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ItemModulating copper metabolism as a strategy to treat neurodegenerative tauopathiesMcKenzie-Nickson, Simon ( 2017)Transition metals such as iron and copper are essential for life and health and yet can cause toxicity through oxidative damage. Therefore, regulation of the levels and location of transition metals is of critical importance to both cellular and organism health. Dyshomeostasis of transition metals has been associated with age-related neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and forms of Frontotemporal dementia (FTD) and thus correcting this dyshomeostasis is an attractive therapeutic target. Previous research from our laboratory has shown that a class of compounds, the CuIIbis(thiosemicarbazones), are efficacious in correcting the pathology in animal models of both AD and PD. The work outlined in this thesis focuses on gaining insight into the mechanism of action of the CuIIbis(thiosemicarbazone), glyoxalbis [N4-methylthiosemicarbazonato]Cu(II) (CuII(gtsm)), in treating an animal model of AD. Additionally, this work aimed to build on these findings by testing the efficacy of CuII(gtsm) in treating an animal model of FTD. Treatment of AD transgenic mice with CuII(gtsm) improved the behavioural deficit seen in the animals in both the Morris water maze and in the Y-maze measures of spatial memory. Quantification of levels of amyloid-β in the brains of these mice revealed no changes in any detectable species. Treatment did however, decrease the levels of phosphorylated forms of Tau, one of the hallmarks of the disease. Analysis of Tau phosphatases and kinases revealed no changes in glycogen synthase kinase 3β, but did reveal an increase in the structural subunit of the Tau phosphatase, protein phosphatase 2A (PP2A). Based on these findings, efficacy of CuII(gtsm) in treating the AD mice in this study is thought to be through an amyloid-β independent reduction in phosphorylated Tau through an increase in PP2A. Additionally, this study supports the concept of AD being an amyloid-β mediated tauopathy. Treatment of an FTD mouse model with CuII(gtsm) improved the spatial memory deficit seen in the Morris water maze performance of these mice. Additionally, treatment reduced the strong hyperactivity phenotype and produced an anxiolytic effect in transgenic mice. Biochemically, treatment reduced Tau tangle load in the hippocampus and reduced a 100 kDa dimer of Tau that was strongly correlated with behavioural deficits. As with the AD model, treatment increased the levels of the same subunit of PP2A. It was hypothesised that the efficacy of CuII(gtsm) in treating FTD was again a reduction in pathological Tau via an increase in PP2A levels. Due to the ability of CuII(gtsm) to increase cellular bioavailable copper, the compounds ability to treat the childhood disease, Menkes disease (MD), was also tested. Utilising the Mottled-Brindled (Mo/Br) mouse model (a naturally occurring mouse model with limited copper transporting ability due to mutant ATP7a) of MD, my work demonstrated that CuII(gtsm) was a strong candidate for treating the disease. Treatment with CuII(gtsm) both orally and via injection increased the levels of brain copper significantly more than copper salt treatment. The findings from this thesis suggest that increasing bioavailable copper has a similar mechanism of action in treating related tauopathies such as AD and FTD. Furthermore, the improvement in behavioural deficits over these two tauopathies suggests this compound could be effective in treating these diseases and validates increasing cellular copper as a clinical therapeutic strategy. Furthermore, the compound has shown promise in the treatment of MD which currently has no effective treatment.
