Clinical and experimental studies have reported biological rhythms impairments (fast/feeding cycles) in obese subjects but also memory alterations. This is particularly worrisome for adolescents as the brain is still maturing. Our hypothesis is that deregulation of the circadian clock, induced by obesogenic diets, impacts the oscillatory expression of genes involved in synaptic plasticity and in memory formation in specific brain regions. The project aims at testing this hypothesis in juvenile mice fed an obesogenic diet using behavioral, cellular and molecular approaches. We will also test the therapeutic benefit of chrono-nutrition (forced synchronization of food intake) on these various levels of analyses in mice. The beneficial effect of chrononutrition on memory performances will also be tested in obese adolescents in a clinical trial. The results of this project should help to better treat the cognitive consequences of adolescent obesity

Obesity and its metabolic consequences (type-2 diabetes, cardiovascular, gastrointestinal and reproductive disorders, as well as certain cancers) have become major causes of morbidity and mortality in developed countries. In France today, 50% of adults (22 million) are overweight, of which 7 million are obese, and the past 30 years have witnessed an alarming 7-fold increase in the incidence of childhood obesity. The development of effective preventive and therapeutic measures for these disorders and the reduction of the associated medical, familial and socioeconomic burdens is impossible without an improved understanding of the pathways and mechanisms leading to obesity. Among these mechanisms is the transport of peripheral metabolic hormones into the brain, a critical limiting step whose deregulation leads to these disorders. Current evidence indicates that the brain is a key site processing information from “adiposity signals” such as the anorexigenic hormone leptin, which circulates in proportion to body fat mass and instructs the individual to stop feeding. Leptin receptors are expressed in regions of the hypothalamus involved in the control of food intake and energy homeostasis, and injections of leptin into the brain of leptin-deficient mice eliminate overfeeding. Conversely, the deletion of leptin receptors or the blockade of leptin entry into the brain results in obesity and resistance to this hormone. During previous work supported by the ANR (GLIODIABESITY, 2009-2013), we raised the groundbreaking notion that tanycytes, a specific type of hypothalamic glial cells, act as “gatekeepers” that regulate the access of blood-borne signals to the hypothalamus (Langlet et al., Cell Metab 2013), and in particular, its vesicular transport into the cerebrospinal fluid, from where it enters other leptin-sensitive regions (Balland et al., Cell Metab 2014). We have shown that this tanycyte-mediated transport of leptin is suppressed in both genetic and diet-induced obesity (Balland et al., Cell Metab 2014). Reversing leptin resistance by restoring tanycytic leptin transport holds important therapeutic potential (Patent WO 2014141124 A1, PI: Prevot V), as shown by previous positive reviews by the ANR, as well as two Marie Sklodowska Curie postdoctoral fellowships in 2015 to pursue this work. However, the molecular mechanisms involved in the tanycytic shuttling of blood-borne leptin into the hypothalamus are still largely unknown. The overall objective of this proposal is to further develop this highly original angle by developing state-of-the-art approaches to i) characterize leptin transporters and the trans-tanycytic route used by peripheral leptin to enter the metabolic brain (Aims 1 and 2), ii) assess whether endoplasmic reticulum stress, which is involved in leptin resistance, alters tanycytic leptin transport (Aim 3), and iii) develop novel models and cutting-edge tools to probe this tanycytic barrier in vitro (Aim 4). To carry out this innovative project in a highly competitive field of biomedical research, we are now teaming up with a group of internationally renowned experts in leptin signaling who have contributed to the aforementioned discoveries (Partner 2: Ralf Jockers & Julie Dam), and a second partner expert in intracellular vesicular trafficking (Partner 3: Stéphane Gasman). We are convinced that the successful outcome of the proposed research plan will have important implications for public health by providing essential clues about how the peripheral hormone leptin, which carries metabolic information to the CNS both during development and adulthood, enters the brain. More broadly, this research promises to shed new light on the cellular and molecular mechanisms used by the hypothalamus to integrate endocrine signals that coordinate energy homeostasis. The results will pave the way for the development of new treatment strategies to overcome hormone resistance in human obesity and associated metabolic syndromes.

