Mitochondrial Apoptosis
Mitochondrial Apoptosis
Mitochondrial Apoptosis
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APOPTOSIS SPECIAL SECTION I
can induce mitochondrial damage and cell death even when caspases
are inactivated (10). Such experimental observations argue that a
A variety of key events in apoptosis focus on mitochondria, caspase-independent mechanism for commitment to death exists. This
including the release of caspase activators (such as cyto- mechanism is likely to involve mitochondria, as we will see.
chrome c), changes in electron transport, loss of mitochon-
drial transmembrane potential, altered cellular oxidation-re- Understudy for the Executioner: How Mitochondria Trigger
duction, and participation of pro- and antiapoptotic Bcl-2 Apoptotic and Nonapoptotic Cell Death
family proteins. The different signals that converge on mi- If mitochondria are pivotal in controlling cell life and death, then how
tochondria to trigger or inhibit these events and their down- do these organelles kill? At least three general mechanisms are
stream effects delineate several major pathways in physio- known, and their effects may be interTelated, including (i) disruption
logical cell death. of electron transport, oxidative phosphorylation, and adenosine
triphosphate (ATP) production; (ii) release of proteins that trigger
About 2 billion years ago, the cells destined to become the ancestors activation of caspase family proteases; and (iii) alteration of cellular
of all eukaiyotes entered into a partnership with an ancestor of today's reduction-oxidation (redox) potential.
purple bacteria. This partnership promised huge benefits to both Disrulption of electron transport and ener gy metabolism. For
parties: it allowed them to exploit the energy opportunities inherent in decades disruption of electron transport has been recognized as an
the emerging oxygen atmosphere, which was toxic to most other life early feature of cell death. y-Irradiation induces apoptosis in thymo-
formlls. The result was a protoeukaryotic cell, and its new endosym- cytes and a disruption in the electron transport chain, probably at the
biotic bacteria were to become mitochondria (1). cytochrome b-c1/cytochrome c (cyto c) step (11). Ceramide (a "sec-
This alliance was probably a shaky one, and catastrophic conflicts ond messenger" implicated in apoptosis signaling) disirLpts electron
in selection between the two genomes undoubtedly occurTed (2). Once transport at the same step in cells as well as in isolated mitochondria
the new symbiotic,organism moved into the aerobic world, life and (12). Ligation of Fas also leads to a disruption in cyto c finction in
death were controlled by the protomitochondria, which provided not electron transport (13).
only critical antioxidants but also a source of reactive oxygen species One consequence of the loss of electron transport should be a drop
(ROS) as a by-product of oxidative phosphorylation. Conditions that in ATP production. Although such a drop has been observed during
favored the protomitochondria over the host cell would lead to death apoptosis, it often occurs relatively late in the process (14). Indeed,
of the cell and release of the free-living endosymbiont. Therefore, the ATP appears to be required for downstream events in apoptosis (15).
symbiosis was perilously unstable until essential genes for mitochon- Thus, although loss of mitochondrial ATP production can kill a cell,
drial metabolism and biogenesis transferred to the nuclear genome, it is unlikely that this is a mechanism for induction of apoptosis.
resulting in an obligate symbiosis. Release of caspase-activating proteins. The importance of mito-
Several investigators have hypothesized that the endosymbiotic chondria in apoptosis was suggested by studies with a cell-free system
origins of mitochondria and the evolution of aerobic metabolism in in which spontaneous, Bcl-2-inhibitable nuclear condensation and
eukaiyotes formed the basis for evolution of active cell death, which DNA fragmentation were found to be dependent on the presence of
manifests predominantly as apoptosis in metazoans (3, 4). Although mitochondria (16). Subsequently, studies in another cell-free system
apoptosis is independent of oxidative phosphoiylation, lacking a showed that induction of caspase activation by addition of deoxyade-
requirement even for mitochondrial DNA (5), central roles for mito- nosine triphosphate depended on the presence of cyto c released from
chondria as the orchestrators of apoptosis have been firmily estab- mitochondria during extract preparation (17). During apoptosis (in
lished in many systems. vitro and in vivo) cyto c is released from mitochondria and this is
inhibited by the presence of Bcl-2 on these organelles (18, 19).
