Organelle-specific initiation of cell death - Nature

REVIEW

Organelle-specific initiation of cell death Lorenzo Galluzzi, José Manuel Bravo-San Pedro and Guido Kroemer In a majority of pathophysiological settings, cell death is not accidental — it is controlled by a complex molecular apparatus. Such a system operates like a computer: it receives several inputs that inform on the current state of the cell and the extracellular microenvironment, integrates them and generates an output. Thus, depending on a network of signals generated at specific subcellular sites, cells can respond to stress by attemptinwg to recover homeostasis or by activating molecular cascades that lead to cell death by apoptosis or necrosis. Here, we discuss the mechanisms whereby cellular compartments — including the nucleus, mitochondria, plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosomes, cytoskeleton and cytosol — sense homeostatic perturbations and translate them into a cell-death-initiating signal.

In response to extreme conditions (for example, freeze–thaw cycles and steep osmotic gradients), cells lose their physical integrity and die in a virtually unpreventable manner. Fortunately, such an accidental form of cell death is rare in pathophysiological scenarios, which are generally affected by relatively mild homeostatic perturbations. In these conditions, cells die following the activation of specialized, genetically encoded molecular machineries1. Whereas accidental cell death invariably manifests with necrotic morphological features, its regulated counterpart can assume a spectrum of phenotypes that ranges from necrotic to fully apoptotic2. According to current understanding, apoptosis is not the only genetically encoded cell death subroutine — it shares this feature with necroptosis and at least two other forms of regulated necrosis3 (Box 1). Macroautophagy (hereafter referred to as autophagy) is an evolutionarily old cytoprotective mechanism generally involved in the maintenance of cellular and organismal homeostasis. Autophagy can also promote cell death in response to stress4, but the underlying mechanisms remain elusive. The systems that regulate cell death operate like computers: they integrate bits of data (‘inputs’) into biological responses (‘outputs’). These inputs are generated at distinct subcellular sites by specific sensors, and inform the cell death machinery on key variables including ATP levels and nutrient availability (Fig. 1). Notably, in most (but not all) instances of cell death, the integration of such pro-death and pro-survival inputs takes place at mitochondria. Here, we discuss the apical molecular mechanisms through which cellular organelles sense homeostatic fluctuations to initiate cell death. The signal transduction pathways that connect such signals to cell death regulation and the central role of Bcl-2 proteins in this setting have been extensively reviewed in refs 1,3,5,6.

Nuclear pathways Apoptosis and regulated necrosis can both be initiated in the nucleus in response to DNA damage or mitotic problems7,8. Double-strand DNA breaks generally activate ATM (ataxia telangiectasia mutated), a kinase that phosphorylates several substrates including the oncosuppressor protein p53. The consequent stabilization of p53 enables a transcriptional response that initially favours DNA repair. If genomic integrity cannot be re-established, p53 transactivates pro-apoptotic BCL-2-like proteins, thus inducing mitochondrial outer membrane permeabilization (MOMP)9. Moreover, cytosolic p53 seems to physically interact with and activate the pro-apoptotic Bcl-2 family member BAX (ref. 10); to engage in inhibitory liaisons with its anti-apoptotic counterpart BCL-XL (ref. 11); and to trigger mitochondrial permeability transition (MPT) after binding to peptidylprolyl isomerase F (PPIF; best known as cyclophilin D, CYPD)12. Interestingly, the transition from an adaptive to a lethal DNA damage response involves the dephosphorylation of histone 2AX (an ATM substrate) by the tyrosine phosphatase EYA1 (eyes absent homolog 1)13. Moreover, the ability of ATM to initiate cell death reportedly relies on the transcriptional factor FOXO3 (forkhead box O3)14. The protein kinase ATR (ataxia telangiectasia and Rad3 related) activates p53 in response to single-strand DNA breaks, following direct phosphorylation as well as via checkpoint kinase 1 (CHEK1)15. In p53-deficient cells, CHEK1 has pro-survival functions as it inhibits a caspase-2-dependent, BCL-2-insensitive apoptotic program ignited by DNA damage16. This observation argues against previous findings suggesting that the activation of caspase-2 by DNA damage critically relies on a multiprotein complex known as PIDDosome, which is assembled around CRADD (CASP2 and RIPK1 domain containing adaptor with

Lorenzo Galluzzi1–3, José Manuel Bravo-San Pedro1,3,4 and Guido Kroemer1,2,4–6 are at 1Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France, 2Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France, 3Gustave Roussy Comprehensive Cancer Center, Villejuif, France, 4 INSERM, U1138, Villejuif, France, 5Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France, 6Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France. e-mail: [email protected]; [email protected]

