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. 2010 Oct 13;134(1):220–234. doi: 10.1093/brain/awq276

Oestrogens ameliorate mitochondrial dysfunction in Leber’s hereditary optic neuropathy

Carla Giordano 1,*, Monica Montopoli 2,*, Elena Perli 1, Maurizia Orlandi 1, Marianna Fantin 3, Fred N Ross-Cisneros 4, Laura Caparrotta 2, Andrea Martinuzzi 3, Eugenio Ragazzi 2, Anna Ghelli 5, Alfredo A Sadun 4, Giulia d’Amati 1, Valerio Carelli 6,✉
PMCID: PMC3025718  PMID: 20943885

Abstract

Leber’s hereditary optic neuropathy, the most frequent mitochondrial disease due to mitochondrial DNA point mutations in complex I, is characterized by the selective degeneration of retinal ganglion cells, leading to optic atrophy and loss of central vision prevalently in young males. The current study investigated the reasons for the higher prevalence of Leber’s hereditary optic neuropathy in males, exploring the potential compensatory effects of oestrogens on mutant cell metabolism. Control and Leber’s hereditary optic neuropathy osteosarcoma-derived cybrids (11778/ND4, 3460/ND1 and 14484/ND6) were grown in glucose or glucose-free, galactose-supplemented medium. After having shown the nuclear and mitochondrial localization of oestrogen receptors in cybrids, experiments were carried out by adding 100 nM of 17β-oestradiol. In a set of experiments, cells were pre-incubated with the oestrogen receptor antagonist ICI 182780. Leber’s hereditary optic neuropathy cybrids in galactose medium presented overproduction of reactive oxygen species, which led to decrease in mitochondrial membrane potential, increased apoptotic rate, loss of cell viability and hyper-fragmented mitochondrial morphology compared with control cybrids. Treatment with 17β-oestradiol significantly rescued these pathological features and led to the activation of the antioxidant enzyme superoxide dismutase 2. In addition, 17β-oestradiol induced a general activation of mitochondrial biogenesis and a small although significant improvement in energetic competence. All these effects were oestrogen receptor mediated. Finally, we showed that the oestrogen receptor β localizes to the mitochondrial network of human retinal ganglion cells. Our results strongly support a metabolic basis for the unexplained male prevalence in Leber’s hereditary optic neuropathy and hold promises for a therapeutic use for oestrogen-like molecules.

Keywords: LHON, oestrogen, mitochondrial disorders, oestrogen receptors, oxidative stress

Introduction

More than 20 years ago, the first point mutation in the mitochondrial genome was associated with a maternally inherited disease, Leber’s hereditary optic neuropathy (LHON; Wallace et al., 1988). LHON is characterized by the selective degeneration of retinal ganglion cells, in particular those contributing to the papillomacular bundle, leading to optic atrophy and loss of central vision (Carelli et al., 2004). LHON is now recognized as the most frequent mitochondrial disorder (Man et al., 2003) and over the last two decades intense work has been carried out to elucidate its clinical and molecular basis (Carelli et al., 2004; Man et al., 2009). However, crucial features of this disease still remain elusive. In particular, it is difficult to explain the incomplete penetrance when all individuals in a maternal lineage carry a homoplasmic mutation (100% of mitochondrial genomes are mutant in each cell). Furthermore, male prevalence also remains unexplained.

To date, three mitochondrial DNA point mutations at positions 11778/ND4, 3460/ND1 and 14484/ND6 are found in >90% of patients with LHON (Carelli et al., 2004; Man et al., 2009). There is also well-established evidence that the non-synonymous population polymorphisms found in mitochondrial DNA haplogroups J1c and J2b increase the penetrance of LHON mutations 11778/ND4 and 14484/ND6, respectively (Carelli et al., 2006; Hudson et al., 2007b). It is also assumed that further genetic (Carelli et al., 2003; Phasukkijwatana et al., 2010) and environmental (Sadun et al., 2003; Kirkman et al., 2009) factors play a role in modulating the variability of penetrance and possibly gender prevalence.

The possibility that nuclear genes on chromosome X play a role has repeatedly been pursued with controversial results (Vilkki et al., 1991; Chalmers et al., 1996). The hypothesis of an X-linked modifying gene is particularly attractive (two-loci hypothesis) as it would explain both features of variable penetrance and male prevalence (Bu et al., 1991). Recently, linkage analysis identified two loci on chromosome X (Hudson et al., 2005; Shankar et al., 2008), suggesting multiple modifying genes that remain unidentified.

However, some data suggest a different scenario. Assuming that an X-linked gene plays a major modifying role in the expression of LHON in males, in the case of affected females homozygosity or skewed inactivation favouring the modifying allele should be assumed (Bu et al., 1991). Comprehensive examination of affected females by independent studies failed to document any excess of skewed inactivation of the X-chromosome (Hudson et al., 2007a). Thus, based on the different hormonal metabolism between genders, it may be that in females, oestrogens play a protective role in modifying the severity of the mitochondrial defect (Carelli et al., 2004, 2007), which is characterized by the combination of defective ATP synthesis driven by complex I substrates (Baracca et al., 2005), increased oxidative stress (Beretta et al., 2004; Floreani et al., 2005) and enhanced sensitivity to apoptotic cell death (Ghelli et al., 2003; Zanna et al., 2005).

Oestrogens may modify this scenario, given the increasing body of evidence of their direct action on the mitochondrial respiratory chain, oxidative stress and mitochondrial biogenesis (Simpkins et al., 2008; Chen et al., 2009), and the suggestion that they are responsible for female longevity (Viña et al., 2006). Oestrogen receptors are present on mitochondria (Chen et al., 2004a; Yang et al., 2004) and oestrogen-responsive elements are localized within the D-loop, a major regulatory region for human mitochondrial DNA transcription and replication (Chen et al., 2004b). These observations suggest that oestrogens may influence mitochondrial functions both by a classic long-term genomic mechanism and also by rapid non-genomic signalling involving membrane-associated oestrogen receptors, including direct effects on mitochondria (Chen et al., 2009).

The current study uses the cybrid cell model to investigate whether oestrogens modify the mitochondrial dysfunction in LHON and provides new insights on the gender bias of this still-elusive disease and possible new therapeutic approaches.

