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S O U R C E : Nature (epdf) (Warning: Paywall activates after some minutes).
Abstract
Through their many and varied metabolic functions, mitochondria power life. Paradoxically , mitochondria also have a central role in apoptotic cell death. Upon induction of mitochondrial apoptosis, mitochondrial outer membrane permeabilization (MOMP) usually commits a cell to die. Apoptotic signalling downstream of MOMP involves cytochrome c release from mitochondria and subsequent caspase activation. As such, targeting MOMP in order to manipulate cell death holds tremendous therapeutic potential across different diseases, including neurodegenerative diseases, autoimmune disorders and cancer. In this Review , we discuss new insights into how mitochondria regulate apoptotic cell death. Surprisingly , recent data demonstrate that besides eliciting caspase activation, MOMP engages various pro- inflammatory signalling functions. As we highlight, together with new findings demonstrating cell survival following MOMP, this pro- inflammatory role suggests that mitochondria- derived signalling downstream of pro- apoptotic cues may also have non- lethal functions. Finally , we discuss the importance and roles of mitochondria in other forms of regulated cell death, including necroptosis, ferroptosis and pyroptosis. Collectively , these new findings offer exciting, unexplored opportunities to target mitochondrial regulation of cell death for clinical benefit.
Mitochondria are essential for life. Positioned at the heart of cellular metabolism, they serve a key role in ATP generation via oxidative phosphorylation. Beyond their many core metabolic functions, mitochondria are implicated in an expanding array of cellular processes, ranging from inflammation to regulation of stem cell generation1,2. In what may seem a paradox,mitochondria are often essential for cell death.
Regulated cell death underpins health; for example, inhibition of cell death promotes cancer and auto-immunity whereas excessive cell death contributes to neurodegenerative diseases, including Parkinson dis-ease, Alzheimer disease, amyotrophic lateral sclerosis and Huntington disease. Consequently, considerable interest has centred upon targeting of mitochondria to manipulate cell death in disease. Validating this rationale, recently developed anticancer drugs called BH3- mimetics sensitize cells to mitochondria- dependent death, displaying potent antitumour activity3,4. The role of mitochondria in cell death is unequivocally established in apoptosis, where mitochondrial outer membrane permeabilization (MOMP) driven by effector pro- apoptotic members of the B cell lymphoma 2 (BCL-2) family of proteins (prominently BAX and BAK; BOX1) initiates a signalling cascade that leads to cell death; although, as we have now come to appreciate, induction of MOMP is not synonymous with apoptosis and the commitment of a cell to die is not definitive downstream of MOMP. In addition, MOMP has other consequences beyond execution of cell death, including induction of pro- inflammatory signalling. Finally, while apoptosis is a major form of regulated cell death, it is by no means the only one. More recently described types of regulated cell death include necroptosis, pyroptosis and ferroptosis. Mitochondria have also been implicated in these addi-tional modalities of regulated cell death, but their roles are still poorly defined and appear less conspicuous.
In this Review we discuss how mitochondria con-tribute to regulated cell death, placing this contribution in the context of health and disease. Specifically, we highlight new insights into how mitochondria initiate apoptosis, and discuss their parallel role in eliciting pro- inflammatory signalling activity with important con-sequences for physiology. Taken together with recent studies showing heterogeneity in MOMP between mitochondria within a cell treated with pro- apoptotic stimuli, we highlight that mitochondrial permeabiliza-tion can exert various non- lethal signalling functions. We then discuss the contribution of mitochondria to more recently described types of regulated cell death, highlighting mitochondria as a central nexus between different cell death modalities.
Mechanisms of mitochondrial apoptosis
Apoptotic cell death is a major form of regulated cell death that has central roles in many processes ranging from embryonic development to immune homeostasis5. As we now discuss, in many instances, mitochondria are crucial for the initiation of apoptosis.
Apoptotic signalling. There are two main apoptotic signalling pathways: the extrinsic (also called death receptor) pathway of apoptosis and the intrinsic, or mito-chondrial, pathway of apoptosis (FIG.1). Both converge upon activation of caspase 3 and caspase 7. As proteases, these executioner caspases cleave hundreds of different proteins causing the biochemical and morpho-logical hallmarks of apoptosis6. The extrinsic pathway is activated at the plasma membrane by death receptor ligands binding to their cognate receptors, leading to activation of caspase 8 (a component of a complex known as the death- inducing signalling complex (DISC))7. Active caspase 8 propagates apoptosis by cleaving pro- caspase 3 and pro- caspase 7, causing their activation (FIG.1).
