Rôle de l’interaction Neurexine-1β/Neuroligine-1 dans l’assemblage des post-synapses glutamatergiques et le recrutement des récepteurs AMPA

Dans le système nerveux central, la synaptogenèse est un processus complexe multi-étapes qui se déroule aux contacts axones/dendrites. Les molécules d’adhérence neurexines/neuroligines jouent un rôle essentiel dans ce processus, en créant un lien physique entre les compartiments pré- et post-synaptiques et en participant au recrutement des complexes macromoléculaires essentiels à la fonction synaptique. Plus spécifiquement, le complexe neurexine-1β/neuroligine-1 induit la formation de post-synapses excitatrices, en recrutant des molécules d’échafaudage telles que PSD-95 et des récepteurs du glutamate.Mon travail de thèse a consisté à étudier les mécanismes moléculaires mis en jeu par les adhésions neurexines/neuroligines lors de la formation des post-synapses glutamatergiques. En utilisant des systèmes biomimétiques (neurexine purifiée fixée sur des billes, ou agrégée par des anticorps réticulés), nous avons induit des adhésions spécifiques neurexine-1β/neuroligine-1 sur des neurones d’hippocampe en culture. Nous avons ainsi étudié la distribution dynamique des composants post-synaptiques (récepteurs AMPA, PSD-95) endogènes ou étiquetés avec des protéines fluorescentes, par vidéo-microscopie. Dans un premier article, nous avons montré que la formation de ces contacts induisait un recrutement rapide de PSD-95 ainsi que des récepteurs NMDA et AMPA fonctionnels. En utilisant des récepteurs AMPA recombinants, j’ai montré que ce recrutement était dicté par la sous-unité GluA2. Dans une deuxième étude, en comparant le recrutement de PSD-95 induit par la neurexine avec des anticorps non–activants, nous avons mis en évidence un mécanisme d’activation spécifique de neuroligine-1 induit par la liaison de neurexine-1β. L’utilisation de mutants ponctuels de neuroligine-1 a permis de montrer que cette activation passe probablement par la déphosphorylation d’une tyrosine unique située dans le domaine C-terminal de la neuroligine-1.Enfin, en étudiant la diffusion latérale des rAMPA de surface par suivi de particules uniques fluorescentes (Quantum dots), ainsi qu’une batterie d’outils moléculaires pour moduler les adhésions neurexine/neuroligine (sur-expression, siRNA, souris KO), nous avons montré que les rAMPA sont recrutés aux adhésions neurexine-1β/neuroligine-1 via l’échafaudage PSD-95 et que ce recrutement nécessite la diffusion des récepteurs dans la membrane plasmique. Nous proposons ainsi que les récepteurs AMPA soient recrutés aux contacts naissants via un mécanisme original de diffusion/piégeage.In the central nervous system, synaptogenesis is a multi step process occuring at axo-dendritic contacts. Neurexins/neuroligins adhesions are particularly involved in this process, making a bridge between the pre- and the post-synapse, and participating to the recruitment of macromolecular complexes essential for synaptic function. More precisely neurexin-1β/neuroligin-1 complex is specifically involved in the formation of excitatory synapses, inducing the recruitment of glutamatergic post-synapses components, such as PSD-95, and glutamate receptors.During my PhD, I focused on the molecular mechanisms involved in glutamatergic post-synapses formation triggered by neurexin-1β/neuroligin-1 adhesions. Using biomimetic models (beads coated with purified neurexin, or purified neurexin cross-linked with aggregated antibodies) we induced specific neurexin-1β/neuroligin-1 adhesions on cultured hippocampal neurons. We then studied the dynamic distribution of either endogenous or recombinant post-synaptic components (PSD-95, AMPARs) with live-imaging techniques. First, we showed that the formation of these contacts induced a rapid recruitment of PSD-95 and functional NMDA and AMPA receptors. Using recombinant AMPA receptors, I showed that this recruitment was mediated by GluA2 subunit.In a second study, using systematic comparison between the recruitment of PSD-95 induced either by neurexin-1β or by “non activating” antibody binding on neuroligin-1, we revealed a specific activation mechanism of neuroligin-1 induced by neurexin-1β binding. Using point mutations on neuroligin-1, we showed that this activation mechanism is mediated by a tyrosine dephosphorylation on neuroligin-1 intracellular tail.Finally, we studied AMPA receptor surface diffusion with single particle tracking experiments, using different molecular tools to perturb neurexin-1β/neuroligin-1 adhesions (overexpression, RNA interference, KO mice). We showed that AMPA receptors recruitment at new-formed neurexin-1β/neuroligin-1 adhesions occurs through PSD-95, and involves surface diffusion of AMPA receptors. We proposed an original diffusion/trap mechanism of AMPA receptors at nascent contacts

