Molecular Mechanisms of Necroptosis an Ordered Cellular Explosion

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Molecular mechanisms of necroptosis: an ordered cellular explosion
Peter Vandenabeele*, Lorenzo Galluzzi‡§, Tom Vanden Berghe* and Guido Kroemer‡||

Abstract | For a long time, apoptosis was considered the sole form of programmed cell death during development, homeostasis and disease, whereas necrosis was regarded as an unregulated and uncontrollable process. Evidence now reveals that necrosis can also occur in a regulated manner. The initiation of programmed necrosis, ‘necroptosis’, by death receptors (such as tumour necrosis factor receptor 1) requires the kinase activity of receptorinteracting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3), and its execution involves the active disintegration of mitochondrial, lysosomal and plasma membranes. Necroptosis participates in the pathogenesis of diseases, including ischaemic injury, neurodegeneration and viral infection, thereby representing an attractive target for the avoidance of unwarranted cell death.
Biased by their focus on life, biologists have neglected cell death for a long time. Although the first morphological descriptions of cellular demise date back to the mid-nineteenth century, the notion of ‘programmed cell death’ was formulated by Lockshin as late as 1964 (REF. 1) . In the early 1970s, Kerr, Wyllie and Currie discovered a peculiar type of mammalian cell death that they dubbed ‘apoptosis’ (REF. 2). The stereotyped features of apoptosis (BOX 1) suggested that it would constitute a regulated cell death process, a notion that was elegantly shown in Caenorhabiditis elegans by the Horvitz laboratory in 1980–1990 (reviewed in REF. 3). Textbooks soon thought of apoptosis and necrosis as opposed mechanisms (BOX 1), necrosis being considered as a purely accidental and passive cell death subroutine. The first morphological classification of cell death was proposed by Schweichel and Merker 4 who described, in rat embryos exposed to toxicants, type I cell death associated with heterophagy, type II cell death associated with autophagy and type III cell death without digestion. Today, these cell death modes are referred to as apoptosis, autophagic cell death and necrosis, respectively 5. The purely unregulated nature of necrosis was questioned in 1988, when it was discovered that distinct cell types died in response to the same trigger, tumour necrosis factor (TNF), while manifesting either the ‘classical’ features of apoptosis or a ‘balloon-like’ morphology without nuclear disintegration6. Since then, accumulating evidence has paved the way to the concept of ‘programmed necrosis’ (TIMELINE) , culminating in the introduction, in 2005, of the neologism necroptosis to describe one instance of regulated (as opposed to accidental) necrotic cell death7. Over the past two decades, a plethora of molecules and processes have been characterized as initiators, modulators or effectors of necroptosis. These include (but are not limited to): receptor-interacting protein 1 (RIP1; also known as RIPK1), RIP3 (also known as RIPK3)8–12, caspase inhibitors13, ubiquitin E3 ligases, deubiquitylating enzymes 11,14, reactive oxygen species (ROS) generated by mitochondria or NADPH oxidase  1 (NOX1) 15–17, bioenergetic reactions such as glycogenolysis12 and glutaminolysis12,18, pro-apoptotic B cell lymphoma 2 (BCL-2) family members14, poly(ADP– ribose) polymerase (PARP)19, the mitochondrial perme‑ ability transition pore complex (PTPC)20–22, lysosomal membrane permeabilization (LMP)23,24 and lysosomal, mitochondrial and cytosolic hydrolases23–25. Altogether, it seems that multiple signal transducers and metabolic processes can ignite or mediate cellular demolition by necrosis (Supplementary information S1 (table)), thereby constituting targets for the therapeutic suppression of necroptosis, a possibility that has raised huge expectations26. As the underlying molecular mechanisms have only recently begun to emerge, a comprehensive review on necroptosis is timely and may shed new light on research areas that, until now, have been dominated by apoptosis. Here, we provide a detailed description of the molecular mechanisms of necroptosis and briefly discuss its immunological outcomes and pathophysiological implications.
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*Molecular Signalling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, and Department of Biomedical Molecular Biology, Ghent University, B‑9052 Ghent, Belgium. ‡ INSERM, U848, F‑94805 Villejuif, France. § Institut Gustave Roussy, and Université Paris‑Sud XI, F‑94805 Villejuif, France. || Metabolomics Platform, Institut Gustave Roussy, F‑94805 Villejuif, France; Centre de Recherche des Cordoliers, F‑75,005 Paris, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP‑HP, F‑75908 Paris, France; and Université Paris Descartes V, F‑75270 Paris, France. Correspondence to G.K. and P.V. e‑mails: [email protected]; peter.vandenabeele@dmbr. vib‑ugent.be doi:10.1038/nrm2970 Published online 8 September 2010

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Initiation of necroptosis: the receptors A sizeable fraction of cells dying in vivo in response to ischaemia–reperfusion, physical or chemical trauma, viral or bacterial infection, or neurodegenerative pro cesses exhibit morphological features of necrosis27 (BOX 1). Although necrosis was initially believed to be triggered by excessively harsh microenvironmental conditions, killing cells in an uncontrollable manner, it turned out that the molecular mechanisms of pathological cell loss (in particular ischaemia–reperfusion-induced necrosis) partially overlap with the biochemical cascades that mediate necroptosis (reviewed in REF. 28).
Death receptors in the initiation of necroptosis. Necroptosis can be induced by the ligation of death receptors, including CD95 (also known as FAS; which binds the ligand CD95L (also known as FASL))29, TNF  receptor 1 (TNFR1), TNFR2 (REFS  6,13,30) , TNF-related apoptosis-inducing ligand receptor 1 (TRAILR1) and TRAILR2 (REF. 9.) These receptors usually activate the apoptotic machinery, and their cytotoxicity often requires the presence of transcriptional or translational inhibitors, suggesting the existence of short-lived cytoprotective proteins that are continuously being synthesized (reviewed in REFS 31,32). Nevertheless, in some cell lines and primary cells, the presence of caspase inhibitors (which block apoptosis) unveils a caspase-independent cell death pathway that emanates from death receptors and culminates in a necrotic morphology 33. PRRs in the initiation of necroptosis. Although the underlying molecular mechanisms remain elusive, it seems that necroptosis can also be initiated by members of the pathogen recognition receptor (PRR) family, which include plasma membrane or endosome membrane-associated Toll-like receptors, cytosolic NOD-like receptors and retinoic acid-inducible gene Ilike receptors. All of these are expressed by cells of the innate immune system to sense pathogen-associated molecular patterns (PAMPs), such as viral or bacterial nucleotides, lipoproteins, lipopolysaccharide or peptidoglycans, and respond by triggering inflammation or cell death34. Various PAMPs have been shown to induce necroptosis by activating PRRs in different cell types. For example, viral dsRNA induces necroptotic cell death in human Jurkat T lymphocytes and murine fibrosarcoma L929 cells 35, and lipopolysaccharide does so in macrophages when caspase 8 activity is inhibited36. Similarly, the Gram-negative bacterium Shigella flexneri triggers necroptosis in neutrophils37 and in

Heterophagy
A term of Greek origin indicating the cellular digestion of an exogenous substance, cell or subcellular particle that has been taken up from the extracellular microenvironment.

Autophagy
A pathway for the recycling of cellular contents, in which materials inside the cell are packaged into vesicles and are then targeted to the vacuole or lysosome for bulk turnover. Autophagy is thought to be prominently cytoprotective.

Caspase
A Cys protease that cleaves its substrate after an Asp residue. Caspases play a crucial part in both the initiation (caspase 2, caspase 8, caspase 9 and caspase 10) and execution (caspase 3, caspase 6 and caspase 7) of apoptosis, and they are also required for many processes that are unrelated to cell death, such as the differentiation of several cell types161.

