Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and... Show moreAnalysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies. Show less
Replication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue... Show moreReplication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue the deficits due to Rev1 loss remain unclear. Here, we report that loss of Rev1 leads to intense changes in metabolites and that this can be manipulated by NAD + supplementation. Autophagy decreases in Rev1-/- mouse embryonic fibroblasts (MEFs) and can be restored by supplementing the NAD+ precursor nicotinamide riboside (NR). The abnormal mitochondrial morphology in Rev1-/-MEFs can be partially reversed by NR supplementation, which also protects the mito-chondrial cristae from rotenone-induced degeneration. In nematodes rev-1 deficiency causes sensitivity to oxidative stress but this cannot be rescued by NR supplementation. In conclusion, Rev1 deficiency leads to metabolic dysregulation of especially lipid and nucleotide metabolism, impaired autophagy, and mitochondrial anomalies, and all of these phenotypes can be improved by NR replenishment in MEFs. Show less
The prevalence of cardiovascular diseases has increased in the last decennia. This thesis studied the potential relationship between oxidative stress and cardiovascular diseases. The first part of... Show moreThe prevalence of cardiovascular diseases has increased in the last decennia. This thesis studied the potential relationship between oxidative stress and cardiovascular diseases. The first part of the thesis focused on anti-oxidants, which are scavengers that protect against oxidative damage. We studied the association between several lifestyle factors, diet, physical activity, sleep, alcohol intake and smoking, and antioxidant levels both in blood and urine. Subsequently, we investigated whether a higher concentration of antioxidants leads to a decrease in ischaemic stroke occurrence. Next, we aimed to study a possible cause of oxidative damage and its effect on cardiovascular disease. Mitochondrial dysfunction is one of the mechanisms that may underly this effect. Mitochondria are an important source of Reactive Oxygen Species (ROS), as an inevitable byproduct of their essential role in energy production. A disbalance in ROS production and scavenging might result in oxidative damage. Thus, we investigated the causal association between mitochondrial dysfunction and stroke using the Mendelian Randomization method. Finally, we studied how socio-demographic traits could modify the causal association between CVD risk factors and coronary artery disease. Show less
Stel, W. van der; Yang, H.; Le Dévédec, S.E.; Water, B. van de; Beltman, J.B.; Danen, E.H.J. 2022
Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation,... Show moreCells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals. Show less
Sampadi, B.; Mullenders, L.H.F.; Vrieling, H. 2022
Background: Although cancer risk is assumed to be linear with ionizing radiation (IR) dose, it is unclear to what extent low doses (LD) of IR from medical and occupational exposures pose a cancer... Show moreBackground: Although cancer risk is assumed to be linear with ionizing radiation (IR) dose, it is unclear to what extent low doses (LD) of IR from medical and occupational exposures pose a cancer risk for humans. Improved mechanistic understanding of the signaling responses to LD may help to clarify this uncertainty. Here, we per -formed quantitative mass spectrometry-based proteomics and phosphoproteomics experiments, using mouse embryonic stem cells, at 0.5 h and 4 h after exposure to LD (0.1 Gy) and high doses (HD; 1 Gy) of IR. Results: The proteome remained relatively stable (29; 0.5% proteins responded), whereas the phosphoproteome changed dynamically (819; 7% phosphosites changed) upon irradiation. Dose-dependent alterations of 25 IR-responsive proteins were identified, with only four in common between LD and HD. Mitochondrial metabolic proteins and pathways responded to LD, whereas transporter proteins and mitochondrial uncoupling pathways responded to HD. Congruently, mitochondrial respiration increased after LD exposure but decreased after HD exposure. While the bulk of the phosphoproteome response to LD (76%) occurred already at 0.5 h, an equivalent proportion of the phosphosites responded to HD at both time points. Motif, kinome/phosphatome, kinase-substrate, and pathway analyses revealed a robust DNA damage response (DDR) activation after HD exposure but not after LD exposure. Instead, LD-irradiation induced (de)phosphorylation of kinases, kinase-substrates and phosphatases that predominantly respond to reactive oxygen species (ROS) production. Conclusion: Our analyses identify discrete global proteome and phosphoproteome responses after LD and HD, uncovering novel proteins and protein (de)phosphorylation events involved in the dose-dependent ionizing ra-diation responses. Show less
