Mitochondria: master regulators of danger signalling - Nature

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Mitochondria: master regulators of danger signalling Lorenzo Galluzzi1,2, Oliver Kepp2,3 and Guido Kroemer1,3–7

Abstract | Throughout more than 1.5 billion years of obligate endosymbiotic co‑evolution, mitochondria have developed not only the capacity to control distinct molecular cascades leading to cell death but also the ability to sense (and react to) multiple situations of cellular stress, including viral infection. In addition, mitochondria can emit danger signals that alert the cell or the whole organism of perturbations in homeostasis, hence promoting the induction of cell-intrinsic or systemic adaptive responses, respectively. As such, mitochondria can be considered as master regulators of danger signalling. Pattern recognition receptors (PRRs). Host receptors, including Toll-like receptors and NOD-like receptors, that can sense pathogen-associated molecular patterns and initiate signalling cascades that lead to an innate immune response.

Université Paris V (Paris Descartes), 2 rue de l’École de Médecine, 75006 Paris, France. 2 Institut Gustave Roussy, 114 rue Edouard Vaillant, 94805 Villejuif, France. 3 INSERM, U848, Pavillon de Recherche 1, 39 rue Camille Desmoulins, 94805 Villejuif, France. 4 Université Paris XI (Paris Sud), 63 rue Gabriel Péri, 94270 Le Kremlin-Bicêtre, France. 5 Metabolomics Platform, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France. 6 Centre de Recherche des Cordeliers, 15 rue de l’École de Médecine, 75006 Paris, France. 7 Pôle de Biologie, Hôpital Européen Georges Pompidou, AP‑HP, 20 rue Leblanc, 75015 Paris, France. Correspondence to G.K. e‑mail: [email protected] doi:10.1038/nrm3479 1

Programmed cell death may have evolved together with the endosymbiotic incorporation of aerobic alpha‑ proteobacteria (the precursors of mitochondria) into eukaryotic cell precursors1. One of the first indica‑ tions that mitochondria participate in the regulation of apoptosis stemmed from the observation that in Caenorhabditis elegans CED‑9 (the worm orthologue of human B cell lymphoma 2 (BCL‑2)), a component of the core apoptotic machinery, is tethered to the mito‑ chondrial outer membrane2. Although mitochondria were initially thought to operate as mere signalling scaffolds in this context, it soon became clear that they actively regulate programmed cell death in a wide array of organisms, ranging from lower eukaryotes like yeast to humans3. Indeed, mitochondrial outer membrane permeabilization (MOMP) represents a near-universal event that marks the point of no return of multiple signal transduction cascades leading to cell death, including apoptosis and regulated necrosis4,5. Thus, mitochondria occupy a central position in the regulation and execution of cell death3,5. Accordingly, defects that alter the capa city of mitochondria to undergo MOMP are associated with a large array of human pathologies, including infec‑ tious diseases, ischaemic conditions, neurodegenerative disorders and cancer 3,5. Recently, mitochondria have also been shown to orchestrate the adaptive response of cells to diverse per‑ turbations of intracellular homeostasis6. For instance, together with the activation of autophagy (BOX 1), nutri‑ ent deprivation induces an extensive remodelling of the mitochondrial network, resulting in the formation of elongated organelles that are selectively spared from degradation7. Such a mitochondrial ‘hyperfusion’ de facto sustains cell survival and has been observed in response to other stress conditions, including ultraviolet (UV)

light irradiation, serum deprivation and exposure to protein synthesis inhibitors 8. Similarly, mitochon‑ dria exert cytoprotective functions during viral infec‑ tion, as they actively regulate antiviral signalling via retinoic acid-inducible protein I (RIG‑I) and melanoma differentiation-associated protein 5 (MDA5), two mem‑ bers of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family of pattern recognition receptors (PRRs)9. Furthermore, it has recently become clear that mitochondria are capable of emitting a range of intra cellular danger signals to alert the cell of perturbations in mitochondrial homeostasis. These include, but are not limited to, mitochondrial DNA (mtDNA), metabolic by‑products such as reactive oxygen species (ROS) and specific nucleus-encoded proteins10. Along similar lines, in conditions of massive cell damage, multiple mitochon drial products enter the bloodstream, where they are recognized by the innate immune system and elicit a local or systemic response10. Such mitochondrial damageassociated molecular patterns (DAMPs), including mtDNA, N‑formyl peptides (NFPs) and mitochondrial lipids such as cardiolipin, have recently been implicated in clinically relevant conditions such as traumatic shock and heart failure11,12. Thus, mitochondria not only actively participate in danger signalling but also constitute a major source of DAMPs that operate at both the cell-intrinsic and the cell-extrinsic (local or systemic) levels. In this Review, we describe how mitochondria operate as master regulators of danger signalling, on the one hand decoding organelle-extrinsic signals of stress to activate adaptive responses or execute cell death and, on the other hand emitting danger signals in response to perturba‑ tions in their homeostasis, leading to the activation of cell-intrinsic or organismal adaptive programmes.

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REVIEWS Type I interferon A group of pro-inflammatory cytokines structurally and functionally related to interferon-α that are produced in high levels by cells of the innate immune system in response to viral infection and promote the establishment of a widespread antiviral status.

