Increasing evidence implicates mitochondrial dysfunction in the pathogenesis of inflammatory bowel disease (IBD), with the IBD-associated pathobiont adherent-invasive E. coli-LF82 (AIEC) promoting epithelial mitochondrial depolarization and fragmentation and a concomitant reduction in barrier function. We hypothesized that the anti-inflammatory cytokine interleukin-10 (IL-10), essential for enteric homeostasis, would protect against AIEC-induced mitochondrial disruption. Mitochondrial fragmentation in colonic organoids following infection with E. coli-LF82 was reduced by IL-10 pretreatment + cotreatment. IL-10 significantly reduced the E. coli-LF82 induced mitochondrial dysfunction in the human colon-derived T84 epithelial cell line as measured by mitochondrial network analysis, membrane potential, permeability transition pore opening, oxidative phosphorylation (via oxygen consumption rate), and epithelial barrier integrity. IL-10-treated T84 cells and organoids exhibited increased phosphorylation of STAT3 at serine 727 (a requirement for STAT3 activity at mitochondria), which was abrogated by pharmacological inhibition of ERK (but not JAK) activation, leading to loss of protection against E. coli-LF82-induced mitochondrial dysfunction. Re-expression of wild-type STAT3, but not the S727A mutant, in HT-29 (human colon epithelial cell line) STAT3 knockout cells restored mitochondrial depolarization and ATP levels, underscoring the role of ERK-driven STAT3S727 phosphorylation in IL-10's protective mechanism. In contrast, IL-22, which primarily activates STAT3Y705 and not STAT3S727, failed to prevent AIEC-induced mitochondrial dysfunction. Thus, in the context of exposure to an E. coli pathobiont, IL-10 supports gut epithelial homeostasis via ERK-dependent STAT3S727 maintenance of mitochondrial integrity, contributing to preservation of epithelial barrier function.

There is a shared hallmark of defective differentiation across genetic myopathies, a process that has been extensively described in Duchenne muscular dystrophy and also observed in Emery-Dreifuss muscular dystrophy. In this article, we broaden the discussion on myopathies associated with differentiation defects, examining their implications in less characterized muscle conditions that can have onset in adulthood, including facioscapulohumeral muscular dystrophy (FSHD), oculopharyngeal muscular dystrophy (OPMD), and myotonic dystrophies (DM), as well as myopathies caused by genetic variants in FHL1, GNE, DES, CAPN3, and members of the HNRNP family. Muscle damage can result from injury, exercise, or disease, necessitating a highly coordinated repair process to restore normal strength and function. Resident satellite cells are activated, differentiate, and fuse with the damaged tissue to facilitate this repair. This overview emphasizes the importance of muscle differentiation in the pathogenesis of myopathies with diverse etiologies and a broad range of underlying molecular mechanisms. These insights highlight differentiation as a potential convergent therapeutic target.

Pathogenic variants in the mitochondrial protein MFN2 are typically associated with a peripheral neuropathy phenotype, but can also cause a variety of additional pathologies including myopathy. Here, we identified an uncharacterized MFN2 variant, Q367H, in a patient diagnosed with late-onset distal myopathy, but without peripheral neuropathy. Supporting the hypothesis that this variant contributes to the patient's pathology, patient fibroblasts and transdifferentiated myoblasts showed changes consistent with impairment of several MFN2 functions. We also observed mtDNA outside of the mitochondrial network that colocalized with early endosomes, and measured activation of both TLR9 and cGAS-STING inflammation pathways that sense mtDNA. Re-expressing the Q367H variant in MFN2 KO cells also induced mtDNA release, demonstrating this phenotype is a direct result of the variant. As elevated inflammation can cause myopathy, our findings linking the Q367H MFN2 variant with elevated TLR9 and cGAS-STING signalling can explain the patient's myopathy. Thus, we characterize a novel MFN2 variant in a patient with an atypical presentation that separates peripheral neuropathy and myopathy phenotypes, and establish a potential pathomechanism connecting MFN2 dysfunction to mtDNA-mediated inflammation.