Diverse cellular stresses, for instance growth- factor deprivation or DNA damage, kill via the mitochon-drial pathway of apoptosis. The mitochondrial pathway requires MOMP to release soluble proteins from the mito-chondrial intermembrane space, leading to cell death (FIG.1). Amongst these intermembrane space proteins, cytochrome c — an essential component of the electron transport chain — binds the adaptor molecule apoptotic peptidase activating factor 1 (APAF1), forming a com-plex called the apoptosome8. The apoptosome, in turn, binds to and activates the initiator caspase 9, which sub-sequently cleaves and activates the executioner caspases. MOMP also causes the release of proteins including SMAC and OMI that block the caspase inhibitor XIAP, facilitating apoptosis. The extrinsic apoptotic pathway crosstalks to the mitochondrial pathway by caspase8- mediated cleavage of BID, a pro- apoptotic BH3-only member of the BCL-2 family (BOX1), which generates tBID that potently induces MOMP (FIG.1).
With some notable exceptions that we will later dis-cuss, MOMP typically commits cells to death, even in the absence of caspase activity (this phenomenon is known as caspase- independent cell death). Thus, MOMP is con-sidered a point of no return in apoptosis execution9–11. Consistent with MOMP being the point of commitment to cell death, mice deficient in caspase activity associ-ated with the mitochondrial pathway of apoptosis (for example, APAF1–/– and caspase-9–/–) display much milder developmental defects than MOMP- inhibited (BAX–/–, BAK–/–) mice12–17. The reason for MOMP being able to mediate caspase- independent cell death is overall meta-bolic catastrophe, related to the fact that often all mito-chondria undergo MOMP during apoptosis18 and their progressive dysfunction following MOMP causes wide-spread ATP loss19. Because MOMP serves to commit a cell to die, it is tightly regulated, primarily by members of the BCL-2 protein family (BOX1).
Mechanisms of MOMP. During mitochondrial apoptosis, activation of the pro- apoptotic effectors BAX and BAK is usually essential for MOMP and cell death20. BAX and BAK are largely considered redundant because only upon their combined loss are cells resistant to mitochon-drial apoptosis and extensive developmental defects are observed15,16,20. Nevertheless, differences for BAX versus BAK in mitochondrial apoptosis have been reported in some studies21,22. For example, BAX and BAK display a differential requirement for the mitochondrial porin voltage- dependent anion- selective channel 2 (VDAC2) in their ability to induce apoptosis: while VDAC2 asso-ciates with both proteins, VDAC2 is required for BAX, but not BAK, to induce apoptosis23–25. Importantly, such differences in apoptotic requirement for BAX or BAK can govern the effectiveness of chemotherapy responses that often require mitochondrial apoptosis21.
In healthy cells, BAX localizes to the cytoplasm and BAK to the mitochondria; however, both can shut-tle between the mitochondria and the cytoplasm26–28 (FIG.2). Under basal conditions, BAX and BAK are inactive. Following activation, BAX accumulates at the mitochondria. BAX and BAK can be directly activated by binding a subclass of BH3-only proteins called direct activators (BID, PUMA and BIM)29. Structural studies have demonstrated that the direct activator BH3 domain binds within the hydrophobic groove of BAX and BAK, leading to extensive conformational changes, allowing activation30–32. This structural information has guided the development of modified BH3 peptides derived from BH3-only proteins that block BAK activation, providing a proof- of-concept for therapeutic targeting of this step to block cell death33.
Experiments with chemically stabilized BH3 peptides also enabled the discovery of a second BH3-binding site in BAX34. This second BH3-binding site is distant from the BAX hydrophobic groove, located in the amino terminus of the protein, and promotes BAX activa-tion through an allosteric conformational change34,35. Notably, BAX- activating small molecules that target this amino- terminal site and promote BAX activation display potent antitumour activity36. Reconciling a requirement for two activation sites, recent data support a sequential model of BAX activation in which BH3-proteins first bind the amino- terminal site, facilitating BH3 bind-ing to the hydrophobic groove for full BAX activity37. Of note, there is evidence that BH3-only proteins are not absolutely essential for BAX and BAK activation (see BOX1). During activation, BAX and BAK expose their BH3 domains, which can further propagate their own activity35,38. Once activated, BAX and BAK homo-di merize and these dimers form higher- order oligomers that are essential for MOMP39–43 (FIG.2).
