The glycoprotein clusterin (CLU) is involved in cell proliferation and DNA damage repair and is highly expressed in tumor cells. Here, we aimed to investigate the effects of CLU dysregulation on two human astrocytic cell lines: CCF-STTG1 astrocytoma cells and SV-40 immortalized normal human astrocytes. We observed that suppression of CLU expression by RNA interference inhibited cell proliferation, triggered the DNA damage response, and resulted in cellular senescence in both cell types tested. To further investigate the underlying mechanism behind these changes, we measured reactive oxygen species, assessed mitochondrial function, and determined selected markers of the senescence-associated secretory phenotype. Our results suggest that CLU deficiency triggers oxidative stress-mediated cellular senescence associated with pronounced alterations in mitochondrial membrane potential, mitochondrial mass, and expression levels of OXPHOS complex I, II, III and IV, indicating mitochondrial dysfunction. This report shows the important role of CLU in cell cycle maintenance in astrocytes. Based on these data, targeting CLU may serve as a potential therapeutic approach valuable for treating gliomas.

• MeSH
• Astrocytes * metabolism   pathology   MeSH
• Clusterin * metabolism   genetics   MeSH
• Humans MeSH
• Membrane Potential, Mitochondrial * physiology   MeSH
• Mitochondria * metabolism   MeSH
• Cell Line, Tumor MeSH
• Oxidative Stress physiology   MeSH
• Oxidative Phosphorylation MeSH
• DNA Damage MeSH
• Cell Proliferation * MeSH
• Reactive Oxygen Species metabolism   MeSH
• Cellular Senescence * physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Mitochondria are key to cellular energetics, metabolism, and signaling. Their dysfunction is linked to devastating diseases, including mitochondrial disorders, diabetes, neurodegenerative diseases, cardiac disorders, and cancer. Here, we present a knockout mouse model lacking the complex IV assembly factor SMIM20/MITRAC7. SMIM20-/- mice display cardiac pathology with reduced heart weight and cardiac output. Heart mitochondria present with reduced levels of complex IV associated with increased complex I activity, have altered fatty acid oxidation, and display elevated levels of ROS production. Interestingly, mutant mouse ventricular myocytes show unphysiological Ca2+ handling, which can be attributed to the increase in mitochondrial ROS production. Our study presents an example of a tissue-specific phenotype in the context of OXPHOS dysfunction. Moreover, our data suggest a link between complex IV dysfunction and Ca2+ handling at the endoplasmic reticulum through ROS signaling.

• MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Myocytes, Cardiac metabolism   MeSH
• Membrane Proteins * metabolism   genetics   MeSH
• Mitochondrial Proteins * metabolism   genetics   MeSH
• Myocardium * metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Oxidative Phosphorylation MeSH
• Zebrafish Proteins MeSH
• Reactive Oxygen Species metabolism   MeSH
• Electron Transport Complex IV * metabolism   MeSH
• Mitochondria, Heart metabolism   MeSH
• Calcium metabolism   MeSH
• Calcium Signaling * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Cardiovascular diseases are associated with an altered cardiomyocyte metabolism. Because of a shortage of human heart tissue, experimental studies mostly rely on alternative approaches including animal and cell culture models. Since the use of isolated primary cardiomyocytes is limited, immortalized cardiomyocyte cell lines may represent a useful tool as they closely mimic human cardiomyocytes. This study is focused on the AC16 cell line generated from adult human ventricular cardiomyocytes. Despite an increasing number of studies employing AC16 cells, a comprehensive proteomic, bioenergetic, and oxygen-sensing characterization of proliferating vs. differentiated cells is still lacking. Here, we provide a comparison of these two stages, particularly emphasizing cell metabolism, mitochondrial function, and hypoxic signaling. Label-free quantitative mass spectrometry revealed a decrease in autophagy and cytoplasmic translation in differentiated AC16, confirming their phenotype. Cell differentiation led to global increase in mitochondrial proteins [e.g. oxidative phosphorylation (OXPHOS) proteins, TFAM, VWA8] reflected by elevated mitochondrial respiration. Fatty acid oxidation proteins were increased in differentiated cells, whereas the expression levels of proteins associated with fatty acid synthesis were unchanged and glycolytic proteins were decreased. There was a profound difference between proliferating and differentiated cells in their response to hypoxia and anoxia-reoxygenation. We conclude that AC16 differentiation leads to proteomic and metabolic shifts and altered cell response to oxygen deprivation. This underscores the requirement for proper selection of the particular differentiation state during experimental planning.NEW & NOTEWORTHY Proliferating and differentiated AC16 cell lines exhibit distinct proteomic and metabolic profiles with critical implications for experimental design. Proliferating cells predominantly utilize glycolysis and are highly sensitive to hypoxia, whereas differentiated cells display enhanced mitochondrial biogenesis, oxidative phosphorylation, and resistance to anoxia-reoxygenation. These findings provide novel insights into the metabolic adaptations during differentiation and highlight the necessity of selecting the appropriate cellular stage to ensure accurate experimental outcomes.

