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Time lapse showing cell aggregation during neurosphere formation in control condition.

Supplementary figures providing additionnal information concerning: - The cells glycome before and after PST3.1a treatment as measured by mass spectrometry (Supp. Figure 1) or Glycoprofile (Supp. Figure 2) - The absence of effect of PST3.1a on O-Glycosylation (Supp. Figure 3) - Western blot quantifications (Supp. Figure 4) - The inhibition of cell invasion in matrigel by PST3.1a (Supp. Figure 5) - The absence of cytotoxicity on non-proliferating PBMC cells (Supp. Figure 6).

Time lapse showing cell aggregation during neurosphere formation in PST3.1a-treated condition. In this condition, less cell aggregation is visible as compared to the control condition.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and accounts for a significant proportion of all primary brain tumors. Median survival after treatment is around 15 months. Remodeling of N-glycans by the N-acetylglucosamine glycosyltransferase (MGAT5) regulates tumoral development. Here, perturbation of MGAT5 enzymatic activity by the small-molecule inhibitor 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST3.1a) restrains GBM growth. In cell-based assays, it is demonstrated that PST3.1a alters the β1,6-GlcNAc N-glycans of GBM-initiating cells (GIC) by inhibiting MGAT5 enzymatic activity, resulting in the inhibition of TGFβR and FAK signaling associated with doublecortin (DCX) upregulation and increase oligodendrocyte lineage transcription factor 2 (OLIG2) expression. PST3.1a thus affects microtubule and microfilament integrity of GBM stem cells, leading to the inhibition of GIC proliferation, migration, invasiveness, and clonogenic capacities. Orthotopic graft models of GIC revealed that PST3.1a treatment leads to a drastic reduction of invasive and proliferative capacity and to an increase in overall survival relative to standard temozolomide therapy. Finally, bioinformatics analyses exposed that PST3.1a cytotoxic activity is positively correlated with the expression of genes of the epithelial–mesenchymal transition (EMT), while the expression of mitochondrial genes correlated negatively with cell sensitivity to the compound. These data demonstrate the relevance of targeting MGAT5, with a novel anti-invasive chemotherapy, to limit glioblastoma stem cell invasion. Mol Cancer Res; 15(10); 1376–87. ©2017 AACR.

PDF file - 86KB, A, Mitochondrial cross-section area bins after saline or DOX injection (top and bottom panels, respectively). A total of 250 mitochondria from each condition were analyzed from 5 electrons micrographs taken at 3000 X magnifications in 4 to 6 different sections. Two animals were used for each condition. B, Electron microscopy quantification of mitochondria numbers in heart right ventricle after saline or DOX injections. Five electron micrographs taken at 3000 X magnification in 4 to 6 different sections. Two representative animals were used for each condition. Between 1500 to 2700 mitochondria were counted.

Purpose: Doxorubicin is one of the most effective chemotherapeutic agents. However, up to 30% of the patients treated with doxorubicin suffer from congestive heart failure. The mechanism of doxorubicin cardiotoxicity is likely multifactorial and most importantly, the genetic factors predisposing to doxorubicin cardiotoxicity are unknown. On the basis of the fact that mtDNA lesions and mitochondrial dysfunctions have been found in human hearts exposed to doxorubicin and that mitochondrial topoisomerase 1 (Top1mt) specifically controls mtDNA homeostasis, we hypothesized that Top1mt knockout (KO) mice might exhibit hypersensitivity to doxorubicin.Experimental Design: Wild-type (WT) and KO Top1mt mice were treated once a week with 4 mg/kg doxorubicin for 8 weeks. Heart tissues were analyzed one week after the last treatment.Results: Genetic inactivation of Top1mt in mice accentuates mtDNA copy number loss and mtDNA damage in heart tissue following doxorubicin treatment. Top1mt KO mice also fail to maintain respiratory chain protein production and mitochondrial cristae ultrastructure organization. These mitochondrial defects result in decreased O2 consumption, increased reactive oxygen species production, and enhanced heart muscle damage in animals treated with doxorubicin. Accordingly, Top1mt KO mice die within 45 days after the last doxorubicin injection, whereas the WT mice survive.Conclusions: Our results provide evidence that Top1mt, which is conserved across vertebrates, is critical for cardiac tolerance to doxorubicin and adaptive response to doxorubicin cardiotoxicity. They also suggest the potential of Top1mt single-nucleotide polymorphisms testing to investigate patient susceptibility to doxorubicin-induced cardiotoxicity. Clin Cancer Res; 20(18); 4873–81. ©2014 AACR.