How do active BAX and BAK permeabilize the mitochondrial outer membrane, initiating cell death? Consensus about this long- standing question centres on activated BAX and BAK inducing lipidic (toroidal) pores in the mitochondrial outer membrane (FIG.2). Such lipidic pores are formed by fusion of the inner and outer leaflets of membranes, which is promoted and stabilized by protein insertion. Indeed, studies using syn-thetic liposomes and mitochondrial outer membrane- derived vesicles demonstrate that BAX can induce large (>100 nm) membrane pores visible by cryo- electron microscopy that grow over time44,45. Moreover, BAX pores are tuneable in size dependent on the BAX con-centration45. Importantly, super- resolution microscopy has enabled direct visualization of BAX- mediated pores in apoptotic cells46,47. On apoptotic mitochondria, BAX localizes in heterogeneous ring- like structures, roughly approximating in size to holes observed in mitochon-drial outer membrane- derived vesicles. Formation of such rings on apoptotic mitochondria was associated with membrane permeabilization, further supporting permeabilization of the mitochondrial outer membrane via lipidic pore formation46.
Extensive genetic and biochemical data firmly establish BAX and BAK as central effectors of MOMP. However, other proteins can also cause MOMP. Particular interest has focused on BOK, a BAX/BAK- like BCL-2 protein, since recent studies have demonstrated that BOK can induce MOMP and cell death in the absence of BAX and BAK48,49. Genetic support for this observation comes from the finding that BOK deficiency exacerbates the developmental defects observed in Bax–/–Bak–/– double- knockout mice15. Nevertheless, BOK- induced MOMP differs from classical BAX/BAK- dependent MOMP in many ways. For instance, unlike BAX and BAK, the pro- apoptotic activity of BOK does not appear to be regulated by BCL-2 proteins in any way48,50. Invitro liposome and mitochondrial permeabilization assays demonstrate that BOK is inherently active48,51. This constitutive activity relates to the intrinsic instability of the BOK hydropho-bic core such that it can mediate MOMP independently of BH3-only proteins51. Consistent with BOK having constitutive pro-apoptotic activity, in healthy cells BOK undergoes endoplasmic reticulum- associated degradation, which maintains the protein at low levels48. However, because BOK is expressed in many healthy tissues, additional regulatory mechanisms must exist to counter its pro- apoptotic activity52.
Non- BCL-2 family proteins can also induce MOMP. Specific members of the gasdermin protein family exhibit pore- forming activity upon cleavage. As we will discuss later, cleavage of gasdermin D (GSDMD) is essential for an inflammatory type of cell death called pyroptosis. During mitochondrial apoptosis, caspase 3- mediated cleavage of gasdermin E (GSDME; also known as DFNA5) liberates a pore- forming amino- terminal fragment that can promote plasma membrane perme-abilization during apoptotic cell death53,54. GSDME- mediated plasma membrane permeabilization induces a necrotic- like cell death that has been proposed to con-tribute to the chemotherapy- associated toxicity53. This GSDME amino- terminal cleavage fragment can also localize to the mitochondria and cause MOMP55. In this manner, GSDME is proposed to elicit a feedforward mechanism that enhances caspase activation during apoptosis. In an analogous manner, during pyroptosis, the GSDMD amino- terminal cleavage fragment can also induce MOMP55 (see also below). Although requir-ing further investigation, given their established pore- forming properties, the amino- terminal fragments of gasdermins likely directly permeabilize mitochondria independently of BAX and BAK.
Dynamics of MOMP. Independent of apoptotic stress, MOMP is usually rapid and complete — all mito-chondria undergo MOMP over a 10-min window18,56. Emphasizing an earlier point, the extensive nature of MOMP is likely central to it being a point of no return in apoptotic commitment. High- speed imaging of mitochondrial apoptosis has shown that MOMP can initiate in a discrete subpopulation of mitochondria, before progressing in a wave- like manner across all of the mitochondria in the cell57–59. Using frog egg extracts invitro, MOMP has been found to propagate between mitochondria as a trigger wave, maintaining constant speed and amplitude over a long distance; this may facilitate the execution of apoptosis in large cells such as neurons60.
