molecular mechanisms of necroptosis an ordered cellular explosion

Biased by their focus on life, biologists have neglected cell death for a long time. Although the first morpho-logical 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 con-sidered as a purely accidental and passive cell death subroutine. The first morphological classification of cell death was proposed by Schweichel and Merker4 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, respectively5.

The purely unregulated nature of necrosis was questioned in 1988, when it was discovered that dis-tinct 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 neo-logism necroptosis to describe one instance of regulated (as opposed to accidental) necrotic cell death7. Over the past two decades, a plethora of molecules and pro-cesses 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, deubiquit-ylating enzymes11,14, reactive oxygen species (ROS) generated by mitochondria or NADPH oxidase 1 (NOX1)15–17, bioenergetic reactions such as glyco-genolysis12 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 therapeu-tic suppression of necroptosis, a possibility that has raised huge expectations26. As the underlying molecu-lar 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.

*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]; [email protected]‑ugent.bedoi:10.1038/nrm2970Published online 8 September 2010

Molecular mechanisms of necroptosis: an ordered cellular explosionPeter 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 receptor-interacting 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.

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Nature Reviews | Molecular Cell Biology

a b

5 µm 5 µm

HeterophagyA 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.

AutophagyA 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.

CaspaseA 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.

GlutaminolysisThe 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 transitionLong‑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.

Initiation of necroptosis: the receptorsA 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 usu-ally activate the apoptotic machinery, and their cyto-toxicity often requires the presence of transcriptional or translational inhibitors, suggesting the existence of short-lived cytoprotective proteins that are con-tinuously 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 morphology33.

PRRs in the initiation of necroptosis. Although the underlying molecular mechanisms remain elusive, it seems that necroptosis can also be initiated by mem-bers 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 I- like 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 peptido-glycans, and respond by triggering inflammation or cell death34.

Various PAMPs have been shown to induce necrop-tosis by activating PRRs in different cell types. For example, viral dsRNA induces necroptotic cell death in human Jurkat T lymphocytes and murine fibro-sarcoma L929 cells35, and lipopolysaccharide does so in macrophages when caspase 8 activity is inhib-ited36. Similarly, the Gram-negative bacterium Shigella flexneri triggers necroptosis in neutrophils37 and in

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.

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 assessments24,162,163.

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InflammasomeA 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.

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

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

human monocyte-derived macrophages38. The lethal response of macro phages 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 features30, 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.

Initiation of necroptosis: TNFR1 decidesThe most extensively characterized pathway lead-ing to necroptosis is initiated by ligation of TNFR1 (TABLE 1). Depending on the cell type, cell activation state and microenvironment factors, TNF administra-tion 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 inter-fere 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 require-ment for NF-κB activation49. In some experimental scenarios, complex I constitutes the molecular plat-form that recruits the ROS-generating NADPH oxi-dase 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 com-plex I regulates an intricate network of pro-survival and pro-death signalling pathways.

Timeline | Evolution of the concept of programmed necrosis

1972 1988 1992 1996 1998 1999 2000 2003 2004 2005 2006 2008 2009

Discovery that TNF can induce both apoptosis and necrosis6.

Chan et al. introduce the term ‘programmed necrosis’ (REF. 30).

Discovery of the role of CYLD in TNFR1 complex I167,168.

Kerr et al. introduce the term apoptosis2.

ROS shown to be involved in TNF-induced cytotoxicity85.

Discovery that caspase inhibition favours necrosis13.

Description of a regulated form of necrosis activated by DNA damage19.

Discovery that RIP1 mediates TNFR1-induced caspase-independent cell death9.

ANT implicated in necroptosis22.

Identification of RIP3 as a crucial modulator of necroptosis10–12.

Gln metabolism implicated in necrosis18.

Discovery that TNFR1 recruits RIP1 (REF. 8).

Molecular characterization of RIP3 (REF. 171).

Characterization of CYPD-deficient mice20,21.

Degterev et al. identify necrostatin 1 and introduce the term ‘necroptosis’7.

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.

