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Synapse stabilization Early in brain development, neurons connect to each other enthusiastically. With development, an overabundance of synapses is winnowed down to refine efficiently connected circuits. Inactive synapses are prime targets for elimination, whereas active synapses tend to be retained. Gomez-Castro et al . took a closer look at how those choices are made (see the Perspective by Blum and Lopes). When postsynaptic adenosine receptors are muted or do not find enough extracellular adenosine, synapses get eliminated. Neurotransmitter-dependent signaling pathways drive protein kinase A to phosphorylate the postsynaptic scaffolding molecule gephyrin. Together with a partner synaptogenic membrane protein, gephyrin is required for the stabilization of γ-aminobutyric acid receptors. Adenosine receptors thus detect synaptic activity and in turn drive the stabilization of synapses that produce such activity. —PJH

International audience; The serotonin 2B (5-HT2B) 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-HT2B 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-HT2B 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-HT2B receptor, and the NR1 subunit of the NMDA receptor form a macromolecular complex. CIPP increased 5-HT2B 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-HT2B receptor stimulation in turn dispersed NR1 clusters colocalized with 5-HT2B receptors and increased the density and maturation of dendritic spines. Collectively, our results suggest that the 5-HT2B receptor, the NMDA receptor, and CIPP may form a signaling platform by which serotonin can influence structural plasticity of excitatory glutamatergic synapses.

The fundamental role of the brain-specific myelin transcription factor 1-like (MYT1L) gene in cases of intellectual disability and in the aetiology of neurodevelopmental disorders is increasingly recognised. 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 (i) MYT1L is required for neuronal differentiation and identified ID1, a HLH inhibitor of premature neurogenesis, as a target. (2) While 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.Neuropsychopharmacology accepted article preview online, 04 May 2017. doi:10.1038/npp.2017.91.

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.SIGNIFICANCE STATEMENTChanges in the expression of neuronal chloride transporters, such as KCC2, occur during postnatal development and are induced in a variety of neurological and psychiatric conditions. Such changes are expected to primarily impact GABA signaling because GABAA receptors are predominantly permeable to chloride ions. However, the KCC2 transporter forms clusters near glutamatergic synapses and interacts with several actin-related proteins. We show that KCC2 is strictly required for LTP expression at hippocampal excitatory synapses. This effect is due to KCC2 interaction with the Rac1/PAK signaling pathway that controls actin polymerization. Suppressing this interaction promotes actin polymerization thereby hindering AMPA receptor traffic upon KCC2 suppression. Alterations of KCC2 expression therefore impact not only GABAergic signaling but also glutamatergic synaptic function and long term plasticity.

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, Ca2+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.

Listeria monocytogenes is an intracellular pathogen that causes severe central nervous system infection in humans and animals. The ability of this bacterium to penetrate nerve cells was investigated by using rat spinal cell cultures. Entry into distinct cell types, i.e., glial cells and neurons, was monitored by a differential immunofluorescence technique with antibodies against cell type-specific markers and the bacterial pathogen. L. monocytogenes was detected predominantly within macrophages constituting the microglia. Astrocytes and oligodendrocytes, the major components of macroglia, were infected to a lesser extent. Surprisingly, Listeria innocua , a noninvasive and nonpathogenic species, also has the capacity to enter into these three types of glial cells. Entry into neurons was a very rare event. In contrast, we found that L. monocytogenes could efficiently invade neurons when these latter cells were cocultivated with Listeria -infected mouse macrophages. In this case, infection of neurons occurs by cell-to-cell spread via an actA -dependent mechanism. These data support the notion that infected phagocytes can be vectors by which L. monocytogenes gains access to privileged niches such as the central nervous system.