Materials and methods

Cell lines and reagents

Control and LHON cybrids (11778/ND4, 3460/ND1 and 14484/ND6) were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% dialysed foetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.1 mg/ml bromodeoxyuridine (referred to as DMEM-glucose). Experiments were performed both in DMEM-glucose and in glucose-free DMEM, supplemented with 10% dialysed foetal bovine serum, 5 mM galactose and 110 mg/ml sodium pyruvate (referred to as DMEM-galactose). 17β-Oestradiol was purchased from Sigma-Aldrich (St Louis, MO, USA) and the oestrogen receptor antagonist ICI 182780 from Tocris Bioscience (Bristol, UK). 17β-Oestradiol and ICI 182780 were dissolved in ethanol at a final concentration of 100 µM and diluted to appropriate concentrations in culture medium as required. Untreated cells were maintained at the same final ethanol concentration. The oestrogen receptor antagonist ICI 182780 was added 30 min before 17β-oestradiol.

Immunostaining and western blot analysis

For immunocytochemistry, cells grown on coverslips were fixed with 4% formaldehyde freshly prepared from paraformaldehyde in phosphate-buffered saline (pH 7.4) with 0.1% Triton X-100. Primary antibodies were visualized using secondary fluorescein isothiocyanate- and Cy3-conjugated antibodies (Jackson Laboratories, Bar Harbor, Maine, USA). Immunofluorescence was performed on formalin-fixed, paraffin-embedded human retinal sections obtained from one patient with LHON (male, aged 52 years) and two control individuals (1 male and 1 female, aged 59 and 70 years, respectively; obtained from the Lions Eye Bank of Oregon, Portland, OR, USA). For western blot analysis, cells were rinsed twice with ice-cold phosphate-buffered saline, lysed in ice-cold radioimmunoprecipitation assay buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1% sodium dodecyl sulphate, 1 mM phenylmethanesulphonylfluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin and 10 mg/ml pepstatin) and centrifuged at 10 000g for 10 min at 4°C. For some experiments mitochondria were isolated from 4 × 106 cybrid cells by standard differential centrifugation. Protein concentration was measured by bicinchoninic acid (Beyotime Biotechnology, Haimen, China). Equal amounts of protein (50 μg) were separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Millipore, Bedford, MO). Primary antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark). Signals were detected by enhanced chemiluminescence (Amersham Biosciences, UK).

The following primary antibodies were used: rabbit polyclonal antibody raised against amino acids 2–185 of human oestrogen receptor α (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal antibody raised against a synthetic peptide (CSSTEDSKNKESSQNLQSQ) corresponding to amino acids 485–503 of rat oestrogen receptor β (ERβ 485-503; Calbiochem, MerckKGaA, Darmstadt, Germany); rabbit polyclonal antibody raised against amino acids 1–150 of human oestrogen receptor β (ERβ H-150, Santa Cruz Biotechnology); rabbit polyclonal antibody anti-superoxide dismutase 2 (Assay Designs; Ann Arbor, MI, USA) mouse monoclonal antibody anti-cytochrome c oxidase subunit IV (COIV) (Mitosciences, Eugene, OR, USA) mouse monoclonal antibody anti-porin and mouse monoclonal antibody anti-ND6 (Invitrogen, Paisley, UK), mouse monoclonal antibody anti-β-actin (Sigma, Saint Luise, MO, USA) and mouse monoclonal antibody anti-mitochondria extract clone MTC (UCS Diagnostic, Morlupo, Italy).

Intracellular level of reactive oxygen species and mitochondrial/transmembrane potential (Δφ)

Direct detection of intracellular steady-state levels of reactive oxygen species was carried out on living cells by cytofluorimetry using 2′,7′-dichlorofluorescin-diacetate (H2-DCF-DA; Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA; Wu et al., 2007). Mitochondrial membrane potential was measured by cytofluorimetry using the cationic lipophilic green fluorochrome rhodamine-123 (Rh123; Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA; Ferlini et al., 2007). Sample fluorescence was analysed by Epics XL Coulter Systems (Fullerton, CA) equipped with a 488 nm Argon laser. Dead cells were excluded by electronically gating data on the basis of forward-versus-side scatter profiles; a minimum of 104 cells/sample were analysed further. Logarithmic detectors were used for the FL1 fluorescence channel necessary for 2′,7′-dichlorofluorescin detection. Mean fluorescence intensity values were obtained by the analysis EXPO 32 software (Coulter Systems, Fullerton, CA, USA).

Superoxide dismutase 2 activity

Superoxide dismutase (SOD) activity was evaluated as reported previously (Oberley et al., 1984), with minor modifications. Briefly, 1 ml of medium consisting of 50 mm KH2PO4 (pH 7.8), 0.1 mM ethylenediaminetetraacetic acid and a superoxide generating system (0.15 mM xanthine plus 0.02 U xanthine oxidase) was used together with 50 µM nitroblue tetrazolium to monitor superoxide formation by following the changes in colorimetric absorbance at 550 nm for 5 min at 25°C. To assess the specific mitochondrial superoxide dismutase 2 (SOD2) activity, cell fractions were pre-incubated for 60 min at 0°C in the presence of 2 mM KCN, which induces inhibition of the cytoplasmic Cu/ZnSOD. The catalytic activities of the samples were evaluated as their ability to inhibit the rate of nitroblue tetrazolium reduction. SOD2 activity was expressed as unit per milligram of protein. One unit of SOD2 is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.

Cell viability and ATP assays

Cell proliferation was measured by the trypan blue dye exclusion assay. Multiple series of 60 mm dishes were seeded with a constant number of cells (106) for 24 and 48 h. Cells were detached by 0.25% trypsin and 0.2% ethylenediaminetetraacetic acid, washed, suspended in phosphate-buffered saline in the presence of trypan blue solution (Sigma, St Louis, MO, USA) at 1:1 ratio and counted using a haematocytometer. Viability was expressed as percentage of untreated cell number in DMEM-glucose. ATP cellular content was measured by using the luciferin/luciferase assay (Zanna et al., 2005).

