Title Promoter Affiliations Abstract "Small heat shock proteins (HSPBs) sequestration at the outer mitochondrial membrane abrogates oxidative stress induced intrinsic apoptosis by preventing the release of cytochrome c from the mitochondria." "Vincent Timmerman" "Peripheral Neuropathies Group" "Small heat shock proteins (HSPBs) are ATP-independent chaperones that are involved in maintaining the proteome integrity by preventing aberrant protein aggregation. They form highly dynamic, polydisperse oligomeric ensembles, and contain long intrinsically disordered regions. Experimental challenges posed by these aforementioned properties have greatly hindered our understanding of HSPBs. Unpublished work from our lab has shown that HSPBs execute an unexpected dual role in mitochondrial protein quality control. We found out that HSPBs are able to translocate to the mitochondrial intermembrane space (IMS) under basal condition where they prevent protein aggregation; however, after heat shock (42°C for 1 hour) to induce protein misfolding, they immediately translocate to the outer mitochondrial membrane (OMM) for a reason that is unknown. Remarkable is that a P182L mutant of HSPB1 (causing peripheral neuropathy) exhibits the enrichment of the HSPB1 protein on the OMM even under the basal condition, triggering mis-signaling of an unknown pathway that subsequently leads to mitochondrial dysfunctions. In this PhD proposal, I will systematically characterize the function of HSPBs on the OMM, identify their upstream regulators for immediate translocation to the OMM post heat shock and the turn of events following it, which is particularly relevant under different cellular states." "Mitochondrial dysfunction in LRRK2-linked Parkinson's disease: analysis of the role of the LRRK2 substrate Rab10 in mitochondrial quality control" "Wim Vandenberghe" "Laboratory for Parkinson Research" "Parkinson’s disease (PD) is a highly prevalent, disabling neurodegenerative disorder for which no cure exists yet. In this project we will use PD patient fibroblasts and dopaminergic neurons differentiated from patient fibroblast-derived iPSCs to dissect the pathogenic effects of mutations in the LRRK2 gene, the most common cause of autosomal dominant PD. LRRK2 encodes an enzyme with a kinase and a GTPase domain. The small GTPase Rab10 was recently identified as a physiological substrate of the kinase activity of LRRK2. We found that LRRK2 mutations impair mitophagy, a mitochondrial quality control pathway that is also disrupted in recessive forms of PD. In addition, we have evidence that Rab10 actively contributes to mitophagy and that this role of Rab10 is disturbed by mutant LRRK2. Using a variety of biochemical and cell biological approaches we will determine the mechanistic basis for the involvement of Rab10 in mitochondrial quality control. This work may help define new targets for disease-modifying therapies for PD." "The role of peroxisomes in hepatocytes and pancreatic Beta-cells in mouse. Relationship of peroxisomes with mitochondrie" "Myriam Baes" "Cell Metabolism, Laboratory of Peroxisome Biology and Intracellular Communication" "Peroxisomes are plastic organelles, present in almost all eukaryotic cells, which play an essential role in intermediary lipid metabolism. Inherited diseases with defects in peroxisomal function give rise to multiple organ defects which have been mimicked in mouse models for these diseases. However, their particular function in different cell types and tissues remains largely obscure. In this thesis we investigated the consequences of peroxisome inactivity in hepatocytes and in pancreatic β-cells. Whereas, historically, liver was the tissue in which peroxisomal function was first and most extensively studied, only scarce information is hitherto available on peroxisomes in β-cells of the pancreas.In early reports on Zellweger syndrome patients, lacking functional peroxisomes, hepatic mitochondrial abnormalities were documented which were later recapitulated in mouse models in which the peroxisome biogenesis factor PEX5 was deleted from hepatocytes. In the latter, the mitochondrial ultrastructural changes were accompanied by a reduced mitochondrial membrane potential and reduced activities of the respiratory complexes I, III and V. Our aim was to search for the mechanisms linking peroxisome dysfunction to mitochondrial disruption. We showed that the mitochondrial anomalies are associated with increased reactive oxygen species. Furthermore, as the ultrastructure of mitochondria and activity of complex I are unchanged in brain, muscle and heart lacking functional peroxisomes these mitochondrial abnormalities appeared to be hepatocyte selective. We further investigated whether peroxisomal metabolites that are specifically enriched in hepatocytes could be a causative factor by either manipulating their levels or comparing their levels in different mouse models of peroxisomal β-oxidation deficiency and correlating them with complex I activity. The severe reduction in the levels of docosahexaenoic acid (DHA) in phospholipids of mitochondria from Pex5-/- hepatocytes could be restored by administering DHA orally to L-Pex5-/- mice. However, no improvements were seen in the complex I activity after the treatment. Furthermore, wild type mice treated with phytol diet accumulated significant amounts of branched chain fatty acids (BCFAs) – phytanic and pristanic acid but did not show any impairment in complex I activity in liver. Moreover, dicarboxylic acids (DCAs) were not found to accumulate in the livers of L-Pex5-/- mice. Also complex I activity in the livers of Mfp1-/- mice fed coconut diet which were previously shown to accumulate DCAs was not decreased. Lastly, we measured the levels of the presumed mito-toxic bile acid intermediates, DHCA and THCA in the livers of adult and prenatal mice lacking either PEX5 or the peroxisomal β-oxidation enzyme MFP2. However, the levels did not correlate with the observed mitochondrial dysfunction. In summary, although we could not find the exact link between absence of peroxisomes and mitochondrial problems, we could exclude the role of depletion of DHA and accumulation of BCFAs, DCAs or bile acid intermediates in mediating the mitochondrial abnormalities.Previous literature supported contrasting ideas about peroxisomal metabolism being either harmful or beneficial for the functioning of β-cells. Therefore, the second aim of this thesis was to investigate the role of peroxisomes in pancreatic β-cells by generating and phenotyping β-cell specific Pex5 knockout mice (Rip-Pex5-/-). We found that glucose homeostasis in these mutant mice is disturbed which was characterized by increased fed as well as fasted blood glucose levels and glucose intolerance. The circulating insulin levels were reduced after a bolus of glucose in Rip-Pex5-/- mice which can be attributed to reduction in total pancreatic insulin content as well as β-cell mass. No changes were found in the glucose stimulated insulin secretion ex vivo, insulin content per islet and cytoplasmic as well as mitochondrial ROS production in cultured islet cells. However, the mitochondrial membrane potential was increased in cultured islet cells of mutant mice. Taken together, these results suggest that peroxisomes contribute to the normal functioning of healthy β-cells, however at this moment we were unable to unravel the mechanisms behind this relationship.Overall, this work pointed towards an essential role of peroxisomes in the normal functioning of hepatocytes and pancreatic β-cells, two cell types of utmost importance in metabolic processes." "Design, synthesis and screening of tRNA stabilising and antigenomic compounds for the treatment of mitochondrial diseases." "Annemieke Madder" "Department of Organic and Macromolecular Chemistry" "Mitochondria are referred to as the powerhouse or batteries of the cell, this is because they produce energy for the cell. To generate this energy (ATP) mitochondria build their own machinery, using mitochondrial DNA (mtDNA) as a blueprint. Errors in the mtDNA are rare, but do occur, which can lead to severe and progressive diseases often with fatal outcome. There is currently no cure for mitochondrial diseases, and since the mtDNA is well defended by 2 layers of membranes, gene therapy is not as accessible as for other genetic diseases. In this project we will explore 2 different routes towards a treatment for mitochondrial diseases. In the first route, we will design chaperone molecules able to stabilise critical but defective nucleic acid components of the mitochondrial machinery involved in translating the blueprints for normal protein synthesis in the mitochondrion (the cause of the more common mitochondrial diseases), thus restoring ATP production. In the second route, we will attempt to remove the faulty DNA from the mitochondria in a specific way, leaving only healthy DNA. To do this, we will design a special molecule to target the mutant mtDNA, and inhibit its replication, while any remaining healthy mtDNA can replicate undisturbed. The discoveries made in this project will serve as a platform, from which we can further develop possible therapies for mitochondrial diseases." "Mitochondrial DNA content in cardiovascular ageing and disease: a population study." "Tatiana Kouznetsova" "Hypertension and Cardiovascular Epidemiology, Environment and Health" "A major burden of modern society is the progressive increase in age-associated diseases such as heart failure (HF). The genetic and environmental drivers of left ventricular (LV) dysfunction progression remain to be elucidated. LV dysfunction is associated with changes in cardiac energy metabolism. Mitochondria play a central role in a variety of cardiac cell functions. The mitochondrial DNA (mtDNA) content correlates with the size and number of mitochondria, which change under pathological conditions, including HF. Hence, biomarkers of mitochondria might be important in disease prediction and to date its association with the progression of LV dysfunction has not been studied. Understanding the role of mitochondrial function in the transition from LV dysfunction to HF necessitates a multidisciplinary approach crossing the borders between clinical and laboratory medicine. We address the objectives by adding a prospective dimension to the unique epidemiological resources on LV structure and function available at the Studies Coordinating Centre, KU Leuven. This project also relies on cutting-edge technologies for measurement of mtDNA content, telomeres, oxidative gene expression and exposure assessment of particulate air pollution available within the Centre for Environmental Sciences, Hasselt University. The objectives of this study are: (1)To measure mtDNA content and