Rôle de l’interaction Neurexine-1β/Neuroligine-1 dans l’assemblage des post-synapses glutamatergiques et le recrutement des récepteurs AMPA

Dans le système nerveux central, la synaptogenèse est un processus complexe multi-étapes qui se déroule aux contacts axones/dendrites. Les molécules d’adhérence neurexines/neuroligines jouent un rôle essentiel dans ce processus, en créant un lien physique entre les compartiments pré- et post-synaptiques et en participant au recrutement des complexes macromoléculaires essentiels à la fonction synaptique. Plus spécifiquement, le complexe neurexine-1β/neuroligine-1 induit la formation de post-synapses excitatrices, en recrutant des molécules d’échafaudage telles que PSD-95 et des récepteurs du glutamate.Mon travail de thèse a consisté à étudier les mécanismes moléculaires mis en jeu par les adhésions neurexines/neuroligines lors de la formation des post-synapses glutamatergiques. En utilisant des systèmes biomimétiques (neurexine purifiée fixée sur des billes, ou agrégée par des anticorps réticulés), nous avons induit des adhésions spécifiques neurexine-1β/neuroligine-1 sur des neurones d’hippocampe en culture. Nous avons ainsi étudié la distribution dynamique des composants post-synaptiques (récepteurs AMPA, PSD-95) endogènes ou étiquetés avec des protéines fluorescentes, par vidéo-microscopie. Dans un premier article, nous avons montré que la formation de ces contacts induisait un recrutement rapide de PSD-95 ainsi que des récepteurs NMDA et AMPA fonctionnels. En utilisant des récepteurs AMPA recombinants, j’ai montré que ce recrutement était dicté par la sous-unité GluA2. Dans une deuxième étude, en comparant le recrutement de PSD-95 induit par la neurexine avec des anticorps non–activants, nous avons mis en évidence un mécanisme d’activation spécifique de neuroligine-1 induit par la liaison de neurexine-1β. L’utilisation de mutants ponctuels de neuroligine-1 a permis de montrer que cette activation passe probablement par la déphosphorylation d’une tyrosine unique située dans le domaine C-terminal de la neuroligine-1.Enfin, en étudiant la diffusion latérale des rAMPA de surface par suivi de particules uniques fluorescentes (Quantum dots), ainsi qu’une batterie d’outils moléculaires pour moduler les adhésions neurexine/neuroligine (sur-expression, siRNA, souris KO), nous avons montré que les rAMPA sont recrutés aux adhésions neurexine-1β/neuroligine-1 via l’échafaudage PSD-95 et que ce recrutement nécessite la diffusion des récepteurs dans la membrane plasmique. Nous proposons ainsi que les récepteurs AMPA soient recrutés aux contacts naissants via un mécanisme original de diffusion/piégeage.In the central nervous system, synaptogenesis is a multi step process occuring at axo-dendritic contacts. Neurexins/neuroligins adhesions are particularly involved in this process, making a bridge between the pre- and the post-synapse, and participating to the recruitment of macromolecular complexes essential for synaptic function. More precisely neurexin-1β/neuroligin-1 complex is specifically involved in the formation of excitatory synapses, inducing the recruitment of glutamatergic post-synapses components, such as PSD-95, and glutamate receptors.During my PhD, I focused on the molecular mechanisms involved in glutamatergic post-synapses formation triggered by neurexin-1β/neuroligin-1 adhesions. Using biomimetic models (beads coated with purified neurexin, or purified neurexin cross-linked with aggregated antibodies) we induced specific neurexin-1β/neuroligin-1 adhesions on cultured hippocampal neurons. We then studied the dynamic distribution of either endogenous or recombinant post-synaptic components (PSD-95, AMPARs) with live-imaging techniques. First, we showed that the formation of these contacts induced a rapid recruitment of PSD-95 and functional NMDA and AMPA receptors. Using recombinant AMPA receptors, I showed that this recruitment was mediated by GluA2 subunit.In a second study, using systematic comparison between the recruitment of PSD-95 induced either by neurexin-1β or by “non activating” antibody binding on neuroligin-1, we revealed a specific activation mechanism of neuroligin-1 induced by neurexin-1β binding. Using point mutations on neuroligin-1, we showed that this activation mechanism is mediated by a tyrosine dephosphorylation on neuroligin-1 intracellular tail.Finally, we studied AMPA receptor surface diffusion with single particle tracking experiments, using different molecular tools to perturb neurexin-1β/neuroligin-1 adhesions (overexpression, RNA interference, KO mice). We showed that AMPA receptors recruitment at new-formed neurexin-1β/neuroligin-1 adhesions occurs through PSD-95, and involves surface diffusion of AMPA receptors. We proposed an original diffusion/trap mechanism of AMPA receptors at nascent contacts

