Professors P. Motta & T. Naguro/Photo Researchers
Each of our cells hangs in a delicate balance between life and death. Which path the cell takes depends on a dense web of signaling pathways that all converge on a single cellular switch, the mitochondrion. Most of the time, pro-life signals keep the mitochondrial membrane intact and encourage the organelle to churn out ATP. But when signals from the outside or accumulated toxins within the cell tip the scales, mitochondria push the cell down a carefully orchestrated pathway to death. To paraphrase the old saw, mitochondria are 99% respiration, 1% expiration.
This death march, dubbed apoptosis in the 1970s, plays roles in both development and disease. It sculpts our hands and feet, destroys cells that contain damaged DNA, and helps maintain the appropriate number of cells throughout our lives. It also has been implicated in cancer, neurodegenerative disorders, and infection, as well...
THE DEATH ORGANELLE
For years, research on mitochondria was largely limited to their role in metabolism. But in the 1990s a flurry of papers highlighted a new task, that of an organellar Cerberus, the gatekeeper of cell death. One of the first reports came from Kroemer, who noticed that shortly before the onset of apoptosis the mitochondrial membrane's carefully sustained polarization broke down.1 Without the membrane potential the mitochondria cannot produce ATP through oxidative phosphorylation.
Soon after, Xiaodong Wang at the University of Texas Southwest Medical Center in Dallas described cytochrome c release from the mitochondria as an initial apoptotic step.2 Apoptosis aficionados were initially skeptical, Wang recalls. Cytochrome c was well known as an electron-shuttling protein in the electron transport chain, sitting between the inner and outer mitochondrial membranes, tethered to the inner membrane via cardiolipin. According to Wang, its role in apoptosis struck many in the field as unlikely.
But a pair of papers by Wang and Donald Newmeyer at the La Jolla Institute for Allergy and Immunology brought people around.34 Newmeyer had previously shown that mitochondria are needed for the apoptotic pathway that's blocked by Bcl-2. This protein is commonly mutated or misregulated in B-cell lymphoma, an alteration that helps protect the cell from normal death signals. Both groups found that Bcl-2 blocks apoptosis by preventing cytochrome c release from the mitochondria.
Further research established that, once in the cytoplasm, cytochrome c and another mitochondrial protein released at the same time, Apaf-1, bind the cysteine protease procaspase-9 to form a complex called the apoptosome. This assemblage cleaves procaspase-3 to its active form, touching off the chain reaction leading to chromatin condensation and fragmentation, organelle disintegration, changes in cell shape, and eventually death.56
'WE DON'T AGREE ON EVERYTHING'
In simplest terms this series of events appears straightforward: The mitochondrial membrane depolarizes and releases cytochrome c, caspases become activated, and death results. In reality, however, competing and conflicting signals from the cell surface, cytoplasm, and the mitochondria itself all converge on a host of proteins to regulate this process. How, exactly, those proteins control apoptosis is a touchy subject among apoptosis experts. "We don't agree on everything. Sometimes we don't agree on anything," says Doug Green, a colleague of Newmeyer at the La Jolla Institute.
Less contentious, though, is how the apoptotic proteins are positioned throughout the cell. In most nonapoptotic cells, Bcl-2 sits on the outer mitochondrial membrane where it prevents cytochrome c release.56 A related protein, the pro-apoptotic Bak, is also normally dispersed across the mitochondrial membrane, perhaps in association with the anion transporter VDAK.
Or perhaps not, says Richard Youle, biochemistry section chief at the National Institute of Neurological Disorders and Stroke (NINDS), who studies Bak and its apoptosis-promoting homolog Bax. This association would make sense given hints that VDAK and the inner mitochondrial membrane adenine-nucleotide transporter (ANT) pair up to form the pore through which mitochondrial proteins exit early in apoptosis. But Youle says no good data exists to validate this association.
