Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in -1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1 and there is no proven prophylactic treatment

Al-Dosary, Mazhor

Blakely, Emma L

Carver, Janet

Diot, Alan

Dombi, Eszter

Downes, Susan M

Fratter, Carl

Hargreaves, Iain

Hickman, Simon J

Jayawant, Sandeep

Liao, Chunyan

Lodge, Tiffany

Morten, Karl

Muir, Rebecca

Ng, Yi Shiau

Poulton, Joanna

Sarkar, Gopa

Taylor, Robert W

Yu-Wai-Man, Patrick

English

PubMed

Maternally-inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber’s hereditary optic neuropathy (LHON) in~ 1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects and there is no proven prophylactic treatment. We identified two families (Figure 1A) and one singleton case (Appendix e-1) harbouring the m.13051G>A pathogenic mtDNA mutation. This mutation was homoplasmic (Figure e-1) but no respiratory chain defect was apparent in skeletal muscle (Figure e-2, Table e-1). Three children were severely affected with lactic acidosis, two with Leigh syndrome (Patient 1 and 2; Figure 1B) and one with a Leigh-like phenotype (Patient 5). Previous authors have shown that mtDNA and mitochondrial mass are increased in individuals harbouring LHON mutations. They suggested that an up-regulation of mitochondrial biogenesis is protective, as the highest mitochondrial content was found in symptom-free carriers. We believe this increase in biogenesis reflects heightened mitochondrial turnover and therefore investigated mitophagy, a cellular mechanism whereby redundant or dysfunctional mitochondria are recycled

Morten, Karl J,

Carver, Janet G.

Lodge, Tiffany A.

Ng, Yi

Blakely, Emma L.

Hickman, Simon J.

Downes, Susan M.

Taylor, Rob W.

ORA - Oxford University Research Archive

The m.13051G&gt;A mitochondrial DNA mutation results in variable neurology and activated mitophagy

Eszter Dombi

Alan Diot

Karl Morten

Janet Carver

Tiffany Lodge

Carl Fratter

Yi Shiau Ng

Chunyan Liao

Rebecca Muir

Emma L. Blakely

Iain Hargreaves

Mazhor Al-Dosary

Gopa Sarkar

Simon J. Hickman

Susan M. Downes

Sandeep Jayawant

Patrick Yu-Wai-Man

Robert W. Taylor

Joanna Poulton

Crossref

Neurology

Apollo (Cambridge)