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NATURE CELL BIOLOGY VOLUME 16 | NUMBER 8 | AUGUST 2014 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEW BOX 1 Molecular mechanisms of regulated cell death Apoptosis represents the form of regulated cell death most intensively characterized so far. The distinctive trait of apoptosis at the biochemical level is the activation of a peculiar class of cysteine proteases, called caspases1. Some, such caspase-8 and -9, operate as ‘initiators’ and transmit a pro-apoptotic signal by activating their ‘executioner’ counterparts, such as caspase-3, -6 and -7. In turn, executioner caspases cleave a wide range of substrates that are required for cell survival150. Such a caspase cascade can be initiated by extracellular stimuli as well as by perturbations of intracellular homeostasis, most often via pathways that converge at mitochondria5,6. Irrespective of the precise molecular mechanisms leading to caspase activation, apoptotic cells exhibit several stereotypical features, including (but not limited to) chromatin condensation, nuclear fragmentation, pyknosis and plasma membrane blebbing, eventually breaking down into discrete corpses (the so-called apoptotic bodies) that are engulfed by phagocytes2. Importantly, while caspase inhibitors abolish the morphological manifestations of apoptosis, in most instances they merely delay, rather than prevent, cell death. This suggests that pro-apoptotic stimuli can induce caspase-independent cell death subroutines3. Of note, apoptosis not only contributes to the aetiology of several acute and chronic disorders characterized by the unwarranted loss of post-mitotic cells, but also plays a major role in embryonic and post-embryonic development, as well as in adult tissue homeostasis150, thus constituting a major form of programmed cell death1. The notion that necrosis can also occur in a regulated manner has emerged relatively recently, implying that the underlying molecular mechanisms are much less characterized than their apoptotic counterparts. Necroptosis, a specific variant of regulated necrosis that obligatorily relies on RIPK3 and MLKL, is actively blocked by a supramolecular complex involving active caspase-8, the apoptotic adaptor FAS (TNFRSF6)-associated via death domain (FADD) and FLIPL (ref. 3). Necroptosis differs from apoptosis in that it does not rely on the obligate contribution of mitochondria43. Recent data suggest that MLKL responds to RIPK3-dependent phosphorylation by forming oligomers that translocate to the inner leaflet of the plasma membrane, hence compromising its ability to preserve ionic homeostasis31. The precise molecular mechanisms underlying this process, however, are still elusive. MPT-driven regulated necrosis proceeds following the structural and functional breakdown of mitochondria, thereby resembling apoptosis3. In both apoptotic and necrotic scenarios, MPT critically relies on CYPD. However, MPT-driven regulated necrosis relies on caspaseindependent executioner mechanisms, including the bioenergetic crisis caused by the dissipation of the mitochondrial transmembrane potential as well as the latent chromatinolytic activity of AIFM1 (apoptosis-inducing factor mitochondrion-associated 1; best known as AIF)3. Both these events are involved in a third signal transduction cascade leading to necrotic cell death, which has been named parthanatos. Of note, necroptosis, MPT-driven regulated necrosis and parthanatos contribute to the demise of post-mitotic cells in the context of ischaemic insults, neurodegeneration or viral infection3. Conversely, the actual pathophysiological relevance of other molecular cascades that have been suggested to drive regulated necrosis, such as a recently identified Fe2+-dependent pathway dubbed ferroptosis109, remains to be conclusively proven.

death domain; best known as RAIDD) and PIDD (p53-induced death domain protein)17,18. Accumulating evidence suggests that caspase-2 does indeed respond to DNA damage independently from PIDD and RAIDD, perhaps owing to an extranuclear caspase-activation platform normally involved in extracellular apoptosis (see below), the so-called death inducing signalling complex (DISC)19. Notably, both the ATMdependent phosphorylation of PIDD and its autoproteolysis within the PIDDosome might play a central role in converting adaptive responses to genotoxic stress into a lethal signal that is optionally propagated by caspase-2 (refs 20,21). It remains unclear whether these two events are functionally interconnected. Alkylating agents (that is, drugs that cause DNA bridges) promote cell death following the hyperactivation of poly(ADP-ribose) (PAR) polymerase 1 (PARP1)3. PARP1 catalyses the NAD+-dependent addition of PAR moieties to several substrates, including histones, and can hence induce the recruitment of factors involved in DNA repair 22. If damage cannot be repaired, however, PARP1 becomes hyperactivated and triggers parthanatos3. The nuclear compartment also initiates the demise of cells that cannot complete mitosis, most often owing to gross chromosomal aberrations or mitotic spindle defects. Such an oncosuppressive mechanism, which avoids the propagation of mitosis-incompetent cells via apoptosis, regulated necrosis or cell senescence, is commonly known as mitotic catastrophe8. Together, these observations suggest that the initiation of cell death by nuclear pathways follows an unsuccessful attempt to re-establish genomic integrity (Fig. 2).

Plasma-membrane-triggered pathways Permanent plasma membrane permeabilization is currently considered as the most reliable marker of cell death23. In addition, the plasma membrane initiates all instances of cell death elicited by cell-impermeant stimuli1. All cells express on their surface several receptors that can trigger apoptosis or necrosis. These include death receptors, such as FAS (also known as CD95) and other members of the tumour necrosis factor receptor (TNFR) superfamily 24, as well as so-called dependence receptors, such as DCC (deleted in colorectal carcinoma), PTCH1 (patched 1) and NTRK3 (neurotrophic tyrosine kinase receptor type 3; best known as TRKC)25. Unlike death receptors, which initiate cell death after engagement with their agonists, dependence receptors dispatch a lethal signal once the concentration of their ligands falls below a critical threshold level24,25. In caspase-proficient cells, death-receptor-elicited cell death proceeds via the DISC-dependent activation of caspase-8 (or caspase-10) at the inner leaflet of the plasma membrane24. Active caspase-8 is sufficient to initiate the demise of some cells (type I cells, such as lymphocytes), but not others (type II cells, such as hepatocytes), owing to the differential expression levels of XIAP (X-linked inhibitor of apoptosis), a cytosolic inhibitor of caspases26. In type II cells, death-receptor-driven apoptosis requires BID, a caspase-8-activated BH3-only protein that triggers MOMP (ref. 27). MOMP promotes the activation of caspase-9 through the so-called apoptosome, as well as via the release of XIAP inhibitors such as DIABLO into the cytosol5,6. Notably, the proteins that assemble at the cytoplasmic tail of different death receptors vary significantly, endowing DISCs with distinct functional profiles1,24. Thus, FAS reportedly promotes the growth and invasive potential of multiple cancer


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REVIEW Organelle-specific sensor 1

Adaptive response 1

Homeostatic perturbations

Organelle-specific sensor 2

Adaptive response 2

Survival Yes

CASPs

Apoptosis

Homeostasis?

