Dysregulation of dopaminergic homeostasis has been established as the primary source of numerous neurological disorders including Parkinson’s and drug addiction. A tonic increase of dopamine (DA) in the nucleus accumbens is required for associating everyday events and behaviors with rewards. Yet many addictive exogenous compounds such as amphetamine (AMPH) and cocaine (COC) produce a much greater augmentation of synaptic DA levels that are linked to euphoria and a shift in behavior towards drug seeking. The protein responsible for maintaining extracellular levels of DA is the dopamine transporter (DAT). It is primarily located in the perisynaptic area at terminals of pre-synaptic neurons where its main function is to sequester DA from the extracellular space and to transport it back into the cell, a process that is electrogenic. AMPH and COC directly interact with DAT and alter its ionic currents. Not much is known about the effect of psychostimulant-induced DAT currents on neuronal excitability and neurotransmitter release. We use synthetic chemistry, molecular biology, and biophysics in heterologous expression systems to decipher the actions of drugs of abuse on DAT. Furthermore we demonstrate drug-induced DAT currents can activate Ca2+ channels associated with dopaminergic excitability. Lastly, we focused on investigating drug effects on excitability in a human midbrain dopaminergic cell line. Understanding how psychostimulants interact with DAT to produce the dysfunctional states of the transporter may facilitate the development of unique therapeutic strategies to treat psychostimulant dependence.

