Neto1 and Neto2 are CUB domain-containing transmembrane proteins that are expressed in the mammalian brain. Previous studies showed that Neto1 is a NMDAR-associated protein with important roles in synaptic plasticity and learning/memory (Ng et al., 2009). To establish the functions of Neto2, I first searched for its binding partners. Using yeast two-hybrid analysis, GST pull-down and co-immunoprecipitation studies, I found that Neto2 can bind to the PDZ domain-containing protein GRIP. In the brain, GRIP regulates the synaptic trafficking and stability of AMPA and kainate receptors (KARs) (Hirbec et al., 2003). To determine whether Neto2 is required for the synaptic expression of KARs and/or AMPARs, I examined whether Neto2 was associated with these receptors at the postsynaptic membrane. Coimmunoprecipitation studies showed that while Neto2 is a component of postsynaptic KAR protein complexes, it is not associated with AMPARs. In the cerebellum, Neto2-null mice showed a 44% (n=3;p<0.01) decrease in the abundance of postsynaptic KARs with no change in the level of total KARs, thus suggesting a specific deficit in KAR synaptic localization. Unexpectedly, loss of Neto2 had no effect on the abundance of hippocampal KARs (n=3; p>0.05), or on neurotransmission by them (n=12; p>0.05). To determine whether this normal KAR function might be due to compensation by Neto1, which also interacts with KARs, I examined KAR abundance in Neto1-null, and Neto1/2-double null hippocampus. Loss of Neto1 resulted in a 53% decrease in postsynaptic levels of GluK2-KARs (n=3;p<0.01). However, in double null animals, the reduction was indistinguishable from Neto1 single null mice, suggesting that Neto2 is not involved in the postsynaptic localization of hippocampal KARs. In Neto1-null mice, KAR-mediated currents showed smaller amplitude (61% of wild-type;n=14;p<0.01), and faster decay kinetics (40% of wild-type;n=14;p<0.001). Together, these findings establish both Neto1 and Neto2 as auxiliary proteins of native KARs: Neto1 regulates the synaptic abundance and kinetics of KARs in the hippocampus, while Neto2 mediates the synaptic localization of cerebellar KARs. Additionally, the results presented here, in conjunction with previous findings, reveal a unique ability of Neto1 in controlling synaptic transmission by serving as an auxiliary protein for two different classes of ionotropic glutamate receptors.

Neto1 and Neto2 are CUB domain-containing transmembrane proteins that are expressed in the mammalian brain. Previous studies showed that Neto1 is a NMDAR-associated protein with important roles in synaptic plasticity and learning/memory (Ng et al., 2009). To establish the functions of Neto2, I first searched for its binding partners. Using yeast two-hybrid analysis, GST pull-down and co-immunoprecipitation studies, I found that Neto2 can bind to the PDZ domain-containing protein GRIP. In the brain, GRIP regulates the synaptic trafficking and stability of AMPA and kainate receptors (KARs) (Hirbec et al., 2003). To determine whether Neto2 is required for the synaptic expression of KARs and/or AMPARs, I examined whether Neto2 was associated with these receptors at the postsynaptic membrane. Coimmunoprecipitation studies showed that while Neto2 is a component of postsynaptic KAR protein complexes, it is not associated with AMPARs. In the cerebellum, Neto2-null mice showed a 44% (n=3;p<0.01) decrease in the abundance of postsynaptic KARs with no change in the level of total KARs, thus suggesting a specific deficit in KAR synaptic localization. Unexpectedly, loss of Neto2 had no effect on the abundance of hippocampal KARs (n=3; p>0.05), or on neurotransmission by them (n=12; p>0.05). To determine whether this normal KAR function might be due to compensation by Neto1, which also interacts with KARs, I examined KAR abundance in Neto1-null, and Neto1/2-double null hippocampus. Loss of Neto1 resulted in a 53% decrease in postsynaptic levels of GluK2-KARs (n=3;p<0.01). However, in double null animals, the reduction was indistinguishable from Neto1 single null mice, suggesting that Neto2 is not involved in the postsynaptic localization of hippocampal KARs. In Neto1-null mice, KAR-mediated currents showed smaller amplitude (61% of wild-type;n=14;p<0.01), and faster decay kinetics (40% of wild-type;n=14;p<0.001). Together, these findings establish both Neto1 and Neto2 as auxiliary proteins of native KARs: Neto1 regulates the synaptic abundance and kinetics of KARs in the hippocampus, while Neto2 mediates the synaptic localization of cerebellar KARs. Additionally, the results presented here, in conjunction with previous findings, reveal a unique ability of Neto1 in controlling synaptic transmission by serving as an auxiliary protein for two different classes of ionotropic glutamate receptors.