MLKL

Necroptosis

No

CYPD

MPT-driven necrosis

PARP1

Parthanatos

Figure 1 General organization of organelle-specific responses to stress. Each subcellular compartment is endowed with sensors that continuously monitor a number of variables. Generally, such sensors respond to homeostatic perturbations by activating an organelle-specific or cell-wide adaptive response aimed at the re-establishment of intracellular homeostasis. When

this objective cannot be attained, for instance when the initiating stimulus is excessively severe or cannot be removed, failing adaptive responses actively trigger cell death. CASPs, caspases; MLKL, mixed-lineage kinase domain-like; CYPD, cyclophilin D; MPT, mitochondrial permeability transition; PARP1, poly(ADP-ribose) polymerase 1. Cell-death-inhibitory factors are shown in blue.

cells, in vitro and in vivo28, whereas TNFR1 initially mediates robust pro-inflammatory and anti-apoptotic functions (by activating NF-κB)29. The switch between the pro-inflammatory/anti-apoptotic and pro-apoptotic activity of TNFR1 is controlled by two independent mechanisms: the proteasomal processing of FLIPL, a short-lived molecule that inhibits caspase-8, and the autodegradation of BIRC2 (baculoviral IAP repeat containing 2) and BIRC3, two ubiquitin ligases that prevent the dissociation of DISC components from the cytoplasmic tails of TNFR1 (an event that is required for caspase-8 activation)30. In caspase-incompetent cells, various death receptors including FAS, TNFR1 and multiple TNF-related apoptosis-inducing ligand receptors (TRAILRs) initiate necroptosis3. In these conditions, RIPK1 (receptorinteracting protein kinase 1) recruits and engages in mutually regulatory interactions with RIPK3, resulting in the stabilization of a RIPK1- and RIPK3-containing complex that delivers a mixed-lineage kinase domainlike (MLKL)-dependent lethal signal, the so-called necrosome31. When the concentration of their agonists falls below a specific threshold, dependence receptors reportedly deliver lethal signals following the cleavage of their intracellular tails by caspases, a process that would occur very soon after ligand withdrawal25. At least three distinct signalling cascades have been proposed to transduce lethal cues elicited by dependence receptors32–34. However, the molecular mechanisms through which dependence receptors promote cell death are a matter of debate. In particular, it remains unclear which specific caspases would be involved and how they would be activated. Plasma membrane ion channels including purinergic receptor P2X, ligand-gated ion channel 7 (P2RX7)35 and transient receptor potential cation channel subfamily V member 1 (TRPV1)36 have a central role in both the initiation and execution of apoptosis and necrosis31,37. However, it is as yet unknown whether the pro-apoptotic effects of ion channels exclusively rely on the activation of ionic fluxes. T cell receptors (TCRs)38, CD4 (ref. 39) and multiple Toll-like receptors (TLRs)40, as well as specific G-protein-coupled receptors (GPCRs)41, can also elicit cell death. That said, most GPCRs exert anti-apoptotic effects, mainly by inhibiting p53 and/or stimulating NF-κB signalling 42. A precise description of the pro-apoptotic cascades elicited by these receptors, which exhibit a high degree of variability, is beyond the scope of this Review. Taken together, these observations indicate that the plasma membrane is a critical site for the initiation of cell death by a plethora of cell-impermeant stimuli (Fig. 3).

