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Deficit of in vivo mitochondrial ATP production in OPA1-related dominant optic atrophy.

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Deficit of In Vivo
Mitochondrial ATP
Production in OPA1-Related
Dominant Optic Atrophy
Raffaele Lodi, MD,1 Caterina Tonon, MD,1
Maria Lucia Valentino, MD,2 Stefano Iotti, PhD,1
Valeria Clementi, PhD,1 Emil Malucelli, PhD,1
Piero Barboni, MD,3 Lora Longanesi, MD,4
Simone Schimpf, MS,5 Bernd Wissinger, PhD,5
Agostino Baruzzi, MD,2 Bruno Barbiroli, MD,1
and Valerio Carelli, MD, PhD2
Dominant optic atrophy has been associated with mutations in the OPA1 gene, which encodes for a dynaminrelated GTPase, a mitochondrial protein implicated in
the formation and maintenance of mitochondrial network and morphology. We used phosphorus magnetic
resonance spectroscopy to assess calf muscle oxidative
metabolism in six patients from two unrelated families
carrying the c.2708-2711delTTAG deletion in exon 27 of
the OPA1 gene. The rate of postexercise phosphocreatine
resynthesis, a measure of mitochondrial adenosine
triphosphate production rate, was significantly delayed in
the patients. Our in vivo results show for the first time to
our knowledge a deficit of oxidative phosphorylation in
OPA1-related DOA.
Ann Neurol 2004;56:719 –723
Dominant optic atrophy (DOA),1 the most common
form of hereditary optic neuropathy with a disease
prevalence ranging between 1 to 10,000 and 1 to
50,000 in different populations,2 is a progressive disorder characterized by early-onset insidious bilateral loss
of central vision. There is a wide variability, even
within the same family, in severity of the visual defect,
which may range from asymptomatic carriers to legally
blind patients.1,2 In DOA patients, pathological examination shows retinal ganglion cell degeneration associ-
From the 1Dipartimento di Medicina Clinica e Biotecnologia Applicata, 2Dipartimento di Scienze Neurologiche, Universita di Bologna, Bologna; 3Centro di Oftalmologia Salus, Bologna; 4Ophthalmic Operating Unit, Lugo Hospital, Ravenna, Italy; and
Molekulargenetisches Labor, Universitats-Augenklinik, Tuebingen,
Received Mar 4, 2004, and in revised form Jul 2. Accepted for
publication Jul 22, 2004.
Published online Oct 28, 2004, in Wiley InterScience
( DOI: 10.1002/ana.20278
Address correspondence to Dr Lodi, Dipartimento di Medicina
Clinica e Biotecnologia Applicata “D. Campanacci,” Universita di
Bologna, Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna,
Italy. E- mail:
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
ated with loss of myelinated fibers and gliosis in the
optic nerves.3,4
Recently, mutations in the OPA1 gene on chromosome 3q28 have been shown to be pathogenic for
DOA,5,6 with the c.2708-2711delTTAG microdeletion
in exon 27 being the most predominant OPA1 mutation in white DOA patients.2 OPA1 encodes a ubiquitously expressed, large, dynamin-related GTPase that
is anchored to the mitochondrial membranes and implicated in the formation and maintenance of mitochondrial network and morphology.2 Most mutations
induce truncation of the OPA1 protein, and haploinsufficiency is believed to be the major disease mechanism in DOA. Downregulation of OPA1 gene expression by RNA interference experiments in HeLa cells
was recently reported to induce fragmentation of the
mitochondrial network, dissipation of the mitochondrial membrane potential, drastic disorganization of the
cristae, cytochrome c release and caspase-dependent apoptotic cell death.7
In view of evidence of the critical role for mitochondrial functions of the OPA1 gene product, we used in
vivo phosphorus magnetic resonance spectroscopy (31PMRS) to test for the presence of mitochondrial dysfunction in skeletal muscle of six OPA1-related DOA
patients. Skeletal muscle is an ideal tissue to assess in
vivo mitochondrial adenosine triphosphate (ATP) production rate by means of 31P-MRS as it can be studied
conveniently at rest, during exercise, and in the subsequent aerobic recovery phase.8,9 We document that the
presence of a mutant OPA1 allele induces an oxidative
phosphorylation defect.