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TNFR1 complex II promotes apoptosis or necroptosis. Ligand-bound TNFR1 is internalized, leading to a shift in the molecular composition of the TNFR1 inter-actome (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 poly-ubiquitylation not only affects NF-κB activation but also influences the transition from complex I to com-plex II (REFS 10,11,51). On deubiquitylation of RIP1 by the Lys63-deubiquitylating enzyme cylindromatosis

(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

Table 1 | The functional interactome of TNFR1 in necroptosis

Factor* Localization Roles in necroptosis Outcome Refs

A20 (TNFAIP3)

Cytoplasm and plasma membrane‡

RIP1-deubiquitylating enzyme Inhibits the NF-κB system to favour necroptosis

52

Caspase 8 Cytoplasm TNFR1-interacting protein in complex II Cleaves and inactivates RIP1 and RIP3 11,55

Ceramidase Mitochondria and plasma membrane

Converts ceramide into sphingosine on TNFR1 ligation

Sphingosine induces lysosomotropic LMP 112,113

Cezanne (OTUD7B)



53

cIAPs Cytoplasm and plasma membrane‡

RIP1-ubiquitylating enzymes Facilitates NF-κB activation and inhibits necroptosis

11,47

cPLA2

Cytoplasm Produces arachidonic acid in response to TNFR1 ligation

Induces lysosomotropic LMP 111

CYLD Cytoplasm and plasma membrane‡


14

FADD Cytoplasm TNFR1-interacting protein in complex II Adaptor for TNF-induced necroptosis in some cells

9,43

JNK1 Cytoplasm and mitochondria§

Degrades ferritin on TNFR1 ligation Favours ROS overgeneration downstream of RIP1

97

LOX Cytoplasm Converts cPLA2-generated arachidonic acid

into lipid hydroperoxidesInduces lysosomotropic LMP 23

NOX1 Plasma membrane‡ NAPDH oxidase that generates O2– in a TRADD-

and RIP1-dependent manner on TNFR1 ligationInduces pro-necrotic ROS generation 16

RFK Cytoplasm and plasma membrane‡

TNFR1-interacting protein in complex I Couples TNFR1 to NOX1 17

RIP1 (RIPK1)

Cytoplasm, plasma membrane and possibly mitochondria

Crucial component of the necrosome Triggers necroptosis (which requires RIP1 kinase activity)

9,14,30, 62

RIP3 (RIPK3)

Cytoplasm, mitochondria and plasma membrane

Crucial component of the necrosome Triggers necroptosis (which requires RIP3 kinase activity)

10–12,55

SMases Lysosomes and plasma membrane

Transform sphingomyelin into ceramide in response to TNF

Trigger ROS generation and lipid peroxidation to induce lysosomotropic LMP

96,112, 115

TNF Extracellular milieu and plasma membrane‡

Pleiotropic pro-inflammatory cytokine I Activates necroptosis in the absence of caspase activity

6,13,30

TNFR2 Plasma membrane Death receptor that potentiates RIP1 recruitment at TNFR1 complex

Triggers necroptosis by facilitating RIP1 activation

30

TRADD Cytoplasm and plasma membrane‡

TNFR1-interacting protein in complex I and II Adaptor for TNF-induced necroptosis in some cells

43,58

TRAF2 and TRAF5


TNFR1-interacting proteins in complex I Promotes NF-κB activation, which inhibits necroptosis

30,57

USP21 Cytoplasm and plasma membrane‡


54

cIAPs, cellular inhibitor of apoptosis proteins; cPLA2, cytosolic phospholipase A

2; 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.

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Caspase 8

TRADDFADD

RIP1TRADD

RIP1FADD


X?

RIP3 Caspase 8RIP3

b

a

c

ROS

NOX1

Activekinase

Caspase 8inhibitorCaspase 8 activity

TNF

TNFR1

P

Caspase-independentexecutioner mechanisms

Caspase-dependentexecutioner mechanisms

Apoptosis Necroptosis

NADPHFAD

p22phox

RFK RFFMN

Complex I

RIP1

TRADD TRAF2 and TRAF5RIP1

cIAPs

CYLD

TAK1–TAB2–TAB3complex

NF-κBactivationLys63-linked

polyubiquitin

Cytosolic formation of complex II

Plasma membrane

Cytoplasm

USP21, A20 or cezanne ?

