Les données accumulées par notre laboratoire montrent une action pro-tumorale des neurotrophines dans le cancer du sein via notamment des effets anti-apoptotiques du NGF, du BDNF et de la NT4/5. Ces effets sont tous relayés par leur récepteur commun p75NTR, je me suis donc appliquée à préciser le rôle de ce récepteur et sa signalisation dans le cancer du sein. Pour cela, nous avons établi une lignée cellulaire surexprimant de manière inductible p75NTR. Ce modèle d’étude a permis de montrer que la survie induite par le récepteur était associée à une inhibition de la voie intrinsèque de l’apoptose. De plus, p75NTR ralentit la prolifération en provoquant une accumulation des cellules dans les phases G0/G1 du cycle cellulaire. In vitro, ces deux effets passent par une augmentation de p21Waf1 car son invalidation par siRNA, non seulement restore la prolifération, mais abolit totalement la survie, faisant de p21Waf1 une protéine clé dans la signalisation de p75NTR. In vivo, la surexpression de p75NTR conduit à une augmentation du volume tumoral et à une résistance accrue à l’apoptogène TRAIL. Enfin, l’analyse de la protéolyse de p75NTR montre qu’il subit deux clivages successifs dans les cellules cancéreuses mammaires. Le premier clivage réalisé par l’enzyme ADAM17/TACE semble indispensable à son effet anti-apoptotique alors que le second clivage n’intervient pas, suggérant plutôt un processus de dégradation du récepteur. Ces travaux ont permis d’approfondir les mécanismes d’action de p75NTR dans les cellules cancéreuses mammaires et suggèrent que ce récepteur pourrait contribuer à la croissance tumorale en favorisant la résistance aux drogues anticancéreuses. / Our laboratory has shown a pro-tumoral action of the neurotrophins in breast cancer especially by the anti-apoptotic effects of NGF, BDNF and NT4/5. These effects are all mediated by their common receptor p75NTR. My thesis work has therefore been to elucidate further the role of p75NTR in breast cancer cells and its signaling. For this purpose, we established breast cancer cells which stably overexpress p75NTR in an inducible manner. This model allowed us to show that its pro-survival effect is associated with an inhibition of the intrinsic pathway of apoptosis. Moreover, p75NTR slows down the cell growth through a cell accumulation in G0/G1 phases. Interestingly, these both effect are mediated by p21Waf1 since its inhibition by siRNA not only restores proliferation but also abolishes the pro-survival effect of p75NTR, indicating the key role of p21waf1 in the biological functions of p75NTR. In a SCID mice xenograft model, p75NTR overexpression favors tumor growth and strongly increases tumor resistance to TRAIL treatment. Finally, the study of p75NTR proteolysis has shown that this receptor undergoes two sequential cleavages in breast cancer cells. The first cleavage by the enzyme ADAM17/TACE is essential to its anti-apoptotic effect. Meanwhile, the second cleavage by the γ-secretase complex doesn’t seem to be involved rather suggesting a degradation process. Together, our findings have improved the knowledge of p75NTR functions in breast cancer cells and suggest that p75NTR overexpression in breast tumor cells could favor tumor survival and contribute to tumor resistance to drugs.