Control of basal autophagy rate by vacuolar peduncle

International audienceBasal autophagy is as a compressive catabolic mechanism engaged in the breakdown of damaged macromolecules and organelles leading to the recycling of elementary nutrients. Thought essential to cellular refreshing, little is known about the origin of a constitutional rate of basal autophagy. Here, we found that loss of Drosophila vacuolar peduncle (vap), a presumed GAP enzyme, is associated with enhanced basal autophagy rate and physiological alterations resulting in a wasteful cell energy balance, a hallmark of overactive autophagy. By contrast, starvation-induced autophagy was disrupted in vap mutant conditions, leading to a block of maturation into autolysosomes. This phenotype stem for exacerbated biogenesis of PI(3)P-dependent endomembranes, including autophagosome membranes and ectopic fusions of vesicles. These findings shed new light on the neurodegenerative phenotype found associated to mutant vap adult brains in a former study. A partner of Vap, Sprint (Spri), acting as an endocytic GEF for Rab5, had the converse effect of leading to a reduction in PI(3)P-dependent endomembrane formation in mutants. Spri was conditional to normal basal autophagy and instrumental to the starvation-sensitivity phenotype specific of vap. Rab5 activity itself was essential for PI(3)P and for pre-autophagosome structures formation. We propose that Vap/Spri complexes promote a cell surface-derived flow of endocytic Rab5-containing vesicles, the traffic of which is crucial for the implementation of a basal autophagy rate

Starvation-sensitivity assays define the range of autophagy defects in <i>vap</i> flies.

<p>The survival rate of 3 day-old adult males of indicated genotypes was recorded at 25°C in condition of complete food deprivation (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.s004" target="_blank">S4A–S4C Fig</a></b> for initial characterization). (A) The <i>vap</i>-dependent starvation sensitivity (white arrow) was compared to weak (<i>Atg8a</i>^<i>1</i>) and strong (<i>Agt8a</i>^<i>2</i>) alleles of <i>Atg8</i>a. <i>Atg8a</i>² flies showed slightly altered development that might contribute to its greater sensitivity to starvation. (B-B’) Starvation sensitivity effect, as assayed at 25°C, is partially recapitulated by flies that were ectopically expressing an <i>UAS-myc</i>:<i>Atg1</i> transgene (Materials and Methods) along fat cell development performed at 25°C (white arrow in B) when driven by <i>cg-Gal4</i>. As a control, there is no detectable starvation sensitivity (as assayed at 25°C) using identical flies (<i>UAS-myc</i>:<i>Atg1</i> /<i>cg-Gal4</i>) that developed at 18°C to minimized transgene expression (white arrow in B’). Ectopic expression of <i>Atg1</i> during development is therefore responsible for the sensitivity effect found in B. (C) The <i>vap</i>-dependent starvation sensitivity is suppressed (white arrow) by co-expressed <i>Atg5(RI)</i> using the broadly expressed <i>arm-Gal4</i> driver. Genotypes. (A) Control: <i>w</i>^<i>1118</i><i>/Y</i>. Assay <i>vap</i>^<i>1</i><i>/Y</i>. <i>Atg8a</i>^<i>1</i><i>/Y</i>. <i>Atg8a</i>^<i>2</i><i>/Y</i>. (B, B’) Control: <i>UAS-myc</i>:<i>Atg1/+</i> and <i>vap</i>^<i>1</i><i>/Y</i> and <i>vap</i>^<i>1</i><i>/Y; cg-GAL4/+</i>. Assay: <i>vap</i>^<i>1</i><i>/Y</i>. <i>cg-GAL4/ UAS-myc</i>:<i>Atg1(RI)/+</i>. (C) Control: <i>arm-GAL4/+</i> and <i>vap</i>^<i>1</i><i>/Y; arm-GAL4/+</i> and <i>arm-GAL4/ UAS-Atg5(RI)/+</i>. Assay: <i>vap</i>^<i>1</i><i>/Y; arm-GAL4/ UAS-Atg5(RI)/+</i>.</p