In the adult brain, synaptic inhibition is mainly mediated by the activity of GABA type A receptors (RGABAA). This inhibition depends on the electrochemical chloride gradient, which is mainly regulated by the chlorine cotransporters KCC2 and NKCC1. KCC2 transports chlorine out of the neuron, while NKCC1 is responsible for its influx. Little is known about the functions of NKCC1 in the adult brain and its regulation. Nevertheless, in pathological cases related to altered GABAergic transmission, NKCC1 is of definite functional importance, in contrast to its apparently discrete role in chlorine homeostasis. This is particularly the case in the context of epilepsy, both experimental and clinical.I have demonstrated, using single particle tracking, immunofluorescence and STORM imaging approaches, that the membrane stability of NKCC1 is rapidly regulated by lateral diffusion in an activity-dependent manner at the surface of mature hippocampal neurons. Basal glutamatergic activity regulates NKCC1 membrane dynamics in the axon but not in the somato-dendritic compartment. In the somato-dendritic compartment, NKCC1 is regulated by RGABAA-mediated signalling. I also assessed the relevance of inhibition of this pathway in the context of ictogenesis. I used a PTZ-induced ictogenesis test in SPAK243A/243A mice, which express a non-active form of SPAK. Behavioural indicators (Racine scale) and electrocorticograms showed that the effects of the mutation on these variables were mild.; Dans le cerveau adulte, l’inhibition synaptique passe majoritairement par l’activité des récepteurs GABA de type A (RGABAA). Cette inhibition dépend du gradient électro-chimique du chlorure, qui est principalement régulé par les co-transporteurs du chlore KCC2 et NKCC1. KCC2 transporte le chlore hors du neurone, tandis que NKCC1 est responsable de son influx. Peu de données existent sur les fonctions de NKCC1 dans le cerveau adulte, et sur sa régulation. Néanmoins dans des cas pathologiques en lien avec une altération de la transmission GABAergique, NKCC1 revêt une importance fonctionnelle certaine qui tranche avec son rôle apparemment discret dans l’homéostasie du chlore. Ceci est notamment le cas dans le contexte de l’épilepsie, expérimentale ou clinique.J'ai démontré, à l'aide d'approches de suivi de particules uniques, d'immunofluorescence et d'imagerie STORM, que la stabilité membranaire de NKCC1 est rapidement régulée par diffusion latérale d’une manière dépendante de l'activité à la surface des neurones matures de l'hippocampe. L'activité glutamatergique basale régule la dynamique membranaire de NKCC1 dans l'axone mais pas dans le compartiment somato-dendritique. Dans le compartiment somato-dendritique, NKCC1 est réglé par une signalisation médiée par le RGABAA. J'ai également évalué la pertinence de l'inhibition de cette voie dans le contexte de l'ictogenèse. J'ai utilisé un test d’ictogénese induite par PTZ dans des souris SPAK243A/243A, qui expriment une forme non active de SPAK. Les indicateurs comportementaux (échelle de Racine) et les électrocorticogrammes ont montré que les effets de la mutation sur ces variables étaient légers.

In the adult brain, synaptic inhibition is mainly mediated by the activity of GABA type A receptors (RGABAA). This inhibition depends on the electrochemical chloride gradient, which is mainly regulated by the chlorine cotransporters KCC2 and NKCC1. KCC2 transports chlorine out of the neuron, while NKCC1 is responsible for its influx. Little is known about the functions of NKCC1 in the adult brain and its regulation. Nevertheless, in pathological cases related to altered GABAergic transmission, NKCC1 is of definite functional importance, in contrast to its apparently discrete role in chlorine homeostasis. This is particularly the case in the context of epilepsy, both experimental and clinical.I have demonstrated, using single particle tracking, immunofluorescence and STORM imaging approaches, that the membrane stability of NKCC1 is rapidly regulated by lateral diffusion in an activity-dependent manner at the surface of mature hippocampal neurons. Basal glutamatergic activity regulates NKCC1 membrane dynamics in the axon but not in the somato-dendritic compartment. In the somato-dendritic compartment, NKCC1 is regulated by RGABAA-mediated signalling. I also assessed the relevance of inhibition of this pathway in the context of ictogenesis. I used a PTZ-induced ictogenesis test in SPAK243A/243A mice, which express a non-active form of SPAK. Behavioural indicators (Racine scale) and electrocorticograms showed that the effects of the mutation on these variables were mild., Dans le cerveau adulte, l’inhibition synaptique passe majoritairement par l’activité des récepteurs GABA de type A (RGABAA). Cette inhibition dépend du gradient électro-chimique du chlorure, qui est principalement régulé par les co-transporteurs du chlore KCC2 et NKCC1. KCC2 transporte le chlore hors du neurone, tandis que NKCC1 est responsable de son influx. Peu de données existent sur les fonctions de NKCC1 dans le cerveau adulte, et sur sa régulation. Néanmoins dans des cas pathologiques en lien avec une altération de la transmission GABAergique, NKCC1 revêt une importance fonctionnelle certaine qui tranche avec son rôle apparemment discret dans l’homéostasie du chlore. Ceci est notamment le cas dans le contexte de l’épilepsie, expérimentale ou clinique.J'ai démontré, à l'aide d'approches de suivi de particules uniques, d'immunofluorescence et d'imagerie STORM, que la stabilité membranaire de NKCC1 est rapidement régulée par diffusion latérale d’une manière dépendante de l'activité à la surface des neurones matures de l'hippocampe. L'activité glutamatergique basale régule la dynamique membranaire de NKCC1 dans l'axone mais pas dans le compartiment somato-dendritique. Dans le compartiment somato-dendritique, NKCC1 est réglé par une signalisation médiée par le RGABAA. J'ai également évalué la pertinence de l'inhibition de cette voie dans le contexte de l'ictogenèse. J'ai utilisé un test d’ictogénese induite par PTZ dans des souris SPAK243A/243A, qui expriment une forme non active de SPAK. Les indicateurs comportementaux (échelle de Racine) et les électrocorticogrammes ont montré que les effets de la mutation sur ces variables étaient légers.

