The aim of the work described in this thesis is to investigate the nature and mechanisms of action of repellent cues for growing axons. In particular I try to resolve the controversy in the literature regarding the need for protein synthesis in the growth cone in response to external guidance cues. My results resolve the conflicting data in the literature on Semaphorin-3A signalling, where differing labs had shown that inhibiting protein synthesis either blocks or has no effect upon repulsion. They demonstrate the presence of at least two independent pathways, protein synthesis-dependent mTOR activation and -independent GSK3? activation. The higher sensitivity of the synthesis-dependent pathway, and its redundancy at higher concentrations where synthesis-independent mechanisms can evoke a full collapse response alone, resolve the apparent conflict. My experiments also demonstrated that Nogo-?20, a domain of Nogo-A, requires local protein synthesis to cause collapse. Unlike Semaphorin-3A, the dependence of collapse upon protein synthesis is concentration-independent and does not involve guanylyl cyclase, but it does share a dependence upon mTOR activity and the synthesis of RhoA, sufficient to cause collapse downstream of Semaphorin-3A. The other axon-repelling domain of Nogo-A, Nogo-66, is partially dependent upon the proteasome instead. It does not share a common pathway with Nogo-?20, except that both are RhoA-dependent. I further attempted to identify the nature of a repulsive activity found in grey matter, ruling out a previously suggested candidate identity. Finally, I examined the phenomenon of nitric oxide-induced growth cone collapse. My experiments revealed that S-nitrosylated glutathione causes growth cone collapse through the activity of protein disulphide isomerase. This mechanism shows only a partial dependence upon soluble guanylyl cyclase, but I argue that it has total dependence upon an S-nitrosylated donor. Coupled with its apparent relation to S-palmitoylation, the reciprocal of S-nitrosylation, I propose that nitric oxide causes collapse by crossing the cell membrane to inhibit S-palmitoylation-determined localisation of proteins. These results reveal some of the many pathways involved in growth cone collapse, whose further characterisation may provide new targets for the treatment of injuries of the central nervous system.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis. Dysregulation of mTOR signaling occurs in many human cancers and its inhibition causes arrest at the G1 cell cycle stage. However, mTOR’s impact on mitosis (M-phase) is less clear. Here, suppressing mTOR activity impacted the G2-M transition and reduced levels of M-phase kinase, Aurora A. mTOR inhibitors did not affect Aurora A mRNA levels. However, translational reporter constructs showed that mRNA containing a short, simple 5’-untranslated region (UTR), rather than a complex structure, is more responsive to mTOR inhibition. mTOR inhibitors decreased Aurora A protein amount whereas blocking proteasomal degradation rescues this phenomenon, revealing that mTOR affects Aurora A protein stability. Inhibition of protein phosphatase, PP2A, a known mTOR substrate and Aurora A partner, restored mTOR-mediated Aurora A abundance. Finally, a non-phosphorylatable Aurora A mutant was more sensitive to destruction in the presence of mTOR inhibitor. These data strongly support the notion that mTOR controls Aurora A destruction by inactivating PP2A and elevating the phosphorylation level of Ser51 in the “activation-box” of Aurora A, which dictates its sensitivity to proteasomal degradation. In summary, this study is the first to demonstrate that mTOR signaling regulates Aurora-A protein expression and stability and provides a better understanding of how mTOR regulates mitotic kinase expression and coordinates cell cycle progression. The involvement of mTOR signaling in the regulation of cell migration by its upstream activator, Rheb, was also examined. Knockdown of Rheb was found to promote F-actin reorganization and was associated with Rac1 activation and increased migration of glioma cells. Suppression of Rheb promoted platelet-derived growth factor receptor (PDGFR) expression. Pharmacological inhibition of PDGFR blocked these events. Therefore, Rheb appears to suppress tumor cell migration by inhibiting expression of growth factor receptors that in turn drive Rac1-mediate actin polymerization.

