Cell death is a fundamental cellular response that has a crucial role in shaping our bodies during develop- ment and in regulating tissue homeostasis by elimi- nating unwanted cells. The first form of regulated or programmed cell death (PCD) to be characterized was apoptosis, which was described in Caenorhabitis��elegans in the early 1990s1. Subsequent genetic analysis of mam- malian apoptosis presented a more complex picture, in which individual apoptosis genes from C.��elegans have expanded into large multi-protein families (FIG.��1). These findings suggest that redundancy, functional special- ization and compensatory regulation of mammalian apoptotic signalling and execution might be important features of mammalian apoptosis. Because of its conserved and uniform nature, apop- tosis is frequently defined mechanistically as a pathway of regulated cell death that involves the sequential activa- tion of caspases, a family of Cys proteases, and that is controlled both positively and negatively by B-cell lym- phoma protein-2 (BCL2) family members. Assays have now been developed for multiple steps of the pathway, allowing the characterization of apoptotic death in��vitro and in��vivo. Apoptotic cell death is characterized by distinctive morphological features, including nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies (see ReF.��2 for further details), that can be used to identify apoptotic cell death events. Genetic studies have shown that apoptosis has a sig- nificant role during normal mammalian development3, especially in the central nervous system where genetic deficiency of apoptotic genes (such as caspase-9, apoptotic protease-activating factor-1 (APAF1), or BCL2-associated X protein (BAX) and BCL2-antagonist/killer-1 (BAK) double mutant mice) results in significant abnormalities4���6. Apoptosis also functions to maintain homeostasis, especially in the immune system, because it eliminates unwanted cells. Dysregulation of apoptosis leads to vari- ous human diseases, such as cancer and autoimmunity. Inappropriate activation of cell death is also the leading cause of tissue injury and functional decline in a large number of acute diseases (such as stroke, myocardial infarction and brain trauma) and chronic diseases (such as diabetes and neurodegeneration). However, effective cytoprotective therapies for these diseases remain a major unmet medical need. To a significant extent, the limited success of cyto- protective drug development can be traced to the simpli- fied view that cell death is either intrinsically regulated by apoptosis or that it is unregulated, caused by over- whelming stress (so-called necrosis). Necrosis possesses characteristic features, such as organelle swelling, mito- chondrial dysfunction, massive oxidative stress and rapid plasma-membrane permeabilization, that are thought to be indicative of the catastrophic nature of cell death, rather than a result of cellular regulation. The general view of the relationship between apoptosis and necrosis is that milder insults to the cell cause apoptosis, whereas more intense insults induce uncontrollable necrosis7. It is thought that such an apparently unregulated ��� and hence untarg- etable ��� process accounts for the bulk of cell death events in acute pathologies. However, in the past few years, evidence has emerged for a number of regulated non-apoptotic cell death pathways, including some with morphological features that were previously attributed to necrosis8. *Tufts University, School of Medicine, Department of Biochemistry, 136 Harrison Ave., Boston, Massachusetts 02111, USA. ���Harvard Medical School, Department of Cell Biology, 200 Longwood Ave., Boston, Massachusetts 02115, USA. Correspondence to J.Y. e���mail: jyuan@hms.harvard.edu doi:10.1038/nrm2393�� Published��online��16��April��2008 Expansion and evolution of cell death programmes Alexei Degterev* and Junying Yuan��� Abstract | Cell death has historically been subdivided into regulated and unregulated mechanisms. Apoptosis, a form of regulated cell death, reflects a cell���s decision to die in response to cues and is executed by intrinsic cellular machinery. Unregulated cell death (often called necrosis) is caused by overwhelming stress that is incompatible with cell survival. Emerging evidence, however, suggests that these two processes do not adequately explain the various cell death mechanisms. Recent data point to the existence of multiple non- apoptotic, regulated cell death mechanisms, some of which overlap or are mutually exclusive with apoptosis. Here we examine how and why these different cell death programmes have evolved, with an eye towards new cytoprotective therapeutic opportunities. REVIEWS 378 | MAy 2008 | voLuMe 9 www.nature.com/reviews/molcellbio �� 2008 Nature Publishing Group

