ERC FUNDED PROJECTS

New neurons are continuously generated in certain regions of adult mammalian brain. One of those regions is the dentate gyrus, a subregion of hippocampus, which is essential for memory formation. Although these new neurons in the adult dentate gyrus are thought to have an important role in learning and memory, it is largely unclear how new neurons are involved in information processing and storage underlying memory. Because new neurons constitute a minor portion of intermingled local neuronal population, simple application of conventional techniques such as multi-unit extracellular recording and pharmacological lesion are not suitable for the functional analysis of new neurons. In this proposed research program, I will combine multi-unit recording and behavioral analysis with virus mediated, cell-type-specific genetic manipulation of neuronal activity, to investigate computational roles of new neurons in learning and memory. Specifically, I will determine: 1) specific memory processes that require new neurons, 2) dynamic patterns of activity that new neurons express during memory-related behavior, 3) influence of new neurons on their downstream structure. Further, based on the information obtained by these three lines of studies, we will establish causal relationship between specific memory-related behavior and specific pattern of activity in new neurons. Solving these issues will cooperatively provide important insight into the understanding of computational roles performed by adult neurogenesis. The information on the function of new neurons in normal brain could contribute to future development of efficient therapeutic strategy for a variety of brain disorders.

1 991 743 €

Start date: 2009-01-01, End date: 2013-12-31

Angiogenesis research has focused on the sprouting of new capillaries. The mechanisms of vessel maturation are much less well understood. Yet, the maintenance of a mature, quiescent, and organotypically-differentiated layer of endothelial cells (ECs) lining the inside of all blood vessels is vital for human health. The goal of ANGIOMATURE is to identify, validate, and implement novel mechanisms of vascular maturation and organotypic EC differentiation that are active during development, maintenance of vascular stability in adults, and undergo changes in aging. We recently identified previously unrecognized gene expression signatures of vascular maturation in a genome-wide screen of ECs isolated from newborn and adult mice. Epigenetic mechanisms were identified that control the EC transcriptome through gain and loss of DNA methylation as well as EC differentiation and signaling specification. These findings pave the way for groundbreaking novel opportunities to study vascular maturation. By characterizing functionally diverse types of blood vessels, including continuous ECs in lung and brain and sinusoidal ECs in liver and bone marrow, the ANGIOMATURE project will (1) determine up to single cell resolution the transcriptional and epigenetic program(s) of vascular maturation and organotypic differentiation during adolescence, (2) analyze the functional consequences of such program(s) in differentiated ECs and their adaptation to challenge, and (3) study changes of maturation and differentiation program(s) and vascular responses during aging. We will towards this end employ an interdisciplinary matrix of approaches involving omics, systems biology, conditional gene targeting, organoid cell culture, and experimental pathology to create a high-resolution structural and functional organotypic angioarchitectural map. The project will thereby yield transformative mechanistic insights into vital biological processes that are most important for human health and healthy aging.

2 338 918 €

Start date: 2018-08-01, End date: 2023-07-31

Despite improved therapy, cardiovascular diseases remain the most prevalent diseases in the European Union and the incidence is rising due to increased obesity and ageing. The fine-tuned regulation of vascular functions is essential not only for preventing atherosclerotic diseases, but also after tissue injury, where the coordinated growth and maturation of new blood vessels provides oxygen and nutrient supply. On the other hand, excessive vessel growth or the generation of immature, leaky vessels contributes to pathological angiogenesis. Thus, the regulation of the complex processes governing vessel growth and maturation has broad impacts for several diseases ranging from tumor angiogenesis, diabetic retinopathy, to ischemic cardiovascular diseases. MicroRNAs (miRs) are small noncoding RNAs, which play a crucial role in embryonic development and tissue homeostasis. However, only limited information is available regarding the role of miRs in the vasculature. MiRs regulate gene expression by binding to the target mRNA leading either to degradation or to translational repression. Because miRs control patterns of target genes, miRs represent an attractive and promising therapeutic target to interfere with complex processes such as neovascularization and repair of ischemic tissues. Therefore, the present application aims to identify miRs in the vasculature, which regulate vessel growth and vessel remodelling and may, thus, serve as therapeutic targets in ischemic diseases. Since ageing critically impairs endothelial function, neovascularization and vascular repair, we will specifically identify miRs, which are dysregulated during ageing in endothelial cells and pro-angiogenic progenitor cells, in order to develop novel strategies to rescue age-induced impairment of neovascularization. Beyond the specific scope of the present application, the principle findings may have impact for other diseases, where deregulated vessel growth causes or accelerates disease states.

2 375 394 €

Start date: 2009-03-01, End date: 2014-02-28

Atherosclerosis is characterized by chronic inflammation of the arterial wall. Mononuclear cell recruitment is driven by chemokines that can be deposited e.g. by activated platelets on inflamed endothelium. Chemokines require oligomerization and immobilization for efficient function, and recent evidence supports the notion that heterodimer formation between chemokines constitutes a new regulatory principle amplifying specific chemokine activities while suppressing others. Although crucial to inflammatory disease, this has been difficult to prove in vivo, primarily as chemokine heterodimers exist in equilibrium with their homodimer counterparts. We introduce the paradigm that heteromerization of chemokines provides the combinatorial diversity for functional plasticity and fine-tuning, coining this interactome. Given the relevance of chemokine heteromers in vivo, we aim to exploit this in an anti-inflammatory approach to selectively target vascular disease. In a multidisciplinary project, we plan to generate covalently-linked heterodimers to establish their biological significance. Obligate heterodimers of CC and CXC chemokines will be designed using computer-assisted modeling, chemically synthesized and cross-linked, structurally assessed using NMR spectroscopy and crystallography, and subjected to functional characterization in vitro and reconstitution in vivo. Conversely, we will develop cyclic beta-sheet-based peptides binding chemokines to specifically disrupt heteromers and we will generate mice with conditional deletion or knock-in of chemokine mutants with defects in heteromerization or proteoglycan binding to be analyzed in models of atherosclerosis. Peptides will be used for molecular imaging and chemokine heteromers will be quantified in cardiovascular patients.

2 500 000 €

Start date: 2010-04-01, End date: 2016-03-31

B-cell malignancies are characterized by high levels of genomic instability, which critically contribute to their pathogenesis and evolution. Recently, the fundamental role of the ubiquitin proteasome system (UPS) in maintaining genome integrity has been appreciated. Two major new therapeutic modalities in B-cell malignancies, proteasome inhibitors and imunomodulatory drugs (IMiDs), target the UPS and demonstrate particular efficacy in multiple myeloma (MM) and mantle cell lymphoma (MCL), two incurable entities with poor prognosis. This suggests the presence of aberrant ubiquitylation events, whose identities have however remained mostly elusive. Our recent studies identify fundamental roles of orphan ubiquitin ligases of the Cullin Ring ligase family (CRLs) and their counterparts, the deubiquitylating enzymes (DUBs) in the cellular DNA damage response machinery, and characterize these candidates as novel oncogenes or tumour suppressors in MM and MCL. These findings provide the foundation for our hypothesis that deregulated ubiquitylation events involving CRLs and DUBs have a far reaching impact on the pathogenesis of B-cell malignancies and can serve as new therapeutic targets and biomarkers. We therefore propose a multistep strategy in which we will (1) characterize previously orphan CRLs and DUBs, which we have distinguished as candidate oncogenes and tumour suppressors in MM (FBXO3, USP24), MCL (FBXO25), or MM and MCL (CRBN), respectively; (2) decipher the global role of CRLs and DUBs in MM and MCL using defined genetic screens; (3) identify relevant substrates of CRLs/DUBs discovered in (2) using mass spectrometry; and (4) validate CRL/DUB candidates in preclinical mouse models and defined patient cohorts as to their disease relevance. We expect that our interdisciplinary approach will unravel the overall role of the UPS in the pathophysiology, evolution and treatment of B-cell malignancies.

1 973 255 €

Start date: 2016-09-01, End date: 2021-08-31

The heart is one of the first and most complex organs formed during human embryogenesis. While its anatomy and physiology have been extensively studied over centuries, the normal development of human heart and dysregulation in disease still remain poorly understood at the molecular/cellular level. Stem cell technologies hold promise for modelling development, analysing disease mechanisms, and developing potential therapies. By combining multidisciplinary approaches centred on human induced pluripotent stem cells (hiPSCs), BIOCARD aims at decoding the cellular and molecular principles of human cardiogenesis and developing advanced inter-chimeric human-pig models of cardiac development and disease. State-of-the-art genetic modification techniques and functional genomics will be used to establish a molecular atlas of cell type intermediates of human cardiogenesis in vitro and unravel how their proliferation, differentiation and lineage choice are regulated in health and disease. This in vitro approach will be complemented by detailed analyses of how distinct hiPSC-derived cardiac progenitor populations commit and contribute to specific cardiac compartments in interspecies chimeric hearts in vivo. Finally, we will capitalize on the novel concept that combinations of different well-defined hiPSC-derived cardiac progenitor pools with timely-matched, native extracellular matrix from embryonic hearts will accomplish for the first time the realization of human heart organoids as 3D culture systems of developing heart structures. Clearly, BIOCARD will open game-changing opportunities for devising novel biomedical applications, such as human heart chamber-specific disease modelling, large-scale drug testing in appropriate human 3D cardiac bio-mimics, and regenerative cell therapies based on functional ventricular-muscle patches and direct cell conversion in vivo.