P-MRS investigations were performed in a 1.5T GE
Medical Systems Signa Horizon LX whole-body scanner, as
previously described.9 Subjects lay supine with a 6cmdiameter surface coil centered on the maximal circumference
of the right calf muscle. Spectra were acquired, with a repetition time of 5 seconds, at rest (one 64-scan spectrum), during an aerobic incremental exercise (12-scan spectra), and
during the following recovery (32 two-scan spectra). The
muscle was exercised by plantar flexion at 0.66Hz against a
pedal using a pneumatic ergometer.10 The force resistance of
the pedal was 10% of lean body mass.8 After two spectra, the
resistance was increased by 5% of lean body mass for each
subsequent spectral acquisition. As soon as the last 12-scan
exercise spectrum was collected, the exercise was stopped,
and data were collected for 5⬘20⬘⬘. Spectra were postprocessed by a time-domain fitting program (AMARES/MRUI),
and the concentrations of inorganic phosphate (Pi) and
phosphocreatine (PCr) were calculated by assuming a normal
ATP concentration of 8mM.9 Intracellular pH was calculated
from the chemical shift of Pi relative to PCr.9 For statistical
purposes, the phosphorylation potential (PP)11 was expressed
as its reciprocal, ([Pi] ⫻ [ADP])/[ATP], where [ADP] (adenosine diphosphate) was calculated from the creatine kinase
The rate of PCr recovery was calculated from the monoexponential equation best fitting the experimental points, reported as time constants (TCs) as a function of the minimum cytosolic pH10 and then normalized to pH ⫽ 7.00.9
Data are presented as means ⫾ SD. Statistical significance,
determined by the Student t test for unpaired data, was
taken as p ⬍ 0.05.
Patients and Methods
We studied six patients (four women aged 43 ⫾ 20 years;
mean ⫾ SD [standard deviation]; range, 18 –70 years) from
two Italian families with OPA1-related DOA (Fig 1), six unrelated healthy subjects (four women; mean age, 41 ⫾ 17
years; range, 20 – 69 years), and two patients (one man, 55
years old, and one woman, 64 years old) from a third Italian
family with DOA negative for mutations in the OPA1 gene.
Ophthalmological examination showed bilateral optic atrophy in all patients with temporal to completely pale optic
discs and centrocecal visual field defect. Visual acuity was
0.05 in Case II-4, 0.16 in Case IV-2, and 0.63 in Cases II-8
and III-1 of Family A, and it was 0.05 in Cases II-3 and
III-2 of Family B. Visual acuity was 0.2 and 0.32, respectively, in the two patients with OPA1-unrelated DOA. In all
eight DOA patients, the centrocecal localization of the visual
defect did not interfere substantially with their working or
their daily physical activity that was similar to that of healthy
controls. Blood DNA was extracted by standard phenolchloroform method, and direct sequencing of polymerase
chain reaction–amplified genomic DNA covering exons 1 to
28 of the OPA1 gene was performed as previously described.6 Informed consent for this study was obtained from
each patient and normal volunteer.
Annals of Neurology
Vol 56
No 5
November 2004
Fig 1. Pedigrees of the two OPA1-related dominant optic
atrophy (DOA) families investigated. The full black symbols indicate the affected individuals. In Family A, no clinical information was available on visual function of the
parents in first generation. The arrows indicate the six individuals who underwent 31P-MRS.
All patients from Families A and B were heterozygotes
for the pathogenic four-base deletion c.27082711delTTAG in exon 27, the most common mutant
allele in OPA1-related DOA.2 The two patients from
the third DOA pedigree (not shown) did not bear mutations in the OPA1 gene, which was also excluded by
linkage analysis (V. Carelli and B. Wissinger, unpublished results).