Why is MOMP rapid and extensive? One model proposes that MOMP initiates a caspase- dependent feedforward loop, possibly by caspase- mediated BID cleavage that promotes further MOMP. However, while caspase activity supports MOMP trigger- wave propagation invitro, blocking caspase activity following a mitochondrial apoptotic stimulus affects neither the kinetics nor the extent of MOMP in cells18. Furthermore, inhibiting caspase activity following a mitochondrial apoptotic stimulus usually does not protect against cell death. These findings argue against an important role for caspase activity in amplifying MOMP. Other proposed mechanisms include reactive oxygen species (ROS)-dependent feedforward propagation of MOMP, although how ROS promote this remains unclear61. Perhaps the most likely explanation centres on the ability of active BAX and BAK to activate other BAX and BAK molecules35,38. Akin to falling dominoes, this would be predicted to rapidly and extensively drive MOMP.
Inner mitochondrial membrane remodelling during apoptosis. Soluble mitochondrial intermembrane space proteins are released following MOMP irre-spective of protein size62. However, some studies have shown that the release of cytochrome c can be fur-ther regulated even following MOMP, affecting caspase activation and apoptosis63–67. This is because themajority of cytochrome c resides within mitochondrial cristae — dynamic inner mitochondrial membrane folds that harbour electron transport chain components. Cristae accessibility to the intermembrane space is regulated by cristae junctions68. As such, cytochrome c has been proposed to be trapped within cristae in healthy cells, necessitating widening of the cristae junctions in order to allow efficient cytochrome c release. Indeed, following MOMP, extensive cristae remodelling has been observed. But how is this remodelling regulated? Mitochondria are dynamic organelles that constantly undergo cycles of fission and fusion. Immediately fol-lowing MOMP, extensive mitochondrial fragmentation occurs at mitochondria–endoplasmic reticulum contact sites69, which requires the mitochondrial fission protein dynamin related protein 1 (DRP1)58,66. Although dispen-sable for MOMP70,71, DRP1 promotes cristae remodel-ling, which has been proposed to facilitate cytochrome c release. Several reports suggest that remodelling occurs via the effect of DRP1 on the GTPase OPA1. In the intermembrane space, OPA1 regulates inner mitochon-drial membrane fusion and the cristae junction size: oli-gomers of OPA1 keep junctions narrow, whereas OPA1 oligomer disassembly widens the junctions70. Following MOMP, OPA1 is cleaved by different intermembrane space proteases including OMA1, leading to oligomer disassembly and junction opening72–74. During apopto-sis, DRP1 is modified with the ubiquitin- like protein SUMO, leading to stabilization of the mitochondria–endoplasmic reticulum membrane contact sites. This promotes calcium influx into the mitochondria from the endoplasmic reticulum, which has been shown to be required for cristae remodelling69. However, it has also been shown that cristae remodelling mediated by DRP1 during apoptosis is independent of OPA1 and that OPA1 oligomers can disassemble even in the absence of DRP1 (REF.75).Regardless of the exact mechanism, the importance of inner membrane remodelling for mitochondrial apoptosis is controversial. For instance, some studies have shown that inhibiting components of the cristae remodelling machinery (for example, DRP1) has min-imal effect upon the release of cytochrome c, caspase activation and apoptosis70,71. Second, inner mitochon-drial membrane remodelling has been reported to occur as a secondary consequence of caspase activation76. Irrespective of caspase activity, inner mitochondrial membrane remodelling occurs subsequent to MOMP. Thus, similar to caspase inhibition, blocking inner mitochondrial membrane remodelling would not be expected to prevent cell death unless cells can somehow survive MOMP — an area we now discuss.
Surviving MOMP
Although MOMP is considered the point of no return in mitochondrial apoptosis, some exceptions exist where, downstream of apoptotic stimuli, MOMP occurs to vary-ing degrees with wide- ranging effects, beyond lethality. It is also now evident that cells are able to survive MOMP, which can have an important impact on physiology. Our discussion centres on how cells can survive MOMP in three distinct settings: widespread MOMP under caspase- inhibited conditions; limited MOMP; and wide-spread mitochondrial permeabilization accompanied by effector caspase activity.
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