RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.

Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.

Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

Distinctive Patterns of Transcription and RNA Processing for Human lincRNAs.

Numerous long intervening noncoding RNAs (lincRNAs) are generated from the mammalian genome by RNA polymerase II (Pol II) transcription. Although multiple functions have been ascribed to lincRNAs, their synthesis and turnover remain poorly characterized. Here, we define systematic differences in transcription and RNA processing between protein-coding and lincRNA genes in human HeLa cells. This is based on a range of nascent transcriptomic approaches applied to different nuclear fractions, including mammalian native elongating transcript sequencing (mNET-seq). Notably, mNET-seq patterns specific for different Pol II CTD phosphorylation states reveal weak co-transcriptional splicing and poly(A) signal-independent Pol II termination of lincRNAs as compared to pre-mRNAs. In addition, lincRNAs are mostly restricted to chromatin, since they are rapidly degraded by the RNA exosome. We also show that a lincRNA-specific co-transcriptional RNA cleavage mechanism acts to induce premature termination. In effect, functional lincRNAs must escape from this targeted nuclear surveillance process.

Mitochondrial double-stranded RNA triggers antiviral signalling in humans.

Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.

Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo.

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.

Perampanel, a potent AMPA receptor antagonist, protects against tetramethylenedisulfotetramine-induced seizures and lethality in mice: comparison with diazepam.

Tetramethylenedisulfotetramine (TETS), a noncompetitive GABAA receptor antagonist, is a potent, highly lethal convulsant that is considered to be a chemical threat agent. Here, we assessed the ability of the AMPA receptor antagonist perampanel to protect against TETS-induced seizures and lethality in mice when administered before or after treatment with the toxicant. For comparison, we conducted parallel testing with diazepam, which is a first-line treatment for chemically induced seizures in humans. Pre-treatment of mice with either perampanel (1-4 mg/kg, i.p.) or diazepam (1-5 mg/kg, i.p.) conferred protection in a dose-dependent fashion against tonic seizures and lethality following a dose of TETS (0.2 mg/kg, i.p.) that rapidly induces seizures and death. The ED50 values for protection against mortality were 1.6 mg/kg for perampanel and 2.1 mg/kg for diazepam. Clonic seizures were unaffected by perampanel and only prevented in a minority of animals by high-dose diazepam. Neither treatment prevented myoclonic body twitches. Perampanel and diazepam also conferred protection against tonic seizures and lethality when administered 15 min following a 0.14 mg/kg, i.p., dose of TETS and 5 min following a 0.2 mg/kg, i.p., dose of TETS. Both posttreatments were highly potent at reducing tonic seizures and lethality in animals exposed to the lower dose of TETS whereas greater doses of both treatments were required in animals exposed to the larger dose of TETS. Neither treatment was as effective suppressing clonic seizures. In an experiment where 0.4 mg/kg TETS was administered by oral gavage and the treatment drugs were administered 5 min later, perampanel only partially protected against lethality whereas diazepam produced nearly complete protection. We conclude that perampanel and diazepam protect against TETS-induced tonic seizures and lethality but have less impact on clonic seizures. Both drugs could have utility in the treatment of TETS intoxication but neither eliminates all seizure activity.

Persistent behavior deficits, neuroinflammation, and oxidative stress in a rat model of acute organophosphate intoxication.

Current medical countermeasures for organophosphate (OP)-induced status epilepticus (SE) are not effective in preventing long-term morbidity and there is an urgent need for improved therapies. Rat models of acute intoxication with the OP, diisopropylfluorophosphate (DFP), are increasingly being used to evaluate therapeutic candidates for efficacy in mitigating the long-term neurologic effects associated with OP-induced SE. Many of these therapeutic candidates target neuroinflammation and oxidative stress because of their implication in the pathogenesis of persistent neurologic deficits associated with OP-induced SE. Critical to these efforts is the rigorous characterization of the rat DFP model with respect to outcomes associated with acute OP intoxication in humans, which include long-term electroencephalographic, neurobehavioral, and neuropathologic effects, and their temporal relationship to neuroinflammation and oxidative stress. To address these needs, we examined a range of outcomes at later times post-exposure than have previously been reported for this model. Adult male Sprague-Dawley rats were given pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), which was immediately followed by atropine sulfate (2 mg/kg, im) and pralidoxime (25 mg/kg, im). This exposure paradigm triggered robust electroencephalographic and behavioral seizures that rapidly progressed to SE lasting several hours in 90% of exposed animals. Animals that survived DFP-induced SE (~70%) exhibited spontaneous recurrent seizures and hyperreactive responses to tactile stimuli over the first 2 months post-exposure. Performance in the elevated plus maze, open field, and Pavlovian fear conditioning tests indicated that acute DFP intoxication reduced anxiety-like behavior and impaired learning and memory at 1 and 2 months post-exposure in the absence of effects on general locomotor behavior. Immunohistochemical analyses revealed significantly increased expression of biomarkers of reactive astrogliosis, microglial activation and oxidative stress in multiple brain regions at 1 and 2 months post-DFP, although there was significant spatiotemporal heterogeneity across these endpoints. Collectively, these data largely support the relevance of the rat model of acute DFP intoxication as a model for acute OP intoxication in the human, and support the hypothesis that neuroinflammation and/or oxidative stress represent potential therapeutic targets for mitigating the long-term neurologic sequelae of acute OP intoxication.

