The heterozygous R445H mutation in OPA1 was foundin five patients with optic atrophy and deafness. Audiom-etry suggested that the sensorineural deafness resultedfrom auditory neuropathy. Skin fibroblasts showed hy-perfragmentation of the mitochondrial network, de-creased mitochondrial membrane potential, and adeno-sine triphosphate synthesis defect. In addition, OPA1 wasfound to be widely expressed in the sensory and neuralcochlear cells of the guinea pig. Thus, optic atrophy anddeafness may be related to energy defects due to a frag-mented mitochondrial network.
Ann Neurol 2005;58:958–963
Autosomal dominant optic atrophy (ADOA, or Kjer’sdisease; OMIM 165500) generally starts in childhoodand is characterized by a progressive decrease in visualacuity, color vision defect in the blue-yellow hues, lossof sensitivity in the central visual field, and optic nervepallor.1 ADOA, which generally appears as an isolated
disorder, is sometimes associated with deafness.2,3 Mu-tations in the Optic Atrophy 1 (OPA1) gene have beenfound in a majority of patients affected withADOA.4–8 OPA1 encodes a dynamin-related GTPase,located in the mitochondrial intermembrane space,which plays a key role in controlling the balance ofmitochondrial fusion and fission. It has been recentlyhypothesized that the R445H mutation of OPA1, al-tering a highly conserved residue in the GTPase do-main, could be involved in ADOA and moderate pro-gressive deafness (ADOAD)9,10 or in a more complexsyndrome including ADOA, deafness, ptosis, and oph-thalmoplegia.11 In this article, we confirm this specificgenotype–phenotype correlation by presenting fournew cases of ADOAD associated with the mutation.We performed audiological investigations in these pa-tients, analyzed the mitochondrial metabolism andmorphology of their skin fibroblasts, and studied theOPA1 expression in the cochlear cells of the guineapig.
Subjects and MethodsPatientsThe case of Patient 1, a 37-year-old French woman, has beenreported briefly elsewhere.9 In this patient, with an ADOAphenotype, a slight sensorineural deafness had appeared atage 6 years, with progressive degradation of hearing (50dB atage 15 years). Audiological examination at age 37 years con-firmed the diagnosis of a sensorineural hearing loss with aU-shaped audiometric curve on the conversational frequen-cies. Whereas auditory brainstem responses (ABRs) were ab-sent, both ears presented normal evoked otoacoustic emis-sions. Because evoked otoacoustic emissions reflect thefunctional state of presynaptic elements (the outer hair cells),and the ABRs reflect the integrity of the auditory pathwayfrom the auditory nerve to the inferior colliculus, the pres-ence of evoked otoacoustic emissions and the lack of ABRssupport the diagnosis of auditory neuropathy. Inner ear com-puted tomography scan examination was normal. Physicaland neurological examinations, as well as brain magnetic res-onance imaging, were normal. These investigations suggestedthat the hearing loss was due to an alteration of either thespiral ganglion (endocochlear) or the auditory nerve (retro-cochlear).
