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Calcium dynamics control synaptic transmission. Calcium triggers synaptic vesicle fusion, determines release probability, modulates vesicle recycling, participates in long-term plasticity and regulates cellular metabolism. Mitochondria, the main source of cellular energy, serve as calcium signaling hubs. Mitochondrial calcium transients are primarily determined by the balance between calcium influx, mediated by the mitochondrial calcium uniporter (MCU), and calcium efflux through the sodium/lithium/calcium exchanger (NCLX). We identified a human recessive missense SLC8B1 variant that impairs NCLX activity and is associated with severe mental retardation. On this basis, we examined the effect of deleting NCLX in mice on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity. Neuronal mitochondria exhibited basal calcium overload, membrane depolarization, and a reduction in the amplitude and rate of calcium influx and efflux. We observed smaller cytoplasmic calcium transients in the presynaptic terminals of NCLX-KO neurons, leading to a lower probability of release and weaker transmission. In agreement, synaptic facilitation in NCLX-KO hippocampal slices was enhanced. Importantly, deletion of NCLX abolished long term potentiation of Schaffer collateral synapses. Our results show that NCLX controls presynaptic calcium transients that are crucial for defining synaptic strength as well as short- and long-term plasticity, key elements of learning and memory processes., Stavsky et al. examined the effects of deleting the mitochondrial sodium/lithium/calcium exchanger, NCLX, on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity in mice. Having identified a human mutation that impairs NCLX activity and is associated with mental retardation, they show that NCLX is crucial for defining synaptic strength and plasticity, which are pivotal elements of learning and memory.

Background & Aims Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism. Methods We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests. Results We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation. Conclusion Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition. Lay summary The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.

PTEN-induced kinase 1 (PINK1) is a short-lived protein required for the removal of damaged mitochondria through Parkin translocation and mitophagy. Because the short half-life of PINK1 limits its ability to be trafficked into neurites, local translation is required for this mitophagy pathway to be active far from the soma. The Pink1 transcript is associated and cotransported with neuronal mitochondria. In concert with translation, the mitochondrial outer membrane proteins synaptojanin 2 binding protein (SYNJ2BP) and synaptojanin 2 (SYNJ2) are required for tethering Pink1 mRNA to mitochondria via an RNA-binding domain in SYNJ2. This neuron-specific adaptation for the local translation of PINK1 provides distal mitochondria with a continuous supply of PINK1 for the activation of mitophagy.

Treatment of cardiac arrhythmia remains challenging due to severe side effects of common anti-arrhythmic drugs. We previously demonstrated that mitochondrial CaHerewe screened 727 compounds with a history of use in human clinical trials in a three-step screening approach. As a primary screening platform we used a permeabilized HeLa cell-based mitochondrial CaWe identifiedtwo candidate compounds, the clinically approved drugs ezetimibe and disulfiram, which stimulate SR-mitochondria CaTaken together we identified ezetimibe and disulfiram as novel MiCUps and efficient suppressors of arrhythmogenesis and as such as, promising candidates for future preclinical and clinical studies.

Calcium dynamics control synaptic transmission. Calcium triggers synaptic vesicle fusion, determines release probability, modulates vesicle recycling, participates in long-term plasticity and regulates cellular metabolism. Mitochondria, the main source of cellular energy, serve as calcium signaling hubs. Mitochondrial calcium transients are primarily determined by the balance between calcium influx, mediated by the mitochondrial calcium uniporter (MCU), and calcium efflux through the sodium/lithium/calcium exchanger (NCLX). We identified a human recessive missense SLC8B1 variant that impairs NCLX activity and is associated with severe mental retardation. On this basis, we examined the effect of deleting NCLX in mice on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity. Neuronal mitochondria exhibited basal calcium overload, membrane depolarization, and a reduction in the amplitude and rate of calcium influx and efflux. We observed smaller cytoplasmic calcium transients in the presynaptic terminals of NCLX-KO neurons, leading to a lower probability of release and weaker transmission. In agreement, synaptic facilitation in NCLX-KO hippocampal slices was enhanced. Importantly, deletion of NCLX abolished long term potentiation of Schaffer collateral synapses. Our results show that NCLX controls presynaptic calcium transients that are crucial for defining synaptic strength as well as short- and long-term plasticity, key elements of learning and memory processes.

The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.

