A guide to cell death pathways

Junying Yuan  1,2 & Dimitry Ofengeim 3

Abstract

Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Apoptosis, necroptosis and pyroptosis are three such programmed cell death modalities. The caspase family of cysteine proteases serve as key regulators of programmed cell death. During apoptosis, a cascade of caspase activation mediates signal transduction and cellular destruction, whereas pyroptosis occurs when activated caspases cleave gasdermins, which can then form pores in the plasma membrane. Necroptosis, a form of caspase-independent programmed necrosis mediated by RIPK3 and MLKL, is inhibited by caspase-

8mediated cleavage of RIPK1. Disruption of cellular homeostatic mechanisms that are essential for cell survival, such as normal ionic and redox balance and lysosomal fux, can also induce cell death without invoking programmed cell death mechanisms. Excitotoxicity, ferroptosis and lysosomal cell death are examples of such cell death modes. In this Review, we provide an overview of the major cell death mechanisms, highlighting the latest insights into their complex regulation and execution, and their relevance to human diseases.

Introduction

Cell death is an essential component of organismal development and adult homeostasis. Elimination of superfluous cells in development is important to ensure normal morphogenesis and organogenesis. In adult life, elimination of auto-reactive immune cells, cancerous cells and damaged cells is essential to homeostasis. Either too much or too little cell death can lead to human disease — neurodegenerative diseases involve the death of neurons that should have been long-lived to maintain neurological functions, and one of the hallmarks of cancer is the failure to eliminate cells that carry cancerous mutations1.Different types of cell death can be classified on the basis of morphology. For example, apoptosis and necrosis have been described for their distinct morphological characteristics2. Apoptosis is characterized by cytoplasmic and nuclear condensation without compromising the integrity of the cytoplasmic membrane. In contrast, early loss of membrane integrity and the consequential expansion of the cytoplasmic compartment due to ionic imbalance are hallmarks of necrosis. The term ‘programmed cell death’ was proposed by Richard Lockshin and Carroll M. Williams in the 1960s to describe the degeneration of intersegmental muscles in Silkmoth during ecdysis (exoskeleton moulting), which implied genetic regulation of muscle degeneration3,4, but insights into the molecular mechanisms of cell death came much later. Research in the last three decades has uncovered the dedicated molecular machinery that mediates apoptosis, necroptosis, and pyroptosis and identified the mammalian genes encoding its components. We propose to collectively refer to apoptosis, necroptosis and pyroptosis as ‘programmed cell death’.In addition to cell death resulting from the activation of programmed cell death pathways — as for apoptosis, necroptosis and pyroptosis — cells can die when key cell survival mechanisms that are required to maintain normal cellular homeostasis are inactivated or disrupted. Such examples may include the loss of cytoplasmic and/or intracellular membrane integrity, accumulation of misfolded proteins, excitotoxicity, oxidative stress and lipid peroxidation. Excitotoxicity results from the toxic accumulation of specific amino acids, such as glutamate, and occurs mainly in neurons5. Ferroptosis results from the inactivation of a cellular redox mechanism in defence against lipid peroxidation6,7. Pathological release of lysosomal acidic hydrolases, whose normal function is to break down macromolecules for cellular recycling, may lead to lysosomal cell death8. A cell may also be eaten alive by another cell in a process known as entosis9.As it is increasingly recognized that dysregulated cell death processes serve important roles in human diseases, substantial effort is being devoted to identifying the mechanisms of cell death involved in specific human pathological conditions — whether cell death results from the activation of programmed cell death mechanisms or the loss of cell survival mechanisms, or both. In this Review, we first provide an overview of the triggers, mechanisms and regulation of programmed cell death, including apoptosis, pyroptosis and necroptosis. We then describe the mechanisms underlying entosis, excitotoxicity, ferroptosis, lysosomal cell death and mitotic catastrophe, which are mediated by the disruption of different cellular homeostatic pro-survival mechanisms. We also discuss connections between these cell death modalities resulting from disruption of cell survival and programmed cell death. Lastly, we briefly discuss the latest insights into the involvement of cell death in human diseases.

Apoptosis

The machinery that mediates cell death was first characterized in Caenorhabditis elegans, and homologues of the C. elegans cell death effector proteins, which include caspases and BCL-2 proteins, were later identified in mammals (Box 1). Apoptosis of mammalian cells can be divided into intrinsic apoptosis and extrinsic apoptosis, which differ in how the apoptotic triggers are sensed and integrated by cells. Extrinsic apoptosis is mediated by the activation of death receptors localized on the plasma membrane, whereas intrinsic apoptosis is initiated without the involvement of death receptors (Fig. 1).

Intrinsic apoptosis

Intrinsic apoptosis can be activated following cellular alterations such as DNA damage, withdrawal of growth factors and mitochondrial damage (Fig. 1). Anoikis is also a form of intrinsic apoptosis that can be activated in certain cells by the loss of integrin-mediated cellular attachment to the extracellular matrix10.The BCL-2 family of proteins are the key upstream regulators of intrinsic apoptosis. BCL-2, a mammalian homologue of the C. elegansCED-9, was first isolated as an oncogene in human follicular lymphomas with the t(14;18) translocation and promotes cell survival rather than oncogenic cellular proliferation11. The mammalian BCL-2 family includes both anti-apoptotic as well as pro-apoptotic members, similar to that of CED-9 and EGL-1 in C. elegans12,13. The defining feature of the BCL-2 protein family, which includes both pro-apoptotic and anti-apoptotic family members, is the presence of one to four BCL-2 homology (BH) domains12,14. High levels of anti-apoptotic BCL-2 proteins suppress the activation of this cell death pathway. Apoptosis is crucial during animal development and adult life as genetic ablation of the anti-apoptotic proteins BCL-2, BCL-xL or MCL-1 can be lethal or lead to deleterious phenotypes in mutant mice15–17. The BH domains in the BCL-2 family mediate the interaction between pro-apoptotic and anti-apoptotic family members to inhibit or activate apoptosis13. In response to DNA damage, oxidative stress or nutrient deprivation, both transcriptional and post-translational activation of the pro-apoptotic BCL-2 family are critical in transducing the pro-apoptotic signal to drive mitochondrial outer membrane permeabilization (Fig. 1). In this process, pro-apoptotic BH3-only BCL-2 proteins, such as BIM, BID, BAD, NOXA and PUMA, utilize their BH3 domains to both actively inhibit anti-apoptotic BCL-2 proteins, such as BCL-2, BCL-xL, MCL-1 and BCL-W, and directly activate pro-apoptotic BAX and BAK to drive the formation of oligomeric pores in the mitochondrial outer membrane. The rupture of the outer mitochondrial membrane lead to the release of cytochrome c and second mitochondria-derived activator of caspase (SMAC, also known as DIABLO) to promote downstream caspase activation14. APAF1, the mammalian homologue of CED-4 and a member of the NOD family18, forms a heptameric apoptosome19,20 to promote the activation of caspase-9, which in turn mediates the cleavage of other downstream caspases, such as caspase-3 and caspase-7, that can cleave hundreds of protein substrates to promote cellular destruction21 (Fig. 1). However, the view of a simple upstream and downstream cascade of caspases should be modified as the double knockout of caspase-3 and caspase-7 in mice can also delay mitochondrial damage and cell death22. Thus, apoptosis may be viewed as a circular cascade with the capability of self-amplification.Activation of intrinsic apoptosis by DNA damage following ionizing irradiation or chemotherapeutic agents, such as etoposide and taxol, forms the basis of conventional cancer radiation therapies and chemotherapies. However, research over the past decade has uncovered redundancy in mammalian cell death mechanisms. Knockout of both Bax and Bak1 in Bax–/–Bak1–/– cells or Bax–/–Bak1–/–double-knockout mice results in high resistance to apoptosis23 and yet a small percentage of the mutant mice can still be born and survive for months24. This suggests that other cell death mechanisms may be activated and/or selected for in Bax–/–Bak1–/– cells to eliminate unwanted cells when apoptosis fails during development. In addition, although DNA damage-induced cell death can activate apoptosis by promoting mitochondrial damage and downstream caspase activation, DNA damage-induced apoptosis may constitute only a part of the cell death response as severe DNA damage can lead to cell death by mitotic catastrophe (see below). Thus, when the programmed cell death mechanism is blocked, disruption of cellular homeostatic pro-survival mechanisms may contribute to cell elimination in mammals.

Extrinsic apoptosis

Extrinsic apoptosis is mediated by the activation of cytoplasmic membrane-localized death receptors that are characterized by the presence of an intracellular protein–protein interaction domain known as the Death domain (DD), such as Fas (also known as CD95), TNFR1, TRAIL (DR4) and TRAIL-R2 (DR5), by their cognate ligands25,26 (Figs. 1 and 2). These pathways are relevant to human disease as genetic defects in Fas and its ligand FasL lead to a rare human autoimmune condition known as autoimmune lymphoproliferative syndrome, which can be phenocopied by Fas and FasL mutant mice27–30. FasL expressed in activated T cells induces apoptosis by promoting the recruitment of adaptor protein FADD and caspase-8 by interacting with the DD on Fas in target cells. Autoimmune lymphoproliferative syndrome-associated mutations in the Fas DD resulted in the loss of FADD recruitment and inhibition of caspase-8 activation. These findings underscore the importance of caspase-8 activation in Fas-mediated apoptosis31.Another example of death receptor-mediated extrinsic apoptosis that has important medical significance is TNF-mediated activation of TNFR1. Activation of TNFR1 by TNF leads to the rapid formation of a transient intracellular signalling complex, known as complex I, which plays a critical role in making the cellular life-or-death decision. The intracellular DD of activated TNFR1 recruits the DD-containing adaptor protein TRADD and RIPK1, a DD-containing kinase32,33 (Fig. 2). TRADD, in turn, mediates the recruitment of TRAF2, TRAF5, cIAP1 and cIAP2, which catalyse K63-linked, K48-linked and K11-linked poly-ubiquitination on complex I34–36. The polyubiquitylated chains generated by cIAP1 and cIAP2 serve to recruit other complex I components such as the linear ubiquitin chain assembly complex (LUBAC). LUBAC comprises the catalytic components HOIP (RNF31), HOIL1 and SHARPIN that catalyse M1-linked ubiquitination on components of complex I such as RIPK1, NEMO, A20, TRADD and TNFR137–39. The ubiquitin chains on complex I also serve to recruit key kinases, including the TAK1–TAB1–TAB2–TAB3 complex and NEMO–IKKα–IKKβ kinase complex, to activate NF-κB as well as to perform inhibitory phosphorylation on RIPK140–42. In living cells, correctly assembled and ubiquitylated complex I is crucial for the activation of canonical NF-κB, a pro-survival and pro-inflammatory transcriptional pathway that controls the expression of CFLAR and TNFAIP3, which encode key regulators of apoptosis and inflammation43–46. A20, encoded by TNFAIP3, is a ubiquitin chain-editing enzyme that modifies the ubiquitination pattern of RIPK1 to control its kinase activity44. CFLAR encodes multiple c-FLIP isoforms, with similarity in amino acid sequence to caspase-8 but lacking enzymatic activity, that can directly bind to caspase-8 and modulate its activation47.Activation of TNFR1 by TNF may promote RIPK1-independent apoptosis or RIPK1-dependent apoptosis (RDA). Treatment with TNF and cycloheximide, which blocks the translation downstream of the NF-κB pathway, can induce RIPK1-independent apoptosis, while dysregulation of RIPK1 ubiquitination and phosphorylation can promote RDA. The loss of cIAP1, cIAP12, LUBAC or NEMO, inhibition of TAK1, TBK1 or IKKs, or mutating a Ub acceptor site in RIPK1 (K377R) results in the hyperactivation of RIPK1 kinase activity48–53. Activated RIPK1 mediates the formation of a cytoplasmic complex, known as complex IIa, that comprises RIPK1, FADD and caspase-8, to mediate the activation of caspase-8 to promote RDA42,53–55 (Fig. 2). Thus, key mediators of NF-κB activation, including TAK1, IKKs, TBK1, NEMO, cIAP1, cIAP2 and LUBAC, also serve important roles in suppressing and modulating RDA. The activation of RIPK1 functions as a key checkpoint in the formation of complex IIa and inhibition of the kinase activity of RIPK1 is highly effective in blocking RDA42,52,56.

