ABSTRACT
Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and multifunctional CSA or CSB protein, but only CS patients display a progeroid and neurodegenerative phenotype, providing a unique conceptual and experimental paradigm. As DNA methylation (DNAm) remodelling is a major ageing marker, we performed genome-wide analysis of DNAm of fibroblasts from healthy, UVSS and CS individuals. Differential analysis highlighted a CS-specific epigenomic signature (progeroid-related; not present in UVSS) enriched in three categories: developmental transcription factors, ion/neurotransmitter membrane transporters and synaptic neuro-developmental genes. A large fraction of CS-specific DNAm changes were associated with expression changes in CS samples, including in previously reported post-mortem cerebella. The progeroid phenotype of CS was further supported by epigenomic hallmarks of ageing: the prediction of DNAm of repetitive elements suggested an hypomethylation of Alu sequences in CS, and the epigenetic clock returned a marked increase in CS biological age respect to healthy and UVSS cells. The epigenomic remodelling of accelerated ageing in CS displayed both commonalities and differences with other progeroid diseases and regular ageing. CS shared DNAm changes with normal ageing more than other progeroid diseases do, and included genes functionally validated for regular ageing. Collectively, our results support the existence of an epigenomic basis of accelerated ageing in CS and unveil new genes and pathways that are potentially associated with the progeroid/degenerative phenotype.
Subject(s)
Cockayne Syndrome , Humans , Cockayne Syndrome/genetics , Cockayne Syndrome/metabolism , Epigenomics , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA Repair , Aging/genetics , MutationABSTRACT
Skeletal muscle regeneration relies on muscle stem (satellite) cells. We previously demonstrated that satellite cells efficiently and accurately repair radiation-induced DNA double-strand breaks (DSBs) via the DNA-dependent kinase DNA-PKcs. We show here that DNA-PKcs affects myogenesis independently of its role in DSB repair. Consequently, this process does not require the accumulation of DSBs and it is also independent of caspase-induced DNA damage. We report that in myogenic cells DNA-PKcs is essential for the expression of the differentiation factor Myogenin in an Akt2-dependent manner. DNA-PKcs interacts with the p300-containing complex that activates Myogenin transcription. We show also that SCID mice that are deficient in DNA-PKcs, and are used for transplantation and muscle regeneration studies, display altered myofiber composition and delayed myogenesis upon injury. These defects are exacerbated after repeated injury/regeneration events resulting in reduced muscle size. We thus identify a novel, caspase-independent, regulation of myogenic differentiation, and define a differentiation phase that does not involve the DNA damage/repair process.
Subject(s)
DNA Repair , Proto-Oncogene Proteins c-akt , Animals , Mice , Myogenin/genetics , Myogenin/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Mice, SCID , DNA Damage , Muscle Development , Caspases/metabolism , DNAABSTRACT
Preeclampsia (PE) is a high-prevalence pregnancy disease characterized by placental insufficiency, gestational hypertension, and proteinuria. Overexpression of the A isoform of the STOX1 transcription factor (STOX1A) recapitulates PE in mice, and STOX1A overexpressing trophoblasts recapitulate PE patients hallmarks in terms of gene expression and pathophysiology. STOX1 overexpression induces nitroso-redox imbalance and mitochondrial hyper-activation. Here, by a thorough analysis on cell models, we show that STOX1 overexpression in trophoblasts alters inducible nitric oxide synthase (iNOS), nitric oxide (NO) content, the nitroso-redox balance, the antioxidant defense, and mitochondrial function. This is accompanied by specific alterations of the Krebs cycle leading to reduced l-malate content. By increasing NOS coupling using the metabolite tetrahydrobiopterin (BH4) we restore this multi-step pathway in vitro. Moving in vivo on two different rodent models (STOX1 mice and RUPP rats, alike early onset and late onset preeclampsia, respectively), we show by transcriptomics that BH4 directly reverts STOX1-deregulated gene expression including glutathione metabolism, oxidative phosphorylation, cholesterol metabolism, inflammation, lipoprotein metabolism and platelet activation, successfully treating placental hypotrophy, gestational hypertension, proteinuria and heart hypertrophy. In the RUPP rats we show that the major fetal issue of preeclampsia, Intra Uterine Growth Restriction (IUGR), is efficiently corrected. Our work posits on solid bases BH4 as a novel potential therapy for preeclampsia.