Mitochondrial pathways Mitochondria are dispensable for necroptosis43, yet are crucial for intrinsic apoptosis, some instances of extrinsic apoptosis (see above), MPT-driven regulated necrosis and parthanatos5,6,44. In addition, several perturbations of intracellular homeostasis are specifically detected by mitochondria, thereby involving them in the initiation of cell death. Hypoxia and respiratory chain inhibitors such as rotenone (blocking complex I), antimycin A (blocking complex III) and oligomycin (blocking ATP production by the F1FO–ATP synthase) promote the overgeneration of mitochondrial reactive oxygen species (ROS), which exert lethal effects as they alter the relative abundance of pro- and anti-apoptotic proteins and directly trigger MPT (refs 45,46). The observation that the glycolytic inhibitor 2-deoxyglucose can deplete ATP as efficiently as respiratory chain inhibitors in the absence of major cytotoxic effects47 suggests that the lethal activity of the latter is not simply a mere consequence of declining ATP levels. Notably, the optimal demise of cells undergoing oxidative stress relies on p53, which promotes not only MPT (ref. 12) but also ROS-elicited parthanatos48. In conditions that imbalance protein homeostasis at specific organelles, cells mount a local unfolded protein response (UPR)49. As is true for other adaptive mechanisms, the mitochondrial UPR is biphasic. In the first phase, mitochondrial chaperones such as HSPD1 (heat shock 60 kDa protein 1) and proteases such as CLPP (caseinolytic mitochondrial matrix peptidase proteolytic subunit) are synthetized in an attempt to restore homeostasis49. If unsuccessful, such an adaptive response is followed by the delivery of lethal signals. In mammalian cells (which are poorly characterized in this respect), both phases of the mitochondrial UPR seem to be centred around mitogen-activated protein kinase 9 (MAPK9, best known as JNK2) and the transcription factors JUN, DDIT3 (DNA-damage-inducible transcript 3; best known as CHOP) and C/EBP-β (CCAAT/enhancer binding protein β)49,50. In Caenorhabditis elegans, a central player in this process is ATFS1 (activating transcription factor associated with stress 1), a transcription factor that can translocate into the nucleus (where it promotes a broad cytoprotective response) only when the mitochondrial import machinery is inhibited by the accumulation of misfolded proteins51. Whether ATFS1 is involved in the lethal phase of the mitochondrial UPR and whether a similar mechanism operates in mammalian cells remains to be determined. Mitochondria are also involved in the initiation of cell death by microbial stimuli. This occurs at special sites of physical and functional

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REVIEW Chromosomal aberrations

DNA damage

Spindle defects

M

Repair?

G0 /G1

S

YES

G2 PARP1

ATM

Necrosis

ATR p53

PARP1 Parthanatos

Senescence

AIF No

Repair?

Homeostasis

Yes

No

MOMP

Necrosis

MPT

BAX

BCL-2

PIDD

CYPD

CASP-3 CASP-2

YES

BAX

RAIDD Repair? PUMA

CASP-9

BID

Nucleus

Mitochondria

Apoptosis

NF-κB

p53

Apoptosis

Mitotic catastrophe

No

SAC

No

MOMP

BCL-2

Nucleus

Figure 2 Major pathways of cell death initiation by the nucleus. Doublestrand and single-strand DNA breaks are mainly sensed by ATM and ATR, respectively. The consequent stabilization of p53 induces a temporary cell cycle arrest that allows for the re-establishment of homeostasis. If this cannot be attained, p53 promotes the transactivation of several pro-apoptotic proteins, including BAX, PUMA and PIDD, coupled to the transcriptional downregulation of key anti-apoptotic factors such as BCL-2, thus favouring mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Cytoplasmic p53 has also been shown to mediate lethal effects, not only as it promotes MOMP by physically interacting with BAX, BCL-2 and BCL-XL, but also as it stimulates mitochondrial permeability transition (MPT)-driven regulated necrosis by binding to CYPD. Together with RAIDD, PIDD generates a supramolecular platform (the so-called PIDDosome) that can either activate

NF-κB, hence inhibiting cell death, or ignite a caspase-2-dependent, BIDmediated signalling axis leading to MOMP. In response to alkylating agents, PARP1 recruits proteins of the DNA repair system. If damage persists, however, PARP1 becomes hyperactivated and, among other effects, initiates a signal transduction cascade that results in the translocation of apoptosisinducing factor (AIF) to the nucleus. In this case, the chromatinolytic activity of AIF marks the execution of a caspase-independent cell death instance known as parthanatos. When gross chromosomal alterations or mitotic spindle defects are sensed in the course of mitosis, the so-called spindle-assembly checkpoint (SAC) is not turned off to allow for the metaphase-to-anaphase transition, resulting in the generation of a signal leading to apoptosis, necrosis or cell senescence. This oncosuppressive mechanism is generally known as mitotic catastrophe. Cell-death-inhibitory factors are shown in blue.

interaction between mitochondria and the endoplasmic reticulum (ER) known as mitochondria-associated ER membranes (MAMs)52. Several sensors of viral RNA including DDX58 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 58; best known as RIG-I) and IFIH1 (interferon-induced with helicase C domain 1; known best as MDA5) can promote an apoptotic response that is independent of type I interferon secretion and p53, but relies on mitochondrial antiviral signalling protein (MAVS)53,54. Such a response reportedly originates from a MAVS–caspase-8 complex and proceeds independently of BAX and its pro-apoptotic homologue BAK1 (ref. 55). Notably, the plasmid-driven overexpression of MAVS (which preferentially localizes at MAMs) stimulates apoptosis per se, and Mavs–/– cells are less prone to succumb to viral infection than their wild-type counterparts56. In summary, mitochondria occupy a central position in the regulation of cell death and emit primary pro-apoptotic stimuli in response to specific homeostatic perturbations.

