Molecular mechanisms of mitochondrial DNA release and activation of the cGAS-STING pathway

Abstract

In addition to constituting the genetic material of an organism, DNA is a tracer for the recognition of foreign pathogens and a trigger of the innate immune system. cGAS functions as a sensor of double-stranded DNA fragments and initiates an immune response via the adaptor protein STING. The cGAS-STING pathway not only defends cells against various DNA-containing pathogens but also modulates many pathological processes caused by the immune response to the ectopic localization of self-DNA, such as cytosolic mitochondrial DNA (mtDNA) and extranuclear chromatin. In addition, macrophages can cause inflammation by forming a class of protein complexes called inflammasomes, and the activation of the NLRP3 inflammasome requires the release of oxidized mtDNA. In innate immunity related to inflammasomes, mtDNA release is mediated by macropores that are formed on the outer membrane of mitochondria via VDAC oligomerization. These macropores are specifically formed in response to mitochondrial stress and tissue damage, and the inhibition of VDAC oligomerization mitigates this inflammatory response. The rapidly expanding area of research on the mechanisms by which mtDNA is released and triggers inflammation has revealed new treatment strategies not only for inflammation but also, surprisingly, for neurodegenerative diseases such as amyotrophic lateral sclerosis.

Fig. 1. Overview of the cGAS-STING signaling pathway.

The host immune response is initiated by the recognition of cytosolic DNA, such as pathogen-derived nucleic acids (e.g., viruses and bacteria) and self-DNA (e.g., from dying cells, tumor cells or mtDNA). Upon recognition of cytosolic DNA, cGAS catalyzes the formation of cGAMP and thereby activates STING. STING then activates TBK1, which phosphorylates IRF3. Phosphorylated IRF3 activates the expression of type I IFNs in the nucleus. STING also activates NF-κB by phosphorylating the kinase IKK. NF-κB then activates the transcription of genes encoding proinflammatory cytokines.

Fig. 2. cGAS activation by DNA binding.

cGAS binds to dsDNA ≥18 bp and can form dimers when the DNA sequence is sufficiently long or is bent by other factors (e.g., TFAM). Liquid phase condensation leads to the stable ladder/net interaction of cGAS and enhances its catalytic activity.

Fig. 3. Intracellular cGAMP transmission.

Intracellular cGAMP can be transferred to neighboring or adjacent cells through gap junctions, formed by the connexin protein, or via plasma-membrane transporters, including SLC19A1, P2X7R, and LRRC8. Degradation of extracellular cGAMP by ENPP1 modulates extracellular cGAMP levels.

Fig. 4. Regulation of STING and the downstream signaling pathway.

Upon binding to cGAMP, STING undergoes a conformational change, and then, the dimers oligomerize. STING oligomerization recruits TBK1 and promotes autotransphosphorylation of TBK1 at the ERGIC. Activated TBK1 phosphorylates the CTT of STING to generate IRF3 docking sites and then phosphorylates the recruited IRF3.

Fig. 5. Posttranslational modification in the cGAS-STING pathway.

Mitochondrial stress induces the release of mtDNA and subsequently activates the cGAS-STING pathway. This signaling pathway can be regulated by various posttranslational modifications and can also be suppressed by caspases during apoptosis.

Fig. 6. mtDNA release via permeabilization of the mitochondrial outer membrane.

(Left) During BAK/BAX-mediated apoptosis, mitochondria undergo hyperfragmentation, and mitochondrial matrix components, including mtDNA, are released into the cytosol through BAK/BAX macropores. mPTP opening is not required for MIM disruption. The released mtDNA does not activate the type-I IFN pathway because of caspase activation. (Right) Mitochondrial stress triggers the formation of VDAC-oligomer pores and activates the type-I IFN pathway. mPTP opening is required for MIM disruption.