Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction.

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells.

Lamins as mediators of oxidative stress.

The nuclear lamina defines both structural and functional properties of the eukaryotic cell nucleus. Mutations in the LMNA gene, encoding A-type lamins, lead to a broad spectrum of diseases termed laminopathies. While different hypotheses have been postulated to explain disease development, there is still no unified view on the mechanistic basis of laminopathies. Recent observations indicate that laminopathies are often accompanied by altered levels of reactive oxygen species and a higher susceptibility to oxidative stress at the cellular level. In this review, we highlight the role of reactive oxygen species for cell function and disease development in the context of laminopathies and present a framework of non-exclusive mechanisms to explain the reciprocal interactions between a dysfunctional lamina and altered redox homeostasis.

Rosiglitazone Protects Endothelial Cells From Irradiation-Induced Mitochondrial Dysfunction.

BACKGROUND AND PURPOSE: Up to 50-60% of all cancer patients receive radiotherapy as part of their treatment strategy. However, the mechanisms accounting for increased vascular risks after irradiation are not completely understood. Mitochondrial dysfunction has been identified as a potential cause of radiation-induced atherosclerosis. MATERIALS AND METHODS: Assays for apoptosis, cellular metabolism, mitochondrial DNA content, functionality and morphology were used to compare the response of endothelial cells to a single 2 Gy dose of X-rays under basal conditions or after pharmacological treatments that either reduced (EtBr) or increased (rosiglitazone) mitochondrial content. RESULTS: Exposure to ionizing radiation caused a persistent reduction in mitochondrial content of endothelial cells. Pharmacological reduction of mitochondrial DNA content rendered endothelial cells more vulnerable to radiation-induced apoptosis, whereas rosiglitazone treatment increased oxidative metabolism and redox state and decreased the levels of apoptosis after irradiation. CONCLUSION: Pre-existing mitochondrial damage sensitizes endothelial cells to ionizing radiation-induced mitochondrial dysfunction. Rosiglitazone protects endothelial cells from the detrimental effects of radiation exposure on mitochondrial metabolism and oxidative stress. Thus, our findings indicate that rosiglitazone may have potential value as prophylactic for radiation-induced atherosclerosis.

Oral microbiota reduce wound healing capacity of epithelial monolayers, irrespective of the presence of 5-fluorouracil.

Oral mucositis is still one of the most painful side effects of chemotherapeutic treatment and a mounting body of evidence suggests a key role for the oral microbiome in mucositis development. However, the underlying mechanisms remain elusive. In this work, we have investigated the interactions between the host, the microbiome, and chemotherapeutic treatments in more detail. The effect of 5-fluorouracil, commonly inducing mucositis, was assessed on a co-culture model that consists of an epithelial cell layer and a biofilm derived from oral microbiota from different types of samples (saliva, buccal swabs and tongue swabs) and donors (healthy individuals and patients suffering from mucositis). After 24 h co-incubation, all oral microbial samples were found to reduce wound healing capacity with 26 ± 15% as compared with untreated condition. Compared with saliva and tongue samples, buccal samples were characterized by lower bacterial cell counts and hence higher wound healing capacity. For samples from healthy individuals, an inverse correlation was observed between bacterial cell counts and wound healing capacity, whereas for patients suffering from mucositis no correlation was observed. Moreover, patient-derived samples had a less diverse microbial community and higher abundances of pathogenic genera. No major impact of 5-fluorouracil on wound healing capacity or the composition of the microbiome was seen at physiologically relevant concentrations in the mouth. In conclusion, bacterial cell count is inversely correlated with wound healing capacity, which emphasizes the importance of oral hygiene during oral wound healing in healthy individuals. However, future research on extra measures besides oral hygiene is needed to assure a good wound healing during mucositis, as for patients the bacterial composition seems also crucial. The direct effect of 5-fluorouracil on both the microbiome and wound healing is minimal, pointing to the importance of the host and its immune system in chemotherapy-induced microbial shifts. Impact statement Chemotherapy-induced oral mucositis has a major impact on the quality of life of patients. The additional costs and treatment time associated with this pathology are significant. Although the pathology of the disease is well understood, the role and importance of oral microbiota currently are less clear. In this study, we focused on the effect of oral microbiota on wound healing, the final phase of oral mucositis, during 5-FU exposure. We show that the bacterial load and composition have a major impact on the healing process in contrast to 5-FU which only marginally slows down healing. This emphasizes the importance of good oral health care during oral mucositis to minimize bacterial load around the oral lesions. However, since we show that also the composition of the oral microbiome plays a role in wound recovery, the identification of specific pathogenic species or their metabolites might be worthwhile to allow proper treatment.

Cellular Redox Profiling Using High-content Microscopy.

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (ΔΨm) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (ΔΨm and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins.

The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the lamina's major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.

Sustained accumulation of prelamin A and depletion of lamin A/C both cause oxidative stress and mitochondrial dysfunction but induce different cell fates.

The cell nucleus is structurally and functionally organized by lamins, intermediate filament proteins that form the nuclear lamina. Point mutations in genes that encode a specific subset of lamins, the A-type lamins, cause a spectrum of diseases termed laminopathies. Recent evidence points to a role for A-type lamins in intracellular redox homeostasis. To determine whether lamin A/C depletion and prelamin A accumulation differentially induce oxidative stress, we have performed a quantitative microscopy-based analysis of reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in human fibroblasts subjected to sustained siRNA-mediated knockdown of LMNA and ZMPSTE24, respectively. We measured a highly significant increase in basal ROS levels and an even more prominent rise of induced ROS levels in lamin A/C depleted cells, eventually resulting in Δψm hyperpolarization and apoptosis. Depletion of ZMPSTE24 on the other hand, triggered a senescence pathway that was associated with moderately increased ROS levels and a transient Δψm depolarization. Both knockdowns were accompanied by an upregulation of several ROS detoxifying enzymes. Taken together, our data suggest that both persistent prelamin A accumulation and lamin A/C depletion elevate ROS levels, but to a different extent and with different effects on cell fate. This may contribute to the variety of disease phenotypes witnessed in laminopathies.