Poster session 1

Non-small cell lung cancer (NSCLC) metastasis is the leading cause of cancer-related death. Resistance to detachment-induced apoptosis (anoikis) is involved in metastasis. Previous study demonstrated that the mitochondrial protein BCL2L13 regulates apoptosis and ceramide synthesis and our data showed that its expression is reduced in lung cancer lymph node metastasis. Here we investigated BCL2L13 role in anoikis resistance and NSCLC metastasis. In a series of gain and loss of function in rodent and human NSCLC, we demonstrated that BCL2L13 deletion induced anoikis resistance. Additionally, we confirmed BCL2L13 interaction with ceramide synthase (CerS) 2/6 and observed that BCL2L13 deletion significantly altered lipid profile in tumour-attached/pre-metastatic and detached/metastatic conditions, especially of ceramide by-products. Based on these observations, we conclude that BCL2L13 potentially regulates NSCLC metastasis through CerS 2/6 activity via anoikis. Therefore, our work allows us to understand the molecular mechanisms of NSCLC metastasis to determine reliable early metastasis biomarkers.  

The emergence of microproteins smaller than 100 amino acids encoded by small open reading frame (sORF) is rapidly expanding the known proteome at the lower end of the size distribution. We recently demonstrated that the mitochondrial proteome, particularly the electron transport chain (ETC), is enriched for such sORF-encoded proteins (mito-SEPs). Brawnin is a 73 amino acid mito-SEP that is required for ETC Complex III assembly via stabilization of nascent mitochondrial Cytochrome b. Deletion of Brawnin in zebrafish results in lethal mitochondrial disease. Surprisingly, deletion of mammalian Brawnin activates a compensatory supercomplex that is absent in zebrafish, which completely rescues Complex III deficiency in mice. As such, Brawnin knockout mice unexpectedly have enhanced protection against exercise stress and viral infection. Our work demonstrates that mito-SEPs play critical roles in organismal physiology despite their small size, and underscores the potential of SEP functionalization in unveiling new paradigms of metabolic regulation.

Mitochondrial morphology is dynamically changed in conjunction with spatiotemporal functionality. Although considerable efforts have been made to understand why abnormal mitochondrial morphology occurs in various diseases, the biological significance of mitochondrial morphology in states of health and disease remains to be elucidated owing to technical limitations. In the present study, we developed a novel method, termed inducible Counter Mitochondrial Morphology (iCMM), to purposely manipulate mitochondrial morphological patterns on a minutes timescale, using a chemically inducible dimerization system. Using iCMM, we showed that mitochondrial morphological changes rapidly lead to the characteristic reconstitution of various biological information, which is difficult to investigate by conventional genetic engineering. The manipulation of mitochondrial morphology using iCMM can improve our understanding of the interplay between mitochondrial morphology and cellular functions. 

The mitochondrial network is subjected to dramatic morphological changes during cell cycle transitions through fusion and fission events along with cytoskeleton-based transport. During mitosis, mitochondrial fission ensures equal distribution of mitochondria to daughter cells. If and how this process can actively drive mitotic progression remains largely unknown. Here we discover a novel regulatory mechanism upstream of the mitochondrial fission factor (MFF) that relies on its phosphorylation by Protein Kinase D (PKD) specifically during mitosis. PKD-mediated MFF phosphorylation does not interfere with the ability of MFF to induce mitochondrial fragmentation in interphase, but represents the main signaling trigger for mitotic mitochondrial fission and is directly coupled to fidelity of chromosome segregation. Cells with a defective PKD-MFF pathway are characterized by an extensively fused mitochondrial network, premature anaphase initiation and mitotic slippage, polyploidy and reduced long-term proliferation capacity. The PKD/MFF mitotic pathway appears to shift the scientific paradigm demonstrating that mitochondrial dynamics can directly drive progression of mitosis and control mitotic surveillance mechanisms.

Mitochondrial dynamics include regulated mechanisms of fission/fusion, MT-based motility, and organelle turnover that are essential for quality control. Any alterations can cause mitochondrial diseases/neuropathies. Here we examine the physiological relevance of α-syn in mitochondria. We found that excess human α-syn causes fragmented mitochondria. Truncation of the C-terminus (α-syn^1-120) or deletion of the NAC region did not eliminate fragmentation. α-syn also caused oxidation measured live using three mitochondrial quality control reporters. However, fragments induced by α-syn^1-120 were healthy. α-syn-mediated oxidized fragments showed biased retrograde motility, but α-syn^1-120-mediated non-oxidized fragments did not. Depletion/inhibition/excess DRP1 rescued α-syn-mediated fragmentation and oxidation, but excess MFN had no effect, indicating a DRP-dependent mechanism for fragmentation. Intriguingly, excess PINK1/Parkin rescued α-syn-mediated fragmentation and membrane depolarization, likely via functional associations with α-syn C-terminus. Together, we postulate that α-syn N- and C-terminal regions have distinct physiological roles during mitochondrial dynamics, opening up a previously unknown mitochondrial pathogenic pathway for PD.

Coenzyme Q (CoQ) is a redox-active lipid with a prominent role in the mitochondrial respiratory chain. Still, it is also involved in other redox processes as an electron acceptor for specific dehydrogenases. Primary CoQ deficiencies are rare mitochondrial conditions, clinically highly heterogeneous and biochemically characterised by a reduction in CoQ, caused by biallelic mutations in any of the -at least- 11 COQ genes participating in its biosynthesis. Remarkably, patients show a broad spectrum of manifestations, severity, and age of onset, but a clear genotype-phenotype correlation is still lacking. Transcriptional analysis of a cohort of COQ4 patients shows specific regulation of pathways that will contribute to unveiling the pathogenesis of these deficiencies and establish specific markers for the severity of the disease. Genes involved in mitochondrial metabolism, development and the cell cycle are modulated in these patients. We hypothesise that the disease unfolding due to defects in specific COQ genes could be different during development and would determine severity, the age of onset and the affected tissues. Modelling rare diseases is a promising approach to overcome the lack of epidemiological studies. That is why we have generated CRISPR-RfxCas13d knockdown and CRISPR-Cas9 loss-of-function Danio rerio (zebrafish) mutant models defective in coq4 and coq6 genes as a paradigm of genes showing a different clinical set of manifestations and severity. Our work will contribute to closing the gap in the knowledge of the regulation of CoQ biosynthesis during development and its coordination with mitochondrial biogenesis. The functional and physiological characterisation of the animals will help to better understand the disease triggered by coq4 and coq6 defects. Furthermore, the models have eventually the potential to serve as drug screening platforms for preclinical assays.

Mitochondrial dynamics play a critical role in regulating mitochondrial homeostasis. This process is controlled by large GTPases of the Dynamin family, which are required for mitochondrial fission and fusion. In addition to these core components, the fission process also requires the presence of an endoplasmic reticulum (ER) tubule and actin at the site of mitochondrial division. In contrast, the precise mechanism or presence of factors facilitating the fusion process are not well understood. Here, we show that actin is recruited at the site of mitochondrial fusion and is required for the process. To study the role of actin in mitochondrial fusion, we first measured the presence of actin at fusion sites using fluorescent protein-tagged actin nanobodies targeted to mitochondrial and ER membranes. Fusion events occurred in the presence of ER and polymerized actin. Specifically, actin was present on the receiving, immobile mitochondrion, but not the incoming, fusing mitochondria. Importantly, disruption of actin polymerization through inhibition or knockdown of the actin-related protein Arp2/3 complex, which initiates branched actin polymerization, greatly reduced the total number of fusion events. Altogether, our results indicate that actin polymerization and the ER are required for mitochondrial fusion.

Mitochondrial dysfunction in dopaminergic neurons (DaNs) is central in Parkinson’s disease (PD) and is caused by the accumulation of mtDNA indels, as shown post-mortem in PD patients. To study the underlying pathomechanisms, we generated mice expressing a dominant-negative variant of Twinkle (K320E) in DaNs, which leads to mtDNA deletions in patients. K320E-Twinkle^DaN caused reduced activity and levels of COX before the onset of neuron loss. Surprisingly, 20-month-old mice showed no motor symptoms, although they lost 75% of DaNs, in stark contrast to MitoPark mice, in which - upon TFAM ablation - a similar 75% loss leads to severe symptoms at 20 weeks. Intriguingly, the innervation area still possessed ~80% of dopaminergic projections, explaining the absence of motor defects in K320E-Twinkle^DaN. Thus, DaNs are able to adapt to accumulating mtDNA indels. RNAseq data of laser-dissected DaNs, compensatory axonal sprouting and additional recruitment of striatal synapses will be shown and explain the phenotype.

Mitochondrial dysfunction caused by mtDNA alterations has been associated with skeletal muscle atrophy and myofiber loss. However, whether such defects occurring in myofibers are sufficient to cause sarcopenia is unclear. Also, the impact of mtDNA alterations in muscle stem cells (MuSCs) in the context of sarcopenia remain to be investigated. To approach this, we used mouse models with accelerated accumulation of mtDNA alterations in myofibers (K320E-skm mice) or MuSCs (K320E-msc mice) only. We show that despite progressive accumulation of myofibers with mitochondrial deficiency, K320E-skm mice have no loss of muscle mass, strength or physical performance. In contrast, 7 days after cardiotoxin-induced muscle regeneration, K320E-msc mice showed 30% of fibers with mitochondrial deficiency, increased fibrosis, fat infiltration, and reduced muscle mass. Taken together, our results suggest that the accumulation of mtDNA alterations in myofibers activates regeneration during aging, which leads to sarcopenia if such alterations have expanded in MuSCs as well.

The regulation of adult stem cell quiescence is essential for stem cell maintenance, longevity and sustained tissue regeneration, however, the upstream regulatory mechanisms are not fully understood. Our studies have uncovered that changes in stem cell mitochondrial shape serve as an upstream signaling mechanism that communicates retrogradely with the nucleus to regulate the quiescent state of stem cells. Here, transient mitochondrial fragmentation and decreased expression of the OPA1 fusion protein occur in response to a microenvironmental stimulus via an HGF/mTOR mechanism. This transitory change in mitochondrial shape drives the exit of stem cells from quiescence, and cell cycle re-entry, by activating an intracellular reactive oxygen species (ROS) and glutathione (GSH) mediated signaling pathway that initiates a nuclear transcriptional program to suppress quiescence and self-renewal genes and promote myogenic gene expression. We provide evidence that excessive mitochondrial fragmentation, commonly observed in myopathies, degenerative diseases and aging,  leads to premature stem cell activation and depletion. Together, our data demonstrate that mitochondrial plasticity and redox balance are essential upstream regulators of stem cell function and longevity. Together, our data demonstrate that mitochondrial plasticity and redox balance are essential upstream regulators of stem cell quiescence and longevity.

Subjects carrying the mitochondrial DNA pathogenic variant m.3243A>G exhibit low bone mineral density. However, the underlying mechanisms remain unknown. Here, we show that bone-marrow mesenchymal stem cells (BM-MSC) from 10 m.3243A>G carriers have lower mitochondrial respiration(FC=0.45,p<0.01) and mitochondrial ATP production(FC=0.74,p<0.01), together with a compensatory increase in glycolysis(FC=1.49,p<0.01) and glycolytic ATP production(FC=1.27,p=0.02), leading to unchanged total ATP production. This metabolic reprogramming is associated with fewer colony-forming units, lower cell proliferation rates, and decreased in vivo heterotopic bone formation capacity(50% lower,p=0.03). Carriers´ osteoclasts show no metabolic reprogramming. Accordingly, carriers´ osteoclasts show lower heteroplasmy (16-30%) than carriers´ BM-MSC (12-82%). These bone cells´ characteristics are reflected in bone biopsies as 50% lower bone volume to total volume(p=0.01), driven by decreased bone formation(FC=0.6,p=0.05) and not the osteoclasts resorptive activity. These data indicate that mitochondria are key for bone formation and can be a new therapeutical target for bone formation.

Tissue homeostasis is maintained via a fine balance between pro- and anti-inflammatory signals, a balance that is lost when mitochondrial metabolism is compromised in T cells, resulting in impaired immunity, multimorbidity and inflammation. Nevertheless, how different tissue specific microenvironments and immune cells crosstalk determining the damage to the tissue and the systemic outcome of a T cell-specific metabolic dysfunction is poorly understood. Here, by using a mouse model of cardiolipin synthesis deficiency in T cells (PTPMT1 ΔT mice), we show that these mice are characterized by extreme gut inflammation and reduced lifespan. The phenotype is independent of both T cell-generated Type I and Type II interferons but relies on reduced survival and impaired immunosuppressive activity of regulatory T (T_REG) cells in the lamina propria of the small intestine and colon. This, in turns, drives unopposed recruitment and activation of myeloid cells and amplification of inflammatory signals.

The singular mitochondrion of trypanosomatids has been at the forefront of revolutionary discoveries concerning molecular biology, such as an intercatenated mitochondrial genome and complex post-transcriptional RNA editing. The TrypTag project represents an effort to localise every protein of T. brucei via endogenous gene tagging, the first protozoan organism to be investigated in such a manner. Using fluorescent protein tagging against both N and C termini, we have generated a definitive mitochondrial proteome in procyclic T. brucei, consisting of approximately 1200 sequences. We additionally utilise tagging phenomena associated with the T. brucei kinetoplast structure to designate sub-organelle distribution of mitochondrial proteins for the first time. Import characteristics of mitochondrial proteins are equally illuminated through this comprehensive tagging approach. We investigate novel and unexplored mitochondrial pathways, including fucose synthesis and one-carbon metabolism. The knowledge gained from this study represents a conclusive step to determining the true mitochondrial proteome of T. brucei.

Thyroid hormones act as master regulators of cellular metabolism. Thereby, the biologically active triiodothyronine (T3) induces the expression of genes to enhance mitochondrial metabolic function. Notably, Ca2+ ions are necessary for the activity of dehydrogenases of the tricarboxylic acid cycle and, thus, mitochondrial respiration. 

Live-cell imaging revealed that T3 boosts a mitochondrial Ca2+ uptake route through the mitochondrial Ca2+ uniporter (MCU) that gets modulated by uncoupling protein 2 (UCP2) and protein arginine methyltransferase 1 (PRMT1). Enhanced mitochondrial Ca2+ uptake was essential for increased mitochondrial ATP production after T3 treatment. Besides, increased mitochondrial Ca2+ and ATP levels correlated with enhanced reactive oxygen species (ROS) production in mitochondria. 

Based on these results, we assume that thyroid hormones adjust mitochondrial Ca2+ homeostasis by modulating the UCP2- and PRMT1-balanced mitochondrial Ca2+ to convey their impact on mitochondrial ATP and ROS production. 

Background: The metabolic reprogramming in cancer cells is linked to the structural abnormalities of mitochondrial network (MN) and mitochondrial associated membranes (MAM). The understanding of the structural and functional characteristics of mitochondrial network (MN) and mitochondria-associated membranes (MAM) in gliomas is essential for the design of future therapeutic. Materials & Methods: The MN and MAM ultrastructure in surgical specimens from human astrocytic neoplasms were studied. Results: Disarrangement of cristae and partial or total cristolysis are the hallmark of mitochondria in human gliomas .Variations in MAM ultrastructure are observed with respect to density, length, and width. Conclusion: These findings imply that a majority of glioma cells would be incapable of producing adequate amounts of energy through OxPhos and would thus require compensatory mitochondrial substrate level phosphorylation for ATP production. Metabolic therapies that restrict availability of glucose and glutamine to the tumor cells can represent a non-toxic approach for gliomas.

Heart failure is a major public health burden with high morbidity and mortality. Identifying novel approaches towards regenerating heart tissue has significant therapeutic potential for heart failure patients. Neonatal mice can regenerate their hearts following injury for a brief window after birth. The metabolic switch from glycolysis to fatty acid oxidation in the postnatal heart contributes to cardiomyocyte cell cycle exit and loss of endogenous cardiac regeneration potential. Succinate dehydrogenase (SDH), also known as mitochondrial complex II, plays a central role in regulating cellular metabolism. We demonstrate that inhibition of SDH by malonate treatment of adult mice following myocardial infarction stimulates cardiomyocyte proliferation, revascularization, and results in restoration of cardiac structure and function following infarction. Remarkably, SDH inhibition promotes dynamic metabolic changes towards glycolysis in the adult heart. Our overarching hypothesis is that SDH inhibition metabolically reprograms the adult heart to a regenerative state at the cellular and molecular level.

Mitochondrial proteins often dynamically assemble with different partners in functionally diverse high molecular weight (HMW) complexes. Here we validate two searchable compendia of the dynamic composition of mitochondrial HMW complexes based on blue-native electrophoresis and mass spectrometry. By comparing HMW complexes in unperturbed, cristae remodeled and outer membrane permeabilized mitochondria we generated MARIGOLD, a searchable Mitochondrial Apoptotic RemodelInG cOmpLexome Database of 589 mitochondrial proteins that elucidates the dynamic organization of HMW complexes. From MARIGOLD we developed mito-CIAO, a searchable mitochondrial Complexes InterActome tOol that uses a statistical correlation approach to predict protein co-occurrence in HMW complexes. We validated mito-CIAO by verifying its ability to predict biologically confirmed interactions among MICOS and Opa1 complexes components and used it to functionalize two HMW complexes of Atad3a, an essential yet elusive protein. One complex contained Opa1 and regulated mitochondrial ultrastructure; the second, genetically distinguishable complex contained ribosomal proteins and was essential for mitoribosome stability. Our compendia reveal the dynamic nature of mitochondrial HMW complexes and facilitates their functionalization.

Mutations in mitochondrial DNA are associated to several maternally inherited genetic conditions which main feature is mitochondrial dysfunction. Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS) syndrome is a rare mitochondrial disorder mainly caused by the m.3243A>G mutation in the mt-tRNA^Leu(UUR) (MT-TL1) gene. This mutation affects mitochondrial protein translation, decreasing mitochondrial function. MELAS syndrome has phenotypical manifestations such as stroke-like episodes or epilepsy. Due to the lack of proper animal models for this disease, several cellular models have been developed. However, they do not fully represent the most affected tissues in the disease such as the brain. In this work, we study the cellular pathophysiology of MELAS syndrome using patient-derived fibroblasts and we directly convert those fibroblasts into induced neurons (iNs). Additionally, we demonstrate that MELAS iNs show pathophysiological features such as decreased bioenergetics. Therefore, direct reprogramming is a promising approach to obtain neuronal models for studying mitochondrial disorders.