Box 1 | Morphological aspects of necrosis versus apoptosis
In 1972, Kerr and colleagues introduced the term ‘apoptosis’ (a Greek word describing falling leaves) to indicate a type of cell death that is morphologically distinct from necrosis2. For more than three decades, apoptosis was considered the sole mechanism of developmental and homeostatic cell death, as well as the only outcome of the activation of a specific class of proteases, caspases161. Now, multiple types of cell death have been classified according to morphological, biochemical or functional aspects, generating a rather diversified nomenclature5. Although biochemical definitions are expected to gradually replace the current vocabulary, the terms apoptosis and necrosis are firmly established in scientific literature5. Apoptosis exhibits peculiar morphological traits, including pseudopod retraction, the rounding up of cells, decreased cellular volume (pyknosis), chromatin condensation and nuclear fragmentation (karyorrhexis), blebbing of the intact plasma membrane, shedding of vacuoles containing cytoplasmic portions and apparently unchanged organelles (known as apoptotic bodies), and the in vivo uptake of apoptotic corpses by neighbouring cells or professional phagocytes (see the figure, part a). When phagocytosis is inefficient, apoptotic bodies progressively lose integrity and their content spills into the extracellular milieu (secondary necrosis). Dying cells were initially catalogued as necrotic in a negative manner; that is, when they failed to display the morphology of apoptotic or autophagic cell death5. However, necrotic cells exhibit some common morphological features, including an increasingly translucent cytoplasm, swelling of organelles, minor ultrastructural modifications of the nucleus (specifically, dilatation of the nuclear membrane and condensation of chromatin into small, irregular, circumscribed patches) and increased cell volume (oncosis), culminating in the disruption of the plasma membrane (see the figure, part b). Necrotic cells do not fragment into discrete corpses as their apoptotic counterparts do. Moreover, their nuclei remain intact and can aggregate and accumulate in necrotic tissues. a b Importantly, although the signalling pathways and/or biochemical events leading to necroptosis, accidental necrosis and secondary necrosis are clearly distinct, these cell death modes are accompanied by similar end-stage degradation and disintegration processes, implying that it is impossible to discriminate among them based on single end-point morphological 5 µm 5 µm assessments24,162,163.
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Glutaminolysis
The bioenergetic pathway by which Glu or Gln is converted to α‑ketoglutarate, an intermediate of the Krebs cycle. Thus, glutaminolysis can provide substrates for ATP generation by oxidative phosphorylation or stimulate the generation of pyruvate through malate decarboxylation.

Mitochondrial permeability transition
Long‑lasting openings of the PTPC lead to an abrupt increase in the inner mitochondrial membrane’s permeability to ions and low‑molecular‑mass solutes, thus provoking osmotic swelling of the mitochondrial matrix and rupture of the mitochondrial outer membrane.

Apoptotic body
A membrane‑surrounded vesicle that is shed from dying cells during the late stages of apoptosis and that may include portions of the nucleus and/or apparently normal organelles.

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Timeline | Evolution of the concept of programmed necrosis
ROS shown to be involved in TNF-induced cytotoxicity 85. Discovery that caspase inhibition favours necrosis13. Discovery that RIP1 mediates TNFR1-induced caspase-independent cell death9. Description of a regulated form of necrosis activated by DNA damage19. Degterev et al. identify necrostatin 1 and introduce the term ‘necroptosis’7. Characterization of CYPD-deficient mice20,21. Identification of RIP3 as a crucial modulator of necroptosis10–12.

Kerr et al. introduce the term apoptosis2.

1972

1988

1992

1996

1998

1999

2000

2003

2004

2005

2006

2008

2009

Discovery that TNF can induce both apoptosis and necrosis6.

Gln metabolism implicated in necrosis18. Discovery that TNFR1 recruits RIP1 (REF. 8).

Molecular characterization of RIP3 (REF. 171).

Chan et al. introduce the term ‘programmed necrosis’ (REF. 30). Discovery of the role of CYLD in TNFR1 complex I167,168.

ANT implicated in necroptosis22.

First systems biology study on necroptosis14 Identification of RIP1 as a specific molecular target of necrostatins63.

ANT, adenine nucleotide translocase; CYLD, cylindromatosis; RIP, receptor-interacting protein (also known as RIPK); ROS, reactive oxygen species; TNF, tumour necrosis factor; TNFR1, TNF receptor 1.

Inflammasome
A supramolecular complex comprising a pattern recognition receptor (such as NLRP3) and an adaptor protein (such as ASC) that is required for the autocatalytic activation of pro‑caspase 1. Active caspase 1 catalyses the proteolytic maturation of interleukin‑1β, a potent pro‑inflammatory cytokine.

human monocyte-derived macrophages38. The lethal response of macrophages to S. flexneri depends on the inflammasome component NACHT, LRR and PYD domains-containing protein 3 (NLRP3; also known as NALP3 and cryopyrin) and shares features with the excessive necrosis seen in monocytes of patients affected by autoinflammatory disorders caused by NLRP3 gain-of-function mutations39. Viral infections have repeatedly been reported to promote cell death with necrotic features 30, although this often results from supraphysiologically high viral loads that directly perturb the plasma membrane40. Infection by vaccinia virus, which encodes the caspase inhibitor B13R (also known as Spi2), has been shown to shift to necroptosis the otherwise apoptotic demise of T cells succumbing to activation‑induced cell death and of mouse embryonic fibroblasts (MEFs) killed by TNF10. Similarly, whereas the infection of pig kidney cells by strains of cowpox virus expressing cytokine response modifier protein A (CrmA; a potent and specific inhibitor of caspase 8) resulted in cytopathic effects consistent with necrotic death, CrmA-deficient viruses generated an apoptotic cell death phenotype41. These examples underscore the notion that viral infection and PAMP-activated PRRs can facilitate necroptosis. However, our Review will focus on TNFR1-initiated necroptosis, as this is the most extensively studied model of programmed necrosis to date.

Activation-induced cell death
After an adaptive immune response, superfluous lymphocytes are eliminated on T cell receptor re‑stimulation by a mechanism that may involve the CD95–CD95L system.

Polyubiquitylation
The attachment of chains of the small protein ubiquitin to Lys residues of proteins, often as a tag for rapid cellular degradation.

Initiation of necroptosis: TNFR1 decides The most extensively characterized pathway leading to necroptosis is initiated by ligation of TNFR1 (TABLE 1). Depending on the cell type, cell activation state and microenvironment factors, TNF administration can result in cell survival, apoptosis or necroptosis, reflecting an intricate network of signals that operate downstream of TNFR1 and that can ‘switch’ between different patterns of response32 (FIG. 1). In particular, the ubiquitin-editing system and initiator caspases such as caspase 8 modulate the molecular switches that dictate the biological response to TNFR1 activation.

TNFR1 complex I promotes cell survival. In the absence of TNF, TNFR1 subunits spontaneously assemble at the plasma membrane to generate trimeric receptors owing to the so-called pre-ligand assembly domain (PLAD), which is localized in the extracellular Cys-rich domain 1 (CRD1) of the protein42. On ligand binding, TNFR1 trimers undergo a conformational change that allows the cytosolic portion of the receptor to recruit multiple proteins, including TNFR-associated death domain (TRADD), RIP1, cellular inhibitor of apoptosis 1 (cIAP1), cIAP2, TNFR-associated factor 2 (TRAF2) and TRAF5. This membrane-proximal supramolecular structure has been named complex I43 (FIG. 1a). cIAPs — E3 ubiquitin ligases that were previously known as apoptosis inhibitors owing to their ability to interfere with caspase activation44 — are recruited (by an amino-terminal domain that contains baculovirus IAP repeats) to complex I by TRAF2, which stabilizes them by preventing their polyubiquitylation45,46. cIAPs catalyse the addition of Lys63-linked polyubiquitin moieties to Lys377 of RIP1 (REF. 47). Lys63-polyubiquitylated RIP1 provides a docking site for transforming growth factor-β-activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB2) and TAB3, which together (the TAK1–TAB2–TAB3 complex) constitute the apical stimulator of the canonical nuclear factor-κB (NF-κB) activation pathway (reviewed in REF. 31; BOX 2). NF-κB transactivates cytoprotective genes and facilitates cell survival. Recent results48 challenge the common notion that RIP1 constitutes an absolute requirement for NF-κB activation 49. In some experimental scenarios, complex I constitutes the molecular platform that recruits the ROS-generating NADPH oxidase NOX1 to the plasma membrane, an event that might be involved in the execution of necroptosis16,17 (see below). Depending on cell type and lethal trigger, complex I might therefore exert either cytoprotective or cytotoxic functions (through NF-κB activation or NOX1 recruitment, respectively), suggesting that complex I regulates an intricate network of pro-survival and pro-death signalling pathways.
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Table 1 | The functional interactome of TNFR1 in necroptosis
Factor*
A20 (TNFAIP3) Caspase 8 Ceramidase Cezanne (OTUD7B) cIAPs cPLA2 CYLD FADD JNK1 LOX NOX1 RFK RIP1 (RIPK1) RIP3 (RIPK3) SMases TNF TNFR2 TRADD TRAF2 and TRAF5 USP21