Patients 2 and 3 were respectively a mother and herdaughter from a family originating from Spain. In Patient 2,ADOA had been diagnosed at age 13 years. A mild to mod-erate sensorineural hearing loss (30–50dB) had been diag-nosed at age 30 years and had worsened thereafter. The firstaudiogram performed at age 33 years showed a predomi-nantly high-frequency hearing loss. In the right ear, the hear-ing loss was 20dB at 250Hz and 40dB at the highest fre-quencies. A more severe hearing loss was found in the left ear(40dB at 250Hz and 80dB at 8,000Hz). The second audio-gram, performed at age 55 years, showed a further degrada-tion of hearing. Rinne’s test showed a 30dB difference be-tween air and bone conduction, supporting a bilateral,slightly asymmetrical, mixed (conductive and neurosensorial)
From the 1Institut National de la Sante et de la Recherche MedicaleU694, Laboratoire de Biochimie et Biologie Moleculaire; 2Service deGenetique Medicale, Centre Hospitalier Universitaire, Angers;3Laboratoire de Biologie Cellulaire et Moleculaire du Controle de laProliferation Unite-Mixte de Recherche-Centre National de la Re-cherche Scientifique 5088, Universite Paul Sabatier; 4Service d’ORLet Oto-Neurologie, Centre Hospitalier Universitaire, Toulouse,France; 5Servicio de Genetica, Fundacion Jimenez Diaz, Madrid,Spain; 6Service de Genetique Medicale, Centre Hospitalier Univer-sitaire, Rennes; 7Service d’Otologie et d’Oto-Neurologie, CentreHospitalier Universitaire, Lille; 8Departement de Neurologie, Cen-tre Hospitalier Universitaire, Angers; and 9Institut National de laSante et de la Recherche Medicale U583, Hopital Saint-Eloi, Mont-pellier, France.
Received Apr 16, 2005, and in revised form Jul 12 and Aug 25.Accepted for publication Aug 26, 2005.
Published online Oct 20, 2005, in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.20681
Address correspondence to Dr Reynier, INSERM U694, Labora-toire de Biochimie et Biologie Moleculaire, Centre Hospitalier Uni-versitaire, F-49033 Angers, France. E-mail: [email protected]
deafness predominant at the high frequencies. In Patient 3,optic atrophy and hearing loss started at age 9 years. Herhearing loss had then been moderate, but she had declinedfurther audiological evaluations.
Patient 4, a 12-year-old girl of French origin, presentedbilateral optic atrophy that had been diagnosed at age 6years. Hearing loss, first found at age 7 years, progressivelyworsened. At age 12 years, pure-tone audiometry indicated abilateral perceptive hearing loss of 70dB from 125 to 750Hzand of 20dB at higher frequencies. Speech audiometry testsdemonstrated an absence of word recognition, with a scoreof 0% at a presentation level of 60 and 70dB. Electrophys-iological investigation showed abnormal and desynchronizedABR responses.
Patient 5, a 20-year-old man of French origin, presentedbilateral hearing loss that had first appeared at age 17 yearsand progressively worsened. Visual impairment started sec-ondarily to the deafness at age 19 years. At age 20 years,pure-tone audiometry showed a bilateral sensorineural hear-ing loss with a deafness of 30dB predominantly affecting thelow frequencies. Speech audiometry produced a word recog-nition score of 0% at a presentation level of 60 and 70dB.ABR showed desynchronized responses in the left ear and norecordable responses in the right ear. Acoustic otoemissionswere present bilaterally.
MethodsBlood samples and skin biopsies were taken from patientsafter obtaining informed consent. OPA1 gene analysis wasperformed by direct sequencing of the entire coding region.Skin fibroblast primary cultures were obtained from threecontrol subjects and from three patients carrying the R445Hmutation (Patients 1–3). Mitochondrial network morphol-ogy and mitochondrial membrane potential (��m) were ex-plored as described elsewhere.12 The activity of mitochon-drial respiratory chain complexes II, III, and IV, as well asthe activity of citrate synthase were determined according toRustin and colleagues.13 Adenosine triphosphate (ATP) mea-surements and ATP synthesis were performed according toOuhabi and colleagues.14 Oxygen consumption was mea-sured by polarography using the Hansatech oxygraph withthe Clark electrode. Analysis of variance, Mann–Whitney U,and Student’s t tests were used for statistical comparisons(p � 0.05). The cellular and subcellular localization ofOPA1 in cochlear cells of the guinea pig was performed oncryostat sections (10�m) from normal cochlear turns of theguinea pig incubated with three primary antibodies, that is,rabbit polyclonal anti-OPA115 (1:200), mouse monoclonalanti–cytochrome c antibody (1:200; Pharmingen Interna-tional, San Jose, CA), and goat polyclonal anti-calbindin(1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA).Fluorescence was visualized with a confocal microscope (Bio-Rad, Hercules, CA).
