Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance

Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1–DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1–dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1–dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1–induced axon outgrowth are impaired in Fyn−/− CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1

Tubulin Tyrosination Is Required for the Proper Organization and Pathfinding of the Growth Cone

International audienceBACKGROUND: During development, neuronal growth cones integrate diffusible and contact guidance cues that are conveyed to both actin and microtubule (MT) cytoskeletons and ensure axon outgrowth and pathfinding. Although several post-translational modifications of tubulin have been identified and despite their strong conservation among species, their physiological roles during development, especially in the nervous sytem, are still poorly understood. METHODOLOGY/FINDINGS: Here, we have dissected the role of a post-translational modification of the last amino acid of the alpha-tubulin on axonal growth by analyzing the phenotype of precerebellar neurons in Tubulin tyrosin ligase knock-out mice (TTL(-/-)) through in vivo, ex vivo and in vitro analyses. TTL(-/-) neurons are devoid of tyrosinated tubulin. Their pathway shows defects in vivo, ex vivo, in hindbrains open-book preparations or in vitro, in a collagen matrix. Their axons still orient toward tropic cues, but they emit supernumerary branches and their growth cones are enlarged and exhibit an emission of mis-oriented filopodia. Further analysis of the TTL(-/-) growth cone intracellular organization also reveals that the respective localization of actin and MT filaments is disturbed, with a decrease in the distal accumulation of Myosin IIB, as well as a concomitant Rac1 over-activation in the hindbrain. Pharmacological inhibition of Rac1 over-activation in TTL(-/-) neurons can rescue Myosin IIB localization. CONCLUSIONS/SIGNIFICANCE: In the growth cone, we propose that tubulin tyrosination takes part in the relative arrangement of actin and MT cytoskeletons, in the regulation of small GTPases activity, and consequently, in the proper morphogenesis, organization and pathfinding of the growth cone during development

Mechanisms controlling neuromuscular junction stability.

International audienceThe neuromuscular junction (NMJ) is the synaptic connection between motor neurons and muscle fibers. It is involved in crucial processes such as body movements and breathing. Its proper development requires the guidance of motor axons toward their specific targets, the development of multi-innervated myofibers, and a selective synapse stabilization. It first consists of the removal of excessive motor axons on myofibers, going from multi-innervation to a single innervation of each myofiber. Whereas guidance cues of motor axons toward their specific muscular targets are well characterized, only few molecular and cellular cues have been reported as clues for selecting and stabilizing specific neuromuscular junctions. We will first provide a brief summary on NMJ development. We will then review molecular cues that are involved in NMJ stabilization, in both pre- and post-synaptic compartments, considering motor neurons and Schwann cells on the one hand, and muscle on the other hand. We will provide links with pathologies and highlight advances that can be brought both by basic research on NMJ development and clinical data resulting from the analyses of neurodegeneration of synaptic connections to obtain a better understanding of this process. The goal of this review is to highlight the findings toward understanding the roles of poly- or single-innervations and the underlying mechanisms of NMJ stabilization

Facteurs moléculaires impliqués dans la survie et la migration des neurones précérébelleux

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Formation de la frontière entre cerveaux moyen et postérieur : implication des gènes Otx2 et Gbx2

Nous avons étudié la subdivision neuromérique de la région mésencéphalique-météncéphalique (mes- met) du tube neural, en utilisant des transplantations hétérospécifiques entre les embryons de Poulet et de Caille et en analysant, chez l’embryon de poulet normal et les embryons chimères, l’étendue de l’expression de plusieurs gènes régulateurs qui s’expriment dans cette région. Des greffes homotopiques ont montré la présence d’une limite interneuromérique entre les ébauches du mésencéphale et du cervelet (frontière mes-met ou entre cerveaux moyen et postérieur). Cette frontière est formée par la confrontation des domaines d’expression Otx2-Wnt1 et Gbx2-Fgf8. En revanche, l’expression des gènes En2 et Pax2 s’étend de part et d’autre de cette frontière. Nous avons montré que la localisation de la limite Otx2/Gbx2 évolue par rapport à la vésicule mésencéphalique et la constriction mes-met entre les stades HH 10 et HH20, ce qui nous a permis de proposer une nouvelle nomenclature pour ces deux entités morphologiques au stade HH10. En utilisant des transplantations ectopiques entre prosencéphale et le territoire mes- met, nous avons montré que l’expression de la cascade génique menant à un phénotype mes-met peut être induite dans les deux prosomères caudaux. Cette cascade se déclenche sélectivement à la frontière entre le prosencéphale, Otx2-positif, et le territoire cérébelleux de l’hôte, fortement Gbx2-positif. Cette cascade implique l’induction d’une nouvelle frontière Otx2lGbx2 à l’intérieur du transplant. Aucun changement phénotypique n’est observé quand le prosencéphale contacte des territoires Otx2 positifs de l’hôte. Bien que Fgf8 soit probablement impliqué dans le déclenchement de l’induction, l’ensemble de nos données suggère fortement que la confrontation des domaines Otx2IGbx2 joue un rôle primordial comme organisateur phénotypique du territoire mesmet

Differential roles of Netrin-1 and its receptor DCC in inferior olivary neuron migration.

International audienceNetrin-1 was previously shown to be required for the tangential migration and survival of neurons that will form the inferior olivary nucleus (ION). Surprisingly, the compared analysis of mutant mice lacking either Netrin-1 or its major receptor DCC reveals striking phenotypic differences besides common features. Although ectopic stops of ION cell bodies occur in the same positions along the migratory stream in both mutants, the ION neurons' number is not affected by the lack of DCC whereas it is reduced in Netrin-1 mutant mice. Thus, cell death results from the absence of Netrin-1 and not from neuron mis-routing, arguing for a role of Netrin-1 as a survival factor in vivo. The secretion of Netrin-1 by the floor plate (FP) is strictly required - whereas DCC is not - to avoid ION axons' repulsion by the FP and allows them to cross it. Leading processes of neurons of other caudal precerebellar nuclei (PCN) cannot cross the FP in either mutant mouse, suggesting differential sensitivity or mechanism of action of Netrin-1 for leading processes of ION and other PCN neurons

Trio GEF mediates RhoA activation downstream of Slit2 and coordinates telencephalic wiring

International audienceTrio, a member of the Dbl family of guanine nucleotide exchange factors, activates Rac1 downstream of netrin 1/DCC signalling in axon outgrowth and guidance. Although it has been proposed that Trio also activates RhoA, the putative upstream factors remain unknown. Here, we show that Slit2 induces Trio-dependent RhoA activation, revealing a crosstalk between Slit and Trio/RhoA signalling. Consistently, we found that RhoA activity is hindered in vivo in T rio mutant mouse embryos. We next studied the development of the ventral telencephalon and thalamocortical axons, which have been previously shown to be controlled by Slit2. Remarkably, this analysis revealed that Trio knockout (KO) mice show phenotypes that bear strong similarities to the ones that have been reported in Slit2 KO mice in both guidepost corridor cells and thalamocortical axon pathfinding in the ventral telencephalon. Taken together, our results show that Trio induces RhoA activation downstream of Slit2, and support a functional role in ensuring the proper positioning of both guidepost cells and a major axonal tract. Our study indicates a novel role for Trio in Slit2 signalling and forebrain wiring, highlighting its role in multiple guidance pathways as well as in biological functions of importance for a factor involved in human brain disorders

Distinct roles of Rac1/Cdc42 and Rho/Rock for axon outgrowth and nucleokinesis of precerebellar neurons toward netrin 1

International audienc