The resting [PCr] in the OPA1-related DOA patients (mean ⫾ SD, 27.9mM ⫾ 2.36) was significantly lower than in controls (31.3 ⫾ 1.53; p ⬍ 0.05;
Fig 2), whereas [Pi] and cytosolic pH were similar in
patients and controls (data not shown). A reduction
in skeletal muscle [PCr] is frequently present in patients carrying mtDNA mutations8,9,12 and is an indicator of altered cellular bioenergetic state. Moreover,
phosphorylation potential (PP; 15mM ⫾ 4, expressed
as 1/PP ⫻ 106) compared with controls (10 ⫾ 2; p ⬍
0.05). However, the rate of muscle oxidative metabolism is very low at rest, and a better assessment can
be obtained by stimulating mitochondrial respiration
with an in-magnet aerobic exercise protocol. In these
experimental conditions, the rate of oxidative ATP
production can be precisely assessed from the postexercise PCr resynthesis rate, which is entirely oxidative.9,10 In the OPA1 patients, the time constant of
postexercise phosphocreatine resynthesis (28 ⫾ 7 seconds) was significantly higher than in controls (19 ⫾
4 seconds, p ⫽ 0.01; Fig 3), indicating a reduced rate
of mitochondrial ATP production in the patients. In
Fig 2. Calf muscle 31P-MRS spectra at rest (64 free-induction
decays) from Case II-3: Family B (right) and a matched control (left). In the patient, the phosphocreatine (PCr) peak is
decreased (relative to the ␤-ATP peak). Pi ⫽ inorganic phosphate; ␥, ␣, and ␤ are the three phosphate groups of ATP.
The abscissa reports the chemical shift in parts per million
(ppm) and ordinate the relative signal intensity in arbitrary
Fig 3. Mean phosphocreatine (PCr) postexercise recovery time
constant (TC) in controls and patients with the c.27082711delTTAG mutation in the OPA1 gene. *p ⬍ 0.05 versus controls.
the two patients lacking an OPA1 gene mutation,
P-MRS variables at rest were all normal (data not
shown). Similarly, the postexercise time constant of
phosphocreatine resynthesis (21.5 and 21.6 seconds)
was within 1 SD of the normal mean.
We show for the first time to our knowledge that the
c.2708-2711delTTAG four-base deletion in exon 27 of
the OPA1 gene is associated with a reduced rate of mitochondrial ATP synthesis in the skeletal muscle of
DOA patients. This OPA1 mutation induces a frameshift at amino acid residue Val903 with the introduction of a premature stop at codon 905.5 This results in
a truncated protein lacking 56 C-terminal amino acids,
which eventually leads to haploinsufficiency. Immunolabeling experiments of monocytes from DOA patients
carrying the c.2708-2711delTTAG mutation showed
an abnormal structure of the mitochondrial network
compared with control monocytes.5 The dynaminrelated GTPase encoded by the OPA1 gene may occur
in different isoforms as a result of splicing variants.13
According to one study, the OPA1 protein localizes to
the mitochondrial inner membrane, facing the intermembrane space.14 However, a subsequent study reported that, depending on the isoform considered, the
OPA1 protein may predominantly associate with the
inner (93kDa isoform) or the outer (88kDa isoform)
mitochondrial membrane, with both isoforms facing
the intermembrane space.15
OPA1-targeting RNA interference experiments in
HeLa cells resulted in fragmentation of the mitochondrial network, drastic changes of the cristae morphology, and dissipation of mitochondrial membrane potential.7 Destabilization of the inner mitochondrial
membrane and disorganization of the mitochondrial
network are hypothesized to occur also with the OPA1
mutations in DOA, and they may result in abnormal
Lodi et al: Mitochondrial Dysfunction in DOA
assembly and function of the respiratory complexes as
well as downstream ATP synthesis. Inactivation of the
Msp1 gene, a fission yeast ortholog of OPA1, consistently resulted in a profound reduction of mitochondrial respiratory capacity as a result of loss of
mtDNA.16 However, the putative involvement of
OPA1 in mtDNA maintenance has not been studied
yet in DOA patients. Northern analysis of human tissues showed that the highest transcript level of the
OPA1 gene was detected in retina, brain, testis, and
cardiac and skeletal muscle.6 Our novel finding of in
vivo impairment of ATP synthesis in skeletal muscle
from DOA patients carrying the c.27082711delTTAG mutant allele points to the central role
of mitochondrial dysfunction in the pathophysiology
of OPA1-related DOA.
