The cerebellum plays a major role in sensory motor adaptation and non-motor tasks. Initially, the cerebellar cortex was considered as a homogeneous structure with a “crystal-like” organization at the level of the cytoarchitecture and neuronal connectivity. However, it is now apparent that the cerebellar cortex is organized in modules found at the functional and molecular levels leading to a large diversity of information processing. A high heterogeneity in the neuronal populations forming this network is starting to be deciphered. But, while neurochemical and functional heterogeneity in Purkinje cells (PC) have been well described and its relationship to the diversity of functional properties for PCs is better understood, diversity of other neuronal populations in the cerebellar cortex is very poorly studied. Some markers have been found to be expressed heterogeneously in granule cells (GC), especially along the antero-posterior axis and single cell transcriptomic analysis has defined at least five types of GCs. The goal of this project is therefore to determine whether the molecular heterogeneity of GCs underlie a functional and computational heterogeneity in the cerebellar cortex. In task 1, we will assess the spatial and functional organization of two different GC lineages that are organized in two different antero-posterior gradients. Thanks to two genetically modified mouse lines already available and characterized as labeling these two GC lineages, we will describe their relationships to specific cerebellar modules defined by Purkinje cells markers, by combining anatomical, electrophysiological, optogenetic and imaging techniques. We will then determine the specific surfaceome for anterior versus posterior GCs using proteomic techniques and identify relevant markers for their specific connectivity and functional properties. Functional analysis of some of those markers will be performed using the Crispr/Cas9 technology to assess the relevance of GC molecular heterogeneity for their functional diversity. In task 2, using an intersectional approach, we will test how two parameters, GC genetic lineage and GC date of birth, interact and influence local molecular heterogeneity and functional synaptic properties. For this we will combine our two genetically modified mouse lines with electroporation of Cre dependent reporter constructs at different postnatal timepoints. Using sophisticated imaging and optogenetic techniques, we will describe how these two parameters interact to influence GC network integration and the diversity of their synaptic properties. In task 3, we will address the influence of the afferent mossy fibers on GC diversity, In a manner similar to how PC afferents influence PC diversity. We will study how specific mossy fibers target GC subgroups, defined by their lineage and the markers that will be identified in our work? We will also invalidate some markers with a potential role in regulating connectivity to test whether MF connectivity pattern is modified. Finally, we will test whether removal (using Diphteria toxin receptor expression) or activation (using chemogenetic tools) of mossy fibers during development modify GC diversity at the molecular and functional level. In task 4, we will assess how these results influence our existing models. Altogether our project will combine the complementary expertise of two groups internationally recognized for their work on the synaptic and functional organization of the cerebellar cortex to address key issues about the diversity of cerebellar synaptic organization and computation. The results will impact our understanding of brain development and function. Our results will also be of interest for the treatment of brain diseases since the cerebellum is also involved in several types of brain disorders such as autism spectrum disorders.

The central machinery by which hormones, proteins and neurotransmitters are released from exocytosis vesicles is well known. Nevertheless, we are still far from having elucidated the regulatory mechanisms that prevent random secretion events from occurring. Subtle contacts between organelles have been observed and it is increasingly established that their major roles are to harbor intensive exchanges of lipids and to control the flow of calcium through specific channels between intracellular organelles or between these organelles and the limiting membrane of the cell. Surprisingly, although the dynamics of calcium and lipids are crucial for the regulation of exocytosis and the subsequent reuptake of certain constituents of exocytosis vesicles by compensatory endocytosis, studies on the contribution of membrane contact sites in these mechanisms show default. Our proposal, based on strong preliminary evidence and novel chemobiology tools supporting the presence of three different types of contact sites between the endoplasmic reticulum, secretory granules, and plasma membrane, is to define how these are formed, what are their dynamics, and their role in the regulation of secretion. From a mechanistic perspective, we will focus on SOC-type calcium channels and the transfer of key lipids that define sites of exocytosis. Our project should provide completely new and unanticipated data to better understand the regulation of a major biological process.

