Mutations Causative of CPEO Differentially Engage Innate Immunity Sensors

Chronic Progressive External Ophthalmoplegia (CPEO) is a primary mitochondrial disorder (PMD) caused by mutations in nuclear genes encoding mitochondrial DNA (mtDNA) maintenance proteins. CPEO is characterized by mtDNA depletion and deletions, and patients primarily present with ocular and muscular features (isolated CPEO). However, additional encephalomyopathy, neurological complications, and Parkinsonism can drive a more severe disease form, CPEO-plus. The evolution from isolated CPEO to CPEO-plus remains poorly understood. Inflammatory and innate immune processes are emerging as strong disease modifiers and may underlie this heterogeneity. Instability of mitochondrial DNA is a major driver of organellar stress and release of mitochondrial contents into the cytosol. Mutations in several genes involved in mtDNA replication and maintenance have been implicated in triggering the escape of mitochondrial nucleic acids from the mitochondrial matrix. Once exposed to cytosolic innate immune sensors, mtDNA and mitochondrial double-stranded RNA (mt-dsRNA) act as potent immunogens, with more than 10 innate immune sensors capable of recognizing them. Therefore, mtDNA and mt-dsRNA release are likely pathological mechanisms in CPEO, yet the list of CPEO-related genes that can trigger inflammatory processes is far from complete. Here, we use patient-derived fibroblasts from individuals with CPEO carrying mutations in RNASEH1 and Twinkle, and provide - for the first time - evidence that their mutations drive innate immune activation through the release of different mitochondrial nucleic acids. RNASEH1 mutations lead to the accumulation and subsequent release of mt-dsRNA, while mtDNA remains protected. On the other hand, mutations in Twinkle cause the release of mtDNA without triggering mt-dsRNA production, or leakage. Supporting this notion, the POLRMT inhibitor IMT-1, and the STING inhibitor H-151, reduced interferon stimulated genes expression downstream of RNASEH1 and Twinkle mutations, respectively. Further, when we analyzed a unique compound patient line carrying mutations in both genes simultaneously, we detect both species of nucleic acids in its cytosol, indicating that both pathways can be engaged simultaneously in the same cell. Lastly, we show that cytosolic sensing triggers paracrine signaling to activate bystander microglia - the resident macrophages of the retina and brain - with potential implications to the neurological progression of CPEO. Overall, our findings reveal a new role for RNASEH1 and Twinkle in driving aberrant innate immunity and paracrine inflammation in CPEO. Our data support a model in which innate immunity is a universal feature of mutations causing mtDNA instability; yet different mutations engage distinct sensing pathways, and in complex scenarios multiple pathways can be triggered at the same time. Given the clinical heterogeneity observed in patients with PMDs, our findings that different signaling pathways are triggered in patient-specific manners might have direct implications for precision medicine approaches aimed at targeting specific innate immunity.