Publications by authors named "Sabine Lévi"

29 Publications

  • Page 1 of 1

Serotonin 2B Receptor by Interacting with NMDA Receptor and CIPP Protein Complex May Control Structural Plasticity at Glutamatergic Synapses.

ACS Chem Neurosci 2021 04 19;12(7):1133-1149. Epub 2021 Mar 19.

INSERM UMR-S 1270, F75005 Paris, France.

The serotonin 2B (5-HT) receptor coupled to Gq-protein contributes to the control of neuronal excitability and is implicated in various psychiatric disorders. The mechanisms underlying its brain function are not fully described. Using peptide affinity chromatography combined with mass spectrometry, we found that the PDZ binding motif of the 5-HT receptor located at its C-terminal end interacts with the scaffolding protein channel interacting PDZ protein (CIPP). We then showed, in COS-7 cells, that the association of the 5-HT receptor to CIPP enhanced receptor-operated inositol phosphate (IP) production without affecting its cell surface and intracellular levels. Co-immunoprecipitation experiments revealed that CIPP, the 5-HT receptor, and the NR1 subunit of the NMDA receptor form a macromolecular complex. CIPP increased 5-HT receptor clustering at the surface of primary cultured hippocampal neurons and prevented receptor dispersion following agonist stimulation, thus potentiating IP production and intracellular calcium mobilization in dendrites. CIPP or 5-HT receptor stimulation in turn dispersed NR1 clusters colocalized with 5-HT receptors and increased the density and maturation of dendritic spines. Collectively, our results suggest that the 5-HT receptor, the NMDA receptor, and CIPP may form a signaling platform by which serotonin can influence structural plasticity of excitatory glutamatergic synapses.
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http://dx.doi.org/10.1021/acschemneuro.0c00638DOI Listing
April 2021

Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in mice.

Mol Psychiatry 2021 02 8;26(2):629-644. Epub 2020 Jan 8.

Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, F-33000, Bordeaux, France.

ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.
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http://dx.doi.org/10.1038/s41380-019-0641-8DOI Listing
February 2021

Exacerbation of C1q dysregulation, synaptic loss and memory deficits in tau pathology linked to neuronal adenosine A2A receptor.

Brain 2019 11;142(11):3636-3654

University of Lille, Inserm, CHU Lille, UMR-S 1172 - JPArc, LabEx DISTALZ, F Lille, France.

Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer's disease, but underlying mechanisms remain ill-defined. Interestingly, ageing and Alzheimer's disease have been associated with an abnormal upregulation of adenosine A2A receptor (A2AR), a fine tuner of synaptic plasticity. However, the link between A2AR signalling and tau pathology has remained largely unexplored. In the present study, we report for the first time a significant upregulation of A2AR in patients suffering from frontotemporal lobar degeneration with the MAPT P301L mutation. To model these alterations, we induced neuronal A2AR upregulation in a tauopathy mouse model (THY-Tau22) using a new conditional strain allowing forebrain overexpression of the receptor. We found that neuronal A2AR upregulation increases tau hyperphosphorylation, potentiating the onset of tau-induced memory deficits. This detrimental effect was linked to a singular microglial signature as revealed by RNA sequencing analysis. In particular, we found that A2AR overexpression in THY-Tau22 mice led to the hippocampal upregulation of C1q complement protein-also observed in patients with frontotemporal lobar degeneration-and correlated with the loss of glutamatergic synapses, likely underlying the observed memory deficits. These data reveal a key impact of overactive neuronal A2AR in the onset of synaptic loss in tauopathies, paving the way for new therapeutic approaches.
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http://dx.doi.org/10.1093/brain/awz288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821333PMC
November 2019

KCC2 membrane diffusion tunes neuronal chloride homeostasis.

Neuropharmacology 2020 06 11;169:107571. Epub 2019 Mar 11.