involved in anterograde vesicle transport57, resulting in a severe secretory impairment, as well as degradation of enzymes and transporters that participate in ceramide metabolism59. Although the cleavage of ER and Golgi proteins often amplifies apoptotic signals60, these alterations are generally not involved in the apical regulation of cell death and hence will not be discussed further. In response to the intrareticular accumulation of unfolded proteins, the ER activates a UPR based on three main sensors: ERN1 (endoplasmic reticulum to nucleus signalling 1; best known as IRE1α), ATF6 (activating transcription factor 6) and EIF2AK3 (eukaryotic initiation factor 2α (EIF2α) kinase 3; best known as PERK)61. The reticular ER initially attempts to re-establish homeostasis through a cell-wide adaptive response orchestrated around a general arrest of translation (mediated by phosphorylated EIF2α) coupled to the selective synthesis of ER chaperones such as GRP78 (glucose-regulated protein 78 kDa)61. If reticular homeostasis cannot be restored, persistent IRE1α, ATF6 and PERK signalling activates an apoptotic response that involves multiple, sometimes concomitant, mechanisms50. These include the MAPKdependent activation of CHOP (ref. 62), the release of Ca2+ ions into the cytoplasm63, the JNK-dependent activating phosphorylation of BH3only proteins64 and the downregulation of anti-apoptotic Bcl-2 family members65, as well as the IRE1α-ignited, TRAF2 (TNFR-associated factor 2)-dependent activation of caspase-12 (ref. 66) and MAP3K5 (mitogen-activated protein kinase kinase kinase 5; best known as ASK1)67. The IRE1α-elicited, microRNA-dependent translational de-repression

Reticular pathways The ER and Golgi apparatus mediate the synthesis, folding and maturation of proteins for secretion or insertion into the plasma membrane, are intimately involved in Ca2+ homeostasis, and detoxify various xenobiotics57. Both these membranous organelles undergo significant rearrangements during apoptosis, owing in part to the caspase-dependent cleavage of multiple proteins that ensure their structural integrity 58. Some instances of apoptosis also coincide with the degradation of proteins NATURE CELL BIOLOGY VOLUME 16 | NUMBER 8 | AUGUST 2014

© 2014 Macmillan Publishers Limited. All rights reserved

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REVIEW of caspase-2 has also been attributed a central role in the induction of apoptosis by ER stress68, yet these data have not been confirmed in an independent study 69. Interestingly, the optimal activation of IRE1α relies on BAX and BAK1 (ref. 70), and is inhibited by the anti-apoptotic protein TMBIM6 (transmembrane BAX inhibitor motif containing 6; best known as BI-1)71, all of which (at least partially) localize to the ER membrane. Thus, the executioner machinery of MOMP physically and functionally interacts with the sensors that initiate cell death in response to ER stress. Notably, the transition between the adaptive and lethal phase of the ER stress response relies, at least partially, on EIF2α dephosphorylation72, allowing CHOP and ATF4 to cause a lethal boost in protein synthesis73. As the dephosphorylation of EIF2α also marks the re-establishment of reticular homeostasis74, additional signals must be involved in the initiation of cell death by ER stress. One of these cues may originate from the ER-stress-dependent secretion of the oncosuppressor protein PAR4, resulting in the activation of caspase-8-dependent apoptosis following the binding of PAR4 to surface-exposed GRP78 (ref. 75). Another cue may involve CRK (v-crk avian sarcoma virus CT10 oncogene homolog), an enzymatically inactive adaptor that promotes MOMP (perhaps owing to a putative BH3-only domain)76. Alternatively, the ER stress response may become lethal following the activation of a cyclin-dependent kinase 5 (CDK5)–MAP3K1–JNK1 signalling pathway. Indeed, the knockdown of any of these kinases limits the ER-stress-elicited demise of retinal cells in Drosophila melanogaster while leaving the cytoprotective branch of the UPR unaffected77. It remains to be explored whether these latter findings also apply to mammalian cells. MAMs represent crucial points of control for Ca2+ homeostasis (hence influencing the magnitude of some apoptotic responses)78 and privileged scaffolds for the assembly of multiprotein complexes52. The MAVSinteracting partner transmembrane protein 173 (TMEM173, best known as STING) mediates distinct biological responses from within several such complexes79. Thus, STING not only elicits the secretion of type I interferon in response to bacterial nucleic acids80, but also can transduce an interferon-regulatory factor 3 (IRF3)-dependent apoptotic signal following ER stress81. MAMs also host an ER-stress-triggered pro-apoptotic cascade relying on CDIP1 (cell-death-inducing p53 target 1). In this setting, CDIP1 recruits BCAP31 (B cell receptor-associated protein 31; a protein involved in anterograde vesicle transport) at the ER membrane to form a complex that promotes the activation of caspase-8 and the sequestration of mitochondrial BCL-2, thereby stimulating MOMP (ref. 82). The Golgi apparatus is also involved in the apical regulation of several paradigms of cell death. For instance, both the conversion of ceramide into ganglioside GD3 (which preferentially occurs at the Golgi apparatus)83 and the cleavage of Golgi reassembly stacking protein 1 (GORASP1, also known as GRASP65)84 are important for the initiation of extrinsic apoptosis by FAS and TNFR1. Moreover, the Golgi apparatus reportedly regulates the cellular sensitivity to FAS ligand (FASL) and TRAIL by controlling the trafficking of their receptors (FAS and TRAILR1, respectively) to the plasma membrane85. Along similar lines, the Golgi apparatus seems to inhibit the spontaneous death of human embryonic stem cells by preventing a constitutively active pool of BAX from promoting MOMP86. Finally, the Golgi apparatus can respond to reticular stress with signals that initiate cell death, such as the apical activation of a local pool of caspase-287. The molecular mechanisms underlying the activation of caspase-2 in this setting have not yet been elucidated, but may 732

Death receptor activation NF-κB

RIPK1

?