Annexin V/propidium iodide staining for apoptotic cells

Cells were seeded at 1.5 × 105 cells/well, incubated overnight, treated according to experimental protocol and incubated for 24 h. Cells were harvested by quick trypsinization to minimize potentially high annexin V background levels in adherent cells, were washed and then stained with Alexa 488/annexin V/propidium iodide (Molecular Probes, Invitrogen UK). Cells were analysed on an Epics XL-flow cytometer using the Analysis software (both hardware and software were from Beckman Coulter, Miami, FL, USA), with the laser excitation wavelength set at λ = 488 nm. The green signal from Alexa 488/annexin V was measured at λ = 525 nm and the red signal from propidium iodide was measured at λ = 620 nm. Cells staining negative for both annexin V and propidium iodide are viable, cells that are annexin V-positive/propidium iodide-negative are in early apoptosis, whereas cells that are annexin V-positive/propidium iodide-positive are necrotic or in late apoptosis (Stadelmann, et al., 2000).

Mitochondrial morphology

Cells were stained with MitoTracker Orange (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 30 min at 37°C, then fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min, counterstained with 4',6-diamidino-2-phenylindole and mounted on glass slides by using Mowiol 40-88 (Sigma, St Louis, MO, USA). Images were acquired with a Nikon C1 confocal microscope and analysed using Nikon EZ-C1 software (version 2.10; NIKON Corporation, Tokyo, Japan).

Quantification of mitochondrial DNA by quantitative real-time polymerase chain reaction

Total DNA was isolated by Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). Evaluation of mitochondrial DNA copy number by quantitative real-time polymerase chain reaction (PCR) was performed as previously described (Mussini et al., 2005). Briefly, a mitochondrial DNA fragment (nt 4625–4714) and a nuclear DNA fragment (FasL gene) were co-amplified by using TaqMan® probe system and Platinum® Quantitative PCR SuperMix-UDG (Invitrogen, Life Technologies, Carlsbad, CA, USA). With each assay, a standard curve for mitochondrial and nuclear DNA was generated using serial known dilutions of a vector (a kind gift from Dr Andrea Cossarizza) in which the regions used as template for the two amplifications were cloned tail to tail to have a ratio of 1:1 of the reference molecules. The absolute mitochondrial DNA copy number per cell was obtained by the ratio of mitochondrial to nuclear DNA values multiplied by two (as two copies of the nuclear gene are present in a cell). PCR was carried out in an iCycler Thermal cycler (BioRad, Hercules, CA, USA) and at least three measurements were obtained for each sample.

Gene expression by quantitative real-time polymerase chain reaction

Total RNA was isolated by using SV Total RNA Isolation System (Promega, Madison, WI, USA) and measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc. Wilmington, DE, USA). One microgram of total RNA was reverse-transcribed to complementary DNA using random hexamer primers (AMV Reverse Transcriptase, Promega, Fitchburg, WI, USA). Relative expression of each gene was determined by quantitative real-time PCR using the Platinum SYBR Green qPCR Super Mix-UDG (Invitrogen, Life Technologies, Paisley, UK). Primers for PGC1-α, PGC1-β, NRF1, NRF2 and Tfam were as previously reported (Sebastiani et al., 2008). Primers for COI, COIV and ND6 are reported in Supplementary Table 1. They were carefully designed with Beacon Designer Software (Biorad) and checked by using Blast software (http://www.ncbi.nlm.nih.gov/BLAST/) to avoid cross-homology. The specificity of the amplicons was confirmed by direct sequencing using an ABI Prism 310 Genetic analyser following standard procedures. Quantitative real-time PCR was performed in triplicate using 1 μl of complementary DNA template in a 50-μl reaction. Linearity and efficiency of PCR amplifications were assessed using standard curves generated by serial dilution of complementary DNA; melt-curve analysis was used to confirm the specificity of amplification and absence of primer dimers. In all samples, the relative expression of each target gene with respect to one control (reference sample) was evaluated with the comparative threshold cycle (ΔCt) method. All values were normalized to the 18S ribosomal RNA housekeeping gene.

Oxygen consumption

Oxygen consumption was measured in intact cells (5 × 106) using a Clark type oxygen electrode, in 1.85 ml DMEM lacking glucose supplemented with 5% dialysed foetal bovine serum at 37°C, as previously described (Carelli et al., 2002).

Statistical analysis

All data are expressed as mean ± SEM. Standard ANOVA procedures followed by multiple pair-wise comparison adjusted with Bonferroni corrections were performed for continuous variables, chi-square test was used for frequency data. Multi-way ANOVA procedures were performed to analyse SOD2 activity and total cellular ATP content. Paired t-tests were used to analyse the rate of oxygen consumption. Significance was considered at P < 0.05.

Results

Characterization of cybrid cells

We investigated the effects of 17β-oestradiol on the cellular phenotype of cybrids obtained from four unrelated patients carrying one of the three classic LHON pathogenic mutations and compared these with two control cybrids (Supplementary Table 2). Osteosarcoma-derived (143B.TK-) cybrid cell lines were previously established and characterized (Ghelli et al., 2003; Baracca et al., 2005; Floreani et al., 2005; Zanna et al., 2005), and the complete mitochondrial DNA sequence had been determined for all the clones used in this study (Pello et al., 2008; Ghelli et al., 2009). The LHON pathogenic mutations were homoplasmic in all cell lines. Furthermore, we established by immunofluorescence that both oestrogen receptor α and oestrogen receptor β were localized to 143B.TK-nucleus, whereas immunofluorescence and western blot analyses showed localization of the oestrogen receptor β to cybrid-derived mitochondria (Fig. 1A and B). This characterization for oestrogen receptors was carried out prior to the cells’ exposure to 17β-oestradiol to verify their constitutive expression.

Figure 1.