INSERM UMR-S 1270, 75005, Paris, France; Sorbonne Université, 75005, Paris, France; Institut du Fer à Moulin, 75005, Paris, France. Electronic address:

Neuronal Cl homeostasis is regulated by the activity of two cation chloride co-transporters (CCCs), the K-Cl cotransporter KCC2 and the Na-K-Cl cotransporter NKCC1, which are primarily extruding and importing chloride in neurons, respectively. Several neurological and psychiatric disorders including epilepsy, neuropathic pain, schizophrenia and autism are associated with altered neuronal chloride (Cl) homeostasis. A current view is that the accumulation of intracellular Cl in neurons as a result of KCC2 down-regulation and/or NKCC1 up-regulation may weaken inhibitory GABA signaling and thereby promote the development of pathological activities. CCC activity is determined mainly by their level of expression in the plasma membrane. Furthermore, CCCs undergo "diffusion-trapping" in the membrane, a mechanism that is rapidly adjusted by activity-dependent post-translational modifications i.e. phosphorylation/dephosphorylation of key serine and threonine residues. This represents probably the most rapid cellular mechanism for adapting CCC function to changes in neuronal activity. Therefore, interfering with these mechanisms may help restoring Cl homeostasis and inhibition under pathological conditions. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.
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http://dx.doi.org/10.1016/j.neuropharm.2019.03.014DOI Listing
June 2020

Reciprocal Regulation of KCC2 Trafficking and Synaptic Activity.

Front Cell Neurosci 2019 20;13:48. Epub 2019 Feb 20.

INSERM UMR-S 1270, Paris, France.

The main inhibitory neurotransmitter receptors in the adult central nervous system (CNS) are type A γ-aminobutyric acid receptors (GABARs) and glycine receptors (GlyRs). Synaptic responses mediated by GlyR and GABAR 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.
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http://dx.doi.org/10.3389/fncel.2019.00048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391895PMC
February 2019

Activity-Dependent Inhibitory Synapse Scaling Is Determined by Gephyrin Phosphorylation and Subsequent Regulation of GABA Receptor Diffusion.

eNeuro 2018 Jan-Feb;5(1). Epub 2018 Jan 18.

INSERM UMR-S, Paris, 75005, France.

Synaptic plasticity relies on the rapid changes in neurotransmitter receptor number at postsynaptic sites. Using superresolution photoactivatable localization microscopy imaging and quantum dot-based single-particle tracking in rat hippocampal cultured neurons, we investigated whether the phosphorylation status of the main scaffolding protein gephyrin influenced the organization of the gephyrin scaffold and GABA receptor (GABAR) membrane dynamics. We found that gephyrin phosphorylation regulates gephyrin microdomain compaction. Extracellular signal-regulated kinase 1/2 and glycogen synthase kinase 3β (GSK3β) signaling alter the gephyrin scaffold mesh differentially. Differences in scaffold organization similarly affected the diffusion of synaptic GABARs, suggesting reduced gephyrin receptor-binding properties. In the context of synaptic scaling, our results identify a novel role of the GSK3β signaling pathway in the activity-dependent regulation of extrasynaptic receptor surface trafficking and GSK3β, protein kinase A, and calcium/calmodulin-dependent protein kinase IIα pathways in facilitating adaptations of synaptic receptors.
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http://dx.doi.org/10.1523/ENEURO.0203-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780843PMC
January 2019

A Simple and Powerful Analysis of Lateral Subdiffusion Using Single Particle Tracking.

