* Serono Pharmaceutical Research Institute, Ares Serono International S.A., CH-1228 Plan-les-Ouates, Geneva, Switzerland;
and Center of Electron Microscopy, University of Lausanne, CH-1005 Lausanne, Switzerland
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Abstract |
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During apoptosis induced by various stimuli, cytochrome c is released from mitochondria into the cytosol where it participates in caspase activation. This process has been proposed to be an irreversible consequence of mitochondrial permeability transition pore opening, which leads to mitochondrial swelling and rupture of the outer mitochondrial membrane. Here we present data demonstrating that NGF-deprived sympathetic neurons protected from apoptosis by caspase inhibitors possess mitochondria which, though depleted of cytochrome c and reduced in size, remained structurally intact as viewed by electron microscopy. After re-exposure of neurons to NGF, mitochondria recovered their normal size and their cytochrome c content, by a process requiring de novo protein synthesis. Altogether, these data suggest that depletion of cytochrome c from mitochondria is a controlled process compatible with function recovery. The ability of sympathetic neurons to recover fully from trophic factor deprivation provided irreversible caspase inhibitors have been present during the insult period, has therapeutical implications for a number of acute neuropathologies.
Key words: apoptosis; Bax; cytochrome c; mitochondria; caspases ![]() |
Introduction |
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MITOCHONDRIA have been found to be essential in
controlling at least certain apoptosis pathways
(Green and Reed, 1998). The mechanisms by
which they exert this function include release of caspase
activators as cytochrome c (Liu et al., 1996
) and apoptosis-inducing factor (Susin et al., 1996
), and disruption of electron transport and oxidative phosphorylation (Hockenbery et al., 1993
; Kane et al., 1993
; Sarafian and Bredesen,
1994
; Greenlund et al., 1995
; Zamzami et al., 1995
; Adachi
et al., 1997
; Garcia-Ruiz et al., 1997
). Cytochrome c has
been reported to be released from mitochondria into the
cytosol of many cell types undergoing apoptosis (Chauhan et al., 1997
; Kluck et al., 1997
; Yang et al., 1997
). Moreover, mitochondrial cytochrome c release has been shown
to be required for apoptosis to occur in sympathetic neurons deprived of NGF (Neame et al., 1998
). In the cytosol,
cytochrome c participates in caspase activation through
binding to Apaf-1 and caspase 9 (Liu et al., 1996
; Li et al.,
1997
). The redistribution of cytochrome c during apoptosis
can be prevented by overexpression of the anti-apoptotic protein Bcl-2 (Kluck et al., 1997
; Yang et al., 1997
). In contrast, just overexpression of the pro-apoptotic protein Bax
has been shown to trigger cytochrome c efflux from mitochondria (Eskes et al., 1998
; Rossé et al., 1998
). Altogether, these results suggest that the release of mitochondrial cytochrome c is tightly regulated by Bcl-2 family
members. However, the mechanisms by which cytochrome
c is released from mitochondria is still unclear. It has been
proposed that opening of the permeability transition pore
(PTP)1 (Zoratti and Szabo, 1995
; Bernardi and Petronilli,
1996
; Beutner et al., 1996
; Nicolli et al., 1996
; Halestrap
et al., 1997
) which leads to mitochondrial swelling and possibly to rupture of the mitochondrial outer membrane
(Vander Heiden et al., 1997) allows the passive release of
caspase-activating proteins from the intermembrane space
of mitochondria into the cytosol (Marchetti et al., 1996
; Susin et al., 1996
; Green and Reed, 1998
). However, it has
also been reported that, in many cell types, the release of
cytochrome c occurs before or in the absence of a change
in mitochondrial permeability suggesting that this process
involves mechanisms other than (or in addition to) opening of the PTP (Kluck et al., 1997
; Yang et al., 1997
; Bossy-Wetzel et al., 1998
; Green and Reed, 1998
). Here we report that in apoptosis of sympathetic neurons induced by
NGF deprivation, cytochrome c is released from mitochondria in the absence of mitochondrial swelling. Moreover, we show that addition of NGF back to neurons rescued by Boc-aspartyl(Ome)-fluoromethylketone (BAF)
leads to restoration of normal cytochrome c content by mitochondria.