2 285 625 €

Start date: 2018-09-01, End date: 2023-08-31

Cardiac connective tissue is regarded as passive in terms of cardiac electro-mechanics. However, recent evidence confirms that fibroblasts interact directly with cardiac muscle cells in a way that is likely to affect their beat-by-beat activity. To overcome limitations of traditional approaches to exploring these interactions in native tissue, we will build and explore murine models that express functional reporters (membrane potential, Vm; calcium concentration, [Ca2+]i) in fibroblasts, to identify how they are functionally integrated in native heart (myocyte => fibroblast effects). Next, we will express light-gated ion channels in murine fibroblast, to selectively interfere with their Vm (fibroblast => myocyte effects). Fibroblast-specific observation and interference will be conducted in normal and pathologically remodelled tissue, to characterise fibroblast relevance for heart function in health & disease. Based on these studies, we will generate 2 transgenic rabbits (fibroblast Vm reporting / interfering). Rabbit cardiac structure-function is more amenable to translational work, e.g. to study fibroblast involvement in normal origin & spread of excitation across the heart, in pathological settings such as arrhythmogenicity of post-infarct scars (a leading causes of sudden death), or as a determinant of therapeutic outcomes such as in healing of atrial ablation lines (interfering with a key interventions to treat atrial fibrillation). The final ‘blue-skies’ study will assess whether modulation of cardiac activity, from ‘tuning’ of biological pacemaker rates to ‘unpinning’ / termination of re-entrant excitation waves, can be achieved by targeting not myocytes, but fibroblasts. The study integrates basic-science-driven discovery research into mechanisms and dynamics of biophysical myocyte-fibroblast interactions, generation of novel transgenic models useful for a broad range of studies, and elucidation of conceptually new approaches to heart rhythm management.

2 498 612 €

Start date: 2013-07-01, End date: 2019-06-30

Contraction and relaxation of cardiac myocytes, and thus the whole heart, are critically dependent on dyads. These functional junctions between t-tubules, which are invaginations of the surface membrane, and the sarcoplasmic reticulum allow efficient control of calcium release into the cytosol, and also its removal. Dyads are formed gradually during development and break down during disease. However, the precise nature of dyadic structure is unclear, even in healthy adult cardiac myocytes, as are the triggers and consequences of altering dyadic integrity. In this proposal, my group will investigate the precise 3-dimensional arrangement of dyads and their proteins during development, adulthood, and heart failure by employing CLEM imaging (PALM and EM tomography). This will be accomplished by developing transgenic mice with fluorescent labels on four dyadic proteins (L-type calcium channel, ryanodine receptor, sodium-calcium exchanger, SERCA), and by imaging tissue from explanted normal and failing human hearts. The signals responsible for controlling dyadic formation, maintenance, and disruption will be determined by performing high-throughput sequencing to identify novel genes involved with these processes in several established model systems. Particular focus will be given to investigating left ventricular wall stress and stretch-dependent gene regulation as controllers of dyadic integrity. Candidate genes will be manipulated in cell models and transgenic animals to promote dyadic formation and maintenance, and reverse dyadic disruption in heart failure. The consequences of dyadic structure for function will be tested experimentally and with mathematical modeling to examine effects on cardiac myocyte calcium homeostasis and whole-heart function. The results of this project are anticipated to yield unprecedented insight into dyadic structure, regulation, and function, and to identify novel therapeutic targets for heart disease patients.

2 000 000 €

Start date: 2015-07-01, End date: 2020-06-30

Tetralogy of Fallot (TOF) is the most common congenital heart disease (CHD) occurring 1 in 3000 births. Genetic studies have identified numerous genes that are responsible for inherited and sporadic forms of TOF, most of which encode key molecules that are part of regulatory networks controlling heart development. The identification of two populations of cardiac precursors, one exclusively forming the left ventricle and the second the outflow tract, the right ventricle and the atria, has suggested a new approach to interpret CHDs, in particular in TOF, not as a defect in a specific gene, but rather as a defect in the formation, expansion, and differentiation of defined subsets of embryonic cardiac precursors. The LIM-homeodomain transcription factor ISL1 marks the second population of cardiac progenitors, but little is known about its downstream targets, and how causative genes of CHDs affect cell-fate decisions in the ISL1 lineage. The main goals of this research program are: (1) to decipher the functional role of Isl1 downstream targets identified by a genome-wide ChIP-Seq approach; (2) to generate induced pluripotent stem (iPS) cells from controls and patients affected by severe forms of TOF characterized by defects in heart compartments known to derive from ISL1 cardiac progenitors; (3) to direct these iPS cells to ISL1+ cardiovascular precursors and identify cell-surface makers enabling their antibody-based purification; and (4) to use TOF-iPS-derived ISL1+ progenitors as an unique in vitro model system for deciphering molecular mechanisms that govern the fates and differentiation of this progenitor lineage and determine the pathological phenotype seen in TOF. This work will shed light on the molecular mechanisms of ISL1+ cardiac progenitor lineage specification and will give important new insights into the mechanisms of how alterations in transcriptional and epigenetic programs translate to a distinct structural defect during cardiogenesis.

1 499 996 €

Start date: 2011-03-01, End date: 2017-02-28

Heart failure has become a worldwide epidemic with more than 23 million people affected resulting in devastating consequences for patients and an enormous burden on health care systems. One in five heart failure patients dies within a year of diagnosis and survival estimates after diagnosis are 50% and 10% at 5 and 10 years, respectively. Despite intensive investigation, the molecular mechanisms leading to heart failure remain poorly understood. We will narrow this critical gap in knowledge by proposing a previously unattainable, comprehensive approach to define the genomic architecture and functional consequences of newly identified micropeptides from multiple classes of RNAs that previously were classified to be non-coding in cardiac biology and heart failure. Our approach is unique and novel in several ways. Thematically, our studies focus on novel classes of orphan peptides and their role in heart failure that have not been discovered previously. Our approach relies on innovative interdisciplinary efforts of scientists working in molecular genetics, genomics, computational biology, and cardiovascular research to identify and characterize pathophysiological pathways that converge on these novel peptides. We will identify these short peptides by using genome-wide measures of active translation and will harness unique clinical resources to ensure human relevance. Analysis of animal and cell models coupled with state-of-the-art biochemical and genetic tools will elucidate the function of newly identified micropeptides within the molecular and cellular pathways of cardiac biology and failure. Through these efforts we will discern fundamental causes of maladaptive responses in the heart and strategies to monitor and limit these.

2 319 514 €

Start date: 2019-01-01, End date: 2023-12-31

The regulation of ion concentrations in the cytoplasm and in the lumen of intracellular vesicles provides suitable environments for biochemical reactions, gradients for signal transduction, and generates osmotic gradients for the regulation of the volume of cells and intracellular organelles. Changes in the ion homeostasis and volume of cells and organelles may in turn influence processes like cell division and migration or the budding of vesicles from cellular membranes. Volume changes of cells, and possibly also of intracellular organelles, in turn regulate ion transport across their membranes. Whereas several swelling-activated plasma membrane ion transporters and channels are known, the molecular identity of a key player, the swelling-activated anion channel VRAC, and its impact on cellular functions remain elusive. Only sketchy information is available on ion homeostasis and volume regulation of intracellular organelles like endosomes and lysosomes, in spite of their importance for several diseases. We propose to perform a genome-wide RNAi screen to finally identify the long-sought swelling-activated Cl- channel VRAC at the molecular level. This screen will also identify genes involved in the regulation of VRAC. The network involved in cell volume regulation will be investigated at the structural, biochemical and cellular level as well as with genetically modified mice. In parallel we will examine the ion homeostasis of endosomes and lysosomes. Until recently only the regulation of luminal H+ and Ca++ concentration was studied, but our recent work demonstrated a crucial role of luminal Cl- and hinted at an important role of cations. A combination of proteomics, siRNA screens, candidate approaches, and mouse models will be used to elucidate the ion homeostasis of endosomes/lysosomes and the impact on organellar function and associated pathologies. We expect that our work will break new ground in ion transport physiology, pathology, and cell biology.

2 499 600 €

Start date: 2012-04-01, End date: 2017-03-31

Current estimates place the prevalence of obesity beyond 1 billion by the year 2030. As a critical risk factor for heart disease, diabetes and stroke, obesity represents one of the chief socio-economic challenges of our day. While studies have mapped a genetic framework for understanding obesity, the etiological contribution of several regulatory layers, and in particular epigenetic regulation, remain poorly understood. A perfect example, we know that isogenic C57Bl6/J mice can vary by as much as 100% in body weight upon high fat feeding; currently, we have no mechanistic explanation for the emergence of such phenotypic variation. Here, I propose three aims dedicated towards understanding the (epi)genetic control of phenotypic variation and disease susceptibility. First, we will catalogue epigenome and phenome variation to an unprecedented depth and resolution in the isogenic context. Next, we will examine two completely novel models of epigenetically sensitized bi-stable obesity and thus begin a mechanistic dissection of phenotypic variation. Finally, we will map a series of gene-gene and gene-environment epistasis interactions including eight models of developmental plasticity and approximately a dozen chromatin regulator mutants. The latter epistasis matrix will identify the molecular mechanisms that trigger, amplify and buffer phenotypic variation and stochastic obesity in mice. The functional (epi)phenomics approach is unique. It builds the first unbiased framework against which to understand developmental plasticity and phenotypic variation, and at the same time generates powerful resources for disease researchers worldwide.