Our current in vivo 31P-MRS results present striking similarities with our findings in patients with
Leber’s hereditary optic neuropathy (LHON), a mitochondrial disorder caused by mtDNA point mutations in complex I subunits, characterized by a deficit
of oxidative phosphorylation and retinal ganglion cell
degeneration.17 In the skeletal muscle of subjects carrying one of the most common LHON pathogenic
mutations (at position 11778, 14484, or 3460), we
found, as in OPA1-related DOA patients, reduced
[PCr] and PP at rest and slow postexercise PCr resynthesis rate.8,9,18
Despite the distinct genetic defect in DOA and
LHON, and the remarkable difference in clinical evolution, the end point of the pathological process is
indistinguishable.16 The few histopathological reports
in DOA and LHON demonstrate an identical selective loss of retinal ganglion cells in the retina, with
prevalent involvement of those serving central vision.3,4,16 The ultimate result of OPA1 suppression in
HeLa cells has been shown to be the release of cytochrome c and the activation of a caspase-dependent
apoptotic cell death.7 It has been reported recently
that transmitochondrial cytoplasmic hybrid (cybrid)
cells carrying the LHON mtDNA point mutations
underwent cytochrome c release and apoptotic cell
death once glucose was replaced by galactose in the
culture medium.19 This is a well-established method
to force cells to rely only on oxidative phosphorylation for ATP synthesis. Thus, a final path of mitochondrial dysfunction in DOA and LHON may be a
common predisposition of neuronal cells to apoptotic
death.7,19 This and other possible common consequences of mitochondrial dysfunction in DOA and
LHON, such as reactive oxygen species overproduction or the organization of mitochondrial network in
the energy-dependent unmyelinated portion of the
optic nerve, need to be carefully explored to unwrap
the pathogenetic peculiarities of these mitochondrial
optic neuropathies.17
Annals of Neurology
Vol 56
No 5
November 2004
Future studies will show whether treatments aimed
at enhancing mitochondrial function are an effective
disease-modifying strategy in OPA1-related DOA.
This work was supported of Fondazione Gino Galletti (V.C.) and
the Interdisciplinary Center of Clinical Research Tübingen
(01KS9602, B.W.).
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6. Alexander C, Votruba M, Pesch UE et al. OPA1, encoding a
dynamin-related GTPase, is mutated in autosomal dominant
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Prolongation of Levodopa
Responses by GlycineB
Antagonists in Parkinsonian
Stella M. Papa, MD,1 Yves P. Auberson, PhD,2
and J. Timothy Greenamyre, MD, PhD1
To examine the antiparkinsonian effects of blocking
glycineB receptors, we designed a pilot study testing the
potent and selective antagonist, PAMQX, in 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine-treated primates.
PAMQX had no intrinsic effects but markedly potentiated the antiparkinsonian action of levodopa. In a dosedependent fashion, coadministration of the glycineB antagonist with levodopa extended the response duration
by nearly 60%. It is noteworthy that PAMQX, within a
considerable dose range, did not cause ataxia or other
side effects. These data indicate that blocking N-methylD -aspartate receptors selectively to manipulate
dopaminergic-mediated motor responses may be produced effectively by glycineB antagonists.
Ann Neurol 2004;56:723–727
In Parkinson’s disease, nigrostriatal dopamine denervation leads to basal ganglia functional alterations that
are largely mediated by glutamate transmission. Likewise, pharmacological manipulation of glutamatergic
activity has proved effective in various animal models,
particularly by the antiparkinsonian action of
N-methyl-D-aspartate (NMDA) antagonists.1–3 However, the therapeutic potential of these agents often declines as a result of significant toxicity.4 –7 Noncompetitive and competitive NMDA antagonists have a
narrow therapeutic window because most effective
doses produce marked adverse reactions such as psychiatric symptoms, ataxia, and sedation (anesthetic-like effects).
Heteromeric NMDA receptors require the binding
of glutamate and glycine for channel activation. As opposed to glutamate-site antagonists, those selective for
From the 1Department of Neurology, Emory University, Atlanta,
Georgia; and 2Novartis Institutes for Biomedical Research, Novartis
Pharma AG, Basel, Switzerland.
Received May 12, 2004, and in revised form Jul 19. Accepted for
publication Jul 22, 2004.
Published online Oct 6, 2004, in Wiley InterScience
( DOI: 10.1002/ana.20279
Address correspondence to Dr. Papa, Department of Neurology,
Emory University, 6000 WMRB, 101 Woodruff Circle, Atlanta,
GA 30322. E-mail:
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
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production, atp, atrophy, optics, dominantly, vivo, opa1, related, deficit, mitochondria
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