Endosomal removal and disposal of dysfunctional, immunostimulatory mitochondrial DNA

Abstract

Maternally inherited mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation. This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts. Cells experiencing mtDNA stress due to depletion of the mtDNA-packaging protein, Transcription Factor A, Mitochondrial (TFAM), or HSV-1 infection exhibit elongated mitochondria, mtDNA depletion, enlargement of nucleoids (mtDNA-protein complexes), and activation of cGAS/STING innate immune signaling via mtDNA released into the cytoplasm. However, the relationships between altered mitochondrial dynamics and mtDNA-mediated activation of the cGAS-STING pathway remain unclear. Here, we show that entire enlarged nucleoids are released from mitochondria that remain bound to TFAM and colocalize with cGAS. These nucleoids arise at sites of mtDNA replication due to a block in mitochondrial fission at a stage when endoplasmic reticulum (ER) actin polymerization would normally commence, which we propose is a fission checkpoint to ensure that mtDNA has completed replication and is competent for segregation into daughter mitochondria. Released nucleoids also colocalize with the early endosomal marker RAB5 as well as the late endosomal marker RAB7 in TFAM-deficient cells and in response to mtDNA stress caused by the HSV-1 UL12.5 protein. Loss of RAB7 increases interferon stimulated gene (ISG) expression. Thus, we propose that defects in mtDNA replication and/or segregation enact a late mitochondrial fission checkpoint that, if persistent, leads to selective removal of dysfunctional nucleoids by a mitochondrial-endosomal pathway. Early steps in this pathway are prone to mtDNA release and cGAS-STING activation, but the immunostimulatory mtDNA is ultimately disposed of through a mechanism involving RAB7-containing late endosomes to prevent excessive innate immune signaling. This mtDNA quality control pathway might represent a therapeutic target to prevent mtDNA-mediated inflammation and associated pathology.

Mitochondrial DNA Release in Innate Immune Signaling

“…In a similar vein to mitophagy, several lines of evidence link defective autophagy and lysosomal function to mtDNA leakage into the cytoplasm ( 4 , 42 – 44 ). Impaired autophagy prevents the degradation of mtDNA, enabling its escape from lysosomes ( 43 , 44 ).…”

A novel genetic fluorescent reporter to visualize mitochondrial nucleoids

“…Nonetheless, we propose that the novel mt-HI-NESS fluorescent proteins developed and described here will be valuable tools to study the dynamics of mtDNA nucleoids in live cell imaging experiments. These reporters will allow researchers to address questions about how nucleoids are distributed throughout the mitochondrial network, how mtDNA replication is coordinated with mitochondrial fission and fusion events, and to study release of mtDNA into the cytosol or its endosomal/vesicle-mediated transport to autophagosomes for degradation [56][57][58] .…”

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT

“…Cells grown on fibronectin-coated coverslips (Fisher Scientific #12-545-81) were fixed at 37°C using a solution of 4% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO4, pH 6.8) for 15 minutes, and then permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 minutes at room temperature. FISH was then performed as previously described[54, 55], using the mREP probe conjugated to Atto633, which was purchased from Integrated DNA Technologies. For immunofluorescence, coverslips were blocked with filtered PBS containing 1% (w/v) BSA at room temperature for at least an hour, following either permeabilization or FISH.…”