequences of these two MFN2 mutations are puzzling and remain unexplained.142 Disrupted mitochondrial axonal transport Mitochondrial transport and localization are central to the survival of highly specialized cells, especially for long



REVIEWS Box 1 | OPA1 disease models Drosophila melanogaster Homozygous Opa1 mutations are embryonically lethal. Eye-specific homozygous Opa1 mutants generated by somatic mutagenesis develop abnormal ommatidial phenotypes (rough and glossy eyes) secondary to increased apoptosis, increased levels of reactive oxygen species and mitochondrial fragmentation.205 Heterozygous Opa1 mutants do not show any gross ommatidial morphology, but there is evidence of disturbed visual function on electroretinography. The mutant heterozygotes also have a decreased heart rate, increased heart arrhythmia, and poor stress tolerance.206,207

Zebrafish Knockdown of the opa1 gene was achieved with translation-blocking antisense morpholinos. Eyes of opa1 morphants are small, but with no observable retinal ganglion cell loss or optic nerve degeneration. Embryos also exhibit abnormal blood circulation and heart defects, developmental delay, small pectoral fin buds, and impaired locomotor activity. Depletion of Opa1 protein levels causes bioenergetic defects without impairing mitochondrial efficiency.208 Mouse Homozygous mutant mice (Opa1–/–) die in utero during early embryogenesis, confirming a critical role for Opa1 in early development. Heterozygous mutant mice (Opa1+/–) develop a slowly progressive bilateral optic neuropathy and reduced visual function. Optic nerve degeneration has been documented as early as 6 months, but is much more striking by 2 years of age.209–213 Loss of dendritic arborization precedes the onset of retinal ganglion cell loss, and the surviving axons have abnormal morphologies, with segmental areas of demyelination and myelin aggregation. The mitochondrial network is fragmented in the tissues that have been examined. Some extraocular features are also noted in older mice, including decreased locomotor activity, abnormal body fat distribution, and cardiomyopathy. No cytochrome c oxidase-negative or ragged red muscle fibres, or multiple mitochondrial DNA deletions, have been detected.214–216 Abbreviation: Opa1, optic atrophy 1.

Box 2 | MFN2 disease models Drosophila melanogaster Mfn2 has been knocked down in the eye, heart tubes and the posterior compartment of the developing wing in different Drosophila models. Depletion of Mfn2 results in mitochondrial network fragmentation, disturbed calcium homeostasis, and induction of apoptosis. A number of developmental abnormalities have also been observed, including small dysmorphic eyes, dilated cardiomyopathy, and a striking loss of adult wing tissue.217–220 Zebrafish Loss of mfn2 results in severe alterations affecting both motor neurons and muscles fibres. Mutant zebrafish show progressive loss of swimming ability. At the cellular level, mitochondrial axonal transport is disrupted, suggesting that the latter is a key disease mechanism in Charcot–Marie–Tooth disease type 2A.221,222 Mouse Several knockout mouse models have been developed, some of which are tissue-specific (dopaminergic neurons, liver and skeletal muscle). Generalized depletion of Mfn1 and Mfn2 results in embryonic lethality secondary to placental insufficiency, further reinforcing the idea that these pro-fusion proteins are indespensable in early development. The mitochondrial network is severely fragmented in cultured embryonic fibroblasts from these knockout mice, and a dramatic loss of membrane potential has been observed in a subpopulation of these fragmented mitochondria.113,114,223–225 Transgenic mouse models of Charcot–Marie–Tooth disease type 2A harbouring pathogenic Mfn2 mutations have also been created that replicate the human disease phenotype. The following abnormalities have been reported in mutant Mfn2 mice: a combined respiratory chain defect involving mitochondrial complexes II and V; mitochondrial proliferation and impaired mitochondrial distribution along axons; and overrepresentation of smaller axons within myelinated peripheral nerves.226–228 Abbreviation: Mfn2, mitofusin 2.

