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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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.
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ItemTherapeutic implications in genetic prion disease( 2011)Prion diseases are fatal neurodegenerative diseases caused by the misfolding of the prion protein (PrP). Of the three subsets of prion diseases (sporadic, genetic, acquired), genetic prion disease is associated with a mutation of PrP, leading to an increased disposition for the mutant PrP to misfold, and adopt a disease associated conformation (PrPD ). The following project investigated how a mutation of the prion protein, the P102L mutation, affected the disease associated properties of PrP and the impact of this mutation upon therapeutic outcome. The 102L and 101L mutation were introduced into human and mouse PrP respectively and expressed in rabbit kidney epithelial (RK13) cells to further investigate the effect of this mutation on the prion protein, and the outcome of prion infection. The 102L and 101L mutant PrP produced by RK13 cells was found to increase the disease associated characteristics of human and mouse PrP. Mutant PrP was also found to enhance the binding of PrP to glycosaminoglycans, a cofactor potentially involved in conversion of PrPC to PrPD . The human and mouse PrP expressing RK13 cell with or without the 102L/101L mutation were exposed to several human and mouse strains of prions to determine the susceptibility of the cells to prion infection. Prion infection of RK13 cells expressing 102P or 102L human PrP (huPrP) was not observed. RK13 cells expressing 101L mouse PrP (moPrP) were more susceptible to infection from two strains of prions, compared with 101P moPrP expressing RK13 cells. The enhanced susceptibility of 101L moPrP expressing RK13 (moRK13) cells was determined to be due to increased uptake of protease resistant PrP (PrPres ), mediated aberrant glycosaminoglycan binding. The therapeutic efficacy of glycosaminoglycan based therapy was investigated in the 101L model of genetic prion disease to determine if the enhanced binding of 101L moPrP affected the therapeutic action. Investigation of the membrane localisation of 101P and 101L moPrP by subcellular fractionation identified a subtle difference between 101P and 101L moPrP. A reduction in the proportion of 101L moPrP was detected in the lipid raft associated fractions compared with 101P moPrP, suggesting that a population of 101L moPrP is aberrantly located in another membrane domain or intracellular compartment. The results of the study suggest that the enhanced binding of 101L moPrP to glycosaminoglycans mediates the association of 101L PrP in an aberrant membrane domain, and mediates conversion of 101L moPrP to PrPres in spite of treatment with glycosaminoglycan or lipid raft altering therapeutics. The prophylactic treatment of persons found to have pathogenic mutations associated with genetic prion disease provides the best opportunity of intervening in prion disease progression, more-so than sporadic and acquired forms of prion disease which are treatable only after transmission or disease is detected. The results generated in the current study suggest that the generation of PrPres in genetic forms of prion disease occurs in additional, or different cellular sites to other forms of prion disease, where, potentially, current therapeutics have little or no effect. Therefore further testing of potential therapeutics developed for treatment and prevention of prion disease is required before use in genetic forms of prion disease is granted.
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ItemInvestigating the cellular uptake, efflux and trafficking of metal complexes: implications for the therapy of neurodegeneration( 2012)Alzheimer’s disease (AD) and other neurodegenerative diseases such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are characterized by altered biometal homeostasis. Low intracellular copper (Cu) levels have been observed in both AD and PD affected brain regions, while abnormal Cu metabolism by superoxide dismutase (SOD) may form the basis of some cases of ALS. Despite a surge in interest, the role of abnormal biometal balance and, in particular, homeostasis of Cu in these neurodegenerative diseases remains unclear. Growing evidence suggests that these changes in Cu homeostasis are an important early step in neurodegenerative processes. Restoring normal biometal metabolism may therefore offer a unique therapeutic opportunity. Supporting this, it has previously been shown that a bis(thiosemicarbazonato)copper(II) complex (CuII(btsc)) called CuII(gtsm) was capable of increasing intracellular Cu bio-availability and inhibited the accumulation of trimeric amyloid-beta (A) and phosphorylated tau in the brains of AD model mice and restored their cognitive function. Another CuII(btsc), CuII(atsm), has also produced positive effects in preliminary studies in multiple animal and cell models of PD and ALS. These Cu-complexes were initially investigated for use in cancer therapy and as imaging agents for hypoxic (low oxygen) tissues such as tumors, and the rationale for their application in neurodegenerative disease