Bcl-2, Bak, and Bax all are members of a family of proteins that regulate apoptosis, some positively, others negatively. Bax resides in the cytoplasm, while the caspases are kept in check by a host of inhibitor-of-apoptosis proteins (IAPs).56 An additional set of proteins that share only a single domain homology with Bcl-2 (so-called BH3-only proteins) helps to initiate apoptosis by activating Bak and Bax. These proteins are normally tied up by Bcl-2 and other homologs, including Bcl-XL.
BRINGING DOWN THE HOUSE
With the BH3-only proteins sequestered and the caspases muzzled, the apoptotic house of cards is relatively stable and the mitochondria are free to carry out their usual role in respiration. But it doesn't take much to make that precarious house tumble.
If the mitochondria depolarize, for instance, such as during a stroke when calcium floods the mitochondria, the permeability transition pore opens and the organelle swells due to osmotic pressure. Reactive oxygen species that accumulate in the mitochondria may also oxidize cardiolipin to release cytochrome c, which then escapes when the mitochondrial membrane eventually ruptures.56 The result is the mitochondrial equivalent of faulty wiring causing a fire.
At the same time, two other proteins also leave the safe confines of the mitochondria. One of these, SMAC/DIABLO, binds the IAPs and frees procaspase-9 and procaspase-3 to be activated by cytochrome c release.56 Another protein discovered by Kroemer and his group heads straight to the nucleus, where it plays a role in DNA condensation and fragmentation. This protein, called apoptosis-inducing factor (AIF), is one of the few apoptotic proteins to completely bypass the caspases.7
Most other mechanisms for toppling the status quo involve signals from outside the cell. When death stimuli such as tumor necrosis factor (TNF) superfamily members bind the FasL receptor, caspases-8 and 10 become active. These then activate caspase-3 and caspase-7, and the pieces are in place for the cell to self-destruct.
In addition to activating caspases, this pathway has a branch heading directly to the mitochondria. Activated caspase-8 truncates the BH3-only protein Bid into tBid, which then moves to the mitochondrial membrane. There, it works in conjunction with Bax to make the membrane permeable, causing release of cytochrome c and other apoptotic proteins. How these proteins work that feat is still a subject of debate.56
THE APOPTOTIC BLACK BOX
These different ways of signaling a cell to die converge on one point: Bax uniting with Bak on the mitochondrial membrane. Even when the death trigger is DNA damage, p53 alone can activate Bax and release the sequestered BH3-only proteins.8 How and why Bax translocates and what it does when it reaches the membrane remains mysterious, but the event is an essential step in apoptosis. In separate experiments Newmeyer and Stan Korsmeyer at Harvard University knocked out Bax and Bak and found that no amount of apoptosis-inducing damage could inspire the cell to kill itself.
"If Bax doesn't translocate, cells don't die, but it's not at all clear what induces that," Youle says. It's also not at all clear how Bak and Bax cause the cell to die once they are united on the mitochondrial membrane. Most people agree that the proteins open the membrane to release pro-apoptotic proteins, but even this step is subject to debate. The proteins might associate with VDAK and hijack that anion pore to release cytochrome c and related proteins.
But letting proteins out of the mitochondria can't be the only thing Bak and Bax do, says Green, adding that it's still unclear why making the membrane permeable leads to cell death. "It's clear that permeabilization leads to cell death in vertebrates whether or not caspase proteases are activated, but we don't know how or why. There are mediators that come from the mitochondria, but in no case have these been shown to be essential for death," he says.
If depolarizing the mitochondrial membrane isn't the primary task of Bax and Bak, Youle has an idea as to what their true calling may be. Mitochondrial fragmentation is a well-known event during apoptosis. Under normal conditions Bak is distributed around the mitochondria, but when apoptosis begins the proteins congregate at the sites where the organelle will eventually pull apart. When Bax moves to the mitochondrial membrane it is initially dispersed, but it quickly joins Bak at the fission sites, Youle says. "Maybe the pore may result from perturbation of mitochondrial division," he says.