neurons with axons that span considerable anatomical distances.143,144 This fundamental requirement is dramatically exemplified by a number of neurodegenerative disorders, which—though distinct—share a number of common molecular and phenotypic themes. A remarkable disease paradigm emerged with the identification of spastic paraplegia 7 (SPG7) and AFG3L2 mutations in patients with clinical manifestations ranging from isolated optic atrophy and spastic paraplegia, to morecomplex neurological phenotypes that were strikingly reminiscent of the ‘plus’ features reported with OPA1 and MFN2 mutations.21,145 Hereditary spastic paraplegia (HSP) is a slowly progressive neurological disorder marked by the development of lower limb spasticity and weakness.143 The classification into pure and complicated forms is based on the presence of additional clinical features besides spastic paraplegia, such as optic atrophy, ataxia, peripheral neuropathy, extrapyramidal deficits and cognitive decline.146 Genetically, HSP is highly heterogeneous with over 40 mapped loci reported to date.143,144 In a subgroup of patients with a type of autosomal recessive HSP, pathogenic mutations were identified in the SPG7 gene, which codes for paraplegin.147 Analogous to the human form of the disease, now called HSP7 (OMIM #607259), paraplegin-deficient mice developed a distal axonopathy of spinal and peripheral axons secondary to impaired anterograde transport of organelles and neurofilaments.148 Interestingly, axonal swelling and neuronal degeneration were preceded in the earliest stages by mitochondrial morphological abnormalities within synaptic terminals and in the more distal nerve segments. The optic nerves of Spg7–/– mice were also abnormal, with swelling of RGC axons, but marked degeneration was not identified, indicating a later and milder degree of i nvolvement than in the spinal cord.148 Paraplegin is a major component of the mitochondrial AAA+ protease family, which is involved in the posttranslational processing of OPA1. Although the mol ecular link remains to be formally established, it is unlikely to be merely coincidental that SPG7 mutations can also result in an expanded neuro-ophthalmological phenotype that includes optic atrophy and CPEO, in addition to classic features of spastic paraplegia.149–151 Even more interestingly, SPG7 mutations are thought to cause CPEO through disordered mtDNA maintenance, similar to OPA1 and MFN2 mutations.152,153 Some SPG7 mutations can also behave dominantly, and in one large multigenerational family with DOA, a novel hetero zygous SPG7 mutation (c.1232A>C, p.Asp411Ala) was shown to segregate convincingly with pure optic atrophy and visual failure.149 AFG3L2 can homo-oligomerize or form functional hetero-oligomeric complexes with paraplegin. 21,67 AFG3L2 mutations cause autosomal dominant spino cerebellar ataxia type 28 (SCA28; OMIM #604581), a juvenile-onset syndrome marked by slowly progressive gait ataxia, dysarthria, limb hyperreflexia, nystagmus and ophthalmoplegia.154 Homozygous AFG3L2 missense mutations were also found, by whole-exome sequencing,



REVIEWS in a consanguineous family with a novel severe spastic ataxia–neuropathy syndrome marked by lower limb spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy.155 The loss of AFG3L2 led to mitochondrial respiratory chain dysfunction, decreased mitochondrial calcium uptake, and mitochondrial network fragmentation.156 A similar cascade of deleterious cellular events secondary to defective mitochondrial ribosomal protein synth esis was observed in a conditional Afg3l2 mouse model that allowed restricted deletion of the gene in cerebellar Purkinje cells.157 Depletion of AFG3L2 also impaired anterograde axonal transport of mitochondria in cortical neurons via a mechanism that is thought to involve reactive oxygen species signalling and hyperphosphorylation of the microtubule-associated protein tau.158 Disturbed axonal mitochondrial transport is not limited to disorders related to SPG7 and AFG3L2 mutations. In 2014, two elegant knockout mouse models highlighted the central importance of MIRO1 with regard to mammalian neuronal function and main tenance. MIRO1 anchors mitochondria to the motor proteins responsible for their anterograde and retrograde transport along neurons. In these two knockout mouse models, suppression of retrograde transport in MIRO1-deficient neurons led to mitochondrial depletion along corticospinal tract axons, and a progressive pattern of neurological deficits analogous to the human form of upper motor neuron disease.159 Mitochondrial dynamics and neurodegeneration The pathological spectrum associated with disturbed mitochondrial dynamics has expanded to encompass a more heterogeneous group of human disorders than initi ally envisaged. Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2), huntingtin (HTT) and sacsin molecular chaperone (SACS) genes result in distinct neurodegenerative phenotypes: respectively, autosomal dominant PD, 160 Huntington disease, 161 and auto somal recessive spastic ataxia of Charlevoix–Saguenay (ARSACS).162 All three protein products have been shown to potentiate the pro-fission activity of DRP1, both in vitro and in vivo, indicating a strong consistent link between disturbed mitochondrial dynamics and neurodegeneration.163–168 Impaired mitochondrial biogenesis, defective axonal transport of mitochondria, and increased DRP1mediated mitochondrial fission also seem to underlie the synaptic degeneration seen in neurons from patients with AD and from an established mouse model.169–171 One of the neuropathological hallmarks of AD is the accumulation of amyloid‑β (Aβ) plaques in the brain secondary to aberrant processing of amyloid precursor protein (APP) by the γ‑secretase complex. Intriguingly, presenilin‑1 (PS‑1) and presenilin‑2 (PS‑2), which are the key catalytic components of the γ‑secretase complex, are concentrated at MAM interfaces, and evidence is mounting that dysregulated crosstalk between the endoplasmic reticulum and mitochondrial compartments contributes to the development and progression

of the pathological process that underlies AD.38,172,173 Recent evidence also implicates disturbed mitochondrial dynamics in patients with frontotemporal dementia plus amyotrophic lateral sclerosis who harbour pathogenic mutations within the coiled-coil-helix-coiled-coil-helix domain containing 10 gene (CHCHD10), which encodes a mitochondrial intermembrane space protein enriched at cristae junctions.174,175