The chemical convulsant diisopropylfluorophosphate (DFP) causes persistent neuropathology in adult male rats independent of seizure activity.

Organophosphate (OP) threat agents can trigger seizures that progress to status epilepticus, resulting in persistent neuropathology and cognitive deficits in humans and preclinical models. However, it remains unclear whether patients who do not show overt seizure behavior develop neurological consequences. Therefore, this study compared two subpopulations of rats with a low versus high seizure response to diisopropylfluorophosphate (DFP) to evaluate whether acute OP intoxication causes persistent neuropathology in non-seizing individuals. Adult male Sprague Dawley rats administered DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and pralidoxime (25 mg/kg, im) were monitored for seizure activity for 4 h post-exposure. Animals were separated into groups with low versus high seizure response based on behavioral criteria and electroencephalogram (EEG) recordings. Cholinesterase activity was evaluated by Ellman assay, and neuropathology was evaluated at 1, 2, 4, and 60 days post-exposure by Fluoro-Jade C (FJC) staining and micro-CT imaging. DFP significantly inhibited cholinesterase activity in the cortex, hippocampus, and amygdala to the same extent in low and high responders. FJC staining revealed significant neurodegeneration in DFP low responders albeit this response was delayed, less persistent, and decreased in magnitude compared to DFP high responders. Micro-CT scans at 60 days revealed extensive mineralization that was not significantly different between low versus high DFP responders. These findings highlight the importance of considering non-seizing patients for medical care in the event of acute OP intoxication. They also suggest that OP intoxication may induce neurological damage via seizure-independent mechanisms, which if identified, might provide insight into novel therapeutic targets.

Natural polyphenols in preclinical models of epilepsy.

Natural polyphenols are being tested both in preclinical and clinical settings for the treatment of different neurological disorders. The article describes the outcome of three polyphenols, resveratrol, epigallocatechin gallate, and quercetin, in preclinical animal models of epilepsy (both acute and chronic) and epileptogenesis. In theory, the antioxidant and neuroprotective properties of these natural polyphenols might be valuable in the management of acute seizures and the prevention of epileptogenesis. It is fascinating to observe that these polyphenols have a capacity to alter various signaling processes involved in the pathogenesis of epilepsy. The antiepileptic or antiseizure potential with these molecules delivers a mixed outcome. Some studies have demonstrated the usefulness of these molecules in preclinical models of epilepsy; however, contrary to the findings also exist. These molecules have poor bioavailability that may remain as the limiting factor in their clinical effects. The use of nanotechnology and other techniques have been tested to enhance bioavailability and brain penetration. There are no randomized double-blinded clinical studies establishing their antiepileptic effects in humans. It is concluded that more preclinical mechanism-based studies are needed to deliver a more certain picture regarding the use of natural polyphenols in the treatment of epilepsy.

Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection.

TDP-43 is a critical RNA-binding factor associated with pre-mRNA splicing in mammals. Its expression is tightly autoregulated, with loss of this regulation implicated in human neuropathology. We demonstrate that TDP-43 overexpression in humans and mice activates a 3' untranslated region (UTR) intron, resulting in excision of the proximal polyA site (PAS) pA(1). This activates a cryptic PAS that prevents TDP-43 expression through a nuclear retention mechanism. Superimposed on this process, overexpression of TDP-43 blocks recognition of pA(1) by competing with CstF-64 for PAS binding. Overall, we uncover complex interplay between transcription, splicing, and 3' end processing to effect autoregulation of TDP-43.

Determination of minimal steady-state plasma level of diazepam causing seizure threshold elevation in rats.