BACKGROUND AND PURPOSE Treatment of cardiac arrhythmia remains challenging due to severe side effects of common anti-arrhythmic drugs. We previously demonstrated that mitochondrial Ca2+ uptake in cardiomyocytes represents a promising new candidate structure for safer drug therapy. However, druggable agonists of mitochondrial Ca2+ uptake suitable for preclinical and clinical studies are still missing. EXPERIMENTAL APPROACH Herewe screened 727 compounds with a history of use in human clinical trials in a three-step screening approach. As a primary screening platform we used a permeabilized HeLa cell-based mitochondrial Ca2+ uptake assay. Hits were validated in cultured HL-1 cardiomyocytes and finally tested for anti-arrhythmic efficacy in three translational models: a Ca2+ overload zebrafish model and cardiomyocytes of both a mouse model for catecholaminergic polymorphic ventricular tachycardia (CPVT) and induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. KEY RESULTS We identifiedtwo candidate compounds, the clinically approved drugs ezetimibe and disulfiram, which stimulate SR-mitochondria Ca2+ transfer at nanomolar concentrations. This is significantly lower compared to the previously described mitochondrial Ca2+ uptake enhancers (MiCUps) efsevin, a gating modifier of the voltage-dependent anion channel 2, and kaempferol, an agonist of the mitochondrial Ca2+ uniporter. Both substances restored rhythmic cardiac contractions in a zebrafish cardiac arrhythmia model and significantly suppressed arrhythmogenesis in freshly isolated ventricular cardiomyocytes from a CPVT mouse model as well as induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. CONCLUSION AND IMPLICATIONS Taken together we identified ezetimibe and disulfiram as novel MiCUps and efficient suppressors of arrhythmogenesis and as such as, promising candidates for future preclinical and clinical studies.

Calcium (Ca2+) influx into mitochondria occurs through a Ca2+-selective uniporter channel, which regulates essential cellular processes in eukaryotic organisms. Previous evolutionary analyses of its pore-forming subunits MCU and EMRE, and gatekeeper MICU1, pinpointed an evolutionary paradox: the presence of MCU homologs in fungal species devoid of any other uniporter components and of mt-Ca2+ uptake. Here, we trace the mt-Ca2+ uniporter evolution across 1,156 fully-sequenced eukaryotes and show that animal and fungal MCUs represent two distinct paralogous subfamilies originating from an ancestral duplication. Accordingly, we find EMRE orthologs outside Holoza and uncover the existence of an animal-like uniporter within chytrid fungi, which enables mt-Ca2+ uptake when reconstituted in vivo in the yeast Saccharomyces cerevisiae. Our study represents the most comprehensive phylogenomic analysis of the mt-Ca2+ uptake system and demonstrates that MCU, EMRE, and MICU formed the core of the ancestral opisthokont uniporter, with major implications for comparative structural and functional studies., The mitochondrial calcium uptake system, crucial for cellular processes, evolved in ancient eukaryotes. Here, authors perform a phylogenomic analysis across 1,156 eukaryotes, and show that previously identified animal and fungal genes in this system originated from an ancestral duplication.