Neurexin-1β Binding to Neuroligin-1 Triggers the Preferential Recruitment of PSD-95 versus Gephyrin through Tyrosine Phosphorylation of Neuroligin-1

Adhesion between neurexin-1β (Nrx1β) and neuroligin-1 (Nlg1) induces early recruitment of the postsynaptic density protein 95 (PSD-95) scaffold; however, the associated signaling mechanisms are unknown. To dissociate the effects of ligand binding and receptor multimerization, we compared conditions in which Nlg1 in neurons was bound to Nrx1β or nonactivating HA antibodies. Time-lapse imaging, fluorescence recovery after photobleaching, and single-particle tracking demonstrated that in addition to aggregating Nlg1, Nrx1β binding stimulates the interaction between Nlg1 and PSD-95. Phosphotyrosine immunoblots and pull-down of gephyrin by Nlg1 peptides in vitro showed that Nlg1 can be phosphorylated at a unique tyrosine (Y782), preventing gephyrin binding. Expression of Nlg1 point mutants in neurons indicated that Y782 phosphorylation controls the preferential binding of Nlg1 to PSD-95 versus gephyrin, and accordingly the formation of inhibitory and excitatory synapses. We propose that ligand-induced changes in the Nlg1 phosphotyrosine level control the balance between excitatory and inhibitory scaffold assembly during synapse formation and stabilization

NRG1 signalling regulates the establishment of Sertoli cell stock in the mouse testis

Testis differentiation requires high levels of proliferation of progenitor cells that give rise to two cell lineages forming the testis, the Sertoli and the Leydig cells. Hence defective cell cycling leads to testicular dysgenesis that has profound effects on androgen production and fertility. The growth factor NRG1 has been implicated in adult Leydig cell proliferation, but a potential function in the fetal testis has not been analysed to date. Here we show that Nrg1 and its receptors ErbB2/3 are already expressed in early gonadal development. Using tissue-specific deletion, we further demonstrate that Nrg1 is required in a dose-dependent manner to induce proliferation of Sertoli progenitor cells and then differentiated Sertoli cells. As a result of reduced numbers of Sertoli cells, Nrg1 knockout mice display a delay in testis differentiation and defects in sex cord partitioning. Taken together Nrg1 signalling is essential for the establishment of the stock of Sertoli cells and thus required to prevent testicular hypoplasia

R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth

International audienceAbstract R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/β-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility

Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/gamma-secretase at the cell surface

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.status: publishe

miR-92a regulates expression of synaptic GluA1-containing AMPA receptors during homeostatic scaling

We investigated whether microRNAs could regulate AMPA receptor expression during activity blockade. miR-92a strongly repressed the translation of GluA1 receptors by binding the 3' untranslated region of rat GluA1 (also known as Gria1) mRNA and was downregulated in rat hippocampal neurons after treatment with tetrodotoxin and AP5. Deleting the seed region in GluA1 or overexpressing miR-92a blocked homeostatic scaling, indicating that miR-92a regulates the translation and synaptic incorporation of new GluA1-containing AMPA receptors