Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. / Ph. D.

Le virus de l’immunodéficience humaine de type 1 (VIH-1), est un rétrovirus dont le génome est composé de deux molécules d’ARN simple brin. La transcriptase inverse codée par le VIH-1 utilise l’ARNt3Lys de la cellule hôte pour amorcer la réplication de son génome ARN en ADN proviral. L’ARNt3Lys est encapsidé dans les virions lors de l’assemblage; la lysyl-ARNt synthétase (LysRS) cellulaire est impliquée dans ce mécanisme et sert de co-transporteur à l’ARNt3Lys.Chez l’homme, il existe deux formes de LysRS, une forme cytoplasmique (cLysRS) et une forme mitochondriale (pmLysRS) qui donnera la forme mature (mLysRS) après translocation dans la mitochondrie. Les deux LysRS sont issues d’un même gène par épissage alternatif. Il a été démontré que seule la forme mitochondriale est présente dans les particules virales.Nous avons établi un modèle des interactions protéine-protéine impliquées dans la formation du complexe d’encapsidation de l’ARNt3Lys. En recherchant les interactions des précurseurs Gag et GagPol avec les LysRS et leurs domaines, nous avons démontré que seul le domaine Pol du précurseur GagPol a la capacité de s’associer à la LysRS. Ce sont les sous-domaines transframe TF et intégrase IN du domaine Pol qui permettent l’association entre LysRS et GagPol. Cette association se fait via le domaine catalytique de l’enzyme. La sélectivité de l'encapsidation de la forme mitochondriale de LysRS aux dépens de sa forme cytoplasmique pourrait résider dans la stricte compartimentation cellulaire de ces deux formes enzymatiques. Nous avons voulu établir à quel stade l’encapsidation de la LysRS mitochondriale a lieu, soit avant sa translocation mitochondriale sous forme de précurseur pmLysRS, soit après sous forme mLysRS maturée. Nous avons déterminé le site de maturation du précurseur pmLysRS puis caractérisé les deux formes mitochondriales de la LysRS, en déterminant leurs paramètres cinétiques et leur affinité pour l’ARNt3Lys. Alors que la forme pmLysRS ne forme pas de complexe stable avec l’ARNt, la forme maturée mLysRS est la plus apte à interagir avec l’ARNt3Lys. Ce serait donc la mLysRS qui serait impliquée dans le transport de l’ARNt3Lys dans les particules virales lors du bourgeonnement.Comme l'interaction GagPol:LysRS n'est pas spécifique in vitro de la forme mLysRS qui est la seule espèce de LysRS encapsidée, nous avons recherché si d’autres protéines virales pouvaient intervenir dans la formation du complexe d’encapsidation et conférer la spécificité pour la seule mLysRS. Nous avons montré que les protéines auxiliaires Rev et Vpr ont la capacité à s’associer à la LysRS sans distinction d'origine, mais ne peuvent interagir dans le contexte du complexe d'encapsidation GagPol:mLysRS:ARNt3Lys. Les différentes formes de LysRS pourraient ainsi réguler l'activité de Vpr et Rev à d'autres étapes du cycle viral. / The Human immunodeficiency virus type 1 (HIV-1) is a retrovirus with a genome composed of two molecules of single stranded RNA. The reverse transcriptase encoded by HIV-1 uses the cellular tRNA3Lys to prime the replication of its RNA genome into a proviral DNA. The tRNA3Lys is packaged into the viral particles during their assembly; the cellular lysyl-tRNA synthetase (LysRS) is involved in this mechanism as a co-carrier of tRNA3Lys.In human, there are two forms of LysRS, a cytoplasmic form (cLysRS) and a mitochondrial form (pmLysRS) that will be maturated into mLysRS after translocation into the mitochondrion. Both LysRS arise from the same gene by alternative splicing. It was demonstrated that only the mitochondrial species is present in the viral particles.We established a model of the protein-protein interactions which are implied in the formation of the packaging complex of tRNA3Lys. By searching for interactions of the viral precursors Gag and GagPol with the LysRS species and their domains, we demonstrated that only the Pol domain of the GagPol precursor has the capacity to interact with LysRS. The transframe (TF) and integrase (IN) domains of the Pol region of the polyprotein GagPol are required for association of LysRS with GagPol. This association is mediated by the catalytic domain of the enzyme. The selectivity of the packaging of the mitochondrial species of LysRS but not of its cytoplasmic species would rest on the cellular compartmentalization of these two enzyme forms. To establish at which step the mitochondrial LysRS is packaged, either as the pmLysRS precursor before its mitochondrial translocation, or after as the mature mLysRS, we determined the site of maturation of the pmLysRS precursor, then we characterized both mitochondrial forms of LysRS, by determining their kinetic parameters and their affinity for tRNA3Lys. Whereas the pmLysRS species did not form a stable complex with tRNA, the mature pmLysRS species did. Thus, mLysRS is the only LysRS species which could be implied in the transport of tRNA3Lys into the viral particles during the budding step. In vitro, the interaction GagPol:LysRS is not specific for the mLysRS species, but only the mitochondrial LysRS is packaged into the viral particles. We determined if another viral protein could impact the specificity of mLysRS packaging. We showed that the auxiliary proteins Rev and Vpr have the capacity to interact with LysRS but this intercation is not recovered in the context of the GagPol:mLysRS:tRNA3Lys packaging complex. These data suggest that the different forms of LysRS might regulate the activity of Vpr and Rev at other steps of the viral cycle.