Cellular proteostasis describes all processes, which are involved in synthesis, folding and degradation of proteins. The balance between protein translation and degradation is strictly regulated in the cell to ensure its viability. For this purpose, the activity of the protein synthesis and degradation machinery can be adapted according to cellular needs. Disturbance of proteostasis leads for example to accumulation of misfolded or damaged proteins in the cell and is linked to aging and conformational diseases (proteinopathies) such as neurodegenerative, cardiovascular and pulmonary diseases. The ubiquitin-proteasome system plays a central role for the balanced protein turnover in the cell as it is responsible for the degradation of up to 80 % of all cellular proteins. The proteasome is a large protein complex, with catalytically active cleavage sites located within the 20S core proteasome. Ubiquitin-dependent degradation of folded proteins is mainly performed by the 26S proteasome, which is formed by the assembly of 20S core particle and 19S regulatory particles. Assembly and activity of the 26S proteasome are fine-tuned according to cellular needs such as growth and differentiation. Regulation of protein synthesis via the mammalian target of rapamycin (mTOR) has for example direct effects on 26S proteasome function. As 26S proteasome function is strictly dependent on energy in form of ATP, mitochondria – the powerhouses of the cell – are also involved in the regulation of protein degradation by the proteasome. Additionally, reactive oxygen species (ROS), which are mainly produced by dysfunctional mitochondria, negatively influence 26S proteasome assembly and activity. However, mitochondria are not only the main source of cellular ATP and ROS but also provide important metabolites and precursors generated by the tricarboxylic acid cycle (TCA), which are involved in central cellular processes such as proliferation. While a variety of regulatory mechanisms for protein translation and 26S proteasome mediated protein degradation have already been described, a metabolic regulation of cellular proteostasis mediated by mitochondria has not been demonstrated so far. Therefore, the main focus of the present study was to dissect a possible connection between mitochondrial metabolism and cellular proteostasis. For that three different models for mitochondrial respiratory chain dysfunction were used: mouse embryonic fibroblasts (MEFs) derived from the so-called mtDNA mutator mouse model, primary human skin fibroblasts with a mutation in the ND5 subunit of respiratory chain complex I and primary human skin and lung fibroblasts treated with the complex I inhibitor and anti-diabetic drug metformin. The different models are all characterized by respiratory chain complex I deficiency in the absence of increased ROS production. Cells maintained cellular viability and did not show signs of severe stress despite respiratory chain dysfunction. Mitochondrial complex I deficiency in mutator MEFs caused metabolic reprogramming of the TCA cycle resulting in diminished aspartate biosynthesis. Reduced aspartate levels caused downregulated proteostasis as both protein translation and 26S proteasome assembly and activity was decreased in respiration deficient cells but could be rescued by supplementation of aspartate. Furthermore, aspartate supplementation induced mTORC1 mediated protein synthesis and mTORC1-dependent transcriptional activation of defined proteasome assembly factors, which were involved in activation of 26S proteasome assembly and activity in cells with complex I deficiency. Similar data were obtained in ND5 mutant skin fibroblasts and upon metformin treatment. In contrast to diminished proteasome function, chronic respiratory chain impairment in mutator MEFs led to strongly induced immunoproteasome expression and activity. Upregulation of the immunoproteasome was accompanied by increased MHC class I antigen presentation during chronic mitochondrial dysfunction representing a so far unknown stress response which may probably serve to alert the immune system. This finding requires further analysis. These results thus uncover a novel concept of how mitochondrial metabolism adaptively adjusts protein synthesis and degradation by the proteasome to the metabolic condition of the cell. These data extend the knowledge about proteasomal regulation in the cell and have therapeutic implications for diseases and drug-targeted mitochondrial reprogramming.

Protein homeostasis involving protein synthesis and degradation by the proteasome system is intricately balanced in the cell and fine-tuned according to cellular needs and upon stress. Metabolic regulation of protein synthesis and proteasome activity by respiratory chain dysfunction has not been investigated so far. In our study, we used mouse embryonic fibroblasts (MEFs) isolated from the mitochondrial (mt)DNA mutator mouse model that is characterized by genetic respiratory chain dysfunction. Cellular proteostasis including protein synthesis and proteasome activity was significantly decreased in mutator MEFs. In particular, mTOR signaling was downregulated and the formation of 26S proteasome complexes was impaired. Electron microscopy and proteomic analysis of isolated mitochondria revealed massive respiratory chain complex I deficiency in mutator mitochondria resulting in the accumulation of NADH and altered Krebs cycle metabolism. Targeted metabolomics of mutator MEFs identified impaired biosynthesis of aspartate as a consequence of disturbed Krebs cycle function. Notably, supplementation with aspartate restored both mTOR mediated protein translation and proteasome activity in mutator cells. Furthermore, we delineated impairment of respiratory chain complex I activity as the driving factor of this metabolic regulation in primary human lung fibroblasts treated with the complex I inhibitor Metformin. Metformin downregulated both protein synthesis and proteasome activity, a phenotype that was rescued by aspartate supplementation. Our data thus unravel a previously unrecognized adaptation of proteostasis to metabolic alterations of the cell, which might be of therapeutic relevance for the treatment of lung diseases.

Mitochondria participate in metabolism and signaling. They adapt to the requirements of various cell types. Publicly available expression data permit to study expression dynamics of genes with mitochondrial function (mito-genes) in various cell types, conditions and organisms. Yet, we lack an easy way of extracting these data for mito-genes. Here, we introduce the visual data mining platform mitoXplorer, which integrates expression and mutation data of mito-genes with a manually curated mitochondrial interactome containing ∼1200 genes grouped in 38 mitochondrial processes. User-friendly analysis and visualization tools allow to mine mitochondrial expression dynamics and mutations across various datasets from four model species including human. To test the predictive power of mitoXplorer, we quantify mito-gene expression dynamics in trisomy 21 cells, as mitochondrial defects are frequent in trisomy 21. We uncover remarkable differences in the regulation of the mitochondrial transcriptome and proteome in one of the trisomy 21 cell lines, caused by dysregulation of the mitochondrial ribosome and resulting in severe defects in oxidative phosphorylation. With the newly developed Fiji plugin mitoMorph, we identify mild changes in mitochondrial morphology in trisomy 21. Taken together, mitoXplorer (http://mitoxplorer.ibdm.univ-mrs.fr) is a user-friendly, web-based and freely accessible software, aiding experimental scientists to quantify mitochondrial expression dynamics.