Localization
Cytoplasm and plasma membrane‡ Cytoplasm Mitochondria and plasma membrane Cytoplasm and plasma membrane‡ Cytoplasm and plasma membrane‡ Cytoplasm Cytoplasm and plasma membrane‡ Cytoplasm Cytoplasm and mitochondria§ Cytoplasm Plasma membrane‡ Cytoplasm and plasma membrane‡ Cytoplasm, plasma membrane and possibly mitochondria

Roles in necroptosis
RIP1-deubiquitylating enzyme TNFR1-interacting protein in complex II Converts ceramide into sphingosine on TNFR1 ligation RIP1-deubiquitylating enzyme RIP1-ubiquitylating enzymes Produces arachidonic acid in response to TNFR1 ligation RIP1-deubiquitylating enzyme TNFR1-interacting protein in complex II Degrades ferritin on TNFR1 ligation Converts cPLA2-generated arachidonic acid into lipid hydroperoxides NAPDH oxidase that generates O2– in a TRADDand RIP1-dependent manner on TNFR1 ligation TNFR1-interacting protein in complex I Crucial component of the necrosome

Outcome
Inhibits the NF-κB system to favour necroptosis Cleaves and inactivates RIP1 and RIP3 Sphingosine induces lysosomotropic LMP Inhibits the NF-κB system to favour necroptosis Facilitates NF-κB activation and inhibits necroptosis Induces lysosomotropic LMP Inhibits the NF-κB system to favour necroptosis Adaptor for TNF-induced necroptosis in some cells Favours ROS overgeneration downstream of RIP1 Induces lysosomotropic LMP Induces pro-necrotic ROS generation Couples TNFR1 to NOX1 Triggers necroptosis (which requires RIP1 kinase activity) Triggers necroptosis (which requires RIP3 kinase activity) Trigger ROS generation and lipid peroxidation to induce lysosomotropic LMP Activates necroptosis in the absence of caspase activity Triggers necroptosis by facilitating RIP1 activation Adaptor for TNF-induced necroptosis in some cells Promotes NF-κB activation, which inhibits necroptosis Inhibits the NF-κB system to favour necroptosis

Refs
52 11,55 112,113 53 11,47 111 14 9,43 97 23 16 17 9,14,30, 62 10–12,55 96,112, 115 6,13,30 30 43,58 30,57 54

Cytoplasm, mitochondria Crucial component of the necrosome and plasma membrane Lysosomes and plasma membrane Extracellular milieu and plasma membrane‡ Plasma membrane Cytoplasm and plasma membrane‡ Cytoplasm and plasma membrane‡ Cytoplasm and plasma membrane‡ Transform sphingomyelin into ceramide in response to TNF Pleiotropic pro-inflammatory cytokine I Death receptor that potentiates RIP1 recruitment at TNFR1 complex TNFR1-interacting protein in complex I and II TNFR1-interacting proteins in complex I RIP1-deubiquitylating enzyme

cIAPs, cellular inhibitor of apoptosis proteins; cPLA2, cytosolic phospholipase A2; CYLD, cylindromatosis; FADD, FAS-associated protein with a death domain; JNK1, JUN N-terminal kinase 1; LMP, lysosomal membrane permeabilization; LOX, lipoxygenase; NF-κB, nuclear factor κB; NOX1, NADPH oxidase 1; O2–, superoxide anion; RFK, riboflavin kinase; RIP, receptor-interacting protein; ROS, reactive oxygen species; SMases, sphingomyelinases; TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, TNFR-associated death domain; TRAF, TNFR-associated factor; USP21, ubiquitin-specific peptidase 21. *Alternative names are provided in brackets. ‡Associated with TNFR. §Associated with the mitochondrial outer membrane. 

TNFR1 complex II promotes apoptosis or necroptosis. Ligand-bound TNFR1 is internalized, leading to a shift in the molecular composition of the TNFR1 interactome (TABLE 1) and to the formation of a cytosolic death-inducing signalling complex (DISC), better known as complex II (REFS 43,50) (FIG. 1). RIP1 polyubiquitylation not only affects NF-κB activation but also influences the transition from complex I to complex II (REFS 10,11,51). On deubiquitylation of RIP1 by the Lys63-deubiquitylating enzyme cylindromatosis
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(CYLD)14, RIP1 (together with its cognate kinase RIP3) is recruited to a supramolecular complex that includes TRADD, FAS-associated protein with a death domain (FADD) and caspase 8 (REF. 43). In line with this model, RNA interference (RNAi)-mediated knockdown of CYLD inhibits TNF-induced necroptosis14. It remains unclear whether other deubiquitylating enzymes, including A20 (also known as TNFAIP3)52, cezanne (also known as OTUD7B) 53 and ubiquitin-specific peptidase 21 (USP21)54, all of which inhibit NF-κB
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ROS NOX1 FAD p22phox NADPH TNF TNFR1 Plasma membrane

a
Complex I Lys63-linked polyubiquitin

FMN

RFK

RF

Cytoplasm

TRADD TRAF2 and TRAF5 RIP1 cIAPs

TAK1–TAB2–TAB3 complex

NF-κB activation

USP21, A20 or cezanne

?

CYLD RIP1

Cytosolic formation of complex II

b
TRADD RIP1 FADD RIP3 Caspase 8

c
Active kinase X? TRADD RIP1 P Caspase 8 inhibitor Caspase-independent executioner mechanisms Necroptosis FADD RIP3 Caspase 8

Caspase 8 activity Caspase-dependent executioner mechanisms Apoptosis

and TNFR1 ligation results (at least in some cell types) in programmed necrosis9,13. Whether FADD or TRADD are strictly required to assemble the necroptosissignalling complex, or ‘necrosome’, is less clear. The absence of FADD sensitizes some cells, including Jurkat lymphocytes, to necrotic cell death9,35. In contrast, MEFs isolated from FADD-deficient mice are resistant to TNF-induced necroptosis57. Both TNF-induced apoptosis and necroptosis (obtained in the presence of the chemical pan-caspase inhibitor Z-VAD.fmk) are blocked in TRADD-deficient cells58, suggesting that, at least in some experimental settings, TRADD (which is also part of TNFR complex I; see above) constitutes an indispensable cell deathinducing adaptor protein43. In contrast to these observations, TRADD cannot be detected in complex II formed on TNFR ligation in the presence of second mitochondria-derived activator of caspase (SMAC; also known as Diablo) mimetics (chemical agents that block cIAPs by mimicking the activity of SMAC, a mitochondrial cIAP inhibitor). Moreover, RNAi-mediated knockdown of TRADD stimulates (rather than inhibits) the formation of complex II in some cell types, suggesting that TRADD is not required for the assembly and function of complex II (REFS 56,59).