In the adult brain, adenosine signaling facilitates or inhibits neurotransmitter vesicular release mainly through activation of type 2A or 1 adenosine receptors (A2AR or A1R), respectively. However, its role in development remains to be elucidated. During my PhD, I addressed the role of A2AR-mediated signalling in GABAergic synaptogenesis in the hippocampus. We found (i) a larger activity-dependent release of ATP and adenosine during the period of synaptogenesis in the hippocampus, (ii) a peak of expression of the ecto-5’-nucleotidase, the rate-limiting enzyme for the formation of adenosine from extracellular ATP in synapses during this critical period, and (iii) a peak of peri/post-synaptic expression of A2AR concomitant with the period of synaptogenesis. This developmental expression of the key molecules of the adenosine A2AR signalling pathway correlated with a role of A2AR in the stabilization of nascent GABA synapses, a regulation restricted to the period of synaptogenesis. Furthermore, suppressing A2AR with a shRNA approach in isolated neurons led to a loss of synapses equivalent to that seen upon A2AR activity blockade, reporting that the A2AR-mediated synapse stabilization is a cell autonomous process that requires A2AR activation in the postsynaptic cell. ATP/adenosine can be secreted by both glia and neurons; however, we found that activity-dependent release of neuronal adenosine is sufficient to stabilize newly formed GABA synapses in vitro. Using live cell imaging, we showed adenosine signalling stabilizes active synapses. We then characterized the molecular mechanism downstream postsynaptic A2AR. We report the contribution of adenylyl cyclase/cyclic adenosine monophosphate/protein kinase A signalling cascade and we identified a key target, the postsynaptic scaffolding molecule gephyrin. We further showed the A2AR-mediated stabilization of the presynaptic compartment most probably requires the trans-synaptic Slitrk3-PTPd complex. Since GABA exerts a similar function during development and GABA and adenosine are co-released at some synapses, I further investigated the interplay between these two pathways. My results support the hypothesis that GABA signalling converge onto the adenosine signalling pathway by potentiating calcium-sensitive adenylyl cyclases through the activation of calmodulin.Altogether these results let us propose that, during a key developmental period, postsynaptic A2ARs act as sensors of the activity of GABAergic presynaptic terminals to stabilize active nascent GABAergic synapses. In absence of activity and therefore secretion of adenosine/ATP, synapses will be eliminated., Dans le cerveau adulte, la signalisation liée à l’adénosine facilite ou inhibe la libération vésiculaire de neurotransmetteurs suite à l’activation des récepteurs de l’adénosine de type 2A ou 1 (A2AR ou A1R), respectivement. Cependant, son rôle dans le développement est mal connu. Au cours de ma thèse, j’ai étudié le rôle de la signalisation adénosine dans la synaptogenèse GABAergiques de l’hippocampe. Nous avons mis en évidence (i) une sécrétion activité-dépendante accrue d’adénosine et d’ATP pendant la période de synaptogenèse, (ii) un pic d’expression de l’enzyme limitant la formation de l’adénosine à partir de l’ATP extracellulaire, l’ecto-5’-nucleotidase, aux synapses pendant cette période critique, et (iii) un pic d’expression péri/post- synaptique du A2AR concomitant de la période de synaptogenèse. Cette expression développementale des molécules clés de la signalisation adénosine dépendante du A2AR corrélait avec un rôle de ce récepteur dans la stabilisation des synapses GABAergiques naissantes, une régulation restreinte à la période de synaptogenèse. De plus, la suppression de A2AR par une approche shRNA dans des neurones isolés conduisait à une perte de synapses GABAergiques équivalente à celle observée après un blocage pharmacologique de l’activité du A2AR, signifiant que la stabilisation synaptique médiée par le A2AR est un processus « cellule autonome » indépendant de l’activité du réseau neuronal et qu’elle requiert l’activation du A2AR dans la cellule post-synaptique.L’ATP et l’adénosine sont secrétés par la glie et les neurones ; cependant, nous avons montré in vitro que la libération neuronale activité-dépendante suffit à stabiliser les synapses GABAergiques naissantes. En utilisant la vidéomicroscopie sur cellules vivantes, nous avons montré que la signalisation adénosine stabilise les synapses actives. Puis, nous avons caractérisé le mécanisme moléculaire sous-jacent. Nous rapportons la contribution de la cascade adénylate cyclase/adénosine monophosphate cyclique/protéine kinase A et nous avons identifié une ciblé clé, la géphrine, la molécule d’ancrage postsynaptique des récepteurs GABAA. Enfin, nous avons mis en évidence que la stabilisation de l’élément présynaptique requiert probablement le complexe trans-synaptique Slitrk3-PTPd.Puisque le GABA exerce une fonction similaire au cours du développement et que le GABA et l’adénosine sont co-libérés à certaines synapses, j’ai étudié l’interaction entre ces deux voies de signalisation. Mes résultats favorisent l’hypothèse que la signalisation GABA, en activant la calcium-calmoduline, converge vers la signalisation adénosine en potentiant les adenylates cyclases sensibles au calcium. Mon travail m’a permis de proposer que, au cours d’une période clé du développement, les A2ARs postsynaptiques agissent comme des senseurs de l’activité des terminaisons présynaptiques GABAergiques pour stabiliser les synapses actives. En absence d’activité et donc de libération d’adénosine/ATP, les synapses seraient éliminées.