Pathology - Theses
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ItemCuproenzyme dysfunction in the pathogenesis of amyotrophic lateral sclerosis and multiple sclerosis( 2016)Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the selective death of motor neurons within the spinal cord and brain. Although the aetiology of the disease is not well understood, inherited genetic mutations account for a small proportion of cases, with Cu,Zn-superoxide dismutase (SOD1) mutations being the most extensively studied. Effective treatment options for ALS do not exist, however, pre-clinical outcomes indicate that therapeutically modulating copper bioavailability in the central nervous system (CNS) may be a feasible treatment strategy for ALS. Therefore, the initial objective of this study was to investigate the significance of copper dyshomeostasis in the progression of a mutant SOD1 mouse model of ALS. We hypothesised that age-related changes to cuproenzymes progress with disease symptoms in SOD1G37R mice compared to age-matched non-transgenic littermates and mice overexpressing wild-type human SOD1. To test this hypothesis, locomotor performance was assessed to track disease progression, then CNS and peripheral tissues were collected at distinct stages of disease for biochemical analyses. Data presented in Chapter 3 provide evidence for copper malfunction in the CNS of ALS mice and indicate that copper malfunction is an early feature of the disease which worsens as symptoms progress. Specifically, a disconnect exists between the abundance and copper-dependent activity of cuproenzymes SOD1 and ceruloplasmin. Next, the therapeutic significance of these changes to SOD1 and ceruloplasmin were assessed. In Chapter 4, data show that overexpressing CTR1 or treating ALS model mice with the copper compound CuII(atsm) extends survival and improves copper bioavailability to SOD1 and ceruloplasmin in the CNS. To ascertain the relevance of outcomes in a broader disease context, we next assessed human cases of sporadic ALS. Data presented in Chapter 5 show that SOD1 and ceruloplasmin dysfunction detected in mice is also evident in sporadic ALS. Significantly, changes to ceruloplasmin are associated with changes to iron homeostasis, where diminished copper- dependent ceruloplasmin activity may contribute to iron overload in the ALS-affected motor cortex and decreased transferrin bound iron in the cerebrospinal fluid. As such, we propose that changes to copper-dependent ceruloplasmin activity in ALS may be the mechanistic basis for two ALS biomarkers and represent the first biochemical evidence for the feasibility of treating ALS, including sporadic ALS, by therapeutically improving copper bioavailability to CNS cuproenzymes. Multiple sclerosis (MS) is a disease characterised by CNS demyelination, with evidence suggesting a link between demyelination and limited copper bioavailability. This is supported by data presented in Chapter 7 from both ALS model mice and MS-affected CNS tissue. We also show that changes to copper, SOD1, ceruloplasmin and myelin-associated proteins are common to ALS and MS, and that modulating copper bioavailability may provide a therapeutic intervention. Overall, data presented in this thesis indicate that: copper malfunction is a feature of ALS and MS; copper malfunction evident in sporadic cases of ALS are recapitulated in mutant SOD1 mouse models of familial ALS; and perturbations to the copper-dependent ceruloplasmin activity may be important to iron accumulation in the ALS-affected motor cortex. The therapeutic implications of these observations are discussed.
