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SummaryAdaptive metabolic switches are proposed to underlie conversions between cellular states during normal development as well as in cancer evolution, where they represent important therapeutic targets. However, the full spectrum, characteristics and regulation of existing metabolic switches are unknown. We propose that metabolic switches can be recognised by locating large alternating gene expression patterns and associate them with specific metabolic states. We developed a method to identify interspersed genesets by massive correlated biclustering (MCbiclust) and to predict their metabolic wiring. Testing the method on major breast cancer transcriptome datasets we discovered a series of gene sets with switch-like behaviour, predicting mitochondrial content, activity and central carbon fluxes in tumours associated with different switch positions. The predictions were experimentally validated by bioenergetic profiling and metabolic flux analysis of13C-labelled substrates and were ultimately extended by geneset analysis to link metabolic alterations to cellular states, thus predicting tumour pathology, prognosis and chemosensitivity. The method is applicable to any large and heterogeneous transcriptome dataset to discover metabolic and associated pathophysiological states.

Heterozygous mutations of the lysosomal enzyme glucocerebrosidase (GBA1) represent the major genetic risk for Parkinson’s disease (PD), while homozygous GBA1 mutations cause Gaucher disease, a lysosomal storage disorder, which may involve severe neurodegeneration. We have previously demonstrated impaired autophagy and proteasomal degradation pathways and mitochondrial dysfunction in neurons from GBA1 knockout (gba1−/−) mice. We now show that stimulation with physiological glutamate concentrations causes pathological [Ca2+]c responses and delayed calcium deregulation, collapse of mitochondrial membrane potential and an irreversible fall in the ATP/ADP ratio. Mitochondrial Ca2+ uptake was reduced in gba1−/− cells as was expression of the mitochondrial calcium uniporter. The rate of free radical generation was increased in gba1−/− neurons. Behavior of gba1+/− neurons was similar to gba1−/− in terms of all variables, consistent with a contribution of these mechanisms to the pathogenesis of PD. These data signpost reduced bioenergetic capacity and [Ca2+]c dysregulation as mechanisms driving neurodegeneration.

Summary The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions., Graphical abstract, Highlights • We screen an FDA-approved drug library for mitochondrial Ca2+ uptake modulators • Amorolfine and benzethonium modulate MCU activity • Amorolfine increases MCU-dependent mitochondrial metabolism and muscle size • Benzethonium decreases MCU-dependent cancer cell growth and migration, In search of mitochondrial Ca2+ uptake modulators, De Mario et al. identify amorolfine and benzethonium as positive hits of a 1,600 FDA drug library high-throughput screen. Amorolfine increases mitochondrial metabolism and muscle size in an MCU-dependent manner. Benzethonium negatively regulates MCU activity, mROS formation, and cancer cell growth and migration.

Alzheimer’s disease (AD) is the most common form of dementia and the most prevalent neurodegenerative disease. Genome-wide association studies have linked PICALM to AD risk. PICALM has been implicated in Aβ42 production and turnover, but whether it plays a direct role in modulating Aβ42 toxicity remains unclear. We found that increased expression of the Drosophila PICALM orthologue lap could rescue Aβ42 toxicity in an adult-onset model of AD, without affecting Aβ42 level. Imbalances in the glutamatergic system, leading to excessive, toxic stimulation, have been associated with AD. We found that Aβ42 caused the accumulation of presynaptic vesicular glutamate transporter (VGlut) and increased spontaneous glutamate release. Increased lap expression reversed these phenotypes back to control levels, suggesting that lap may modulate glutamatergic transmission. We also found that lap modulated the localization of amphiphysin (Amph), the homologue of another AD risk factor BIN1, and that Amph itself modulated postsynaptic glutamate receptor (GluRII) localization. We propose a model where PICALM modulates glutamatergic transmission, together with BIN1, to ameliorate synaptic dysfunction and disease progression., Graphical Abstract Graphical Abstractlap and Amph in glutamate metabolism. Aβ affects the localization of both VGlut and GluRIIA, leading to increased glutamate signalling. Lap and Amph restore glutamate signalling, potentially by promoting presynaptic endocytosis of VGlut via lap and/or Rab5/EndoA, and postsynaptic retrival of GluRIIA via Amph.