treatment was based on their ability to deliver metals into cells. In the current project, mechanisms of uptake, intracellular distribution and efflux of two CuII(btsc) complexes in neuronal and glial-like cells were examined. The aim was to more thoroughly understand their cellular accumulation and trafficking profiles and potential use in therapy for neurodegeneration. A combination of inductively-coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS), microscopic analyses and additional techniques were employed. This study found that no single mechanism was clearly responsible for the uptake of the CuII(btsc)s. Instead, a combination of passive diffusion and ATP-independent, facilitated uptake most likely mediated accumulation of the CuII(btsc)s in the U87MG glioblastoma and M17 neuroblastoma cell lines. Efflux of the CuII(btsc) complexes appeared to be dependent upon the ligand backbone of the complex and the data supported a rapid, ATP-dependent efflux process that was difficult to delineate temporally from uptake. To investigate the intracellular distribution of CuII(btsc) complexes, a fluorescent derivative of CuII(atsm) (termed CuIIL1) was used. Upon entry into cells, CuIIL1 localized to organelles of a lysosomal and possibly autophagic origin in the M17 cell line. This appeared to be associated with a robust down-regulation of BiP protein expression indicative of a possible role for the CuII(btsc)s in cellular ER stress. Confocal microscopic studies were also performed using ‘copper sensor 1’ (CS1) in an attempt to establish the subcellular localization of Cu ions delivered by CuII(btsc) complexes in the M17 cell line. Although CS1 was previously reported to be a selective fluorescent sensor for intracellular Cu(I), the current data indicated that CS1 was unlikely to compete with native intracellular Cu-sequestering molecules, and was sensitive to changes resulting from altered cellular pH. In addition, CS1 also localized to lysosomes in M17 cells. Therefore, CS1 was found to be unsuitable to identify the subcellular localization of Cu(I) delivered by CuII(btsc)s. Alternative approaches will need to be investigated to determine spatial distribution of Cu(I) from CuII(btsc) complexes. This was the first comprehensive examination of the transport and distribution of the CuII(btsc)s in different cell types relevant to neurodegeneration. This research has demonstrated that potentially therapeutic CuII(btsc) complexes have complicated cellular uptake mechanisms, are actively effluxed (either as Cu ions or intact Cu-complex) and upon entry to cells may be associated with lysosomal, autophagic and ER-associated cellular processes. These data may have important implications for the future development of CuII(btsc) complexes as potential therapeutic compounds for treatment of neurodegenerative diseases.
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ItemTherapeutic effects of copper bis(thiosemicarbazone) complexes in Alzheimer’s and Parkinson’s diseases( 2010)Neurodegeneration is a complex process and one that involves a myriad of physiological changes leading to chronic pathological states. Examples of neurodegenerative disorders include Alzheimer’s disease (AD) and Parkinson’s disease (PD). In this thesis, a group of compounds, CuIIbis(thiosemicarbazones) or CuII(btsc), was investigated for their ability as therapeutic agents in AD and PD. The CuII(btsc) compounds include CuII(atsm) and CuII(gtsm), and have been identified to be good candidates for CNS drugs due to their ability to be bioavailable and cross the blood brain barrier. In addition, they also possess unique properties that help target pathologies in both AD and PD. CuII(gtsm) administration to AD transgenic mice increased intracellular copper bio-availability and inhibited glycogen synthase kinase-3β (GSK-3β) through activation of an Akt signalling pathway. CuII(gtsm) also decreased the abundance of Aβ trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvements in Y-maze cognition correlated directly with decreased Aβ trimer levels. Therefore, therapeutic ability of CuII(gtsm) in the transgenic mice demonstrated that increasing intracellular copper bio-availability can restore cognitive function by inhibiting the accumulation of neurotoxic Aβ trimers and phosphorylated tau. CuII(atsm), on the other hand, was shown to be therapeutic in PD models by inhibiting nitrosative stress, in particular peroxynitrite (ONOO-) driven α-synuclein aggregation. Treatment of CuII(atsm) in four different PD animal models resulted in significant reductions in α-synuclein nitration and oligomerisation within the substantia nigra. In all models a significant increase in the survival of dopaminergic neurons was observed, indicating treatment was neuroprotective. Motor function was also improved in all models; this was consistent with improved dopamine metabolism as indicated by increased levels of tyrosine hydroxylase within the substantia nigra and vesicular monoamine transporter2 in the striatum. The findings from this thesis establish the therapeutic effects of CuII(btsc) compounds in animal models and suggest that these compounds could be effective disease modifying therapeutic agents for neurodegeneration in clinical studies.