1 997 853 €

Start date: 2017-01-01, End date: 2021-12-31

Dynamic signalling networks in Diabetic Nephropathy (DN) – New avenues to a personalized therapy.- We have developed an exquisite experimental platform that facilitates the systematic unravelling of the signalling networks leading to (1) the initiation, (2) the progression and (3) the potential regeneration of podocytes in DN, paving the way to novel therapeutic strategies: (1) DN initiation: Identification of signalling cascades leading to microalbuminuria: Molecular By combining transgenic Drosophila lines carrying secreted fluorescent proteins to monitor the barrier function in vivo with a genome-wide siRNA screen we will establish a unique system to directly identify gene networks contributing to microalbuminuria. (2a) DN progression: Molecular fingerprinting of podocyte degeneration: Based on a transgenic fluorescent mouse model, we have pioneered a highly efficient podocyte purification method from type1 and type 2 diabetic mice allowing us to develop a precise molecular genetic, quantitative proteomic and micro RNA fingerprint from freshly isolated podocytes from diabetic and non-diabetic mice. (2b) DN progression: We established a proteomic approach to measure site-specific phosphorylation dynamics in primary podocyte cultures originating from transgenic mice that are TORC1 deficient, TORC2 deficient or TORC1 hyperactive (TSC1 KO) solely in the podocytes. (3) Potential role of podocyte regeneration in DN: Finally, to target mechanisms that could potentially reverse the disease process (by repopulating lost podocytes), we invented a strategy to quantitatively monitor podocyte turnover from different stem cell niches allowing us to precisely assess and potentially manipulating the capacity of podocyte regeneration in DN.

1 999 920 €

Start date: 2014-06-01, End date: 2019-05-31

The bone marrow (BM) represents the prime location in which cancer cells survive aggressive treatments. This poses a major health challenge because the mortality rate of patients with curable cancer doubles if tumor cells persist in the BM. One unresolved question is why the immune system fails to eradicate cancer cells from this microenvironment even though it represents a lymphatic organ. Surprisingly, despite the ongoing revolution in immune oncology, the regulation and potential therapeutic activation of the immune response in the BM still remains largely unexplored. In this grant application a new line of research is proposed with the overall objective of understanding the cellular and molecular mechanisms, which control anti-cancer immune responses in the BM. The originality of this proposal relates to the hypothesis that innate and adaptive immune cells are suppressed by stroma cells in the BM. Therefore, we will conduct a comprehensive phenotypic and functional profiling of immune and stroma cells in the BM of cancer patients with and without persisting tumor cells. Based on these insights we will develop novel strategies to harness the immune system to eliminate malignant cells from the BM. The ground-breaking nature of the project is that it will shed light on the unappreciated immune microenvironment in the BM. Its specific strength lies in the multidisciplinary design encompassing informative patient cohorts, state-of-the-art mouse models and cutting-edge technologies including Next-Generation-Sequencing as well as innovative drug candidates. Hereby I can build on my internationally recognized expertise in the BM microenvironment field, which has already led to the successful development of a clinical-stage drug. Novel strategies to eliminate malignant cells from the bone marrow are of utmost medical importance because they would increase the cure rate of cancer patients.

1 490 825 €

Start date: 2018-02-01, End date: 2023-01-31

Social neuroscientists study the neural mechanisms underlying our capacity to understand our own and other people’s feelings. Despite neuroscientists’ advances in plasticity research and empathy research, little is known about cortical and behavioural plasticity in emotion understanding and empathy. Clearly, in today’s world, acquiring the capacity to effectively enhance empathy and prosocial behaviour is of the utmost importance. In the present project, we will investigate the malleability of empathy via training. We will adopt a multimethod and interdisciplinary approach, combining techniques and paradigms from the fields of neuroscience, (bio-)psychology, and economics. Studies 1-3 will provide a cross-sectional look at structural and functional differences in the brains of individuals scoring high vs. low on empathy, of those with pathological deficits in empathy (psychopaths, alexithymics), and of individuals starting vs. finishing a three-year training program in Carl Rogers’ person-centred therapy, which aims to increase emotional capacity and empathy. Study 4 will examine brain plasticity using real-time fMRI: Participants will learn to self-regulate brain activity through the use of immediate feedback from emotion-related brain areas while practicing certain mental techniques. In Study 5, a small-scale longitudinal study, healthy individuals will receive extensive training by professional instructors in either empathy- or memory-enhancing techniques previously developed in the East and the West. We will measure training-related changes in brain structure and functioning, in hormone levels, and in behaviour. Evidence for emotional brain plasticity in adults and children would not only have important implications for the implementation of scientifically validated, effective training programs for schools and for economic and political organizations, but also for the treatment of the marked social deficits in autistic and psychopathic populations.

1 499 821 €

Start date: 2008-09-01, End date: 2014-08-31

The diseases of older age are a major challenge to human societies. Despite the complexity of ageing, both reduced food intake (dietary restriction) and simple genetic alterations can greatly increase lifespan and provide broad-spectrum protection against diseases of ageing in laboratory animals. Furthermore, there is strong evolutionary conservation of mechanisms. For instance the nutrient-sensing insulin/IGF and TOR signalling network modulates lifespan in yeast, invertebrates and rodents. There is thus a major scientific opportunity to use model organisms to discover how to ameliorate ageing and hence to protect against ageing-related disease in humans. Our recent findings on dietary amino acid balance in the fruit fly Drosophila imply that consumption of nutrients irrelevant to metabolism is life-shortening. Using a novel genomic approach, we shall determine if the same is true in mice and measure the role of dietary imbalance in extension of lifespan by dietary restriction. Late life dietary restriction in invertebrates can increase future survival as much as permanent restriction, implying that chemical mimetics administered late in life could also be fully effective. We shall determine if dietary restriction in mice has similarly acute effects, and use dietary switches to identify candidate mechanisms of increased health and lifespan. Recent evidence has pointed to particular components of nutrient-sensing pathways as promising drug targets for prevention of age-related disease, and we shall investigate two candidates. The work will break new ground in understanding how ageing is modulated by diet and signaling pathways and point to interventions that could protect against the effects of ageing to reduce the burden of ageing-related disease in humans.

2 489 200 €

Start date: 2011-06-01, End date: 2016-05-31

Epigenetic reprogramming is a hallmark of brain cancer. Remarkably, driver mutations of the histone H3.3 variant and its loading machinery have been recently found in paediatric glioblastoma multiforme (GBM), a devastating neoplasm originating from transformed neural precursors. Thus, the very basic building blocks of chromatin can be mutated in cancer. The present challenge is to define at which level altered H3.3 loading influences GBM pathogenesis and provide clues into the underlying mechanisms. Based on work from our group and others, we hypothesise that alterations of H3.3 function/deposition would lead to epigenetic changes, deregulated transcription at bivalent loci and other genomic regions, and alterations of telomere maintenance mechanisms, in turn contributing to tumourigenesis. The main objectives of this proposal are to: 1. Examine the impact of H3.3 mutations on brain cancer pathogenesis, by determining the effect of mutant H3.3 expression on neural precursor cell transformation (A), and tumour maintenance (B). 2. Define the molecular changes caused by incorporation of H3.3 mutants into the genome and their involvement in tumourigenesis, by A. determining the genome-wide distribution of WT and mutant H3.3 proteins, B. identifying mutant H3.3-driven transcriptional and epigenetic changes, C. defining effects on telomere maintenance mechanisms, and D. connecting mutant H3.3-driven molecular changes to the biological phenotypes. The discovery of mutations in histones and their loading machinery represents a paradigm change in the field of cancer epigenetics. We anticipate this study to provide key insights into the role of these alterations in chromatin regulation and cancer pathogenesis. More broadly, this work will increase our understanding of the fundamental mechanisms governing chromatin modification in mammalian cells.

1 999 998 €

Start date: 2014-05-01, End date: 2019-04-30

Obesity is a metabolic disorder leading to various health risks and reduced life expectancy. Insulin resistance is a major obesity related disorder, and a main cause for the onset of type 2 diabetes. During cold exposure or caloric restriction (CR), brown adipocytes emerge within the white fat (known as “beige” cells). This process, referred to as fat browning, increases the metabolic capacity of the adipose tissues to combust energy and is seen as promising anti-obesity and anti-diabetic strategy. The intestinal microbiota co-develops with the host; microbiota depletion, or cold-induced shift of its composition are sufficient to improve insulin sensitivity and glucose metabolism, in part mediated by the innate immune system-mediated fat browning. The microbial signals and composition, critical for our understanding of the microbiota-host mutualism and metabolic improvements during cold and CR, remain unclear. By integrating expertise from several areas including physiology, bioinformatics, immunology, microbiology and developmental biology; and by developing computational approaches for comparing the metagenomics, metabolomics and transcriptomics data from the CR- and the cold-exposed mice with cohorts of human subjects, we will establish the microbiota role in orchestrating the CR-induced metabolic improvements and innate immune response, and provide mechanistic explanations on the microbiota-host mutualism during CR and cold. Finally, by using lineage-tracing studies and developing transgenic mouse models, we will determine the importance of the beige fat in the CR-induced beneficial effects on the host, and the importance of the microbiota in mediating this process. Manipulating the gut microbiota and exploiting the mechanistic links revealed by this study would be of conceptual importance for our understanding of microbiota-host mutualism in the metabolic homeostasis, and could lead to development of novel therapeutics for improving metabolic health.

