Bioscience Reports, Vol. 17, No. 1, 1997
REVIEW
Role of the Mitochondrial Permeability
Transition Pore in Apoptosis
Tamara Hirsch,1 Isabel Marzo,1 and Guido Kroemer1*2
Received October 21, 1996
Mitochondrial permeability transition (PT) involves the formation of proteaceous, regulated pores,
probably by apposition of inner and outer mitochondrial membrane proteins which cooperate to form
the mitochondria! megachannel (= mitochondria! PT pore). PT has important metabolic consequences, namely the collapse of the mitochondria! transmembrane potential, uncoupling of the
respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis,
outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins. Recent
evidence suggests that PT is a critical, rate limiting event of apoptosis (programmed cell death): (i)
induction of PT suffices to cause apoptosis; (ii) one of the immediate consequences of PT, disruption
of the mitochondria! transmembrane potential (AWm), is a constant feature of early apoptosis; (iii)
prevention of PT impedes the Wm collapse as well as all other features of apoptosis at the levels of
the cytoplasma, the nucleus, and the plasma membrane; (iv) PT is modulated by members of the
apoptosis-regulatory bcl-2 gene family. Recent data suggest that the acquisition of the apoptotic
phenotype, including characteristic changes in nuclear morphology and biochemistry (chromatin
condensation and DNA fragmentation), depends on the action of apoptogenic proteins released from
the mitochondria! intermembrane space.
KEY WORDS: Apoptosis; necrosis; mitochondria; megachannel; permeability transition; programmed cell death; poteases.
INTRODUCTION
Programmed cell death (apoptosis) is commonly defined as being in opposition
with accidental cell death (necrosis). In contrast to necrosis, apoptosis involves a
regular pattern of action of catabolic enzymes (proteases and nucleases) within
the limits of a near-to-intact plasma membrane [1-5]. Apoptosis is commonly
accompanied by a characteristic change of nuclear morphology (chromatin
condensation, pyknosis, karyorhexis) and of chromatin biochemistry (step-wise
DNA fragmentation culminating in the formation of mono- and/or oligomers of
200 base pairs) [6,7]. Such alterations are not seen in necrosis. It is important to
1
2
CNRS-UPR420, 19 rue Guy Moquet, B.P. 8, F-94801 Villejuif, France.
To whom correspondence should be addressed.
67
0144-8463/97/0200-0067$12.50/0© 1997 Plenum Publishing Corporation
68
Hirsch, Marzo and Kroemer
note, however, that the nucleus is not necessary for the apoptotic process to
occur. Anucleate cells (cytoplasts) produced in the laboratory can be induced to
undergo programmed cell death, indicating that non-nuclear (= cytoplasmic)
events must control the apoptotic process [8-11].
Several authors have speculated that a cytoplasmic "central executioner"
would allow for the integration of very different apoptosis-inducing stimuli
(absence of trophic supply, anoxy, lack of growth factors, cellular damage,
presence of "death signals" etc.) into one common pathway [4,5,8,9,12-14].
The induction phase of apoptosis would thus involve rather heterogeneous,
stimulus-dependent signaling steps. In contrast, once the "central executioner" is
activated, an ordered, regular sequence of biochemical events would ensue. The
phase during which the cytoplasmic "central executioner" is activated (the
effector phase of apoptosis) would be still subject to regulatory effects, whereas,
once the "point-of-no-return" has been passed and the degradation phase of
apoptosis has been initiated, death would be the cell's ineludible fate. The nuclear
features of apoptosis would be acquired during this late degradation phase.
It has recently become clear that the delimitation between apoptosis and
necrosis is not as neat as it appeared initially. First, many agents that induce
necrosis (detergents, anoxia, certain toxins, physical damage by heat etc.) can
induce apoptosis, provided that they are applied at a low (subnecrotic) dose [15].
Second, the apoptosis-inhibitory protooncogene bcl-2 can prevent death from
necrosis in a series of models [16-18], suggesting that, at least in some cases,
necrosis and apoptosis involve similar rate-limiting events. Third, cells that
undergo apoptosis will eventually undergo secondary necrosis, that is disruption
of the plasma membrane. This latter phenomenon probably accounts for the fact
that necrosis has been diagnosed in many pathologies that nowadays are thought
to primarily involve apoptosis [19, 20].
