The non-adrenergic imidazoline-1 receptor protein nischarin is a key regulator of astrocyte glutamate uptake

Astrocytic GLT-1 is the main glutamate transporter involved in glutamate buffering in the brain, pivotal for glutamate removal at excitatory synapses to terminate neurotransmission and for preventing excitotoxicity. We show here that the surface expression and function of GLT-1 can be rapidly modulated through the interaction of its N-terminus with the nonadrenergic imidazoline-1 receptor protein, Nischarin. The phox domain of Nischarin is critical for interaction and internalization of surface GLT-1. Using live super-resolution imaging, we found that glutamate accelerated Nischarin-GLT-1 internalization into endosomal structures. The surface GLT-1 level increased in Nischarin knockout astrocytes, and this correlated with a significant increase in transporter uptake current. In addition, Nischarin knockout in astrocytes is neuroprotective against glutamate excitotoxicity. These data provide new molecular insights into regulation of GLT-1 surface level and function and suggest new drug targets for the treatment of neurological disorders

Mécanisme d'adressage dendritique du récepteur 5-HT1A de la sérotonine : identification de Yif1B, protéine d'échafaudage essentielle au recrutement de Rab6, Yip1A et de la tubuline

The serotoninergic 5-HT1A receptors localized at the plasma membrane of the soma and dendrites of the raphe neurons play a key role in the onset of antidepressant action. The aim of my work was to identify partner proteins involved in the polarized 5-HT1A receptor targeting in neurons. Thus, these proteins could be news targets for innovative therapeutic strategies. Following the discovery in the Lab of Yif1B, the first 5-HT1A receptor partner protein implicated in its dendritic targeting, the first part of my thesis work has resulted in the characterization of the interaction between the 5-HT1A receptor and Yif1B. By combining directed mutagenesis, GST-Pull Down and Surface Plasmon Resonance (or Biacore), I have highlighted the role of a tribasic motif in the C-tail of the 5-HT1A receptor, that is in direct interaction with three aspartic acid residues in the N-tail of Yif1B. In the second part of my work, I have characterized the mechanism of this specific trafficking by a multidisciplinary approach combining proteomic, biochemistry, topological analysis on primary culture of hippocampal neurons and videomicroscopy on live neurons. Thus, I have identified a new dendritic targeting pathway involving Yif1B as a scaffold protein assembling the 5-HT1A receptor with Yip1A and Rab6, two other partners of the vesicular cargo complex transporting the receptor along the dendritic microtubules. Finally, the identification of the molecular motors involved in this addressing process complete the description of this new pathway.Les autorécepteurs 5-HT1A présents à la membrane somatodendritique des neurones du raphé jouent un rôle clé dans le délai d’action des antidépresseurs inhibiteurs sélectifs de la recapture de la sérotonine (ISRS) administrés en chronique. Ils constituent donc une cible envue d’accélérer l’efficacité thérapeutique de ces psychotropes. A cette fin, je me suis appliquée à identifier et caractériser les protéines partenaires impliquées dans l’adressage membranaire dendritique du récepteur 5-HT1A et sur lesquelles il devrait être possible d’agir pour moduler son activité. Des études antérieures menées au laboratoire ont montré le rôle de l’extrémité C-terminale cytosolique du récepteur 5-HT1A dans son adressage polarisé et ont permis, par la technique de double hybride chez la levure, l’identification de Yif1B, première protéine interagissant avec cette extrémité. Mon travail de thèse a comporté deux volets : le premier a abouti à la caractérisation de l’interaction entre le récepteur 5-HT1A et sa protéine partenaire Yif1B. En combinant des approches de mutagénèse dirigée et de biochimie : GST-Pull Down et technologie de résonance plasmonique de surface (ou Biacore), j’ai pu mettre en évidence, dans l’extrémité C-terminale du récepteur 5-HT1A, un motif tribasique en interaction directe avec trois résidus aspartate de l’extrémité N-terminale cytosolique de Yif1B. En parallèle, des résultats de l’équipe ont démontré le rôle déterminant de cette protéine Yif1B dans l’adressage dendritique du récepteur 5-HT1A. Pour le deuxième volet de mon travail, je me suis appliquée à tenter d’élucider les mécanismes de ce routage spécifique. Ainsi, par une approche multidisciplinaire mêlant protéomique et biochimie, analyses topologiques sur des cultures primaires de neurones d’hippocampe et vidéomicroscopie sur des neurones vivants, j’ai pu identifier une nouvelle voie d’adressage dendritique dans laquelle Yif1B est la protéine d’échafaudage qui assemble le récepteur 5-HT1A avec Yip1A et Rab6, les autres partenaires du complexe cargo vésiculaire qui transporte le récepteur le long des microtubules dendritiques. L’identification des moteurs moléculaires impliqués dans le transport bidirectionnel du récepteur 5-HT1A dans les dendrites distales constitue le dernier objectif de mon travail de thèse. Les perspectives ouvertes par ce travail concernent d’une part la généralisation éventuelle du rôle de Yif1B dans le trafic intracellulaire d’autres récepteurs, et d’autre part la conception d’un peptide mimétique inhibant spécifiquement l’interaction « récepteur 5-HT1A/Yif1B ». Un tel peptide pourrait, en réalité, être un premier outil pharmacologique pour affecter l’activité du récepteur 5-HT1A via une perturbation de son adressage

