Pathology - Theses
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ItemInvestigating the mechanistic link between neuroinflammation and biometal homeostasis in neurodegenerative diseases( 2016)Neuroinflammation and biometal dyshomeostasis are two pathogenic features underlying a number of neurodegenerative diseases, however the mechanistic link between these two pathways has yet to be delineated. This study examined the hypothesis that impaired biometal homeostasis is associated with neuroinflammatory changes. To test this hypothesis I aimed to investigate the effects of key biometals on inflammatory processes in cultured microglia, and in turn, investigate how inflammatory activation of microglia affects homeostasis of biometals. These relationships were further examined in vivo to determine the effects of the type 1 interferon (IFN) pathway on biometal homeostasis in the CNS. In my in vitro study, primary murine microglial cultures were treated for 24h with maximal sub-toxic doses of biometals, delivered as ferric ammonium chloride (FAC), ZnCl2 and CuCl2 and the biometal chelators, diamsar or N,N,N_,N_-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) with and without concurrent interferon-_ (IFN_) and tumour necrosis factor-_ (TNF_) stimulation. Non-stimulated and IFN_/TNF_ stimulated microglia served as negative and positive controls for inflammatory activated microglia, respectively. I measured the levels of a number of key inflammatory cytokines to assess microglial inflammatory response to biometal and biometal chelator treatments. I found that FAC and CuCl2 treatment, significantly induced Fe and Cu uptake respectively, in both non-stimulated and stimulated microglia and that all biometal treatments, significantly reduced the expression of MCP-1 in stimulated and non-stimulated microglia, indicative of an anti-inflammatory role. In contrast, FAC treatment also induced TNF_ mRNA expression in these cultures, suggesting Fe may play a dual role in neuroinflammation. In addition, to investigate how inflammatory activation of microglia affects biometal homeostasis, the gene expression of the metal-binding protein, metallothionein-1 (MT-1) and the biometal transporter, ZRT/IRT-like transporter protein (Zip7) were also measured. I also found that IFN_/TNF_ stimulation inhibited Fe-induced MT-1 and Zip7 expression in microglia. These findings demonstrate that sub-toxic levels of key biometals have multiple modulatory actions on cultured microglia, with both inhibitory and stimulatory effects on cytokines. These changes may be associated with induction or inhibition of major metal response proteins, such as MT-1 and transporters. To examine the effects of the type 1 IFN pathway on biometal homeostasis in the CNS, I performed a spatio-temporal analysis of Fe, Zn, Cu and Mn levels in the CNS of interferon _ receptor-1 (Ifnar1) knock-out (-/-) mice and wild type (WT) mice at 6 and 10 months of age using ICP-MS analysis. A subset of 6-month-old Ifnar1-/- mice was also stimulated with lipopolysaccharide (LPS) treatment for 6h to determine the effects of Ifnar1-/- on biometals homeostasis under inflammatory conditions. I found reduced Cu and Mn levels in the cerebellum of aged (10-month-old) Ifnar1-/- mice, however, expression of key Cu and Mn transporter and regulatory proteins remained unchanged. I also found no significant alterations to biometals between WT and Ifnar1-/- mice at 6-month of age, however, when mice were challenged with LPS, I found a significant decrease in Fe levels in the cerebellum and cerebrum of WT mice and a significant decrease in Zn levels in the cerebrum of Ifnar1-/- mice compared to naïve mice of their respective genotypes. A significant increase and an upward trend in transferrin receptor1 (TfR1) levels in the cerebrum of LPS-challenged and naïve Ifnar1-/- mice, respectively was also observed. These data demonstrate that the type 1 IFN pathway is involved in the regulation of CNS biometal homoeostasis. The studies provide further evidence to support a major role for biometals in neuroinflammatory pathways, with important implications for neurodegenerative disease in which brain biometal homeostasis is altered.
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ItemCuII(atsm) as a potential therapeutic for neurodegenerative diseases( 2013)Neurodegenerative disease is caused by a progressive deterioration of cells in the central nervous system. Commonly known neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal lobar degeneration (FTLD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). These diseases are all fatal and do not have effective therapeutics. Through the use of various models of neurodegenerative disease, metallocomplexes of bis(thiosemicarbazones) have been identified with a strong therapeutic potential. Of these compounds, diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)) has demonstrated the best potential as a treatment for a broad range of neurodegenerative diseases. Treating with CuII(atsm) has thus far shown to attenuate disease symptoms in multiple mouse models of PD and a mouse model of ALS. This project therefore aimed to determine the broader therapeutic activity of CuII(atsm), by examining its therapeutic effects in additional mouse models of neurodegenerative disease. Treatment with CuII(atsm) at a daily dose of 30mg per kg of body weight to the SOD1G37R mouse model of ALS, the TDP-43A315T mouse model of FTLD/ALS and the R6/1 mouse model of HD, showed CuII(atsm) was only able to improve the disease phenotype of the SOD1G37R mice. Treatment with CuII(atsm) improved the survival of the SOD1G37R mice by 16% and their locomotor deficit, however there was no phenotypic change of symptoms with CuII(atsm) treatment in the TDP-43A315T or R6/1 mice. Biochemically, treating with CuII(atsm) decreased markers of inflammation and/or oxidative damage in all of the mouse models analysed. Given that CuII(atsm) treatment only improved the phenotype of one of the animal models analysed, suggest the suppression of the inflammatory and oxidative damage pathways in isolation in these animal models of disease are insufficient for CuII(atsm) to mediate its therapeutic effects. Although treatment with CuII(atsm) in the SOD1G37R mice attenuated disease symptoms and improved survival, the treatment also paradoxically increased levels of the ALS-causing mutant form of the copper/zinc containing superoxide dismutase 1 (SOD1). To investigate this paradox, mass spectrometry was utilised to analyse how increasing levels of the putative pathogenic mutant SOD1 could decrease disease symptoms in the SOD1G37R mice. For its normal function, SOD1 requires one copper and one zinc ion per subunit, but analysis of the ALS model mice spinal cord showed a majority of the SOD1 was copper-deficient. Treatment with CuII(atsm) increased the copper content of the mutant SOD1 and consequently, increased the fully-metallated, non-toxic and stable form of mutant SOD1. Thus, increasing the bioavailability of metals and improving the aberrant metallation states of pathogenic metalloproteins may be a part of the therapeutic activity of CuII(atsm). Overall, outcomes from this thesis indicate treatment with CuII(atsm) suppresses markers of oxidative stress and/or inflammation in distinct animal models of ALS, FTLS/ALS and HD. However, the inability of CuII(atsm) to alter the phenotype of the TDP-43A315T and R6/1 mice suggest the alteration of these pathogenic pathways alone is insufficient for CuII(atsm) to have therapeutic efficacy. If the protective activity of CuII(atsm) observed in the SOD1G37R mice is related to the ability of the compound to improve the metal state of mutant SOD1, this may indicate the therapeutic potential for CuII(atsm) is best suited to diseases in which pathogenesis is related to disrupted metal homeostasis.