OBJECTIVE: Diazepam, administered by the intravenous, oral, or rectal routes, is widely used for the management of acute seizures. Dosage forms for delivery of diazepam by other routes of administration, including intranasal, intramuscular, and transbuccal, are under investigation. In predicting what dosages are necessary to terminate seizures, the minimal exposure required to confer seizure protection must be known. Here we administered diazepam by continuous intravenous infusion to obtain near-steady-state levels, which allowed an assessment of the minimal levels that elevate seizure threshold. METHODS: The thresholds for various behavioral seizure signs (myoclonic jerk, clonus, and tonus) were determined with the timed intravenous pentylenetetrazol seizure threshold test in rats. Diazepam was administered to freely moving animals by continuous intravenous infusion via an indwelling jugular vein cannula. Blood samples for assay of plasma levels of diazepam and metabolites were recovered via an indwelling cannula in the contralateral jugular vein. RESULTS: The pharmacokinetic parameters of diazepam following a single 80-µg/kg intravenous bolus injection were determined using a noncompartmental pharmacokinetic approach. The derived parameters Vd , CL, t1/2α (distribution half-life) and t1/2ß (terminal half-life) for diazepam were, respectively, 608 mL, 22.1 mL/min, 13.7 minutes, and 76.8 minutes, respectively. Various doses of diazepam were continuously infused without or with an initial loading dose. At the end of the infusions, the thresholds for various behavioral seizure signs were determined. The minimal plasma diazepam concentration associated with threshold elevations was estimated at approximately 70 ng/mL. The active metabolites nordiazepam, oxazepam, and temazepam achieved levels that are expected to make only minor contributions to the threshold elevations. SIGNIFICANCE: Diazepam elevates seizure threshold at steady-state plasma concentrations lower than previously recognized. The minimally effective plasma concentration provides a reference that may be considered when estimating the diazepam exposure required for acute seizure treatment.

Curcumin in epilepsy disorders.

Curcumin, a principal curcuminoid present in turmeric, has an antioxidant, anti-inflammatory and neuroprotective properties. Preclinical studies have indicated its beneficial effect for the treatment of epilepsy disorders. The molecule has an anti-seizure potential in preclinical studies, including chemical and electrical models of acute and chronic epilepsy. Curcumin also possesses an anti-epileptogenic activity as it reduces spontaneous recurrent seizures severity in a kainate model of temporal lobe epilepsy. The antioxidant and anti-inflammatory nature of curcumin might be responsible for its observed anti-seizure effects; nevertheless, the exact mechanism is not yet clear. The poor availability of curcumin to the brain limits its use in clinics. The application of nanoliposome and liposome technologies has been tested to enhance its brain availability and penetrability. Unfortunately, there are no randomized, double-blinded controlled clinical trials validating the use of curcumin in epilepsy. The present article analyzes different preclinical evidence illustrating the effect of curcumin in seizure models. The review encourages carrying out clinical trials in this important area of research. In conclusion, curcumin might be beneficial in patients with epilepsy disorders, if its bioavailability issues are resolved.

Optimal antisense target reducing INS intron 1 retention is adjacent to a parallel G quadruplex.

Splice-switching oligonucleotides (SSOs) have been widely used to inhibit exon usage but antisense strategies that promote removal of entire introns to increase splicing-mediated gene expression have not been developed. Here we show reduction of INS intron 1 retention by SSOs that bind transcripts derived from a human haplotype expressing low levels of proinsulin. This haplotype is tagged by a polypyrimidine tract variant rs689 that decreases the efficiency of intron 1 splicing and increases the relative abundance of mRNAs with extended 5' untranslated region (5' UTR), which curtails translation. Co-expression of haplotype-specific reporter constructs with SSOs bound to splicing regulatory motifs and decoy splice sites in primary transcripts revealed a motif that significantly reduced intron 1-containing mRNAs. Using an antisense microwalk at a single nucleotide resolution, the optimal target was mapped to a splicing silencer containing two pseudoacceptor sites sandwiched between predicted RNA guanine (G) quadruplex structures. Circular dichroism spectroscopy and nuclear magnetic resonance of synthetic G-rich oligoribonucleotide tracts derived from this region showed formation of a stable parallel 2-quartet G-quadruplex on the 3' side of the antisense retention target and an equilibrium between quadruplexes and stable hairpin-loop structures bound by optimal SSOs. This region interacts with heterogeneous nuclear ribonucleoproteins F and H that may interfere with conformational transitions involving the antisense target. The SSO-assisted promotion of weak intron removal from the 5' UTR through competing noncanonical and canonical RNA structures may facilitate development of novel strategies to enhance gene expression.

TDP-43 regulates its mRNA levels through a negative feedback loop.

TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3' UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3' UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.

The intronic splicing code: multiple factors involved in ATM pseudoexon definition.

