Mitochondrial DNA in cell death and inflammation

Abstract

Cytosolic DNA is recognized by the innate immune system as a potential threat. During apoptotic cell death, mitochondrial DNA (mtDNA) release activates the DNA sensor cyclic GMP-AMP synthase (cGAS) to promote a pro-inflammatory type I interferon response. Inflammation following mtDNA release during apoptotic cell death can be exploited to engage anti-tumor immunity and represents a potential avenue for cancer therapy. Additionally, various studies have described leakage of mtDNA, independent of cell death, with different underlying cues such as pathogenic infections, changes in mtDNA packaging, mtDNA stress or reduced mitochondrial clearance. The interferon response in these scenarios can be beneficial but also potentially disadvantageous, as suggested by a variety of disease phenotypes. In this review, we discuss mechanisms underlying mtDNA release governed by cell death pathways and summarize release mechanisms independent of cell death. We further highlight the similarities and differences in mtDNA release pathways, outlining gaps in our knowledge and questions for further research. Together, a deeper understanding of how and when mtDNA is released may enable the development of drugs to specifically target or inhibit mtDNA release in different disease settings.

Keywords: apoptosis; cell death; mitochondria; mtDNA; pyroptosis.

Figure 1.. Role of mitochondria in cell death.

Apoptosis: Mitochondrial apoptosis is held in check by the balance of pro- and anti-apoptotic proteins. An apoptotic trigger promotes BAX BAK oligomerization and lipid pore formation in the mitochondrial outer membrane — mitochondrial outer membrane permeabilisation (MOMP), apoptotosome formation (cyto c, APAF1 and caspase-9) in the cytosol and activation of executioner caspases-3 and -7 enforcing cell death. The intermembrane space protein SMAC binds to and inhibits XIAP thereby relieving the brake on caspase-9 and -3 activity. Activation of caspase-8 results in caspase-3 activation and in type II cells, as well as after activation of the ripoptosome and necrosome; apoptotic cell death requires caspase-8 mediated cleavage of BID, mitochondrial outer membrane permeabilization and mitochondrial apoptosis. Pyroptosis: mtDNA and mtROS can act as ligands for the inflammasome receptor NLRP3. Activation of the inflammasome platform (receptor, ASC, caspase-1), allows autoproteolytic cleavage and activation of caspase-1, thus promoting cleavage of GSDMD. The N-terminal domain of GSDMD (GSDMD^NT) inserts into the plasma membrane — promoting pyroptotic cell death. GSDMD^NT also forms pores in the mitochondrial membrane. While caspase-3 cleaves GSDMD in its N-terminus (amino acid D87 mouse) deactivating the N-terminal fragment, caspase-8 cleavage of GSDMD results in an active GSDMD^NT. Activation of caspase-3 can additionally cleave GSDME yielding an active N-terminal fragment promoting pore formation in the mitochondrial and plasma membrane. Necroptosis: Mitochondria are not required for the execution of necroptosis, however, mtROS can promote RIP1 kinase phosphorylation and thus promote the initial steps of necroptosis. Ferroptosis: Mitochondria impact ferroptosis in at least two ways; firstly, promoting ferroptosis due to mtROS oxidizing mitochondrial lipids and secondly dampening ferroptosis by chelating iron (Abbreviations: MOMP, mitochondrial outer membrane permabilization; APAF1, apoptotic protease-activating factor1; cyto c, cytochrome c; SMAC, second mitochondria-derived activator of caspases; XIAP, X-linked inhibitor of apoptosis protein; BID, BH3-interacting domain death agonist; mtDNA, mitochondrial DNA; mtROS, mitochondrial reactive oxygen species; NLRP3, NACHT, LRR and PYD domains-containing protein 3; ASC, apoptosis-associated speck-like containing a card; GSDMD, gasdermin-D; RIP1, receptor-interacting protein 1).

Figure 2.. Signaling pathways activated upon mitochondrial DNA recognition in the cytosol.

(a) Cytosolic mtDNA binds and activates cGAS inducing the production of the second messenger cGAMP and dimerization of STING. STING recruits TBK1 to phosphorylate STING, itself as well as the transcription factor IRF3, thus enabling transcription of pro-inflammatory target genes via NF-κB and IRF3 in the nucleus. (b) MtDNA activates the NLRP3 and potentially AIM2 inflammasome receptor which act as a platform for caspase-1 activation and induction of pyroptotic cell death via GSDMD pore formation in the plasma membrane. (c) TLR9 recognizes mtDNA in the endosomal compartment and relays the signaling to the adaptor protein MyD88-IRAK1/IRAK4-TRAF6 and IRF7, TAK1-IκKBα or TAK1-MAPK1 which in turn promotes pro-inflammatory gene expression via activation of the transcription factor IRF7, NF-κB or AP-1 (Abbreviations: TLR9, Toll-like receptor 9; mtDNA, mitochondrial DNA; MyD88, myeloid differentiation primary response protein; IRAK, interleukin-1 receptor-associated kinase; TRAF6, TNF receptor-associated factor 6; MAPK, mitogen-activated protein kinase 1; IRF7, interferon regulatory factor 7; cGAMP, cyclic GMP–AMP; cGAS, cyclic GMP–AMP synthase; STING, stimulator of interferon genes protein; IRF3, Inteferon regulatory factor 3; AIM2, absent in melanoma 2; NLRP3, NACHT, LRR and PYD domains-containing protein 3).

Figure 3.. Overview of how mtDNA is released during cell death.

Apoptosis: Extrinsic or intrinsic apoptosis signals result in BAX/BAK pore formation activation of the apoptotic pathway, extrusion of the inner mitochondrial membrane and release of mtDNA into the cytosol. DNA is recognized by cGAS and the cGAS-STING pathway is activated resulting in IRF3 activation and a type I IFN response. This gene expression is counteracted by caspase-3 cleaving and inactivating cGAS and IRF3. Pyroptosis: Activation of gasdermins (GSDMD, GSDME, Gasdermin A3) promotes pore formation in the mitochondria inducing mtDNA release and activation of the cGAS-STING signaling cascade resulting in a type I interferon response. However, the type I IFN reponse is prohibited by caspase-1 and -3 by inactivating cleavage of cGAS and IRF3 (Abbreviations: cGAS, cyclic GMP–AMP synthase; STING, stimulator of interferon genes protein; IRF3, inteferon regulatory factor 3; ISG, interferon stimulatory genes; GSDMD, gasdermin-D; GSDME, gasdermin-E; mtDNA, mitochondrial DNA).

Figure 4.. Overview of factors inducing mtDNA release.

mtDNA release has been reported in cell death during apoptosis and pyroptosis but also independent of cell death pathways. The non-cell death cues can be categorized in exterior signals, mitochondrial stress, mtDNA stress, inefficient mitochondrial clearance and pathogenic infections. Factors in gray induce a type I IFN response possibly mediating by mtDNA release.