The development, maintenance, and survival of neurons depend on the function of neurotrophins such as nerve growth factor (NGF). One population of neurons that rely heavily on NGF for axonal growth and survival is the postganglionic sympathetic neurons. Trauma or disease resulting in injury to the peripheral nervous system causes an increase in the levels of this neurotrophin. This augmentation promotes the collateral sprouting of postganglionic sympathetic axons into those tissues having elevated levels of NGF. Often, NGF-induced sympathetic sprouting occurs in tissues that are normally innervated by these fibers however, high levels of NGF can also promote sprouting of axons into tissues that are normally devoid of sympathetic fibers, such as the sensory ganglia. When postganglionic sympathetic axons grow into the environment of sensory ganglia, they can converge and wrap around a subset of somata (i.e., cell bodies) belonging to primary sensory neurons. This phenomenon, referred to as sympathosensory plexuses is observed in adult mice and rats following peripheral nerve injury, and is also seen in adult transgenic mice that ectopically over express NGF. The overall aim for this project is to examine the morphological and neurochemical features, as well as the overall consequence of sympathosensory plexuses in nerve-injured adult mice and in adult transgenic mice that over express NGF. We hope that this novel information will add to our understanding of the underlying mechanisms associated with the formation of sympathosensory plexuses that occur following injury. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2013-07-23 18:51:47.902

The development, maintenance, and survival of neurons depend on the function of neurotrophins such as nerve growth factor (NGF). One population of neurons that rely heavily on NGF for axonal growth and survival is the postganglionic sympathetic neurons. Trauma or disease resulting in injury to the peripheral nervous system causes an increase in the levels of this neurotrophin. This augmentation promotes the collateral sprouting of postganglionic sympathetic axons into those tissues having elevated levels of NGF. Often, NGF-induced sympathetic sprouting occurs in tissues that are normally innervated by these fibers however, high levels of NGF can also promote sprouting of axons into tissues that are normally devoid of sympathetic fibers, such as the sensory ganglia. When postganglionic sympathetic axons grow into the environment of sensory ganglia, they can converge and wrap around a subset of somata (i.e., cell bodies) belonging to primary sensory neurons. This phenomenon, referred to as sympathosensory plexuses is observed in adult mice and rats following peripheral nerve injury, and is also seen in adult transgenic mice that ectopically over express NGF. The overall aim for this project is to examine the morphological and neurochemical features, as well as the overall consequence of sympathosensory plexuses in nerve-injured adult mice and in adult transgenic mice that over express NGF. We hope that this novel information will add to our understanding of the underlying mechanisms associated with the formation of sympathosensory plexuses that occur following injury. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2013-07-23 18:51:47.902

The common neurotrophin receptor, p75NTR, has been implicated in diverse responses of sensory neurons including a role in nociception following nerve injury, suggesting that it may serve a similar role in intact sensory neurons and their satellite glial cells (SGCs). To examine the impact of suppressing neuronal p75NTR expression on known molecular modulators/regulators of the nociceptive state namely, the sodium channels NaV1.8 and NaV1.9, the nerve growth factor receptor TrkA, the potassium channel Kir4.1, glial fibrillary acidic protein (GFAP), SGC p75NTR, connexin 43, we intrathecally infused p75NTR anti-sense oligonucleotides (AS OGN), previously shown by Obata et al. (2006) to effectively suppress p75NTR expression in intact neurons. Male, Wistar rats were divided into three groups, receiving either no treatment (non-infused), seven day intrathecal infusion of p75NTR AS OGN or sense control (SC OGN) via an osmotic pump. Serial L4 and L5 DRG sections were processed for immunohistochemistry to detect alterations in NaV1.8, NaV1.9, TrkA, Kir4.1, p75NTR, GFAP and connexin-43 protein expression. Sciatic nerve sections were also processed for immunohistochemistry to detect NaV1.8, NaV1.9, TrkA and GFAP protein expression. Infusion of p75NTR AS OGNs resulted in a significant decrease in neuronal p75NTR expression, however no significant change was observed in neuronal NaV1.8, NaV1.9 or TrkA expression relative to SC OGN treated or non-infused controls. On the contrary, SGC expression of phenotypic markers normally associated with the reactive state that is induced in these cells in response to peripheral nerve axotomy was dramatically altered. More specifically, in response to p75NTR AS OGN infusion, there was a significant increase in SGC protein expression of the cytoskeletal protein GFAP and p75NTR, along with a significant decrease in expression of the inward rectifying potassium channel Kir4.1. Preliminary data also revealed this induced reactive state in SGCs to be associated with an increase in the number of SGCs surrounding individual neurons as well as increased SGC expression of the gap junction protein, connexin 43. In conclusion, reductions in neuronal p75NTR expression and potentially reduced neurotrophin signaling lead to alterations in neuron/glial or axon/glial communication that results in induction of a reactive phenotype in the associated SGCs. With our ever increasing understanding of the role of SGCs modulating pain states, elucidation of the pathways leading to adoption of pathological phenotypes can help in the identification of novel therapeutic targets.

No description available.

Le facteur neurotrophique dérivé du cerveau (BDNF) synthétisé sous la forme d'un précurseur (proBDNF) qui peut être clivé pour donner sa forme mature (mBDNF). Le mBDNF et le proBDNF produisent des réponses physiologiques opposées par l'activation de deux classes distinctes de récepteurs transmembranaires : respectivement, le récepteur TrkB et p75NTR. Le ratio mature/pro-BDNF est un élément important impliqué dans la plasticité synaptique, la formation des circuits neuronaux et in fine les fonctions cognitives. Les altérations dans ce clivage peuvent ainsi expliquer l’émergence de conditions pathologiques post-lésionnelles, comme la mort cellulaire induite par un état de mal épileptique. Au cours de ma thèse, j'ai montré que l'altération de la maturation du BDNF in vitro, provoquait, via le récepteur p75NTR, une altération de l’activité GABAergique. Par ailleurs, au cours des crises d'épilepsies, les réponses dépolarisantes et excitatrices du GABA, soutenus par la baisse d’expression et d’activité du co-transporteur KCC2, ont été rapportées. Ainsi, in vivo, j’ai montré que la voie proBDNF/p75NTR module l'homéostasie chlore au cours du développement et dans des processus d’épileptogenèse. Pendant le développement, l’activation de la voie proBDNF/p75NTR contrôle le passage d’un GABA immature dépolarisant à un GABA mature hyperpolarisant via KCC2. Pendant l’épileptogenèse, le proBDNF via p75NTR contribuerait à l’hyperexcitabilité des réseaux neuronaux. De plus, le blocage de p75NTR permet de réduire le nombre de crises épileptiques. En conclusion, proBDNF/p75NTR est un facteur clé dans la séquence maturative du GABA et dans la mise en place de l’épilepsie du lobe temporal. / The brain-derived neurotophic factor (BDNF) is synthesized as a precursor (proBDNF) that can be processed intracellularly to the mature form (mBDNF). mBDNF and proBDNF are assumed to produce opposing physiological responses mediated by the activation of two distinct classes of transmembrane receptors, the TrkB and the p75NTR respectively. The proteolysis of proBDNF is crucial for cognitive functions; its impairment may account for the emergence of brain disorders such as epilepsy. During my thesis, I showed that alteration in BDNF maturation in vitro triggers an up-regulation of p75NTR, inducing a disruption of GABAergic transmission. Moreover, in epilepsy, depolarizing and excitatory GABAergic responses, due to alteration of KCC2, have been reported. Interestingly, I described novel insights into the proBDNF/p75NTR mechanisms and function in vivo in modulating chloride homeostasis during the development of neuronal networks and in the pathogenesis of epilepsy. In physiological conditions, p75NTR activation by proBDNF may be a key regulator in shaping neural circuitry and synaptic plasticity. Moreover, I have shown that proBDNF/p75NTR to mBDNF/TrkB ratio may control the timing of the developmental shift of GABA depolarizing to hyperpolarizing. During epileptogenesis, proBDNF via p75NTR alters the excitatory/inhibitory equilibrium thereby enhancing neuronal activity through the inhibition of KCC2 function. Hence, blockade of p75NTR can prevent some of the epileptogenic mechanisms. Altogether, these data provide the first compelling evidence that proBDNF disrupts the GABA excitatory/inhibitory developmental sequence, which then favors the emergence of epileptic disorders.

Neurotrophins are a family of growth factors necessary for the development and maintenance of the nervous system. They produce their effects through receptor mediated signaling mechanisms that are highly regulated by sophisticated intracellular transport networks. The impairment of intracellular trafficking of neurotrophins in motor neurons has been identified as one possible factor in the development of motor neuron diseases, but remains inadequately studied. Aided by advances in imaging technology and the development of more powerful and sensitive detection tools for in-vitro studies, the dynamics of intracellular transport of neurotrophins are beginning to be unraveled. However, a primary limiting factor in the study of neurotrophin-transport dynamics in motor neurons has been the lack of alternative and easily available in-vitro systems able to substitute the often difficult and costly primary motor neuron cultures. The aim of this project was to develop a suitable motor neuron model using the NSC-34 cell line for the study of receptor mediated trafficking events through endosomal transport pathways. Successful evaluation and characterization of NSC-34 cells for motor neuron specific markers would result in the investigation of the p75 neurotrophin receptor (p75NTR) trafficking pathways in the presence of exogenous neurotrophins, with a variety of confocal imaging techniques. Chapter 3 describes the optimisation of NSC-34 cell culture conditions through media modification and the development of a suitable growth substrate matrix, which significantly improved cell adhesion, differentiation and the ability to culture the cells for extended time periods in serum free conditions. Quantitative measurements of cell proliferation, culture viability, cell-body size and neurite length are described to highlight the increased value of the cell line for long-term culture and experiments examining a broad range of issues relevant to motor neurons. In Chapter 4, multiple experimental approaches were used to extensively screen the NSC-34 cell line for the presence of motor neuron-specific markers, neurotrophin receptors and proteins involved in regulation of endosomal transport. This characterization established the presence of a developing motor neuron-like