Chemical-induced organ toxicity is a major concern in the development and use of chemicals, including drugs, pesticides, industrial chemicals and cosmetics. In the last decades, the onset and... Show moreChemical-induced organ toxicity is a major concern in the development and use of chemicals, including drugs, pesticides, industrial chemicals and cosmetics. In the last decades, the onset and progression of chemical-induced organ toxicity has been linked amongst others to perturbation of mitochondria.In this work, we set out to further unravel the mechanisms behind mitochondrial toxicity initiated by a chemical insult. More in-depth and biology driven assessment of the onset and progression of mitochondrial perturbation supports the development of relevant prediction tools. We aimed to stimulate the integration of these in vitro and in silico prediction tools in the safety assessment of existing and new chemicals, to allow detailed assessment of mitochondrial fitness upon chemical exposure. Show less
Neurodegenerative diseases, including Parkinson’s disease (PD), are increasing in prevalence due to the aging population. Despite extensive study, these diseases are still not fully understood and... Show moreNeurodegenerative diseases, including Parkinson’s disease (PD), are increasing in prevalence due to the aging population. Despite extensive study, these diseases are still not fully understood and the lack of personalised treatment options that can target the cause of the diseases, rather than the symptoms, has led to a greater demand for improved disease understanding, therapies and diagnostic procedures. In this thesis, we use systems biology approaches to construct disease-specific models intended for biomarker discovery, therapeutic treatment strategy identification and drug repurposing in PD. Systems biology is a mathematical field of research that analyses biological systems via construction of a computational model using experimental data. This is achieved by integration of omics data, including genomics, proteomics, transcriptomics and metabolomics. A specific approach used to identify the physico- and biochemical bounds within a biological system is constraint-based modelling, which requires the input of absolute quantitative metabolomics data. To improve our absolute quantitative coverage of the metabolome, we developed and improved new quantitative metabolomics methods using a targeted mass spectrometry workflow to obtain data intended to be integrated into constraint-based metabolic models for the study of PD. Show less
Purpose: This study was designed to identify mitochondrial (mt) DNA variations in primary and metastatic uveal melanoma (UM) cell lines and their relation with cell metabolism to gain insight into... Show morePurpose: This study was designed to identify mitochondrial (mt) DNA variations in primary and metastatic uveal melanoma (UM) cell lines and their relation with cell metabolism to gain insight into metastatic progression.Method: The entire mtDNA genomes were sequenced using Sanger sequencing from two primary UM cell lines (92.1 and MEL270) and two cell lines (OMM2.3 and OMM2.5) derived from liver metastases of the MEL270 patient. The mtDNA copy numbers determined by the ratio of nDNA versus mtDNA. qRT-PCR was used to evaluate expression levels of mitochondrial biogenesis genes.Results: Sequencing showed that cell line MEL270 and metastases-derived OMM2.3 and OMM2.5 cell lines had homoplasmic single nucleotide polymorphisms (SNPs) representing J1c7a haplogroup, whereas 92.1 cells had mtDNA H31a haplogroup. mtDNA copy numbers were significantly higher in primary cell lines. The metastatic UM cells showed down-regulation of POLG, TFAM, NRF-1 and SIRT1 compared to their primary MEL270 cells. PGC-1 alpha was downregulated in 92.1 and upregulated in MEL270, OMM2.3 and OMM2.5.Conclusions: Our finding suggests that within metastatic cells, the heteroplasmic SNPs, copy numbers and mitochondrial biogenesis genes are modulated differentially compared to their primary UM cells. Therefore, investigating pathogenic mtDNA variants associated with cancer metabolic susceptibility may provide future therapeutic strategies in metastatic UM. (C) 2021 Published by Elsevier Inc. Show less
Metabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile... Show moreMetabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile technique for the elaboration of physiology and disease, studying metabolism using standard cell culture protocols is profoundly interfered by the Crabtree effect. This phenomenon refers to the adaptation of cultured cells to a glycolytic phenotype, away from oxidative phosphorylation in glucose-containing medium, and questions the applicability of cell culture in certain fields of research. In this systematic review we aim to provide a comprehensive overview and critical appraisal of strategies reported to circumvent the Crabtree effect. Show less
Kok, M.J.C. de; Schaapherder, A.F.; Wüst, R.C.I.; Zuiderwijk, M.; Bakker, J.A.; Lindeman, J.H.N.; Le Dévédec S.E. 2021
Metabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile... Show moreMetabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile technique for the elaboration of physiology and disease, studying metabolism using standard cell culture protocols is profoundly interfered by the Crabtree effect. This phenomenon refers to the adaptation of cultured cells to a glycolytic phenotype, away from aoxidative phosphorylation in glucose-containing medium, and questions the applicability of cell culture in certain fields of research. In this systematic review we aim to provide a comprehensive overview and critical appraisal of strategies reported to circumvent the Crabtree effect. Show less
Chondrosarcoma is the second most common primary bone malignancy, representing one fourth of all primary bone sarcomas. It is typically resistant to radiation and chemotherapy treatments. However,... Show moreChondrosarcoma is the second most common primary bone malignancy, representing one fourth of all primary bone sarcomas. It is typically resistant to radiation and chemotherapy treatments. However, the molecular mechanisms that contribute to cancer aggressiveness in chondrosarcomas remain poorly characterized. Here, we studied the role of mitochondrial transporters in chondrosarcoma aggressiveness including chemotherapy resistance. Histological grade along with stage are the most important prognostic biomarkers in chondrosarcoma. We found that high-grade human chondrosarcoma tumors have higher expression of the mitochondrial protein, translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20), compared to low-grade tumors. TOMM20 overexpression in human chondrosarcoma cells induces chondrosarcoma tumor growth in vivo. TOMM20 drives proliferation, resistance to apoptosis and chemotherapy resistance. Also, TOMM20 induces markers of epithelial to mesenchymal transition (EMT) and metabolic reprogramming in these mesenchymal tumors. In conclusion, TOMM20 drives chondrosarcoma aggressiveness and resistance to chemotherapy. Show less
Calcium is the third most abundant metal on earth, and the fundaments of its homeostasis date back to pre-eukaryotic life forms. In higher organisms, Ca2+ serves as a cofactor for a wide array of ... Show moreCalcium is the third most abundant metal on earth, and the fundaments of its homeostasis date back to pre-eukaryotic life forms. In higher organisms, Ca2+ serves as a cofactor for a wide array of (enzymatic) interactions in diverse cellular contexts and constitutes the most important signaling entity in excitable cells. To enable responsive behavior, cytosolic Ca2+ concentrations are kept low through sequestration into organellar stores, particularly the endoplasmic reticulum (ER), but also mitochondria and lysosomes. Specific triggers are then used to instigate a local release of Ca2+ on demand. Here, communication between organelles comes into play, which is accomplished through intimate yet dynamic contacts, termed membrane contact sites (MCSs). The field of MCS biology in relation to cellular Ca2+ homeostasis has exploded in recent years. Taking advantage of this new wealth of knowledge, in this Review, we invite the reader on a journey of Ca2+ flux through the ER and its associated MCSs. New mechanistic insights and technological advances inform the narrative on Ca2+ acquisition and mobilization at these sites of communication between organelles, and guide the discussion of their consequences for cellular physiology. Show less