Fig. 1 | Intrinsic and extrinsic apoptosis. Extrinsic apoptosis (right) is mediated by the activation of plasma membrane-localized death receptors (such as TNFR1, Fas and TRAIL receptors DR4 and DR5) by their cognate ligands (such as TNF, FasL and TRAIL, respectively). Intrinsic apoptosis (left) can be activated by growth factor withdrawal, mitochondrial damage, DNA damage and chemotherapeutic drugs such as taxol. Activation of BH3-only members of the BCL-2 family, such as transcriptional induction of NOXA and PUMA by p53, post-translational modification of BAD and BIM, and cleavage of BID by caspase-8, induces mitochondrial damage by inactivating pro-survival BCL-2 family members, such as BCL-2, BCL-xL and MCL-1, and activating oligomerization of pro-death BCL-2 family members BAX and BAK. Mitochondrial damage leads to the release of cytochrome c and second mitochondria-derived activator of caspase (SMAC; also known as DIABLO) to promote the activation of caspase-9 mediated by APAF1. Activated caspase-9 in turn cleaves downstream caspases, caspase-3 and caspase-7, to mediate the execution of intrinsic apoptosis. Activated caspase-3 and caspase-7 can also exert feedback activation of upstream caspases to allow amplification of caspase cascades. ER, endoplasmic reticulum; UV, ultraviolet.

Necroptosis

Necrosis was traditionally believed to be a passive form of cell death and therefore to be unregulated. The groundbreaking discoveries made since the mid-2000s identified RIPK1, RIPK3 and MLKL, encoded in the genomes of mammals and higher vertebrates, to mediate a necrotic programmed cell death pathway known as necroptosis48,57–62. Necroptosis represents the first discovered programmed cell death mechanism that mediates necrosis57. The activation of death receptors, including TNFR1, Fas, DR4 and DR5, by their cognate ligands in cells under apoptosis-deficient conditions can promote necroptosis. Necroptosis was first defined by the inhibition of caspase-independent necrosis upon treatment with necrostatin-1 (Nec-1)57. Nec-1 and its improved small-molecule analogues, such as Nec-1s, were later found to be inhibitors of RIPK1 kinase activity48,63. The activation of RIPK1 can mediate RDA under apoptosis-competent conditions and necroptosis under apoptosis-deficient conditions34,42,64–68. Thus, RIPK1 activation plays an important role in mediating multiple deleterious responses downstream of TNFR1 depending on genetic background, cell type and disease condition.The caspase 8-mediated cleavage of RIPK1 in the intermediate domain (after D325 in mouse RIPK1 and D324 in human RIPK1) suppresses the activation of RIPK1 kinase activity by separating the N-terminal kinase domain from the intermediate domain and C-terminal DD, which mediates the dimerization of RIPK1 to promote its kinase activation69,70. Rare missense mutations in RIPK1 that alter the cleavage site by caspase 8 define a class of early-onset autoinflammatory diseases in humans characterized by periodic fevers71,72.

Activation of RIPK1 in caspase-deficient cells promotes the for mation of complex IIb (necrosome), which includes RIPK3 and MLKL, to mediate necroptosis59–61 (Fig. 2). During necroptosis, the interaction of activated RIPK1 with RIPK3 is mediated by their respective homotypic RIP homotypic interaction motifs (RHIM), which form a RHIM-mediated amyloid-like structure, to promote the activation of RIPK373,74. Activated RIPK3, marked by the phosphorylation of Thr231 and Ser232 in murine RIPK3 and Ser227 in human RIPK3 (ref. 75), in turn mediates the phosphorylation of a pseudokinase MLKL at Ser358 in human MLKL or Ser345 in murine MLKL. This phosphorylation promotes MLKL oligomerization and the interaction of charged amino acids in its N-terminal four-helix bundle (4HB) domain with phosphatidylinositol phosphates in the plasma membrane. As a result, oligomerized MLKL inserts into the plasma membrane and forms pores that lead to necrosis76–78. Multiple lines of Ripk1 kinase-dead knock-in mutant mice do not show developmental defects and have normal adult phenotypes but are highly resistant to TNF injection, which induces rapid lethality in wild-type mice69,79–84. Ripk3-null mice and Mlkl-null mice also show resistance to TNF injection62. These results indicate the importance of necroptosis in mediating TNF-mediated sepsis.Similar to that of Fas and TNFR1, the activation of DD-receptors DR4 and DR5 by their cognate ligand TRAIL in cells lacking caspase activity can activate necroptosis85,86. Additionally, necroptosis can result from the activation of pattern-recognition receptors (PRRs), such as Toll-like receptor 3 (TLR3), TLR4 and Z-DNA-binding protein 1 (ZBP1), in cells lacking caspase activity87–90 (Fig. 2). Necroptosis mediated by TLR3 and TLR4 is driven through TIR domain-containing adaptor-inducing IFNβ (TRIF, also known as TICAM1) by a RHIM-dependent engagement of RIPK3. Activation of ZBP1 by viral Z-RNA or Z-DNA with interferon stimulation can promote necroptosis as part of a host defence response, independent of RIPK1 and TNFR188,89. ZBP1 is a sensor of cytoplasmic nucleic acids and can bind with viral Z-DNA and Z-RNA to promote the activation of necroptosis in response to viral infection by binding with RIPK3 via its two RHIM domains to form a necrosome91. Activation of these RHIM adaptor proteins can also lead to phosphorylation, oligomerization and membrane targeting of the necroptosis effector protein MLKL. The RHIM-dependent binding of ZBP1 to RIPK3 is inhibited by the scaffold function of RIPK150,92. Thus, RIPK1 and ZBP1 may compete for their binding to RIPK3. Knockout of either Zbp1 or core interferon signalling components prolongs the survival of Ripk1-null mice, suggesting the activation of a ZBP1-dependent interferon-mediated response upon the loss of RIPK1.Metabolic stress and hypoxia can also promote the activation of RIPK1 and necroptosis independent of TNF or TNFR185,93. Energy-stress activation of AMPK serves as a metabolic checkpoint94. Whilst activated AMPK mediates inhibitory phosphorylation of S416 in human RIPK1 and S415 in murine RIPK1, sustained glucose deprivation can promote the activation of RIPK1, RIPK3 and MLKL to induce cell death85. Moreover, under normoxic conditions, activation of RIPK1 is controlled by prolyl hydroxylation at multiple proline residues, such as Pro195, by EGLN proteins93. This leads to the recognition and suppression of RIPK1 activation by binding with phosphorylated von Hippel–Lindau disease tumour suppressor (pVHL) protein (an E3 ubiquitin ligase that is known to target the transcriptional factor hypoxia-inducible factor 1α, a key regulator of cellular response to oxygen concentration) for proteasomal degradation93,95. Prolonged hypoxia inactivates EGLN proteins and the loss of proline hydroxylation on RIPK1 as well as binding with pVHL1, which can promote the activation of RIPK1 without affecting its degradation. The interactions of RIPK1 with key mediators of metabolism and hypoxia, such as AMPK and pVHL, may contribute to the underlying mechanism for RIPK1 in mediating ischaemic damage in multiple organs, including brain, heart and kidney57,96–98.

Fig. 2 | Necroptosis mediated by DD-containing receptors and pattern-recognition receptors.

Upon activation by TNF, the cytoplasmic Death domain (DD) of trimerized TNFR1 mediates the formation of a transient intracellular complex, named complex I, which recruits adaptor protein TRADD, the DD-containing protein kinase RIPK1 and several E3 ubiquitin ligases, including TRAF2, cIAP1, cIAP2, the linear ubiquitin chain assembly complex (LUBAC), ubiquitin editing enzyme A20 and deubiquitylation enzyme cylindromatosis complex (CYLD and SPATA2) as well as multiple ubiquitin-binding proteins, including NEMO, ABIN1 and OPTN. In complex I, RIPK1 is rapidly polyubiquitylated by Lys63-linked and linear Met1-linked ubiquitin chains, which mediate the recruitment and activation of TAK1, TBK1 and IKK complexes. When RIPK1 kinase is not activated in living cells, the phosphorylation and subsequent ubiquitin–proteasome system-mediated degradation of IκB leads to the activation of NF-κB in the nucleus (not shown). Dysregulation of complex I promotes the activation of RIPK1 (as marked by pS166 RIPK1), which leads to the formation of two alternative cytosolic complexes, complex IIa or complex IIb, to mediate RIPK1-dependent apoptosis and necroptosis, respectively. Complex IIa includes the adaptor FADD protein, caspase-8 and RIPK1 to promote the activation of caspase-8, which in turn cleaves downstream caspases such as caspase-3, ultimately leading to apoptosis. When the activation of caspase-8 is inhibited, activated RIPK1 kinase binds to RIPK3 to form complex IIb. Activated RIPK3 in turn phosphorylates MLKL to mediate the execution of necroptosis by disrupting the integrity of the plasma membrane. The activation of Toll-like receptor (TLR3) and TLR4 by their cognate ligands, viral nucleic acids and bacterial lipopolysaccharides (LPS), respectively, in cells lacking caspase function can promote the binding of TIR domain-containing adaptor-inducing IFNβ (TRIF) to RIPK3 to mediate necroptosis. When activated by viral Z-DNA or Z-RNA, the binding of Z-DNAbinding protein 1 (ZBP1; also known as DAI) to RIPK3, mediated by their respective RHIMs (receptor-interacting protein homotypic interaction motifs), can also promote necroptosis in the absence of caspase function. The activation of DR4 or DR5 by TRAIL, Fas by FasL, and TLR4 by LPS has also been shown to promote necroptosis under certain conditions.

