Precise control of the transition from self-renewal to terminal differentiation in stem cells is critical to maintain a balance between cell populations: an excess of stem cell self-renewal can lead to tumourigenesis, whereas an excess of differentiation can deplete the stem-cell pool. In the adult Caenorhabditis elegans germline, Notch signals emanate from the somatic distal tip cell to maintain germline stem cells (GSCs) in a proliferative state by repressing the translation of meiotic promoting factors. We have uncovered a novel pathway regulating the decision between GSC renewal and meiotic differentiation that involves the ubiquitin-proteolytic system (UPS). Using a novel temperature-sensitive allele of the cul-2 gene, we found that the CUL-2 RING E3 ubiquitin ligase, in combination with the Leucine Rich Repeat 1 substrate recognition subunit (CRL2LRR-1), negatively regulates the transition from the mitotic zone of the germline to the meiotic programme of chromosome pairing, synapsis, and recombination. More specifically, we find that CRL2LRR-1 regulates in stem cells the stability of the HORMA domain-containing protein HTP-3, which is required for loading structural proteins onto meiotic chromosomes and for the formation of the double-strand breaks that initiate meiotic recombination. Furthermore, we found that cyclin E/Cdk2 kinase, which is specifically activated in GSCs but repressed upon meiotic differentiation, phosphorylates HTP-3 and regulates its stability. Besides HTP-3, CUL-2 targets other factors for degradation to prevent precocious meiotic entry and to promote germline stem cell proliferation. Herein, we propose to use a unique combination of genetics, cell biology, biochemical and quantitative proteomic approaches to elucidate the role of protein degradation in germ cell biology. In particular, we propose to identify CUL-2 targets in the germline and the molecular mechanisms controlling their degradation in space. The role of the UPS and CUL-2 in germline stem cell biology has not been studied so far. CUL-2 is evolutionarily conserved in metazoans and appears to regulate germ cell divisions in Drosophila. Therefore emerging paradigms provided by the study of germ cell biology in C. elegans should be directly applicable in other systems and possibly also in humans.

The present ANR project aims at fabricating and exploring the properties of a special class of capsules called “ethosomes”, resulting from the self-assembly of phospholipids in the presence of water and ethanol. The main objective of the project is to develop the underpinning science required to address challenges in designing ethosomes based on phospholipids rich in n-3 Poly-Unsaturated Fatty Acids (PUFA). Ethosomes can be used in nutrition applications because of the substantial benefits of PUFA in human health. Moreover, due to the presence of ethanol, ethosomes present membrane fluidity properties that make them interesting delivery systems by the topical route. So far, the phospholipids used to fabricate conventional liposomes or ethosomes are extracted from soya or egg yolk sources. The food industry is generating large amounts of by-products, most of them remaining under valorised. In this project, we propose to produce phospholipids either from animal marine or vegetable sources using supercritical CO2 as a “green” extraction solvent. Ethanol will be mixed with CO2 in order to improve the extraction yield. Delivering phospholipids rich in n-3 PUFA is really challenging due to their susceptibility to chemical degradation. Indeed, polyunsaturated lipids are sensitive to heat, light and oxygen exposure. This causes product deterioration in terms of aroma, texture, shelf life and colour. Within this project, we will examine the impact of ethanol on the physical and chemical stability of liposomes containing a large fraction of n-3 PUFA chains under well-defined storage conditions. In addition to the high-resolution techniques already available, we will develop a novel technique to follow lipid oxidation based on micro-calorimetry. Indeed, non-destructive, simple and efficient technique to follow the complex oxidation process is highly sought-after and not presently available.

