ORIGINAL CONTRIBUTION Severe Impairment of Complex I–Driven Adenosine Triphosphate Synthes
ORIGINAL CONTRIBUTION
Severe Impairment of Complex I–Driven Adenosine Triphosphate Synthesis in Leber Hereditary Optic Neuropathy Cybrids
Alessandra Baracca,PhD;Giancarlo Solaini,PhD;Gianluca Sgarbi,PhD;Giorgio Lenaz,MD;Agostino Baruzzi,MD; Anthony H.V.Schapira,MD;Andrea Martinuzzi,MD,PhD;Valerio Carelli,MD,PhD
Background:Leber hereditary optic neuropathy(LHON) is a maternally inherited form of central vision loss as-sociated with mitochondrial DNA point mutations that affect the ND subunits of complex I.
Objective:To elucidate the bioenergetic conse-quences of complex I dysfunction in LHON.
Design:The biochemical phenotypes of LHON muta-tions have been investigated using the transmitochon-drial cytoplasmic hybrid(cybrid)cell model derived from the osteocarcoma parental cell https://www.wendangku.net/doc/7c1270731.html,?.
Setting:Research laboratories at neuroscience and bio-chemistry departments at the University of Bologna,Sci-entific Institute“E.Medea,”and University of College Medical School.
Participants:Fibroblast cell lines were obtained from patients affected with LHON,as defined by the pres-ence of1pathogenic mutation,and from healthy volun-teers as controls to construct cybrid cell lines.
Main Outcome Measures:Complex I(glutamate-malate)–and complex II(succinate)–dependent adeno-sine triphosphate(ATP)synthesis,their respective res-piratory rates,and total cellular ATP content were investigated using digitonin permeabilized cybrid cells. Multiple cybrid cell lines were constructed,introducing into osteosarcoma-derived rho0cells either wild-type or LHON mutant mitochondria carrying each of the3com-mon mutations at positions11778/ND4,3460/ND1,and 14484/ND6.
Results:All3LHON mutations impaired ATP synthe-sis and the respiratory control ratio driven by complex I substrates.In contrast,succinate-driven ATP synthesis, respiration rates,and respiratory control ratios were not affected.However,the defective ATP synthesis with com-plex I substrates did not result in reduced ATP cellular content,indicating a compensatory mechanism.
Conclusions:The LHON pathogenic mutations pro-foundly impair complex I–dependent synthesis of ATP, providing a common biochemical feature that may play a major role in LHON pathogenesis.Stratification of the results by mutation suggests that the11778/ND4muta-tion may induce an uncoupling of cybrid respiration, whereas the other2mutations impair the oxygen con-sumption rate.
Arch Neurol.2005;62:730-736
L EBER HEREDITARY OPTIC NEU-
ropathy(LHON)is a mater-
nally inherited form of reti-
nal ganglion cell degeneration
that leads to optic atrophy and loss of central vision prevalently in young males.1Leber hereditary optic neu-ropathy was the first human disease to be linked to a mitochondrial DNA(mtDNA) point mutation,and it is now considered to be the most common mitochondrial disor-der.2,3Three prevalent and a few rare mu-tations are established as pathogenic,and they all affect ND subunits of complex I.4 Biochemical investigations4,5of the3 most common mutations at positions 11778/ND4,3460/ND1,and14484/ND6 revealed different biochemical signatures that generally induce modest or subtle changes in measurable aspects of complex I function.Only the3460/ND1mutation showed a consistent reduction in com-plex I activity(60%-80%),6-10whereas 11778/ND4and14484/ND6mutations had
normal or slightly reduced activities(0%-25%for the11778/ND4).7-9,11-13These re-sults were essentially reproduced in the cy-brid cellular model,where only the mutant mitochondria from the patient’s cells are transferred in a perennial host cell line pre-viously depleted of mtDNA.14-17In gen-For editorial comment
see page711
Author Affiliations are listed at the end of this article.