Le genre des Deltaretrovirus est composé des virus de la leucose bovine (BLV) et des virus T-lymphotropes de primates (PTLV-1, -2, -3 et -4) qui regroupent les virus humains T-lymphotropes (HTLV-1, -2, -3 et -4) et les virus simiens apparentés (STLV-1, -2, -3 et -4). Seules les infections par les PTLV-1 et BLV sont associées à des pathologies : une lymphoprolifération maligne appelée ATLL chez l'homme et le singe ou une maladie neurologique appelée HAM/TSP chez l'homme infectés par les PTLV-1 et une lymphoproliferation maligne chez les bovins infectés par BLV. L'infection par HTLV-2 n'est associée qu'à une lymphocytose et le pouvoir pathogène d'HTLV-3 n'est pas caractérisé à ce jour. Les lentivirus, parmi lesquels on trouve les agents étiologiques du SIDA VIH-1 et VIH-2, et les Deltaretrovirus, sont des rétrovirus complexes. Ils codent donc, en plus des protéines structurales et enzymatiques, pour des protéines régulatrices ainsi que pour des protéines auxiliaires qui seront au centre des travaux présentés. Chez HTLV-1 et BLV, les protéines auxiliaires jouent un rôle primordial dans l'infectiosité in vivo. Or ces protéines n'avaient pas encore été décrites chez les PTLV-3. Leur caractérisation composait donc le premier objectif de ces travaux de thèse. Nous avons ainsi identifié les ARN messagers codant 3 nouvelles protéines putatives in vitro. Nous avons étudié les caractéristiques de ces protéines, notamment leur rôle dans le cycle des PTLV-3 in vitro et leur expression in vivo. Dans un second travail, nous avons essayé de comprendre si les différents domaines fonctionnels déjà identifiés dans la protéine Vpx, une protéine auxiliaire de VIH-2 influençaient sa capacité à interagir avec le facteur de restriction cellulaire SAMHD-1. Nous avons voulu déterminer le compartiment cellulaire dans lequel Vpx induisait la dégradation de SAMHD-1 et la cinétique de ce phénomène, qui permet à ce virus de se répliquer dans les lignées myéloïdes. Ces travaux apportent des éléments nouveaux dans la compréhension du rôle des protéines auxiliaires sur la régulation fine du cycle rétroviral et l'échappement au système immunitaire inné / Deltaretroviruses include bovine leukemia viruses (BLV) and primate Tlymphotropic viruses (PTLV-1, -2, -3 and -4) which are composed of human Tlymphotropic (HTLV-1, -2, -3 and -4) and of their simian counterparts (STLV-1, -2, -3 and -4). PTLV-1 and BLV are the only ones associated to pathologies: a lymphoproliferative disorder named ATLL in humans and non human primates and a neurological disorder named HAM/TSP in humans in the case of PTLV-1 and a Bmalignant lymphoproliferation in BLV infected cattle. HTLV-2 has not been associated with any disease so far and the pathogenic potential of HTLV-3 remains unknown. Lentiviruses, including HIV-1 and -2 the AIDS etiological agents, and Deltaretroviruses, are complex retroviruses. Therefore, in addition to structural and enzymatic proteins they encode regulatory proteins and also auxiliary proteins, the main subject of this work. HTLV-1 and BLV auxiliary proteins play key roles in viral infection in vivo. Whether the genome of PTLV-3 encodes such proteins was not determined yet. Therefore their characterization was the first goal of my PhD work. We identified in vitro messenger RNAs encoding 3 new putative proteins. Their impact on the PTLV-3 viral life cycle in vitro and their expression in vivo were then investigated. As a second part of this work, we examined the relationship existing between the Vpx HIV-2 auxiliary protein and its ability to interact with a restriction factor named SAMHD-1. Vpx induces SAMHD-1 degradation and the kinetic of such degradation allows the virus to replicate in myeloid cells. Altogether, these projects provide new insights into the understanding of the roles played by retroviral auxiliary proteins in connection with a tight regulation of viral life cycle and an escape from innate immunity