Clinical/ScientificNotesEszter Dombi, BScAlan Diot, PhDKarl Morten, PhDJanet Carver, BScTiffany Lodge, BScCarl Fratter, FRCPathYi Shiau Ng, MRCPChunyan Liao, PhDRebecca MuirEmma L. Blakely, PhD,FRCPathIain Hargreaves, PhDMazhor Al-Dosary, PhDGopa Sarkar, MRCPCHSimon J. Hickman, PhD,FRCPSusan M. Downes, MDSandeep Jayawant,FRCPCHPatrick Yu-Wai-Man,PhD, FRCOphthRobert W. Taylor, PhD,FRCPathJoanna Poulton,DMFRCPSupplemental dataat Neurology.orgTHE M.13051G.A MITOCHONDRIAL DNAMUTATION RESULTS IN VARIABLE NEUROLOGYAND ACTIVATED MITOPHAGYMaternally inherited mitochondrial DNA (mtDNA)mutations cause symptoms of Leber hereditary opticneuropathy (LHON) in ;1 in 30,000 individuals.Most of the affected individuals lack respiratorychain defects1 and there is no proven prophylactictreatment.We identified 2 families (figure 1A) and 1 sin-gleton case (appendix e-1 on the Neurology® Website at Neurology.org) harboring the m.13051G.Apathogenic mtDNA mutation.2 This mutation washomoplasmic (figure e-1) but no respiratory chaindefect was apparent in skeletal muscle (figure e-2,table e-1). Three children were severely affected bylactic acidosis: 2 with Leigh syndrome (patients 1and 2; figure 1B) and 1 with a Leigh-like phenotype(patient 5). Previous authors have shown thatmtDNA and mitochondrial mass are increased inindividuals harboring LHON mutations.3 Theysuggested that an upregulation of mitochondrialbiogenesis is protective, as the highest mitochon-drial content was found in symptom-free carriers.3We believe this increase in biogenesis reflectsheightened mitochondrial turnover and thereforeinvestigated mitophagy, a cellular mechanismwhereby redundant or dysfunctional mitochondriaare recycled.Methods. We used IN Cell1000, a previouslydeveloped high-throughput imaging method forquantifying mitophagy and mtDNA4 in culturedfibroblasts from patients compared with culturesderived from karyotypically normal disease controlsaged 0–20 years and healthy volunteers aged 21–80years. Cells were immunostained for the autophagymarker LC3 and the mitochondrial protein TOM20.Mitophagy was assessed as colocalization of LC3punctae with TOM20-positive mitochondria.Results. We found that the m.13051G.Amutationoccurred on the background of 3 different clades(table e-2), suggesting that this has arisen on multipleoccasions. We established that fibroblasts from allpatients have fragmented mitochondrial network(figure 1C) along with elevated levels of mitophagy(figure 1D) when compared to controls. Mitochon-drial volume was also increased, as was reactive oxy-gen species (ROS) production, accompanied by anincrease in the mitochondrial antioxidant manganesesuperoxide dismutase (figures e-3 and e-4).Changing cell culture substrates from glucose toglucose-free (galactose) media forced cells to use oxidativephosphorylation and further increased levels of mitoph-agy (figure 1D). An increase in LC3 punctae colocalizedwith mitochondria does not distinguish between sloweddegradation of autophagosomes and increased flux unlessit is validated, for example by mtDNA content. Theincrease in mitophagy was accompanied by a dropin mtDNA content (figure 1E), suggesting increasedturnover. Treatment of m.13051G.A patient cells withidebenone attenuated the increase in mitophagy(figure 1F).Discussion. Combining our data with 2 previouslypublished cases,2 the m.13051G.A mtDNA muta-tion appears to have arisen independently severaltimes, cosegregating with clinical features of eitherclassical LHON or a complicated early-onset Leigh-like neurodegenerative phenotype. This specificmtDNA variant was not detected in 990 controlmtDNA sequences. Taken together, our geneticand functional in vitro assays firmly establisha pathogenic role for the m.13051G.A mutationin causing mitochondrial disease.Interestingly, patient-derived fibroblasts had a frag-mented mitochondrial network pointing towards animbalance between fusion and fission. In keepingwith this observation, mitochondrial mass wasincreased in the mutant cell lines, which is notsurprising given that activation of mitochondrialbiogenesis is a well-reported compensatory cellularmechanism. Furthermore, there was a significantincrease in the levels of ROS production at baseline(figure e-3). Mitophagy was robustly increased in allthe fibroblast cultures tested that carried them.13051G.A mutation (figure 1D). Previouslypublished data showed increased mitophagy in pa-tients with complex I deficient mtDNA disease.5We also have additional data supporting the sameeffect in fibroblasts from other classical mitochondrialNeurology 86 May 17, 2016 1921Figure 1 Clinical data and evidence of activated mitophagy in patients with m.13051G.A mutation(A) Family trees of patients with the m.13051G.A mutation. All maternally related individuals harbored the m.13051G.A mito-chondrial DNA (mtDNA) mutation, but only those shaded black are clinically affected. Three children were severely affected withlactic acidosis: 2 with Leigh syndrome (patients 1 and 2) and one with a Leigh-like phenotype (patient 5). For further clinical details,see appendix e-1. (B) T2-weighted axial brainMRI scan head of patient 2. Arrows show established, bilateral, and symmetrical areasof hyperintensity in the lentiform nuclei, consistent with Leigh disease. (C) Mean mitochondrial length (measured using the INCell1000 Analyzer, GE Life Sciences, Piscataway, NJ) was significantly decreased in the patient cell lines compared with controls.(D) Number ofmitophagic eventswas significantly increased in all patient cell lines carrying them.13051G.AmtDNAmutation. Aneven more marked degree of mitophagy activation was observed when the cells were stressed under conditions of energeticdeprivation induced by culture in galactose media; this was not significant in the control. Cells were grown in glucose or galactosemedia (represented by blue or gray bars, respectively) and measured using in Cell1000 Analyzer. (E) MtDNA content was mildlyincreased in the patient cell lines compared with controls (n 5 3) under glucose media conditions (NS). There was a significantreduction in mtDNA content when the cells were grown in galactosemedia. (F) We investigated the effect of idebenone (a syntheticanalogue of coenzyme Q10) on mitophagy in both control and patient cells (n5 4) by adding 100 mL of idebenone (ID, 1 mM finalconcentration) to the growthmedia. Idebenone led to a significant reduction in the levels ofmitophagy in them.13051G.Amutantcell lines, suggestive of a beneficial effect on overall mitochondrial function (*p# 0.05, **p# 0.01, ***p# 0.001, ****p# 0.0001;one-way analysis of variance with multiple comparison, error bars are SEM).1922 Neurology 86 May 17, 2016optic neuropathies caused by the m.11778A.G(n 5 2) and m.3460G.A (n 5 1) mtDNA muta-tions and from a patient who is a compound hetero-zygote for pathogenic ACAD9 mutations (figure e-5).In the absence of an overt mitochondrial respiratorychain defect, documenting increased mitophagy infibroblasts could be a useful functional assay thatwould further support the pathogenic nature of a spe-cific mtDNA variant.The energetic stress induced by forcing the cellsto use oxidative phosphorylation leads to an increasein mitophagy and a decrease in mtDNA content.This could be explained by an increase in ROS pro-duction that overcomes the antioxidative defensesresulting in mitochondrial damage and increasedmitophagy. Idebenone attenuates this mitophagyand seems to improve cell viability (not shown),most likely by ameliorating respiratory chain dys-function and limiting the production of ROS.Patients with LHON may benefit from treatmentwith idebenone.6We have shown that mitophagy is increased incells from patients with Leigh/LHON phenotypessecondary to the m.13051G.A mtDNA mutation.Furthermore, idebenone attenuates the increased mi-tophagy. Drugs that modulate mitophagy are there-fore potentially useful treatments for mitochondrialand other neurodegenerative disorders.7From the University of Oxford (E.D., A.D., K.M., J.C., T.L., C.L.,R.M., S.M.D., J.P.); Churchill Hospital (C.F.), Oxford; NewcastleUniversity (Y.S.N., E.L.B., M.A.-D., P.Y.-W.-M., R.W.T.), New-castle upon Tyne; National Hospital for Neurology and Neurosurgery(I.H.), UCLH, Queen Square, London; Stoke Mandeville Hospital(G.S.), Aylesbury; Royal Hallamshire Hospital (S.J.H.), Sheffield;John Radcliffe Hospital (S.J.), Oxford; Royal Victoria Infirmary(P.Y.-W.-M.), Newcastle upon Tyne; and Moorfields Eye Hospitaland UCL Institute of Ophthalmology (P.Y.-W.-M.), London, UK.Author contributions: Eszter Dombi: laboratory work, data analysis,and conceptualization of study. Alan Diot: laboratory work and anal-ysis of data. Karl Morten: laboratory work and analysis of data. JanetCarver: responsible for cell lines. Tiffany Lodge: laboratory work. CarlFratter: laboratory work. Yi Shiau Ng: clinical description of patient.Chunyan Liao: laboratory work. RebeccaMuir: paper construction anddata analysis. Emma Blakely: acquisition and analysis of data. IainHargreaves: laboratory work. Mazhor Al-Dosary: laboratory work.Gopa Sarkar: clinical description of patient. Simon J. Hickman: clin-ical description of patient. Susan M. Downes: clinical description ofpatient. Sandeep Jayawant: clinical description of patient. Patrick Yu-Wai-Man: clinical description of patient and revision of the manu-script. Robert W. Taylor: critical revision of the manuscript. JoannaPoulton: supervisor role, design and conceptualization of the study.Acknowledgment: Teams in Oxford and Newcastle acknowledge thesupport of the NHS Highly Specialized Rare Mitochondrial Disordersof Adults and Children Service. The authors thank the patients andtheir families and clinicians for participating and Aviva Tolkovskyand Stephen Kennedy for their support.Study funding: This work was financed by the Lily Foundation,NewLife (SG/14-15/11), the MRC (MR/J010448/1), the WellcomeTrust (0948685/Z/10/Z), and the Angus Memorial MitochondrialFund. J.P., J.C., and C.F. have salary support from the NHS Spe-cialized Services Rare Mitochondrial Disorders Service.Disclosure: E. Dombi was funded by the Lily Foundation, the AngusMemorial Mitochondrial Fund, and the NewLife Foundation.A. Diot is funded by the MRC (MR/J010448/1) and the NewLifeFoundation. K. Morten was funded by the Williams Foundation.J. Carver was funded by the MRC (MR/J010448/1). T. Lodge wasfunded by the Wellcome Trust (0948685/Z/10/Z). C. Fratter andY. Ng report no disclosures relevant to the manuscript. C. Liao wasfunded by the Wellcome Trust (0948685/Z/10/Z). R. Muir wasfunded by the Angus Memorial Mitochondrial Fund. E. Blakely,I. Hargreaves, M. Al-Dosary, G. Sarkar, S. Hickman, S. Downes,and S. Jayawant report no disclosures relevant to the manuscript.P. Yu-Wai-Man is a Medical Research Council (MRC) ClinicianScientist and receives funding from Fight for Sight (UK) and the UKNational Institute of Health Research (NIHR) as part of the RareDiseases Translational Research Collaboration. R. Taylor is sup-ported by a Wellcome Trust Strategic Award (096919/Z/11/Z),the Medical Research Council (UK) Centre for Translational MuscleDisease Research (G0601943), and The Lily Foundation. J. Poultonis supported by the MRC (MR/J010448/1), the Wellcome Trust(0948685/Z/10/Z), NewLife (SG/14-15/11), the Lily Foundation,and the Angus Memorial Mitochondrial Fund, and has salary sup-port from the NHS Specialized Services Rare Mitochondrial Disor-ders Service. Go to Neurology.org for full disclosures. The ArticleProcessing Charge was paid by MRC and Wellcome Trust.Received June 9, 2015. Accepted in final form January 25, 2016.Correspondence to Dr. Poulton: joanna.poulton@obs-gyn.ox.ac.ukThis is an open access article distributed under the terms of theCreative Commons Attribution License 4.0 (CC BY), which permitsunrestricted use, distribution, and reproduction in any medium, pro-vided the original work is properly cited.© 2016 American Academy of NeurologyREFERENCES1. Yu-Wai-Man P, Turnbull DM, Chinnery PF. Leber hered-itary optic neuropathy. J Med Genet 2002;39:162–169.2. Howell N, Oostra RJ, Bolhuis PA, et al. Sequence analysisof the mitochondrial genomes from Dutch pedigrees withLeber hereditary optic neuropathy. Am J Hum Genet 2003;72:1460–1469.3. Giordano C, Iommarini L, Giordano L, et al. Efficientmitochondrial biogenesis drives incomplete penetrance inLeber’s hereditary optic neuropathy. Brain 2014;137:335–353.4. Diot A, Hinks-Roberts A, Lodge T, et al. A novel quanti-tative assay of mitophagy: combining high content analysisfluorescence with mitochondrial DNA mutant load to iden-tify novel pharmacological modulators of mitophagy. Phar-macol Res 2015;100:24–35.5. Granatiero V, Giorgio V, Cali T, et al. Reduced mitochon-drial Ca transients stimulate autophagy in human fibroblastscarrying the 13514A.G mutation of the ND5 subunitof NADH dehydrogenase. Cell Death Differ 2015;23:231–241.6. Klopstock T, Yu-Wai-Man P, Dimitriadis K, et al. Arandomized placebo-controlled trial of idebenone inLeber’s hereditary optic neuropathy. Brain 2011;134:2677–2686.7. Nixon RA. The role of autophagy in neurodegenerativedisease. Nat Med 2013;19:983–997.Neurology 86 May 17, 2016 1923

The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy.

Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in ;1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1 and there is no proven prophylactic treatment. We identified 2 families (figure 1A) and 1 singleton case (appendix e-1 on the Neurology® Web site at Neurology.org) harboring the m.13051G.A pathogenic mtDNA mutation.2 This mutation was homoplasmic (figure e-1) but no respiratory chain defect was apparent in skeletal muscle (figure e-2, table e-1). Three children were severely affected by lactic acidosis: 2 with Leigh syndrome (patients 1 and 2; figure 1B) and 1 with a Leigh-like phenotype (patient 5). Previous authors have shown that mtDNA and mitochondrial mass are increased in individuals harboring LHON mutations.3 They suggested that an upregulation of mitochondrial biogenesis is protective, as the highest mitochondrial content was found in symptom-free carriers.3 We believe this increase in biogenesis reflects heightened mitochondrial turnover and therefore investigated mitophagy, a cellular mechanism whereby redundant or dysfunctional mitochondria are recycled

Dombi, E

Diot, A

Morten, K

Carver, J

Lodge, T

Fratter, C

Ng, YS

Liao, C

Muir, R

Blakely, EL

Hargreaves, IP

Al-Dosary, M

Sarkar, G

Hickman, SJ

Downes, SM

Jayawant, S

Yu-Wai-Man, P

Taylor, RW

Poulton, J

LJMU Research Online (Liverpool John Moores University)