ABSTRACT
Mitochondrial DNA (mtDNA) damaged by reactive oxygen species (ROS) triggers so far poorly understood processes of mtDNA maintenance that are coordinated by a complex interplay among DNA repair, DNA degradation, and DNA replication. This study was designed to identify the proteins involved in mtDNA maintenance by applying a special long-range PCR, reflecting mtDNA integrity in the minor arc. A siRNA screening of literature-based candidates was performed under conditions of enforced oxidative phosphorylation revealing the functional group of polymerases and therein polymerase ζ (POLZ) as top hits. Thus, POLZ knockdown caused mtDNA accumulation, which required the activity of the base excision repair (BER) nuclease APE1, and was followed by compensatory mtDNA replication determined by the single-cell mitochondrial in situ hybridization protocol (mTRIP). Quenching reactive oxygen species (ROS) in mitochondria unveiled an additional, ROS-independent involvement of POLZ in the formation of a typical deletion in the minor arc region. Together with data demonstrating the localization of POLZ in mitochondria, we suggest that POLZ plays a significant role in mtDNA turnover, particularly under conditions of oxidative stress.
Subject(s)
DNA, Mitochondrial , Mitochondria , DNA Repair/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Nucleotidyltransferases/genetics , Reactive Oxygen Species/metabolismABSTRACT
BACKGROUND: Cockayne syndrome (CS) is a rare autosomal recessive disorder caused by mutations in ERCC6/CSB or ERCC8/CSA that participate in the transcription-coupled nucleotide excision repair (TC-NER) of UV-induced DNA damage. CS patients display a large heterogeneity of clinical symptoms and severities, the reason of which is not fully understood, and that cannot be anticipated in the diagnostic phase. In addition, little data is available for affected siblings, and this disease is largely undiagnosed in North Africa. METHODS: We report here the clinical description as well as genetic and functional characterization of eight Tunisian CS patients, including siblings. These patients, who belonged to six unrelated families, underwent complete clinical examination and biochemical analyses. Sanger sequencing was performed for the recurrent mutation in five families, and targeted gene sequencing was done for one patient of the sixth family. We also performed Recovery RNA Synthesis (RRS) to confirm the functional impairment of DNA repair in patient-derived fibroblasts. RESULTS: Six out of eight patients carried a homozygous indel mutation (c.598_600delinsAA) in exon 7 of ERCC8, and displayed a variable clinical spectrum including between siblings sharing the same mutation. The other two patients were siblings who carried a homozygous splice-site variant in ERCC8 (c.843+1G>C). This last pair presented more severe clinical manifestations, which are rarely associated with CSA mutations, leading to gastrostomy and hepatic damage. Impaired TC-NER was confirmed by RRS in six tested patients. CONCLUSIONS: This study provides the first deep characterization of case series of CS patients carrying CSA mutations in North Africa. These mutations have been described only in this region and in the Middle-East. We also provide the largest characterization of multiple unrelated patients, as well as siblings, carrying the same mutation, providing a framework for dissecting elusive genotype-phenotype correlations in CS.