Les récepteurs du glutamate de type AMPA (rAMPA) sont les acteurs principaux de la transmission synaptique excitatrice rapide. Leur abondance au niveau de la densité postsynaptique est essentielle pour l'établissement et le maintien de la fonction synaptique, et est le résultat d'un trafic hautement dynamique. De nombreuses études ont permis de caractériser les mécanismes de diffusion membranaire impliqués dans l’adressage des rAMPA jusqu’à la synapse. Le rôle majeur des protéines auxiliaires des rAMPA dans la modulation de cette étape de trafic a été démontré. Par ailleurs, il est suggéré que la localisation synaptique des rAMPA est aussi régulée lors des phases plus précoces du trafic intracellulaire, c’est-à-dire de l'appareil de Golgi vers la membrane plasmique via les vésicules post-Golgiennes. Cependant le trafic vésiculaire post-Golgien des rAMPA n'a jamais été visualisé et reste donc encore très mal compris. En collaboration avec l'équipe de Guus Smit (Amsterdam), j’ai participé à la caractérisation d’une nouvelle protéine auxiliaire des rAMPA, appelée Shisa6. Dans le cadre de ce projet, j’ai pu étudier le rôle de cette protéine sur la diffusion membranaire des rAMPA en utilisant une technique de suivi de particule unique (Quantum dot) développée au laboratoire. Mon projet de thèse principal a consisté à étudier le trafic vésiculaire post-Golgien des rAMPA par le développement d’une nouvelle méthode d’étude. En effet, l'échec dans la visualisation dynamique du trafic vésiculaire des récepteurs pourrait être expliqué par un faible rapport signal/bruit, conséquence d'une faible concentration vésiculaire en rAMPA combinée à un bruit de fond important dû aux marquages provenant du réticulum endoplasmique (RE) et de la membrane plasmique. Dans le but de surpasser cette difficulté, nous avons mis au point un outil ingénieux (système ARIAD) afin de bloquer les rAMPA dans le RE et contrôler, par l'ajout d'un ligand, leur sécrétion du RE jusqu'à la membrane plasmique. Grâce à cet outil, nous avons non seulement augmenté considérablement la concentration des rAMPA dans les vésicules post-Golgiennes, mais aussi éliminé le bruit de fond membranaire. Par la technique de FRAP nous avons pu éliminer le bruit de fond provenant du RE. Une telle approche, combinée à des techniques d'imagerie sur neurones vivants, nous a permis de visualiser pour la première fois le trafic vésiculaire post-Golgien des rAMPA et de l’étudier. / AMPA-type glutamate receptors (AMPAR) are the main actors of the fast excitatory synaptic transmission. Their abundance at the postsynaptic density is essential for the establishment and maintenance of synaptic function, and is the result of a highly dynamic trafficking. Many studies have characterized the membrane diffusion mechanisms involved in the AMPAR synaptic localization, and revealed the critical role of the AMPAR auxiliary proteins in the modulation of this trafficking. Furthermore, it is suggested that AMPAR synaptic localization is also regulated during the early steps of the intracellular trafficking, from the Golgi apparatus to the plasma membrane via the post-Golgi vesicles. However, the post-Golgi vesicular trafficking of AMPAR has never been visualized and therefore remains poorly understood. In collaboration with the Guus Smit team (Amsterdam), I participated in the caracterization of a novel AMPAR auxiliary protein called Shisa6. As part of this project, I studied the role of this protein on the AMPAR membrane diffusion, using a method of single particle tracking (Quantum dot) developed in the laboratory. My main thesis project was to study the post-Golgi vesicular trafficking of AMPAR through the development of a new experimental protocol. Indeed, the failure in the dynamic visualization of the receptor vesicular trafficking could be explained by a low signal/noise ratio resulting of a poor AMPAR vesicular concentration, combined with a high background noise due to receptors localized both in the endoplasmic reticulum (ER) and at the plasma membrane. In order to overcome this difficulty, we have used an ingenious tool (ARIAD system) so as to block AMPAR into the ER and, by adding a ligand, control their trafficking from the ER to the plasma membrane. Thanks to this tool we have not only significantly increased the AMPAR concentration in the post-Golgi vesicles, but also eliminated the plasma membrane background noise. The FRAP imaging technique was used in order to remove the ER background noise. Such methodological approach combined with imaging techniques in living neurons, allowed us to clearly visualize for the first time the post-Golgi vesicular trafficking of AMPAR, and to study the mechanisms involved in this trafficking.