Figure 1

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Oestrogen receptor localization to osteosarcoma-derived (143B. TK-) cybrid cell lines. (A) Fluorescence microscopy localization of oestrogen receptors to the nucleus and mitochondria of cybrid cells. Oestrogen receptor β is stained red by oestrogen receptor β BIO1974 antibody (a) and oestrogen receptor β H150 antibody (e); oestrogen receptor α is stained red by H-184 antibody (i); mitochondria are stained green using anti-mitochondria extract antibody (b, f and l); merged image of oestrogen receptors and mitochondria (c, g and m); negative controls loaded with 4',6-diamidino-2-phenylindole (blue) (d, h and n). Overlap is seen in bright green (original magnification ×40). (B) Western blot analysis of mitochondrial preparation from 143B.TK-derived wild-type cybrids and MCF7 oestrogen–dependent breast cancer cells used as positive control. The oestrogen receptor β (ERβ) protein (identified with oestrogen receptor β 485–503 antibody) is constitutively over-expressed in the mitochondrial fraction of MCF7 cells, whereas oestrogen receptor α (ERα) is present at lower levels, confirming previous data (Pedram et al., 2006). In wild-type cybrids, oestrogen receptor β is less abundant than in MCF7 cells, whereas oestrogen receptor α is not expressed.

17β-Oestradiol decreases levels of reactive oxygen species

Previous results have shown that production of mitochondrial reactive oxygen species was significantly increased in cybrids bearing LHON mutations (Beretta et al. 2004; Floreani et al., 2005). We first confirmed that in glucose medium, 11778/ND4 cybrids present an ∼3-fold increase in levels of reactive oxygen species as compared with controls (Fig. 2A). After 1 h of incubation in glucose-free medium containing galactose, a condition forcing cells to rely predominantly on oxidative phosphorylation for ATP synthesis, production of reactive oxygen species further increased by ∼2-fold in both cell lines. We then added 17β-oestradiol to the galactose medium at increasing concentrations ranging from 10 to 200 nM, and evaluated levels of reactive oxygen species after 1 h of incubation. As shown in Fig. 2A, addition of 17β-oestradiol caused a decrease in levels of reactive oxygen species in both LHON and control cybrids, in a concentration-dependent manner. The lowest levels of reactive oxygen species were reached with 100 nM 17β-oestradiol, and this concentration was chosen for the further experiments. This experiment was replicated in all the LHON cybrid cell lines. As shown in Fig. 2A, supplementation of medium with 17β-oestradiol induced significant decrease in levels of reactive oxygen species in all cell lines. This phenomenon was evident both in cells incubated in glucose and in galactose medium. Finally, we looked as to whether the observed effects were mediated by oestrogen receptors. We pre-incubated cells with the oestrogen receptor antagonist ICI 182780, and found that the antioxidant effect of 17β-oestradiol was fully abolished (Fig. 2A). As a control experiment, cells were also exposed to ICI 182780 alone without significant effect (data not shown). Similar results were obtained after 3 h of incubation in galactose medium (Supplementary Fig. 1). In all of these experiments, the LHON cybrids carrying the 11778/ND4 mutation displayed the highest levels of reactive oxygen species compared with the other cell lines.

Figure 2.

Figure 2

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17β-Oestradiol decreases levels of reactive oxygen species and induces SOD2 activity. (A) Left: Control and 11778/ND4 cybrids were incubated for 1 h in glucose (glu) or galactose (gal) medium ± 10–200 nM 17β-oestradiol (E2), then the intracellular steady-state levels of reactive oxygen species was evaluated. Right: In a further experiment control and LHON cybrids were incubated for 1 h in glucose (glu) or galactose (gal) medium ± 100 nM 17β-oestradiol (E2). The oestrogen receptor antagonist ICI 182780 (I) was added 30 min before 17β-oestradiol. Untreated cells were maintained at the same final ethanol concentration. Data are expressed as mean fluorescence intensity (MFI) (± SEM) and are from three experiments. °P < 0.05; °°P < 0.01; °°°P < 0.001 LHON versus control; †P < 0.05 for glu + E2 versus glu; *P < 0.05, ***P < 0.001 for gal versus glu; +P < 0.05; ++P < 0.01 for gal + E2 versus gal and versus gal + E2+ I (see Supplementary Fig. 1). (B) Time course of SOD2 activity of control and 11778/ND4 (HPE9) cybrids incubated in glucose or galactose medium ± 100 nM 17β-oestradiol (E2). Incubation in galactose medium induced a significant increase of SOD2 activity in control cybrids (P < 0.001) that was not observed in LHON. SOD2 activity is expressed as unit per milligram of protein. Each data point is the mean of quadruplicate replicates. °°°P < 0.001 for LHON versus control; +++P < 0.001 for E2 versus ethanol. (C) Left: The relative expression of mitochondrial SOD2 gene was evaluated by real-time PCR in control (HGA and HP27) and 11778/ND4 (HPE9 and HFF3) cybrids incubated for 6 h in glucose (glu) or galactose (gal) medium ± 100 nM 17β-oestradiol (E2). The oestrogen receptor antagonist ICI 182780 was added 30 min before 17β-oestradiol. Data represent mean arbitrary units (± SEM) normalized to control values in glucose medium and are from three experiments. †P < 0.05 and ††P < 0.01 for glu + E2 versus glu; ***P < 0.001 for gal versus glu; +++P < 0.001 for gal+E2 versus gal. Right: Western blot analysis of SOD2 protein performed on extract from control (HGA) and 11778/ND4 (HPE9) cybrids incubated for 24 h in glucose or galactose medium ± 100 nM 17β-oestradiol. A representative blot from three is shown.

17β-Oestradiol induces superoxide dismutase 2 activity

Manganese SOD2 is the main mitochondrial antioxidant enzyme. Oestrogens can modulate SOD2 activity both by a rapid, non-genomic action (Pedram et al., 2006, Gottipati and Cammarata, 2008) and by long-term regulation of gene transcription (Borrás et al., 2005). We previously showed that LHON cybrids have a significant reduction of SOD2 activity as compared with controls, despite an increased amount of protein (Floreani et al., 2005). To shed light on the mechanism involved in the 17β-oestradiol-dependent decrease in levels of reactive oxygen species described above, we evaluated SOD2 activity in a time-course experiment (1, 6, 12 and 24 h) using 11778/ND4 and control cybrids grown in glucose and galactose medium supplemented with 100 nM 17β-oestradiol. We first confirmed that the SOD2 activity of 11778/ND4 cybrids maintained in glucose medium was significantly lower than in controls (Fig. 2B). Supplementation with 17β-oestradiol led to a significant increase of SOD2 activity both in control and 11778/ND4 cybrids. This increase was more marked in LHON cybrids and remained stable for 24 h. Gene expression of SOD2 was enhanced by 17β-oestradiol in both control and 11778/ND4 cybrids after 6 h of incubation, and a significant SOD2 protein increase was observed after 24 h (Fig. 2C).