PDF file - 43KB, A, Histograms show Top2B expression levels quantified by Western blotting and the right panel shows representative Western blotting images. B, Quantification of TOP2B mRNA levels in heart tissue from WT and Top1mt KO mice.

PDF file - 118KB, A, Representative Western blots of respiratory chain proteins from skeletal muscle (left panel) and heart tissue (right panel) after saline or DOX injections. Animals from similar litters were used for each condition. B, Complex IV, III, I protein quantification from Western blot of heart tissues. n = 5, *, p

PDF file - 108KB, Distribution of the mutations along the Top1mt polypeptide. Red: Deleterious missense variants. Blue: Essential amino acids (aa) for Top1mt activity. Green: High frequent missense variants.

Impaired spermatogenesis and male infertility are common manifestations of mitochondrial diseases, but the underlying mechanisms are unclear. Here we show that mice deficient for PARL, the mitochondrial rhomboid protease, a recently reported model of Leigh syndrome, develop postpubertal testicular atrophy caused by arrested spermatogenesis and germ cell death independently of neurodegeneration. Genetic modifications of PINK1, PGAM5, and TTC19, three major substrates of PARL with important roles in mitochondrial homeostasis, do not reproduce or modify this phenotype. PARL deficiency in testis mitochondria leads to severe mitochondrial electron transfer chain defects, alterations in Coenzyme Q biosynthesis and redox status, and abrogates GPX4 expression specifically in spermatocytes leading to massive ferroptosis, an iron-dependent regulated cell death modality characterized by uncontrolled lipid peroxidation. Thus, mitochondrial defects can initiate ferroptosisin vivoin specific cell types by simultaneous effects on GPX4 and Coenzyme Q. These results highlight the importance of ferroptosis and cell-type specific downstream responses to mitochondrial deficits with respect to specific manifestations of mitochondrial diseases.

Cancer/Testis Antigens (CTAs) represent a group of proteins whose expression under physiological conditions is restricted to testis but activated in many human cancers. Also, it was observed that co-expression of multiple CTAs worsens the patient prognosis. Five CTAs were reported acting in mitochondria and we recently reported 147 transcripts encoded by 67 CTAs encoding for proteins potentially targeted to mitochondria. Among them, we identified the two isoforms encoded by CT55 for whom the function is poorly understood. First, we found that patients with tumors expressing wild-type CT55 are associated with poor survival. Moreover, CT55 silencing decreases dramatically cell proliferation. Second, to investigate the role of CT55 on mitochondria, we first show that CT55 is localized to both mitochondria and endoplasmic reticulum (ER) due to the presence of an ambiguous N-terminal targeting signal. Then, we show that CT55 silencing decreases mtDNA copy number and delays mtDNA recovery after an acute depletion. Moreover, demethylation of CT55 promotor increases its expression, which in turn increases mtDNA copy number. Finally, we measured the mtDNA copy number in NCI-60 cell lines and screened for genes whose expression is strongly correlated to mtDNA amount. We identified CT55 as the second highest correlated hit. Also, we show that compared to siRNA scrambled control (siCtrl) treatment, CT55 specific siRNA (siCT55) treatment down-regulates aerobic respiration, indicating that CT55 sustains mitochondrial respiration. Altogether, these data show for first time that CT55 acts on mtDNA copy number, modulates mitochondrial activity to sustain cancer cell proliferation.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells., Graphical Abstract Graphical Abstract, By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, Charif et al. report 61 new cases harbouring variants in ACO2, among whom 50 with dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy.

Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.

International audience; Anticipating very long space trips, da Silveira et al. performed pan-omic analyses on in-flight samples from astronauts, mice, and cells. Results revealed major mitochondrial dysfunctions responsible for alterations in metabolism, immunity, and circadian rhythm, which should prompt the evaluation of countermeasures to reduce the risks of future space odysseys, especially toward the planet Mars.

International audience; Cancer/Testis Antigens (CTAs) genes are expressed only during spermatogenesis and tumorigenesis. Both processes share common specific metabolic adaptation related to energy supply, with a glucose to lactate gradient, leading to changes in mitochondrial physiology paralleling CTAs expression. In this review, we address the role of CTAs in mitochondria (mitoCTAs), by reviewing all published data, and assessing the putative localization of CTAs by screening for the presence of a mitochondrial targeting sequence (MTS). We evidenced that among the 276 CTAs, five were already shown to interfere with mitochondrial activities and 67 display a potential MTS.

