Anatomy and Neuroscience - Theses
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ItemImaging and neuropsychological correlates of neurodegeneration and cerebrovascular disease in elderly Australians: vascular risk factors, vascular pathology and β-amyloid positron emission tomography (PET)( 2016)Vascular risk factors increase risk for dementia, however whether they contribute to the dementia phenotype through the pathology of Alzheimer’s disease (AD) or cerebrovascular disease (CVD) is unclear. This thesis utilizes multimodal neuroimaging with 3-Tesla Magnetic Resonance Imaging, 11C-PiB and 18F-NAV4694 Positron Emission Tomography (β-amyloid PET), and Carotid Artery Ultrasound (Intima-Media Thickness) in a cohort of older Australians to characterize associations between vascular risk, vascular pathology and AD-biomarkers, and their relative contributions to cognitive change over time. The broad aims of the thesis are to determine the associations between vascular risk factors and vascular pathology with molecular and structural neuroimaging markers of Alzheimer’s disease in a cohort of pre-symptomatic older Australians, and to identify potentially-modifiable factors associated with increased AD-pathology or cognitive loss which could represent potential targets for intervention studies in the future. This thesis comprises three areas of study. In the first (Chapter 6 and 7), Susceptibility- Weighted Magnetic Resonance Imaging (SWI) was used to assess prevalence and incidence of asymptomatic cerebral microhaemorrhage, and their relationship with Aβ- burden, other imaging biomarkers, and cognitive changes. In older cognitively-normal controls, lobar microhaemorrhage were common (19.1%) and associated with presence of Aβ (PiB+ status) and age, but not vascular risk factors. In this cohort, microbleeds were not independently associated with poorer cognition at baseline, nor incident cognitive decline, after adjustment for covariates including baseline Aβ-burden. Longitudinally, incident microbleeds were associated with Aβ burden, and markers of vascular disease (baseline microbleeds, white matter hyperintensity severity and lacunar infarction). The second area (Chapters 8 and 9) examined the relationship between vascular risk factors and vascular disease (cerebrovascular disease, microhaemorrhage and CIMT) with in vivo neuroimaging biomarkers of AD (Aβ-burden and brain atrophy), both cross-sectionally, and over six years’ follow-up. Burden of vascular risk was associated with the presence of Aβ, and vascular risk burden and genetic risk (Apolipoprotein [APOE] ε4 status) interacted significantly to influence Aβ most strongly in APOE ε4 carriers. Important independent relationships with Aβ also emerged for hypertension, hypercholesterolaemia, insulin resistance, and body mass index, some of which were also influenced by genetic risk (APOE ε4 status). An interaction between APOE ε4 and CVD was associated with greater Aβ, such that APOE ε4 carriers with CVD had greater Aβ burden. However, as this finding was negated by controlling for presence of microbleeds, the finding may be explained by presence of cerebral amyloid angiopathy in these participants. Carotid artery disease (high CIMT) was not associated with higher Aβ overall or in APOE ε4 carriers, however high CIMT was associated with greater Aβ accumulation over time in APOE ε4 non-carriers. The final area of study (Chapter 10) sought to determine the relative contributions between (MRI-defined) cerebrovascular disease and Aβ (PET imaging) biomarkers to change in cognitive performance and incident dementia in preclinical and prodromal AD. In cognitively-normal controls, presence of Aβ and CVD were each independently associated with longitudinal performance in memory, visuospatial function, attention and processing speed, however an interaction between Aβ and CVD was seen for change in executive function. Second, in a non-demented cohort of controls and MCI participants, risk of incident cognitive decline and dementia were most strongly influenced by presence of Aβ, rather than CVD, with an additive, rather than synergistic association seen between the two pathologies. These chapters describe the relationships between markers of increased vascular risk, evidence of peripheral and cerebral vascular pathology, Alzheimer’s disease biomarkers, and cognitive changes. Taken together, the findings suggest that several risk factors for vascular disease may also be associated with Alzheimer’s disease pathology, and that genetic risk (APOE ε4 status), and timing of assessment may influence observed associations. Interventions to mitigate vascular risk are likely to prove beneficial to reduce the imposte of dementia on future generations and intervention trials targeting modifiable lifestyle factors will be vital in this regard. The thesis also confirms the benefit of multimodal neuroimaging in combination with longitudinal clinical assessment in providing new insights into the natural history of dementia due to Alzheimer’s disease, particularly its presymptomatic stages.
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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.
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ItemPyroglutamate-Aß in the pathogenesis of Alzheimer's disease( 2013)Background: Amyloid-beta (Aβ) peptides are central contributors to Alzheimer’s disease (AD) pathogenesis. Although Aβ peptides are present in all human brains, the AD brain is particularly enriched for oligomeric Aβ species and Aβ peptides containing post-translational modifications such as oxidation, amino-truncation and pyroglutamate (pE). Metal interactions are a critical aspect of Aβ-induced neurotoxicity, however the effects of pE formation on Aβ-metal reactions such as nucleated oligomerisation, redox cycling and the production of reactive oxygen species (ROS) have not been investigated. Recent reports have indicated that pE-Aβ peptides are more neurotoxic than full-length Aβ, although a mechanistic difference in the toxic properties of these peptides has yet to be established. Increased levels of glutaminyl cyclase (QC) are thought to be responsible for the abundance of pE-Aβ in the AD brain, via the cyclisation of exposed N-terminal glutamate to pyroglutamate on amino-truncated Aβ. The relative levels of QC protein and mRNA are reported to be elevated in the temporal cortex and peripheral blood of individuals with AD compared to healthy controls. However, there are no published values of QC enzymatic activity in human central nervous system tissues. The involvement of QC in pE-Aβ formation and AD pathogenesis has led to the recent generation of QC inhibitors as a potential therapeutic intervention for AD. For this reason, there is a critical need to establish standardised levels of QC protein and activity in populations of healthy individuals and people with AD. Furthermore, there is a lack of animal models of pE-Aβ expression, thus the generation of simple pE-Aβ expression models may facilitate the study of potential QC inhibitors as an AD therapeutic. Objectives: I aimed to assess potential differences between synthetic pE-Aβ and full-length Aβ peptides in terms of their oligomerisation rate, fibril ultrastructure, cellular life-span and neurotoxicity. I also sought to compare the Aβ variants for their capacity to undergo nucleated polymerisation in the presence of Cu2+ or Zn2+, in addition to the generation of ROS and oxidative modifications such as dityrosine via redox cycling reactions with Cu2+ and ascorbate. To determine whether changes in soluble QC (sQC) expression and activity are a feature of AD pathogenesis, I aimed to establish standardised ranges of sQC protein and activity in the human brain through analysis of post-mortem cortical tissue samples from a cohort of AD and control brains. Finally, I sought to generate a Caenorhabditis elegans nematode model of pE-Aβ expression for in vivo comparisons of Aβ variant cytotoxicity and cellular interactions. Results: Vastly different rates of fibrilisation and fibril ultrastructures were observed for amino-truncated and pE-Aβ peptides compared with full-length peptides. Amino-truncated Aβ showed accelerated fibril seeding compared to full-length Aβ, while further addition of Cu2+ inhibited fibrilisation and produced aggregates of different ultrastructures between the seeded mixtures. In contrast, Zn2+ promoted fibrilisation but was also found to rapidly and reversibly aggregate Aβ peptides in short incubation periods. Redox-cycling reactions of Aβ, Cu2+ and ascorbate demonstrated significant differences between full-length Aβ and pE-Aβ peptides in the profiles of oligomers produced as well as the rate of hydroxyl radical production and dityrosine formation. The reaction of Aβ1-40 with Cu2+ and ascorbate was further found to cause amide-bond hydrolysis and the formation of amino-truncated Aβ peptides. Both the Aβ1-42 and Aβ3pE-42 peptides were toxic to cortical neurons and inhibited hippocampal long-term potentiation, however methodological differences in the preparation of peptides were found to significantly alter the relative Aβ neurotoxicity. Aβ1-42 was the only peptide to significantly increase neuronal ROS levels, suggesting that the toxicity observed for Aβ3pE-42 was ROS-independent. The levels of Aβ3pE-42 were much higher than Aβ1-42 following 48 h treatment of the peptides on cortical neurons, indicating that Aβ3pE-42 is highly resistant to proteolysis in neurons. Mean levels of sQC protein were modestly, though significantly, elevated in the frontal cortex of individuals with AD compared with healthy controls. No significant difference in the mean levels of total sQC activity or specific activity were observed between AD and control subjects. Gel electrophoresis and mass-spectrometry analyses of a C. elegans strain designed to express Aβ1-42 (CL2120) unexpectedly revealed that the predominant peptide expressed was actually Aβ3-42 – a precursor substrate for pE-Aβ formation. Genetic manipulation of this strain to co-express human sQC resulted in production of an additional Aβ species in these worms with hydrophobic properties consistent with Aβ3pE-42. Conclusions: Previous studies have established that amino-truncation and pE formation greatly enhance the oligomerisation and fibrilisation of Aβ peptides. The data presented here demonstrate that these modifications also affect the capacity of Aβ to undergo facile redox cycling with Cu2+, thus altering the relative production of cytotoxic ROS and oxidative protein modifications such as dityrosine. Aβ3pE-42 showed either comparable or enhanced toxicity to cortical neurons compared with Aβ1-42, although different peptide dissolution methods were seen to skew the relative toxicity of each peptide. Aβ1-42 greatly increased cytosolic ROS in neurons, whereas pE-Aβ peptides did not, suggesting that pE-Aβ induced neurotoxicity is ROS-independent. Furthermore, pyroglutamate formation renders Aβ resistant to proteolysis in neurons, indicating that pE-Aβ peptides are both neurotoxic and biologically persistent. The reported abundance of pE-Aβ in AD brains is not however a function of increased QC activity in the frontal cortex, suggesting that the rate of pE-Aβ formation is either due to regional-specific changes in QC activity or increased production of amino-truncated Aβ precursors, or both. The process of Aβ amino-truncation in vivo may also be due to multiple processes such as aminopeptidase activity and the interactions of Aβ with Cu2+. C. elegans models of pE-Aβ expression may facilitate further studies into the biological properties of these amyloidogenic peptides and the screening of potential therapeutics to inhibit their formation.