The necrosome signalling complex. The term necrosome refers to a multiprotein complex containing RIP1 and RIP3 that stimulates necroptosis59. The formation Figure 1 | TNFR1‑elicited signalling pathways. a | On tumour necrosis factor (TNF) Nature Reviews | Molecular Cell Biology binding, TNF receptor 1 (TNFR1) undergoes a conformational change, allowing for the of the necrosome is highly regulated by ubiquitylation intracellular assembly of the so-called TNFR complex I, which includes TNF receptor(see above) and mutual RIP1 and RIP3 phosphorylaassociated death domain (TRADD), receptor-interacting protein 1 (RIP1; also known as tion (see below). Whereas many cell lines are protected RIPK1), cellular inhibitor of apoptosis proteins (cIAPs), TNF receptor-associated factor 2 against TNF-induced apoptosis by Z-VAD.fmk, others (TRAF2) and TRAF5. On cIAP-mediated Lys63-ubiquitylation, RIP1 can serve as a scaffold respond to TNF plus Z-VAD.fmk by activating necropfor the recruitment of transforming growth factor-β activated kinase 1 (TAK1), tosis60, a phenomenon that has recently been correlated TAK1-binding protein 2 (TAB2) and TAB3, which initiate the canonical nuclear factor-κB with the expression of RIP3 (REF. 11). RIP3 contains an (NF-κB) activation pathway (BOX 2). Riboflavin kinase (RFK) physically bridges the TNFR1 N-terminal kinase domain and a C-terminal RIP homodeath domain to p22phox (also known as CYBA), the common subunit of multiple typic interaction motif (RHIM), which mediates its NADPH oxidases, including NADPH oxidase 1 (NOX1), which also contributes to interaction with RIP1 (REF. 61). Necroptosis induced by TNFα-induced necroptosis by generating reactive oxygen species (ROS). Conversely, on deubiquitylation by cylindromatosis (CYLD; and perhaps also by A20 (also known as CD95L, TRAIL or TNF in combination with Z-VAD.fmk TNFAIP3), cezanne (also known as OTUD7B) or ubiquitin-specific peptidase 21 (USP21)), is abrogated in RIP1-deficient T cells9, and enforced RIP1 exerts lethal functions, which can be executed by two distinct types of cell death. dimerization of RIP1 can induce necroptosis in FADDb | The internalization of TNFR1 is accompanied by a change in its binding partners that deficient Jurkat lymphocytes7. Consistent with a role for leads to the cytosolic assembly of TNFR complex II, which often (but not invariably) RIP1 in NF-κB-mediated pro-survival signalling, mice contains TRADD, FAS-associated protein with a death domain (FADD), caspase 8, RIP1 lacking RIP1 display extensive apoptosis in lymphoid and RIP3 (also known as RIPK3). Normally, caspase 8 triggers apoptosis by activating and adipose tissues and die 1–3 days after birth62. In conthe classical caspase cascade. It also cleaves, and hence inactivates, RIP1 and RIP3. trast to RIP1, RIP3 is not involved in NF-κB activation10. c | If caspase 8 is blocked by pharmacological or genetic interventions, RIP1 and RIP3 Recent experiments with cells that have been stably or become phosphorylated (perhaps by an unidentified kinase) and engage the effector mechanisms of necroptosis. FAD, flavin adenine nucleotide; FMN; flavin mononucleotide. temporarily depleted of RIP3 showed that this kinase is required for necroptosis and revealed the existence of a RIP1- and RIP3-containing complex that is assembled activation, also stimulate the lethal functions of RIP1. In in response to TNF and is stabilized in the presence of complex II, caspase 8 inactivates RIP1 and RIP3 by pro- SMAC mimetics or caspase inhibitors10–12. teolytic cleavage and initiates the pro-apoptotic caspase In 2005, Yuan and colleagues identified necrostatin 1 activation cascade11,55. Moreover, genetic or pharmaco- and necrostatin 3, small molecules that allosterically logical inhibition of cIAPs prevents RIP1 ubiquitylation block the kinase activity of RIP1, thereby inhibiting Necrostatin 1 A Trp‑based molecule and favours the formation of complex II, thus sensitiz- necroptosis but leaving RIP1-mediated activation of (5‑(1H‑indol‑3‑ylmethyl)‑ ing cells to RIP1-dependent activation of caspase 8 and NF-κB, mitogen-activated protein kinase p38 and JUN 3‑methyl‑2‑thioxo‑4‑ apoptosis47,56. By contrast, when caspase 8 is deleted, N-terminal kinase 1 (JNK1) unaffected7,63. Several imidazolidinone) that was first depleted or inhibited by CrmA or pharmacological other necrostatins have been identified by virtue of identified as a specific and potent inhibitor of necroptosis7. agents, complex II cannot enter the ‘apoptotic mode’ their capacity to suppress necrosis induced by TNF
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Box 2 | The NF-κB system
Nuclear factor-κB (NF-κB) refers to a heterogeneous group of dimeric transcription factors belonging to the REL protein family, which can be activated by tumour necrosis factor receptor 1 (TNFR1), pathogens, toxins, drugs and oxidants. In mammals, five NF-κB subunits share a highly conserved REL homology domain (RHD), which mediates dimerization, DNA binding and the interaction with inhibitor of NF-κB (IκB) proteins. These subunits are NFKB1 (p50 and its precursor p105), NFKB2 (p52 and its precursor p100), cREL, RELA (p65) and RELB164. NF-κB homodimers or heterodimers are normally sequestered in the cytoplasm by IκB proteins. In the canonical pathway (see the figure, part a), the IκB kinase (IKK) complex (composed of one regulatory subunit, IKKγ (also known as NEMO), and two catalytic subunits, IKKα and IKKβ) responds to specific signals (including TNFR1 ligation) or nonspecific stress by phosphorylating IκB to target it for destruction by E1–E2–E3-mediated ubiquitylation and proteasomal degradation31. IκB degradation unmasks the RHD and a nuclear localization signal (NLS; which is common to all REL proteins) on associated NF-κB dimers, allowing them to access the nucleus and bind DNA31. The IKK complex is stabilized by the linear ubiquitin chain assembly complex (LUBAC), which linearly adds ubiquitin (Ub) moieties to IKKγ165. In the non-canonical pathway (see the figure, part b), which responds to a specific set of differentiating or developmental stimuli, the IKK complex comprises IKKα dimers and is activated by NF-κB-inducing kinase (NIK)-mediated phosphorylation. In turn, active IKKα phosphorylates p100 to promote its proteolytic processing to p52, which can dimerize with other NF-κB subunits and enter the nucleus. Once bound to nuclear DNA, NF-κB dimers regulate the expression of genes implicated in a plethora of pathophysiological processes, including innate and adaptive immune responses, inflammation, cell proliferation, cell death and cell survival. Alterations of the IKK–NF-κB signalling module (most often resulting in constitutive NF-κB activation) contribute to oncogenesis and tumour development in many solid or haematopoietic malignancies166. One example is provided by the negative NF-κB regulator cylindromatosis (CYLD), a deubiquitylating enzyme, the loss-of-function mutation of which leads to familial cylindromatosis167,168. Moreover, NF-κB activation reduces the apoptotic potential of anticancer chemotherapeutics, thereby favouring resistance169. Pharmacological inhibitors of the NF-κB system might therefore directly target oncogene addiction170 or sensitize tumour cells to chemotherapy. Multiple NF-κB inhibitors are being evaluated in clinical trials, alone or in combination with radiotherapy or chemotherapy169.

a Canonical pathway
IκB p65 IκB p50 IKK complex IKKγ

b Non-canonical pathway
IKK complex IKKα IKKα NIK

Linear ubiquitylation Ub

Kγ

IK

Ub Ub Ub Ub Ub
IκB IκB

0 p5 0 p5

p65 p50

p6 p6 5 5

Ub Ub

P

P

Degradation
Oncogene addiction
An expression coined by Weinberg in 2002 (REF. 170) to describe the observation that tumour maintenance often depends on the continued activity of some oncogenes.