Abundance of pseudo splice sites in introns can potentially give rise to innumerable pseudoexons, outnumbering the real ones. Nonetheless, these are efficiently ignored by the splicing machinery, a process yet to be understood completely. Although numerous 5' splice site-like sequences functioning as splicing silencers have been found to be enriched in predicted human pseudoexons, the lack of active pseudoexons pose a fundamental challenge to how these U1snRNP-binding sites function in splicing inhibition. Here, we address this issue by focusing on a previously described pathological ATM pseudoexon whose inhibition is mediated by U1snRNP binding at intronic splicing processing element (ISPE), composed of a consensus donor splice site. Spliceosomal complex assembly demonstrates inefficient A complex formation when ISPE is intact, implying U1snRNP-mediated unproductive U2snRNP recruitment. Furthermore, interaction of SF2/ASF with its motif seems to be dependent on RNA structure and U1snRNP interaction. Our results suggest a complex combinatorial interplay of RNA structure and trans-acting factors in determining the splicing outcome and contribute to understanding the intronic splicing code for the ATM pseudoexon.

Characterization of seizures induced by acute and repeated exposure to tetramethylenedisulfotetramine.

Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral, and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures, and death. The CD50 values for clonic and tonic seizures after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 µg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity ("kindling") as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days after a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.

Seizure protection by intrapulmonary delivery of propofol hemisuccinate.

The lung provides a portal of entry for drug delivery that could be used to administer anticonvulsant substances to prevent or abort seizures. Here, we demonstrate that intrapulmonary propofol hemisuccinate (PHS) rapidly confers seizure protection in various rodent chemoconvulsant models. Propofol is a powerful anticonvulsant substance at subanesthetic doses, but it is a viscous, water-immiscible oil that is not suitable for intrapulmonary administration. We found that PHS can be formulated as an aqueous solution that is well tolerated when instilled into the lung. High-dose intraperitoneally administered PHS induced loss-of-righting reflex in rats and mice. The onset of action of PHS was delayed in comparison with propofol, suggesting that conversion to propofol is required for activity. A lower dose of PHS (40 mg/kg i.p.) did not cause general anesthesia but protected against pentylenetetrazol (PTZ)-induced seizures in rats. Intrapulmonary administration of an aqueous PHS solution via a tracheal cannula at lower doses (5 and 10 mg/kg) conferred equivalent seizure protection without acute motor toxicity. In mice, intraperitoneal PHS (60-80 mg/kg) was associated with an elevation in PTZ, bicuculline, picrotoxin, and kainic acid seizure thresholds. Intratracheal PHS was markedly more potent, producing seizure threshold elevations at doses of 10 to 15 mg/kg. In the PTZ threshold model in mice, PHS was active at 5 min, maximally active at 10 min, and no longer active at 20 min. Intratracheal PHS also prolonged the onset of 4-aminopyridine-induced convulsions but did not affect the threshold for N-methyl-D-aspartate-induced convulsions. We conclude that intratracheal administration of an aqueous solution of PHS, a putative propofol prodrug, provides potent seizure protection of rapid onset and brief duration. Intrapulmonary PHS may be useful for preventing the spread of seizures or aborting seizure clusters without causing prolonged sedation.

Nitric oxide and major depression.

Nitric oxide has been known to play a significant role in the pathophysiology of various disorders of the body. Despite its very short half-life, nitric oxide is known to modulate various neurotransmitter system(s) in the body and thus is speculated to play an imperative role in the pathogenesis of neurological disorders. This "wonder" molecule has been often found to possess a "dual role" in many neurological disorders of the body. Evidences have shown its prominent role in the pathogenesis of major depression. Nitric oxide modulates norepinephrine, serotonin, dopamine, glutamate, the major neurotransmitters involved in the neurobiology of major depression. The nitric oxide modulatory activity of various new generations of antidepressants has been demonstrated. Clinical studies have also confirmed the nitric oxide modulatory activity of various antidepressants particularly belonging to the class of selective serotonin reuptake inhibitors. The present review attempts to discuss the role of nitric oxide in the pathophysiology of major depression. Further, the involvement of nitric oxide system in the mechanism of various antidepressants has been discussed in detail. Nitric oxide based antidepressants can be the future drugs of choice for major depression, particularly in the treatment of pharmacoresistant depression.

Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A.

Mitochondrial DNA (mtDNA) has been suggested to drive immune system activation, but the induction of interferon signaling by mtDNA has not been demonstrated in a Mendelian mitochondrial disease. We initially ascertained two patients, one with a purely neurological phenotype and one with features suggestive of systemic sclerosis in a syndromic context, and found them both to demonstrate enhanced interferon-stimulated gene (ISG) expression in blood. We determined each to harbor a previously described de novo dominant-negative heterozygous mutation in ATAD3A, encoding ATPase family AAA domain-containing protein 3A (ATAD3A). We identified five further patients with mutations in ATAD3A and recorded up-regulated ISG expression and interferon α protein in four of them. Knockdown of ATAD3A in THP-1 cells resulted in increased interferon signaling, mediated by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Enhanced interferon signaling was abrogated in THP-1 cells and patient fibroblasts depleted of mtDNA. Thus, mutations in the mitochondrial membrane protein ATAD3A define a novel type I interferonopathy.