Biophys J 2017 Dec;113(11):2452-2463

École Normale Supérieure, PSL Research University, CNRS, INSERM, Institute of Biology (IBENS), Paris, France. Electronic address:

In biological membranes, many factors such as cytoskeleton, lipid composition, crowding, and molecular interactions deviate lateral diffusion from the expected random walks. These factors have different effects on diffusion but act simultaneously, so the observed diffusion is a complex mixture of diffusive behaviors (directed, Brownian, anomalous, or confined). Therefore, commonly used approaches to quantify diffusion based on averaging of the displacements such as the mean square displacement, are not adapted to the analysis of this heterogeneity. We introduce a parameter-the packing coefficient Pc, which gives an estimate of the degree of free movement that a molecule displays in a period of time independently of its global diffusivity. Applying this approach to two different situations (diffusion of a lipid probe and trapping of receptors at synapses), we show that Pc detected and localized temporary changes of diffusive behavior both in time and in space. More importantly, it allowed the detection of periods with very high confinement as well as their frequency and duration, and thus it can be used to calculate the effective k and k of scaffolding interactions such as those that immobilize receptors at synapses.
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http://dx.doi.org/10.1016/j.bpj.2017.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738498PMC
December 2017

GABA receptor dependent synaptic inhibition rapidly tunes KCC2 activity via the Cl-sensitive WNK1 kinase.

Nat Commun 2017 11 24;8(1):1776. Epub 2017 Nov 24.

Inserm UMR-S 839, 75005, Paris, France.

The K-Cl co-transporter KCC2 (SLC12A5) tunes the efficacy of GABA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl]. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABA receptor (GABAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl-sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl] to GABAR activity.
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http://dx.doi.org/10.1038/s41467-017-01749-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701213PMC
November 2017

Associations of the Intellectual Disability Gene MYT1L with Helix-Loop-Helix Gene Expression, Hippocampus Volume and Hippocampus Activation During Memory Retrieval.

Neuropsychopharmacology 2017 Dec 4;42(13):2516-2526. Epub 2017 May 4.

Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK.

The fundamental role of the brain-specific myelin transcription factor 1-like (MYT1L) gene in cases of intellectual disability and in the etiology of neurodevelopmental disorders is increasingly recognized. Yet, its function remains under-investigated. Here, we identify a network of helix-loop-helix (HLH) transcriptional regulators controlled by MYT1L, as indicated by our analyses in human neural stem cells and in the human brain. Using cell-based knockdown approaches and microarray analyses we found that (1) MYT1L is required for neuronal differentiation and identified ID1, a HLH inhibitor of premature neurogenesis, as a target. (2) Although MYT1L prevented expression of ID1, it induced expression of a large number of terminal differentiation genes. (3) Consistently, expression of MYT1L in the human brain coincided with neuronal maturation and inversely correlated with that of ID1 and ID3 throughout the lifespan. (4) Genetic polymorphisms that reduced expression of MYT1L in the hippocampus resulted in increased expression of ID1 and ID3, decreased levels of the proneural basic HLH (bHLH) transcriptional regulators TCF4 and NEUROD6 and decreased expression of genes involved in long-term potentiation and synaptic transmission, cancer and neurodegeneration. Furthermore, our neuroimaging analyses indicated that MYT1L expression associated with hippocampal volume and activation during episodic memory recall, as measured by blood-oxygen-level-dependent (BOLD) signals. Overall, our findings suggest that MYT1L influences memory-related processes by controlling a neuronal proliferation/differentiation switch of ID-bHLH factors.
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http://dx.doi.org/10.1038/npp.2017.91DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549840PMC
December 2017

Bidirectional Control of Synaptic GABAAR Clustering by Glutamate and Calcium.

Cell Rep 2015 Dec 17;13(12):2768-80. Epub 2015 Dec 17.

Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute (BSI), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Electronic address:

GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.
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http://dx.doi.org/10.1016/j.celrep.2015.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700050PMC
December 2015

KCC2 Gates Activity-Driven AMPA Receptor Traffic through Cofilin Phosphorylation.