DISC

Inflammation FLIPL

BID MOMP

FADD

Type II cells Yes DIABLO

XIAP

Proliferation

BIRCs

FLIPL Caspase-8 FADD ? Yes Type I CASP-3 cells

No RIPK1 RIPK3 MLKL

CASP-9 CASP-3 Apoptosis

Necroptosis

Figure 3 Major pathways of cell death initiation by the plasma membrane. In response to their ligands, death receptors recruit several proteins to their cytoplasmic tails to form a highly dynamic multiprotein complex that can dispatch either mitogenic or lethal signals, depending on several variables. For instance, FAS can stimulate proliferation (via FADD and other hitherto uncharacterized transducers), whereas TNFR1 (tumour necrosis factor receptor 1) can mediate anti-apoptotic and pro-inflammatory effects as it activates NF-κB through RIPK1 (receptor-interacting protein kinase 1). The lethal activity of death receptors is robustly inhibited by FLIPL and several members of the BIRC (baculoviral IAP repeat containing) protein family. Following the proteasomal degradation of FLIPL and the autodegradation of BIRCs, the death-inducing signalling complex (DISC) acquires the ability to initiate a caspase-8 to caspase-3 proteolytic cascade. In type I cells, which express low levels of XIAP (X-linked inhibitor of apoptosis), this is sufficient to induce extrinsic apoptosis. In type II cells (which express high levels of XIAP), caspase-8 must proteolitically activate BID and hence trigger MOMP (resulting in the release of DIABLO and other XIAP antagonists in the cytosol) to promote apoptosis. In caspaseincompetent cells, TNFR1 triggers a RIPK3-dependent signal transduction cascade that stimulates the oligomerization and consequent translocation to the plasma membrane of MLKL, resulting in the loss of ionic homeostasis. This instance of regulated necrosis, which is known as necroptosis, is actively inhibited by a complex involving proteolitically active caspase-8, FADD and FLIPL. Cell-death-inhibitory factors are shown in blue.

involve a Golgi-apparatus-resident pool of RIG-I (ref. 88). Serine/threonine kinase 25 (STK25, best known as SOK1) may also be involved in the initiation of cell death at the Golgi apparatus. The caspase-dependent translocation of SOK1 from the Golgi apparatus to the nucleus is required for cell death in response to chemical anoxia, which promotes reticular stress as a consequence of ROS overgeneration89. Moreover, SOK1 reportedly phosphorylates the chaperone 14-3-3ζ in response to oxidative stress, hence releasing its inhibitory interaction with the pro-apoptotic kinase ASK1 (ref. 90). Whether it is caspase-2 that releases SOK1 from the Golgi apparatus in response to reticular stress remains an open question. Altogether, these observations suggest that the ER and Golgi apparatus are sensitive to conditions that promote protein unfolding. If the adaptive arm of the UPR fails to re-establish homeostasis, both these organelles can elicit cell death through multiple mechanisms. Lysosomal pathways To fulfil their key catabolic functions, lysosomes contain a large array of hydrolases. These catabolic enzymes are activated by the highly acidic NATURE CELL BIOLOGY VOLUME 16 | NUMBER 8 | AUGUST 2014

© 2014 Macmillan Publishers Limited. All rights reserved

REVIEW Lysosomotrophic agents Cathepsins

BCL-2

Serpins

CASP-9 CASP-2 Sphingosine

LAMP1

Cholesterol

LAMP2

HSP70

BAX

p53

Calpains

Necroptosis

Calpains

LMP RIPK1 CASP-8

Serpins

XIAP

Apoptosis

Cathepsins

ROS

BID BCL-2

MOMP

Fe2+

Ferroptosis

MPT

Necrosis

Figure 4 Major pathways of cell death initiation by lysosomes. Lysosomal membrane permeabilization (LMP) constitutes a very apical event in the signal transduction cascades ignited by lysosomotropic agents, oxidative stress and RIG-I agonists. Massive LMP results in the liberation (and allows for the activation) of acid hydrolases in the cytosol, which in turn catalyse the (activatory or inhibitory) processing of several substrates involved in cell death regulation. Cathepsins stimulate both mitochondrial outer membrane permeabilization (MOMP) and downstream events of the apoptotic cascade. Moreover, in dendritic cells cathepsin D seems to inactivate caspase-8 while promoting the recruitment of RIPK1 to mitochondria-associated ER membranes (MAMs), hence triggering necroptosis. LMP also compromises iron homeostasis, possibly inducing ferroptosis. Owing to its catastrophic consequences, multiple factors inhibit LMP, including HSP70 (which prevents the accumulation of sphingosine), serpins (which also inhibit cathepsins), LAMP1 (lysomal-associated membrane protein-1), LAMP2, cholesterol and possibly a lysosomal pool of BCL-2. The LMP-promoting activity of calpains at least in part stems from their ability to degrade HSP70 and LAMP2. Cell-death-inhibitory factors are shown in blue.