ResultsThe R445H OPA1 mutation was found in all five pa-tients with the ADOAD phenotype that we investi-gated. Patients 2, 3, and 4 belonged to families withADOAD, whereas Patients 1 and 5 harbored a de novomutation. The mutation was excluded in 400 control
chromosomes, and a splicing defect associated with theR445H mutation was excluded by reverse transcriptasepolymerase chain reaction. We examined the cellularand subcellular localization of OPA1 in cochlear cellsusing antibodies directed against OPA1 and cyto-chrome c (Fig 1). OPA1 immunostaining was ubiqui-tously distributed in the cochlear cells of the guineapig. The fluorescence associated with OPA1 and thecytochrome c proteins, although colocalized, differedsignificantly according to the cell type. In particular,the sensory and neuronal cells showed high OPA1 lev-els. We found an alteration of the mitochondrial net-work in skin fibroblasts from patients (Fig 2). Mito-tracker labeling showed that more than 90% of thecells carrying the R445H mutation possessed a highlyfragmented mitochondrial network. JC-1 fluorescencemeasurement in fibroblasts from patients, in compari-son with control subjects, showed a reduction of ��m
(Fig 3A). Measurement of the respiratory chain activi-ties performed in these fibroblasts did not show anydeficiency (data not shown), although we found re-duced ATP content (see Fig 3B) and ATP synthesis(see Fig 3D). The impairment of ATP synthesis wasassociated with increased oxygen consumption (see Fig3C) and mitochondrial mass, estimated by citrate syn-thase activity (see Fig 3E).
DiscussionWe present further evidence that the heterozygousR445H mutation in OPA1 is specifically involved insensorineural deafness. This mutation appearsstrongly linked with the ADOAD phenotype becauseit was found in all five patients examined in thisstudy. Moreover, deafness has never been described inpatients harboring other OPA1 mutations. However,the nature of this rare genetic disorder remains heter-ogeneous because OPA1 mutations were excluded ina large ADOAD pedigree reported previously.3 Au-diological investigations performed in three of ourfive patients indicate that the sensorineural deafnessprobably results from an auditory neuropathy. Thehigh expression level of OPA1 in the sensory andneuronal cells of guinea pig cochlea is compatiblewith this finding.
The correlation between ADOAD and the R445Hmutation in OPA1 raises the question of the mecha-nism responsible for this condition. In most celltypes, mitochondria are fused together and form theinterconnected dynamic network required for normalmitochondrial and cellular functions.15 Inactivationof the OPA1 gene expression leads to a dramatic per-turbation of the mitochondrial inner membranestructure, mitochondrial network fragmentation, andapoptosis.16 Our results demonstrate that the frag-mentation of the mitochondrial network is detectablein the skin fibroblasts of ADOAD patients and is as-
Amati-Bonneau et al: OPA1 Mutation in ADOAD 959
sociated with impaired oxidative phosphorylation,confirming data obtained in vivo in OPA1-relatedADOA patients.17 Indeed, phosphorus magnetic res-onance spectroscopy showed that the rate ofATP production was significantly impaired in skeletalmuscle.
