In cells and organelles transporters are responsible for translocation of amino acids, sugars and nucleotides among others. In the central nervous system (CNS), amino acid transporters can function as neurotransmitter transporters and nutrient sensors. The Solute carrier (SLC) superfamily is the largest family of transporters with 395 members divided in 52 families. The system A and system N amino acid transporter family, SLC38, consists of 11 members, SNAT1-11 (SLC38A1-11). The members are expressed in the brain, exclusively in neurons or astrocytes and some in both. Amino acid signaling is mainly regulated via two pathways, the amino acid responsive (AAR) pathway and the mechanistic/mammalian target of rapamycin complex 1 (mTORC1) pathway. These pathways regulate the protein synthesis in opposite directions depending on the amino acid availability. SLC38 members along with other SLCs have been identified to participate in these pathways. In paper I, the regulation of SLC genes after complete amino acid starvation in mouse hypothalamic cells have been studied with microarray and we found that 47 SLC genes were significantly altered at five hours of starvation. Interestingly, we found that Slc38a1 and Slc38a7 were upregulated along with the known starvation responding gene, Slc38a2. A complementary starvation study for the SLC38 genes was performed using primary mouse embryonic cortex cells. We found that Slc38a1, Slc38a2, Slc38a5, Slc38a6 and Slc38a8 were upregulated while Slc38a3, Slc38a7 and Slc38a11 were downregulated. Three members from the SLC38 family, SNAT8 (paper IV), SNAT9 (paper III) and SNAT10 (paper II) have been histologically characterized in mouse brain and all these transporters are exclusively neuronal. SNAT8 and SNAT10 were also functionally characterized and shown to be transporters for alanine and glutamine among others. SNAT8 was shown to mediate sodium dependent transport and was classified to system A. SNAT10 was shown to be a sodium independent bidirectional transporter and displayed characteristics for system A and N. SNAT9 is a lysosomal component of the Ragulator-Rag complex which senses amino acid availability and activates mTORC1. In paper III we also found that Slc38a9 gene expression was upregulated following starvation and downregulated following high-fat diet in mouse brain.

Lors d’une infection microbienne, la défense de l’hôte comprend deux facettes complémentaires. Premièrement, le système immunitaire cible les pathogènes dans le but de les éliminer, une attaque correspondant à la résistance. Dans un second temps, l’organisme doit réparer les dégâts causés par le pathogène ou par la réponse immunitaire de l’hôte, un mécanisme appelé résilience. J’ai étudié les effets d’une infection intestinale par la bactérie Serratia marcescens chez la drosophile. Nous avons mis en évidence un processus de purge dans l’intestin, lors duquel les enterocytes -les cellules principales de l’intestin- se vident partiellement de leur contenu. L’épithélium intestinal devient alors très fin mais se régénère rapidement, protégeant ainsi la mouche des effets délétères de l’infection. J’ai identifié un transporteur d’acides aminés, CG1139, qui est nécessaire à la régénération de l’intestin. CG1139 est requis pour la mobilisation de certaines réserves métaboliques de la drosophile et pour le transport rétrograde de ces dernières vers l’intestin. / Upon microbial infections, host defenses comprise two complementary facets. First, immune effectors target and kill the invading pathogen, an attack referred to as resistance. Second, the infected host must repair the damages inflicted by microbes or by the immune response itself, a mechanism called resilience. I have studied the effects of an intestinal infection with the bacterium Serratia marcescens in Drosophila. We have discovered a purge mechanism in the intestine, where enterocytes -the main cell type in the gut- extrude some of their internal contents. The intestinal epithelium thus becomes very thin but rapidly recovers its shape, thereby protecting the fly against the deleterious effects of infection. I have identified an amino acid transporter, CG1139, which is required for the intestinal recovery. CG1139 is necessary to mobilize the fly’s internal metabolic reserves and to transport some these metabolic stores back to the gut, in a retrograde manner.

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Autism is a lifelong neurodevelopmental disorder. The etiology of autism still remains unclear due to the heterogeneous and complex nature of the disorder, however synergistic actions between genetic components and environmental factors have been suggested. Acetaminophen (APAP) is one of the most popular over-the-counter drugs that possess antipyretic and analgesic effects. It is considered a relatively safe and effective within therapeutic doses. Recently, early exposure to APAP has been suggested to be one of the underlying cause of autism. Children are often prescribed APAP to lessen fever or irritability after vaccination during the first year, and APAP may adversely affect the normal brain development. In order to better understand the association with APAP and autism, we used an inbred mouse strain BTBR T+tf/J (BTBR). BTBR exhibits behavioral deficits that mimic the core behavioral deficits of human autism. In the study, investigated 1) if BTBR mice showed differences in thiol biochemistry and EAAT3 levels in brain compared with C57BL/6J (C57) mice, 2) if early exposure to APAP induced behavioral changes worsening the autistic phenotypes of BTBR in adolescence, and 3) if APAP exposure in neonatal mice induced possible toxicity at various doses. As a result, we observed that BTBR mice have significantly lower plasma sulfate levels and EAAT expression levels in the frontal cortex compared to C57 mice. Surprisingly, neonatal therapeutic dose of APAP administration did not induce behavioral changes in both C57 and BTBR in adolescence. However, we showed that a supratheraputic dose of APAP significantly elevated levels of oxidative stress marker in the brain. Overall, the results suggested that BTBR mice would be a useful mouse model to investigate effects of various environmental factors that have been associated with autism. In addition, early exposure to APAP at supratherapeutic doses may negatively affect normal brain development.

