emerging therapeutic approaches to mitochondrial diseases

EMERGING THERAPEUTIC APPROACHES TOMITOCHONDRIAL DISEASES

Tina Wenz,1 Sion L. Williams,1 Sandra R. Bacman,1 and Carlos T. Moraes1,2*1Department of Neurology, University of Miami School of Medicine, Miami, Florida

2Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida

Mitochondrial diseases are very heterogeneous and can affect dif-ferent tissues and organs. Moreover, they can be caused by geneticdefects in either nuclear or mitochondrial DNA as well as by environmen-tal factors. All of these factors have made the development of therapiesdifficult. In this review article, we will discuss emerging approaches tothe therapy of mitochondrial disorders, some of which are targeted tospecific conditions whereas others may be applicable to a more diversegroup of patients. ' 2010 Wiley-Liss, Inc.Dev Disabil Res Rev 2010;16:219–229.

Key Words: mitochondrial diseases; gene therapy; heteroplasmy; oxida-

tive phosphorylation defects

Mitochondrial disorders are no longer considered tobe rare diseases. Recent estimates predict that 1 in5,000 children will develop a mitochondrial disease

[Schaefer et al., 2004]. Moreover, a recent study found that 1in 200 adults carry a common mtDNA mutation and thus canpotentially transmit a mitochondrial disease [Cree et al.,2009]. In the last decade, diagnosing and deciphering themechanism of the broad spectrum of mitochondrial disordershas proceeded at a fast pace. However, this heterogeneousgroup of disorders is still faced with limited therapeuticoptions. It is therefore exciting that in recent years several dif-ferent strategies to overcome the initial defect and alleviate itsup- and downstream effects have been developed and tested incell and animal models of different mitochondrial disorders.In this review, we will discuss these emerging therapeuticadvances.

ENTRY POINTS FOR THERAPEUTIC STRATEGIESDefects in the oxidative phosphorylation system

(OXPHOS) can be caused by mutations in the nuclear (n) andthe mitochondrial (mt) DNA. Mutations in mtDNA aremostly heteroplasmic, meaning that there is a mixture of wild-type and mutated mtDNA molecules. Depending on the per-centage of mutated DNA, human subjects are either asymp-tomic or develop a mitochondrial disease when the number ofmutated mtDNA molecules exceeds a certain threshold(threshold effect). Alterations in both genomes ultimatelyresult in stalled electron transfer within the respiratory chainand disrupted ATP synthesis. These effects also influence othercellular metabolic process, which are linked to OXPHOS such

as citric acid cycle and glycolysis. Hence, novel therapeuticstrategies target genomic, protein or cellular metabolic levelsto overcome the mitochondrial defect:

1. Preventing transmission of mtDNA defects.2. Shifting the heteroplasmy level.3. Replacement of the defective mitochondrial genes,tRNAs and proteins.

4. Scavenging of toxic intermediates.5. Optimizing ATP synthetic capacity.6. Bypassing defective OXPHOS components.

Different strategies focusing on these issues will be dis-cussed in the following text.

PREVENTING TRANSMISSION OF mtDNADEFECTS—GERMLINE THERAPY

Mutations in mtDNA are transmitted through thematernal lineage by women who, despite being asymptomatic,are carriers of mtDNA mutations. Asymmetrical cell divisionresults in oocytes with different mutational load (ratio muta-ted—wild-type mtDNA), which theoretically can result inchildren with different degrees of mitochondrial dysfunction.This unpredictability contrasts to the Mendelian pattern of in-heritance for nuclear gene mutation and makes genetic coun-seling difficult for women with a mtDNA mutation. Substitu-tion of the mutated mtDNA by wild-type mtDNA is the besttherapeutic option to prevent transmission of mitochondrialdisease caused by mtDNA mutation from mother to child.Two options are conceptually possible to perform this substi-tution: Cytoplasmic transfer and pronuclear transfer.

Present address of Tina Wenz: Institute for Genetics, University of Cologne,Zulpicher Str. 47, 50674 Cologne, Germany.Grant sponsor: PHS; Grant numbers: EY10804, NS041777, CA85700.; Grantsponsors: The Parkinson Disease Foundation, the Muscular Dystrophy Associa-tion, United Mitochondria Disease Foundation.*Correspondence to: Carlos Moraes, Department of Neurology, University of MiamiSchool of Medicine, 1095 NW 14th Terrace, Miami, FL 33136.E-mail: [email protected] 16 April 2010; Accepted 29 June 2010View this article online at wileyonlinelibrary.com.DOI: 10.1002/ddrr.109

DEVELOPMENTAL DISABILITIESRESEARCH REVIEWS 16: 219 – 229 (2010)

' 2010Wiley -Liss, Inc.

Cytoplasmic TransferIn the cytoplasmic transfer, nor-

mal mitochondria (in cytoplasts) wouldbe transferred into the oocyte, and thusdilute the effect of any mtDNA defect.Cytoplasmic transfer has been carriedout in the past used to improve the suc-cess rate of assisted Reproduction,especially with old donors where itcan ‘‘rejuvenate’’ the oocyte [Cohenet al., 1997]. The technique has beenalso used with mitochondrial diseasepatients. Nevertheless, some of the chil-dren born were still heteroplasmic withlow levels of mtDNA from the donoroocyte [Brown et al., 2006; Fulka et al.,2007]. Experiments in mice suggest thatthe amount of mtDNA that can betransferred by this techniques is less thana third of total mtDNA explaining therelatively little change observed in thechildren [Thorburn and Dahl, 2001].Hence, this technique will probablyhave little value in patients withmtDNA mutations.