J Neurosci 2015 Dec;35(48):15772-86

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F-75005, Paris, France, Sorbonne Universités, Université Pierre et Marie Curie Université Paris 06, Unité Mixte de Recherche-S 839, F-75005, Paris, France, Institut du Fer a Moulin, F-75005, Paris, France, and

Expression of the neuronal K/Cl transporter KCC2 is tightly regulated throughout development and by both normal and pathological neuronal activity. Changes in KCC2 expression have often been associated with altered chloride homeostasis and GABA signaling. However, recent evidence supports a role of KCC2 in the development and function of glutamatergic synapses through mechanisms that remain poorly understood. Here we show that suppressing KCC2 expression in rat hippocampal neurons precludes long-term potentiation of glutamatergic synapses specifically by preventing activity-driven membrane delivery of AMPA receptors. This effect is independent of KCC2 transporter function and can be accounted for by increased Rac1/PAK- and LIMK-dependent cofilin phosphorylation and actin polymerization in dendritic spines. Our results demonstrate that KCC2 plays a critical role in the regulation of spine actin cytoskeleton and gates long-term plasticity at excitatory synapses in cortical neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.1735-15.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605455PMC
December 2015

Benzodiazepine ligands rapidly influence GABAA receptor diffusion and clustering at hippocampal inhibitory synapses.

Neuropharmacology 2015 Jan 12;88:199-208. Epub 2014 Jun 12.

INSERM UMR-S 839, 75005, Paris, France; Université Pierre et Marie Curie, 75005, Paris, France; Institut du Fer a Moulin, 75005, Paris, France. Electronic address:

Benzodiazepines (BZDs) are widely used in the treatment of a variety of neurological and psychiatric conditions including anxiety, insomnia and epilepsy. BZDs are thought to act predominantly by affecting the gating of GABAA receptor channels, resulting in enhanced GABA-mediated currents in neurons. However, mutations mimicking the effect of BZDs on GABAAR channel gating have been shown to also impact the membrane dynamics and synaptic anchoring of the receptors. Here, using single molecule tracking combined with electrophysiological recordings, we show that BZD ligands rapidly influence the dynamic behavior of GABAARs in hippocampal neurons. Application of the inverse BZD agonist DMCM rapidly increased the diffusion and reduced the clustering of GABAARs at synapses, resulting in reduced postsynaptic currents. Conversely, the BZD full agonist diazepam had little effect at rest but reduced lateral diffusion and increased synaptic stabilization and clustering of GABAARs upon sustained neuronal activity, resulting in enhanced potency of inhibitory synapses. These effects occurred in the absence of detectable changes in gephyrin clusters, suggesting they did not reflect a rapid dispersion of the synaptic scaffold. Thus, alterations of the diffusion and synaptic anchoring of GABAARs represent a novel, unsuspected mechanism through which BZDs rapidly modulate GABA signaling in central neurons.
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http://dx.doi.org/10.1016/j.neuropharm.2014.06.002DOI Listing
January 2015

Activity-dependent regulation of the K/Cl transporter KCC2 membrane diffusion, clustering, and function in hippocampal neurons.

J Neurosci 2013 Sep;33(39):15488-503

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Recherche en Santé 839, F-75005 Paris, France, Université Pierre et Marie Curie, F-75005 Paris, France, Institut du Fer a Moulin, F-75005 Paris, France, and Institut de Biologie de l'Ecole Normale Supérieure, INSERM, Unité 1024, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, F-75005 Paris, France.

The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2-actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca(2+) influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.5889-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618451PMC
September 2013

Diffusion barriers constrain receptors at synapses.

PLoS One 2012 13;7(8):e43032. Epub 2012 Aug 13.

Institut de Biologie de l'Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale U1024, Centre National de la Recherche Scientifique UMR8197, Paris, France.

