Myocardial infarction remains a frequent and disabling disease with still no therapeutic strategy to mitigate the risk of developing heart failure. The mitochondrial Ca2+ uniporter represents the key structure which controls Ca2+ entry inside mitochondria and therefore a relevant target upstream of the mitochondrial Ca2+ overload to modulate not only cell death but also mitochondrial bioenergetics and Ca2+ homeostasis. The PI recently identified the interaction site between the Ca2+-sensing regulator MICU1, with MCU, the pore forming protein of the uniporter, required to control the Ca2+ flux and in fine cell survival. Based on this evidence, we postulate that a loss of mitochondrial Ca2+ uptake regulation by MICU1 would be the key trigger of mitochondrial Ca2+ overload-induced cell death during ischemia-reperfusion. In this project, we aim at determining the molecular mechanisms controlling the MICU1-MCU interaction during ischemia-reperfusion in order to mimic the regulation of MCU by MICU1 as a new therapeutic target in mitochondrial Ca2+ overload diseases, such as myocardial infarction. Our hypothesis will be tested through three original tasks from molecular to whole animal scale, up to the translational level in human cells: Task 1: Decipher the mechanistic role of the MICU1-MCU interaction during ischemia-reperfusion (IR) Task 2: Examine the modulation of the MICU1/MCU interaction during IR Task 3: Investigate the medical relevance of MICU1 mimicking as a therapeutic strategy We believe that the MitoCaRe research program will provide new potential protective molecules against mitochondrial Ca2+ overload-induced pathologies such as myocardial infarction and stroke, which could further be extended to the neuromuscular degenerative diseases field.

The most common primary form of familial hypobetalipoproteinemia, known as familial hypobetalipoproteinemia due to type 2 secretion defect (FHBL-SD2, MIM: 61558), results from deleterious variants in the APOB gene, transmitted in an autosomal semi-dominant manner. Four types of mutations have been identified, primarily nonsense mutations, splicing site mutations, frame-shift mutations leading to truncation of ApoB from 2% to 89% of ApoB-100, as well as some missense variants. The presence of truncated ApoB increases the risk of hepatic steatosis by six-fold, and patients carrying small truncated ApoB variants appear to exhibit pronounced non-alcoholic fatty liver disease (NAFLD). However, the association with type 2 diabetes remains controversial. Additionally, although the alteration of contact points between the endoplasmic reticulum and mitochondria (called MAMs for mitochondria-associated ER membranes) has emerged as an early, causal, and reversible trigger for insulin resistance and hepatic steatosis, its role in FHBL-SD2 is unknown. The FABULOUS project aims to elucidate the potential link between FHBL2 and associated hepatic steatosis and insulin resistance, focusing on the role of APOB variant types and organelle miscommunication leading to mitochondrial dysfunction as a causal mechanism. Furthermore, in this project, we will explore the interaction between deleterious APOB variants and the rs738409 polymorphism in the PNPLA3 gene, previously associated with an increased risk of hepatic steatosis, fibrosis, and insulin resistance. By combining multicellular approaches in humans (Huh7, hiPSCs, PBMCs), the objectives of the FABULOUS project are: 1) to identify in Huh7 clones the variations in the APOB of FHBL-SD2 patients which lead to a reduction of ApoB secretion and confirm the pathogenicity of the most relevant variations in hiPSCs cells; 2) to study the consequences of these mutations on hepatic signaling and metabolism and to confirm them in patient PBMCs; and 3) to study the interaction between APOB mutations and PNPLA3 polymorphism on hepatic signaling and metabolism. This approach should contribute to a better understanding of the pathophysiological mechanisms and the identification of new therapeutic targets to address hepatocyte dysfunction and metabolic disorders associated with FHBL-SD2.