Management and treatment strategies Supportive measures Genetic counselling is central to the management of patients carrying pathogenic OPA1 or MFN2 mutations.176 Testing for these mutations raises sensitive ethical issues that are best addressed by specialist clinicians and trained genetic counsellors, who can provide patients and their families with the accurate information that would allow them to make an informed decision. Currently, diseasemodifying interventions for DOA and CMT2A are limited—a situation that places even higher importance on physical and occupational rehabilitation for affected patients.3,177 As OPA1 and MFN2 mutations can result in progressive multisystemic involvement, a multidisciplin ary team approach is essential to maximize the patient’s quality of life and minimize the long-term morbidity associated with chronic disease. Gene therapy A uniform treatment strategy for disorders related to OPA1 and MFN2 is not realistic given the genetic complexity involved, with some mutations resulting in haploinsufficiency and others thought to exert a dominant-negative effect. Furthermore, the group of tissues affected can be highly variable even between patients carrying the same pathogenic mutation.92,103 Gene replacement therapy is being explored for patients with DOA and visual failure, as the eye is an easily accessible organ that has the distinct advantage of providing robust outcome measures for judging treatment efficacy.178 The intraocular safety and the efficiency of RGC transfection after intravitreal injection of an adeno-associated virus vector (AAV2-pOPA1) are currently being investi gated in heterozygous mice harbouring a pathogenic OPA1 deletion (c.2708–2711delTTAG), which is commonly seen in patients with DOA (G. Lenaers, personal communication). Classic gene therapy paradigms will prove much more challenging for pathogenic mutations that result in gain of function, and for the correction of the underlying defects in tissues that are anatomically poorly defined or difficult to access, for example, the CNS. Modulating mitochondrial dynamics As disturbed mitochondrial dynamics are a prominent pathological feature in a number of late-onset neuro degenerative disorders, it is biologically plausible that restoration of physiological equilibrium between mitochondrial fusion and fission could help salvage the remaining neuronal populations, thereby halting further clinical deterioration.179 Tractable means of rectifying



REVIEWS this imbalance are currently rudimentary, and future approaches will require a high level of sophistication given the myriad of accessory factors that regulate mitochondrial shape, and the complex reciprocal interactions with other cellular processes such as mitochondrial transport and mitophagy.180–182

Neuroprotective drugs Mitochondrial antioxidants are a promising approach for the management of patients with Leber hereditary optic neuropathy (LHON), a classic primary mtDNA disorder, which, like DOA, is characterized by the preferential destruction of the RGC layer, leading to cata strophic visual loss in at-risk mutation carriers.183 Two compounds have recently been investigated for LHON. The first is idebenone, a short-chain benzoquinone structurally related to coenzyme Q10, which promotes mitochondrial ATP synthesis in addition to having antioxidant properties.184–186 The second is EPI‑743, another quinone analogue, which is reported to be more potent than idebenone, at least in vitro.187 These two drugs are obvious candidates for patients with progressive visual failure secondary to optic nerve degeneration and, if properly conducted, future clinical trials could provide preliminary data on the possible benefits of idebenone and EPI‑743 for the neurological features associated with OPA1 and MFN2 mutations. Clearly, these are only the first tentative steps towards modulation of disease progression for this group of disorders intrinsically linked to mitochondrial dysfunction. Several groups are actively pursuing the identification of novel neuroprotective agents by using complementary approaches that combine the power of high-throughput genomics with in vitro screening of small molecule libraries, and with further in vivo validation using existing animal models. This ambitious strategy will also be directly relevant to the broader field of mitochondrial dynamics, with implications for late-onset neurodegenerative disorders such as PD and AD.54,182 1.

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Conclusions

The genetic basis of several common neurodegenerative disorders has been clarified in recent years, and the pace of discovery will accelerate exponentially with the advent of next-generation whole-genome sequen cing and more-powerful bioinformatics technology. A remarkable element of the past decade of research has been the increasing realization that disturbed mitochondrial dynamics have a fundamental role in the pathophysiology of disease groups as diverse as DOA, CMT, HSP and the inherited spinocerebellar ataxias, reflecting to a large extent the broader phenotypic spectrum observed in classic mitochondrial syndromes. Treatment options for these progressive neurodegenerative dis orders remain limited, but the future looks promising given the availability of established disease models to test both the in vitro and the in vivo efficacy of potential new agents. Prospective deep-phenotyping studies that make use of existing national patient networks will also be essential to better define the natural history of these diseases, which in turn will reveal the clinical biomarkers that are most likely to predict disease progression, and the outcome measures that will prove most sensitive in detecting a treatment effect. Review criteria The articles included in this Review were primarily selected from the extensive personal bibliographies of the authors, and following discussions with experts in the field at national and international meetings. Additional studies were also identified on PubMed, MEDLINE and Web of Science using combinations of the following keywords: “AFG3L2”, “dominant optic atrophy”, “Charcot– Marie–Tooth disease”, “Drosophila model”, “gene therapy”, “mitochondrial disease”, “mitochondrial DNA instability”, “mitochondrial dynamics”, “mitochondriaassociated membranes”, “mitophagy”, “mouse model”, “neurodegenerative disease”, “neuroprotection”, “MFN2”, “OPA1”, “SPG7” and “zebrafish model”.

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