eral,no differences were detectable between cells derived from patients with LHON and cells from unaffected car-riers of LHON mutations.6,8,9,11,13
Polarographic assessment of complex I–dependent(py-ruvate or glutamate-malate)respiration in isolated mi-tochondria from muscle,11lymphoblasts,7,17,18and LHON cybrids15,17showed variable impairment with the11778/ ND4and3460/ND1mutations.In the most extended study,17this impairment was quantified as a30%to36% decrease for the11778/ND4mutation,as a20%to28% decrease for the3460/ND1mutation,and as a10%to15% decrease for the milder14484/ND6mutation.Thus,an apparent discrepancy is observed between spectropho-tometric evaluation of complex I–specific activity and complex I–dependent respiration.9,13,17Moreover,no com-mon feature seems to characterize the3most common LHON mutations.5
Further investigations9,13,18,19that assessed the sensi-tivity of complex I to different specific inhibitors con-sistently suggested that all3LHON mutations interfere with the interaction of complex I with the ubiquinone substrate(coenzyme Q10).These results have been in-terpreted as an indication that ubiquinone binding may be affected by the LHON mutations and hence that ubi-semiquinone intermediates may have reduced stabil-ity.20At least3major downstream consequences may de-rive from complex I dysfunction in LHON:(1)respiratory function may be disturbed at the level of quinol product release because of impaired electron flow;(2)proton pumping through complex I may be defective and affect energy conservation;and(3)unstable ubisemiquinone radicals may rapidly dismutate,reacting with oxygen to increase reactive oxygen species production.
The first2hypothesized mechanisms would possibly result in a decrease in net adenosine triphosphate(ATP) synthesis driven by complex I.This issue has been poorly investigated so far.Direct measurements of ATP synthe-sis in fibroblasts that carry the3460/ND1mutation were reported as normal in1study,10whereas2recent stud-ies21,22documented that osteosarcoma-derived cybrids that carry the11778/ND4mutation had an approximately60% reduction in ATP synthesis with complex I–dependent substrates.Another study23of the ATP cellular content in lymphocytes that carry the11778/ND4mutation did not detect any difference from controls.
In the present study,we investigated ATP synthesis and ATP cellular content and cell respiration in mul-tiple osteosarcoma-derived cybrids obtained from unre-lated probands with LHON carrying each of the3com-mon LHON pathogenic mutations.
METHODS
CYBRID CELL LINES AND CULTURE
CONDITIONS
Cybrid cell lines were constructed using enucleated fibro-blasts from3control individuals and6unrelated probands with LHON as mitochondria donors and the osteosarcoma (https://www.wendangku.net/doc/7c1270731.html,–)-derived206cell line as an acceptor rho0cell line (https://www.wendangku.net/doc/7c1270731.html,–and rho0206cell lines were provided by Giuseppe Attardi,MD,and Michael King,MD).All fibroblast cell lines were established from skin biopsy samples or from umbilical cord specimens after having obtained the informed consent of patients with LHON and controls.Cell fusions of fibroblast-derived cytoplasts(enucleated fibroblasts)with the rho0206cells were performed as previously reported else-where.24Definition of the mtDNA haplogroup and identifica-tion of the LHON pathogenic mutations were performed us-ing the polymerase chain reaction/restriction fragment length polymorphism method carried out as previously reported else-where.25,26Parental and cybrid cell lines were grown in Dul-becco Modified Eagle Medium supplemented with10%fetal bovine serum,2mM levoglutamine,penicillin G sodium(100 U/mL),streptomycin sulfate(100μg/mL),and bromodeoxy-uridine(0.1mg/mL).
ATP AND CITRATE SYNTHASE ASSAYS
The ATP synthesis rate was assayed by incubating cells(5?106/ mL)permeabilized by digitonin exposure according to the method described by Ouhabi et al.27The reaction was started by adding20mM succinate(plus4μM rotenone)or10mM glu-tamate–10mM malate(plus0.6mM malonate)and0.5mM aden-osine diphosphate(ADP).Incubation was conducted for5min-utes at30°C,and the reaction was stopped by the addition of 80%(vol/vol)dimethylsulfoxide.The ATP content of the vial was measured using the luciferin-luciferase chemilumines-cent method.28The same method was used to assay cellular ATP content after extraction with80%(vol/vol)dimethylsulfoxide added to cell samples(0.5-1.0?106/mL).Citrate synthase ac-tivity was assayed essentially according to the method of Trounce et al29by incubating cell samples with0.02%(vol/vol)Triton X-100and following the reaction spectrophotometrically by mea-suring the rate of free coenzyme A release.
