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

Abstract

The recognition that cytosolic mtDNA activates cGAS-STING innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early-onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels, and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

Mitochondrial DNA Release in Innate Immune Signaling

“…The TFAM paradigm suggested that other forms of mtDNA stress might also lead to mtDNA-release-mediated cGAS–STING signaling, which has turned out to be the case. Cells either lacking TOP1MT (EC 5.6.2.1) or expressing a pathogenic variant associated with lupus exhibit mtDNA release and activation of the cGAS–STING pathway ( 73 ). TOP1MT knockout cells have reduced mtDNA copy number and mitochondrial transcripts, defects in nucleoid organization, elongated mitochondria, and lower OXPHOS.…”

Measuring mtDNA turnover, synthesis, and supercoiling via selective bromodeoxyuridine incorporation

“…In addition, our method is robust enough to not only measure total levels of mtDNA for synthesis and turnover, but also separate linear, supercoiled and relaxed forms of mtDNA for subsequent quantification and analysis. As a result, our protocols allow for pinpointing the molecular mechanisms underlying changes in mtDNA copy number, as we have shown in models of mitochondrial dysfunction such as loss of TOP1MT 1,2 , or reduced TFAM expression 3 .…”

“…Here we describe a protocol that selectively incorporates bromodeoxyuridine into mtDNA for the measurement of mtDNA turnover, synthesis or supercoiling via an adapted immunoblot and Southern blot protocol. For complete use and execution of this protocol see Al Khatib et al 1,2 and Lei et al 3…”

The role of cGAS-STING signaling in pulmonary fibrosis and its therapeutic potential

“…Furthermore, an increasing body of research has indicated that aberrant activation of the cGAS-STING pathway can lead to excessive and sustained production of inflammatory factors, including type I IFNs, contributing to the development of various diseases (74). The undeniable role of the cGAS-STING signaling pathway in innate immune diseases is supported by Al Khatib et al, who demonstrated that TOP1MT influences the activation of the mtDNA-mediated cGAS-STING innate immune response, and that the P193L variant may contribute to the autoimmune phenotype observed in patients (75). The inflammatory response induced by activation of the cGAS-STING signaling pathway is a significant factor in the progression of pulmonary fibrosis.…”