Thèse Régulation du Développement Synaptique dans les Circuits Corticaux par le Récepteur Atypique du Glutamate Delta-2 H/F

Doctorat.Gouv.Fr

Établissement : Ecole normale supérieure - PSL École doctorale : Sciences du Vivant Laboratoire de recherche : Institut de Biologie de l'École Normale Supérieure Direction de la thèse : Cécile CHARRIER ORCID 0000000290531715 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-06-10T23:59:59 Le code moléculaire qui régit la connectivité et la spécialisation fonctionnelle des circuits corticaux reste largement méconnu. Les récepteurs ionotropiques du glutamate de la sous-famille delta (GluD1 et GluD2) sont des récepteurs non conventionnels qui, au lieu de participer à la neurotransmission excitatrice rapide, établissent des interactions trans-synaptiques. Les récepteurs GluD agissent comme des organisateurs synaptiques dans le cervelet, mais leur fonction dans le néocortex est soit énigmatique (GluD1), soit totalement inconnue (GluD2). Ce projet vise à étudier le rôle de GluD2 dans le développement des circuits corticaux et à élucider les mécanismes sous-jacents aux niveaux moléculaire, cellulaire et des circuits.

Plus précisément, nous :
(1) caractériserons le rôle de GluD2 dans le développement des dendrites et des synapses, en modulant son expression dans les neurones pyramidaux corticaux des couches 2/3 in vivo ;
(2) étudierons l'interaction entre GluD2 et les voies du métabolisme lipidique, en utilisant des techniques de biochimie et d'imagerie FRAP ;
(3) identifierons quels circuits corticaux sont affectés par la manipulation de GluD2, grâce à des approches de traçage trans-synaptique.

Ce travail apportera un éclairage nouveau sur les mécanismes fondamentaux du développement neuronal et de l'assemblage des circuits corticaux, avec des répercussions potentielles sur notre compréhension de la plasticité cérébrale et des neuropathologies. The establishment of precise cortical circuits during postnatal development is essential for sensory processing and cognitive function. Glutamatergic synapse formation, refinement, and stabilization are tightly regulated processes involved in this cortical wiring. The glutamate delta-2 receptor (GluD2), encoded by the Grid2 gene, is known for its role in synapse formation and plasticity in cerebellar Purkinje cells, but its functional role in the neocortex is largely unexplored. GluD2 is a member of the ionotropic glutamate receptor family but does not function as a conventional ligand-gated ion channel. Instead, it acts as a synaptic organizer through trans-synaptic interactions with presynaptic proteins such as neurexins, mediated by cerebellin (Cbln) family ligands. In the cerebellum, GluD2 is essential for parallel fiber-Purkinje cell synapse formation, synaptic plasticity, and motor learning1. Emerging data suggest that GluD2 is also expressed in most cortical neurons2,3. Single-cell RNA sequencing studies indicate that Grid2 is expressed in subsets of cortical excitatory neurons, including L2/3 pyramidal neurons (L2/3 CPNs)4. L2/3 CPNs play a central role in intracortical communication, integrating thalamocortical inputs and mediating horizontal and interlaminar connectivity, and disruptions in the development of these key circuits have been identified in neurodevelopmental disorders. Interestingly, GRID2 mutations are linked to autism spectrum disorders, schizophrenia, or delayed cognitive development and intellectual disability5,6,7,8, pointing to a possible role of GluD2 dysfunction in cortical circuits impairment.

The aim of this project is to determine the role of GluD2 in the development and plasticity of cortical circuits. Preliminary results from the candidate and other members of the Charrier's team suggest that GluD2 is required for the elaboration of complex dendritic arbors and that its loss of function leads to a major rearrangement of excitatory cortical circuits. At the synaptic level it alters synaptic density and dendritic spine morphology. By combining sparse and global knockout experiments in L2/3 CPNs in mouse somatosensory cortex and analyzing key developmental time points, we will further establish how GluD2 regulates neuronal and synaptic development. Aim1 - Compare the role of GluD2 in sparse vs global KO models. Unpublished results of the lab show that following suppression of GluD2 in L2/3 CPNs, changes appear both at the global level of the cell (modification in the branching of the dendritic tree) and at subcellular levels (altered dendritic spines numbers and spine morphology). The first stage of the project will be to complete the extensive dataset already available for IUE experiments, regarding quantification of synapse density and spine morphology changes, as well as dendritic tree reconstructions in ShGluD2, rescue, and overexpression (OE) conditions at P7, P21 and P75. In a second step, analysis of these parameters will also need to be done on the GluD2 KO mouse line. We will then be able to compare changes induced by the manipulation of Grid2 in sparse vs. global KO models.

