Transmissible spongiform encephalopathies (TSEs) are rare, uniformly fatal neurodegenerative disorders that can affect many mammalian species, including humans. A hallmark of these diseases is the conversion of cellular prion protein (PrPC) into an abnormally folded form. This misfolded PrPC is infectious, since it can provide a template for pathogenic conversion of PrPC in a new host. In addition to any toxicity of the misfolded protein, loss of normal PrPC function could be involved in the neurodegenerative processes. However, the physiological role of PrPC is still poorly understood and this project has aimed to address that lack of knowledge. Out of the many putative functions ascribed to PrPC, the most commonly proposed is that it protects cells from stress. In contrast, I have found that stable transfection of the prion protein gene into SH-SY5Y neuroblastoma cells increases cell death in response to serum removal from the culture medium. Following treatment with several chemical toxins, two out of four stably transfected clones did, generally, display greater viability than untransfected cells that do not express detectable levels of PrPC. However, knockdown of PrPC expression by RNA interference had no effect on this stress resistance, indicating that it may not have been mediated directly by PrPC. Given the lack of robust stress protection afforded by PrPC transfection, proteomic analyses of the cells were carried out to identify alternative processes that were perturbed as a result of PrPC expression. The results obtained suggested roles for PrPC in cytoskeletal organisation and cell cycle regulation. Various proteins involved in cytoskeletal organisation were confirmed by western blotting to be differentially expressed in some or all of the stably transfected clones. Additionally, the expression changes to proteins involved in cell cycle regulation resulted in slower proliferation of the clones compared with untransfected cells, a difference that was reduced following RNA interference-mediated knockdown of PrPC. Taken together, these data suggested that specific growth factor-activated pathways were differentially regulated in the stably transfected clones. One candidate pathway was nerve growth factor (NGF) signalling, which promotes neuronal survival and differentiation as well as regulating various processes outside of the nervous system. PrPC-transfection resulted in altered expression of receptors for NGF, suggesting that the stably transfected clones were, indeed, responding differently to NGF stimulation. However, the molecular mechanism responsible for these expression changes remains to be determined, since co-immunoprecipitation experiments did not identify any physical interactions between PrPC and the NGF receptors. Nonetheless, a role for PrPC in modulating NGF signalling has the potential to explain many of the diverse phenotypic observations in PrPC-null mice and might indicate that loss of PrPC function is an important part of TSE pathogenesis.

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.

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.