Mitochondria house anabolic and catabolic processes that must be balanced and adjusted to meet cellular demands. The RNA‐binding protein CLUH (clustered mitochondria homolog) binds mRNAs of nuclear‐encoded mitochondrial proteins and is highly expressed in the liver, where it regulates metabolic plasticity. Here, we show that in primary hepatocytes, CLUH coalesces in specific ribonucleoprotein particles that define the translational fate of target mRNAs, such as Pcx, Hadha, and Hmgcs2, to match nutrient availability. Moreover, CLUH granules play signaling roles, by recruiting mTOR kinase and the RNA‐binding proteins G3BP1 and G3BP2. Upon starvation, CLUH regulates translation of Hmgcs2, involved in ketogenesis, inhibits mTORC1 activation and mitochondrial anabolic pathways, and promotes mitochondrial turnover, thus allowing efficient reprograming of metabolic function. In the absence of CLUH, a mitophagy block causes mitochondrial clustering that is rescued by rapamycin treatment or depletion of G3BP1 and G3BP2. Our data demonstrate that metabolic adaptation of liver mitochondria to nutrient availability depends on a compartmentalized CLUH‐dependent post‐transcriptional mechanism that controls both mTORC1 and G3BP signaling and ensures survival., CLUH‐containing ribonucleoprotein particles promote turnover of mitochondria and metabolic rewiring in murine hepatocytes to ensure cell survival upon starvation.

International audience; Angiogenesis is a complex process leading to the growth of new blood vessels from existing vasculature, triggered by local proangiogenic factors such as VEGF. An excess of angiogenesis is a recurrent feature of various pathologic conditions such as tumor growth. Phostines are a family of synthetic glycomimetic compounds that exhibit anticancer properties, and the lead compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST 3.1a) shows antiglioblastoma properties both in vitro and in vivo. In the present study, we assessed the effect of PST 3.1a on angiogenesis and endothelial metabolism. In vitro, PST 3.1a (10 µM) inhibited all steps that regulate angiogenesis, including migration, proliferation, adhesion, and tube formation. In vivo, PST 3.1a reduced intersegmental vessel formation and vascularization of the subintestinal plexus in zebrafish embryos and also altered pathologic angiogenesis and glioblastoma progression in vivo. Mechanistically, PST 3.1a altered interaction of VEGF receptor 2 and glycosylation-regulating protein galectin-1, a key component regulating angiogenesis associated with tumor resistance. Thus, these data show that use of PST 3.1a is an innovative approach to target angiogenesis.-Bousseau, S., Marchand, M., Soleti, R., Vergori, L., Hilairet, G., Recoquillon, S., Le Mao, M., Gueguen, N., Khiati, S., Clarion, L., Bakalara, N., Martinez, M. C., Germain, S., Lenaers, G., Andriantsitohaina, R. Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Mitochondrial topoisomerase IB (TOP1MT) is a nuclear-encoded topoisomerase, exclusively localized to mitochondria, which resolves topological stress generated during mtDNA replication and transcription. Here, we report that TOP1MT is overexpressed in cancer tissues and demonstrate that TOP1MT deficiency attenuates tumor growth in human and mouse models of colon and liver cancer. Due to their mitochondrial dysfunction, TOP1MT-KO cells become addicted to glycolysis, which limits synthetic building blocks and energy supply required for the proliferation of cancer cells in a nutrient-deprived tumor microenvironment. Mechanistically, we show that TOP1MT associates with mitoribosomal subunits, ensuring optimal mitochondrial translation and assembly of oxidative phosphorylation complexes that are critical for sustaining tumor growth. The TOP1MT genomic signature profile, based on Top1mt-KO liver cancers, is correlated with enhanced survival of hepatocellular carcinoma patients. Our results highlight the importance of TOP1MT for tumor development, providing a potential rationale to develop TOP1MT-targeted drugs as anticancer therapies., TOP1MT is a topoisomerase that is localised to mitochondria. Here, the authors show that TOP1MT has a tumor promoting role in hepatocellular carcinoma by supporting mitochondrial translation and that its deficiency limits tumorigenicity.

