Exploring the role of A-type lamins in cellular oxidative stress

Every cell contains the genetic information needed to create an entire organism. This blueprint is stored in the cell nucleus. The nucleus continuously regulates the accessibility of this information based on ever-changing intra- and extracellular stimuli. Therefore, proper functioning of the nucleus is crucial for cellular and organismal survival. The nuclear lamina, a perinuclear network composed of type V intermediate filaments called lamins, is emerging as key regulator in nuclear organization. It physically shapes the nucleus, influences gene expression and modulates cell differentiation. A recent addition to the expanding list of functions of the nuclear lamina is an apparent involvement in cellular redox homeostasis. Indeed, cells from patients suffering from various laminopathies display increased levels of intracellular reactive oxygen species (ROS) and often show a higher susceptibility towards induced ROS. The underlying pathways however, remain poorly understood. The goal of this PhD dissertation was to obtain a better insight in this novel putative pathogenic feature. Chapter 1 comprises a general introduction into lamin biology and the state of the art with respect to their involvement in redox biology, next to a guide into fluorescence microscopy of redox-related processes. In Chapter 2, a quantitative comparison and characterization is presented of various experimental perturbations to interfere with lamin A metabolism in primary fibroblast cells. Based on the results shown in this chapter, perturbations were selected to create the models that were used in the experiments in chapters 4 & 5. In chapter 3, the development and benchmarking of a novel high-content microscopy method for the simultaneous measurement of intracellular ROS levels and mitochondrial function is outlined, together with a complementary automated analysis pipeline. The application of the newly developed method from Chapter 3 on the selected models from Chapter 2 culminated in the discovery that distinct lamin variants induce divergent oxidative responses, eventually resulting in different cell fates (Chapter 4), and pointed to the involvement of perturbed protein degradation pathways as a causal factor for oxidative stress (Chapter 5)

In silico synchronization reveals regulators of nuclear ruptures in lamin A/C deficient model cells

The nuclear lamina is a critical regulator of nuclear structure and function. Nuclei from laminopathy patient cells experience repetitive disruptions of the nuclear envelope, causing transient intermingling of nuclear and cytoplasmic components. The exact causes and consequences of these events are not fully understood, but their stochastic occurrence complicates in-depth analyses. To resolve this, we have established a method that enables quantitative investigation of spontaneous nuclear ruptures, based on co-expression of a rmly bound nuclear reference marker and a uorescent protein that shuttles between the nucleus and cytoplasm during ruptures. Minimally invasive imaging of both reporters, combined with automated tracking and in silico synchronization of individual rupture events, allowed extracting information on rupture frequency and recovery kinetics. Using this approach, we found that rupture frequency correlates inversely with lamin A/C levels, and can be reduced in genome- edited LMNA knockout cells by blocking actomyosin contractility or inhibiting the acetyl-transferase protein NAT10. Nuclear signal recovery followed a kinetic that is co-determined by the severity of the rupture event, and could be prolonged by knockdown of the ESCRT-III complex component CHMP4B. In conclusion, our approach reveals regulators of nuclear rupture induction and repair, which may have critical roles in disease development

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

Quantitative proteomics reveals altered expression of actin binding proteins after LMNA knockdown in human dermal fibroblasts

The nuclear lamina physically supports the cell nucleus and has a central role in gene regulation. Mutations in the LMNA gene, which encodes A-type lamins, cause a wide spectrum of tissue-specific and systemic diseases collectively called laminopathies. To elucidate the molecular mechanisms underlying this phenotypic diversity, we set out to identify changes in the proteome upon specific lamin perturbations. More specifically, mature lamin A was reduced by sustained knockdown of LMNA in human dermal fibroblasts. To quantitatively compare protein composition, we made use of SILAC-based shotgun proteomics. The expression of 48 proteins was altered after LMNA knockdown. Gene Ontology analysis of the most significant hits revealed that the largest fraction of the differentially produced proteins in lamin A/C depleted cells were cytoskeletal proteins, more specifically those involved in actin cytoskeleton organization, such as ACTR2 and ACTR3 which are components of the ARP2/3 complex and FSCN1 which plays a critical role in cell migration, motility, adhesion and cellular interactions. Indeed, impaired wound healing and focal adhesion was observed in lamin A/C depleted fibroblasts. Furthermore, decreased expression of FSCN1 was correlated with a downregulation of IL6 and STAT3, which might be the instigators of the altered FSCN1 expression

Perturbation of lamin A maturation alters oxidative stress response in human fibroblasts

The nuclear lamina is located on the inside of the nuclear envelope and plays a key role in nuclear shape and -organization. Mutations in the LMNA gene, encoding A-type lamins, lead to a broad spectrum of diseases termed laminopathies. Many of these diseases are associated with a reduction in the total amount of mature lamin A and an accumulation of intermediate immature prelamin A isoforms. 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 certain laminopathies are accompanied by altered levels of reactive oxygen species (ROS) and a higher susceptibility to oxidative stress on the cellular level (Caron et al., 2007; De Vos et al., 2011). However, to date, the underlying molecular mechanisms and functional consequences for the cell remain elusive. Moreover, it is not yet known how widespread this phenomenon is. To provide more insights in this pathway, we have implemented a dynamic, multiparametric image-based assay to measure oxidative stress in human fibroblasts after directed perturbation of lamin A metabolism. To this end, cells were either treated with drugs that induce accumulation of specific prelamin A isoforms or they were transfected with silencing or overexpression constructs. Using live cell imaging in conjunction with high content cytometry, a quantitative comparison was made of the levels of reactive oxygen species between different conditions. Significant differences were detected between control cells and treated cells, suggesting the involvement of ROS as a widespread phenomenon in the development of laminopathies

High-content analysis of cellular oxidative stress

Reactive oxygen species (ROS) are small, short lived molecules that mediate various cellular responses including cell proliferation, differentiation, gene expression and migration. Excessive accumulation of ROS however, can lead to DNA damage and the build-up of irreversibly oxidized proteins. To counter the potential damaging effects of ROS, cells have evolved several antioxidant systems, including ROS defusing enzymes and vitamins. An imbalance between production of oxidative radicals and antioxidant mechanisms induces a state of oxidative stress, a phenomenon associated with various systemic diseases and aging. One of the major sources of ROS is the mitochondrion, which harbours the electron transport chain, handling highly active electrons in close proximity to oxygen. To study the relationship between ROS levels and mitochondrial (dys-) function in various contexts, we have developed an integrated workflow for measurement of intracellular ROS levels, mitochondrial potential and mitochondrial morphology. To this end, human dermal fibroblasts, cultured in glass-bottom 96-well plates, were stained with a fluorescent ROS-sensitive dye (pan-cellular (CM-H2DCFDA) or mitochondrially targeted (MitoSOX)), or a mitochondrial membrane potential-sensitive dye (TMRM), after which they were acquired in a fully automated manner and analysed using home-written image analysis protocols (RedoxMetrics and MitoMetrics). In fluxo chemical perturbation allowed determining the dynamic range and sensitivity towards the applied stressors. In case of mitochondrial analysis, mitochondria were specifically enhanced using a multiscale Laplacian operator and an exhaustive morphological and textural feature extraction was performed. Using this approach, we showed that specific chemical perturbations selectively affect cellular ROS levels or mitochondrial morphology