During obesity, macrophages infiltrate the breast tissue leading to low‐grade chronic inflammation, a factor considered responsible for the higher risk of breast cancer associated with obesity. Here, we formally demonstrate that breast epithelial cells acquire malignant properties when exposed to medium conditioned by macrophages derived from human healthy donors. These effects were mediated by the breast cancer oncogene IKKε and its downstream target—the serine biosynthesis pathway as demonstrated by genetic or pharmacological tools. Furthermore, amlexanox, an FDA‐approved drug targeting IKKε and its homologue TBK1, delayed in vivo tumour formation in a combined genetic mouse model of breast cancer and high‐fat diet‐induced obesity/inflammation. Finally, in human breast cancer tissues, we validated the link between inflammation–IKKε and alteration of cellular metabolism. Altogether, we identified a pathway connecting obesity‐driven inflammation to breast cancer and a potential therapeutic strategy to reduce the risk of breast cancer associated with obesity., In this article a new pathway has been identified that links macrophage‐driven inflammation and cellular metabolism to the acquisition of malignant traits in breast epithelium providing new insight into the well‐established association between breast cancer and obesity and offering a new preventive strategy.

IκB kinase ε (IKKε) is a key molecule at the crossroads of inflammation and cancer. Known to regulate cytokine secretion via NFκB and IRF3, the kinase is also a breast cancer oncogene, overexpressed in a variety of tumours. However, to what extent IKKε remodels cellular metabolism is currently unknown. Here, we used metabolic tracer analysis to show that IKKε orchestrates a complex metabolic reprogramming that affects mitochondrial metabolism and consequently serine biosynthesis independently of its canonical signalling role. We found that IKKε upregulates the serine biosynthesis pathway (SBP) indirectly, by limiting glucose‐derived pyruvate utilisation in the TCA cycle, inhibiting oxidative phosphorylation. Inhibition of mitochondrial function induces activating transcription factor 4 (ATF4), which in turn drives upregulation of the expression of SBP genes. Importantly, pharmacological reversal of the IKKε‐induced metabolic phenotype reduces proliferation of breast cancer cells. Finally, we show that in a highly proliferative set of ER negative, basal breast tumours, IKKε and PSAT1 are both overexpressed, corroborating the link between IKKε and the SBP in the clinical context., The IκB kinase IKKε inhibits mitochondrial metabolism by limiting pyruvate oxidation, in turn activating the serine biosynthesis pathway via ATF4 in a NFκB and IRF3‐independent manner.

Mitochondrial Ca2+ uptake has a key role in cellular Ca2+ homeostasis. Excessive matrix Ca2+ concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH2-linked substrates. The functional consequences of Ca2+-induced mPTP opening were assessed by Ca2+ retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca2+ handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca2+ overload.

The potential to understand fundamental biological processes from gene expression data has grown parallel with the recent explosion of the size of data collections. However, to exploit this potential, novel analytical methods are required, capable of handling massive data matrices. We found current methods limited in the size of correlated gene sets they could discover within biologically heterogeneous data collections, hampering the identification of multi-gene controlled fundamental cellular processes such as energy metabolism, organelle biogenesis and stress responses. Here we describe a novel biclustering algorithm called Massively Correlated Biclustering (MCbiclust) that selects samples and genes from large datasets with maximal correlated gene expression, allowing regulation of complex pathway to be examined. The method has been evaluated using synthetic data and applied to large bacterial and cancer cell datasets. We show that the large biclusters discovered, so far elusive to identification by existing techniques, are biologically relevant and thus MCbiclust has great potential use in the analysis of transcriptomics data to identify large scale unknown effects hidden within the data. The identified massive biclusters can be used to develop improved transcriptomics based diagnosis tools for diseases caused by altered gene expression, or used for further network analysis to understand genotype-phenotype correlations.

