Mitochondrial temperature homeostasis resists external metabolic stresses.

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

A century of mitochondrial research, 1922-2022.

Although recognized earlier as subcellular entities by microscopists, mitochondria have been the subject of functional studies since 1922, when their biochemical similarities with bacteria were first noted. In this overview I trace the history of research on mitochondria from that time up to the present day, focussing on the major milestones of the overlapping eras of mitochondrial biochemistry, genetics, pathology and cell biology, and its explosion into new areas in the past 25 years. Nowadays, mitochondria are considered to be fully integrated into cell physiology, rather than serving specific functions in isolation.

Alternative oxidase causes cell type- and tissue-specific responses in mutator mice.

Energetic insufficiency, excess production of reactive oxygen species (ROS), and aberrant signaling partially account for the diverse pathology of mitochondrial diseases. Whether interventions affecting ROS, a regulator of stem cell pools, could modify somatic stem cell homeostasis remains unknown. Previous data from mitochondrial DNA mutator mice showed that increased ROS leads to oxidative damage in erythroid progenitors, causing lifespan-limiting anemia. Also unclear is how ROS-targeted interventions affect terminally differentiated tissues. Here, we set out to test in mitochondrial DNA mutator mice how ubiquitous expression of the Ciona intestinalis alternative oxidase (AOX), which attenuates ROS production, affects murine stem cell pools. We found that AOX does not affect neural stem cells but delays the progression of mutator-driven anemia. Furthermore, when combined with the mutator, AOX potentiates mitochondrial stress and inflammatory responses in skeletal muscle. These differential cell type-specific findings demonstrate that AOX expression is not a global panacea for curing mitochondrial dysfunction. ROS attenuation must be carefully studied regarding specific underlying defects before AOX can be safely used in therapy.

AOX delays the onset of the lethal phenotype in a mouse model of Uqcrh (complex III) disease.

The alternative oxidase, AOX, provides a by-pass of the cytochrome segment of the mitochondrial respiratory chain when the chain is unavailable. AOX is absent from mammals, but AOX from Ciona intestinalis is benign when expressed in mice. Although non-protonmotive, so does not contribute directly to ATP production, it has been shown to modify and in some cases rescue phenotypes of respiratory-chain disease models. Here we studied the effect of C. intestinalis AOX on mice engineered to express a disease-equivalent mutant of Uqcrh, encoding the hinge subunit of mitochondrial respiratory complex III, which results in a complex metabolic phenotype beginning at 4-5 weeks, rapidly progressing to lethality within a further 6-7 weeks. AOX expression delayed the onset of this phenotype by several weeks, but provided no long-term benefit. We discuss the significance of this finding in light of the known and hypothesized effects of AOX on metabolism, redox homeostasis, oxidative stress and cell signaling. Although not a panacea, the ability of AOX to mitigate disease onset and progression means it could be useful in treatment.

Nuclear genetic background influences the phenotype of the Drosophila tko25t mitochondrial protein-synthesis mutant.

The Drosophila tko25t point mutation in the gene encoding mitoribosomal protein S12 produces a complex phenotype of multiple respiratory chain deficiency, developmental delay, bang-sensitivity, impaired hearing, sugar and antibiotic sensitivity, and impaired male courtship. Its phenotypic severity was previously shown to be alleviated by inbreeding and to vary with mitochondrial genetic background. Here, we show similarly profound effects conferred by nuclear genetic background. We backcrossed tko25t into each of 2 standard nuclear backgrounds, Oregon R and w1118, the latter used as recipient line in many transgenic applications requiring selection for the white minigene marker. In the w1118 background, tko25t flies showed a moderate developmental delay and modest bang-sensitivity. In the Oregon R background, males showed longer developmental delay and more severe bang-sensitivity, and we were initially unable to produce homozygous tko25t females in sufficient numbers to conduct a meaningful analysis. When maintained as a balanced stock over 2 years, tko25t flies in the Oregon R background showed clear phenotypic improvement though were still more severely affected than in the w1118 background. Phenotypic severity did not correlate with the expression level of the tko gene. Analysis of tko25t hybrids between the 2 backgrounds indicated that phenotypic severity was conferred by autosomal, X-chromosomal, and parent-of-origin-dependent determinants. Although some of these effects may be tko25t specific, we recommend that, in order to minimize genetic drift and confounding background effects, the genetic background of nonlethal mutants should be controlled by regular backcrossing, even if stocks are usually maintained over a balancer chromosome.