 Dombi, E, Diot, A, Morten, K, Carver, J, Lodge, T, Fratter, C, Ng, YS, Liao, C, Muir, R, Blakely, EL, Hargreaves, IP, Al-Dosary, M, Sarkar, G, Hickman, SJ, Downes, SM, Jayawant, S, Yu-Wai-Man, P, Taylor, RW and Poulton, J The m.13051G > A mitochondrial DNA mutation results in variable neurology and activated mitophagyhttp://researchonline.ljmu.ac.uk/id/eprint/13682/ArticleLJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain.The version presented here may differ from the published version or from the version of the record. Please see the repository URL above for details on accessing the published version and note that access may require a subscription. For more information please contact researchonline@ljmu.ac.ukhttp://researchonline.ljmu.ac.uk/Citation (please note it is advisable to refer to the publisher’s version if you intend to cite from this work) Dombi, E, Diot, A, Morten, K, Carver, J, Lodge, T, Fratter, C, Ng, YS, Liao, C, Muir, R, Blakely, EL, Hargreaves, IP, Al-Dosary, M, Sarkar, G, Hickman, SJ, Downes, SM, Jayawant, S, Yu-Wai-Man, P, Taylor, RW and Poulton, J (2016) The m.13051G > A mitochondrial DNA mutation results in variable LJMU Research Onlinehttp://researchonline.ljmu.ac.uk/Clinical/ScientificNotesEszter Dombi, BScAlan Diot, PhDKarl Morten, PhDJanet Carver, BScTiffany Lodge, BScCarl Fratter, FRCPathYi Shiau Ng, MRCPChunyan Liao, PhDRebecca MuirEmma L. Blakely, PhD,FRCPathIain Hargreaves, PhDMazhor Al-Dosary, PhDGopa Sarkar, MRCPCHSimon J. Hickman, PhD,FRCPSusan M. Downes, MDSandeep Jayawant,FRCPCHPatrick Yu-Wai-Man,PhD, FRCOphthRobert W. Taylor, PhD,FRCPathJoanna Poulton,DMFRCPSupplemental dataat Neurology.orgTHE M.13051G.A MITOCHONDRIAL DNAMUTATION RESULTS IN VARIABLE NEUROLOGYAND ACTIVATED MITOPHAGYMaternally inherited mitochondrial DNA (mtDNA)mutations cause symptoms of Leber hereditary opticneuropathy (LHON) in ;1 in 30,000 individuals.Most of the affected individuals lack respiratorychain defects1 and there is no proven prophylactictreatment.We identified 2 families (figure 1A) and 1 sin-gleton case (appendix e-1 on the Neurology® Website at Neurology.org) harboring the m.13051G.Apathogenic mtDNA mutation.2 This mutation washomoplasmic (figure e-1) but no respiratory chaindefect was apparent in skeletal muscle (figure e-2,table e-1). Three children were severely affected bylactic acidosis: 2 with Leigh syndrome (patients 1and 2; figure 1B) and 1 with a Leigh-like phenotype(patient 5). Previous authors have shown thatmtDNA and mitochondrial mass are increased inindividuals harboring LHON mutations.3 Theysuggested that an upregulation of mitochondrialbiogenesis is protective, as the highest mitochon-drial content was found in symptom-free carriers.3We believe this increase in biogenesis reflectsheightened mitochondrial turnover and thereforeinvestigated mitophagy, a cellular mechanismwhereby redundant or dysfunctional mitochondriaare recycled.Methods. We used IN Cell1000, a previouslydeveloped high-throughput imaging method forquantifying mitophagy and mtDNA4 in culturedfibroblasts from patients compared with culturesderived from karyotypically normal disease controlsaged 0–20 years and healthy volunteers aged 21–80years. Cells were immunostained for the autophagymarker LC3 and the mitochondrial protein TOM20.Mitophagy was assessed as colocalization of LC3punctae with TOM20-positive mitochondria.Results. We found that the m.13051G.Amutationoccurred on the background of 3 different clades(table e-2), suggesting that this has arisen on multipleoccasions. We established that fibroblasts from allpatients have fragmented mitochondrial network(figure 1C) along with elevated levels of mitophagy(figure 1D) when compared to controls. Mitochon-drial volume was also increased, as was reactive oxy-gen species (ROS) production, accompanied by anincrease in the mitochondrial antioxidant manganesesuperoxide dismutase (figures e-3 and e-4).Changing cell culture substrates from glucose toglucose-free (galactose) media forced cells to use oxidativephosphorylation and further increased levels of mitoph-agy (figure 1D). An increase in LC3 punctae colocalizedwith mitochondria does not distinguish between sloweddegradation of autophagosomes and increased flux unlessit is validated, for example by mtDNA content. Theincrease in mitophagy was accompanied by a dropin mtDNA content (figure 1E), suggesting increasedturnover. Treatment of m.13051G.A patient cells withidebenone attenuated the increase in mitophagy(figure 1F).Discussion. Combining our data with 2 previouslypublished cases,2 the m.13051G.A mtDNA muta-tion appears to have arisen independently severaltimes, cosegregating with clinical features of eitherclassical LHON or a complicated early-onset Leigh-like neurodegenerative phenotype. This specificmtDNA variant was not detected in 990 controlmtDNA sequences. Taken together, our geneticand functional in vitro assays firmly establisha pathogenic role for the m.13051G.A mutationin causing mitochondrial disease.Interestingly, patient-derived fibroblasts had a frag-mented mitochondrial network pointing towards animbalance between fusion and fission. In keepingwith this observation, mitochondrial mass wasincreased in the mutant cell lines, which is notsurprising given that activation of mitochondrialbiogenesis is a well-reported compensatory cellularmechanism. Furthermore, there was a significantincrease in the levels of ROS production at baseline(figure e-3). Mitophagy was robustly increased in allthe fibroblast cultures tested that carried them.13051G.A mutation (figure 1D). Previouslypublished data showed increased mitophagy in pa-tients with complex I deficient mtDNA disease.5We also have additional data supporting the sameeffect in fibroblasts from other classical mitochondrialNeurology 86 May 17, 2016 1921Figure 1 Clinical data and evidence of activated mitophagy in patients with m.13051G.A mutation(A) Family trees of patients with the m.13051G.A mutation. All maternally related individuals harbored the m.13051G.A mito-chondrial DNA (mtDNA) mutation, but only those shaded black are clinically affected. Three children were severely affected withlactic acidosis: 2 with Leigh syndrome (patients 1 and 2) and one with a Leigh-like phenotype (patient 5). For further clinical details,see appendix e-1. (B) T2-weighted axial brainMRI scan head of patient 2. Arrows show established, bilateral, and symmetrical areasof hyperintensity in the lentiform nuclei, consistent with Leigh disease. (C) Mean mitochondrial length (measured using the INCell1000 Analyzer, GE Life Sciences, Piscataway, NJ) was significantly decreased in the patient cell lines compared with controls.(D) Number ofmitophagic eventswas significantly increased in all patient cell lines carrying them.13051G.AmtDNAmutation. Aneven more marked degree of mitophagy activation was observed when the cells were stressed under conditions of energeticdeprivation induced by culture in galactose media; this was not significant in the control. Cells were grown in glucose or galactosemedia (represented by blue or gray bars, respectively) and measured using in Cell1000 Analyzer. (E) MtDNA content was mildlyincreased in the patient cell lines compared with controls (n 5 3) under glucose media conditions (NS). There was a significantreduction in mtDNA content when the cells were grown in galactosemedia. (F) We investigated the effect of idebenone (a syntheticanalogue of coenzyme Q10) on mitophagy in both control and patient cells (n5 4) by adding 100 mL of idebenone (ID, 1 mM finalconcentration) to the growthmedia. Idebenone led to a significant reduction in the levels ofmitophagy in them.13051G.Amutantcell lines, suggestive of a beneficial effect on overall mitochondrial function (*p# 0.05, **p# 0.01, ***p# 0.001, ****p# 0.0001;one-way analysis of variance with multiple comparison, error bars are SEM).1922 Neurology 86 May 17, 2016optic neuropathies caused by the m.11778A.G(n 5 2) and m.3460G.A (n 5 1) mtDNA muta-tions and from a patient who is a compound hetero-zygote for pathogenic ACAD9 mutations (figure e-5).In the absence of an overt mitochondrial respiratorychain defect, documenting increased mitophagy infibroblasts could be a useful functional assay thatwould further support the pathogenic nature of a spe-cific mtDNA variant.The energetic stress induced by forcing the cellsto use oxidative phosphorylation leads to an increasein mitophagy and a decrease in mtDNA content.This could be explained by an increase in ROS pro-duction that overcomes the antioxidative defensesresulting in mitochondrial damage and increasedmitophagy. Idebenone attenuates this mitophagyand seems to improve cell viability (not shown),most likely by ameliorating respiratory chain dys-function and limiting the production of ROS.Patients with LHON may benefit from treatmentwith idebenone.6We have shown that mitophagy is increased incells from patients with Leigh/LHON phenotypessecondary to the m.13051G.A mtDNA mutation.Furthermore, idebenone attenuates the increased mi-tophagy. Drugs that modulate mitophagy are there-fore potentially useful treatments for mitochondrialand other neurodegenerative disorders.7From the University of Oxford (E.D., A.D., K.M., J.C., T.L., C.L.,R.M., S.M.D., J.P.); Churchill Hospital (C.F.), Oxford; NewcastleUniversity (Y.S.N., E.L.B., M.A.-D., P.Y.-W.-M., R.W.T.), New-castle upon Tyne; National Hospital for Neurology and Neurosurgery(I.H.), UCLH, Queen Square, London; Stoke Mandeville Hospital(G.S.), Aylesbury; Royal Hallamshire Hospital (S.J.H.), Sheffield;John Radcliffe Hospital (S.J.), Oxford; Royal Victoria Infirmary(P.Y.-W.-M.), Newcastle upon Tyne; and Moorfields Eye Hospitaland UCL Institute of Ophthalmology (P.Y.-W.-M.), London, UK.Author contributions: Eszter Dombi: laboratory work, data analysis,and conceptualization of study. Alan Diot: laboratory work and anal-ysis of data. Karl Morten: laboratory work and analysis of data. JanetCarver: responsible for cell lines. Tiffany Lodge: laboratory work. CarlFratter: laboratory work. Yi Shiau Ng: clinical description of patient.Chunyan Liao: laboratory work. RebeccaMuir: paper construction anddata analysis. Emma Blakely: acquisition and analysis of data. IainHargreaves: laboratory work. Mazhor Al-Dosary: laboratory work.Gopa Sarkar: clinical description of patient. Simon J. Hickman: clin-ical description of patient. Susan M. Downes: clinical description ofpatient. Sandeep Jayawant: clinical description of patient. Patrick Yu-Wai-Man: clinical description of patient and revision of the manu-script. Robert W. Taylor: critical revision of the manuscript. JoannaPoulton: supervisor role, design and conceptualization of the study.Acknowledgment: Teams in Oxford and Newcastle acknowledge thesupport of the NHS Highly Specialized Rare Mitochondrial Disordersof Adults and Children Service. The authors thank the patients andtheir families and clinicians for participating and Aviva Tolkovskyand Stephen Kennedy for their support.Study funding: This work was financed by the Lily Foundation,NewLife (SG/14-15/11), the MRC (MR/J010448/1), the WellcomeTrust (0948685/Z/10/Z), and the Angus Memorial MitochondrialFund. J.P., J.C., and C.F. have salary support from the NHS Spe-cialized Services Rare Mitochondrial Disorders Service.Disclosure: E. Dombi was funded by the Lily Foundation, the AngusMemorial Mitochondrial Fund, and the NewLife Foundation.A. Diot is funded by the MRC (MR/J010448/1) and the NewLifeFoundation. K. Morten was funded by the Williams Foundation.J. Carver was funded by the MRC (MR/J010448/1). T. Lodge wasfunded by the Wellcome Trust (0948685/Z/10/Z). C. Fratter andY. Ng report no disclosures relevant to the manuscript. C. Liao wasfunded by the Wellcome Trust (0948685/Z/10/Z). R. Muir wasfunded by the Angus Memorial Mitochondrial Fund. E. Blakely,I. Hargreaves, M. Al-Dosary, G. Sarkar, S. Hickman, S. Downes,and S. Jayawant report no disclosures relevant to the manuscript.P. Yu-Wai-Man is a Medical Research Council (MRC) ClinicianScientist and receives funding from Fight for Sight (UK) and the UKNational Institute of Health Research (NIHR) as part of the RareDiseases Translational Research Collaboration. R. Taylor is sup-ported by a Wellcome Trust Strategic Award (096919/Z/11/Z),the Medical Research Council (UK) Centre for Translational MuscleDisease Research (G0601943), and The Lily Foundation. J. Poultonis supported by the MRC (MR/J010448/1), the Wellcome Trust(0948685/Z/10/Z), NewLife (SG/14-15/11), the Lily Foundation,and the Angus Memorial Mitochondrial Fund, and has salary sup-port from the NHS Specialized Services Rare Mitochondrial Disor-ders Service. Go to Neurology.org for full disclosures. The ArticleProcessing Charge was paid by MRC and Wellcome Trust.Received June 9, 2015. Accepted in final form January 25, 2016.Correspondence to Dr. Poulton: joanna.poulton@obs-gyn.ox.ac.ukThis is an open access article distributed under the terms of theCreative Commons Attribution License 4.0 (CC BY), which permitsunrestricted use, distribution, and reproduction in any medium, pro-vided the original work is properly cited.© 2016 American Academy of NeurologyREFERENCES1. Yu-Wai-Man P, Turnbull DM, Chinnery PF. Leber hered-itary optic neuropathy. J Med Genet 2002;39:162–169.2. Howell N, Oostra RJ, Bolhuis PA, et al. Sequence analysisof the mitochondrial genomes from Dutch pedigrees withLeber hereditary optic neuropathy. Am J Hum Genet 2003;72:1460–1469.3. Giordano C, Iommarini L, Giordano L, et al. Efficientmitochondrial biogenesis drives incomplete penetrance inLeber’s hereditary optic neuropathy. Brain 2014;137:335–353.4. Diot A, Hinks-Roberts A, Lodge T, et al. A novel quanti-tative assay of mitophagy: combining high content analysisfluorescence with mitochondrial DNA mutant load to iden-tify novel pharmacological modulators of mitophagy. Phar-macol Res 2015;100:24–35.5. Granatiero V, Giorgio V, Cali T, et al. Reduced mitochon-drial Ca transients stimulate autophagy in human fibroblastscarrying the 13514A.G mutation of the ND5 subunitof NADH dehydrogenase. Cell Death Differ 2015;23:231–241.6. Klopstock T, Yu-Wai-Man P, Dimitriadis K, et al. Arandomized placebo-controlled trial of idebenone inLeber’s hereditary optic neuropathy. Brain 2011;134:2677–2686.7. Nixon RA. The role of autophagy in neurodegenerativedisease. Nat Med 2013;19:983–997.Neurology 86 May 17, 2016 1923

The m.13051G > A mitochondrial DNA mutation results in variable neurology and activated mitophagy

Oxford University Research Archive

Clinical/ScientificNotesEszter Dombi, BScAlan Diot, PhDKarl Morten, PhDJanet Carver, BScTiffany Lodge, BScCarl Fratter, FRCPathYi Shiau Ng, MRCPChunyan Liao, PhDRebecca MuirEmma L. Blakely, PhD,FRCPathIain Hargreaves, PhDMazhor Al-Dosary, PhDGopa Sarkar, MRCPCHSimon J. Hickman, PhD,FRCPSusan M. Downes, MDSandeep Jayawant,FRCPCHPatrick Yu-Wai-Man,PhD, FRCOphthRobert W. Taylor, PhD,FRCPathJoanna Poulton,DMFRCPSupplemental dataat Neurology.orgTHE M.13051G.A MITOCHONDRIAL DNAMUTATION RESULTS IN VARIABLE NEUROLOGYAND ACTIVATED MITOPHAGYMaternally inherited mitochondrial DNA (mtDNA)mutations cause symptoms of Leber hereditary opticneuropathy (LHON) in ;1 in 30,000 individuals.Most of the affected individuals lack respiratorychain defects1 and there is no proven prophylactictreatment.We identified 2 families (figure 1A) and 1 sin-gleton case (appendix e-1 on the Neurology® Website at Neurology.org) harboring the m.13051G.Apathogenic mtDNA mutation.2 This mutation washomoplasmic (figure e-1) but no respiratory chaindefect was apparent in skeletal muscle (figure e-2,table e-1). Three children were severely affected bylactic acidosis: 2 with Leigh syndrome (patients 1and 2; figure 1B) and 1 with a Leigh-like phenotype(patient 5). Previous authors have shown thatmtDNA and mitochondrial mass are increased inindividuals harboring LHON mutations.3 Theysuggested that an upregulation of mitochondrialbiogenesis is protective, as the highest mitochon-drial content was found in symptom-free carriers.3We believe this increase in biogenesis reflectsheightened mitochondrial turnover and thereforeinvestigated mitophagy, a cellular mechanismwhereby redundant or dysfunctional mitochondriaare recycled.Methods. We used IN Cell1000, a previouslydeveloped high-throughput imaging method forquantifying mitophagy and mtDNA4 in culturedfibroblasts from patients compared with culturesderived from karyotypically normal disease controlsaged 0–20 years and healthy volunteers aged 21–80years. Cells were immunostained for the autophagymarker LC3 and the mitochondrial protein TOM20.Mitophagy was assessed as colocalization of LC3punctae with TOM20-positive mitochondria.Results. We found that the m.13051G.Amutationoccurred on the background of 3 different clades(table e-2), suggesting that this has arisen on multipleoccasions. We established that fibroblasts from allpatients have fragmented mitochondrial network(figure 1C) along with elevated levels of mitophagy(figure 1D) when compared to controls. Mitochon-drial volume was also increased, as was reactive oxy-gen species (ROS) production, accompanied by anincrease in the mitochondrial antioxidant manganesesuperoxide dismutase (figures e-3 and e-4).Changing cell culture substrates from glucose toglucose-free (galactose) media forced cells to use oxidativephosphorylation and further increased levels of mitoph-agy (figure 1D). An increase in LC3 punctae colocalizedwith mitochondria does not distinguish between sloweddegradation of autophagosomes and increased flux unlessit is validated, for example by mtDNA content. Theincrease in mitophagy was accompanied by a dropin mtDNA content (figure 1E), suggesting increasedturnover. Treatment of m.13051G.A patient cells withidebenone attenuated the increase in mitophagy(figure 1F).Discussion. Combining our data with 2 previouslypublished cases,2 the m.13051G.A mtDNA muta-tion appears to have arisen independently severaltimes, cosegregating with clinical features of eitherclassical LHON or a complicated early-onset Leigh-like neurodegenerative phenotype. This specificmtDNA variant was not detected in 990 controlmtDNA sequences. Taken together, our geneticand functional in vitro assays firmly establisha pathogenic role for the m.13051G.A mutationin causing mitochondrial disease.Interestingly, patient-derived fibroblasts had a frag-mented mitochondrial network pointing towards animbalance between fusion and fission. In keepingwith this observation, mitochondrial mass wasincreased in the mutant cell lines, which is notsurprising given that activation of mitochondrialbiogenesis is a well-reported compensatory cellularmechanism. Furthermore, there was a significantincrease in the levels of ROS production at baseline(figure e-3). Mitophagy was robustly increased in allthe fibroblast cultures tested that carried them.13051G.A mutation (figure 1D). Previouslypublished data showed increased mitophagy in pa-tients with complex I deficient mtDNA disease.5We also have additional data supporting the sameeffect in fibroblasts from other classical mitochondrialNeurology 86 May 17, 2016 1921Figure 1 Clinical data and evidence of activated mitophagy in patients with m.13051G.A mutation(A) Family trees of patients with the m.13051G.A mutation. All maternally related individuals harbored the m.13051G.A mito-chondrial DNA (mtDNA) mutation, but only those shaded black are clinically affected. Three children were severely affected withlactic acidosis: 2 with Leigh syndrome (patients 1 and 2) and one with a Leigh-like phenotype (patient 5). For further clinical details,see appendix e-1. (B) T2-weighted axial brainMRI scan head of patient 2. Arrows show established, bilateral, and symmetrical areasof hyperintensity in the lentiform nuclei, consistent with Leigh disease. (C) Mean mitochondrial length (measured using the INCell1000 Analyzer, GE Life Sciences, Piscataway, NJ) was significantly decreased in the patient cell lines compared with controls.(D) Number ofmitophagic eventswas significantly increased in all patient cell lines carrying them.13051G.AmtDNAmutation. Aneven more marked degree of mitophagy activation was observed when the cells were stressed under conditions of energeticdeprivation induced by culture in galactose media; this was not significant in the control. Cells were grown in glucose or galactosemedia (represented by blue or gray bars, respectively) and measured using in Cell1000 Analyzer. (E) MtDNA content was mildlyincreased in the patient cell lines compared with controls (n 5 3) under glucose media conditions (NS). There was a significantreduction in mtDNA content when the cells were grown in galactosemedia. (F) We investigated the effect of idebenone (a syntheticanalogue of coenzyme Q10) on mitophagy in both control and patient cells (n5 4) by adding 100 mL of idebenone (ID, 1 mM finalconcentration) to the growthmedia. Idebenone led to a significant reduction in the levels ofmitophagy in them.13051G.Amutantcell lines, suggestive of a beneficial effect on overall mitochondrial function (*p# 0.05, **p# 0.01, ***p# 0.001, ****p# 0.0001;one-way analysis of variance with multiple comparison, error bars are SEM).1922 Neurology 86 May 17, 2016optic neuropathies caused by the m.11778A.G(n 5 2) and m.3460G.A (n 5 1) mtDNA muta-tions and from a patient who is a compound hetero-zygote for pathogenic ACAD9 mutations (figure e-5).In the absence of an overt mitochondrial respiratorychain defect, documenting increased mitophagy infibroblasts could be a useful functional assay thatwould further support the pathogenic nature of a spe-cific mtDNA variant.The energetic stress induced by forcing the cellsto use oxidative phosphorylation leads to an increasein mitophagy and a decrease in mtDNA content.This could be explained by an increase in ROS pro-duction that overcomes the antioxidative defensesresulting in mitochondrial damage and increasedmitophagy. Idebenone attenuates this mitophagyand seems to improve cell viability (not shown),most likely by ameliorating respiratory chain dys-function and limiting the production of ROS.Patients with LHON may benefit from treatmentwith idebenone.6We have shown that mitophagy is increased incells from patients with Leigh/LHON phenotypessecondary to the m.13051G.A mtDNA mutation.Furthermore, idebenone attenuates the increased mi-tophagy. Drugs that modulate mitophagy are there-fore potentially useful treatments for mitochondrialand other neurodegenerative disorders.7From the University of Oxford (E.D., A.D., K.M., J.C., T.L., C.L.,R.M., S.M.D., J.P.); Churchill Hospital (C.F.), Oxford; NewcastleUniversity (Y.S.N., E.L.B., M.A.-D., P.Y.-W.-M., R.W.T.), New-castle upon Tyne; National Hospital for Neurology and Neurosurgery(I.H.), UCLH, Queen Square, London; Stoke Mandeville Hospital(G.S.), Aylesbury; Royal Hallamshire Hospital (S.J.H.), Sheffield;John Radcliffe Hospital (S.J.), Oxford; Royal Victoria Infirmary(P.Y.-W.-M.), Newcastle upon Tyne; and Moorfields Eye Hospitaland UCL Institute of Ophthalmology (P.Y.-W.-M.), London, UK.Author contributions: Eszter Dombi: laboratory work, data analysis,and conceptualization of study. Alan Diot: laboratory work and anal-ysis of data. Karl Morten: laboratory work and analysis of data. JanetCarver: responsible for cell lines. Tiffany Lodge: laboratory work. CarlFratter: laboratory work. Yi Shiau Ng: clinical description of patient.Chunyan Liao: laboratory work. RebeccaMuir: paper construction anddata analysis. Emma Blakely: acquisition and analysis of data. IainHargreaves: laboratory work. Mazhor Al-Dosary: laboratory work.Gopa Sarkar: clinical description of patient. Simon J. Hickman: clin-ical description of patient. Susan M. Downes: clinical description ofpatient. Sandeep Jayawant: clinical description of patient. Patrick Yu-Wai-Man: clinical description of patient and revision of the manu-script. Robert W. Taylor: critical revision of the manuscript. JoannaPoulton: supervisor role, design and conceptualization of the study.Acknowledgment: Teams in Oxford and Newcastle acknowledge thesupport of the NHS Highly Specialized Rare Mitochondrial Disordersof Adults and Children Service. The authors thank the patients andtheir families and clinicians for participating and Aviva Tolkovskyand Stephen Kennedy for their support.Study funding: This work was financed by the Lily Foundation,NewLife (SG/14-15/11), the MRC (MR/J010448/1), the WellcomeTrust (0948685/Z/10/Z), and the Angus Memorial MitochondrialFund. J.P., J.C., and C.F. have salary support from the NHS Spe-cialized Services Rare Mitochondrial Disorders Service.Disclosure: E. Dombi was funded by the Lily Foundation, the AngusMemorial Mitochondrial Fund, and the NewLife Foundation.A. Diot is funded by the MRC (MR/J010448/1) and the NewLifeFoundation. K. Morten was funded by the Williams Foundation.J. Carver was funded by the MRC (MR/J010448/1). T. Lodge wasfunded by the Wellcome Trust (0948685/Z/10/Z). C. Fratter andY. Ng report no disclosures relevant to the manuscript. C. Liao wasfunded by the Wellcome Trust (0948685/Z/10/Z). R. Muir wasfunded by the Angus Memorial Mitochondrial Fund. E. Blakely,I. Hargreaves, M. Al-Dosary, G. Sarkar, S. Hickman, S. Downes,and S. Jayawant report no disclosures relevant to the manuscript.P. Yu-Wai-Man is a Medical Research Council (MRC) ClinicianScientist and receives funding from Fight for Sight (UK) and the UKNational Institute of Health Research (NIHR) as part of the RareDiseases Translational Research Collaboration. R. Taylor is sup-ported by a Wellcome Trust Strategic Award (096919/Z/11/Z),the Medical Research Council (UK) Centre for Translational MuscleDisease Research (G0601943), and The Lily Foundation. J. Poultonis supported by the MRC (MR/J010448/1), the Wellcome Trust(0948685/Z/10/Z), NewLife (SG/14-15/11), the Lily Foundation,and the Angus Memorial Mitochondrial Fund, and has salary sup-port from the NHS Specialized Services Rare Mitochondrial Disor-ders Service. Go to Neurology.org for full disclosures. The ArticleProcessing Charge was paid by MRC and Wellcome Trust.Received June 9, 2015. Accepted in final form January 25, 2016.Correspondence to Dr. Poulton: joanna.poulton@obs-gyn.ox.ac.ukThis is an open access article distributed under the terms of theCreative Commons Attribution License 4.0 (CC BY), which permitsunrestricted use, distribution, and reproduction in any medium, pro-vided the original work is properly cited.© 2016 American Academy of NeurologyREFERENCES1. Yu-Wai-Man P, Turnbull DM, Chinnery PF. Leber hered-itary optic neuropathy. J Med Genet 2002;39:162–169.2. Howell N, Oostra RJ, Bolhuis PA, et al. Sequence analysisof the mitochondrial genomes from Dutch pedigrees withLeber hereditary optic neuropathy. Am J Hum Genet 2003;72:1460–1469.3. Giordano C, Iommarini L, Giordano L, et al. Efficientmitochondrial biogenesis drives incomplete penetrance inLeber’s hereditary optic neuropathy. Brain 2014;137:335–353.4. Diot A, Hinks-Roberts A, Lodge T, et al. A novel quanti-tative assay of mitophagy: combining high content analysisfluorescence with mitochondrial DNA mutant load to iden-tify novel pharmacological modulators of mitophagy. Phar-macol Res 2015;100:24–35.5. Granatiero V, Giorgio V, Cali T, et al. Reduced mitochon-drial Ca transients stimulate autophagy in human fibroblastscarrying the 13514A.G mutation of the ND5 subunitof NADH dehydrogenase. Cell Death Differ 2015;23:231–241.6. Klopstock T, Yu-Wai-Man P, Dimitriadis K, et al. Arandomized placebo-controlled trial of idebenone inLeber’s hereditary optic neuropathy. Brain 2011;134:2677–2686.7. Nixon RA. The role of autophagy in neurodegenerativedisease. Nat Med 2013;19:983–997.Neurology 86 May 17, 2016 1923DOI 10.1212/WNL.00000000000026882016;86;1921-1923 Published Online before print April 22, 2016Neurology Eszter Dombi, Alan Diot, Karl Morten, et al. activated mitophagyThe m.13051G>A mitochondrial DNA mutation results in variable neurology andThis information is current as of April 22, 2016ServicesUpdated Information & http://www.neurology.org/content/86/20/1921.full.htmlincluding high resolution figures, can be found at:Supplementary Material 002688.DC1http://www.neurology.org/content/suppl/2016/04/24/WNL.0000000000Supplementary material can be found at: References http://www.neurology.org/content/86/20/1921.full.html##ref-list-1This article cites 7 articles, 1 of which you can access for free at: Citations http://www.neurology.org/content/86/20/1921.full.html##otherarticlesThis article has been cited by 1 HighWire-hosted articles: Subspecialty Collections http://www.neurology.org//cgi/collection/optic_nerveOptic nerve http://www.neurology.org//cgi/collection/mitochondrial_disordersMitochondrial disorders http://www.neurology.org//cgi/collection/all_clinical_neurologyAll Clinical Neurologyfollowing collection(s): This article, along with others on similar topics, appears in the  Permissions & Licensing http://www.neurology.org/misc/about.xhtml#permissionsits entirety can be found online at:Information about reproducing this article in parts (figures,tables) or in  Reprints http://www.neurology.org/misc/addir.xhtml#reprintsusInformation about ordering reprints can be found online:rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.1951, it is now a weekly with 48 issues per year. Copyright © 2016 American Academy of Neurology. All ® is the official journal of the American Academy of Neurology. Published continuously sinceNeurology 