Mitochondrial control of cell death The oversimplified view that mitochondria are purely metabolic organelles was abandoned with the discovery that the dissipation of the mitochondrial transmembrane potential (Δψm) constitutes an early and irreversible step in the cascade of events that leads to apoptotic cell death13. Since then, the signalling pathways bridging MOMP to apoptosis execution have been extensively characterized. Thus, the interplay between pro- and anti-apoptotic members of the BCL‑2 protein family has been found to control MOMP5. In addition, several proteins that have key roles within mitochondria (including cytochrome c, OMI (also known as HTRA2) and SMAC (also known as DIABLO)) have been shown to activate caspases once they are released into the cytoplasm4,14. Similarly, the mitochondrial release of apoptosis-inducing factor (AIF), an NADH oxidoreductase required for the stab ility of respiratory complex I, has been causally linked to caspase-independent cell death4,14. Mitochondria not only modulate apoptosis triggered by alterations of intracellular homeostasis (intrinsic apoptosis) but also participate, at least in some instances, in the regulation of the apoptotic response to external stimuli (extrinsic apoptosis)3,5. Thus, in hepatocytes, the engagement of death receptors such as CD95 (also known as FAS) leads to apoptosis via a signalling path‑ way that involves the caspase 8‑mediated proteolytic activation of BH3‑interacting domain death agonist (BID), BID-induced MOMP and the consequent release of SMAC and OMI15.

Box 1 | Autophagy Macroautophagy, most often referred to as autophagy, is a catabolic pathway that results in the lysosomal degradation of intracellular components, including portions of the cytoplasm and damaged organelles83. Autophagy is mediated by specialized vesicles known as autophagosomes and occurs at baseline levels in all mammalian cells, thus contributing to the maintenance of intracellular homeostasis48. In addition, the autophagic flow is upregulated in response to a wide range of stress conditions, including nutrient deprivation, hypoxia and exposure to cytotoxic agents6. Whether autophagy is a cytoprotective or a cytotoxic process has been the subject of intense debate. The existence of bona fide autophagic cell death (a cell death subtype that is mediated, rather than merely accompanied, by autophagy) is generally accepted, even though this seems to occur in a limited number of, mostly developmental, scenarios4. Inhibition of the autophagic flow indeed accelerates cell death triggered by multiple distinct stimuli, indicating that autophagy most often exerts cytoprotective rather than cytotoxic functions6. Autophagy is regulated by a complex signal transduction machinery involving autophagy-related (ATG) gene products and other proteins that sense the intracellular energy status, such as AMP-dependent protein kinase (AMPK) and mammalian target of rapamycin (mTOR). Moreover, the signalling pathways that control autophagy intersect at multiple levels with those that regulate apoptosis and often operate in a coordinate manner to mediate a switch between these two responses83. Thus, at least initially, autophagy inhibits apoptosis by various mechanisms, whereas the full-blown activation of caspases near‑to‑invariably coincides with the degradation of essential autophagic regulators such as Beclin 1 (REF. 83). Of note, although in some instances autophagy functions in a relatively nonspecific manner, the autophagic machinery can also respond to specific perturbations by targeting well-defined intracellular entities, including mitochondria (mitophagy) and invading pathogens (xenophagy)84. Owing to its crucial role in the maintenance of intracellular homeostasis and in the adaptation to stress, defects in autophagy contribute to the development of multiple human pathophysiological conditions, including infectious diseases, cancer and ageing84.

Importantly, mitochondria also control non-apoptotic types of programmed cell death, including regulated necrosis16,17. The execution of regulated necrosis has indeed been associated with an oxidative and metabolic burst that is mainly mediated by mitochondria16. In addi‑ tion, it has recently been shown that pro-necrotic signals transduced by the RIPK1 (receptor-interacting protein kinase 1)–RIPK3‑containing complex, commonly known as the necrosome, activate a mechanism that depends on MLKL (mixed lineage kinase domain-like) and PGAM5 (phosphoglycerate mutase family member 5) and ulti‑ mately results in mitochondrial fragmentation and necrotic cell death18. In summary, mitochondria have a key role in dis‑ tinct signalling pathways that ultimately lead to cell death (FIG. 1), including (but not limited to) intrinsic and extrinsic apoptosis as well as regulated necrosis.

Mitochondria, sentinels of danger Mitochondria stand at the hub of a complex system of sensors that detect perturbations of intracellular homeo‑ stasis, including oxidative stress, growth factor withdrawal and viral infection. From such a central position, mito‑ chondria respond to relatively mild degrees of stress by orchestrating adaptive responses aimed at re‑establishing homeostasis19. Conversely, when damage is beyond recovery, mitochondria translate danger signals into the execution of cell death (see above). The scenario that perhaps best exemplifies the com‑ plex role of mitochondria as sensors of danger is provided by the antiviral signalling cascades that are activated by RIG-I and MDA5 and transduced by mitochondrial anti‑ viral signalling (MAVS)9. RIG-I and MDA5 specifically recognize single-stranded RNA harbouring a triphos‑ phate group at the 5ʹ end (5ʹ pppRNA) and long doublestranded RNA, respectively, two structures that are generated during viral replication and are normally not found in host RNA20,21. The binding of viral RNA to the carboxyl terminus of RIG‑I presumably induces a confor‑ mational change that exposes its CARDs (caspase activa‑ tion and recruitment domains) and allows them to bind unanchored Lys63‑linked polyubiquitin chains22. This potently activates RIG-I, promoting its CARD-dependent association with the mitochondrial adaptor MAVS22,23. Recent data indicate that the relocalization of RIG-I to mitochondria is aided by the cytosolic ubiquitin ligase TRIM25 (tripartite motif-containing protein 25) and the mitochondrial targeting chaperone 14‑3‑3ε24. Upon inter‑ action with RIG-I and in the presence of Lys63‑linked polyubiquitin chains, MAVS forms self-perpetuating prion-like polymers that actively recruit TNF-associated factor 2 (TRAF2) and TRAF6, which, together with RIPK1, activate nuclear factor-κB (NF‑κB)‑dependent innate immune responses25,26. In addition, MAVS physi‑ cally binds to TRAF3, an interaction that is relevant for the production of type I interferon (IFN) in response to viral infection27. Besides being localized at mitochondria, MAVS and its central role in antiviral signalling are regulated by several mitochondrial proteins (TABLE 1), including the NLR family member X1 (NLRX1), C1QBP (complement