1 999 999 €

Start date: 2019-06-01, End date: 2024-05-31

Overweight and metabolic syndrome are reaching pandemic dimensions in industrialized countries and are rising in developing countries. Clinically these diseases can manifest in non-alcoholic fatty liver disease (NAFLD), the most frequent liver disease world-wide. A significant number of NAFLD patients develop non-alcoholic steatohepatitis (NASH), fibrosis and hepatocellular carcinoma (HCC), making NASH-driven HCC the most rapidly increasing cancer in the USA, with a similar trend in Europe. While HCC is the second most common cause of cancer related death, the mechanisms triggering NASH and subsequent HCC are poorly understood and efficacious therapies are lacking. My group has strong expertise in inflammation-driven HCC (e.g. by Hepatitis B, C viruses). Recently, we have established a mouse model of NASH-driven HCC recapitulating human pathology in the context of metabolic syndrome. We demonstrated for the first time that CD8+ T- and natural killer T (NKT)-cells become activated during metabolic syndrome, cross-talk with hepatocytes and alter hepatic lipid metabolism causing NASH and HCC. We found an identical profile of CD8+T and NKT-cell activation in human NASH underlining the clinical relevance of our model. As the mechanisms of immune cell activation in NASH and transition to HCC remain unknown, this research proposal aims to (1) Identify the priming cell types in metabolic CD8+ T-, NKT-cell activation and the molecular mechanisms of immune cell-hepatocyte crosstalk. (2) Determine the role of antigen recognition and danger- or pathogen-associated molecular patterns in NASH/HCC. (3) Identify the environmental and genetic determinants of NASH to HCC transition. Our findings will enhance the understanding of NASH and HCC development by identifying the underlying mechanisms of immune cell activation. We will identify genetic changes facilitating NASH to HCC transition and whether metabolic normalization of former NASH patients suffices to significantly reduce HCC.

1 995 860 €

Start date: 2016-07-01, End date: 2021-06-30

Despite educational, political and biomedical research efforts, obesity, type 2 diabetes and related metabolic diseases are increasing worldwide at an alarming rate. Failure to deliver efficient and safe medical therapies is a result of our incomplete understanding of the pathogenesis of the metabolic syndrome. Current knowledge implicates impaired CNS control over appetite, body weight and systemic metabolism as a key pathogenic process leading to obesity and type 2 diabetes. Yet, years of intense study focused on neuronal signalling have produced no transformative breakthroughs. Control centers located in the hypothalamic arcuate nucleus (ARC) serve as primary targets of afferent hormone signals regulating systemic control of body weight and metabolism and appear to be most affected by high fat high sugar (HFHS) diets. Recently, we discovered that in the early stages of the metabolic syndrome induced by consumption of a HFHS diet, significant changes beyond neuronal pathologies occur in hypothalamic nuclei responsible for metabolic control, such as the ARC. Specifically, we observe in hypothalami of mice, rats and humans increased reactive microgliosis and astrocytosis as well as a decline in regulatory T-cell presence. We hypothesize that hypothalamic inflammatory processes triggered by hypercaloric environments impair hormone sensing, disrupt glial homeostasis and incapacitate these hypothalamic control centers, ultimately contributing to development of obesity and diabetes. Building on a considerable body of preliminary data, we will apply an array of advanced technologies to A) develop a functional understanding of the pathophysiology of diet-induced hypothalamic inflammation, B) test if hypothalamic inflammation plays a critical role in the development of the metabolic syndrome, and C) attempt to target these novel pathogenic processes for the first time using novel targeted therapeutic approaches.

2 402 280 €

Start date: 2016-09-01, End date: 2021-08-31

We have previously demonstrated that cellular senescence opposes tumorigenesis thereby opening up new potential opportunities for cancer treatment. Senescence and tumor immunity in cancer are tightly interconnected. Tumor-infiltrating immune cells promote the clearance of senescent tumor cells thereby contributing to the tumor suppressive function of senescence. Moreover, T lymphocytes can drive senescence in cancers by secreting different cytokines in the tumor microenvironment. We have also recently reported that GR1+ myeloid cells antagonize treatment-induced senescence (TIS) and that compounds that block the tumor recruitment of GR1+ cells enhance TIS. Major objective of this proposal is to characterize the immune landscape of different prostate cancer mouse models in order to develop novel treatment modalities that combine pro-senescence compounds with immunotherapy. Using proteomics and bioinformatics approaches, we will assess how the genetic background of prostate tumors, shapes the tumor microenvironment and immune response during TIS. Next, we will define the mechanisms that regulate the recruitment and activation of myeloid derived suppressive cells, macrophages and B-lymphocytes in Pten deficient prostate tumors by focusing on a novel class of secreted factors identified in these tumors. We will also assess in vivo whether the secretome of tumor cells can transmit senescence to TILs and compounds that interfere with the secretome can prevent immunosenescence. Finally, we will develop monoclonal antibodies directed towards senescent tumors cells that we will use as diagnostic and therapeutic tools. These antibodies will be used as biomarkers to detect senescent tumor cells in prostate cancers and will be tested in pre-clinical trials to assess whether they improve tumor clearance during TIS. Our findings will form the basis for future clinical trials in prostate cancer patients.

2 000 000 €

Start date: 2016-07-01, End date: 2021-06-30

Glucose sensing cells constantly monitor glucose absorption from food and variations in blood glycemic levels. They control the secretion of GLP-1, insulin and glucagon, and the activity of the autonomic nervous system. These hormonal and nervous signals coordinate glucose utilization by liver, fat and muscle, and endogenous glucose production as well as feeding and energy expenditure. Type 2 diabetes, a disease that afflicts an increasing proportion of the world population, is characterized by insufficient insulin production by pancreatic beta-cells, abnormal secretion of GLP-1 and glucagon, and is often associated with imbalance between feeding and energy expenditure. Type 2 diabetes can thus be considered a disease of glucose sensing. Here, I propose a research program using cell biological, genetic, genomic and physiology techniques to investigate three aspects of this integrated glucose sensing network: 1. The identification of novel molecular pathways activated by GLP-1 and that control adult beta-cell proliferation, glucose competence and apoptosis in order to maintain sufficient insulin secretion capacity. 2. The identification and molecular characterization of brain glucose sensors, which share functional similarities with pancreatic beta-cells, and which control glucose homeostasis and pancreatic islet mass and function. 3. The discovery by unbiased genetic-genomic analysis of loci, genes, and gene networks involved in central hypoglycemia detection and the secretion of glucagon, a process whose deregulation is a major limitation in insulin treatment of both type 1 and type 2 diabetes. Together these investigations will bring new knowledge on the integrated control of glucose homeostasis that may lead to novel strategies to control diabetes.

2 499 421 €

Start date: 2011-08-01, End date: 2016-07-31

Many reports indicate the number of people with diabetes will exceed 350 million by the year 2030. Both type 1 and type 2 diabetes are characterized by the deterioration and impaired function of pancreatic b-cells. While transplantation is a promising strategy to replace lost tissue, several obstacles remain in the pathway to its clinical application. Whether b-cells are derived from patient samples or differentiated from embryonic stem cells, a major concern facing these strategies is how a recipient will respond to transplanted foreign tissue. Since the native environment for pancreatic islets is comprised of neural and vascular networks, successful integration may depend upon signals received from these neighboring cell types. Using a multidisciplinary approach, the principal investigator plans to elucidate molecular mechanisms underlying the interactions between pancreatic islet cells and their neighboring endothelial cells. Developing an understanding of how these interactions change during the pathogenesis of disease will provide insight into how islet growth and insulin release is dependent upon signals received from adjacent cell types. Emphasis will be placed on genetic mouse models to measure changes in gene expression in both isolated pancreatic b-cells and endothelial cells to identify genes that mediate the interaction between these cell types. In addition, it is of great interest to identify secreted factors that may constitute autocrine or paracrine signalling mechanisms that influence growth and function between these cell types. Furthermore, it will be determined whether current protocols for the differentiation of mouse stem cells into insulin producing cells are improved by restoring the expression of genes which facilitate communication to endothelial cells. This project aims to identify genes essential to the vascular context of pancreatic b-cells to improve transplantation protocols and facilitate the development of therapeutic strategies for diabetes.

1 496 257 €

Start date: 2010-11-01, End date: 2015-10-31

This project aims at exploring the roles or KRAB/KAP1-mediated gene regulation in mammalian physiology and the possible impact of its dysfunctions on human health. The proper control of gene expression is paramount to all biological events, and is orchestrated through a sophisticated balance of activating and repressing influences. The mouse and human genomes contain around four hundred genes encoding KRAB-containing zinc finger proteins (KRAB-ZFPs), a family of tetrapod-restricted sequence-specific DNA-binding transcriptional repressors. Even though these KRAB-ZFPs represent the single largest group of transcriptional regulators encoded by higher vertebrates, their functions remain largely unknown. Nevertheless, it has been established that they share an essential cofactor, the histone methyltransferase- and histone deacetylase-recruiting KAP1, and act by triggering the formation of heterochromatin. KAP1 is ubiquitous, and KRAB-ZFPs are present in most if not all cells, albeit along distinctly cell type-, stage- and state-specific patterns, suggesting that KRAB/KAP1 gene regulation influences a very large number of physiological events. A few years ago, we launched a program aimed at addressing this hypothesis through a combination of genetic, functional and molecular studies focused on two paradigmatic organs, the lympho-hematopoietic system and the liver. Our preliminary results confirm that KRAB/KAP1-mediated transcriptional control is a master regulator of mammalian homeostasis. Accordingly, we now propose to dissect the regulatory networks orchestrated by KAP1 and KRAB-ZFPs in these two systems, to identify their gene targets and the mechanisms of their control, and to probe their possible implication in human pathologies targeting these organs.