Subject(s)
Cockayne Syndrome , Cockayne Syndrome/diagnosis , Cockayne Syndrome/genetics , DNA Repair/genetics , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Homozygote , Humans , Mutation/genetics , Poly-ADP-Ribose Binding Proteins/genetics , Siblings , Transcription Factors/geneticsABSTRACT
Significance: Reactive species have been classically considered causative of age-related degenerative processes, but the scenario appears considerably more complex and to some extent counterintuitive than originally anticipated. The impact of reactive species in precocious aging syndromes is revealing new clues to understand and perhaps challenge the resulting degenerative processes. Recent Advances: Our understanding of reactive species has considerably evolved, including their hormetic effect (beneficial at a certain level, harmful beyond this level), the occurrence of diverse hormetic peaks in different cell types and organisms, and the extended type of reactive species that are relevant in biological processes. Our understanding of the impact of reactive species has also expanded from the dichotomic damaging/signaling role to modulation of gene expression. Critical Issues: These new concepts are affecting the study of aging and diseases where aging is greatly accelerated. We discuss how notions arising from the study of the underlying mechanisms of a progeroid disease, Cockayne syndrome, represent a paradigm shift that may shed a new light in understanding the role of reactive species in age-related degenerative processes. Future Issues: Future investigations urge to explore established and emerging notions to elucidate the multiple contributions of reactive species in degenerative processes linked to pathophysiological aging and their possible amelioration. Antioxid. Redox Signal. 37, 208-228.
Subject(s)
Aging , Cockayne Syndrome , Reactive Nitrogen Species , Reactive Oxygen Species , Sulfur , Animals , Antioxidants/therapeutic use , Cockayne Syndrome/physiopathology , Down Syndrome/physiopathology , Humans , Mitochondria , Oxidative Stress , Progeria/physiopathology , Reactive Nitrogen Species/chemistry , Reactive Oxygen Species/chemistry , Signal Transduction , Sulfur/chemistry , Werner Syndrome/physiopathologyABSTRACT
Cockayne syndrome (CS) is a rare disease caused by mutations in ERCC6/CSB or ERCC8/CSA. We report here the clinical, genetic, and functional analyses of three unrelated patients mutated in ERCC6/CSB with a severe phenotype. After clinical examination, two patients were investigated via next generation sequencing, targeting seventeen Nucleotide Excision Repair (NER) genes. All three patients harbored a novel, c.3156dup, homozygous mutation located in exon 18 of ERCC6/CSB that affects the C-terminal region of the protein. Sanger sequencing confirmed the mutation and the parental segregation in the three families, and Western blots showed a lack of the full-length protein. NER functional impairment was shown by reduced recovery of RNA synthesis with proficient unscheduled DNA synthesis after UV-C radiations in patient-derived fibroblasts. Despite sharing the same mutation, the clinical spectrum was heterogeneous among the three patients, and only two patients displayed clinical photosensitivity. This novel ERCC6 variant in Tunisian patients suggests a founder effect and has implications for setting-up prenatal diagnosis/genetic counselling in North Africa, where this disease is largely undiagnosed. This study reveals one of the rare cases of CS clinical heterogeneity despite the same mutation. Moreover, the occurrence of an identical homozygous mutation, which either results in clinical photosensitivity or does not, strongly suggests that this classic CS symptom relies on multiple factors.
Subject(s)
Cockayne Syndrome/genetics , DNA Helicases/genetics , DNA Repair Enzymes/genetics , Mutation , Poly-ADP-Ribose Binding Proteins/genetics , Blotting, Western , Cells, Cultured , Child , Child, Preschool , Cockayne Syndrome/diagnostic imaging , Cockayne Syndrome/physiopathology , Consanguinity , DNA Repair/genetics , Female , Fibroblasts/radiation effects , Homozygote , Humans , Magnetic Resonance Imaging , Male , Pedigree , Ultraviolet RaysABSTRACT
Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.
Subject(s)
DNA, Mitochondrial/chemistry , Mitochondria/genetics , Mitochondrial Diseases/genetics , Single-Cell Analysis/methods , Animals , Humans , In Situ Hybridization, Fluorescence , Mice , RNA, Mitochondrial/chemistry , Transcription, GeneticABSTRACT
Cellular senescence has causative links with ageing and age-related diseases, however, it remains unclear if progeroid factors cause senescence in normal cells. Here, we show that depletion of CSB, a protein mutated in progeroid Cockayne syndrome (CS), is the earliest known trigger of p21-dependent replicative senescence. CSB depletion promotes overexpression of the HTRA3 protease resulting in mitochondrial impairments, which are causally linked to CS pathological phenotypes. The CSB promoter is downregulated by histone H3 hypoacetylation during DNA damage-response. Mechanistically, CSB binds to the p21 promoter thereby downregulating its transcription and blocking replicative senescence in a p53-independent manner. This activity of CSB is independent of its role in the repair of UV-induced DNA damage. HTRA3 accumulation and senescence are partially rescued upon reduction of oxidative/nitrosative stress. These findings establish a CSB/p21 axis that acts as a barrier to replicative senescence, and link a progeroid factor with the process of regular ageing in human.