In addition, we found that defective ATP synthesiswas associated with an increased rate of oxygen con-sumption. We therefore hypothesize that the frag-mentation of the mitochondrial network could impairthe coupling efficiency of oxidative phosphorylation,thus leading to weak ATP production. Mitochondrialcristae, which play a central role in oxidative phos-phorylation, could also be involved in this couplingdefect because these structures were found to be dras-tically disorganized in HeLa cells downregulated forOPA1.16
Defective ATP synthesis also is believed to be re-
sponsible for the mitochondrial DNA–related Leber’shereditary optic neuropathy18; it is therefore temptingto hypothesize that the energetic defect could drivea pathogenic mechanism common to these mito-chondrial hereditary optic neuropathies. Besides theenergetic defect, the fission of the mitochondrial net-work also could be an important event during theearly stages of apoptotic cell death.19 An increasedsensitivity to apoptosis has been hypothesized to ex-plain the pathogenesis of a hereditary optic atrophyassociated to cataract due to mutations in the OPA3gene.12 More generally, the alteration of the mito-chondrial network could be an important mechanismin neurodegenerative diseases. The recent finding ofmutations in mitofusin 2, a mitochondrial fusion pro-tein, in type 2A Charcot–Marie–Tooth neuropathy,20
as well as our observations, strongly support this hy-pothesis.
Fig 1. Confocal micrographs of transverse cryostat sections through the organ of Corti (oC) and the spiral ganglion (sg), stainedwith antibodies against OPA1 (1/200, red), cytochrome c (1/200, green), and calbindin (1/50, blue). (A) Low magnification ofthe upper basal turn of cochlea showing the spiral ganglion and the organ of Corti. (B) Higher magnification of the spiral gan-glion. (C) Higher magnification of the organ of Corti showing the inner hair cells (IHC) and the outer hair cells (OHCs). Notethat an intense, punctuated pattern of OPA1 (red) and cytochrome c (green) colocalized (yellow) in the IHCs and the OHCs, thesupporting cells (sc) of the oC, the spiral ganglion neurons (in the sg), and their nerve fibers (nf). No immunolabeling was seen inthe nuclei. Cochlear hair cells and one subpopulation of type I neurons were counterstained (blue) with the antibody against calbi-ndin. bm � basilar membrane; sl � spiral lamina; tc � tunnel of Corti; tm � tectorial membrane. Scale bars � 40�m (A);25�m (B); 20�m (C).
960 Annals of Neurology Vol 58 No 6 December 2005
Fig 2. Mitochondrial network morphology. Fibroblasts from three control subjects (three top lines) and from three autosomal domi-nant optic atrophy and moderate progressive deafness (ADOAD) patients (three bottom lines) carrying the R445H OPA1 muta-tion. Mitotracker labeling of control cultured fibroblasts showed that most of the cells displayed a finely interconnected mitochondrialnetwork spread uniformly in the cytoplasm. In contrast, more than 90% of the cells carrying the OPA1 R445H mutation possesseda highly fragmented mitochondrial network with a punctuated aspect.
Amati-Bonneau et al: OPA1 Mutation in ADOAD 961
This work was supported by Institut National de la Sante et de laRecherche Medicale, Groupement d’intenet Scientifique-Institut desMaladies Rares (P.A.B.), Association Francaise Centre les Myopa-thies, (C.H.), the University Hospital of Angers (Centre HospitalierUniversitaire), the University of Angers, and Retina France, (D.B.).
We are grateful to the technicians of the Molecular Biology Labo-ratory of Angers, and to K. Malkani for critical reading and com-ments on the manuscript.
References1. Kjer P. Infantile optic atrophy with dominant mode of
inheritance: a clinical and genetic study of 19 Danish families.Acta Ophtalmol Scand 1959;37(suppl 54):1–147.2.
2. Konigsmark BW, Knox DL, Hussels IE, Moses H. Dominantcongenital deafness and progressive optic nerve atrophy. ArchOphthalmol 1974;91:99–103.
3. Ozden S, Duzcan F, Wollnik B, et al. Progressive autosomaldominant optic atrophy and sensorineural hearing loss in aTurkish family. Ophthalmic Genet 2002;23:29–36.