Pronuclear TransferIn pronuclear transfer, a woman

with known mtDNA mutations couldhave the cytoplasm (and with it most ofthe mitochondria) of her fertilizedoocytes removed in vitro. The nakednucleus could then be transferred to anormal enucleated host oocyte from adonor with wild-type mtDNA andimplanted into the woman uterus[Brown et al., 2006; Fulka et al., 2007].Very recently, success of this techniquehas been demonstrated in monkeys:Healthy monkeys were developed fromembryos in which the egg contains thenuclear DNA from one donor and themitochondrial DNA from another do-nor [Kuehn, 2009; Tachibana et al.,2009]. In September 2005, the UKHuman Fertilization and EmbryologyAuthority approved a research licenseapplication to determine whether pro-nuclear transfer would be a feasibleoption for the prevention of transmis-sion of mtDNA disease in humanembryos. Although this technique mayone day prevent the inheritance of dis-ease-related mitochondrial gene muta-tions from mothers to their children,the method is ethically controversial:While the nuclear traits are from fatherand mother, which carries the mtDNAmutations, the usage of an oocyte withwild-type mtDNA basically introduces athird parent. Future studies will showwhether pronuclear transfer is a feasibleand acceptable procedure for humans.

REDUCTIONS IN MUTANTLOAD THROUGH‘‘HETEROPLASMY SHIFT’’

Novel therapeutic strategies aim toshift the ratio of mutated to wild-typemtDNA through so-called heteroplasmyshift. Since the pathological thresholdlevels for heteroplasmic mtDNA muta-tions tend to be relatively high, typicallyin the range of 80–90%, reducing themutant load of affected tissues only afew percent might be curative. Toachieve this goal, the mutated mtDNAcan be either selectively degraded or thewild-type mtDNA enriched.

Removal of Mutated mtDNA UsingMitochondria-Targeted RestrictionEndonucleases

Restriction endonucleases (RE)selectively cleave double stranded DNA atspecific restriction sites. When a mutation

Since the pathologicalthreshold levels for

heteroplasmic mtDNAmutations tend to berelatively high, typicallyin the range of 80–90%,reducing the mutant loadof affected tissues only afew percent might be

curative.

in mtDNA creates a novel restrictionsite, the expression of mitochondria-targeted RE that recognize the novelrestriction site can reduce the propor-tion of mutated mtDNA in heteroplas-mic cells and induce heteroplasmy shift.This occurs through digestion ofcleaved mtDNA molecules and concur-rent proliferation of residual mtDNA[Bayona-Bafaluy et al., 2005]. Mito-chondria-targeted RE were initiallyused in xenomitochondrial cybrid cells.The expression of mitochondrial PstI-RE (mito-PstI) was able to shift heter-oplasmy in cells containing both mouseand rat mtDNA, as the latter lacks PstIsites [Srivastava and Moraes 2001].Using a well-characterized asymptom-atic mouse model containing two hap-lotypes of murine mtDNA (BALB andNZB) [Jenuth et al., 1997], ex vivo andin vivo experiments showed that

expression of REs targeted to mito-chondria using recombinant viral vec-tors was effective in changing mtDNAheteroplasmy. These two mtDNA hap-lotypes can be differentiated by the REApaLI, which recognizes a single site inthe BALB mtDNA, but none in theNZB mtDNA. Focal injection ofrecombinant adenovirus, coding for amitochondrial targeted ApaLI RE(mito-ApaLI), in muscle and brain ofthese heteroplasmic mice showed arapid, directional and complete shift inmtDNA heteroplasmy in the targetedtissues as predicted, since only theBALB mtDNA variant contains aunique ApaLI site [Bayona-Bafaluyet al., 2005]. This approach was alsoshown to reduce the mutation load ofthe T8993G transversion in the mito-chondrial ATP6 gene in cybrid cellscarrying the mutation, which creates aunique SmaI-site in the humanmtDNA [Alexeyev et al., 2008; Tanakaet al., 2002]. T8993G is associated withneuropathy, ataxia, and retinitis pig-mentosa (NARP), or when present athigher levels (>90%), with a moresevere maternally inherited syndrome(MILS) [DiMauro and Schon,2003].Organ-specific shifts in mtDNAheteroplasmy following intravenoussystemic delivery of a mitochondria-targeted ApaLI-RE was shown in heartand liver using cardiotropic AAV6 andhepatotropic adenovirus as viral vectors[Bacman et al., 2010]. As the mito-ApaLI only cleaves BALB mtDNA, andthe remaining NZB mtDNA replicatesto maintain normal mtDNA copynumbers, no significant mtDNA deple-tion was observed [Bacman et al.,2010].

The degree and efficiency ofmtDNA heteroplasmy shift depends onthe presence of a residual population ofuncut mtDNA. In a ‘‘differential multi-ple cleavage site model,’’ we expressed amito-ScaI that recognizes multiple siteson both NZB/BALB mtDNA haplo-types. However, there are 5 ScaI sites inNZB but only 3 in BALB mtDNA).After transient expression of mito-ScaIwe observed only small changes in het-eroplasmy. As expected, a transientmtDNA depletion, caused by the sus-ceptibility of the entire mtDNA popu-lation in cells or mouse tissue, was moreprominent than when one mtDNApopulation was completely resistant tothe RE [Bacman et al., 2007]. Theremay be a use for mitochondria-targetedRE in a differential multiple cleavagesite model if expression can be tightlycontrolled.

220 Dev Disabil Res Rev � NOVEL THERAPIES IN MITOCHONDRIA DISORDERS � WENZ ET AL.

Removal of Mutated mtDNA UsingMitochondria-Targeted Zinc-FingerNucleases

A major limitation for the use ofmitochondria-targeted REs as a means toinduce heteroplasmy shift (see above) isthat fact that few clinically relevantmutations create suitable restrictionsites. Zinc-finger nucleases (ZFNs) arerecombinant endonucleases that canbe engineered to target specific DNAsequences [Klug 2010], offering a solu-tion to the limited applicability of mito-chondria-targeted RE. ZFNs haveproved to be robust tools for nuclear ge-nome modification [Carroll, 2008]through the production of double strandbreaks that induce modification via ho-mologous recombination [Urnov et al.,2005] or nonhomologous end joining[Perez et al., 2008].

