Stress signaling and cellular proliferation reverse the effects of mitochondrial mistranslation.

Translation fidelity is crucial for prokaryotes and eukaryotic nuclear-encoded proteins; however, little is known about the role of mistranslation in mitochondria and its potential effects on metabolism. We generated yeast and mouse models with error-prone and hyper-accurate mitochondrial translation, and found that translation rate is more important than translational accuracy for cell function in mammals. Specifically, we found that mitochondrial mistranslation causes reduced overall mitochondrial translation and respiratory complex assembly rates. In mammals, this effect is compensated for by increased mitochondrial protein stability and upregulation of the citric acid cycle. Moreover, this induced mitochondrial stress signaling, which enables the recovery of mitochondrial translation via mitochondrial biogenesis, telomerase expression, and cell proliferation, and thereby normalizes metabolism. Conversely, we show that increased fidelity of mitochondrial translation reduces the rate of protein synthesis without eliciting a mitochondrial stress response. Consequently, the rate of translation cannot be recovered and this leads to dilated cardiomyopathy in mice. In summary, our findings reveal mammalian-specific signaling pathways that respond to changes in the fidelity of mitochondrial protein synthesis and affect metabolism.

Murine cytomegalovirus infection exacerbates complex IV deficiency in a model of mitochondrial disease.

The influence of environmental insults on the onset and progression of mitochondrial diseases is unknown. To evaluate the effects of infection on mitochondrial disease we used a mouse model of Leigh Syndrome, where a missense mutation in the Taco1 gene results in the loss of the translation activator of cytochrome c oxidase subunit I (TACO1) protein. The mutation leads to an isolated complex IV deficiency that mimics the disease pathology observed in human patients with TACO1 mutations. We infected Taco1 mutant and wild-type mice with a murine cytomegalovirus and show that a common viral infection exacerbates the complex IV deficiency in a tissue-specific manner. We identified changes in neuromuscular morphology and tissue-specific regulation of the mammalian target of rapamycin pathway in response to viral infection. Taken together, we report for the first time that a common stress condition, such as viral infection, can exacerbate mitochondrial dysfunction in a genetic model of mitochondrial disease.

Previous Influenza Infection Exacerbates Allergen Specific Response and Impairs Airway Barrier Integrity in Pre-Sensitized Mice.

In this study we assessed the effects of antigen exposure in mice pre-sensitized with allergen following viral infection on changes in lung function, cellular responses and tight junction expression. Female BALB/c mice were sensitized to ovalbumin and infected with influenza A before receiving a second ovalbumin sensitization and challenge with saline, ovalbumin (OVA) or house dust mite (HDM). Fifteen days post-infection, bronchoalveolar inflammation, serum antibodies, responsiveness to methacholine and barrier integrity were assessed. There was no effect of infection alone on bronchoalveolar lavage cellular inflammation 15 days post-infection; however, OVA or HDM challenge resulted in increased bronchoalveolar inflammation dominated by eosinophils/neutrophils or neutrophils, respectively. Previously infected mice had higher serum OVA-specific IgE compared with uninfected mice. Mice previously infected, sensitized and challenged with OVA were most responsive to methacholine with respect to airway resistance, while HDM challenge caused significant increases in both tissue damping and tissue elastance regardless of previous infection status. Previous influenza infection was associated with decreased claudin-1 expression in all groups and decreased occludin expression in OVA or HDM-challenged mice. This study demonstrates the importance of the respiratory epithelium in pre-sensitized individuals, where influenza-infection-induced barrier disruption resulted in increased systemic OVA sensitization and downstream effects on lung function.

Mutation in MRPS34 compromises protein synthesis and causes mitochondrial dysfunction.

The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The Mrps34 mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The Mrps34 mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age.

Route of exposure alters inflammation and lung function responses to diesel exhaust.

CONTEXT: Mice are commonly used in studies investigating the effects of diesel exhaust exposure on respiratory health. A plethora of studies in this field has resulted in a range of exposure protocols, from inhalation of diesel exhaust, to the administration (via various routes) of diesel exhaust particles in solution. OBJECTIVE: In this study, we compared the physiological consequences of short-term exposure to diesel exhaust via inhalation to those due to exposure to the same diesel exhaust particles suspended in solution and delivered intranasally. MATERIALS AND METHODS: Adult BALB/c mice were exposed to diesel exhaust via inhalation for 2 hours per day for 8 days. A representative, simultaneous sample of particles was collected and a second group of mice then exposed to them suspended in saline. A low and a high-dose were studied, with these matched based on respiratory parameters. Six and twenty-four hours after the last exposure we measured bronchoalveolar inflammation, lung volume, lung function and the amount of elemental carbon in alveolar macrophages. RESULTS: Exposure via either route elicited pulmonary inflammation and changes in lung function. We identified significant differences in response between the two routes of exposure, with mice exposed via inhalation generally displaying more realistic dose-response relationships. Mice exposed via intranasal instillation responded more variably, with little influence of dose. CONCLUSIONS: Our results suggest that selection of the route of exposure is of critical importance in studies such as this. Further, inhalation exposure, while more methodologically difficult, resulted in responses more akin to those seen in humans.