Friedreich’s ataxia is a cardio-neurodegenerative, autosomal recessive disease considered a rare disease. It is caused by a hyperextension of the GAA triplet located in the first intron of the frataxin gene. This mutation leads to a decrease in frataxin gene transcription and a deficiency of this protein. Frataxin is a mitochondrial matrix protein with different roles that prevent oxidative stress. Using patient-derived fibroblasts, we have shown that there is a negative correlation between the GAA expansion and the frataxin protein expression. We have observed a decrease in iron metabolism and mitochondrial proteins expression involved in iron-sulfur cluster biosynthesis which promotes iron accumulation and mitochondrial oxidative stress. As consequence, there is a mitochondrial dysfunction in patient-derived fibroblasts reducing parameters related to oxygen consumption rate. Finally, performing direct reprogramming from fibroblasts to neurons we can also study the effect of frataxin deficiency in a more accurate cellular model.

Human Tim8a and Tim8b are paralogous mitochondrial proteins that participate in a chaperone network within the intermembrane space of mitochondria. Both isoforms are presumed to enable import of nascent membrane proteins, following studies of fungal homologues. However, the isoforms are differentially expressed in human tissues and only hTim8a mutations manifest clinically – as neurodegenerative Mohr-Tranbjærg syndrome. We interrogate the cell-specific functions of hTim8a and hTim8b with knockout HEK293 and SH-SY5Y cell lines, finding misassembly of Complex IV and increased H₂O₂ levels specific to loss of the dominant isoform in each cell type. We show hTim8a, hTim8b and hTim13 interact together and with cysteine-rich intermembrane space proteins, including Complex IV assembly factors. Only hTim8a^KO cells are apoptotic and neither paralogue can rescue loss of the other. We conclude hTim8 isoforms have unique but complementary functions in assembly of Complex IV (COX2 module), which maintains intermembrane space proteostasis and protects against mitochondrial disease.

Psychosocial stress is a common risk factor for anxiety disorders. The cellular mechanism for the anxiogenic effect of psychosocial stress is largely unclear. Here, we show that chronic social defeat (CSD) stress in mice causes mitochondrial impairment, which triggers the PINK1-Parkin mitophagy pathway selectively in the amygdala. This mitophagy elevation causes excessive mitochondrial elimination and consequent mitochondrial deficiency. Mitochondrial deficiency in the basolateral amygdalae (BLA) causes weakening of synaptic transmission in the BLA-BNST (bed nucleus of the stria terminalis) anxiolytic pathway and increased anxiety. The CSD-induced increase in anxiety-like behaviors is abolished in Pink1-/- and Park2-/- mice and alleviated by optogenetic activation of the BLA-BNST synapse. This study identifies an unsuspected role of mitophagy in psychogenetic-stress-induced anxiety elevation and reveals that mitochondrial deficiency is sufficient to increase anxiety and underlies the psychosocial-stress-induced anxiety increase. Mitochondria and mitophagy, therefore, can be potentially targeted to ameliorate anxiety.

Macrophages are a functionally heterogeneous cell population that plays a vital role in inflammatory responses and host defense against pathogenic infections. Previously, we described that recombinant human growth hormone (rhGH) triggers the reprogramming of GM-macrophages towards an anti-inflammatory phenotype. Based on these data, we have now performed a GSEA analysis which suggested a decrease in glycolysis that was confirmed by Seahorse methods. Subsequently, we analyzed metabolic key points in glycolysis, the tricarboxylic acid cycle (TCA) and fatty acid synthesis in macrophages. The results allow us to conclude that rhGH-treated GM-MØ shift their metabolism towards a more active Oxphos profile that correlates with the acquisition of an anti-inflammatory phenotype.

We have studied in fixed cells and ex vivo how these metabolic changes affect the number, dynamics and 2D morphology of mitochondria and characterized the 3D ultrastructure by correlative cryogenic fluorescence microscopy and cryo-FIB-SEM. GM-MØ treated with rhGH undergo a decrease in the number of mitochondria, in addition to increases in biogenesis, fusion and mitophagy. We also observed an increase in mitochondrial volume, cristae density and elongation in rhGH-treated GM-MØ. Altogether our data indicated that GH acts as a metabolic modulator of GM- MØ that correlates with GM- MØ reprogramming.

Macrophage metabolism, in particular their metabolic repolarization, offers new therapeutic opportunities for the treatment of inflammatory diseases and cancer.

Hyperandrogenism and insulin resistance constitute the central pathophysiological mechanisms that contribute to the reproductive dysfunctions such as miscarriage seen in women with polycystic ovary syndrome (PCOS). In our studies, pregnant rats chronically treated with 5α-dihydrotestosterone (DHT) and insulin exhibited hyperandrogenism and insulin resistance, as well as increased fetal loss, and these features are strikingly similar to those observed in pregnant PCOS patients. We found that exposure with DHT and insulin decreased uterine and placental mtDNA copy number. This finding is similar to the clinical observation that circulating mtDNA copy number is reduced in PCOS patients compared to non-PCOS patients. By transmission electron microscopy, the mitochondria showed swelling and collapsed and poorly defined tubular cristae in the gravid rat uterus and placenta exposed to DHT and insulin. Further, DHT+insulin-exposed pregnant rats exhibited abnormal expression of genes that are involved in mitochondrial fusion, fission, biogenesis, and mitophagy in the gravid uterus and placenta. Collectively, our findings suggest that defective mitochondria are responsible PCOS-induced fetal loss.

Dynamic regulation of mitochondrial morphology provides cells with the flexibility required to adapt and respond to toxins and mitochondrial DNA-linked disease mutations, yet the mechanisms underpinning the regulation of mitochondrial dynamics machinery by these stimuli is poorly understood. Here we show that pyruvate dehydrogenase kinase 4 (PDK4) is genetically required for cells to undergo rapid mitochondrial fragmentation when challenged with mitochondrial toxins. Moreover, PDK4 overexpression was sufficient to promote mitochondrial fission even in the absence of mitochondrial stress. Phosphoproteomic screen for PDK4 substrates, followed by mutational analysis of the PDK4 site revealed cytoplasmic GTPase, Septin 2 (SEPT2), as the key effector molecule that acts as a receptor for DRP1 in the outer mitochondrial membrane to promote mitochondrial fission. PDK4-mediated mitochondrial reshaping limits mitochondrial bioenergetics and supports cancer cell growth. Our results identify the PDK4-SEPT2-DRP1 axis as a regulator of mitochondrial function at the interface between cellular bioenergetics and mitochondrial dynamics.

The maintenance of a proper mitochondrial ultrastructure is crucial for cellular metabolism and viability. Our group has recently dissected the role of the AAA+ ATPase ATAD3A in cristae biogenesis and mitoribosomal stability, raising the question about the participation of ATAD3B, the other member of this protein family, in mitochondrial function. Despite the high similarity with ATAD3A, ATAD3B is present only in primates and humans, and shows an extra 62 aa extension at the C-terminus. Furthermore, ATAD3A is constitutively expressed, while ATAD3B is only present in undifferentiated and cancer cells, suggesting a possible involvement in the metabolic signature of high-proliferative cell types. Our data show that MAFs expressing ATAD3B exhibit impaired mitochondrial respiration while maintaining a regular proliferation and viability. However, they poorly proliferate in glucose-depleted conditions. These results suggest that ATAD3B expression might act as a metabolic switcher towards a more glycolytic metabolism, typically found in embryonic and cancer cells.

Cancer cachexia (CC) affects 50-80% of patients with cancer and is characterized by metabolic and immunological alterations related to loss of muscle mass and adipose tissue. The loss of muscle mass is directly linked to reduced quality of life, treatment toxicity, and lower survival. With no pharmacological treatment options, key to improving life expectancy is to understand the molecular determinants of CC, however, these are poorly defined. Mitochondrial dysfunction and inflammation are associated with muscle atrophy in CC. In this regard, we have recently described that primary mitochondrial alterations in skeletal muscle activate inflammatory signals through mitochondrial (mt) DNA recognition by intracellular DNA sensors, resulting in muscle atrophy and reduced physical performance. Yet, the implication of muscle mitochondrial dysfunction in promoting mtDNA-dependent inflammation in the context of CC remains unknown. Here we have characterized the mitochondrial homeostasis and inflammation in an in vitro model for CC. Reduced diameter and upregulation of atrogenes in cachectic differentiated muscle cells (myotubes) validated the model. We observed increased mitochondrial mass and altered morphology in cachectic myotubes, accompanied by increased membrane potential and ROS production, and reduced mitochondrial respiration. Under these conditions, a remarkable 10-fold upregulation of IL6 was observed, suggesting a role for IL6 in driving muscle inflammation in conditions of cachexia. Current work is being directed to assess the involvement of mtDNA recognition in the induction IL6-specific responses and to validate our observations in in vivo models and in human CC.

Mitochondrial ATP synthase is a key enzyme of energy metabolism and signaling hub whose activity is regulated by its physiological inhibitor IF1. IF1 binds the ATP synthase under phosphorylating conditions to regulate the hydrolytic and synthetic activities of the enzyme. Moreover, IF1 also regulates the oligomeric state of the enzyme to promote inactive ATP synthase tetramers. IF1 is expressed in stem cells and in some differentiated cells, being murine colon a tissue with highest expression. Overexpression of IF1 in colon was previously reported to promote an anti-inflammatory phenotype supporting a tight link between the ATP synthase/IF1 axis and the immune system. Herein, we have developed a conditional IF1 knockout mouse model in colon. Ablation of IF1 causes structural and functional alterations in mitochondria. Moreover, triggers the activation of purine anabolism and a proinflammatory phenotype, highlighting the relevance of IF1 in non-cell autonomous programs.

IF1, the physiological inhibitor of the mitochondrial ATP synthase, inhibits the synthetic and hydrolytic activities of the enzyme, also playing an important role in the oligomerization of the enzyme. Hence, it is a master regulator of mitochondrial metabolism and cristae architecture. Murine macrophages greatly express IF1 in all its functional phenotypes (M0, M1 and M2). Ablation of IF1 in bone marrow derived macrophages promote metabolic rewiring to an enhanced respiration, alteration of mitochondrial structure and activation of a nuclear inflammatory response as assessed by an increased production of interferon gamma. On the contrary, quiescent CD4+ mouse T lymphocytes express negligible levels of IF1, but its expression bursts when activated or fully differentiated into Th1 subset. For that, ablation of IF1 in CD4+ lymphocytes compromises Th1 responses. These results point out the relevance of the ATP synthase/IF1 axis as potential target of aberrant immune responses.  

From transient caloric excess to obesity, adipose tissue macrophages (ATMs) adapt to changes in their microenvironment by transitioning between oxidative and inflammatory states. We demonstrate that the Interferon Regulatory Factor (IRF)-5 is a key regulator of macrophage oxidative capacity in response to caloric excess. ATMs from mice with genetic-deficiency of IRF5 are characterised by increased oxidative respiration and mitochondrial membrane potential. This phenotype is inducible in mature macrophages and reversible by adenoviral reconstitution of IRF5 expression. Using public ChIP-sequencing and in-house RNA-sequencing, we found that the highly oxidative nature of IRF5-deficient macrophages results from derepression of the Growth Hormone Inducible Transmembrane Protein (GHITM), important for stabilizing mitochondrial cristae. Coregulated expression of IRF5 and GHITM, and associated cellular energetic phenotype, is conserved in humans. We shed light on a mechanism by which the inflammatory transcription factor IRF5 alters mitochondrial architecture in macrophages, limiting capacity to physiologically adapt to caloric excess.

Sofosbuvir is a NS5B nucleos(t)ide polymerase inhibitor that interferes with the Hepatitis C viral RNA replication and used during the last decade for mass treatment of HCV in the endemic countries like Egypt which may increase the exposure of young women at childbearing age to Sofosbuvir. In this study, we aimed to explore the long-lasting consequences of preconception exposure of young female rats to Sofosbuvir on the ovarian tissues of F1-offspring and to explore the pathway of mitochondrial biogenesis as the possible molecular mechanism of these transgenerational effects. The ovarian tissues of the F1-offspring showed modulation of estrogen receptors and Kiss1 expression and its receptor, and imbalance in mitochondrial homeostasis which is associated with lipid peroxidation, oxidative DNA damage, and inflammation. So, the preconception maternal exposure to Sofosbuvir may have a detrimental transgenerational effect on the F1 ovarian tissues

Sofosbuvir is a nucleotide analog inhibitor of hepatitis C virus polymerase essential for viral replication. Environment and diet influence spermatogenesis with known consequences on male fertility. We aimed to investigate the possible effects of pre-conception maternal exposure to Sofosbuvir on the mitochondrial biogenesis of the testicular tissues of the F1-offspring. Preconception maternal exposure to Sofosbuvir impairs mitochondrial biogenesis as indicated by the decline in mitochondrial DNA copy number and impairs the mitochondrial functions of F1-offspring through increased lipid peroxidation marker, DNA oxidation marker, and nuclear factor kappa B, decreased nuclear erythroid-2 related factor-2. The present finding demonstrates that Sofosbuvir has transgenerational effects through modulation of mitochondrial biogenesis and function of testicular tissues. These adverse effects may be mediated by an increase in oxidative stress and inflammation.

Contacts between the mitochondrial IM and OM participate in mitochondrial biogenesis and in transduction of signals from the extramitochondrial space. Our knowledge of mediators of IM-OM contacts is scant, besides the interaction between TOM40 and TIM23 complexes and of the MIB complex. Here we show that processing of the mitochondria-shaping protein OPA1 controls the extent of contacts between OM and IM. Reintroduction of a mutant of OPA1 that is not processed into its short (S) form in an Opa1-/- cell line resulted in decreased OM-IM juxtaposition. These ultrastructural changes were paralleled by defects in MICOS and import complexes. Molecularly, OPA1 interacted with Tim29, member of the TIM22 complex essential for mitochondrial biogenesis and respiration. Indeed, growth and respiration of these cells were impaired. Our data indicate a role for OPA1 processing in the stability of the mitochondrial protein import complex TIM22, linking the core mitochondrial dynamics machinery to mitochondrial biogenesis.

Mitochondria contain by two distinct membrane systems. The outer membrane (OM) mediates communication with the cytosol and other organelles. The inner membrane (IM) is particularly protein-rich and harbors the machinery for ATP synthesis by oxidative phosphorylation. Intimate cooperation of both membranes is required for key functions of mitochondria, like lipid synthesis, channeling of metabolites an ions, like calcium, and apoptosis. We and others have identified and  initially described a direct OM-IM contact site in yeast mitochondria formed by the Mitochondrial Contact Site and Cristae Organizing System (MICOS) in the IM and the Sorting and Assembly Machinery (SAM) in the OM. Our new studies on this Mitochondrial Intermembrane Bridging (MIB) super-complex in human mitochondria have revealed a novel mechanism for the biogenesis of OM beta-barrel proteins, like VDACs, that requires the Hsp40 co-chaperone DNAJC11 at the MIB.

Macrophages can accelerate tumor growth in several types of cancer by dampening anti-tumor immunity, preventing effective immune checkpoint blockade therapy, and negatively influencing the efficacy of antitumor drugs. The mechanisms that underpin these functions have not been fully elucidated, however, Arginase 1 (ARG1) expression is believed to play an important role. ARG1 is induced in macrophages by interleukin-4 and/or lactic acid, which can be present in tumors. We have shown that acute exposure to IL-4 induces sustained ARG1 expression for up to 6 months in long lived macrophages, leading to substantial alterations to the mitochondrial function of these cells, including persistent changes in mitochondrial mass and regulation of arginine-derived ornithine metabolism, which we have previously shown to be critical for maintaining mitochondrial function. Our data suggest that sustained ARG1 expression allows macrophages to survive and accumulate in the tumor by rewiring mitochondrial function.

Tissue,cellular and molecular adjustment to hypoxia elicits variety of responses, suggesting the existence of an O2 sensor capable of triggering physiological adaptation.The mitochondria regulate response to O2 deprivation remains poorly understood.Hypoxia triggers mitochondrial complex I transition from its active to deactive form,was associated with several pathological scenarios.We show that lack of a mitochondrial protein,impairs the hypoxia-induced complex I active-deactive transition increasing the proportion of deactive complex I  undernormoxia.Mitochondrial hypoxic is triggered resulting in basal hypoxicactivation mediated by the ROS/mTOR/Hypoxia-inducible factor1(HIF-1α)axis,which protects against chronic hypoxia-induced pulmonary hypertension,cardiac and pulmonary.We demonstrated that blocking O2 sensing mechanism,by silencing a mitochondrial protein in cardiomyocytes,confers cardio protection against hypoxia-induced right ventricular dysfunction.Our results demonstrated the importance of this protein as a target for hypoxia-related diseases,also provide new therapeutics tools against the hypoxic heart,a organ that need to be treated in pulmonary hypertension.

Mitochondria are double membrane-bound organelles present in most eukaryotic cells. They are highly dynamic and continually remodel their morphologies by fusion and fission processes. During remodelling, mitochondrial membranes, including their lipids, undergo substantial changes. Although the protein players involved have been extensively studied, little is known about the roles of different lipids in this process, in part due to a lack of techniques to visualize lipids and perturb their cellular levels. In this project, we used an siRNA screen targeting 260 different lipid biosynthetic enzymes to uncover potential lipid species that may have functions in mitochondrial remodelling. The most interesting candidates were selected to study the lipidomic changes they bring to cells and their influence on mitochondrial functions such as ATP production. After this study, we will establish a general understanding of the roles that lipids play in mitochondrial remodelling, including unique functions of different lipids or/and lipid species.

Metastatic progression in patients with triple negative breast cancer (TNBC) occurs in approximately half of patients, reducing median overall survival. Metastasis may be facilitated through the epithelial to mesenchymal transition (EMT), generating cancer cells with enhanced self-renewal and chemotherapeutic resistance, which are partially mediated by alterations in metabolic pathways and mitochondrial function. We show that a drug-like small molecule possesses EMT-specific cytotoxic activity through effects on metabolic and mitochondrial functions. The fungus-derived sesterterpenoid, Ophiobolin A (OpA), possesses nanomolar cytotoxic activity and high therapeutic index, though its target and mechanism of action remain unknown. Analysis indicates OpA acts in a mitochondria-specific manner to cause loss of membrane potential in EMT-positive, but not EMT-negative, cells with specific effects on complex III of the electron transport chain, calcium homeostasis, and TCA cycle. Therefore, we conclude that EMT imparts alterations in mitochondrial function and metabolic pathways, conferring sensitivity to the cytotoxic effects of OpA.

Infection with the SARS-CoV-2 virus causes coronavirus disease 2019 (COVID-19). Largely driven by hyper immune-activation, a pronounced acute phase reaction with high CRP-levels characterizes severe COVID-19. The cellular metabolic correlates of the immune pathology driving COVID-19 remain ill-understood. We have previously found that bulk immune cells from patients with severe COVID-19 display glycolytic- and oxidative phosphorylation gene signatures during early and late phases of the disease, respectively. Here, we performed mass cytometry-based deep immunometabolic phenotyping among patients stratified according to CRP dynamics and clinical disease course. Immunometabolic phenotypes of B and T cells from young and elderly patients will be presented, emphasizing alterations in the tricarboxylic acid cycle, electron transport chain, and mitochondrial biogenesis. Our phenotyping strategy has potential to (i) identify metabolic markers predictive of patient susceptibility towards developing severe acute and/or long COVID-19, (ii) uncover metabolic pathways mechanistically linked to immune pathology, and (iii) point at druggable metabolic targets.