PROTEIN DETERMINATION AND RESPIRATION
MEASUREMENTS
The protein concentration of cybrid samples was assessed us-ing the Lowry colorimetric method in the presence of0.3%(wt/ vol)sodium deoxycholate.30Bovine serum albumin was used as the standard.
Respiratory rates of digitonin(40μg/mL)-permeabilized cell samples were measured at30°C using a Clark-type oxygen elec-trode as previously reported by Aicardi and Solaini.31The res-piratory control ratio(RCR)(respiration rate in state3–respi-ration rate in state4,where state3refers to maximal respiration, ADP induced)was evaluated using either glutamate-malate or succinate as substrate.
STATISTICAL ANALYSIS
The data are presented as mean±SD.Differences in measure-ments were evaluated using1-way analysis of variance fol-lowed by the Bonferroni posttest using a statistical software pro-gram(SigmaStat;Systat Software Inc,Point Richmond,Calif).
A value of P?.05was considered statistically significant.
RESULTS
CYBRID CELL LINES INVESTIGATED
We established cybrid cell lines from probands with LHON belonging to unrelated families carrying each of the most common pathogenic mutations at positions 11778/ND4,3460/ND1,and14484/ND6(Table1).Two
cell lines for each mutation were available for experi-ments,and the cybrid clones used herein are the same as those used in previous studies.14,21,25,26,32Each cybrid cell line was stably homoplasmic for the LHON patho-genic mutation,as verified by regular checks of the mu-tation-specific polymerase chain reaction/restriction frag-ment length polymorphism pattern.Furthermore,LHON and control cybrid cell lines were defined for their mtDNA haplogroup(Table1).
ATP SYNTHESIS
We tested the efficiency of oxidative phosphorylation in LHON and control cybrids by assaying the ATP syn-thase activity driven by complex I(glutamate-malate)and complex II(succinate)substrates.The rate of ATP syn-thesis was sharply reduced(?10%residue)in cells car-rying the3460/ND1and14484/ND6mutations with nico-tinamide adenine dinucleotide–dependent substrates, which implies electron transfer through complex I, whereas a milder reduction(35%residue)was observed with the11778/ND4mutation(Figure1A).When the substrate was succinate,implying electron transfer through complex II,the ATP synthesis rate was not af-fected in LHON cybrids even if a nonsignificant ten-dency toward a reduction was observed with the11778/ ND4and14484/ND6mutations(Figure1A).
The assay of citrate synthase in LHON cybrids,used as a well-known marker of mitochondrial mass content, indicated a significant decrease(–21%)for the11778/ ND4mutation compared with control cybrids(Figure1B). This finding was somewhat surprising given that slight mitochondrial proliferation has previously been re-ported in patient-derived tissues,such as platelets,6,8lym-phoblasts,7,18fibroblasts,10and skeletal muscle.11We then corrected the ATP synthesis rate for citrate synthase ac-tivity(ATP synthase–citrate synthase activity ratio),con-firming the result of dramatic reductions with the glu-tamate-malate–driven oxidative phosphorylation for all LHON mutations(Figure1C).The11778/ND4muta-tion remained the least affected in ATP synthesis(–60%). Correcting for citrate synthase activity,the succinate-driven oxidative phosphorylation did not substantially change the previous results(Figure1C).
TOTAL CELLULAR ATP CONTENT
To assess the bioenergetics of LHON cybrids,we also de-termined the cellular ATP content.Figure2shows a normal level of ATP in the11778/ND4cybrid,whereas the14484/ND6and3460/ND1cybrids were slightly re-duced compared with control cybrids.These reductions did not reach statistical significance(P=.36for3460/ ND1;P?.99for14484/ND6,with respect to controls).