Incubation in galactose medium induced a significant increase of SOD2 activity in control cells (Fig. 1B), which was not observed in LHON cybrids, despite the induction of gene expression and increased protein (Fig. 2C). Supplementation of galactose medium with 17β-oestradiol led to a further rapid (1 h) upregulation of SOD2 activity, both in control and 11778/ND4 cybrids, which remained stable for 24 h, and was not linked with increased protein levels despite increased gene expression. The latter increase was fully abolished by the oestrogen antagonist ICI 182780.

17β-Oestradiol ameliorates cell viability and reduces apoptosis in galactose medium

Previous work by our group showed that the growth of LHON cybrids in galactose medium suffered a marked decrease in cell number compared with controls, secondary to apoptotic cell death (Ghelli et al., 2003; Zanna et al., 2005). As shown in Fig. 3A, in both LHON and control cybrids, supplementation of medium with 100 nM 17β-oestradiol significantly reduced the galactose-dependent loss of viability, as evaluated by the trypan blue dye assay. As an additional marker of cell viability, we measured the mitochondrial membrane potential (mtΔψ). After 24-h incubation in galactose medium, LHON cybrids displayed a significant decrease (35%) of mtΔψ compared with control cybrids. Supplementation with 100 nM 17β-oestradiol prevented the mtΔψ reduction and this effect was lost by pre-incubation with the oestrogen receptor antagonist ICI 182780 (Fig. 3B).

Figure 3.

Figure 3

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17β-Oestradiol ameliorates cell viability in galactose medium by reducing apoptosis. (A) Growth curves of control and LHON cybrids maintained in glucose (glu) or galactose (gal) medium ± 100 nM 17β-oestradiol (E2). ***P < 0.001 for gal versus glu; +P < 0.05, ++P < 0.01, +++P < 0.001 for gal + E2 versus gal. Data are expressed as % of untreated cell number in glucose medium, and are mean ± SEM from four different experiments in duplicate. Growth curves for control and 11778/ND4 are from HP27 and HFF3 clones. Similar results were obtained with HPE9 and HGA clones (data not shown). (B) Mitochondrial membrane potential (mtΔφ) of control and LHON cybrids incubated for 24 h in glucose (glu) or galactose (gal) medium ± 100 nM 17β-oestradiol (E2). In a subset of experiments, cells were pre-incubated with ICI 182780 (I). Data are expressed as mean fluorescence intensity (MFI; ± SEM) (% of values of untreated cells in glucose medium) and are from three independent experiments. Control and 11778/ND4 values are the mean from two clones. *P < 0.05, ***P < 0.001 for gal versus glu; +P < 0.05, +++P < 0.001 for gal + E2 versus gal and versus gal + E2+ I. (C) Percentages of apoptotic cells in control and LHON cybrids incubated in glucose (glu) or galactose (gal) medium ± 100 nM 17β-oestradiol (E2), as evaluated by labelling the cells with annexin V. Data are the mean ± SEM of the percent number of apoptotic cells from three repeated experiments. Control and 11778/ND4 values are the mean values from two clones. °°°P < 0.001 LHON versus control; ***P < 0.001 for gal versus glu; ++P < 0.01 for gal + E2 versus gal (see Supplementary Fig. 2).

The next set of experiments was designed to examine whether protection of cell viability by 17β-oestradiol was related to reduction of the apoptotic cell death rate by labelling the cells with annexin V. Annexin V is an early marker of apoptosis and binds phosphatidylserine exposed on the cytoplasmic surface of the cell membrane of apoptotic cells (Stadelmann and Lassmann, 2000). After incubation in galactose medium for 24 h, a significant number of LHON cybrids shifted from the viable to the apoptotic state, thus confirming our previous results (Ghelli et al., 2003; Zanna et al., 2005). Incubation with 17β-oestradiol prevented the shift towards apoptosis, corroborating the increase in cell viability reported above (Supplementary Fig. 2). Figure 3C summarizes the percentages of apoptotic cells in control and LHON cybrids, and demonstrates the statistically significant decrease in cell death following treatment with 17β-oestradiol.

17β-Oestradiol reduces mitochondrial network fragmentation

The importance of the mitochondrial network organization in both mitochondrial respiratory chain defects and apoptosis is now well recognized (Bernard et al., 2007). We therefore investigated the morphology of the mitochondrial network under the conditions used in the previous experiments. Control and 11778/ND4 LHON cybrids were stained with Mitotraker Orange, counterstained with 4′,6-diamidino-2-phenylindole and scored into three categories based on mitochondrial morphology as previously reported (Zanna et al., 2008). Briefly, Class I cells showed a typical filamentous network, Class II cells showed filamentous mitochondria containing balloon-like structures and Class III cells showed complete fragmentation resulting in only isolated mitochondrial balloons (Fig. 4A). In the present study, we added Class IV to take into account the presence of shrunken cells with dense, fluorescent cytoplasm and fragmented nucleus, representing end-stage, dying apoptotic cells. In galactose medium, the percentage of the four classes was significantly different between control and LHON cybrids. In fact, control cybrids exhibited a combination of Class I (∼47%) and Class II (∼31%) cells, with about the same limited amount of Class III and IV cells (∼10%). In contrast, incubation of the 11778/ND4 cybrids in galactose medium for 24 h led to a dramatic increase of Class III (∼20%) and IV (∼47%) cells, with parallel reduction of Class I and II cells. This phenomenon was significantly compensated by incubation of the 11778/ND4 cybrids with 17β-oestradiol (Fig. 4B). The results of these experiments are compatible and complementary to those obtained from cell growth rates and the annexin assay.

Figure 4.