LAT1, also named SLC7A5, is a human plasma membrane transporter covalently associated with the glycoprotein CD98 (SLC3A2). It catalyses the uptake of several essential amino acids, including leucine, thereby contributing to the activation of the mTORC1 (mechanistic Target of Rapamycin Complex 1) kinase complex. This control of mTORC1 by LAT1 is notably important for the rapid proliferation of tumour cells. Moreover, LAT1 overexpression has been observed in numerous cancers, and is often associated with a poor prognosis. Despite the importance of LAT1 in normal and tumour cells, the mechanisms controlling the intracellular traffic of this transporter, and particularly its endocytosis, are still poorly known.Many plasma membrane transporters are downregulated by endocytosis, in response to various signals. In yeast, endocytosis of plasma membrane permeases is triggered by their ubiquitylation mediated by the ubiquitin ligase Rsp5, acting in association with adaptors of the α-arrestin family. Ubiquitin also plays an important role in endocytosis of many human transporters. The main objective of this thesis work was to characterize the mechanisms mediating endocytosis of LAT1 in HeLa tumour cells.In the first part of this work, we studied the effect of FTY720, an analog of sphingoid bases with antitumor properties, on a HeLa T-Rex stable cell line expressing a LAT1-GFP construct. We also analysed the effect of FTY720 on several yeast plasma membrane permeases. Our results show that FTY720 induces a loss of activity and the endocytosis of permeases in yeast and of LAT1 in HeLa cells. Yeast permease internalization is associated with their ubiquitylation by the Rsp5 ubiquitin ligase. Moreover, FTY720 decreases TORC1/mTORC1 activity. Based on these results, we propose a model for the effect of FTY720 in yeast: this drug, by decreasing the activity of numerous nutrient transporters, leads to TORC1 inhibition. This loss of TORC1 activity promotes, via a still unknown mechanism, the Rsp5-dependent ubiquitylation and endocytosis of permeases. A similar mechanism could be responsible for FTY720-induced endocytosis of LAT1 in HeLa cells.In the second part of this work, we further investigated the mechanisms involved in LAT1 endocytosis. We found that LAT1 is internalized and degraded upon activation of protein kinase C (PKC) by the phorbol ester PMA. This treatment also elicited the ubiquitylation of LAT1 via the Nedd4-2 ubiquitin ligase. Finally, we identified the lysines 19, 25 and 30 in the N-terminal tail of LAT1 as the major sites of its ubiquitylation in response to PMA, and we showed that these lysines are important for PMA-induced downregulation. Future work will determine the importance of this control mechanism of LAT1 in the context of normal and tumour cells. / Ce travail porte sur l’étude du transporteur d’acides aminés humain LAT1, également appelé SLC7A5. LAT1, situé à la membrane plasmique où il est associé à la protéine CD98 (SLC3A2), est le transporteur principal de leucine dans les cellules et joue un rôle central dans l’activation de mTORC1 par cet acide aminé. Ce transporteur est surexprimé dans les cellules de nombreuses tumeurs, dans lesquelles il est souvent associé à un mauvais pronostic. Le trafic intracellulaire de LAT1 et, en particulier, son endocytose sont très peu connus. Mon travail de thèse a eu pour but d’étudier l’endocytose de LAT1 et les facteurs impliqués dans ce mécanisme. Dans la première partie de cette recherche, nous avons étudié l’effet du FTY720, un analogue de bases sphingoïdes, sur une lignée stable de cellules HeLa TRex exprimant LAT1-GFP, et nous l’avons comparé à son effet dans la levure. Nous avons observé que ce composé induisait une diminution de l’activité et l’endocytose de nombreux transporteurs de nutriments chez la levure ainsi que de LAT1 dans les cellules HeLa. De plus, l’internalisation des perméases de levure en réponse au FTY720 est associée à leur ubiquitylation, via l’ubiquitine ligase Rsp5. Nous avons également montré que ce composé diminue l’activité du complexe kinase TOR1/mTOR1. Ces différents résultats nous ont amené à l’hypothèse suivante :le FTY720, en induisant une perte d’activité de nombreux transporteurs de nutriments chez la levure, inhibe TORC1. Cette perte d’activité de TORC1 activerait, via un mécanisme encore inconnu, l’ubiquitylation et l’endocytose Rsp5-dépendante des perméases. Un mécanisme similaire pourrait être responsable de l’endocytose de LAT1 dans les cellules HeLa.Dans une seconde partie, nous avons étudié les facteurs impliqués dans l’endocytose de LAT1 en réponse à un second composé, le PMA, un activateur de la PKC. Nous avons montré que ce composé induit l’internalisation et l’ubiquitylation de LAT1 via l’activation de la PKC. Nous avons trouvé que cette modification dépend de l’ubiquitine ligase Nedd4-2. De plus, les lysines 19, 25 et 30 semblent être les cibles principales de l’ubiquitine ligase. En effet, une diminution importante de l’ubiquitylation et de la localisation intracellulaire de LAT1 en réponse au PMA est observée dans un mutant LAT1K19R,K25R,K30R. D’autres études seront nécessaires pour évaluer l’importance de ce mécanisme de régulation de LAT1 dans les cellules saines et cancéreuses. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Cancer cells utilise glycolysis to produce lactate, even in the presence of sufficient levels of oxygen. Excess lactate is removed from cancer cells by MCT1 and MCT4, to prevent intracellular acidosis, apoptosis and to aid the continuous glycolytic flux. MCT1 and MCT4 are over-expressed in many types of human cancers, which correlates with reduced overall survival and increased treatment resistance. The potential role of MCT1 and MCT4 in two EOC cell lines (Skov3 and OV90) was investigated in this study. MCT1 was expressed at similar levels in Skov3 and OV90 cells. Therefore, stable cell lines over-expressing MCT1 were produced using both cell lines. MCT4 was expressed at high levels in Skov3 cells, but very low levels in OV90 cells. Therefore, a stable cell line with MCT4 silencing under the inducible control of doxycycline was produced using Skov3 cells, and a stable cell line to over-express MCT4 was produced using OV90 cells. The consequences of these genetic modifications on the metabolic phenotype, metastatic abilities and the sensitivity of cell lines to treatment with Carboplatin and Paclitaxel, were assessed in normoxia and 1 % hypoxia and 0.1 % hypoxia. Over-expressing MCT1 in Skov3 cells, had no effect on their metabolic phenotype or the sensitivity to treatment with Carboplatin and Paclitaxel. However, over-expressing MCT1 in Skov3 cells significantly enhanced their metastatic abilities, which correlated with reduced focal adhesion size. Silencing MCT4 in Skov3 cells, had no effect on the use of glycolysis, sensitivity to treatment with Carboplatin and Paclitaxel, or their metastatic abilities. However, following MCT4 silencing there was a significant increase in the levels of intracellular ROS. Over-expressing MCT1 in OV90 cells, had no effect on lactate levels or intracellular ROS. However, there was a significant reduction in both their glycolytic activity and mitochondrial mass. Furthermore, over-expressing MCT1 in OV90 cells, increased their resistance to treatment with Paclitaxel, which correlated with increased Pgp and LDHA expression. Over-expressing MCT4 in OV90 cells, caused an increase in the use of glycolysis and increased cell survival in hypoxia. There was also a significant enhancement in the metastatic abilities of these cells following the over- expression of MCT4, which correlated with reduced focal adhesion size. Furthermore, over-expressing MCT4 in OV90 cells, increased their resistance to treatment with Paclitaxel, which correlated with an increase in the expression of Pgp and LDHA.In summary, the findings of this study revealed that MCT1 and MCT4 play a significant role in the biological function of Skov3 and OV90 cells. High expression levels of MCT4 correlated with an increase in glycolysis and cell survival in hypoxia. Whereas high expression levels of MCT1 and MCT4 in correlated with an increase in the metastatic abilities, as well as with Paclitaxel resistance and increased Pgp expression.

The transport of glucose across the plasma membrane is a fundamental mechanism to provide cells with its basic requirements for energy yielding processes. It is also vital for clearing glucose from blood into tissues, a process normally stimulated by the hormone insulin in mammals. The sympathetic nervous system, normally activated during stress, also regulates glucose transport. The sympathetic neurotransmitter noradrenaline, acts on the family of adrenergic receptors (ARs). An important subtype of the AR family is the β-AR, which is subdivided into the β1, β2, and β3-AR. Glucose is transported across the plasma membrane by the family of glucose transporters (GLUT1-12, and HMIT). In this thesis, I have investigated the β-AR regulation of GLUT1 and 4, and glucose uptake, in skeletal muscle cells and brown adipocytes in culture, model systems which correspond to metabolically active, sympathetically innervated and insulin-sensitive tissues. In brown adipocytes, activation of the β3-ARs induced the expression of GLUT1, resulting in a large increase of glucose uptake. In skeletal myotubes, we postulate there is a possible mechanism where β2-ARs can regulate the intrinsic activity of GLUT1. We found that insulin signaling, but not β-adrenergic signaling, mediated glucose uptake through class I phosphatidylinositol 3-kinase (PI3K). The β-adrenergic signaling to glucose uptake appeared to involve a PI3K related kinase (PIKK), in both skeletal myotubes and brown adipocytes. Furthermore, the increase of glucose uptake by β-ARs in brown adipocytes is partially mediated by AMP-activated protein kinase (AMPK). However, in an artificially constructed system, with cells expressing GLUT4 and β2-ARs, both insulin and β-adrenergic activation translocated GLUT4 and increased glucose uptake. These results show that β-adrenergic signaling increase glucose uptake by regulating glucose transporters through distinct pathways, in skeletal myotubes and brown adipocytes.

The integrated Membrane Yeast Two-Hybrid technology (iMYTH) was employed to screen the ABCD family of ABC Transporters in S. cerevisiae. The two ABCD members, Pxa1p and Pxa2p were screened against yeast libraries which detected many interactors involved in expected biological processes in accordance with Pxa1p and Pxa2p’s annotated function (i.e. fatty acid metabolism), and unexpected new interactors that may elucidate a new role for the transporters (e.g. oxidative protection). Members of the glutaredoxins and thioredoxins, and associated proteins were found to interact with Pxa1p and Pxa2p. This may indicate that Pxa1p and Pxa2p also play a role in managing the redox environment, protecting against reactive oxygen species. The iMYTH technology was also used to show that Pxa2p is able to form homodimers, and that Pxa1p localization is directly or indirectly dependent on Pxa2p.

Non-alcoholic fatty liver disease is affecting an increasing population worldwide. NAFLD is closely associated with obesity and diabetes. Research has shown that the expression of some important hepatic transporters and enzymes are altered under inflammatory conditions. We examined the effect of NAFLD on the gene expression of several hepatic transporters and enzymes, as well as the impact of exercise in attenuating the effect of NAFLD. We have demonstrated that the mRNA expression of several hepatic transporters and enzymes, as well as FXR were significantly downregulated in liver of rats treated with a HFHCD. We concluded that HFHCD-induced hepatic steatosis, together with the reduced expression of FXR, contributed to the downregulation of expression of hepatic transporters and enzymes. The mRNA expression of TNF-α and IL-1β were unaffected. Interestingly, exercise was found to improve the expression levels of some transporters and enzymes.

Non-alcoholic fatty liver disease is affecting an increasing population worldwide. NAFLD is closely associated with obesity and diabetes. Research has shown that the expression of some important hepatic transporters and enzymes are altered under inflammatory conditions. We examined the effect of NAFLD on the gene expression of several hepatic transporters and enzymes, as well as the impact of exercise in attenuating the effect of NAFLD. We have demonstrated that the mRNA expression of several hepatic transporters and enzymes, as well as FXR were significantly downregulated in liver of rats treated with a HFHCD. We concluded that HFHCD-induced hepatic steatosis, together with the reduced expression of FXR, contributed to the downregulation of expression of hepatic transporters and enzymes. The mRNA expression of TNF-α and IL-1β were unaffected. Interestingly, exercise was found to improve the expression levels of some transporters and enzymes.

The integrated Membrane Yeast Two-Hybrid technology (iMYTH) was employed to screen the ABCD family of ABC Transporters in S. cerevisiae. The two ABCD members, Pxa1p and Pxa2p were screened against yeast libraries which detected many interactors involved in expected biological processes in accordance with Pxa1p and Pxa2p’s annotated function (i.e. fatty acid metabolism), and unexpected new interactors that may elucidate a new role for the transporters (e.g. oxidative protection). Members of the glutaredoxins and thioredoxins, and associated proteins were found to interact with Pxa1p and Pxa2p. This may indicate that Pxa1p and Pxa2p also play a role in managing the redox environment, protecting against reactive oxygen species. The iMYTH technology was also used to show that Pxa2p is able to form homodimers, and that Pxa1p localization is directly or indirectly dependent on Pxa2p.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 164-188).

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