Pathology - Theses
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ItemDevelopment of a candidate for PET imaging of BACE1 in Alzheimer's diseaseDOWN, RUSSELL ( 2015)Alzheimer's disease (AD) is the most common form of dementia but there is currently no cure or disease modifying treatment. The pathological processes which lead to AD begin up to 20 years before the onset of symptoms, offering a large window for diagnosis and potential therapeutic intervention. β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartyl protease involved in the pathogenesis of AD. BACE1 levels are increased in AD patients and this elevation is known to occur early in the course of the disease; however, there is currently no method for measuring cortical BACE1 levels in vivo. It is hypothesised that the development of a BACE1 positron emission tomography (PET) imaging agent could have clinical utility for AD diagnosis as well as offering other benefits to AD research. A series of analogues of the hit compound (E)-2-(5-(3,4-dimethoxyphenyl)thiazol-2-yl)-3-(4-hydroxy-3-methoxyphenyl)acrylonitrile, identified from a screen of 70 compounds against BACE1 activity, was synthesised in order to generate a structure activity relationship. Initial attempts to radiolabel the general scaffold of the compounds with the 18F isotope by direct labelling were unsuccessful due to the instability of the scaffold under radiofluorination conditions; however, a route to radiolabel the scaffold was achieved by utilising [18F]-fluorobenzaldehyde as a prosthetic labelling group. The ability of the compounds to bind to BACE1 was assessed by a surface plasmon resonance (SPR) inhibition in solution assay. While a small number of compounds displayed limited activity, IC50 values could not be generated and most compounds were inactive. In addition, most of the compounds were too insoluble to be assessed for membrane permeability using the parallel artificial membrane permeability assay (PAMPA), although a small number of more soluble compounds were shown to have high permeability. Due to the slow progress and problems encountered in developing a de novo BACE1 PET imaging candidate, it was decided to modify an existing BACE1 inhibitor to incorporate the 18F radiolabel. MK-8931 (Merck) has a high affinity for BACE1 and is the most advanced clinical candidate for BACE1 inhibition and a synthetic route was developed to introduce a fluorine atom to the molecule. Following the synthesis of MK-8931, achieved by modification of a literature procedure, a radiofluorinated analogue, (S)-2-amino-6-(4-(5-(3-[18F]-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one, was prepared; unfortunately, the amount of labelled material was low and purification was not attempted. Analysis of (S)-2-amino-6-(4-(5-(3-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one by SPR showed an IC50 of 2.05 nM compared to 5.87 nM for that of MK-8931, and the passive membrane permeability of both compounds as measured by PAMPA was very similar. These results suggest that should the radiosynthesis be optimised, (S)-2-amino-6-(4-(5-(3-[18F]-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one would be a good candidate for PET imaging of BACE1.
ItemThe Role of metals and Aβ in excitotoxicity and Alzheimer’s diseaseJOHANSSEN, TIMOTHY ( 2015)Background: N-methyl-d-aspartate receptors (NMDARs) are ionotropic channels gated by the excitatory amino acid, glutamate. They play an essential role in synaptic plasticity, enhancing synaptic signal strength through long-term potentiation (LTP), a process thought to underlie learning and memory. At the synapse, NMDARs mediate neuroprotective signaling pathways including the regulation of calcineurin activity and inhibition of glycogen synthase kinase (GSK3). Under pathological conditions the prolonged and enhanced exposure of NMDARs to glutamate results in an excessive flux of calcium (Ca2+) into the cell. This triggers a range of responses resulting in cell death, including increased oxidative stress, inappropriate activation of proteases such as calpain, dysregulation of Ca2+- related pathways, mitochondrial damage and an apoptotic cascade. This process, termed excitotoxicity, contributes significantly to the acute neurodegeneration in ischemia and traumatic brain injury (TBI) and is believed to underlie the chronic neurodegeneration in Huntington’s disease (HD) and more recently, Alzheimer’s disease (AD). Alzheimer’s disease (AD) is characterised by progressive cognitive impairment resulting from synaptic degeneration and neuronal loss. A proposed key event in its aetiology is the formation of oligomeric species of the beta amyloid (Aβ) peptide. Recent work has demonstrated that the soluble Aβ oligomers induce excessive calcium influx across the cell membrane resulting in neuronal death by excitotoxicity. It is believed these toxic species of Aβ oligmomerise in the synaptic cleft between neurons in the hippocampus due to high levels of zinc and copper. These metals are released upon NMDAR activity from the pre- and post-synapse, respectively and can bind Aβ, increasing its rate of oligomerisation. Subsequent excitotoxic interactions between Aβ and NMDARs are copper (Cu2+)-dependent. In contrast, Cu2+ is also neuroprotective against excitotoxicity demonstrating the crucial role of metal homeostasis in specific regions of the brain affected by neurodegenerative diseases. Objectives: This PhD project has sought to determine the contribution of metals in excitotoxicity and whether modulating their levels could provide a mechanism to protect against this form of cell death. As excitotoxicity is strongly implicated in the aetiology of Alzheimer’s disease subsequent research aimed to describe the involvement of excitotoxicity in Aβ-mediated cell death in cortical neural model and to establish whether metals played a necessary role in this process. The final goal of the research presented here was the development of a neural-based assay, which could be employed to screen various forms of Aβ to detect more toxic forms of the peptide. Results: In experiments with the metal chaperone PBT2, a therapeutic in clinical trials for chronic neurodegenerative diseases, neurons were protected against excitotoxic cell death by pretreatment with the drug. Subsequent experiments demonstrated that this was a metal-mediated effect that required zinc. Pretreatment with this drug induced preconditioning in neurons by moderate increases in intracellular levels of calcium that activated survival pathways and inhibited activation of calcineurin and GSK3 preventing cell death. In further investigations the parameters for Aβ-induced excitotoxicity in cortical neurons were determined. In the presence of non-toxic levels of glutamate, Aβ induced significant toxicity that was dependent on the presence of metals, as demonstrated by metal chelation. These findings translated to the development of a calcium flux assay, which provided a functional readout of Aβ toxicity. Finally, this assay was validated by screening species of Aβ with varied degrees of toxicity to neurons. Conclusions: This work highlights the importance of metals in neurodegenerative disease and demonstrates modulation of both Cu2+ and Zn2+ levels in hippocampal synapses provide valid targets for future therapeutic approaches by preventing the formation of toxic oligomeric species. A concurrent finding has iii been the identification of the parameters required for Aβ-induced excitotoxicity, which provides the tools to screen an array of both in vivo and in vitro Aβ species to determine their toxicity. This knowledge will enable targeted clearance of these forms of the Aβ peptide, which, along with therapies preventing oligomer formation, will show significant therapeutic affects in the treatment of Alzheimer’s Disease.
ItemThe use of SELDI-TOF MS in the search for biological markers of Alzheimer’s diseaseWatt, Andrew David ( 2013)Alzheimer’s disease (AD) is an insidious, neurodegenerative disease characterised by progressive decline in memory and cognitive performance. However, despite more than a century passing since the discovery of AD, two major challenges in the field remain unmet: the ability to identify individuals with preclinical AD using large-scale population screens and the ability to delay or halt its progression. The advent of more sensitive technologies, such as surface enhanced laser desorption / ionisation time-of-flight mass spectrometry (SELDI-TOF MS), has enabled a more thorough investigation of Aβ profiles in AD-affected tissue. These investigations have revealed that the AD brain is besieged by an array of N-terminally truncated monomeric Aβn-42 peptides. However, despite the ability to detect synthetic Aβ dimers using this method, spectral evidence for the naturally occurring low-order oligomeric Aβ observable using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was not found. The formation of low-order oligomers of Aβ is believed to be a central component of AD pathogenesis; however, further examination using SELDI-TOF MS and SDS-PAGE revealed that the dimeric Aβ observable using SDS-PAGE was an SDS induced artefact. Attempts to regulate Aβ oligomerisation and aggregation within the AD brain and CSF have formed the basis of AD therapeutics and led to the development of a number of monoclonal anti-Aβ antibodies, such as bapineuzumab, solanezumab and crenezumab. All three antibodies demonstrated selective affinity for synthetic Aβ peptides, corresponding to their respective epitopes, when analysed using surface plasmon resonance (SPR). However, SELDI-TOF MS analysis of AD brain using these antibodies indicated that the selectivity of all three compounds was lost in complex biological samples, with each antibody binding a comparable array of Aβ species. These findings highlight the difficulty of targeting Aβ in AD, as complex interactions between different Aβ isoforms makes selective targeting of individual peptides inherently difficult. The central role of Aβ in AD has led to expansive research into its viability as a peripheral marker of disease. In this thesis, an investigation of Aβ levels in blood samples obtained from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Flagship Study of Aging was conducted using the WO2 antibody in conjunction with SELDI-TOF MS. This investigation revealed that observable levels of Aβ in the blood cellular fraction deteriorated over time, with variations in basic sample collection and handling procedures significantly affecting the stability of the peptide. An examination of the prevailing literature revealed a lack of consensus regarding the optimal pre-analytical methodology for blood-based investigations and an overwhelming failure amongst researchers in the field to adequately report basic methodologies, thus preventing accurate comparisons across investigations. Despite the instability of Aβ in the AIBL samples, further analysis was conducted using immobilised metal affinity capture and found that peripheral levels of α-defensin were elevated in AD. In the absence of Aβ levels, the α-defensin levels did not have the necessary power to produce a predictive model; however, the finding did act to support the growing body of literature reporting increases in peripheral inflammation as an early indicator of AD.