Figure 4

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17β-Oestradiol reduces mitochondrial network fragmentation. (A) Representative images of the four classes of cells (see ‘Results’ section) as observed in 11778/ND4 cybrids incubated for 24 h in galactose medium, loaded with MitoTracker Orange and counterstained with 4',6-diamidino-2-phenylindole. The inset shows the corresponding nuclear morphology. (B) Bar graphs showing quantification of the four categories by blind test. Cybrids analysed were from 30 photos obtained from two controls (HGA, HP27) and two 11778/ND4 (HFF3, HPE9) cybrid cell lines grown in galactose medium (gal) ± 100 nM 17β-oestradiol (E2). °°°P < 0.001, °°P < 0.05 for LHON versus WT; +++P < 0.001 for gal + E2 versus gal.

17β-Oestradiol induces mitochondrial biogenesis

Recent studies showed oestrogen modulation of mitochondrial biogenesis by transcriptional regulation of nuclear and mitochondrial genes (Mattingly et al., 2008). Thus, we investigated whether induction of mitochondrial biogenesis and respiration occurs in LHON cybrids grown in glucose and galactose medium when treated with 100 nM 17β-oestradiol. First, we evaluated, as a marker of mitochondrial biogenesis, the mitochondrial DNA content of cells. Control and LHON cybrids showed a similar increase in mitochondrial DNA amount after 3–6 h of incubation in galactose medium. A further increase in mitochondrial DNA content was observed when glucose or galactose medium was supplemented with 17β-oestradiol (Fig. 5A). This event was evident after 15–30 min of incubation with 17β-oestradiol, reached a plateau (up to 2.5-fold increase) after ∼3 h and did not change over the following 72 h (Supplementary Fig. 3). The increase in mitochondrial DNA content was inhibited by pre-incubation with ICI 182780 (Fig. 5A). Among LHON cybrids, the one carrying the 3460/ND1 mutation displayed the highest increase of mitochondrial DNA content after 17β-oestradiol treatment, so we chose this cell line for the further experiments. To gain insight into the mechanism involved in the 17β-oestradiol-mediated mitochondrial DNA copy number increase, we evaluated the gene expression level of the master regulators of mitochondrial biogenesis: PPAR-γ coactivator 1-α (PGC1-α) and its homologue PGC-1β; nuclear respiratory factor 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (Tfam). We observed a coordinated gene induction (∼up to 12-fold) after 6 h of treatment both in control and LHON cybrids (Fig. 5B). The induction was completely inhibited by the 17β-oestradiol antagonist ICI 182780. The increase in mitochondrial DNA content and the induction of the key regulators of mitochondrial biogenesis were paralleled by the upregulation of two mitochondrial-encoded messenger RNAs: cytochrome c oxidase (COX) subunits I (MTCOI), and NADH dehydrogenase subunit 5 (MTND5; Fig. 5B). A postponed upregulation (after 12–24 h of 17β-oestradiol treatment) was observed for the nuclear-encoded COX subunit IV (COIV; Fig. 5C). The induction of nuclear and mitochondrial genes was inhibited by ICI 182780. Despite gene induction, control and LHON cybrids grown in galactose medium revealed a decrease of COIV and ND6 protein, with the LHON cybrids showing the lowest protein amount. Supplementation of medium with 17β-oestradiol partially prevented the galactose-dependent COIV and ND6 protein reduction and led to a remarkably increased amount of protein when cells were maintained in glucose medium (Fig. 5C).

Figure 5.

Figure 5

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17β-Oestradiol induces mitochondrial biogenesis. (A) Amount of mitochondrial DNA in control and LHON cybrids maintained for 3 h in glucose (glu) or galactose (gal) medium ± 100 nM 17β-oestradiol (E2). In a subset of experiments, cells were pre-incubated with ICI 182780 (I). Bar graph represents the mean plus SEM from three experiments. †††P < 0.001 for glu + E2 versus glu and versus glu + E2+ I; **P < 0.01 for gal versus glu; +++P < 0.001 for gal + E2 versus gal (see Supplementary Fig. 3). (B) Control and LHON cybrids were incubated for 6 h in glucose (glu) or galactose (gal) medium ± 100 nM 17β-oestradiol (E2). In a subset of experiments, cells were pre-incubated with ICI 182780 (I). The relative expression of the following genes was evaluated by real-time PCR analysis: PGC1-α and its homologue PGC-1β, NRF1, NRF2, Tfam, MTCOI, MTND5. (C) In a subsequent experiment, cells were incubated for 24 h in glucose (glu) or galactose (gal) medium ± 100 nM 17β-oestradiol (E2), and the relative gene and protein expression of the nuclear encoded respiratory chain subunits COIV evaluated by real-time PCR (top) and western blot analysis of mitochondrial fraction (bottom) along with the protein expression of the mitochondrial encoded complex I subunit ND6. Data represent mean arbitrary units (± SEM) normalized to control values in glucose medium and are from three experiments. A representative blot out of three is shown. †††P < 0.001, ††P < 0.01, †P < 0.05 for glu + E2 versus glu and versus glu + E2+ I; ***P < 0.001, **P < 0.01, *P < 0.05 for gal versus glu; +++P < 0.001 for gal + E2 versus gal.

17β-Oestradiol enhances energetic competence

To assess whether oestrogens improved the energetic competence of cybrid cell lines, we measured the rate of oxygen consumption and the total cellular ATP content in the presence or absence of 17β-oestradiol. Supplementation of glucose medium with 17β-oestradiol led to a small although significant increase in the rate of oxygen consumption both in LHON and control cybrids after 24 h of incubation (Fig. 6A). Similar results were obtained after 48 h of incubation (not shown). Similarly, supplementation of glucose medium with 100 nM 17β-oestradiol led to a ∼20% increase of cellular ATP content in both 11778/ND4 and control cybrids (Fig. 6B). After 24 h of incubation with galactose medium, the ATP content significantly decreased in both control and LHON cybrids as previously described (Zanna et al., 2005), the LHON cybrids performing slightly worse. Supplementation of medium with 100 nM 17β-oestradiol partially prevented the galactose-induced ATP-content decrease. This phenomenon was particularly evident in LHON cybrids (<40% increase in total ATP content in galactose medium plus oestrogens compared with galactose medium plus vehicle; Fig. 6B).

Figure 6.