Mitochondria are pivotal organelles in calcium (Ca2+ ) handling and signalling, constituting intracellular checkpoints for numerous processes that are vital for cell life. Alterations in mitochondrial Ca2+ homeostasis have been linked to a variety of pathological conditions and are critical in the aetiology of several human diseases. Efforts have been taken to harness mitochondrial Ca2+ transport mechanisms for therapeutic intervention, but pharmacological compounds that direct and selectively modulate mitochondrial Ca2+ homeostasis are currently lacking. New avenues have, however, emerged with the breakthrough discoveries on the genetic identification of the main players involved in mitochondrial Ca2+ influx and efflux pathways and with recent hints towards a deep understanding of the function of these molecular systems. Here, we review the current advances in the understanding of the mechanisms and regulation of mitochondrial Ca2+ homeostasis and its contribution to physiology and human disease. We also introduce and comment on the recent progress towards a systems-level pharmacological targeting of mitochondrial Ca2+ homeostasis.

Reactive oxygen species (ROS) are generated by virus-infected cells; however, the physiological importance of ROS generated under these conditions is unclear. Here we found that the inflammation and cell death induced by exposure of mice or cells to sources of ROS were not altered in the absence of canonical ROS-sensing pathways or known cell-death pathways. ROS-induced cell-death signaling involved interactions among the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5 -/- mice showed exacerbated lung inflammation and proinflammatory cytokines in an ozone-exposure model. Similarly, challenge with influenza A virus led to increased infiltration of the virus, lymphocytic bronchiolitis and reduced survival of Pgam5 -/- mice. This pathway, which we have called 'oxeiptosis', was a ROS-sensitive, caspase independent, non-inflammatory cell-death pathway and was important for protection against inflammation induced by ROS or ROS-generating agents such as viral pathogens.

BackgroundMitochondria produce cellular energy in the form of ATP and are involved in various metabolic and signaling processes. However, the cellular requirements for mitochondria are different depending on cell type, cell state or organism. Information on the expression dynamics of genes with mitochondrial functions (mito-genes) is embedded in publicly available transcriptomic or proteomic studies and the variety of available datasets enables us to study the expression dynamics of mito-genes in many different cell types, conditions and organisms. Yet, we lack an easy way of extracting these data for gene groups such as mito-genes.ResultsHere, we introduce the web-based visual data mining platform mitoXplorer, which systematically integrates expression and mutation data of mito-genes. The central part of mitoXplorer is a manually curated mitochondrial interactome containing ∼1200 genes, which we have annotated in 35 different mitochondrial processes. This mitochondrial interactome can be integrated with publicly available transcriptomic, proteomic or mutation data in a user-centric manner. A set of analysis and visualization tools allows the mining and exploration of mitochondrial expression dynamics and mutations across various datasets from different organisms and to quantify the adaptation of mitochondrial dynamics to different conditions. We apply mitoXplorer to quantify expression changes of mito-genes of a set of aneuploid cell lines that carry an extra copy of chromosome 21. mitoXplorer uncovers remarkable differences in the regulation of the mitochondrial transcriptome and proteome due to the dysregulation of the mitochondrial ribosome in retinal pigment epithelial trisomy 21 cells which results in severe defects in oxidative phosphorylation.ConclusionsWe demonstrate the power of the visual data mining platform mitoXplorer to explore expression data in a focused and detailed way to uncover underlying potential mechanisms for further experimental studies. We validate the hypothesis-creating power of mitoXplorer by testing predicted phenotypes in trisomy 21 model systems. MitoXplorer is freely available athttp://mitoxplorer.ibdm.univ-mrs.fr. MitoXplorer does not require installation nor programming knowledge and is web-based. Therefore, mitoXplorer is accessible to a wide audience of experimental experts studying mitochondrial dynamics.

The development of fluorescence-based oxygen sensors coupled with microplate-based assays for quantitative bioenergetics analyses enables screening multiple experimental conditions at once with small biological material and in a timely manner. In this chapter, we outline detailed protocols and practical tips to design and perform controlled measurements of (a) respiratory and glycolytic metabolism of intact cells, (b) substrate-dependent respiration in permeabilized cells and isolated mitochondria, and (c) calcium-dependent regulation of mitochondrial bioenergetics with Seahorse XF Flux Analyzers.