Courtesy of Roche
At first glance apoptosis is a simple concept. Once the signal to die is given, the mitochondrial membrane depolarizes, cytochrome c is released, caspases come to life, and the cell dies. But as this segment of an apoptotic signaling pathway map illustrates, the situation is actually much more complicated. Now scientists are developing new tools to help them untangle these cellular circuits – a goal that could benefit basic researchers and drug developers alike.
Most apoptosis research takes place in cell culture or cell-free systems, which are poor mimics of normal cellular conditions. Until recently, creating knockout mice was the only way to study the absence of particular proteins in a normal environment. But animals lacking Apaf-1, caspase-9, or caspase-3 die before birth due to defects in brain development.9 These shortfalls have blocked both basic research and drug-development efforts.
RNAi could help move apoptosis research forward. With this technology researchers can knock out proteins in specific tissues or at specific times in development. "Knocking things out in mammalian cells has helped clarify a lot," Youle says. This clarification has benefits in addition to shedding light on the Bcl-2 family black box; it's also key to developing drugs that block or activate apoptosis, depending on the disease.
Just about every protein in the apoptosis pathways has been found mutated, underexpressed, overexpressed, or otherwise misregulated in cancerous cells. Overexpression of Bcl-2 in lymphoma was among the first known regulatory misfires, but the caspase-binding IAP proteins are also commonly upregulated in cancer cells. The most famous IAP family member, survivin, is overexpressed in most cancer cells. For this reason, these proteins are also sought-after targets for chemotherapy. With RNAi it's now possible to validate a likely protein as a target before developing drugs.
How the lipid cardiolipin binds and releases cytochrome c is another aspect of apoptosis that has been hard to study, according to Sten Orrenius at the Karolinska Institute in Stockholm. He says that in several apoptosis pathways cardiolipin must be modified before it can release cytochrome c, which can happen either by oxidation when the mitochondrial antioxidant defenses are worn down, or perhaps by tBid when it targets cardiolipin on the mitochondrial membrane. "Cardiolipin modification ties these various pathways together, whether you start from the outside or inside," he says.
But even if cardiolipin is a unifying feature of apoptosis, Youle says therapies targeting this interaction are not easily developed because lipid technology is so primitive. "Part of the problem is that we don't have good techniques for dealing with lipids in situ. We don't know precisely where they are. We can't readily knock them out. We can't follow their movements in living cells as we can with GFP [green fluorescent protein] fusion proteins," he says.
Green adds that another difficulty lies in precisely monitoring cytochrome c release and caspase activation. Many methods exist for detecting caspase activation, but he says none are sensitive enough to detect exactly which caspase activates when. What's more, he says recent papers suggest that some caspases can become active by proximity rather than through cleavage. Since most methods detect cleavage, this throws some earlier results into doubt, according to Green.
Working out how mitochondria regulate apoptosis has repercussions beyond cancer. Orrenius points out that many viruses and bacteria hijack the mitochondrial pathways to either destroy cells or keep them alive long enough to harbor viral reproduction. Manipulating apoptosis could eliminate the viral safe haven. Kroemer adds that preventing apoptosis could extend the life of organs due to be transplanted or perhaps save brain cells after a stroke. "In this area I think there will be applications in the next years that may have economic applications," Kroemer says.
LIFE AND DEATH DECISIONS
The multiple, intertwining pathways regulating apoptosis have stymied researchers trying to unravel them. But this complexity seems appropriate to Orrenius. "Usually important decisions in nature are regulated in a complex way," he says.
It turns out that although the process of apoptosis is conserved in all eukaryotes, the pathways involved in regulating cell death became more complicated throughout evolution. Kroemer says that as organisms themselves become more complex, so does their need to balance life and death. One example is in the brain; because most brain cells don't divide, the body needs to weigh carefully any decision to destroy a neuron. "It might be of interest to your well-being that some neurons die," he says, noting that malfunctioning cells sometimes are worse than dead ones. "There is a subtle balance."
With this increasing complexity the mitochondrion has moved from the periphery of cell death in lower organisms to its central role in mammalian cells. It's only in vertebrates, with all their intricacies, that mitochondria take center stage in both maintaining the life of the cell and taking it away.