neurotrophin receptor profile (p75NTR, TrkB and TrkC), a genetic marker of developing motor neurons, cholinergic markers, proteins regulating transport within the endosomal pathway, and additional proteins previously shown to directly interact with neurotrophin receptors, including sortilin, and the lipid raft associated ganglioside GT1b. Furthermore, evidence is provided that NSC-34 cells undergo apoptosis in response to exogenous nerve growth factor (NGF) or neurotrophin-3 (NT-3), but not brain derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4). In addition characterization of mouse specific p75NTR antibodies is presented to establish their suitability for internalization studies without altering the binding of exogenous neurotrophins to the receptor. Subsequent confocal microscopy examination focusing on p75NTR trafficking in Chapter 5 revealed that internalization and intracellular transport of this receptor is regulated by exogenous neurotrophins at the cell surface where ligand binding and internalization occur, and in endosomal compartments where the bulk of receptors and ligands are targeted to their specific destinations. Evidence is provided showing that p75NTR internalization is altered in the presence of NGF, NT-3, or NT-4, but not BDNF, and the receptor is diverted into non-clathrin mediated endosomal pathways in response to NGF but not BDNF. Immunofluorescence confocal microscopy suggests that p75NTR recycles to the plasma membrane in a Rab4 GTPase dependent manner in the absence of neurotrophins. Addition of neurotrophins diverted p75NTR from the recycling Rab4 positive pathway, into EEA-1 positive sorting endosomes in the presence of NGF or NT-3, or lysosomal degradation in the presence of BDNF or NT-4. This study clearly demonstrates the suitability of the NSC-34 cell line as an alternate in-vitro system for the study of motor neuron biology, particularly the study of neurotrophin receptor trafficking. Taken together the results represented in this study suggest for the first time, that the fate of the p75NTR receptor depends on which neurotrophin is bound. These findings have important implications for understanding the dynamic mechanisms of action of p75NTR in normal neuronal function, and may also offer further insight into the potential role of neurotrophins in the treatment of neurodegenerative diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22496号 / 農博第2400号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5276(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Cette thèse a pour objectif spécifique d’explorer les événements précoces pouvant être à l’origine du bourgeonnement aberrant des fibres moussues (FM) du gyrus denté, une réorganisation majeure dans l’Epilepsie du Lobe Tempora (ELT). Nous avons utilisé le modèle pilocarpine d’ELT chez le rat afin de montrer que la transmission GABAergique jouait un rôle prépondérant dans la formation des FM aberrantes au cours de l’épileptogenèse. Ceci étant due à une altération de l’homéostasie chlore, suite à une augmentation de l’expression du co-transporteur NKCC1 et une diminution du co-transporteur KCC2. Nos résultats ont démontré que le récepteur aux neurotrophines p75NTR était un médiateur de l’action trophique de la réponse GABAergique dépolarisante sur le bourgeonnement aberrant des FM. Le blocage de l’action dépolarisante de la transmission GABAergique via l’utilisation de la bumétanide, a permis de réduire le bourgeonnement aberrant des MF en réduisant l’expression de p75NTR. Enfin, l’application transitoire de la bumétanide au cours de l’épileptogenèse a abouti à la réduction du nombre de crises récurrentes et spontanées au cours de la phase chronique d’ELT chez le rat. Ce travail a permis de dévoiler les mécanismes moléculaires sous-jacents de la réorganisation du réseau neuronal glutamatergique consécutif à une crise inaugurale dans un modèle d’ELT. Dans l'ensemble, cette thèse apporte un éclairage nouveau sur l’importance de l’interaction de la signalisation GABAergique avec les neurotrophines afin d’orchestrer la plasticité réactive au sein de l’hippocampe dans TLE. / The present dissertation undertakes to investigate the early triggering events of the mossy fiber sprouting (MFS) in the dentate gyrus, a hallmark of hippocampal reactive plasticity in Temporal Lobe Epilepsy (TLE). We used the rat pilocarpine model of TLE to show that altered GABAA receptor-mediated transmission play a key role in the formation of early ectopic MFS during epileptogenesis. This is likely due to a compromised chloride homeostasis, as a result of increased expression of chloride loader NKCC1 and downregulation of the neuronal chloride extruder KCC2. We next addressed the mechanistic action of depolarizing GABAAR responses with regard to neurotrophin signaling. Our findings uncovered that the pan neurotrophin receptor p75 (p75NTR) mediated the sculpting action of depolarizing GABAAR responses on the ectopic MFS. Blockade of depolarizing GABAAR responses using the loop diuretic bumetanide reduced abnormal p75NTR subsequently decreased the ectopic MFS. Finally, transitory application of bumetanide during epileptogenesis resulted in reduction of spontaneous and recurrent seizures during the chronic phase of TLE. The rationale of this work is that unveiling the molecular mechanisms underlying the hippocampal post-seizure glutamatergic network rewiring will help to drive future novel therapeutic avenues involving chloride homeostasis and neurotrophin interplay. Overall, this dissertation shed a new light on how GABAergic transmission and neurotrophin signaling crosstalk can orchestrate reactive hippocampal plasticity in TLE.

Le NGF est reconnu, et le signal qu’il véhicule est donc médié, par deux récepteurs membranaires : p75NTR et TrkA. Il a été démontré qu’au niveau de la membrane, p75NTR et TrkA sont localisées dans les radeaux membranaires, des microdomaines caractérisés par la présence de protéines cavéolines (Cav-1 et/ou Cav-2). Dans le présent travail, nous avons constaté que la surexpression de Cav-1 dans les neurones des ganglions de la racine dorsale diminue l’extension des neurites. De la même manière, la surexpression de Cav-1 dans les cellules PC12 inhibe les réponses cellulaires déclenchées par l’exposition au NGF. L’activation des effecteurs situés en aval de TrkA n’est pas inhibée. L’expression de Cav-1 provoque une inhibition de la sortie du récepteur des radeaux accompagné par la rétention au niveau de la surface cellulaire, des effecteurs situés en aval incluant Rsk2 phosphorylé. Dans le même temps, la présence de formes phosphorylées de CREB n’est plus détectable. En revanche, la surexpression de Cav-2 potentialise la différenciation des cellules induite par le NGF, ce qui est associé à une activation prolongée des effecteurs situés en aval et à une internalisation des récepteurs. Ces différents effets pourraient être dû à la localisation des cavéolines, qui résulte en une perturbation du microenvironnement des cellules et donc de la signalisation du NGF. En outre, l’expression d’une Cav-1 mutée sur la sérine 80 (S80V) dans des cellules PC12, ne gêne ni le trafic ni la signalisation de TrkA. Au contraire elles se comportent de façon semblable à des cellules Cav-2. Ces études soulignent également le rôle potentiel de Cav-1 et ses mutations dans des cancers NGF-dépendantes. / At the plasma membrane, both NGF receptors have been shown to localized to lipid rafts, specific subdomains that are enriched in cholesterol, sphingolipids and the presence of caveolin proteins (Cav1 and/or Cav2). The focus of this work is on this membrane microenvironment mediated modulation of NGF signaling which via two receptors: p75NTR and TrkA. In the present work we found that overexpression of Cav-1 in mouse dorsal root ganglia neurons significantly impacted neurite extension. Similarly, overexpression of Cav-1 in PC12 cells strongly inhibits their ability to grow neurites in response to NGF. It inhibits NGF signaling without, impairing transient MAPK pathway activation. Rather, it does so by sequestering NGF receptors in lipid rafts, which correlates with the cell surface localization of downstream effectors, and phosphorylated-Rsk2, resulting in the prevention of the phosphorylation of CREB. By contrast, overexpression of Cav-2 potentiates NGF induced differentiation, which is accompanied by sustained activation of downstream effectors, and standard internalization of the receptors. This differential effect could be due to the different localization of Caveolins, that modifies the microenvironment, thereby affecting NGF signaling. Furthermore, PC12 cells expressing the non-phosphorylatable Cav-1 mutant (S80V), neither TrkA trafficking or CREB phosphorylation are inhibited and the response resembles that observed in Cav-2 expressing PC12 cells. These studies underline the interplay between caveolins and NGF signalling, offering insight into the potential impact of Caveolin-1 and mutations thereof in certain cancers where NGF signaling is involved.