At the last examination six months later, the patient, still amaurotic, was given a poor prognosis for further recovery of the visual defects. Fundus examination revealed severe optic disc atrophy in both eyes (Figure 1). The clinical examination and the MRI confirmed the diagnosis of isolated bilateral optic neuropathy. We searched for a genetic etiology. The patient's parents were dead; she had no brothers or sisters; but she had a 28-year-old son who had no ophthalmic problems.
Blood samples were taken from the patient after obtaining informed consent. Genomic DNA was extracted from blood samples using the BioRobot EZ1 and the EZ1 DNA Blood kit (Qiagen, Courtaboeuf, France). Next, 30 primer pairs (Appendix 1) were used to amplify the 30 OPA1 coding exons, including exon-intron junctions. PCR amplifications of the DNA were conducted under standard protocols. The purified PCR products were sequenced using a Ceq2000 DNA sequencer (CEQ DTCS-Quick Start kit; Beckman Coulter, Fullerton, CA). The OPA1 mutation is described according to the OPA1 transcript variant 1 (RefSeq: NM_015560).
To exclude the presence of any rare mitochondrial DNA mutation, we sequenced entirely the mitochondrial genome. The mtDNA was PCR amplified in eight fragments using the protocol 96  C for 10 min, followed by 30 cycles of 96  C for 45 s, 58  C for 30 s, 72  C for 3 min, and a last extension at 72  C for 10 min (Appendix 2). PCR products were purified and sequenced as described in the previous section.
The research followed the tenets of the Declaration of Helsinki. Fibroblast cells were obtained from skin biopsies taken after obtaining written consent from the patient, as described elsewhere. Fibroblasts, obtained by explants from skin punch biopsy, were maintained in DMEM (Invitrogen, Carlsbad, CA) with 10% bovine calf serum at 37  C in a humidified atmosphere with 5% CO2. All fibroblast cultures were mycoplasma-free, as shown by the DAPI/Hoechst in situ coloration and by PCR (Venor Gem, BioValley, Marne-la-Vallee, France). All experiments were performed on cells with similar passage numbers, ranging from 5 to 15, so as to avoid artifacts due to senescence, known to occur at passage numbers greater than 30.
The rate of mitochondrial ATP synthesis and the ATP/O ratio were determined in cells permeabilized by exposure to digitonin, as described below. Cells were resuspended in the respiratory buffer, which contained 10 mM KH2PO4, 300 mM mannitol, 10 mM KCl, and 5 mM MgCl2, pH 7.4. This buffer was supplemented with 2 mM iodoacetate and 2 mM EDTA so as to prevent glycolytic ATP synthesis and ATP hydrolysis by cellular ATPases. The respiratory rates (O2/min/mg of proteins) of 3-5x106 cells were recorded at 37  C in 2 ml glass chambers using a two-channel, high-resolution Oxygraph respirometer (Oroboros, Innsbruck, Austria). ATP synthesis was started by addition of 5 mM malate, 5 mM pyruvate, and 10 mM succinate, followed by addition of 1.5 mM ADP. Five aliquots were sampled each minute during mitochondrial ATP synthesis, quenched with an equal volume of 1%(W/V) TCA solution and neutralized by adding a 25 mM HEPES, 2 mM EDTA, pH 7.75 buffer. Three aliquots were also sampled after addition of 8 microg/ml oligomycine (an ATP synthase inhibitor) to check for residual nonmitochondrial ATP synthesis. The ATP synthesized in situ was measured using the Enliten ATP assay (Promega, Madison, WI). Luminescence was measured on a multidetection reader for microplates Xenius XML (SAFAS, Monaco, Monaco) using a 10-s integration period. Standardization was performed with known quantities of ATP measured under the same conditions. Coupling efficiency (ATP/oxygen [ATP/O]) was measured by polarography as the number of nanomoles of ATP produced from ADP+Pi per nanomole of oxygen consumed by permeabilized cells. The efficiency of the respiratory chain was tested using malate, pyruvate, and succinate as substrates.
