Role of the neuronal K-Cl co-transporter KCC2 in inhibitory and excitatory neurotransmission

The K-Cl co-transporter KCC2 plays multiple roles in the physiology of central neurons and alterations of its function and/or expression are associated with several neurological conditions. By regulating intraneuronal chloride homeostasis, KCC2 strongly influences the efficacy and polarity of the chloride-permeable γ-aminobutyric acid (GABA) type A and glycine receptor (GlyR) mediated synaptic transmission. This appears particularly critical for the development of neuronal circuits as well as for the dynamic control of GABA and glycine signaling in mature networks. The activity of the transporter is also associated with transmembrane water fluxes which compensate solute fluxes associated with synaptic activity. Finally, KCC2 interaction with the actin cytoskeleton appears critical both for dendritic spine morphogenesis and the maintenance of glutamatergic synapses. In light of the pivotal role of KCC2 in the maturation and function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. These include development and activity-dependent modifications both at the transcriptional and post-translational levels. We emphasize the importance of post-translational mechanisms such as phosphorylation and dephosphorylation, oligomerization, cell surface stability, clustering and membrane diffusion for the rapid and dynamic regulation of KCC2 function

Involvement of the K/Cl cotransporter KCC2 in glutamatergic transmission and regulation of its surface expression by excitatory transmisssion

Dans les neurones matures du système nerveux central, la polarité et l efficacité de la transmission inhibitrice GABAAR-dépendante repose sur la concentration intracellulaire de chlore, principalement régulée par le cotransporteur KCC2. Dans des pathologies telles l épilepsie, où la balance excitation-inhibition est altérée, l expression et la fonction de KCC2 sont fortement réduites, entrainant une perte d inhibition. Cependant, KCC2 est enrichi au niveau des synapses excitatrices, ce qui pose la question de sa fonction à cet endroit. Nos résultats révèlent un rôle inattendu du transporteur qui, de par son interaction avec le cytosquelette d actine, contraint la diffusion latérale de protéines transmembranaires dans les épines dendritiques. Nos résultats démontrent également que KCC2 est nécessaire au maintient de l efficacité de la transmission excitatrice. Le suivi de particule unique révèle que la diffusion de KCC2 est contrainte par son interaction avec le cytosquelette via la protéine d ancrage 4.1N aux synapses excitatrices, et représente la première caractérisation de la diffusion latérale d un transporteur ionique dans des neurones. Nous montrons également qu une augmentation de la transmission excitatrice déstabilise rapidement les agrégats membranaires de KCC2 en augmentant sa diffusion via l activation des récepteurs NMDA et la signalisation calcique; et se traduit par une perte de transport de chlore. Ces résultats suggèrent que KCC2 pourrait participer au cross-talk entre synapses excitatrices et inhibitrices, et que la régulation de sa diffusion par l activité neuronale sont des mécanismes clés permettant le contrôle rapide de l homéostasie du chlore.In mature neurons of the central nervous system, the polarity and efficacy of GABAAR-mediated inhibition is determined by intra-neuronal chloride concentrations, which are mainly regulated by the potassium chloride co-transporter, KCC2. Pathological conditions such as epilepsy, where inhibitory to excitatory balance is impaired, are associated with a dramatic downregulation of KCC2 expression and function leading to a loss of inhibition. However, KCC2 is enriched in the vicinity of excitatory synapses raising the question of its function at this particular location. Our results revealed an unsuspected role for this transporter in hindering lateral diffusion of transmembrane proteins in dendritic spines through structural interactions with the actin cytoskeleton, and demonstrate that KCC2 is required for the maintenance of glutamatergic efficacy in mature neurons. Single particle tracking of KCC2 further revealed constrained diffusion via actin tethering of the transporter at/near excitatory synapses, by the adaptor protein 4.1N, and provides the first characterization of an ion transporter s lateral diffusion in neurons. Sustained glutamatergic activity induced a rapid NMDAR- and Ca2+- dependent destabilization of KCC2 membrane clusters, through increased diffusion, and resulted in altered chloride transport. These results thus demonstrate that KCC2 is particularly positioned to mediate a cross-talk between excitatory and inhibitory synapses. Activity-dependent regulation of KCC2 by lateral diffusion and clustering appears as a key mechanism to control chloride homeostasis, and might as well influence excitatory synaptic strength through structural interactions.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation

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MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior

International audienceMDGA molecules can bind neuroligins and interfere with trans-synaptic interactions to neurexins, thereby impairing synapse development. However, the subcellular localization and dynamics of MDGAs, or their specific action mode in neurons remain unclear. Here, surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons reveal that MDGAs are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation. Knocking-down/out MDGAs using shRNAs and CRISPR/Cas9 strategies increases the density of excitatory synapses, the membrane confinement of neuroligin-1, and the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, MDGA silencing reduces the mobility of AMPA receptors, increases the frequency of miniature EPSCs (but not IPSCs), and selectively enhances evoked AMPA-receptor-mediated EPSCs in CA1 pyramidal neurons. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

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TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering

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Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques

Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules

International audienceDesigning highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules

The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors

The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here, we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1-knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1-deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer disease, suggesting that perturbed receptor trafficking contributes to synaptic-composition and -function defects underlying synaptopathies.publisher: Elsevier articletitle: The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors journaltitle: Neuron articlelink: http://dx.doi.org/10.1016/j.neuron.2015.08.007 content_type: article copyright: Copyright © 2015 Elsevier Inc. All rights reserved.status: publishe