Subject(s)
Cellular Senescence/physiology , Cockayne Syndrome/metabolism , DNA Helicases/metabolism , DNA Repair Enzymes/metabolism , Histones/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Cell Line , Cellular Senescence/genetics , Cockayne Syndrome/genetics , Cockayne Syndrome/pathology , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA/metabolism , DNA/radiation effects , DNA Damage , DNA Helicases/genetics , DNA Repair , DNA Repair Enzymes/genetics , Down-Regulation , Epigenomics , Fibroblasts , Gene Expression Regulation , Gene Knockdown Techniques , HEK293 Cells , Humans , Mitochondria/metabolism , Oxidative Stress , Poly-ADP-Ribose Binding Proteins/genetics , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Transcriptome , Ultraviolet Rays/adverse effectsABSTRACT
Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.
Subject(s)
DNA, Mitochondrial/metabolism , Mitochondria/metabolism , Neoplasms/metabolism , Oxidoreductases Acting on CH-CH Group Donors/physiology , Pyrimidines/metabolism , Animals , Cell Line, Tumor , Cell Respiration , Dihydroorotate Dehydrogenase , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Oxidative Phosphorylation , Ubiquinone/metabolismABSTRACT
[This corrects the article DOI: 10.18632/oncotarget.24822.].
ABSTRACT
During growth, homeostasis and regeneration, stem cells are exposed to different energy demands. Here, we characterise the metabolic pathways that mediate the commitment and differentiation of mouse skeletal muscle stem cells, and how their modulation can influence the cell state. We show that quiescent satellite stem cells have low energetic demands and perturbed oxidative phosphorylation during ageing, which is also the case for cells from post-mortem tissues. We show also that myogenic fetal cells have distinct metabolic requirements compared to those proliferating during regeneration, with the former displaying a low respiration demand relying mostly on glycolysis. Furthermore, we show distinct requirements for peroxisomal and mitochondrial fatty acid oxidation (FAO) in myogenic cells. Compromising peroxisomal but not mitochondrial FAO promotes early differentiation of myogenic cells. Acute muscle injury and pharmacological block of peroxisomal and mitochondrial FAO expose differential requirements for these organelles during muscle regeneration. Taken together, these observations indicate that changes in myogenic cell state lead to significant alterations in metabolic requirements. In addition, perturbing specific metabolic pathways impacts on myogenic cell fates and the regeneration process.
Subject(s)
Muscle Development , Muscle, Skeletal/growth & development , Stem Cells/cytology , Stem Cells/metabolism , Animals , Cell Proliferation , Fatty Acids/metabolism , Mice , Mitochondria/metabolism , Muscle, Skeletal/metabolism , Oxidation-Reduction , Peroxisomes/metabolism , Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/metabolismABSTRACT
Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis.
ABSTRACT
Skeletal muscle stem cells in the adult display heterogeneity that has been functionally linked to their behavior, self-renewal capacity, and resistance to stress in hostile environments. Behavioral heterogeneity emerges also during developmental myogenesis. Muscle stem cell diversity may be functionally linked to the changing needs of skeletal muscle regeneration. Intriguingly, dramatic reduction of stem cell diversity, the "clonal drift", that implies loss of stem cells and related expansion of clonally related stem cells has been reported for tissue replacement in several adult tissues and suggested in the zebrafish embryo. A recent study shows clonal drift of muscle stem cells in the zebrafish embryo caused by inhibition of the cell cycle and directed by the homeobox protein Meox1. Although stem cell quiescence is associated with inhibition of the transition phase G0/G1 of the cell cycle, Meox1 triggers the muscle stem cell fate by an arrest in G2 phase. Why efficient muscle growth in the zebrafish embryo requires sacrificing stem cell heterogeneity in favor of a small number of dominant clones has not been elucidated. The significance of G2-halted stem cells, which are generally associated with robust regeneration capacity, is also intriguing. These processes are relevant for understanding organ growth and the mechanisms that govern stem cell quiescence.