The DNA mismatch repair (MMR) pathway is responsible for the repair of base–base mismatches and insertion/deletion loops that arise during DNA replication. MMR deficiency is currently estimated to be present in 15–17% of colorectal cancer cases and 30% of endometrial cancers. MLH1 is one of the key proteins involved in the MMR pathway. Inhibition of a number of mitochondrial genes, including POLG and PINK1 can induce synthetic lethality in MLH1-deficient cells. Here we demonstrate for the first time that loss of MLH1 is associated with a deregulated mitochondrial metabolism, with reduced basal oxygen consumption rate and reduced spare respiratory capacity. Furthermore, MLH1-deficient cells display a significant reduction in activity of the respiratory chain Complex I. As a functional consequence of this perturbed mitochondrial metabolism, MLH1-deficient cells have a reduced anti-oxidant response and show increased sensitivity to reactive oxidative species (ROS)-inducing drugs. Taken together, our results provide evidence for an intrinsic mitochondrial dysfunction in MLH1-deficient cells and a requirement for MLH1 in the regulation of mitochondrial function.

Altered metabolism is a hallmark of cancer, both resulting from and driving oncogenesis. The NAD and NADP redox couples play a key role in a large number of the metabolic pathways involved. In their reduced forms, NADH and NADPH, these molecules are intrinsically fluorescent. As the average time for fluorescence to be emitted following excitation by a laser pulse, the fluorescence lifetime, is exquisitely sensitive to changes in the local environment of the fluorophore, imaging the fluorescence lifetime of NADH and NADPH offers the potential for label-free monitoring of metabolic changes inside living tumors. Here, we describe the biological, photophysical, and methodological considerations required to establish fluorescence lifetime imaging (FLIM) of NAD(P)H as a routine method for profiling the metabolism of living cancer cells and tissues.

Transcription of a large set of nuclear-encoded genes underlies biogenesis of mitochondria, regulated by a complex network of transcription factors and co-regulators. A remarkable heterogeneity can be detected in the expression of these genes in different cell types and tissues, and the recent availability of large gene expression compendiums allows the quantification of specific mitochondrial biogenesis patterns. We have developed a method to effectively perform this task. Massively correlated biclustering (MCbiclust) is a novel bioinformatics method that has been successfully applied to identify co-regulation patterns in large genesets, underlying essential cellular functions and determining cell types. The method has been recently evaluated and made available as a package in Bioconductor for R. One of the potential applications of the method is to compare expression of nuclear-encoded mitochondrial genes or larger sets of metabolism-related genes between different cell types or cellular metabolic states. Here we describe the essential steps to use MCbiclust as a tool to investigate co-regulation of mitochondrial genes and metabolic pathways.

Cytoplasmic and mitochondrial Ca

BACKGROUNDMitochondrial metabolism involves oxidative phosphorylation (OXPHOS) via the respiratory chain and is required for the maintenance of tumour cell proliferation and regulation of epithelial–mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study we interrogate the role of CHCHD4-regulated respiratory chain activity and metabolism in tumour cell proliferation and EMT-related phenotypes.RESULTSWe show that CHCHD4 is essential for the proliferation of tumour cells irrespective of their aetiology. In human tumours, elevated CHCHD4 expression is correlated with a mitochondrial OXPHOS gene signature and with a proliferative gene signature associated with the mTORC1 signalling pathway. Elevated CHCHD4 increases tumour cell proliferation, in a manner that is dependent on complex I (CI) activity, glutamine consumption and mTORC1 activation. CHCHD4 expression is inversely correlated with EMT gene expression both in vitro and in vivo. Finally, we show CHCHD4 regulates the intracellular distribution of the EMT marker vimentin, in a CI-mediated manner.CONCLUSIONSCHCHD4 regulates tumour cell proliferation and metastatic (EMT-related) phenotypes through its control of CI-mediated mitochondrial metabolism.

Altered metabolism is a hallmark of cancer, both resulting from and driving oncogenesis. The NAD and NADP redox couples play a key role in a large number of the metabolic pathways involved. In their reduced forms, NADH and NADPH, these molecules are intrinsically fluorescent. As the average time for fluorescence to be emitted following excitation by a laser pulse, the fluorescence lifetime, is exquisitely sensitive to changes in the local environment of the fluorophore, imaging the fluorescence lifetime of NADH and NADPH offers the potential for label-free monitoring of metabolic changes inside living tumors. Here, we describe the biological, photophysical, and methodological considerations required to establish fluorescence lifetime imaging (FLIM) of NAD(P)H as a routine method for profiling the metabolism of living cancer cells and tissues.

Transcription of a large set of nuclear-encoded genes underlies biogenesis of mitochondria, regulated by a complex network of transcription factors and co-regulators. A remarkable heterogeneity can be detected in the expression of these genes in different cell types and tissues, and the recent availability of large gene expression compendiums allows the quantification of specific mitochondrial biogenesis patterns. We have developed a method to effectively perform this task. Massively correlated biclustering (MCbiclust) is a novel bioinformatics method that has been successfully applied to identify co-regulation patterns in large genesets, underlying essential cellular functions and determining cell types. The method has been recently evaluated and made available as a package in Bioconductor for R. One of the potential applications of the method is to compare expression of nuclear-encoded mitochondrial genes or larger sets of metabolism-related genes between different cell types or cellular metabolic states. Here we describe the essential steps to use MCbiclust as a tool to investigate co-regulation of mitochondrial genes and metabolic pathways.

Background Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved. Results Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes. Conclusions CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells. Electronic supplementary material The online version of this article (10.1186/s40170-019-0200-4) contains supplementary material, which is available to authorized users.

Mitochondria drive cellular adaptation to stress by retro-communicating with the nucleus to promote cytoprotective mechanisms. This process of inter-organelles interplay is known as the Mitochondrial Retrograde Response (MRR) and is enacted by alterations in the mitochondrial function which result into the nuclear stabilization of transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Here, we demonstrate that this adaptive response is relying on the formation of contact sites between mitochondria and nucleus. These hot-spots of communication allow for the redistribution of cholesterol to the nucleus which favours nuclear residency and activity of the NF-kB. The expression level of the cholesterol Translocator Protein (TSPO), resident on the Outer Mitochondrial Membrane (OMM), is key to the formation of these contact sites and the implementation of the cytoprotective response. This work provides therefore a new paradigm in the intracellular communication during stress by reporting the first evidence for a functional and biochemical tethering between mitochondria and nucleus which governs cellular adaptation.

Figure S4. CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Chart shows extracellular levels of glutamine measured in culture medium from control U2OS cells, and cells expressing wild-type CHCHD4 (WT.cl1). Representative of 2 experiments. ±SD. n = 5. (PDF 61 kb)

Cytoplasmic and mitochondrial Ca2+ signals couple cellular ATP production to activity-related energy demand. In order to accurately determine the bioenergetic effect of Ca2+ signals, cellular energy charge, i.e., the compound ratio of the phosphorylated adenine nucleotides AMP, ADP, and ATP, should be estimated. Reversed-phase high-performance liquid chromatography (RP-HPLC) allows the rapid separation and quantitation of these molecules. Here we describe a protocol applied in our laboratories to quantify ATP, ADP, and AMP nucleotides in cellular extracts.

Mitochondrial permeability transition, as the consequence of opening of a mitochondrial permeability transition pore (mPTP), is a cellular catastrophe. Initiating bioenergetic collapse and cell death, it has been implicated in the pathophysiology of major human diseases, including neuromuscular diseases of childhood, ischaemia-reperfusion injury, and age-related neurodegenerative disease. Opening of the mPTP represents a major therapeutic target, as it can be mitigated by a number of compounds. However, clinical studies have so far been disappointing. We therefore address the prospects and challenges faced in translating in vitro findings to clinical benefit. We review the role of mPTP opening in disease, discuss recent findings defining the putative structure of the mPTP, and explore strategies to identify novel, clinically useful mPTP inhibitors, highlighting key considerations in the drug discovery process.