The flux of neurotransmitter receptors in and out of synapses depends on receptor interaction with scaffolding molecules. However, the crowd of transmembrane proteins and the rich cytoskeletal environment may constitute obstacles to the diffusion of receptors within the synapse. To address this question, we studied the membrane diffusion of the γ-aminobutyric acid type A receptor (GABA(A)R) subunits clustered (γ2) or not (α5) at inhibitory synapses in rat hippocampal dissociated neurons. Relative to the extrasynaptic region, γ2 and α5 showed reduced diffusion and increased confinement at both inhibitory and excitatory synapses but they dwelled for a short time at excitatory synapses. In contrast, γ2 was ~3-fold more confined and dwelled ~3-fold longer in inhibitory synapses than α5, indicating faster synaptic escape of α5. Furthermore, using a gephyrin dominant-negative approach, we showed that the increased residency time of γ2 at inhibitory synapses was due to receptor-scaffold interactions. As shown for GABA(A)R, the excitatory glutamate receptor 2 subunit (GluA2) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had lower mobility in both excitatory and inhibitory synapses but a higher residency time at excitatory synapses. Therefore barriers impose significant diffusion constraints onto receptors at synapses where they accumulate or not. Our data further reveal that the confinement and the dwell time but not the diffusion coefficient report on the synapse specific sorting, trapping and accumulation of receptors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043032PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418229PMC
May 2013

The 22nd ion channel meeting, september 2011, france.

Channels (Austin) 2012 May-Jun;6(3):149-53. Epub 2012 May 1.

Neurobiologie des Canaux Ioniques, INSERM-Université de la Méditerranée, Marseille, France.

The 22(nd) Ion Channel Meeting was organized by the French Ion Channel Society (Association Canaux Ioniques) from the 25(th) to the 28(th) of September 2011 on the French Riviera (Giens). This year again, more than one hundred researchers from France, Europe and extra-European countries gathered to present and discuss their recent advances and future challenges in the ion channels and transporters field. The scientific committee organized a plenary lecture and five thematic symposia by inviting international researchers to present their recent outstanding work on themes as diverse as muscular channelopathies, regulation of channels by extracellular matrix, receptor-channels interactions, localization and distribution of ion channels, their involvement in the cell life and death, and finally how they participate in the evolution and adaptability of cellular excitability. These presentations are summarized in this meeting report. Two sessions of oral communications selected from submitted abstracts and two poster sessions were also organized to present the ongoing work of young researchers worldwide.
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http://dx.doi.org/10.4161/chan.20795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431583PMC
December 2012

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

Front Cell Neurosci 2012 21;6. Epub 2012 Feb 21.

INSERM, UMR-839 Paris, France.

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.
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http://dx.doi.org/10.3389/fncel.2012.00005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282916PMC
October 2012

A human mutation in Gabrg2 associated with generalized epilepsy alters the membrane dynamics of GABAA receptors.

Cereb Cortex 2012 Jul 9;22(7):1542-53. Epub 2011 Sep 9.

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche en Santé 839, 75005 Paris, France.

Neuronal activity modulates the membrane diffusion of postsynaptic γ-aminobutyric acid (GABA)(A) receptors (GABA(A)Rs), thereby regulating the efficacy of GABAergic synapses. The K289M mutation in GABA(A)Rs subunit γ2 has been associated with the generalized epilepsy with febrile seizures plus (GEFS+) syndrome. This mutation accelerates receptor deactivation and therefore reduces inhibitory synaptic transmission. Yet, it is not clear why this mutation specifically promotes febrile seizures. We show that upon raising temperature both the number of GABA(A)Rs clusters and the frequency of miniature inhibitory postsynaptic currents decreased in neurons expressing the K289M mutant but not wild-type (WT) recombinant γ2. Single-particle tracking experiments revealed that raising temperature increases the membrane diffusion of synaptic GABA(A)Rs containing the K289M mutant but not WT recombinant γ2. This effect was mediated by enhanced neuronal activity as it was blocked by glutamate receptor antagonists and was mimicked by the convulsant 4-aminopyridine. Our data suggest the K289M mutation in γ2 confers GABA(A)Rs with enhanced sensitivity of their membrane diffusion to neuronal activity. Enhanced activity during hyperthermia may then trigger the escape of receptors from synapses and thereby further reduce the efficacy of GABAergic inhibition. Alteration of the membrane diffusion of neurotransmitter receptors therefore represents a new mechanism in human epilepsy.
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http://dx.doi.org/10.1093/cercor/bhr225DOI Listing
July 2012

The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines.