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Materials and Methods |
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Culture of Sympathetic Neurons
Sympathetic neurons from newborn rat cervical superior ganglia were cultured as previously described (Martinou et al., 1995). For electron microscopy studies, neurons were fixed with 1% glutaraldehyde in PBS for 48 h
at 4°C and washed in PBS before pre-embedding.
Subcellular Fractionation
Sympathetic neurons (2 × 105 in 3.5-cm-diam Petri dish) were harvested
in 100 µl of isotonic buffer (210 mM mannitol, 70 mM sucrose, 1 mM
EDTA, and 10 mM Hepes, pH 7.5) supplemented with protease inhibitors
cocktail Complete (Boehringer Mannheim) and homogenized with a
Dounce homogenizer. Samples were transferred to Eppendorf centrifuge
tubes, centrifuged at 900 g for 5 min to remove nuclei, and then followed
by centrifugation at 10,000 g for 30 min at 4°C to obtain the heavy membrane pellet (HM) enriched in mitochondria. The HM material was resuspended in 20 µl PBS, 0.2% Triton X-100. The protein concentration was
determined by the method of Bradford (1976) in both the HM and soluble
fractions. 5 µg (HM fraction) and 8 µg (soluble fraction) were used for
Western blotting.
Isolation of Mouse Liver Mitochondria and Incubation with Bax
Mitochondria were isolated by sucrose density gradient centrifugation as
previously described (Eskes et al., 1998). Mitochondria were incubated
with 5 µM Bax
Tm for 30 min at 30°C in a buffer containing 125 mM KCl,
4 mM MgCl2, 5 mM Na2HPO4, 5 mM succinate, 5 µM rotenone, 0.5 mM
EGTA, 15 mM Hepes-KOH, pH 7.4. For electron microscopy, pellets of
mitochondria were fixed in 1.5% glutaraldehyde in Sorensen phosphate
buffer for 1 h at 4°C and processed as indicated.
Electron Microscopy Studies
Pellets of glutaraldehyde-fixed neurons and isolated mitochondria were pre-embedded into low viscosity agarose, washed with Sorensen phosphate buffer, and then postfixed in 2% OsO4 in phosphate buffer for 1 h at room temperature. Then the samples were washed again in phosphate buffer, dehydrated in alcohol and propylene oxide, and then embedded in Epon. Ultrathin sections of comparable thickness were prepared with a Leica Ultracut ultramicrotome and placed on formvar carbon-coated copper grids. The grids were stained with uranyl acetate and lead citrate and observed with a Philips CM10 transmission electron microscope at 80 kV using a 30-40-µm objective aperture.
Immunocytochemistry
Neurons were fixed with 4% paraformaldehyde in PBS, permeabilized for 10 min with PBS containing 0.2% Triton X-100, incubated for 2 h with an anti-cytochrome c monoclonal antibody (dilution 1:15 in PBS with 5% normal goat serum; PharMingen), and then revealed with a fluorescein-labeled goat anti-mouse antibody.
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Results |
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Cytochrome C Release from NGF-deprived Sympathetic Neurons
We have studied the distribution of cytochrome c in cultured sympathetic neurons from rat superior cervical ganglia (SCG) undergoing apoptosis induced by NGF deprivation. Cytochrome c was analyzed by Western blotting in both soluble cytosolic and the mitochondria enriched heavy membrane (HM) fractions obtained from SCG neurons at 8, 15, and 24 h after NGF. Fig. 1, a and b, highlights a significant decrease of cytochrome c in the HM fraction of neurons deprived of NGF for 8, 15, and 24 h (Fig. 1 b), accompanied by an increase of cytochrome c in the cytosolic fraction (Fig. 1 a). This result was confirmed by immunostaining studies (Fig. 2, a and b). Although all neurons cultured in the presence of NGF displayed a punctate staining (Fig. 2 a), ~50-75% of those deprived of NGF for 15 h displayed a diffuse cytosolic pattern (Fig. 2 b) and >95% of the latter had a condensed apoptotic nucleus (data not shown). Immunocytochemistry studies revealed that mitochondria present in neurites preserved their cytochrome c content longer than mitochondria present in soma (data not shown).