Maternally-inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber’s hereditary optic neuropathy (LHON) in~ 1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects and there is no proven prophylactic treatment.
We identified two families (Figure 1A) and one singleton case (Appendix e-1) harbouring the m.13051G>A pathogenic mtDNA mutation. This mutation was homoplasmic (Figure e-1) but no respiratory chain defect was apparent in skeletal muscle (Figure e-2, Table e-1). Three children were severely affected with lactic acidosis, two with Leigh syndrome (Patient 1 and 2; Figure 1B) and one with a Leigh-like phenotype (Patient 5). Previous authors have shown that mtDNA and mitochondrial mass are increased in individuals harbouring LHON mutations. They suggested that an up-regulation of mitochondrial biogenesis is protective, as the highest mitochondrial content was found in symptom-free carriers. We believe this increase in biogenesis reflects heightened mitochondrial turnover and therefore investigated mitophagy, a cellular mechanism whereby redundant or dysfunctional mitochondria are recycled.</p

Morten, KJ

Carver, JG

Lodge, TA

Hargreaves, I

Oxford University Research Archive (ORA)

The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy

Dombi, E.

Diot, A.

Morten, K.

Carver, J.

Lodge, T.

Fratter, C.

Ng, Y.S.

Liao, C.

Muir, R.

Blakely, E.L.

Hargreaves, I.

Al-Dosary, M.

Sarkar, G.

Hickman, S.J.

Downes, S.M.

Jayawant, S.

Yu-Wai-Man, P.

Taylor, R.W.

Poulton, J.