During the last two years, evidence has been accumulating that mitochondrial alterations, which previously only had been implicated in the necrotic mode
of cell death, are also involved in apoptosis. In particular, it has become clear that
opening of mitochondrial permeability transition pores (also called "megachannels") might constitute a critical event of apoptosis and form part of the "central
executioner" [5, 11, 21-31]. This review will focus on the role of mitochondrial
permeability transition in apoptosis.
THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE: A
TARGET OF MULTIPLE APOPTOSIS-INDUCING AGENTS
It has been known since the fifties that isolated mitochondria exhibit
characteristic changes in their behaviour (large amplitude swelling, non-specific
increase in inner membrane permeability for solutessl500Da with dissipation of
the A^) in determined in vitro conditions (for review see Ref. [32, 33]). This
phenomenon, which initially was considered as a sign of mitochondrial
Permeability Transition in Apoptosis
69
Table 1. Inducers of permeability transition that are also inducers of apoptosis
Substance and probable active principle
References
Calcium elevation (conformational effects on proteins)
Protoporphyrin IX (ligand of the PRB)
Diamide (thiol crosslinking of vicinal thiols in the mitochondrial matrix)
Pro-oxidants (matrix thiols?)
Protonophores (inner mitochondrial membrane)
[32,38]
[29,57]
[31,58,59]
[32,41]
[24,32]
Abbreviations: diamide, diazenedicarboxylic acid bis 5N,N-dimethylamide; PRB, peripheral benzodiazepine receptor.
membrane damage, has later been baptized "permeability transition" (PT). PT is
now thought to be due to the formation of dynamic multiprotein ensembles (the
so-called "PT pores" or "mitochondrial megachannels") at inner/outer membrane contact sites. These complexes are currently believed to involve outer
membrane proteins (peripheral benzodiazepin receptor [PBR]; porin, also called
voltage-dependent anion channel [VDAC]), intermembrane proteins (hexokinase, kreatine kinase), at least one inner membrane protein (the adenine
nucleotide translocator, ANT), and at least one matrix protein (cyclophilin D)
[32-36].
Although the physiological function of the PT pore remains elusive [32, 33],
it has become clear that a variety of different drugs and experimental conditions
can induce PT in vitro. Among these PT inducers, there are non-specific
compounds such as calcium or pro-oxidants, which are well-known for their
apoptosis-inducing potential [37-39]. In addition, a few membrane-permeable
substances are capable of inducing PT via acting specifically on mitochondrial
structures (Table 1). This is true for protoporphyrin IX, a ligand of the PBR, as
well as for protonophores (which dissipate the proton gradient built up on the
inner mitochondrial membrane). We have recently shown that such
mitochondrion-targeted PT inducers do trigger apoptosis in thymocytes and cell
lines [24, 29]. Induction of apoptosis by these agents is precluded when
mitochondrial PT is prevented by specific inhibitors such as bongkrekic acid (a
ligand of the ANT) [24, 29]. Altogether, this set of data clearly indicates that PT
suffices to trigger apoptosis.
MITOCHONDRIAL PERMEABILITY TRANSITION: AN EARLY
EVENT OF PHYSIOLOGICAL APOPTOSIS
Apoptosis induced via physiological stimuli, for instance glucocorticoid
receptor agonists, growth factor withdrawal or ligation of TNF or
Fas/APO-l/CD95 receptors, is accompanied by disruption of the AWm [5, 11,
21-31]. To determine the mechanism of this tf¥m collapse, we have performed a
series of experiments in which cells were first labeled with AWm-sensitive
fluorochromes and then purified in a fluorocytometer, based on their AW m . This
Hirsch, Marzo and Kroemer
70
experimental strategy allows for the purification of cells with low AWm values and
a still normal ultrastructure (=pre-apoptotic cells) or, alternatively, of cells with a
still high A^ that will lose their A*^ upon a short-term (30 to 120 min) culture
period [21, 22, 26]. We have used this system to show that PT-inhibitory drugs
[32, 33] inhibit the &Wm loss of AWm high cells. Such drugs comprise cyclosporin
A (CsA), N-methyl-4-valine-CsA (a CsA analogue lacking immunosuppressive
effects), and bongkrekic acid (BA) [21, 26, 27]. Thus, these data suggests that the
PT accounts for the ^Vm collapse which precedes apoptosis.