CYBRID OXYGEN
CONSUMPTION RATE
Our results on ATP synthesis driven by complex I–de-pendent substrates showed a profound defect in LHON cybrids,with the11778/ND4mutation being the least af-fected.This functional defect in oxidative phosphoryla-tion may imply that the enzyme is incapable of either transporting electrons and/or translocating protons through the inner mitochondrial membrane.20To clarify which of the2proposed mechanisms was effective,the
Figure1.Adenosine triphosphate(ATP)synthesis rates with complex I (glutamate-malate)–and complex II(succinate)–dependent substrates.A, Mean rate of ATP synthesis of digitonin-permeabilized cybrids homoplasmic for the3common Leber hereditary optic neuropathy mutations and control cybrids.B,Mean citrate synthase–specific activity of the cybrids investigated.C,The ATP synthase activity reported as the mean ATP–citrate synthesis rate ratio to correct the enzyme activity for the mitochondrial mass of cybrid samples.Asterisk indicates P?.01;dagger,P?.05.Details of the methods are given in the“Methods”section.Error bars represent standard deviations.
Abbreviation:LHON,Leber hereditary optic neuropathy.
malate as substrates were all statistically significantly re-duced compared with the control cybrids(Figure3C and Table2).The RCR faithfully expresses mitochondrial function and hence cellular ATP synthesis capability through oxidative phosphorylation.In our hands,the RCR values reflected the results of ATP synthesis driven by complex I–dependent substrates,with3460/ND1and
14484/ND6cybrids being similarly reduced and the 11778/ND4cybrid being the least affected.
When the respiratory substrate was succinate,the oxy-gen consumption rate was barely affected in either state 3or state4respirations(Figure4A and B and Table3), and the RCR values seem to be affected only to a minor extent,with no statistical significance(state4respira-tion rate:P?.99for3460/ND1,P=.26for11778/ND4, and P?.99for14484/ND6;state3repsiration rate:P?.99 for3460/ND1,P?.99for11778/ND4,and P?.99for 11484/ND6;RCR:P?.99for3460/ND1,P=.42for11778/ ND4,and P=.89for14484/ND6,with respect to the cor-responding controls(Figure4C).
COMMENT
Expanding on previous results limited to the11778/ ND4mutation,21,22this study shows that all the com-mon LHON pathogenic mutations severely impair com-plex I–driven ATP synthesis compared with control cybrids.The reduction was less pronounced with the 11778/ND4mutation.This impairment was not com-pensated efficiently by the parallel succinate pathway,as frequently seen in patient-derived tissues or cell lines.6,7,11,33 In fact,complex II–driven ATP synthesis was essen-tially unaffected by all3LHON mutations.However,total cellular ATP content of LHON cybrids was not signifi-cantly decreased.Thus,despite a potentially harmful de-fective ATP synthesis through oxidative phosphoryla-tion,a metabolic balance seems to be maintained by LHON cybrids in glucose medium,mostly by glycolytic ATP pro-duction.34This was also confirmed by the lack of mito-chondrial proliferation in LHON cybrids,as indicated by normal citrate synthase activity.Variable levels of mito-chondrial proliferation are normally seen in patient-derived tissues.6-8,10,11,18
Figure3.Mean rates of state4(A)and state3(B)respiration and respiratory control ratios(RCRs)(C)of cybrid cells with complex
I–dependent substrates.Respiration rates of state4and state3are reported after normalization of the polarographic oxygen consumption rates to citrate synthase activity of the corresponding cell samples.The substrates for respiration were10mM glutamate plus10mM malate,and0.6mM malonate was present to inhibit complex II.Asterisk indicates P?.01;dagger,P?.05. Error bars represent standard deviations.