Multi-omic profiling reveals an RNA processing rheostat that predisposes to prostate cancer.

Prostate cancer is the most commonly diagnosed malignancy and the third leading cause of cancer deaths. GWAS have identified variants associated with prostate cancer susceptibility; however, mechanistic and functional validation of these mutations is lacking. We used CRISPR-Cas9 genome editing to introduce a missense variant identified in the ELAC2 gene, which encodes a dually localised nuclear and mitochondrial RNA processing enzyme, into the mouse Elac2 gene as well as to generate a prostate-specific knockout of Elac2. These mutations caused enlargement and inflammation of the prostate and nodule formation. The Elac2 variant or knockout mice on the background of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model show that Elac2 mutation with a secondary genetic insult exacerbated the onset and progression of prostate cancer. Multiomic profiling revealed defects in energy metabolism that activated proinflammatory and tumorigenic pathways as a consequence of impaired noncoding RNA processing and reduced protein synthesis. Our physiologically relevant models show that the ELAC2 variant is a predisposing factor for prostate cancer and identify changes that underlie the pathogenesis of this cancer.

Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan.

Changes in the rate and fidelity of mitochondrial protein synthesis impact the metabolic and physiological roles of mitochondria. Here we explored how environmental stress in the form of a high-fat diet modulates mitochondrial translation and affects lifespan in mutant mice with error-prone (Mrps12ep/ep ) or hyper-accurate (Mrps12ha/ha ) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high-fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue-specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12ep/ep mice were protected against heart defects but sensitive towards lipid accumulation in the liver, activating genes involved in steroid and amino acid metabolism. In contrast, enhanced translational accuracy in Mrps12ha/ha mice protected the liver from a high-fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post-mitotic (heart) and highly proliferative (liver) tissues. We show trade-offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue-specific outcomes due to commonly encountered stressful environmental conditions or aging.

A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance.

Mitochondrial energy metabolism plays an important role in the pathophysiology of insulin resistance. Recently, a missense N437S variant was identified in the MRPP3 gene, which encodes a mitochondrial RNA processing enzyme within the RNase P complex, with predicted impact on metabolism. We used CRISPR-Cas9 genome editing to introduce this variant into the mouse Mrpp3 gene and show that the variant causes insulin resistance on a high-fat diet. The variant did not influence mitochondrial gene expression markedly, but instead, it reduced mitochondrial calcium that lowered insulin release from the pancreatic islet ß cells of the Mrpp3 variant mice. Reduced insulin secretion resulted in lower insulin levels that contributed to imbalanced metabolism and liver steatosis in the Mrpp3 variant mice on a high-fat diet. Our findings reveal that the MRPP3 variant may be a predisposing factor to insulin resistance and metabolic disease in the human population.

Reduced mitochondrial translation prevents diet-induced metabolic dysfunction but not inflammation.

The contribution of dysregulated mitochondrial gene expression and consequent imbalance in biogenesis is not well understood in metabolic disorders such as insulin resistance and obesity. The ribosomal RNA maturation protein PTCD1 is essential for mitochondrial protein synthesis and its reduction causes adult-onset obesity and liver steatosis. We used haploinsufficient Ptcd1 mice fed normal or high fat diets to understand how changes in mitochondrial biogenesis can lead to metabolic dysfunction. We show that Akt-stimulated reduction in lipid content and upregulation of mitochondrial biogenesis effectively protected mice with reduced mitochondrial protein synthesis from excessive weight gain on a high fat diet, resulting in improved glucose and insulin tolerance and reduced lipid accumulation in the liver. However, inflammation of the white adipose tissue and early signs of fibrosis in skeletal muscle, as a consequence of reduced protein synthesis, were exacerbated with the high fat diet. We identify that reduced mitochondrial protein synthesis and OXPHOS biogenesis can be recovered in a tissue-specific manner via Akt-mediated increase in insulin sensitivity and transcriptional activation of the mitochondrial stress response.

Fidelity of translation initiation is required for coordinated respiratory complex assembly.

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle-specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.

PTCD1 Is Required for 16S rRNA Maturation Complex Stability and Mitochondrial Ribosome Assembly.

The regulation of mitochondrial RNA life cycles and their roles in ribosome biogenesis and energy metabolism are not fully understood. We used CRISPR/Cas9 to generate heart- and skeletal-muscle-specific knockout mice of the pentatricopeptide repeat domain protein 1, PTCD1, and show that its loss leads to severe cardiomyopathy and premature death. Our detailed transcriptome-wide and functional analyses of these mice enabled us to identify the molecular role of PTCD1 as a 16S rRNA-binding protein essential for its stability, pseudouridylation, and correct biogenesis of the mitochondrial large ribosomal subunit. We show that impaired mitoribosome biogenesis can have retrograde signaling effects on nuclear gene expression through the transcriptional activation of the mTOR pathway and upregulation of cytoplasmic protein synthesis and pro-survival factors in the absence of mitochondrial translation. Taken together, our data show that impaired assembly of the mitoribosome exerts its consequences via differential regulation of mitochondrial and cytoplasmic protein synthesis.