Pseudoxanthoma elasticum (PXE) is a rare genetic condition primarily caused by hepatic ABCC6 transporter dysfunction. Most clinical manifestations of PXE are due to premature calcification of elastic fibers. However, the vascular impact of PXE is pleiotropic and remains ill defined. ABCC6 expression has recently been associated with cellular nucleotide export. We studied the impact of ABCC6 deficiency on blood levels of aden-osine triphosphate and related metabolites and on soluble nucleotidase activities in PXE patients and Abcc6(−/−) mice. In addition, we investigated the expression of genes encoding ectocellular purinergic signaling proteins in mouse liver and aorta. Plasma adenosine triphosphate and pyrophosphate levels were significantly reduced in PXE patients and in Abcc6(−/−) mice, whereas adenosine concentration was not modified. Moreover, 5′-nucleotidase/CD73 activity was increased in the serum of PXE patients and Abcc6(−/−) mice. Consistent with alterations of purinergic signaling, the expression of genes involved in purine and phosphate transport/metabolism was dramatically modified in Abcc6(−/−) mouse aorta, with much less impact on the liver. ABCC6 deficiency causes impaired vascular homeostasis and tissue perfusion. Our findings suggest that these alterations are linked to changes in extracellular nucleotide metabolism that are remote from the liver. This opens new perspectives for the understanding of PXE pathophysiology.

Introduction It has been suggested that mitochondria play a crucial role in sustaining pregnancy and foetal growth. The aim of the study was to assess the influence of mitochondrial functions and genetics on placental insufficiency diseases. Methods A total of 115 patients were recruited, subdivided into 74 placenta samples and 41 maternal blood samples: placental insufficiency diseases including intra uterine growth restriction (IUGR) (n = 35), preeclampsia (PE) (n = 13), IUGR associated to PE (PER) (n = 25); and controls (n = 42). Haplogroups were determined for all patients. Eighty-six placenta samples were studied for quantitative and qualitative analyses of mtDNA: IUGR (n = 25), PE (n = 1), PER (n = 18) and controls (n = 42). Sixteen placenta samples were selected for functional analysis: IUGR (n = 6), PER (n = 2) and controls (n = 8). Results Mitochondrial DNA copy numbers and rearrangements and haplogroup distribution were not significantly altered in the patient group. Enzyme activity and expression of respiratory chain complexes were also comparable between both groups. Discussion Our results do not argue in favour of a mitochondrial involvement in placental insufficiency, suggesting that the glycolytic pathway may represent a key energetic source in placental insufficiency diseases.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

International audience; Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and accounts for a significant proportion of all primary brain tumors. Median survival after treatment is around 15 months. Remodeling of N-glycans by the N-acetylglucosamine glycosyltransferase (MGAT5) regulates tumoral development. Here, perturbation of MGAT5 enzymatic activity by the small-molecule inhibitor 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST3.1a) restrains GBM growth. In cell-based assays, it is demonstrated that PST3.1a alters the β1,6-GlcNAc N-glycans of GBM-initiating cells (GIC) by inhibiting MGAT5 enzymatic activity, resulting in the inhibition of TGFβR and FAK signaling associated with doublecortin (DCX) upregulation and increase oligodendrocyte lineage transcription factor 2 (OLIG2) expression. PST3.1a thus affects microtubule and microfilament integrity of GBM stem cells, leading to the inhibition of GIC proliferation, migration, invasiveness, and clonogenic capacities. Orthotopic graft models of GIC revealed that PST3.1a treatment leads to a drastic reduction of invasive and proliferative capacity and to an increase in overall survival relative to standard temozolomide therapy. Finally, bioinformatics analyses exposed that PST3.1a cytotoxic activity is positively correlated with the expression of genes of the epithelial–mesenchymal transition (EMT), while the expression of mitochondrial genes correlated negatively with cell sensitivity to the compound. These data demonstrate the relevance of targeting MGAT5, with a novel anti-invasive chemotherapy, to limit glioblastoma stem cell invasion. Mol Cancer Res; 15(10); 1376–87. ©2017 AACR.

Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status.

Lamellarin D (Lam-D) is a hexacyclic pyrole alkaloid isolated from marine invertebrates, whose biologic properties have been attributed to mitochondrial targeting. Mitochondria contain their own DNA (mtDNA), and the only specific mitochondrial topoisomerase in vertebrates is mitochondrial topoisomerase I (Top1mt). Here, we show that Top1mt is a direct mitochondrial target of Lam-D. In vitro Lam-D traps Top1mt and induces Top1mt cleavage complexes (Top1mtcc). Using single-molecule analyses, we also show that Lam-D slows down supercoil relaxation of Top1mt and strongly inhibits Top1mt religation in contrast to the inefficacy of camptothecin on Top1mt. In living cells, we show that Lam-D accumulates rapidly inside mitochondria, induces cellular Top1mtcc, and leads to mtDNA damage. This study provides evidence that Top1mt is a direct mitochondrial target of Lam-D and suggests that developing Top1mt inhibitors represents a novel strategy for targeting mitochondrial DNA.

Topoisomerases are critical for replication, DNA packing and repair, as well as for transcription by allowing changes in DNA topology. Cellular DNA is present both in nuclei and mitochondria, and mitochondrial topoisomerase I (Top1mt) is the only DNA topoisomerase specific for mitochondria in vertebrates. Here, we report in detail the generation of TOP1mt knockout mice, and demonstrate that mitochondrial DNA (mtDNA) displays increased negative supercoiling in TOP1mt knockout cells and murine tissues. This finding suggested imbalanced topoisomerase activity in the absence of Top1mt and the activity of other topoisomerases in mitochondria. Accordingly, we found that both Top2α and Top2β are present and active in mouse and human mitochondria. The presence of Top2α-DNA complexes in the mtDNA D-loop region, at the sites where both ends of 7S DNA are positioned, suggests a structural role for Top2 in addition to its classical topoisomerase activities.

The development of drug delivery nanocarriers is emerging as a promising therapeutic tool to transport anti-cancer agents to tumors. In this contribution, preparation of nanoparticles (NPs) highly loaded with cisplatin using the bio-inspired hybrid nucleoside-lipids has been reported. The construction of these NPs using a “layer-by-layer’’ approach allows surface functionalization with polyethylene glycol and targeting moieties. Uridine moieties were pegylated for folic acid (FA) functionalisation to render specificity for active targeting. The uridine moieties at the surface of the nanoparticle act as ligands for 99mTc radiolabeling, whereas the lipid chains maintain the structure of the nanoparticle. In vitro, these hybrid NPs are stable and actively internalize in two different cell lines overexpressing the folic acid receptor. In vivo scintigraphy shows that nucleolipid functionalized NPs notably improved the pharmacokinetic profile of cisplatin (enhanced blood circulation time) and accumulated in the tumor xenografted mice model.

International audience; Via whole-exome sequencing, we identified rare autosomal-recessive variants in UBA5 in five children from four unrelated families affected with a similar pattern of severe intellectual deficiency, microcephaly, movement disorders, and/or early-onset intractable epilepsy. UBA5 encodes the E1-activating enzyme of ubiquitin-fold modifier 1 (UFM1), a recently identified ubiquitin-like protein. Biochemical studies of mutant UBA5 proteins and studies in fibroblasts from affected individuals revealed that UBA5 mutations impair the process of ufmylation, resulting in an abnormal endoplasmic reticulum structure. In Caenorhabditis elegans, knockout of uba-5 and of human orthologous genes in the UFM1 cascade alter cholinergic, but not glutamatergic, neurotransmission. In addition, uba5 silencing in zebrafish decreased motility while inducing abnormal movements suggestive of seizures. These clinical, biochemical, and experimental findings support our finding of UBA5 mutations as a pathophysiological cause for early-onset encephalopathies due to abnormal protein ufmylation.

Tyrosyl‐DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2(S)) expressed from an alternative transcription start site. TDP2(S) contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full‐length TDP2 contains a nuclear localization signal and the ubiquitin‐associated domain in the N‐terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild‐type (WT) and TDP2 knockout (TDP2 (−/−/−)) DT40 cells shows that TDP2 (−/−/−) cells are hypersensitive to mitochondrial‐targeted doxorubicin (mtDox), and that complementing TDP2 (−/−/−) cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2 (−/−/−) cells. Using CRISPR‐engineered human cells expressing only the TDP2(S) isoform, we show that TDP2(S) also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2(S) isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

International audience; The liver has an exceptional replicative capacity following partial hepatectomy or chemical injuries. Cellular proliferation requires increased production of energy and essential metabolites, which critically depend on the mitochondria. To determine whether Top1mt, the vertebrate mitochondrial topoisomerase, is involved in this process, we studied liver regeneration after carbon tetrachloride (CCl4) administration. TOP1mt knockout (KO) mice showed a marked reduction in regeneration and hepatocyte proliferation. The hepatic mitochondrial DNA (mtDNA) failed to increase during recovery from CCl4 exposure. Reduced glutathione was also depleted, indicating increased reactive oxygen species (ROS). Steady-state levels of ATP, O2 consumption, mtDNA, and mitochondrial mass were also reduced in primary hepatocytes from CCl4-treated KO mice. To further test whether Top1mt acted by enabling mtDNA regeneration, we tested TOP1mt KO fibroblasts and human colon carcinoma HCT116 cells and measured mtDNA after 3-d treatment with ethidium bromide. Both types of TOP1mt knockout cells showed defective mtDNA regeneration following mtDNA depletion. Our study demonstrates that Top1mt is required for normal mtDNA homeostasis and for linking mtDNA expansion with hepatocyte proliferation.

Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin OH, in the ribosomal genes (12S and 16S) and at the light strand replication origin OL. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.

Purpose: Doxorubicin is one of the most effective chemotherapeutic agents. However, up to 30% of the patients treated with doxorubicin suffer from congestive heart failure. The mechanism of doxorubicin cardiotoxicity is likely multifactorial and most importantly, the genetic factors predisposing to doxorubicin cardiotoxicity are unknown. On the basis of the fact that mtDNA lesions and mitochondrial dysfunctions have been found in human hearts exposed to doxorubicin and that mitochondrial topoisomerase 1 (Top1mt) specifically controls mtDNA homeostasis, we hypothesized that Top1mt knockout (KO) mice might exhibit hypersensitivity to doxorubicin. Experimental Design: Wild-type (WT) and KO Top1mt mice were treated once a week with 4 mg/kg doxorubicin for 8 weeks. Heart tissues were analyzed one week after the last treatment. Results: Genetic inactivation of Top1mt in mice accentuates mtDNA copy number loss and mtDNA damage in heart tissue following doxorubicin treatment. Top1mt KO mice also fail to maintain respiratory chain protein production and mitochondrial cristae ultrastructure organization. These mitochondrial defects result in decreased O2 consumption, increased reactive oxygen species production, and enhanced heart muscle damage in animals treated with doxorubicin. Accordingly, Top1mt KO mice die within 45 days after the last doxorubicin injection, whereas the WT mice survive. Conclusions: Our results provide evidence that Top1mt, which is conserved across vertebrates, is critical for cardiac tolerance to doxorubicin and adaptive response to doxorubicin cardiotoxicity. They also suggest the potential of Top1mt single-nucleotide polymorphisms testing to investigate patient susceptibility to doxorubicin-induced cardiotoxicity. Clin Cancer Res; 20(18); 4873–81. ©2014 AACR.

A novel nucleoside lipid derived from dioleyl ketal was synthesized from uridine in three steps starting from dioleyl ketone. Electronic microscopy studies show that Ketals Nucleoside Lipids (KNL) self-assemble to form liposome-like structures in aqueous solutions. KNL is able to bind siRNA as demonstrated by electrophoresis experiment and standard ethidium bromide fluorescence displacement assay. Transfection assays of stable hepatic cell lines HupIRF, carrying a luciferase reporter gene demonstrate that KNL is able to transfect siRNA and exhibits protein knockdown more efficiently than its diester analog (DOTAU) and lipofectamine. Herein, we also report that KNLs are suitable transfecting reagents for the development of novel therapeutic approaches involving either siRNA or antisense oligonucleotide against human prostate cancer PC-3 cells resistant to chemotherapy.

10 pages; International audience; Langmuir monolayers have been extensively investigated by various experimental techniques. These studies allowed an in-depth understanding of the molecular conformation in the layer, phase transitions, and the structure of the multilayer. As the monolayer is compressed and the surface pressure is increased beyond a critical value, usually occurring in the minimal closely packed molecular area, the monolayer fractures and/or folds, forming multilayers in a process referred to as collapse. Various mechanisms for monolayer collapse and the resulting reorganization of the film have been proposed, and only a few studies have demonstrated the formation of a bilayer after collapse and with the use of a Ca2+ solution. In this work, Langmuir isotherms coupled with imaging ellipsometry and polarization modulation infrared reflection absorption spectroscopy were recorded to investigate the air−water interface properties of Langmuir films of anionic nucleolipids. We report for these new molecules the formation of a quasi-hexagonal packing of bilayer domains at a low compression rate, a singular behavior for lipids at the air−water interface that has not yet been documented.

The use of delivery vehicles to selectively transport anticancer agents to tumors is very attractive to address both toxicity and efficacy issues. We report a novel approach based on hybrid nucleoside-lipids allowing the efficient encapsulation and delivery of cisplatin. We demonstrate that the nucleoside polar heads guide the self-assembly of the aggregates into highly loaded and stable nanoparticles. The nanoparticles, which are efficient vehicles for the delivery of cisplatin into different sensitive and resistant cancer cell lines, can overcome the disadvantages and limitations of drug delivery systems previously reported.

A new non-ionic nucleoside based lipid (DOU-SS-PEG(2000), 5'-PEG(2000)-2',3'-dioleoyluridine) featuring uridine (U) as nucleoside and 2',3'-dioleyl (DO), as lipid moieties and a poly(ethylene glycol) (PEG) thiolytic cleavable spacer for in vitro delivery of drugs is described. The PEG detachable nucleotide lipid (DOU-SS-PEG(2000)) was prepared via a convergent synthesis starting from HS-PEG-OMe and uridine. The reduction-triggered delivery using the PEG detachable nucleoside lipid DOU-SS-PEG(2000) was evaluated on both liposomal and micellar objects. The liposomes were prepared from of a mixture of DOTAU (N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium tosylate), the PEG detachable nucleoside lipid DOU-SS-PEG(2000) and DOPE-rhodamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl ammonium salt) (60/40/0.1), whereas a mixture of 99.9% of DOU-SS-PEG(2000) and 0.1% of DOPE-rhodamine was used to prepare micelles. In addition, the supramolecular systems underwent a reduction-induced morphology transition from a micellar to vesicular states, which was characterized by DLS, zeta potential and TEM. The disulfide bond of the PEG chain was cleaved, by adding a reducing agent such as dithiothreitol (DTT), to expose the cationic surface of the liposome. The internalization of the resulting liposomes was facilitated as shown by the enhanced fluorescence signal observed in ovarian cancer cells (SKOV3) compared to the pegylated liposome. Likewise, when DTT was added to the mixture of cells incubated in the presence of DOU-SS-PEG(2000)/DOPE-rhodamine micelle, the fluorescence was observed in almost 100% of the SKOV3 cells.

Cationic nucleoside lipids (CNLs) derived from 5-nitroindole and 4-nitroimidazole bases were prepared from d-ribose by using a straightforward chemical synthesis. TEM experiments indicate that these amphiphilic molecules self-assemble to form supramolecular organizations in aqueous solutions. Electrophoresis and standard ethidium bromide (EB) fluorescence displacement assay shows that CNLs are able to bind siRNA. We demonstrated that both the nature of the universal bases and the stereochemistry of the anomeric position (alpha, beta) have an impact on the CNLs-siRNA complex formation. Correlations among chemical structure, stereochemistry, siRNA knockdown effect, and binding affinities for all the compounds were shown and analyzed with a simple molecular modeling study. The best binding affinities for siRNA were found for the beta anomer of the 5-nitroindole CNL which exhibits protein knockdown activity similar to the standard siPORT NeoFX positive control. It is noteworthy that no significant cytotoxicity at the tested concentration was observed for the novel CNLs.

8 pages; International audience; A family of new anionic nucleotide based lipids featuring thymidine-3′-monophosphate as nucleotide and 1,2- diacyl-sn-glycerol as lipid moiety for in Vitro delivery of nucleic acids is described. The nucleotide lipids were prepared in three steps starting from 1,2-diacyl-sn-glycerols and 2′-deoxythymidine-3′-phosphoramidite. Gel electrophoresis experiments show that nucleotide-based lipid-DNA complexes are observed at Ca2+ concentration higher than 1 mM. The transfection experiments carried out on mammalian Hek cell lines clearly demonstrate that the nucleotide moiety enhances the transfection efficacy of the natural anionic DPPA and DPPG lipids. SAXS studies indicate that the enhancement in transfection for nucleotide-based lipid formulations compared to those of the abasic natural derivative (DPPA) is likely due to the presence of the 2D columnar inverted hexagonal phase (HII) with a unit cell parameter a ) 69.1 Å in the nucleotide lipid formulations. The cytotoxicity studies of lipoplexes, evaluated against Hek cells using an MTS assay, revealed that palmitoyl nucleotide derivative complexes were not toxic even after 4 h of incubation, thus indicating that the anionic nucleotide lipids presented in this work offer an alternative to cationic transfection reagents.

Mitochondrial dynamics and distribution are critical insupplying ATP in response to energy demands. CLUHis a highly conserved protein involved in mitochondrial distribution, whose dysfunction leads to mitochondrial clustering around the nucleus. To gain insight into the role of CLUH in cellular metabolism, we generated CLUH knockout cells using CRISPR/Cas9. We show that mitochondrial clustering is associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in OXPHOS defects. This energetic impairment was found to be due to the alteration of mitochondrial translation, leading to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectrometry disclosed a dysfunctional Krebs cycle and an alteration of fatty acidoxidation. Thus, we established a clear function of CLUH in coupling mitochondrial distribution to the control of cellular energetic and metabolic status. To further analyze CLUH function, we performed in silico the prediction of the functional domains of this protein, disclosing 5 evolutionary conserved domains within the CLUH primary sequence. We reveal an oligomerization of CLUH into tetramers and octamers, and show a dominant negative effect associated to the expression of CLUH truncated forms missing Clu-N or TPR domains. Taken together, our studies reveal the importance of CLUH in maintaining cellular metabolism homeostasis and the potential regulation of its function through oligomerization.; La dynamique et la distribution mitochondriale sont essentielles pour l’homéostasie énergétique cellulaire. CLUH est une protéine indispensable à la distribution mitochondriale, dont la déplétion provoque une agrégation mitochondriale périnucléaire. Afin de comprendre le rôle de CLUH dans le métabolisme cellulaire, nous avons généré des cellules knockout CLUH par la méthode CRISPR-cas9. Nos résultats montrent que l’agrégation mitochondriale est associée à la diminution de la taille cellulaire et à la réduction quantitative des complexes de la chaîne respiratoire, menant ainsi à des défauts de la phosphorylation oxydative. Cette déficience énergétique est due à la perturbation de la traduction mitochondriale, et provoque un shift métabolique vers la glycolyse. Le profil métabolique des cellules KO montre un dysfonctionnement du cycle de Krebs et une altération de l’oxydation des acides gras. Dans ce sens, nous avons déterminé une fonction cruciale de CLUH dans le couplage de la distribution mitochondriale au contrôle de l’état cellulaire énergétique et métabolique. Pour approfondir l’analyse de la fonction de CLUH, nous avons effectué une étude de prédiction des domaines fonctionnels in silico, et avons identifié cinq domaines évolutivement conservés au sein de la séquence primaire de CLUH. De plus, nous démontrons que CLUH oligomérise en tétramères et en octomères, qui sont déstabilisés par l’expression ectopique de formes tronquées de CLUH dépourvues des domaines Clu-Nou TPR, par un effet dominant négatif. En résumé, nos résultats montrent l’importance de CLUH dans le maintien de l’homéostasie métabolique cellulaire, et une régulation potentielle de ses fonctions par oligomérisation.

Mitochondrial dynamics and distribution are critical insupplying ATP in response to energy demands. CLUHis a highly conserved protein involved in mitochondrial distribution, whose dysfunction leads to mitochondrial clustering around the nucleus. To gain insight into the role of CLUH in cellular metabolism, we generated CLUH knockout cells using CRISPR/Cas9. We show that mitochondrial clustering is associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in OXPHOS defects. This energetic impairment was found to be due to the alteration of mitochondrial translation, leading to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectrometry disclosed a dysfunctional Krebs cycle and an alteration of fatty acidoxidation. Thus, we established a clear function of CLUH in coupling mitochondrial distribution to the control of cellular energetic and metabolic status. To further analyze CLUH function, we performed in silico the prediction of the functional domains of this protein, disclosing 5 evolutionary conserved domains within the CLUH primary sequence. We reveal an oligomerization of CLUH into tetramers and octamers, and show a dominant negative effect associated to the expression of CLUH truncated forms missing Clu-N or TPR domains. Taken together, our studies reveal the importance of CLUH in maintaining cellular metabolism homeostasis and the potential regulation of its function through oligomerization., La dynamique et la distribution mitochondriale sont essentielles pour l’homéostasie énergétique cellulaire. CLUH est une protéine indispensable à la distribution mitochondriale, dont la déplétion provoque une agrégation mitochondriale périnucléaire. Afin de comprendre le rôle de CLUH dans le métabolisme cellulaire, nous avons généré des cellules knockout CLUH par la méthode CRISPR-cas9. Nos résultats montrent que l’agrégation mitochondriale est associée à la diminution de la taille cellulaire et à la réduction quantitative des complexes de la chaîne respiratoire, menant ainsi à des défauts de la phosphorylation oxydative. Cette déficience énergétique est due à la perturbation de la traduction mitochondriale, et provoque un shift métabolique vers la glycolyse. Le profil métabolique des cellules KO montre un dysfonctionnement du cycle de Krebs et une altération de l’oxydation des acides gras. Dans ce sens, nous avons déterminé une fonction cruciale de CLUH dans le couplage de la distribution mitochondriale au contrôle de l’état cellulaire énergétique et métabolique. Pour approfondir l’analyse de la fonction de CLUH, nous avons effectué une étude de prédiction des domaines fonctionnels in silico, et avons identifié cinq domaines évolutivement conservés au sein de la séquence primaire de CLUH. De plus, nous démontrons que CLUH oligomérise en tétramères et en octomères, qui sont déstabilisés par l’expression ectopique de formes tronquées de CLUH dépourvues des domaines Clu-Nou TPR, par un effet dominant négatif. En résumé, nos résultats montrent l’importance de CLUH dans le maintien de l’homéostasie métabolique cellulaire, et une régulation potentielle de ses fonctions par oligomérisation.