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Thèse Steve Dos Santos Carvalho

Morpho-functional impact of Vangl2 on hippocampus development

Le 30 novembre 2016

En thèse avec Mireille Montcouquiol, dans l'equipe "Polarité Planaire et Plasticité", Neurocentre Magendie


 Planar Cell Polarity (PCP) is a signaling pathway originally known for its role in the establishment of cellular asymmetry perpendicular to the apico-basal axis, in the plane of an epithelium. PCP signaling has been shown to be crucial for many tissue patterning, including epithelial and mesenchymal tissue, but also cardiac, lung, bone, or kidney tissues, to cite a few. PCP signaling controls the regulation of cellular movement via the control of adhesion turnover and cytoskeleton reorganization.

Vangl2 is one of the most upstream core PCP proteins that has been implicated in the recent years in various neuronal mechanisms, such as axonal guidance, dendrite morphogenesis or synaptogenesis. However, most of these studies rely on acute downregulation of the gene in vitro or in the use of a mouse presenting a spontaneous mutation of this gene, called Loop-tail (Vangl2Lp) which causes the death of the embryo at birth. Moreover, the Vangl2Lp form of this protein has been described has a dominant-negative form, making it difficult to untangle the molecular mechanism leading to the many phenotypes (included neuronal ones) reported in homozygotes Looptail mice.


To bypass this problem we created a conditional knockout (cKO) mouse in which vangl2 is deleted in the telencephalon during early embryogenesis. First, I analyzed the profile of expression of the protein during the first 3 weeks after birth, and I show that Vangl2 is specifically targeted to the arborization of granular cells (GC) of the dentate gyrus (DG) of the hippocampus, and excluded from cell bodies. Also, the protein was highly enriched in immature neurons of the subgranular zone of the DG, and in the stratum lucidum, a region of high-density contacts between the GC and the CA3. In this region, a special type of synapse is formed: the Mossy Fiber Bouton (MfB) / Thorny Excrescence (TE) synapse. These synapses are bigger and more complex than conventional synapses.

I then performed a structural and ultrastructural analysis of the DG/CA3 circuit in the Vangl2 cKO mice in order to understand the role of Vangl2 in the hippocampus maturation. For this, I used stereotaxic mice infection viruses, and Serial block face scanning electron microscopy (SBFsEM) with 3D reconstruction. Results show that in cKO mice, Mfs fasciculation is mildly impacted, and that the enlargement and complexification of the MfB/TE synapse is arrested, with TEs almost absent. I was able to link these morphological abnormalities to deficits in complex hippocampal-dependent learning tasks. This work demonstrates for the first time the importance of PCP signaling for the in vivo maturation of a specific hippocampal circuit and its specific cognitive consequences.

Next, I attempted to identify the functional consequences of vangl2 deletion on young hippocampal neuron maturation. My results confirm that Vangl2 is expressed in young hippocampal neurons and that the deletion of the gene affected neurite outgrowth on N-cadherin substrate. I used spt-PALM-TIRF super-resolution microscopy to show that this increased neurite outgrowth was inversely proportional to a decrease in actin retrograde flow and to a decrease in the number of directed actin trajectories. These results strongly suggest that N-cadherin adhesions are affected by Vangl2 deletion. FRAP experiments demonstrated that in Vangl2 cKO neurons the recovery of N-cadherin molecules engaged in homophilic bindings (adhesion) was decreased, suggesting that the turnover of N-cadherin involved in adhesion is reduced. Altogether, I propose that Vangl2 controls the turnover/stability of N-cadherin proteins at adhesion sites to regulate local actin dynamics and consequently neuronal outgrowth.

Jury

Olivier Thoumine
(CNRS Bordeaux)Président
Rapporteurs:
Dr. Fanny Mann
( Unviersité Aix Marseille),
Dr. Froylan Calderon de Anda
(UKE, Hamburg);
Examinateur
Prof. Esther Stoeckli
(Université de Zurich);
Directrice de thèse
Mireille Montcouquiol
(INSERM Bordeaux).