From the Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération, UMR 5088, and the Laboratoire de Biologie Moléculaire des Eucaryotes, IFR109, Université Paul Sabatier, Bât. 4R3-B1, 118 route de Narbonne, F-31062 Toulouse cedex 04, France
Received for publication, December 6, 2002
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ABSTRACT |
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OPA1 encodes a large GTPase related
to dynamins, anchored to the mitochondrial cristae inner membrane,
facing the intermembrane space. OPA1
haplo-insufficiency is responsible for the most common form of
autosomal dominant optic atrophy (ADOA, MIM165500), a neuropathy
resulting from degeneration of the retinal ganglion cells and optic
nerve atrophy. Here we show that down-regulation of OPA1 in HeLa cells
using specific small interfering RNA (siRNA) leads to
fragmentation of the mitochondrial network concomitantly to the
dissipation of the mitochondrial membrane potential and to a drastic
disorganization of the cristae. These events are followed by cytochrome
c release and caspase-dependent apoptotic nuclear events. Similarly, in NIH-OVCAR-3 cells, the OPA1 siRNA induces
mitochondrial fragmentation and apoptosis, the latter being
inhibited by Bcl2 overexpression. These results suggest that OPA1 is a
major organizer of the mitochondrial inner membrane from which the
maintenance of the cristae integrity depends. As loss of OPA1 commits
cells to apoptosis without any other stimulus, we propose that OPA1 is
involved in the cytochrome c sequestration and might be a
target for mitochondrial apoptotic effectors. Our results also suggest
that abnormal apoptosis is a possible pathophysiological process
leading to the retinal ganglion cells degeneration in ADOA patients.
Large GTPases from the dynamin family are involved in various
processes related to membrane dynamic (6, 7). Two dynamin subfamilies
are specifically involved in the mitochondrial network morphology. One
comprises the Dnm1/Drp1 protein that is located on the cytoplasmic side
of the outer mitochondrial membrane
(OM)1 to control the network
fission process (8, 9). The second one includes the intra-mitochondrial
Mgm1p/Msp1/OPA1 protein, which is required for the maintenance of the
mitochondrial structure and genome in yeasts (10-12). Localization of
this dynamin in the different species remains controversial and so does
its precise function on the mitochondrial membranes. Mgm1 has been
first localized anchored to the OM facing the cytoplasm (12), then in
the intermembrane space (IMS) associated to the inner membrane (IM)
(13). Msp1 has been located in the matrix anchored to the IM (11).
Finally OPA1 has been recently located to the IMS anchored on the
cristae IM (1). We have previously shown that mutations in the OPA1 gene induce the most common form of autosomal dominant optic atrophy (2, 3) (ADOA, MIM165500) and affect the structure of the mitochondrial
network in monocytes from ADOA patients (3). The possible function of
OPA1 as a mechano-enzyme (14) or as a molecular switch (15) acting on
the IM suggests that it might be involved in the structure and dynamic
of the cristae (16) and therefore on mitochondrial activity. In
metazoan vertebrates, cristae structures have a crucial role as they
define intra-cristae volumes that segregate the majority of the
cytochrome c (cyt c) from the IMS (17). In
vitro, upon addition of the pro-apoptotic factor tBid on purified
mitochondria, this pool of cyt c is released in the IMS by a
major cristae remodeling process, which allows in vivo the
extensive release of the cyt c in the cytoplasm to trigger
apoptosis (18). To address OPA1 function on the mitochondrial membranes and gain insight into the pathophysiological mechanism of
ADOA, we have analyzed the OPA1 knock-down in culture cells and found
that it disrupts the mitochondrial IM structure and functional
integrity and leads to cyt c release and apoptosis.
Antibodies--
OPA1 antibodies have been described previously
(1). Commercial antibodies were from the sources indicated: anti-HSP60
(LK2, Sigma), anti-Actin (Chemicon), anti-cytochrome c
(Santa Cruz), anti-cleaved PARP (Promega), Alexa-594 anti-rabbit IgG
and Alexa-488 anti-mouse IgG (Molecular Probes), and anti-rabbit
IgG-HRP and anti-mouse IgG-HRP (New England Biolabs).
Cell Culture, RNA Interference (siRNA), and Western
Blots--
HeLa cells were cultured in DMEM, 10% fetal calf
serum, 5% CO2. NIH-OVCAR-3 cells (19) were cultured in
RPMI, 5% fetal calf serum, 5% CO2. siRNA experiments were
carried out as described previously (20). The sequence of the region
targeted by the OPA1-siRNA (Dharmacond Research),
5'-AAGTTATCAGTCTGAGCCAGGTT-3', corresponds to nucleotides 1810-1833 of
OPA1 open reading frame (GenBankTM accession number
AB011139) and is present in all OPA1 alternate splicing variants (21).