The development of fluorescence-based oxygen sensors coupled with microplate-based assays for quantitative bioenergetics analyses enables screening multiple experimental conditions at once with small biological material and in a timely manner. In this chapter, we outline detailed protocols and practical tips to design and perform controlled measurements of (a) respiratory and glycolytic metabolism of intact cells, (b) substrate-dependent respiration in permeabilized cells and isolated mitochondria, and (c) calcium-dependent regulation of mitochondrial bioenergetics with Seahorse XF Flux Analyzers.

Human mitochondria are complex and highly dynamic biological systems, comprised of over a thousand parts and evolved to fully integrate into the specialized intracellular signaling networks and metabolic requirements of each cell and organ. Over the last two decades, several complementary, top-down computational and experimental approaches have been developed to identify, characterize and modulate the human mitochondrial system, demonstrating the power of integrating classical reductionist and discovery-driven analyses in order to de-orphanize hitherto unknown molecular components of mitochondrial machineries and pathways. To this goal, systematic, multiomics-based surveys of proteome composition, protein networks, and phenotype-to-pathway associations at the tissue, cell and organellar level have been largely exploited to predict the full complement of mitochondrial proteins and their functional interactions, therefore catalyzing data-driven hypotheses. Collectively, these multidisciplinary and integrative research approaches hold the potential to propel our understanding of mitochondrial biology and provide a systems-level framework to unraveling mitochondria-mediated and disease-spanning pathomechanisms.

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer's disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum-mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.

Mitochondria vary in morphology and function in different tissues, however little is known about their molecular diversity among cell types. To investigate mitochondrial diversity in vivo, we developed an efficient protocol to isolate cell type-specific mitochondria based on a new MitoTag mouse. We profiled the mitochondrial proteome of three major neural cell types in cerebellum and identified a substantial number of differential mitochondrial markers for these cell types in mice and humans. Based on predictions from these proteomes, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neurons. Moreover, we identified Rmdn3 as a major determinant of ER-mitochondria proximity in Purkinje cells. Our novel approach enables exploring mitochondrial diversity on the functional and molecular level in many in vivo contexts.

SUMMARYThe mitochondrial calcium uniporter is a highly selective ion channel composed of species-and tissue-specific structural and regulatory subunits. However, the contribution of each component to uniporter-mediated activity still remains unclear. Here, we employ an evolutionary and synthetic biology approach to investigate the functional inter-dependence between the pore-forming subunit MCU and the EF-hand protein MICU1. Using phylogenetic profiling and genetic complementation analyses, we show that MCU and MICU1 constitute the minimal eukaryotic unit of the uniporter, pointing towards a strong selective pressure behind their co-occurrence. Heterologous reconstitution of MCU-mediated and MICU1-gated mitochondrial calcium entryin vivoin yeast cells demonstrates that MICU1per seis essential to protect yeast from MCU-dependent manganese cytotoxicity. Accordingly, MICU1 deletion significantly sensitizes human HEK-293 cells to manganese-induced stress. Our study identifies a critical role of MICU1 in the regulation of MCU ion selectivity, with potential implications for patients with MICU1 deficiency.

The mitochondrial calcium uniporter is a highly selective ion channel composed of species- and tissue-specific subunits. However, the functional role of each component still remains unclear. Here, we establish a synthetic biology approach to dissect the interdependence between the pore-forming subunit MCU and the calcium-sensing regulator MICU1. Correlated evolutionary patterns across 247 eukaryotes indicate that their co-occurrence may have conferred a positive fitness advantage. We find that, while the heterologous reconstitution of MCU and EMRE in vivo in yeast enhances manganese stress, this is prevented by co-expression of MICU1. Accordingly, MICU1 deletion sensitizes human cells to manganese-dependent cell death by disinhibiting MCU-mediated manganese uptake. As a result, manganese overload increases oxidative stress, which can be effectively prevented by NAC treatment. Our study identifies a critical contribution of MICU1 to the uniporter selectivity, with important implications for patients with MICU1 deficiency, as well as neurological disorders arising upon chronic manganese exposure. We acknowledge support from the German Research Foundation (DFG) under the Emmy Noether Programme (PE 2053/1-1 to F.P. and J.W.), the Munich Center for Systems Neurology (SyNergy EXC 1010 to F.P.), the Juniorverbund in der Systemmedizin “mitOmics” (FKZ 01ZX1405B to V.G. and A.L.), The Bert L & N Kuggie Vallee Foundation (to F.P. and D.M.A.), the DFG (MO1944/1-2 to D.M.), the Spanish Ministry of Economy, Industry, and Competitiveness (MEIC; BFU2015-67107), the European Union’s Horizon 2020 research and innovation program under grant agreement ERC-2016-724173 (to T.G. and A.A.P.), and the NIH (RO1 GM102724 to G.H.).

Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300 mM SM for 1 h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50 mu M BER for 24 h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies.

Global or local ischemia contributes to the pathogenesis of acute kidney injury (AKI). Currently there are no specific therapies to prevent AKI. Potentiation of glycolytic metabolism and attenuation of mitochondrial respiration may decrease cell injury and reduce reactive oxygen species generation from the mitochondria. Meclizine, an over-the-counter anti-nausea and -dizziness drug, was identified in a ‘nutrient-sensitized’ chemical screen. Pretreatment with 100 mg/kg of meclizine, 17 h prior to ischemia protected mice from IRI. Serum creatinine levels at 24 h after IRI were 0.13 ± 0.06 mg/dl (sham, n = 3), 1.59 ± 0.10 mg/dl (vehicle, n = 8) and 0.89 ± 0.11 mg/dl (meclizine, n = 8). Kidney injury was significantly decreased in meclizine treated mice compared with vehicle group (p, Graphical abstract, Highlights • Pretreatment with meclizine protected mice from kidney ischemia–reperfusion injury. • Meclizine reduced mitochondrial oxygen consumption and upregulated glycolysis. • Meclizine caused accumulation of phosphoethanolamine, a mitochondrial respiration inhibitor. • Phosphoethanolamine recapitulated meclizine's effects on metabolism and renal protection. Kishi et al. demonstrate that meclizine, an over-the-counter anti-nausea drug, protects mouse against kidney ischemia. Pretreatment with 100 mg/kg of meclizine, 17 or 24 h prior to ischemia showed kidney protection in mice. Meclizine reduced mitochondrial oxygen consumption by directly inhibiting the Kennedy pathway of phosphatidylethanolamine biosynthesis and up-regulated glycolysis.

ProtPhylo is a web-based tool to identify proteins that are functionally linked to either a phenotype or a protein of interest based on co-evolution. ProtPhylo infers functional associations by comparing protein phylogenetic profiles (co-occurrence patterns of orthology relationships) for more than 9.7 million non-redundant protein sequences from all three domains of life. Users can query any of 2048 fully sequenced organisms, including 1678 bacteria, 255 eukaryotes and 115 archaea. In addition, they can tailor ProtPhylo to a particular kind of biological question by choosing among four main orthology inference methods based either on pair-wise sequence comparisons (One-way Best Hits and Best Reciprocal Hits) or clustering of orthologous proteins across multiple species (OrthoMCL and eggNOG). Next, ProtPhylo ranks phylogenetic neighbors of query proteins or phenotypic properties using the Hamming distance as a measure of similarity between pairs of phylogenetic profiles. Candidate hits can be easily and flexibly prioritized by complementary clues on subcellular localization, known protein-protein interactions, membrane spanning regions and protein domains. The resulting protein list can be quickly exported into a csv text file for further analyses. ProtPhylo is freely available at http://www.protphylo.org.

Mitochondria are pivotal organelles in calcium (Ca(2+)) handling and signalling, constituting intracellular checkpoints for numerous processes that are vital for cell life. Alterations in mitochondrial Ca(2+) homeostasis have been linked to a variety of pathological conditions and are critical in the aetiology of several human diseases. Efforts have been taken to harness mitochondrial Ca(2+) transport mechanisms for therapeutic intervention, but pharmacological compounds that direct and selectively modulate mitochondrial Ca(2+) homeostasis are currently lacking. New avenues have, however, emerged with the breakthrough discoveries on the genetic identification of the main players involved in mitochondrial Ca(2+) influx and efflux pathways and with recent hints towards a deep understanding of the function of these molecular systems. Here, we review the current advances in the understanding of the mechanisms and regulation of mitochondrial Ca(2+) homeostasis and its contribution to physiology and human disease. We also introduce and comment on the recent progress towards a systems‐level pharmacological targeting of mitochondrial Ca(2+) homeostasis. [Image: see text]