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

VOLUME 13 | DECEMBER 2012 | 781 © 2012 Macmillan Publishers Limited. All rights reserved

Homeostatic perturbations Death signals

is ptos apo

Survival signals

E sic rin xt

Intrins ic a po pt os is

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MOMP

Effector mechanisms

Cell death

R eg u

lated necrosis

Figure 1 | Mitochondrial control of cell death. Nature Reviews | Molecular Cell Biology Perturbations of intracellular homeostasis and extracellular stress conditions near‑to‑invariably result in the activation of both pro-survival and pro-death signal transduction cascades that often converge at mitochondria. When lethal stimuli predominate, mitochondrial membranes become permeabilized, which results not only in the functional breakdown of the organelle but also in the activation of caspase-dependent and caspase-independ‑ ent cell death effector mechanisms. Thus, mitochondria have a prominent role in the regulation and in the execution of distinct cell death modalities, including extrinsic and intrinsic apoptosis and regulated necrosis. MOMP, mitochondrial outer membrane permeabilization.

Mitochondria-associated membranes (MAMs). Sites of anatomical and functional interconnection between mitochondria and the endoplasmic reticulum.

component 1q subcomponent-binding protein) and the mitochondrial protein import receptor TOM70 (trans locase of outer mitochondrial membrane 70 kDa sub unit). In Nlrx1−/− mice, MAVS and RIG-I constitutively interact, which results in increased expression of anti viral molecules, including IFNβ and interleukin‑6 (IL‑6) in response to influenza virus infection28. Interestingly, NLRX1 also binds the mitochondrial protein Tu trans‑ lation elongation factor (TUFM)29. Via TUFM, which physically interacts with essential autophagic mediators such as autophagy-related 16-like 1 (ATG16L1) and the ATG5–ATG12 complex, NRLX1 stimulates autophagy during viral infection. Nlrx1−/− cells seem to control the replication of vesicular stomatitis virus better than their wild-type counterparts29, reflecting a prominent role for type I IFN induction in this setting and/or the fact the some (but not all) viruses co‑opt the autophagic machinery for their benefit 30. C1QBP, which is a pleio‑ tropic protein that is involved in innate immunity as part of the complement system, has been reported to accumulate at the mitochondrial outer membrane in response to viral infection, thus inhibiting RIG-I-like receptor family (RLR) signalling upon interaction with MAVS31. In contrast to NLRX1 and C1QBP, TOM70 binds MAVS during viral infection to promote type I IFN production by favouring the recruitment of TANKbinding kinase 1 (TBK1) and IFN regulatory factor 3

(IRF3)32, which under normal conditions are constitu‑ tively associated with heat shock protein 90 (HSP90) in the cytoplasm33. MAVS has been found to interact with proteins that play a crucial part in mitochondrial dynamics, including mitofusin 1 (MFN1) and MFN2 (REFS 34,35). However, the functional implications of these interactions are unclear, as MFN1 and MFN2 may have opposite roles in the regulation of innate immunity despite the fact that they both promote mitochondrial fusion34,35. That said, dissipation of the Δψm as triggered by overexpres‑ sion of uncoupling protein 2 (UCP2; which favours proton leakage from the respiratory chain) or by the administration of the chemical uncoupler carbonyl cyanide m‑chlorophenylhydrazone (CCCP) abolishes MAVS-mediated antiviral responses36. Thus, there seems to be a crucial link between mitochondrial functions and MAVS‑mediated antiviral signalling. MAVS functions are finely tuned not only by mito‑ chondrial proteins but also by several non-mitochondrial interactors (for a recent review, see REF. 37) (TABLE 1). Among these, stimulator of IFN genes (STING) is particu‑ larly interesting as it is enriched at mitochondria-associated membranes (MAMs)38, which may constitute the prefer ential site of RIG-I recruitment to mitochondria39. In response to RIG-I (but not MDA5) signalling, STING binds MAVS to activate a TBK1- and IRF3‑dependent cascade that ultimately results in type I IFN production40. To be functional, STING must interact with components of the translocon-associated protein (TRAP) complex, which is required for the post-translational transloca‑ tion of proteins across the endoplasmic reticulum (ER) membrane40. Upon ubiquitylation-dependent dimer ization, STING is crucial for type I IFN responses to DNA pathogens, including multiple viruses and bacteria, via a mechanism that involves the TBK1‑mediated activating phosphorylation of IRF3 (REFS 41,42). This suggests that MAMs delineate a signalling synapse (involving both mitochondrial and ER components) that is essential for optimal antiviral responses. Of note, several MAVS interactors, including RIPK1, FAS-associated with a death domain (FADD), TNFR1‑associated death domain (TRADD) and voltagedependent anion channel 1 (VDAC1)37, also regulate cell death3. In this regard, MAVS was initially suggested to promote a cell death subtype that requires caspase activ‑ ity and displays morphological features of apoptosis43. More recently, however, MAVS has been proposed to mediate anti-apoptotic functions by binding to, and thus inhibiting, VDAC1 (REF. 44). Therefore, the exact role of MAVS in cell death regulation remains to be elucidated. Irrespective of this, multiple viruses have evolved a broad array of strategies to hijack MAVS functions39,45,46, further underscoring the notion that MAVS-transduced signals are integral for adaptive responses to viral infection. Thus, mitochondria (both as self-standing organelles and as MAM components) operate as fundamental hubs in the signalling pathways that link the detection of intracellular danger to adaptive responses, by ‘housing’ crucial signal transducers and by providing a structural scaffold that regulates their activity.