ZFNs are composed of DNA-binding domains containing 3-9 tandemzinc-fingers and FokI nonspecific endo-nuclease domains. DNA binding speci-ficity is determined by the tandem zincfinger domains, with each zinc fingerbinding approximately three bases ofDNA [Klug, 2010]. The relationshipbetween the primary sequence of eachzinc finger and triplet binding specific-ity is relatively well understood. Thus,by combining different zinc fingers intandem it is possible to tailor DNAbinding to specific sequences. DNAcleavage by ZFNs requires dimerizationof the FokI domains, which determinesthe different structural forms of ZFNs.The most widely used form function asheterodimers [Carroll, 2008]. In thisform each monomer is composed of asingle N-terminal domain of tandemzinc-fingers and a single C-terminalFokI domain. Monomers are designedto bind target sequences in close prox-imity ‘‘tail-tail’’ on opposing strands, en-abling dimerization of FokI domainsand DNA cleavage. ZFNs have alsobeen designed as functional monomerswith single DNA binding domainsN-terminal to pairs of FokI domainsseparated by flexible linkers [Minczuket al., 2008] and as ‘‘sandwich’’ ZFNswhich contain an N-terminal DNAbinding domain, a pair of FokI domainsand a C-terminal DNA binding domaindesigned to bind the same strand as theC-terminal DNA-binding domain[Mori et al., 2009].

Initial proof of concept that ZFNsmight function in mitochondria was pro-vided by Minczuk et al. who successfullytargeted a monomeric zinc-finger meth-ylase to mitochondria and demonstratedsite specific methylation of mtDNA

[Minczuk et al., 2006]. This work wasfollowed by the delivery of a monomericZFN to cells carrying an 85% mutantload of the m.8993T>G NARP muta-tion [Minczuk et al., 2008]. Transientexpression of ZFNs that targeted 8993Gled to a stable doubling of the proportionof wild-type mtDNA with only a mildmtDNA depletion at 48hrs postexpres-sion. In contrast, expression of ZFNsthat targeted wild-type sequence in theD-loop produced no change in 8993Gmutant load and a severe mtDNA deple-tion at two days postexpression, confirm-ing that reduction in 8993G levels wasdue to clearance by the mutation specificZFNs in a similar manner to mitochon-dria-targeted RE.

Optimization of mitochondrial tar-geting remains an important area of mi-tochondrial ZFN research. Proteins con-taining tandem zinc-finger domains tendto localize to the nucleus even if theycarry mitochondrial import signals [Min-czuk et al., 2006], posing a hurdle fortheir use in mtDNA gene therapy. Theaddition of nuclear export signals to mi-tochondria-targeted ZFNs can amelio-rate the problem, although there is evi-dence that certain ZFNs are moredifficult to direct to mitochondriathan others [Minczuk et al., 2006]. Thisimplies that more complex modificationmay be required for universal delivery ofZFNs to mitochondria. Detailed proto-cols for the production and testing of mi-tochondrial ZFNs have recently beenpublished [Minczuk et al., 2010] whichwill likely stimulate research in the field.

Antigenome TherapyPeptide nucleic acid (PNA)

oligomers are mitochondrially targetednucleic acid analogues that still retain theability to form Watson-Crick base pairsand show binding selectivity at physio-logical conditions [Chinnery et al.,2000]. As such, they can theoreticallyblock mtDNA transcription/translation.In in vitro experiments, an 11-mer PNAcaused selective termination of aMERRF template close to the 8344position [Taylor et al., 1997]. However,PNAs could not get across the innermitochondrial membrane to targetmtDNA. The general concept of PNAswas further developed and optimized asmolecules named ‘‘cell membranecrossing oligomers’’ (CMCO). Withgreater polarity than PNAs, CMCOswere designed to improve penetrationthrough membranes and mitochondriallocalization [Kyriakouli et al., 2008].The potential of these optimized drugshas yet to be determined.

Enrichment of Wild-Type mtDNATwo main strategies have been

examined to specifically inhibit themaintenance of the mutated mitochon-drial genomes and enrich for the wild-type mtDNA: Ketogenic diet and stim-ulation of satellite cells.

Ketogenic DietThe basis of the use of the keto-

genic diet in gene shifting relies on find-ings in cell lines from patients with heter-plasmic mtDNA mutations [Santra et al.,2004]. Exposure of these cell lines har-boring single large-scale mtDNA dele-tions to ketone bodies resulted in a shifttowards higher wild-type mtDNA ratios[Santra et al., 2004]. Ketogenic diet is al-ready used to control epilepsy in mito-chondrial disease patients [Kang et al.,2007]. It might have some additionalbeneficial effects such as being neuropro-tective [Noh et al., 2008] and decreasingROS production [Maalouf et al., 2007].Ketogenic diet treatment of mice with adefective twinkle helicase, which accu-mulate deleted mtDNA molecules, ame-liorated the OXPHOS deficiency andimproved the phenotype of the mito-chondrial myopathy associated with theloss of twinkle function. However, theketogenic diet did not affect mtDNAquantity and quality [Ahola-Erkkilaet al., 2010]. These results suggest thatthe ketogenic diet might be useful for thetreatment of mitochondrial myopathies,but the mechanisms of this therapeuticeffect still remains to be elucidated.