Abstract Background The Crohn’s disease-associated adherent invasive E. coli (AIEC) (strain LF82) decreases mitochondrial membrane potential and fragments the epithelial mitochondrial network. Other groups have also reported activation of NF-κB-mediated inflammation and generation of reactive oxygen species in AIEC-LF82 infected epithelia. It remains unclear how these findings integrate over the course of AIEC-LF82 infection and how they influence each other over the course of infection. Aims To characterize the sequence of events following AIEC-LF82 infection in a non-cancer derived epithelial cell model focusing on mitochondrial disruption, cell death, mtDNA release, and inflammatory gene expression. Methods The fetal derived human colon epithelial cell line (FHC) were infected with E. coli-LF82 or the commensal E. coli-HB101 at multiplicity of infection (MOI) of 100 or uninfected over the course of various experiments. FHCs were stained with SYTOX green and imaged on a CellCyte X system for 24h to measure cell death or stained with MitoTracker Deep Red and imaged on a spinning disc-confocal microscope for 16h to assess mitochondrial morphology. Also, in fixed-cell preparations mitochondrial networks were imaged by staining with anti-TOMM20 antibodies. Imaging of cytosolic DNA was performed via immunofluorescent staining of mtDNA and the mitochondrial network. As markers of inflammation, CXCL10 and IL-6 mRNA were assessed via qRT-PCR. Results Epithelia infected with E. coli-LF82 but not E. coli-HB101 displayed dramatic fragmentation of their mitochondrial network at 6h (p < 0.0001) (n=6), while SYTOX green imaging revealed significant cell death beginning at 9h (p < 0.001) (n=3). Furthermore, our preliminary data indicates that transcription of inflammatory cytokines including CXCL10, and IL-6 occurs at 8h while cytosolic escape of mtDNA occurs at 6h, after mitochondrial network disruption, but prior to cell death. Conclusions The data begins to unravel the sequence of events in a non-cancer derived epithelium after infection with E. coli-LF82, integrating mitochondrial function, inflammatory gene expression, and cell viability and providing a more fulsome understanding of the course of E. coli-LF82 infection. Together, these data advance understanding of relationship between mitochondrial fragmentation and cell death in the context of a Crohn’s disease-relevant pathobiont, AIEC-LF82. Funding Agencies TRIANGLE

Clozapine is the only approved pharmacotherapy for treatment-resistant schizophrenia. However, widespread utilization of clozapine is constrained due to the potential for severe adverse effects, including myocarditis. Multiple mechanisms have been suggested to account for the cardiotoxic effects of clozapine, yet these investigations have not used cells derived from clozapine treated patients. In this study, cardiomyocytes that were derived from induced pluripotent stem cells generated from four patients with treatment-resistant schizophrenia with (n = 2) and without (n = 2) a history of clozapine-induced myocarditis were used to assess mitochondria- and NLRP3 inflammasome-mediated mechanisms of this severe adverse drug reaction. We found treatment of cardiomyocytes with a physiologically-relevant dose (2.8 mu M) of clozapine for 24 h: (1) induced cardiac dysfunction, increased cytotoxicity, and apoptosis, (2) induced oxidative stress by elevating the level of reactive oxygen species and mitochondrial fragmentation, and (3) elevated levels of proinflammatory cytokines and activated the NLRP3 inflammasome. These effects were more pronounced in cardiomyocytes derived from individuals with a history of clozapine-induced myocarditis. Furthermore, pharmacological targeting of the mitochondria (elamipretide) and inflammasome (ustekinumab) attenuated these clozapine-induced cardiotoxic effects. Collectively, these results suggest a mitochondria- and NLRP3 inflammasome-mediated mechanism in the development of myocarditis associated with clozapine and support further evaluation of therapeutics that target mitochondria and NLRP3 signaling.

