Bio21 - Theses
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ItemMechanisms of inclusion assembly in Huntington’s disease( 2017)Formation of microscopically visible intracellular aggregates (inclusions) containing exon 1 fragments of mutant Huntingtin (mHttex1) is a hallmark of Huntington’s Disease (HD) pathology. The role of inclusions in HD pathogenesis is not fully understood and is described by two alternative models. One model considers Huntingtin inclusions being adaptive by sequestering toxic soluble mHttex1 and prolonging cellular survival. In the other, inclusions are toxic by coaggregating with growth factors and proteins crucial for normal cellular activity. Through a fortuitous discovery of early-formed and mature types of mHttex1 inclusions by applying a genetically-encoded biosensor of mHttex1 conformation, we describe a mechanism that ties these two models together. Cells with soluble mHttex1 lived for a shorter period than the ones with inclusions and exclusively died via apoptosis. Early-formed inclusions contained ribonucleoproteins, mRNA splicing and processing machinery. As aggregation progressed, chaperones and aggregation-prone proteins were recruited into mature inclusions that converted into amyloid state. Cells with mature inclusions became metabolically inactive and died exclusively via necrosis. Our data suggests inclusions arise via mHttex1 halting protein translation. Soluble mHttex1 appears to be the trigger for apoptosis in the system. Inclusion formation sequesters soluble mHttex1 and correlates with a reduced risk of apoptosis. The prolonged survival of cells with inclusions correlates with progressive metabolic dysfunction, a failed attempt to clear inclusions and ultimate switch from apoptosis to necrosis.
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ItemModulating copper metabolism as a strategy to treat neurodegenerative tauopathies( 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.