L’infarctus du myocarde (IM) survenant après l’obstruction de l’artère coronaire est la cause principale des décès cardiovasculaires. Après l’IM, le coeur endommagé répond à l’augmentation du stress hémodynamique avec une cicatrice et une hypertrophie dans la région non-infarcie du myocarde. Dans la région infarcie, la cicatrice se forme grâce au dépôt du collagène. Pendant formation de la cicatrice, les cardiomyocytes ventriculaires résidant dans la région non-infarcie subissent une réponse hypertrophique après l’activation chronique due au système sympathique et à l’angiotensine II. La cicatrisation préserve l’intégrité structurale du coeur et l'hypertrophie des cardiomyocytes apporte un support ionotropique. Le canal CaV1.2 joue un rôle dans la réponse hypertrophique après l’IM. L’activation du CaV1.2 déclenche la signalisation dépendante de Ca2+ induisant l’hypertrophie. Cependant, il est rapporté que l’ouverture des canaux potassiques (KATP) ATP sensitifs joue un rôle sélectif dans l’expansion de la cicatrice après IM. Malgré leur expression dans les coeurs mâles, les KATP fournissent une cardioprotection sexe dépendante limitant l’expansion de la cicatrice chez les femelles. L’administration de rapamycine aux rates ayant subi un infarctus produit l’expansion de la cicatrice, soutenant la relation possible entre la cible de rapamycine, mTORC1 et les KATP dans la cardioprotection sexe spécifique. Effectivement, dans les cellules pancréatiques α, la signalisation mTORC1 était couplée à l'activation du KATP. Cependant, le lien entre mTORC1 et les canaux KATP dans le coeur reste inconnu. L'objectif de la thèse est d’examiner le rôle des canaux ioniques dans le remodelage cardiaque post-IM, surtout des canaux calciques dans l'hypertrophie et d'élucider la relation entre les KATP et mTORC1. L’hypothèse première teste que l’hypertrophie médiée par le système sympathique des cardiomyocytes ventriculaires des rats néonataux (NRCM) produit une augmentation de l’influx calcique après une augmentation des sous-unités du CaV1.2. Le traitement de norépinéphrine (NE) quadruple l’amplitude du courant calcique type L et double l’expression protéique des sous unités de CaVα2δ1 et CaVβ3. L’hypertrophie des NRCM au NE s’associe à une augmentation de la phosphorylation de la Kinase ERK 1/2. Le β1-bloqueur metoprolol et l’inhibiteur ii de ERK1/2 diminuent l’effet de NE sur CaVα2δ1. Cependant, l’augmentation de CaVβ3 et de la réponse hypertrophique persiste. Ainsi, le signal β1-adrenergique à travers ERK augmente les sous-unités CaVα2δ1 outre l’hypertrophie. L’autre hypothèse examine la spécificité du sexe sur l’expansion cicatricielle médiée par rapamycine et l’influence de mTOR sur l’expression de KATP. Rapamycin augmente la surface de la cicatrice et inhibe la phosphorylation de mTOR chez les coeurs de femelles. Dans les coeurs des deux sexes, la phosphorylation de mTOR et l’expression de KATP, Kir6.2 et SUR2A sont similaires. Cependant, une grande inactivation de la tubérine et une faible expression de raptor sont détectées chez les femelles. Le traitement à l’ester de phorbol des NRCM induit l’hypertrophie, augmente la phosphorylation de p70S6K et l’expression SUR2A. Le prétraitement par Rapamycine atténue chacune des réponses. Rapamycin démontre un patron d’expansion cicatriciel sexe spécifique et une régulation de phosphorylation de mTOR dans IM. Aussi, l’augmentation de SUR2A dans les NRCM traités par PDBu révèle une interaction entre mTOR et KATP. / Myocardial infarction (MI) secondary to the obstruction of the coronary artery is the main cause of cardiovascular death. Following MI, the damaged heart adapts to the increased hemodynamic stress via formation of a scar and a hypertrophic response of ventricular cardiomyocytes in the non-infarcted myocardium. In the infarcted region, a scar is formed via the rapid deposition of collagen. With ongoing scar formation, ventricular cardiomyocytes in the non-infarcted myocardium undergo a hypertrophic response secondary to the chronic activation by the sympathetic system and angiotensin II. Collectively, scar formation and cardiomyocyte hypertrophy preserve the structural integrity of the heart and provide inotropic support, respectively. CaV1.2 channels play a significant role in the hypertrophic response post-MI. Notably, the activation of CaV1.2 channel triggers Ca2+-dependent signaling that induces hypertrophy. By contrast, the opening of ATP-sensitive potassium (KATP) channels was shown to partake in selective scar expansion following MI. Notwithstanding its expression in male hearts, KATP channels endow a sex-dependent cardioprotection limiting scar expansion selectively in females. Moreover, administration of the macrolide rapamycin to the infarcted female rat heart led to scar expansion, supporting the possible relationship between the target of rapamycin, mTORC1 and KATP channels in providing sex-specific cardioprotection. Indeed, in pancreatic-α cells, mTORC1 signaling was coupled to KATP channel activation. However, whether mTORC1 targets KATP channels in the heart remains unknown. Thus, the AIM of the thesis was to explore the role of ion channels in cardiac remodeling post-MI by specifically addressing the role of Ca channels in cardiomyocyte hypertrophy and elucidate the potential relationship between KATP channels and mTORC1 signaling. The first study tested the hypothesis that hypertrophied neonatal rat ventricular cardiomyocytes (NRVMs) following sympathetic stimulation translated to an increase in calcium influx secondary to the augmentation of CaV1.2 channel subunits. NE treatment led to a 4-fold increase of L-type Ca2+ peak current associated with a 2-fold upregulation of CaVα2δ1 and CaVβ3 protein subunits in hypertrophied NRVMs. The hypertrophic response of NNVMs to NE was associated with the increased phosphorylation of extracellular regulated kinase (ERK1/2). The β1-blocker metoprolol and the ERK1/2 inhibitor suppressed NE-mediated protein upregulation of CaVα2δ1 whereas CaVβ3 upregulation and the hypertrophic response persisted. Therefore, sympathetic mediated β1-adrenergic signaling via ERK selectively upregulated the CaVα2δ1 subunit independent of NRVM hypertrophy. The second study tested the hypothesis that rapamycin-mediated scar expansion was sexspecific and mTOR influenced KATP channel subunit expression. Rapamycin administration translated to scar expansion and inhibited mTOR phosphorylation exclusively in females. In normal adult male and female rat hearts, mTOR phosphorylation and protein levels of KATP channel subunits Kir6.2 and SUR2A were similar. However, greater tuberin inactivation and reduced raptor protein levels were detected in females. NRVMs treated with a phorbol ester induced hypertrophy, increased p70S6K phosphorylation and SUR2A protein levels and rapamycin pretreatment attenuated each response. Thus, rapamycin administration to MI rats unmasked a sex-specific pattern of scar expansion and highlighted the disparate regulation of mTOR phosphorylation. Moreover, rapamycin-dependent upregulation of SUR2A in PDButreated NRVMs revealed a novel interaction between mTOR and KATP channel subunit expression

La adaptación de la levadura Saccharomyces cerevisiae a condiciones de alta osmolaridad está mediada por la vía de HOG ((high-osmolarity glycerol). La activación de esta vía induce una serie de respuestas que van a permitir la supervivencia celular en respuesta a estrés. La regulación génica constituye una respuesta clave para dicha supervivencia. Se han descrito cinco factores de transcripción regulados por Hog1 en respuesta a estrés osmótico. Sin embargo, éstos no pueden explicar la totalidad de los genes regulados por la MAPK Hog1. En el presente trabajo describimos cómo el complejo transcripcional formado por las proteínas Rtg1 y Rtg3 regula, a través de la quinasa Hog1, la expresión de un conjunto específico de genes. Hog1 fosforila Rtg1 y Rtg3, aunque ninguna de estas fosforilaciones son esenciales para regulación transcripcional en respuesta a estrés. Este trabajo también muestra cómo la deleción de proteínas RTG provoca osmosensibilidad celular, lo que indica que la integridad de la vía de RTG es esencial para la supervivencia celular frente a un estrés osmótico. / In Saccharomyces cerevisiae the adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway, which elicits different cellular responses required for cell survival upon osmostress. Regulation of gene expression is a major adaptative response required for cell survival in response to osmotic stress. At least five transcription factors have been reported to be controlled by the Hog1 MAPK. However, they cannot account for the regulation of all of the genes under the control of the Hog1 MAPK. Here we show that the Rtg1/3 transcriptional complex regulates the expression of specific genes upon osmostress in a Hog1-dependent manner. Hog1 phosphorylates both Rtg1 and Rtg3 proteins. However, none of these phosphorylations are essential for the transcriptional regulation upon osmostress. Here we also show that the deletion of RTG proteins leads to osmosensitivity at high osmolarity, suggesting that the RTG-pathway integrity is essential for cell survival upon stress.

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.