Genetics - Theses
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ItemNo Preview AvailableCopper homeostasis and the Alzheimer's disease amyloid precursor proteinBellingham, Shayne Anthony. (University of Melbourne, 2005)The transition metal copper is both essential for normal cellular function and potentially highly toxic, thus it is critical that all living organisms, from bacteria to humans, have developed copper homeostasis mechanisms. Alzheimer's disease is characterised by the accumulation of amyloid-? peptide, which is cleaved from the copper-binding Amyloid Precursor Protein. The normal biological function of the Amyloid Precursor Protein is poorly understood. Amyloid Precursor Protein is a member of a multi-gene family, including Amyloid Precursor like Proteins-1 and -2. The copper-binding domain is similar among Amyloid Precursor Protein family members, suggesting an overall conservation in its function or activity. This study investigated the hypothesis that the Amyloid Precursor Protein functions in copper homeostasis and that copper levels may regulate Amyloid precursor protein gene expression. To investigate the hypothesis that Amyloid Precursor Protein has a functional role in cellular copper homeostasis, mammalian cell culture systems in both neuronal and non-neuronal cells with varied Amyloid Precursor Protein expression were characterised for copper transport utilising a radio-copper assay. Over-expression of Amyloid Precursor Protein increased intracellular copper accumulation in non-neuronal and neuronal mammalian cells. Studies utilising mouse primary neuronal cortical cultures, demonstrated a gene dosage-dependent effect of Amyloid Precursor Protein expression on increasing cellular copper levels in cells that have a genetic ablation of Amyloid Precursor-like Protein-2 expression. In contrast, overexpression of the Swedish mutant of Amyloid Precursor Protein in primary cortical neurons reduces cellular copper levels. These findings provide evidence for the role of Amyloid Precursor Protein in neuronal copper homeostasis as a copper detoxification and/or efflux protein. To investigate the hypothesis that copper may regulate Amyloid Precursor Protein gene expression, a novel cell culture system was utilised. In this system, intracellular copper levels were genetically manipulated through altered expression of the Menkes protein, a major mammalian copper efflux protein. Cells lacking the Menkes protein show high intracellular copper levels due to reduced copper efflux, while restoration of Menkes protein function by over-expression restores copper efflux ability, resulting in dramatically decreased intracellular copper levels. Data presented in this study show that depletion of intracellular copper results in significantly reduced Amyloid Precursor Protein levels and a significant reduction in Amyloid Precursor Protein gene expression. In addition. Amyloid Precursor Protein promoter analysis suggests that putative metal regulatory elements, in the region -490 to +104 of the Amyloid Precursor Protein promoter, may be involved in mediating the response to copper depletion to regulate Amyloid Precursor Protein gene expression. This demonstrates a previously uncharacterised aspect of human Amyloid Precursor Protein gene regulation and supports the hypothesis that copper can regulate Amyloid Precursor Protein gene expression. Furthermore, these data support a role for the Amyloid Precursor Protein in copper homeostasis as a copper detoxification and/or efflux protein. Overall, this thesis presents strong evidence for the role of the Amyloid Precursor Protein in copper detoxification and/or efflux and the role of copper in the regulation of Amyloid Precursor Protein gene expression. Characterisation of copper homeostasis mechanisms of Amyloid Precursor Protein in copper detoxification and/or efflux and the elucidation of the copper-regulation mechanisms of the Amyloid Precursor Protein gene may provide potential therapeutic targets towards the treatment of Alzheimer's disease.