These observations demonstrate that physiological, receptor-induced apoptosis involves a PT-mediated l^Vm disruption. Recent experiments indicate that
non-physiological apoptosis inducers (-/-irradiation, topoisomerase inhibition by
etoposide or camptothecin, antimetabolites used for chemotherapy, inhibition of
glycolysis etc.) also provoke a &Wm collapse that precedes nuclear apoptosis [11,
22, 23, 25]. Independently from the cell types and the apoptosis inducers that we
have studied, we have always found that the AWm collapse is an early feature of
the apoptotic cascade in the sense that it antecedes other manifestations of
apoptotis such as chromatin condensation, DNA fragmentation, hyperproduction
of reactive oxygen species, oxidative damage of cell membranes, as well as
apoptotis-associated changes in the plasma membrane (Ref. [5, 11, 21-31] and
Table 2).
Extensive studies involving cytofluorometric methods indicate the following
sequence of events during the apoptotic process. At a first level, cells lose their
AWm. At this stage, that we have baptized "pre-apoptosis", cells are still
morphologically normal although they are irreversibly committed to death [22].
Subsequently, cells aberrantly expose phosphatidylserine residues at the plasma
membrane surface. Since phagocytes possess receptors for phosphatidylserine
residues, cells thus can be removed by heterophagy at this early stage of
apoptosis, i.e. they become "palatable" [11]. It is only after this stage that the
complete picture of full-blown apoptosis becomes manifest: enhanced generation
of superoxide anion radicals on the uncoupled respiratory chain [22], shrinkage of
the cell, and chromatin condensation/fragmentation [11, 22, 23].
Table 2. Chronology of apoptotic changes in murine thymocytes"
Stage of apoptosis
Simultaneous or near-to-simultaneous alterations
(flurochrome used for detection)
Pre-apoptosis
Early apoptosis
(palatable cells)
Late apoptosis
AVm collapse (DiOQo), CMXRos, JC-1)"
Phosphatidylserine exposure on plasma membrane
(Annexin-V-FITC conjugate)
Enhanced generation of superoxide anion (HE, NAO)
Shrinkage
DNA fragmentation (Tunel technique)
Loss of viability (PI)
Secondary necrosis
" Data from References [5, 11, 21-24, 27].
* Abbreviations of fluorochromes. CMXRos, chloromethyl-X-rosamine; DiOC6(3h 3,3'
dihexyloxacarbocyanine iodide; HE, hydroethidine; JC-1, 5,5',6,6'-tetrachloro-l,l'3,3'tetraethylbenzimidazolcarbocyanine iodide; MCB, monochorobimane; NAO, nonylacridine orange; PI, propidium iodide.
Permeability Transition in Apoptosis
71
In summary, AW disruption is a constant feature of the apoptotic effector
phase and precedes all other manifestations of apoptosis.
MITOCHONDRIAL PERMEABILITY TRANSITION: A CRITICAL
EVENT OF APOPTOSIS
The findings detailed above indicate that PT is sufficient to trigger apoptosis
and that PT constitutes (one of) the earliest steps of the common pathway of
apoptosis. But does this mean that PT is really necessary for apoptosis to occur?
To answer this question, we have investigated a variety of experimental
conditions in which the induction of apoptosis is prevented by genetic or
pharmacological manipulations. In all these experiments, we have found an
absolute correction between the PT-mediated AVm disruption and the posterior
nuclear apoptosis. Thus for instance, inactivation of the p53 gene by homologous
recombination prevents induction of thymocyte apoptosis via DNA damage
(•y-irradiation, etoposide) but not by other stimuli (glucocorticoids, anti-Fas
antibodies etc.). Only in those situations in which p53 inactivation prevents cell
death, it also prevents AW disruption, indicating that p53 regulates the initiation
pathway of apoptosis upstream of PT [11, 23, 27]. Similar results have been
obtained using a number of interventions on specific apoptosis induction
pathways, for instance inhibition of protein synthesis (which prevents
glucocorticoid-induced cell death but not Fas-induced cell death) or inhibition of
interleukin-lS converting enzyme (ICE, which blocks Fas-induced cell death but
not glucorticoid-induced apoptosis) [11, 21-23, 25, 27].