Necrostatin 1A Trp‑based molecule (5‑(1H‑indol‑3‑ylmethyl)‑3‑methyl‑2‑thioxo‑4‑ imidazolidinone) that was first identified as a specific and potent inhibitor of necroptosis7.

activation, also stimulate the lethal functions of RIP1. In complex II, caspase 8 inactivates RIP1 and RIP3 by pro-teolytic cleavage and initiates the pro-apoptotic caspase activation cascade11,55. Moreover, genetic or pharmaco-logical inhibition of cIAPs prevents RIP1 ubiquitylation and favours the formation of complex II, thus sensitiz-ing cells to RIP1-dependent activation of caspase 8 and apoptosis47,56. By contrast, when caspase 8 is deleted, depleted or inhibited by CrmA or pharmacological agents, complex II cannot enter the ‘apoptotic mode’

and TNFR1 ligation results (at least in some cell types) in programmed necrosis9,13. Whether FADD or TRADD are strictly required to assemble the necroptosis- signalling 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 set-tings, TRADD (which is also part of TNFR complex I; see above) constitutes an indispensable cell death-inducing adaptor protein43. In contrast to these obser-vations, 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 mito-chondrial cIAP inhibitor). Moreover, RNAi-mediated knockdown of TRADD stimulates (rather than inhibits) the formation of complex II in some cell types, suggest-ing that TRADD is not required for the assembly and function of complex II (REFS 56,59).

The necrosome signalling complex. The term necro-some refers to a multiprotein complex containing RIP1 and RIP3 that stimulates necroptosis59. The formation of the necrosome is highly regulated by ubiquitylation (see above) and mutual RIP1 and RIP3 phosphoryla-tion (see below). Whereas many cell lines are protected against TNF-induced apoptosis by Z-VAD.fmk, others respond to TNF plus Z-VAD.fmk by activating necrop-tosis60, a phenomenon that has recently been correlated with the expression of RIP3 (REF. 11). RIP3 contains an N-terminal kinase domain and a C-terminal RIP homo-typic interaction motif (RHIM), which mediates its interaction with RIP1 (REF. 61). Necroptosis induced by CD95L, TRAIL or TNF in combination with Z-VAD.fmk is abrogated in RIP1-deficient T cells9, and enforced dimerization of RIP1 can induce necroptosis in FADD-deficient Jurkat lymphocytes7. Consistent with a role for RIP1 in NF-κB-mediated pro-survival signalling, mice lacking RIP1 display extensive apoptosis in lymphoid and adipose tissues and die 1–3 days after birth62. In con-trast to RIP1, RIP3 is not involved in NF-κB activation10. Recent experiments with cells that have been stably or 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 in response to TNF and is stabilized in the presence of SMAC mimetics or caspase inhibitors10–12.

In 2005, Yuan and colleagues identified necrostatin 1 and necrostatin 3, small molecules that allosterically block the kinase activity of RIP1, thereby inhibiting necroptosis but leaving RIP1-mediated activation of NF-κB, mitogen-activated protein kinase p38 and JUN N-terminal kinase 1 (JNK1) unaffected7,63. Several other necrostatins have been identified by virtue of their capacity to suppress necrosis induced by TNF

Figure 1 | TNFR1‑elicited signalling pathways. a | On tumour necrosis factor (TNF) binding, TNF receptor 1 (TNFR1) undergoes a conformational change, allowing for the intracellular assembly of the so-called TNFR complex I, which includes TNF receptor-associated death domain (TRADD), receptor-interacting protein 1 (RIP1; also known as RIPK1), cellular inhibitor of apoptosis proteins (cIAPs), TNF receptor-associated factor 2 (TRAF2) and TRAF5. On cIAP-mediated Lys63-ubiquitylation, RIP1 can serve as a scaffold for the recruitment of transforming growth factor-β activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB2) and TAB3, which initiate the canonical nuclear factor-κB (NF-κB) activation pathway (BOX 2). Riboflavin kinase (RFK) physically bridges the TNFR1 death domain to p22phox (also known as CYBA), the common subunit of multiple NADPH oxidases, including NADPH oxidase 1 (NOX1), which also contributes to TNFα-induced necroptosis by generating reactive oxygen species (ROS). Conversely, on deubiquitylation by cylindromatosis (CYLD; and perhaps also by A20 (also known as TNFAIP3), cezanne (also known as OTUD7B) or ubiquitin-specific peptidase 21 (USP21)), RIP1 exerts lethal functions, which can be executed by two distinct types of cell death. b | The internalization of TNFR1 is accompanied by a change in its binding partners that leads to the cytosolic assembly of TNFR complex II, which often (but not invariably) contains TRADD, FAS-associated protein with a death domain (FADD), caspase 8, RIP1 and RIP3 (also known as RIPK3). Normally, caspase 8 triggers apoptosis by activating the classical caspase cascade. It also cleaves, and hence inactivates, RIP1 and RIP3. c | If caspase 8 is blocked by pharmacological or genetic interventions, RIP1 and RIP3 become phosphorylated (perhaps by an unidentified kinase) and engage the effector mechanisms of necroptosis. FAD, flavin adenine nucleotide; FMN; flavin mononucleotide.