REVIEWS Table 1 | Functional and physical interactors of MAVS Interactor

Localization*

Notes

Refs

ATG5–ATG12, ATG16L1

Forming autophagosomes

A TUFM- and NRLX1‑containing complex organized around MAVS that stimulates autophagy in response to viral infection

29

C1QBP

Pleiotropic

Accumulates at mitochondria during viral infection, thus inhibiting RLR signalling upon interaction with MAVS

31

AZI2

Cytoplasm

Involved in a MAVS-dependent antiviral pathway that is elicited by respiratory syncytial virus infection

37

ABL

Cytoplasm, nucleus

Phosphorylates MAVS, thus regulating innate immune responses following viral infection

37

SRC

Cytoplasm, nucleus

Interacts with both RIG-I and MAVS, exacerbating the activation of IRF3 in response to viral infection

37

CYLD

Cytoplasm

Negatively regulates RLR signalling by removing Lys63‑linked ubiquitin chains

22,37

DDX3X

Cytoplasm, nucleus

RNA helicase that promotes MAVS signalling upon infection

23,26

DHX58

Cytoplasm

RNA helicase that initiates MAVS signalling upon infection

23,26

FADD

Cytoplasm

Involved in a TRADD- and RIPK1‑containing MAVS complex

IKKα, IKKβ, IKKγ, IKKε

Cytoplasm, nucleus

Upstream regulators of TBK1 activation

26,37

IRFs

Cytoplasm, nucleus

Dimerize upon phosphorylation by IKKε and TBK1, enter the nucleus and transactivate genes coding for type I IFNs

23,37

ITCH

Cytoplasm, nucleus Recruited by PCBPs; targets MAVS to proteasomal degradation

MDA5

Cytoplasm

RNA helicase that promotes MAVS signalling upon infection

23,26

MFNs

Mitochondria

MAVS binds MFN1 and MFN2, with diverging functional outcomes

34,35

NLRX1

Mitochondria

Negatively regulates the interaction between MAVS and RIG-I

28

PCBPs

Cytoplasm

Recruit ITCH to mediate MAVS ubiquitylation and degradation

37

PLK1

Cytoplasm, nucleus

Irrespective of phosphorylation, PLK1 binding to MAVS displaces TRAF3, thereby inhibiting type I IFN production

37

PSMA7

Cytoplasm

Binds to MAVS and targets it for proteasomal degradation

37

RIG‑I

Cytoplasm

RNA helicase that initiates MAVS signalling upon infection

23,26

RIPK1

Cytoplasm

Involved in a FADD- and TRADD-containing MAVS complex

37

RNFs

Mitochondria, MAMs

RNF5 and RNF125 negatively regulate RLR signalling by targeting MAVS, RIG-I and MDA5 for degradation

37

STING

MAMs

Binds to MAVS to recruit TBK1 during RIG-I signalling

40

TANK

Cytoplasm

Upstream regulator of IKKε and TBK1 activation

37

TBK1

Cytoplasm

Phosphorylates, and therefore activates, IRF3 and IRF7

37

TICAM1

Cytoplasm

Interacts with TANK, thus indirectly regulating TBK1 activation

37

TOM70

Mitochondria

Binds MAVS during viral infection, thereby facilitates the recruitment of TBK1 and IRF3

32

TRADD

Cytoplasm

Involved in a FADD- and RIPK1‑containing MAVS complex

37

TRAFs

Cytoplasm, mitochondria

TRAF3 is an upstream regulator of IKKε and TBK1 activation, TRAF2 and TRAF6 bridge MAVS to NF‑κB signalling

TUFM

Mitochondria

Connects the MAVS interactor NRLX1 to the autophagic machinery

29

VDAC1

Mitochondria

Inhibited by MAVS, which results in anti-apoptotic effects

44

WDR5

Cytoplasm

Indicated as essential for the assembly of the MAVS complex

37

37

37

25,27

ATG, autophagy-related; AZI2, 5‑azacytidine-induced 2; C1QBP, complement component 1q subcomponent-binding protein; DDX3X, DEAD (Asp-Glu-Ala-Asp) box protein 3 X‑linked; DHX58, DEXH (Asp-Glu‑X‑His) box protein 58; FADD, FAS-associated with a death domain; IFN, interferon; IKK, inhibitor of NF‑κB kinase; IRF, IFN-regulatory factor; ITCH, itchy E3 ubiquitin protein ligase; MAM, mitochondria-associated membrane; MAVS, mitochondrial antiviral signalling; MDA5, melanoma differentiation-associated protein 5; MFN, mitofusin; NF‑κB, nuclear factor-κB; NLRX1, NLR family member X1; PCBP, poly(rC)-binding protein; PLK1, Polo-like kinase 1; PSMA7, proteasome subunit, α-type 7; RIG-I, retinoic acid-inducible protein I; RIPK1, receptor-interacting protein kinase 1; RLR, RIG‑I‑like receptor; RNF, ring finger; STING, stimulator of IFN genes; TANK, TRAF family member-associated NF‑κB activator; TBK1, TANK-binding kinase 1; TICAM1, TIR domain-containing adaptor molecule 1; TOM70, translocase of outer mitochondrial membrane 70 kDa subunit; TRADD, TNFR1‑associated death domain; TRAF, TNF-associated factor; TUFM, mitochondrial Tu translation elongation factor; VDAC1, voltage-dependent anion channel 1; WDR5, WD repeat domain-containing protein 5. *Predominant localization when not engaged in interactions with MAVS.