Transfections of HeLa cells were performed with Oligofectamine®
reagent (Invitrogen). Final concentration of the siRNA duplex in
culture medium was 100 nM. Control experiments included
treatments of cells with 100 µM z-VAD-fmk (Calbiochem) for 48 h or 5 µM CDDP (Sigma) for 24 h. To
detect OPA1 and actin by Western blot, transfected cells were
trypsinized, washed once in ice-cold PBS, and harvested. Equal
amounts of cells were solubilized in 50 µl of Laemmeli sample
buffer and boiled for 10 min. Samples were run on 8% polyacrylamide
gels and transferred to nitrocellulose. Immunodetection (anti-OPA1:
1/300; anti-actin: 1/10000; IgG-HRP: 1/10,000) was carried out using
ECL (Amersham Biosciences). PARP detection was performed
according to the protocol from Promega (anti-PARP: 1/1000).
Microscopy and FACS Analysis--
Cells grown on glass
coverslips were fixed in PBS, 3.7% paraformaldehyde (30 min, 4 °C),
permeated in 100% methanol (1 min, Loss of OPA1 Induces the Fragmentation of the Mitochondrial
Network--
We have designed a specific human OPA1 siRNA that
corresponds to a sequence situated downstream of the GTPase coding
sequence and analyzed its effect in HeLa cells comparatively with a
control scramble siRNA (Dharmacond Research). After transfection, cells were collected every day for 3 days and Western blots performed. The
different forms of OPA1 (1, 21) were slightly affected at 24 h,
declined at 48 h, and were almost not detectable at 72 h
(Fig. 1a). In parallel, the
mitochondrial morphology was monitored on cells stained with the
Mitotracker® Red (Fig. 1b). 24 h after transfection
of the OPA1 siRNA, cells retained a filamentous mitochondrial network
as control cells (Fig. 1b, top left). At 48 h, when OPA1 levels started to decline, half of the cells showed a
filamentous mitochondrial network and half showed a fragmented one
(Fig. 1b, bottom left). At 72 h, when OPA1
levels were barely detectable, the filamentous phenotype had virtually
disappeared to the benefit of the fragmented phenotype (60%), and of
an additional phenotype (35%) that we described in Fig. 4.
Fragmentation of the mitochondrial network induced by the loss of OPA1
was further visualized by immunofluorescence experiments using HSP60
antibodies and confocal microscopy three-dimensional reconstructions
(Fig. 1c).
Loss of OPA1 Dissipates the Mitochondrial Membrane
Potential--
As a correlation between the IM potential dissipation
and the fragmentation of the mitochondrial network has been recently established (23), we checked the effect of OPA1 down-regulation on the
mitochondrial membrane potential ( Electron Microscopy Observations Show Major Disruption
of the Cristae Structures--
To gain insight on the possible
function of OPA1 on the mitochondrial IM, we performed transmission
electron microscopic observations on cells transfected or not by the
OPA1 siRNA and grown for 72 h. Classical long tubular cylinders
(mean mitochondrial surface area: 0.66 ± 0.49 µm2,
n = 69) were observed in control cells (Fig.
3a), while circular vesicles
of reduced diameter (0.115 ± 0.059 µm2,
n = 194) predominated in treated cells (Fig. 3,
b and c), confirming the photonic observations.
Mitochondria from cells devoid of OPA1 further presented electron
denser matrix and cristae completely unstructured, adopting unusual
shapes consisting of vesicle-like structures with abnormal increased
space between the membranes (Fig. 3, b, c, and
b'), providing evidence suggesting that OPA1 has a major
function in structuring the cristae membranes.
Loss of OPA1 Triggers a Bcl2-inhibitable Apoptosis--
Our
electron microscopy pictures are consistent with descriptions of
mitochondrial pyknosis (24) and of the switch from wild-type to
apoptotic mitochondria in liver cells (18). In addition, our previous
photonic observations, revealed 72 h after transfection of the
OPA1 siRNA a high proportion (35%) of cells, with aggregated
mitochondria, condensed chromatin, and fractionated nucleus reminiscent
to apoptotic phenotype (Fig.
4a). To assert apoptosis, we
estimated the amounts of the cleaved PARP protein (25) by Western blot
(Fig. 4a) and found that they correlated with the percentage
of apoptotic cells. PARP cleavage did not occur in the presence of the
caspases inhibitor z-VAD-fmk, but well in cells treated with CDDP (Fig.