2 499 996 €

Start date: 2011-04-01, End date: 2016-03-31

Hepatocellular carcinoma (HCC) is caused by chronic hepatitis and is the third most common cause of cancer-related death worldwide, with a rising incidence in first world countries. To date no effective therapies other than liver transplantation are available for this disease. Previous studies have provided evidence that inflammatory signalling pathways (e.g. the NF-b pathway) are crucial modulators of liver cancer development. However, the exact mechanisms driving hepatitis-induced liver damage and cancer formation remain elusive. Among others, aberrant expression of cytotoxic cytokines is thought to be critically involved. We have recently shown that the inflammatory cytokines lymphotoxin (LT)  and  are specifically upregulated in livers of patients suffering from hepatitis C and B virus-induced liver inflammation or HCC and that liver specific expression of LT and  in mice (AlbLT) suffices to induce inflammation-induced liver cancer development. We could further demonstrate that this depended on the presence of functional lymphocytes. My proposal is pillared by three main approaches that all aim to elucidate the exact cellular and molecular mechanisms underlying chronic liver damage and HCC development in humans as well as in mouse models of inflammation- or carcinogen-induced liver cancer. First, we will identify the particular immune cell type(s) (e.g. B- or T-lymphocytes; macrophages; NK-T cells) involved in HCC development. Although inflammatory signalling and immune cells appear to be important in HCC development it remains elusive, which immune cell type(s) contribute to inflammation induced liver cancer development. Secondly, we will investigate how inflammatory signalling pathways induce chromosomal aberrations. It is known that inflammatory signalling cascades cause chromosomal aberrations; however, the detailed mechanisms by which this occurs are not fully understood. Additionally, we will determine how inflammatory signalling influences the pathways involved in DNA repair, replication and chromosomal segregation culminating in chromosomal aberrations and cancer. Finally, we will examine the role of oval cells in liver cancer formation. Oval cells, which are putative liver-cancer stem cells, differentiate into either hepatocytes or cholangiocytes, proliferate under inflammatory conditions, and are found within HCC. However, their exact functional role in liver carcinogenesis is unknown. We will biochemically characterize proliferating and HCC-associated ovals cells in mouse models of inflammation-induced HCC and in diseased human liver tissues. This will pave the way for the development of the first genetic tools to deplete or express genes in an oval cell-specific manner. The new scientific knowledge gained by these studies investigating how immune cells and inflammatory signalling induce chronic liver damage and cancer on a mechanistic level, and how oval cells contribute to HCC will provide the basis for future novel pharmacological approaches to treating inflammatory liver diseases and HCC in humans.

1 212 190 €

Start date: 2010-10-01, End date: 2015-09-30

"The identification of the first histone demethylase lysine-specific demethylase 1 (LSD1) established not only the concept of reversible histone methylation in epigenetic regulation but also translated this fundamentally novel biological observation into understanding the molecular mechanisms regulation stemness, differentiation, proliferation, and pathological growth. To unravel in an unbiased and comprehensive manner the biological function of LSD1 in physiology and pathology, we developed LSD1-deficient and LSD1-transgenic mouse models. LSD1-transgenic animals develop prostate tumours demonstrating that increased expression of LSD1 suffices for oncogenic growth in vivo. In addition, LSD1-transgenic animals exhibit a metabolic shift towards overt obesity in adulthood. LSD1-deficiency causes early embryonic lethality around day 7.5 of development. However, deletion of LSD1 is not essential for the development of the embryo proper until the onset of gastrulation, suggesting that the early embryonic lethality is caused by trophoblast defects. Indeed, our data demonstrate that LSD1 is crucial for maintaining trophoblast stem cells in their niche and required for the specification of trophoblast stem cell fate during initial steps of differentiation. To identify the underlying mechanisms that allow LSD1 to control a wide range of biological systems such as trophoblast stem cell fate in the early embryo, obesity, and prostate tumourigenesis in the adult, we propose to a) identify LSD1-associated protein complexes and b) LSD1 target genes establishing these phenotypes in the mouse. In addition, we shall uncover c) signalling pathways that modify LSD1 in these phenotypes allowing us to explore the therapeutic potential of targeting these signalling pathways."

2 488 800 €

Start date: 2013-04-01, End date: 2018-03-31

Primary cancers can induce lymphatic vessel growth (lymphangiogenesis), enhancing metastasis to draining lymph nodes (LNs). We found that tumors also induce lymphangiogenesis in draining LNs, leading to increased cancer spread to distal LNs and beyond. Very recently, we found that lymphatic vessel activation in peripheral tissues and draining LNs also plays a previously unanticipated role in the control of chronic inflammatory diseases. This proposal aims at a comprehensive characterization of the function of lymphatic vessels in inflammation, using a variety of genetic mouse models for enhanced or reduced lymphatic function, novel quantitative techniques for the in vivo imaging of lymphatic function, and a miniaturized 3-dimensional in vitro platform for the high-throughput phenotypic screening of libraries of small, drug-like molecules for modulators of lymphatic function. A genome-wide analysis of the gene expression profile shall be made from lymphatic vessels isolated by high-speed cell sorting and by immuno-laser capture microdissection from tumors and their lymph node metastases, inflamed tissue and its draining lymph nodes, and normal tissues, followed by functional characterization of potential therapeutic targets and diagnostic markers. Finally, we will establish novel genetically fluorescent mouse models for the in vivo real-time imaging of lymphatic activation, and we will develop an innovative approach for the in vivo detection of early micrometastases, using antibody-based PET and near-infrared imaging of tumor-induced stromal changes. These studies will improve our understanding of lymphatic involvement in inflammation and cancer metastasis, and will provide the basis for completely novel approaches to treat and detect inflammation and cancer metastasis.

2 493 300 €

Start date: 2011-05-01, End date: 2016-04-30

The last decade has seen an explosion of research into the epigenetic regulation of cell biology. Indeed, great efforts have been made to elucidate epigenetic regulation in stem cell biology, development, and within the plastic states of cancer. Current estimates place the prevalence of obesity in the range of 300 million to beyond 1 billion by the year 2030. As a critical risk factor for heart disease, diabetes and stroke, obesity currently represents one of the world’s chief economic and health care challenges. While studies have established a genetic framework for our current understanding of obesity, the contribution of several critical regulatory layers, in particular epigenetic regulation, remains poorly understood. We recently performed the first genome-wide RNAi screen for obesity regulators in the adult fly. Intriguingly, developmental regulators scored among the most enriched pathways (Pospisilik, Cell 2010). Systematic interrogation of the 500 candidate obesity genes by tissue-specific knockdown has now identified the Polycomb-Trithorax system (PcG-Trx) as the most enriched obesity-altering pathway. Here, we propose to translate these findings to the mammalian context through completion of three aims: i. generation and characterization of 8 PcG conditional knockout mouse lines, ii. dissection of the functional roles of PcG in adipose tissue differentiation, function and disease, and iii. building of a functionally interrogated unbiased epigenetic map of the PcG-system for murine and human obesity. To achieve these goals we will combine targeted mouse genetics, complex phenotyping and state-of-the-art integrative bioinformatics. Using this unique functional-genetics-to-epigenomics approach we will provide an unprecedented functionally validated genomics resource for obesity research worldwide, unravel an entire regulatory niveau in obesity, and if we are lucky, highlight novel therapeutic strategies for metabolic disease.

1 654 430 €

Start date: 2011-12-01, End date: 2016-11-30

Ageing is a complex physiological process that affects almost all species, including humans. Despite its importance for all of us, the biology of ageing is insufficiently understood. To uncover the molecular underpinnings of ageing, I propose an interdisciplinary research program that will identify and investigate metabolic and genetic regulators of ageing. Progressive loss of cellular homeostasis causes ageing and an age-associated decline in protein quality control has been implicated in numerous diseases, including neurodegeneration. Seeking for ways to improve protein quality, I have identified a novel longevity pathway in Caenorhabditis elegans. In a forward genetic screen, I found a link between metabolites in the hexosamine pathway and cellular protein quality control. Hexosamine pathway activation extends C. elegans lifespan, suggesting modulation of ageing by endogenous molecules. In a first step, I will explore the mechanism by which hexosamine metabolites improve protein quality control in mammals, using cultured mammalian cells and a mouse model for neurodegeneration. Preliminary data show that hexosamine pathway metabolites enhance proteolytic capacity in cells and reduce protein aggregation, suggesting conservation. Second, I will investigate molecular mechanisms that activate the hexosamine pathway to promote protein homeostasis and counter ageing. Third, I will perform a direct forward genetic screen for modulators of ageing in C. elegans. For the first time, mutagenesis and next generation sequencing can be paired in forward genetic screens to interrogate the whole genome for lifespan-extending mutations in a truly unbiased manner. This innovative approach has the potential to reveal novel modulators of the ageing process. Taken together, this work aims to understand molecular mechanisms that maintain cellular homeostasis to slow the ageing process, and to develop a new technology to identify yet unknown genetic modulators of ageing.