Aim2 - Investigate the interaction between GluD2 and lipid metabolism pathways. Proteomic data from the team identified phosphatidylinositol transfer protein membrane-associated 2 (Pitpnm2) and phospholipase D2 (Pld2) as potential GluD2 partners, suggesting that GluD2 operates by regulating lipid metabolism. Inhibition of Pitpnm2 expression leads in our model to changes in synaptic density and spine morphology similar to those observed in shGluD2 experiments, pointing to a possible shared signaling pathway. Both Pitpnm2 and Pld2 are key regulators of phosphoinositide signaling and membrane lipid remodeling, processes that are critical for synapse formation, spine morphogenesis, and receptor trafficking10. We will test the hypothesis that GluD2 regulates the development of L2/3 CPNs by modulating local lipid signaling at synapses.
Goal 1_ Validation of the functional association between GluD2 and lipid metabolism-related proteins. By performing epistasic experiments with different combined deletions of GluD2, Pitpnm2 and Pld2 in sparse or global Glud2 KO L2/3 CPNs, and analyzing changes in synaptic spines and dendritic morphology, we will be able to demonstrated which protein is located upstream of the signaling pathway. Complementary biochemistry approaches (co-IP, pull-down) would establish if there is a direct interaction between GluD2-Pitpnm2 and GluD2-Pld2.
Goal 2_ Examination of the consequences of the disruption of GluD2-Pitpnm2 and GluD2-Pld2 interactions on the lipid exchange dynamics. FRAP imaging with a genetically-encoded fluorescent lipid sensor for phosphatidylinositol phosphate PIP2 (PLC1-PH-GFP) or PIP4 (SidM-P4M-GFP) will be used to monitor local lipid signaling dynamics between the endoplasmic reticulum and the plasma membrane in cortical dendrites. Depending on the experimental design, brain slices either from the Glud2 KO mouse line, or from brains IUE with shGluD2, and shPitpnm2 or shPld2 will be used.
Together, these experiments will help unravel the link between GluD2 function and lipid metabolic signaling in the developing cortex, providing mechanistic insight into how membrane dynamics contribute to synapse maturation.

Aim 3 - Identify the neuronal circuits impacted by the manipulation of GluD2 expression. The analysis of the neuronal networks to which L2/3 CPNs belong will help define which connections are modified and in which circuits GluD2 is specifically involved. In GluD2-deficient neurons, the loss of dendritic spines together with the strong increase in the size of dendritic spine heads suggests that some connections are lost while others are strengthened. Also, a major default in the reduction of the apical dendritic tree is observed, indicating connectivity impairment. Excitatory synapses on L2/3 CPNs are mainly established by other L2/3 CPNs (via local and long-range connections) and L4 neurons (via vertical connections). To identify the origin of the different projections, we will use a trans-synaptic tracing strategy with genetically encoded GFP-rabies virus expressed in L2/3 CPNs (IUE experiments) and subsequent imaging of whole brain slices to locate the regions from which axons are projected11. In addition, projections arising from L2/3 CPNs or L4 neurons express different isoforms of Cbln (the extracellular scaffolding protein binding GluD2) at their target contact sites, namely Cbln2 and Cbln4, respectively12. To determine which circuits are regulated by GluD2, we will prepare Cbln2 and Cbln4 AAVs12 and use them to inactivate Cbln2 and Cbln4 and quantify spine density and morphology in sparse shGluD2-neurons. We will record excitatory synaptic currents evoked by input-specific electrical in brain slices. Complementary experiments could be carried on the GluD2 global KO mouse. These experiments will establish the role of GluD2 in the formation of specific excitatory connections formed onto L2/3 CPNs and determine if GluD2 is a circuit-specific or a general excitatory synaptic organizer in CPNs.