4a). Cytochrome c release was further monitored
by immunofluorescence using cyt c antibodies 72 h after
the OPA1 siRNA transfection. Barely half of the cell population had
released cyt c in the cytoplasm (Fig. 4b), while no cyt c release or apoptotic figure was observed in control
cells. To address the issue whether the anti-apoptotic Bcl2 protein
could counteract the OPA1 induced apoptotic process, the OPA1 and
scramble siRNA were transfected in an ovarian adenocarcinoma cell line (NIH-OVCAR-3) expressing constitutively or not Bcl2 (19) (Fig. 5d). Loss of OPA1 (Fig.
5d) triggered fragmentation of the mitochondrial network in
both cell lines (Fig. 5, a and b), while no
morphological change was found using the scramble siRNA. In addition,
72 h after transfection, apoptosis (Fig. 5c) reached
27% in the OVCAR cells, but was reduced by a 3-fold ratio in cells
overexpressing Bcl2 (Fig. 5d). Thus, in a second transformed
cell line, loss of OPA1 triggers fragmentation of the mitochondrial
network and an apoptotic process that can be inhibited by Bcl2
overexpression.
Results presented here have shown that loss of OPA1 induces
structural and functional modifications of the mitochondria. Indeed, down-regulation of OPA1 expression changes the mitochondrial network fusion-fission balance toward fission, as already noticed for its
orthologs Mgm1p (13) and
Msp1 2 in yeast. Thus OPA1
could either contributes as a fusion effector or as a fission
inhibitor. Alternatively, the fragmentation of the mitochondrial
network could be the consequence of the dissipation of the IM potential
induced by loss of OPA1. In this respect, we further observed that the
cristae were completely disorganized in the absence of OPA1. This later
phenomenon could be the central process that would first dissipate the
IM potential, then open the cristae junctions, allowing mobilization of
cyt c and its release in the cytoplasm to trigger
caspase-dependent apoptosis. How in this situation the cyt
c crosses the OM remains unclear, but this process and the
downstream apoptotic events are inhibited by the overexpression of
Bcl2. These data converge to propose that OPA1 participates in
structuring the cristae to maintain their functional integrity.
Therefore, OPA1 might be a key protein that modulates the IM dynamic to
either maintain cell homeostasis or commit them to apoptosis and,
consequently, could be a target for pro- or anti-apoptotic effectors.
Thus coordinate structural changes of both mitochondrial membranes
during apoptosis could be performed by both families of mitochondrial
dynamin, as Drp1 was recently shown to be involved in the fragmentation
of the mitochondrial network during apoptosis (26).
Finally, the link between OPA1 and apoptosis gives clues on the
possible pathophysiological effect of mutations in the OPA1 gene found in ADOA patients that leads to the neurodegeneration of the
retinal ganglion cells (5). Indeed, this cell population undergoes two
rounds of apoptotic process during normal life: one during development
when setting up the retina, which eliminates 70% of them, and one
during every day life, which accounts for a loss of few thousands of
them per year (27). Haplo-insufficiency generated by the dysfunction of
one OPA1 allele (4) might increase their susceptibility to
apoptogen stimuli and in particular the one generated by the daily
exposure to the UV light.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C), then in PBS, 0.2%
Triton X-100 (10 min, room temperature) and immunolabeled in PBS, 2%
bovine serum albumin, using the following antibodies (HSP60: 1/100;
cytochrome c: 1/100; Alexa-594 anti-rabbit IgG: 1/500;
Alexa-488 anti-mouse IgG: 1/500) and stained with DAPI (0.1 µg/ml).
To quantify phenotypes, cells were stained directly in the culture
using 100 nM CMXros Mitotracker® Red (Molecular Probes)
for 30 min, then fixed and DAPI-stained. To analyze the
m, cells were incubated 20 min with 5 µg/ml JC-1
(Molecular Probes) in culture medium and observed by confocal
microscopy. Fluorescence images were captured and processed using a
Leica DMIRE-2 microscope or a LSM-410 Zeiss confocal microscope. For FACS analyses of the
m, cells were removed from the
culture dishes and then pelleted by low centrifugation, resuspended in culture medium with 50 nM TMRE (Molecular Probes) (22), and analyzed 15 min later by flow cytometry (FACS-Calibur, BD
Biosciences) using the Cellquest software. Control experiments included
treatment with 50 mM DNP (Sigma) for 60 min or 2 µM Oligomycin (Sigma) for 30 min or 0.1% Nonidet P-40
for 30 min. For transmission electron microscopy, cells were fixed for
2 h with 4% glutaraldehyde in sodium cacodylate buffer,
post-fixed for 1 h with 1% osmium tetroxide, dehydrated, and
embedded in Epon (EMS). Thin sections adsorbed onto nickel grids
were stained with 1% uranyl acetate and 0.3% lead citrate and imaged
in a JEOL-1200 EX electron microscope at 80 kV. Mitochondria surfaces
were assessed with the Image Tools 3.0 software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Loss of OPA1 induces fragmentation of the
mitochondrial network. HeLa cells were transfected with
either the scramble siRNA (control/ ) or the OPA1 siRNA (+) and grown
for 24, 48, and 72 h. a, OPA1 and actin detection by
Western blot. b, analysis of Mitotracker® Red and DAPI
staining by fluorescence microscopy. Top left, filamentous
mitochondrial network; bottom left, punctuated mitochondrial
network; right, percentages of both phenotypes in the cell
population. c, three-dimensional confocal imaging of cells
labeled with HSP60 antibody 72 h after transfection.
