> Stages de M2 > Liste des stages proposés pour l année 2018-2019 > Dissect the role of ADCY6 in Schwann cell (SC) myelination.

Dissect the role of ADCY6 in Schwann cell (SC) myelination.

proposé par Marcel TAWK , U1195, Inserm, Université Paris Saclay, Hôpital Kremlin Bicêtre, Bâtiment Pincus, 94276 le Kremlin Bicêtre

Projet de stage :

Aim. What are the molecular bases of ADCY6 function ? Is ADCY6 essential for GPR126 function in peripheral myelination ? It has been shown that the G protein-coupled receptor 126 is essential for PNS myelination in zebrafish, mice and humans (Monk K et al., 2009 ; Monk K et al., 2011 ; Ravenscroft G et al., 2015). This is highlighted by the complete lack of myelin specifically in the PNS. Moreover, several results point to a role of GPR126 protein in activating G proteins and elevating cAMP within SCs in order to drive SC myelination. What is intriguing is the fact that the adcy6 mutation in humans leads to the exact same phenotype observed in gpr126 patients. Furthermore, given the fact that adcy6 is an adenylate cyclase required for cAMP synthesis, and that GPR126 function is dependent on cAMP elevation, we hypothesise that these two proteins are part of the signalling pathway required for peripheral myelination. Our aim is to understand i) how does ADCY6 impact SC myelination ? ii) how these proteins (GPR126 and ADCY6), that are both essential for PNS myelination, are related ? Is ADCY6 the only protein that is able to relay GPR126 function in SCs ?

Techniques mises en œuvre par le stagiaire : In order to answer these questions, we will use two different models : zebrafish and mice. The zebrafish is a unique vertebrate model in which one can watch cells behave in real time in an intact organism and is easily amenable to drug treatment. We will use zebrafish to study genetic interactions between GPR126 and ADCY6 proteins. We will manipulate adcy6 expression, by using antisens morpholinos, in order to reduce adcy6 expression in controls and gpr126 mutants. By doing so, we will be able to tell whether these two proteins work together to achieve myelination. We will also test the downstream signalling of ADCY6 using several drugs. If ADCY6 function is to drive cAMP synthesis in SCs, then activating the downstream effector of cAMP, in this case the Protein Kinase A (PKA), by incubating embryos at 2dpf with 8-CPT-cAMP that activates PKA should rescue the phenotype. Moreover, if the ADCY6 function to drive SC myelination is mediated by cAMP, then treating adcy6 morphant embryos by adding forskolin should not rescue the phenotype since forskolin elevates cAMP levels by binding to ADCY proteins. Finally, if GPR126 function is dependent on ADCY6, then treating gpr126 mutants with forskolin, that normally rescues the GPR126 myelin mutant phenotype, will fail to do so if adcy6 is knocked-down in gpr126 mutants. We will proceed by embryo injections, in situ hybridisation, whole mount immunostaining, Transmitted Electron Microscopy (TEM) and time-lapse imaging in order to answer these questions. We will also use a mouse model of floxed adcy6 mutant that specifically knockout adcy6 function in SCs when crossed with the Mpz-Cre mouse, in order to test whether adcy6 function is autonomous to SCs ; this will be achieved by using TEM and measuring cAMP levels in mutant nerves. We will also take advantage of this mouse model to co-culture control Dorsal Root Ganglia (DRG) neurons with adcy6 deficient SCs in order to study the signalling pathway downstream of adcy6 in mice too, by using the same strategy and set of drugs described for zebrafish.

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