1 500 000 €

Start date: 2015-08-01, End date: 2020-07-31

"Mammals have two types of fat: brown and white, with opposing functions. The white adipose tissue (WAT) is an important regulator of the whole body homeostasis that also serves to store energy in form of triglycerides (TGs). The main function of the brown adipose tissue (BAT) is to catabolize lipids in order to produce heat, a function that can be induced by cold exposure or diet. Disruption of the normal differentiation or development of the WAT causes ectopic lipid storage and severe pathology in both humans and experimental animals. Increased BAT development leads to increased energy expenditure without causing dysfunction in other tissues, and is associated with a lean and healthy phenotype, outlining the manipulation of the fat stores as an obvious therapeutic objective. With the proposed research we will identify miRNAs and other factors that regulate BAT and WAT differentiation and function. We will distinguish the miRNAs that specifically regulate brown or white adipogenesis and are expressed in the respective precursors, and establish them as signatures for either cell type. We will also identify the molecular mechanisms of action of the identified miRNAs in regulation of adipose tissue differentiation and metabolism. Using in vitro and in vivo systems, linage tracing studies, transgenic animals, as well as cohorts of human patients, we will determine the origin of the beige cells within the SAT, establish their importance in the regulation of metabolism in vivo, and develop novel strategies to induce the brown fat differentiation and function. Finally, we will discover ways to exclusively silence miRNAs in the brown fat will that will allow us not only to investigate the miRNAs function specifically in the brown fat, but also to develop new strategies for treatment of dyslipedaemia, diabetes and obesity."

1 400 014 €

Start date: 2014-02-01, End date: 2019-01-31

"Cells use sensor pathways compartmentalized in subcellular organelles to recognize stress conditions, including aberrant protein folding, and in response activate gene expression programs aimed at maintaining cell survival and restoring homeostasis. Fine-tuning of the protein-folding environment in organelles like mitochondria is important for adaptive homeostasis and may participate in development of human diseases and ageing. Work in cultured mammalian cells and more recently in Caenorhabditis elegans has highlighted the importance of mechanisms linking perturbations in the protein-folding environment in the mitochondrial matrix to the expression of nuclear genes encoding mitochondrial proteins. This mitochondrial stress pathway is named mitochondrial unfolded protein response. Much of our knowledge regarding the organelle unfolded protein response (UPR) signalling comes from studies of the endoplasmatic reticulum stress response machinery. In contrast, a potential role of mitochondria in UPR pathway is far less defined, and physiologic regulators of this pathway have not been defined. Here I propose three complementary strategies to identify molecular mechanisms and signalling pathways of the mitochondrial unfolded protein response (UPRmt) under different stress conditions and during ageing. My laboratory has experience in using transgenic mice and C. elegans as experimental tools and both of these powerful model systems will be used in this project. Specific aims of this proposal are: Aim 1. To identify specific substrates of UPRmt Aim 2. To define mechanisms regulating mammalian UPRmt Aim 3. To elucidate the role of UPRmt signalling in ageing"

1 500 000 €

Start date: 2013-01-01, End date: 2017-12-31

Obesity has become a leading medical disorder, which is associated with life threatening conditions such as glucose intolerance, insulin resistance (IR) and type 2 diabetes (T2D). In the maintenance of glucose homeostasis, muscle is a critical organ and current health recommendations include regular physical activity as a cornerstone in the prevention and treatment of IR/T2D. The development of exercise mimetics has been proposed as a novel therapeutic strategy, but this has failed so far. This is because we still do not completely understand the etiology of glucose intolerance and how exercise improves glucose tolerance. In particular, angiogenesis – the growth of new blood vessels from existing ones – is an early adaptive event following exercise training, but the role of the muscle vasculature in the regulation of muscle metabolism and glucose tolerance has been largely overlooked. In this project, I will investigate the metabolic crosstalk between the vasculature and the muscle to increase our understanding on how the endothelium contributes to muscle metabolism and glucose homeostasis. First, I will evaluate whether and how vessels need to reprogram their metabolism to promote angiogenesis following exercise training. Second, I will explore whether this metabolic reprogramming that results into enhanced angiogenesis is required for the muscle to allow training adaptations. I pose the novel and unexplored hypothesis that endothelial cells and the muscle intensely communicate to ensure optimal muscle function and to orchestrate muscle adaptations to exercise training via metabolic signaling. I will combine in vitro, ex vivo, and in vivo techniques using targeted and untargeted approaches to answer these exciting questions. Ultimately, I will investigate whether this communication is affected during the development of T2D. And if so, whether this interaction can be exploited to prevent IR/T2D.

1 498 823 €

Start date: 2017-06-01, End date: 2022-05-31

Stimulated Emission Depletion (STED) microscopy is one of the most important recent developments in light microscopy (Willig et al., 2006, Nature 440:935-9). STED allows for imaging cellular elements with diffraction-unlimited resolution; in practical terms, the resolution (normally limited to ~200-300 nm) is improved down to 30-60 nm. Together with the development of two-color STED microscopy (Donnert et al., 2007, Biophys J. 92:L67-9), this technique allows experimenters to pinpoint the position of various cellular elements with nanometer precision. Obtaining a cellular nanomap is not feasible with conventional light microscopy, due to its low resolution. Electron microscopy cannot be applied, as its labeling efficiency it too low. I propose here to use STED microscopy to characterize the positions of the major components of the synapse. The preparation will be cultured hippocampal neurons, which have numerous small (about one micron in diameter) synaptic nerve terminals. I will determine the locations of synaptic proteins involved in neurotransmitter release, in membrane retrieval and in pre- and post-synaptic active zone structure. Less specialized elements such as the cytoskeleton, mitochondria and endosomes of the synapse will also be investigated. The work will provide answers for a number of questions in the neuroscience field, such as how and where the synaptic vesicles get retrieved, how pre- and post-synaptic active zone elements correlate, and what the role of cytoskeletal elements is in synaptic transmission. The small size and relatively low complexity (compared to whole cells) of the synaptic boutons will allows the work to be completed within a reasonable timeframe. Successful completion of the project will encourage researchers to perform larger scale cellular nano-maps, which would eventually replace the largely erroneous cellular fractionation techniques currently used nowadays to determine the location of various proteins.

1 670 000 €

Start date: 2008-09-01, End date: 2013-08-31

Chemosensory systems permit organisms to perceive diverse chemicals in the environment signalling the presence of food, dangers, kin or mates. How a specific chemical stimulus is recognised and converted into neural activity that provokes the appropriate behaviour is a fundamental problem in neuroscience. I investigate this question in the olfactory system of the fruit fly, Drosophila melanogaster, which exhibits sophisticated odour-driven behaviours under the control of a simple and genetically accessible nervous system. I recently discovered a novel Drosophila olfactory receptor family, the Ionotropic Receptors (IRs). IRs are expressed in sensory neurons distinct from the previously described Odorant Receptor (OR) family. Strikingly, IRs are structurally similar to ionotropic glutamate receptors (iGluRs), a conserved family of ligand-gated ion channels present in animals, plants and bacteria. iGluRs are best characterised for their role in mediating synaptic communication in the mammalian brain as receptors for the neurotransmitter glutamate, but IRs have divergent ligand-binding domains. The proposed project investigates the function of the IRs and their sensory circuits in the recognition of, and behavioural responses to, olfactory stimuli through four specific aims. Aim 1: Defining the molecular basis of IR/odour interactions. Aim 2: Visualising the mechanisms of IR trafficking. Aim 3: Mapping IR sensory circuits in the brain. Aim 4: Exploring the behavioural responses mediated by IR olfactory pathways. By combining genetic, cell biological, electrophysiological and behavioural approaches, this project will provide an integrated understanding of the function and evolution of these novel olfactory receptors and circuits. This knowledge will be of significance to chemical detection mechanisms across diverse sensory systems in eukaryotes and prokaryotes, and of interest to chemical ecologists, neuroscientists, evolutionary biologists and biomedical researchers.

1 500 000 €

Start date: 2008-07-01, End date: 2013-06-30

p73 is a transcription factor of the p53 tumor suppressor family. In approximately 50% of all cancer patients the tumor suppressor function of p53 is irreversibly disabled by point mutations, which makes p53 one of the most frequently mutated genes in cancer. This is entirely different for p73 and much of my previous work in this field has been devoted to research on the role of p73 in cancer. In sharp contrast to p53, p73 is often highly expressed in its wild-type form in solid tumors compared to the surrounding normal tissue. This suggests the rather challenging hypothesis that p73 has oncogenic functions in cancer cells, which promote tumor progression and therapy resistance. This concept is supported by clinical data demonstrating p73 overexpression to be correlated with advanced tumor stage, metastasis, therapy resistance and poor overall survival in multiple tumor entities including the ‘major killers’: breast, lung and colorectal cancer. When p73 is depleted from cancer cell lines, tumor cell proliferation and tumorigenicity are reduced, indicating that tumor cells with high p73 expression are p73-dependent. This places p73 in line with oncogenes like Myc or mutant Ras, which are similarly essential for the tumorigenic phenotype. However, tumor cells are not only addicted to a particular oncogene but in many cases codependent on other cellular factors - a phenomenon termed ‘non-oncogene addiction’. Since p73 also has tumor suppressive functions, p73-dependent tumor cells are likely to be critically dependent on cooperating factors, which keep the proapoptotic and tumor suppressive functions of p73 in check. Inhibition of these factors would be ‘synthetically lethal’ with overexpression of p73. From a clinical point-of-view it would be extremely valuable, if we knew these factors and were able to block them in order to reactivate p73’s tumor suppressor activity and trigger growth inhibition or cell death. This approach promises to be specifically effective in the therapeutically challenging p73-dependent tumors with little or no side effects in normal tissues with low p73 expression. The goal of this project is therefore the identification and validation of such synthetic lethal interactions with p73 using different functional genomics approaches. In the first part of the project we will characterize the impact of p73-dependence on gene expression using genome-wide expression and global chromatin state profiling. This will enhance our molecular understanding why cancer cells rely on high-level expression of p73. In addition, this will pinpoint genes and pathways, which are co-expressed and activated together with p73 and which are therefore candidate genes for therapeutic targeting of p73-dependent cancer cells. In the second part of the project we will use RNAi screening techniques on a genome-wide scale to identify in an unbiased manner cellular factors, which enable cancer cells to tolerate high-level expression of p73. These genes are essential for long-term proliferation and survival in the context of p73 overexpression and could be ideal drug targets for a tumor-selective therapy of the prognostically dismal class of p73-dependent tumors