Mitochondria and Commitment to Cell Death Cytosolic cyto c forms an essential part of the veitebrate "apopto-
When is a dying cell actually dead and when is a cell irTevocably some," which is composed of cyto c, Apaf-1, and procaspase-9 (20).
committed to death? In recent years, we have come to understand that The result is activation of caspase-9, which then processes and
the effectors of apoptosis are represented by a family of intracellular activates other caspases to orchestrate the biochemical execution of
cysteine proteases kniown as caspases. Inhibiting caspases, however, cells.
does not always inihibit cell death induced by proapoptotic stimuli. Significantly, caspase inhibitors do not prevent cyto c release
Although caspase inhibitors block some or all of the apoptotic mor- induced by several apoptogenic agents, including UV irradiation,
phology induced by growth factor withdrawal, etoposide, actinomycin staurosporine, and overexpression of Bax (14, 21, 22). An exception
D, ultraviolet (UV) radiation, staurosporine, enforced c-Myc expres- is cyto c release from mitochondria induced by the tumor necrosis
sion, or glucocorticoids, they do not necessarily maintain replicative factor receptor family member Fas, in which cyto c release is pre-
or clonogenic potential; ultimately, the cells die despite inactivation of vented by inhibition of caspases (primarily caspase-8) recirLited to the
caspases by way of a slower, nonapoptotic cell death (6-9). In cytosolic domain of ligated Fas (21). Neveitheless, cyto c release can
sometimes contribute to Fas-mediated apoptosis by amplifying the
contrast, antiapoptotic proteins such as Bcl-2, BCl-XL, and oncogenic
Abl can maintain survival and clonogenicity in the face of these effects of caspase-8 on activation of downstream caspases (23).
treatments. Conversely, some proapoptotic proteins such as Bax, a The emergent view is that once cyto c is released, this commits the
mammalian cell death protein that targets mitochondrial membranes, cell to die by either a rapid apoptotic mechanism involving Apaf-1-
mediated caspase activation or a slower necrotic process due to
collapse of electron transport, which occurs when cyto c is depleted
D. R. Green is at the La Jotta Institute for Attergy and Immunology, 10355 Science
Center Drive, San Diego, CA 92121, USA. J. C. Reed is at the Burnham Institute, from mitochondria, resulting in a variety of deleterious sequelae
10901 North Torrey Pines Road, La Jolla, CA 92037, USA. including generation of oxygen free radicals and decreased production
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S P E C I A L S E CTI O N APOPTOSIS
of ATP (Fig. 1). dria is apoptosis-inducing factor (AIF), which apparently processes
The consequences of cyto c release may depend on the cell type. purified procaspase-3 in vitro (26, 27). Its activity is blocked by
In those cells where cyto c is available in excess, caspases can be zVAD-fmk, a general caspase inhibitor, raising the possibility that
activated and enough cyto c may remain docked by its high-affinity AIF is another caspase.
binding sites to cytochrome b-cl and cyto c oxidase to maintain Reactive oxygen species and cellular redox. Mitochondria are the
electron transport. In this case, oxygen consumption and ATP pro- major source of superoxide anion production in cells. During transfer
duction may continue unabated while caspases unleash their attack on of electrons to molecular oxygen, an estimated 1 to 5% of electrons in
cytosolic and nuclear substrates, resulting in apoptosis. Alternatively, the respiratory chain lose their way, most participating in formation of
in cells that contain large quantities of endogenous caspase inhibitors, 02- Anything that decreases the coupling efficiency of electron
release of cyto c may fail to induce caspase-dependent apoptosis and chain transport can therefore increase production of superoxides.
instead the eventual loss of electron chain transport may drive the cell Superoxides and lipid peroxidation are increased during apoptosis
toward a necrotic demise (Fig. 1). induced by myriad stimuli (28). However, generation of ROS may be
Other apoptosis mediators are also released from mitochondria. a relatively late event, occurring after cells have embarked on a
Mitochondria of some cells contain procaspase-3 (24) that is liberated process of caspase activation. In this regard, attempts to study apo-
into the cytosol during apoptosis, although it remains unclear whether ptosis under conditions of anoxia have demonstrated that at least some
it becomes activated before release. The possibility that an intrami- proapoptotic stimuli function in the absence or near absence of
tochondrial pool of procaspase-3 participates in apoptosis of some oxygen, which implies that ROSs are not the sine qua non of apoptosis
primary cells is intriguing and, if prevalent in neurons, could provide
(29, 30). However, ROSs can be generated under conditions of virtual
an explanation for the profound impact of murine caspase-3 gene anaerobiosis (31), and thus their role in apoptosis cannot be excluded
disruption on neuronal but not other forms of apoptosis (25). solely on this basis.
Another caspase-activating protein that is released from mitochon-
PT Pore: Assassin or Accomplice?