Mécanisme d'adressage dendritique du récepteur 5-HT1A de la sérotonine (identification de Yif1B, protéine d'échafaudage essentielle au recrutement de Rab6, Yip1A et de la tubuline)

PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

La géphyrine interagit avec le co-transporteur K-Cl KCC2 pour réguler son expression de surface et sa fonction dans les neurones corticaux.

International audienceThe K+-Cl- cotransporter KCC2, encoded by the Slc12a5 gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABAA receptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.SIGNIFICANCE STATEMENT Fast synaptic inhibition in the brain is mediated by chloride-permeable GABAA receptors (GABAARs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABAARs, gephyrin, and KCC2 act to regulate synaptic GABA signaling

KCC2 Regulates Neuronal Excitability and Hippocampal Activity via Interaction with Task-3 Channels

International audienceKCC2 regulates neuronal transmembrane chloride gradients and thereby controls GABA signaling in the brain. KCC2 downregulation is observed in numerous neurological and psychiatric disorders. Paradoxical, excitatory GABA signaling is usually assumed to contribute to abnormal network activity underlying the pathology. We tested this hypothesis and explored the functional impact of chronic KCC2 downregulation in the rat dentate gyrus. Although the reversal potential of GABAA receptor currents is depolarized in KCC2 knockdown neurons, this shift is compensated by depolarization of the resting membrane potential. This reflects downregulation of leak potassium currents. We show KCC2 interacts with Task-3 (KCNK9) channels and is required for their membrane expression. Increased neuronal excitability upon KCC2 suppression altered dentate gyrus rhythmogenesis, which could be normalized by chemogenetic hyperpolarization. Our data reveal KCC2 downregulation engages complex synaptic and cellular alterations beyond GABA signaling that perturb network activity thus offering additional targets for therapeutic intervention

A New Vesicular Scaffolding Complex Mediates the G-Protein-Coupled 5-HT1A Receptor Targeting to Neuronal Dendrites

International audienceAlthough essential for their neuronal function, the molecular mechanisms underlying the dendritic targeting of serotonin G-protein-coupled receptors are poorly understood. Here, we characterized a Yif1B-dependent vesicular scaffolding complex mediating the intracellular traffic of the rat 5-HT1A receptor (5-HT1AR) toward dendrites. By combining directed mutagenesis, GST-pull down, and surface plasmon resonance, we identified a tribasic motif in the C-tail of the 5-HT1AR on which Yif1B binds directly with high affinity (K-D approximate to 37 nM). Moreover, we identified Yip1A, Rab6, and Kif5B as new partners of the 5-HT1AR/Yif1B complex, and showed that their expression in neurons is also crucial for the dendritic targeting of the 5-HT1AR. Live videomicroscopy revealed that 5-HT1AR, Yif1B, Yip1A, and Rab6 traffic in vesicles exiting the soma toward the dendritic tree, and also exhibit bidirectional motions, sustaining their role in 5-HT1AR dendritic targeting. Hence, we propose a new trafficking pathway model in which Yif1B is the scaffold protein recruiting the 5-HT1AR in a complex including Yip1A and Rab6, with Kif5B and dynein as two opposite molecular motors coordinating the traffic of vesicles along dendritic microtubules. This targeting pathway opens new insights for G-protein-coupled receptors trafficking in neurons