Bar = 1 µm.
m) using JC1 and
TMRE dyes. 72 h after OPA1 siRNA transfection, fluorescence
microscopy observations revealed that the fragmented cells were all
devoid of the JC1
m-sensitive red fluorescence (Fig.
2a), as cells treated with the
uncoupling drug DNP, and in deep contrast to cells treated with
oligomycine, that increases the
m and consequently the amount of red fluorescence. FACS analyses performed with TMRE dye
revealed that the
m in cells treated with the OPA1
siRNA was decreased to a level similar to that estimated for cells
treated with the DNP drug and slightly higher than that observed for
cells treated with the Nonidet P-40 detergent (Fig. 2b).
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Fig. 2.
Loss of OPA1 induces mitochondrial membrane
potential reduction. m was assessed
72 h after cell transfection with scramble siRNA
(control) or OPA1 siRNA, or on cells treated with various
drugs (oligomycin, DNP, or Nonidet P-40
(NP40)). a, confocal pictures of cells stained
with JC-1 dye. b, FACS analyses of TMRE incorporation.
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Fig. 3.
Loss of OPA1 causes a disorganization of the
cristae structure. Electron microscopic micrographs of a thin
section of HeLa cells showing the structure of mitochondria 72 h
after transfection without (a) or with OPA1 siRNA
(b, c). Drawing of two representative
mitochondria are presented (a' and b').
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Fig. 4.
Loss of OPA1 triggers apoptosis.
a, kinetic of appearance of the apoptotic phenotype
in cells transfected with the scramble ( ) or the OPA1 (+) siRNA
stained with DAPI and Mitotracker® Red. A Western blot detecting the
PARP-cleaved form (PARP p89) and actin, 24, 48, and 72 h after transfection. As control, OPA1 siRNA-transfected cells were
treated with z-VAD-fmk, or untransfected cells were treated with CDDP.
b, fluorescence microscopy using DAPI and Mitotracker® Red
or cytochrome c antibodies on cells, 72 h after
transfection with scramble (control) or OPA1 siRNA.
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Fig. 5.
Apoptosis induced by the OPA1 siRNA is
inhibited by Bcl-2 in NIH-OVCAR-3 cells. NIH-OVCAR-3 cells
expressing constitutively (Bcl2) or not (Neo)
Bcl2 were transfected with the scramble (control) or the OPA1 siRNA.
a-c, pictures showing the filamentous (a),
punctuated (b), and apoptotic (c) phenotypes of
the OVCAR-Neo cells stained with Mitotracker® Red and DAPI.
d, percentage of apoptotis and Western blots revealing OPA1,
actin, and Bcl2 in both cell lines, 72 h after transfection.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are indebted to the IFR109 technical resources and Brice Roncin for help in electron and confocal microscopy and members of the UMR5088 for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from the Centre National pour la Recherche Scientifique, Université Paul Sabatier, Ministère de la Recherche et de l'Education, Rétina France, Fondation pour la Recherche Médicale, Association pour la Recherche sur le Cancer, Fondation Electricité de France, and l'Association Française contre les Myopathies.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 33-561-55-62-38;
Fax: 33-561-55-81-09; E-mail: belengue@cict.fr.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.C200677200
2 E. Guillou, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: OM, outer mitochondrial membrane; IM, inner mitochondrial membrane; IMS, intermembrane space; cyt c, cytochrome c; HRP, horseradish peroxidase; z-VAD-fmk, z-Val-Ala-Asp-fluoromethylketone; CDDP, cis-diamine dichloroplatinum; PBS, phosphate-buffered saline; PARP, poly(ADP-ribose) polymerase; FACS, fluorescence-activated cell sorter; DAPI, 4',6-diamidino-2-phenylindole; TMRE, tetramethyl rhodamine ethyl ester; DNP, dinitrophenol; siRNA, small interfering RNA.
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