its dynamic in a longitudinal population study; (2) To investigate the impact on the mtDNA of telomere biology and oxidative genes; (3) To determine the influence of mtDNA content and its dynamic on LV structure and function; (4) To study the role of environmental exposure to particulate air pollution on the previously mentioned associations." "Endothelial mitochondria: enigmatic function and potential target for tumor anti-angiogenesis?" "Peter Carmeliet" "Laboratory of Angiogenesis and Vascular Metabolism (VIB-KU Leuven)" "Blood vessel formation (angiogenesis) promotes tumor growth and metastasis. Current antiangiogenic therapies target endothelial growth factors, but suffer from limited efficacy. Also, since tumor vessels are structurally and functionally abnormal (which impairs perfusion), pruning them by anti-angiogenic therapy may create a more hostile nutrient-deprived milieu, promoting cancer cell escape and dissemination. A novel paradigm is to “normalize” the tumor vessels to improve perfusion, which reduces metastasis and improves chemotherapy. The host lab pioneered the novel concept that endothelial cells (ECs) reprogram their metabolism to form new blood vessels. They discovered an important role of glycolysis and fatty acid oxidation in angiogenesis, and that blocking these pathways inhibits pathological angiogenesis. However, the role of mitochondria in ECs during angiogenesis remains unknown. Using a multidisciplinaryapproach involving EC cultures and genetic mouse models with dysfunctional mitochondria in ECs, I will study the role of mitochondria in tumor angiogenesis and vessel normalization, characterize the underlying cellular and metabolic mechanisms, and explore which metabolites are altered upon EC mitochondrial dysfunction, as this may identify novel metabolic targets for future antiangiogenesis approaches in cancer therapy. This project will yield the first conclusive in vivo genetic evidence about the role and importance of mitochondria for (tumor) angiogenesis." "Mitochondrial homeostasis and its importance for neurodegenerative disorders." "Bart De Strooper" "Laboratory for the Research of Neurodegenerative Diseases (VIB-KU Leuven)" "Parkinson Disease is the second most important neurodegenerative disorder. Research has shown that mutations in a particular gene (PINK1) can cause the disease by affecting the mitochondria, the energy factories of the brain. PINK1 is cleaved by a molecular scissor, PARL, which is sitting in the mitochondria. A second protein, called PGAM5 is also cleaved by PARL. We think that these three proteins together are very important to maintain the health of mitochondria. We have seen that in mice, in which we have deleted the molecular scissor PARL, a severe degeneration of the brain occurs, and this is paralleled by a strong accumulation of the two substrates PGAM5 and PINK1. We wonder whether the accumulation of these substrates causes the neurodegeneration in those mice, and we will therefore lower the expression of these proteins in these mice without PARL to see whether this improves the neurodegeneration. We propose experiments in mice and in cell cultures to understand how these three proteins work together. The fact that mutations in PINK1 (substrate), and possibly in PARL (the protease) can cause Parkinson Disease, indicates that our work will teach us not only the importance of the interplay of these proteins to keep mitochondria healthy, but will also provide important novel insights in a pathway important for Parkinson Disease." "'The role of ATP13A2 in mitochondrial functionality'" "Peter Vangheluwe" "Laboratory of Cellular Transport Systems, Translational Research in GastroIntestinal Disorders, Molecular Biotechnology of Plants and Micro-organisms" "ATP13A2 is a late endolysosomal P5B-type transport ATPase that exports polyamines from the late endolysosome to the cytosol. Loss-of-function mutations in this transporter lead to a range of neurodegenerative disorders, which are all characterized by increased oxidative stress and a flawed mitochondrial-lysosomal axis at the cellular level. In addition, overexpression of ATP13A2 in cell models has been reported to provide protection against rotenone, an environmental Parkinson’s disease risk factor and mitochondrial toxin. By exporting polyamines from the lysosome, ATP13A2 prevents lysosomal swelling, rupture and subsequent cathepsin B-dependent cell death, thereby contributing to its neuroprotective effect. However, it remained unclear how ATP13A2 provides mitochondrial protection and whether this involves lysosomal polyamine transport.In this PhD project, we found that polyamines transported by ATP13A2 complement polyamine synthesis in the mitigation of mitochondrial-generated reactive oxygen species (mitoROS). This is a conserved pathway, as key findings were recapitulated in patient fibroblasts with ATP13A2 mutations and in vivo in a C. elegans model. Cells deficient for ATP13A2 or overexpressing a catalytically dead ATP13A2 mutant were sensitized to rotenone, represented by increased mitochondrial superoxide generation, the induction of a mitoROS-dependent stress response, and cell death. We showed that this mitochondrial protective effect of ATP13A2 is at least in part independent of the lysosomal phenotype, since endocytosis of acidic nanoparticles - which rescues the lysosomal pH and functionality - was unable to decrease mitoROS. We also revealed the cellular route of polyamines taken up via ATP13A2, displaying their redistribution to mitochondria in an ATP13A2-dependent manner, indicating that these polyamines may exert their ROS-scavenging effect locally inside mitochondria.Moreover, in this PhD project we investigated the impact of disease-associated variants on ATP13A2 activity and on other Parkinson’s disease linked proteins with a role in mitochondrial-lysosomal communication. We documented that several ATP13A2 variants affected the ATPase activity, polyamine uptake potential and subcellular location demonstrating their pathogenic effect.Finally, we generated novel tools to further dissect the role of ATP13A2 in mitochondrial-lysosomal interplay. Organelle immunoprecipitation techniques allowed us to isolate highly pure and intact mitochondria and lysosomes, which uncovered subcellular lipid changes in ATP13A2 deficient cells. We also developed and validated photocrosslinkable polyamine probes to determine other polyamine handling proteins and transporter(s) under control of ATP13A2 in the future.In conclusion, we uncovered a highly conserved antioxidative pathway mediated by ATP13A2’s polyamine transport function that protects against mitochondrial oxidative stress. In addition, we described the functional impact of ATP13A2 disease variants, developed organelle- and substrate-specific tools to identify new players under control of ATP13A2, and we revealed alterations in the subcellular lipidome of ATP13A2 deficient cells." "Identification of proteases that activate membrane-bound transcription factors during mitochondrial retrograde regulation" "Inge De Clercq" "Department of Plants and Crops, Department of Plant Biotechnology and Bioinformatics" "Plants are our main food and feed source, but they often get threatened by adverse environments in which they must adapt to survive. These adverse environments due to (a)biotic factors cause major yield losses, that are expected to become worse due to climate change, raising the demand for stress tolerant crops. Mitochondria contain their own genome whose expression needs to be coordinated with the nuclear genome. This resulted in an extensive mitochondria-to-nucleus signalling network: ‘mitochondrial retrograde regulation’ (MRR). Plant mitochondria have a major function in energy production but altering mitochondrial function also has drastic effects on the plant’s resistance to various (a)biotic stresses, indicating that mitochondria play a crucial role in sensing and reporting stress signals to the nucleus. The host lab previously identified a novel MRR pathway mediated by NAC transcription factors (TF) that are stored at ER membranes. Upon mitochondrial perturbation by stresses, these TFs are released from the ER membranes and translocated to the nucleus, but the underlying molecular mechanisms of how mitochondria signal to activate these TFs in the ER are not understood. This project aims to identify the proteases responsible for NAC cleavage using state-of-the-art proteomics and chemical-biology approaches and thereby aims to expand our fragmentary understanding of how mitochondria communicate to other organelles and eventually the nucleus during plant stress responses." "Mitochondrial adaptations in the adjacent and remote myocardium of a chronic myocardial infarction" "Experimental Cardiology, Cardiovascular Imaging and Dynamics" "Scarring and remodelling of the left ventricle (LV) after myocardial infarction (MI) results in ischemic cardiomyopathy with reduced contractile function. Regional differences related to persisting ischemia may exist. We investigated the hypothesis that mitochondrial function and structure is altered in the myocardium adjacent to MI with reduced perfusion (MI adjacent ) and less so in the remote, nonischemic myocardium (MI remote). We used a pig model of chronic coronary stenosis and MI (n = 13). Functional and perfusion MR imaging 6 wk after intervention showed reduced ejection fraction and increased global wall stress compared with sham-operated animals (Sham; n = 14). Regional strain in MI adjacent was reduced with reduced contractile reserve; in MI remote strain was also reduced but responsive to dobutamine and perfusion was normal compared with Sham. Capillary density was unchanged. Cardiac myocytes isolated from both regions had reduced basal and maximal oxygen consumption rate, as well as through complex I and II, but complex IV activity was unchanged. Reduced respiration was not associated with a detectable reduction of mitochondrial density. There was no significant change in AMPK or glucose transporter expression levels, but glycogen content was significantly increased in both MI adjacent and MI remote. Glycogen accumulation was predominantly perinuclear; mitochondria in this area were smaller but only in MI adjacent where also subsarcolemmal mitochondria were smaller. PN mitochondria also have different behaviour. They can show oscillation independently from the IMF mitochondria population, suggesting they have a separate network. In conclusion, after MI reduction of mitochondrial respiration and glycogen accumulation occur in all LV regions suggesting that reduced perfusion does not lead to additional specific changes and that increased hemodynamic load is the major driver for changes in mitochondrial function."