Triple‐negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca2+ uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca2+ uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU‐silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia‐inducible factor‐1α (HIF‐1α) is reduced, suggesting a signaling role for mROS and HIF‐1α, downstream of mitochondrial Ca2+. Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF‐1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca2+ uptake is a potential novel therapeutic target for clinical intervention.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mitochondria drive cellular adaptation to stress by retro-communicating with the nucleus. This process is known as Mitochondrial Retrograde Response (MRR) and is induced by mitochondrial dysfunctions which perturb cell signalling. MRR results in the nuclear stabilization and activation of pro-survival transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Here we demonstrate that MRR is facilitated by the formation of contact sites between mitochondria and the nucleus which establish microdomains of communication between the two organelles. The 18kD Translocator Protein (TSPO), which de-ubiquitylates and stabilizes the mitochondrial network preventing its mitophagy-mediated segregation, is required for this interaction. The tethering TSPO enacts is mediated by the complex formed with the Protein Kinase A via the A-kinase anchoring protein Acyl-CoA Binding Domain Containing 3 (ACBD3) and allows the redistribution of cholesterol which sustains the pro-survival response by blocking NF-kB de-acetylation. This work proposes a new paradigm in the mitochondrial retro-communication by revealing the existence of contact sites between mitochondrial and the nucleus and a signalling role for cholesterol.

Despite reported sightings over many years, certain mitochondrial-specific channels have proven to be elusive beasts, evading molecular identification. However, combining modern genetics with a wave of their ion-sensing wand, researchers have managed to capture first the mitochondrial calcium uniporter, and now that semi-mythological beast, the mitochondrial ATP-sensitive potassium (mitoKATP) channel.

Designing novel inverse agonists of NR RORγt still represents a challenge for the pharmaceutical community to develop therapeutics for treating immune diseases. By exploring the structure of NRs natural ligands, the representative arotenoid ligands and RORs specific ligands share some chemical homologies which can be exploited to design a novel molecular structure characterized by a polycyclic core bearing a polar head and a hydrophobic tail. Compound MG 2778 (8), a cyclopenta[a]phenantrene derivative, was identified as lead compound which was chemically modified at position 2 in order to obtain a small library for preliminary SARs. Cell viability and estrogenic activity of compounds 7, 8, 19a, 30, 31 and 32 were evaluated to attest selectivity. The selected 7, 8, 19a and 31 compounds were assayed in a Gal4 UAS-Luc co-transfection system in order to determine their ability to modulate RORγt activity in a cellular environment. They were evaluated as inverse agonists taken ursolic acid as reference compound. The potency of compounds was lower than that of ursolic acid, but their efficacy was similar. Compound 19a was the most active, significantly reducing RORγt activity at low micromolar concentrations.

Core myopathies are a group of childhood muscle disorders caused by mutations of the ryanodine receptor (RyR1), the Ca2+ release channel of the sarcoplasmic reticulum. These mutations have previously been associated with elevated inositol trisphosphate receptor (IP3R) levels in skeletal muscle myotubes derived from patients. However, the functional relevance and the relationship of IP3R mediated Ca2+ signalling with the pathophysiology of the disease is unclear. It has also been suggested that mitochondrial dysfunction underlies the development of central and diffuse multi-mini-cores, devoid of mitochondrial activity, which is a key pathological consequence of RyR1 mutations. Here we used muscle biopsies of central core and multi-minicore disease patients with RyR1 mutations, as well as cellular and in vivo mouse models of the disease to characterize global cellular and mitochondrial Ca2+ signalling, mitochondrial function and gene expression associated with the disease. We show that RyR1 mutations that lead to the depletion of the channel are associated with increased IP3-mediated nuclear and mitochondrial Ca2+ signals and increased mitochondrial activity. Moreover, western blot and microarray analysis indicated enhanced mitochondrial biogenesis at the transcriptional and protein levels and was reflected in increased mitochondrial DNA content. The phenotype was recapitulated by RYR1 silencing in mouse cellular myotube models. Altogether, these data indicate that remodelling of skeletal muscle Ca2+ signalling following loss of functional RyR1 mediates bioenergetic adaptation.