REVIEWS Sending cell-intrinsic danger signals Mitochondria not only decode incoming signals of dan‑ ger by activating adaptive responses or by promoting cell death, but they also emit danger signals that alert the cell of situations of mitochondrial stress. Indeed, as a result of more than one billion years of co‑evolution, most of which as obligatory endosym‑ bionts, mitochondria and eukaryotic cells have devel‑ oped an intricate signalling system that allows them to coordinately adapt to changing microenvironmental con‑ ditions47. Alongside, reflecting the crucial importance of mitochondria for metabolism, host cells have evolved a series of mechanisms whereby they sense (and attempt a ↑O2 ↓ROS

e

↓O2 ↑ROS

Mitochondrion HIF1α

to correct) perturbations in mitochondrial homeostasis (FIG. 2). Thus, well before igniting a cell-wide wave of MOMP that would seal the fate of the cell, mitochon‑ dria react to stress conditions by delivering specific danger signals that cells translate into adaptive responses aimed at re‑establishing homeostasis10,48,49. These sig‑ nals include mitochondrial products such as mtDNA and ROS (FIG. 2a,b), as well as nucleus-encoded proteins that have been integrated into refined signalling circuit‑ ries that bridge mitochondria and their hosts (FIG. 2c,d). Of note, when homeostasis cannot be restored, excess mitochondrial danger signals contribute to the activation of cell death mechanisms10,48,49.

HIF1α

HIF

1α

1α

HIF

F

HI

VEGF

HIF1α

1α

HIF1α

Nucleus

HIF1α

Proteasome

b ↓ROS

IκB NF-κB

f

↑ROS

NF-κB

NF

IκB

IκB NF-κB

-κ B

BCL2

mtDNA

BCL2 Inflammasome activation

c

↑Δψ

g ↓Δψ

↑ATP

↓ATP

↑AMP

1

PINK

Parkin

PIN

K1

PINK1 PINK1 PARL PINK1

P ULK1

MFN1 MFN2 BCL-2 VDAC1

PARL

h mtUPR on

mtUPR off

P

PINK1

PINK1

d

AMPK

Ub

Mitophagy

PKR

JUN P

ATFS1

ATFS1 LONP1 ATFS1

P

ATFS1

ATFS1

ATFS1

P

elF2α ATFS1

Translational block

FOS P AP-1 HSPD1 HSPD1

LONP1

ATFS1

Unfolded protein

Nature Reviews | Molecular Cell Biology 784 | DECEMBER 2012 | VOLUME 13


REVIEWS Mitochondria constantly generate ROS as a byproduct of respiration, a physiological process that is normally kept in check by a diversified set of antioxidant defences50. However, in response to stimuli such as hypoxia, viral infection and perturbations of mitochon‑ drial metabolism (including Δψm dissipation; see below), the intracellular levels of free ROS increase and convey danger signals. Mitochondria have recently been shown to cluster around the nucleus during hypoxia, induc‑ ing oxidative modifications of the vascular-endothelial growth factor (VEGF) promoter that are required for the optimal binding of hypoxia-inducible factor 1α (HIF1α) and gene transactivation51 (FIG. 2e). Similarly, mitochondrial ROS are needed or the activation of the pro-survival NF‑κB pathway 52 and of the NLRP3 (NLR family, pyrin domain-containing 3) inflamma‑ some (FIG. 2f), a supramolecular platform that mediates the proteolytic maturation and secretion of potent proinflammatory cytokines such as IL‑1β and IL‑18 (REF. 53). Of note, mtDNA is not only released by mitochondria in response to ROS-mediated inflammasome activation54 but also operates as a bona fide inflammasome activa‑ tor (in its oxidized form) following MOMP55. Thus, ROS and mtDNA may participate in a feedforward signal‑ ling circuitry whereby mitochondrial danger signals are first translated into adaptive responses and then, if homeostasis cannot be re‑established, execute cell death. Intriguingly, NLRP3 is particularly enriched at MAMs, and the NLRP3‑dependent secretion of IL‑1β is inhibited by the knockdown of either VDAC1 or VDAC2 (REF. 53). These observations lend further support to the hypothesis that MAMs constitute essential intracellular synapses linking mitochondrial functions to danger signalling. ◀ Figure 2 | Intracellular danger signals emitted by mitochondria. a–d | In physiological conditions, mitochondria maintain a high transmembrane potential (Δψm), which is used to generate ATP and for the import of proteins, including PTEN-induced putative kinase 1 (PINK1) and activating transcription factor associated with stress 1 (ATFS1). In this setting, reactive oxygen species (ROS) are kept to a minimum (a,b), hypoxia-inducible factor 1α (HIF1α) is degraded by an O2-dependent, proteasome-mediated mechanism (a), inhibitor of NF‑κB (IκB) proteins sequester nuclear factor-κB (NF‑κB) subunits in the cytoplasm (b), PINK1 (c) and ATFS1 (d) are degraded within mitochondria by presenilinassociated rhomboid-like (PARL) and LON peptidase 1 (LONP1), respectively, and the mitochondrial unfolded protein response (mtUPR) is off (d). e–h | During hypoxia, HIF1α accumulates, dimerizes, gains access to the nucleus and transactivates genes such as the one encoding vascular endothelial growth factor (VEGF). This process is facilitated by oxidative modifications of the VEGF promoter that accompany the hypoxia-induced perinuclear clustering of mitochondria (e). High levels of ROS promote the proteasomal degradation of IκB, allowing NF‑κB subunits to dimerize, enter the nucleus and transactivate pro-survival genes such as B cell lymphoma‑2 (BCL2). In addition, ROS stimulate the activation of the inflammasome, a phenomenon that both facilitates and is facilitated by the release of mitochondrial DNA (mtDNA) (f). Upon Δψm dissipation, PINK1 accumulates at the mitochondrial outer membrane and recruits parkin, which ubiquitylates proteins such as BCL‑2, voltage-dependent anion channel 1 (VDAC1) and mitofusins (MFN1 and MFN2). Concomitantly, increased AMP levels promote the activation of AMP-activated protein kinase (AMPK), which phosphorylates unc‑51‑like kinase 1 (ULK1). Together, these events stimulate mitophagy (g). During the mtUPR, mitochondrial import of ATFS1 is blocked, resulting in its dimerization, nuclear translocation and hence in the upregulation of mitochondrial chaperones like heat shock protein 60 (HSP60; encoded by HSPD1). Alongside, the mtUPR is sensed by PKR (protein kinase, RNA-activated), which promotes a translational block by phosphorylating eukaryotic translation initiation factor 2α (eIF2α) and the activation of the transcriptional factor AP‑1, resulting in HSP60 upregulation (h). Ub, ubiquitin.