To better clarify how the ATP synthesis defect is gen-erated by complex I dysfunction,we also studied respi-ratory rates using complex I–and complex II–dependent substrates.The respiratory behavior of the11778/ND4mutation seems to differ from the other2mutations, suggesting a possible uncoupling effect on complex I function.This interpretation is based on the observation that ADP-stimulated respiration(state3)with the 11778/ND4mutation is normal,whereas state4respira-tion is significantly enhanced.On the contrary,state3 respiration was reduced with3460/ND1and14484/ND6 mutations,and state4respiration was similar to that of controls.Our results are consistent with those previ-ously reported in a different WAL-2A–derived cybrid cell system.17Overall,the best fit between our polaro-graphic respiration experiments and the luminometric ATP synthesis rate was observed considering the RCR with complex I–dependent substrates.In fact,all3 LHON mutant cybrids were significantly reduced com-pared with controls,with the11778/ND4mutation being the least affected,paralleling the ATP synthesis results.
These results obtained in LHON cybrids do not match the severity of the disease in patients and the results of other biochemical measurements.Considering the rate of spontaneous visual recovery in patients with LHON, the phenotypic severity would decrease from the11778/ ND4?3460/ND4?14484/ND6mutation.1,4However, considering the impairment of complex I activity,as mea-sured spectrophotometrically in patient-derived tis-sues,cell lines,or cybrids,the severity would decrease from the3460/ND1?11778/ND4?14484/ND6muta-tion.5,17Based on the present results on complex I–driven ATP synthesis,the severity of the biochemical phe-notype would decrease from the14484/ND6?3460/ ND1?11778/ND4mutation.These latter data match our results on the rate of apoptotic cell death of LHON cy-brids when glucose is replaced by galactose in the me-dium,with the11778/ND4mutation being the least se-vere.32In line with these findings,in vivo assessment by phosphorus31magnetic resonance spectroscopy of the maximum rate of ATP production in skeletal muscle also revealed a53%residual rate with the14484/ND6muta-tion compared with controls.35In contrast,the3460/ ND1mutation had a normal rate,whereas the11778/ ND4mutation was more severely reduced to27%of controls.36
The biochemical effect of LHON pathogenic muta-tions has been studied for more than a decade and still remains controversial.37This study clarifies that the ATP synthesis defect that depends on complex I substrates is severe with all3common LHON mutations.However, there is also evidence that the cells may effectively com-pensate for this impairment in most human tissues,as also happens in our cell system.This leads to the ab-sence of any evident pathologic features in most indi-viduals with the homoplasmic LHON mutation.How-ever,the compensation is probably an unstable equilibrium that may be upset by various factors,such as the frequently suggested nuclear modifying genes and environmental triggers.On the other hand,the rare re-ports of LHON cases complicated by a syndromic neu-rologic involvement,such as Leigh disease,38multiple scle-rosis–like features,39cerebellar atrophy,40or dystonia associated with basal ganglia lesions,11all support the pos-sibility that the energetic failure may spread.
*P?.01.
?P?.05.
Figure4.Mean rates of state4(A)and state3(B)respiration and respiratory control ratios(RCRs)(C)of cybrid cells with complex
II–dependent substrate.Respiration rates of state4and state3are reported after normalization of the polarographic oxygen consumption rates to citrate synthase activity of the corresponding cell samples.The substrate for respiration was20mM succinate,and4μM rotenone was present to inhibit complex I.Error bars represent standard deviations.
There is increasing evidence that the nuclear gene ex-pression may profoundly modify the pathogenic expres-sion of the LHON mutations.This has been shown in cel-lular studies16and with phosphorus31magnetic resonance spectroscopy investigation of different tis-sues in the same patients carrying the3460/ND1muta-tion.36Thus,incongruent results among different stud-ies performed in different cell types or patient-derived tissues may depend on nuclear gene expression.We also noted this feature having assayed the complex I–depen-dent ATP synthesis of lymphocyte mitochondria from the same patient homoplasmic for the3460/ND1mutation used to generate1of the cybrid cell lines reported herein
(HMM in Table1;also,patient II-1in the study by Lodi et al36).The reduction amounted to approximately30% compared with controls,which was a much milder de-fect than that observed in its cybrids(A.B.,G.Solaini, G.Sgarbi,and V.C.,unpublished data,2003).
