Abstract: We have previously shown that increased mitochondrialbiogenesis by activation of Peroxisome proliferator-activated receptorgamma coactivator-1-alpha (PPAR-γ), PGC-1α, delayed the onset ofmitochondrial myopathies in mouse models. Endurance exercise isknown to induce PGC-1α expression, and accordingly a similar delaywas observed when the myopathy mouse model was exercisedregularly. AICAR, an allosteric activator of AMPK, and GW1516, aperoxisome proliferator-activated receptor agonist (PPARδ), have beenshown to be active in wild-type mice and to increase endurance.Moreover AICAR treatment during 1 month, partially correct a mousemodel with partial Complex IV deficiency.

In the present work we treated a mitochondrial myopathy mousemodel, associated with a muscle-specific cytochrome oxidase defect,with AICAR and GW1516 for 3 months. Our results showed thatAICAR treatment alone, but not GW1516 could rescue the muscleendurance phenotype of the mitochondrial myopathy mouse model.The beneficial effects were evident after 4 weeks of treatment andwere maintained during the treatment period. The detailed mecha-nisms of AICAR effect in this model are currently under investigation.

doi:10.1016/j.mito.2013.07.049

55Specific reduction of mutant mitochondrial genomes loadin patient-derived cells by mitoTALENsPresenter: Sandra R. BacmanSandra R. Bacmana, Sion L. Williamsa, Milena Pintoa,Susana Peraltaa, Carlos T. Moraesa,baUniversity of Miami Miller School of Medicine,Departments of Neurology, Miami, FL, USAbUniversity of Miami Miller School of Medicine,Departments of Cell Biology, Miami, FL, USA

Body of Abstract: Mitochondrial diseases are commonly causedby high levels of heteroplasmy mutations in the mitochondrial DNA(mtDNA). In an attempt to reduce the mutant mtDNA load, we haveengineered TAL-effector nucleases (TALEN) to localize to mitochon-dria. We designed two different mitochondrial-targeted TALENs(mitoTALENs) to bind and cleave two different mtDNA pathogenicmutations: the “common deletion of 5 kbp” (associated with ocularmyopathies and Kearns–Sayre syndrome) and a G14459A pointmutation in the ND6 subunit of Complex I (associatedwith optic atrophyand dystonia). The mitoTALENs included a mitochondrial localizationsignal, a tag in the N-terminus of the mature protein, a 3′UTR from amitochondrial gene, and a fluorescent marker to select for doubletransfected cells. All these elements were assembled in a single plasmid.Cybrid cells harboring heteroplasmic levels of themutatedmtDNAsweretransfected with the mitoTALENs. MitoTALENs were able to effectivelylocalize to mitochondria and express the expected size protein. Cybridcells were transfected and flow sorted for the fluorescent markers after48 h. MitoTALENs were able to reduce the levels of the pathogenicmtDNA common deletion (5 kb deletion) and the levels of the pointmutationG14,459A in the respective cybrids, in both cases increasing thelevels of WTmtDNA. No significant depletion of total mtDNA was foundwhen quantified by RT-PCR normalized to genomic ß actin. Functionalassays for Complex I activity in cybrid lysates showed that the cellsharboring the point mutation G14458A that originally were defective inComplex I, were able to recover the activity to control levels. Thesefindings raise the possibility that mitoTALENs can be beneficial to a largesubgroup of patients with mitochondrial diseases associated withheteroplasmic mtDNA andmay be a viable genetic therapy in the future.

doi:10.1016/j.mito.2013.07.050

56Deciphering the respiratory Complex I assembly pathwayPresenter: Rasika VartakRasika Vartak, Janice Deng, Yidong BaiDepartment of Cell and Structural Biology, University of Texas HealthScience Center, San Antonio, USA