Neurons and neuroendocrine cells release neurotransmitters, neuropeptides and hormones through calcium-regulated exocytosis. The ultimate step of exocytosis involves fusion of secretory vesicles with the plasma membrane, leading to the merging of these two compartments. Following exocytosis, the excess of membrane supply is compensated through membrane fission process by endocytosis. Although much has been learned concerning the proteins regulating vesicle fission and fusion at donor and acceptor compartments, relatively little attention has been paid to the necessary role of lipids. In particular, how membrane phospholipids asymmetry is regulated during exo-endocytosis remain a fundamental yet unresolved question. This aspect is particularly important in neurosecretory cells that display intense membrane trafficking and mixing between organelle membranes to support crucial neuro-physiological functions such as neurotransmission. One of the key features of the plasma and the exo-endo membranes is the asymmetric distribution of lipids between the leaflets with for example, the aminophospholipids (APLs) phosphatidylserine and phosphatidylethanolamine primarily restricted to the cytosolic leaflet. This selective asymmetry is involved in many cellular processes and is controlled by proteins like the P4-type ATPases, flippases that catalyze the transport of APLs from the exoplasmic to the cytoplasmic leaflet of membranes. On the contrary, proteins named “scramblases” catalyze the bidirectional transbilayer movement of a broad range of phospholipids resulting in the disruption of lipid asymmetry. Preliminary data from our consortium indicate that i) APLs scrambling occurs at the close vicinity of the secretory vesicle fusion sites in neuroendocrine cells, ii) scramblase protein is somehow important to control exo-endocytic processes in neurons and neuroendocrine cells and iii) that P4-ATPases control various endocytic processes. However, the nature of the flippases and scramblases involved in neuro-secretory processes, their functional role and the importance of transbilayer lipid movement remains unknown. Therefore, our project’s central aim is to delineate which, why and how P4-ATPases and scramblases controls hormone secretion in neuroendocrine cells and neurotransmission in neurons. Investigating lipids dynamic is clearly a challenge and to succeed in this ambitious task, we have built a consortium able to lift the main technical limitations away. We indeed propose here a unique collaborative strategy that combines the generation of CRISPR/Cas9-engineered neuroendocrine cell lines knock-in or knock out for various scramblase and P4-ATPases, ultrastructural electron microscopy imaging on freeze-fracture replica of membrane layer fragments and the super-resolution imaging STED and SMLM methods with the use of specific lipid probes, membrane deformation assay and in cellulo exploration of secretory exo-endocytic events. Such synergistic combination will allow us to unravel for the first time the importance of lipid transbilayer transport regulation during vesicular membrane trafficking events in neuro-secretory cells. Moreover, we believe that this project will not only provide new insight into exo- and endocytic processes, but will create general and novel tools and methods for understanding complex mechanisms of lipid biology and biomembrane dynamics.

Schizophrenia is a multifactorial disease with a strong genetic component that is characterized by positive symptoms, negative symptoms and cognitive impairment. It is currently hypothesized that schizophrenia is a neurodevelopmental disorder that affects neuronal dendritogenesis and synaptogenesis. The Brain Angiogenesis Inhibitor family of receptors is a new family of seven-transmembrane receptors whose role in the brain is still poorly described. Several studies have shown the association of polymorphisms or copy number variations in the gene coding for BAI3 with schizophrenia. The characteristics of these receptors and data obtained by the coordinator’s team suggest an important role for BAI receptors in the formation of neuronal networks. In particular, they might coordinate the development of neuronal architecture with synatogenesis during brain development. The proposed project presents a multidisciplinary approach that will bridge basic research with translational research by providing a better understanding of the role of the BAI3 signaling pathway during brain development and of its direct involvement in the symptoms of schizophrenia. The BAI3 receptor is expressed in diverse brain regions including in cerebellar Purkinje cells. Because increasing evidence implicate cerebellar deficits in schizophrenia, especially in patients with high scores of neurological soft signs and stronger cognitive dysfunction, and because of the well-described cellular, physiological and behavioral characteristics of the olivo-cerebellar network, we will focus on the role of BAI3 in Purkinje cell development for our studies of BAI3 signaling and function. First, we will generate genetically modified mouse models with deficits of BAI3 signaling specifically in Purkinje cells and characterize the induced deficits in neuronal network organization at the morphological level, at the physiological level in vitro and in vivo, and at the level of behavior using a neurophenotyping strategy. This study will thus provide a description of the morphological and physiological correlates of behaviors relevant to schizophrenia and identify the symptoms that are due to cerebellar deficits. Second, we will combine genetic tagging in mice, biochemistry and mass spectrometry to identify the synaptic partners of the BAI3 receptor in neurons in vivo, and use a knockdown approach in vivo to validate the role of these candidates in the formation and function of neuronal networks. Finally, we will combine genetic association studies in patients with schizophrenia and functional studies of identified polymorphisms in cultured neurons to provide direct evidence of the role of the BAI3 signaling pathway in schizophrenia. This study will help us identify patients with relevant polymorphisms. We will then be able to transdifferentiate cells from these selected patients into “neurons” so as to confirm the effects of deficits in the BAI3 pathway on neuronal development and more importantly to test whether these deficits can be rescued by genetic modifications. Overall, this project takes advantage of the complementarities of the three partners not only to provide a comprehensive study of the role of a new signaling pathway in schizophrenia, but also new animal models for this disease and functional confirmation of genetic association studies using cellular models established directly from patients. Thus, our study will provide a better understanding of the etiology of schizophrenia, open new avenues of research and provide new potential therapeutic targets for this disease.