Stel W. van der, Carta G., Eakins J., Darici S., Delp J., Forsby A., Bennekou S.H., Gardner I., Leist M., Danen E.H.J., Walker P., Water B. van de, Jennings P. 2020
Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic... Show moreEvidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition. Show less
Pluijm, I. van der; Burger, J.; Heijningen, P.M. van; Ijpma, A.; Vliet, N. van; Milanese, C.; ... ; Essers, J. 2018
Aim Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms underlying... Show moreAim Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms underlying aneurysm formation.Methods and results In Fibulin-4(R/R) mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed proteomics and genomics studies on Fibulin-4(R/R) mouse aortas. Intriguingly, we observed alterations in mitochondrial protein composition in Fibulin-4(R/R) aortas. Consistently, functional studies in Fibulin-4(R/R) vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates. Yet, mitochondria in Fibulin-4(R/R) VSMCs showed no aberrant cytoplasmic localization. We found similar reduced mitochondrial respiration in Tgfbr-1(M318R/+) VSMCs, a mouse model for Loeys-Dietz syndrome (LDS). Interestingly, also human fibroblasts from Marfan (FBN1) and LDS (TGFBR2 and SMAD3) patients showed lower oxygen consumption. While individual mitochondrial Complexes I-V activities were unaltered in Fibulin-4(R/R) heart and muscle, these tissues showed similar decreased oxygen consumption. Furthermore, aortas of aneurysmal Fibulin-4(R/R) mice displayed increased reactive oxygen species (ROS) levels. Consistent with these findings, gene expression analyses revealed dysregulation of metabolic pathways. Accordingly, blood ketone levels of Fibulin-4(R/R) mice were reduced and liver fatty acids were decreased, while liver glycogen was increased, indicating dysregulated metabolism at the organismal level. As predicted by gene expression analysis, the activity of PGC1 alpha, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4(R/R) VSMCs. Increased TGF beta reduced PGC1 alpha levels, indicating involvement of TGF beta signalling in PGC1 alpha regulation. Activation of PGC1 alpha restored the decreased oxygen consumption in Fibulin-4(R/R) VSMCs and improved their reduced growth potential, emphasizing the importance of this key regulator.Conclusion Our data indicate altered mitochondrial function and metabolic dysregulation, leading to increased ROS levels and altered energy production, as a novel mechanism, which may contribute to thoracic aortic aneurysm formation. Show less
Pluijm, I. van der; Burger, J.; Heijningen, P.M. van; Ijpma, A.; Vliet, N. van; Milanese, C.; ... ; Essers, J. 2018
This thesis provides insights into the mechanisms of renal I/R injury based on human kidney transplantation (i.e. the status of delayed graft function: DGF). A severe energetic crisis... Show moreThis thesis provides insights into the mechanisms of renal I/R injury based on human kidney transplantation (i.e. the status of delayed graft function: DGF). A severe energetic crisis differentiates DGF kidneys from adequately functioning controls. Although intact beta-oxidation, aerobic glycolysis and glutaminolysis provide Krebs Cycle intermediates, these intermediates are not able to enter the mitochondrial Krebs cycle. Hence, dysfunctional mitochondria disable efficient ATP production leading to the metabolic incompetence that causes DGF and underlies renal I/R injury. This finding sheds a whole new light on I/R injury and explains why ATP-dependent therapeutics are ineffective as treatment for I/R injury. A major difference in the vulnerability of mitochondria to ischemia and reperfusion between rodents and humans was found. This could explain the current differences in effectiveness of therapies in the experimental versus the clinical setting. Big cohort studies give insights in donor, recipient and transplant-procedure variables and challenge the reluctance towards the use of DCD donor kidneys. New preventive strategies could limit I/R injury by preserving mitochondria (hypothetically with peptide SS-31 or activation of mitochondrial aldehyde dehydrogenase). This will overcome the detrimental effects of I/R injury on graft function and survival - thereby increasing the success rate of kidney transplantation. Show less