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration.

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the gene's numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

Cyanide resistant respiration and the alternative oxidase pathway: A journey from plants to mammals.

In a large number of organisms covering all phyla, the mitochondrial respiratory chain harbors, in addition to the conventional elements, auxiliary proteins that confer adaptive metabolic plasticity. The alternative oxidase (AOX) represents one of the most studied auxiliary proteins, initially identified in plants. In contrast to the standard respiratory chain, the AOX mediates a thermogenic cyanide-resistant respiration; a phenomenon that has been of great interest for over 2 centuries in that energy is not conserved when electrons flow through it. Here we summarize centuries of studies starting from the early observations of thermogenicity in plants and the identification of cyanide resistant respiration, to the fascinating discovery of the AOX and its current applications in animals under normal and pathological conditions.

Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation.

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.

What physiological role(s) does the alternative oxidase perform in animals?

Although the alternative oxidase, AOX, was known to be widespread in the animal kingdom by 2004, its exact physiological role in animals remains poorly understood. Here we present what evidence has accumulated thus far, indicating that it may play a role in enabling animals to resist various kinds of stress, including toxins, abnormal oxygen or nutrient levels, protein unfolding, dessication and pathogen attack. Much of our knowledge comes from studies in model organisms, where any benefits from exogenously expressed AOX may be masked by its unregulated expression, which may itself be stressful. The further question arises as to why AOX has been lost from some major crown groups, namely vertebrates, insects and cephalopods, if it plays important roles favouring the survival of other animals. We conclude by presenting some speculative ideas addressing this question, and an outline of how it might be approached experimentally.

Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active.

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.

Effects on Dopaminergic Neurons Are Secondary in COX-Deficient Locomotor Dysfunction in Drosophila.

Dopaminergic (DA) neurons have been implicated as key targets in neurological disorders, notably those involving locomotor impairment, and are considered to be highly vulnerable to mitochondrial dysfunction, a common feature of such diseases. Here we investigated a Drosophila model of locomotor disorders in which functional impairment is brought about by pan-neuronal RNAi knockdown of subunit COX7A of cytochrome oxidase (COX). Despite minimal neuronal loss by apoptosis, the expression and activity of tyrosine hydroxylase was decreased by half. Surprisingly, COX7A knockdown specifically targeted to DA neurons did not produce locomotor defect. Instead, using various drivers, we found that COX7A knockdown in specific groups of cholinergic and glutamatergic neurons underlay the phenotype. Based on our main finding, the vulnerability of DA neurons to mitochondrial dysfunction as a cause of impaired locomotion in other organisms, including mammals, warrants detailed investigation.

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1.

In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing.

Mitochondria play an important role in sensing both acute and chronic hypoxia in the pulmonary vasculature, but their primary oxygen-sensing mechanism and contribution to stabilization of the hypoxia-inducible factor (HIF) remains elusive. Alteration of the mitochondrial electron flux and increased superoxide release from complex III has been proposed as an essential trigger for hypoxic pulmonary vasoconstriction (HPV). We used mice expressing a tunicate alternative oxidase, AOX, which maintains electron flux when respiratory complexes III and/or IV are inhibited. Respiratory restoration by AOX prevented acute HPV and hypoxic responses of pulmonary arterial smooth muscle cells (PASMC), acute hypoxia-induced redox changes of NADH and cytochrome c, and superoxide production. In contrast, AOX did not affect the development of chronic hypoxia-induced pulmonary hypertension and HIF-1α stabilization. These results indicate that distal inhibition of the mitochondrial electron transport chain in PASMC is an essential initial step for acute but not chronic oxygen sensing.

Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia.

Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.

Correction to: Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila.

The article Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila, written by Dmytro V. Gospodaryov. Olha M. Strilbytska. Uliana V. Semaniuk. Natalia V. Perkhulyn. Bohdana M. Rovenko. Ihor S. Yurkevych. Ana G. Barata. Tobias P. Dick. Oleh V. Lushchak and Howard T. Jacobs, was originally published electronically on the publisher's internet portal on 20 November 2019 without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed on 27 January 2020 to © The Author(s) 2020 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The original article has been corrected.