Pyroptosis

Caspases were found to mediate a form of programmed necrosis known as pyroptosis99–102. Thus, pyroptosis represents another type of programmed necrotic cell death regulated by dedicated genetic pathways in mammalian cells. The activation of the caspase 1 subfamily, including caspase-1 and caspase-11 in mouse and the corresponding caspase-1, caspase-4 and caspase-5 in human, promotes an inflammatory response in which mature IL-1β is generated by the cleavage of pro-IL-1β as well as other downstream caspases such as caspase-3103–105 (Fig. 3). Activated caspase-1 and caspase-11 can mediate the cleavage of gasdermin D (GSDMD) to remove an autoinhibitory carboxy-terminal domain from GSDMD, enabling the N-terminal fragment to form pores on the plasma membrane and resulting in pyroptosis101,106–108. The pores formed by 16 GSDMD N-domain protomers have an inner diameter of 10–14 nm, which can mediate the release of cytokines without signal peptides such as mature IL-1β in living cells as well as necrosis109,110. Pyroptosis occurs predominantly in professional phagocytic cells, including macrophages, monocytes and dendritic cells, upon stimulation by pathogens, such as bacteria or viruses, or induced by pathogen-derived material such as lipopolysaccharide and viral DNA.Release of pro-inflammatory factors, such as mature IL-1β, through gasdermin pores occurs via a charge-based mechanism independently of cell death111,112. The mechanism that mediates the release of cytokines without a signal peptide, such as mature IL-1β, has long been the subject of studies and debate. In human postmortem pathological samples, apparently live microglia and astrocytes that showed positive staining for cleaved GSDMD were found in close proximity to amyloid-β plaques, suggesting that the formation of pores by the GSDMD N-terminal domain triggers an inflammatory response 113. Of note, both the inflammatory response and cell death are crucial for organismal survival in the presence of severe inflammation. For example, Casp11-null mice are highly resistant to lipopolysaccharide-induced lethality103, which may be attributed to the inhibition of GSDMD pore-mediated release of pro-inflammatory cytokines such as mature IL-1β and IL-18, the inhibition of pyroptosis, or both. Future biomarker-based studies are needed to better understand the mechanism underlying reduced lethality.Other members of the gasdermin family, GSDMA to GSDME and pejvakin, encoded in the human genome can also be cleaved by bacterial proteases or mammalian granzyme A. The bacterial SpeB-mediated cleavage of GSDMA in keratinocytes infected by group A Streptococcus(GAS) promotes pyroptosis114. Mutant mice with triple knockout of the three homologs of human GSDMA are sensitized to hypervirulent GAS strain invasion, suggesting a role of pyroptosis as a host defence mechanism. Granzyme A-mediated activation of GSDMB is part of the bacteriocidic activity of natural killer cells, which is targeted by IpaH7.8 in Shigella115. Interestingly, certain bacteria were found to encode gasdermin homologues with pore-forming activity used in defending against phage infection116, suggesting that gasdermins may be an evolutionarily conserved ancient host defence mechanism.

Dead cell removal by efferocytosis

Elimination of dead cell bodies is important to prevent the release of intracellular content that may function as damage-associated molecular patterns to activate an inflammatory response and possibly lead to autoimmunity117. Efficient removal of apoptotic bodies by a cleansing mechanism, known as efferocytosis, mediated by professional phagocytes such as macrophages and dendritic cells, is important for avoiding such auto-immunogenicity118–120. The caspase-mediated exposure of phosphatidylserine (PtdSer) on the surface of apoptotic cells serves as a recognition motif for efferocytosis. PtdSer is normally maintained in the inner leaflet of the plasma membrane in living cells by flippases, including ATP11A and ATP11C120. PtdSer becomes exposed on the surface of apoptotic cells because of caspase-3-dependent cleavage and inactivation of the flippases as well as simultaneous activation of the phospholipid scramblase XKR8, a homologue of CED-8 in C. elegans121,122. Efferocytosis by macrophages involves the recognition of apoptotic cells by various PtdSer receptors such as TIM4, TAM tyrosine-kinase receptors, MFGE8, GAS6, and Protein S. Similar to what is observed in C. elegans, small G proteins of the Rho and Rab family and their guanine nucleotide exchange factors in mammalian cells are involved in the execution of efferocytosis by regulating the formation of a phagocytic cup and maturation of phagosomes for the degradation of apoptotic bodies118.Consistent with the activation of caspases in pyroptosis, pyroptotic cells also display phosphatidylserine ‘eat-me’ signals on their outer plasma membrane and the dead cell bodies are rapidly removed by phagocytic cells123,124. These data suggest that both apoptotic and pyroptotic cells can be eliminated by phagocytes in vivo. Since necroptosis does not involve the activation of caspases, it is still unclear how necroptotic cell bodies are removed in vivo. Understanding the mechanism of efferocytosis for programmed necrosis, including necroptosis and pyroptosis, is important as the presence of a highly efficient efferocytosis mechanism might lead to the engulfment, and therefore the removal, of dying cells, before cell lysis and the release of intracellular DAMPs can occur.

Fig. 3 | Pyroptosis induction and mature pro-inflammatory cytokine (IL-1β)

release. Pathogenic infection by bacteria and viruses can promote the activation of caspase-1 and caspase-11 in mouse and caspase-1, caspase-4 and caspase-5 in human to mediate the cleavage of gasdermin D (GSDMD) to release the pore-forming N-terminal domain of GSDMD (GSDMD-NT) from its inhibitory C-terminal domain (GSDMD-CT). Activated caspase-8, caspase-3 and caspase-7 as well as pathogenic proteases can also mediate the cleavage of GSDMD. The pore formed by GSDMD-NT can mediate the secretion of mature IL-1β and IL-18 in living cells to promote inflammation and also promotes pyroptosis by cell lysis. LPS, lipopolysaccharides.

Membrane rupture after cell death

Necrosis or failure in the efficient removal of apoptotic cell bodies may lead to the rupture of the plasma membrane in a process involving the membrane protein NINJ1125. In dying cells, NINJ1 proteins form filamentous assemblies with tightly packed fence-like arrays of transmembrane α-helices126. Application of anti-NINJ1 antibodies can limit tissue damage in animal models of liver injury, suggesting that targeting membrane damage may ameliorate pathology in diseases127. NINJ1 may be normally involved in the maintenance of functional synapses as Ninj1-null mice exhibit compulsive grooming-induced hair loss and self-made lesions as well as increased anxiety-like behaviours128.NINJ1 has been implicated in mediating cell–cell adhesion in the peripheral nervous system and central nervous system (CNS) after injury129. The levels of NINJ1 are increased in dorsal root ganglion neurons upon axonal injury and anterogradely transported to the site of injury130. NINJ1 may also be involved in inflammatory responses by promoting the activation of immune cells and transmigration of immune cells across the blood–brain barrier in models of multiple sclerosis and stroke131,132. Further studies are required to understand if the role of NINJ1 in regulating these conditions involves the disruption of cell membranes and cell death133,134. NINJ1-mediated plasma membrane rupture may be involved in pathologies involving a massive amount of cell death following acute neurological and tissue damage, which could ‘overwhelm’ dead cell clearance mechanism. Alternatively, genetic defects in efferocytosis may reduce the efficiency of dead cell removal leading to the accumulation of dead cells with ruptured cell membranes.

Interaction of programmed cell death pathways

While apoptosis, pyroptosis and necroptosis are executed by genetically designated cell death machineries, the evolutionary origins and functions of apoptosis, pyroptosis and necroptosis in organisms are dramatically different. The genes that regulate apoptosis are found in primitive organisms such as nematodes and flies, whereas the genes that regulate necroptosis, such as RIPK1, RIPK3 and MLKL, are only found in a subset of highly evolved vertebrates and mammals such as mouse and human58,135. The embryonic developmental defects seen in Apaf1-null mice, Casp9-null mice as well as Casp3 and Casp7double-null mice demonstrate the functional importance of apoptosis in development22. In contrast, Casp1-null mice and Casp11-null mice are healthy and display resistance to inflammatory apoptosis and pyroptosis and do not release mature IL-1β and IL-18103,107,136. In addition, while Ripk1-null mice die perinatally137, multiple mutant mice lines expressing catalytically inactive RIPK1 kinase are normal69,79,80. Ripk3-null mice and Mlkl-null mice are also normal138. Furthermore, blocking RIPK1 kinase and necroptosis can suppress early embryonic lethality of Casp8-null mice, Fadd-null mice, Abin1-null mice and Tbk1-null mice, suggesting that necroptosis may be involved in the removal of defective developing embryos52,67,139,140. Thus, apoptosis is involved in mediating development whilst necroptosis may have evolved to safeguard normal development by aborting defective embryos in higher vertebrates58.Distinct roles of apoptosis and necroptosis have been found in cancers. Amplification and increased expression of FADD are associated with the progression of many types of cancers, including breast cancer, ovarian cancer and lung cancers141. Conversely, many cancers and cancer cell lines show decreased or loss of RIPK3 expression associated with necroptosis inhibition142–144. These findings indicate that apoptosis supports organismal development, whereas necroptosis functions as a checkpoint to block the progression of diseased states when caspases are dysregulated during embryonic development and in adult life. In particular, the prevalent loss of RIPK3 expression in cancer cells suggests the role of necroptosis as a checkpoint in cancer development.

