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

The Multifaceted Roles of KCC2 in Cortical Development

KCC2, best known as the neuron-specific chloride-extruder that sets the strength and polarity of GABAergic currents during neuronal maturation, isa multifunctional molecule that can regulate cytoskeletal dynamics via its C-terminal domain (CTD). We describe the molecular and cellular mechanisms involved in the multiple functions of KCC2 and its splice variants, ranging from developmental apoptosis and the control of early network events to the formation and plasticity of cortical dendritic spines. The versatility of KCC2 actions at the cellular and subcellular levels is also evident in mature neurons during plasticity, disease, and aging. Thus, KCC2 has emerged as one of the most important molecules that shape the overall neuronal phenotype.Peer reviewe

Reciprocal Regulation of KCC2 Trafficking and Synaptic Activity

The main inhibitory neurotransmitter receptors in the adult central nervous system (CNS) are type A γ-aminobutyric acid receptors (GABAARs) and glycine receptors (GlyRs). Synaptic responses mediated by GlyR and GABAAR display a hyperpolarizing shift during development. This shift relies mainly on the developmental up-regulation of the K+-Cl− co-transporter KCC2 responsible for the extrusion of Cl−. In mature neurons, altered KCC2 function—mainly through increased endocytosis—leads to the re-emergence of depolarizing GABAergic and glycinergic signaling, which promotes hyperexcitability and pathological activities. Identifying signaling pathways and molecular partners that control KCC2 surface stability thus represents a key step in the development of novel therapeutic strategies. Here, we present our current knowledge on the cellular and molecular mechanisms governing the plasma membrane turnover rate of the transporter under resting conditions and in response to synaptic activity. We also discuss the notion that KCC2 lateral diffusion is one of the first parameters modulating the transporter membrane stability, allowing for rapid adaptation of Cl− transport to changes in neuronal activity

Pyk2 modulates hippocampal excitatory synapses and contributes to cognitive deficits in a Huntington's disease model.

The structure and function of spines and excitatory synapses are under the dynamic control of multiple signalling networks. Although tyrosine phosphorylation is involved, its regulation and importance are not well understood. Here we study the role of Pyk2, a non-receptor calcium-dependent protein-tyrosine kinase highly expressed in the hippocampus. Hippocampal-related learning and CA1 long-term potentiation are severely impaired in Pyk2-deficient mice and are associated with alterations in NMDA receptors, PSD-95 and dendritic spines. In cultured hippocampal neurons, Pyk2 has autophosphorylation-dependent and -independent roles in determining PSD-95 enrichment and spines density. Pyk2 levels are decreased in the hippocampus of individuals with Huntington and in the R6/1 mouse model of the disease. Normalizing Pyk2 levels in the hippocampus of R6/1 mice rescues memory deficits, spines pathology and PSD-95 localization. Our results reveal a role for Pyk2 in spine structure and synaptic function, and suggest that its deficit contributes to Huntington's disease cognitive impairments

Vezatin is essential for dendritic spine morphogenesis and functional synaptic maturation.

International audienceVezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. Here, we show that Vezatin localizes in spines in mature mouse hippocampal neurons and codistributes with PSD95, a major scaffolding protein of the excitatory postsynaptic density. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear-conditioning memory response. Vezatin knock-down in cultured hippocampal neurons and Vezatin conditional knock-out in mice led to a significantly increased proportion of stubby spines and a reduced proportion of mature dendritic spines. PSD95 remained tethered to presynaptic terminals in Vezatin-deficient hippocampal neurons, suggesting that the reduced expression of Vezatin does not compromise the maintenance of synaptic connections. Accordingly, neither the amplitude nor the frequency of miniature EPSCs was affected in Vezatin-deficient hippocampal neurons. However, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results suggest a role of Vezatin in dendritic spine morphogenesis and functional synaptic maturation

Prenatal Activation of Microglia Induces Delayed Impairment of Glutamatergic Synaptic Function

BACKGROUND: Epidemiological studies have linked maternal infection during pregnancy to later development of neuropsychiatric disorders in the offspring. In mice, experimental inflammation during embryonic development impairs behavioral and cognitive performances in adulthood. Synaptic dysfunctions may be at the origin of cognitive impairments, however the link between prenatal inflammation and synaptic defects remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that prenatal alteration of microglial function, including inflammation, induces delayed synaptic dysfunction in the adult. DAP12 is a microglial signaling protein expressed around birth, mutations of which in the human induces the Nasu-Hakola disease, characterized by early dementia. We presently report that synaptic excitatory currents in mice bearing a loss-of-function mutation in the DAP12 gene (DAP12(KI) mice) display enhanced relative contribution of AMPA. Furthermore, neurons from DAP12(KI) P0 pups cultured without microglia develop similar synaptic alterations, suggesting that a prenatal dysfunction of microglia may impact synaptic function in the adult. As we observed that DAP12(KI) microglia overexpress genes for IL1beta, IL6 and NOS2, which are inflammatory proteins, we analyzed the impact of a pharmacologically-induced prenatal inflammation on synaptic function. Maternal injection of lipopolysaccharides induced activation of microglia at birth and alteration of glutamatergic synapses in the adult offspring. Finally, neurons cultured from neonates born to inflamed mothers and cultured without microglia also displayed altered neuronal activity. CONCLUSION/SIGNIFICANCE: Our results demonstrate that prenatal inflammation is sufficient to induce synaptic alterations with delay. We propose that these alterations triggered by prenatal activation of microglia provide a cellular basis for the neuropsychiatric defects induced by prenatal inflammation

Multiple Mechanisms for the Potentiation of AMPA Receptor- Mediated Transmission by -Ca2 /Calmodulin-Dependent Protein Kinase II