The present results are not in contrast with the cur-rently most widely accepted hypothesis that besides an ATP defect,overproduction of reactive oxygen species may rep-resent a major element in the pathogenesis of LHON.4,5 In the long term,bioenergetically compensated cells may experience chronic reactive oxygen species overproduc-tion,and the compromised oxidative phosphorylation in LHON may play a major role once the threshold for cell death is crossed.The present model of apoptotic cell death of LHON cybrids grown in galactose32shows a rapid course of cellular ATP depletion,which precipitates a caspase-independent mode of apoptosis.41The exact understand-ing of the reciprocal role of ATP defective synthesis and reactive oxygen species production will be crucial to de-sign appropriate therapeutic approaches,and the current evidence of some differences in the pathophysiologic mechanism of LHON mutations may prove to be impor-tant for the administration of agents such as coenzyme Q10 or the quinone analog idebenone.
Accepted for Publication:July7,2004.
Author Affiliations:Dipartimento di Biochimica(Drs Baracca,Solaini,and Lenaz)and Dipartimento di Sci-enze Neurologiche(Drs Baruzzi and Carelli),Univer-sity of Bologna,Bologna,Italy;Scuola Superiore di Studi Universitari e di Perfezionamento S Anna,Pisa,Italy(Dr Sgarbi);University Department of Clinical Neurosci-ences,Royal Free and University College Medical School, London,England(Dr Schapira);and Scientific Institute “E.Medea,”Conegliano,Treviso,Italy(Dr Martinuzzi). Correspondence:Valerio Carelli,MD,PhD,Diparti-mento di Scienze Neurologiche,Universitàdi Bologna, Via Ugo Foscolo7,40123Bologna,Italy(carelli@neuro .unibo.it).
Author Contributions:Study concept and design:Baracca, Solaini,Lenaz,Baruzzi,Schapira,Martinuzzi,and Carelli. Acquisition of data:Baracca and Sgarbi.Analysis and in-terpretation of data:Baracca,Solaini,Sgarbi,and Carelli. Drafting of the manuscript:Carelli.Critical revision of the manuscript for important intellectual content:Baracca,So-laini,Sgarbi,Lenaz,Baruzzi,Schapira,and Martinuzzi. Statistical analysis:Baracca and Sgarbi.Obtained fund-ing:Carelli.Study supervision:Solaini,Lenaz,Baruzzi, Schapira,and Martinuzzi.Funding/Support:This study was funded by grant GGP02323from Telethon Fondazione Onlus,Rome,Italy (Dr Carelli).
REFERENCES
1.Newman NJ.Leber’s optic neuropathy.In:Miller NR,Newman NJ,eds.Walsh
and Hoyt’s Clinical Neuro Ophthalmology.Baltimore,Md:Williams&Wilkins, 1998:742-753.
2.Wallace DC,Singh G,Lott MT,et al.Mitochondrial DNA mutation associated with
Leber’s hereditary optic neuropathy.Science.1988;242:1427-1430.
3.Man PY,Griffiths PG,Brown DT,Howell N,Turnbull DM,Chinnery PF.The epi-
demiology of Leber hereditary optic neuropathy in the North East of England.
Am J Hum Genet.2003;72:333-339.
4.Carelli V,Ross-Cisneros FN,Sadun AA.Mitochondrial dysfunction as a cause of
optic neuropathies.Prog Retin Eye Res.2004;23:53-89.
5.Brown MD.The enigmatic relationship between mitochondrial dysfunction and
Leber’s hereditary optic neuropathy.J Neurol Sci.1999;165:1-5.
6.Howell N,Bindoff LA,McCullough DA,et al.Leber hereditary optic neuropathy:
identification of the same mitochondrial ND1mutation in six pedigrees.Am J Hum Genet.1991;49:939-950.
7.Majander A,Huoponen K,Savontaus ML,Nikoskelainen E,Wikstrom M.Elec-
tron transfer properties of NADH:ubiquinone reductase in the ND1/3460and the ND4/11778mutations of the Leber hereditary optic neuroretinopathy(LHON).