Adult-onset obesity is triggered by impaired mitochondrial gene expression.

Mitochondrial gene expression is essential for energy production; however, an understanding of how it can influence physiology and metabolism is lacking. Several proteins from the pentatricopeptide repeat (PPR) family are essential for the regulation of mitochondrial gene expression, but the functions of the remaining members of this family are poorly understood. We created knockout mice to investigate the role of the PPR domain 1 (PTCD1) protein and show that loss of PTCD1 is embryonic lethal, whereas haploinsufficient, heterozygous mice develop age-induced obesity. The molecular defects and metabolic consequences of mitochondrial protein haploinsufficiency in vivo have not been investigated previously. We show that PTCD1 haploinsufficiency results in increased RNA metabolism, in response to decreased protein synthesis and impaired RNA processing that affect the biogenesis of the respiratory chain, causing mild uncoupling and changes in mitochondrial morphology. We demonstrate that with age, these effects lead to adult-onset obesity that results in liver steatosis and cardiac hypertrophy in response to tissue-specific differential regulation of the mammalian target of rapamycin pathways. Our findings indicate that changes in mitochondrial gene expression have long-term consequences on energy metabolism, providing evidence that haploinsufficiency of PTCD1 can be a major predisposing factor for the development of metabolic syndrome.

A pathogenic role for the integrin CD103 in experimental allergic airways disease.

The integrin CD103 is the αE chain of integrin αEß7 that is important in the maintenance of intraepithelial lymphocytes and recruitment of T cells and dendritic cells (DC) to mucosal surfaces. The role of CD103 in intestinal immune homeostasis has been well described, however, its role in allergic airway inflammation is less well understood. In this study, we used an ovalbumin (OVA)-induced, CD103-knockout (KO) BALB/c mouse model of experimental allergic airways disease (EAAD) to investigate the role of CD103 in disease expression, CD4+ T-cell activation and DC activation and function in airways and lymph nodes. We found reduced airways hyper-responsiveness and eosinophil recruitment to airways after aerosol challenge of CD103 KO compared to wild-type (WT) mice, although CD103 KO mice showed enhanced serum OVA-specific IgE levels. Following aerosol challenge, total numbers of effector and regulatory CD4+ T-cell subsets were significantly increased in the airways of WT but not CD103 KO mice, as well as a lack of DC recruitment into the airways in the absence of CD103. While total airway DC numbers, and their in vivo allergen capture activity, were essentially normal in steady-state CD103 KO mice, migration of allergen-laden airway DC to draining lymph nodes was disrupted in the absence of CD103 at 24 h after aerosol challenge. These data support a role for CD103 in the pathogenesis of EAAD in BALB/c mice through local control of CD4+ T cell and DC subset recruitment to, and migration from, the airway mucosa during induction of allergic inflammation.

Geogenic PM10 exposure exacerbates responses to influenza infection.

Particulate matter (PM) exposure has been linked epidemiologically to exacerbations of lung disease, including respiratory infections. We investigated the effects of geogenic (earth-derived) PM10 (PM<10 µm diameter) on the response to a respiratory viral infection. Geogenic dust was sampled from four communities in arid environments in Western Australia. Adult female BALB/c mice were intranasally exposed to chronic doses of PM10 (10 µg/day for 10 days), and/or infected with influenza (A/Mem/1/71) virus. Inflammation (cells, IL-6, IFN-γ) was measured in bronchoalveolar lavage. Lung mechanics were measured using the forced oscillation technique. Geogenic PM10 induced lung inflammation (neutrophils, macrophages) with additive effects in mice also infected with influenza. PM10 also modified the influenza-induced IL-6 and IFN-γ responses. Geogenic PM10 increased airway resistance, and increased hysteresivity in those exposed to both insults. Viral titres were significantly higher after PM10 exposure. Iron concentration was inversely associated with IFN-γ and positively associated with viral titre and hysteresivity. Geogenic PM10 exposure increases inflammation, impairs lung function and increases viral load, exacerbating the response to respiratory viral infection. Iron in the particles may be a driver of these responses. This has important implications for respiratory health in communities exposed to high geogenic PM10, such as those in arid environments.

House dust mite induced lung inflammation does not alter circulating vitamin D levels.

Low circulating levels of 25-hydroxyvitamin D [25(OH)D] are associated with chronic lung diseases such as asthma. However, it is unclear whether vitamin D is involved in disease pathogenesis or is modified by the inflammation associated with the disease process. We hypothesized that allergic inflammation decreases the level of circulating 25(OH)D and tested this using a mice model of house dust mite (HDM) induced allergic airway inflammation. Cellular influx was measured in bronchoalvelar lavage (BAL) fluid, and allergic sensitization and 25(OH)D levels were measured in serum. Exposure to HDM caused a robust inflammatory response in the lung that was enhanced by prior influenza infection. These responses were not associated with any change in circulating levels of 25(OH)D. These data suggest that alterations in circulating 25(OH)D levels induced by Th-2 driven inflammation are unlikely to explain the cross-sectional epidemiological association between vitamin D deficiency and asthma.