Figure 6

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17β-Oestradiol ameliorates energetic competence of cybrid cells. (A) Control, 11778/ND4, 3460/ND1 and 14484/ND6 cybrids were incubated for 24 h in glucose (glu) ± 100 nM 17β-oestradiol (E2) and the rate of oxygen consumption measured. Data are mean ± SEM from three to four separate experiments. For each clone, the rate of oxygen consumption in glucose medium supplemented with 17β-oestradiol has been normalized to the rate of oxygen consumption in glucose medium plus ethanol. *P < 0.03; **P < 0.003 for glu + E2 versus glu. (B) Control and 11778/ND4 LHON cybrids were incubated for 24 h in glucose (glu) ± 100 nM 17β-oestradiol and the total ATP cellular content measured by luciferin/luciferase assay. Data are mean ± SEM from four separate experiments. For each clone, values are normalized for the total ATP content in glucose medium. +++P < 0.001 for E2 versus ethanol; *** for galactose (gal) versus glucose.

Oestrogen receptor β localizes to retinal ganglion cells

Several studies have localized both oestrogen receptor α and oestrogen receptor β to mitochondria of many cell types, including rat primary neurons, human lens epithelial cells and human foetal cortical neurons (for a review see Simpkins et al., 2008 and Chen et al., 2009). To evaluate whether oestrogen receptors localize to human retinal ganglion cells, the main target of LHON, we performed immunoperoxidase staining on formalin-fixed, paraffin-embedded retinal sections obtained post-mortem from one patient with LHON and two control individuals of both genders. A punctuate staining typical of mitochondrial pattern was observed in the somata of retinal ganglion cells and the unmyelinated portion of the axons in the retinal nerve fibre layer, as well as in the inner and outer plexiform layers (Fig. 7A and B). Residual retinal ganglion cells in the patient with LHON retained a similar expression pattern. A punctuate cytoplasm staining was also observed in neoplastic cells from a paraffin breast cancer section, similar to what has been reported in BRC7 cells (Pedram et al., 2006; Fig. 7C). In addition, double immunofluorescence with anti-mitochondrial and anti-oestrogen receptor β antibodies demonstrated co-localization of mitochondria and oestrogen receptor β in the somata of retinal ganglion cells (Fig. 7D). Immunostaining with oestrogen receptor α antibodies gave negative results (not shown).

Figure 7.

Figure 7

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Oestrogen receptor β localizes to retinal ganglion cells. (A) Immunoperoxidase stain with anti-oestrogen receptor β antibodies (ERβ-H150) on horizontal retinal sections from a normal individual (left: male, 59-years-old) and a patient with LHON with the 11778/ND4 mutation (Middle: male, 52-years-old). In the normal individual, a positive stain is observed in the somata of retinal ganglion cells (between arrows) and the unmyelinated portion of the axons in the retinal nerve fibre layer, as well as in the inner and outer plexiform layers. Residual retinal ganglion cells in the patient with LHON (arrows) show a similar expression pattern. Similar results were obtained with the antibody ERβ 485-503 both on male and female control individuals (not shown). (B) Higher magnification showing a typical mitochondrial punctuate pattern of oestrogen receptor β in the somata of retinal ganglion cells. (C) Immunoperoxidase stain with anti-oestrogen receptor β antibodies (ERβ-H150) on a formalin fixed, paraffin embedded section of breast cancer used as a positive control. Note the faint nuclear and the strong cytoplasm staining, similar to that reported in BRC7 breast cancer cells (Pedram et al., 2006). (D) Double immunofluorescence stain with anti-mitochondrial (green) and anti-oestrogen receptor β (red) antibodies. The negative control (right) shows a non-specific background fluorescence induced by fixation media, yet on top of this background is the evident co-localization of mitochondria and oestrogen receptor β in the somata of retinal ganglion cells. Overlap is seen in yellow. NFL = nerve fiber layer; IPL = inner plexiform layer; OPL = outer plexiform layer.

Discussion

The current study, part of a long-standing investigation to characterize the cellular behaviour of mitochondrial DNA mutations in LHON using the cybrid cell model, had the dual aim of investigating the reasons for the higher prevalence of LHON in males and exploring the potential compensatory effects of oestrogens on mutant cell metabolism. Firstly, we provided evidence that both oestrogen receptor α and oestrogen receptor β are present in the nuclei of 143B.TK-cells, whereas only the oestrogen receptor β localized to the mitochondria of osteosarcoma-derived cybrids. These results extend previous studies on osteosarcoma-derived cell lines (Solakidi et al., 2005) and confirm that oestrogen receptor β is enriched in the mitochondria of different cell types (Yang et al., 2004). We then showed that oestrogens present a multilayer effect on complex I-defective LHON cybrids, leading to reduced production of reactive oxygen species, partially rescuing cell viability in galactose medium by restoring membrane potential and limiting apoptotic cell death. In addition, we also documented a coordinated activation of mitochondrial biogenesis and a small but significant improvement in the energetic competence of cybrids induced by oestrogens. Finally, we showed that oestrogen receptor β is localized to the mitochondrial network of human retinal ganglion cells and the unmyelinated portion of their axons in the retinal nerve fibre layer, and their expression is retained in the surviving retinal ganglion cells of patients with LHON. Thus, our results on the cybrid cell model apply to this target tissue. Our study provides an explanatory framework for male prevalence in LHON and a potential new avenue for therapeutic interventions.

Previous work by our group with cybrids has shown that mitochondrial DNA point mutations in LHON affecting different subunits of complex I essentially lead to increased oxidative stress (Beretta et al., 2004; Floreani et al., 2005), defective complex I-driven bioenergetics (Baracca et al., 2005) and an enhanced predisposition to apoptotic cell death (Ghelli et al., 2003; Zanna et al., 2005). Most of these pathologic cell phenotypes become full blown when cybrids are grown in glucose-free, galactose-supplemented medium, a condition forcing cells to rely on oxidative phosphorylation for ATP production (Robinson et al., 1992). In the present study, we further showed that under this stressful metabolic condition, cybrids undergo a rapid increase in production of reactive oxygen species. This was associated with early increase in mitochondrial DNA copy number and, over time, with a general upregulation of mitochondrial biogenesis, as revealed by the increased expression of both mitochondrial and nuclear genes coding for components of the mitochondrial respiratory chain. These changes are tightly linked to and driven by the induction of the upstream master regulators of mitochondrial biogenesis, PGC1-α and PGC1-β and their targets, confirming that these transcription co-activators can be powerfully induced by reactive oxygen species (St-Pierre et al., 2006). The forced use of oxidative phosphorylation in galactose medium ultimately led to a massive increase of reactive oxygen species in LHON cybrids, significantly higher than controls, and reduced cell viability due to an increased rate of apoptotic cell death. Using this metabolic paradigm, we investigated the effects of oestrogen treatment.