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Ub Ub

IKKα IKKβ P P E1 E2 E3 IκB p65 IκB p50 P P100 RELB

P P

IKKα IKKα P P100 RELB

LUBAC Ub Ubiquitylation

Processing

RELB NF-κB p52 dimers
REL p52 B

cREL cREL

cREL cREL

p65 p50 p52 RELB

Immunity

Inflammation

Cell death

Cell survival

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plus Z-VAD.fmk, but they inhibit RIP1 indirectly by interfering with upstream signals that are yet to be elucidated63–65. Necrostatin 1 abolishes the assembly of the RIP1–RIP3 complex, suggesting that the kinase activity of RIP1 is required for necrosome formation10,11. Necroptosis depends on a tightly regulated mutual relationship between RIP1 and RIP3 kinase activities, involving the autophosphorylation of RIP1 on Ser161 and direct or indirect RIP3-mediated phosphorylation of RIP1 (REFS 11,63). Recently, murine cytomegalovirus infection was shown to induce RIP3-dependent but RIP1-independent necroptosis66. Moreover, overexpression of catalytically active RIP3 can trigger necroptosis irrespective of the presence of RIP1 (REF. 12). Thus, at least in some cases, RIP1 may not constitute an absolute requirement for necroptosis induction. Altogether, these results point to the existence of a highly complex, tightly regulated signal transduction pathway that connects death receptors to pro-inflammatory, apoptotic or necrotic signal transducers. PARP1 hyperactivation not only causes mitochondrial dysfunction but also activates JNKs, two processes that have been shown to enhance necrotic cell death in some experimental setups80,81. A direct link between RIP1 and decreasing ATP concentrations (which occur during necroptosis) was postulated when the existence of a RIP1-dependent signal that results in the inhibition of adenine nucleotide translocase (ANT) was uncovered22. In physiological circumstances, ANT, an integral protein of the inner mitochondrial membrane, exchanges mitochondrially neosynthesized ATP with cytosolic ADP25. Inhibition of ANT by RIP1 can be expected to reduce intramitochondrial ADP levels, leading first to the inhibition of F1–FO ATP synthase (as ADP is its substrate) and then to the reversal of F1–FO ATP synthase activity, which causes the ATP hydrolysis-driven extrusion of protons from the mitochondrial matrix, resulting in a net increase in the mitochondrial transmembrane potential (Δψm). This model is apparently corroborated by the fact that mitochondria show transiently increased Δψm during the early phases of necroptosis15,24. ANT has also been suggested to interact with the voltage-dependent anion channel (VDAC; present on the outer mitochondrial membrane) and cyclophilin D (CYPD; present in the mitochondrial matrix) to generate the PTPC (reviewed in REF. 25). In response to some lethal triggers, including oxidative stress and Ca2+ overload, the PTPC adopts a high conductance conformation, permitting the unregulated entry of solutes and water into the mitochondrial matrix, a phenomenon that has been dubbed the mitochondrial permeability transition25. The PTPC is a highly dynamic entity that interacts with multiple proteins, including pro- and anti-apoptotic members of the BCL-2 protein family 82. However, it is not known whether BCL-2-modifying factor (BMF), a BH3-only protein required for TNF-induced necroptosis14, functionally or physically interacts with the PTPC. Both pharmacological and genetic interventions aimed at inhibiting backbone components of the PTPC, including VDAC, ANT and CYPD, mediate cytoprotective effects against numerous insults in vitro and in vivo (reviewed in REFS 25,78). As it stands, CYPD seems to be the leading player of the PTPC, as genetic ablation of peptidylprolyl isomerase F (Ppif; the CYPD-encoding gene), but not of the genes coding for all known VDAC and ANT isoforms83,84, consistently protects mice against ischaemic injury of the brain and heart in vivo20,21. These results underscore the importance of mitochondrial events in pathological necroptosis. ROS and RNS contribute to the execution of necroptosis. Mitochondrial energy metabolism was first linked to the execution of necrosis in the early 1990s, when the Fiers group showed that ROS production by mitochondrial respiratory complex I is crucial for the necrotic response of L929 cells to TNF85. Mitochondrial ROS also mediate cell death-associated ultrastructural changes of the mitochondria and endoplasmic reticulum (ER)85,86. Although ROS production is not essential for all instances of TNFinduced necrosis11,86, the kinase activity of RIP3 may link TNFR1 signalling, mitochondrial bioenergetics
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Execution of necroptosis Several distinct molecular mechanisms contribute to the execution of TNFR1-initiated necroptosis. Some of these effectors can also be activated by other necroptotic triggers, including PAMPs and DNA damage (see above).
Bioenergetic aspects of the execution of necroptosis. During apoptosis, ATP-consuming processes including PARP1 activity 67, translation68 and proteasome-mediated degradation69 are rapidly shut off by caspases. By contrast, during TNF-induced necroptosis, these processes persist and hence may contribute to the lethal decline in intracellular ATP70. PARP1 is a nuclear enzyme involved in DNA repair and transcriptional regulation71. The overactivation of PARP1, perhaps due to ROS-mediated DNA damage, is critically involved in the necroptotic response of L929 fibrosarcoma cells to TNF72 and eventually results in the depletion of ATP and NAD (FIG. 2). In response to DNA alkylation, PARP1 activation and the consequent NAD depletion and/or PAR accumulation stimulates the release of apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space, a process that reportedly depends on calpains — Ca2+activated non-caspase Cys proteases73–75. Cytosolic AIF rapidly relocalizes to the nuclear compartment, where it mediates caspase-independent, large-scale DNA fragmentation25, which in turn can further stimulate PARP activation, thereby initiating a vicious cycle. Harlequin mice, which bear a hypomorphic mutation of Aifm1 and therefore express reduced amounts of AIF, are protected against several necrotic stimuli, including ischaemia– reperfusion injury of the brain76–78. Similarly, pharmacological and genetic inhibition of PARP1 has consistent cytoprotective effects79. Surprisingly, both RIP1-deficient and TRAF2-deficient MEFs are resistant to PARP1-induced cell death in response to DNA alkylating agents80, indicating that RIP1 activation can also occur downstream of PARP1, at least in specific experimental settings. In line with this notion,

Mitochondrial transmembrane potential (Δψm)

The electrochemical gradient built across the inner mitochondrial membrane by the proton pumps associated with the respiratory chain. The Δψm creates a proton‑moving force that is required for mitochondrial ATP generation by the F1–FO ATP synthase, and its permanent dissipation is considered an early sign of apoptosis.

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UV or ROS Nucleus

DNA damage 8

PARP activation Glycogenolysis Glycogen PYGL G1P PGM G6P Glycolysis

PARP activation Methylglyoxal↑ 6 AGEs↑ ROS Calpains AIF

1 Glu + NH4+ Glutaminolysis Gln GLUL Glutaminase

? RIP1–RIP3 necrosome P Ser161 RIP1 P X? 3 JNK Ferritin↓ Labile iron pool↑ ROS [ATP]↓
ANT

Pyruvate

Glu + NH4+ Pyruvate

P Ser199 RIP3 Active kinase

TCA 2 cycle Fumarate CYPD Succinate ADP ATP T H+ NADH NAD+ SDH ATP AN T ½O2 H2O AC AN V VD AC II I VD IV III + PTPC ∆ψm ↑ ROS H Lipid 7 H+ H+ Mitochondrion peroxidation

GLUD1 α-Ketoglutarate arate rat te

5 Lipid peroxidation Phospholipids

4 SMases

Ca2+

cPLA2 Arachidonic acid Lipoxygenase Lipid hydroperoxides

Ceramide Calpains Sphingosine

[ATP]↓

Respiratory complex component Coenzyme Q10 Cytochrome c Necroptosis

Lipid peroxidation LMP

Lysosomes

Figure 2 | Execution of necroptosis. When caspase activation is prevented, receptor-interacting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3) are phosphorylated and elicit necroptosis. The RIP1–RIP3 necrosome Nature Reviews | Molecular Cell Biology stimulates glycogenolysis and glutaminolysis by enhancing glycogen phosphorylase (PYGL), glutamate–ammonia ligase (GLUL) and glutamate dehydrogenase 1 (GLUD1) activity (1), inhibits the mitochondrial adenine nucleotide translocase (ANT) to deplete cytosolic ATP (2), activates JUN N-terminal kinase (JNK)-mediated degradation of ferritin, thus increasing the labile iron pool (3), and favours sphingomyelinase (SMase)-mediated generation of ceramide, which is converted into sphingosine by ceramidase and promotes a cytosolic Ca2+ wave that activates calpains and cytosolic phospholipase A2 (cPLA2; 4). cPLA2 triggers lipid peroxidation by mobilizing the lipoxygenase substrate arachidonic acid and may be required for SMase-mediated ceramide generation (5). Sphingosine, calpains and lipid hydroperoxides induce lysosome membrane permeabilization (LMP), resulting in the leakage of cytotoxic hydrolases into the cytosol.  Oxidative metabolism favours the generation of reactive oxygen species (ROS) by the mitochondrial respiratory chain and the formation of redox-active advanced glycation end products (AGEs; 6). ROS (which also derive from NADPH oxidase 1 (NOX1; see FIG. 1), ceramide metabolism and labile iron pool elevation), initiate vicious cycles of damage by exacerbating mitochondrial uncoupling and lipid peroxidation and favour the opening of the permeability-transition pore complex (PTPC; 7). This results in the permeabilization of mitochondrial membranes and the translocation of cytotoxic proteins, including apoptosis-inducing factor (AIF), from the mitochondrial intermembrane space to the cytosol. Alternatively, AIF release can follow a poly(ADP-ribose) polymerase 1 (PARP1)–calpain cascade triggered by DNA damage (8). As cytosolic AIF enters the nucleus to exert endonucleolytic functions, and PARP1 overactivation rapidly depletes cytosolic ATP, DNA damage can initiate a feed-forward signalling loop towards necroptosis. Notably, RIP1 can also operate downstream of PARP1 to execute necroptosis. Δψm, mitochondrial transmembrane potential; CYPD, cyclophilin D; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; PGM, phosphoglucomutase; SDH, succinate dehydrogenase; TCA, tricarboxylic acid; VDAC, voltage-dependent anion channel.