Proc Natl Acad Sci U S A 2011 Sep 30;108(37):15474-9. Epub 2011 Aug 30.

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France.

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.
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http://dx.doi.org/10.1073/pnas.1107893108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174661PMC
September 2011

Labeling neuronal membrane receptors with quantum dots.

Cold Spring Harb Protoc 2011 Mar 1;2011(3):prot5580. Epub 2011 Mar 1.

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http://dx.doi.org/10.1101/pdb.prot5580DOI Listing
March 2011

Activity-dependent tuning of inhibitory neurotransmission based on GABAAR diffusion dynamics.

Neuron 2009 Jun;62(5):670-82

Biologie Cellulaire de la Synapse N&P, Ecole Normale Supérieure Paris, INSERM U789, 46 Rue d'Ulm 75005 Paris, France.

An activity-dependent change in synaptic efficacy is a central tenet in learning, memory, and pathological states of neuronal excitability. The lateral diffusion dynamics of neurotransmitter receptors are one of the important parameters regulating synaptic efficacy. We report here that neuronal activity modifies diffusion properties of type-A GABA receptors (GABA(A)R) in cultured hippocampal neurons: enhanced excitatory synaptic activity decreases the cluster size of GABA(A)Rs and reduces GABAergic mIPSC. Single-particle tracking of the GABA(A)R gamma2 subunit labeled with quantum dots reveals that the diffusion coefficient and the synaptic confinement domain size of GABA(A)R increases in parallel with neuronal activity, depending on Ca(2+) influx and calcineurin activity. These results indicate that GABA(A)R diffusion dynamics are directly linked to rapid and plastic modifications of inhibitory synaptic transmission in response to changes in intracellular Ca(2+) concentration. This transient activity-dependent reduction of inhibition would favor the onset of LTP during conditioning.
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http://dx.doi.org/10.1016/j.neuron.2009.04.023DOI Listing
June 2009

Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion.

Neuron 2008 Jul;59(2):261-73

Biologie Cellulaire de la Synapse, INSERM U789, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France.

In the spinal cord, most inhibitory synapses have a mixed glycine-GABA phenotype. Using a pharmacological approach, we report an NMDAR activity-dependent regulation of the mobility of GlyRs but not GABA(A)Rs at inhibitory synapses in cultured rat spinal cord neurons. The NMDAR-induced decrease in GlyR lateral diffusion was correlated with an increase in receptor cluster number and glycinergic mIPSC amplitude. Changes in GlyR diffusion properties occurred rapidly and before the changes in the number of synaptic receptors. Regulation of synaptic GlyR content occurred without change in the amount of gephyrin. Moreover, NMDAR-dependent regulation of GlyR lateral diffusion required calcium influx and calcium release from stores. Therefore, excitation may increase GlyR levels at synapses by a calcium-mediated increase in postsynaptic GlyR trapping involving regulation of receptor-scaffold interactions. This provides a mechanism for a rapid homeostatic regulation of the inhibitory glycinergic component at mixed glycine-GABA synapses in response to increased NMDA excitatory transmission.
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http://dx.doi.org/10.1016/j.neuron.2008.05.030DOI Listing
July 2008

Imaging the lateral diffusion of membrane molecules with quantum dots.

Nat Protoc 2006 ;1(6):2628-34

INSERM U789, Biologie Cellulaire de la Synapse N&P, Ecole Normale Supérieure Paris, 46, Rue d'Ulm 75005 Paris, France.

This protocol describes a sensitive approach to tracking the motion of membrane molecules such as lipids and proteins with molecular resolution in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane molecules of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and organic dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The experimental procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.
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http://dx.doi.org/10.1038/nprot.2006.429DOI Listing
November 2007

Single quantum dot tracking of membrane receptors.

Methods Mol Biol 2007 ;374:81-91

Laboratoire Kastler Brossel, Physics Department, Ecole Normale Supérieure, Paris, France.