Steroid hormones are important signaling molecules that are primarily produced by specialized cells. The ability to culture steroidogenic cells is critical to study this important process. While a few steroidogenic cell lines are available for study, they are typically derived from cancer patients and do not reflect genetic alterations in steroidogenic genes that cause human disease. In this regard, iPSCs are a powerful tool for modeling human disease, as patient-derived iPSCs can be differentiated into a variety of different cell types. While other approaches exist to differentiate iPSCs into steroidogenic cells, they are typically complex and time consuming. In contrast, a simple approach based on lentivirus transduction of SF1, the master regulator of steroidogenesis, can differentiate multiple types of cultured cells into a steroidogenic state. However, we show here that this approach does not work for iPSCs. To circumvent this limitation, we report a simple adaptation that first differentiates iPSCs to embryoid bodies prior of SF1 transduction. With this modified approach, we provide a straightforward cost-effective approach to differentiate iPSCs into actively steroidogenic cells. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Background The IBD-associated pathobiont attaching-invading Escherichia coli (AIEC) strain LF82 induces mitochondrial fragmentation and depolarization in epithelial cells, resulting in impaired ATP production and reduced barrier function. Interleukin (IL)-6 and IL-22 are omnipresent in the inflamed gut, can directly affect epithelial function and both mobilize STAT-3, the serine727 phosphorylated form of which can translocate to mitochondria. Thus, we posed the question, will IL-6 or IL-22 rescue or exaggerate the mitochondrial damage evoked by exposure to E. coli-LF82? Aims 1. To determine if IL-6 and IL-22 modulate mitochondrial function during E. coli-LF82 infection 2. To assess the mechanisms by which IL-6 and IL-22 affect mitochondrial function in model gut epithelia Methods Human colon-derived T84 epithelial cells were infected with E. coli-LF82 (108 CFU/mL, 4h) ± a 18h pre-treatment and then co-treatment with IL-6 or IL-22 (both 10 ng/mL). Bacterial internalization was evaluated, and mitochondrial network morphology visualized with MitoTracker Red and confocal microscopy. Mitochondrial membrane potential was evaluated using TMRE staining and flow cytometry, and ATP measured by a luminescence assay. STAT3 phosphorylation (S727 and Y705) in whole-cell extracts was determined via western blot in response to IL-6 or IL-22 during E. coli-LF82 infection. Results IL-6 inhibited E. coli-LF82 evoked mitochondrial fragmentation, with significantly more cells showing a fused tubular pattern: mitochondrial membrane potential was restored by ~50% and ATP depletion was less severe (n=5). IL-6 elicited increased pS727-STAT3. In contrast, IL-22 failed to prevent any of the mitochondrial changes caused by E. coli-LF82, did not cause increased pS727-STAT3 but bioactivity was confirmed by increased pY705-STAT3. IL-6, but not IL-22, reduced the number of viable intracellular bacteria without affecting bacterial growth and increased markers of autophagy activation. Conclusions We have uncovered divergent effects of the STAT3 activators, IL-6 and IL-22, in the control of epithelial mitochondrial dynamics and function when challenged with a bacterial pathobiont. We speculate that IL-6 limits the effects of E. coli-LF82 via mitochondrially-directed pS727-STAT3 signaling and upregulation of autophagy. Understanding the precise role of STAT3- activating cytokines signaling in mitochondrial regulation could reveal new target to enhance epithelial barrier function. Funding Agencies CIHR

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) – comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI – is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that pharmacologic activation of the ISR reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions, including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

As we have learned more about mitochondria over the past decades, including about their essential cellular roles and how altered mitochondrial biology results in disease, it has become apparent that they are not just powerplants pumping out ATP at the whim of the cell. Rather, mitochondria are dynamic information and energy processors that play crucial roles in directing dozens of cellular processes and behaviors. They provide instructions to enact programs that regulate various cellular operations, such as complex metabolic networks, signaling and innate immunity, and even control cell fate, dictating when cells should divide, differentiate or die. To help current and future generations of cell biologists incorporate the dynamic, multifaceted nature of mitochondria and assimilate modern discoveries into their scientific framework, mitochondria need a 21st century 'rebranding'. In this Opinion article, we argue that mitochondria should be considered as the 'Chief Executive Organelle' - the CEO - of the cell.

Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth type 2A (CMT2A). These mutations can disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR)-the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2-deficient cells. These results demonstrate the potential for pharmacologic ISR activation through the OMA1-DELE1-HRI signaling pathway as a potential strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.