New findings What is the topic of this review? This paper overviews the links between Ca2+ and Na+ signalling in various types of cells. What advances does it highlight? This paper highlights the general importance of ionic signalling and overviews the molecular mechanisms linking Na+ and Ca2+ dynamics. In particular, the narrative focuses on the molecular physiology of plasmalemmal and mitochondrial Na+ -Ca2+ exchangers and plasmalemmal transient receptor potential channels. Functional consequences of Ca2+ and Na+ signalling for co-ordination of neuronal activity with astroglial homeostatic pathways fundamental for synaptic transmission are discussed. Abstract Transmembrane ionic gradients, which are an indispensable feature of life, are used for generation of cytosolic ionic signals that regulate a host of cellular functions. Intracellular signalling mediated by Ca2+ and Na+ is tightly linked through several molecular pathways that generate Ca2+ and Na+ fluxes and are in turn regulated by both ions. Transient receptor potential (TRP) channels bridge endoplasmic reticulum Ca2+ release with generation of Na+ and Ca2+ currents. The plasmalemmal Na+ -Ca2+ exchanger (NCX) flickers between forward and reverse mode to co-ordinate the influx and efflux of both ions with membrane polarization and cytosolic ion concentrations. The mitochondrial calcium uniporter channel (MCU) and mitochondrial Na+ -Ca2+ exchanger (NCLX) mediate Ca2+ entry into and release from this organelle and couple cytosolic Ca2+ and Na+ fluctuations with cellular energetics. Cellular Ca2+ and Na+ signalling controls numerous functional responses and, in the CNS, provides for fast regulation of astroglial homeostatic cascades that are crucial for maintenance of synaptic transmission.

Mitochondrial functions depend on the steep H+ electrochemical gradient (ΔμH+) across their inner membrane. The available tools for controlling this gradient are essentially limited to inhibitors of the respiratory chain or of the H+ ATPase or to uncouplers, poisons plagued by important side effects and that lack both cell and spatial specificity. We show here that, by transfecting cells with the cDNA encoding channelrhodopsins specifically targeted to the inner mitochondrial membrane, we can obtain an accurate and spatially confined, light-dependent control of mitochondrial membrane potential and, as a consequence, of a series of mitochondrial activities ranging from electron transport to ATP synthesis and Ca2+ signaling.

Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH+) across the inner membrane. Although several drugs can modulate ΔμH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondriatargeted opsinswere used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

Mitochondria serve as the center stage for a number of cellular processes, including energy production, apoptosis, ion homeostasis, iron and copper processing, steroid metabolism, de novo pyrimidine, and heme biosynthesis. The study of mitochondrial function often requires the purification of intact and respiratory-competent organelles. Here, we provide detailed protocols to isolate functional mitochondria from various types of mammalian cells and mouse tissues, in both crude and pure forms. We introduce the use of nitrogen cavitation for the disruption of plasma membrane and the reproducible isolation of mitochondria-enriched fractions of high yield. Mitochondria that are isolated by these procedures are intact and coupled and can directly be used for several downstream analyses, such as measurements of oxygen consumption and calcium buffering capacity.

Mitochondrial calcium ([Ca(2+)]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca(2+)]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca(2+) measurements with the Ca(2+)-binding mitochondria-targeted aequorin and FRET-based [Ca(2+)]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca(2+)]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.Cell Death and Differentiation advance online publication, 10 March 2017; doi:10.1038/cdd.2017.2.

The mixed-valent oxo-bridged ruthenium complex [(HCO2)(NH3)4Ru(μ-O)Ru(NH3)4(O2CH)]3+, known as Ru360, is a selective inhibitor of mitochondrial calcium uptake. Although this compound is useful for studying the role of mitochondrial calcium in biological processes, its widespread availability is limited because of challenges in purification and characterization. Here, we describe our investigations of three different synthetic methods for the preparation of a functional analogue of this valuable compound. We demonstrate that this analogue, isolated from our procedures, exhibits potent mitochondrial calcium uptake inhibitory properties in permeabilized HeLa cells and in isolated mitochondria.

Mitochondria serve as the center stage for a number of cellular processes, including energy production, apoptosis, ion homeostasis, iron and copper processing, steroid metabolism, de novo pyrimidine, and heme biosynthesis. The study of mitochondrial function often requires the purification of intact and respiratory-competent organelles. Here, we provide detailed protocols to isolate functional mitochondria from various types of mammalian cells and mouse tissues, in both crude and pure forms. We introduce the use of nitrogen cavitation for the disruption of plasma membrane and the reproducible isolation of mitochondria-enriched fractions of high yield. Mitochondria that are isolated by these procedures are intact and coupled and can directly be used for several downstream analyses, such as measurements of oxygen consumption and calcium buffering capacity.

