Myotonic dystrophy type I (DM1) is a rare inherited neuromuscular disease affecting multiple organs. The genetic mutation is located on chromosome 19 and consists of dynamic expansion of a trinucleotide motif (CTG) in the 3’ untranslated region of DM protein kinase gene (DMPK). The aetiology of DM1 relies essentially on a toxic gain of function of RNAs transcribe from the mutated gene. Thus, RNAs bearing the long CUG expansion are retained in cell nuclei, associate to RNA-binding proteins, form proteoribonuclear inclusions named foci, ultimately leading to a defective splicing of several target transcripts. The defective splicing of each transcripts can be related to a specific symptom. Central nervous system involvement in DM1 was reported long ago, but systematic studies to analyze implicated brain structures, neuropathological hallmarks and molecular mechanisms, have only been initiated. We showed an altered splicing of RNA transcripts coding for a neuronal protein essential for maintaining the architecture and plasticity of neurons, the microtubule-associated Tau. This mis splicing is associated with the development of neurofibrillary degeneration. Neurofibrillary degeneration (NFD) is a neuropathological hallmark common to more than twenty neurological disorders named Tauopathies, to which DM1 belongs. Altogether, a growing body of evidences including our data suppose that a modified splicing of Tau is likely instrumental to NFD, and myotonic dystrophy is a good model to consolidate the proof of concept of the relationship between the indirect mis splicing of Tau, brain function and the development of NFD. Therefore, our global objective is to determine the contribution of a modified splicing of Tau to the development of NFD, to reverse the mis splicing of Tau and we intend to model, study and reverse this pathophysiological mechanism in novel transgenic mouse models of NFD in DM1. To that purpose, we propose to cross DM1 transgenic mice expressing the mutated DM1 human locus with mice transgenic for the human TAU gene but invalidated for the endogenous murine Tau gene. The resulting transgenic offspring will enable to address our hypothesis. Our preliminary results demonstrated an altered splicing of Tau, hyperphosphorylation of endogenous Tau and altered synaptic plasticity in the DM1 transgenic mouse model. However, the endogenous splicing of the murine Tau is different from that of the human TAU splicing pattern. In contrast, the human TAU transgenic model is expressing the six Tau isoforms alike in the human brain. By multidisciplinary approaches and complementary expertises of the two laboratories implicated in this project, we expect to develop and characterize DM1 x human TAU transgenic mouse model that will reproduce a NFD process similar to the human pathophysiology, associated with Tau splicing defects. Moreover, our preliminary results have established that Tau mis splicing directly result from the loss of function of a splicing factor that is MBNL. We have engineered a chimeric splicing factor that enables to correct the mis splicing of Tau in vitro. Moreover, in control condition, ectopic expression of this construct is not toxic and does not regulate the splicing of Tau and other targets. We are currently producing the lentiviral vector in order to infect DM1 mice as well as the future hTAU x DM1 transgenic models. Therefore, besides being essential to address fundamental questions, the DM1 model of NFD we will develop will be very useful to test drugs and to test viral gene transfer in order to rescue in vivo the mis splicing of tau and most likely the NFD and associated symptoms. Altogether, our project aim to develop a completely novel transgenic mouse model of NFD and to rescue the pathophysiology of DM1 using a viral gene transfer therapy.

Reproduction in mammals is under the control of the hypothalamic neuropeptide GnRH-1. GnRH-1-secreting neurons, like olfactory neurons, originate in the olfactory placode, which will later develop into the olfactory epithelium. During normal development, olfactory neurons send their axons to the olfactory bulb, while GnRH-1 neurons migrate from nose to brain along these axons. Developmental alterations in this migratory process result in reproductive failure as demonstrated by patients affected by the human genetic disease known as Kallmann’s syndrome (KS) which results in anosmia (reduced or absent odor perception) and hypogonadism (lack of gonadal development). Only approximately 30% of all KS cases are attributable to mutations of known genes, suggesting that other genetic pathways might be relevant for this pathogenesis. Even though, many progresses have been made, especially during the last 15 years, in the field of developmental neuroendocrinology, the elaboration of new therapeutic strategies for reproductive disorders requires the identification of the molecules orchestrating migration and differentiation of GnRH-1 neurons. The goal of this project is to gain a comprehensive view on the molecular events governing GnRH-1 cell migration from nose to brain and its role in the establishment of neural circuits leading to reproductive functions. Combining mouse genetics, ex vivo manipulations and imaging techniques, I propose to investigate: i) the molecular mechanisms controlling the specification and migratory routes of GnRH-1 in vivo; ii) the nature of GnRH-1 cells interactions with olfactory axons as well as their role in the correct targeting to their final destination areas. iii) In parallel, we will investigate how adult GnRH-1 axons terminals rearrange in the median eminence under the influence of different chemotropic molecules. Our combination of approaches will generate a novel framework to understand how the hypothalamic-pituitary-gonadal axis connectivity is established during normal and pathological development.