White Rose Research Online

Clinical/ScientificNotesEszter Dombi, BScAlan Diot, PhDKarl Morten, PhDJanet Carver, BScTiffany Lodge, BScCarl Fratter, FRCPathYi Shiau Ng, MRCPChunyan Liao, PhDRebecca MuirEmma L. Blakely, PhD,FRCPathIain Hargreaves, PhDMazhor Al-Dosary, PhDGopa Sarkar, MRCPCHSimon J. Hickman, PhD,FRCPSusan M. Downes, MDSandeep Jayawant,FRCPCHPatrick Yu-Wai-Man,PhD, FRCOphthRobert W. Taylor, PhD,FRCPathJoanna Poulton,DMFRCPSupplemental dataat Neurology.orgTHE M.13051G.A MITOCHONDRIAL DNAMUTATION RESULTS IN VARIABLE NEUROLOGYAND ACTIVATED MITOPHAGYMaternally inherited mitochondrial DNA (mtDNA)mutations cause symptoms of Leber hereditary opticneuropathy (LHON) in ;1 in 30,000 individuals.Most of the affected individuals lack respiratorychain defects1 and there is no proven prophylactictreatment.We identified 2 families (figure 1A) and 1 sin-gleton case (appendix e-1 on the Neurology® Website at Neurology.org) harboring the m.13051G.Apathogenic mtDNA mutation.2 This mutation washomoplasmic (figure e-1) but no respiratory chaindefect was apparent in skeletal muscle (figure e-2,table e-1). Three children were severely affected bylactic acidosis: 2 with Leigh syndrome (patients 1and 2; figure 1B) and 1 with a Leigh-like phenotype(patient 5). Previous authors have shown thatmtDNA and mitochondrial mass are increased inindividuals harboring LHON mutations.3 Theysuggested that an upregulation of mitochondrialbiogenesis is protective, as the highest mitochon-drial content was found in symptom-free carriers.3We believe this increase in biogenesis reflectsheightened mitochondrial turnover and thereforeinvestigated mitophagy, a cellular mechanismwhereby redundant or dysfunctional mitochondriaare recycled.Methods. We used IN Cell1000, a previouslydeveloped high-throughput imaging method forquantifying mitophagy and mtDNA4 in culturedfibroblasts from patients compared with culturesderived from karyotypically normal disease controlsaged 0–20 years and healthy volunteers aged 21–80years. Cells were immunostained for the autophagymarker LC3 and the mitochondrial protein TOM20.Mitophagy was assessed as colocalization of LC3punctae with TOM20-positive mitochondria.Results. We found that the m.13051G.Amutationoccurred on the background of 3 different clades(table e-2), suggesting that this has arisen on multipleoccasions. We established that fibroblasts from allpatients have fragmented mitochondrial network(figure 1C) along with elevated levels of mitophagy(figure 1D) when compared to controls. Mitochon-drial volume was also increased, as was reactive oxy-gen species (ROS) production, accompanied by anincrease in the mitochondrial antioxidant manganesesuperoxide dismutase (figures e-3 and e-4).Changing cell culture substrates from glucose toglucose-free (galactose) media forced cells to use oxidativephosphorylation and further increased levels of mitoph-agy (figure 1D). An increase in LC3 punctae colocalizedwith mitochondria does not distinguish between sloweddegradation of autophagosomes and increased flux unlessit is validated, for example by mtDNA content. Theincrease in mitophagy was accompanied by a dropin mtDNA content (figure 1E), suggesting increasedturnover. Treatment of m.13051G.A patient cells withidebenone attenuated the increase in mitophagy(figure 1F).Discussion. Combining our data with 2 previouslypublished cases,2 the m.13051G.A mtDNA muta-tion appears to have arisen independently severaltimes, cosegregating with clinical features of eitherclassical LHON or a complicated early-onset Leigh-like neurodegenerative phenotype. This specificmtDNA variant was not detected in 990 controlmtDNA sequences. Taken together, our geneticand functional in vitro assays firmly establisha pathogenic role for the m.13051G.A mutationin causing mitochondrial disease.Interestingly, patient-derived fibroblasts had a frag-mented mitochondrial network pointing towards animbalance between fusion and fission. In keepingwith this observation, mitochondrial mass wasincreased in the mutant cell lines, which is notsurprising given that activation of mitochondrialbiogenesis is a well-reported compensatory cellularmechanism. Furthermore, there was a significantincrease in the levels of ROS production at baseline(figure e-3). Mitophagy was robustly increased in allthe fibroblast cultures tested that carried them.13051G.A mutation (figure 1D). Previouslypublished data showed increased mitophagy in pa-tients with complex I deficient mtDNA disease.5We also have additional data supporting the sameeffect in fibroblasts from other classical mitochondrialNeurology 86 May 17, 2016 1921Figure 1 Clinical data and evidence of activated mitophagy in patients with m.13051G.A mutation(A) Family trees of patients with the m.13051G.A mutation. All maternally related individuals harbored the m.13051G.A mito-chondrial DNA (mtDNA) mutation, but only those shaded black are clinically affected. Three children were severely affected withlactic acidosis: 2 with Leigh syndrome (patients 1 and 2) and one with a Leigh-like phenotype (patient 5). For further clinical details,see appendix e-1. (B) T2-weighted axial brainMRI scan head of patient 2. Arrows show established, bilateral, and symmetrical areasof hyperintensity in the lentiform nuclei, consistent with Leigh disease. (C) Mean mitochondrial length (measured using the INCell1000 Analyzer, GE Life Sciences, Piscataway, NJ) was significantly decreased in the patient cell lines compared with controls.(D) Number ofmitophagic eventswas significantly increased in all patient cell lines carrying them.13051G.AmtDNAmutation. Aneven more marked degree of mitophagy activation was observed when the cells were stressed under conditions of energeticdeprivation induced by culture in galactose media; this was not significant in the control. Cells were grown in glucose or galactosemedia (represented by blue or gray bars, respectively) and measured using in Cell1000 Analyzer. (E) MtDNA content was mildlyincreased in the patient cell lines compared with controls (n 5 3) under glucose media conditions (NS). There was a significantreduction in mtDNA content when the cells were grown in galactosemedia. (F) We investigated the effect of idebenone (a syntheticanalogue of coenzyme Q10) on mitophagy in both control and patient cells (n5 4) by adding 100 mL of idebenone (ID, 1 mM finalconcentration) to the growthmedia. Idebenone led to a significant reduction in the levels ofmitophagy in them.13051G.Amutantcell lines, suggestive of a beneficial effect on overall mitochondrial function (*p# 0.05, **p# 0.01, ***p# 0.001, ****p# 0.0001;one-way analysis of variance with multiple comparison, error bars are SEM).1922 Neurology 86 May 17, 2016optic neuropathies caused by the m.11778A.G(n 5 2) and m.3460G.A (n 5 1) mtDNA muta-tions and from a patient who is a compound hetero-zygote for pathogenic ACAD9 mutations (figure e-5).In the absence of an overt mitochondrial respiratorychain defect, documenting increased mitophagy infibroblasts could be a useful functional assay thatwould further support the pathogenic nature of a spe-cific mtDNA variant.The energetic stress induced by forcing the cellsto use oxidative phosphorylation leads to an increasein mitophagy and a decrease in mtDNA content.This could be explained by an increase in ROS pro-duction that overcomes the antioxidative defensesresulting in mitochondrial damage and increasedmitophagy. Idebenone attenuates this mitophagyand seems to improve cell viability (not shown),most likely by ameliorating respiratory chain dys-function and limiting the production of ROS.Patients with LHON may benefit from treatmentwith idebenone.6We have shown that mitophagy is increased incells from patients with Leigh/LHON phenotypessecondary to the m.13051G.A mtDNA mutation.Furthermore, idebenone attenuates the increased mi-tophagy. Drugs that modulate mitophagy are there-fore potentially useful treatments for mitochondrialand other neurodegenerative disorders.7From the University of Oxford (E.D., A.D., K.M., J.C., T.L., C.L.,R.M., S.M.D., J.P.); Churchill Hospital (C.F.), Oxford; NewcastleUniversity (Y.S.N., E.L.B., M.A.-D., P.Y.-W.-M., R.W.T.), New-castle upon Tyne; National Hospital for Neurology and Neurosurgery(I.H.), UCLH, Queen Square, London; Stoke Mandeville Hospital(G.S.), Aylesbury; Royal Hallamshire Hospital (S.J.H.), Sheffield;John Radcliffe Hospital (S.J.), Oxford; Royal Victoria Infirmary(P.Y.-W.-M.), Newcastle upon Tyne; and Moorfields Eye Hospitaland UCL Institute of Ophthalmology (P.Y.-W.-M.), London, UK.Author contributions: Eszter Dombi: laboratory work, data analysis,and conceptualization of study. Alan Diot: laboratory work and anal-ysis of data. Karl Morten: laboratory work and analysis of data. JanetCarver: responsible for cell lines. Tiffany Lodge: laboratory work. CarlFratter: laboratory work. Yi Shiau Ng: clinical description of patient.Chunyan Liao: laboratory work. RebeccaMuir: paper construction anddata analysis. Emma Blakely: acquisition and analysis of data. IainHargreaves: laboratory work. Mazhor Al-Dosary: laboratory work.Gopa Sarkar: clinical description of patient. Simon J. Hickman: clin-ical description of patient. Susan M. Downes: clinical description ofpatient. Sandeep Jayawant: clinical description of patient. Patrick Yu-Wai-Man: clinical description of patient and revision of the manu-script. Robert W. Taylor: critical revision of the manuscript. JoannaPoulton: supervisor role, design and conceptualization of the study.Acknowledgment: Teams in Oxford and Newcastle acknowledge thesupport of the NHS Highly Specialized Rare Mitochondrial Disordersof Adults and Children Service. The authors thank the patients andtheir families and clinicians for participating and Aviva Tolkovskyand Stephen Kennedy for their support.Study funding: This work was financed by the Lily Foundation,NewLife (SG/14-15/11), the MRC (MR/J010448/1), the WellcomeTrust (0948685/Z/10/Z), and the Angus Memorial MitochondrialFund. J.P., J.C., and C.F. have salary support from the NHS Spe-cialized Services Rare Mitochondrial Disorders Service.Disclosure: E. Dombi was funded by the Lily Foundation, the AngusMemorial Mitochondrial Fund, and the NewLife Foundation.A. Diot is funded by the MRC (MR/J010448/1) and the NewLifeFoundation. K. Morten was funded by the Williams Foundation.J. Carver was funded by the MRC (MR/J010448/1). T. Lodge wasfunded by the Wellcome Trust (0948685/Z/10/Z). C. Fratter andY. Ng report no disclosures relevant to the manuscript. C. Liao wasfunded by the Wellcome Trust (0948685/Z/10/Z). R. Muir wasfunded by the Angus Memorial Mitochondrial Fund. E. Blakely,I. Hargreaves, M. Al-Dosary, G. Sarkar, S. Hickman, S. Downes,and S. Jayawant report no disclosures relevant to the manuscript.P. Yu-Wai-Man is a Medical Research Council (MRC) ClinicianScientist and receives funding from Fight for Sight (UK) and the UKNational Institute of Health Research (NIHR) as part of the RareDiseases Translational Research Collaboration. R. Taylor is sup-ported by a Wellcome Trust Strategic Award (096919/Z/11/Z),the Medical Research Council (UK) Centre for Translational MuscleDisease Research (G0601943), and The Lily Foundation. J. Poultonis supported by the MRC (MR/J010448/1), the Wellcome Trust(0948685/Z/10/Z), NewLife (SG/14-15/11), the Lily Foundation,and the Angus Memorial Mitochondrial Fund, and has salary sup-port from the NHS Specialized Services Rare Mitochondrial Disor-ders Service. Go to Neurology.org for full disclosures. The ArticleProcessing Charge was paid by MRC and Wellcome Trust.Received June 9, 2015. Accepted in final form January 25, 2016.Correspondence to Dr. Poulton: joanna.poulton@obs-gyn.ox.ac.ukThis is an open access article distributed under the terms of theCreative Commons Attribution License 4.0 (CC BY), which permitsunrestricted use, distribution, and reproduction in any medium, pro-vided the original work is properly cited.© 2016 American Academy of NeurologyREFERENCES1. Yu-Wai-Man P, Turnbull DM, Chinnery PF. Leber hered-itary optic neuropathy. J Med Genet 2002;39:162–169.2. Howell N, Oostra RJ, Bolhuis PA, et al. Sequence analysisof the mitochondrial genomes from Dutch pedigrees withLeber hereditary optic neuropathy. Am J Hum Genet 2003;72:1460–1469.3. Giordano C, Iommarini L, Giordano L, et al. Efficientmitochondrial biogenesis drives incomplete penetrance inLeber’s hereditary optic neuropathy. Brain 2014;137:335–353.4. Diot A, Hinks-Roberts A, Lodge T, et al. A novel quanti-tative assay of mitophagy: combining high content analysisfluorescence with mitochondrial DNA mutant load to iden-tify novel pharmacological modulators of mitophagy. Phar-macol Res 2015;100:24–35.5. Granatiero V, Giorgio V, Cali T, et al. Reduced mitochon-drial Ca transients stimulate autophagy in human fibroblastscarrying the 13514A.G mutation of the ND5 subunitof NADH dehydrogenase. Cell Death Differ 2015;23:231–241.6. Klopstock T, Yu-Wai-Man P, Dimitriadis K, et al. Arandomized placebo-controlled trial of idebenone inLeber’s hereditary optic neuropathy. Brain 2011;134:2677–2686.7. Nixon RA. The role of autophagy in neurodegenerativedisease. Nat Med 2013;19:983–997.Neurology 86 May 17, 2016 1923DOI 10.1212/WNL.00000000000026882016;86;1921-1923 Published Online before print April 22, 2016Neurology Eszter Dombi, Alan Diot, Karl Morten, et al. activated mitophagyThe m.13051G>A mitochondrial DNA mutation results in variable neurology andThis information is current as of April 22, 2016ServicesUpdated Information & http://www.neurology.org/content/86/20/1921.full.htmlincluding high resolution figures, can be found at:Supplementary Material 002688.DC1.htmlhttp://www.neurology.org/content/suppl/2016/04/24/WNL.0000000000Supplementary material can be found at: References http://www.neurology.org/content/86/20/1921.full.html##ref-list-1This article cites 7 articles, 3 of which you can access for free at: Subspecialty Collections http://www.neurology.org//cgi/collection/optic_nerveOptic nerve http://www.neurology.org//cgi/collection/mitochondrial_disordersMitochondrial disorders http://www.neurology.org//cgi/collection/all_clinical_neurologyAll Clinical Neurologyfollowing collection(s): This article, along with others on similar topics, appears in the  Permissions & Licensing http://www.neurology.org/misc/about.xhtml#permissionsits entirety can be found online at:Information about reproducing this article in parts (figures,tables) or in  Reprints http://www.neurology.org/misc/addir.xhtml#reprintsusInformation about ordering reprints can be found online:rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.1951, it is now a weekly with 48 issues per year. Copyright © 2016 American Academy of Neurology. All ® is the official journal of the American Academy of Neurology. Published continuously sinceNeurology 

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The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy.

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