Les acides aminés ont de nombreuses fonctions dans l’organisme en plus de leur rôle comme constituants élémentaires des protéines. Ils peuvent par exemple servir de neurotransmetteur ou de signal pour l’activation de voies de signalisation intracellulaires. Leur passage à travers la membrane plasmique est facilité par des transporteurs de la famille des protéines SLC. Les transporteurs hétérodimériques d’acides aminés HAT appartiennent aux SLC. Les HAT sont constitués d’une chaîne légère SLC7 assurant la spécificité de transport et d’une chaîne lourde SLC3 impliquée dans l’adressage du complexe protéique à la membrane. Ma thèse a porté sur l’étude du rôle d’un homologue de SLC7 chez la drosophile, Minidiscs (Mnd), dans le fonctionnement du système nerveux. Mnd appartiendrait aux transporteurs du système L, principalement connus pour leur rôle dans la prolifération cellulaire. Mes travaux de thèse ont permis de mettre en évidence la localisation de Mnd dans le cerveau de drosophile dans certains neurones (corps pédonculés, neurones dopaminergiques) et dans certaines cellules gliales (glie corticale). La présence de Mnd dans le cerveau semble intervenir dans la modulation de certains comportements, tels que le réflexe de géotaxie négative. Ces travaux ont aussi montré que, comme les HAT de mammifères, Mnd s’associe de façon covalente à un partenaire protéique. Les expériences de transport semblent par ailleurs confirmer l’appartenance de Mnd au système L.Ces résultats suggèrent que Mnd est probablement impliqué dans la régulation de l’activité neuronale et donc dans le fonctionnement du système nerveux, ce qui n’avait encore jamais été décrit pour un transporteur du système L. / Amino acids have many functions in the body in addition to their role as basic constituents of proteins. They can for example serve as a neurotransmitter or signal for the activation of intracellular pathways. Carriers of the SLC protein family facilitate their path through the plasma membrane. The heterodimeric amino acid transporters HAT belong to SLC proteins. HAT are composed of a light chain SLC7 ensuring the specificity of transport and a heavy chain SLC3 involved in the addressing of the protein complex to the plasma membrane. My thesis focused on studying the role of a SLC7 homologue in drosophila, Minidiscs (Mnd), in the functioning of the nervous system. Mnd might belong to system L carriers, mainly known for their role in cell proliferation. My thesis work led to highlight the location of Mnd in the drosophila brain in some neurons (mushroom bodies, dopaminergic neurons) and some glial cells (cortical glia). The presence of Mnd in the brain seems to be involved in the modulation of some behaviors such as negative geotaxis reflex. This work also showed that, as for mammal HAT, Mnd is associated covalently to a protein partner. Transport experiments seem also to confirm the belonging of Mnd to the system L. These results suggest that Mnd is probably involved in the regulation of neuronal activity and thus in the functioning of the nervous system, which had never been described for a system L carrier.

Summary: Many primary human tumors and tumor cell lines highly express human L-type amino acid transporter 1 (hLAT1); cancerous cells in vivo are strongly linked to LAT1 expression. Synthetic chemistry and in vitro screening efforts have afforded a variety of novel and highly hLAT1 selective compounds, such as JPH203 1. In a recent report, we demonstrated that 1 has potent in vitro and in vivo activity. JPH203 was intravenously administered to produce significant growth inhibition against HT-29 tumors transplanted in nude mice. The current work develops a robust LC/MS-MS method to monitor 1 and its major Phase II metabolite N-acetyl-JPH203 2 from biological samples. We have conducted in vitro and in vivo experiments and the major scientific findings are: i) the major route of biotransformation of 1 is Phase II metabolism to produce 2; ii) metabolite 2 is formed in various organs/tissues (i.e. blood, liver, kidney); and iii) as dogs, which are deficient in NAT genes, do not produce 2, the dog will not be an appropriate toxicological model to evaluate 1.

No / The L-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Forster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques. In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies. A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods. A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed. Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane. Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.