Stimulation of Satellite CellsSatellite cells are undifferentiated,

myogenic cells that reside within theskeletal muscle [Morgan and Partridge2003]. They can be activated by micro-traumas, which can be induced mechan-ically (e.g., by resistance exercise) orchemically, and are able to replace orrepair damaged muscle fibers [Hawkeand Garry, 2001]. Important in the con-text of mitochondrial disorders is thefact that satellite cells appear to have alower mutational load than maturedmuscle fibers [Clark et al., 1997]. Afteractivation, the satellite cells mature andare incorporated within the muscle,where due to their lower mutationalload shift the heteroplasmy in favor ofthe wild-type genome. Resistance exer-cise leads to segmental muscle necrosisand has been proven to induce sufficienttraumatic injury to stimulate prolifera-tion of satellite cells promoting hetero-plasmic shift [Murphy et al., 2008].Muscle necrosis with following regener-ation can be also induced by injections

Dev Disabil Res Rev � NOVEL THERAPIES IN MITOCHONDRIA DISORDERS � WENZ ET AL. 221

of substances such as bupivacaine intomuscle. This strategy has been employedin one patient carrying a nonsensemutation in the COX I gene. Recurr-ent chemically induced muscle micro-trauma led to satellite cell proliferationand resulted in improved biochemicalOXPHOS activities and an reductionfibers with mutant mtDNA [Andrewset al., 1999] .

REPLACEMENT OF THEDEFECTIVE MITOCHONDRIALGENES, tRNAs, AND PROTEINS

Protein transduction/ProtofectionMitochondrial proteins encoded

by the mtDNA are very hydrophobic,hence it can be challenging to devise asuccessful import strategy. A novel strat-egy to deliver proteins regardless of theirphysical characteristics is protein trans-duction. The discovery of protein trans-duction domains with cell-penetratingproperties offers the possibility to over-come many of these problems [Wagstaffand Jans, 2006]. One of the most com-monly used protein transductiondomains is TAT, a short cationic peptidederived from the larger TAT protein ofHIV that is able to cross cell membranes[Becker-Hapak et al., 2001; Vocero-Akbani et al., 2001]. TAT-fusion proteinscoupled to either a nuclear localizationsignal (NLS) or a mitochondrial target-ing sequence (MTS) can transduce into acell and localize and remain in the sub-cellular compartment [Vyas and Payne,2008]. Successful mitochondrial target-ing was reported for a TAT-mediateddelivery of lipoamide dehydrogenase(LAD) using the intrinisic MTS restor-ing mitochondrial pyruvate dehydrogen-ase complex activity in cell lines frompatients with LAD deficiency [Rapoportet al., 2008]. In mice, TAT-mediateddelivery of purine nucleoside phospho-rylase corrected its deficiency withoutincreasing immune response [Toro andGrunebaum, 2006]. A TAT fusion wasalso used to generate a mitochondriallocalized GFP (MTS from malate dehy-drogenase) [Del Gaizo et al., 2003; DelGaizo and Payne, 2003]. The advantageof protein transduction is that the trans-port across the membrane is apparentlyindependent of TIM and TOM com-plexes [Yamada et al., 2007]. However, ithas the disadvantage of being a shortlived correction.

In recent years, the group of JamesBennett used the general principle ofprotein transduction to develop a mito-chondrial protein transfection strategy,dubbed protofection. They used a fusion

of a protein transduction domain and amitochondrial localization signal to con-struct a mitochondrial transduction do-main (MTD). Fusion of MTD to themitochondrial transcription factor A didnot interfere with the DNA bindingabilities of TFAM. MTD-TFAM wastransported across membranes and local-ized to mitochondria [Keeney et al.,2009]. In cybrid cells from Parkinsondisease and cells from LHON patients, amixture of MTD-TFAM with labeledmtDNA lead to rapid localization of thelabeled mtDNA to mitochondria sug-gesting that MTD-TFAM shuttledbound mtDNA into mitochondria.This protofection resulted in increasedmtDNA content, cell respiration andmitochondrial movement velocity in

Mitochondrial proteinsencoded by the mtDNAare very hydrophobic,hence it can be

challenging to devise asuccessful import strategy.

both cell types [Keeney et al., 2009].Improvements of bioenergetic charac-teristics were also evident when theMTD-TFAM/mtDNA complex wasadministered in wild-type mice [Iyeret al., 2009]. Long-term fate and intra-molecular localization of the MTD-TFAM/mtDNA complex are an un-solved issue and need to be addressed toevaluate the potential of this technique.The future will show if protofection isbeneficial in mouse models of mito-chondrial disease and last in human sub-jects, including tissue specificity.

rAAV Mediated Gene TransferIn addition to protein transduc-

tion, replacement of the defective pro-tein can be in principle also achievedby virus-mediated expression of thewild-type form of the protein. Thisapproach was studied using a mousewith a knockout of the mitochondrialATP/ADP translocator (ANT1), whichdevelops among other symptoms a mi-tochondrial myopathy [Graham et al.,1997]. The rAAV-ANT1 vector wasused to transduce ANT1 activity intothe skeletal muscle of ANT1-deficientmice. The transgene ANT1 protein wastargeted to the mitochondrion, was

inserted into the mitochondrial innermembrane, formed a functional ADP/ATP carrier, increased the mitochon-drial export of ATP and reversed thehistopathological changes associatedwith the mitochondrial myopathy [Flierlet al., 2005]. However, ANT1 expres-sion could only be observed in closevicinity of the injection site. Injectionof rAAV-ANT1 virus into neonat-al ANT1-deficient muscle resulted intransgene expression in a much greaternumber of muscle fibers due to a largernumber of precursor cells [Flierl et al.,2005]. Future work will show if virus-mediated protein replacement is a usefulapproach.

tRNA ImportThe vast majority of mtDNA

mutations concerns mitochondrialtRNAs. While import of tRNA intomitochondria is well established in yeastcells, this process is still challenging inthe mammalian system [Tarassov et al.,2007]. Yeast tRNA-Lys could be suc-cessfully imported into mitochondrialcybrid and patient cell lines harboringthe MERRF mutation. The importedyeast tRNA-Lys partially restored mito-chondrial translation and partiallyrestored OXPHOS function [Kolesni-kova et al., 2004]. However, the experi-ments suffered from low efficiency, vari-ability, and toxicity of the transfectionvehicle. In another approach, the Leish-mania RNA import complex (RIC)was employed. This complex couldenter human cells, successfully inducedimport of tRNA-Lys and restored mito-chondrial translation and OXPHOS ac-tivity in cybrid MERRF cells. All othercytosolic tRNAs could also be importedinto mitochondria opening the possibil-ity for correction of any other RNAmutation [Mahata et al., 2006]. TheLeishmania RIC could also import anti-sense RNA into the mitochondrial ma-trix resulting in impairment of the pro-tein synthesis and degradation of thetargeted RNA species [Mukherjeeet al., 2008]. This finding suggests thatwith the use of Leishmania RICmutated mtDNA genes or tRNAscould be targeted and specificallysilenced. Future work in animals is nec-essary to assess if the effects seen in cellstranslates to the use in vivo.