Exposure of LHON and control cybrids to 100 nM of 17β-oestradiol led to a rapid decrease in levels of reactive oxygen species, both in glucose and galactose medium, which was particularly remarkable for LHON cells. This decline in reactive oxygen species was paralleled by an increase in SOD2 activity, which was rapid and not linked with higher protein levels in the first hours of 17β-oestradiol incubation. After 24 h, SOD2 protein increase was evident only in cells maintained in glucose medium. These effects also applied to control cells and were oestrogen receptor dependent. Our results are compatible with previous studies showing that oestrogens may activate SOD2 both by a direct effect and by increased transcription (Borrás et al., 2004; Pedram et al., 2006; Gottipati and Cammarata, 2008). Similarly, LHON cybrid viability, loss of membrane potential and rate of apoptotic cell death in galactose medium were all significantly rescued by 17β-oestradiol treatment. These results were mirrored by changes in mitochondrial network dynamics of LHON cybrids in galactose. In fact, the hyper-fragmented mitochondrial morphology, typically associated with pre-apoptotic or apoptotic cell morphology, was clearly reduced by 17β-oestradiol treatment.

The administration of 17β-oestradiol also resulted in the powerful activation of mitochondrial biogenesis, orchestrated by the upstream transcription machinery including transcriptional co-activators PGC1α and PGC1β, and transcription factors NRF1, NRF2 and Tfam. The timing of this activation indicates early events, possibly induced by direct action of oestrogen on mitochondria, and long-term events due to genomic effects of oestrogen (Mattingly et al., 2008). In fact, the increase in mitochondrial DNA copy number occurred as early as 15 min after treatment. This is most probably driven by a direct effect of oestrogen on mitochondrial DNA replication as suggested by the described oestrogen responsive elements in the D-loop region (Chen et al., 2004b). However, this increase in mitochondrial biogenesis ultimately resulting in increased respiratory chain complexes was not mirrored by an equivalent improvement in cell bioenergetics as measured by oxygen consumption and total ATP content in this model system.

Overall, our results using cybrid cells demonstrate that oestrogens can improve the mitochondrial dysfunction in LHON, most prominently by counteracting the excess of reactive oxygen species production, which leads to rapid loss of viability and apoptotic cell death in the time-course experiments after switching to galactose medium. In fact, under this condition of over-imposed metabolic stress, administration of oestrogens improved all parameters measured. In contrast, the beneficial effect of oestrogens did not seem to be equally efficient in ameliorating the bioenergetic competence of LHON cybrids. All results obtained after oestrogen treatment were mediated by oestrogen receptors as counteracted by the use of the oestrogen receptor antagonist ICI 182780.

Our observations in the cell model of cybrids most probably apply to the in vivo target tissue, i.e. retinal ganglion cells and their axons. We demonstrate here for the first time that oestrogen receptors are expressed in human retina, particularly abundant in retinal ganglion cells. The oestrogen receptor β was shown by immunofluorescence to decorate the mitochondrial network co-localizing with mitochondrial directed antibodies. The expression of oestrogen receptor β was also retained in the surviving retinal ganglion cells from patients with LHON. There is clinical evidence that pathologic expression of subclinical LHON differs in male and female unaffected carriers of the mitochondrial DNA mutation. In particular, optical coherence tomography measurement of nerve-fibre-layer swelling in the temporal–inferior quadrants (papillomacular bundle) of unaffected mutation carriers was significantly thicker in males compared with females (Savini et al., 2005). Similarly, subclinical colour vision defects observed in asymptomatic carriers were prevalent in males (Ventura et al., 2007). A further supporting element is the slightly older age of onset in females compared with males (Carelli et al., 2004). This is possibly due to a subgroup of females developing LHON after menopause. Furthermore, cybrids exposure to testosterone did not show relevant changes in cell growth and respiration (Andrea Martinuzzi, data not shown), pointing to a specific role of oestrogen on mitochondrial metabolism.

In conclusion, our study provides new insights for LHON, strongly supporting a metabolic basis for the observed and, to date, unexplained male prevalence. The different exposure to oestrogens between males and females is sufficient to modify the severity of mitochondrial dysfunction induced by mitochondrial DNA mutations affecting complex I in LHON, as clearly demonstrated by our results. Furthermore, these observations hold promise for a therapeutic use of molecules with oestrogen-like activities, such as phyto-oestrogens (Kuiper et al., 1997) that preferentially bind to oestrogen receptor β, thus limiting their oestrogenic activity to cells expressing these receptors, such as retinal ganglion cells.

Funding

This work was supported by Telethon Grant GGP06233 (V.C.), Associazione Serena Talarico per i giovani nel mondo and Fondazione Giuseppe Tomasello O.N.L.U.S. (C.G.), Research to Prevent Blindness (F.N.R.-C. and A.A.S.), Struggling Within Leber’s Foundation (F.N.R.-C. and A.A.S.), Eierman Foundation (F.N.R.-C. and A.A.S.) and National Institutes of Health grant EY03040 (F.N.R.-C. and A.A.S.).

Supplementary material

Supplementary material is available at Brain online.

Supplementary Data
supp_134_1_220__index.html (1.1KB, html)

Acknowledgements

The authors wish to thank Claudia Travaglini, Mariangela Sebastiani, Enrico Secchi, Daniela Catanzaro and Silvia Vidali for their excellent technical assistance.

Glossary

Abbreviations

COIV 

 cytochrome oxidase c subunit IV

DMEM 

 Dulbecco’s modified eagle medium

LHON 

 Leber’s hereditary optic neuropathy

PCR 

 polymerase chain reaction

SOD2 

 superoxide dismutase 2

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