Iron dyshomeostasis contributes to aging, but little information is available about the molecular mechanisms. Here, we provide evidence that, in Saccharomyces cerevisiae, aging is associated with altered expression of genes involved in iron homeostasis. We further demonstrate that defects in the conserved mRNA-binding protein Cth2, which controls stability and translation of mRNAs encoding iron-containing proteins, increase lifespan by alleviating its repressive effects on mitochondrial function. Mutation of the conserved cysteine residue in Cth2 that inhibits its RNA-binding activity is sufficient to confer longevity, whereas Cth2 gain-of-function shortens replicative lifespan. Consistent with its function in RNA degradation, we demonstrate that Cth2 deficiency relieves Cth2-mediated post-transcriptional repression of nuclear-encoded components of the electron transport chain. Our findings uncover a major role of the RNA-binding protein Cth2 in the regulation of lifespan and suggest that modulation of iron starvation signaling can serve as a target for potential aging interventions.

MT-ND1 encodes the core subunit of mitochondrial NADH dehydrogenase. We discovered that m.3937T>C mutation is responsible for MELAS. The patient carrying this mutation suffers from recurrent episodes of recurrent stroke, refractory epilepsy, vision loss, and lactic acidemia. Cranial MRI and MRS showed large lesions, magnanimous lactic peak, and cerebral atrophy. Mitochondrial functional assays revealed a declined complex I activity, ATP production, and respiratory capacity. “Mitochondrial cocktails” and a combination of AEDs were administered with minimal improvement. To test the therapeutic potential of niacin for this disorder, we administered niacin to this patient in a dose-escalation manner. The patient’s seizures were ceased, and he has been free of hospitalization for more than one year. We further investigated the impact of niacin supplements on his fibroblasts. Seahorse and ATP, ROS production assay showed 100uM was the most favorable concentration while overdosing might be detrimental. Moreover, niacin was found to inhibit apoptosis.

The mitochondrial phospholipid cardiolipin promotes bioenergetics via oxidative phosphorylation (OXPHOS). Three tightly bound cardiolipins have been evolutionarily identified in the ADP/ATP carrier (Aac in yeast, ANT in mammals) which resides in the inner mitochondrial membrane and exchanges ADP and ATP to enable OXPHOS. We investigated the role of these buried cardiolipins in the carrier using yeast Aac2 as a model. We introduced negatively charged mutations into each cardiolipin-binding site of Aac2 to disrupt the cardiolipin interactions via electrostatic repulsion. The mutations disrupted the lipid-protein interaction, destabilized Aac2 monomeric structure, and impaired its transport activity. In human ANT1, a missense mutation L141F corresponding to one yeast Aac2 cardiolipin-binding mutant was identified from a mitochondrial myopathy patient. The patient mutation compromised the structure and transport activity of ANT1, resulting in OXPHOS defects. Our findings highlight the conserved significance of cardiolipin in Aac/ANT structure and function, directly tied to specific lipid-protein interactions.

Mitochondrial ribosomes (mitoribosomes) have undergone substantial structural remodelling throughout evolution. Compared to their prokaryotic counterparts, mitoribosomes show a substantial loss of ribosomal RNA, whilst acquiring unique protein subunits located on the periphery of the ribosomal subunit structures. We set out to investigate the functional properties of all 14 unique (mitochondrial-specific or supernumerary) human mitoribosomal proteins in the small subunit. Using genome editing with CRISPR-Cas9, we made knockouts for each subunit in HEK293 cells to study the effect on mitoribosome assembly and function in protein synthesis. Unexpectedly, we show that each supernumerary knockout leads to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Our data demonstrates that all supernumerary subunits are essential structural components except mS37. Surprisingly, we found the stability of mS37 was reduced in all our supernumerary knockouts of the small and large ribosomal subunits as well as patient-derived lines with mitoribosome assembly defects. We identified that a redox regulated CX9C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.

Mitochondrial augmentation technology (MAT) is an ex-vivo method enriching cells with exogenous mitochondria.  In particular, hematopoietic stem and progenitor cells (HSPCs) require high levels of mitochondrial activity to differentiate and proliferate into the various hematopoietic lineages. Intriguingly, recent evidence suggests that immunological factors may play a stronger role in non-hematopoietic PMD pathology than originally envisioned.

Single-cell methodologies assessing protein synthesis capacity as a proxy for bioenergetic potential have demonstrated that higher levels of augmentation of patient-derived cells correlate with higher protein synthesis levels. Using banked placenta-derived mitochondria from a variety of donors, we are exploring parameters affecting the persistence of exogenous mtDNA and the effect on cell function after MAT. 

Our studies aim to ensure optimization of augmentation of HSPCs to provide significant and durable benefit to patients with primary mitochondrial disorders.

Many anti-cancer chemotherapeutic agents evoke cellular senescence not only in cancer cells but also in stromal cells such as fibroblasts, called therapy-induced senescence (TIS). Mitochondria have been found to play roles in senescent cells and their secretory phenotype. To define how mitochondria contribute to the TIS, we monitored the adaptation of the mitochondrial proteome upon TIS induction in a time-dependent fashion. IMR90 human lung fibroblasts were induced to senesce by two chemotherapeutic agents, doxorubicin and decitabine. Time-resolved proteomic analyses of mitochondria upon the induction of TIS revealed rewiring of mitochondrial metabolism and increased branched-chain amino acids (BCAA) degradation, which was confirmed by metabolic flux analyses. An enhanced BCAA catabolism supports the synthesis of non-essential amino acids by transamination and thereby amino acid homeostasis in senescent cells. In addition, TIS cells exhibited an altered one carbon and serine metabolism in mitochondria resulting in the depletion of purines and thymidylates. The reduction of serine catabolism in the mitochondria was accompanied by an attenuated mitochondrial translation. The analysis of the mitochondrial volume in senescent cells combined with bioenergetic profiling revealed a profound accumulation of bioenergetically hypoactive mitochondria in the TIS cells, consistent with the observed decrease in mitochondrial translation. These data collectively demonstrate the mitochondrial reprogramming in the TIS stromal cells and its potential implications in the tumor microenvironment possibly through regulating the secretory phenotype.

Mammalian genomes harbor three RAS genes, HRAS, NRAS and KRAS that encode four RAS proteins. We recently found that KRAS4A directly regulates hexokinase I, providing the first evidence of a RAS isoform-specific regulation of a metabolic enzyme. Searching for other pathways differentially controlled by RAS isoforms, we affinity purified proteins that interact with RAS and found the mitochondrial leucine-rich PPR-motif-containing protein (LRPPRC). LRPPRC is a posttranscriptional regulator of mtDNA-coded mRNAs that is overexpressed in many cancers. Mutations in the LRPPRC gene are known to induce mitochondrial Complex IV deficiency and ATP synthase defects. Co-immunoprecipitation of GFP-tagged RAS proteins and endogenous LRPPRC validated the result of the affinity purification. The interaction with endogenous LRPPRC was both isoform-specific (HRAS ≫ NRAS > KRAS4A = KRAS4B) and palmitoylation dependent. Because LRPPRC is a mitochondrial matrix protein, our data suggest that the interaction occurs in the mitochondria. To test this hypothesis, we expressed GFP-tagged HRAS(G12V) in HEK293 cells and enriched mitochondria from cell homogenates.  Both GFP-tagged HRAS(G12V) and endogenous RAS were detected in the mitochondrial enriched fraction. Co-immunoprecipitation of RAS and LRPPRC derived from mitochondria further supported our hypothesis. Mitochondrial STAT3 has been reported to facilitate HRAS(G12V)-driven transformation by promoting oxidative phosphorylation. We found that mitochondrial STAT3 interacts with both endogenous LRPPRC and Flag-tagged HRAS(G12V) and that silencing LRPPRC significantly decreased the interaction between STAT3 and HRAS(G12V), suggesting a trimolecular complex.  Future work will focus on the role of these interactions on mitochondrial function and cancer cell metabolism.

The Mitochondrial Rho GTPase MIRO1 regulates mitochondrial trafficking along microtubules. When Ca2+ binds to its EF hands, MIRO1 dissociates from microtubules, leading to mitochondrial arrest. The role of MIRO1 in proliferating cells in contrast to neurons is poorly understood.

MIRO1 deletion abolished proliferation and inhibited cell cycle progression in G1/S phase in cultured smooth muscle cells and impaired cristae ultrastructure. After application of growth factors, mitochondrial translocation to the cell periphery was inhibited. Cell cycle-dependent increases in ATP production, mitochondrial length and membrane potential were reduced with MIRO1 deletion. Expression of WT MIRO1 but not of a mutant lacking the EF hands restored ATP production and mitochondrial mobility in MIRO1-/- cells. In mice lacking MIRO1 in smooth muscle cells, proliferation with formation of vascular stenosis was inhibited.

Our data highlight a key function for MIRO1 in mitochondrial adaptation during cell cycle progression.  

Mitochondrial dysfunction imposed by deficiencies in the electron transport chain (ETC) rewire cellular metabolism creating a strong reliance on glucose uptake and consumption. While the established paradigm postulate that glucose is mainly used to support high levels of glycolysis in order to generate ATP in OXPHOS-deficient cells, the contribution of glucose to other metabolic pathways have been largely overlooked. Here, we discovered that Pentose Phosphate Pathway (PPP) inhibition is synthetic lethal with mitochondrial dysfunction. Upon PPP inhibition, mitochondrial function becomes essential to maintain NADPH levels and counteract oxidative stress, which involves mitochondrial-derived malate production and consumption by the Malic Enzyme 1 (ME1) to generate NADPH through a non-canonical TCA cycle. This adaptive mechanism is conserved across multiple cell lines and xenograft experiments demonstrated that can be exploited to target a specific subset of tumors that rely on ME1 with clinical-grade small molecule inhibitors of complex I.

The brain regularly experiences glucose shortage over the course of normal function. Bouts of high neuronal activity can quickly deplete available glucose in the brain, which also lacks glucose storage in the form of glycogen.  Mitochondria are essential for neuronal utilization of oxidative fuels during glucose scarcity. But it is not known how mitochondrial metabolism is regulated in response to fuel availability. Here, we show that glucose deprivation, both in vitro and in vivo, activates the CREB transcriptional program which induces the neuronal expression of PGC-1α and its target gene, Sirtuin 3 (Sirt3), a mitochondrial deacetylase enzyme. We quantatively examined mitochondrial function in indvidual synaptic terminals using optical sensors and demonstrated that Sirt3 stimulates mitochondrial ATP synthesis and sustains neurotransmission during glucose deprivation. We further determined that Sirt3 stimulates mitochondrial pyruvate uptakethrough deacetylation of the mitochondrial pyruvate carrier (MPC). Thus Sirts ensures neuronal metabolic fitness and sustains neurotransmission during glucose deprivation. 

Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway to control mitochondrial homeostasis and organ size, of which dysregulation yields to organomegaly or tumorigenesis. Upon activation, YAP/TAZ translocate into the nucleus and bind to TEAD transcription factors to promote transcriptional programs for proliferation or mitochondrial quality control. Immediate early genes, represented by AP-1 complex, are rapidly induced and control later-phase transcriptional program to play key roles in tumorigenesis and organ maintenance. Here, we report that YAP/TAZ directly promote FOS transcription that in turn contributes to the biological function of YAP/TAZ. YAP/TAZ bind to the promoter region of FOS to stimulate its transcription. Deletion of YAP/TAZ blocks the induction of immediate early genes in response to mitogenic stimuli. FOS induction contributes to expression of YAP/TAZ downstream target genes. Genetic deletion or chemical inhibition of AP-1 suppresses growth of YAP-driven cancer cells, such as Lats1/2-deficient cancer cells as well as Gαq/11-mutated uveal melanoma. Furthermore, AP-1 inhibition almost completely abrogates the hepatomegaly induced by YAP overexpression. These reveal a feedforward interplay between immediate early transcription of AP-1 and Hippo pathway function, which may be important for mitochondrial quality control.

Mitochondrial aging, which results in mitochondrial dysfunction, is strongly linked to many age-related diseases. Aging is associated with mitochondrial enlargement and transport of cytosolic proteins into mitochondria. The underlying homeostatic mechanisms that regulate mitochondrial morphology and function, and breakdown during aging, remain unclear.  Here we identify a pathway for mitochondrial protein trafficking in Drosophila melanogaster, involving the mitochondria-associated protein Dosmit. Dosmit induces mitochondrial enlargement and the formation of double-membraned vesicles which contains cytosolic protein within mitochondria, that the rate of vesicle formation increases with age. These vesicles originated from the outer mitochondrial membrane by tracking Tom20 localization, and this trafficking process is mediated by the mitochondria-associated Rab32 protein. Dosmit expression level is closely linked to the rate of aggregation of ubiquitinated proteins, which are themselves associated with age-related diseases. This mitochondrial protein trafficking route mediated by Dosmit offers a promising target for future therapies targeting age-related mitochondrial diseases.

Mitochondrial metabolism is critical for the function of beta-cells and is known to affect stem cell fate decisions. We therefore aimed to investigate the role of mitochondrial age and function during pancreatic progenitor cell differentiation in vitro. For this purpose, we created a pluripotent reporter cell line which allows for the temporal live-cell labelling of mitochondria. Using this system pancreatic progenitors differentiated from stem cells can be sorted into populations of differing mitochondrial ages. These populations can then be replated and differentiated further to mature stem cell derived islets, which can be assessed for cell composition and transcriptomic profiles as well as glucose stimulated insulin secretion capacity of beta-cells. Ongoing work will uncover whether the mitochondrial age differences seen in the pancreatic progenitors influence the fate and function of the given populations.

Chromatin remodellers contribute to cell lineage determination. Here we report that the SWI/SNF/BAF complex directs neural progenitor cell (NPC) differentiation toward neurons by priming mitochondrial oxidative phosphorylation. We identify BCL7A as a critical subunit of the SWI/SNF/BAF complex that stimulates the genome-wide occupancy of the ATPase BRG1. We demonstrate that BCL7A is dispensable for SWI/SNF/BAF complex integrity, whereas it is essential to potentiate Notch/Wnt pathway and mitochondrial bioenergetics in differentiating NPCs. Activation of the Wnt pathway restores mitochondrial respiration and attenuates the defective neurogenic patterns observed in mouse- and iPSC-derived NPCs lacking BCL7A. Using conditional KO mice, we reveal that BCL7A expression in NPCs and postmitotic neurons ensures neuronal plasticity and supports behavioral and cognitive performance. Together, our findings uncover the unique mechanistic contribution of BCL7A-containing SWI/SNF/BAF complexes in mitochondria-driven NPC commitment, thereby providing a better understanding of the cell-intrinsic transcriptional processes that connect metabolism, neuronal morphogenesis and cognitive flexibility.

Diagnostic yields from exome (ES) or genome sequencing (GS) in suspected mitochondrial disease are typically ~50%. Large numbers of potential candidate genes with variable genotype-phenotype correlations complicate variant curation. Undiagnosed patients can have zero to tens of candidate variants that may warrant functional follow-up to determine disease causation. However, functional tests often lack specificity and sensitivity. Quantitative proteomics (QP) has potential utility in identifying affected gene products and the functional impact of variants by analysing thousands of proteins in a single test. We developed a QP pipeline that achieved 80% diagnostic rate in a retrospective cohort (N=10) of patients suspected of mitochondrial disease where ES/GS were inconclusive. QP was also able to detect OXPHOS defects in patients with unremarkable respiratory chain enzymology. Including this cohort, our QP pipeline has guided the genetic diagnosis of over 28 patients and the identification of 4 new disease genes for future diagnostic investigations.

The human mitochondrial genome contains information for the transcription and translation of 13 vital proteins that assemble into the respiratory chain. Mutations in the mitochondrial DNA are the cause of several diseases and current treatment options are largely palliative. Gene therapy can be a viable curative option if the conditions for the efficient exogenous expression for these genes can be delineated. Utilizing the codon optimization tool to synchronize the mtDNA genes to the nuclear background, we showed that allotopic expression for the 13 genes is robust with their protein products associating with the organelle. Here, we describe an in vivo model for one of the genes namely ATP8. We expressed a single copy of the allotopic ATP8 gene from the mROSA26 safe harbor locus in the polymorphic mtFVB mice (mt.7778G/T). We show that the exogenous protein is well tolerated and stably expressed across tissues over four generations. The biochemical properties of the allotopic protein and its capability in competing with the endogenous mutant protein using mass spectrometry are discussed.

The translocator protein (TSPO) is an evolutionarily conserved outer mitochondrial membrane protein. Although TSPO is considered a multifunctional protein its precise role is still elusive. Using the CRISPR/Cas9 technology, we generated TSPO knock-out variants of human induced pluripotent stem cells (hiPSCs) to unravel TSPO's functional roles.
We investigated parameters of cellular and mitochondrial functions in hiPSCs-derived neural progenitor cells (NPCs). In the TSPO knock-out NPCs we found altered Ca²⁺-levels, a depolarized mitochondrial membrane potential and increased levels of reactive oxygen species, indicating increased oxidative stress. While the mitochondrial content seems to be unaltered, TSPO knock-out led to reduced basal and maximal respiration. Increased mitochondrial DNA copy numbers might be part of a cellular strategy to cope with oxidative stress and to compensate for reduced mitochondrial respiration.
Our findings point consistently towards impairments of mitochondrial function in TSPO knock-outs and suggest a regulatory role of TSPO for mitochondrial homeostasis.

As appreciation of tissue-specific mitochondrial biology advances, there is an increased need for novel approaches to rapidly isolate and analyse mitochondrial function without disrupting the mitochondrial membrane with shear stress or mechanical force. Here we describe a method for the intact recovery of mitochondria from skeletal muscle cells and tissue using nitrogen cavitation. This protocol results in pure and respiring mitochondria without the need for purification gradients and is complete in under 45 minutes. Western immunoblotting and transmission electron microscopy show enrichment of mitochondria with their ultrastructure intact. Functional analysis via the seahorse XFe24 and Oroboros-O2K show mitochondria extracted from murine cells and tissue have a respiratory control ratio (state 3/state 4) of 4.9 ± 0.8 and 4.6 ± 0.2 respectively, indicative of preserved respiratory function. Our method successfully demonstrates the rapid isolation of mitochondria using nitrogen cavitation and differential centrifugation from skeletal muscle cells and tissue for high-resolution respiratory studies.