Abstract Background The Crohn’s disease-associated adherent invasive E. coli (AIEC) (strain LF82) decreases mitochondrial membrane potential and fragments the epithelial mitochondrial network. Due to the evolutionary history of mitochondria as former bacteria, they present a variety of molecular patterns known to trigger innate immune responses. Specifically, mitochondrial DNA (mtDNA) released following mitochondrial damage causes upregulation of type I interferons, priming inflammatory signalling. Thus, we hypothesized that AIEC-induced mitochondrial dysfunction results in mtDNA release to activate innate immune signalling that could contribute to disease pathogenesis in the gut. Aims We aimed determine if an inflammatory response occurs downstream of mitochondrial damage evoked by E. coli-LF82 infection. Methods Control T84 epithelial cells, T84s infected E. coli-LF82 (108 cfu, MOI=100, 4h) and T84s infected with the commensal E. coli-HB101 (108 cfu for an MOI=100, 4h) were collected for protein and RNA extraction. RNA was used to measure IFNα and IFNβ expression following infection, while protein was used to measure upstream mediators including phosphorylation of TANK Binding Kinase-1 (TBK1) via Western blot. Results Epithelia infected with E. coli-LF82 but not E. coli-HB101 displayed dramatic fragmentation of their mitochondria network and increased levels of phosphorylated-TBK1 compared to control T84 cells (pampersand:003C0.01). Assessment of IFNα and IFNβ mRNA revealed significant up-regulation E. coli-LF82 infected T84 cells and human colon organoids. Conclusions Our findings identify a novel role for E. coli-LF82 in activating type I interferon response and for the first time show AIEC-induced activation of TBK1 and type I interferons, which may occur through mitochondrial damage, rather than through response to bacterial molecular patterns. As a result, our findings suggest that the AIEC colonization seen in Crohn’s disease may dysregulate inflammatory signalling through mitochondrial damage. Ultimately, we believe this dysregulation of inflammatory signalling may sensitize the host to further inflammatory signalling, contributing to the pathogenesis of inflammatory bowel disease. Funding Agencies CIHR

Mitochondria are pleiotropic organelles central to an array of cellular pathways including metabolism, signal transduction, and programmed cell death. Mitochondria are also key drivers of mammalian immune responses, functioning as scaffolds for innate immune signaling, governing metabolic switches required for immune cell activation, and releasing agonists that promote inflammation. Mitochondrial DNA (mtDNA) is a potent immunostimulatory agonist, triggering pro-inflammatory and type I interferon responses in a host of mammalian cell types. Here we review recent advances in how mtDNA is detected by nucleic acid sensors of the innate immune system upon release into the cytoplasm and extracellular space. We also discuss how the interplay between mtDNA release and sensing impacts cellular innate immune endpoints relevant to health and disease.

Pathogenic variants in the mitochondrial fusion protein Mitofusin2 typically cause axonal Charcot-Marie-Tooth disease type 2A (CMT2A), a progressively degenerative peripheral neuropathy. Here, we present two siblings with a lethal disorder of neonatal onset who carried a novel homozygous MFN2 variant R334K. Given the severe clinical presentation, which is atypical of MFN2 variants, further functional investigations were warranted to confirm the pathogenicity of the R334K variant, which was deemed to be likely pathogenic. Characterization of patient fibroblasts showed severe disruptions all MFN2-related functions that were assayed, including reduced mitochondrial respiration, altered mito-ER contacts, decreased mtDNA copy number and size, as well as increased abundance of cellular lipid droplets. We also observed reduced Complex I activity, which is noted in cells lacking MFN2, but is not standard for pathogenic MFN2 variants that cause CMT2A. These functional deficits, combined with the fact that loss of MFN2 is lethal in model organisms (e.g. mice/fruit-flies), support the notion that the MFN2 R334K is a loss of function variant that is responsible for patient phenotype. Thus, we present a novel pathogenic MFN2 variant causing severe fatal neonatal disease. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by funds provided by the Canadian Institutes of Health Research (TES) and the Alberta Children's Hospital Research Institute (Owerko Center) (MZ). MZ was supported by a Hotchkiss Brain Institute International Recruitment Scholarship. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of the University of Calgary gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript

In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, endoplasmic reticulum-associated degradation (ERAD), autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM- and ATR-mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons, sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict.