FEBS Lett.1991;292:289-292.
8.Smith PR,Cooper JM,Govan GG,Harding AE,Schapira AH.Platelet mitochon-
drial function in Leber’s hereditary optic neuropathy.J Neurol Sci.1994;122: 80-83.
9.Carelli V,Ghelli A,Ratta M,et al.Leber’s hereditary optic neuropathy:biochemi-
cal effect of11778/ND4and3460/ND1mutations and correlation with the mi-tochondrial genotype.Neurology.1997;48:1623-1632.
10.Cock HR,Cooper JM,Schapira AHV.Functional consequences of the3460-bp
mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy.
J Neurol Sci.1999;165:10-17.
https://www.wendangku.net/doc/7c1270731.html,rsson NG,Andersen O,Holme E,Oldfors A,Wahlstrom J.Leber’s hereditary
optic neuropathy and complex I deficiency in muscle.Ann Neurol.1991;30: 701-708.
12.Cock HR,Cooper JM,Schapira AHV.The14484ND6mtDNA mutation in Leber
hereditary optic neuropathy does not affect fibroblast complex I activity[letter].
Am J Hum Genet.1995;57:1501-1502.
13.Carelli V,Ghelli A,Bucchi L,et al.Biochemical features of mtDNA14484(ND6/
M64V)point mutation associated with Leber’s hereditary optic neuropathy.Ann Neurol.1999;45:320-328.
14.Vergani L,Martinuzzi A,Carelli V,et al.MtDNA mutations associated with Leb-
er’s hereditary optic neuropathy:studies on cytoplasmic hybrid(cybrid)cells.
Biochem Biophys Res Commun.1995;210:880-888.
15.Hofhaus G,Johns DR,Hurko O,Attardi G,Chomyn A.Respiration and growth
defects in transmitochondrial cell lines carrying the11778mutation associated with Leber’s hereditary optic neuropathy.J Biol Chem.1996;271:13155-13161.
16.Cock HR,Tabrizi SJ,Cooper JM,Schapira AHV.The influence of nuclear back-
ground on the biochemical expression of3460Leber’s hereditary optic neuropathy.
Ann Neurol.1998;44:187-193.
17.Brown MD,Trounce IA,Jun AS,Allen JC,Wallace DC.Functional analysis of lym-
phoblast and cybrid mitochondria containing the3460,11778,or14484Leb-er’s hereditary optic neuropathy mitochondrial DNA mutation.J Biol Chem.2000;
275:39831-39836.
18.Majander A,Finel M,Savontaus ML,Nikoskelainen E,Wikstrom M.Catalytic ac-
tivity of complex I in cell lines that possess replacement mutations in the ND genes in Leber’s hereditary optic neuropathy.Eur J Biochem.1996;239:201-207.
19.Ghelli A,Degli Esposti M,Carelli V,Lenaz G.Changes in mitochondrial complex
I activity and coenzyme Q binding site in Leber’s hereditary optic neuropathy
(LHON).Mol Aspects Med.1997;18(suppl):S263-S267.
20.Degli Esposti M,Carelli V,Ghelli A,et al.Functional alterations of the mitochon-
drially encoded ND4subunit associated with Leber’s hereditary optic neuropathy.
FEBS Lett.1994;352:375-379.
21.Guy J,Qi X,Pallotti F,et al.Rescue of a mitochondrial deficiency causing Leber
Hereditary Optic Neuropathy.Ann Neurol.2002;52:534-542.
22.Komaki H,Akanuma J,Iwata H,et al.A novel mtDNA C11777A mutation in Leigh
syndrome.Mitochondrion.2003;2:293-304.
23.Yen MY,Lee JF,Liu JH,Wei YH.Energy charge is not decreased in lymphocytes
of patients with Leber’s hereditary optic neuropathy with the11778mutation.
J Neuroophthalmol.1998;18:84-85.
24.King MP,Attardi G.Human cells lacking mtDNA:repopulation with exogenous
mitochondria by complementation.Science.1989;246:500-503.
25.Danielson SR,Wong A,Carelli V,Martinuzzi A,Schapira AH,Cortopassi GA.
Cells bearing mutations causing Leber’s hereditary optic neuropathy are sensi-tized to Fas-induced apoptosis.J Biol Chem.2002;277:5810-5815.
26.Carelli V,Vergani L,Bernazzi B,et al.Respiratory function in cybrid cell lines
carrying European mtDNA haplogroups:implications for Leber’s hereditary op-tic neuropathy.Biochim Biophys Acta.2002;1588:7-14.
27.Ouhabi R,Boue-Grabot M,Mazat JP.Mitochondrial ATP synthesis in permeabi-
lized cells:assessment of the ATP/O values in situ.Anal Biochem.1998;263: 169-175.
28.Stanley PE,Williams https://www.wendangku.net/doc/7c1270731.html,e of the liquid scintillation spectrometer for deter-
mining adenosine triphosphate by the luciferase enzyme.Anal Biochem.1969;
29:381-392.
29.Trounce IA,Kim YL,Jun AS,Wallace DC.Assessment of mitochondrial oxida-
tive phosphorylation in patient muscle biopsies,lymphoblasts,and transmito-chondrial cell lines.Methods Enzymol.264.1996:484-509.30.Lowry OH,Rosebrough NJ,Lewis Farr A,Randall RJ.Protein measurement with
the Folin phenol reagent.J Biol Chem.1951;193:265-275.
31.Aicardi G,Solaini G.Effects of niridazole and5-nitroimidazoles on heart mito-
chondrial respiration.Biochem Pharmacol.1982;31:3703-3705.
32.Ghelli A,Zanna C,Porcelli AM,et al.Leber’s hereditary optic neuropathy(LHON)
pathogenic mutations induce mitochondrial-dependent apoptotic death in trans-mitochondrial cells incubated with galactose medium.J Biol Chem.2003;278: 4145-4150.
33.Yen MY,Lee HC,Liu JH,Wei https://www.wendangku.net/doc/7c1270731.html,pensatory elevation of complex II activity
in Leber’s hereditary optic neuropathy.Br J Ophthalmol.1996;80:78-81. 34.Gajewski CD,Yang L,Schon EA,Manfredi G.New insights into the bioenerget-
ics of mitochondrial disorders using intracellular ATP reporters.Mol Biol Cell.
2003;14:3628-3635.
35.Lodi R,Taylor DJ,Tabrizi SJ,et al.In vivo skeletal muscle mitochondrial func-
tion in Leber’s hereditary optic neuropathy assessed by31P magnetic reso-nance spectroscopy.Ann Neurol.1997;42:573-579.
36.Lodi R,Carelli V,Cortelli P,et al.Phosphorus MR spectroscopy shows a tissue
specific in vivo distribution of biochemical expression of the G3460A mutation in Leber’s hereditary optic neuropathy.J Neurol Neurosurg Psychiatry.2002;
72:805-807.
37.Howell N.LHON and other optic nerve atrophies:the mitochondrial connection.
Dev Ophthalmol.2003;37:94-108.
38.Funalot B,Reynier P,Vighetto A,et al.Leigh-like encephalopathy complicating
Leber’s hereditary optic neuropathy.Ann Neurol.2002;52:374-377.
39.Harding AE,Sweeney MG,Miller DH,et al.Occurrence of a multiple sclerosis–
like illness in women who have a Leber’s hereditary optic neuropathy mitochon-drial DNA mutation.Brain.1992;115:979-989.
40.Funakawa I,Kato H,Terao A,et al.Cerebellar ataxia in patients with Leber’s he-
reditary optic neuropathy.J Neurol.1995;242:75-77.
41.Zanna C,Ghelli A,Porcelli AM,Carelli V,Martinuzzi A,Rugolo M.Apoptotic cell
death of cybrid cells bearing Leber’s hereditary optic neuropathy mutations is caspase independent.Ann N Y Acad Sci.2003;1010:213-217.