Increasing MitochondrialtRNA Stability

Mutations in tRNAs reduce thelevel and fraction of aminoacetylatedtRNAs and hence result in mitochon-drial translation defects and respiratory


chain defects [Enriquez et al., 1995;Ling et al., 2007; Park et al., 2003;Sasarman et al., 2008]. Recent work incell lines carrying tRNA mutationsindicates that the respiratory chaindefects can be suppressed by overexpression of tRNA synthases. Interest-ingly, the rates of oxygen consumptionin suppressed cells were directly propor-tional to the levels of leucyl-tRNA syn-thetase in A3243G mutant cells andcould even reach wild-type respiratoryfunction. The suppressed cells hadincreased steady-state levels of tRNA(Leu(UUR)) and up to threefold highersteady-state levels of mitochondrialtranslation products, but rates of proteinsynthesis did not differed from those inparental mutant cells [Li and Guan,2010; Park et al., 2008].

Allotopic Expression ofMitochondrial Genes

Protein replacement of mtDNA-encoded proteins follows the natural ev-olutionary process, where mtDNAgenes migrated to the nucleus. The nu-clear expression of mtDNA genes isreferred to as allotopic expression. Forproper expression, the mtDNA geneneeds to be recoded from the mito-chondrial code to the universal geneticcode. Also, a cleavable mitochondrialtargeting sequence needs to be attachedto the preprotein to ensure import intomitochondria. The problem with thisstrategy is that the proteins thatremained encoded by the mtDNA arehighly hydrophobic, hence expressionand targeting is challenging. Allotopicexpression was pioneered in yeast. Thefirst allotopic expressed proteins wasATP8 fused to a mitochondrial target-ing sequence, incorporated andexpressed in the nucleus. The newlysynthesized protein was successfullyexpressed, imported and assembled intocomplex V and rescued ATP8 KO yeastcells [Gray et al., 1996]. In the mamma-lian system, cybrid osteosarcoma cellsharboring mt-ATP6 mutation weretransformed with rAAV-ATP6 vectorand resulted in low, but detectableexpression and significant restoration ofATP production [Manfredi et al., 2002].In contrast, allotopically expressed apoc-ytochrome b and ND4 could not befully imported into mitochondria. Ei-ther full-length or truncated proteinsformed aggregates or clogged mito-chondrial import pores, leading to a lossof mitochondrial membrane potential intransfected cells. The latter studyshowed the difficulties of allotopicexpression of the highly hydrophobic

mtDNA encoded proteins [Oca-Cossioet al., 2003].

Recent studies suggest that inclu-sion of the 30UTR of a transcript codingfor a mitochondrial protein maximizeimport of the highly hydrophobic pro-teins by sorting of the mRNA to themitochondrial surface [Sylvestre et al.,2003]. Corral-Debrinski used thisapproach to express engineered ND1 orND4 in the nucleus of human skinfibroblasts with LHON ND1 or ND4mutations. OXPHOS function was

However, it has so farnot been clearlydemonstrated that

allotopically expressedgenes are actuallyassembled into

OXPHOS complexes norwhat is the molecular

mechanism for the rescue.

significantly restored indicated by ATPsynthesis and complex I activity [Bonnetet al., 2008]. The same group alsoshowed that optimized allotopic expres-sion of human mitochondrial ND4 pre-vented blindness in a LHON rat model[Ellouze et al., 2008]. The LHONmutation was introduced in vivo byelectroporation and caused retinal gan-glion cell degeneration. Electroporationwith the wild-type ND4 preventedthe neurodegeneration and preservedvisual function. Similarly, AAV2-medi-ated expression of human mitochondrialND4 lead to functional expression inmouse eyes [Guy et al., 2009]. However,it has so far not been clearly demon-strated that allotopically expressed genesare actually assembled into OXPHOScomplexes nor what is the molecularmechanism for the rescue. Furtherexperiments are necessary to assess theefficacy and therapeutic value of theoptimized allotopic expression.

SCAVENGING OF TOXICINTERMEDIATES

Defective OXPHOS and theresulting stalled electron flow lead toover-reduction of the quinone andNADH/NAD pool. This over-reduc-tion results in the accumulation of

intermediates at several steps in themetabolic pathways leading and con-nected to OXPHOS. Some of thoseintermediates are toxic and thus aremajor contributors to the symptoms ofmitochondrial disease. Removal of thesetoxic intermediates can help to preventa fatal course of the disease and is idealto support other therapeutic strategiesthat target the OXPHOS deficiencies.

Buffering LactateLactic acidosis is a typical finding

in mitochondrial diseases. Because ofthe stalled OXPHOS system, pyruvateis further metabolized to lactate, whichaccumulates in the blood. Several strat-egies have been employed to reduce se-rum lactate such as buffering with bi-carbonate or treating patients withdichloroacetate [Warner and Vaziri,1981]. However, the latter treatmentcan lead to a severe toxic neuropathy.

Allogenic Stem CellTransplantation and PlateletInfusion

Mitochondrial gastrointestinalencephalopathy (MNGIE) is a mito-chondrial disease caused by a primarythymidine phosphorylase (TP) defi-ciency. The reduced TP activity resultsin increased dThd and dUrd levels inthe blood stream [Walia et al., 2006].Recently, two strategies have beendescribed to scavenge the nucleosides inthe blood stream: allogenic stem celltransplantation and platelet infusion.

Allogenic stem cell (bone mar-row) transplantation (alloSCT) partiallyrestored TP activity in the recipient,and lowered plasma dThd and dUrdlevels [Hirano et al., 2006]. The patientstill continues to improve, indicatinglong-term restoration of TP activity[Dimauro and Rustin 2009]. Infusion ofplatelets from healthy donors to patientswith MNGIE also restored transientlycirculating TP and reduced plasmadThd and dUrd levels [Lara et al.,2006]. While both strategies provideproof of principle that toxic nucleosidescan be removed in vivo by infusing bio-logically active agents from healthyhuman donors, their clinical efficiencyand therapeutic value remain to be eval-uated.

Quinone SpeciesOXPHOS defects may lead to an

increased production of reactive oxygenspecies and damage to proteins, lipidand mtDNA [Balaban et al., 2005; Fin-kel 2005]. Evidence of oxidative stresshas been proposed not only in mito-


chondrial disease, but also in many neu-rodegenerative disorders such as Frie-dreich’s ataxia, Huntington’s disease,ALS, and Parkinson’s disease [Mancusoet al., 2006; Rego and Oliveira 2003].In an attempt to scavenge ROS, anti-oxidants have been administered topatients [Murphy and Smith, 2000].One prominent class of antioxidantsthat have been aggressively developed inthe recent years are quinones.

CoQ10Quinones have a dual role in RC

disorders. First, they are an electron car-riers between complexes I/II and III.Second, quinones are powerful ROSscavengers. In its first role, administra-tion of quinone is able to fully restorequinone deficiency and is therefore theonly existing cure for mitochondrialdiseases due to CoQ10 deficiency [DiGiovanni et al., 2001]. Their secondrole as a ROS scavenger seems to bemore applicable for the vast majority ofmitochondrial diseases. Experimentalevidence suggests that CoQ10 supple-mentation in patients with different mi-tochondrial disorders increases ATPsynthesis [Barbiroli et al., 1997]. A largeclinical trial is needed but still missing.Detailed data on bioavailibity of exoge-neously administered CoQ10 are alsomissing. The symptomatic improvementin patients with CoQ10 deficiency,who received orally administeredCoQ10 suggest some bioavailibity [DiGiovanni et al., 2001]. At present, de-spite the lack of adequate data fromclinical trials in mitochondrial disease,most practitioners treat their patientswith CoQ10 supplementation [Haas,2007].

IdebenoneThe delivery of CoQ10 to cells

and mitochondria is fraught with diffi-culty, because of its lipophilic properties[Ernster and Dallner, 1995]. Idebenoneis a synthetic form of CoQ10 that hasan optimized penetrance through theblood brain barrier and an overallhigher bioavailability [Artuch et al.,2004; Nitta et al., 1994]. As CoQ10,idebenone is an antioxidant and anelectron carrier. In the latter function,idebenone can be a substrate forcomplexes I, II, and III and for gly-ceraldehyde-3-phosphate dehydrogenase[Esposti et al., 1996; James et al., 2005].Idebenone has been used in clinical tri-als for Friedrich’s ataxia, where it showsa dose-dependent beneficial effect while

being well tolerated [Meier and Buyse,2009]. In pediatric cases, idebenone alsoimproves neurological function whengiven in high doses [Schulz et al.,2009].

MitoQA novel player in the quinone

field is mitochondrial-localized quinone,called Mito-Q. This quinone species isspecifically targeted to mitochondriaby conjugation of the quinone moietyto a lipophilic cation such as triphenyl-phosphonium (TPP) [Cocheme et al.,2007]. TPP was designed to shuttle sub-strates to the mitochondrial matrix andhas been employed with ubiqinone, spintraps, and lipoic acid among others

MitoQ prevented lipidperoxidation andprotected against

ischemia-reperfusioninduced decreases in

respiratory control ratio,damage to complex I anddecreases in aconitase

activity

[Abu-Gosh et al., 2009; Brown et al.,2007; James et al., 2003]. The lipophiliccations can pass through the phospho-lipid bilayer without the requirement ofa specific uptake mechanism and accu-mulate within mitochondria due to thehigh membrane potential [Ross 2005],where the quinone moiety protectsfrom oxidative stress and can function aselectron carrier. In contrast, CoQ10accumulates in mitochondria only to alimited extent [Ernster and Dallner,1995]. Since TPP cations pass easilythrough phospholipid bilayers, theycross from the gut to the bloodstreamand from there reach most tissues,including the brain through the bloodbrain barrier and hence have improvedbioavailibity [Smith et al., 2003].MitoQ has been extensively studied inisolated mitochondria, cells, and in vivo,both in animal models and in humans[Cocheme et al., 2007]. The high bioa-vailability was demonstrated in miceafter oral MitoQ administration in sev-eral tissues including heart, liver, kidney,brain and white adipocytes [Rodriguez-

Cuenca et al., 2010]. Importantly, thesestudies showed that administration ofMitoQ prevented lipid peroxidation andprotected against ischemia-reperfusioninduced decreases in respiratory controlratio, damage to complex I anddecreases in aconitase activity [Adlamet al., 2005]. In humans, the drug canbe successfully delivered after oraladministration. MitoQ is currentlybeing developed as a pharmaceuticalproduct and has been shown to beeffective in phase I trials when adminis-tered orally [Cocheme et al., 2007].

MitoQ is can accept electronsfrom complex II, but not from com-plexes I or III [James et al., 2005]. Thissuggests that the positive effects observedfor MitoQ are most likely due to itsantioxidant defects. The next years willshow whether MitoQ is beneficial formitochondrial disease patients as part ofa mitochondria-protective therapy.

OPTIMIZING ATP SYNTHESISCAPACITY

The energy crisis caused by de-fective OXPHOS is considered to bethe main pathological mechanism inmitochondrial disease. Optimizing thecellular ATP supply has been the focusof several studies, which augment ATPsynthesis at the level of substrate phos-phorylation or oxidative phosphory-lation.

Optimizing Substrate LevelPhosphorylation

Glycolysis is one major source ofanaerobic ATP production. However, inthe case of stalled OXPHOS, sustainedanaerobic glycolysis results in increasedlactate levels due to a surplus of pyru-vate. Another source of anerobic ATPproduction has been exploited recentlyto increase substrate level phosphoryla-tion: The succinyl coenzyme A synthasein the TCA cycle catalyzes substrate-level phosphorylation of ADP or GDP.In a novel approach, exogenous sub-strates capable of stimulating the Krebscycle flux were used to activate theenzymes [Haller and Vissing, 2009]. Atthe same time excess of reduced pyrim-idine nucleotides (nicotinamide adeninedinucleotide [NADH]) were removedby reversal of the normal flux of theKrebs cycle with oxaloacetate convertedto malate to avoid accumulation of suc-cinyl-CoA [Sgarbi et al., 2009]. Usingthis approach, cells with defective aero-bic ATP synthesis, either by oligomy-cin-inhibition or caused by mutation inATP6, maintained their viability andcellular ATP levels in glucose-free


media, when they were incubated witha-ketoglutarate (the precursor of succi-nyl coenzyme A) and aspartate (the pre-cursor, via transamination, of oxaloace-tate). Increasing cellular ATP supply byimproving substrate-level phosphoryla-tion without increased toxic byproductssuch as lactic acid might offer an alter-native therapeutic approach.

Optimizing OXPHOS CapacityThis strategy takes advantage of

the fact that mitochondrial disorders,which are compatible with life, stillretain a residual, even if minimalOXPHOS capacity. Several recent stud-ies took advantage of this fact andshowed that boosting of this residualOXPHOS activity maximizes overallATP synthesis and is thus beneficial forcell fate. To date, three different strat-egies to boost OXPHOS capacity havebeen devised:

1. Modulation of mitochondrialCa2þ levels.

2. Increased mitochondrial bio-genesis.

3. Allosteric activation.

Modulation of Mitochondrial CalciumBesides performing ATP synthesis, mi-tochondria are important buffers forcalcium. Recent work indicates that thecytosolic Ca2þ signal in response tohormonal stimulation is impaired insome cell lines from mitochondrial dis-ease patients [Brini et al., 1999]. It wasshown that an increase in mitochondrialCa2þ activates Ca2þ-sensitive dehydro-genases in the citric acid cycle andtherefore increases ATP synthesis [Vischet al., 2004]. Accordingly, in some celllines from mitochondrial disease patients(MERRF, G3664A) treatment withdrugs that affect organellar Ca2þ-trans-port stimulated ATP synthesis [Briniet al., 1999; Visch et al., 2004]. Theseresults underscore the potential of cal-cium modulation in mitochondrial dis-ease with disturbed Ca2þ homeostasis.

Stimulation of MitochondrialBiogenesis

We and others recently showedthat increasing the number of mitochon-dria in a cell could improve cellular ATPsynthesis in OXPHOS deficient cellsand tissues by elevating competentOXPHOS units per cell [Bastin et al.,2008; Srivastava et al., 2009; Wenz et al.,2008, 2009]. Mitochondrial biogenesis isgoverned by the key regulator PGC-1a,a transcriptional coactivator that stimu-lates a group of transcription factors

involved in the expression of mitochon-drial proteins [Scarpulla, 2008]. Trans-genic over-expression of PGC-1a inskeletal muscle restored ATP synthesis ina mouse model of mitochondrial myop-athy caused by COX deficiency andgreatly improved symptoms and courseof the disease [Wenz et al., 2008].Importantly, we could show that thisimprovement by transgenic PGC-1aexpression can be mimicked by exter-nally activating PGC-1a and hencemitochondrial biogenesis. A very well-studied activator is endurance exercise.Here, PGC-1a is activated through thecellular fuel gauge AMPK as well as theincrease in cytosolic Ca2þ levels throughthe muscle contractions [Benton et al.,2008; Ojuka 2004; Wu et al., 2002]. En-durance exercise improves the physio-logical and biochemical features inmuscles of patients with mitochondrialdisease [Taivassalo and Haller 2004,2005]. In our mouse model with mito-chondrial myopathy, a regular enduranceregimen partially ameliorated theOXPHOS defect in the affected skeletalmuscle and resulted in a slower progres-sion of the disease [Wenz et al., 2009].In addition to exercise, many pharma-ceuticals are available that regulate PGC-1a and hence mitochondrial biogenesis.PGC-1a activity is mainly controlled bythe PPARs, AMPK and Sirt1 [Wenz,2009]. Pharmacological activators forthese proteins include fibrates and rosi-glitazone (PPAR), metformin andAICAR (AMPK) as well as resveratrol(Sirt1) [Wenz, 2009]. Induction of mito-chondrial biogenesis has been demon-strated for most of these substances[Dong et al., 2007; Wu et al., 2006].Treatment of a mouse model with mito-chondrial myopathy with bezafibratestimulated increased ATP generatingcapacity by increasing mitochondrialbiogenesis and resulted in a less aggres-sive course of the disease [Wenz et al.,2008]. While these studies demonstratethe potential of induced mitochondrialbiogenesis in mitochondrial disease, itremains to be seen whether pharmaceu-tical activation of PGC-1a will be bene-ficial for mitochondrial disease patients.

Allosteric ActivationThe group of Giovanni Manfredi

showed that mitochondria contain a sig-naling pathway that controls OXPHOSactivity by phosphorylation of severalenzyme subunits [Acin-Perez et al.,2009b]. Protein kinase A (PKA) phos-phorylates the OXPHOS subunits andis activated by cyclic AMP (cAMP),which is generated within mitochondria

by the dioxide/bicarbonate-regulatedsoluble adenylyl cyclase (sAC) inresponse to metabolically generated car-bon dioxide. Elevated cAMP levelsresult in increased phosphorylation ofcomplexs I and IV subunits and higherenzyme activity [Acin-Perez et al.,2009b]. This mt-sAC/PKA pathway issensitive to metabolic conditions thataffect CO2 conditions. Bicarbonatefluctuates inside mitochondria in directproportion to the CO2 generated viathe TCA cycle and b-oxidation andhence provide a regulatory link betweenOXPHOS and other cellular metabolicprocesses.