Fragile X syndrome (FXS) is a monogenic pathology responsible of the main cause of inherited intellectual disability (ID) and autism. FXS affects 1/4000 men and 1/8000 women. There is no treatment yet validated. FXS is due to the absence or the loss of function of the FMRP (Fragile X Mental Retardation Protein). The Fmr1 knock-out mouse model (Fmr1-KO) recapitulates the symptoms of FXS and demonstrates that the lack of FMRP leads to alterations of synaptic plasticity underlying neuronal troubles of FXS. FMRP is an RNA binding protein whose absence causes an excessive translation of hundreds of proteins in neurons of several brain regions. This neuronal protein synthesis excess leads to the alteration of several forms of translation-dependent synaptic plasticity. Thus, FMRP appears as a key regulator of the mechanisms underlying the inter-neuronal communication. The precise molecular function of FMRP in this process is however still not fully understood. One outstanding question that remains unresolved despite extensive research efforts is how quasi-ubiquitous FMRP controls the translation of hundreds of mRNAs specifically in neurons. In this context, understand how the absence of FMRP leads to synaptic alterations remains a major goal to define the molecular basis of FXS and identify a treatment. Towards this goal, this project is based on our recent discovery that the loss of FMRP, besides leading to protein translation excess in neurons, is also leading to diacylglycerol (DAG) and phosphatidic acid (PA) lipid signaling deregulation. In neurons, FMRP is mostly associated with diacylglycerol kinase kappa (DGKk) mRNA and positively controls its translation. DGK enzymes are the master regulators of the switch between DAG- and PA- signaling pathways that control protein translation and actin filament stability, respectively, and that are proposed to orchestrate synaptic plasticity. The loss of DGKk is sufficient to reproduce FXS associated symptoms in the mouse. These data lead to a change of paradigm in the pathological mechanism of FXS and the function of FMRP: DGKk is a primary target of FMRP in neurons and the excess of DAG and a lack of PA signaling consecutive to DGKk deregulation contributes predominantly to the pathology. These data open new avenues of research towards understanding of FMRP function, and suggests novel therapeutic means. The scientific program, based on our newly identified pathomechanism, aims at understanding the molecular basis of Fragile X syndrome by following two main axes: understand the molecular function of the FMRP protein and identify a novel way of intervention. The program is organized to achieve four distinct goals: 1) define the molecular mechanism of FMRP translation control of Dgkk in neurons, 2) determine the functional consequences of Dgkk deregulation in the mouse and humans, or its absence (new Dgkk-KO mouse model) and demonstrate that its deregulation is critical for FXS condition, 3) validate DGKk as a novel therapeutic target for FXS in the Fmr1-KO mouse model. The program will be performed by a consortium that combines a panel of expertise spanning RNA/protein interactions, lipid signaling, neuronal electrophysiology, and animal behavior. The main expected outcome will be a detailed molecular description of the molecular mechanism by which FMRP contributes to the control of local protein translation within neurons, the deregulation of which is causing the well-defined neurological alterations of the Fragile X syndrome. A second main expected outcome is the validation of a proof of concept for a novel therapeutic mean in the FXS mouse model.