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a Canonical pathway b Non-canonical pathway

Ub

Ub

Ub

Ub IKK complex

IKK complexIκB p65IκB p50

IκB p65IκB p50

p52RELB

p52

RELB

IKKγ IKKα

IKKβ

IKKαIKKα

IKKαIKKα

Ub

Ub

IKKγ

NIK

Linearubiquitylation

P

P P

P

Ubiquitylation

Degradation

IκBIκB

UbUbUbUbUb

E1E2

E3

P

PP P

P100P100

RELB

cREL

cREL

cRELcREL

RELB

Processing

NF-κBdimers

Cell death Cell survival ProliferationInflammationImmunity

LUBAC

p65

p65p65

p50p50

p50

p65p50

p52RELBOncogene 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.

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 patho-physiological 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.

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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.

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, overexpres-sion 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 abso-lute requirement for necroptosis induction. Altogether, these results point to the existence of a highly complex, tightly regulated signal transduction pathway that con-nects death receptors to pro-inflammatory, apoptotic or necrotic signal transducers.

Execution of necroptosisSeveral distinct molecular mechanisms contribute to the execution of TNFR1-initiated necroptosis. Some of these effectors can also be activated by other necrop-totic triggers, including PAMPs and DNA damage (see above).

Bioenergetic aspects of the execution of necroptosis. During apoptosis, ATP-consuming processes including PARP1 activity67, translation68 and proteasome-mediated degradation69 are rapidly shut off by caspases. By con-trast, 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 even-tually 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 accumula-tion 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 frag-mentation25, 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, pharmaco-logical 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,

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 dur-ing 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 mito-chondrially neosynthesized ATP with cytosolic ADP25. Inhibition of ANT by RIP1 can be expected to reduce intramitochondrial ADP levels, leading first to the inhi-bition 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 gener-ate the PTPC (reviewed in REF. 25). In response to some lethal triggers, including oxidative stress and Ca2+ over-load, the PTPC adopts a high conductance conforma-tion, permitting the unregulated entry of solutes and water into the mitochondrial matrix, a phenomenon that has been dubbed the mitochondrial permeability transi-tion25. The PTPC is a highly dynamic entity that interacts with multiple proteins, including pro- and anti-apoptotic members of the BCL-2 protein family82. However, it is not known whether BCL-2-modifying factor (BMF), a BH3-only protein required for TNF-induced necrop-tosis14, 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 cytoprotec-tive 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 mito-chondria and endoplasmic reticulum (ER)85,86. Although ROS production is not essential for all instances of TNF-induced necrosis11,86, the kinase activity of RIP3 may link TNFR1 signalling, mitochondrial bioenergetics

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II

ANT

ANT

VDAC

VDAC

H+

IIII

NADH NAD+

H+

H+

IV

H+

V

AN

T

ATPATP

ADP

H2O½O2SDH

JNK SMases Ca2+

Ferritin↓ Ceramide Calpains

cPLA2

Lipoxygenase

Arachidonicacid

Lipidhydroperoxides

Phospholipids

Labileiron pool↑

Sphingosine

Lipid peroxidation

ROS

ROS

ROS

ROS NucleusUV or

Succinate

α-Ketoglutarate

Glu + NH4+

Glu + NH4+

Gln

Glutaminase

Fumarate

Pyruvate

Pyruvate

G6P

Glycogen

PARP activation

PARP activationDNA damage

G1P

AGEs↑

Methylglyoxal↑

araaraararatttteteteteGLUD1

GLUL

PYGL

PGM

[ATP]↓[ATP]↓

RIP1–RIP3 necrosome

RIP1

X?

