Brain Advance Access published October 13, 2010
doi:10.1093/brain/awq276
Brain 2010: Page 1 of 15
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BRAIN
A JOURNAL OF NEUROLOGY
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’Amati1 and Valerio Carelli6
1
2
3
4
5
6
Dipartimento di Medicina Sperimentale e Patologia, Sapienza, Università di Roma, 00161 Rome, Italy
Dipartimento di Farmacologia ed Anestesiologia, Università di Padova, 35131 Padova, Italy
IRCCS ‘E. Medea’, 31015 Conegliano, Treviso, Italy
Departments of Ophthalmology and Neurosurgery, Keck School of Medicine at University of Southern California, Los Angeles, 90033 CA, USA
Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, 40123 Bologna, Italy
Dipartimento di Scienze Neurologiche, Universita’ di Bologna, 40123 Bologna, Italy
*These authors contributed equally to this work.
Correspondence to: Dr Valerio Carelli,
Dipartimento di Scienze Neurologiche,
Università di Bologna,
Via Ugo Foscolo 7,
40123 Bologna, Italy
E-mail: valerio.carelli@unibo.it
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 17b-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 17b-oestradiol significantly rescued these pathological features and led to the activation of the antioxidant
enzyme superoxide dismutase 2. In addition, 17b-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 b 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
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
Received March 11, 2010. Revised July 27, 2010. Accepted August 9, 2010.
ß The Author (2010). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
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Oestrogens ameliorate mitochondrial dysfunction
in Leber’s hereditary optic neuropathy
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| Brain 2010: Page 2 of 15
Introduction
(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 mg/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).
17b-Oestradiol was purchased from Sigma-Aldrich (St Louis, MO,
USA) and the oestrogen receptor antagonist ICI 182780 from Tocris
Bioscience (Bristol, UK). 17b-Oestradiol and ICI 182780 were dissolved
in ethanol at a final concentration of 100 mM 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
17b-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 isothiocyanateand 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
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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 490% 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
C. Giordano et al.
LHON and oestrogens
Intracellular level of reactive oxygen
species and mitochondrial/
transmembrane potential (j)
Direct detection of intracellular steady-state levels of reactive oxygen
species was carried out on living cells by cytofluorimetry using
20 ,70 -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 20 ,70 -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 mM 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.
| 3
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 = 488 nm. The green signal from Alexa 488/annexin V
was measured at = 525 nm and the red signal from propidium iodide
was measured at = 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 TaqManÕ
probe system and PlatinumÕ 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
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amounts of protein (50 mg) 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 a (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 b (ERb 485-503; Calbiochem, MerckKGaA,
Darmstadt, Germany); rabbit polyclonal antibody raised against amino
acids 1–150 of human oestrogen receptor b (ERb 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-b-actin (Sigma, Saint Luise, MO, USA) and mouse
monoclonal antibody anti-mitochondria extract clone MTC (UCS
Diagnostic, Morlupo, Italy).
Brain 2010: Page 3 of 15
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| Brain 2010: Page 4 of 15
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
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 P50.05.
Results
Characterization of cybrid cells
We investigated the effects of 17b-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 a and oestrogen receptor b
were localized to 143B.TK-nucleus, whereas immunofluorescence
and western blot analyses showed localization of the oestrogen
receptor b to cybrid-derived mitochondria (Fig. 1A and B). This
characterization for oestrogen receptors was carried out prior to
the cells’ exposure to 17b-oestradiol to verify their constitutive
expression.
17b-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 17b-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 17b-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 17b-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 17b-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 17b-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.
17b-Oestradiol induces superoxide
dismutase 2 activity
Manganese SOD2 is the main mitochondrial antioxidant enzyme.
Oestrogens can modulate SOD2 activity both by a rapid,
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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 reversetranscribed 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-, PGC1-, 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 ml of complementary DNA template
in a 50-ml 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 (Ct) method. All values were normalized to the 18S ribosomal
RNA housekeeping gene.
C. Giordano et al.
LHON and oestrogens
Brain 2010: Page 5 of 15
| 5
of oestrogen receptors to the nucleus and mitochondria of cybrid cells. Oestrogen receptor b is stained red by oestrogen receptor b
BIO1974 antibody (a) and oestrogen receptor b H150 antibody (e); oestrogen receptor a 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 b (ERb) protein (identified with oestrogen
receptor b 485–503 antibody) is constitutively over-expressed in the mitochondrial fraction of MCF7 cells, whereas oestrogen receptor a
(ERa) is present at lower levels, confirming previous data (Pedram et al., 2006). In wild-type cybrids, oestrogen receptor b is less abundant
than in MCF7 cells, whereas oestrogen receptor a is not expressed.