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.

Disorders affecting social behaviors, such as autism spectrum disorders (ASD), are complex neurodevelopmental disorders involving deficits in social interaction and communication. To date, no pharmacological treatment that improve social symptoms exists for ASD, and the only therapeutic options rely on costly behavioral intervention programs with limited beneficial effects. Oxytocin and its receptor are key determinant players of social behaviors with therapeutic potential for social interaction deficits. Oxytocin receptor is expressed in specific brain structures and cell types in the central nervous system. Here, we propose i) to dissect the role of oxytocin receptor in these structures, spanning from olfactory neurons to glial cells and neurons in interconnected central brain areas, and ii) establish a therapeutic framework of exogenous intranasal oxytocin administration in a mouse model of autism. We will identify how oxytocin receptor in the mouse olfactory system modulates social interactions, determine the modulatory role of oxytocin receptor centrally, in both neurons and astrocytes and use administration of exogenous oxytocin in a well-established and mouse model of ASD and Fragile X syndrome. Overall, our project will provide essential novel information on how oxytocin modulate social signals and brain activity to enable social interactions, providing therapeutic levers in the context of sociability disorders.

The control of female reproduction has become a major societal and economic concern, and thus a better understanding of the central mechanisms acting on the reproductive axis is necessary. Early studies on the neural pathways involved in the control of the reproductive axis have highlighted the pivotal role of GnRH (Gonadotropin Releasing Hormone) neurons located in the rostral hypothalamus. GnRH release into the portal blood stimulates the secretion of the pituitary gonadotropins which are critical to trigger puberty and to regulate reproductive fonction. In recent years, however, studies have revealed that hypothalamic neurons producing peptides of the RF-amide (Arg-Phe-NH2) family, especially kisspeptins (Kp) and RFamide-related peptide-3 (RFRP-3), play a key role in the control of reproduction. These neuropeptides appear to act upstream of GnRH neurons, and current findings in this domain are leading to a new model for the neuroendocrine control of reproduction. Kp neurons, located in the arcuate and anteroventral periventricular nuclei, project to GnRH cell bodies and nerve terminals. There, Kp binds to its specific receptor Kiss1R (or GPR54) to potently stimulate GnRH release. Notably, Kp neurons are the main central targets for the positive and negative feedback effects of sex steroids. Moreover, Kp neurons sense metabolic signals to tune reproductive activity according to the energetic status of the organism. In seasonal breeders, Kp expression is regulated by photoperiod and the peptides synchronise reproduction with seasons. Altogether, current data have demonstrated that Kp is a key regulator of the gonadotropic axis in all mammalian species studied so far. On the other hand, RFRP-3, expressed in neurons of the dorsomedial hypothalamus, was first reported to inhibit reproductive activity by reducing GnRH neuron activity and pituitary gonadotropin release. However, recent data report that RFRP-3 activity depends on species, gender and physiological status, indicating complex mechanisms of action. The distribution and pharmacology of the receptor for RFRP-3, GPR147, are poorly known and the lack of selective pharmacological and genetic tools severely limits the study of the RFRP-3/GPR147system. To complicate the picture, a few studies suggest that GPR147 may bind other endogenous RFamide peptides, including Kp. In this highly competitive context of a renovated understanding of the neuroendocrine control of reproduction in mammals, the main aim of the REPRAMIDE proposal is to elucidate the role and to describe the mechanisms underpinning RFRP-3/GPR147 influence on mammalian reproduction. Our specific objectives are 1) to determine the physiological effect of RFRP-3 on female reproductive activity using three complementary animal models in order to clarify species-specific differences; 2) to generate suitable tools to refine the pharmacological and biochemical knowledge of GPR147; 3) to evaluate the putative interaction between GPR147 and other RF-amide receptors, particularly Kiss1R, both at the pharmacological and physiological levels. The findings obtained in the course of our studies will contribute to the development of better strategies to treat human fertility problem and to manage reproduction in livestock.