Summary Mitochondrial Ca 2+ uptake via the uniporter is central to cell metabolism, signaling, and survival. Recent studies identified MCU as the uniporter's likely pore and MICU1, an EF-hand protein, as its critical regulator. How this complex decodes dynamic cytoplasmic [Ca 2+ ] ([Ca 2+ ] c ) signals, to tune out small [Ca 2+ ] c increases yet permit pulse transmission, remains unknown. We report that loss of MICU1 in mouse liver and cultured cells causes mitochondrial Ca 2+ accumulation during small [Ca 2+ ] c elevations but an attenuated response to agonist-induced [Ca 2+ ] c pulses. The latter reflects loss of positive cooperativity, likely via the EF-hands. MICU1 faces the intermembrane space and responds to [Ca 2+ ] c changes. Prolonged MICU1 loss leads to an adaptive increase in matrix Ca 2+ binding, yet cells show impaired oxidative metabolism and sensitization to Ca 2+ overload. Collectively, the data indicate that MICU1 senses the [Ca 2+ ] c to establish the uniporter's threshold and gain, thereby allowing mitochondria to properly decode different inputs.

Small conductance calcium-activated potassium (SK2/K(Ca)2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K(+) currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction.

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction incancer cells. We investigated the mechanism ofinflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial ComplexI. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component ofthis inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanismfor NLRP3 activation and identify targets of imiquimod that might be clinically relevant.

Mitochondrial calcium uptake has a central role in cell physiology by stimulating ATP production, shaping cytosolic calcium transients and regulating cell death. The biophysical properties of mitochondrial calcium uptake have been studied in detail, but the underlying proteins remain elusive. Here we use an integrative strategy to predict human genes involved in mitochondrial calcium entry based on clues from comparative physiology, evolutionary genomics and organelle proteomics. RNA interference against 13 top candidates highlighted one gene, CBARA1, that we call hereafter mitochondrial calcium uptake 1 (MICU1). Silencing MICU1 does not disrupt mitochondrial respiration or membrane potential but abolishes mitochondrial calcium entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic calcium transients and activation of matrix dehydrogenases. MICU1 is associated with the mitochondrial inner membrane and has two canonical EF hands that are essential for its activity, indicating a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high-capacity mitochondrial calcium uptake. Its discovery may lead to the complete molecular characterization of mitochondrial calcium uptake pathways, and offers genetic strategies for understanding their contribution to normal physiology and disease.

Most cells have the inherent capacity to shift their reliance on glycolysis relative to oxidative metabolism, and studies in model systems have shown that targeting such shifts may be useful in treating or preventing a variety of diseases ranging from cancer to ischemic injury. However, we currently have a limited number of mechanistically distinct classes of drugs that alter the relative activities of these two pathways. We screen for such compounds by scoring the ability of >3,500 small molecules to selectively impair growth and viability of human fibroblasts in media containing either galactose or glucose as the sole sugar source. We identify several clinically used drugs never linked to energy metabolism, including the antiemetic meclizine, which attenuates mitochondrial respiration through a mechanism distinct from that of canonical inhibitors. We further show that meclizine pretreatment confers cardioprotection and neuroprotection against ischemia-reperfusion injury in murine models. Nutrient-sensitized screening may provide a useful framework for understanding gene function and drug action within the context of energy metabolism.

Systematic screens for human disease genes have emerged in recent years, due to the wealth of information provided by genome sequences and large scale datasets. Here we review how integration of genomic data in yeast and human is helping to elucidate the genetic basis of mitochondrial diseases. The identification of nearly all yeast mitochondrial proteins and many of their functional interactions provides insight into the role of mitochondria in cellular processes. This information enables prioritization of the candidate genes underlying mitochondrial disorders. In an iterative fashion, the link between predicted human candidate genes and their disease phenotypes can be experimentally tested back in yeast.

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K

According to the endosymbiotic theory, mitochondria originate from a free-living alpha-proteobacteria that established an intracellular symbiosis with the ancestor of present-day eukaryotic cells. During the bacterium-to-organelle transformation, the proto-mitochondrial proteome has undergone a massive turnover, whereby less than 20 % of modern mitochondrial proteomes can be traced back to the bacterial ancestor. Moreover, mitochondrial proteomes from several eukaryotic organisms, for example, yeast and human, show a rather modest overlap, reflecting differences in mitochondrial physiology. Those differences may result from the combination of differential gain and loss of genes and retargeting processes among lineages. Therefore, an evolutionary signature, also called "phylogenetic profile", could be generated for every mitochondrial protein. Here, we present two evolutionary biology approaches to study mitochondrial physiology: the first strategy, which we refer to as "comparative physiology," allows the de novo identification of mitochondrial proteins involved in a physiological function; the second, known as "phylogenetic profiling," allows to predict protein functions and functional interactions by comparing phylogenetic profiles of uncharacterized and known components.