The dynamics of membrane proteins in living cells has become a major issue to understand important biological questions such as chemotaxis, synaptic regulation, or signal transduction. The advent of semi-conductor quantum dots (QDs) has opened new perspectives for the study of membrane properties because these new nanomaterials enable measurements at the single molecule level with high signal-to-noise ratio. Probes used until now indeed encounter significant limitations: organic fluorophores and fluorescent proteins rapidly photobleach, whereas gold particles and latex beads, although more stable, are bulky and usually stain only one protein per experiment. In comparison, QDs are bright and photostable fluorescent probes with a size on the order of 10 nm can be used with standard immunochemical methods. We present the experimental protocols and methods of analysis which we used to investigate the dynamics of individual GABAA receptors in the axonal growth cone of spinal neurons in culture. Single QD tracking is nevertheless a general method, suitable to study many transmembrane proteins.
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http://dx.doi.org/10.1385/1-59745-369-2:81DOI Listing
December 2007

Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane.

J Neurosci 2006 Aug;26(33):8502-11

Laboratoire de Biologie Cellulaire de la Synapse, Institut National de la Santé et de la Recherche Médicale, Unité 789, Ecole Normale Supérieure, 75005 Paris, France.

Lateral diffusion of neurotransmitter receptors in and out of synapses has been postulated as a core mechanism for rapid changes in receptor number at synapses during plastic processes. In this study, we have used single particle tracking to investigate how changes in glycine receptor (GlyR) lateral diffusion properties might account for changes in receptor number at synapses after disruption of the cytoskeleton in dissociated spinal cord neurons. We found that pharmacological disruption of F-actin and microtubules decreased the amount of GlyR and gephyrin, the backbone of the inhibitory postsynaptic scaffold, at synapses. F-actin and microtubule disruption increased GlyR exchanges between the synaptic and extrasynaptic membranes and decreased receptor dwell time at synapses. GlyR lateral diffusion was predominantly controlled by microtubules in the extrasynaptic membrane and by actin at synapses. Both diffusion coefficients and confinement at synapses were affected after F-actin disruption. Our results indicate that receptor exchanges between the synaptic and extrasynaptic compartments depend on the properties of both the postsynaptic differentiation and the extrasynaptic membrane. Consequently, GlyR number at synapses may be rapidly modulated by the cytoskeleton through the regulation of lateral diffusion in the plasma membrane and of receptor stabilization at synapses.
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http://dx.doi.org/10.1523/JNEUROSCI.1758-06.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6674337PMC
August 2006

Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons.

J Neurosci 2004 Jan;24(1):207-17

Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

The role of the scaffolding protein gephyrin at hippocampal inhibitory synapses is not well understood. A previous study (Kneussel et al., 1999) reported a complete loss of synaptic clusters of the major GABA(A)R subunits alpha2 and gamma2 in hippocampal neurons lacking gephyrin. In contrast, we show here that GABA(A)R alpha2 and gamma2 subunits do cluster at pyramidal synapses in hippocampal cultures from gephyrin-/- mice, albeit at reduced levels compared with control neurons. Synaptic aggregation of GABA(A)R alpha1 on interneurons was identical between the culture types. Furthermore, we recorded miniature IPSCs (mIPSCs) from gephyrin-/- neurons. Although the mean mIPSC amplitude was reduced (by 23%) compared with control, the frequency of these events was unchanged. Cell surface labeling experiments indicated that gephyrin contributes, in part, to aggregation but not to insertion or stabilization of GABA(A)R alpha2 and gamma2 in the plasma membrane. Thus, a major gephyrin-independent component of hippocampal inhibitory synapse development must exist. We also report that glycine receptors cluster at GABAergic synapses in a subset of hippocampal interneurons and pyramidal neurons. Unlike GABA(A)Rs, synaptic clustering of glycine receptors was completely abolished in gephyrin-/- neurons. Finally, artificial extrasynaptic aggregation of GABA(A)R was able to redistribute and cocluster gephyrin by a mechanism requiring a neuron-specific modification or intermediary protein. We propose a model of hippocampal inhibitory synapse development in which some GABA(A)Rs cluster at synapses by a gephyrin-independent mechanism and recruit gephyrin. This clustered gephyrin may then recruit glycine receptors, additional GABA(A)Rs, and other signal-transducing components.
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http://dx.doi.org/10.1523/JNEUROSCI.1661-03.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6729579PMC
January 2004

Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking.