Autophagy and lysosomal cell death

In the following sections, we discuss well-characterized examples of cell death due to the disruption of homeostatic pro-survival mechanisms that do not involve genetically programmed cell death mechanisms. Cells that die due to disruptions in cellular homeostasis may also exhibit certain hallmarks of apoptosis or necroptosis. However, inhibition of apoptosis or necroptosis per se may not be sufficient to rescue cell survival due to the loss of key cellular homeostatic pro-survival mechanisms under these conditions.The term ‘autophagic cell death’ was first used to describe a form of cell death morphologically distinct from apoptosis in embryonic tissues145. Autophagic cell death has been described during meta morphosis of Drosophila146. However, autophagy is a normal physiological mechanism that delivers cytoplasmic contents to the lysosome for degradation and is important in promoting cell survival under stress147,148. Inhibition of autophagy can lead to necrosis149. Moreover, morphological evidence of autophagy activation is often associated with cell death processes such as necroptosis57. Blocking autophagy has minimal effect on cell death but increases the accumulation of cellular debris associated with necroptosis, which suggests that activation of autophagy in necroptosis promotes the degradation of cellular debris but not cell death. In the nervous system, autophagy is crucial for maintaining cellular homeostasis by promoting the degradation of aggregate-prone proteins and dysfunctional organelles such as damaged mitochondria. Defects in autophagy or lysosomal function are associated with ageing-related neurodegenerative diseases150,151.The lysosome is an important intracellular recycling centre filled with different hydrolases, such as lysosomal cathepsins, that can degrade cellular macromolecules to produce amino acids and other metabolic products for recycling and, thus, the lysosome is vital for cell survival. However, this organelle is also known as the ‘suicide bag’ as the rupture of lysosomal membranes has deadly consequences due to the leakage of lysosomal cathepsins that can lead to lysosomal cell death8,152 (Fig. 4). Lysosomal cell death is executed by cathepsin-mediated cleavage and activation of key regulators of apoptosis, such as caspases and BCL-2 family members, as well as by the direct action of cathepsins on key pro-survival mechanisms153. Gaucher disease, caused by mutations in the GBA gene (encoding glucocerebrosidase (GBA)), is the most common lysosomal storage disease. Conditional loss of GBA in neurons in mice leads to increased expression of RIPK1 and RIPK3 and non-apoptotic cell death, which can be attenuated by Ripk3 knockout154. In addition, lysosomal damage induced by spinal cord injury may mediate the recruitment of RIPK1 to the lysosome and promote the activation of RIPK1 and RIPK3155. Lysosomal permeabilization induced by the accumulation of lipofuscin, the cause for the degeneration of retinal pigment epithelial cells in Stargardt and dry age-related macular degeneration, can lead to atypical necroptosis by activating MLKL156. Furthermore, there is a large group of lysosomal storage diseases that are caused by altered function in this organelle and have severe manifestations in multiple organs, in particular the CNS157,158. A loss of lysosomal homeostatic cell survival mechanism may be a main driver of cell death in these patients.

Entosis

Entosis occurs when one cell engulfs and digests another living cell; thus, it may be considered a form of cellular cannibalism9,159. The engulfment process is triggered by cell detachment (of the cell being engulfed) from the extracellular matrix and involves the contractile activity generated by actin, myosin II, Rho-GTPase and Rho-associated protein kinase ROCK160. The internalized cell is eventually degraded by an autophagic lipidation-mediated lysosomal process, which shares similarities with the degradation of internalized pathogens such Listeria monocytogenes, GAS and Shigella161–164. Activation of AMPK under a glucose starvation condition can enhance the frequency of entosis, suggesting a role for metabolic stress in regulating entosis165. In addition, the activation of DR4 and DR5 by TRAIL can also stimulate entosis driven by the scaffold function of caspase-8 but not its protease cleavage activity. Entosis is found in human cancers, but the role of entosis in cancer needs further investigation166.

Ferroptosis

Ferroptosis is mediated by iron-dependent lipid peroxidation and was originally defined as cell death that can be activated by small molecules such as erastin, an irreversible inhibitor of cystine/glutamate antiporter xCT, or RSL3, a GPX4 inhibitor6,167 (Fig. 5). Ferroptosis differs morphologically from apoptosis or necrosis and does not involve the cellular machinery that mediates apoptosis or necroptosis6. Maintenance of a cellular homeostatic redox balance is critical for cell and animal survival168 (Fig. 5). Antiporter xCT, encoded by the gene SLC7A11, and GPX4 are two key regulators of cellular redox mechanisms that protect cells against lipid peroxidation. xCT is a heterodimeric amino acid antiporter specific for exporting glutamate in exchange for importing cystine169. Reduction of cystine generates cysteine, the rate-limiting precursor in the synthesis of glutathione that protects cells from oxidative stress. GPX4, a selenoprotein with glutathione-dependent peroxidase activity, is the major cellular defence mechanism against lipid peroxidation. Inactivation of GPX4 leads to the production of excessive amounts of reactive oxygen species (ROS), inducing lipid peroxidation, disruption of the plasma membrane and mitochondrial membrane and, consequently, activation of cell death. Inducible inactivation of GPX4 in mice leads to lipid oxidation-induced acute renal failure and animal death170. Lipid peroxidation of poly-unsaturated fatty acids, one of the consequences of increased cellular ROS, generates reactive aldehydes, such as 4-hydroxynonenal (4-HNE), which can form adducts with proteins, lipids and DNA in a cell type-dependent and concentration-dependent manner171–173.Lipid peroxidation can occur as a normal consequence of cell metabolism when oxidants attack lipids, especially poly-unsaturated fatty acids, which can be modulated by cellular redox mechanisms174. Increased lipid peroxidation is observed in living cells during inflammation and in diseased conditions. Ageing may lead to increased production of oxidized products, exceeding the detoxification capacity of a biological system171. Increased levels of 4-HNE-modified proteins detected by a HNEJ-1 monoclonal antibody were found in different organs of ageing mice when compared to young mice, suggesting that ageing is associated with increased lipid peroxidation175. Oxidative stress and lipid peroxidation have long been associated with a variety of ageing-related human diseases, including diabetes and neurodegeneration176,177. However, because we still do not have a specific biomarker for ferroptosis, caution should be exercised to differentiate lipid peroxidation (without cell death) and ferroptosis (with cell death).The levels of lipid peroxidation are modulated by cellular redox mechanisms as a reversible process(Fig. 5). Accumulation of HNE-modified proteins can be reduced by increasing NRF2-mediated antioxidant defence gene expression178. Notably, Freidrich ataxia, caused by a mutation in the gene encoding frataxin leads to alterations in iron handling within mitochondria and impairment of mitochondrial respiration, which can be treated with NRF2 activators179. The production of cellular radical-trapping antioxidants, including vitamin E and vitamin K, can also provide defence against lipid peroxidation and ferroptosis. In addition to GPX4, lipid peroxidation is regulated by coenzyme Q10 oxidoreductase FSP1, which catalyses the regeneration of the reduced form of ubiquinone (ubiquinol) with free radical-trapping antioxidant activity180,181. FSP1 can function to reduce vitamin K to hydroquinone, which has radical-trapping antioxidant activity182. In addition, induction of the phospholipid-modifying enzymes MBOAT1 and MBOAT2 by sex hormone receptors, such as oestrogen receptor and androgen receptor, can also modulate lipid peroxidation183.Excess accumulation of lipid peroxidation may promote inflammation independent of cell death. Iron is a key regulator of cellular homeostasis and survival as it is involved in mitochondrial respiration. The loss of transferrin receptor 1, which is involved in iron uptake in the brain, leads to neuronal iron deficiency and progressive loss of dopaminergic neurons184. Mutations in genes for ferritin light chain (FTL), which has an important function in iron storage, lead to rare Parkinson-like diseases known as neuroferritinopathies due to brain iron overload185. An inflammatory response to iron accumulation in microglial cells can be coupled to ferroptosis186. Thus, inactivating key cellular redox pro-survival mechanisms in defence against lipid peroxidation may include activation of inflammation as a consequence.

Fig. 5 | Lipid peroxidation and ferroptosis.

Cellular mechanisms that protect against lipid peroxidation include (1) xCT (encoded by the gene SLC7A11), which is a heterodimeric amino acid antiporter specific for importing cystine, the oxidized form of cysteine, in 1:1 exchange for exporting glutamate. Reduction of cystine generates cysteine, the rate-limiting precursor in glutathione (GSH) synthesis that protects cells from oxidative stress. (2) GPX4, which protects against lipid peroxidation by converting lipid hydroperoxides into lipid alcohols. (3) Plasma membrane-localized FSP1 (previously known as AIFM2), which functions as an oxidoreductase that reduces coenzyme Q10 (CoQ) to generate a lipophilic radical-trapping antioxidant to block lipid peroxides. Lipid peroxidation of poly-unsaturated fatty acids generates reactive aldehydes, such as 4-hydroxynonenal (4-HNE), which can form relatively stable adducts with protein, lipid and DNA. Electrons may escape from cellular oxidation–reduction reactions and be captured by oxygen to form peroxides (H2O2). The Fenton reaction mediates oxidation by Fe2+ and H2O2, producing hydroxyl radicals to react with lipids to form lipid radicals, which initiate lipid peroxidation. Ferroptosis can be induced by the chemical inhibition of GPX4 by RSL3 or inhibition of xCT by erastin, which defeats cellular defence mechanisms against lipid peroxidation to induce cell death by massive iron-dependent lipid peroxidation. Ferrostatins and liproxstatin 1 inhibit lipid peroxidation by functioning as radical-trapping antioxidants. ROS, reactive oxygen species; TFR1, transferrin receptor 1.

Excitotoxicity

Disruption of intracellular ionic balance after overactivation of specific ion channels can lead to a type of necrosis known as excitotoxicity (Fig. 6). Genetic studies on mechanosensitivity in C. elegans provide an elegant prototypical example of cell death induced by the loss of a cellular homeostatic pro-survival mechanism mediated by overactivation of ion channels187,188. Missense mutations in the genes mec-4and deg-1, which encode proteins similar to subunits of the vertebrate amiloride-sensitive epithelial Na+ channel, lead to ionic imbalance, swelling and necrotic degeneration of touch-receptor neurons in C. elegans. The cell death mediated by mutant MEC-4 and DEG-1 is independent of CED-3 but partially mediated by aspartyl and calpain proteases, which are activated by increased intracellular Ca2+ (refs. 189,190).Glutamate is a key excitatory neurotransmitter in the mammalian nervous system that drives Ca2+ influx into neurons191 (Fig. 6). In a pathological state, altered Ca2+ signalling can result in excessive levels of this second messenger and lead to excitotoxicity from ionic imbalance. The cells are unable to survive with a cellular Ca2+ overload. The downstream cell death effectors of excitotoxicity may include both apoptotic and necroptotic processes, and potentially depend on the magnitude of this homeostatic disruption192,193. The clearance or recycling of glutamate by astrocytes may also determine the availability of glutamate as a neurotransmitter for proper signalling and prevention of neuronal hyperexcitation194. Excessive or persistent activation of glutamate-gated ion channels, such as the NMDA receptor, has been implicated in mediating necrotic cell death in the CNS after acute neuronal injury such as cerebral ischaemia, status epilepticus, traumatic CNS injury and hypoglycaemia176. The cytotoxicity of glutamate may be mediated by the ability of increased Ca2+ influx to promote the activation of neuronal nitric oxide synthase, which is also known to modulate many physiological functions such as synaptic plasticity, learning, memory and neurogenesis195–197. Excitotoxic events in neurons can also alter the oxidative state, which has been shown to drive s-nitrosylation198.