Our project aims at challenging the classic view of the “neurocentric” concept in a brain function such as sleep regulation. This view postulates that rhythmic activities recorded during sleep are only based on neuronal properties and circuits. However, glial cells that represent the most numerous populations in the mammalian brain establish dynamic interactions with neurons. In particular, astrocytes are tightly associated with pre- and post-synaptic elements on which they exert a modulatory action through different mechanisms such as: i) the uptake of ions and neurotransmitters, ii) the release of bioactive substances including energy substrates and the so-called “gliotransmitters” whose involvement in sleep homeostasis was recently shown, and iii) the control of the extracellular volume. In addition, astrocytes express numerous gap junctions and thus are organized as networks of communicating cells. These gap junctions are constituted by aggregates of intercellular channels composed by the apposition of two hexameres of membrane proteins named connexins (Cxs) in vertebrates and innexins (Inxs) in invertebrates. Glial networks are involved in the homeostasis of ions and neurotransmitters, intercellular calcium signalling and in the removal of toxic substances. They also contribute to the intercellular trafficking of energy metabolites delivered to neurons and sustain synaptic activity. While there is increasing evidence for a participation of glial Cxs and gap junctions in brain diseases, much less is know about their role in brain functions. The objective of this project is to determine the contribution of this typical feature of glial cells to neuroglial interactions occurring in a complex brain function such as sleep regulation. Addressing this question is justified by: i) the high level of expression of Cxs/Inxs in glia that confers to these cells a network organization; ii) preliminary data suggesting changes in the expression and function of these proteins following sleep disruption in mouse and fly models; iii) the modification in the pattern of neuronal slow oscillations characteristic of deep phases of sleep in animal lacking astroglial Cxs. The proposed objectives are: i) to decipher the impact of the impairment of Cx and Inx functions on the quantity and quality of sleep-wake in mice and flies, respectively, ii) to establish in vitro and in vivo the contribution of astroglial Cx-based communication to the rhythmic activity of cortical neurons typical of slow waves sleep, iii) to identify changes in expression and function of astroglial Cxs triggered by behavioral or pharmacological treatments altering sleep and iv) to use the Drosophila model to rapidly screen and identify genes and molecular pathways linking glial gap junction communication to sleep regulation. This project will be conducted by a partnership associating expertise and know-how in several domains including brain gap junctions and neuroglial interactions, in vivo and in vitro recording/analysis of neuronal rhythmic activity, sleep physiology and fly genetics of sleep. Such interdisciplinarity provides the best chance of success for this translational project. Moreover, the involvement of a partner who has a recognized knowledge in neuroglial metabolic interactions and in sleep regulation, represents an essential added value to this application. Once completed, this program should provide a full overview of the contribution of glial networking to sleep regulation at the molecular, cellular and behavioral levels as well as its potential involvement in sleep disorders.

One of the most remarkable features of vertebrate morphology is the complexity of the head that harbours a “big” brain, many sensory organs, and an assemblage of bones, cartilages and muscles organized in a much-elaborated way, and its evolution remains an unanswered question. Concerning muscles, their developmental origins are diverse, (some derive from the somites (tongue), others from the paraxial unsegmented pharyngeal mesoderm, whereas the extraocular muscles develop from the prechordal plate). Vertebrates belong to the chordate phylum, with the tunicates and the cephalochordates. Both tunicates and cephalochordates (i. e. amphioxus) are filter feeders and share a pharynx adapted to this mode of nutrition, little sensory organs and reduced anterior centralized nervous system, as well as relatively simple oropharyngeal/atrial muscles. It has been proposed that vertebrates acquired their complex head during evolution as an adaptation to a predatory life style and Partner1 proposed a scenario for the evolution of the head muscles of vertebrates from a chordate ancestor with a mesoderm organization similar to that of cephalochordates. However, how anterior mesoderm derived muscles and associated motoneurons (MNs) co-evolved is still a major question. Thus, the aim of this project is to provide clues about the evolution of the motoneurons associated with anterior mesoderm derived muscles. In other words: are the motoneurons contacting non-myomeric mesodermal anterior muscles of the three chordate clades homologous? To achieve this, we will use cutting-edge techniques (scRNA-seq/ATAC-seq, transgenesis, retrograde labelling, etc.) applied to three chordate models (amphioxus, ascidian and mouse) to provide the information needed for an exhaustive comparison of anterior muscle/neuron systems, in order to define the co-evolution scenario of these structures in relation to the appearance of the complex vertebrate head.