A Beckman DU 640 spectrophotometer (Beckman Coulter, Fullerton, CA) was used to measure the activity of the mitochondrial respiratory chain complexes on cell homogenates at 37  C in a cell buffer that contained 250 mM saccharose, 20 mM tris[hydroxymethyl]aminomethane, 2 mM EGTA, and 1 mg/ml BSA, pH 7.2. Complex IV (cytochrome c oxidase, COX), complex V (F1-ATPase), and citrate synthase activities were recorded following Rustin et al.. Complex IV activity was measured in a 50 mM KH2PO4 buffer, using 15 microM reduced cytochrome c on 105 cells permeabilized by 2.5 mM beta-D-dodecylmaltoside. To measure the activity of complex V, we first disrupted cells by freezing in liquid nitrogen, followed by rapid thawing at 37  C. The cells were then centrifuged for 2 min at 800x g, resuspended in cell buffer (250 microl/106 cells). This was followed by a sonication step (6x5 s with an MSE sonicator). Complex V activity was immediately assayed on this cell lysate (0.5x106 cells) in a Tris/KCl buffer, composed of 50 mM Tris and 10 mM KCl, and containing 2 mM Phosphoenolpyruvate (PEP), 0.5 mM ATP, 5 mM MgCl2, 1.5 microM FCCP, 2.5 microg/microl antimycin A, 0.1 U lactate dehydrogenase, and 0.1 U pyruvate kinase (Roche Diagnostics, Meylan, France), 5 mg/ml BSA, pH 8. After incubation for 3 min, the reaction was started by adding 0.1 mM NADH and the rate of disappearance of NADH was monitored at 340 nm. In addition, 10 microM oligomycin was added to determine the background rate (nonspecific for complex V activity). Citrate synthase was assayed by standard procedures, using 0.15 mM 5-5-dithiobis(2-nitrobenzoic acid) (DTNB), 0.5 mM oxaloacetate, 0.3 mM acetyl-CoA, and 0.1% (V/V) Triton X-100. Specific enzymatic activities were expressed in mIU (e.g., nanomoles of cytochrome c, NADH or DTNB/min/mg protein, respectively). Enzymatic activities of complexes IV, V and citrate synthase are listed in Table 1.
Cells cultured in a two-well chamber slide (Labtek, Nunc International, Naperville, IL) were incubated with 100 nM Mitotracker  to label the mitochondrial network (Molecular Probes, Carlsbad, CA) according to manufacturer's instructions. Z-Stack fluorescent images were acquired with a Leica (DMI6000B; Microsystems GmbH, Wetzlar, Germany) and a Roper CoolSnap HQ2 camera. MetaMorph software (Molecular Devices, Sunnyvale, CA) was used to analyze images. Mitochondrial length and number were determined using integrated morphometric analysis of the regions created around mitochondria after deconvolution (Metamorph , Molecular Devices), as described above by Cassereau et al.. Around 50 deconvolved images were used to quantify the mitochondrial length. Measurements of mitochondria length distribution were divided into four groups: <1 microm, 1 to 5 microm, 5 to 10 microm, and >10 microm.
Pellets of 5x106 fibroblasts from the patient and controls were stored at -80  C until used for western blot analysis. Next, 40 microg protein were solubilized in Laemmli buffer and heated for 5 min at 50  C. Proteins were separated on an 8% SDS-polyacrylamide gel and electroblotted to a PVDF membrane (Amersham Biosciences, Buckinghamshire, UK). Membranes were saturated overnight at 4  C with 5% nonfat milk dissolved in TBS-Tween-0.1%, pH 7.4, which contained 137 mM NaCl, 2.7 mM KCl, 23 mM Tris, and 0.1% Tween-20. Membranes were then incubated 2 h at room temperature with monoclonal mouse anti-OPA1 antibody (BD Bioscience PharMingen, Milan, Italy) and mouse monoclonal anti-Hsp60 antibody (Stressgene, Victoria, Canada). Membranes were then washed three times in TBS-Tween-0.1% and incubated with 1:10,000 horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody for 1 h at room temperature. The immunoreactive proteins were visualized with enhanced chemiluminescence (ECL Plus Western Blotting Detection Reagents; Amersham Biosciences). Band intensities were quantified with Quantity One software (Bio-Rad, Hercules, CA).