and ROS overproduction (FIG. 2). RIP3 physically interacts with and allosterically activates several metabolic enzymes, including glycogen phosphorylase (PYGL), glutamate–ammonia ligase (GLUL) and glutamate dehydrogenase 1 (GLUD1). RNAi-mediated knockdown of any of these enzymes attenuates TNF plus Z-VAD.fmk-mediated ROS production and necroptosis12. PYGL catalyses the breakdown of glycogen
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into glucose-1-phosphate (glycogenolysis), which can be converted into the glycolytic substrate glucose-6phosphate87, thereby stimulating glycolysis (which eventually contributes to ROS generation). The RIP3-mediated necrotic boost on glycogenolysis can also favour the production of methylglyoxal, a cytotoxic compound for which the synthetic rate is proportional to glycolytic flux 88. Methylglyoxal covalently binds to proteins and
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forms advanced glycation end products (AGEs), which alter protein function and constitute new centres of sustained ROS generation88. Mitochondrial proteins seem to be particularly prone to methylglyoxal-mediated post-translational modifications89. Thus, inhibition of glycolysis reportedly attenuates cell death by apoptosis and necroptosis (as both these processes are stimulated by ROS), whereas the blockage of methylglyoxaldetoxifying pathways accelerates it 88. Both GLUL, a cytosolic enzyme that condensates glutamate and free ammonia into Glu, and GLUD1, a mitochondrial enzyme that converts glutamate to α-ketoglutarate, are required for glutaminolysis (reviewed in REF. 90). Glutaminolysis results in the generation of α-ketoglutarate, which feeds into the Krebs cycle to generate reduced equivalents and pyruvate (by malate decarboxylase), in turn favouring lactate accumulation90. Moreover, mitochondrial Glu catabolism increases the local concentration of ammonia, thus facilitating ROS generation by the respiratory chain91. Altogether, there seem to be several mechanisms by which enhanced glycogenolysis, glycolysis and glutaminolysis can contribute to the respiratory burst that characterizes necrotic cell death. Non-mitochondrial ROS production by the plasma membrane NADPH oxidase NOX1 (which is recruited by RIP1) also contributes to TNF-induced necrotic cell death16. NOX1 activation is dependent on riboflavin kinase, which physically bridges the TNFR1 death domain and p22phox (also known as CYBA), the common subunit of multiple NADPH oxidases17. NOX1 is activated within minutes of the administration of TNF, and it is possible that NOX1-generated ROS trigger or sustain the subsequent production of ROS by the mitochondrial respiratory chain92. ROS generation is auto-amplified through several reactions. For instance, interaction of hydrogen peroxide with the superoxide anion in the Haber–Weiss reaction or with ferrous (Fe2+) ions in the Fenton reaction generates the highly reactive hydroxyl radical, further promoting lipid peroxidation28. Interestingly, whereas low levels of ROS can favour a mild mitochondrial uncoupling that is detrimental to ATP synthesis but exerts cytoprotective effects93, ROS overgeneration engages the respiratory chain in a potentially lethal vicious cycle that also entails the generation of reactive nitrogen species (RNS)94 (see below). Similar to mitochondrial ROS, NOX1-derived ROS are not a requisite for necroptosis, as shown by the fact that small interfering RNA-mediated downregulation of NOX1 almost abrogates TNF-induced ROS generation but only marginally rescues L929 fibrosarcoma cells from necroptosis16. Accordingly, ROS scavengers such as tert-butyl4-hydroxyanisole (BHA) exert anti-necroptotic effects in some (but not all) experimental settings95,96. Thus, the relative contribution of ROS from distinct sources to necroptosis may be dictated by the cell type. TNF also stimulates ROS formation by favouring JNK1-dependent degradation of the ubiquitous ironbinding protein ferritin, resulting in an increase in the labile iron pool97. Redox-active iron is a threat to cells and needs to be cautiously transported and stored in an inactive form98. Ferritin-deficient cells (in which the iron storage capacity is reduced) are more resistant to TNF-induced labile iron pool elevation, ROS generation and necroptosis than their wild-type counterparts99. Intriguingly, RIP1-deficient MEFs also failed to elevate the labile iron pool on TNF administration99, suggesting that RIP1 might modulate the induction of ROS through an effect on ferritin. However, the exact molecular mechanisms underlying this phenomenon and the possible implication of RIP1 remain to be elucidated. At low intracellular concentrations, nitric oxide functions as a second messenger in a myriad of signalling pathways. If overproduced, nitric oxide is highly toxic and leads to the generation of RNS with distinctive chemical and biological properties100. Similar to ROS, RNS are potent oxidants and can initiate or propagate lipid101 and protein oxidation and peroxidation102. Recently, nitration has been shown to elicit RIP1- and RIP3-mediated necroptosis, with respiratory complex I subunit NDUFB8 being involved103. This is apparently in contrast with the well-known cytoprotective effects of nitrite, which attenuates oxidative stress, mitochondrial damage and dysfunction, hypothermia, tissue infarction and organismal death in a murine model of TNF-induced shock104. Nitrites also confer protection against ischaemia–reperfusion injuries in vivo, presumably owing to nitrite-dependent inhibition of mitochondrial ROS generation105 or to an effect on the soluble guanylate cyclase α1 subunit, one of the main intracellular receptors for nitric oxide and signal transducers in the cardiovascular system104. Involvement of LMP in the execution of necroptosis. ROS can react with polyunsaturated fatty acids in cellular membranes to generate reactive aldehydes (such as 4-hydroxynonenal), which in turn can attack protein and lipid moieties in membranes, thereby compromising their integrity 106. In mitochondria, the products of lipid peroxidation inhibit oxidative phosphorylation, compromise the permeability of the inner membrane, dissipate the Δψm and reduce the Ca2+ buffering capacity, thus contributing to necrosis107. Lipid peroxidationmediated destabilization of cellular membranes (including the plasma, lysosomal and ER membranes) results in a leakage of proteases or an elevation of cytosolic Ca2+ concentrations, two phenomena that participate in necrotic cell death. Lysosomes are the only intracellular compartment in which redox-active iron temporarily resides before it is incorporated into the catalytic centre of specific enzymes or stored in ferritin108. Typically, the Fenton reaction is favoured in the lumen of lysosomes, not only because lysosomes are enriched in reduced iron (Fe2+) and reducing equivalents (provided by Cys, ascorbic acid and reduced glutathione), but also because they are permeable to hydrogen peroxide and lack hydrogen peroxidedetoxifying enzymes, such as catalases and glutathione peroxidases108. Accordingly, oxidative stress-induced lipid peroxidation, LMP and cell death can be prevented by the iron chelator desferrioxamine 24,109. Cytosolic phospholipase A2 (cPLA2) and ceramide also act upstream of lipid peroxidation to stimulate LMP. PLA2 is an esterase that produces arachidonic acid from
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Advanced glycation end product (AGE)
The product of a chain of chemical reactions that most often is initiated by non‑enzymatic protein glycosylation. Increased extracellular glucose favours the accumulation of AGEs, which interact with specific receptors on the plasma membrane to stimulate the generation of intracellular ROS.

Haber–Weiss reaction
The generation of hydroxyl radicals from hydrogen peroxide and superoxide (H2O2 + O•2– → OH•+ HO– + O2). The reaction is very slow, but is catalysed by ferric ions (Fe3+).

Fenton reaction
The ferrous ion (Fe2+)‑dependent decomposition of dihydrogen peroxide, generating the highly reactive hydroxyl radical (Fe2+ + H2O2 → Fe3+ + OH• + OH–).

Lipid peroxidation
The biochemical reaction whereby free radicals ‘steal’ electrons from lipids in cell membranes, resulting in ultrastructural damage to organelles.

Labile iron pool
A cytosolic fraction of iron ions loosely bound to macromolecules (for example, ferritin) — also known as a chelatable iron pool — that harbours the metabolically active (and hence potentially toxic) forms of ferrous (Fe2+) and ferric (Fe3+) ions.

Oxidative phosphorylation
The process whereby respiratory chain complexes embedded in the inner mitochondrial membrane catalyse a series of redox reactions that provide the free energy to generate the Δψm.

