Genetics - Theses
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ItemThe role of presenilin in metal homeostasis and Alzheimer's disease( 2012)Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and there is currently no effective disease-modifying treatment available. One of the pathological features of AD is the accumulation of amyloid deposits in the brain. These extracellular amyloid deposits (or plaques) are primarily composed of misfolded amyloid beta (Aβ) peptide aggregates but also contain high levels of copper (Cu) and zinc (Zn). Both Aβ and its parent molecule, the amyloid precursor protein (APP), are metal-binding proteins. The formation of Aβ from APP is carried out by the sequential cleavage by β-secretase (BACE1) and γ-secretase. γ-secretase is a multi-protein aspartyl protease that contains the protein presenilin (PS, PSEN) as its catalytic component. Studies carried out almost two decades ago revealed that both Zn and Cu can cause Aβ to aggregate in vitro and subsequent studies in mice demonstrated that Zn and Cu augment Aβ pathology in vivo. Based on this pathological phenomenon the “Metal Hypothesis of Alzheimer’s Disease” was proposed whereby a disease-associated metal dyshomeostasis facilitates the pooling of Zn and Cu in synaptic junctions, the very site where Aβ accumulates. The flipside to this hypothesis is that as extracellular Zn and Cu levels rise, cells such as neurons can become deficient in these metals. This has important consequences, as Cu and Zn are essential co-factors for numerous cellular enzymes. Consistent with this hypothesis, there is evidence of reduced Cu/Zn dependent superoxide dismutase 1 (SOD1) activity in AD patient brains and AD animal models. However, there is still limited knowledge about the molecular mechanisms that can potentiate the Cu and Zn dyshomeostasis that is evident in AD. An inherited form of AD, called familial AD (FAD), is predominantly caused by mutations in PSEN1 and PSEN2, the genes that encode PS1 and PS2 respectively in mammals. In general, FAD mutations are associated with an increase in longer, more pathogenic forms of Aβ that are prone to aggregate. However, PS/γ-secretase cleaves numerous substrates other than APP, including Notch. Hence, PSEN1 and PSEN2 mutations that alter γ-secretase activity may also lead to loss-of-function phenotypes. In addition, PS has been demonstrated to function independently of γ-secretase, affecting cellular processes such as protein trafficking and calcium homeostasis. The primary aim of this PhD project was to determine whether PS expression could affect Cu or Zn homeostasis. Initial experiments focused on utilizing cultured PS knockout (PS KO) mouse embryonic fibroblast (MEF) cells and 64Cu and 65Zn radioisotope uptake assays. These experiments revealed that PS is required for a significant proportion of cellular Cu and Zn uptake in these cells. RNAi of PSEN1 or PSEN2 in cultured human embryonic kidney (HEK293) cells confirmed that 64Cu and 65Zn transport was affected by PS expression in multiple cell types. Interestingly, total Cu and Zn levels were unchanged in PS KO MEFs and 64Cu and 65Zn retention studies indicated that turnover of Cu and Zn was reduced in these cells. Consistent with a role for PS in Cu and Zn uptake, brain and other tissues from PS1 heterozygous knockout mice had lower Cu and Zn levels compared to littermate control mice. The activity of Cu/Zn dependent SOD1 was lower in both PS deficient cultured MEF cells and PS1-deficient mouse brain, a potential consequence of disturbed Cu and/or Zn homeostasis in these cells/tissue. Tellingly, knock-in mice that harbour a PS1 mutation that causes FAD in humans also had lower brain Cu and SOD1 activity. Cell surface biotinylation and confocal microscopy immuno-localization experiments indicated reduced expression of several Cu and Zn transporters at the cell surface of cultured PS deficient cells. A model of PS-dependent trafficking of these transporters is proposed whereby PS is required for the delivery or retention of specific metal transporters at the cell surface. In this model PS could influence trafficking at one or more sites along the biosynthetic or endocytic pathways, consistent with the roles for PS in protein trafficking that have been reported in the literature. These studies demonstrate that as a consequence of loss of PS function, either by loss of expression or mutation, Cu and Zn homeostasis is altered. This has important implications in terms of AD, and in particular the familial form of the disease, whereby altered PS function may modulate Cu and Zn re-uptake in cells and promote Cu and Zn interaction with extracellular Aβ. These novel findings should stimulate further studies in neuronal cells and thereby facilitate a better understanding of the involvement of PS in Cu and Zn homeostasis in the brain.
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ItemIdentification and characterisation of copper homeostasis genes( 2010)Copper is essential for life, yet also potentially toxic in excess. Copper homeostasis is therefore regulated at the cellular, tissue and organismal levels. Studies with eukaryotic model systems, primarily yeast and mammals, have identified conserved mechanisms for copper uptake, distribution, sequestration and efflux. Nevertheless, there is much we do not know about how copper levels are sensed and regulated. Additional proteins involved in copper homeostasis under both ‘normal’ and ‘diseased’ conditions remain to be identified. The emergence of Drosophila melanogaster as a bona fide model system for the study of copper homeostasis has coincided with research aiming to identify and characterise novel copper regulatory genes in this organism. Whereas others have focused on in vivo studies, the studies reported in this thesis are focussed on cultured D. melanogaster S2 cells. The first aim of this project was to use cDNA microarrays to identify genes transcriptionally regulated by copper levels in S2 cells. The second and third aims were to characterise the function and localisation of novel copper homeostasis genes in vitro and in vivo respectively. Initial characterisation of D. melanogaster S2 cells found these cells express orthologues of key mammalian copper regulatory genes. Copper uptake primarily occurs via Ctr1A and Ctr1B, the orthologues of human Ctr1. Copper efflux occurs via DmATP7, the sole D. melanogaster orthologue of the mammalian P-type ATPases, ATP7A and ATP7B. S2 cells are highly copper tolerant and primarily rely on metallothionein-mediated copper sequestration and copper efflux to maintain homeostasis. Whereas ATP7A and ATP7B undergo copper-induced trafficking between the trans-Golgi network and plasma membrane of mammalian cells, this does not appear to occur with endogenous DmATP7 in cultured D. melanogaster cells. Interestingly, when expressed in mammalian cells DmATP7 does undergo copper-induced trafficking to the plasma membrane and can facilitate copper efflux, demonstrating functional conservation of localisation and trafficking motifs in these P-type ATPases. Malvolio, the orthologue of Divalent Metal ion Transporter 1, also contributes to copper uptake in S2 cells and D. melanogaster, with impaired function associated with sensitivity to copper limitation. D. melanogaster therefore utilises this general metal transporter in addition to the copper-specific Ctr1 pathway. cDNA microarrays were used to identify genes transcriptionally regulated by copper in S2 cells, with RNA interference used to determine whether candidate genes could affect copper homeostasis. Several components of the COPI vesicle trafficking pathway, including ADP-ribosylation factor 1 (Arf1), were found to affect copper levels in S2 cells. Arf1 was found to have a conserved role in regulating copper uptake in cultured mammalian and D. melanogaster cells, and this is likely to be mediated via the localisation of Ctr1 at the plasma membrane. Taken together these studies demonstrate the value of D. melanogaster S2 cells for the study of copper homeostasis, thereby complementing the D. melanogaster model system. These novel findings should stimulate additional research in both D. melanogaster and mammalian systems and facilitate a greater understanding of copper homeostasis.