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We also analyzed the cytochrome c distribution in sympathetic neurons rescued from NGF-deprivation with the
caspase peptide inhibitor BAF. In the presence of 100 µM
BAF, >80% neurons were able to survive after NGF deprivation for 5 d, although they had markedly smaller somas
and atrophic neurites (Fig. 3 c). In agreement with previous data (Deshmukh et al., 1996), we found that BAF was able to inhibit caspase activity as assessed by immunoblot analysis of poly-(ADP ribose) polymerase (data not
shown). A major decrease in cytochrome c levels was detected in the HM fraction of those neurons (Fig. 1 c), a
finding confirmed by cytochrome c immunostaining (Fig. 2
c). These data indicate that, during apoptosis, the release
of cytochrome c from mitochondria of sympathetic neurons does not require caspase activity.
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Cytochrome C Recovery by Mitochondria of NGF-rescued Neurons Re-exposed to NGF
The major objective of this article was to test whether mitochondria from BAF-rescued neurons could recover their
cytochrome c content following re-exposure of neurons to
NGF. Cytochrome c analysis in HM fraction of neurons
deprived of NGF in the presence of BAF for 2 d and then
re-exposed to NGF for 3 d, revealed a significant increase
in the level of mitochondrial cytochrome c compared with
neurons cultured in the absence of NGF and the presence
of BAF alone for 5 d (Fig. 1 c). A kinetic analysis revealed a progressive recovery of cytochrome c by mitochondria
which was almost complete after a 4-d NGF treatment
(Fig. 1 d). Consistent with this finding, immunocytochemistry studies showed reappearance of a punctate mitochondrial cytochrome c staining in almost all neurons (>80%,
n = 3) re-exposed to NGF for 24 h (Fig. 2 d). The effect of
re-exposure to NGF was blocked by cycloheximide (Fig. 1
c and Fig. 2 f), suggesting the requirement of de novo protein synthesis for its action. It is possible that synthesis of
cytochrome c precursor apocytochrome c was a prerequisite for cytochrome c recovery by mitochondria as only
apocytochrome c has been shown to be imported into mitochondria (Mayer et al., 1995). In contrast, the recovery
of normal cytochrome c levels by mitochondria was not
prevented by the microtubule disrupting agent colchicine
(Fig. 2 e), therefore excluding the possibility that reappearance of a normal cytochrome c staining pattern in the
cell body was due to microtubule associated migration of
still intact mitochondria from neurites to the soma.
In agreement with previous data (Deshmukh et al.,
1996), we observed that addition of NGF back to BAF-protected SCG neurons caused an increase in soma diameter and neurite extension (Fig. 3 d). Altogether, these
results indicate that the NGF receptors TRKA (tyrosine
kinase receptor) and their signaling components remained
functional in neurons deprived of NGF for at least 5 d. Interestingly, addition of NGF alone (without BAF) back to BAF-rescued neurons was sufficient to promote mitochondria recovery, regrowth of neurons, and long-term
survival, suggesting that the caspases which had been activated during apoptosis had been irreversibly inhibited
by BAF.
Ultrastructure of Mitochondria
The reappearance of cytochrome c in mitochondria of
BAF-rescued neurons re-exposed to NGF suggested that
the ultrastructure of mitochondria had been preserved.