ABSTRACT
Mitochondrial DNA (mtDNA), which is essential for mitochondrial and cell function, is replicated and transcribed in the organelle by proteins that are entirely coded in the nucleus. Replication of mtDNA is challenged not only by threats related to the replication machinery and orchestration of DNA synthesis, but also by factors linked to the peculiarity of this genome. Indeed the architecture, organization, copy number, and location of mtDNA, which are markedly distinct from the nuclear genome, require ad hoc and complex regulation to ensure coordinated replication. As a consequence sub-optimal mtDNA replication, which results from compromised regulation of these factors, is generally associated with mitochondrial dysfunction and disease. Mitochondrial DNA replication should be considered in the context of the organelle and the whole cell, and not just a single genome or a single replication event. Major threats to mtDNA replication are linked to its dependence on both mitochondrial and nuclear factors, which require exquisite coordination of these crucial subcellular compartments. Moreover, regulation of replication events deals with a dynamic population of multiple mtDNA molecules rather than with a fixed number of genome copies, as it is the case for nuclear DNA. Importantly, the mechanistic aspects of mtDNA replication are still debated. We describe here major challenges for human mtDNA replication, the mechanistic aspects of the process that are to a large extent original, and their consequences on disease.
Subject(s)
Cell Nucleus/genetics , DNA Replication , DNA, Mitochondrial/genetics , Mitochondria/genetics , Oxidative Stress , Genome, Mitochondrial , Humans , Mitochondria/metabolismABSTRACT
Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , Cell Transformation, Neoplastic/pathology , Electron Transport Complex I/metabolism , Gene Expression Regulation, Neoplastic , Mitochondria/pathology , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Movement , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Electron Transport Complex I/genetics , Genome, Mitochondrial , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/genetics , Mitochondria/metabolism , Neoplasm Invasiveness , Oxidative Stress , Proto-Oncogene Proteins c-mdm2/genetics , Signal Transduction , Transcription, Genetic , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor AssaysABSTRACT
Targeting mitochondria is a powerful strategy for pathogens to subvert cell physiology and establish infection. Helicobacter pylori is a bacterial pathogen associated with gastric cancer development that is known to target mitochondria directly and exclusively through its pro-apoptotic and vacuolating cytotoxin VacA. By in vitro infection of gastric epithelial cells with wild-type and VacA-deficient H. pylori strains, treatment of cells with purified VacA proteins and infection of a mouse model, we show that H. pylori deregulates mitochondria by two novel mechanisms, both rather associated with host cell survival. First, early upon infection VacA induces transient increase of mitochondrial translocases and a dramatic accumulation of the mitochondrial DNA replication and maintenance factors POLG and TFAM. These events occur when VacA is not detected intracellularly, therefore do not require the direct interaction of the cytotoxin with the organelle, and are independent of the toxin vacuolating activity. In vivo, these alterations coincide with the evolution of gastric lesions towards severity. Second, H. pylori also induces VacA-independent alteration of mitochondrial replication and import components, suggesting the involvement of additional H. pylori activities in mitochondria-mediated effects. These data unveil two novel mitochondrial effectors in H. pylori-host interaction with links on gastric pathogenesis.