Science 2003 Oct;302(5644):442-5

Laboratoire Kastler Brossel, CNRS UMR 8552, Ecole Normale Supérieure and Université Pierre et Marie Curie, 24 rue Lhomond, 75005 Paris, France.

Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biological imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was observed and further confirmed by electron microscopy imaging of QD-tagged receptors.
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http://dx.doi.org/10.1126/science.1088525DOI Listing
October 2003

Gamma protocadherins are required for survival of spinal interneurons.

Neuron 2002 Dec;36(5):843-54

Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

The murine genome contains approximately 70 protocadherin (Pcdh) genes. Many are expressed in the nervous system, suggesting that Pcdhs may specify neuronal connectivity. Here, we analyze the 22 contiguous genes of the Pcdh-gamma cluster. Individual neurons express subsets of Pcdh-gamma genes. Pcdh-gamma proteins are present in most neurons and associated with, but not confined to, synapses. Early steps in neuronal migration, axon outgrowth, and synapse formation proceed in mutant mice lacking all 22 Pcdh-gamma genes. At late embryonic stages, however, dramatic neurodegeneration leads to neonatal death. In mutant spinal cord, many interneurons are lost, but sensory and motor neurons are relatively spared. In cultures from mutant spinal cord, neurons differentiate and form synapses but then die. Thus, Pcdh-gamma genes are dispensable for at least some aspects of connectivity but required for survival of specific neuronal types.
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http://dx.doi.org/10.1016/s0896-6273(02)01090-5DOI Listing
December 2002

Dystroglycan is selectively associated with inhibitory GABAergic synapses but is dispensable for their differentiation.

J Neurosci 2002 Jun;22(11):4274-85

Washington University, Department of Anatomy and Neurobiology, St. Louis, Missouri 63110, USA.

The dystrophin glycoprotein complex (DGC) is a multimolecular complex that links the extracellular matrix to the cytoskeleton. The DGC is present at the skeletal neuromuscular junction and required for its maturation and maintenance. Members of the DGC are also expressed in brain. We used cultured hippocampal neurons to analyze the distribution, regulation, and role in synaptogenesis of the major transmembrane component of the DGC, dystroglycan; one of its extracellular ligands, agrin; and one of its cytoskeletal binding partners, dystrophin. alpha-Dystroglycan, beta-dystroglycan, and dystrophin clustered at a subset of inhibitory synapses containing GABA(A)R subunits alpha1, alpha2, and gamma2, and the inhibitory receptor anchoring protein gephyrin. DGC components were not detected at excitatory glutamatergic synapses. Dystroglycan is the first identified adhesive macromolecule at mature GABA synapses. Developmentally, dystroglycan clustered at synaptic loci after synaptic vesicles, GABA(A)R, and gephyrin, the latter being closely associated with GABA(A)R at all stages of synaptogenesis analyzed. Analysis of gephyrin -/-, agrin -/-, and mdx mouse hippocampal neurons in culture indicated that synaptic clustering of dystroglycan occurs independently of gephyrin, agrin, and dystrophin. In dystroglycan-deficient neurons, cultured from a conditional mutant strain, GABAergic synapses differentiated with clusters of gephyrin and GABA(A)R apposed to synaptic terminals, but these synapses did not contain detectable dystrophin. Thus the DGC is not essential for GABAergic synaptogenesis but is likely to function in modulating inhibitory synapses or conferring specialized properties on a subset of them.
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http://dx.doi.org/20026440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6758826PMC
June 2002