pH of the lysosomal lumen, which is generated by an ATP-dependent proton pump of the lysosomal membrane. Owing to their enzymatic equipment as well as to their elevated oxidative potential, lysosomes can initiate cell death in response to many conditions that disrupt their structural integrity, a process known as lysosomal membrane permeabilization (LMP)91. Such stimuli include multiple lysosomotropic agents (that is, molecules that preferentially accumulate within lysosomes)92, RIG-I agonists93 and oxidative stress94. In addition, LMP participates in both apoptotic and necrotic responses initiated at extralysosomal sites, including the nucleus95, plasma membrane96, cytoskeleton97, ER98 and cytosol99. Interestingly, it has been suggested that caspase-8 (as activated by death receptors) proteolytically processes caspase-9, rendering it capable of triggering LMP, but not of promoting the maturation of caspase-3100. The pathophysiological relevance of this lethal signalling pathway remains unclear. Although lysosomal hydrolases are virtually inactive at neutral pH, widespread LMP results in a partial increase in cytosolic acidity that allows for the activation of toxic proteases including cathepsins91. These enzymes reportedly promote apoptosis by cleaving a variety of substrates101, including other cathepsins102, many members of the Bcl-2 protein family 103,104, PARP1 (ref. 105), sphingosine kinase 1 (ref. 106),

XIAP (ref. 104) and perhaps some caspases107. Interestingly, LMP as induced by the RIG-I agonist polyinosinic-polycytidylic acid commits a fraction of dendritic cells to necroptosis. This results from the interaction of cathepsin D with MAVS, promoting the degradation of caspase-8 concomitantly with the recruitment of RIPK1 to MAMs93. Moreover, LMP often compromises iron homeostasis (as lysosomes are involved in the release of iron from ferritin stores)108 and may therefore induce ferroptosis109. In fact, several paradigms of regulated necrosis have been shown to rely on LMP in model organisms as diverse as worms110, rodents111 and primates112,113. The enzymatic activity of cathepsins is blocked by endogenous protease inhibitors known as serpins114. In addition, the structural integrity of lysosomes is preserved by cytoprotective factors, including: (1) specific serpins that inhibit both LMP and its consequences114; (2) heat shock 70kDa protein 1A (HSPA1A, best known as HSP70), which prevents the accumulation of the endogenous lysosomotropic sphingolipid sphingosine115; (3) lysosomal-associated membrane protein 1 (LAMP1) and LAMP2, which are required for the fusion of the lysosome with other vesicular compartments116; (4) lysosomal cholesterol, which possibly operates as a local antioxidant117; and perhaps (5) a lysosomal pool of BCL-2 (ref. 118). Lysosomal stability is relatively sensitive to declines in the levels of all these factors. This occurs, for instance, following the activation of calpains, which are Ca2+-activated proteases that promote LMP at least in part by degrading HSP70 and LAMP2 (refs 99,113). Calpains play a major aetiological role in several models of neuronal cell death in vivo119, and have recently been implicated in the LMP-dependent death of epithelial cells in the involuting mammary gland99,120. In this setting, LMP is controlled by the transcription factor STAT3 (signal transducer and activator of transcription 3), which promotes the synthesis of cathepsins B and L, while decreasing that of serpin 2A (ref. 120). This indicates that LMP is involved in both regulated and programmed paradigms of cell death. In summary, LMP generates lethal signals in response to primary lysosomal perturbations and marks the involvement of lysosomes in cell death subroutines that are initiated at extralysosomal sites (Fig. 4). Cytoskeletal pathways Similarly to the ER and Golgi apparatus, the cytoskeleton undergoes dramatic structural changes in the course of cell death. Plasma membrane permeabilization, the execution step of regulated necrosis, coincides with an ionic imbalance that drives the osmotic breakdown of the cell, hence disrupting the cytoskeletal apparatus3. Moreover, several cytoskeletal components are actively cleaved by caspases121. This not only underlies most of the cell-wide morphological manifestations of apoptosis, including blebbing 122, but also promotes a catastrophic auto-amplification of lethal signals123,124. In addition, several components of the cytoskeletal apparatus deliver a pro-apoptotic signal in response to perturbations of the microtubular or actin networks125. The prototypic sensors of cytoskeletal damage are the BH3-only proteins BCL2L11 (BCL-2-like 11; best known as BIM)126 and BMF (BCL2modifying factor)127. In physiological conditions, BIM and BMF are inactive as they physically interact with the cytoskeletal components DYNLL1 (dynein light chain LC8-type 1; best known as DLC1) and/or DYNLL2 (DLC2)126,127. Such inhibitory liaisons are disrupted in the presence of cytoskeleton-targeting agents128, allowing BIM and BMF to translocate to mitochondria and promote MOMP (refs 126,127). BIM and


