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The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.How the mitochondrial electron transport chain (ETC) interacts with the NLRP3 inflammasome is somewhat unclear. Here the authors use individual complex inhibitors and new genetic models to show that ETC is critical in providing ATP via the phosphocreatine shuttle to activate the NLRP3 inflammasome.

T cell activation requires extensive metabolic reprogramming, but the specific requirement for mitochondrial respiration (MR) remains unresolved. While most studies have focused on aerobic glycolysis as the primary driver of proliferation and effector function, the role of MR has not been completely defined. To isolate MR from proton pumping by cytochrome c oxidase (COX), we expressed the non-proton-pumping alternative oxidase (AOX) in activated COX-deficient T cells. AOX restored electron flow, membrane potential, and mitochondrial ATP production, ultimately rescuing proliferation, effector and memory differentiation, and antiviral immunity. These improvements required upstream electron input, particularly from Complex I, with Complex II and DHODH contributing more modestly. Despite restored MR, glycolysis remained elevated, likely due to altered redox signaling. These findings demonstrate that MR, normally mediated by COX, is necessary and can be sufficient to support T cell activation and function, independent of proton translocation, provided upstream electron input is maintained. Mitochondrial respiration provides reducing power to the electron transport chain (ETC), driving proton pumping and ATP synthesis required for T cell activation and differentiation. Here, the authors use alternative oxidase (AOX) as a mechanistic probe to bypass cytochrome c oxidase deficiency and thereby isolate the role of respiration and demonstrate that intact mitochondrial respiration is important for T cell proliferation, effector function, memory formation, and regulation of apoptotic and metabolic signaling pathways.

Alternative oxidase (AOX) is a non‐mammalian enzyme that can bypass blockade of the complex III‐IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)‐deficient Bcs1l p.S78G knock‐in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue‐specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies. Synopsis Bcs1l mutant mice show respiratory chain cIII dysfunction resulting in poor electron and proton transfer, and quinol oxidation. This study makes use of alternative oxidase (AOX), an enzyme that shunts electrons from quinols directly to oxygen, thus restoring electron flow upstream of cIII. Broadly expressed transgenic AOX prevents lethal cardiomyopathy in Bcs1lp.S78G knock‐in mice and extends their median survival from 210 to 590 days. Restoration of electron flow upstream of cIII by AOX is sufficient to prevent the cardiac decompensation. AOX prevents pathological changes in the expression of central transcriptional regulators of substrate utilization and metabolic stress in the heart. Analyses of reactive oxygen species (ROS) production and damage suggest that ROS are not instrumental in the rescue. Graphical Abstract Bcs1l mutant mice show respiratory chain cIII dysfunction resulting in poor electron and proton transfer, and quinol oxidation. This study makes use of alternative oxidase (AOX), an enzyme that shunts electrons from quinols directly to oxygen, thus restoring electron flow upstream of cIII.

Plants and many lower organisms, but not mammals, express alternative oxidases (AOX) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell-lines, Drosophila disease models and, most recently, in the mouse, where multiple, lentivector-AOX transgenes conferred substantial expression in specific tissues. Here we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello, whilst animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.

The alternative oxidase (AOX) from Ciona intestinalis was previously shown to be expressible in mice and to cause no physiological disturbance under unstressed conditions. Because AOX is known to become activated under some metabolic stress conditions, resulting in altered energy balance, we studied its effects in mice subjected to dietary stress. Wild‐type mice (Mus musculus, strain C57BL/6JOlaHsd) fed a high‐fat or ketogenic (high‐fat, low‐carbohydrate) diet show weight gain with increased fat mass, as well as loss of performance, compared with chow‐fed animals. Unexpectedly, AOX‐expressing mice fed on these metabolically stressful, fat‐rich diets showed almost indistinguishable patterns of weight gain and altered body composition as control animals. Cardiac performance was impaired to a similar extent by ketogenic diet in AOX mice as in nontransgenic littermates. AOX and control animals fed on ketogenic diet both showed wide variance in weight gain. Analysis of the gut microbiome in stool revealed a strong correlation with diet, rather than with genotype. The microbiome of the most and least obese outliers reared on the ketogenic diet showed no consistent trends compared with animals of normal body weight. We conclude that AOX expression in mice does not modify physiological responses to extreme diets. AOX‐expressing mice reared on high‐fat or ketogenic diet show similar patterns of weight gain and altered body composition as wild‐type mice. Prolonged ketogenic diet impairs cardiac function in both AOX and control mice. Microbiome analysis shows stronger correlations with ketogenic diet, rather than with AOX expression or extent of weight gain per se.