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ItemThe mechanism of action of CuII(atsm) for the treatment of amyotrophic lateral sclerosis( 2015)Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the progressive loss of motor neurons in the spinal cord, motor cortex and brain stem leading to complete paralysis and death, usually within 2-3 years of diagnosis. There is currently no cure for ALS and the only approved therapeutic is riluzole. However, its clinical efficacy is marginal with an average extension in survival of 3 months. A subset of ALS cases (~10%) can be attributed to genetically inherited mutations in a number of different genes (familial ALS). Mutations in the gene for Cu,Zn superoxide dismutase (SOD1) – an antioxidant enzyme – were the first to be identified. These mutations lead to a toxic gain of function but the exact nature of this toxicity remains largely unknown. There is evidence to suggest that mutations may cause incorrect metallation of SOD1 leading to aberrant catalytic chemistry and misfolding. Over-expression of the mutant forms of the human protein in mice gives rise to a phenotype that recapitulates many of the symptoms of the human condition including progressive paralysis and premature death. The PET imaging agent, diacetyl-bis(4-methylthiosemicarbazonato)Cu(II) [CuII(atsm)] has been shown to have therapeutic potential in one of these models – SOD1G93A mice. In addition, CuII(atsm) has also been shown to be protective in multiple models of Parkinson's disease. The purpose of this thesis was to further characterise the therapeutic potential of CuII(atsm) in a second model of ALS and to determine if its therapeutic mechanism involves modulation of Cu bioavailability in disease affected tissue. CuII(atsm) was shown to have similar therapeutic potential in the SOD1G37R model as in the SOD1G93A model. Survival extension and improvement in locomotor symptoms were dependent on the dose administered with the highest dose administered proving to be the most effective. No apparent therapeutic ceiling was reached. CuII(atsm) was also co-administered with riluzole with no apparent additive or detrimental effects. When administered alone, riluzole was not as effective at attenuating symptoms and survival as CuII(atsm). Additionally, CuII(atsm) was therapeutic even when given post-onset of a locomotor deficit. Even though severity of disease symptoms in these mice is dependent on mutant SOD1 expression levels, treatment with CuII(atsm) was shown to paradoxically increase the concentration of mutant SOD1 in the spinal cord of these mice. This was due to an increase in fully metallated holo SOD1 – the stable, non-toxic form of the enzyme. The holo SOD1 pool was increased by incorporation of Cu from CuII(atsm) into the Cu-deficient, Zn-containing SOD1 pool. Several other proteins also incorporated Cu from CuII(atsm) however, not all detectable cuproproteins were targets of CuII(atsm)-mediated Cu delivery. Preliminary results suggest that the cuproprotein targets of CuII(atsm) are involved in oxidative stress, metal homeostasis and Cu delivery to SOD1, potentially inhibiting the toxic action of metal-deficient SOD1 on mitochondria. The clinical and pathological similarities between familial and sporadic ALS suggest that similar pathological processes occur in both forms of the disease. There is evidence to suggest that SOD1 can cause disease in the absence of mutations and there is ample evidence implicating mitochondrial dysfunction in sporadic ALS as well as familial ALS. CuII(atsm) is therefore a promising therapeutic for the treatment of ALS and the results presented and mechanism proposed in this thesis position CuII(atsm) as an excellent candidate for translation into human clinical trials.
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ItemProteomic profiling of altered copper homeostasis( 2014)Altered copper (Cu) metabolism is often the hallmark of some diseases of neurodegeneration and cancers. The primary aim of this thesis was to identify proteins that may be associated with Cu metabolism using a high-throughput antibody microarray. We examined the global changes in protein expression in human fibroblast cell lines resulting from altered Cu homeostasis. We identified a number of novel proteins that showed altered expression levels in response to changes in cellular Cu that have not been previously reported. In the process of analysing and validating the array data, we have applied techniques in signal processing, validation and statistical analysis not previously reported in the analysis of antibody arrays. We analysed a panel of 11 proteins identified as being highly ranked as differentially expressed by the antibody array and found that 8 of the 11 proteins could be confirmed by an alternative detection method (Western blotting). Moreover, the richness of the information of the Ab array data supported evidence for cellular processes of cell DNA replication and repair processes that were underpinning the changes observed in protein expression. We identified Ku80 as a protein of interest in relation to altered Cu homeostasis due to its previously known functions and confirmed observations that its metabolism is associated with Cu homeostasis in vitro. We gained insight into the mechanism of Ku80 metabolism in response to altered cellular Cu levels and observed that changes to Ku80 occurred mainly in the cytosol of the cell. We showed that Ku80 expression was inducible by Cu as well as other oxidative stress inducers and that this induction of Ku80 by Cu in these cells showed protective effects against oxidative cell damage in terms of oxidised DNA. We hypothesised that cytosolic interactions with intermediary proteins such as p53 and ATOX1 were involved in the Ku80 response to Cu because of compensatory metabolism to accommodate increased Cu levels and potential protein-protein binding interactions. This thesis demonstrated the progression from using of a multiplexed Ab microarray to identify candidate proteins of interest, to validating of the proteins that were changed, and then exploring a protein of interest (Ku80) in vitro to understand the molecular mechanisms behind the changes observed due to altered cellular Cu levels. We demonstrated the reliability and specificity of the techniques in determining protein expression changes in the cell. Ku80 and some of these proteins identified may have potential for further studies to determine their viability as biomarkers of the Cu associated diseases.
