Pharmacology and Therapeutics - Theses
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ItemDevelopment and application of advanced bioimaging techniques to investigate stress pathways and drug action in neurodegeneration( 2019)Neurodegenerative diseases such as Frontotemporal Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS), also known as Motor Neuron Disease (MND), have limited therapeutics available, placing a high demand on healthcare systems and overall economic and social burden. Recently, it has been found that pathological mutations in RNA binding proteins such as TDP-43 (Tar DNA-binding Protein, 43kDa), FUS (Fused in Sarcoma), and hnRNP (heterogenous nuclear RiboNucleoProteins) can be causative of ALS or FTD. Cell stress has been implicated in ALS and FTD, particularly oxidative stress, mitochondrial dysfunction, ATP depletion and metal dyshomeostasis. In cell culture models, it has been shown that TDP-43 and hnRNP proteins can incorporate into stress granules in conditions of cell stress, including during oxidative stress, metal dyshomeostasis or conditions of ATP depletion, however this is not well defined. Furthermore, there is limited evidence of FUS incorporating within stress granules. The study described here has enhanced the understanding of RNA binding proteins in vitro. Utilising neurodegeneration-associated cell stress pathways in conjunction with advanced bioimaging techniques, we show that both non-mutated FUS and non-mutated TDP-43 can occur within the same stress granule. Furthermore, we show different populations of TDP-43 and FUS within stress granules from different stress types. This study also enhanced our understanding of the sub-cellular distribution of a neuroprotective metal-complex therapeutic, utilising fluorescence lifetime imaging on live cells; this therapeutic has previously been shown to be neuroprotective against TDP-43 aggregation. To extend the study, a novel metal complex was utilised in attempt to enhance the field of techniques available to study RNA binding proteins in vitro. Overall, this thesis has advanced our understanding of disease pathways, therapeutic action, and new investigative tools for neurodegeneration.
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ItemInvestigating Aβ toxicity and binding to neurons from differentiated human stem cells( 2019)Alzheimer’s disease (AD) is a neurodegenerative disease that is pathologically characterized by abnormal deposition of extracellular amyloid plaques and intraneuronal neurofibrillary tangles. The deposition of these aggregated proteins causes progressive brain atrophy resulting from gradual synaptic loss and neuronal cell death. Although the aetiology of AD remains elusive, studies have shown that amyloid beta (Aβ) peptide, a cleavage product from amyloid precursor protein (APP), is a key protein causing AD pathogenesis. Recent studies have identified how the presence of soluble low molecular weight Aβ oligomers in the brain correlate best with synaptic loss, and they are a better predictor of disease progression compared to the presence of amyloid plaques or neurofibrillary tangles. In the AD brain, neuronal subpopulations appear to exhibit different levels of vulnerability to Aβ, particularly the basal forebrain cholinergic and hippocampal glutamatergic neurons, while GABAergic neurons appear to remain unaffected till later disease stages. Current treatments based on knowledge gathered from mouse models targeting the cholinergic and glutamatergic systems only alleviate symptoms and are ineffective in halting disease progression. Therefore, we hypothesize that Aβ exerts its neurotoxic effect by binding to a subpopulation of mature neurons. To address this, human embryonic stem cells (hESCs) were differentiated into mature glutamatergic and GABAergic neurons and cultured up to 12 weeks. These cultures were treated fortnightly with soluble synthetic Aβ peptide for 96 hrs. We found that Aβ bound to neurites in culture, altered gene expression and neurotoxicity was more pronounced in 6-week old glutamatergic than GABAergic cultures. Further investigations in determining the specific toxic species of Aβ oligomers revealed that 12 week old cultures were more susceptible to Aβ oligomer induced toxicity, with Aβ dimers being most toxic to glutamatergic neurons while GABAergic neurons were most susceptible to Aβ tetramers. In summary, we successfully established a simple hESC based model to study Aβ toxicity. Our findings also highlight the importance of using relevant human cell-based models to study AD pathogenesis as well as identify potential AD modifying therapeutic strategies.
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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.
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ItemOn type-1 interferons, neuro-inflammation and Alzheimer's disease( 2015)Alzheimer’s disease (AD) is the most common form of dementia worldwide. Hyper-phosphorylation of tau, leading to intracellular neurofibrillary tangles, and accumulation of the amyloid-β (Aβ) peptide, leading to formation extracellular plaques, are the two key brain proteinopathies associated with the progressive neuro-degenerative disease. To date, therapeutics targeting these pathologies have proven ineffective in the treatment of AD and highlights the need for new lines of investigation into disease mechanisms. Neuro-inflammation is evident in AD patients, comprising of enhanced gliosis surrounding Aβ deposits and pro-inflammatory cytokine load. This dysregulated innate inflammatory response is deleterious and facilitates neuro-degeneration. Identifying critical mediators controlling this neuro-inflammation will prove beneficial in developing anti-inflammatory therapies for the treatment of AD. The type-1 interferons (IFNs) are pleiotropic cytokines that control pro-inflammatory cytokine secretion and are master regulators of the innate immune response. This thesis carries the hypothesis that the type-1 IFNs play a critical role in the exacerbation of neuro-inflammation and actively contribute to the progression of AD. This thesis aimed to characterise the role of type-1 IFNs in the neuro-inflammatory response to soluble Aβ1-42 in CNS cell types, evaluate the effect of removing type-1 IFN signalling in the APPSWE/PS1ΔE9 mouse model of AD and hence identify a role for type-1 IFNs in the progression of AD. Soluble Aβ1-42 triggers a type-1 IFN neuro-inflammatory response in primary cultured neurons. Removal of type-1 IFN signalling (IFNAR1-/-) in these cultures attenuated pro-inflammatory responses to Aβ1-42 affording protection against neurotoxicity via attenuation of pro-apoptotic caspase-3 activation. The use of Myd88-/-, IRF7 and IRF3 knockdown cultures, critical in toll-like receptor-dependent signalling, identified that neurons utilise this receptor family to detect Aβ1-42 and initiate a neuro-degenerative type-1 IFN response. Primary IFNAR1-/- mixed astrocyte and microglial cultures display an attenuated type-1 IFN and pro-inflammatory cytokine response to Aβ1-42. These cultures adopt an anti-inflammatory and neuro-protective M2-like polarisation state, opposing the wildtype neuro-degenerative M1-like response to soluble Aβ1-42. To investigate the role of in vivo type-1 IFN signalling in the progression of AD, APPSWE/PS1ΔE9 mice lacking type-1 IFN signaling were generated. APPSWE/PS1ΔE9 x IFNAR1-/- were partially rescued from spatial learning and memory deficits as assessed by the Morris water maze. These mice displayed no alterations in amyloid plaque load but reduced soluble Aβ monomer concentrations were detected. The type-1 IFN response was attenuated in APPSWE/PS1ΔE9 x IFNAR1-/- mice displaying altered pro-inflammatory cytokine expression. Interestingly, cortical astrogliosis was elevated in these mice but microgliosis was attenuated. These microglial populations adopted a neuro-protective M2-like activation state, supporting our in vitro findings. Finally, a type-1 IFN signature was evident in the brains of human post-mortem AD patients Findings from this thesis identify the type-1 IFNs as key mediators of the neuro-inflammatory response in AD. This response is deleterious to disease progression and suggests that targeting type-1 IFN signalling may be therapeutically relevant for anti-inflammatory treatment of AD.