Final Report Summary - KARTRAF (Synaptic trafficking of Kainate receptors in Hippocampal CA3 pyramidal cells in vivo)
During my fellowship, I have directed my attention on different aspects of trafficking and plasticity of KARs at MF to CA3 synapses. During my study, I have used a variety of different experimental approaches such as slice electrophysiology, molecular biology and imaging. In the summary below, I am presenting the results and progress for each one of the aims of the first year of my project.
Molecular interactions that govern targeting of GluR6 to MF synapses through its CTD in vivo
I have characterised the properties of the rescued KAR mediated synaptic current at MF to CA3 synapses by re-expression of wt GluR6a in the GluR6-/- mice background. To quantify the level of synaptic KARs, I have compared the KAR mediated synaptic current with respect the total MF response. In wild type animal, the KAR mediated response resulted to be about 10 % of the total MF response. I have found that after 3-7 days of transfection of GluR6a in slice from GluR6-/- mice, the KAR mediated current was restored to a level comparable to the one observed in slice of wild type animal, about 10 % of the total MF response.
After the characterisation of the properties of the KAR mediated current rescued with GluR6a, I have started to study the possible molecular interactions that govern targeting of GluR6a to MF synapses through its CTD. For this purpose, I have used GluR6a mutants, already present in the host lab, that have sequential deletion of the CTD (54 amino acids). I have used mutants missing the last 39, 29, 14 and 4 amino acids. As expected, in cell transfected with the ?39 mutant (a mutant that does not go to the membrane when expressed in non neuronal cells) no synaptic KAR mediated currents was detected. Transfection with ?29 mutant (that can go to the membrane in non neuronal cells) also failed to rescue the synaptic KAR mediated current, suggesting that the last 29 amino acids are necessary for proper synaptic localization of GluR6a subunit at synaptic location. Interestingly, also transfection with the ?14 mutant (that does not interact with the N-cadherin / ß-catenin complex) fail to completely rescue the synaptic current, suggesting a role of this adhesion complex in stabilising KAR at this synapse. Finally, ?4 mutant (that does not have the PDZ binding domain) fully rescue the synaptic response mediated by KAR, suggesting that PSD95 or other proteins that can interact with PDZ domain are not playing a role major in targeting KAR at MF synapse.
In order to understand the mechanism by which GluR6a is stabilised at MF by N-cadherin / ß-catenin complex, I have transfected slice prepared from wilt type mice with a mutated form N-cadherin (Ncad-?390) that works as a dominant negative. Interestingly, I have found, after this genetic manipulation, reduced KAR mediated responses when compared to not transfected slice. These data confirm our original hypotesis that N-cadherin / ß-catenin plays a fundamental role in the regulation of propper targeting of KAR at Mf-CA3 synapse.
Molecular interactions that govern targeting of GluR6 to MF synapses through its CTD in vivo
I have characterised the properties of the rescued KAR mediated synaptic current at MF to CA3 synapses by re-expression of wt GluR6a in the GluR6-/- mice background. To quantify the level of synaptic KARs, I have compared the KAR mediated synaptic current with respect the total MF response. In wild type animal, the KAR mediated response resulted to be about 10 % of the total MF response. I have found that after 3-7 days of transfection of GluR6a in slice from GluR6-/- mice, the KAR mediated current was restored to a level comparable to the one observed in slice of wild type animal, about 10 % of the total MF response.
After the characterisation of the properties of the KAR mediated current rescued with GluR6a, I have started to study the possible molecular interactions that govern targeting of GluR6a to MF synapses through its CTD. For this purpose, I have used GluR6a mutants, already present in the host lab, that have sequential deletion of the CTD (54 amino acids). I have used mutants missing the last 39, 29, 14 and 4 amino acids. As expected, in cell transfected with the ?39 mutant (a mutant that does not go to the membrane when expressed in non neuronal cells) no synaptic KAR mediated currents was detected. Transfection with ?29 mutant (that can go to the membrane in non neuronal cells) also failed to rescue the synaptic KAR mediated current, suggesting that the last 29 amino acids are necessary for proper synaptic localization of GluR6a subunit at synaptic location. Interestingly, also transfection with the ?14 mutant (that does not interact with the N-cadherin / ß-catenin complex) fail to completely rescue the synaptic current, suggesting a role of this adhesion complex in stabilising KAR at this synapse. Finally, ?4 mutant (that does not have the PDZ binding domain) fully rescue the synaptic response mediated by KAR, suggesting that PSD95 or other proteins that can interact with PDZ domain are not playing a role major in targeting KAR at MF synapse.
In order to understand the mechanism by which GluR6a is stabilised at MF by N-cadherin / ß-catenin complex, I have transfected slice prepared from wilt type mice with a mutated form N-cadherin (Ncad-?390) that works as a dominant negative. Interestingly, I have found, after this genetic manipulation, reduced KAR mediated responses when compared to not transfected slice. These data confirm our original hypotesis that N-cadherin / ß-catenin plays a fundamental role in the regulation of propper targeting of KAR at Mf-CA3 synapse.