Regulation of growth in Drosophila melanogaster: the roles of mitochondrial metabolism.

Mitochondrial functions are often considered purely from the standpoint of catabolism, but in growing cells they are mainly dedicated to anabolic processes, and can have a profound impact on the rate of growth. The Drosophila larva, which increases in body mass ∼200-fold over the course of ∼3 days at 25°C, provides an excellent model to study the underlying regulatory machinery that connects mitochondrial metabolic capacity to growth. In this review, we will focus on several key aspects of this machinery: nutrient sensing, endocrine control of feeding and nutrient mobilization, metabolic signalling, protein synthesis regulation and pathways of steroid biosynthesis and activity. In all these aspects, mitochondria appear to play a crucial role.

Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila.

Mitochondrial alternative NADH dehydrogenase (aNDH) was found to extend lifespan when expressed in the fruit fly. We have found that fruit flies expressing aNDH from Ciona intestinalis (NDX) had 17-71% lifespan prolongation on media with different protein-tocarbohydrate ratios except NDX-expressing males that had 19% shorter lifespan than controls on a high protein diet. NDX-expressing flies were more resistant to organic xenobiotics, 2,4-dichlorophenoxyacetic acid and alloxan, and inorganic toxicant potassium iodate, and partially to sodium molybdate treatments. On the other hand, NDX-expressing flies were more sensitive to catechol and sodium chromate. Enzymatic analysis showed that NDX-expressing males had higher glucose 6-phosphate dehydrogenase activity, whilst both sexes showed increased glutathione S-transferase activity.

Manipulating mtDNA in vivo reprograms metabolism via novel response mechanisms.

Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in Drosophila using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decrease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS.

Hyperoxia but not AOX expression mitigates pathological cardiac remodeling in a mouse model of inflammatory cardiomyopathy.

Constitutive expression of the chemokine Mcp1 in mouse cardiomyocytes creates a model of inflammatory cardiomyopathy, with death from heart failure at age 7-8 months. A critical pathogenic role has previously been proposed for induced oxidative stress, involving NADPH oxidase activation. To test this idea, we exposed the mice to elevated oxygen levels. Against expectation, this prevented, rather than accelerated, the ultrastructural and functional signs of heart failure. This result suggests that the immune signaling initiated by Mcp1 leads instead to the inhibition of cellular oxygen usage, for which mitochondrial respiration is an obvious target. To address this hypothesis, we combined the Mcp1 model with xenotopic expression of the alternative oxidase (AOX), which provides a sink for electrons blocked from passage to oxygen via respiratory complexes III and IV. Ubiquitous AOX expression provided only a minor delay to cardiac functional deterioration and did not prevent the induction of markers of cardiac and metabolic remodeling considered a hallmark of the model. Moreover, cardiomyocyte-specific AOX expression resulted in exacerbation of Mcp1-induced heart failure, and failed to rescue a second cardiomyopathy model directly involving loss of cIV. Our findings imply that mitochondrial involvement in the pathology of inflammatory cardiomyopathy is multifaceted and complex.

Mitochondrial dysfunction generates a growth-restraining signal linked to pyruvate in Drosophila larvae.

The Drosophila bang-sensitive mutant tko25t, manifesting a global deficiency in oxidative phosphorylation due to a mitochondrial protein synthesis defect, exhibits a pronounced delay in larval development. We previously identified a number of metabolic abnormalities in tko25t larvae, including elevated pyruvate and lactate, and found the larval gut to be a crucial tissue for the regulation of larval growth in the mutant. Here we established that expression of wild-type tko in any of several other tissues of tko25t also partially alleviates developmental delay. The effects appeared to be additive, whilst knockdown of tko in a variety of specific tissues phenocopied tko25t, producing developmental delay and bang-sensitivity. These findings imply the existence of a systemic signal regulating growth in response to mitochondrial dysfunction. Drugs and RNAi-targeted on pyruvate metabolism interacted with tko25t in ways that implicated pyruvate or one of its metabolic derivatives in playing a central role in generating such a signal. RNA-seq revealed that dietary pyruvate-induced changes in transcript representation were mostly non-coherent with those produced by tko25t or high-sugar, consistent with the idea that growth regulation operates primarily at the translational and/or metabolic level.