Ser161

RIP3Ser199

Lipidperoxidation

Lipidperoxidation

PTPC

CYPD

Glycolysis

Glycogenolysis

Glutaminolysis

Calpains

?

∆ ↑

Mitochondrion

Lysosomes

LMP

AIF

Cytochrome c

TCAcycle

Coenzyme Q10

Respiratory complexcomponent

Active kinase

1

2 3 4

5

6

7

8

mψ

P

P

P

Necroptosis

and ROS overproduction (FIG. 2). RIP3 physically inter-acts with and allosterically activates several metabolic enzymes, including glycogen phosphorylase (PYGL), glutamate–ammonia ligase (GLUL) and glutamate dehydrogenase 1 (GLUD1). RNAi-mediated knock-down of any of these enzymes attenuates TNF plus Z-VAD.fmk-mediated ROS production and necrop-tosis12. PYGL catalyses the breakdown of glycogen

into glucose-1-phosphate (glycogenolysis), which can be converted into the glycolytic substrate glucose-6- phosphate87, thereby stimulating glycolysis (which even-tually 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 glyco lytic flux88. Methylglyoxal covalently binds to proteins and

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 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 A

2

(cPLA2; 4). cPLA

2 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. Δψ

m, 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.

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

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

Labile iron poolA 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 phosphorylationThe 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.

forms advanced glycation end products (AGEs), which alter protein function and constitute new centres of sus-tained 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 methylglyoxal- detoxifying pathways accelerates it88. 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 glutamino lysis (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 ammo-nia, thus facilitating ROS generation by the respiratory chain91. Altogether, there seem to be several mecha-nisms 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 ribo flavin kinase, which physically bridges the TNFR1 death domain and p22phox (also known as CYBA), the com-mon 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 poten-tially 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 necrop-tosis16. Accordingly, ROS scavengers such as tert-butyl-4-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 iron-binding 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 genera-tion 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 mech-anisms underlying this phenomenon and the possible implication of RIP1 remain to be elucidated.

At low intracellular concentrations, nitric oxide func-tions 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 chem-ical and biological properties100. Similar to ROS, RNS are potent oxidants and can initiate or propagate lipid101 and protein oxidation and peroxidation102. Recently, nitra-tion 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 attenu-ates oxidative stress, mitochondrial damage and dysfunc-tion, 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 inhibi-tion 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 cel-lular membranes to generate reactive aldehydes (such as 4-hydroxynonenal), which in turn can attack protein and lipid moieties in membranes, thereby compromis-ing their integrity106. 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 capac-ity, thus contributing to necrosis107. Lipid peroxidation-mediated destabilization of cellular membranes (includ-ing 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 reduc-ing equivalents (provided by Cys, ascorbic acid and reduced glutathione), but also because they are perme-able to hydrogen peroxide and lack hydrogen peroxide-detoxifying 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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MacropinosomeA large intracellular vesicle filled with extracellular fluids and macromolecules that is formed by macropinocytosis.

arachidonate-containing phospholipids110. Treatment of L929 cells with TNF leads to PLA2 activation, and over-expression of cPLA2 sensitizes TNF-resistant L929 vari-ants to necroptosis111. Arachidonic acid is converted by lipoxygenase into membrane-damaging lipid hydroper-oxides23 (FIG. 2). In response to TNF, both the lysosomal enzyme acid sphingomyelinase (aSMase) and its neutral counterpart at the plasma membrane (nSMase) trans-form 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. Isoform-specific pharmacological inhibition of nSMase protects breast cancer MCF7 cells against TNF-induced apopto-sis115. Notably, both RIPK1–/– human Jurkat lymphocytes and cPLA2-deficient murine L929 cells fail to accumu-late 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 set-tings, 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 integ-rity121. HSP70 specifically interacts with the endo-lysosomal anionic phospholipid bis(monoacylglycero)phosphate121 and may also constitute the lysosomal tar-get of calpain-mediated proteolysis on its ROS-mediated carbony lation118. 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 repre-sents a primary effect of HSP70 on LMP, mitochondrial membranes or iron homeostasis124 is not yet clear.

