Pathology - Theses

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    Investigating the mechanistic link between neuroinflammation and biometal homeostasis in neurodegenerative diseases
    Alukaidey, Lobna ( 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.