The mammalian cerebral cortex is a complex laminar structure with a variety of neuronal and non-neuronal cell types that develop in a finely orchestrated and stereotypic manner. Final laminar position and synaptic specificity of most cortical cell types are well described. Strikingly, any alteration in the developmental unfolding of one of these processes, even for a single cell type among tens, can be sufficient to generate neurodevelopmental disorders. However, how the establishment of this precise cellular architecture is regulated at the molecular level remains largely unknown. Several lines of evidence suggest a role of cell-cell communications via ligand-receptor (LR) interactions. Using a single cell RNA-seq (scRNA-seq) approach in mice, we have generated a bioinformatic atlas that infers LR based cellular communications across all cell types over somatosensory (SS) cortex development. Querying our atlas for known LR interactions has demonstrated its validity, but new LR-mediated cell-cell interactions remain to be discovered to interrogate its power as a hypothesis generator. In parallel, a technique called Multiplexed-Error Robust Fluorescence In Situ Hybridization (MERFISH) has been recently developed and implemented by us, which images single cell transcriptomes in situ and thereby adds precious information about spatial expression. Here, we will: (i) test some LR interactions predicted by our scRNA-seq atlas for a role in SS microcircuit development, (ii) build on the MERFISH technique to complement our atlas with spatial information and to characterize SS cortex cellular development with unprecedented resolution and (iii) use MERFISH to interrogate altered developmental processes in the SS cortex of a mouse model of neurodevelopmental disorder, the Neurod2 KO mouse.

We propose to decipher the mechanisms of action of neuroendothelial N-methyl-D-aspartate receptors (NMDAR). In addition to neurons, where they drive glutamatergic neurotransmission, NMDAR are expressed in a variety of cell types. In particular, brain endothelial cells express NMDAR, which could be involved in blood-brain barrier maintenance and alteration. In a recent paper, we identified an unexpected population of NMDAR in endothelial cells, expressed at the luminal side of microvessels and located at the vicinity of blood/spinal cord barrier-forming tight junctions. We developed a monoclonal antibody (Glunomab®), directed against a specific site of NMDAR (aminoacids 176-180), which blocked the entry of leukocytes to the inflamed spinal cord, thus providing therapeutic effects in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Nevertheless, the downstream targets which link endothelial NMDAR function to leukocyte migration across the blood/brain and blood/spinal cord barriers are not fully understood yet. Interestingly, our current studies show that these receptors have an unconventional composition, including the presence of the rare GluN3 subunit, which provides response to glycine (in addition to glutamate) and metabotropic signaling (in addition to ionotropic function). Interestingly, naturally occurring circulating auto-antibodies against NMDAR are present in ~10% of human subjects and are overexpressed in neuropsychiatric and neurological diseases. Beyond these quantitative data, qualitative information are needed concerning the regions of NMDAR recognized by these antibodies. In fact, circulating autoantibodies against NMDAR could provide either beneficial or deleterious effects, depending on the region that they target. In line with this, we postulate that identifying the regions targeted by NMDAR auto-antibodies could have prognosis value in neurological diseases. Given our recent work concerning NMDAR in animal models of MS, we believe that investigating circulating antibodies would be particularly relevant for prognosis of MS. Thus the goals of this project are i) To characterize the signaling pathways and downstream targets triggered by NMDAR activation in endothelial cells, in relation to leukocyte penetration towards the spinal cord, ii) To identify the repertoire of NMDAR antibodies in MS patients (based on their target regions on NMDAR) and to determine whether different clusters of antibodies are associated with different outcomes, iii) To investigate the effects of these different clusters on the function of endothelial NMDAR and in animal models of MS, and iv) To bridge experimental data and clinical observations.

Membrane contact sites (MCSs) enable specific lipid exchange between organelles. Our recent results on the archetypical OSBP/VAP complex suggest that the architecture and dynamics of MCS are influenced by intrinsically disordered regions (IDRs). These overlooked structural attributes enable formation of MCS of adjustable thickness and reduce protein crowding. These effects are likely to be general given the abundance of IDRs in MCS proteins. We posit that IDRs guarantee the lateral and/or vertical flexibility of proteins. We will dissect the link between these characteristics and the function, dynamics and organization of MCSs, by using a multidisciplinary and multi-scale approach involving quantitative and super-resolution cell imaging, in vitro reconstitution of membrane systems, structural analysis by cryo-electron tomography, and the development of innovative pharmacological tools. This project will offer new perspectives on the efficiency, plasticity and specificity of MCSs.