ItemMolecular characterisation of copper resistance and transport proteins in E. coliUdagedara, Saumya Ruvani ( 2015-03-19)Copper is an essential trace element required for all living organisms except for a few archae bacteria. However, it is potentially toxic when in excess or in its “free” form due to its redox activity and highly competitive binding affinities. Specific metabolic pathways have developed to safeguard its uptake, distribution and efflux within the cells. A disruption at any point in these pathways in humans is manifested in fatal genetic diseases such Menkes’ and Wilson’s and is linked to neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Bacterial cells protect themselves from toxic environmental copper by expressing resistance defenses, similar to antibiotic or drug resistance. The Pco operon in E. coli and Cop operon in Pseudomonas syringae pv tomato are part of the plasmid-encoded copper resistance systems that enable the bacterial cells to survive in millimolar copper concentrations. The pco operon comprises four structural gene products PcoABCD that are equivalent to the cop determinant CopABCD. These proteins are homologues sharing 37-76% amino acid sequence identity between the expressed proteins that are therefore presumed to perform similar functions. The pco determinant contains an extra gene PcoE located downstream on the plasmid pRJ1004. PcoE is a periplasmic protein that may play a role as a copper ‘sponge’, sequestering copper in the periplasm before the pco system is fully active. Overexpression of PcoE alone has little effect on overall copper resistance, but does reduce the time required for E. coli strains to recover from copper stress. This protein shares a high sequence similarity with more than 140 proteins predicted from the gene databases, but only one protein SilE has been isolated and partially characterized. PcoE shares 48% of sequence identity with SilE. This work expressed, isolated and characterized the PcoE protein. Deamidation of amino acids Asn 54 and 103 makes the isolation complicated at alkaline pH and, at neutral pH, its low affinities for either cation- or anion-exchange resins make the isolation difficult. Apo-PcoE is unfolded in vitro but binds multiple equivalents of soft metal ions such as Cu(I) and Ag(I). Binding of these metal ions induces dimerization and partial structural folding. This work also shows that PcoE binds multiple Cu(I) and Ag(I) ions non-cooperatively with varying affinities with the highest affinity for Cu(I) in the picomolar concentration range. Using chromatographic approaches, the transfer of Cu(I) from PcoE to potential partner PcoC has been demonstrated. The cue system in E. coli is part of the chromosomally-encoded system that controls normal nutritional copper homeostasis at micromolar copper concentrations. This system involves two proteins: CopA and CueO. CopA is a P-type ATPase which removes excess Cu(I) ions from the cytoplasm to the periplasm where they are presumably to be oxidized by the enzyme CueO to less toxic Cu(II) ions. P-type ATPases are a superfamily of active transporters that transport a wide range of ions across membranes. This transport is coupled to the hydrolysis of ATP molecules and the Gibbs free energy released creates an electrochemical potential gradient across the membrane. CopA protein in E. coli is similar to those encoding for mammalian Cu(I) pumps ATP7A and ATP7B. An aim of this study is that success in characterization of CopA will expedite study of ATP7A and ATP7B. This work develops a strategy for expression and purification of an active full-length recombinant CopA. A green fluorescent protein (GFP) construct was included in the recombinant gene to assist detection as well as a His-tag to expedite isolation. GFP was useful in identifying and visualizing the position of CopA within bacterial cells under in vivo conditions. This work has expressed and characterized this protein successfully and optimized the conditions required for purification and storage. The most popular model to rationalise ion transport by P-type ATPases is the Albers-Post catalytic cycle and this model has been adapted for the study of CopA in this work. Hence monitoring hydrolysis of ATP directly can monitor the activity. This hydrolysis yields orthophosphate and ADP. Therefore activity can be evaluated by monitoring orthophosphate and/or ADP generation or ATP depletion. This work compares the commonly used colourimetric approach that measures the amount of orthophosphate released to the medium with a new chromatographic approach that monitors the depletion of ATP and/or generation of ADP. This is the first case that a chromatographic approach was employed to evaluate its activity of an ATPase. An option to study the chemistry of the vectorial transport function of a membrane protein involves its incorporation into a closed lipid bilayer. Liposomes are lipid spheres that mimic the actual in vivo environment with hydrophilic and hydrophobic properties. CopA protein has successfully been incorporated into liposomes and preliminary studies in this work detected copper transport across the membrane. In addition, preliminary results are presented for copper probe ligands that may be used for the direct visualization of copper transport in bacterial cells in vivo.