This hypothesis was tested by electron microscopic studies. Fig. 4 a shows that mitochondria from neurons cultured
continuously in the presence of NGF appeared elongated
or oval-shaped, with sparse cristae (mean cross-sectional area ± SEM was 0.110 ± 0.007 µm2, n = 115, Fig. 5 a). 24 h
after NGF deprivation (Fig. 4 b) most mitochondria were
round in shape, smaller than normal (mean cross-sectional area: 0.082 ± 0.004 µm2, mean ± SEM, n = 124, Fig. 5 a)
with a hyperdense matrix. No obvious rupture of the outer
mitochondrial membrane has been observed. Mitochondria from BAF-rescued neurons (Fig. 4 c) were even
smaller (mean cross-sectional area: 0.071 ± 0.005 µm2,
mean ± SEM, n = 80, Fig. 5 a), often forming aggregates
surrounded by lysosomes (data not shown). After addition
of NGF back to BAF-rescued neurons (Fig. 4 d), mitochondria recovered the shape and size typical of neurons
continuously cultured in the presence of NGF (0.134 ± 0.08 µm2, mean ± SEM, n = 121, Fig. 5 a). Lysosomes
containing myelin figures, lipid droplets, as well as images
of autophagy, have also been observed as previously described by Martin et al. (1988).
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Bax Induces Mitochondrial Shrinking and Triggers Cytochrome C Efflux from Mitochondria
Bax is known to play an essential role in neuronal apoptosis. Sympathetic neurons from Bax-deficient mice can survive more than 20 d in the absence of NGF (Deckwerth et al.,
1996). Moreover, Bax is known to trigger cytochrome c release when added directly to isolated mitochondria (Eskes
et al., 1998
; Jürgensmeier et al., 1998
). We therefore wondered whether Bax could also be responsible for the mitochondrial morphological change observed in neurons after
NGF deprivation. This hypothesis was tested by electron microscopy using isolated mitochondria that had been incubated for 15 min in the presence of 5 µM Bax
Tm or,
for comparison, with 100 µM calcium which stimulates
PTP opening (Fig. 5 b and Fig. 6). Although, as previously
reported (Petronilli et al., 1993
), opening of the PTP with
calcium led to mitochondrial swelling, Bax, in contrast,
triggered mitochondrial shrinkage resulting in a morphology similar to that observed in apoptotic neurons (Fig. 5 b
and Fig. 6).
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Discussion |
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We report that in sympathetic neurons undergoing apoptosis triggered by NGF deprivation, mitochondria are
reduced in size, display a hyperdense matrix, and are
depleted of cytochrome c. A similar morphology of mitochondria lacking cytochrome c was observed in neurons
protected from NGF deprivation by the caspase peptide
inhibitor BAF. Interestingly, upon re-exposure to NGF, the mitochondria from BAF-rescued neurons recovered
both a normal size and cytochrome c content. Altogether,
these data do not support the hypothesis according to
which cytochrome c release is the result of opening of the
PTP (Marchetti et al., 1996; Susin et al., 1996
; Green and
Reed, 1998
) and subsequent mitochondrial swelling and
rupture of the outer mitochondrial membrane (Vander Heiden et al., 1997) since neither of these two features
were observed in apoptotic neurons. Instead, our data
agree with previous morphological studies of various cell
types undergoing apoptosis showing preservation of mitochondrial membrane ultrastructure (Wyllie et al., 1980
)
and a reduction in total mitochondrial volume (Mannweiler and Bernhard, 1957
; Rouiller, 1957
; Svoboda and
Higginson, 1963
; Wachstein and Besen, 1964
; Hackenbrock, 1968
). More recently, ultracondensation of mitochondria has also been observed in apoptotic nodal myocytes (James et al., 1993
), in lymphoblastic leukemic cells
undergoing tumor necrosis factor-induced apoptosis (Lia et al., 1997
), in a colon carcinoma cell line treated with
herbimycin A (Mancini et al., 1997
) and in etoposide-treated THP.1 cells (Zhuang et al., 1998
). In some of these
studies, a condensed conformation of mitochondria associated with hyperdensity of the matrix, as described here,
has been postulated to be the result of water and ion loss
from the matrix and to correspond to low-energy states of
mitochondria (Hackenbrock, 1968
).