Life on the earth has evolved in a form suitable for the gravitational force (1 g). Although the pivotal role of gravity in gene expression has been exemplified by the aging-like symptoms of astronauts in space, the molecular details how mammalian cells harness the gravity have been remained unclear. Here we show that mitochondria utilize gravity for activating protein synthesis within the organelle. Genome-wide ribosome profiling unveiled the reduced mitochondrial translation in mammalian cells under microgravity in the Internal Space Station and in the clinostat on earth. In addition, we found that cell adhesion, which is attenuated by microgravity, and the subsequent signaling pathway relayed by FAK, RAC1, PAK1, and Bcl-2 family proteins enhance the mitochondrial protein synthesis. These results indicated the mechanistic insight into how cells convert the gravitational force into biological function in mammals, especially into translation in an energy-producing organelle.

White adipose tissue (WAT) mainly serves as an energy reservoir, storing calories or releasing fatty acids, in response to hormonal and energetic cues. With >2 billion of obese/overweight individual worldwide, decoding the mechanisms of WAT expansion is currently of great importance. 3-mercaptopyruvate sulfurtransferase (MPST), a sulfide generating enzyme, is downregulated in adipose tissue of obese mice and humans. ^Mpst-deficient (Mpst^-/-) mice fed a high fat diet (HFD) exhibited increased body weight, excessive inguinal WAT (iWAT) fat accumulation, decreased metabolic rate and impaired glucose/insulin tolerance. Mpst deficiency activates ΗIF1α and downregulates translocase of outer/inner membrane (ΤΙΜ/ΤΟΜ) components causing β-oxidation/TCA cycle/oxidative phosphorylation suppression, thereby enhancing  lipid accumulation. Therapeutic administration of sulfide donors reversed the phenotypic and molecular changes observed with HFD. Our findings highlight the importance of MPST in adipose tissue biology and metabolic health and indicate sulfide donors as potential therapeutic strategies for obesity.

The mitochondrion is a complex, dynamic organelle, the characterization of which is fundamental to understand human disease and pharmacology. To handle the diverse functionalities, the mitochondria is structurally organized into various sub-compartment, which are spatially arranged to host and execute highly regulated functional complexes, like the mitochondrial contact site and cristae organizing system (MICOS) and the oxidative phosphorylation (OXPHOS) sub-complexes. High-throughput methods such as mass spectrometry (MS)-based proteomics have already provided comprehensive inventories of the mitochondrial proteome and insights into the organization of macro complexes. However, despite decades of intensive studies on mitochondria, critical questions, regarding the spatial arrangement, stoichiometry and assemble of macromolecular complexes within this interaction network, remain to be answered. Here, we present an integrative approach, combining cross-linking mass spectrometry, quantitative proteomics and super-resolution microscopy, to construct a three-dimensional landscape of mitochondrial architecture.

We propose the usage of mass spectrometry to determine the copy numbers and protein densities of mitochondrial proteins and cross-linking mass spectrometry to profile protein interactions and higher-order organizations. Super-resolution microscopy will be used to characterize protein localizations and distributions and together with electron microscopy, to measure the sizes and positions of individual mitochondrial sub-compartments.

Up to this point, we have determined the sub-compartment localization of more than 600 mitochondrial proteins. We have also discovered several new mitochondrial and mitochondrial-associated proteins. For validation, we expressed HA-tagged candidates in HeLa cells, performed confocal fluorescence, stimulated emission depletion (STED) imaging, and assessed their co-localization with mitochondrial marker TOMM20. We also validated the sub-compartment localization of several candidates using STED imaging combined with selective permeabilization of mitochondrial outer membrane. With the completion of this project, we anticipate the obtained rich dataset can serve as a door opener for follow-up studies, which will ultimately provide us a better mechanistic understanding of mitochondrial biology.

Mitochondrial diseases (MD) are complex disorders that arise due to mutations in genes important for mitochondrial function, most common are mutations in complex I of the electron transport chain. However, due to tissue heterogeneity, molecular mechanisms underlying mitochondrial cardiomyopathies (MCM) are still not yet fully understood. We utilised single-cell transcriptomics on heart-specific Ndufs6-deficient mouse hearts exhibiting mild to severe cardiac function with age progression. Unsupervised clustering of integrated datasets unveiled significant heterogeneity and a shift in the cardiomyocytes, as well as unique maladaptive sub-populations correlated to the disease phenotype. Pseudo-time trajectory analysis of these sub-populations revealed dynamic transitioning of cellular states from adapting to severely impaired, facilitated by transient gene expression regulating metabolic homeostasis. Further, we identified a key transcription factor likely involved in advancing MCM to heart failure. In conclusion, single-cell transcriptomics analysis helps understand the pathological mechanism of mitochondrial cardiomyopathy and identify therapeutic targets.

Parkinson’s disease (PD) is a major neurodegenerative disorder, yet the biological mechanisms involved in its aetiology are poorly understood. Evidence links this disorder with mitochondrial dysfunction and impaired lysosomal degradation – key features of mitophagy. We investigated the role of LRRK2, a protein kinase frequently mutated in PD, in this process. Using reporter mice, bearing either knockout of LRRK2 or expressing the pathogenic kinase-activating G2019S LRRK2 mutation, we found that basal mitophagy was specifically altered in clinically relevant cells and tissues. Our data show that basal mitophagy inversely correlates with LRRK2 kinase activity in vivo. Furthermore, use of distinct LRRK2 kinase inhibitors in cells increased basal mitophagy, and a CNS penetrant LRRK2 kinase inhibitor, GSK3357679A, rescued these mitophagy defects. This study provides the first in vivo evidence that pathogenic LRRK2 directly impairs basal mitophagy and demonstrates that pharmacological inhibition of LRRK2 is a rational mitophagy-rescue approach and potential PD therapy.

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the tricarboxylic acid (TCA) cycle. Here we show that DJ-1 (encoded by PARK7, a key familial Parkinson’s disease gene) is a pacemaker regulating PDH activity in CD4+ regulatory T cells (Treg). DJ-1 binds to PDHE1-β (PDHB), inhibiting phosphorylation of PDHE1-α (PDHA), thus promoting PDH activity and oxidative phosphorylation. Park7 (Dj-1) deletion impairs Treg survival starting in young mice and reduces Treg homeostatic proliferation and cellularity only in aged mice. This leads to increased severity in aged mice during the remission of experimental autoimmune encephalomyelitis. Dj-1 deletion also compromises differentiation of inducible Treg especially in aged mice, and the impairment occurs via regulation of PDHB. These findings provide unforeseen insight into the complicated regulatory machinery of the PDH complex. As Treg homeostasis is dysregulated in many complex diseases, the DJ-1–PDHB axis represents a potential target to maintain or re-establish Treg homeostasis.

Recent evidence of mitochondria-based metabolism for tumor growth prompted us to investigate the role of MCJ, an endogenous negative regulator of mitochondrial complex I, in hepatocellular carcinoma (HCC). Reduced MCJ expression was observed in patients at stage IV HCC. Two different experimental models of HCC were used, the diethylnitrosamine (DEN) in whole-body MCJ-/- mice and the MYC-luc;sgp53 plasmid combination in liver specific Mcjsilenced mice. Absence of MCJ increased tumorigenesis and mortality. A strong oxidative phenotype was confirmed in MCJ-/- tumors, as mitochondrial oxygen consumption rate was significantly higher, with increased intracellular ATP and NAD+ levels. Examination of the immune system showed reduced tumor infiltrating T cells, inflammatory cytokine production and increased PDL-1 in MCJ-/- mice. Similar results were observed in Mcj silenced mice. Interestingly, nearly 30% of Mcj-silenced mice developed brain metastases. Overall, decreased hepatic MCJ promotes oxidative respiration and leads to metabolic rewiring that impedes antitumor immune potential.

ATP-citrate lyase is a central integrator of cellular metabolism in the interface of protein, carbohydrate, and lipid metabolism. The physiological consequences as well as the molecular mechanisms orchestrating the response to long-term pharmacologically-induced ATP-citrate lyase inhibition are unknown.

We report here that pharmacologic inhibition of ATP-citrate lyase improves metabolic health and physical strength in high fat diet fed mice, while produces metabolic imbalance in healthy fed mice. By applying untargeted metabolomics, transcriptomics, proteomics and hypothesis-driven experimentation we determined that, in vivo, ATP-citrate lyase inhibitors modulate energy metabolism and mitochondrial function in the absence of global alterations on histone acetylation. At cellular level, ATP-citrate lyase inhibitors decrease maximal oxygen consumption and extracellular acidification.

Our findings indicate a mechanism for regulating molecular pathways that prevents the development of metabolic abnormalities associated with unhealthy dieting. This strategy might be exploited for devising therapeutic approaches to prevent metabolic diseases and to promote healthy aging.

To investigate the metabolic role of FoxO1 in skeletal muscle (SKM), we generated SKM-specific inducible FoxO1 knockout (mFoxO1KO) mice and fed them high-fat diet to induce obesity. FoxO1 deficiency improved insulin resistance and fatty acid oxidation, which was followed by increased mitochondrial fusion and respiratory capacity in SKM of obese mice. Improved mitochondrial function and metabolic flexibility in obese mFoxO1KO mice led to enhanced exercise performance. Transcriptomic analysis, ChIP assay and promoter assay revealed that FoxO1 deficiency increased the transcriptional expression of peroxisome proliferator-activated receptor-δ (PPARδ) and its target genes in SKM. Moreover, FoxO1/PPARδ double-knockout abolished beneficial effects of FoxO1 deficiency in SKM of obese mice. In human SKM, FoxO1 proteins were increased in diabetic patients and were negatively correlated with PPARδ. In conclusion, FoxO1 deficiency improves mitochondrial function and metabolic inflexibility via increased PPARδ in obese mice. Therefore, we propose SKM FoxO1 as potential therapeutic target in metabolic diseases.

Tetraspanins, by generating membrane microdomains, play a crucial role in the regulation of the endosomal network. They regulate endocytosis and recycling of different receptors and adhesion molecules and the formation and maturation of both Multi vesicular bodies, lysosomes and extracellular vesicles. Besides, tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. We showed that in CD9 KO cells there was a deregulation on the endosomal compartments, with a reduction in the number of early endosomes and an increment in the number of multivesicular bodies, and as a consequence, higher secretion of EVs but with reduced protein content. Interestingly, when chemically inhibiting CD9, cells showed a defect in mitochondrial activity, which is recovered in the CD9 KO cell line by increasing the total mitochondrial mass by restraining macroautophagy-driven mitophagy. Single cell metabolic analyses revealed that CD9 KO cells have reduced mitochondrial ROS and cellular ATP levels, and overall reduced mitochondrial metabolic dependence. A selective reduction in the number of MAMs could also be detected in CD9 KO melanoma cells, further proving the connection of tetraspanin CD9 with mitochondrial quality control pathways.

Wolfram Syndrome (WFS) is a rare genetic disease that shares many features with mitochondriopathies as it’s characterised by diabetes mellitus/insipidus and progressive neurodegenerative symptoms like optic atrophy and sensorineural deafness. WFS is related to mutations in WFS1 and CISD2 genes, both encoding ER-membrane resident proteins with largely unclear function. We have demonstrated earlier that WFS1 deficient neurons suffer from mitochondrial dysfunction leading to energy deficiency and axon development retard. Several studies have reported higher resting cytosolic calcium concentrations in different types of WFS1/CISD2-deficient cells, thus linking the lack of these proteins to ER stress and altered Ca²⁺ homeostasis. Here, we demonstrate that in neurons, WFS1/CISD2 deficiency impairs the ER Ca²⁺ uptake through SERCA and release through RyR receptors that leads to increased cytosolic calcium levels and suppressed mitochondrial calcium uptake in MAMs. Furthermore, we identified possible pharmacological approaches to normalize calcium homeostasis and improve the health of neurons.

Mitochondrial fission is a highly regulated process that, when disrupted, can alter in metabolism, proliferation and apoptosis. Dysregulations have been linked to human diseases including neurodegeneration, cardiovascular disease and cancer. The fission processs is orchestrated by a endoplasmic reticulum and actin mediated pre-constriction, before dynamin-related protein 1 (DRP1) binds to the outer mitochondrial membrane via adaptor proteins, to drive scission. In the mitochondrial life cycle, fission enables both biogenesis of new mitochondria but also clearance of dysfunctional mitochondria through mitophagy. Current models of fission regulation fail to explain how the fission machinery is accounting for those opposing functions. Therefore we used live-cell super-resolution micrsocopy to analyze hundreds of fission events in Cos-7 cells and primary mouse cardiomyocytes. We discovered two functionally and mechanistically distinct types of fission: mitochondria divide in their periphery to evacuate damaged material into a smaller daughter mitochondrion that subsequently undergoes mitophagy. Healthy mitochondria proliferate via “midzone division”, which is coupled with mtDNA replication and results in two equal daughter mitochondria. Both types are mediated by DRP1, but endoplasmic reticulum- and actin-mediated pre-constriction and the adaptor MFF govern only midzone fission. Peripheral fission is preceded by lysosomal contact and is regulated by the mitochondrial outer membrane protein FIS1. These distinct molecular mechanisms explain how cells independently regulate fission, leading to distinct mitochondrial fates. Moreover, our preliminary results indicate, that midzone and peripheral fission can dictate cellular fates and that specific manipulations can provide a promising approach for therapy. 

Rab21 is a small GTPase involved in early endosomal trafficking. We recently characterized its associated proteome in vivo using an enzyme-based proximity labeling approach. We identified Rab21-specific neighbors in differentiated intestinal cells, the enterocytes, and revealed unexpected new neighbors for Rab21, among which were mitochondrial proteins. These data led us to investigate Rab21 function at mitochondria.

Using a cell-specific inducible system combined with mitochondrial reporters, we showed that Rab21 localizes at mitochondria in adult fly enterocytes and regulates their sizes. Importantly, we demonstrated that Rab21 mitochondrial function was conserved among species. Using two different human cell lines, we revealed that RAB21 localizes to mitochondria and modulates their sizes, which was specific to RAB21, among other RABs tested. 

Altogether, these data highlight an unexpected function for Rab21 at mitochondria. Although its function remains to be identified, preliminary data suggest a putative involvement in Dosmit-mediated cytosolic protein uptake at mitochondria.

Several studies describe the central role of mitochondrial DNA (mtDNA) defects in multiple diseases. Specifically, certain accumulated variations in mtDNA drive to a wide variety of haplogroups which could represent a higher or lower risk for suffering these mitochondrial-related diseases, including certain cardiopathies. Given this background, the aim of this study was to assess mtDNA haplogroups in patients with coronary artery disease (CAD) and aortic stenosis (AS). Single-base extension assay was employed to identified the most common European mtDNA haplogroups in 598 patients from a region of northern Spain. Results showed that haplogroup H is associated with an increased possibility of developing CAD (OR= 2.12; 95% CI= 1.37-3.27; p= 0.001) and AS (OR= 2.23; 95% CI= 1.53-3.25; p≤ 0.001), in a northern Spanish population, suggesting its role as a risk factor. In this sense, mitochondrial haplogroups could be considered as potential tools for the development of preventive or therapeutic strategies.

Mitochondria form a dynamic network that constantly undergoes fission and fusion events. While the mechanisms underlying outer mitochondrial membrane (OMM) remodelling are well described, how inner mitochondrial membrane (IMM) dynamics are regulated is still under investigation. Due to the fact that nucleoids-containing mtDNA are closely associated with the IMM, elucidating the processes driving IMM dynamics is essential to understand how mtDNA content and distribution are regulated. Using advanced microscopy techniques and precise mtDNA quantifications, we shed light on the processes governing IMM remodelling and mtDNA dynamics by characterizing new factors involved in the control of IMM fusion. Furthermore, we revealed that specific IMM remodelling events are required for the degradation of damaged nucleoids through a previously unidentified mechanism. Finally, our results suggest that the regulation of mtDNA copy number by IMM dynamics manipulation could be beneficial during heteroplasmy, which opens new therapeutic opportunities for the treatment of mitochondrial diseases.

Mitochondrial dysfunction underlies degeneration of neurons in neurodegenerative diseases. The Polg mutator (PolgD257A) mouse model and derived mouse embryonic fibroblasts (MEFs) recapitulate the pathological elements of age-related somatic mitochondrial dysfunction. PolgD257A mice demonstrated increasing locomotive deficits with age compared to littermate wild type mice. At 8-10 month old PolgD257A mice, there was significant elevation of plasma lactate, 3-hydroxybutyrate, TCA metabolites. In CSF, there were decreases in ascorbate, myo-inositol, amino acids, and TCA metabolites.  Concurrent with elevated plasma alanine, alanine-derived deoxydihydroceramides and deoxyceramides  were elevated in Polg brain extracts and exhibited marked age-dependent increases compared to age-matched wild type controls. PolgD257A mutant MEFs show reduced OCR, increased glycolysis, increased lactate production, decreased mitochondrial membrane potential and increased mtDNA copy number, compared to wild type MEFs. This study thus identifies and consolidated in vivo-in vitro aligned sensitive biomarkers which will enable efficacy testing of novel mitochondrial-targeted therapeutics. 

Mitochondrial glutaminase converts glutamine into glutamate fueling TCA cycle and to support oxidative phosphorylation (OXPHOS). Whereas in a healthy liver, glutaminase 2 is predominant, the expression of the isoenzyme glutaminase 1 (GLS) is heightened in metabolic-associated fatty liver disease (MAFLD). By using Seahorse analyzer, we have shown that specific silencing or inhibition of Gls in mouse hepatocytes, with high Gls expression after de-differentiation or stimulation with oleic acid, reduces oxygen consumption rate (OCR), a surrogate of OXPHOS. Likewise, silencing of Gls1 in mouse models of diet induced MAFLD decreases OCR in isolated liver mitochondria. Reduced OXPHOS after Gls silencing both in vitro and in vivo translates into decreased production of reactive oxygen species (ROS). Overall, hepatic GLS controls OXPHOS in the context of MAFLD where targeting GLS is a valuable therapeutic approach to hamper the excessive ROS generation and preventing both structural and functional mitochondrial abnormalities characteristic of advanced MAFLD.

Mitochondrial dysfunction is increasingly recognized as a causative factor in neurodegenerative diseases. Recent analysis of samples from patients with amyotrophic lateral sclerosis (ALS) identified several missense mutations in the conserved metallopeptidase Oma1 – a key controller of mitochondrial physiology; however, the underlying mechanism of pathogenesis remains unclear. 

Here, we investigated one of the ALS-linked Oma1 mutations using cell culture and yeast models. The mutation does not affect the topogenesis of Oma1, nor its substrate-binding properties. Further analyses show the mutation impairs the enzyme’s basal proteolytic activity as well as stability of the Oma1 oligomeric complex. Consequently, mutant Oma1-expressing mitochondria exhibit higher levels of ubiquitination and reduced tolerance to homeostatic insults. 

These results suggest that ALS-linked mutations in Oma1 may be associated with conformation changes that render the Oma1 complex less active and/or sensitive to homeostatic challenges, thereby impinging on mitochondrial fidelity and signaling, and contributing to etiology of ALS.

Within a rapidly aging society, the burden of age-associated diseases is rising. Mild inhibition of ATP generation as well as activation of the redox-sensitive transcriptional regulator Nuclear factor erythroid 2-related factor 2 (NRF2), is known to be instrumental in delaying such diseases. In this study, ATP-attenuating and Nrf2-activating small molecules were identified in a cell-based screening assay. Subsequently, the nematode C. elegans was supplemented with the top candidates and potential effects on lifespan were investigated. As a result, an un-described polyacetylene derived from the root of Daucus carota, hereafter named Compound III, was revealed as potent promotor of longevity. At the molecular level, Compound III affects cellular respiration in cells, C. elegans, and mice, by interacting with the α-subunit of the mitochondrial ATP synthase eventually leading to increased mitochondrial biogenesis. Phenotypically, this results in decreased tumor cell growth and an enhanced performance of C. elegans in various health and disease assays leading to improved motility and stress resistance as well as delayed development of protein accumulation in models of Alzheimer’s & Huntington’s disease. In addition, Compound III supplementation to wild-type C57BL/6N mice on high-fat diet resulted in improved glucose metabolism and increased exercise endurance. Interestingly, a similar potential as anti-diabetic and exercise mimetic was seen in aged mice. Due to these versatile effects on health parameters, Compound III might become a promising substance for further human studies to delay or prevent aging-associated diseases.

Armadillo Repeat Containing, X-linked 1 (Armcx1) is a protein of the outer mitochondrial membrane of unclear function. We previously showed that Armcx1 overexpression increases mitochondrial transport and that this increase promotes axonal regeneration and neuronal survival after an optic nerve injury. Here, we are deciphering the endogenous function of this repair-promoting protein using two approaches. First, using an in vitro system, we discovered that over-expressed Armcx1 localizes in puncta at the ends of mitochondrial tubules. This effect was independent of the tag and could be related to mitochondrial fission. Second, we hypothesize that endogenous Armcx1 controls axonal growth during development in vivo. We generated an Armcx1^f/f mouse and using specific Cre lines are testing this hypothesis in two different neuron types namely the retinal ganglion cells and cortical neurons. Overall, this project aims to uncover the cellular function of a key protein in the neural repair machinery.

Diabetic cardiomyopathy is a major cause of heart failure with preserved ejection fraction (HFpEF) for which there are limited therapeutic options. Cardiomyocyte calcium overload and mitochondrial dysfunction are pathophysiologic mechanisms contributing to the development of diabetic cardiomyopathy. Here we report that, in db/db mice with type 2 diabetes, cBIN1 gene therapy restores the known calcium-handling microdomains of transverse-tubules in failing diabetic cardiomyocytes, which improves diastolic function and exercise capacity. Surprisingly, AAV9-introduced exogenous cBIN1 promoted protective mitophagy in db/db cardiomyocytes to facilitate effective clearance of damaged mitochondria, which otherwise accumulated intracellularly causing impaired respiration and elevated oxidative stress. The mechanisms of mitophagy activation are linked to cBIN1-recruitment of the ESCRT-III family member CHMP4B for the formation of mitophagosomes and subsequent degradation of damaged mitochondria in the endolysosome. As a result, AAV9-cBIN1 rescued diabetic cardiomyopathy and phenotypic HFpEF. cBIN1 gene therapy is a promising treatment for patients with diabetic cardiomyopathy.

Metabolic cardiomyopathy is a disease of the cardiac muscle, which may progress to congestive heart failure. We recently published that the cardio-specific microRNA (miR) 208a regulates the pathological stress markers and mitochondrial metabolism in cultured human cardiomyocytes that are metabolically challenged with diabetogenic conditions. In this study we aimed to characterize the microRNA profile of left ventricles harvested from rodents with metabolic syndrome. Lewis rats fed a high fat diet (HFD) for 18 weeks developed metabolic syndrome characterized by hyperglycemia and hyperinsulinemia, which was associated with an increase in cardiac pathological stress markers. We report a decrease in cardiac miR 208a in rats with high fat diet (HFD)-induced metabolic syndrome, which was associated with cardiac fibrosis and a metabolic remodeling that supports an increased mitochondrial fatty acids (FA) oxidation. This study confirms the role of miR 208a to control cardiac reliance on FA oxidation for ATP generation, which is the bioenergetic signature of the metabolic heart.

The pathways affecting the number and distribution of mtDNA under mtDNA instability is not well understood. To characterize mechanisms of mtDNA maintenance, we introduced mtDNA-specific damage in budding yeast. Under damage, we observed steady decrease in number of mtDNA nucleoids, and, an increase in percentage of cells without any mtDNA foci. This loss was not dependent on mitochondrial loss, mitophagy or mitochondrially-localized endonucleases. Instead, we found that exonuclease activity of the mitochondrial replicative polymerase, Mip1, is essential for the observed damage-dependent loss of mtDNA; perturbations to the same resulted in cell cycle slowdown under damage. Importantly, this DNA degradative action was independent of cell division and was not triggered via differences in dntp levels under stress. Since mtDNA replication and degradation is carried out by the same protein, our results pose an intriguing paradigm for context-dependent, selective regulation of Mip1 function(s) between mtDNA synthesis and its clearance, in the mitochondria.

Aim: PCSK9 (expressed mainly in the liver and to a lesser extent in the heart) regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9^–/– mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells.

Results: CM-Pcsk9^–/– mice had reduced contractile capacity, impaired cardiac function and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and metabolism in CM-Pcsk9^–/– hearts mice versus wildtype littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9^–/– hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in CM-Pcsk9^–/– cardiomyocytes. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9^–/– mice. Circulating lipid levels were unchanged in CM-Pcsk9^–/– mice, but the lipid composition of mitochondrial membranes was altered. We showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism.

Conclusion: PCSK9, despite its low expression in cardiomyocytes, plays a key role in cardiac metabolic function and PCSK9 deficiency is linked to cardiomyopathy, impaired heart function and compromised energy production.

Mitochondrial organization is important for mitochondrial genome maintenance, however, the impact of mtDNA perturbations on mitochondrial dynamics and/or organellar maintenance remains less understood. To address this, we develop a tool to induce mitochondria-specific DNA damage, using a base modifying bacterial toxin, DarT. Following damage induction, we observe dynamic reorganization of the mitochondrial network, which is associated with loss of mtDNA, independent of mitophagy. Unexpectedly, we find that perturbation to exonuclease function of the mtDNA replicative polymerase, Mip1, results in rapid loss of mtDNA. Our data suggest that the partitioning of mtDNA and organelle can be de-coupled, with an emphasis on mitochondrial segregation even in the absence of its DNA.  We also show evidence towards activation of a DNA damage response via Rad53 under mtDNA damage. Phosphorylation of Rad53 is observed only in cells retaining damaged mtDNA, and is not observed in cells devoid of mtDNA. Parallely, loss of mtDNA also appears to be essential for metabolic adaptation of cells, with potential implications on cell growth and survival. Our current efforts are focussed at delineating the mechanism of DDR activation, as well as the mechanism of damage induced metabolic rewiring.

Highly conserved in metazoa, ATPase family AAA+ domain-containing protein 3 (ATAD3) has undergone duplication in hominids giving rise to three highly homologous tandemly-arrayed paralogs: ATAD3A, and the hominid-specific ATAD3B and ATAD3C. Owing to its genomic complexity, the ATAD3 locus is a hotspot for pathogenic gene variants and rearrangements, placing it among the most common known causes for lethal infantile mitochondrial disease. 

Though implicated in numerous mitochondrial processes, the precise roles of the locus remain unresolved. ATAD3 is essential for cell viability so to disentangle individual functions of ATAD3 proteins and their roles in disease pathogenesis, we generated a human knockout cell model lacking the entire ATAD3 locus, with inducible ATAD3A expression. We demonstrate a supportive role for ATAD3 in maintaining mitochondrial ultrastructure through its mediation of F₁F₀ ATP synthase function. Use of this cell model also provides a pathway to delineating the impact of pathogenic variants in disease.

Mitochondria and lysosomes were long considered as independent entities in the cell. Recently, evidence was reported that these organelles are dependent on one another, and that defects in one of these organelles result in perturbations of the other. In models and patients of mitochondrial disease, stalled autophagy and saturated lysosomes are often observed. Nevertheless, the mechanisms mediating the communication between mitochondria and lysosomes remain unclear.

Using mouse models of mitochondrial and lysosomal disease, we have unveiled a bi-directional communication pathway between mitochondria and lysosomes. Mitochondrial dysfunction results in lysosomal saturation and impairment of lysosomal calcium signaling and accumulation of lysosomal substrates, including autophagy intermediates. On the other hand, the impairment of lysosomes results in mitochondrial malfunction via impairment on the homeostasis of iron. Furthermore, mitochondrial and lysosomal dysfunctions have opposite effects on lipid metabolism. 

Mechanistically, we found that the folliculin complex, which regulates the balance between AMPK and mTORC1 and thus the cellular balance between anabolism and catabolism, is a key mediator of mitochondria-lysosome crosstalk and determines how cellular metabolism responds to organelle perturbations. Notably, by manipulating the pathways that affect lysosomes in models of mitochondrial disease, we observed an improvement of the respective phenotypes. We will build on these findings to explore an overall picture of organelle homeostasis in physiology and in mitochondrial diseases.

Aging and myopathies are characterized by a decline in skeletal muscle mitochondrial function. Yet whether age and disease-dependent changes in mitochondria play causal roles in propagating homeostatic adaptation and pathological progression remain unclear. Here we show that aging reduces levels of cardiolipin (CL), a membrane lipid essential for mitochondrial function, and its synthase, CRLS1, in both glycolytic and oxidative muscle. Loss of skeletal muscle Crls1 in young mice is alone sufficient to drive aging hallmarks and trigger adaptive programs through mitonuclear communication. Surprisingly, CL-deficient glycolytic fibers are able to retain respiratory capacity by doubling mitochondrial abundance and globally adopting an oxidative profile. Moreover, all muscles lacking CL boost glucose uptake via increased reactive oxygen species (ROS) and shunt glycolytic intermediates into antioxidant defense pathways, independent of canonical stress factors, FGF21 and GDF15. Thus, disrupted CL synthesis directly contributes to age-associated myopathy and orchestrates both fiber type-specific and universal bioenergetic adaptations.

PINK1 is a nuclear encoded, mitochondria targeted Serine/Threonine kinase, which interacts with several substrates to regulate mitochondrial functions. Interestingly, this protein has a dual role, depending on mitochondria state. In healthy mitochondria, PINK1 regulates ATP production by phosphorylating the Complex I subunit NdufA10. However, when in the presence of depolarized mitochondria, PINK1 phosphorylates ubiquitin and Parkin triggering mitochondria for clearance via mitophagy. Mutations in PINK1 has been linked to early-onset recessive familial forms of Parkinson's disease (PD). Interestingly, deficits in Complex I enzymatic activity and an increase in oxidative damage have been identified in multiple brain regions of PD patients. Unravelling how PINK1 activity regulates mitochondria fate is pivotal. Therefore, we aimed to understand how PINK1-PD related clinical mutants affect PINK1's decision when in the presence of healthy or unhealthy mitochondria. Cell-based assays revealed that PINK1 has critical amino acid residues that are involved in recognition of unhealthy mitochondria. Our findings indicate that the N-terminal portion of PINK1 seems to be crucial for substrate recognition and phosphorylation. Through molecular dynamics we were able to gain mechanistic insight on how these specific clinical mutations alter the tertiary structure of PINK1 leading to modification in substrate docking. Concluding, different residues mutated in PINK1 can impair different mitochondrial pathways, since depending on the residue that is mutated a different cross-talk between PINK1 and its already described substrates will be deteriorated. In disease context, our work strengthened the notion that each PD patient is a particular case, supporting a future personalized medicine approach.

Endothelial cells (ECs) are metabolic gatekeepers of the organism, but their contribution to the development of metabolic disorders remains incomplete. The role of mitochondria in ECs has been overlooked, but recent evidence indicates that this organelle is crucial for proper EC function. We hypothesized that mitochondrial dynamics in ECs influences whole-body metabolic status. In obesity states, ECs showed reduced mitochondrial elongation and expression of pro-fusion determinants Mitofusin (Mfn) 1 and 2. Genetic deletion of Mfn2 in ECs (Mfn2iΔEC) in mice, caused a mitohormetic response leading to advantageous vascular adaptations in terms of antioxidant defenses, mitochondrial fitness and lipid oxidation. Consequently, Mfn2iΔEC mice exhibited improved metabolism, protection against diet-induced obesity and ameliorated healthspan. Here, we uncovered a novel mechanism, named vascular mitohormesis, that influences systemic metabolism and age-related decline.

Age-related loss of skeletal muscle mass and function is associated with disrupted redox signalling and accompanied by a loss of mitochondrial content. Regulation of mitochondrial dynamics is essential to maintain a healthy mitochondrial population and to prevent the accumulation of damaged mitochondria. We have confirmed a loss of mitochondria content and altered expression of mitophagic regulators in skeletal muscle from old mice. A complimentary analysis of microRNA’s reveal a downregulation of miR181a with age. We have confirmed known (Park2) and identified novel targets (p62/SQSTM and Protein DJ-1) of miR181 in a myoblast cell model of mitochondrial dysfunction. Treatment of adult and old mice with miR181a and Anti-miR181a confirmed an altered expression of mitophagic target genes and proteins, with an improvement in muscle mass and function in old mice treated with miR181a. Our results demonstrate the key regulatory role of miR181 in the maintenance of mitochondria in muscle of old mice.

Cristae, invaginations of the mitochondrial inner membrane, are hubs for oxidative phosphorylation. Two antagonistic machineries, the mitochondrial contact site & cristae organizing system (MICOS) and the F₁F_o-ATP synthase, play important roles in controlling cristae architecture. It is unclear whether these machineries coordinate their complementary activities in cristae biogenesis. The MICOS core subunit Mic10 is crucial for membrane shaping by forming oligomers at MICOS. We have found that Mic10 has a second role: It interacts with ATP synthase dimers and regulates their association into oligomeric rows. Unexpectedly, Mic10’s importance for efficient metabolic adaptation and respiration largely depends on its regulatory function at the ATP synthase, not on its MICOS-dependent activity. Mic10 variants that selectively function at the ATP synthase promote efficient inner membrane energization and respiration. Thus, Mic10 plays dual roles as MICOS core subunit and as partner of the F₁F_o-ATP synthase with distinct roles in cristae shaping and respiratory adaptation.

Despite the importance of mitochondria for health, the effects of different exercises on mitochondrial adaptations are unresolved. Twenty-eight men completed one of two 8-week training interventions - Moderate Intensity Continuous Training (MICT) or very high-intensity Sprint Interval Training (SIT). Our RNAseq results suggest a highly coordinated transcriptional response that was largely shared across the two different exercise prescriptions. However, only SIT was characterised by activation of transcriptional pathways associated with mitochondrial stress and the unfolded protein response, combined with structural mitochondrial disturbances (measured via TEM), suggesting increased activation of mitochondrial quality control pathways. Whole-muscle proteomics demonstrated that SIT also increased proteins involved in mitochondrial protein quality control, while MICT increased proteins involved in the TCA cycle and OXPHOS. Our single-fibre proteomics revealed only a few fibre-specific differences. Only SIT led to an increase in mitochondrial respiratory function, while only MICT was characterised by increased mitochondrial content and complex I protein abundance.

Mitochondrial dysfunction is critically driving the degeneration of nigral dopaminergic neurons (DaNs) in Parkinson’s disease, causing the cardinal motor symptoms. MitoPark mice recapitulate both the decline in motor performance and progressive loss of DaNs. The extraordinary vulnerability of DaNs to mitochondrial defects is linked to cytosolic Ca²⁺ rises mediated via voltage-gated Ca²⁺ channels. The presence of the Ca²⁺-buffering protein Cb-D28k, on the contrary, is associated with higher resilience against neurodegeneration. To study the impact of the L-type Ca²⁺ channel Ca_v1.3 as well as the presence of Cb-D28k on DaN survival, we crossed MitoPark mice with animals lacking either Ca_v1.3 or Cb-D28k. While knockout of Ca_v1.3 improved motor performance and decelerated loss of DaNs, absence of Cb-D28k did neither alter motor behavior nor neuron survival in MitoPark mice. These data underline the importance of targeting Ca_v1.3 in Parkinson’s disease and simultaneously question the neuroprotective role of Cb-D28k.

Mitochondrial dysfunction is linked to many neurodegenerative disorders such as optic neuropathies. These blinding diseases are typically caused by the degeneration of retinal ganglion cells (RGCs). RGCs are projecting neurons of the inner retina that relay electrical signal to the brain via their axons in the optic nerve. In RGC axons, little is known about the local function and distribution of mitochondria. The proximal region of the optic nerve is called the glial lamina (GL) in which RGC axons are unmyelinated. Axons in the GL have been shown to be rich in mitochondria and susceptible to insults in glaucoma. Using two independent imaging techniques, we demonstrated that the mitochondrial enrichment in the GL is restricted to larger axons and is established at P6-P8, a stage that precedes myelination. Therefore, our study shows that, unlike previously thought, mitochondrial accumulation in axons of the GL is independent of myelination.

RNA viruses depend on extensive manipulation of subcellular organelles for replication. Viral replication complexes (VRCs) are observed for all types of organelles including mitochondria, which significantly alters their morphology. Little is known about the cellular stress response involved in coping with such stress. Here we uncover the critical role of selective autophagy in promoting survival in cells undergoing active replication. We show that autophagy is robustly induced in cells displaying VRCs and that autophagy mutants are severely intolerant to infection. Hijacking of mitochondria by carmoviruses leads to degradation of soluble mitochondria matrix proteins, and accumulation of abnormal material in the cytosol of autophagy mutants. Finally, we identify and characterize two novel autophagy receptor proteins that are activated during carmovirus infection. We propose that autophagy relieves a proteotoxic burden incurred by excessive mitochondrial stress, which ultimately enhances the survival of the infected plant.

By 2030, half of the US population might have some form of cardiovascular disease (CVD), some of them resulting from anti-cancer treatment induced cardiotoxicity. This highlights the need for new therapeutic strategy or an improvement of existing treatments. In many cardiac diseases, mitochondria can receive harmful signals, dysfunction and then, participate actively in the pathogenesis. In this context, we evaluated the mitochondrial effects of Digoxin and Digitoxigenin, two cardiac glycosides used in clinics for heart failure treatment. We selected these two compounds in a high throughput screening of cell death inhibitors revealing their activity as potent inhibitors of apoptosis and necrosis of cardiomyoblasts. We confirmed their activity in rat neonatal ventricular cardiomyocytes and showed their capacity to induce autophagy. We also analysed their effects on mitochondrial network structure by fluorescent confocal microscopy and on bioenergetics. In conclusion, our study offers new insights into the pharmacological effects of cardiac glycosides.