Fig 3. Biochemical profile of mitochondria. Values are the mean of measurements performed in triplicate in three patients versusthree control fibroblast cultures. (A) ��m assessment with JC-1 dye (p � 0.01). (B) Adenosine triphosphate (ATP) content (p �0.05). ATP measurements were performed using Enliten ATP assay (Promega, Madison, WI). Luminescence was measured on aMiniluma luminometer Berthold with the use of a 10-second integration period. Standard curves, determined with ATP standards,were used to ensure linearity under these conditions. (C) Oxygen consumption (p � 0.05) was measured by polarography on perme-abilized fibroblasts (25�g/ml digitonin) using 5 � 106 cells. Respiratory rates were determined by the addition of 20mM succinateand 1mM adenosine diphosphate. (D) ATP synthesis rate was assayed by incubating fibroblasts (5 � 106 cells/1,500�l) permeabil-ized by digitonin exposure, according to Ouhabi and colleagues14 (p � 0.05). (E) Citrate synthase activity (p � 0.05).
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4. Delettre C, Lenaers G, Griffoin JM, et al. Nuclear gene OPA1,encoding a mitochondrial dynamin-related protein, is mutatedin dominant optic atrophy. Nat Genet 2000;26:207–210.
5. Alexander C, Votruba M, Pesch UE, et al. OPA1, encoding adynamin-related GTPase, is mutated in autosomal dominantoptic atrophy linked to chromosome 3q28. Nat Genet 2000;26:211–215.
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18. Baracca A, Solaini G, Sgarbi G, et al. Severe impairment ofcomplex I-driven adenosine triphosphate synthesis in Leber he-reditary optic neuropathy cybrids. Arch Neurol 2005;62:730–736.
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NonsteroidalAntiinflammatory Drug Useand the Risk for Parkinson’sDiseaseHonglei Chen, MD, PhD1,2 Eric Jacobs, PhD3
Michael A. Schwarzschild, MD, PhD4
Marjorie L. McCullough, ScD3 Eugenia E. Calle, PhD3
Michael J. Thun, MD3 and Alberto Ascherio, MD, DrPH2,5
We investigated whether nonsteroidal antiinflammatorydrug use was associated with a lower risk for Parkinson’sdisease (PD) in a large cohort of US men and women.PD risk was lower among ibuprofen users than nonusers.Compared with nonusers, the relative risks were 0.73 forusers of fewer than 2 tablets/week, 0.72 for 2 to 6.9 tab-lets/week, and 0.62 for 1 or more tablets/day (p trend �0.03). No association was found between the use of aspi-rin, other nonsteroidal antiinflammatory drugs, or acet-aminophen and PD risk. The results suggest that ibupro-fen use may delay or prevent the onset of PD.
Ann Neurol 2005;58:963–967
Evidence from both experimental and postmortemstudies suggests a role of neuroinflammation in thepathogenesis of Parkinson’s disease (PD).1,2 Consis-tently, protective effects of nonsteroidal antiinflamma-tory drugs (NSAIDs) have been observed in animalmodels of PD.3–5 We have reported previously thatnonaspirin NSAID use was associated with a lower riskfor PD.6 Here, by taking advantage of the well-established American Cancer Society’s Cancer Preven-tion Study II (CPS-II) Nutrition Cohort, we were ableto further examine the relation between NSAID useand PD risk with more detailed information on differ-ent types of NSAIDs.
From the 1Epidemiology Branch, National Institute of Environmen-tal Health Sciences, National Institutes of Health, Research TrianglePark, NC; 2Department of Nutrition, Harvard School of PublicHealth, Boston, MA; 3Epidemiology and Surveillance Research De-partment, American Cancer Society, Atlanta, GA; 4Department ofNeurology, Massachusetts General Hospital; and 5Department ofEpidemiology, Harvard School of Public Health, Boston, MA.
Received Mar 21, 2005, and in revised form Jun 2 and Aug 8.Accepted for publication Aug 8, 2005.
Published online Oct 20, 2005, in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.20682
Address correspondence to Dr Ascherio, Department of Nutrition,Harvard School of Public Health, 665 Huntington Avenue, Boston,MA 02115. E-mail [email protected]
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