Aging has been linked to several degenerative processes that, through the accumulation of molecular and cellular damage, can progressively lead to cell dysfunction and organ failure. Human aging is linked with a higher risk for individuals to develop cancer, neurodegenerative, cardiovascular, and metabolic disorders. The understanding of the molecular basis of aging and associated diseases has been one major challenge of scientific research over the last decades. Mitochondria, the center of oxidative metabolism and principal site of reactive oxygen species (ROS) production, are crucial both in health and in pathogenesis of many diseases. Redox signaling is important for the modulation of cell functions and several studies indicate a dual role for ROS in cell physiology. In fact, high concentrations of ROS are pathogenic and can cause severe damage to cell and organelle membranes, DNA, and proteins. On the other hand, moderate amounts of ROS are essential for the maintenance of several biological processes, including gene expression. In this review, we provide an update regarding the key roles of ROS—mitochondria cross talk in different fundamental physiological or pathological situations accompanying aging and highlighting that mitochondrial ROS may be a decisive target in clinical practice.

Calcium signalling is fundamental to the function of the nervous system, in association with changes in ionic gradients across the membrane. Although restoring ionic gradients is energetically costly, a rise in intracellular Ca(2+) acts through multiple pathways to increase ATP synthesis, matching energy supply to demand. Increasing cytosolic Ca(2+) stimulates metabolite transfer across the inner mitochondrial membrane through activation of Ca(2+) -regulated mitochondrial carriers, whereas an increase in matrix Ca(2+) stimulates the citric acid cycle and ATP synthase. The aspartate-glutamate exchanger Aralar/AGC1 (Slc25a12), a component of the malate-aspartate shuttle (MAS), is stimulated by modest increases in cytosolic Ca(2+) and upregulates respiration in cortical neurons by enhancing pyruvate supply into mitochondria. Failure to increase respiration in response to small (carbachol) and moderate (K(+) -depolarization) workloads and blunted stimulation of respiration in response to high workloads (veratridine) in Aralar/AGC1 knockout neurons reflect impaired MAS activity and limited mitochondrial pyruvate supply. In response to large workloads (veratridine), acute stimulation of respiration occurs in the absence of MAS through Ca(2+) influx through the mitochondrial calcium uniporter (MCU) and a rise in matrix [Ca(2+) ]. Although the physiological importance of the MCU complex in work-induced stimulation of respiration of CNS neurons is not yet clarified, abnormal mitochondrial Ca(2+) signalling causes pathology. Indeed, loss of function mutations in MICU1, a regulator of MCU complex, are associated with neuromuscular disease. In patient-derived MICU1 deficient fibroblasts, resting matrix Ca(2+) is increased and mitochondria fragmented. Thus, the fine tuning of Ca(2+) signals plays a key role in shaping mitochondrial bioenergetics.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ?accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. "Regulated cell death" (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Nomenclature Committee on Cell Death. Equipe 11 Apoptose, Cancer et Immunité. Centre de Recherche des Cordeliers; Francia

