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Modelling copper-linked diseases and glutathione deficiency using Drosophila melanogaster
thesisposted on 27.02.2017, 00:04 by Mercer, Stephen William
Copper is an essential micronutrient that is required for a multitude of cellular functions and cellular pathways in the human body, such as energy metabolism, neuropeptide maturation and pigmentation. Multicellular eukaryotes have evolved sophisticated homeostatic mechanisms to circumvent the severe consequences of either too much or too little copper. The consequences of copper dyshomeostasis are evidenced by two hereditary disorders, Menkes and Wilson’s disease, caused by mutations in the copper efflux proteins ATP7A and ATP7B respectively. Although the roles of ATP7A and ATP7B in trafficking copper are well characterised, the differential impact of various Menkes and Wilson’s disease mutations is not well understood. Furthermore, despite the fact that many of the factors involved in intracellular copper distribution have been identified, there is still a lack of in vivo data on the roles of the glutathionylation machinery, mainly glutathione (GSH) and human GRX1 (hGRX1), in copper homeostasis. This project aimed to use Drosophila melanogaster to not only generate an efficient in vivo system for testing various Menkes and Wilson’s disease mutations, but also to further evaluate the roles of GSH and Drosophila orthologue of hGRX1 in copper homeostasis. Here I show that we have generated a single, in vivo model for studying multiple pathogenic mutations in ATP7 proteins, using the model organism Drosophila melanogaster, which has a single orthologue of ATP7A and ATP7B. Three pathogenic ATP7A mutations and one ATP7B mutation were introduced into a genomic ATP7 rescue construct containing an in-frame C-terminal eGFP tag. Analysis of gATP7:eGFP transgenes containing pathogenic mutations showed that the functionality of ATP7 was directly affected by all four of the human mutations investigated in this study. In addition to my study on pathogenic ATP7 mutations, I found that knockdown of the catalytic subunit of Glutamate cysteine ligase (Gclc), the rate limiting enzyme in GSH biosynthesis, in all neurons, caused lethality. The flies were partially rescued by copper supplementation but mortality was increased by additional knockdown of the copper uptake transporter Ctr1A, or overexpression of the copper efflux transporter ATP7. In addition, when Gclc was knocked down in a subset of neuropeptide-producing cells, this resulted in adult progeny with unexpanded wings, a phenotype previously associated with copper dyshomeostasis. Finally, I found that depletion of the putative Drosophila orthologue of hGrx1, CG6852, in both the adult thorax and eye resulted in copper-linked phenotypes that were in both cases exacerbated by co-suppression of copper uptake genes. Overall this study illustrates the usefulness of Drosophila for modelling copper-linked diseases, but also for further investigation of conserved copper homeostasis proteins.