Orme, Mariam Helen; (2002) Regulation of neuritogenesis in neuroblastoma cells by components of the Wnt signalling pathway. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this work was to determine the roles of [beta]-catenin and the Wnt signalling pathway in neurite outgrowth in a model cell system, the Neuro-2a neuroblastoma cell line. The canonical Wnt signalling pathway regulates cytosolic levels of the protein - catenin: activation of Wnt signalling disrupts a multiprotein complex that includes [beta]-catenin, Axin, and glycogen synthase kinase-3 (GSK-3), which would otherwise promote the degradation of [beta]-catenin. Stabilised [beta]-catenin accumulates in the cytosol and in the nucleus. In the nucleus it binds members of the T-cell factor (TCF) family of transcription factors, forming a bipartite transcriptional activator that stimulates transcription of Wnt target genes. Inhibition of GSK-3 by lithium (Li+) or a selective inhibitor induced neurite outgrowth of Neuro-2a cells. Cells that had differentiated in the presence of Li+ showed altered microtubule organisation and altered localisation of GSK-3, [beta]-catenin and Axin compared with cells that had differentiated as a result of serum deprivation. The Li+- induced neurite outgrowth was not due to [beta]-catenin/TCF-dependent transcription of Wnt target genes, nor was stabilisation of [beta]-catenin sufficient to induce neurite outgrowth. Stabilisation of [beta]-catenin had no effect on the morphology of neurites in Neuro-2a cells, other than to cause a slight decrease in the average neurite length. In addition to directly binding GSK-3, Axin is a GSK-3 substrate. Axin was destabilised by treatment of Neuro-2a cells with Li+, and overexpression of Axin inhibited neurite outgrowth. This suggests that Li+-induced destabilisation of Axin is required for neurite outgrowth. Deletion analysis indicated that the GSK-3 binding site of Axin, but not the [beta]-catenin binding site, is required for its ability to inhibit neurite outgrowth. Furthermore, the central region of Axin (amino acids 298-506 of rat Axin) is sufficient to inhibit Li+-dependent neurite outgrowth. These results suggest that a signalling pathway involving Axin and GSK-3, but not [beta]-catenin, regulates Li+-induced neurite outgrowth in Neuro-2a cells.

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