Bax, a pro-apoptotic member of the Bcl-2 family essential for neuronal apoptosis (Deckwerth et al., 1996), can
form ion channels in synthetic lipid membranes (Antonsson et al., 1997
; Schlesinger et al., 1997
) and therefore
could be a likely candidate responsible for these mitochondrial changes during apoptosis. In support of this hypothesis, addition of Bax directly to isolated mitochondria triggers the release of cytochrome c (Eskes et al., 1998
;
Jürgensmeier et al., 1998
) by a mechanism that may involve pore formation. We now show that this effect is accompanied by a reduction in mitochondrial volume. However, the mechanisms of action of Bax are still unclear. It
has been recently reported that Bax can interact with the
adenine nucleotide translocator (ANT), a component of
the mitochondrial PTP (Marzo et al., 1998
). Moreover, in
yeast, the ANT appears to be required for the Bax killing
function (Marzo et al., 1998
). However, the importance of
the ANT in apoptosis of mammalian cells has not yet been demonstrated.
One of the most striking observations reported here is the ability of mitochondria from BAF-rescued neurons to recover a normal size and cytochrome c content after re-exposure of neurons to NGF. The transition from small to large mitochondria induced by NGF deserves particular attention. This may be the result of mitochondrial fusion, an hypothesis that we are currently testing. We cannot exclude the possibility that proliferation of mitochondria may also take part in the complete recovery of neurons associated with the ability of neurons to grow in size, to extend neurites and to survive over long periods after re-exposure to NGF.
Recovery of function after protection by caspase inhibitors does not apply to all cell types. Indeed, it has previously been reported that inhibition of caspases in diverse
types of apoptosis is incompatible with long-term survival,
suggesting that in those cells caspases are activated after
the cells become committed to apoptosis (McCarthy et al.,
1997). Moreover, it has been shown that overexpression of
Bax in Jurkat cells leads to mitochondrial dysfunction and
caspase-independent apoptosis (Xiang et al., 1996
). In the
case of sympathetic neurons undergoing apoptosis induced by NGF deprivation, the situation is different as
caspase activation appears to represent the point at which
cells become committed to die (Deshmukh et al., 1996
).
Consistent with this, caspase inhibitors can inhibit apoptosis induced by overexpression of Bax (Vekrellis et al.,
1997
; Martinou et al., 1998
) or Bak (Martinou et al., 1998
)
in these neurons. The difference between sympathetic neurons and other cell types may reside in intrinsic specificities of their apoptotic pathway, in specific properties of
their mitochondria or could be related, at least in vitro, to
their ability to produce ATP through glycolysis rather
than through oxidative phosphorylation. The ability of mitochondria to recover fully their function when homeostatic conditions are restored may be specific for mitochondria from neurons. This could explain why in both
caspase 3- and caspase 9-deficient mice, only neurons are
protected from apoptosis during development (Kuida et al.,
1996
, 1998
; Kakem et al., 1998
).
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Footnotes |
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Address correspondence to J.-C. Martinou, Serono Pharmaceutical Research Institute, Ares Serono International S.A., 14 chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland. Tel.: (41) 22 706 9822. Fax: (41) 22 794 6965. E-mail: jean-claude.martinou.ch_gva03{at}serono.com
Received for publication 23 October 1998 and in revised form 23 December 1998.
We are very grateful to S. Catsicas (University of Lausanne, Switzerland)
for having made possible the collaboration between S. Fakan (University
of Lausanne) and J.-C. Martinou and for his interest in this work; to S. Arkinstall and K. Maundrell (SPRI, Geneva, Switzerland) for critical reading
of the manuscript; T. Wells for encouraging support; J. Fakan, V. Mamin,
and F. Voinesco (University of Lausanne) for excellent technical assistance; N. Ruchonnet and S. Tapia (University of Lausanne) for help with morphometrical and quantitative evaluation of the results; and C. Hebert
(SPRI, Geneva, Switzerland) for artwork.
Work performed at the Center of Electron Microscopy (University of
Lausanne, Lausanne, Switzerland) was supported by funds from Ares Serono. Part of this work was also supported by grants from the European
Community (Biotech grant BIO4CT96 0774 to J.-C. Martinou).
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Abbreviations used in this paper |
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ANT, adenine nucleotide translocator; BAF, Boc-aspartyl(Ome)-fluoromethylketone; HM, heavy membrane fraction enriched in mitochondria; PTP, permeability transition pore; SCG, sympathetic neurons from newborn rat cervical superior ganglia.
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