Several mitochondrial functions, including the pro‑ duction of ATP by the F1F0-ATP synthase and the import of nucleus-encoded proteins depend on the Δψm (REF. 47). Accordingly, the widespread and permanent dissipa‑ tion of the Δψm, as occurs during MOMP, is sufficient to induce a bioenergetic crisis that is cytotoxic, although with delayed kinetics, even in the absence of caspase activation4,56. Moreover, depolarized mitochondria pro‑ duce ROS at high levels, a setting in which ROS exert cytotoxic rather than signalling functions49. It is there‑ fore not surprising that cells have evolved molecular cascades that rapidly sense (and react to) Δψ m dissi pation as a mitochondrial danger signal. Perhaps the best-characterized of these pathways relies on PTEN induced putative kinase 1 (PINK1) and the ubiquitin ligase parkin, two proteins that are frequently mutated in individuals affected by early-onset parkinsonism57. Whereas PINK1 is efficiently imported into healthy mitochondria and degraded by presenilin-associated rhomboid-like (PARL), the same cannot occur upon Δψm dissipation, which leads to PINK1 accumulation at the mitochondrial surface. In turn, PINK1 favours the recruitment and activation of parkin, which ubiquity‑ lates several mitochondrial outer membrane proteins, including BCL‑2, VDAC1, MFN1 and MFN2, leading to the selective removal of potentially dangerous mitochon‑ dria via mitophagy 48 (FIG. 2g). Intriguingly, mitochondrial dysfunction may relay danger signals even before Δψm dissipation, owing to the accumulation of AMP in con‑ ditions in which respiration occurs at suboptimal rates. This leads to AMP-activated protein kinase (AMPK) activation and mitophagy 58. Similarly to the ER, mitochondria can react to the accumulation of misfolded proteins into the mitochon‑ drial matrix by activating an organelle-specific unfolded protein response (UPR)59. This can be induced experi‑ mentally or occurs spontaneously under pathophysio‑ logical conditions, including obesity 60. The mechanisms underlying the mitochondrial UPR have only recently begun to emerge and seem to involve components of the signalling pathway that is elicited during the ER UPR, such as the phosphorylation of eukaryotic trans‑ lation initiation factor 2α (eIF2α)61,62. Thus, in response to mitochondrial protein overload, both PKR (protein kinase, RNA-activated; also known as EIF2AK2) and GCN2 (general control non-derepressible 2) seem to phosphorylate eIF2α and hence block protein transla‑ tion via a mechanism that (at least in some settings) involves ROS61,62. Moreover, upon the activation of the mitochondrial UPR, PKR reportedly phosphorylates the transcription factor JUN to stimulate the produc‑ tion of the mitochondrial chaperone HSP60 (REF. 62). Intriguingly, both these functions of PKR require the mitochondrial caseinolytic peptidase (CLPP), sug‑ gesting that, in conditions of mitochondrial protein overload, CLPP may generate protein fragments that operate as danger signals. In addition, the mitochondrial UPR has been asso‑ ciated with specific signal transduction pathways. For instance, it has been shown that ATFS1 (activat‑ ing transcription factor associated with stress 1) is