Body of Abstract: An underlying cause of a majority of Complex Idisorders is deficiency of respiratory Complex I. Complex I is a 1MDalarge, 45 subunit complex whose assembly has been particular difficultto study, partly due to the fact that yeast, a frequently used modelorganism lacks Complex I and partly due to its large size. It is an Lshaped complex with a matrix arm protruding in the mitochondrialmatrix and amembrane arm, core subunits of which are encoded by themitochondrial DNA, embedded in the inner mitochondrial membrane.Using mitochondria isolated from patients, it was shown earlier thatpathologic mutations in Complex I subunits ND4 and ND6 abolishComplex I assembly, due to loss of membrane arm. Using cell lines withdifferent mitochondrial DNA mutations, we show that ND6 mutationallows for assembly of an almost complete Complex I. S[35] labeling ofmitochondrial translational products followed by pulse chase analysisand 2D SDS PAGE, showed presence of membrane arm formation inND6 mutant cells and an almost complete assembly of Complex I at anearly time point which clearly redefines the assembly patternpreviously published. Mass spectroscopy analysis shows the presenceof many early as well as late incorporated subunits in the Complex I ofthe ND6mutant cells.We show a similar phenomenon in two other celllines with ND6 mutation. We thus propose that ND6 subunit is notrequired for the membrane arm formation in Complex I but may beassociated with stability and interaction with other respiratorycomplexes to form supercomplexes. This thus opens new avenues tostudying Complex I deficiency due to pathologic ND6 mutation.

doi:10.1016/j.mito.2013.07.051

57Lessons learned from the analyses of 1500 mitochondrialgenomes by NGSPresenter: Victor Wei ZhangVictor Wei Zhang, Fangyuan Li, Guoli Wang, Cavatina K. Truong,Hui Yu, David Chen, Hong Cui, Xia Tian, Hao Wang,Megan Landsverk, Jing Wang, Lee-Jun WongDepartment of Molecular and Human Genetics,Baylor College of Medicine, Houston, TX, United States

Background: Traditional molecular analysis of the mitochondrialgenome is performed by Sanger sequencing of overlapping PCRfragments. This approach does not provide quantitative information ofmutation heteroplasmy anddoes not detect large deletions. The obstaclesare overcome by deep sequencing of the single amplicon of the wholemitochondrial genome (Clin Chem. 2012; 58:1322). We report sequenceresults of 1500 mitochondrial genomes using this approach.

Methods: The whole circular mitochondrial genome was amplifiedas a single amplicon using a back-to-back primer pair, followed bymassively parallel sequencing. More than 1500mitochondrial genomeswere analyzed.

Results: In addition to the advantages of minimizing the interfer-ence fromnuclearmitochondrial DNA homologs and accurate detectionof variant calls, our NGS approach also measures low levels ofheteroplasmy across the entire mitochondrial genome and detectssingle andmultiple deletions. Analysis of 1500mitochondrial genomes,consisting of 73, 24, 2.3 and 0.5% from blood, muscle, fibroblast, andliver respectively, revealed a mutation detection rate of 8% (3.5% pointmutations, 1.2% single deletion and 3.3% multiple deletions). A total of

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55 cases showed heteroplasmic changes and 27 of them are knownmutations. The heteroplasmies detected have awide range from 1.2% to94.5% and more than half of them have heteroplasmies lower than 20%,which may not be reliably detected by Sanger. Three heteroplasmicnovel variants, m.8319ANG (tRNA Lys), m.14982TNC (p.I79T, CYTB),andm.15437GNA (p.G231S, CYTB) are likely to be pathogenic mutations assupported by at least two in silico prediction algorithms, segregationwith disease in family studies, and the correlation of degree ofheteroplasmy with disease severity. About 4.5% of the samples showedsingle deletions (1.2%) and multiple mtDNA deletions (3.3%). Whilepointmutationswere detected in both blood andmuscle samples, so farall mtDNA multiple deletions were detected in muscle samples only.The average age of patients is 42.4 +/− 22.9 years with mtDNAdeletions, compared to 19.2 +/− 15.7 years for all patients with pointmutations, and 15.1+/12.1 for patients with pathogenic mutations atN20% heteroplasmies. In comparison to the experience of wholemitochondrial genome sequencing by Sanger method, the NGS deepsequencing with single amplicon approach resolved the interferencecaused by NUMT and SNPs. In addition, it accurately and reproduciblydetects low levels of heteroplasmies andmtDNAmultiple deletions thatcannot be detected by Sanger method.

Conclusions: Using this NGS approach, the diagnostics yield is 4.5%for blood samples and 13% formuscle samples. Although themajority ofpoint mutations could be detected in blood samples, multiple mtDNAdeletions were only detected in muscle. NGS-based sequencingproduces a diagnostic yield almost double that of traditional Sangersequencingmethod. Despitemany years of experience inmitochondrialwhole genome sequencing, novel pathogenic mtDNA mutationscontinue to be discovered.

doi:10.1016/j.mito.2013.07.052

58Discovery of modulators of oxygen consumption for use inmitochondrial diseasePresenter: Sunil SahdeoSunil Sahdeo, Gino CortopassiUniversity of California, Davis, Molecular Biosciences,School of Veterinary Medicine, Davis, CA 95616, USA