The pentameric type 3 serotonin receptors (5-HT3Rs) are involved in irritable-bowel syndrome and chemo- or radiotherapy-induced vomiting. Since most 5-HT3R agonists and competitive antagonists, aka orthosteric effectors, have low affinity/selectivity and generate critical side effects, the development of allosteric modulators with 5-HT3R-subtype selectivity is of great interest for the pharmaceutical industry. Snake venoms feature vast natural libraries of peptides/proteins displaying many molecular structures and even more pharmacological activities. Many of these “toxins”, which are not always lethal, have been used as tools for studying their receptor at the basic research level and/or as molecular templates for the design of new effectors/drugs with selected pharmacological properties. A proof-of-concept study involving ligand binding on solubilized 5-HT3R and electrophysiology measurements on oocyte-expressed 5-HT3R led us to identify, from a snake venom, a high-affinity binder that seems to act as an allosteric modulator of channel activity. Low resolution negative stain electron microscopy (EM) imaging of the toxin-receptor complex revealed toxin molecules bound far from the orthosteric binding site for agonists and competitive antagonists, consistent with our binding and functional data. To ascertain the binding site and mode of action of this new, peptidic 5-HT3R binder, we now propose to (i) complete/refine our electrophysiology study and complement our data set with binding data on 5-HT3R subtypes embedded in mammalian cell membranes; (ii) identify the precise toxin binding site and orientation at the receptor surface and characterize the interacting molecular determinants in both the toxin and receptor, by solving a high resolution cryo-EM structure of the pentameric complex; (iii) explore possible conformational changes in the extracellular ligand-binding domain and the ion-channel associated with toxin binding, by comparing the receptor conformations in presence versus absence of the bound toxin; (iv) use the same strategy to document the identity and mode of action of a second 5-HT3R binder present in a semi-purified fraction issued from the venom of another snake species. Availability of complementary binding, functional and structural data on these peptidic ligands of the 5-HT3R with atypical binding sites and original modes of action will provide new avenues through their use as pharmacological tools to further explore new pathways for modulation of 5-HT3R activity. Such data could also lead to the structure-based design of new peptidic or organic molecules with tailored pharmacological activities aimed at modulating this receptor allosterically, or to be associated with chemo- or radiotherapy treatments to attenuate/alleviate their undesirable side effects. This proposal involves three French teams with complementary expertise in cell biology, biochemistry, biophysics, pharmacology, toxinology and structural biology, and with established publications and collaboration records in the field.

The pathophysiology of pain remains poorly understood and there is clearly a need for new analgesics. ASICs (Acid-Sensing Ion Channels) have emerged as important players in the pain pathway. They form depolarizing cation channels activated by extracellular protons that are expressed in both sensory and central neurons. Our project aims to provide a better understanding of the mechanisms of pain by exploring with innovative pharmacological tools the role of ASICs in these processes, and to provide at the same time leads in the development of new analgesics. We will use the complementary expertises of the two partners, i.e., ASICs, electrophysiology, toxins and pain behaviour (Team#1), animal pain models and behaviour (Team#2) to achieve the following objectives: 1 – Better characterize the in vivo effects on chronic pain of mambalgins and develop from animal venoms new specific peptide blockers of ASICs with therapeutic potential. Take advantage of these toxins to investigate the structure-function of ASIC channels. Mambalgins are snake peptides recently identified by Team#1 in collaboration with Team#2 that block subtypes of central and peripheral ASIC channels to produce a potent analgesic effect in rodents in acute pain and inflammation. We propose to further characterize the in vivo analgesic activity of these peptides by exploring their effect via different delivery routes (i.t., i.c.v., i.pl., i.v.) on a variety of chronic pain models of clinical relevance like monoarthritis, neuropathy and migraine. We propose in parallel to screen animal venoms for new peptide toxins against ASIC channels lacking a specific pharmacology and/or of potential clinical interest. The most interesting ones will be patented and licensed for further clinical development. In addition, analysis of the interaction of the toxins with the targeted channels and of their mechanisms of inhibition will help to better understand the relationship between ASIC channel structure and function. 2 – Combine toxins with the other available tools to explore the pathophysiological role of ASIC channels in the peripheral and the central nervous system in inflammatory and neuropathic pain. We have previously shown the importance for inflammatory pain of peripheral ASIC channels in sensory neurons. However, a lot still remains to be understood in the regulation of these channels. We propose to explore the role of peripheral ASICs expressed in nociceptors in a context of inflammation and inflammatory pain by identifying new and/or pathology-specific modulators of these channels, and by evaluating the contribution of ASICs to the in vivo effects of these modulators. We will combine studies of the sensitivity of recombinant and native ASIC channels to specific inflammatory mediators (candidate approach) with a global translational approach through the effect of whole human inflammatory exudates isolated from different pathological conditions. The role of central and peripheral ASIC channels in neuropathic pain has never been directly investigated. We propose to take advantage of the specific toxin inhibitors already available and developed in the course of this project, as well as of ASIC-deficient and ASIC knockdown animals to study this role in two models of neuropathic pain of different aetiology (oxaliplatin-induced neuropathy and chronic constriction injury of sciatic nerve).