Some forms of activity-dependent synaptic potentiation require the activation of postsynaptic Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Activation of CaMKII has been shown to phosphorylate the glutamate receptor 1 subunit of the AMPA receptor (AMPAR), thereby affecting some of the properties of the receptor. Here, a recombinant, constitutively active form of alphaCaMKII tagged with the fluorescent marker green fluorescent protein (GFP) [alphaCaMKII(1-290)-enhanced GFP (EGFP)] was expressed in CA1 pyramidal neurons from hippocampal slices. The changes in glutamatergic transmission onto these cells were analyzed. AMPA but not NMDA receptor-mediated EPSCs were specifically potentiated in infected compared with nearby noninfected neurons. This potentiation was associated with a reduction in the proportion of synapses devoid of AMPARs. In addition, expression of alphaCaMKII(1-290)-EGFP increased the quantal size of AMPAR-mediated responses. This effect reflected, at least in part, an increased unitary conductance of the channels underlying the EPSCs. These results reveal that several key features of long-term potentiation of hippocampal glutamatergic synapses are reproduced by the sole activity of alphaCaMKII.This work was supported by the Human Frontier Science Program Organization (J.C.P.) and by the Mathers Foundation and the National Institutes of Health (R.M.). We thank Nancy Dawkins-Pisani for technical assistance, Norbert Ankri for providing event detection and analysis software, and Yasunori Hayashi and Richard Miles for critical reading of this manuscript.Peer reviewe

Altérations de l'homéostasie neuronale du chlore et leur implication dans l'épilepsie

Le GABA assure l inhibition synaptique rapide dans le cerveau en agissant sur des récepteurs de type GABAA. Ces récepteurs formant un canal perméable principalement aux ions chlorure, l efficacité voire la polarité de la transmission GABAergique dépendent de la concentration intracellulaire de ces ions. Plusieurs maladies neurologiques sont associées à l altération de mécanismes d homéostasie des ions chlorure, ce qui agirait en affectant la transmission GABAergique. Je me suis intéressé au canal chlore voltage-dépendant CLC-2, et au cotransporteur potassium/chlore KCC2. Ces protéines sont toutes deux impliquées dans diverses formes d épilepsie sans que les altérations de l activité neuronale associées à leur dysfonctionnement n aient été élucidées. J ai identifié les conséquences fonctionnelles de deux mutations du gène Clcn2, codant pour le canal CLC-2, associées à un syndrome d épilepsie généralisée. Les données obtenues confirment l implication du gène Clcn2 comme facteur de susceptibilité à certaines formes d épilepsie, une question jusque là vivement débattue. Par ailleurs, j ai examiné les conséquences fonctionnelles de la perte d expression du transporteur KCC2 associée à diverses formes d épilepsie humaine et expérimentale. J ai mis en évidence un rôle insoupçonné de ce transporteur dans le maintien de la morphologie des épines dendritiques et de leur contenu en récepteurs postsynaptiques. L ensemble de mon travail a donc révélé des fonctions inattendues de deux partenaires de l homéostasie neuronale des ions chlorure dont les altérations de fonction ou d expression semblent altérer l activité neuronale intrinsèque ou la fonction des synapses excitatricesPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

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

Cotransporteurs cation-chlorure et polarité de la signalisation GABAergique dans les interneurones à parvalbumine de l'hippiocampe chez la souris

International audienceKEY POINTS:Cation-chloride cotransporters (CCCs) play a critical role in controlling the efficacy and polarity of GABAA receptor (GABAAR)-mediated transmission in the brain, yet their expression and function in GABAergic interneurons has been overlooked. We compared the polarity of GABA signalling and the function of CCCs in mouse hippocampal pyramidal neurons and parvalbumin-expressing interneurons. Under resting conditions, GABAAR activation was mostly depolarizing and yet inhibitory in both cell types. KCC2 blockade further depolarized the reversal potential of GABAAR-mediated currents often above action potential threshold. However, during repetitive GABAAR activation, the postsynaptic response declined independently of the ion flux direction or KCC2 function, suggesting intracellular chloride buildup is not responsible for this form of plasticity. Our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal pyramidal neurons and parvalbumin interneurons.ABSTRACT:Transmembrane chloride gradients govern the efficacy and polarity of GABA signalling in neurons and are usually maintained by the activity of cation chloride cotransporters, such as KCC2 and NKCC1. Whereas their role is well established in cortical principal neurons, it remains poorly documented in GABAergic interneurons. We used complementary electrophysiological approaches to compare the effects of GABAAR activation in adult mouse hippocampal parvalbumin interneurons (PV INs) and pyramidal cells (PCs). Loose cell attached, tight-seal and gramicidin-perforated patch recordings all show GABAAR-mediated transmission is slightly depolarizing and yet inhibitory in both PV INs and PCs. Focal GABA uncaging in whole-cell recordings reveal that KCC2 and NKCC1 are functional in both PV INs and PCs but differentially contribute to transmembrane chloride gradients in their soma and dendrites. Blocking KCC2 function depolarizes the reversal potential of GABAAR-mediated currents in PV INs and PCs, often beyond firing threshold, showing KCC2 is essential to maintain the inhibitory effect of GABAARs. Finally, we show that repetitive 10 Hz activation of GABAARs in both PV INs and PCs leads to a progressive decline of the postsynaptic response independently of the ion flux direction or KCC2 function. This suggests intraneuronal chloride buildup may not predominantly contribute to activity-dependent plasticity of GABAergic synapses in this frequency range. Altogether our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal PV INs and PCs and suggest KCC2 downregulation in the pathology may affect the valence of GABA signalling in both cell types