REVIEWS a

pDC

Neutrophil

AGER

mtDNA

NFP

TLR9

TFAM

FPR1

Type I IFN production

Neutrophil activation Multiorgan failure and SIRS

b

ATP

Apoptotic T cell

DC P2RX7

c

Cardiolipin

CD1d

APC

P2Y2 Priming of γδ T cells

Inflammasome activation

Figure 3 | Examples of extracellular danger signals emitted by mitochondria. Nature Reviews | Molecular Cell Biology In response to stress or tissue injury, mitochondria emit danger signals that the immune system translates into local or systemic responses. a | TFAM (mitochondrial transcription factor A) is detected by AGER (advanced glycosylation end product-specific receptor) on pDCs (plasmacytoid dendritic cells), mtDNA (mitochondrial DNA) by TLR9 (Toll-like receptor 9) on pDCs and neutrophils, and NFPs (N‑formyl peptides) by FPR1 (formyl peptide receptor 1) on neutrophils. This leads to type I interferon (IFN) secretion by pDCs and to the activation of neutrophils, resulting in multiorgan failure and systemic inflammatory response syndrome (SIRS). b | ATP, which is released by an autophagydependent pathway under specific circumstances (for instance, during immunogenic cell death), can bind purinergic P2RX7 receptors on DCs, thus activating the inflammasome and promoting immune responses. In addition, ATP secreted during tissue injury functions as a chemoattractant for multiple cell types by binding to P2Y2 receptors. c | Cardiolipin presented in association with the CD1d receptor by antigen-presenting cells (APCs) drives the priming of cardiolipin-specific γδ T cells.

CpG Cytosine-guanosine DNA sequence. Unmethylated CpG sequences are prevalent in bacterial DNA but rare in eukaryotic genomes.

Systemic inflammatory response syndrome (SIRS). A frequently lethal clinical condition that resemble sepsis in its manifestations but does not necessarily involve a microbial component.

Plasmacytoid dendritic cells (pDCs). A subset of DCs that differ from their conventional (also known as myeloid) counterparts in morphology and in their capacity to produce copious amounts of type I interferon in response to viruses and Toll-like receptor ligands.

imported into mitochondria and degraded by LONP1 (LON peptidase 1) under physiological conditions, but not when the mitochondrial UPR is activated 63. In this setting, ATFS1 accumulates in the cytosol and traffics to the nucleus, where it transactivates genes coding for mitochondrial chaperones such as HSP60 (REF. 63) (FIG. 2h). Taken together, these examples demonstrate that, in response to organellar stress, mitochondria emit a set of danger signals that host cells translate into adaptive responses aimed at re‑establishing homeostasis.

Sending systemic danger signals Accumulating evidence indicates that multiple mito‑ chondrial components convey danger signals not only at a cell-intrinsic level but also as paracrine or endocrine modulators, thus eliciting potent biological or immuno‑ logical responses that can involve whole organs or even the entire organism10. In most cases, such mitochondrial components are released into the extracellular space when cell-intrinsic adaptive responses (such as those activated by viral infection) fail to restore homeostasis and cell death ensues, hence alerting the whole organism of an unresolved situation of danger.

mtDNA can be detected in the bloodstream fol‑ lowing massive episodes of necrotic cell death, such as upon trauma, burns, fractures or acetaminopheninduced liver damage11,64,65. As it contains unmethylated CpG domains (similarly to bacterial DNA), circulating mtDNA is detected by Toll-like receptor 9 (TLR9), an endosomal PRR that is expressed by multiple cells of the innate immune system66. Extracellular mtDNA recruits neutrophils and promotes their activation in a TLR9‑dependent manner, resulting in severe organ injury and eventually in the systemic inflammatory response syndrome (SIRS) 11,64 (FIG. 3a) . Furthermore, mtDNA contributes to the pathogenesis of chronic diseases, including heart failure, as recently shown in an autophagy-deficient mouse model of pressure overload12. In this setting, mtDNA seems to set off a cell-autonomous TLR9‑dependent signalling pathway that promotes local inflammation upon the release of IL‑1β (which is indicative of inflammasome activation) and IL‑6 (REF. 12). Thus, autophagy operates as a major brake of mtDNA-mediated inflammation12,54, and it does so both by intercepting damaged mitochondria before they release mtDNA, thus blocking inflam‑ masome activation54 (see above), and by promoting the degradation of mtDNA within endosomes, thus impeding the activation of TLR9 (REF. 12). Intriguingly, mtDNA is also released in a rapid, ‘catapult-like’ man‑ ner by IL‑5- or IFNγ-primed neutrophils that responde to lipopolysaccharide from Gram-negative bacteria67. This phenomenon, which seems to occur via a ROSdependent mechanism, contributes to the generation of extracellular structures that exert antibacterial activity in vitro and in vivo67. Similarly to mtDNA, NFPs, which are specifically produced by mitochondria and closely resemble bacte‑ rial components, are released by dying cells and have recently been shown to be involved in the development of post-traumatic SIRS 11, acetaminophen-induced liver damage64, bone fracture-ensuing lung injury 65 and smoking-triggered lung emphysema68. By binding to formyl peptide receptor 1 (FPR1) on the surface of neutrophils, NFPs stimulate intracellular Ca2+ signalling and the activation of mitogen-activated protein kinases (MAPKs), thus functioning as potent chemoattractants and triggers of degranulation and oxidative bursts 69 (FIG. 3a). Inhibition of FRP1 with cyclosporine H, block‑ ing antibodies or gene knockout suppresses the signal transduction pathway that is elicited by NFPs11,64,65,68 and ameliorates the manifestation of NFP-related conditions in mice64,68. Thus, NFPs constitute pathophysiologically relevant danger signals. TFAM (mitochondrial transcription factor A), a highly abundant mtDNA-binding protein that is encoded by the nuclear genome, has recently been shown to synergize with CpG-containing oligonucleotides and mtDNA in stimulating plasmacytoid dendritic cells (pDCs) to produce type I IFN70 (FIG. 3a). TFAM is functionally and structurally homologous to high mobility group box 1 (HMGB1), a non-histone nuclear protein that is released into the extracellular space upon cell death, conveying danger signals71. Moreover, the stimulatory



REVIEWS CD1d A glycoprotein expressed on the surface of several antigen-presenting cells that is involved in the presentation of lipid antigens to specific subsets of T cells.