These results indicate that AWm disruption and the subsequent apoptosis are
indissociable. More importantly, the use of substances that directly inhibit PT
indicate that PT is necessary for apoptosis to occur, as will be discussed in the
following section.
MITOCHONDRIAL PERMEABILITY TRANSITION: A
COORDINATING EVENT OF APOPTOSIS
We have employed two different inhibitors of PT that are specifically
targeted to mitochondria: bongkreckic acid (BA) and chloromethyl-X-rosamine
(CMXRos). BA is a ligand of the ANT, one of the constituents of the PT pore
complex. Binding of BA favors a conformation of the ANT (the "m" state) that is
incompatible with PT pore opening [40]. CMXRos is a cationic lipophilic
substance that, due to its physicochemical characteristics, distributes to the matrix
side of the inner mitochondrial membrane. By virtue of its chloromethylresidues,
CMXRos reacts with thiols and prevents the formation of disulfide links between
matrix or inner membrane proteins [31]. It is thought that the formation of such
disulfide bridges is necessary for the induction of PT in some instances [41].
Both BA and CMXRos prevent the induction of thymocyte apoptosis in
response to a variety of different stimuli: glucocorticoids, ^-irradiation, and
Hirsch, Marzo and Kroemer
72
Table 3. Inhibitors of permeability transition (PT) that prevent apoptosis
Apoptosis inducer
PPIX
Diamide
Glucocorticoids
DNA damage
(etoposide, irradiation)
PT inhibitor (probable active principle
involved in apoptotis prevention)
bongkrekic acid (NAT ligand)"
MCB, CMXRos (prevention of
thiol crosslinking)
bongkrekic acid, MCB, CMXRos
bongkrekic acid, MCB, CMXRos
References
[29]
[31]
[24,27,31]
[24,27,31]
" Abbreviations: ANT, adenine nucleotide translocator; CMXRos, chloromethyl-X-rosamine;
MCB, monochlorobimane.
topoisomerase II inhibition by etoposide [27,31] (Table 3). It appears that the
prevention of PT blocks the manifestation of all signs of apoptosis, at the level of
the nucleus (chromatin condensation and DNA fragmentation), at the level of the
plasma membrane (exposure of phosphatidylserine residues, loss of cell viability),
at the level of redox regulation (loss of non-oxidized glutathione, hyperproduction of reactive oxygen species, oxidation of inner membrane cardiolipin), and at
the level of the cytoplasma (vacuolization, increase of cytosolic calcium levels)
[27].
These data indicate that PT is a rate-limiting, coordinating event of the
apoptotic cascade.
MITOCHONDRIAL PERMEABILITY TRANSITION: REGULATION BY
Bcl-2
Bcl-2 belongs to a growing family of proteins which can either inhibit (Bcl-2,
Bcl-XL, Bcl-2w, Mcl-1, Bfl-1, Al etc.) or favour (Bax, Bcl-Xs, Bad, Bak, Bik etc.)
apoptosis. Enhanced expression of Bcl-2 or of its apoptosis-inhibitory homologs is
involved in the pathogenesis of numerous human cancers. In contrast, knocking
out of members of the Bcl-2 gene family entails severe developmental alterations
ranging from intrauterind death (Bcl-X~y~) to mutiorgan dysfunctions (Bcl-2~+ ~).
Hence, the members of the Bcl-2 family are involved in the regulation of cell
survival, both in health and in disease (reviewed in Ref. [42, 43]). Bcl-2 is
expressed in the outer mitochondrial membrane, the nuclear envelope, as well as
the endoplasmic reticulum [42, 44]. In quantitative terms, the mitochondrial
localization is the most important one for Bcl-2 [45, 46], as well as for some Bcl-2
homologs such as Bcl-xL [47] and the Epstein-Barr virus gene product BHRF-1
[48]. At least in some experimental models, Bcl-2 exerts its apoptosis-inhibitory
effect only if it is present in the mitochondrion rather than in other subcellular
sites [49-52]. The C terminus of Bcl-2 incorporates into the outer mitochondrial
membrane, whereas the N terminus faces the cytosol. It exhibits a nonhomogeneous, scattered distribution in the proximity of the inner/outer membrane contact site [46]. In lymphoid cells, the level of Bcl-2 expression correlates
in a quasi-stoichiometric fashion with that of the PER, one of the putative PT
Permeability Transition in Apoptosis
73
Table 4. Experimental systems in which BcI-2 prevents the induction of
permeability transition
PT inducer
System
Cells
Cytoplasts
Isolated mitochondria
Reference
protoporphyrin IX
protonophore (mClCCP)
respiratory chain inhibitors
glucocorticoid
etoposide
irradiation
ceramide
ceramide
protoporphyrin IX
protonophore
/er-butylhydroperoxide
atractyloside
calcium
29
24
18
22
22
I"'
[11,22]
11)
29]
24, 28]
24,28]
24,28]
[60]
pore constituents, suggesting that Bcl-2 might associate with the PBR or with a
PBR-associated protein [53].
The above facts suggest that Bcl-2 could exert its apoptosis-regulatory
function by influencing PT. In accord with this hypothesis, we found that Bcl-2
prevents PT in cells [21, 22], cytoplasts (= cells lacking nuclei) [11], as well as in
isolated mitochondria [24, 28, 29, 31] (Table 4). When inhibiting PT, Bcl-2
prevents all metabolic and functional consequences of PT including uncoupling of
the respiratory chain [22], release of small molecules such as calcium from the
mitochondrial matrix [54], and liberation of intermembrane proteins from the
intermembrane space [24, 28]. These data suggest that Bcl-2—and probably other
members of the Bcl-2 family—regulate apoptosis by virtue of their capacity to
modulate PT. The exact molecular mechanisms of this regulation remain elusive.
CONCLUSIONS AND SPECULATIONS
As discussed in this review; our data establish that mitochrondrial PT
constitutes a critical coordinating event of apoptosis and that the oncoprotein
Bcl-2 functions as an inhibitor of PT. Thus, PT may form part of the "central
executioner" of apoptosis. PT has several metabolic consequences that are
self-sufficient to cause cell death: uncoupling of oxidative phosphorylation and a
major disturbance of redox regulation with an increase of superoxide anion
generation. In addition, PT is accompanied by the mitochondrial release of
intermembrane proteins that may be toxic for cells. Thus, cytochrome C released
from mitochondria undergoing PT may cooperate with yet unknown factors to
activate the protease CPP32/Yama/Apopain and to induce nuclear apoptosis
[55]. Mitochondria also contain an apoptogenic protease ("apoptosis-inducing
factor", AIF) that suffices to induce apoptotic changes in isolated nuclei in vitro,
in the absence of additional cytoplasmic components [24, 28]. This latter
Hirsch, Marzo and Kroemer
74
molecule, AIF, is inhibited by a protease inhibitor, W-benzyloxycarbonyl-ValAla-Asp.fluoromethylketone (z-VAD.fmk), which also prevents nuclear apoptosis,
underlining the probable in vivo relevance of AIF [28, 56]. At present, however,
it remains to be determined whether AIF and other proteases activated by
mitochondrial products are really necessary to kill the cell. Moreover, it remains
elusive which of the late manifestations of the apoptotic degradation phase can be
attributed to mitochondrial failure or to PT-dependent activation of proteases. As
a possibility, cells that have been driven to undergo apoptosis may die from
necrosis when apoptogenic proteases fail to come into action. This view of cell
death would be compatible with the fact that Bcl-2 can prevent both necrosis and
apoptosis [16-18]. It would also be compatible with the fact that many substances
induce apoptosis at low doses (when PT is induced smoothly and cells can activate
proteases) but necrosis at higher doses (when PT is caused abruptly and cells
disrupt before proteases come into action). This working hypothesis is currently
under active investigation in our laboratory.
ACKNOWLEDGMENTS
Supported by Association pour la Recherche contre le Cancer, Centre
Nationale de la Recherche Scientifique, Fondation de France, Fondation pour la
Recherche Medicale, Ligue Francaise contre le Cancer, Institut National de la
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