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 1 . This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts 2 . 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 3 . 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.

Endosomal removal and disposal of dysfunctional, immunostimulatory mitochondrial DNA