Body of Abstract: Mitochondrial dysfunction occurs in a broadrange of neurodegenerative diseases such as Friedriech's Ataxia, Leber'sHereditary Neuropathy (LHON) and Parkinson's disease. Therefore,potential therapies for these indications could possibly involve drugsthat increase mitochondrial function or number. Our goal is to discovernew chemical entities that will potentiate the function of themitochondria for use in human diseases. The majority of the energyused by the body is produced through the electron transport chain inthe mitochondria. This process generates ATP and consumes oxygen inthe process. Using 384-well oxygen biosensor plates (BD Biosciences)with afluorescent dye that produces a signal as oxygen is consumed,wehave established a robust and reproducible screening assay capable ofidentifying potentiators of mitochondrial function (%CV: b10, z-prime:0.8) amenable to high-throughput. Using this system in retinal ganglioncells, we screened a library of small molecule drugs that have reachedhuman clinical evaluation (Microsource) and identified multiplepotentiators and inhibitors in the short (2 h) and long term (24 h).After elimination of false positives and confirmation of dose depen-dence, we have identified compounds currently in clinical use withnovel mitochondrial interactions. These drugs were also tested in acybrid cell line containing the LHON mutation 11778. Additionally, wetested the effects of a collection of environmental toxins on oxygenconsumption and ATP turnover. This revealed a potential mitochondrialmechanism of toxicity of the chemicals and highlights the potential of

this assay to reveal off-targetmitochondrial effects of clinical candidatesin drug development. In conclusion, the evaluation of effects onmitochondrial oxygen consumption by drugs currently in clinical usecould potentially reveal novel ‘repurposed’ therapeutic leads for humandiseases of mitochondrial function.

doi:10.1016/j.mito.2013.07.053

59Bendavia, a novel peptide that improves mitochondrial functionand reverses diabetes visual declinePresenter: Peter OatesNazia Alam, Glen PruskyWeill Cornell Medical College, New York, NY 10065, United States

Diabetes is a leading cause of progressive vision loss, for which thereare no effective treatments. Similar to certainmitochondrial disease, theetiology of pathology involves degraded energy metabolism, withgrowing evidence that impaired mitochondrial function plays a keyrole. However, no study has directly investigated whether improvingmitochondrial function will improve visual dysfunction. A potentialefficacious treatment is Bendavia, a compound that selectively targetsmitochondria and improves mitochondrial function by increasing ATPproduction and preventing the formation of reactive oxygen species.Thus, we treated animal models of diabetes with Bendavia, andquantified visual thresholds longitudinally, using a virtual optokineticsystem (Prusky et al., 2004). In a mouse model of diabetic visualdysfunction, in which we combined a diabetic diet with streptozocin(STZ) injections, visual dysfunction emerged 1 week after injections,and was reduced by ~15% within 1 month. Visual decline was haltedwithin 1 week of initiating Bendavia treatment, and gradually improveduntil normal function was restored within 5 months. Groups fed adiabetic diet alone or treatedwith STZ only displayed less visual decline,which was also fully restored with Bendavia. Thus, visual decline inanimalmodels ofmetabolic dysfunction can be limited or reversedwitha peptide that improves mitochondrial function. Since Bendavia hasfavorable pharmacokinetics and is well tolerated in several clinicalstudies, it may be a viable tool to treat human visual loss related togenetic mitochondrial diseases in addition to metabolic dysfunction.

doi:10.1016/j.mito.2013.07.054

60Mitochondrial insufficiency is associated with autonomicnervous system dysfunction as confirmed by physiologic Headup Tilt table test (HUTT)Presenter: Mohammed Numan, MDMohammed Numan, Ian Butler, Rebecca Martinez, Melissa Knight,Nada Memon, Mary K. KoenigUniversity of Texas, Houston, United States

Body of Abstract:Introduction: Autonomic dysfunction has frequently been pro-

posed as a cause of several symptoms in mitochondrial patientsincluding palpitations, orthostatic intolerance, fatigue, GI dysmotilityand syncope; however, this proposal has not previously been supportedby physiologic testing. The aim of our study was to demonstrate thephysiologic changes during HUTT that may explain these symptoms.

Methods: Our physiologic HUTT consists of measuring heart rate,continuous (beat to beat) blood pressure, cardiac stroke volume, cardiacoutput, peripheral resistance, sympathetic and parasympathetic tone,diastolic BP, cerebral brain perfusion [obtained by Near Infra-Red

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