Mitotic catastrophe

Mitotic catastrophe is a type of cell death induced by inappropriate mitotic entry due to chemical or physical stresses199. Biochemically, mitotic catastrophe is characterized by premature condensation of under-replicated or damaged chromosomes with double-stranded DNA breaks at the centromere200. Cancer cells with genetic defects in G2 checkpoint genes, such as ATM, ATR, CHEK1, CHEK2 and polo-like kinase genes (PLK1, PLK2, PLK3), can be induced to undergo mitotic catastrophe by pharmacological agents targeting microtubules, such as taxanes, vinca alkaloids and colchicine, and DNA damaging agents. Cancer cells undergoing mitotic catastrophe may exhibit hallmarks of caspase activation. However, inhibition of caspase activity cannot block defective mitotic chromosome segregation and can thus lead to defective cell division and aneuploidy201.

Cell death in human diseases

In this section, we briefly discuss the involvement of cell death and disruption of cellular homeostasis in human diseases, whereas Box 2discusses the impact of our increased understanding of programmed cell death mechanisms on drug discovery.

Anti-apoptotic BCl-2 family as oncogenes

BCL2 is transcriptionally activated in human follicular lymphomas as a consequence of t(14;18) chromosomal translocation, and overexpression of anti-apoptotic BCL-2 family members is also found in many types of cancer cells13. The pro-oncogenic activity of the anti-apoptotic BCL-2 family functions by prolonging the survival of cancer cells under stressful conditions, rather than promoting cell proliferation11. The critical role of this family in regulating the balance between death and survival is underscored by the successful development of venetoclax (BCL-2-specific inhibitor) and navitoclax (inhibitor of BCL-2, BCL-xL and BCL-W) for the treatment of leukaemias and lymphomas202,203.

Necroptosis in neurodegenerative diseases

Necroptosis has been implicated in mediating the pathogenesis of multiple human diseases characterized by cell death with inflammation, such as ischaemic brain injury, glaucoma, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS)49,66,204. Biomarkers for the activation of RIPK1, RIPK3 and MLKL, including the phosphorylated forms (pRIPK1, pRIPK3 and pMLKL) have been identified in postmortem pathological samples from patients with ALS, MS and Alzheimer disease49,66,205,206. Since RIPK1 is recruited exclusively to TNFR1 via DD-mediated interaction, the activation of RIPK1 specifically mediates TNFR1 signalling. Thus, inhibition of RIPK1 presents an opportunity to selectively inhibit the deleterious responses mediated by TNFR1 without affecting TNFR2.

Inflammation mediated by RIPK1 and caspases

The two main pro-inflammatory pathways include RIPK1-mediated pro-inflammatory cytokine production downstream of TNFR1 and inflammasome-mediated caspase-1 activation. While the upstream activators of these two pathways may show both stimulus and cell specificity, the production and release of downstream cytokines often include a common set of pro-inflammatory factors, such as TNF, IL-1β, IL-6 and IFNγ, although the difference in the levels, timing and tissue specificity of such release may be crucial to determine the consequences on the organism. Inactivation of caspase-8-mediated cleavage of RIPK1 leads to autoinflammatory disease in humans71,72. Activated RIPK1 has been detected in the nucleus under necroptotic and inflammatory conditions such as in ALS207. Nuclear RIPK1 interacts with a striking array of transcriptional activators and coactivators, such as p65 (also known as RELA), SP1 and JUNB, as well as almost all components of the BAF chromatin remodelling complex, to promote a transcriptional induction of pro-inflammatory factors by regulating chromatin dynamics208.Activation of caspase-1, caspase-11 and the related caspases is mediated by multi-molecular complexes, known as inflammasomes, including different PRRs such as NLRP3, NLRP1, NAIPs, NLRC4, AIM2, Pyrin and adaptor proteins like ASC. The downstream signalling of these complexes is determined in both a PRR-dependent and stimulus-dependent manner209. The NLRP3 inflammasome has recently been shown to be activated in the context of neurodegenerative diseases, leading to both inflammation and degeneration210. The cleavage of GSDMD regulates the release of mature IL-1β and IL-18 to promote inflammation as a key pro-inflammatory mechanism in human inflammatory conditions111.

Disruption of homeostasis in diseases

Disruption of protein homeostasis, the proper balance between protein synthesis and degradation, leads to the accumulation of misfolded and aggregated proteins in neurodegenerative diseases211. The restoration of cellular homeostasis is an important mechanism to consider, particularly for neurodegenerative diseases151. Targeting TRADD, an important adaptor in the TNFR1 signalling pathway, presents an opportunity to inhibit RIPK1-dependent apoptosis and to activate autophagy to restore cellular homeostasis by promoting the degradation of misfolded proteins212.Disruption of redox homeostasis leads to the accumulation of ROS, which is common in patients with chronic inflammatory diseases213. In addition, the accumulation of lipid peroxidation, as indicated by the elevated levels of 4-HNE, can lead to cellular damage and ferroptosis. Owing to its consumption of a disproportionally high amount of oxygen (~20%) in relation to only accounting for ~2% of body weight, the brain is highly sensitive to oxidative damage. Lipid peroxidation is an early pathological event in the brain of patients with amnestic mild cognitive impairment and in early-stage Alzheimer disease214. Oxidative stress and lipid peroxidation may be involved in promoting the formation of amyloid-β plaques in animal models of Alzheimer disease215.

Conclusions and perspectives

Genetic studies of programmed cell death in the nematode C. elegansprovided the first insights into the molecular mechanisms (mediated by EGL-1, CED-9, CED-3 and CED-4) that control developmental apoptotic cell death216. This evolutionarily conserved cell death mechanism has been significantly expanded in mammalian cells, presumably through gene duplications, to include genes that encode multiple homologues of CED-3 (caspase family) and CED-9 (BCL-2 family) that mediate not only developmental cell death but also disease-associated cell death and inflammation. Activation of necroptosis, mediated by RIPK1, RIPK3, MLKL and ZBP1, may have been added late in evolution because of additional demands to eliminate cells in complex multi-cellular organisms and during defence against pathogens. In addition, the cleavage of GSDMDs by caspases, including caspase-11, caspase-1, caspase-3 and caspase-8, can promote pyroptosis, a form of necrosis, that can also mediate the release of cytokines without a signal peptide, such as mature IL-1β, to drive inflammation under pathological conditions.The consequences of activating specific cell death and inflammation pathways in vivo may differ depending on cell type and on the expression levels of different mediators of these pathways. For example, activation of RIPK1 has been associated with increased production of pro-inflammatory cytokines rather than cell death in microglia, whereas activation of RIPK1 in oligodendrocytes may result in cell death and dysmyelination due to the loss of mature oligodendrocytes and lead to compensatory regeneration of new oligodendrocytes49,66,205. Furthermore, the activation of caspase-1 and caspase-11 can promote the activation of downstream caspases (caspase-3 and caspase-7) as their specificities are similar to other long-prodomain caspases such as caspase-9104,217. The observation that caspase-1 and caspase-11 as well as caspase-8 and caspase-3 can cleave both apoptotic substrates and pyroptotic substrates is consistent with their cleavage specificities. However, whether the activation of caspases leads to apoptosis or necrosis may depend upon the expression levels of key caspase substrates, among the hundreds that are cleaved, in the dying cells.The involvement of diverse cellular homeostatic mechanisms in mediating cell survival explains the complexity and increasing number of different cell death modalities being discovered. Since inactivation of cellular homeostatic pro-survival mechanisms may provide a wide variety of options to selectively induce the death of cancer cells, targeting selective cancer-preferred pro-survival mechanisms, by chemical or genetic manipulation, may provide exciting new options for the treatment of cancers in the future. Moreover, while many strategies for therapeutic intervention have focused on blocking aberrant activation of pro-death pathways, the possibility of enhancing specific pro-survival mechanisms for the treatment of neurodegenerative diseases should be further explored. Identifying which cell survival signals are disrupted and developing specific therapeutic strategies to restore them may enable cells, for example, neurons or pancreatic β-cells, to survive in the context of a deleterious milieu under specific disease conditions. Thus, understanding the signalling pathways that connect cellular homeostasis and cell death may enable the development of new therapeutic avenues that inhibit cell death and restore cell homeostasis and survival.

Published online: 18 December 2023

References

1. Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57–70 (2000).

2. Yuan, J., Lipinski, M. & Degterev, A. Diversity in the mechanisms of neuronal cell death. Neuron 40, 401–413 (2003).

3. Lockshin, R. A. & Williams, C. M. Programmed cell death–I. Cytology of degeneration in the intersegmental muscles of the pernyi silkmoth. J. Insect Physiol. 11, 123–133 (1965).

4. Lockshin, R. A. Programmed cell death 50 (and beyond). Cell Death Difer. 23, 10–17 (2016).

5. Choi, D. W. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1, 623–634 (1988).

6. Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell149, 1060–1072 (2012).

7. Stockwell, B. R. Ferroptosis turns 10: emerging mechanisms, physiological functions, and therapeutic applications. Cell 185, 2401–2421 (2022).

8. Aits, S. & Jaattela, M. Lysosomal cell death at a glance. J. Cell Sci. 126, 1905–1912 (2013).

9. Overholtzer, M. et al. A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion. Cell 131, 966–979 (2007).

10. Frisch, S. M. & Francis, H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol. 124, 619–626 (1994).

11. Vaux, D. L., Cory, S. & Adams, J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335, 440–442 (1988).

12. Kelekar, A. & Thompson, C. B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol. 8, 324–330 (1998).

13. Cory, S. & Adams, J. M. The Bcl2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer 2, 647–656 (2002).

14. Green, D. R. The mitochondrial pathway of apoptosis part II: the BCL-2 protein family. Cold Spring Harb. Perspect. Biol. 14, a041046 (2022).

15. Motoyama, N. et al. Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science 267, 1506–1510 (1995).

16. Rinkenberger, J. L., Horning, S., Klocke, B., Roth, K. & Korsmeyer, S. J. Mcl-1 deficiency results in peri-implantation embryonic lethality. Genes Dev. 14, 23–27 (2000).

17. Veis, D. J., Sorenson, C. M., Shutter, J. R. & Korsmeyer, S. J. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75, 229–240 (1993).

18. Zou, H., Henzel, W. J., Liu, X., Lutschg, A. & Wang, X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413 (1997).

19. Yuan, S. et al. The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3. Structure 19, 1084–1096 (2011).

20. Li, Y. et al. Mechanistic insights into caspase-9 activation by the structure of the apoptosome holoenzyme. Proc. Natl Acad. Sci. USA 114, 1542–1547 (2017).