Antigen-presenting cells (APCs). Crucial cellular components of the adaptive immune response owing to their capacity to process antigens and present antigenic peptides to T cells in the context of appropriate stimulatory signals.

Immunogenic cell death A functionally unconventional type of apoptosis that results in the activation of an adaptive immune response against antigens from dead cells.

γδ T cells Type of T cell bearing an unconventional T cell receptor composed of one γ-chain and one δ‑chain, rather than one α-chain and one β‑chain. Their antigenic repertoire is not yet well characterized, but γδ T cells seem to play a prominent part in the recognition of lipid antigens.

Sterile inflammation Inflammatory condition that develops in the complete absence of a microbial component.

1. 2. 3.

4.

5. 6. 7.

effect of TFAM on pDCs depends on AGER (advanced glycosylation end product-specific receptor; also known as RAGE), which is one of the main mediators of the pro-inflammatory functions of extracellular HMGB1 (REF. 72). These observations suggest that HMGB1 and perhaps other non-mitochondrial DAMPs may have evolved from bacterial proteins. Additional mitochondrial factors that can operate as danger signals include (but presumably are not limited to) ATP, cardiolipin, SMAC, HSP60 and the β‑subunit of the F1-ATPase10. ATP, the major metabolic product of mitochondria, is actively secreted via an autophagydependent mechanism by cancer cells that succumb to specific cell death inducers73. Extracellular ATP not only functions as a potent chemoattractant 69,74 but also stimulates the activation of the NLRP3 inflammasome by binding to purinergic P2RX7 receptors71 (FIG. 3b). Cardiolipin, a tetra-acylated phospholipid that is nor‑ mally confined to the mitochondrial inner membrane, can be released or exposed to the cell surface following cell death or injury 75. It reportedly binds to the CD1d receptor on the surface of antigen-presenting cells (APCs), which then prime and activate a cardiolipin-specific population of γδ T cells76 (FIG. 3c). The overexpression of a cytosolic variant of SMAC has recently been shown to promote immunogenic cell death71,77, which suggests that cytosolic SMAC-mediated pathways may indirectly relay danger signals to the immune system. Both HSP60 and the F1-ATPase β‑subunit are targeted by autoanti‑ bodies that contribute to (or at least accompany) ath‑ erosclerosis78 and vasculitis79. Interestingly, an ectopic variant of the F1-ATPase‑β subunit may act as the recep‑ tor for the docking of HSP60 at the surface of endothe‑ lial cells, reminiscent of the mitochondrial interaction between these two proteins79. Of note, extracellular and circulating HSP60 seem to stimulate a multi-pronged pro-inflammatory response 80, whereas cell surface F1-ATPase has been suggested to mediate the presenta‑ tion of phosphorylated antigens to a particular subset of γδ T cells81. So, multiple mitochondrial components, once exposed or released in the extracellular microenvironment or in

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8. 9. 10.

11.

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the bloodstream (often upon cell death) convey danger signals that are decoded by the organism to promote local or systemic innate immune responses (FIG. 3).

Concluding remarks In addition to being the major source of intracellular ATP and to having a prominent role in the regulation of cell death, mitochondria are deeply involved in signal‑ ling pathways elicited by perturbations in homeostasis. On the one hand, mitochondria are capable of decoding incoming danger signals (for instance, those triggered by viral infection) and translating them into appropriate adaptive responses (for example, the production of anti‑ viral factors such as type I IFN or the activation of cell death). On the other hand, stressed mitochondria emit intracellular danger signals that are translated by the cell into homeostatic responses and systemic DAMPs that, if homeostasis cannot be restored and cell death ensues, are sensed by the innate immune system to promote sterile inflammation. For these reasons, mitochondria can be viewed as master regulators of danger signalling. Despite more than a billion years of co‑evolution, mitochondria have preserved the ability to elicit potent biological and immunological responses. It has already been shown that some of these evolutionarily ancient sig‑ nalling cascades (such as those mediated by TLRs) can be harnessed to promote therapeutic immune responses (for instance, in the context of cancer immunotherapy)82. Further work is required to assess whether mimicking other mitochondrial danger signals, for example with compounds that activate FPR1, could be used therapeu‑ tically to stimulate the immune system. In addition, it will be interesting to see whether inhibiting pro-inflammatory signalling cascades that are activated by mitochondrial DAMPs represents an efficient therapeutic approach to life-threatening conditions such as SIRS. Of note, the molecular and cellular circuitries underlying the emission of systemic DAMPs by mitochondria and their detection at the systemic level have just begun to emerge. The list of clinically relevant conditions in which mitochondrial DAMPs play a prominent part is, in our opinion, destined to grow significantly in the near future.

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Acknowledgements

The authors are supported by the Ligue contre le Cancer (équipe labellisée), AXA Chair for Longevity Research, Cancéropôle Ile‑de‑France, Institut National du Cancer (INCa), Fondation Bettencourt–Schueller, Fondation de France, Fondation pour la Recherche Médicale, Agence National de la Recherche and the European Commission (Apo-Sys, ArtForce, ChemoRes. Death-Train) and the LabEx Immuno-Oncology.

Competing interests statement

The authors declare no competing financial interests.

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