Pathogenic bacteria have evolved mechanisms to secrete proteins such as enzymes, toxins and adhesins that enable them to interact efficiently with the host environment and to perform their infectious cycle. In Gram-negative bacteria the presence of an outer membrane has required the development of specific systems to address these proteins to their proper location. Understanding the molecular mechanisms of these secretion systems which represent an essential aspect of bacterial pathogenesis is important from a basic science perspective, and also because these systems are potentially new therapeutic targets. The Two-Partner Secretion (TPS) pathway is widespread in Gram-negative bacteria, including important pathogens such as Bordetella pertussis, Neisseria meningitidis or Pseudomonas aeruginosa, and it is devoted to the secretion of large adhesins and cytolysins that fold into long beta helices. The two partners of these systems are the secreted TpsA protein harbouring a conserved, N-terminal TPS domain essential for secretion, and its specific TpsB transporter forming a pore in the outer membrane. The secretion system of the FHA adhesin of B. pertussis by its FhaC partner serves as a model for the TPS pathway. FHA (TpsA partner) is an important virulence factor of this pathogen. FhaC (TpsB partner) belongs to the Omp85/TpsB superfamily which includes notably essential protein transporters of mitochondria and chloroplasts. Our system thus represents also a model to decipher the molecular mechanisms in this superfamily. We obtained in 2007 the X ray structure of FhaC, which has remained thus far the only one of the TpsB/Omp85 superfamily. FhaC forms a 16-stranded transmembrane beta barrel preceded by a periplasmic domain composed of two POTRA domains. Large extracellular loops and short periplasmic turns connect the anti-parallel strands of the barrel. The barrel pore is obstructed by a long N-terminal alpha helix (H1) and an extracellular loop (L6) folded back into the barrel. H1 is followed by a periplasmic linker that connects it to the first POTRA. We have demonstrated the functional role of the two POTRAs for the molecular recognition of the TPS domain of FHA by FhaC and the role of L6 and the linker for the activity of FhaC. Our current model of secretion based on numerous experimental results goes as follows. Following Sec-dependent export of FHA through the cytoplasmic membrane, the TPS domain of FHA in an extended conformation interacts with the POTRAs. This triggers the opening of the pore, leading to the progressive translocation of FHA in an extended conformation through the FhaC pore. FHA folds progressively at the cell surface to form its extended beta helix. The X ray structure of FhaC most likely represent the resting state of the transporter, which will undergo significant conformational changes during the secretion cycle, such as the move of H1, L6 or both out of the pore. The proposed program rests on the structure of FhaC and our experience on this model system. Our goal is to decipher the conformational dynamics of FhaC in the secretion cycle at the molecular level. We will combine X ray crystallography, structural approaches in solution (SANS), biochemical and biophysical methods such as cross-linking in vivo and electronic paramagnetic resonance, to address the conformational changes of FhaC in the course of secretion and to obtain the structure of FhaC in action. In particular, we will analyse the dynamics of important structural elements (POTRAs, H1, linker, L6, other surface loops) and compare them between the resting and active states. We will define the pathway followed by FHA through its transporter by probing its interactions with these various elements. We want to obtain the structure of FhaC in action, by preparing a blocked complex between FhaC and a chimeric protein containing a secretion-competent N-terminal FHA fragment fused to a heterologous, secretion-incompetent globular domain.