Nature Reviews | Molecular Cell Biology BCL2 CED-9 CED-4 CED-3 BAX/BAK Activated BH3-only proteins EGL-1 Apoptosome Mitochondria APAF1 Caspase-9 Cytochrome c release Caspase-3 Cell death Cell death Mammalian Upstream signal (processing, transcription) C. elegans Upstream signal (transcription) BH3-only member Pro-apoptotic��BCL2��family�� member��possessing��only��a�� BCL2��homology-3��(BH3)�� domain. It has become clear that the simple apoptosis���necrosis classification does not adequately represent the complex- ity of endogenous cell death regulation. We are now just beginning to appreciate the important functions of regu- lated non-apoptotic cell death in homeostatic regulation and development of human disease. In this review, we first provide a brief overview of apoptosis and then examine how mammals might have acquired more apoptotic genes and more complex apoptotic regulation through gene duplication. We then describe examples of regulated non-apoptotic cell death pathways and consider the possible relationships and evolutionary origins of the diverse cell death processes. Apoptosis ��� the fundamental mechanism of PCD The mechanism of PCD elucidated by genetic studies in the nematode C.��elegans defines a primordial paradigm of apoptosis9. In C.��elegans, the activation of PCD is controlled by an elegant and simple pathway (FIG.��1). The initiation of apoptosis is regulated by transcriptional upregulation of egl���1, a pro-apoptotic BCL2 homology-3 (BH3)-only��member of the BCL2 protein family10. Binding of eGL-1 to anti- apoptotic CeD-9 relieves the inhibition that CeD-9 exerts on the adaptor protein CeD-4, allowing CeD-4 to bind and activate the Cys protease CeD-3, which in turn cleaves multiple specific cellular substrates to execute cell death11. Analysis of apoptosis in mammalian cells has led to the identification of multiple mammalian homologues for each class of the C.��elegans CeD proteins. extensive studies have revealed that the mechanism of mammalian apoptosis is similar to that of C.��elegans, although it�� has become much more complex (FIG.��1). For example, although transcriptional upregulation of pro-apoptotic members of the BCL2 family can still play a part in the initiation of apoptosis in mammalian cells12, many BH3- only, pro-apoptotic BCL2 family members can be activ- ated in a number of different ways (including cleavage, phosphorylation, myristoylation and ubiquitylation13���16) to regulate the initiation of apoptosis17. In mammalian cells, the activated eGL-1-like BH3- only members of the BCL2 family also inhibit CeD-9-like anti-apoptotic members of the BCL2 family (such as BCL2, BCL-XL and MCL1) by direct interaction17 (BOX��1 FIG.��1). However, a major mechanism by which anti-apoptotic members of the BCL2 family protect cells is not through the direct inhibition of caspase activation at the level of adaptor molecules (for example, the mammalian CeD-4- like APAF1 protein), but by protecting the integrity of the mitochondria (BOX��1 FIG.��1). BH3-only factors bind and antagonize anti-apoptotic BCL2 family members that reside in the outer mitochondrial membrane. Anti- apoptotic BCL2 proteins exert their activity by preventing the pro-apoptotic multidomain BCL2 family members BAX and BAK from causing mitochondrial damage18, and binding of BH3-only proteins relieves this inhibi- tion. Additionally, a subclass of BH3-only factors might directly induce the formation of a BAX���BAK channel18, although how much this direct mechanism contributes to apoptosis remains a subject of debate19. BAX and BAK have been proposed to form an oligomeric (at least tetrameric or larger6) channel that leads to mitochondrial damage and cytochrome c release. In some cases, BAX and BAK can act through interactions with the components of the mitochondrial permeability pore, namely with the voltage-dependent anionic channel (vDAC)20. Mitochon- drial damage might also be caused by BAX- and BAK- independent mechanisms, such as those induced by intra- mitochondrial K+ influx, or caused by the direct action of caspase-2 on mitochondria (which occurs, curiously, independently of the protease activity of caspase-2)20. Mitochondrial damage and the release of mitochon- drial proteins amplifies apoptotic signalling in mam- malian cells, a step that is considered to be less important for apoptosis in lower organisms, including flies and Figure 1 | Evolutionary expansion of C. elegans apoptotic machinery in mammalian cells. Side-by-side comparison of the Caenorhabditis elegans CED protein pathway and the core apoptotic machinery in mammalian cells shows the conservation of the general outline of the pathway. Extension of the apoptotic machinery can also be observed at every step of the pathway, including multiple B-cell lymphoma protein-2 (BCL2) homology-3 (BH3)-only- protein activating signals, complex regulation of the BCL2 family and the addition of mitochondrial cytochrome c release, which drives the formation of an apoptosome and activation of the upstream caspases (first caspase-9 and then the executioner caspases, such as caspase-3 and caspase-7). Added complexity is provided by the existence of multiple family members in each class of the apoptotic regulators, with both redundant and non-redundant functions. These regulators provide ���fail-safe��� apoptosis machinery that can generate specialized responses to various upstream stimuli. Possible direct activation of BAX and BAK by BH3-only proteins is indicated by a dotted line. APAF1, apoptotic protease-activating factor-1 BAK, BCL2-antagonist/killer-1 BAX, BCL2-associated X protein. REVIEWS NATure revIeWS | molEcular cEll biology voLuMe 9 | MAy 2008 | 379 �� 2008 Nature Publishing Group