21. Julien, O. & Wells, J. A. Caspases and their substrates. Cell Death Difer.24, 1380–1389 (2017).

22. Lakhani, S. A. et al. Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311, 847–851 (2006).

23. Lindsten, T. et al. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol. Cell 6, 1389–1399 (2000).

24. Lindsten, T. & Thompson, C. B. Cell death in the absence of Bax and Bak. Cell Death Difer.13, 1272–1276 (2006).

25. Krammer, P. H. CD95’s deadly mission in the immune system. Nature 407, 789–795 (2000).

26. Nagata, S. Apoptosis by death factor. Cell 88, 355–365 (1997).

27. Fisher, G. H. et al. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81, 935–946 (1995).

28. Rieux-Laucat, F. et al. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268, 1347–1349 (1995).

29. Takahashi, T. et al. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76, 969–976 (1994).

30. Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A. & Nagata, S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356, 314–317 (1992).

31. Martin, D. A. et al. Defective CD95/APO-1/Fas signal complex formation in the human autoimmune lymphoproliferative syndrome, type Ia. Proc. Natl Acad. Sci. USA 96, 4552–4557 (1999).

32. Micheau, O. & Tschopp, J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181–190 (2003).

33. Hsu, H., Huang, J., Shu, H. B., Baichwal, V. & Goeddel, D. V. TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4, 387–396 (1996).

34. Bertrand, M. J. et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol. Cell 30, 689–700 (2008).

35. Mahoney, D. J. et al. Both cIAP1 and cIAP2 regulate TNFα-mediated NF-κB activation. Proc. Natl Acad. Sci. USA 105, 11778–11783 (2008).

36. Varfolomeev, E. et al. c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor α (TNFα)-induced NF-κB activation. J. Biol. Chem. 283, 24295–24299 (2008).

37. Haas, T. L. et al. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Mol. Cell 36, 831–844 (2009).

38. Draber, P. et al. LUBAC-recruited CYLD and A20 regulate gene activation and cell death by exerting opposing efects on linear ubiquitin in signaling complexes. Cell Rep. 13, 2258–2272 (2015).

39. Tokunaga, F. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nat. Cell Biol. 11, 123–132 (2009).

40. Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).

41. Ea, C. K., Deng, L., Xia, Z. P., Pineda, G. & Chen, Z. J. Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006).

42. Geng, J. et al. Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis. Nat. Commun. 8, 359 (2017).

43. Daniel, S. et al. A20 protects endothelial cells from TNF-, Fas-, and NK-mediated cell death by inhibiting caspase 8 activation. Blood 104, 2376–2384 (2004).

44. Wertz, I. E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004).

45. He, K. L. & Ting, A. T. A20 inhibits tumor necrosis factor (TNF) alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in Jurkat T cells. Mol. Cell Biol. 22, 6034–6045 (2002).

46. Micheau, O., Lens, S., Gaide, O., Alevizopoulos, K. & Tschopp, J. NF-κB signals induce the expression of c-FLIP. Mol. Cell Biol. 21, 5299–5305 (2001).

47. Kataoka, T. The caspase-8 modulator c-FLIP. Crit. Rev. Immunol. 25, 31–58 (2005).

48. Degterev, A. et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat. Chem. Biol. 4, 313–321 (2008).

49. Ofengeim, D. et al. Activation of necroptosis in multiple sclerosis. Cell Rep. 10, 1836–1849 (2015).

50. Newton, K. et al. RIPK1 inhibits ZBP1-driven necroptosis during development. Nature 540, 129–133 (2016).

51. Zhang, X. et al. Ubiquitination of RIPK1 suppresses programmed cell death by regulating RIPK1 kinase activation during embryogenesis. Nat. Commun. 10, 4158 (2019).

52. Xu, D. et al. TBK1 suppresses RIPK1-driven apoptosis and inflammation during development and in aging. Cell 174, 1477–1491.e19 (2018).

53. Dondelinger, Y. et al. NF-κB-independent role of IKKα/IKKβ in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling. Mol. Cell60, 63–76 (2015).

54. Gerlach, B. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011).

55. Wang, L., Du, F. & Wang, X. TNF-α induces two distinct caspase-8 activation pathways. Cell 133, 693–703 (2008).

56. Jaco, I. et al. MK2 phosphorylates RIPK1 to prevent TNF-induced cell death. Mol. Cell 66, 698–710.e5 (2017).

57. Degterev, A. et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat. Chem. Biol. 1, 112–119 (2005). Refs 48 and 57 provided the first evidence for the existence of necroptosis and the role of RIPK1 in mediating necroptosis by isolating Nec1, which was the first small-molecule RIPK1 inhibitor.

58. Shan, B., Pan, H., Najafov, A. & Yuan, J. Necroptosis in development and diseases. Genes Dev. 32, 327–340 (2018).

59. 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).

60. He, S. et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α. Cell 137, 1100–1111 (2009).

61. Zhang, D. W. et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325, 332–336 (2009).

62. Wu, J. et al. Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis. Cell Res. 23, 994–1006 (2013).

63. Degterev, A., Maki, J. L. & Yuan, J. Activity and specificity of necrostatin-1, small-molecule inhibitor of RIP1 kinase. Cell Death Difer. 20, 366 (2013).

64. Peltzer, N., Darding, M. & Walczak, H. Holding RIPK1 on the ubiquitin leash in TNFR1 signaling. Trends Cell Biol. 26, 445–461 (2016).

65. Li, X. et al. Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners. Nat. Commun. 11, 6364 (2020).

66. Ito, Y. et al. RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS. Science 353, 603–608 (2016).

67. Dziedzic, S. A. et al. ABIN-1 regulates RIPK1 activation by linking Met1 ubiquitylation with Lys63 deubiquitylation in TNF-RSC. Nat. Cell Biol. 20, 58–68 (2018).

68. Vlantis, K. et al. NEMO prevents RIP kinase 1-mediated epithelial cell death and chronic intestinal inflammation by NF-κB-dependent and -independent functions. Immunity 44, 553–567 (2016).

69. Meng, H. et al. Death-domain dimerization-mediated activation of RIPK1 controls necroptosis and RIPK1-dependent apoptosis. Proc. Natl Acad. Sci. USA 115, E2001–E2009 (2018).

70. Lin, Y., Devin, A., Rodriguez, Y. & Liu, Z. G. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 13, 2514–2526 (1999).

71. Tao, P. et al. A dominant autoinflammatory disease caused by non-cleavable variants of RIPK1. Nature 577, 109–114 (2020).

72. Lalaoui, N. et al. Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease. Nature 577, 103–108 (2020).

73. Li, J. et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell 150, 339–350 (2012).

74. Wu, X. et al. The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3. Proc. Natl Acad. Sci. USA 118, e2022933118 (2021).

75. Chen, W. et al. Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J. Biol. Chem. 288, 16247–16261 (2013).

76. Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012). Refs 59, 60, 61 and 76 provided evidence for the roles of RIPK3 and MLKL in mediating necroptosis.

77. Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39, 443–453 (2013).

78. Hildebrand, J. M. et al. Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc. Natl Acad. Sci. USA 111, 15072–15077 (2014).

79. Polykratis, A. et al. Cutting edge: RIPK1 kinase inactive mice are viable and protected from TNF-induced necroptosis in vivo. J. Immunol. 193, 1539–1543 (2014).

80. Berger, S. B. et al. Cutting edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice. J. Immunol. 192, 5476–5480 (2014).

81. Laurien, L. et al. Autophosphorylation at serine 166 regulates RIP kinase 1-mediated cell death and inflammation. Nat. Commun. 11, 1747 (2020).

82. Blanchett, S., Dondelinger, Y., Barbarulo, A., Bertrand, M. J. M. & Seddon, B. Phosphorylation of RIPK1 serine 25 mediates IKK dependent control of extrinsic cell death in T cells. Front. Immunol. 13, 1067164 (2022).

83. Dondelinger, Y. et al. Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation. Nat. Commun. 10, 1729 (2019).

84. Zelic, M. et al. RIP kinase 1-dependent endothelial necroptosis underlies systemic inflammatory response syndrome. J. Clin. Invest. 128, 2064–2075 (2018).

85. Zhang, T. et al. Metabolic orchestration of cell death by AMPK-mediated phosphorylation of RIPK1. Science 380, 1372–1380 (2023).

86. Sun, W. et al. Small molecule activators of TAK1 promotes its activity-dependent ubiquitination and TRAIL-mediated tumor cell death. Proc. Natl Acad. Sci. USA 120, e2308079120 (2023).

87. Kang, K., Park, C. & Chan, F. K. Necroptosis at a glance. J. Cell Sci. 135, jcs260091 (2022).

88. Balachandran, S. & Mocarski, E. S. Viral Z-RNA triggers ZBP1-dependent cell death. Curr. Opin. Virol. 51, 134–140 (2021).

89. He, S., Liang, Y., Shao, F. & Wang, X. Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. Proc. Natl Acad. Sci. USA 108, 20054–20059 (2011).

90. Riebeling, T., Kunzendorf, U. & Krautwald, S. The role of RHIM in necroptosis. Biochem. Soc. Trans. 50, 1197–1205 (2022).

91. Jiao, H. et al. Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation. Nature 580, 391–395 (2020).

92. Ingram, J. P. et al. ZBP1/DAI drives RIPK3-mediated cell death induced by IFNs in the absence of RIPK1. J. Immunol. 203, 1348–1355 (2019).

93. Zhang, T. et al. Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation. Nat. Cell Biol. 25, 950–962 (2023).

94. Hardie, D. G. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat. Rev. Mol. Cell Biol. 8, 774–785 (2007).

95. Ohh, M. et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2, 423–427 (2000).

96. Naito, M. G. et al. Sequential activation of necroptosis and apoptosis cooperates to mediate vascular and neural pathology in stroke. Proc. Natl Acad. Sci. USA 117, 4959–4970 (2020).

97. Linkermann, A. et al. Necroptosis in immunity and ischemia-reperfusion injury. Am. J. Transpl. 13, 2797–2804 (2013).

98. Jouan-Lanhouet, S. et al. Necroptosis, in vivo detection in experimental disease models. Semin. Cell Dev. Biol. 35, 2–13 (2014).

99. Degterev, A., Boyce, M. & Yuan, J. A decade of caspases. Oncogene 22, 8543–8567 (2003).

100. Yuan, J., Najafov, A. & Py, B. F. Roles of caspases in necrotic cell death. Cell 167, 1693–1704 (2016).

101. Newton, K., Dixit, V. M. & Kayagaki, N. Dying cells fan the flames of inflammation. Science374, 1076–1080 (2021).

102. Rathinam, V. A. K., Zhao, Y. & Shao, F. Innate immunity to intracellular LPS. Nat. Immunol.20, 527–533 (2019).

103. Wang, S. et al. Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 92, 501–509 (1998).

104. Kang, S. J. et al. Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under pathological conditions. J. Cell Biol. 149, 613–622 (2000).

105. de Zoete, M. R., Palm, N. W., Zhu, S. & Flavell, R. A. Inflammasomes. Cold Spring Harb. Perspect. Biol. 6, a016287 (2014).

106. Broz, P., Pelegrin, P. & Shao, F. The gasdermins, a protein family executing cell death and inflammation. Nat. Rev. Immunol. 20, 143–157 (2020).

107. Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015).

108. Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).

109. Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111–116 (2016).

110. Liu, X. et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535, 153–158 (2016). Refs 107, 108, 109 and 110 demonstrated the role of caspase-1 and caspase-11 in the cleavage of GSDMD to promote necroptosis and the structural basis of GSDMD-NT pore formation.

111. Xia, S. et al. Gasdermin D pore structure reveals preferential release of mature interleukin-1. Nature 593, 607–611 (2021).

112. He, W. T. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res. 25, 1285–1298 (2015).

113. Moonen, S. et al. Pyroptosis in Alzheimer’s disease: cell type-specific activation in microglia, astrocytes and neurons. Acta Neuropathol. 145, 175–195 (2023).

114. LaRock, D. L. et al. Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes. Nature 605, 527–531 (2022).

115. Hansen, J. M. et al. Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions. Cell 184, 3178–3191.e18 (2021).

116. Johnson, A. G. et al. Bacterial gasdermins reveal an ancient mechanism of cell death. Science 375, 221–225 (2022).

117. Sangiuliano, B., Perez, N. M., Moreira, D. F. & Belizario, J. E. Cell death-associated molecular-pattern molecules: inflammatory signaling and control. Mediators Inflamm.2014, 821043 (2014).

118. Nagata, S. Apoptosis and clearance of apoptotic cells. Annu. Rev. Immunol. 36, 489–517 (2018).

119. deCathelineau, A. M. & Henson, P. M. The final step in programmed cell death: phagocytes carry apoptotic cells to the grave. Essays Biochem. 39, 105–117 (2003).

120. Nagata, S. & Segawa, K. Sensing and clearance of apoptotic cells. Curr. Opin. Immunol.68, 1–8 (2021).

121. Segawa, K. et al. Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure. Science 344, 1164–1168 (2014).

122. Suzuki, J., Denning, D. P., Imanishi, E., Horvitz, H. R. & Nagata, S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science 341, 403–406 (2013). Refs 121 and 122 described the mechanism by which caspase mediates the cleavage of phospholipid flippase to promote phosphatidylserine exposure on apoptotic cells in eferocytosis.

123. Lu, J. et al. Eficient engulfment of necroptotic and pyroptotic cells by nonprofessional and professional phagocytes. Cell Discov. 5, 39 (2019).

124. Zargarian, S. et al. Phosphatidylserine externalization, “necroptotic bodies” release, and phagocytosis during necroptosis. PLoS Biol. 15, e2002711 (2017).

125. Kayagaki, N. et al. NINJ1 mediates plasma membrane rupture during lytic cell death. Nature 591, 131–136 (2021).

126. Degen, M. et al. Structural basis of NINJ1-mediated plasma membrane rupture in cell death. Nature 618, 1065–1071 (2023). Refs 125 and 126 report on the role of NINJ1 in mediating membrane disrupture after cell death and the structure of NINJ1.

127. Kayagaki, N. et al. Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury. Nature 618, 1072–1077 (2023).

128. Le, H. et al. Disruption of ninjurin1 leads to repetitive and anxiety-like behaviors in mice. Mol. Neurobiol. 54, 7353–7368 (2017).

129. Liu, K., Wang, Y. & Li, H. The role of ninjurin1 and its impact beyond the nervous system. Dev. Neurosci. 42, 159–169 (2020).

130. Tomita, Y. et al. Ninjurin 1 mediates peripheral nerve regeneration through Schwann cell maturation of NG2-positive cells. Biochem. Biophys. Res. Commun. 519, 462–468 (2019).

131. Ifergan, I. et al. Role of Ninjurin-1 in the migration of myeloid cells to central nervous system inflammatory lesions. Ann. Neurol. 70, 751–763 (2011).

132. Lee, H. J., Ahn, B. J., Shin, M. W., Choi, J. H. & Kim, K. W. Ninjurin1: a potential adhesion molecule and its role in inflammation and tissue remodeling. Mol. Cell 29, 223–227 (2010).

133. Lee, H. K., Lee, H., Luo, L. & Lee, J. K. Induction of nerve injury-induced protein 1 (ninjurin 1) in myeloid cells in rat brain after transient focal cerebral ischemia. Exp. Neurobiol. 25, 64–74 (2016).

134. Ahn, B. J. et al. Ninjurin1 enhances the basal motility and transendothelial migration of immune cells by inducing protrusive membrane dynamics. J. Biol. Chem. 289, 21926–21936 (2014).

135. Dondelinger, Y., Hulpiau, P., Saeys, Y., Bertrand, M. J. M. & Vandenabeele, P. An evolutionary perspective on the necroptotic pathway. Trends Cell Biol. 26, 721–732 (2016).

136. Kuida, K. et al. Altered cytokine export and apoptosis in mice deficient in interleukin-1β converting enzyme. Science 267, 2000–2003 (1995).

137. Kelliher, M. A. et al. The death domain kinase RIP mediates the TNF-induced NF-κB signal. Immunity 8, 297–303 (1998).

138. Newton, K. et al. RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury. Cell Death Difer. 23, 1565–1576 (2016).

139. Kaiser, W. J. et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 471, 368–372 (2011).

140. Oberst, A. et al. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 471, 363–367 (2011).

141. Liu, Y., Li, X., Zhou, X., Wang, J. & Ao, X. FADD as a key molecular player in cancer progression. Mol. Med. 28, 132 (2022).142. Najafov, A., Chen, H. & Yuan, J. Necroptosis and cancer. Trends Cancer 3, 294–301 (2017).

143. Koo, G. B. et al. Methylation-dependent loss of RIP3 expression in cancer represses programmed necrosis in response to chemotherapeutics. Cell Res. 25, 707–725 (2015).

144. Najafov, A. et al. BRAF and AXL oncogenes drive RIPK3 expression loss in cancer. PLoS Biol. 16, e2005756 (2018).

145. Schweichel, J. U. & Merker, H. J. The morphology of various types of cell death in prenatal tissues. Teratology 7, 253–266 (1973).

146. Anding, A. L. & Baehrecke, E. H. Autophagy in cell life and cell death. Curr. Top. Dev. Biol.114, 67–91 (2015).

147. Levine, B. & Yuan, J. Autophagy in cell death: an innocent convict? J. Clin. Invest. 115, 2679–2688 (2005).

148. Tsukada, M. & Ohsumi, Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 333, 169–174 (1993).

149. White, E. Autophagic cell death unraveled: pharmacological inhibition of apoptosis and autophagy enables necrosis. Autophagy 4, 399–401 (2008).

150. Fleming, A. et al. The diferent autophagy degradation pathways and neurodegeneration. Neuron 110, 935–966 (2022).

151. Menzies, F. M. et al. Autophagy and neurodegeneration: pathogenic mechanisms and therapeutic opportunities. Neuron 93, 1015–1034 (2017).

152. de Duve, C. et al. Commentary. Lysosomotropic agents. Biochem. Pharmacol. 23, 2495–2531 (1974).

153. Xie, Z. et al. Cathepsin B in programmed cell death machinery: mechanisms of execution and regulatory pathways. Cell Death Dis. 14, 255 (2023).

154. Vitner, E. B. et al. RIPK3 as a potential therapeutic target for Gaucher’s disease. Nat. Med.20, 204–208 (2014).

155. Liu, S. et al. Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis. Cell Death Dis. 9, 476 (2018).

156. Pan, C. et al. Lipofuscin causes atypical necroptosis through lysosomal membrane permeabilization. Proc. Natl Acad. Sci. USA 118, e2100122118 (2021).

157. Ryckman, A. E., Brockhausen, I. & Walia, J. S. Metabolism of glycosphingolipids and their role in the pathophysiology of lysosomal storage disorders. Int. J. Mol. Sci. 21, 6881 (2020).

158. Yanez, M. J. et al. Finding pathogenic commonalities between Niemann-Pick type C and other lysosomal storage disorders: opportunities for shared therapeutic interventions. Biochim. Biophys. Acta Mol. Basis Dis. 1866, 165875 (2020).

159. White, E. Entosis: it’s a cell-eat-cell world. Cell 131, 840–842 (2007).

160. Zeng, C., Zeng, B., Dong, C., Liu, J. & Xing, F. Rho-ROCK signaling mediates entotic cell death in tumor. Cell Death Discov. 6, 4 (2020).

161. Florey, O., Kim, S. E., Sandoval, C. P., Haynes, C. M. & Overholtzer, M. Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes. Nat. Cell Biol. 13, 1335–1343 (2011).

162. Rich, K. A., Burkett, C. & Webster, P. Cytoplasmic bacteria can be targets for autophagy. Cell Microbiol. 5, 455–468 (2003).

163. Nakagawa, I. et al. Autophagy defends cells against invading group A Streptococcus. Science 306, 1037–1040 (2004).

164. Ogawa, M. et al. Escape of intracellular Shigella from autophagy. Science 307, 727–731 (2005).

165. Hamann, J. C. et al. Entosis is induced by glucose starvation. Cell Rep. 20, 201–210 (2017).

166. Bozkurt, E. et al. TRAIL signaling promotes entosis in colorectal cancer. J. Cell Biol. 220, e202010030 (2021).

167. Yang, W. S. & Stockwell, B. R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 15, 234–245 (2008).

168. Trachootham, D., Lu, W., Ogasawara, M. A., Nilsa, R. D. & Huang, P. Redox regulation of cell survival. Antioxid. Redox Signal. 10, 1343–1374 (2008).

169. Sato, M. et al. The ferroptosis inducer erastin irreversibly inhibits system xc- and synergizes with cisplatin to increase cisplatin’s cytotoxicity in cancer cells. Sci. Rep. 8, 968 (2018).

170. Friedmann Angeli, J. P. et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat. Cell Biol. 16, 1180–1191 (2014).

171. Zhang, H., Morgan, T. E. & Forman, H. J. Age-related alteration in HNE elimination enzymes. Arch. Biochem. Biophys. 699, 108749 (2021).

172. Vazdar, K., Skulj, S., Bakaric, D., Margetic, D. & Vazdar, M. Chemistry and reactivity of 4-hydroxy-2-nonenal (HNE) in model biological systems. Mini Rev. Med. Chem. 21, 1394–1405 (2021).

173. Gentile, F. et al. DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity. AIMS Genet. 4, 103–137 (2017).

174. Ayala, A., Munoz, M. F. & Arguelles, S. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid. Med. Cell. Longev. 2014, 360438 (2014).

175. Zheng, H., Jiang, L., Tsuduki, T., Conrad, M. & Toyokuni, S. Embryonal erythropoiesis and aging exploit ferroptosis. Redox Biol. 48, 102175 (2021).

