Florey Department of Neuroscience and Mental Health - Theses
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ItemSleep-wake dysfunction in human ischaemic stroke( 2020)Sleep-wake dysfunction is increasingly recognised as a key modifiable risk factor and consequence of stroke. Chronic sleep-wake abnormalities, characterised by excessively long sleep duration or sleep disorders, increase the risk of ischaemic stroke. Following stroke, de novo sleep-wake impairment is common and associated with poor recovery. However, the pathogenesis and evolution of sleep-wake disturbances in stroke have been poorly characterised thus far, largely due to methodological limitations. In this thesis, gold-standard sleep measurement tools and advanced MRI methodologies were applied to investigate the impact of chronic stroke on sleep-wake function. There were three primary research questions for this thesis, and each formed the conceptual framework for three major studies: (1) To what extent is sleep-wake dysfunction associated with both stroke risk and post-stroke evolution? (2) What are the neurodegenerative markers of sleep-wake after stroke? (3) What are the sleep architectural and sleep-respiratory characteristics of chronic stroke patients relative to healthy controls? To address the first question, a scoping systematic review of over 5,000 studies was conducted in order to assess the bidirectional relationship between sleep and circadian rhythm dysfunction in human ischaemic stroke. A qualitative synthesis of the extant literature showed that excessively long sleep duration and sleep disorders significantly increase the risk of ischaemic stroke. On the other hand, acute stroke patients exhibit fragmented sleep architecture in the weeks following the incident event – potentially driven by newfound sleep disorders which may also be associated with post-stroke topography and recovery. These findings support a bidirectional relationship between sleep-wake dysfunction and ischaemic stroke with important clinical implications. To expand on limitations of prior studies identified in the aforementioned systematic review, the associations between regional neurodegeneration and objectively measured sleep were investigated in a cohort of mild-to-moderate stroke patients and healthy controls from the Cognition and Neocortical Volume After Stroke (CANVAS) study. Stroke patients with excessively long sleep duration and poor sleep efficiency exhibited volumetric reductions to the thalamus and amygdala relative to healthy controls. Next, a novel method known as a whole brain fixel-based analysis was utilised to investigate fibre-specific white matter degeneration in stroke patients with poor sleep. Stroke patients with excessively long sleep duration exhibited tract-specific neurodegeneration to the cortico-ponto-cerebellar tract. These findings suggest that poor sleep efficiency or long sleep duration may contribute to neurodegeneration following stroke. The final study in this thesis aimed to characterise hemispheric sleep architecture and sleep-respiratory characteristics in stroke patients >4 years after their incident event using gold-standard polysomnography. In a subsample of patients from the CANVAS study, stroke patients and matched controls underwent overnight ambulatory polysomnography and completed an array of sleep and circadian questionnaires. Over half of all stroke patients in this sample exhibited undiagnosed moderate to severe obstructive sleep apnoea. Stroke patients had nearly 40% less restorative slow-wave sleep and potentially compensatory increases in lighter sleep stages relative to healthy controls. Sleep architectural disturbances were not attenuated by obstructive sleep apnoea. There were no sleep architectural differences in the stroke-affected versus healthy-hemisphere. These findings suggest that sleep impairment post-stroke is unlikely to be driven by comorbid obstructive sleep apnoea or the hemispheric distribution of stroke lesions. Furthermore, these results highlight the importance of formal sleep studies in stroke patients in order to identify undiagnosed obstructive sleep apnoea and fragmented sleep architecture. The overall findings from this thesis offer valuable insight into the potential in vivo pathogenesis of sleep-wake dysfunction after stroke and the evolution of sleep abnormalities in the chronic stages of stroke. The clinical-pathogenic implications of sleep-wake dysfunction in stroke are unravelled, and a research agenda for future studies in this emerging field of medicine is outlined.
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ItemThe role of copper in neurodegenerative ubiquitinopathy( 2019)Ubiquitin (Ub) is one of the most stable and soluble intracellular proteins. Intriguingly, the accumulation of intracellular Ub is associated to protein aggregates that are characteristic features of neurodegenerative diseases. Nevertheless, the factors that promote Ub aggregation and/or accumulation are unknown. Conversely, in vitro studies have shown that the interaction between Ub and cupric copper (Cu(II)) promotes the formation of high molecular weight (HMW), SDS and trypsin resistant, named as Ubres species. There is a general consensus that bioavailable copper only exists as Cu(I) in the cytosol, and there is limited evidence to support the presence of Cu(II) in this compartment. However, in vivo Ub denaturation may require a pool of labile Cu(II) that, to date, has not been identified. Cu(I) is maintained in the cytosol by a complex network of copper ligands and chaperones, and by reducing agents, predominantly glutathione (GSH). Brain glutathione depletion as well as copper dyshomeostasis are features of neurodegeneration. Therefore, we hypothesized that intracellular Cu(II) is elevated by the loss of GSH in these diseases. In this thesis, using XANES and a fluorescent Cu(II)-probe (CBS), we demonstrated that labile Cu(II) pools are present in the cytosol. Moreover, our results showed that decreasing GSH levels in cells, increases intracellular Cu(II) levels. Loss of Cu(I) and the emergence of Cu(II) may exacerbate the metabolic brain lesions in these neurodegenerative disorders. In fact, the increase in intracellular Cu(II) concurred with an increase in the levels of HMW Ub species in a SDS-insoluble cellular fraction, which were resistant to enzymatic proteolysis, characteristic features of Ubres species. We also found the presence of these Ubres species in brain tissue from Alzheimer’s disease and Lewy body dementia diagnosed patients. The outcomes of this research shed lights on the mechanism that may explain the ubiquinopathy of these neurogenerative disorders and may help to develop novel therapeutic strategies for these diseases.
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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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ItemUndetected preclinical neurodegenerative disease in models of normal cognitive aging( 2018)With population aging, understanding cognitive changes that occur in late life is vital to support these increasing numbers of older adults to maintain their wellbeing and independence. Furthermore, accurate estimates of age-related cognitive change will enable the differentiation of early stage neurodegenerative disease from normal aging. Current cognitive aging models describe a pattern of progressive decline in memory,executive function, and processing speed abilities, and retention of experience-based knowledge, with increasing age. However, given that many older adults show signs of neurodegenerative disease, despite not meeting clinical criteria for dementia, it is possible that cognitive aging studies may have over-estimated the nature and magnitude of age-related cognitive decline. The aim of this thesis was to determine the extent to which undetected preclinical neurodegenerative disease could influence models of cognitive aging. Age-related change in cognition was examined in cognitively normal healthy older adults who underwent repeated clinical and neuropsychological assessments, as well as biomarker assessment for neurodegenerative disease. The influence of progression to mild cognitive impairment (MCI) and dementia was also considered. Results indicated that estimates of age-related cognitive decline were inflated by undetected disease. This was also found when the data were reconceptualised as intelligence factors and was confirmed across two cohorts and utilizing a range of analytic methods. Notably, in the absence of disease, increasing age was associated with stability of performance in episodic and working memory, and an attenuated rate of decline in some processing speed and executive functions. Together these results indicate that current expectations about cognitive loss in aging are biased by unrecognized neurodegenerative disease.
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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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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.
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ItemNew insights into molecular and cellular pathways of neurodegeneration in amyotrophic lateral sclerosis models( 2016)Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and paralysing neurological disorder usually fatal within 2-5 years from diagnosis. First described by Jean-Martin Charcot in the late 1860s, ALS still remains a terminal disease with no effective treatments or cure. Riluzole is the only clinically-approved drug for ALS that may extend survival by 2-3 months. Therefore, there is an urgent need to understand the underlying pathogenesis of ALS to better guide development of disease-modifying treatment strategies. This thesis investigates the molecular basis of three inter-related pathogenic mechanisms implicated in motor neuron vulnerability and loss in ALS: defective energy homeostasis; disruption of protein homeostasis and abnormal RNA homeostasis. Two leading mouse models of ALS were implemented in these studies; transgenic SOD1G93A and TDP-43A315T mice in which novel pharmacological and genetic interventions were evaluated for efficacy. To examine whether defective energy metabolism is causal or consequential in the pathological cascade of ALS, the role of the key metabolic and stress sensor; AMP-activated protein kinase (AMPK) was investigated for the first time in two ALS mouse models. AMPK activation in the spinal cord associated with symptom progression, but not onset, in SOD1G93A mice, implicating AMPK activity in mediating disease course. Conversely, AMPK inactivation occurred in spinal cord and brain of pre-symptomatic TDP-43A315T mice by a protein phosphatase 2A-dependent mechanism, identifying a novel regulation of AMPK activity by pathogenic TDP-43. AMPK inactivity may therefore drive disease initiation in this mouse model. Hence, mutant SOD1 and TDP-43 exert contrasting effects on regulation of AMPK activation which may reflect intrinsic differences in energy metabolism and neurodegeneration in these two ALS mouse models. Next, a novel pharmacological strategy to improve protein homeostasis and motor neuron health was developed and evaluated for ALS. The intracellular catabolic pathway, autophagy, particularly macroautophagy, was robustly induced in mutant SOD1 and TDP-43 models of ALS. To potentiate autophagy in ALS, a novel autophagy enhancer rilmenidine was used to stimulate mTOR-independent macroautophagy in mutant SOD1 cell and mouse models. Rilmenidine treatment achieved efficient macroautophagy induction in vitro and in vivo. However, the treatment worsened motor neuron degeneration and survival of male SOD1G93A mice by exacerbating accumulation of insoluble and misfolded SOD1 species and aggregates in spinal cords. Thus, macroautophagy stimulation using rilmenidine may mediate disease progression in this specific mouse model of ALS. Lastly, a new gene therapy strategy to alleviate defects in the RNA binding protein TDP-43 was investigated. Survival motor neuron (SMN) protein deficiency causes progressive motor neuron degeneration in spinal muscular atrophy (SMA) and may be linked to pathology in ALS. SMN overexpression was previously determined to be beneficial in mutant SOD1 models of ALS. To extend these studies to TDP-43 proteinopathy, upregulation and accumulation of endogenous SMN protein into stress granules within motor neurons was demonstrated for the first time in TDP-43A315T mice. The impact of forced SMN overexpression in TDP-43A315T mice was examined, revealing improved SMN nuclear targeting, motor neuron survival, neuroinflammation and metabolic deficits as shown by AMPK activation, in female mice. Furthermore, levels of androgen receptor (AR), mutations of which cause spinal bulbar muscular atrophy (SBMA), were significantly impaired in spinal cords of male TDP-43A315T mice. This provides evidence for shared biochemical pathways in ALS, SMA and SBMA, mediated by deficiency of factors such as SMN and AR which confer motor neuron vulnerability. In summary, in mutant SOD1-linked disease, persistent AMPK signalling and autophagy activation in motor neurons may be key determinants of disease progression. In mutant TDP-43-mediated ALS, AMPK inactivation and cytoplasmic accumulation of SMN in motor neurons may be early events triggering disease onset. In conclusion, this thesis provides novel insights into pathogenic mechanisms underlying disruption of energy, protein and RNA homeostasis within motor neurons and significant clues to therapeutic alleviation of these defective pathways in ALS. In addition, this thesis identifies new links between three main neurological disorders affecting the motor system of humans; ALS, SMA and SBMA, mediated by dysregulation of SMN and AR, suggesting shared pathogenic pathways. Finally, this work importantly extends the spectrum of motor neuron diseases that may benefit from SMN restoration, excitingly paving the way for future therapeutic development and testing of SMN enhancing agents for ALS.