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
17b-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 17b-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 17b-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 17b-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 17b-oestradiol led to a further rapid (1 h)
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Figure 1 Oestrogen receptor localization to osteosarcoma-derived (143B. TK-) cybrid cell lines. (A) Fluorescence microscopy localization
6
| Brain 2010: Page 6 of 15
C. Giordano et al.
were incubated for 1 h in glucose (glu) or galactose (gal) medium 10–200 nM 17b-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 17b-oestradiol (E2). The oestrogen receptor antagonist ICI 182780 (I) was added 30 min before
17b-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. P50.05; P50.01; P50.001 LHON versus control; †P50.05 for
glu + E2 versus glu; *P50.05, ***P50.001 for gal versus glu; +P50.05; ++P50.01 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 17b-oestradiol (E2). Incubation in galactose medium induced a significant increase of SOD2 activity in control cybrids
(P50.001) 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. P50.001 for LHON versus control; +++P50.001 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 17b-oestradiol (E2). The oestrogen receptor antagonist ICI 182780
was added 30 min before 17b-oestradiol. Data represent mean arbitrary units ( SEM) normalized to control values in glucose medium
and are from three experiments. †P50.05 and ††P50.01 for glu + E2 versus glu; ***P50.001 for gal versus glu; +++P50.001 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 17b-oestradiol. A representative blot from three is shown.
Downloaded from brain.oxfordjournals.org at Universit? di Bologna - Sistema Bibliotecario d'Ateneo on October 14, 2010
Figure 2 17b-Oestradiol decreases levels of reactive oxygen species and induces SOD2 activity. (A) Left: Control and 11778/ND4 cybrids
LHON and oestrogens
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.
17b-Oestradiol ameliorates cell viability
and reduces apoptosis in galactose
medium
17b-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 40 ,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
| 7
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 17b-oestradiol
(Fig. 4B). The results of these experiments are compatible and
complementary to those obtained from cell growth rates and
the annexin assay.
17b-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 17b-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 17b-oestradiol (Fig. 5A). This event was evident after
15–30 min of incubation with 17b-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 17b-oestradiol treatment, so we
chose this cell line for the further experiments. To gain insight
into the mechanism involved in the 17b-oestradiol-mediated
mitochondrial DNA copy number increase, we evaluated the
gene expression level of the master regulators of mitochondrial
biogenesis: PPAR- coactivator 1-a (PGC1-) and its homologue
PGC-1; 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 17b-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
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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 17b-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 (mtc).
After 24-h incubation in galactose medium, LHON cybrids
displayed a significant decrease (35%) of mtc compared with
control cybrids. Supplementation with 100 nM 17b-oestradiol
prevented the mtc reduction and this effect was lost by
pre-incubation with the oestrogen receptor antagonist ICI
182780 (Fig. 3B).
The next set of experiments was designed to examine whether
protection of cell viability by 17b-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
17b-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
17b-oestradiol.
Brain 2010: Page 7 of 15
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| Brain 2010: Page 8 of 15
C. Giordano et al.
17b-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 17b-oestradiol partially
prevented the galactose-dependent COIV and ND6 protein
Downloaded from brain.oxfordjournals.org at Universit? di Bologna - Sistema Bibliotecario d'Ateneo on October 14, 2010
Figure 3 17b-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 17b-oestradiol (E2). ***P50.001 for gal versus glu; +P50.05,
++
P50.01, +++P50.001 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 (mtj) of control and
LHON cybrids incubated for 24 h in glucose (glu) or galactose (gal) medium 100 nM 17b-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. *P50.05,
***P50.001 for gal versus glu; +P50.05, +++P50.001 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 17b-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. P50.001 LHON versus control; ***P50.001 for gal versus glu; ++P50.01
for gal + E2 versus gal (see Supplementary Fig. 2).
LHON and oestrogens
Brain 2010: Page 9 of 15
| 9
‘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 17b-oestradiol (E2). P50.001, P50.05 for
LHON versus WT; +++P50.001 for gal + E2 versus gal.
reduction and led to a remarkably increased amount of protein
when cells were maintained in glucose medium (Fig. 5C).