• MeSH
• Cell Differentiation * physiology   MeSH
• Cell Line MeSH
• Energy Metabolism MeSH
• Cell Hypoxia physiology   MeSH
• Myocytes, Cardiac * metabolism   MeSH
• Humans MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria * metabolism   MeSH
• Oxidative Phosphorylation MeSH
• Cell Proliferation MeSH
• Proteomics methods   MeSH
• Signal Transduction * physiology   MeSH
• Mitochondria, Heart * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

In patients with testicular germ cell tumours (TGCT), sperm cryopreservation prior to anti-cancer treatment represents the main fertility preservation approach. However, it is associated with a low sperm recovery rate after thawing. Since sperm is a high-energy demanding cell, which is supplied by glycolysis and oxidative phosphorylation (OXPHOS), mitochondrial dysfunctionality can directly result in sperm anomalies. In this study, we investigated the bioenergetic pattern of cryopreserved sperm of TGCT patients in comparison with normozoospermic samples using two state-of-the-art methods: the Extracellular Flux Analyzer (XF Analyzer) and two-photon fluorescence lifetime imaging microscopy (2P-FLIM), in order to assess the contributions of OXPHOS and glycolysis to energy provision. A novel protocol for the combined measurement of OXPHOS (oxygen consumption rate: OCR) and glycolysis (extracellular acidification rate: ECAR) using the XF Analyzer was developed together with a unique customized AI-based approach for semiautomated processing of 2P-FLIM images. Our study delivers optimized low-HEPES modified human tubal fluid media (mHTF) for sperm handling during pre-analytical and analytical phases, to maintain sperm physiological parameters and optimal OCR, equivalent to OXPHOS. The negative effect of cryopreservation was signified by the deterioration of both bioenergetic pathways represented by modified OCR and ECAR curves and the derived parameters. This was true for normozoospermic as well as samples from TGCT patients, which showed even stronger damage within the respiratory chain compared to the level of glycolytic activity impairment. The impact of cryopreservation and pathology are supported by 2P-FLIM analysis, showing a significant decrease in bound NADH in contrast to unbound NAD(P)H, which reflects decreased metabolic activity in samples from TGCT patients. Our study provides novel insights into the impact of TGCT on sperm bioenergetics and delivers a verified protocol to be used for the assessment of human sperm metabolic activity, which can be a valuable tool for further research and clinical andrology.

• MeSH
• Adult MeSH
• Energy Metabolism * MeSH
• Neoplasms, Germ Cell and Embryonal * metabolism   pathology   MeSH
• Glycolysis * MeSH
• Cryopreservation * methods   MeSH
• Humans MeSH
• Mitochondria metabolism   MeSH
• Oxidative Phosphorylation * MeSH
• Spermatozoa * metabolism   MeSH
• Oxygen Consumption physiology   MeSH
• Testicular Neoplasms * metabolism   pathology   MeSH
• Semen Preservation methods   MeSH
• Fertility Preservation methods   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH

Testicular cancer is the most common form of cancer in young men of reproductive age and its incidence is increasing globally. With the currently successful treatment and 95% survival rate, there is a need for deeper understanding of testicular cancer-related infertility. Most patients with testicular cancer experience semen abnormalities prior to cancer therapy. However, the exact mechanism of the effect of testicular cancer on sperm anomalies is not known. Mitochondria are organelles that play a crucial role in both tumorigenesis and spermatogenesis and their malfunction may be an important factor resulting in sperm abnormalities in testicular cancer patients. Within the scope of this review, we will discuss current knowledge of testicular cancer-related alterations in the ATP production pathway, a possible pathophysiological switch from oxidative phosphorylation (OXPHOS) to glycolysis, as well as the role of oxidative stress promoting sperm dysfunction. In this regard, the review provides a summary of the impact of testicular cancer on sperm quality as a possible consequence of impaired mitochondrial function including the energy metabolic pathways that are known to be altered in the sperm of testicular cancer patients.