In many apoptosis scenarios, the mitochondrial inner transmem-
brane potential (ATm) collapses (32), indicating the opening of a
large conductance channel known as the mitochondrial PT pore
Bax
(33) (Fig. 2). The structure and composition of the PT pore remain
Oxidants Ca-2
only partially defined, but its constituents include both inner
membrane proteins, such as the adenine nucleotide translocator
Ceramide-* <- Caspases <-
(ANT), and outer membrane proteins, such as porin (voltage-
Matrix Swelling Channel(s) Open
dependent anion channel; VDAC), which operate in concert, pre-
outer Iembrane)
(ou rupxures X /{outer membrane)
\ mostly intact)
sumably at inner and outer membrane contact sites, and create a
channel through which molecules ? 1.5 kD pass (32, 34). Opening
of this nonselective channel in the inner membrane allows for an
equilibration of ions within the matrix and intermembrane space of
mitochondria, thus dissipating the H+ gradient across the inner
membrane and uncoupling the respiratory chain. Perhaps more
+ ~~~~~Iactive Apaf - 1 .C
importantly, PT pore opening results in a volume dysregulation of
Cyto c a I* Active Apaf-l /{ mitochondria due to the hyperosmolality of the matrix, which
other causes the matrix space to expand. Because the inner membrane
caspase
Activators with its folded cristae possesses a larger surface area than the outer
Caspase-9 membrane, this matrix volume expansion can eventually cause
A44l outer membrane rupture, releasing caspase-activating proteins lo-
ROSY cated within the intermembrane space into the cytosol (Fig. 1).
ATPl
Inhibitors of PT pore opening, including cyclosporins (which
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APOPToSIS SPECIAL SECTION
bind cyclophilin D, associated with the ANT; Fig. 2) and bongkre- Although speculative, the idea of a protein channel is attractive
kic acid (which also inhibits the ANT), appear to block apoptosis because it obviates the need to postulate mitochondrial swelling-
in some systems, thus providing support for the idea that the PT fitting better with intact mitochondrial morphology in most apoptosis
is central to apoptotic processes (35). Bcl-2 can prevent the PT in vivo (41).
(36), whereas the ANT-activator atractyloside and Bax induce both
apoptosis and PT (36, 37). Other stimuli that affect the PT pore Hotey Mitochondria: Bct-2 Family Proteins As Pores
directly, such as oxidants and pathological elevations in cytosolic Another link between apoptosis and mitochondrial physiology is
Ca +, can induce rupture of the outer membrane of mitochondria suggested by the presence of Bcl-2 family proteins in mitochondrial
and release of caspase-activating proteins (26, 27). Thus, in many membranes (42). The Caenorhabditis elegans Bcl-2 homolog CED-9
cases, the PT appears to be the mastermind that orchestrates is expressed from a bicistronic mRNA that encodes both CED-9 and
apoptosis. cytochrome b (43). This suggests a functional connection between
Pharmacological inhibitors of the PT pore reportedly prevent Bcl-2 family proteins and these organelles and implies that CED-9
apoptosis induction by several direct stimuli (35), implying a general may have originated from the genome of protomitochondrial symbi-
role. However, some studies have provided evidence that cyto c release onts, transferred along with other mitochondrial genes to the nuclear
alnd caspase activation can occur before any detectable loss of ATm, genome.
implying that PT pore opening may occur downstream of apoptosome- Many (but not all) Bcl-2 family proteins reside in the mitochon-
mediated caspase activation (14, 21). The ability of caspases to induce PT drial outer membrane, anchored by a hydrophobic stretch of amino
pore opening (27, 38), which in turn can induce caspase activation (by acids located within their COOH-termini, with the proteins orientated
release of cyto c and AIF), creates opportumities for a feedforward toward the cytosol (42). A wide variety of mitochondrial events have
amplification loop (Fig. 1) and complicates attempts to dissect the normal been reported to be modulated by Bcl-2 and its homologs. These
sequence of cell death events. include some that directly affect mitochondria such as oligomycin,
The release of cyto c before or in the absence of a drop in AP\m which inhibits the FoFl-adenosine triphosphatase (ATPase) proton
in some cells suggests that different regulatory events control perme- pump of the mitochondrial inner membrane; cyanide, which poisons
ability of the inner and outer mitochondrial membranes. A rapid oxidative phosphorylation; and BSO, which inhibits glutathione syn-
opening and closing of the PT pore at its reversible low conductance thesis (21, 35, 44-48). Bcl-2 and Bcl-xL suppress release of seques-
state may allow a repeated, respiration-driven reestablishment of tered matrix Ca2" induced by uncouplers of respiration (49). In
APrm (39) so that outer membrane disruption and cyto c release can isolated mitochondria, Bcl-2 or Bcl-xL enhances proton extrusion
occur before ATm collapse. from mitochondria and increases mitochondrial Ca2" buffering ca-
pacity (26, 50).
More Than One Way to Skin an Organelle One clue to how Bcl-2 family proteins exert their mitochondrial
Another mechanism of outer membrane disiuption has recently been effects has come from determination of the three-dimensional struc-
suggested, and it involves hypeipolarization of the mitochondrial ture of the Bcl-xL protein (51). The BCl-XL protein is composed of
inner membrane (in sharp contrast to the hypopolarization associated seven cx helices joined by flexible loops and shares striking similarity
with PT pore opening). This transient hyperpolarization has been to the pore-forming domains of some types of bacterial toxins-for
observed in several cell types under a variety of conditions (21) and example, diphtheria toxin and the colicins. As predicted by their
is suppressed by Bcl-xL. How the polarization occurs is obscure; structures, Bcl-2, Bcl-xL, and Bax can form ion channels when they
nevertheless, increased export of protons into the intermembrane are added to synthetic membranes (52-55). Deletion of the predicted
space may result in protonation of weak acids. These can then freely pore-forming cx5 and cx6 helices abolishes channel formation by Bcl-2
diffuse across the inner membrane and are trapped when the protons and Bax in synthetic membranes (52).
are lost (and exported). As these metabolites accumulate, osmolality How can a small conductance ion channel created in the outer
increases and water enters, resulting in matrix space expansion and membrane by Bcl-2 or Bcl-xL influence mitochondrial physiology?
eventually rupture of the outer membrane, thereby releasing all con- Even in its closed conformation, VDAC creates pores of an estimated
tents of the intermembrane space. 1.5-nm diameter (56); thus, the outer membrane should be freely
However, nearly all studies of AP\m regulation have relied exclu- permeable to ions and most metabolites. Bcl-2 and Bcl-xL may
sively on cationic lipophilic dyes such as rhodamine 123, DiOC6, and communicate functionally or physically with inner membrane proteins
others that partition across the inner membrane based on charge. that govern ion transport, such as components of the PT pore, or
These are fraught with potential artifacts, including autoquenching of ion-transporting proteins that control volume regulation of the matrix
their fluorescence when the intramitochondrial concentrations are too space independently of the PT (21). Bcl-2 and Bcl-xL may also
high, oxidative inactivation of their fluorescence, and alterations in somehow regulate the pH of the intermembrane space, causing in-
uptake (and hence total fluorescence) caused by changes in mitochon- creased rates of proton extrusion from mitochondria (50).
drial volume rather than ATm. For example, rhodamine 123 can Bax-mediated cytotoxicity in yeast and Bax-induced apoptosis in
indicate a hyperpolarization of the inner membrane after ligation of mammalian cells requires a functional FF-ATPase proton pump in
Fas, whereas other dyes simultaneously reveal a hypopolarization of the inner membrane of mitochondria (57). In vitro evidence suggests
this membrane, suggesting that the apparent hyperpolarization may be that the Bax channel is pH- and voltage-dependent (54, 55). Although
an artifact (40). the outer membrane of mitochondria apparently is not polarized, its
Although rupture of the outer membrane causes release of cyto c, close apposition to the inner membrane at the junctional complexes
caspases, AIF, and sometimes generation of ROSs, only a subset of may affect proteins located there.
mitochondria appear to be affected (21). In contrast, studies have Whatever the biochemical mechanism of Bcl-2 family proteins,
suggested that all (or most) of the cyto c in mitochondria can be their effects cannot be reduced merely to models that envision anti-
released during apoptosis (18, 19), which suggests either that the apoptotic Bcl-2 homologs as suppressors of caspase-activating pro-
methods for detecting disruption of the outer membrane are insensi- teins such as CED-4 family members, because (i) Bcl-2 family
tive or that release of cyto c involves other mechanisms. proteins regulate a variety of mitochondrial events even in the pres-
An alternative to gross disruption of the outer membrane theoret- ence of broad-spectrum caspase inhibitors (zVAD-fink), (ii) Bcl-2 can
ically could involve opening a large outer membrane channel capable inhibit not only caspase-dependent apoptosis but also oxidant and
of liberating cyto c or other proteins from the intermembrane space. hypoxia-induced necrosis, and (iii) Bax can induce cyto c release and
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SPECIAL SECTION APoPToSIS
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