It was also shown that stimulatingthis metabolic sensing signaling pathwayimproves respiration and ATP produc-tion in cells with defective OXPHOSfunction, virtually reducing the effect ofthe OXPHOS defect [Acin-Perez et al.,2009a]. This effect was achieved by ei-ther using pharmacological PKA agonistor by promoting higher levels of sAC inmitochondria. Interestingly, this effectwas greater in COX deficient cells thanin wild-type cells suggesting that defec-tive cells have a better capacity to up-regulate this pathway.

BYPASSING DEFECTIVEOXPHOS COMPONENTS

Defects in individual OXPHOSenzymes, as occurs in many mitochon-drial disorders, result in stalled electrontransfer with an over-reduction of thequinone and NADH/NAD pool. Inplants, such constraints can be bypassedby the use of alternative electron trans-fer proteins, which are not present inhumans. Hence, the alternative enzymesmight be useful to bypass defectiveOXPHOS enzyme, prevent stalled elec-tron transfer and an over-reduced state.Thereby, the accumulation of toxicmetabolites (see above) can be pre-vented, electron transfer is maintainedand improved ATP synthesis mightoccur if a proton gradient is generatedby the remaining functional OXPHOScomplexes. To date, two alternativeenzymes with potential use in mammalshave been described: The rotenone-insensitive NADH dehydrogenase(NDi1) and the cyanide-insensitive al-ternative oxidase (AOX). While bothenzymes can engage in electron transfer,neither of them contributes to mem-brane potential since both lack protonpumping activity. Hence, their contri-bution is probably based on preventingover-reduction by maintaining electronflow and thereby preventing ROS pro-duction and stalling of cell metabolism.


NDi1NDi1 is a rotenone-insensitive

NADH -Q-oxidoreductase from bud-ding yeast, an organism which lacks amembrane-embedded complex I. NDi1can be functionally expressed and local-ized to mitochondria in mammaliancells. The yeast protein renders the cellsrotenone-resistant and thereby maintainsATP-production [Seo et al., 2002] andsuppresses ROS produced by complex Iinhibition [Seo et al., 2006]. NDi1 alsocould partially overcome the bioener-getic defect in human cell lines harbor-ing the G11778A ND4 LHON muta-tion [Park et al., 2007]. Recent worksuggests, that NDi1 can also rescuecomplex I deficiency in vivo [Marellaet al., 2008]. When administered as anrAAV construct into substantial nigra,Ndi1 showed a preserved nigrostriatalpathway and intact dopaminergic cellsafter acute or chronic chemical inhibi-tion of complex I in mice [Marellaet al., 2008]. Importantly, these rAAV-Ndi1 injections provided long-termprotection from chemical insults [Bar-ber-Singh et al., 2009].

Expression of the alternativeenzyme also improved respiration, fit-ness, reproduction, and restored mito-chondrial membrane potential to wildtype levels in a C. elegans model of mi-tochondrial disease due to impairedcomplex I activity. Ndi1p functionallyintegrated into the nematode respiratorychain and mitigated the deleteriouseffects of a complex I deficit [DeCorbyet al., 2007]. Experiments with NDi1in Drosophila are also underway [Rustinand Jacobs, 2009].

AOXAnother alternative electron-trans-

ferring enzyme is the cyanide-insensi-

tive alternative oxidase. AOX transferselectrons from ubiquinol and passesthem directly to molecular oxygen[Siedow and Umbach, 2000]. Thereby,AOX bypasses complexs III and IV andpotentially prevents over-reducing thequinone-pool in case of a complex IIIor complex IV deficiency [Rustin andJacobs 2009]. AOX from C. intestinaliscan be functionally expressed in mam-malian cells and localized to mitochon-dria [Hakkaart et al., 2006]. Here,AOX maintains electron flow in anti-mycin or cyanide inhibited cells,preventing lactate accumulation, ROSproduction, and cell death. Lentiviral-mediated AOX expression could also al-leviate growth defects in cells withpathological mutations in COX10 orCOX15 [Dassa et al., 2009a,b]. Impor-tantly, AOX can be functionallyexpressed in vivo: In Drosophila, AOXexpression maintained a significant cya-nide-resistant respiration, protectedagainst respiratory toxins, decreased mi-tochondrial ROS production and allevi-ated the phenotype of several OXPHOSdisease-equivalent mutations [Fer-nandez-Ayala et al., 2009].

Combination of NDi1 and AOXBoth alternative enzymes, NDi1

and AOX, can be combined to restoreelectron transport in cells devoid ofmtDNA (q0 cells). Expression of the E.nidulans AOX and the yeast NDi1recovered the NADH CoQ oxido-re-ductase and the CoQ oxidase activityand improved NAD recycling. The si-multaneous expression of the two alter-native enzymes also restored uridineand pyruvate auxotrophy in q0 cells[Perales-Clemente et al., 2008].

CONCLUSIONSWhile the diagnosis and decipher-

ing of the mechanisms of many mito-chondrial disorders has rapidly advancedin the last decade, development ofproper treatment is lacking behind. It isthus encouraging that in recent yearsseveral experimental strategies have beendeveloped to target and correct mito-chondrial disorders and their effect on agenetic, metabolic and bioenergetic level(Fig. 1). Some of these therapeutic strat-egies are complementary and might beused in combination to simultaneouslycontrol the defects and effects of the mi-tochondrial dysfunction. Future work incell and animal models, and in somecases in clinical trials, will show the fea-sibility of these approaches, individuallyor in combination, for the treatment ofmitochondrial disease patients. n

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