176. Coyle, J. T. & Puttfarcken, P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 262, 689–695 (1993).

177. Dmitriev, L. F. & Titov, V. N. Lipid peroxidation in relation to ageing and the role of endogenous aldehydes in diabetes and other age-related diseases. Ageing Res. Rev. 9, 200–210 (2010).

178. Luczaj, W., Gegotek, A. & Skrzydlewska, E. Antioxidants and HNE in redox homeostasis. Free Radic. Biol. Med. 111, 87–101 (2017).

179. Lynch, D. R. & Johnson, J. Omaveloxolone: potential new agent for Friedreich ataxia. Neurodegener. Dis. Manag. 11, 91–98 (2021).

180. Doll, S. et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature 575, 693–698 (2019).

181. Bersuker, K. et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 575, 688–692 (2019).

182. Mishima, E. et al. A non-canonical vitamin K cycle is a potent ferroptosis suppressor. Nature 608, 778–783 (2022).

183. Liang, D. et al. Ferroptosis surveillance independent of GPX4 and diferentially regulated by sex hormones. Cell 186, 2748–2764.e22 (2023). Refs 6, 181, 182 and 183 describe the roles of xCT, GPX4, FSP1 and sex hormonemediated control of cellular lipid peroxidation and illustrate how inactivation of cellular defence mechanisms against lipid peroxidation can lead to ferroptosis.

184. Matak, P. et al. Disrupted iron homeostasis causes dopaminergic neurodegeneration in mice. Proc. Natl Acad. Sci. USA 113, 3428–3435 (2016).

185. Cozzi, A. et al. Oxidative stress and cell death in cells expressing L-ferritin variants causing neuroferritinopathy. Neurobiol. Dis. 37, 77–85 (2010).

186. Ryan, S. K. et al. Microglia ferroptosis is regulated by SEC24B and contributes to neurodegeneration. Nat. Neurosci. 26, 12–26 (2023).

187. Chalfie, M. & Wolinsky, E. The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans. Nature 345, 410–416 (1990).

188. Driscoll, M. & Chalfie, M. The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration. Nature 349, 588–593 (1991).

189. Syntichaki, P., Xu, K., Driscoll, M. & Tavernarakis, N. Specific aspartyl and calpain proteases are required for neurodegeneration in C. elegans. Nature 419, 939–944 (2002).

190. Bianchi, L. et al. The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation. Nat. Neurosci. 7, 1337–1344 (2004).

191. Hardingham, G. E., Fukunaga, Y. & Bading, H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-of and cell death pathways. Nat. Neurosci. 5, 405–414 (2002).

192. Hernandez, D. E. et al. Axonal degeneration induced by glutamate excitotoxicity is mediated by necroptosis. J. Cell Sci. 131, jcs214684 (2018).

193. D’Orsi, B. et al. Bax regulates neuronal Ca2+ homeostasis. J. Neurosci. 35, 1706–1722 (2015).

194. Mahmoud, S., Gharagozloo, M., Simard, C. & Gris, D. Astrocytes maintain glutamate homeostasis in the CNS by controlling the balance between glutamate uptake and release. Cells 8, 184 (2019).

195. Dawson, T. M. & Dawson, V. L. Nitric oxide signaling in neurodegeneration and cell death. Adv. Pharmacol. 82, 57–83 (2018).

196. Wang, Y. & Golledge, J. Neuronal nitric oxide synthase and sympathetic nerve activity in neurovascular and metabolic systems. Curr. Neurovasc. Res. 10, 81–89 (2013).

197. Steinert, J. R., Chernova, T. & Forsythe, I. D. Nitric oxide signaling in brain function, dysfunction, and dementia. Neuroscientist 16, 435–452 (2010).

198. Ghatak, S., Nakamura, T. & Lipton, S. A. Aberrant protein S-nitrosylation contributes to hyperexcitability-induced synaptic damage in Alzheimer’s disease: mechanistic insights and potential therapies. Front. Neural Circuits 17, 1099467 (2023).

199. Vakifahmetoglu, H., Olsson, M. & Zhivotovsky, B. Death through a tragedy: mitotic catastrophe. Cell Death Difer. 15, 1153–1162 (2008).

200. Castedo, M. et al. Cell death by mitotic catastrophe: a molecular definition. Oncogene23, 2825–2837 (2004).

201. Castedo, M. et al. Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy. Oncogene 23, 4362–4370 (2004).

202. Brinkmann, K., Ng, A. P., de Graaf, C. A. & Strasser, A. What can we learn from mice lacking pro-survival BCL-2 proteins to advance BH3 mimetic drugs for cancer therapy? Cell Death Difer. 29, 1079–1093 (2022).

203. Smith, W. M. & Reed, D. R. Targeting apoptosis in ALL. Curr. Hematol. Malig. Rep. 17, 53–60 (2022).

204. Miflin, L., Ofengeim, D. & Yuan, J. Receptor-interacting protein kinase 1 (RIPK1) as a therapeutic target. Nat. Rev. Drug Discov. 19, 553–571 (2020).

205. Ofengeim, D. et al. RIPK1 mediates a disease-associated microglial response in Alzheimer’s disease. Proc. Natl Acad. Sci. USA 114, E8788–E8797 (2017).

206. Zelic, M. et al. RIPK1 activation mediates neuroinflammation and disease progression in multiple sclerosis. Cell Rep. 35, 109112 (2021).

207. Li, W. et al. Nuclear RIPK1 promotes chromatin remodeling to mediate inflammatory response. Cell Res. 32, 621–637 (2022).

208. Li, W. & Yuan, J. Targeting RIPK1 kinase for modulating inflammation in human diseases. Front. Immunol. 14, 1159743 (2023).

209. Rathinam, V. A. & Fitzgerald, K. A. Inflammasome complexes: emerging mechanisms and efector functions. Cell 165, 792–800 (2016).

210. Zhan, X., Li, Q., Xu, G., Xiao, X. & Bai, Z. The mechanism of NLRP3 inflammasome activation and its pharmacological inhibitors. Front. Immunol. 13, 1109938 (2022).

211. Sweeney, P. et al. Protein misfolding in neurodegenerative diseases: implications and strategies. Transl. Neurodegener. 6, 6 (2017).

212. Xu, D. et al. Modulating TRADD to restore cellular homeostasis and inhibit apoptosis. Nature 587, 133–138 (2020).

213. Sies, H. & Jones, D. P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat. Rev. Mol. Cell Biol. 21, 363–383 (2020).

214. Markesbery, W. R., Kryscio, R. J., Lovell, M. A. & Morrow, J. D. Lipid peroxidation is an early event in the brain in amnestic mild cognitive impairment. Ann. Neurol. 58, 730–735 (2005).

215. Pratico, D., Uryu, K., Leight, S., Trojanoswki, J. Q. & Lee, V. M. Increased lipid peroxidation precedes amyloid plaque formation in an animal model of Alzheimer amyloidosis. J. Neurosci. 21, 4183–4187 (2001).

216. Yuan, J. & Horvitz, H. R. A first insight into the molecular mechanisms of apoptosis. Cell116, S53–S56 (2004).

217. Thornberry, N. A. et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907–17911 (1997).

218. Cerretti, D. P. et al. Molecular cloning of the interleukin-1β converting enzyme. Science256, 97–100 (1992).

219. Thornberry, N. A. et al. A novel heterodimeric cysteine protease is required for interleukin-1β processing in monocytes. Nature 356, 768–774 (1992).

220. Horvitz, H. R., Shaham, S. & Hengartner, M. O. The genetics of programmed cell death in the nematode Caenorhabditis elegans. Cold Spring Harb. Symp. Quant. Biol. 59, 377–385 (1994).

221. Miura, M., Zhu, H., Rotello, R., Hartwieg, E. A. & Yuan, J. Induction of apoptosis in fibroblasts by IL-1β-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75, 653–660 (1993).

222. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme. Cell 75, 641–652 (1993).

223. Cory, S., Roberts, A. W., Colman, P. M. & Adams, J. M. Targeting BCL-2-like proteins to kill cancer cells. Trends Cancer 2, 443–460 (2016).

224. Nakamura, T. et al. Phase separation of FSP1 promotes ferroptosis. Nature 619, 371–377 (2023).

225. Ryan, S. K. et al. Therapeutic inhibition of ferroptosis in neurodegenerative disease. Trends Pharmacol. Sci. 44, 674–688 (2023).

226. Tonnus, W. et al. Dysfunction of the key ferroptosis-surveilling systems hypersensitizes mice to tubular necrosis during acute kidney injury. Nat. Commun. 12, 4402 (2021).

227. Singh, J., Habean, M. L. & Panicker, N. Inflammasome assembly in neurodegenerative diseases. Trends Neurosci. 65, 885–904 (2023).

228. Fetter, T., de Graaf, D. M., Claus, I. & Wenzel, J. Aberrant inflammasome activation as a driving force of human autoimmune skin disease. Front. Immunol. 14, 1190388 (2023).

229. Vontell, R. T. et al. Identification of inflammasome signaling proteins in neurons and microglia in early and intermediate stages of Alzheimer’s disease. Brain Pathol. 33, e13142 (2023).

230. Parmar, D. V. et al. Safety, tolerability, pharmacokinetics, and pharmacodynamics of the oral NLRP3 inflammasome inhibitor ZYIL1: first-in-human phase 1 studies (single ascending dose and multiple ascending dose). Clin. Pharmacol. Drug Dev. 12, 202–211 (2023).

231. Ambrus-Aikelin, G. et al. JT002, a small molecule inhibitor of the NLRP3 inflammasome for the treatment of autoinflammatory disorders. Sci. Rep. 13, 13524 (2023).

232. Xie, T. et al. Structural basis of RIP1 inhibition by necrostatins. Structure 21, 493–499 (2013).

233. Chen, L. et al. Advances in RIPK1 kinase inhibitors. Front. Pharmacol. 13, 976435 (2022).

234. Feng, X. et al. Receptor-interacting protein kinase 3 is a predictor of survival and plays a tumor suppressive role in colorectal cancer. Neoplasma 62, 592–601 (2015).

235. Yan, J., Wan, P., Choksi, S. & Liu, Z. G. Necroptosis and tumor progression. Trends Cancer8, 21–27 (2022).236. Seifert, L. et al. The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincle-induced immune suppression. Nature 532, 245–249 (2016).

Acknowledgements

The work of J.Y. is supported, in part, by the China National Natural Science Foundation (82188101, 21837004, 91849204 and 92049303), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB39030200), the Shanghai Municipal Science and Technology Major Project (grant no. 2019SHZDZX02), and the Shanghai Key Laboratory of Aging Studies (19DZ2260400).

Author contributions

The manuscript was written and revised by J.Y. with input from D.O.

Competing interests

D.O. is an employee of Sanofi.

Additional information

Peer review information Nature Reviews Molecular Cell Biology thanks Andreas Linkermann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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