Mitochondrial quality control (MQC) mechanisms are required to maintain a functional proteome, which enables mitochondria to perform a myriad of important cellular functions from oxidative phosphorylation to numerous other metabolic pathways. Mitochondrial protein homeostasis begins with the import of over 1000 nuclear-encoded mitochondrial proteins and the synthesis of 13 mitochondrial DNA-encoded proteins. A network of chaperones and proteases helps to fold new proteins and degrade unnecessary, damaged, or misfolded proteins, whereas more extensive damage can be removed by mitochondrial-derived vesicles (MDVs) or mitochondrial autophagy (mitophagy). Here, focusing on mechanisms in mammalian cells, we review the importance of mitochondrial protein import as a sentinel of mitochondrial function that activates multiple MQC mechanisms when impaired.

Prolonged severe hypoxia follows brief seizures and represents a mechanism underlying several negative postictal manifestations without interventions. Approximately 50% of the postictal hypoxia phenomenon can be accounted for by arteriole vasoconstriction. What accounts for the rest of the drop in unbound oxygen is unclear. Here, we determined the effect of pharmacological modulation of mitochondrial function on tissue oxygenation in the hippocampus of rats after repeatedly evoked seizures. Rats were treated with mitochondrial uncoupler 2,4 dinitrophenol (DNP) or antioxidants. Oxygen profiles were recorded using a chronically implanted oxygen-sensing probe, before, during, and after seizure induction. Mitochondrial function and redox tone were measured using in vitro mitochondrial assays and immunohistochemistry. Postictal cognitive impairment was assessed using the novel object recognition task. Mild mitochondrial uncoupling by DNP raised hippocampal oxygen tension and ameliorated postictal hypoxia. Chronic DNP also lowered mitochondrial oxygen-derived reactive species and oxidative stress in the hippocampus during postictal hypoxia. Uncoupling the mitochondria exerts therapeutic benefits on postictal cognitive dysfunction. Finally, antioxidants do not affect postictal hypoxia, but protect the brain from associated cognitive deficits. We provided evidence for a metabolic component of the prolonged oxygen deprivation that follow seizures and its pathological sequelae. Furthermore, we identified a molecular underpinning of this metabolic component, which involves excessive oxygen conversion into reactive species. Mild mitochondrial uncoupling may be a potential therapeutic strategy to treat the postictal state where seizure control is absent or poor.

Increasing work on mitochondria is focused on the dynamic aspects of this organelle, a key part of which is regulation of the mitochondrial genome (mtDNA). 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

Gut bacteria provide benefits to the host and have been implicated in inflammatory bowel disease (IBD), where adherent-invasive E. coli (AIEC) pathobionts (e.g., strain LF82) are associated with Crohn's disease. E. coli-LF82 causes fragmentation of the epithelial mitochondrial network, leading to increased epithelial permeability. We hypothesized that butyrate would limit the epithelial mitochondrial disruption caused by E. coli-LF82. Human colonic organoids and the T84 epithelial cell line infected with E. coli-LF82 (MOI = 100, 4 h) showed a significant increase in mitochondrial network fission that was reduced by butyrate (10 mM) co-treatment. Butyrate reduced the loss of mitochondrial membrane potential caused by E. coli-LF82 and increased expression of PGC-1$\alpha $alpha mRNA, the master regulator of mitochondrial biogenesis. Metabolomics revealed that butyrate significantly altered E. coli-LF82 central carbon metabolism leading to diminished glucose uptake and increased succinate secretion. Correlating with preservation of mitochondrial network form/function, butyrate reduced E. coli-LF82 transcytosis across T84-cell monolayers. The use of the G-protein inhibitor, pertussis toxin, implicated GPCR signaling as critical to the effect of butyrate, and the free fatty acid receptor three (FFAR3, GPR41) agonist, AR420626, reproduced butyrate's effect in terms of ameliorating the loss of barrier function and reducing the mitochondrial fragmentation observed in E. coli-LF82 infected T84-cells and organoids. These data indicate that butyrate helps maintain epithelial mitochondrial form/function when challenged by E. coli-LF82 and that this occurs, at least in part, via FFAR3. Thus, loss of butyrate-producing bacteria in IBD in the context of pathobionts would contribute to loss of epithelial mitochondrial and barrier functions that could evoke disease and/or exaggerate a low-grade inflammation.

The recognition that cytosolic mitochondrial DNA (mtDNA) activates cyclic GMP-AMP synthase-stimulator of interferon genes (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.