Metastatic growth of cancer cells requires extracellular matrix (ECM) production. The current understanding is that transcription factors regulate ECM production and thus metastatic growth by increasing the expression of collagen prolyl 4-hydroxylase (CP4H). In contrast, we recently discovered that metabolism regulates CP4H activity independently of the known transcription factors. Specifically, we found that loss of pyruvate metabolism inhibits CP4H activity and consequently ECM–dependent breast cancer cell growth. Based on this discovery we propose the novel concept that metabolism regulates metastatic growth by increasing ECM production. In this project we will investigate the following questions: 1) What is the mechanism by which pyruvate regulates CP4H activity in breast cancer cells? To address this question we will investigate pyruvate metabolism and ECM production in 3D cultures of various breast cancer cell lines using 13C tracer analysis, metabolomics, and two-photon microscopy based ECM visualization. 2) How can this novel metabolic regulation be exploited to inhibit breast cancer-derived lung metastases growth? To address this question we will inhibit pyruvate metabolism in metastatic breast cancer mouse models using genetically modified cells and small molecules in combination with immuno- and chemotherapy. 3) How can this novel regulation be translated to different metastatic sites and cancers of different origin? To address this question we will determine the in vivo metabolism of breast cancer-, lung cancer-, and melanoma-derived liver and lung metastases (using metabolomics and 13C tracer analysis), and link it to ECM production (using two-photon microscopy based ECM visualization). With this project we will deliver a novel concept by which metabolism regulates metastatic growth. In a long-term perspective we expect that targeting this novel metabolic regulation will pave the way for an unexplored approach to treat cancer metastases.

Plants contribute up to 80% of all biomass on earth. Despite their staggering diversity; dominant land plants share a highly important characteristic: the presence of a vascular system providing physical support and long distance transport. This is however not a simple binary trait, as some non-vascular mosses contain cells with conductive capacity resembling that of vascular plants. Available evidence indeed suggests that conductive tissues of non-vascular plants are functionally homologous to vascular tissues in vascular plants and can even be compared at a molecular level. However, the molecular players involved in conductive tissue development remain almost completely unknown. Moreover, although key molecular regulators of vascular tissue development have been identified in the model plant Arabidopsis, very few are shown to be functionally conserved across vascular plants. Despite their importance for growth and development, we thus have a limited understanding of the evolutionary conserved regulators of plant plumbing systems. In PIPELINES, I will consolidate my expertise in single-cell applications and build a dedicated team to identify conserved molecular players specific to vascular and conductive tissues by combining multi-species comparative single-cell and spatial transcriptomics with gene regulatory network inference; and characterize these factors using loss-of-function approaches. By comparing this data, I will determine the ancestral set of regulators sufficient to trigger specification and differentiation events in plants; and validate these through introduction of single-cell sample multiplexing in a heterologous system. By unravelling the molecular basis of vascular and conductive tissue development and identifying conserved core developmental regulators, the output of PIPELINES will act as a starting point for targeted engineering of vascular tissues; which holds great potential for improving plant biomass and productivity in crop species.

Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. The disease is highly prevalent in countries of the Mediterranean basin, the Caucasus and the Middle East, with 1:1000 up to 1:500 inhabitants being affected in countries like Turkey and Armenia. FMF has further spread to other regions of the world with migrations of these populations in modern history and today. Colchicine therapy is the gold standard for FMF patients, but given the risks involved, correct FMF diagnosis is desired before onset of therapy. Diagnosis of FMF is currently based on clinical presentation, and is further supported by review of ethnic origin, family history and genetic analysis of disease-associated MEFV alleles. However, clinical and genetic diagnosis of FMF are complicated by significant overlap of the clinical picture with other autoinflammatory diseases, and over 280 FMF alleles of MEFV have been described. Common genetic tests focus on the most common mutations in exons 2 and 10 of MEFV, but mutations may also occur in other parts of the MEFV gene. Consequently, genetic analysis of FMF is sometimes inconclusive, and correct diagnosis of FMF may sometimes be delayed for years. Here, we describe an immunological assay that for the first time allows selective identification of FMF patients based on the differential inflammasome activation response of their blood monocytes. The availability of a single robust, affordable and convenient assay that immunologically stratifies FMF patients from other autoinflammatory disease patients will be instrumental for improving timely and correct identification of FMF patients for colchicine therapy, and (where desired) will enable more cost-effective selection of patients for genetic confirmation of MEFV mutations.

Tauopathies (that include Alzheimer’s disease) are characterized by hyperphosphorylation or mutations in the microtubuleassociated protein Tau. This reduces the affinity of Tau to bind microtubules and increases the levels of soluble detached Tau in neurons. Previous ERC-supported work from my lab showed that pathogenic Tau accumulates at pre-synaptic terminals and clusters synaptic vesicles in mouse and fly Tauopathy models and in Alzheimer patient brain samples. This pre-synaptic Tau reduces vesicle mobility and neurotransmitter release (Zhou et al., 2017). The interaction of Tau with synaptic vesicles occurs via the synaptic vesicle-associated protein Synaptogyrin-3. Lowering the levels of Synaptogyrin-3 blocks the ability of Tau to efficiently bind to synaptic vesicles (McInnes et al., 2018). Hence, Synaptogyrin-3 is a promising therapeutic target to tackle tauopathies. In this project I propose to use antisense oligonucleotides (ASOs) to reduce the levels of Synaptogyrin-3 in the brain of Tauopathy mouse models to rescue tau-induced pre-synaptic defects, including cognitive decline. ASO-technology has vastly improved over the past years and was recently approved to treat other degenerative disorders. My goal is to provide in vivo proof-of-concept that targeting Synaptogyrin-3 with an ASO in a therapeutic setting suppresses Tau-induced defects, including cognitive decline.

Although we spend a third of our lives sleeping, the function of sleep remains mysterious. Studies considering neural networks, brain regions and behavior suggest the intriguing hypothesis that sleep is important for synaptic plasticity. However, as prior studies have conducted analyses using broad brain regions or circuit networks, the precise role of sleep in synaptic plasticity remains intensely debated. Progress in this area is hindered by the lack of a genetically-tractable system of sleep-dependent synaptic plasticity. To solve this, I developed a unique fruit fly model. It is the first model of its kind in which the cellular players comprising the synapses can be genetically labelled and manipulated. I will use this model to address a long-running controversy in the field--which form(s) of synaptic plasticity is promoted by sleep--by directly monitoring the effects of sleep on precisely identified synapses at electrophysiological and ultrastructural levels. Then, I will investigate the mechanisms underlying this process by analyzing the effects of sleep on every cellular component (pre and postsynaptic neurons and perisynaptic astrocytes) in this model at molecular and cellular levels. Emerging data suggest that astrocytes play a key role in synaptic plasticity and have further implicated these cells in regulating sleep. However, it has been difficult to directly examine the role of astrocytes in sleep-dependent synaptic plasticity, since no methodology currently exists for reproducibly manipulating local astrocytes enveloping the synapse of interest. For this, I will implement a unique tool, named G-CLAMP, and use it to assess potential glial mechanisms underlying sleep-dependent plasticity. Considered as a whole, my project will produce unique and comprehensive understanding of the role of sleep in synaptic plasticity, an essential question if we aim to understand sleep, and move us towards explaining the evolutionarily origins of this mysterious behavior.