17b-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 17b-oestradiol. Supplementation of glucose medium with
17b-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 17b-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 17b-oestradiol
partially prevented the galactose-induced ATP-content decrease.
This phenomenon was particularly evident in LHON cybrids
(540% increase in total ATP content in galactose medium
plus oestrogens compared with galactose medium plus vehicle;
Fig. 6B).
Oestrogen receptor b localizes to retinal
ganglion cells
Several studies have localized both oestrogen receptor a and
oestrogen receptor b 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
Downloaded from brain.oxfordjournals.org at Universit? di Bologna - Sistema Bibliotecario d'Ateneo on October 14, 2010
Figure 4 17b-Oestradiol reduces mitochondrial network fragmentation. (A) Representative images of the four classes of cells (see
10
| Brain 2010: Page 10 of 15
C. Giordano et al.
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Figure 5 17b-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 17b-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. †††P50.001 for glu + E2 versus glu and versus glu + E2+ I;
**P50.01 for gal versus glu; +++P50.001 for gal + E2 versus gal (see Supplementary Fig. 3). (B) Control and LHON cybrids were
(continued)
LHON and oestrogens
Brain 2010: Page 11 of 15
| 11
Discussion
Figure 6 17b-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 17b-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
17b-oestradiol has been normalized to the rate of oxygen
consumption in glucose medium plus ethanol. *P50.03;
**P50.003 for glu + E2 versus glu. (B) Control and 11778/ND4
LHON cybrids were incubated for 24 h in glucose (glu) 100 nM
17b-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. +++P50.001 for E2
versus ethanol; *** for galactose (gal) versus glucose.
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
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 a and oestrogen receptor b are present in the
nuclei of 143B.TK-cells, whereas only the oestrogen receptor b
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 b 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 b 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
Figure 5 Continued
incubated for 6 h in glucose (glu) or galactose (gal) medium 100 nM 17b-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- and its
homologue PGC-1, 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 17b-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. †††P50.001, ††P50.01, †P50.05
for glu + E2 versus glu and versus glu + E2+ I; ***P50.001, **P50.01, *P50.05 for gal versus glu; +++P50.001 for gal + E2 versus gal.
Downloaded from brain.oxfordjournals.org at Universit? di Bologna - Sistema Bibliotecario d'Ateneo on October 14, 2010
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 b
antibodies demonstrated co-localization of mitochondria and oestrogen receptor b in the somata of retinal ganglion cells (Fig. 7D).
Immunostaining with oestrogen receptor a antibodies gave negative results (not shown).
12
| Brain 2010: Page 12 of 15
C. Giordano et al.
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, galactosesupplemented 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
Downloaded from brain.oxfordjournals.org at Universit? di Bologna - Sistema Bibliotecario d'Ateneo on October 14, 2010
Figure 7 Oestrogen receptor b localizes to retinal ganglion cells. (A) Immunoperoxidase stain with anti-oestrogen receptor b antibodies
(ERb-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 ERb 485-503 both on male and female control individuals (not shown). (B) Higher magnification showing a typical
mitochondrial punctuate pattern of oestrogen receptor b in the somata of retinal ganglion cells. (C) Immunoperoxidase stain with
anti-oestrogen receptor b antibodies (ERb-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 b (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 b in the somata of retinal ganglion cells. Overlap is seen in yellow. NFL = nerve
fiber layer; IPL = inner plexiform layer; OPL = outer plexiform layer.
LHON and oestrogens
| 13
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 b was shown by
immunofluorescence to decorate the mitochondrial network
co-localizing with mitochondrial directed antibodies. The expression of oestrogen receptor b 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 b, thus limiting their oestrogenic activity to cells expressing these receptors, such as retinal ganglion
cells.
Acknowledgements
The authors wish to thank Claudia Travaglini, Mariangela
Sebastiani, Enrico Secchi, Daniela Catanzaro and Silvia Vidali for
their excellent technical assistance.
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
Downloaded from brain.oxfordjournals.org at Universit? di Bologna - Sistema Bibliotecario d'Ateneo on October 14, 2010
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- and PGC1- 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
17b-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 17b-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 17b-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 17b-oestradiol treatment.
The administration of 17b-oestradiol also resulted in the
powerful activation of mitochondrial biogenesis, orchestrated by
the upstream transcription machinery including transcriptional
co-activators PGC1 and PGC1, 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
Brain 2010: Page 13 of 15
14
| Brain 2010: Page 14 of 15
(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.
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