Increased genotoxic E. coli producing the genotoxin colibactin features gut microbiota dysbiosis during intestinal inflammatory diseases. Colibactin can alter host DNA stability, leading to dramatic consequences such as colorectal cancer (CRC) in humans. CRC is the 3rd most diagnosed human cancer, with an estimation of 1.9 million new cases worldwide in 2020, with about 935,000 deaths. The economic burden of CRC is significant, with estimated cost of INT(international) $2.8 trillion globally. A European study estimated the total cost of CRC across Europe to be €19.1 billion per year. These epidemiological data underline the huge clinical relevance and massive societal impact of genotoxic E. coli infection. This scenario is aggravated by the evidence that genotoxic E. coli are spreading to a pandemic rate, especially in urbanized area. We showed that colibactin from genotoxic SP15 E. coli induces gut microbiota dysbiosis and altered microbial functions, in mice offspring. Though gut microbiota dysbiosis is a starting condition prompting gut and systemic inflammation, yet, no mechanistic study links genotoxic E. coli infection, colibactin production, gut microbiota dysbiosis and both gut and systemic inflammation. The lack of data in this context represents the scientific barrier that our project PLEXOMIR wants to lift. To study the inflammatory impact of genotoxic SP15 E. coli vs. a commensal control E. coli, we assessed reduced platelet count, a rapid and reliable index of infection-induced systemic inflammation in humans, in adult male mice, better responders to bacterial infections than females, as reported in humans. Platelets are key cellular mediators of infection-induced systemic inflammation. Our preliminary data show that genotoxic SP15 E. coli reduces platelet count and induces higher IL-6 plasma levels and a strong colon inflammation. In vitro human intestinal epithelial cells infected with E. coli release proinflammatory extracellular vesicles (EVs, <100 nm). Thus, we hypothesize that the colon from mice infected with genotoxic SP15 E. coli release EVs which may target platelets. Key signalling factors in colon EVs are microRNAs (or miRs). In mice with colitis, colon EVs contain miR-21. We validated the presence of miR-21 in our model of infection, suggesting miR-21 as a promising colon EVs effector to modulate platelets during genotoxic SP15 E. coli infection. Thus, we propose: i) to study the role of colibactin on gut microbiota and microbiome, colon and systemic inflammation (work package 1, WP1); ii) to identify new miRs from colon EVs and to study in detail miR-21; we will also realise a targeted lipidomic and a proteomic study to characterise, respectively, those lipids and proteins from EVs that are known to affect platelets functions, i.e. aggregation (WP2); iii) to study the modulation of platelet (from mice and humans) homeostasis (morphology and function) by colon EVs miRs (WP3). For the integration of the overall significant data, P1 will count on an established local network of bioinformaticians. Before integration, data will be converted and structured into a common format to ensure consistency, accuracy and compatibility. We will set a bioinformatic pipeline from data checking, i.e. errors, inconsistencies and data integrity, to data analysis to final data comparison (WP4). This pipeline will allow running altogether the overall data of our project and will serve as a bioinformatic tool to analyse data from future projects. The setting of the bioinformatic pipeline represents the added value of PLEXOMIR in terms of methodological approach. Our experimental strategy is based on in vitro cellular models (human/murine platelets) coupled to in vivo murine models of SP15 E. coli infection. We expect no major experimental risk as all partners master their models. We believe this multi-steps strategy together with the expertise of our consortium will make our project successful.

Old age, sedentary lifestyle and illness are often associated with osteosarcopenia (osteopenia and sarcopenia), which significantly increases morbidity and negatively impacts the quality of life and degree of independence of the affected person. Osteopenia is defined as a moderate loss of bone mass, without the overt clinical manifestations of osteoporosis. Sarcopenia is defined as a loss in muscle mass and strength, often linked to aging and cancer, which can be accelerated by malnutrition and a sedentary lifestyle, leading to a deterioration in muscle strength and physical performance. It is well established that healthy bones and muscles are necessary to maintain a good level of physical exercise and, conversely, that exercise will maintain a functional musculo-skeletal system. In frail, sick and/or aged individuals, osteopenia and sarcopenia establish a vicious circle, where one condition triggers and accelerates the development of the other. At present there are no efficient treatments to slow down or to break that vicious circle. The hormone FGF19, produced by the intestine, has a strong influence on metabolism. Our teams have demonstrated that this enterokine additionally regulates muscle mass and strength in healthy mice and in mice with sarcopenia (Benoit et al, Nature Medicine, 2017). Furthermore, pilot experiments in our groups showed that FGF19 receptors are expressed in bone and that treatment with FGF19 can protect the degradation of the skeleton observed in an animal model of osteoporosis. Our consortium now wants to better understand the relationship between FGF19 and bone physiology and to explore whether FGF19 can efficiently counter both osteopenia and sarcopenia. The proposed project will reveal the role and mode of action of FGF19 in the skeleton of mice of different ages and in various models of osteosarcopenia, where bone and muscle loss is induced by aging, hind limb disuse (mimicking bed rest), or ovarian hormone deficiency (mimicking post-menopausal osteosarcopenia). After identifying the target cells in bones, signalling pathways will be analysed through in vitro studies. To understand the roles played by FGF19 in bone versus muscle in the context of the observed skeletal phenotypes, we will investigate tissue-specific genetic knock-out models of ß-klotho, an essential co-receptor for FGF19 action. Beyond mechanistic studies in animal models, we also want to translate these findings to humans. By determining the levels of FGF19 in the serum in different cohorts of osteosarcopenic individuals, we aim to better understand how FGF19 levels correlate with bone and/or muscle biology and function in humans. To do so, we will assess FGF19 concentrations in cosmonauts and immobilized subjects, as well as in a large cohort of elderly patients, and we will probe possible links with several parameters of their musculoskeletal physiopathology. Our results will lead to a better understanding of the role of FGF19 in bone and muscle physiology and should contribute to the development of new therapeutic strategies to prevent or to fight osteosarcopenia, based on FGF19 or FGF19 analogs.

Cells release information (RNA / Protein / Lipids) into lipid nanovesicles called extracellular vesicles (EVs). We have shown that EVs released by skeletal muscle during the development of insulin resistance had virus-like properties and transferred deleterious information between insulin-sensitive cells. However, the systemic role of EVs released by skeletal muscle has hitherto remained confined to the extrapolation of the results obtained in vitro due to the lack of animal models making it possible to follow their secretion / fate / integration in vivo. This project proposes to use zebrafish to bridge this scientific barrier. This model will express fluorescent proteins involved in the formation of EVs allowing them to be tracked from their secretion to their final destination in order to specify their target tissues and their biological functions, and the impact of obesity on their trafficking and their properties at whole body level.