• MeSH
• Semen Analysis MeSH
• Neoplasms, Germ Cell and Embryonal * MeSH
• Humans MeSH
• Mitochondrial Diseases * metabolism   MeSH
• Semen metabolism   MeSH
• Spermatozoa MeSH
• Testicular Neoplasms * metabolism   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND: High-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive subtype of epithelial ovarian carcinoma. It is primarily diagnosed at stage III or IV when the 5-year survival rate ranges between 20% and 40%. Here, we aimed to validate the hypothesis, based on HGSOC cell lines, that proposed the existence of two distinct groups of HGSOC cells with high and low oxidative phosphorylation (OXPHOS) metabolism, respectively, which are associated with their responses to glucose and glutamine withdrawal. METHODS: We isolated and cultivated primary cancer cell cultures from HGSOC and nontransformed ovarian fibroblasts from the surrounding ovarium of 45 HGSOC patients. We tested the metabolic flexibility of the primary cells, particularly in response to glucose and glutamine depletion, analyzed and modulated endoplasmic reticulum stress, and searched for indices of the existence of previously reported groups of HGSOC cells with high and low OXPHOS metabolism. RESULTS: The primary HGSOC cells did not form two groups with high and low OXPHOS that responded differently to glucose and glutamine availabilities in the cell culture medium. Instead, they exhibited a continuum of OXPHOS phenotypes. In most tumor cell isolates, the responses to glucose or glutamine withdrawal were mild and surprisingly correlated with those of nontransformed ovarian fibroblasts from the same patients. The growth of tumor-derived cells in the absence of glucose was positively correlated with the lipid trafficking regulator FABP4 and was negatively correlated with the expression levels of HK2 and HK1. The correlations between the expression of electron transport chain (ETC) proteins and the oxygen consumption rates or extracellular acidification rates were weak. ER stress markers were strongly expressed in all the analyzed tumors. ER stress was further potentiated by tunicamycin but not by the recently proposed ER stress inducers based on copper(II)-phenanthroline complexes. ER stress modulation increased autophagy in tumor cell isolates but not in nontransformed ovarian fibroblasts. CONCLUSIONS: Analysis of the metabolism of primary HGSOC cells rejects the previously proposed hypothesis that there are distinct groups of HGSOC cells with high and low OXPHOS metabolism that respond differently to glutamine or glucose withdrawal and are characterized by ETC protein levels.

• Publication type
• Journal Article MeSH

Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.

• MeSH
• Cyclooxygenase 1 * MeSH
• DEAD Box Protein 58 MeSH
• DEAD-box RNA Helicases metabolism   genetics   MeSH
• Liver * metabolism   pathology   MeSH
• Humans MeSH
• Membrane Proteins MeSH
• Mitochondrial Proteins metabolism   genetics   MeSH
• Mitochondria metabolism   MeSH
• Mutation MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Oxidative Phosphorylation * MeSH
• Protein Biosynthesis MeSH
• Reactive Oxygen Species * metabolism   MeSH
• Electron Transport Complex IV * metabolism   genetics   MeSH
• RNA, Mitochondrial genetics   metabolism   MeSH
• Inflammation * metabolism   genetics   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Hypoxia is a common feature of many solid tumors and causes radiotherapy and immunotherapy resistance. Pharmacological inhibition of oxidative phosphorylation (OXPHOS) has emerged as a therapeutic strategy to reduce hypoxia. However, the OXPHOS inhibitors tested in clinical trials caused only moderate responses in hypoxia alleviation or trials were terminated due to dose-limiting toxicities. To improve the therapeutic benefit, FDA approved OXPHOS inhibitors (e.g. atovaquone) were conjugated to triphenylphosphonium (TPP+) to preferentially target cancer cell's mitochondria. In this study, we evaluated the hypoxia reducing effects of several mitochondria-targeted OXPHOS inhibitors and compared them to non-mitochondria-targeted OXPHOS inhibitors using newly developed spheroid models for diffusion-limited hypoxia. METHODS: B16OVA murine melanoma cells and MC38 murine colon cancer cells expressing a HIF-Responsive Element (HRE)-induced Green Fluorescent Protein (GFP) with an oxygen-dependent degradation domain (HRE-eGFP-ODD) were generated to assess diffusion-limited hypoxia dynamics in spheroids. Spheroids were treated with IACS-010759, atovaquone, metformin, tamoxifen or with mitochondria-targeted atovaquone (Mito-ATO), PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) or mitochondria-targeted tamoxifen (MitoTam). Hypoxia dynamics were followed and quantified over time using the IncuCyte Zoom Live Cell-Imaging system. RESULTS: Hypoxic cores developed in B16OVA.HRE and MC38.HRE spheroids within 24 h hours after seeding. Treatment with IACS-010759, metformin, atovaquone, Mito-PEG-ATO and MitoTam showed a dose-dependent reduction of hypoxia in both B16OVA.HRE and MC38.HRE spheroids. Mito-ATO only alleviated hypoxia in MC38.HRE spheroids while tamoxifen was not able to reduce hypoxia in any of the spheroid models. The mitochondria-targeted OXPHOS inhibitors demonstrated stronger anti-hypoxic effects compared to the non-mito-targeted OXPHOS inhibitors. CONCLUSIONS: We successfully developed a high-throughput spheroid model in which hypoxia dynamics can be quantified over time. Using this model, we showed that the mitochondria-targeted OXPHOS inhibitors Mito-ATO, Mito-PEG-ATO and MitoTam reduce hypoxia in tumor cells in a dose-dependent manner, potentially sensitizing hypoxic tumor cells for radiotherapy.

• Publication type
• Journal Article MeSH

Mitochondria are central for cellular metabolism and energy supply. Barth syndrome (BTHS) is a severe disorder, due to dysfunction of the mitochondrial cardiolipin acyl transferase tafazzin. Altered cardiolipin remodeling affects mitochondrial inner membrane organization and function of membrane proteins such as transporters and the oxidative phosphorylation (OXPHOS) system. Here, we describe a mouse model that carries a G197V exchange in tafazzin, corresponding to BTHS patients. TAZG197V mice recapitulate disease-specific pathology including cardiac dysfunction and reduced oxidative phosphorylation. We show that mutant mitochondria display defective fatty acid-driven oxidative phosphorylation due to reduced levels of carnitine palmitoyl transferases. A metabolic switch in ATP production from OXPHOS to glycolysis is apparent in mouse heart and patient iPSC cell-derived cardiomyocytes. An increase in glycolytic ATP production inactivates AMPK causing altered metabolic signaling in TAZG197V . Treatment of mutant cells with AMPK activator reestablishes fatty acid-driven OXPHOS and protects mice against cardiac dysfunction.

• MeSH
• Adenosine Triphosphate MeSH
• Barth Syndrome * metabolism   pathology   MeSH
• Glycolysis MeSH
• Cardiolipins metabolism   MeSH
• Fatty Acids metabolism   MeSH
• Mice MeSH
• AMP-Activated Protein Kinases metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Combined inhibition of oxidative phosphorylation (OXPHOS) and glycolysis has been shown to activate a PP2A-dependent signaling pathway, leading to tumor cell death. Here, we analyze highly selective mitochondrial complex I or III inhibitors in vitro and in vivo to elucidate the molecular mechanisms leading to cell death following OXPHOS inhibition. We show that IACS-010759 treatment (complex I inhibitor) induces a reactive oxygen species (ROS)-dependent dissociation of CIP2A from PP2A, leading to its destabilization and degradation through chaperone-mediated autophagy. Mitochondrial complex III inhibition has analogous effects. We establish that activation of the PP2A holoenzyme containing B56δ regulatory subunit selectively mediates tumor cell death, while the arrest in proliferation that is observed upon IACS-010759 treatment does not depend on the PP2A-B56δ complex. These studies provide a molecular characterization of the events subsequent to the alteration of critical bioenergetic pathways and help to refine clinical studies aimed to exploit metabolic vulnerabilities of tumor cells.

• MeSH
• Autoantigens metabolism   MeSH
• Chaperone-Mediated Autophagy * MeSH
• Energy Metabolism MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Neoplasms * pathology   MeSH
• Oxidative Phosphorylation MeSH
• Protein Phosphatase 2 antagonists & inhibitors   metabolism   MeSH
• Electron Transport Complex I * antagonists & inhibitors   MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH