Translational control of gene expression plays a crucial role in the response to stress by blocking translation of most mRNAs. However, alternative translation mechanisms allow enhanced expression of a small group of mRNAs involved in cell survival. This occurs in pathologies such as ischemic heart disease, where tissues are exposed to hypoxia or endoplasmic reticulum (ER) stress. One of the major alternative mechanisms able to overcome this global inhibition of translation depends on internal ribosome entry sites (IRESs), RNA structural elements allowing ribosome recruitment directly on the initiator region of mRNA during stress. In particular, hypoxia and ER stress both induce expression of angiogenic growth factors (FGF and VEGF) leading to tissue revascularization, and of genes involved in cardiomyocyte survival such as insulin-like growth factor 1 receptor (IGF1-R). These mRNAs contain an IRES and are induced by hypoxia. How the expression of these survival genes is regulated during hypoxia remains poorly understood. However, it represents an important biological question to address with both fundamental and medical valorisation perspectives. The idea of specialized ribosomes in mammals emerged as a new concept. Ribosome heterogeneity could include diversity in the composition and post-translational modifications of ribosomal proteins or variations in ribosomal RNA sequence or chemical modifications. Several reports, including our data, show the involvement of ribosome heterogeneity and rRNA modifications in the control of IRES-dependent translation. These observations suggest a crucial role of specialized ribosomes in the control of IRES-dependent translation, but up to now there is a lack of knowledge in the field of hypoxia and ER stress occurring in ischemic diseases. The aim of the RIBOCARD project is to explore the role of ribosomes in the control of IRES-dependent translation in stressed cardiomyocytes, in cell culture and in ischemic heart. The specific objectives are 1) to study the impact of hypoxia and ER stress on ribosome composition and ribosomal protein modifications, 2) to analyse the impact of hypoxia and ER stress on rRNA modifications, 3) to characterize the role of ribosome composition and modifications on IRES-dependent translation, and 4) to determine the structure of an IRES-bound human ribosome complex. To ensure the feasibility of its programme, the RIBOCARD consortium gathers three partners with international reputation in the fields of cardiovascular diseases, cell stress, translational control, rRNA modifications, ribosome composition and high resolution structural analysis, possessing all scientific and technical skills to achieve the project. In addition, our recently published results and preliminary data validate our hypotheses. The three partners have visualized rRNA chemical modifications in the human 80S ribosome, demonstrated the plasticity of rRNA 2'-O-methylation and its impact on ribosome translational capability. In addition, we have shown in hypoxic cardiomyocytes that the major genes involved in angiogenesis and cardiomyocyte survival are induced at the translational level, involving IRES-dependent translation. Also, we have identified specific ribosomal proteins bound to IRES RNA, as well as significant changes in rRNA methylation of hypoxic cells polysomal ribosomes. The RIBOCARD proposal is fully original considering that little information is available regarding translational control through variable ribosome composition, the role of rRNA modifications therein and the molecular mechanisms involved. This project thus addresses a new frontier in gene regulation. The structure of an IRES bound to the human ribosome will provide completely novel mechanistic insights. Thus, RIBOCARD will raise a new concept in the field of gene regulation in ischemic diseases, and will provide new targets to improve both understanding and treatment of these pathologies.

We aim at deciphering the role of Vangl2, a main component of the planar cell polarity (PCP) signaling, in hippocampal network function which is to compute information involving object location, spatial contexts and events relevant for every day memory function. PCP signaling is necessary for proper coordination of development and growth of many tissues, by controlling notably intercellular and intracellular communication, and integrating signaling cues leading to a reorganization of the cytoskeleton. In the brain, growing evidence suggests that PCP signaling is fundamental for neuronal development, including neuronal migration, neuronal polarity and maturation, axonal guidance, but also dendrite morphogenesis, synaptogenesis and synaptic plasticity. But a fundamental and yet unresolved question is why PCP proteins are also expressed at the adult stage in the hippocampus and neuronal circuits critical for cognition and memory. Recent work from our lab (Montcouquiol/Sans) revealed specific behavioral consequences of brain-specific disruptions of the PCP-signaling scrib gene, using mainly deletion or mutation models in mice (Moreau et al, J. Neurosci. 2010; Hilal et al, Cerebral Cortex, 2017; Ezan et al., in preparation). Preliminary results from our lab suggest that early or late deletion of vangl2, restricted to the brain, lead to altered hippocampus-dependent memory functions (Moreau et al., in preparation; Robert et al., SFN 2016 poster #356.09; in preparation). This suggests that unexpectedly, PCP proteins may be essential component of the adult central nervous system and required for proper function of memory-related brain structures such as the hippocampus. Our project is to combine molecular, behavioral and electrophysiological approaches to assess hippocampal function in the adult mouse after manipulating the expression of Vangl2 in specific hippocampal neuronal populations. Here, we will use well-established and new molecular, cellular and genetic tools to manipulate PCP signaling (Montcouquiol-Sans lab), combined with in vivo recording associated with behavioral paradigms (Leinekugel lab) to evaluate the consequences in terms of cellular communication and circuit function. A strong innovative aspect of this proposal is to develop new AAV or lentivirus construction driving the cre-recombinase or other specific activating genes or dominant negative constructs into specific neuronal population. Using these tools, it will be possible to achieve spatial and temporal activation or inhibition of the vangl2-dependent pathway in adult mice. Combined with high-end electrophysiological recordings and opto- and pharmaco-genetic manipulation of the activity of hilar mossy cells, we will also have the very timely opportunity to address the respective roles of dentate mossy and granule cells in hippocampal circuit function. This project will provide novel conceptual insights into the role of Vangl2-dependent signaling and function in the hippocampal circuit, and more generally pave the way for a better understanding of the importance of PCPs in adult neuronal microcircuits.

The overall objective of our project is to understand the role of newly identified apical Planar Cell Polarity (PCP) signaling dependent on Gai-proteins during the maturation of the inner ear of mammals, in physiological and pathophysiological conditions. In the Western world, the proportion of the population that suffers from hearing loss is around 7 to 8%. Statistics collected from different countries show that out of 1,000 births, 1 to 1.5 will suffer profound hearing loss or deafness. Because the mechanoreceptive hair cells which mediate the sensory transduction in the inner ear can be injured or definitively lost after exposure to noise, otoxic drugs, or as part of normal aging, hearing losses are the fastest growing, and one of the most prevalent chronic conditions facing an aging population. Developing knowledge on the genetic and molecular bases of auditory cells differentiation that could guide strategies for regeneration and protection has the potential to lead to the establishment of new tools for prognosis and diagnosis of deafness, but also has the potential to open new avenue of research for inner ear pathologies in the hope to explore opportunities for preventive and therapeutic interventions. Recently, we have identified a new PCP signaling pathway, which we called G-protein-dependent PCP signaling (Ezan et al., 2013).During the course of this original study, we observed that in later stages of maturation, the hair bundles topping the hair cells appeared malformed, shorter and fragmented in two of the studied mice mutants, suggesting the involvement of certain genes of this PCP pathway in the late maturation of the hair cells and more generally in hearing function. As a first step, our proposal will explore this hypothesis, notably through the use of transgenic mouse models and in particular via Cre-lox technology that will allow us to study the postnatal development of the inner ear, its maturation and its function, or disruption of function. As a second step, we will explore the hypothesis that the apical complex Crumbs controls the dynamic of tubulin and actin, at least in part via the recruitment of certain of the apical PCP signaling pathway. For this, we will build on a multidisciplinary and multi-model approach that will bring us the benefits of three species: Xenopus, mouse and Drosophila. The results of our project will lead to the identification of a new family of candidate genes for deafness, to the elucidation of the molecular mechanism leading to these deafnesses and to decipher new protein networks at the crossroads between the apico-basal polarity, the Planar Cell Polarity and the cilium.