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arachidonate-containing phospholipids110. Treatment of L929 cells with TNF leads to PLA2 activation, and overexpression of cPLA2 sensitizes TNF-resistant L929 variants to necroptosis111. Arachidonic acid is converted by lipoxygenase into membrane-damaging lipid hydroperoxides23 (FIG. 2). In response to TNF, both the lysosomal enzyme acid sphingomyelinase (aSMase) and its neutral counterpart at the plasma membrane (nSMase) transform sphingomyelin into ceramide, which in turn can be converted to sphingosine by ceramidase112. Sphingosine has been characterized as a lysosomotropic LMP inducer (see below)113. Depending on the specific experimental setting (the cell type or lethal trigger), ceramide can induce either apoptosis or necroptosis114. Isoformspecific pharmacological inhibition of nSMase protects breast cancer MCF7 cells against TNF-induced apoptosis115. Notably, both RIPK1–/– human Jurkat lymphocytes and cPLA2-deficient murine L929 cells fail to accumulate ceramide on TNF administration and are protected against TNF-induced necroptosis, suggesting that RIP1, as well as cPLA2, might be required for SMase-mediated generation of ceramide and consequent cell death96. A connection between Ca2+ homeostasis and LMP was first suggested by the observation that TNF induces a moderate increase in intracellular Ca2+ concentrations, resulting in the generation of enlarged lysosomes that are particularly prone to LMP116. In some experimental settings, for example in vivo during the neuronal response to ischaemia–reperfusion, lysosomal membranes can be destabilized by calpains78,117. Calpain-mediated LMP results in the cytosolic spillage of lysosomal hydrolases such as proteases of the cathepsin family, which play an important part during necrotic cell death118. Accordingly, pharmacological inhibitors of cathepsins confer consistent neuroprotection in vivo119. Importantly, calpain has also been shown to proteolytically inactivate the plasma membrane Na+–Ca2+ exchanger, thereby engaging a positive feedback loop of self-activation mediated by the irreversible accumulation of cytosolic Ca2+ (REF. 120). Additional evidence showing the important role of LMP in necrotic cell death has been provided by the genetic manipulation of 70 kDa heat shock protein (HSP70), a guardian of lysosomal membrane integrity 121. HSP70 specifically interacts with the endolysosomal anionic phospholipid bis(monoacylglycero) phosphate121 and may also constitute the lysosomal target of calpain-mediated proteolysis on its ROS-mediated carbony lation 118. HSP70 delays LMP and necrosis induced by TNF, heat shock or oxidative stress122–124. In response to an ischaemic insult, mitochondria from HSP70-overexpressing cells exhibit reduced levels of ROS production and lipid peroxidation125. Whether this represents a primary effect of HSP70 on LMP, mitochondrial membranes or iron homeostasis124 is not yet clear. of prior attempts to cope with stress and the biochemical routes leading to death influence the cell surface characteristics while affecting the release of ‘find-me’ signals (for the attraction of phagocytes), the exposure of ‘eat-me’ signals (for corpse engulfment) and the disclosure of ‘danger’ signals (which are often part of otherwise ‘hidden’ molecules). The combination of these cell death-associated molecules (CDAMs) determines which engulfing cells are recruited, how they are activated and how they interpret the dead cell’s antigens. A particular set of CDAMs can be decoded by the microenvironment of dying cells to alternatively trigger silent corpse removal, tissue repair responses, recruitment of additional inflammatory effectors, or full-blown immune responses126,127. So, what impact does programmed necrosis have on the inflammatory or immune system? Apoptotic cells emit a series of well-defined ‘find-me’ (such as soluble lysophosphatidylcholine (LPC)128 and ATP129) and ‘eat-me’ (such as surface-exposed and oxidized phosphatidylserine130) signals, allowing them to engage in synapse-like interactions with macrophages and to be recruited into tight-fitting phagosomes through a zipper-like mechanism131. Often, apoptotic corpses are taken up by phagocytic cells in the absence of inflammatory or immunogenic reactions. In some cases, cells that are en route to necrosis also externalize phosphatidylserine before plasma membrane permeabilization132, thus facilitating their recognition and internalization by phagocytes133,134. However, fully necrotic cells are internalized by macrophages through the formation of spacious macropinosomes135, a process that is accompanied by macrophage ruffling and involves the sorting of fluid-phase macromolecules, as judged by the colocalization of fluid-phase tracers131. Thus, the handling of apoptotic and necrotic cells by the immune system is radically distinct. In spite of this fundamental difference, both apoptotic and necrotic cells are efficiently cleared by professional and non-professional phagocytes and hence are rarely found in tissues. Defective clearance of dying cells, however, may contribute to the persistence of inflammation, excessive tissue injury and the pathogenesis of chronic obstructive pulmonary disease 136, diabetes137, atherosclerosis138 or autoimmune diseases such as systemic lupus erythematosus139. It has been a common paradigm that apoptosis is antiphlogistic (anti-inflammatory) and tolerogenic (producing immunological tolerance) but necrosis triggers inflammation and an immune response. This paradigm must be refined because in some cases, in particular on ER stress, apoptosis can be interpreted by the immune system as immunogenic 140, and the immunogenicity of apoptosis is lost when the same cells undergo necrotic lysis on freeze–thaw cycles141. Moreover, depending on the cell type, necrotic cells can even inhibit inflammatory reactions. Necrotic (ATP depleted or subjected to freeze–thaw cycles) and apoptotic (but not heat-killed) Jurkat lymphocytes have been shown to inhibit Escherichia coli-induced TNF secretion by human macrophages to a similar extent 134. Moreover, macrophages can engulf necrotic L929 cells (which have been killed by TNF) without producing
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Macropinosome
A large intracellular vesicle filled with extracellular fluids and macromolecules that is formed by macropinocytosis.

Disposal of necrotic cells When confronted with cell death, the immune system clears corpses, stimulates the replacement of lost cells, alerts host defences if infectious agents are detected and possibly eliminates cells approaching oncogenic transformation. The type and nature of dying cells, the history

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inflammatory cytokines133. These findings reveal an unexpected complexity in the interaction between necrotic cells and the phagocytic system. In spite of these caveats, it must be noted that necrotic cells can release multiple pro-inflammatory factors, including the alarmin SAP130, heat-shock proteins (such as HSP70, HSP90 and GP96), histones, high mobility group protein B1 (HMGB1) and several nonproteinaceous factors (such as RNA, DNA and monosodium urate microcrystals), all of which act on different PRRs on immune effector cells to activate inflammatory reactions (reviewed in REF. 126). Histones released from necrotic cells have a major pathogenic role in sepsis, and their neutralization by antibodies or activated protein C can prevent organismal lethality 142. Recently, mitochondrial damage-associated molecular patterns (DAMPs), including N-formylated peptides and mitochondrial DNA, were found to be released by necrotic cells into the circulation and to contribute to neutrophil-mediated organ injury similarly to bacterial PAMPs143, underscoring an evolutionarily conserved link between distinct routes to innate immunity. Recent genetic manipulations suggest an important role for necrosis in the outcome of viral infections and immunosurveillance. Ripk3–/– mice fail to control vaccinia virus infection, because virus-elicited necrosis can limit viral replication and/or stimulate the antiviral immune response10. Intriguingly, some viral genomes encode inhibitors of necrotic cell death that may facilitate their propagation and their subversion of the immune response. For example, murine cytomegalovirus (MCMV) expresses the proteins M36 and M45, which inhibit caspase 8-mediated apoptosis144 and RIP1- and RIP3-mediated necroptosis145, respectively. We suspect that multiple virus-encoded necrosis-inhibitory factors will be discovered as the comprehension of signalling events in necroptosis advances. It has not been investigated in detail whether necrotic cell death might exert a tumour-suppressive function like apoptotic cell death does. However, mice lacking CYLD (which is required for TNF-induced necrosis)14 are highly susceptible to developing a wide array of tumours, including skin and colon cancers146,147. It remains to be determined whether CYLD merely acts as a cell-autonomous tumour suppressor or whether it is required for stimulating immunosurveillance, and whether these effects can be attributed to its role in necroptosis or NF-κB signalling. the same timing as cells from wild-type mice would undergo apoptosis150. Nonetheless, necrosis is mostly associated with pathological conditions, including neurodegeneration, ischaemia–reperfusion and infection. Excitotoxicity, oxidative stress and mitochondrial dysfunction, all of which contribute to the execution of necroptosis (see above), are indeed implicated in stroke as well as in Alzheimer’s, Huntington’s and Parkinson’s diseases (reviewed in REF. 151). The ageing brain accumulates iron, copper and zinc, resulting in increased oxidative stress by the Fenton reaction, which contributes to necrotic cell death152. Accordingly, iron chelation and ROS scavenging may delay the manifestation of neurodegenerative diseases109. Intriguingly, necroptosis has recently emerged as a prominent antiviral mechanism, as shown by the fact that Ripk3–/– mice are more susceptible to viral infection than their wild-type counterparts10, and by the existence of viral factors that contain the RHIM domain and interfere with the RIP1–RIP3 interaction66. Ripk1–/– mice are not viable62, and tissue-specific knockout and kinase-dead knock-in models will be required to elucidate the contribution of RIP1 to pathological cell loss. Pharmacological RIP inhibitors, including the RIP1-specific agent necrostatin 1 and geldanamycin (which downregulates RIP1, RIP3 and several other HSP90 client proteins)10,153, exert cytoprotective effects in vitro in several distinct experimental settings (Supplementary information  S2 (table)). Intriguingly, in some cell types, geldanamycin induces a switch from TNF-induced necroptosis to apoptosis154. The inhibition of RIP1 kinase activity also attenuates neurodegenerative diseases155, brain ischaemia7, myocardial infarction 156 and head trauma 157 in vivo (Supplementary information S3 (table)), underscoring the contribution of RIP1 to pathological cell death. Similarly, pharmacological or genetic inhibition of PARP1, CYPD, cPLA2 or RIP3 limits cell loss in vivo in several rodent models of injury (Supplementary information S3 (table) and Supplementary information S4 (table)). Parp1–/– mice are protected from haemorrhagic shock158, acute pancreatitis and consequent lung injury 159. Mice lacking CYPD are more resistant to ischaemia–reperfusion damage of the brain21 and the heart 20 than their wild-type counterparts. Similarly, cPLA2-deficient mice exhibit reduced injury after brain ischaemia160. Finally, cerulein-induced pancreatic acinar cell loss and pancreatitis are greatly reduced in Ripk3–/– mice11,12. Altogether, these results support the idea that specific inhibitors of RIP1, RIP3, PARP1, CYPD and cPLA2 can attenuate pathological cell loss in vivo in rodent models of human disease.

