The role of the ubiquitin protein ligase, Nedd4-2, in neuronal development

Correct ontogeny of the nervous system is reliant upon exquisite regulation of exogenous growth factors and their cellular reception. Integration of a myriad of signalling pathways during the development of the nervous system results in up to 100 billion nerve cells differentiating and migrating to take the their place in the elegant neuronal network that underpins brain homeostasis. The importance of such receptor ligand interactions to the proper initiation of neuronal differentiation, outgrowth and innervation has been demonstrated using a variety of structure- function studies, analysis of knockout animal models, protein-protein interactions and behavioural studies. Despite this, at present, knowledge concerning how such a vast array of signalling pathways are coordinately regulated to achieve the appropriate temporal and spatial regulation of neurogenic proteins is deficient. Modulation of the functional protein output of cell can be regulated by ubiquitination. Attachment of ubiquitin to a target protein is a post-translation modification carried out primarily by ubiquitin protein ligases that bind to consensus motifs on target proteins and conjugate ubiquitin chains to lysine residues within that protein. This process is known to label proteins for recycling or degradation in the proteasome. Thereby providing a mechanism for the removal and attenuation of a proteins signalling potential-when and where it is required. Such a modification is rapid and provides regulation of a protein that is context dependent and easily reversible, therefore providing a on/off switching mechanism, appropriate to the formation complex networks, as seen during neuronal development. Nedd4-2 is an ubiquitin protein ligase that is developmentally regulated; the protein was first identified from a subtractive cDNA library as developmentally down-regulated transcript in the brain. The expression of Nedd4-2 peaks at the time of neurogenesis and is maintained at significantly lower levels in the adult brain. Presently, the knowledge concerning the regulation of Nedd4-2, protein recruitment by Nedd4-2 and how this regulates neuronal development is lacking. Early studies of protein recruitment by Nedd4-2 indicated that it binds to target proteins through a consensus PY motif, despite a small list of proteins being identified and characterized as targets of Nedd4-2 mediated proteolytic degradation; the role of Nedd4-2 has largely not been characterized in the mammalian brain. Therefore, the key focus of this thesis was to examine the role of Nedd4-2, in neuronal development by defining and pinpointing where and how Nedd4-2 influences initiation of neurite development,differentiation and survival. The work presented in this thesis addresses this by establishing the importance of Nedd4-2 expression in regulating neurite outgrowth in PC12 cells. Studies presented here indicate that Nedd4-2 acts to negatively regulate neurite development in PC12 cells, furthermore it is demonstrated that neurogenic proteins such as NGF and Ras differentially regulate Nedd4-2. This work was extended into an in vivo model, made possible by the generation of Nedd4-2 knockout mice. These studies demonstrated the importance of Nedd4-2 in maintaining homeostasis, indicating that complete abrogation of Nedd4-2 expression is peri-natal lethal. Histological assessment of key brain regions developing at this time indicates that the absence of Nedd4-2 is accompanied by deranged development of cortical areas in the brain. To gain insight into molecular mechanisms that mediate the phenotype observed in both the in vitro and in vivo modelling of Nedd4-2 during development an in silico screen was performed to develop a definitive list of putative binding candidates, this revealed the extent of the potential role for Nedd4-2 in regulating biological processes. Most notably, it was shown that PY containing proteins are enriched in pathways that regulate axon guidance receptors and the cytoskeleton. Of this list, it was demonstrated for the first time that Nedd4-2 binds to the axon guidance receptor, Neogenin and that Neogenin is ubiquitinated most likely in a Nedd4-2 dependent manner, highlighting the importance of Nedd4-2 in regulating the cell surface expression of proteins. Furthermore, prediction of the Nedd4-2 interactome led to the identification and characterization of MAP2 as a novel Nedd4-2 binding protein. These studies gave rise to the novel observation that Nedd4-2 is a potent regulator of the neuronally expressed MAP2 and indicate that Nedd4-2 can effect the differentiation and branching behaviour of nascent neurons possibly through destabilization of tubulin polymerization. Taken together this thesis demonstrates that Nedd4-2 is an important negative or rate-limiting regulator of neuronal differentiation and development in the embryonic brain, dorsal root ganglia and in PC12 cells. Furthermore, it is suggested that ubiquitin-mediated regulation of Nedd4-2 ligands such as MAP2 and Neogenin may be the effectors of the phenotype observed in the Nedd4- 2 knockout mice. This thesis also contributes to the broader understanding of how complex and convergent signalling networks are discretely regulated to achieve proper development and homeostasis