Subject(s)
Bacterial Proteins/metabolism , DNA Replication , DNA, Mitochondrial/metabolism , Helicobacter pylori/metabolism , Mitochondria/metabolism , Animals , Cell Line , Cytosol/metabolism , DNA Polymerase gamma/metabolism , DNA-Binding Proteins/metabolism , Helicobacter Infections/metabolism , Helicobacter Infections/microbiology , High Mobility Group Proteins/metabolism , Humans , Mice , Mitochondrial ADP, ATP Translocases/metabolism , Models, Biological , Protein TransportABSTRACT
Sepsis is an acute systemic inflammatory response of the body to microbial infection and a life threatening condition associated with multiple organ failure. Survivors may display long-term disability with muscle weakness that remains poorly understood. Recent data suggest that long-term myopathy in sepsis survivors is due to failure of skeletal muscle stem cells (satellite cells) to regenerate the muscle. Satellite cells impairment in the acute phase of sepsis is linked to unusual mitochondrial dysfunctions, characterized by a dramatic reduction of the mitochondrial mass and hyperactivity of residual organelles. Survivors maintain the impairment of satellite cells, including alterations of the mitochondrial DNA (mtDNA), in the long-term. This condition can be rescued by treatment with mesenchymal stem cells (MSCs) that restore mtDNA alterations and mitochondrial function in satellite cells, and in fine their regenerative potential. Injection of MSCs in turn increases the force of isolated muscle fibers and of the whole animal, and improves the survival rate. These effects occur in the context of reduced inflammation markers that also raised during sepsis. Targeting muscle stem cells mitochondria, in a context of reduced inflammation, may represent a valuable strategy to reduce morbidity and long-term impairment of the muscle upon sepsis.
Subject(s)
Mitochondria, Muscle/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Sepsis/metabolism , Animals , DNA, Mitochondrial/metabolism , Humans , Inflammation/metabolism , Inflammation/mortality , Inflammation/therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Mitochondria, Muscle/pathology , Muscle Strength , Satellite Cells, Skeletal Muscle/pathology , Sepsis/mortality , Sepsis/therapyABSTRACT
Skeletal muscle stem cells or satellite cells are responsible for muscle regeneration in the adult. Although satellite cells are highly resistant to stress, and display greater capacity to repair molecular damage than the committed progeny, their regenerative potential declines with age. During ageing, satellite cells switch to a state of permanent cell cycle arrest or senescence which prevents their activation. A recent study reveals that the senescence of satellite cell relies on defective autophagy, the quality control mechanism that degrades damaged proteins and organelles. Molecular damage is generated by oxidative stress that also promotes epigenetic changes that activate the expression of master genes, in a double-hit mechanism that ensures senescence. Importantly, genetic, and pharmacological correction of defective autophagy reverses satellite cell senescence and restores muscle regeneration in geriatric mice, with perspectives of modulating age-related functional decline of muscle. This study provides new clues to understand stem cell and organismal ageing.
Subject(s)
Cell Proliferation , Cellular Senescence , Stem Cells/physiology , Animals , Autophagy , Epigenesis, Genetic , Humans , Oxidative Stress , Stem Cells/metabolismABSTRACT
Mitochondria autonomously replicate and transcribe their own genome, which is present in multiple copies in the organelle. Transcription and replication of the mitochondrial DNA (mtDNA), which are defined here as mtDNA processing, are essential for mitochondrial function. The extent, efficiency, and coordination of mtDNA processing are key parameters of the mitochondrial state in living cells. Recently, single-cell analysis of mtDNA processing revealed a large and dynamic heterogeneity of mitochondrial populations in single cells, which is linked to mitochondrial function and is altered during disease. This was achieved using mitochondrial Transcription and Replication Imaging Protocol (mTRIP), a modified fluorescence in situ hybridization (FISH) approach that simultaneously reveals the mitochondrial RNA content and mtDNA engaged in initiation of replication at the single-cell level. mTRIP can also be coupled to immunofluorescence or MitoTracker, resulting in the additional labeling of proteins or active mitochondria, respectively. Therefore, mTRIP detects quantitative and qualitative alterations of the dynamics of mtDNA processing in human cells that respond to physiological changes or result from diseases. In addition, we show here that mTRIP is a rather sensitive tool for detecting mitochondrial alterations that may lead to loss of cell viability, and is thereby a useful tool for monitoring sublethal cytotoxicity for instance during chronic drug treatment.