Pathophysiological facets of necroptosis Necrosis can occur in a programmed manner during development (for example, the death of chondrocytes controlling the longitudinal growth of bones)148 and in adult tissue homeostasis (for example, in intestinal epithelial cells)149. Moreover, cells in which apoptosisassociated caspase activation has been blocked often succumb to necrosis in response to the same stimuli that would usually induce apoptosis. Thus, interdigital cells or thymocytes from apoptotic peptidase-activating factor 1 (Apaf1–/–) embryos or adult mice, respectively, undergo necrotic cell death to the same extent and with
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Problems and perspectives Necroptosis can be conceived as a partially programmed event of cellular explosion. Physiological signals or cellular damage are perceived by specific receptors or sensors that ignite a detonator, which in turn activates the blasting agent. It is important to accurately distinguish and molecularly identify the upstream signals — the detonator and the explosives — for several important reasons.
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First, the elucidation of the precise molecular hierarchy involved in different cell death scenarios may clarify whether one ‘core programme’ or several independent pathways of necrosis exist. Undoubtedly, there are different ways to induce necrosis, be it through the activation of specific receptors or by inflicting distinct types of cellular damage. However, it is still debatable whether the core features of necrosis (such as RIP1 activation, bioenergetic and redox crisis, and lysosomal and mitochondrial perturbation) are built in a single interdependent circuit or several independent, mutually stimulatory (self-amplifying) circuits. Understanding this is important for the development of necroptosis-inhibitory cytoprotective drugs. The existence of several distinct pathways that ignite necrosis would imply that they all need to be interrupted simultaneously for cytoprotection, suggesting the need for combination therapies. Although this has not been addressed systematically, it may be beneficial to combine cytoprotective agents that target different lethal subroutines (for example, apoptosis and necroptosis) and processes (for example, LMP and the mitochondrial permeability transition) for optimal therapeutic results. Second, upstream signals (as opposed to downstream effectors) may constitute better targets for the specific pharmacological suppression of unwarranted cell death. The interception of a pro-necrotic signal transduction pathway would be more efficient if it occurred at an early step, for instance at the level of TNF–TNFR1 interaction, rather than downstream. Moreover, the interruption of specific (stimulus-dependent) pro-necrotic signals should decrease negative side effects. As has previously been shown for several components of the apoptotic machinery (reviewed in REF. 161), necrosis-relevant molecules and processes have ‘day jobs’ and hence exert physiological functions, in particular in the response to cell stress and infection and in immune or inflammatory responses, that should not be perturbed. Third, downstream signals, which are usually (but not always) activated late in the pathway (when the initial signalling cascade has already been engaged), are also attractive therapeutic targets, as (at least in some settings) they could be blocked after the primary lesion (such as stroke, trauma, infarction and sepsis). Although intercepting upstream signals is the most desirable therapeutic choice, early interventions are rarely (if ever) achievable in the treatment of, for example, patients with stroke and trauma. Moreover, targeting downstream events is desirable when the upstream signals are not uniform or when they are transduced by multiple, interconnected pathways. These considerations underscore the importance of appropriately dissecting the chronological and functional aspects of necrotic demolition and defining the exact ‘point of no return’ beyond which cytoprotection can no longer be achieved. Fourth, cytotoxic T lymphocytes and natural killer cells, the cell death-inducing activity of which can contribute to the pathophysiology of human diseases, including AIDS and autoimmune disorders, reportedly ‘overkill’ their targets by transferring multiple proteases and membranepermeabilizing proteins into them, thereby triggering both apoptotic and necrotic programmes. Thus, rescuing targets from this type of cytotoxic attack may require a multipronged strategy that is yet to be optimized. Fifth, multiple cancer cell lines display an altered propensity to undergo necroptosis, which, at least partially, correlates with the expression levels of RIP3 (REF. 11). Further work is required to elucidate the importance of this finding in vivo and, in particular, whether it would be possible to stimulate the necrotic demise of RIP3-proficient tumour cells to circumvent apoptosis resistance. It is also unknown whether, and which, necrotic pathways might elicit immunogenic tumour cell death and hence ignite a highly desirable anticancer immune response that would eliminate residual tumour (stem) cells. We anticipate that resolving these questions will help in the design of cytoprotective and cytotoxic therapies, with important implications for neuroprotection, cardioprotection, organ preservation and cancer therapy. Although the molecular exploration of programmed necrosis is still in its infancy, it is clear that interrupting pro-necrotic signals may prevent pathological cell loss in many human diseases. In this respect, the development of necrostatins63 may have paved the way for the development of a new class of potentially powerful therapeutic agents for clinical applications.

1.

2.

3. 4. 5.

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Acknowledgements

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We apologize to our colleagues for not citing all primary research papers owing to space restrictions, and we thank W. Declercq for fruitful discussions. Electron microscopy pictures in Box 1 were kindly provided by D. Krysko, Ghent University, VIB, Belgium. P.V. holds a Methusalem grant from the Flemish Government (BOF09/01M00709) and is supported by the Flanders Institute for Biotechnology (VIB), the Interuniversity Poles of Attraction-Belgian Science Policy (IAP6/18), Fonds voor Wetenschappelijk Onderzoek – Vlaanderen (FWO, G.0133.05 and 3G.0218.06), The Special Research Fund of Ghent University (Geconcerteerde Onderzoekstacties 12.0505.02) and the European Commission (EU Marie Curie Training and Mobility Program, ApopTrain, MRTN-CT-035,624; EU FP7 Integrated Project, APO-SYS, HEALTH-F4-2007-200,767; EU FP6 Integrated Project, Epistem, LSHB-CT-2005-019,067; Marie Curie Training and Mobility Program). L.G. and T.V.B. are financed by APO-SYS and FWO, respectively. G.K. is supported by Ligue Nationale contre le Cancer (Equipe labellisée), Agence Nationale pour la Recherche (ANR), the European Commission (APO-SYS, ChemoRes, ApopTrain, Active p53), Fondation pour la Recherche Médicale (FRM), Institut National du Cancer (INCa) and Cancéropôle Ile-de-France.

Competing interests statement

The authors declare no competing financial interests.

168.

FURTHER INFORMATION
Peter Vandenabeele’s homepage: http://www.dmbr.ugent.be/

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