The intracellular autophagic degradative pathway can have a tumour suppressive or tumour-promoting role depending on the stage of tumour development. Upon starvation or targeting of oncogenic receptor tyrosine kinases (RTKs), autophagy is activated owing to the inhibition of PI3K/AKT/mTORC1 signalling pathway and promotes survival, suggesting that autophagy is a relevant therapeutic target in these settings. However, the role of autophagy in cancer cells where the PI3K/AKT/mTORC1 pathway is constitutively active remains partially understood. Here we report a role for mTORC1-independent basal autophagy in regulation of RTK activation and cell migration in colorectal cancer (CRC) cells. PI3K and RAS-mutant CRC cells display basal autophagy levels despite constitutive mTORC1 signalling, but fail to increase autophagic flux upon RTK inhibition. Inhibition of basal autophagy via knockdown of ATG7 or ATG5 leads to decreased phosphorylation of several RTKs, in particular c-MET. Internalised c-MET colocalised with LAMP1-negative, LC3-positive vesicles. Finally, autophagy regulates c-MET phosphorylation via an mTORC2-dependent mechanism. Overall, our findings reveal a previously unappreciated role of autophagy and mTORC2 in regulation of oncogenic RTK activation, with implications for understanding of cancer cell signalling.

NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzyme-binding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be detected between cell types and altered physiological and pathological states.

DJ-1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinson's disease (PD), but growing evidence suggests involvement of DJ-1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH-SY5Y neuroblastoma cell lines that DJ-1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ-1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ-1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ-1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ-1 complexes. In the KD model, the absence of DJ-1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re-expression of DJ-1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7-related PD. Notably, oxidised DJ-1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease. Correction: Dominik Piston, Lydia Alvarez-Erviti, Vikas Bansal, Daniela Gargano, Zhi Yao, Gyorgy Szabadkai, Mark Odell, M Rhyan Puno, Benny Björkblom, Jodi Maple-Grødem, Peter Breuer, Oliver Kaut, Jan Petter Larsen, Stefan Bonn, Simon Geir Møller, Ullrich Wüllner, Anthony H V Schapira, Matthew E Gegg. DJ-1 is a redox sensitive adapter protein for high molecular weight complexes involved in regulation of catecholamine homeostasis. Human Molecular Genetics. Volume 27, Issue 3, 1 February 2018, Pages 576. DOI: 10.1093/hmg/ddx425

Mitochondria provide the primary source of ATP in the oocyte and early embryo and mitochondrial dysfunction and deficit of mitochondria-derived ATP has been linked to suboptimal developmental competence. We have undertaken a study of ATP in the maturing mouse oocyte using a novel recombinant FRET based probe, AT1.03. We show that AT1.03 can be successfully used to monitor cytosolic ATP levels in single live oocytes over extended time periods. We find that ATP levels undergo dynamic changes associated with specific maturational events and that oocytes display altered rates of ATP consumption at different stages of maturation. Cumulus enclosed oocytes have a higher ATP level during maturation than denuded oocytes and this can be abolished by inhibition of gap junctional communication between the oocyte and cumulus cells. Our work uses a new approach to shed light on regulation of ATP levels and ATP consumption during oocyte maturation.

We are currently witnessing a stream of major discoveries describing the elementary constituents of complex mitochondrial machineries that define the shape, dynamics and intracellular distribution of the organelle. On the basis of these findings the next wave of research holds the promise of unveiling the so far hidden relationships between the fascinating ultrastructural mitochondrial anatomy and its key roles in many areas of cell biology.

The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.

Rationale: The ability of a cell to independently regulate nuclear and cytosolic Ca 2+ signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca 2+ signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca 2+ signals locally has not been explored. Objective: To study the role of perinuclear sarcolemma in selective nuclear Ca 2+ signaling. Methods and Results: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca 2+ signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca 2+ signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca 2+ release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca 2+ buffers—parvalbumin—with cytosolic or nuclear localization demonstrated that the nuclear Ca 2+ handling system is physically and functionally segregated from the cytosolic Ca 2+ signaling machinery. Conclusions: These data reveal the existence of an inositol 1,4,5-trisphosphate–dependent nuclear Ca 2+ toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca 2+ signaling in response to an extracellular ligand.

Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.

Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca2+ uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca2+-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca2+-induced membrane depolarisation and Ca2+ retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature.