Florey Department of Neuroscience and Mental Health - Theses
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Itemα-synuclein, iron and Multiple System Atrophy( 2019)Multiple System Atrophy (MSA) is an atypical parkinsonian disorder characterised by progressive neurodegeneration in substantia nigra, striatum, cerebellum, pons, inferior olives and spinal cord. The presence of protein aggregates primarily composed of misfolded α-synuclein in oligodendrocytes is the pathological hallmark of MSA, classifying it as a synucleinopathy. However, the aetiology of MSA remains poorly understood and due to the lack of identification of potential targets for drug therapy, no disease modifying therapies are available. Brain region-specific changes in the metabolism of biological trace metals – especially iron and copper – have been reported in α-synucleinopathies like Parkinson’s disease but, their contribution in MSA pathogenesis requires further investigation. Hence, in this thesis, I studied the role of iron and copper in the pathogenesis of MSA using post mortem human MSA brains and a mouse model of MSA. Quantification of metal levels using inductively coupled plasma-mass spectrometry (ICP-MS) revealed an increase in cytosolic iron content in putamen and occipital cortex from MSA brains. Since ferritin is a major iron storage protein, the amount of iron bound to ferritin was investigated using size exclusion chromatography-ICP-MS and it was found that ferritin-bound iron remained unchanged in MSA brain. Furthermore, ferritin protein levels were also unchanged in MSA putamen and occipital cortex. In order to better understand how iron and copper levels change through the course of disease progression in MSA, I used a transgenic mouse model of MSA and studied age-dependent changes in these metals. I found increased iron in substantia nigra, putamen and cerebellum in aged MSA mice compared with non-transgenic littermates, and a copper-binding protein with a molecular weight consistent with ceruloplasmin had a significantly decreased copper content. Ceruloplasmin is a copper-dependent protein that is involved in iron export from cells. In addition, the levels of ferritin were found to be decreased. These results indicate that elevated iron in MSA mice may result from ceruloplasmin dysfunction. Decreased copper binding to ceruloplasmin may result into loss of activity and hence, impaired iron export from the cell leading to iron accumulation that could contribute to the ongoing neurodegeneration in MSA. I further investigated if administration of ceruloplasmin or deferiprone alleviated neuronal pathology and motor impairment in MSA mice. Deferiprone is a clinically approved iron chelator and exogenous ceruloplasmin administration has been shown to be therapeutic in animal models. Compared to vehicle treated mice, deferiprone and ceruloplasmin treatments prevented the decline in motor performance, prevented loss of substantia nigra neurons and reduced the number of α-synuclein aggregates in substantia nigra. The results from this proof of concept pre-clinical trial provide evidence that targeting iron in MSA could be a viable therapeutic option.
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ItemStem cells for the treatment of neurodegenerative disorders( 2016)Neurological disorders present a special challenge to medical science, because of their increasing prevalence, and the inability of the brain and spinal cord to repair itself. While the prevention of these conditions is preferable, the reality is that there will most likely always be a heavy dependence on therapies that treat established disease. Cell therapy holds significant promise for the treatment of these disorders, although substantial challenges remain before they can be progressed into mainstream therapies. This thesis explores some of the factors that affect the successful integration of stem cell-derived neural cells into the brain, in a series of experiments presented across four research chapters. Notably, the research has demonstrated that therapeutic neural cells derived from human iPS cells require a period of 12 months after implantation to mature, in a similar manner as what is observed in normal human development. This has implications for preclinical investigations utilising human cells to repair the damaged brain, which may require studies to run for periods of up to 1 year. These studies rely heavily on the transplantation of human therapeutic cells into discordant species, such as rodents and non-human primates. Accordingly, we conducted a systematic evaluation of the ability range of immune-modulating approaches to sustain human xenograft survival in the rat. While robust survival of hESC-derived neural cells was observed in athymic animals, survival of the same cells in immunocompetent adult or neonatal-grafted animals did not exceed 14 weeks. Immunosuppression by pharmaceutical agents resulted in cell-survival beyond 20 weeks, which was associated with a reduction in blood and brain T-cell quantities. These results demonstrate the utility of the athymic rat in xenografting studies, and provide practical information for the design of preclinical studies. When we attempted to utilise pharmacological immunosuppression to bolster grafted-cell survival in the SOD1G93A rat model of motor neuron disease, we observed a significantly detrimental impact associated with this agent on the motor performance of these animals. This highlighted the problem of immunosuppression in models of neurodegeneration, where the disease pathology is influenced by immune-effects. This presents a particular challenge to cell therapies aimed at treating these conditions, and we therefore evaluated the impact of treatment with three commonly used immunosuppressants, cyclosporin A, FK506 and rapamycin, on core disease characteristics in the SOD1G93A rat. Rotarod performance was disrupted by treatment with rapamycin and cyclosporin A, but not by treatment with FK506. The observed impairment occurred despite rapamycin reducing local expansion and recruitment of microglia, and cyclosporin A reducing levels of misfolded SOD1 protein within the spinal cord. In contrast, FK506 appeared to increase astrocyte activation, whilst not impairing behavioural outcomes. This study highlights that immunosuppression to support xenograft survival may directly affect important disease traits in models of neurodegeneration. Thus, the use of immunosuppression in such paradigms should be carefully considered within study design. While the long-term survival of implanted cells is a critical feature of successful stem cell-based therapies for neurodegenerative disorders, understanding the factors that guide the growth and integration of implanted neural cells is equally important. We performed a study that compared the fibre pathways of orthotopically and heterotopically transplanted fetal tissue-, as well as mouse embryonic stem cell- derived neural progenitors, with endogenous fibres of the intact adult murine cortex. Fetal tissue transplanted into the visual and motor cortices projected fibres across the entire dorsoventral axis of the adult brain. Quantification of the innervation of these fibres in specific targets of the cortex did not reveal an overwhelming tendency for grafted cells to target nuclei relevant to their intrinsic identity. While mouse embryonic stem cell-derived neural progenitors survived and expressed markers of mature cortical cells types in vivo, these cells did not demonstrate a high degree of axonal outgrowth. When quantified, there was no substantial difference in the innervation of specific cortical areas when mouse embryonic stem cell-derived neural progenitors were placed in either the motor or visual cortices. This suggests that the innervation patterns of implanted cells cannot be used to assign a particular areal identity to donor cells, as the passive outgrowth of fibres along host white matter tracts cannot be excluded as a possibility. Instead, immunohistochemical analysis of the expression of markers for specific areal identity may assist future studies in this purpose. Taken together, this work enhances our understanding of how certain factors must be managed to ensure the integration of donor cells in preclinical investigations, so that one day these studies can be progressed into therapies that support full patient rehabilitation.