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ItemCellular mechanisms underlying the cognition-enhancing properties of metal-complexes( 2014)Alzheimer’s disease (AD) is caused by a myriad of complex pathological factors that contribute to Amyloid-beta (Aβ) accumulation and oxidative stress as well as synaptic damage and dysfunction that result in cognitive decline. Metal dyshomeostasis is a key factor in these processes and is therefore an attractive therapeutic target. Clioquinol (CQ) was initially investigated as an AD therapeutic due to its copper- and zinc-chelating properties. It inhibited Aβ accumulation and enhanced cognitive performance in an AD mouse model as well as humans with AD in a clinical trial; however, issues with purification led to development of an alternative candidate, PBT2. PBT2 has been tested in AD mouse models and trialled in humans with AD, it improved cognition and reduced cerebrospinal fluid (CSF) Aβ levels. Bis(thiosemicarbazonato) metal-complexes (mII(btsc)s), previously used in applications such as cancer imaging, have been examined as potential treatments for AD as well as other neurodegenerative disorders such as Parkinson’s disease and Amyotrophic Lateral Sclerosis. Their structure contains a Cu or Zn molecule and is able to cross the cell membrane into the cytosol where metals are released, making them bioavailable. Like CQ and PBT2, the mII(btsc) CuII(gtsm) enhanced cognition while lowering CSF Aβ in a mouse model of AD. CQ and PBT2 don’t introduce more metals into the body or cell but may help remove excess metals from outside cells and redistribute them into a metal-depleted intracellular environment. However, mII(btsc)s allow a more controlled delivery of bioavailable metals that has proven to have therapeutic effects in models of AD as well as other neurodegenerative disorders. Despite these advances, little is known of the cellular metal delivery and neurotherapeutic action of these metal-binding compounds. This thesis investigated the potential mechanisms of action of metal delivery agents PBT2 and CuII(gtsm), potential therapeutic compounds for AD. While CQ is not currently being pursued as an AD therapeutic, its effects were also examined. PBT2 enhanced dendritic spine density of Tg2576 mice compared to sham treated controls and had no effect on wild-type controls. Several biomarkers of synaptic plasticity were examined and found to be increased with PBT2 treatment. In vitro, neurite elongation was also increased by exposure to PBT2 with a significantly stronger effect with the addition of equimolar Cu or Zn. When a chelator with high affinity for Cu and Zn was present, the effect of PBT2 on neurite elongation was blocked, indicating that bioavailable Cu and Zn is necessary for this effect. CuII(gtsm) also enhanced neurite elongation in vitro and similar compounds with different Cu-binding affinity or with a Zn molecule instead of Cu required 10-fold higher concentrations to elicit a similar neurogenerative effect. The neurogenerative effect of CuII(gtsm) was examined further and found to require JNK phosphorylation and was associated with inhibition of cellular phosphatase activity, in particular, calcineurin. Furthermore, specific inhibition of calcineurin with FK506 enhanced neurite elongation. While there were subtle differences in the effects of the metal delivery agents examined, this thesis supports the use of metal delivery agents as a potential AD therapeutic and demonstrated that the effects of these agents involves neurogenerative actions requiring the activity of phosphatases such as calcineurin.