1 499 040 €

Start date: 2010-10-01, End date: 2016-09-30

Maintenance and drug resistance of pancreatic ductal adenocarcioma (PDAC) depends on cancer cell intrinsic mechanisms and a stroma that supports tumor growth. Mouse models of human PDAC have provided important insights into the evolution of this highly lethal tumor, but there are no models that allow secondary genetic manipulation of autochthonous tumors, the tumor microenvironment or the metastatic host niche once the tumor has formed. We generated an inducible dual-recombinase system by combining Flp/frt and Cre/loxP. This novel PDAC model permits spatial and temporal control of gene expression enabling unbiased genetic approaches to study the role of tumor cell-autonomous and non-autonomous functions in endogenous cancers. This tool provides unparalleled access to the native biology of cancer cells and their hosting stroma, and rigorous genetic validation of candidate therapeutic targets. We performed tumor cell-autonomous and non-autonomous targeting, uncovered hallmarks of human multistep carcinogenesis, validated genetic tumor therapy, and showed that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are in fact dispensable for tumor formation. In the proposed research program, we will 1) develop and further improve next-generation PDAC models, 2) deploy these systems to identify and target key features of PDAC maintenance in tumor cells and their microenvironment, and 3) discover mechanisms of treatment resistance. The application of cutting edge genetic engineering and screening technologies will allow us to address biological questions that could not be addressed before. The PanCaT project will open new horizons for the functional understanding of pancreatic cancer biology with a strong impact on clinical management and prognosis of PDAC patients. It will also produce a unique set of highly versatile and widely applicable genetic tools that will facilitate the study of PDAC at an organismal level.

2 440 275 €

Start date: 2016-02-01, End date: 2021-01-31

Sarcomas are an extremely heterogeneous group of mesenchymal tumors that arise in a multitude of tissues from many different cell types. Several genetic events have been identified in different sarcoma sub-types, but very few models were developed to study their role in tumorigenesis aiming at exploiting them as therapeutic vulnerabilities. As a result, the treatment of sarcoma has extremely limited advancement in therapeutic options compared to other cancers. Therefore, the generation of faithful in vitro and in vivo models for sarcoma research is urgently needed to provide insights into the pathobiology of these tumors and discover novel vulnerabilities in these lethal but yet understudied disease. Many types of soft tissue sarcomas arising in children and young adults have a unifying underlying genetic mechanism, where chromosomal translocations generate fusion oncoproteins that serve as drivers of the disease. Exploiting this genetic simplicity provides an exceptional opportunity to develop effective and specific therapies. My past research has applied cutting edge technology to define epigenetic vulnerabilities associated with the SS18-SSX gene fusion, the defining event in synovial sarcoma (one subgroup of pediatric sarcomas), and to study its chromatin occupancy genome-wide. In this proposal my team will combine a toolbox consisting of CRISPR/Cas9, RNAi technology and expertise in mouse models to systematically elucidate key genetic and epigenetic mechanisms in the pathobiology of pediatric sarcomas. This work will help to understand key players in epigenetic deregulation in pediatric sarcomas, generate new sarcoma models to assist clinical translation, and identify new therapeutic targets for these deadly diseases.

1 499 375 €

Start date: 2019-01-01, End date: 2023-12-31

Sensory perception has recently emerged as a master regulator of integrative physiology and behavior, including feeding, by controlling fundamental and pleiotropic regulatory processes of energy and glucose homeostasis. Further, sensory perception is altered in obesity and type 2 diabetes, and childhood obesity correlates with early sensory deficit. Along this line, the discovery of the developmental origins of health and diseases revealed that metabolic diseases have recognized roots in the very early stages of life and can be predisposed to by changes in the perinatal hormonal and nutritional environments, such as occur in cases of maternal obesity and unhealthy diet. In this context, an accumulating body of evidence suggests that maternal health and nutrition could negatively impinge on the development of sensory perception, and subsequently, on the lifelong regulation of sensory-dependent control of metabolic, physiological, and behavioral regulatory processes. This innovative research program consists of four autonomous but complementary projects aimed at (1) deciphering the exact central regulatory processes mediating sensory control of feeding behavior and glucose homeostasis, (2) uncovering the influence of maternal health and nutrition on lifelong sensory regulation of metabolism, and (3) & (4) investigating two independent, yet synergistic, mechanisms that could mediate developmental programming of sensory metabolic regulation. This research program will employ a technology framework of physiological, behavioral, and developmental analyses in mice in concert with state-of-the-art systems neuroscience approaches, including optogenetics, chemogenetics, and in vivo calcium imaging. Collectively, the overarching goals of this research program are to provide new insights into the precise regulatory processes of sensory metabolic regulation and to shed light on critical basic mechanisms underlying the developmental programming of metabolic diseases.

1 500 000 €

Start date: 2019-06-01, End date: 2024-05-31

The skeletal system and its vasculature form a functional unit with great relevance in health, regeneration, and disease. Our recent work has provided fundamental insights into the organization of the bone vasculature in mouse, its changes during aging, the heterogeneity and functional specialization of bone capillaries and endothelial cells, the regulation of these properties by Notch and hypoxia-inducible factor signaling, and the crosstalk with osteoblast lineage cells. Most importantly, we found that the manipulation of ECs in the aging animal can trigger the expansion of osteoprogenitors and thereby induce bone formation. PROVEC will now systemically identify and characterize endothelial cell subpopulations, their gene expression and functional properties in the healthy, aging, diseased and regenerating skeletal system. Preclinical models will establish whether endothelial cells are involved in the response to therapeutic treatments aiming at osteoblasts or osteoclasts, or if the modulation of ECs alone is sufficient to generate beneficial effects. Finally, PROVEC will investigate whether cultured mouse and human ECs can be endowed with beneficial properties to enhance bone formation in 3D organoid cultures and after transplantation into mice, which will be monitored by imaging in living animals. To achieve its ambitions aims, PROVEC will use a powerful combination of mouse genetics, disease models, genetic fate mapping, RNA-seq and single cell sequencing, computational biology, confocal and 2-photon microscopy, micro-CT imaging, pharmacological treatments, and cell biology methods to establish if and how vascular endothelial cells can be used to increase bone mineral density in preclinical models. The successful completion of PROVEC would be highly relevant for diseases such as osteoporosis, which affects around 27.5 million patients in the EU, generates annual costs of about 37 billion Euros, and for which we currently lack appropriate treatments.

2 205 875 €

Start date: 2019-02-01, End date: 2024-01-31

"A link between inflammation and cancer has been suspected over a long period of time and in recent years genetic evidence supporting such notion could be obtained. Several signaling pathways, such as NF-κB and STAT3, could be identified by our group as well as other groups to play important roles during tumor promotion and progression. Chronic inflammation leads to the formation of reactive oxygen and nitrogen species, which are known to cause DNA damage and inactivation of DNA repair mechanisms, thereby presumably inducing tumor-initiating mutations. On the other hand, oxidative stress has been documented to trigger apoptosis and cellular senescence. However, in vivo evidence demonstrating the consequences of increased oxidative stress on tumor development in a distinct genetic model is lacking. Selenoproteins of the glutathione peroxidase and thioredoxin reductase family are important anti-oxidant scavenger systems. Using cell type specific inactivation (epithelial cells an myeloid cells) of several of these family members in various well established and relevant models of inflammation-associated and sporadic colon tumorigenesis we will directly examine the role of these anti-oxidant systems. These models will allow us to genetically determine the outcome of increased lipid peroxidation and accumulation of reactive oxygen species in each cell type and to address whether cancer development is supported or inhibited under such conditions and will help to identify which phase of tumor development is affected. Furthermore, we expect to obtain results that will determine whether increased levels of ROS/RNS found during chronic inflammation are capable of initiating tumorigenesis. Our proposed experiments are supposed to provide fundamental insight into the molecular changes in the tumor microenvironment, which will ultimately help to identify novel strategies to prevent and treat colorectal cancer."

1 500 000 €

Start date: 2011-10-01, End date: 2016-09-30

Vertebrate sensory perception and integration depend on the formation of ordered connections between sensory neurons and their target cells that occur along several steps: a- establishment of cellular identity, b- neuronal axon growth, arborization and dendritogenesis, c- somatodendritic contact and synaptic refinement. Although there is a wealth of information about the molecular mechanisms controlling neuronal cell-fate determination and synaptogenesis during embryonic development, very few studies have analyzed the long-term dynamics of sensorineural circuitry in adult animals. We are employing the zebrafish lateral-line system to understand the mechanisms that govern the formation, homeostasis and regeneration of a mechanosensory organ. We have obtained evidence that intrinsic and activity-dependent mechanisms cooperate during the elaboration and refinement of the organ’s innervation. The goal of this research proposal is twofold: to characterize the mechanisms of sensorineural dendritic elaboration and remodeling, and to analyze how sensory organs reinnervate to recover function after damage. The first aim is to perform an exhaustive analysis of the cellular and molecular events leading to the formation of neuronal topography and the kinetics of target recognition. The second aim is to characterize the contribution of sensory function to dendrite arborization, asking whether epigenetic mechanisms shape sensorineural synaptic fields in the periphery. Finally we shall analyze how the architecture of the sensory epithelium relates to dendrite arborization. Achieving these goals will help us understand how animals maintain sensory abilities throughout their entire lives. It may also provide a framework for the development of strategies of regenerative medicine aimed at ameliorating the negative effects of age-related loss of sensory function, peripheral neuropathies, or cerebral stroke in humans.

1 100 000 €

Start date: 2008-09-01, End date: 2014-02-28

Over 380 million people suffer from diabetes worldwide, with majority of cases being attributed to type 2 diabetes (T2D). Obesity is a major risk factor predisposing to the development of this disease. T2D is characterized by peripheral insulin resistance in combination with relative insulin deficiency that results in hyperglycemia and hyperlipidemia. Liver and adipose tissue are central for regulation of glucose and lipids levels. However, during T2D the hepatic glucose uptake is reduced while rates of gluconeogenesis and lipogenesis are increased. In the adipose tissue, T2D leads to decreased glucose uptake, perturbations in secretion of adipokines and increased lipolysis. Importantly, dysfunction of the liver and the adipose tissue during T2D is caused by defective phosphorylation signaling cascades and normalization of these pathways was shown to attenuate the course of T2D. However, the specific roles of different classes of signaling molecules in these organs remain poorly characterized. We hypothesize that the cross-talk of different classes of signaling molecules determines regulation of metabolism. Thus, we aim to identify the signaling networks regulating metabolism. The results generated in my own laboratory suggest that the Pkd family kinases are the crucial regulators of metabolic homeostasis. Specifically, Pkd1 and Pkd2 promote obesity and diabetes while Pkd3 controls liver function. Thus, we plan to characterize the molecular mechanisms controlling Pkds signaling. In parallel, we will utilize screening approaches to identify novel, non-canonical signaling modules (phosphatases and components of the ubiquitin system) regulating abundance, localization and phosphorylation of targets of Pkds and, in the long term, also other kinases implicated in T2D. By identifying and characterizing the essential signaling networks in liver and adipose tissue the project will contribute to more targeted pharmacological strategies for the treatment of T2D.

1 499 128 €

Start date: 2016-06-01, End date: 2021-05-31

By modulating the activity of transcription factors, cofactors influence cellular and organismal metabolic pathways. Recently the role of the sirtuin cofactor gene family received a lot of attention in this context, as their beneficial impact on longevity was linked to effects on metabolic control. Most sirtuins catalyze deacetylation reactions that are tightly linked to cellular energy (NAD+) levels. To ascertain the tissue-specific contributions of sirtuins in metabolism, we propose here to: 1) identify novel deacetylation targets for the sirtuins; 2) generate and characterize genetically engineered mouse models (GEMMs) with somatic mutations in the 7 sirtuin genes in liver, muscle and adipose tissue, key metabolic tissues; 3) study mouse models with natural genetic variation in sirtuin expression, such as found in genetic reference populations (GRPs), like the BxD recombinant inbred mouse lines; and 4) validate the relevance of sirtuin signaling for human metabolism through clinical genetic studies. As the goal is to progress towards the treatment and prevention of metabolic diseases in humans, GEMMs are optimized to study the actions of isolated genetic loci and are thus insufficient to characterize polygenic networks and genetic interactions. Moreover environmental factors can impact on the manifestations of the genotype or phenocopy the genetically produced phenotype. Thus, to dissect complex genetic traits, experimental models, like GRPs, that imitate the genetic structure of human populations provide complimentary information to that obtained from GEMMs. As we will combine data generated using directional genetic strategies in GEMMs with a high-throughput phenogenomic analysis of GRPs, our approach merges the benefits of clear-cut results of single gene perturbations in a given tissue with subtle alterations that result from natural innumerable allelic variants. This will ultimately favor the definition of the role of the sirtuins in metabolic homeostasis.

2 485 000 €

Start date: 2009-02-01, End date: 2014-01-31

Adult stem cells are essential for the lifelong maintenance and regeneration of various organs and tissues. Experimental and clinical data indicate that the functional capacity of adult stem cells in organ regeneration declines during aging. Molecular mechanisms that cause impairments in stem cell function during aging remain to be delineated. Genetic analyses identified a growing number of genes and genetic loci that are associated with longevity and aging in model organisms and humans. For most of these associations the molecular mechanisms and its functional relevance for mammalian aging remain unknown. In many cases of genetic loci associations, the responsible genes have not even been identified. A bottleneck in our understanding of aging remains to identify functionally relevant genes and molecular mechanisms from this growing list of genetic association with aging. Emerging experimental data indicate that aging/longevity-associated genes influence the functional reserve of adult stem cells. Here, I propose to develop and lead a research program analyzing longevity and aging associated genes and gene loci by reverse genetic approaches. In vivo and ex vivo RNAi will identify genes and molecular mechanisms that affect the function of stem cells in aging mice or genetically engineered mice modeling accelerated accumulation of molecular damages and stem cell dysfunction. Analysis of primary human stem cells from young vs. old donors will delineate whether the identified genes and mechanisms are conserved in humans. Reverse genetic approaches of aging/longevity-associated genes have not been conducted in adult mammalian stem cells. Our group gained significant expertise in analyzing molecular mechanisms of stem cell maintenance and function as well as in conducting RNAi screens in different murine stem cell compartments. Our studies will delineate novel mechanisms of stem cell aging and its implication for defects in organ homeostasis and regeneration during aging.

2 498 400 €

Start date: 2013-07-01, End date: 2018-06-30

The control of translation is a key determinant of protein abundance, which in turn defines cellular states. The impact of translational regulation may be even greater during the transition from homeostasis to malignancy, as revealed by the surprisingly low correlations between mRNA and protein levels in human cancer databases. This raises the intriguing possibility that through an ability to generate aberrant downstream networks of translational regulators, oncogenic drivers might impose altered protein synthesis programs that become the driving force for tumor formation and malignant progression. We recently unveiled a hitherto unappreciated role for upstream open reading frame (uORF) translation in tumorigenesis and unearthed a novel switch from conventional EIF2 initiation factor-mediated to alternative EIF2A-mediated uORF translation. These observations suggest that uORFs constitute an exciting new frontier in the field of translational regulation with the potential to fundamentally impact cellular fate. Here, I propose to systematically analyze the function of uORFs during tumorigenesis. First, we will conduct an in vivo CRISPR/CAS9-based screen in mice to elucidate the role of thousands of uORFs in development, differentiation and upon oncogenic transformation. Second, focusing on select uORFs surfacing in the screen, we will document their role during tumor initiation and progression. Third, we will develop novel tools to detect uORF translation in vivo, exploit them to monitor uORF translation during different stages of tumorigenesis, gain mechanistic insight into their function and finally test the relevance of these findings in human cancer. Collectively, these approaches will provide unprecedented and comprehensive insight into the function of uORFs, unravel new paradigms in the control of gene expression and expose novel strategies for cancer diagnostics and treatment.

1 977 148 €

Start date: 2018-08-01, End date: 2023-10-31

Cells must regulate their volume in response to changes in osmolarity and during cell division, migration, apoptosis, and transepithelial transport. Regulated membrane transport of ions and metabolites creates osmotic gradients that secondarily drive water across the membrane. Organic ‘osmolytes’ such as glutamate also serve in extracellular signalling and volume-regulatory ion transporters are often used for other purposes, putting volume regulation into the context of diverse organismal functions. Research on cell volume regulation stagnated because the identity of a key player, the Volume-Regulated Anion Channel VRAC, remained unknown. Very recently we identified LRRC8 heteromers as VRAC components and discovered that VRACs are a heterogeneous group of channels. Their remarkable ability to transport not only Cl-, but also signalling molecules or drugs, depends on their LRRC8 subunit composition. This breakthrough now allows us to search for functionally relevant interactors and to dissect the physiological roles of different VRACs using mouse models. Whereas disruption of Lrrc8a abolishes VRAC function, abrogating other Lrrc8 genes (in total five) will change its transport properties. Conditional KO mice will first focus on epithelia which faces large osmolarity changes, on the brain where VRAC-released signalling molecules are supposed to play important roles in physiology and pathology, and on VRAC’s assumed role in vesicle exocytosis. We expect to discover many surprising novel roles of VRACs. Emboldened by our identification of VRAC, we will use genome-wide siRNA screens to identify two other ‘missing’ ion channels, which have been known physiologically for many years and may have widespread roles in signalling and other physiological processes. Once identified, these channels will be studied at a structural, cellular and organismal level. These projects will break new ground in physiology, cell biology, signalling and pathology.

2 499 991 €

Start date: 2017-10-01, End date: 2022-09-30