Disposal of necrotic cellsWhen 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 trans-formation. The type and nature of dying cells, the history

of prior attempts to cope with stress and the biochemi-cal 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 dis-closure of ‘danger’ signals (which are often part of other-wise ‘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 microenviron-ment of dying cells to alternatively trigger silent corpse removal, tissue repair responses, recruitment of addi-tional 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 oxi-dized 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 inflamma-tory or immunogenic reactions. In some cases, cells that are en route to necrosis also externalize phosphatidyl-serine 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 accompa-nied by macrophage ruffling and involves the sorting of fluid-phase macromolecules, as judged by the colo-calization 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 patho-genesis of chronic obstructive pulmonary disease136, 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 immunogenic140, 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 extent134. Moreover, macrophages can engulf necrotic L929 cells (which have been killed by TNF) without producing

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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 pro-teins (such as HSP70, HSP90 and GP96), histones, high mobility group protein B1 (HMGB1) and several non-proteinaceous factors (such as RNA, DNA and monoso-dium 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 lethality142. Recently, mitochon-drial 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, underscor-ing 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 vac-cinia 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 necro-sis)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.

Pathophysiological facets of necroptosisNecrosis 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 apoptosis-associated 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

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 infec-tion. 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 accu-mulates iron, copper and zinc, resulting in increased oxi-dative stress by the Fenton reaction, which contributes to necrotic cell death152. Accordingly, iron chelation and ROS scavenging may delay the manifestation of neuro-degenerative diseases109. Intriguingly, necroptosis has recently emerged as a prominent antiviral mechanism, as shown by the fact that Ripk3–/– mice are more sus-ceptible to viral infection than their wild-type counter-parts10, 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 cyto-protective 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 attenu-ates neurodegenerative diseases155, brain ischaemia7, myocardial infarction156 and head trauma157 in vivo (Supplementary information S3 (table)), underscor-ing 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 infor-mation S3 (table) and Supplementary information S4 (table)). Parp1–/– mice are protected from haemor-rhagic shock158, acute pancreatitis and consequent lung injury159. Mice lacking CYPD are more resistant to ischaemia–reperfusion damage of the brain21 and the heart20 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.

Problems and perspectivesNecroptosis can be conceived as a partially programmed event of cellular explosion. Physiological signals or cellu-lar damage are perceived by specific receptors or sensors that ignite a detonator, which in turn activates the blast-ing 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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9. Holler, N. et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nature Immunol. 1, 489–495 (2000).The authors discovered that, in some cell types, FAS can trigger non-apoptotic cell death that is independent of caspases but dependent on the adaptor protein FADD and the presence and enzymatic activity of the protein kinase RIP1. This milestone paper is the first report of RIP1-dependent necroptosis.

10. Cho, Y. S. et al. Phosphorylation-driven assembly of the RIP1–RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137, 1112–1123 (2009).

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13. Vercammen, D. et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J. Exp. Med. 187, 1477–1485 (1998).

First, the elucidation of the precise molecular hierar-chy involved in different cell death scenarios may clarify whether one ‘core programme’ or several independent pathways of necrosis exist. Undoubtedly, there are differ-ent 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 pertur-bation) are built in a single interdependent circuit or sev-eral independent, mutually stimulatory (self-amplifying) circuits. Understanding this is important for the develop-ment of necroptosis-inhibitory cytoprotective drugs. The existence of several distinct pathways that ignite necrosis would imply that they all need to be interrupted simulta-neously for cytoprotection, suggesting the need for com-bination therapies. Although this has not been addressed systematically, it may be beneficial to combine cytopro-tective 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 func-tional 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 tar-gets by transferring multiple proteases and membrane-permeabilizing 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 pro-pensity 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 pos-sible 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 thera-pies, with important implications for neuroprotection, cardio protection, 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 develop-ment of a new class of potentially powerful therapeutic agents for clinical applications.

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AcknowledgementsWe 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 pic-tures 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 statementThe authors declare no competing financial interests.

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

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molecular mechanisms of necroptosis an ordered cellular explosion

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