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REVIEW BMF are also involved in anoikis, the apoptotic demise of cells detaching from their substrate129. Anoikis is regulated by other cytoskeletal proteins, including filamin A (FLNA), which inhibits it by stabilizing β1-integrin-dependent cell adhesions130, and tropomyosin 1 (TPM1), which promotes it 131. USP9X (ubiquitin-specific peptidase 9 X-linked) also seems to participate in the initiation of anoikis132. This argues against previous findings indicating that USP9X mediates major pro-survival roles by preventing the proteasomal degradation of the short-lived, antiapoptotic Bcl-2 family member MCL-1 (ref. 133). BIM, BMF and other cytoskeletal proteins also transduce pro-apoptotic signals elicited at extracytoskeletal locations, including the nucleus134, cytosol135, plasma membrane136 and phagosomes137. For instance, FAS is tightly bound to the actin cytoskeleton via ezrin (EZR), an interaction that is necessary (but must be broken) for death-receptor-induced apoptosis to proceed136. Moreover, microtubules and dynein seem to be required for the accumulation of p53 in the nucleus in response to DNA damage138. Perturbations of cytoskeletal dynamics promote the apoptotic demise of T lymphocytes following cytokine deprivation139, a process that is inhibited by the actin-binding protein coronin 1A (CORO1A)124. Another factor that controls actin dynamics, the small ADP-binding protein cofilin 1 (CFL1), is released from actin in response to multiple pro-oxidants, translocates to mitochondria, and promotes MPT-driven apoptosis140,141 or regulated necrosis142, perhaps depending on the cellular context. In summary, the cytoskeleton can initiate cell death in response to perturbations of actin or the microtubular system, as well as following oxidative stress. Other pathways Other organelles have also been shown to initiate cell death in response to homeostatic perturbations or to transmit apical signals following the ignition of cell death at different subcellular locations143. Besides providing a scaffold for the delivery of prominent pro-survival signals144, multiple organelles of the endocytic pathway participate in the initiation of cell death through at least three mechanisms. First, the internalization of ligand-bound death receptors within endosomes is required for the activation of the DISC by FAS and TNFR1 (ref. 24). Second, several endosome-restricted TLRs mediate lethal effects following engagement, including TLR3 (ref. 145) and TLR9 (ref. 146). Third, phagosomes can dispatch lethal signals following formation via BIM (ref.137). Autophagosomes have been proposed to provide a scaffold for the activation of the DISC in response to specific stimuli, including the proteasomal inhibitor bortezomib147. Conversely, the essential autophagic factor ATG7 reportedly binds and inhibits caspase-9148. Finally, sequestosome 1 (SQSTM1; best known as p62), which is another component of autophagosomes, seems to be required for the TRAILR-elicited recruitment of ubiquitinated caspase-8 to cytoplasmic foci and the consequent initiation of apoptosis149. Whether these foci constitute nascent autophagosomes, however, has not yet been determined. These examples corroborate the notion that virtually every subcellular compartment contains damage sensors that can initiate an adaptive or lethal response to homeostatic perturbations. Conclusions Eukaryotic cells have evolved a need to monitor subcellular compartments for potentially dangerous stress conditions. Such a need has 734

created a highly interconnected network of sensors that specifically monitor each organelle and convey these data to downstream integrators and effectors. In this context, organelle-specific stress conditions generally activate a coordinated response that aims at re-establishing homeostasis while actively suppressing cell death. When this objective cannot be fulfilled, the very same sensors dispatch a lethal signal to the cell death machinery (via one or several transducers), eventually resulting in the activation of apoptosis or necrosis. Mitochondria regulate the transition between cell death initiation and execution in a vast majority of settings, with the notable exceptions of extrinsic apoptosis in type I cells and necroptosis. Thus, mitochondria stand out as key controllers of cell death subroutines initiated at most subcellular locations44. Owing to pronounced interconnectivity of the signal transduction cascades that mediate apoptosis and necrosis, the inhibition of post-mitochondrial executioner enzymes such as caspases generally does not mediate bona fide cytoprotective effects, but just delays or changes the morphological manifestations of cell death1. The most effective strategies devised so far for the inhibition of pathological cell death do indeed target pre-mitochondrial and mitochondrial processes119. Future studies will have to elucidate whether strategies aimed at blocking the generation or apical propagation of lethal signals truly mediate optimal effects when the avoidance of unwarranted cell death is the therapeutic goal. ACKNOWLEDGEMENTS We apologise to the scientists working in this area for being unable to cite here the huge amount of top-quality literature dealing with the organelle-specific initiation of cell death. Authors are supported by the Ligue contre le Cancer (équipe labelisée); Agence National de la Recherche (ANR); Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; AXA Chair for Longevity Research; Institut National du Cancer (INCa); Fondation BettencourtSchueller; Fondation de France; Fondation pour la Recherche Médicale (FRM); the European Commission (ArtForce); the European Research Council (ERC); the LabEx Immuno-Oncology; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); and the Paris Alliance of Cancer Research Institutes (PACRI). AUTHOR CONTRIBUTIONS L.G. and J.M.B-S.P contributed equally to this work. L.G. and G.K. jointly supervised this work. ADDITIONAL INFORMATION Reprints and permissions information is available online at www.nature.com/ reprints. Correspondence should be addressed to L.G. or G.K. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Galluzzi, L. et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 19, 107–120 (2012). 2. Kroemer, G. et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ. 16, 3–11 (2009). 3. Vanden Berghe, T., Linkermann, A., Jouan-Lanhouet, S., Walczak, H. & Vandenabeele, P. Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat. Rev. Mol. Cell Biol. 15, 135–147 (2014). 4. Elgendy, M., Sheridan, C., Brumatti, G. & Martin, S. J. Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol. Cell 42, 23–35 (2011). 5. Kroemer, G., Galluzzi, L. & Brenner, C. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 87, 99–163 (2007). 6. Tait, S. W. & Green, D. R. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat. Rev. Mol. Cell Biol. 11, 621–632 (2010). 7. Bouwman, P. & Jonkers, J. The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nat. Rev. Cancer 12, 587–598 (2012). 8. Vitale, I., Galluzzi, L., Castedo, M. & Kroemer, G. Mitotic catastrophe: a mechanism for avoiding genomic instability. Nat. Rev. Mol. Cell Biol. 12, 385–392 (2011). 9. Bieging, K. T. & Attardi, L. D. Deconstructing p53 transcriptional networks in tumor suppression. Trends Cell Biol. 22, 97–106 (2012).


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