Respiratory progenitors can be efficiently generated from pluripotent stem cells (PSCs). However, further targeted differentiation into bronchoalveolar sublineages is still in its infancy, and distinct specifying effects of key differentiation factors are not well explored. Focusing on airway epithelial Clara cell generation, we analyzed the effect of the glucocorticoid dexamethasone plus cAMP-elevating agents (DCI) on the differentiation of murine embryonic and induced pluripotent stem cells (iPSCs) into bronchoalveolar epithelial lineages, and whether keratinocyte growth factor (KGF) might further influence lineage decisions. We demonstrate that DCI strongly induce expression of the Clara cell marker Clara cell secretory protein (CCSP). While KGF synergistically supports the inducing effect of DCI on alveolar markers with increased expression of surfactant protein (SP)-C and SP-B, an inhibitory effect on CCSP expression was shown. In contrast, neither KGF nor DCI seem to have an inducing effect on ciliated cell markers. Furthermore, the use of iPSCs from transgenic mice with CCSP promoter-dependent lacZ expression or a knockin of a YFP reporter cassette in the CCSP locus enabled detection of derivatives with Clara cell typical features. Collectively, DCI was shown to support bronchoalveolar specification of mouse PSCs, in particular Clara-like cells, and KGF to inhibit bronchial epithelial differentiation. The targeted in vitro generation of Clara cells with their important function in airway protection and regeneration will enable the evaluation of innovative cellular therapies in animal models of lung diseases.

The mitochondrial matrix GTPase NOA1 is a nuclear encoded protein, essential for mitochondrial protein synthesis, oxidative phosphorylation and ATP production. Here, we demonstrate that newly translated NOA1 protein is imported into the nucleus, where it localizes to the nucleolus and interacts with UBF1 before nuclear export and import into mitochondria. Mutation of the nuclear localization signal (NLS) prevented both nuclear and mitochondrial import while deletion of the N-terminal mitochondrial targeting sequence (MTS) or the C-terminal RNA binding domain of NOA1 impaired mitochondrial import. Absence of the MTS resulted in accumulation of NOA1 in the nucleus and increased caspase-dependent apoptosis. We also found that export of NOA1 from the nucleus requires a leptomycin-B sensitive, Crm1-dependent nuclear export signal (NES). Finally, we show that NOA1 is a new substrate of the mitochondrial matrix protease complex ClpXP. Our results uncovered an unexpected, mandatory detour of NOA1 through the nucleolus before uptake into mitochondria. We propose that nucleo-mitochondrial translocation of proteins is more widespread than previously anticipated providing additional means to control protein bioavailability as well as cellular communication between both compartments.

Cardiomyocytes continuously generate the contractile force to circulate blood through the body. Imbalances in contractile performance or energy supply cause adaptive responses of the heart resulting in adverse rearrangement of regular structures, which in turn might lead to heart failure. At the cellular level, cardiomyocyte remodeling includes (1) restructuring of the contractile apparatus; (2) rearrangement of the cytoskeleton; and (3) changes in energy metabolism. Dedifferentiation represents a key feature of cardiomyocyte remodeling. It is characterized by reciprocal changes in the expression pattern of “mature” and “immature” cardiomyocyte-specific genes. Dedifferentiation may enable cardiomyocytes to cope with hypoxic stress by disassembly of the energy demanding contractile machinery and by reduction of the cellular energy demand. Dedifferentiation during myocardial repair might provide cardiomyocytes with additional plasticity, enabling survival under hypoxic conditions and increasing the propensity to enter the cell cycle. Although dedifferentiation of cardiomyocytes has been described during tissue regeneration in zebrafish and newts, little is known about corresponding mechanisms and regulatory circuits in mammals. The recent finding that the cytokine oncostatin M (OSM) is pivotal for cardiomyocyte dedifferentiation and exerts strong protective effects during myocardial infarction highlights the role of cytokines as potent stimulators of cardiac remodeling. Here, we summarize the current knowledge about transient dedifferentiation of cardiomyocytes in the context of myocardial remodeling, and propose a model for the role of OSM in this process.

Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.