Our earlier work in Drosophila flight muscles found that mitochondria intercalate during development between the growing chains of sarcomeres called myofibrils which is necessary for muscle type specification (Avellaneda et al. 2021). Here, we are investigating the mechanism of mitochondrial intercalation between myofibrils during development. We have established live in vivo imaging of mitochondria together with the actin and the microtubule cytoskeleton in developing flight muscles. We found that upon myofibril assembly, mitochondria are relocating from an actin-free zone into the assembled myofibril bundles within less than two hours to insulate each myofibril from its neighbours. Using photoactivation of individual mitochondria revealed a dramatic increase in mitochondrial transport dynamics during intercalation. Concomitantly, the microtubule cytoskeleton also reorganises with microtubules surrounding each myofibril suggesting a role for microtubule-based transport of mitochondria. Genetic interventions will be presented to dissect the role of the microtubule cytoskeleton in coordinating mitochondria with myofibril morphogenesis.

Diverse pathways control mitochondrial activity to preserve brain functions. One example is the activation of GPCRs which can modulate brain physiology and function via mitochondria. To address the impact of G proteins inside mitochondria, we generated an engineered muscarinic receptor targeted to mitochondria which couples to endogenous Gαs proteins, named mitoDREADD-Gs. Activation of mitoDREADD-Gs increases mitochondrial membrane potential and oxygen consumption via PKA both in vitro and in vivo. Interestingly, activation of mitoDREADD-Gs prevents the cannabinoid-induced impairment of memory performance and the cannabinoid-induced catalepsy, two processes mediated by the direct inhibition of mitochondrial activity by cannabinoids. MitoDREADD-Gs is thus among the first tool to improve mitochondrial activity and higher brain functions. These findings reveal that the impact of intramitochondrial G proteins signaling on both brain metabolism and physiology could be considered as potential therapeutic target in neurodegenerative disorders and associated cognitive deficits.

During infection, dengue virus (DENV) and Zika virus (ZIKV) NS4B proteins induce the elongation of mitochondria in favor of viral replication suggesting a viral co-opting of mitochondria functions. Here, we performed an extensive transmission electron microscopy-based quantitative analysis to demonstrate that both DENV and ZIKV alter endoplasmic reticulum-mitochondria contacts (ERMC). This correlated at the molecular level with an impairment of ERMC tethering protein complexes located at the surface of both organelles, which was attributed to NS4B. Furthermore, virus infection, as well as NS4B expression modulated the mitochondrial oxygen consumption rate. Consistently, our metabolomic and mitoproteomic analyses revealed a decrease in the abundance of several metabolites of the Krebs cycle and changes in the stoichiometry of the electron transport chain. Most importantly, ERMC destabilization by protein knockdown increased virus replication while dampening ZIKV-induced apoptosis. Overall our results support that flaviviruses hijack ERMCs for the benefit of a sustained and efficient replication.

Alveolar progenitor cells during adaptive stress response, upregulate mtDNA-encoded small ncRNA, mmu-mito-ncR-805, generated from the D-loop of the light strand. During recovery  mmu-mito-ncR-805 appears in the nucleus, correlatively with increased transcripts of nuclear encoded mitochondrial genes (NeMito), and bioenergetics. The first 20nt represent a putative functional bit, which is evolutionary conserved in mammalian mitochondria, with no NUMTS, and no nuclear transcript detected. Forced expression of mouse and human orthologs, is inter-changeable in their ability to increase NeMito transcripts, and mitochondrial respiration. Data suggests, mmu-mito-ncR-805 acts as a retrograde signal that convey mitochondrial status to the nucleus, and via its nuclear function promotes mitochondrial bioenergetics.  Nuclear retention of mito-ncR-805 using SnoVectors, or nuclear targeting by  nanoparticles proved causality between nuclear location and function of mmu-mito-ncR-805, suggesting the role of the Control region of the mitochondrial genome beyond regulation of mitochondrial replication/transcription balance, and opening interventional opportunities for improving mitochondrial bioenergetics.

Muscle diseases and aging are associated with impaired myogenic stem cell self-renewal and fewer proliferating progenitors (MPs). Importantly, distinct metabolic states induced by glycolysis or oxidative phosphorylation have been connected to MP proliferation and differentiation. However, how these energy-provisioning mechanisms cooperate remain obscure. Herein, we describe a novel mechanism in which mitochondrial-localized transcriptional co-repressor p107 regulates MP proliferation. We show that p107 directly interacts with the mitochondrial DNA, repressing mitochondrial-encoded gene transcription. This reduces ATP production by limiting electron transport chain complex formation. ATP output, controlled by the mitochondrial function of p107, is directly associated with the cell cycle rate. p107 subcellular localization is dependent on the glycolysis metabolite NAD⁺. Activation of Sirt1 by NAD⁺ enables direct interaction with p107, impeding its mitochondrial localization and function. The metabolic control of MP proliferation, driven by p107 mitochondrial function, establishes a cell cycle paradigm that might extend to other dividing cell types.

The topology of the inner mitochondrial membrane (IMM) and its cristae is essential for cellular respiration. Several proteins shape the morphology of the IMM, but similar roles for IMM lipids are unresolved. We describe how lipid-encoded mechanical properties dictate the formation of cristae in the IMM. Modulation of lipid saturation in yeast first resulted in an abrupt transition in cristae ultrastructure. This finding motivated the development of a data-derived continuum model for mitochondrial membrane morphology and suggested that cristae are defined by a ‘snap-through’ instability. Simulations predicted that epistasis between bending rigidity, controlled by saturation, and spontaneous curvature, controlled by the phospholipid cardiolipin, is key for cristae assembly. We confirmed this prediction experimentally and found that cardiolipin is essential for mitochondrial function when lipid saturation is increased genetically or through growth in natural yeast environments. Our results show how multiple aspects of lipid chemistry dictate mitochondrial morphology and function.

The endoplasmic reticulum (ER) and mitochondria establish a unique cellular component called mitochondria-associated ER membranes (MAM). Our genome-wide kinase-MAM interactome screening proposed CK2A1, a catalytic subunit of casein kinase 2, as a novel regulator of MAM formation and Ca²⁺ transfer from the ER to mitochondria. CK2A1 interacts with PKD2-PACS2 and re-positions this complex to MAM. In addition, phosphorylation of PACS2 by CK2A1 at S207-213 alters MAM composition and Ca²⁺ transfer between two organelles in PKD2-dependent manner. We further reveal that pathogenic mutations of PACS2 (E209, E211K), associated with a genetic disease named developmental and epileptic encephalopathy 66, affect the recruitment of CK2A1-PACS2-PKD2 complex to MAM, causing the disturbances of MAM integrity and Ca²⁺ homeostasis at pre-synapses of primary neurons. Our findings suggest a novel molecular mechanism whereby alterations of MAM by pathogenic PACS2 mutations modulate Ca²⁺-dependent neurotransmitter release from glutamatergic neurons.

The double-membrane-bound architecture of mitochondria sub-divides the organelle into inter-membrane space (IMS) and matrix. Mitochondrial IMS and matrix possess contrasting protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and specifically targeted to yeast mitochondrial IMS or matrix to impart stress.  Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS.  A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that IMS stress leads to upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-stress lead to upregulation of matrix-chaperones, cytosolic quality control machineries and Vms1.

Fundamental role of mitochondria is production of energy in cells and their dysfunction is involved in various diseases. Here, we demonstrate a novel method of isolation of mitochondria (Q)- with intact mitochondria isolation technology (iMIT), that keeps mitochondrial structural and functional integrity to a greater level when compared with commercial mitochondria isolation kit. Q had higher production of ATP and maintained outer membrane integrity demonstrated by cytochrome c oxidation, this allowed cryopreservation of Q while maintaining their functions. When Q were incubated in multiple cell cultures, they were readily incorporated in recipient cells determined by mtDNA copy number. Confocal microscopy and flow cytometry also confirmed incorporation of Q when co-cultured with recipient cells. Finally, Q were able to  modulate release of IL-6 elicited by LPS in THP-1 cells. iMIT provides a crucial tool in isolation of high-quality mitochondria for research as well as potential as biopharmaceutical agents.

We explored using non-invasive blood tests to diagnose thyroid cancer. We analyzed blood and thyroid tissue from patients undergoing thyroidectomy. Thyroid cancer revealed increased levels of active AKT, alterations in mitochondrial sub-cellular distribution reduced mtDNA and elevated Prx3, suggesting the presence of mitochondrial stress. In plasma, cancer patients showed higher levels of cfDNA and mtDNA. Plasma mtDNA inversely correlated with tissue mtDNA. In PBMCs, cancer patients showed higher levels of PGC-1α, but this increase was not associated with a induction of its target genes, We also observed differences in the prx3/pfkfb3 correlation, between carcinoma and hyperplasia patients, and a correlation of mtDNA levels in tissue and PBMCs. ND1/mtDNA positively correlated in PBMCs and tissue samples. In contrast, ND4 evaluation was informative of tumor development, with ND4/mtDNA specifically altered in tumors. Our data suggest that metabolic dysregulation in thyroid cancer might be exploited for the discrimination of cancer from hyperplasia.

Intake of Second generation Antipsychotics (SGAs) has been related to increased risk of CVD. to investigate if and how impacted on and its role on Cardiovascular Disease (CVD). We tested the mitochondria effects of Olanzapine (Ola) and Aripiprazole (Ari) and its role on CVD. Both accumulate in mitochondrial membranes, and inhibit mitochondrial respiration but only Ola increases mitochondrial ROS. As a result, mitochondria from Ola treated cells recovered more efficiently. In a PGC-1α dependent manner, Ola induced compensatory antioxidant gene expression and mitochondrial turnover, while Ari did not. While both Ari and Ola induced adverse cardiac remodelling in PGC-1α treated mice, the effect of Ari was significantly higher and was apparent at earlier time points. These results support the hypothesis interference with mitochondrial function plays an important role on SGAs CV risk and ROS dependent induction of PGC-1α activity can partially compensate these effects, reducing the net risk.

Mitochondrial oxidative phosphorylation system (complexes I-V; OXPHOS) produces most of cellular ATP. Maintenance of mitochondrial function requires selective intramitochondrial proteolysis. 

We prepared HEK293 cells with deficient ClpXP protease (found within the mitochondrial matrix) due to RNAi-based knockdown of CLPP or CLPX component to characterize mitochondria in these cells and to check for specific ClpXP substrates. In CLPP knockdown, we used ectopic expression of FLAG-tagged, proteolytically active and inactive versions of CLPP followed by immunoprecipitation and Western Blot. Then, these data we correlated with BN-PAGE immunoblots to seek for altered accumulation levels of OXPHOS subassemblies or individual structural subunits. We found increased accumulation of CI- and CIII–specific subcomplexes in addition to virtually normal steady-state level of the whole complexes or supercomplexes.

Our data contributes to the previous studies suggesting the role of ClpXP protease in the maintenance of OXPHOS complexes I and III.

Supported by RVO-VFN64165 and AZV MZCR NV-19-07-00149.

Cytochrome bc₁ is a key component of the electron transport chain coupling electron transfer to proton transport across the membrane. It operates according to the mechanism of the Q-cycle, in which oxidation of ubiquinol at the Q_o catalytic site leads to electron transfer onto two distinct cofactor chains. The uncompleted bifurcation can lead to side reactions resulting in the formation of radical oxygen species (ROS). In this study we explore electron transfer sequences that potentially lead to ROS generation within cytochrome bc₁, benefiting from the newly designed ROS detection method. Towards this goal we analyze heme b Rb. capsulatus mutant strains with impaired electron transfer (at the level of hemes b_L and b_H), and Rb. capsulatus heme c₁ mutant strain mimicking redox equilibration of its mitochondrial equivalent. With this approach we examine how changes in the electron distribution along the cofactor chains impact on ROS production under various conditions.

Molecular oxygen sustains intracellular bioenergetics and is consumed by more than 400 biochemical reactions, making it essential for life on Earth.Reduced oxygen concentration (hypoxia) is a prominent feature of pathological states encountered in inflammation, cardiovascular defects and cancer. Despite the fundamental importance of oxygen in human physiology and disease, we currently lack a complete understanding of how the mitochondrial proteome adapts fluctuations in oxygen tensions. We recently identify a mTORC1-LIPIN1-lipid signalling cascade that activates the i-AAA protease YME1L in hypoxia to ensure pyrimidine synthesis. Here, we determined protein turnover rates of mitochondrial proteins in hypoxia. The mitochondrial m-AAA protease AFG3L2 was found to reshape the mitochondrial matrix and inner mitochondrial membrane  proteome in hypoxia. This proteolytic rewiring regulates proline metabolism, which maintains mitochondrial NAD⁺ levels at low oxygen concentrations. We show that in hypoxia changes  protein turnover, the mitochondrial m-AAA protease AFG3L2 reshapes the mitochondrial proteome to adapt to hypoxia.

Mitochondria exchange their content as an essential component of mitochondrial homeostasis. Fusion can disperse content diffusively throughout the mitochondrial network. Alternatively, mitochondria can disperse more directly via transport on the cytoskeletal network before fusing at different locations. However, it remains unknown how mitochondria balance directed transport over local fusion and diffusion. We spread cells into standardized shapes on protein micropatterns, thereby spatially separating mitochondrial subpopulations. Using photoconvertible proteins, we track single mitochondria originating from different parts of the cell. Interestingly, small, perinuclear mitochondria are rapidly transported throughout cells. Conversely, peripheral mitochondria are less mobile interacting primarily with neighboring organelles. Comparison of fission type and distance to the cells center reveals a concentration of proliferative fissions near the nucleus. Degradative fissions are concentrated near the periphery of the cell. These results suggest the existence of a proliferative mitochondrial population surrounding the nucleus, capable of rapidly replenishing other regions of the cell.

The mitochondrial protein IF1 binds to the catalytic domain of F1Fo ATP synthase and inhibits ATP hydrolysis in ischemic tissues. IF1 is overexpressed in many tumors, although its mechanism(s) of action is still debated. We show that IF1 immunoprecipitates with ATP synthase in mitochondria derived from different cancer cell lines, maintained under State 3 respiratory condition. Since the IF1 binding to the ATP synthase catalytic domain requires ATP hydrolysis, our finding suggests the presence of an additional binding site. The new site has been identified by immunoprecipitation, proximity ligation and NMR spectroscopy. The lack of the IF1 interaction under oxidative phosphorylation does not affect proliferation or respiration, but sensitizes cells to permeability transition, in line with a reduced HeLa colony formation upon disruption of the IF1 gene. Overall, this study indicates that IF1 interacts with an additional site on ATP synthase and desensitizes permeability transition, protecting cells from apoptosis.

Ligase 3 has been previously identified essential for cellular viability. Here, we applied CRISPR/Cas9 genome editing to genetically inactivate LIG3 in HEK293 cells to study effects on mtDNA maintenance and integrity. We observed an about 50% decrease of mtDNA copy number at lowered amounts of its supercoiled form leading to modest functional effects on OxPhos. Many of the mtDNA molecules had single-strand breaks, as detected by ultra-deep long- and short-read sequencing of S1 nuclease-treated mtDNA. The location of major sites was very similar to that of mtDNA double-strand breaks from H₂O₂-treated HEK293 cells, suggesting oxidative stress as potential cause. Control cells recovered mtDNA copy numbers and integrity after H₂O₂ treatment, but LIG3 knockout cells lost most of mtDNA copies. Our data provide direct evidence for the pivotal importance of LIG3 for repair of oxidative lesions, while for replication-related end ligation of de novo synthesized mtDNA LIG3 appears dispensable.

Mitochondria play a crucial role in energy homeostasis regulation during development. Even though, it was shown that the respiratory activity of mitochondria increases during embryogenesis, the molecular mechanisms mediating this rise remain unclear. To investigate mechanisms underlying the mitochondrial activation during embryogenesis, we assessed oxygen consumption and employed blue-native PAGE, proteomics, metabolomics, and imaging in zebrafish embryos. We find that oxygen consumption continuously increases during zebrafish embryogenesis. Metabolomics analyses reveal dynamic changes of metabolites and high level of NADH during early embryogenesis. Using a transgenic fish line with labelled mitochondria, we show that mitochondrial morphology changes during development from spheroid to tubular. Analysis of the mitochondrial proteome throughout embryogenesis revealed several factors that might contribute to the changes in mitochondrial morphology. Our study aims to fill the gaps in our understanding of mitochondrial activation after fertilization and how changes in mitochondrial morphology contribute to the increase in mitochondrial activity.

Dynamin-1- like protein (Drp1) encoded by DNM1L gene, is crucial in the mitochondrial and peroxisomes fission process, during which it forms an oligomerization ring around these organelles, and in the final fission step are separated from each other by GTP hydrolysis. We describe four patients with de novo mutations in the DNM1L gene. In patients´ fibroblasts and control fibroblasts overexpressing mutated Drp1, we observed different profound effects of individual mutations on the mitochondrial network, ultrastructure, impaired peroxisome division and significant enlarged nucleoids, but mitochondrial respiration was not impaired. In cells carrying the mutation in the GTPase domain, the average nucleoid size was up to 20% larger than in the control, while in cells with the mutation in the oligomerization domain, nucleoids were up to 82% larger. Supported by research projects: SVV 260516; AZV 17-30965A, AZV NU22-07-00614, and RVO VFN640165.

Hematopoietic stem cells (HSCs) are responsible for lifelong balanced production of myeloid and lymphoid leukocytes, erythrocytes and platelets. Previously I discovered that mitochondrial membrane potential (MMP) of HSCs is a determinant of lineage potential that is pharmacologically perturbable in vivo with consequences for stem cell function. Next, we explored clinical scenarios that might benefit from mitochondrial-targeted treatment, including hematopoietic recovery from myelo-ablative chemotherapy. Common chemotherapeutic agents also kill the rapidly dividing healthy blood cells, resulting in periods of myelo-suppression in which patients are susceptible to infection and anaemia. Prolonged myelo-suppression is a serious side effect that can necessitate treatment adjustment or discontinuation. We treated wild-type mice with chemotherapy (5-FU; Fluorouracil) with or without follow-up treatment with mitoquinol (Mito-Q). Initial data reveals that Mito-Q accelerates multi-lineage recovery in 5-FU-treated mice. We are now exploring if aged chemotherapy-treated mice similarly benefit from Mito-Q treatment, before aiming to model this in a human context.

We have previously linked single-nucleotide polymorphisms in the anoctamin 7 (ANO7) gene to the risk of aggressive prostate cancer. To gain information into what pathways that ANO7 is affecting, we performed RNA-sequencing and pathway analysis. Interestingly, we found enrichment of mitochondrial genes participating in oxidative phosphorylation in cells overexpressing ANO7. We measured oxidative phosphorylation capability and showed that the maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced compared to control cells. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result indicates that ANO7 cells are not using glucose as effectively as control cells. Furthermore, preliminary results from metabolites screening suggests that ANO7 overexpressing cells have decreased levels of TCA cycle intermediates. This study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism.

Post-translational modifications (PTMs) represent an efficient way to modulate the activity of eukaryotic proteins. Phosphorylation, as the most common PTM, covalently attaches a phosphate group to serine, threonine or tyrosine residues. Particularly in humans, more than one third of proteins, including mitochondrial, is phosphorylated. Human mitochondrial ATP-dependent protease LON, which secures degradation of misfolded and damaged proteins, was found phosphorylated at multiple sites. Interestingly, these phosphorylations were associated with various cancers, including endometrial, stomach, or lung cancer. To better understand the effects of LON phosphorylation, we prepared several point mutations. The corresponding Tyr codons were mutated to Phe, Glu and amber stop codons and a non-hydrolysable analog of phosphotyrosine was incorporated into particular sites. We found that phosphorylated LON mostly retained its hexameric form, but its other activities were highly altered. Still, further experiments are needed to find out more about the mechanism of mitochondrial proteostasis and its further implications.

Reversible oxidation of methionine plays a key role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can obliterate protein functionality. Methionine sulfoxide reductases (MSRs) reduces back oxidized methionine in proteins, thus restores their function. Deletion and mutation in MSRs lead to several age-related human diseases and abrogates non-fermentable growth in yeast. But there is a lack of knowledge about their physiological substrates. In this study, we show that, Mxr2 interacts with Atg19 and Ape1, two vital proteins of a specific autophagy pathway, Cvt pathway and protects them from early degradation. Further, we found that Mxr2 specifically involved in the early stage of Cvt pathway as it interacts with premature Ape1 and Atg19. By identifying Atg19 and Ape1 as substrates of Mxr2, our study provides much deeper insights into the physiological importance of Mxr2 and its probable role in autophagy pathways.

The actin-based motor myosin 19 (Myo19) localizes to mitochondria and regulates their intracellular distribution and inheritance during cytokinesis. The outer mitochondrial membrane proteins Miro1/2 interact with Myo19 and stabilize it. In absence of Myo19, mitochondrial cristae architecture is disturbed with impaired OXPHOS. Cristae structure is regulated by the MICOS complex- part of Mitochondrial Intermembrane Bridging (MIB) supercomplex which connects the outer and inner membranes of mitochondria. A Turbo-ID screen with Myo19 demonstrated proximity biotinylation of metaxin-3 (Mtx3) in addition to Miro1/2. Immunoprecipitation showed that Mtx3 and Mic60, a MICOS component, co-precipitate with Myo19 supporting its linkage to the MIB complex. To elucidate further how Myo19 couples to the inner mitochondrial membrane, we generated Miro1/2KO cells to assess their potential involvement in connecting Myo19 to the MIB complex. In summary, Myo19 transduces directed force produced along actin filaments collectively to the outer and inner membranes of mitochondria.

Ion fluxes across the inner mitochondrial membrane control mitochondrial volume, energy production, and apoptosis. TMBIM5, a highly-conserved protein with homology to putative pH-dependent ion channels, is involved in the maintenance of mitochondrial cristae architecture, ATP production, and apoptosis. To identify the in vivo consequences of TMBIM5 dysfunction, we generated mice carrying a mutation in the channel pore and a knockout fly. Mutant mice display increased embryonic or perinatal lethality and a skeletal myopathy which strongly correlates with tissue-specific disruption of cristae architecture, early opening of the mitochondrial permeability transition pore, reduced calcium uptake capability, and mitochondrial swelling. Knockout flies have a reduced lifespan and ATP production. Using the fly model, we interrogated the interaction with the mitochondrial Ca²⁺ machinery. Our results demonstrate that TMBIM5 is an essential and important part of the mitochondrial ion transport system machinery with particular importance for embryonic development and muscle function.

The heavy chain of ferritin (FTH) plays a vital non-redundant role and its constitutive deletion is embryonically lethal. We found that Tamoxifen-inducible deletion of Fth in adult mice compromises mitochondrial function leading to impaired organismal energy homeostasis, and is associated with liver damage, heart failure, loss of thermoregulation, depletion of white adipose tissue, and death. We report that Fth-deleted mice are rescued by circulating FTH-competent bone marrow-derived macrophages. Reconstitution of lethally irradiated FTH-deficient mice with FTH-competent bone marrow restored organismal energy expenditure and prevented death. This was associated with improved mitochondrial function, restoring liver and heart function as well as thermoregulation and white adipose tissue. RNAseq analysis of rescuing macrophages revealed the induction of many mitochondrial genes which were not expressed in FTH-deficient macrophages, suggesting that FTH-competent macrophages restore homeostasis in Fth-deleted mice via a mechanism associated with mitochondrial biogenesis and arguably mediated via mitochondria transfer to stressed tissues.

Loss of mitochondrial homeostasis is well documented in neurodegenerative conditions, and depends on the pathological decline in the proteolytic activity of the ubiquitin-proteasome and the lysosome-autophagy system. Indeed, promoting proteasome or autophagy activity increases lifespan, and rescues the pathological phenotype of animal models of neurodegeneration presumably by enhancing the degradation of misfolded proteins and dysfunctional organelles, which are known to accumulate in these models. While many studies investigate the effect of potentiating proteostasis to scavenge intracytoplasmic neurotoxic aggregates, very little attention has yet been paid to explore the potential link between alteration in mitochondrial homeostasis and (in)stability of core components of the circadian clock. Thus, we exploited two drosophila models of neurodegeneration, and investigated the beneficial effect of enhancing proteostasis on circadian rhythmicity. We found that inhibition of USP14, which is known to enhance proteasome and mitophagy activity, ameliorates sleep disturbances and circadian defects in these models of neurodegeneration.

Prohibitins (PHB) form a multimeric structure at the mitochondrial inner membrane. PHB deficiency shortens the lifespan of wild type Caenorhabditis elegans nematodes, but dramatically extends that of insulin signalling receptor (daf-2) mutants. This phenotype is accompanied by a differential induction of the mitochondrial Unfolded Protein Response (UPRmt) that is attenuated in daf-2 mutants. We identified Heterochromatin Protein Like 1 (HPL-1) as a new regulator of the UPRmt and mediator of the opposing longevity phenotype caused by PHB depletion. We report functional and structural impairments of mitochondria when HP1 is depleted from worm and human cells, showing a conservation of function. We uncovered ~70% of differently bound genes by HPL-1 upon mitochondrial stress and determined HPL-1-dependent tissue-specific alterations in gene expression in hypodermal cells. Our data shows for the first time a role for HP1 proteins in controlling gene expression in response to mitochondrial dysfunction to modulate lifespan.

Nonyl acridine orange (NAO) is a fluorescent molecule widely used for mitochondrial staining due to its affinity for cardiolipin (CL). At micromolar concentrations, NAO has the ability to stack into antiparallel H-aggregates that promote the membrane remodeling of mitochondria eventually triggering cell apoptosis. To understand the cytotoxic mechanism of NAO, we use giant unilamellar vesicles as mitochondrial membrane models and incubate with NAO and derivatives. We report that NAO promote membrane fusion of CL-containing vesicles. To unveil the molecular mechanism of membrane fusion, we run coarse-grained molecular simulations under MARTINI3 framework. Simulations revealed that NAO act as lipid-hooks providing a hydrophobic environment for lipid nucleation at the intermembrane space. To achieve this process, tertiary amine arms and hydrocarbonated tail play a crucial role, as seen for different acridine derivatives. This remodelling allows the contact between apposing bilayers yielding to a hemifusion state, essential for full fusion of membranes.

Cbs is important for mitochondrial function through its rate-limitation of the transsulfuration pathway and production of H₂S. H₂S plays multiple roles, e.g. as gasotransmitter, antioxidant and most importantly mitochondrial modulator.
To study the local protective role of Cbs/H₂S in the development of liver-disease, we have created a liver-specific Cbs^-/- mouse and investigated its response to high-fat, high-cholesterol diet(HFD).

Cbs^-/- rendered mice susceptible to and exacerbated liver-disease in mice on HFD. Fatty-acid metabolism/synthesis were strongly dysregulated in Cbs^-/- (down-regulation of mRNA of FAS, HmgCoa, SCD-1, ACC-1 and SREBP-1C and ABC-G5; upregulation of SR-B1) with inhibition of FA shuttling through reduced acyl-carnitines. Disease progression was associated with increased MitoRos and decreased glutathione. FAO and ETC were unaffected, beta oxidation was fully functional (respirometry). Similarly, ER-mitochondrial interaction was unaffected.

Taken together, development of fatty-liver-disease and lipid dysregulation in Cbs-/- are associated with increased MitoRos, rather than dysfunctional beta oxidation and ETC-complexes.

Ovarian clear cell carcinoma (OCCC) patients respond poorly to platinum-based therapy owing to intrinsic chemo-resistance. There is therefore an urgent need to develop alternative therapeutic strategies for OCCC. In this study, we found that OCCC exhibited profound dependence on the amino acid cysteine for survival and the modes of cell death induced by cysteine deprivation in OCCC are determined by their innate metabolic profiles. Cysteine-deprived glycolytic OCCC is abolished primarily by oxidative stress-dependent necrosis and ferroptosis, which can otherwise be prevented by pretreatment with antioxidative agents. However, in respiring OCCC, cellular cytotoxicity was a result of the impediment of iron-sulfur cluster (Fe-S) synthesis in the mitochondria. Respiring OCCC responds to Fe-S cluster deficit by increasing iron influx into the mitochondria, which leads to iron overload, mitochondria damage, and eventual cell death. This study highlights the importance of cysteine availability in OCCC growth and presents cysteine limitation as a potential therapeutic strategy for OCCC.

Mitochondrial contact with the endoplasmic reticulum (ER) influences numerous cellular processes such as mitochondrial fission and fusion, mitophagy, and calcium signaling. Dysfunction of these contact sites is implicated in several disease processes such as type II diabetes and Parkinson’s disease. While current open-source applications can provide colocalization measurements between two subcellular structures, these methods offer limited insight into the details of these contact sites. Here, we present a new open-source data analysis pipeline, MitER, which couples the three-dimensional rendering capabilities of Mitograph with the powerful animation software Blender to allow for detailed quantitative analysis of mitochondrial-ER contact in addition to organelle morphology and distribution. Using MitER, we found increased mitochondrial surface area, mitochondrial-ER contacts, and mitochondrial symmetry as Saccharomyces cerevisiae cells switch from fermentation to respiration. This novel method addresses the growing need for tools to analyze inter-organelle contacts in addition to mitochondrial morphology and distribution.

Cells control the size and function of the mitochondrial network through their conserved ability to degrade mitochondria in a targeted manner by selective autophagy (mitophagy). We have started to dissect MRS networks for functional redundancy and specialization to define how different MRS may contribute to size and quality control of mitochondrial networks in response to inherent physiological signals.

We have discovered that the membrane-bound mitophagy receptor Atg32 mediates a homeostatic form of mitophagy, which scales mitochondrial networks in non-dividing yeast cells during starvation. Importantly, KD of the mammalian Atg32 homologue Bcl2l13 significantly increases mitochondria network size in MEFs, supporting an evolutionarily conserved function of Atg32/Bcl213-mediated mitophagy in size control. Intriguingly, in contrast to yeast, Bcl2l13 KD MEFs display elevated mitophagy levels, suggesting induction of compensatory mitophagy. 

Our systematic analysis across mammalian MRS points towards a clear specialization of the two classes of MRS with significant cooperative behavior.

We have recently described a new class of small molecule activators of the mitochondrial protease ClpP (“TR compounds”), demonstrating their ability to inhibit triple-negative breast cancer cell growth at greater potency than the related compound ONC201. One selected compound (TR-107) demonstrated ClpP-dependent reduction of mitochondrial proteins (including OXPHOS and TCA cycle components), and Seahorse XF analysis confirmed inactivation of OXPHOS and increased glycolysis following TR-107 treatment. Pharmacokinetic properties of TR-107 were investigated and compared to other known ClpP activators (e.g. ONC201, ONC212). TR-107 showed excellent exposure and serum t_1/2 following oral administration. MDA-MB-231 xenografts were used to investigate the anti-tumor response of TR-107 in vivo, and demonstrated reduced tumor volume and extension of survival in TR-107 treated mice. In summary, we have identified highly potent ClpP agonists with improved efficacy against TNBC through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

We have previously described the mechanism by which complex I deactivation can be transduced into superoxide production during the first minutes of hypoxia, through mitochondrial sodium/calcium/lithium exchanger (NCLX) activation and, as a consequence, mitochondrial Na⁺ import that affects oxidative phosphorylation by altering coenzyme Q diffusion (Hernansanz-Agustín et al., Nature 2020 586:287). It is known that NCLX can transport Li⁺ instead of Na⁺. Thus, we wondered what would be the effect of lithium in acute hypoxia. We have shown that the presence of lithium increases ROS production in normoxia and inhibits the superoxide burst production induced by acute hypoxia. Also, lithium ions do not alter the fluidity of the inner mitochondrial membrane in normoxia and hypoxia. However, lithium induces a slightly break of the mitochondrial network, affecting the maximal respiratory capacity. Thus, lithium can alter the effect of NCLX-mediated Na⁺ import into mitochondria, putatively changing acute response to hypoxia.

Iron represents a crucial biological catalyst required for cell replication and metabolism. Furthermore, a relationship between iron and cancer has been evident due to increased requirement of iron for rapidly proliferating cells. Iron chelation has shown anti-cancer effect in preclinical experiments. Therefore, we designed a mitochondrially targeted derivative of the iron chelator deferasirox (mitoDFX), which shows robust cytostatic and cytotoxic effects in vitro at nM concentrations and significantly suppresses tumor growth and metastasis in vivo. The underlying molecular mechanism includes impairment of iron-containing enzymes resulting in induction of mitophagy, alterations in TCA cycle and amino acid metabolism, decrease of reduced and total glutathione, and induction of tumor suppressor protein NDRG1. Collectively, these results indicate the importance of mitochondrial iron metabolism for cancer cells and illustrate the novel concept of repurposing iron chelators via mitochondrial targeting to serve as anti-cancer agents. This work was supported by GAUK (1310420) and GACR (18-13103S).

Recent studies have indicated that the vertebrate mitogenomes are not as conserved as previously thought. Likewise, hundreds of mtDNA mutations associated with human pathologies have been mapped showing a possible relationship between these two features. Therefore, we performed a comparative genomic analysis of the 2831 vertebrate mitogenomes available in the NCBI database. By using a combination of bioinformatics methods, higher rearrangement rates per gene and taxonomic class with higher values observed in Actinopteri, Amphibia and Reptilia, and distinct hotspots in the vertebrate mitogenome were found. By comparing the mtDNA mutations associated with human pathologies available in the database such as Hypertrophic cardiomyopathy, different types of cancer, among others; overlaps in the same genetic regions mainly located in tRNAs and D-loop Region were observed. The methodology presented here, in addition to the explanation of the vertebrate mitogenome dynamics, postulates a possible evolutionary origin of fragile regions in the human mitogenome.

Bacterioruberin (BR) is a xanthophyll purified from Haloterrigena sp. SGH1, an extreme halophilic archaeon strain isolated from endolithic microbial consortia colonizing halites in the Atacama Desert, northern Chile. Previous results showed us that BR decreased the mitochondrial membrane potential (Dym) at non-toxic levels in THP-1 cells. This novel and previously unknown effect of archaeal carotenoid on human mitochondria prompted us to an in-depth exploration. Recently, we have evaluated the  antioxidant activity against Antimycin A-induced ROS in THP-1 cells using flow cytometry and confocal microscopy. We can now demonstrate that BR crosses the cellular membrane and has a strong intracellular antioxidant activity compared with Trolox, probably preventing ROS production by decreasing the Dym in human monocytes. BR stabilizes trimeric archaerhodopsins, proteins involved in archaeal light-driven proton pumping; then, we propose that BR stabilizes mitochondrial uncoupling proteins and activates its intrinsic activity, the proton leak in human mitochondria.

OGG1 is proposed to be a major DNA glycosylase in base excision repair pathway of mitochondria, removing 8-oxoguanine (8-oxoG), previously reported as the main oxidative mtDNA lesion. Our goal was to study the mechanistic detail of formation of single-strand breaks (SSBs) in presence of H₂O₂. Using long-read ultra-deep PacBio sequencing, we show in S1-nuclease treated mtDNA of CRISPR-Cas9 generated OGG1 knock-out HEK-293 cells after H₂O₂-treatment the presence of abundant SSBs, which then decrease after 24 hours. This was also the case in control HEK-293 cells. SSBs were likewise visible using Southern blotting in OGG1 knock-out cells upon H₂O₂-treatment, but were two-fold less upon KBrO₃-treatment. This suggests that the formation of SSBs is caused by direct attack of the hydroxyl radical (^•OH) on sugar phosphate backbone and that OGG1 is dispensable for repair of mtDNA lesions caused by H₂O₂. But OGG1 is indispensable for repair of 8-oxoG lesions.

An association between embryonic gastrulation and metabolic shifts in mitochondrial respiration and glycolysis was suggested 100 years ago. We seek to understand whether these shifts in metabolism are required for the onset of dynamic cell rearrangements at gastrulation, such as the migration of mesendoderm tissue. Monopolar directed protrusions in collectively migrating mesendoderm cells are regulated by cell-ECM traction forces and cell-cell adhesive stresses. Mitochondrial activity (TMRE staining) and clustering are greatest at the leading edge of collectively migrating mesendoderm explants. Distinct patterns of metabolic activity are observed in mesendoderm cells and tissue explants plated on fibronectin fusion protein substrates, which support distinct conformational states of α5β1 integrin. These data suggest that integrin activation and signaling are associated with changes in mitochondrial metabolism and organelle localization in leading row mesendoderm cells. Ongoing studies are exploring potential links between mitochondrial processes and the adhesive and mechanical signals involved in collective cell migration.

Genetic defects of mitochondrial oxidative phosphorylation (OxPhos) cause multi-system disorders and reduced lifespan. Recent work suggests OxPhos dysfunction induces transcript-level and hormonal stress signaling that increases whole-body energy expenditure, contributing to disease. Our meta-analysis investigating whole-body energy expenditure in mitochondrial disease (690 mitochondrial disease patients, 225 healthy controls) revealed that patients exhibit markers of elevated metabolic rate, including elevated VO²/kg body mass (+30%, p<0.0001) and lower BMI (-9.8%, p<0.05). Likewise, OxPhos-deficient (SURF1^Mut) patient-derived fibroblasts exhibited shorter lifespan (-53%, p<0.072) and greater total energy expenditure (+91%, p<0.001) compared to controls. RNAseq and DNA methylation data suggest specific cellular pathways for hypermetabolism, investigated through current analyses, and an ongoing human study is directly quantifying total and resting whole-body metabolism in patients with mitochondrial disease. Integrating in vitro and whole-body energy expenditure measurements can lead to mechanistic insights into the origin of evolved energetic constraints on organismal metabolism and influence on health processes.

Hypoxia increases histone methylation by inhibiting O2- and α-ketoglutarate-dependent histone lysine demethylases (KDMs). This study is the first to demonstrate how the hypoxic increment of methylated histones cross-talks with other epigenetic changes, such as histone clipping, and heterochromatin redistribution (senescence-associated heterochromatin foci, SAHF) found during oncogene-induced senescence (OIS). Raf activation in primary human fibroblasts IMR90 increased cathepsin L (CTSL)-mediated clipping of histone 3 (H3), H2B and H4. In addition, OIS and hypoxia oppositely changed the mitochondria contents. Thus, hypoxia protects chromatin features and mitochondria from dramatic changes during senescence. [This study has been supported by NRF-2019M3A9D5A01102794]

Little information exists on the mechanisms governing mitochondrial ubiquitination, including the activity of prototypical mitochondrial E3 Ub ligase MARCH5, under conditions where mitochondria are the primary source of ATP. We developed a model in which cells entirely rely on oxidative phosphorylation (OXPHOS) for ATP generation. The shift from high glucose glycolytic medium to OXPHOS-only growth conditions induces mitochondrial and peroxisomal biogenesis. While mitochondria were not affected by the knockout of MARCH5, peroxisomal biogenesis was stalled in MARCH5-/- cells. Imaging of ATP generation with cytosolic ATP sensor revealed inhibition of ATP generation in MARCH5-/- cells supplemented with lipid OXPHOS substrate palmitoyl-L-carnitine. We also identified a peroxisomal substrate of MARCH5-mediated ubiquitination that suggests that MARCH5 is a critical factor for lipid-induced peroxisome biogenesis.  The data indicate that MARCH5 serves as a metabolic sensor and its activity is critical for the utilization of lipids as the OXPHOS substrates through mitochondrial and peroxisomal pathways.

Background: “Oxidative stress” has often been suggested to be a secondary insult which underlies progression from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). To date, however, a molecular mechanism behind this has not been demonstrated. 

Hypothesis: Mitochondria and peroxisomes generate hydrogen peroxide as a side product of long chain fatty acid oxidation. Lipid overload induced dysregulation of mitochondrial or peroxisomal metabolism could therefore increase capacity for hepatic reactive oxygen species (ROS) generation in NASH, impairing metabolic processes, and leading to disease progression. 

Methods & Results: TEM, fluorescence microscopy and proteomics demonstrated greater peroxisome numbers, yet lower expression of the peroxisomal “antioxidant” enzyme catalase, in NASH. This was associated with enhanced ROS generation as measured by electron paramagnetic resonance. Novel transgenic probes for measuring peroxisomal and mitochondrial oxidation state further demonstrated greater mitochondrial and peroxisomal ROS production in both lipid-loaded hepatocytes and precision-cut NASH liver slices. A newly developed proteomic technique for determining cysteine oxidation sites showed disproportionate oxidation of key mitochondrial and peroxisomal metabolism related proteins in NASH. Adenoviral reintroduction of catalase to NASH livers lowered ROS generation and hepatic lipid accumulation in vitro and in vivo.

Insights: Differential peroxisome proliferation and mitochondrial dysregulation in NASH underlies increased ROS production, impairment of fat metabolism enzymes, and disease progression.

The electronegative subfraction of low-density lipoprotein (L5-LDL) is highly atherogenic and could be a novel biomarker for atherosclerotic cardiovascular disease. L5-LDL is not recognized by LDL receptors. Instead, it damages endothelial cells (ECs) through lectin-like oxidized LDL receptor-1 (LOX-1). In the present study, we aimed to investigate the intracellular trafficking of L5-LDL and its after-effects on mitochondrial dysfunction and cell apoptosis. Human aortic endothelial cells (HAECs) were challenged with control or L5-LDL. By confocal microscopy, we demonstrated that L5-LDL colocalized with mitochondria. Furthermore, L5-LDL-induced mitochondrial fission was observed by electron microscopy. The mitochondrial fusion protein mitofusin (MFN1/2) and mitochondrial dynamin-like GTPase (Opa1) were expressed downregulated. Manganese superoxide dismutase (MnSOD) was significantly overexpressed; however, the functions of MnSOD need further investigation. Besides, DNA was fragmented during the treatment of L5-LDL for 24 hours. In summary, L5-LDL can be trafficked into mitochondria through LOX-1 and cause mitochondrial impairment, leading to endothelial dysfunction.

The most electronegative subfraction of low-density lipoprotein (L5-LDL) is atherogenic and highly elevated in patients with cardiometabolic diseases. The well-known chemical feature of L5-LDL is apolipoprotein E (apoE) glycosylation. However, detailed mechanisms of L5-LDL formation remain unclear. We aimed to investigate where and how it originates. V5-VLDL and L5-LDL were isolated from patients with metabolic syndrome. ApoE glycosylation and lipid components were analyzed by liquid chromatography/mass spectrometry (LC/MS^E). Glycosylation mechanisms were investigated in vitro and livers of leptin-deficient (ob/ob) mice. Results showed that lysophosphatidylcholines (LPCs) were elevated in the livers of ob/ob mice. LPC induced apoE glycosylation and upregulated the expression of glycosylation-associated enzymes, polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2) and ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1). Co-treatment with hypoxia-inducible factor-1α (HIF-1α) inhibitor can attenuated the LPC effects on apoE glycosylation. In summary, we suggest LPC involves the mechanism of L5-LDL formation through enhancing HIF-1α signaling.

Our understanding of mitochondrial respiration extends to its connection with key metabolic processes such as ATP production, redox signaling and ion homeostasis. Rendering respiration fundamental, high-resolution respirometry coupled with the simultaneous measurement of other mitochondrial parameters (e.g., mitochondrial membrane potential) has provided novel insights into the role of mitochondria in cell metabolism. For example, a recent paper [Flockhart et al., Cell Metab 33:957-970 (2021)] combined respirometry with the AmplexRed assay to link excessive exercise training with muscle mitochondrial impairment and reduced glucose tolerance in humans ― only to be criticized for the use of isolated mitochondria for respirometry [Hawley & Bishop Nat Rev Endocrinol 7:385-386 (2021)]. Arguments against isolated mitochondria centered around a premise that they do not accurately reflect mitochondrial function in vivo and that using permeabilized fibers would be a better approach. Directly addressing this topic, here, we show that mitochondrial respiratory capacities are quantitatively comparable when assessed in properly prepared permeabilized fibers, isolated mitochondria, and tissue homogenate. Using beef heart permeabilized muscle fibers, tissue homogenate, and isolated mitochondria, we assessed respiratory capacity in different pathway and coupling control states. Relative respiratory flux control ratio profiles were similar in the three preparations, indicating preserved functional quality. Furthermore, in permeabilized fibers and tissue homogenates prepared from mouse heart the respiratory capacities normalized to tissue wet mass were nearly identical. We conclude that isolated mitochondria, tissue homogenate, and permeabilized fibers are all appropriate for the assessment of mitochondrial respiratory capacities ex vivo. The most appropriate depends on the specific experimental question being investigated and additional mitochondrial parameters being measured simultaneously. We anticipate that our findings will be a reference point for researchers when choosing mitochondrial preparations to address their specific scientific question of interest.

To save energy and protect the cell from the aggregation of nascent hydrophobic proteins, evolution developed a controlled system to regulate translation in response to stress. Hence, when cells are facing reactive oxygen species (ROS) or other mitochondria-associated stresses, the energy-consuming process of translation is attenuated. Protein synthesis is shut down quickly and recovered efficiently within a short time window upon stress relief. Still, it remains elusive what are the alternations in the nascent proteome when global translation is attenuated and when reactivated. The translatome is highly dynamic and proteomic methods routinely used for nascent protein analysis often do not provide the sensitivity to detect the most dynamic part of the nascent proteome at a sufficiently high temporal resolution. To monitor translation in response to mitochondrial stress we first optimized conditions in which global translation in HEK293 cells is largely but not entirely inhibited by ROS or by brief uncoupling of the mitochondrial proton gradient - and rapidly recovered during the wash. Next, we used Biorthogonal Non-Canonical Aminoacid tagging (BONCAT) to purify the nascently translated proteins at short intervals within the treatment and wash. Purified proteins were digested, azidohomoalanine-containing peptides enriched via affinity purification after click reaction with a biotinylating agent, and then labeled with tandem mass tags prior to mass spectrometry. Our data suggest that global translation inhibition is characterized by quick, temporary specific changes in the nascent proteome with up to half of the significantly regulated proteins being specific for a single assayed time window. We describe the widespread regulation of translation of ribosomal proteins as well as differential regulation of constituents of OXPHOS complexes and their assembly factors. The regulation of translation of poly-adenine binding proteins suggests that specific changes in the nascent proteome in response to stress may serve to fine-tune subsequent adaptive RNA metabolism during recovery.

Beyond glucose control T2D patients are at high risk of developing CVD. Mitochondrial dysfunction has been shown to drive CVD and is one of the hallmarks of T2D. We aimed to evaluate if non-invasive blood tests could be used to determine mitochondrial activity and be used for CVD risk assessment in T2D patients. We found that subjects with intima-media thickening of the caroid artery had lower PGC-1alpha and TFAM levels, increased correlation of PRX3 with MCAD and reduced with IL-4, along with increased ccgDNA, reduced mtDNA/gDNA ratio and increased deleted mtDNA fragments. Our data suggests that loss of mitochondrial plasticity in T2D can be evaluated in PBMCs from T2D patients and used for risk estratification.

Neurodegenerative diseases are often defined by a marked anatomical and cellular specificity. Among them, Amyotrophic Lateral Sclerosis (ALS) results from the selective loss of corticospinal motor neurons in the motor cortex and lower motor neurons of the spinal cord eventually leading to paralysis and death. Despite significant progress in identifying the genetic and molecular factors of ALS, little is known about the exact mechanisms that contribute to the selective vulnerability of motor neurons. A major anomaly that remains is many ALS-causing gene mutations are ubiquitously expressed throughout the body, yet only a subset of neurons is vulnerable. Mitochondrial dysfunction has emerged as a common and early phenomenon in both familial and sporadic ALS suggesting an important role for loss of mitochondrial integrity in the etiology of ALS. To date, studies of ALS-associated mitochondria dysfunction have relied on whole brain or regions of brain tissue for analysis. The usefulness of this information is limited given the cellular heterogeneity in the cortex which diminishes the ability to detect and distinguish meaningful changes in the relatively rare, corticospinal neurons. Here, we developed and applied a strategy to facilitate the isolation of mitochondria in a cell type specific manner. The use of our novel retroAAV-mediated approach, term TOM-TAG, for the cell type specific immunopurification of mitochondria by magnetic beads from whole tissue is a powerful tool for the rapid assessment of mitochondria across cell populations in complex tissues like the cortex. Using this strategy in combination with proteomic profiling and real-time metabolic analysis, we have identified stark differences in isolated mitochondria from ALS “resilient” versus “vulnerable” populations at baseline and in a relevant animal model. Taken together, our findings indicate intrinsic differences in mitochondrial physiology of these distinct neuronal populations that may contribute to disease susceptibility.

Years of chronic insult to the lungs result in excessive oxidative stress, mitochondrial dysfunction and senescence. These cellular states are implicated as drivers of accelerated aging diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The constant cellular stress response (increased protein synthesis for example) requires increased energy supply which leads to enhanced mitochondrial ROS production. In this situation mitochondrial DNA (mtDNA) damage can occur at much higher rate than that of nuclear DNA, resulting in defective transcription and a decline in mitochondrial function, leading in turn to enhanced ROS production and further damage to mtDNA. Therapeutic agents are required to enhance cellular oxidative stress response and reduce mitochondrial dysfunction  in COPD and IPF.

Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. Mitochondrial redox cycler MitoPQ produces superoxide at complex I, selectively increasing superoxide production within mitochondria. In this work, we studied the effects of oxidative stress and etoposide (a molecule that causes dsDNA breaks, precipitating cell cycle arrest and senescence) on mitochondrial morphology in human bronchial epithelial cells (HBECs) and alveolospheres (a iPSC-derived 3D in vitro model of human alveoli). Using confocal imaging combined with data analysis based on deep learning networks, we could show that senescence induction using etoposide was associated with hyperfused mitochondrial networks, whereas H2O2 treatment induced both mitochondrial fusion and fragmentation. High doses of MitoPQ caused severe fragmentation.

Overall, we have demonstrated that the oxidative stress and etoposide treatment affects mitochondrial networks in primary human lung cells and a 3D alveolar organoid model. These in vitro models can be used to explore mitochondrial function under stress conditions as well as response to pharmacological intervention.

Active absorption and secretion during plasma filtration and urine formation make renal tubular cells a highly energy-demanding cell type. Fatty acid oxidation (FAO) is the main source of tubular energy which is rapidly impaired during kidney injury without substantial compensatory glycolytic capacity. Lipid chaperons FABPs mediate lipid trafficking and lipid-mediated metabolic pathways, among them FAO. Although inhibition of induced FABPs is beneficial in several disease conditions, including kidney damage, their functional implication in renal metabolic homeostasis remains elusive. Therefore, we approached this issue in cultured renal tubular cells and kidney tissue. Kidneys from mice with AKI exhibited early lowered FABPs tubular expression. In serum-depleted murine tubular cells, pharmacological FABPs inhibition with BM309403 (BMS) led to necrosis, whereas, in non-depleted cells, BMS induced apoptosis, increased the mRNA expression of proinflammatory cytokines and oxidative stress-responsive genes, and restrained proliferation. BMS also rapidly decreased ATP levels, which correlated with increased mitochondrial oxidative stress and concomitant adaptive upregulation of FAO genes (although without further glycolytic gene expression changes) consistent with the observed AMPK phosphorylation/activation. Oil-red staining revealed ATGL-dependent progressive lipid droplet (LD) voiding in serum-depleted or BMS-treated cells. In pulse-chase and live confocal image assays, the fluorescent fatty acid RedC12 colocalized with BODIPY493/503-stained LDs in a serum-containing medium, while it colocalized only with mitotracker-stained mitochondria under serum depletion. In BMS-treated cells in a serum-containing medium, despite the lowered ATP content (and the transcriptional FAO upregulation), RedC12 neither localizes with LDs nor with mitochondria, suggesting a low-energy state by impairment of both lipid transit and FAO. Accordingly, TLC assessment of BMS-treated cells did not show lipid breakdown-derived products, which otherwise did appear in serum-depleted cells doing FAO.In brief, FABP inhibition disrupts tubular energetic status and favors cell stress. Thus, protecting FABP activity could help to preserve tubular metabolic homeostasis during early kidney damage.

Background: Our prior work in human cancer cell lines shows that, on a systems level, a cell’s requirement for respiratory and glycolytic genes depends on the metabolic substrate. However, the differential roles of glycolysis and respiratory metabolism in supporting tumor growth and metastases formation in vivo remain poorly understood.  Method: To determine how metabolic substrate and local environment impact the requirement for respiratory chain genes in vitro and in vivo, we performed parallel functional screens in multiple human lung cancer cell lines expressing a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory) impacting ATP. Individual sgRNAs associated with significant growth effects were analyzed using in vitro and in vivo metabolomics and RNA Seq, and then validated in an orthotopic xenograft model of lung cancer that develops metastases.  Results: While knockdown of mitochondrial ribosomal protein and respiratory chain genes had little impact on growth in vitro, silencing these genes significantly decreased growth of the same cells grown as either flank or primary orthotopic lung tumor xenografts.  RNA-Seq and metabolomics analysis showed that knockdown of mito-respiratory genes led to overexpression of glycolytic genes and increased sensitivity to glycolytic inhibition in vitro. While in vivo these cells demonstrated decreased primary tumor growth, metastasis formation was maintained or even increased compared to controls.  Conclusion: Respiration and ATP production may enable tumor cells to survive metabolically-driven selection as they grow in diverse in vivo contexts.  This suggests that metastases may be metabolically targetable on the basis of locally-shaped dependencies.

Microbiomes have a profound impact on animal physiology including mitochondrial homeostasis that is linked to major human diseases, but our knowledge regarding functions associated with individual microbial metabolites is limited. Using sensitive genetic assays, we discovered unexpected beneficial roles of certain peptidoglycan (PG) fragments and the siderophore enterobactin, both known for their involvement in pathogenicity and immune responses. Specifically, we showed that these PG fragments enter mitochondria in intestinal cells to repress mitochondrial and oxidative stress and support animal development. Mechanistically, PG binds to and promotes ATP synthase activity and this role as a rare ATP synthase agonist is conserved in mammals, presenting an important advance regarding microbe-host mitochondrial interactions. In the other project, we found that enterobactin promotes mitochondrial iron uptake in animals and does so through a novel mechanism involving the ATP synthase α subunit. Our recent data in mammals indicate potential therapeutic usage to treat iron deficiency anemia.

Reversible acetylation of mitochondrial proteins is a regulatory mechanism central to adaptive metabolic responses. Yet, how the specificity for functionally relevant protein acetylation in mitochondria is achieved remains unexplored. Here, we report an unprecedented role of the MYST lysine acetyltransferase MOF, an epigenetic regulator, in functional mitochondrial protein acetylation. Loss of MOF-KANSL complex members leads to impaired mitochondrial ultrastructure, respiratory supercomplex assembly and complex IV (CIV) activity. We identify COX17, a CIV assembly factor, as a prime mitochondrial acetylation target of MOF. COX17 acetylation is sufficient to restore mitochondrial defects observed upon MOF loss. Fibroblasts derived from MOF syndrome patients manifest metabolic catastrophe associated with CIV dysfunction irrespective of MOF-driven histone H4 lysine 16 acetylation. Impaired CIV activity is restored by ectopic expression of Ciona intestinalis alternative oxidase, highlighting mtETC cytochrome segment as an evolutionary conserved target. Thus, MOF-KANSL complex emerges as an epimetabolic regulator of mitochondrial integrity in mammals.

Ferroptosis is an iron-dependent inflammatory cell death modality associated with lipid peroxidation. Glutathione peroxidase 4 and coenzyme Q (CoQ) oxidoreductase FsP1 are part of the two cellular pathways which limit lipid peroxidation and confer ferroptotic resistance. CoQ is a hydrophobic redox active lipid that is synthesized in the mitochondria and is exported from mitochondria in order to limit lipid peroxidation in the plasma membrane. However, the mechanism of CoQ distribution in the cell is not known. In this study, we have identified the lipid transfer protein STARD7 as a novel regulator of intracellular CoQ distribution. STARD7 processing by rhomboid protease PARL during mitochondrial import allows kinetic partitioning and dual localization of STARD7 to the cytosol and to mitochondria. We show that mitochondrial STARD7 is required for CoQ biosynthesis, whereas cytosolic-STARD7 is necessary for CoQ transport from the mitochondria to the plasma membrane and serves as a ferroptosis suppressor.