ItemIroning out the involvement of tau protein in neurodegenerative diseasesLEI, PENG ( 2012)Tau protein has been extensively implicated in Alzheimer’s disease (AD), Parkinson’s disease (PD), and other neurodegenerative diseases which exhibit tau depositions, termed tauopathies. Brain iron accumulation is a cooccurred pathological feature of many tauopathies and hypothesized to contribute to neurodegeneration by engendering oxidative stress. It is currently unknown what, if any, link exists between tau and iron accumulation in these diseases. The aims of this thesis were to understand, 1) how does tau participate in neurodegeneration; 2) whether tau is involved in brain iron homeostasis; and 3) whether this putative interaction contributes to neurodegeneration in the tauopathies. The normal function of tau has remained elusive, partly owing to the fact that tau knockout mice (tau KO) have been reported to be viable and fertile without behavioural deficits or neurodegeneration. These mice, however, have not extensively been investigated older than 7-months of age. Therefore, an analysis of aged (12-24 months) tau KO mice was undertaken in this thesis. Aged tau KO mice exhibited features of dementia (reduction of brain wet weight, neural cortical atrophy and cognitive impairment) and parkinsonism (L-DOPA responsive motor disability, neuron loss in substantia nigra [SN], striatal dopamine reduction and dopaminergic terminal atrophy). These observations may have implications for AD, which have been reported to exhibit reduced soluble tau in affected regions, and also PD, which was shown to have a similar reduction in this thesis. Iron accumulation was observed in the brains of aged tau KO mice. This specific brain-iron accumulation was prevented by chronic, orally administration of clioquinol, a moderate iron chelator previously reported to prevent MPTP-induced parkinsonism. When administration commenced before disease onset (6-months-of-age), chronic iron chelation prevented the onset of disease phenotype and neuronal loss in tau KO mice at advanced age. Likewise, treatment with clioquinol after the onset of disease (12-months-of-age) prevented further atrophy, and ameliorated behavioural disability. Therefore, tau depletion (as also observed in AD and PD) may engender pro−oxidant neuronal Fe2+ elevation preceding neurodegeneration. The mechanism of tau-induced iron accumulation was investigated in this thesis, which revealed a previously overlooked functional interaction between tau and the AD-implicated, amyloid precursor protein (APP). Immature APP undergoes several post-translational modifications before it is transported to the cell surface where it functions as an iron-export ferroxidase. Deletion of tau decreased the presence of mature APP presented on the cell surface which prevented the efficient neural export of iron. Disrupted trafficking of APP could therefore explain iron accumulation in the tau KO mouse, and in diseases exhibiting soluble tau reduction. Tau reduction may also be pharmacologically induced by the cation, lithium, which is a drug for bipolar disorder. In this thesis, lithium chloride caused intracellular iron accumulation and did not affect copper or zinc. This accumulation was abolished when treated to neurons that lacked tau or APP, demonstrating that lithium-induced iron accumulation was mediated by disruption to the tau-APP axis presented in this thesis. Lithium treatment has been previously associated with Parkinsonian side-effects; in this thesis, oral administration of lithium (in an upper-therapeutic range) to background mice caused brain-iron accumulation accompanying Parkinsonian neurodegeneration (motor disability, neuronal loss in SN, dopamine reduction in striatum). Given these striking findings, an MRI-analysis of iron in the brains of individuals who were treated with lithium (for three months) was performed, which revealed evidence of reversible iron elevation in selected regions upon treatment. This thesis, which investigated the function of tau and its loss-of-function phenotype, concluded that the reduction of soluble tau primes neurons for age-dependent neurodegeneration by decreasing APP-mediated iron export. Therefore, strategies that maintain tau solubility, or reduce iron content, may be promising therapeutic strategies for diseases featuring soluble tau loss such as AD and PD.
ItemMolecular basis of amyloid-β formation: focus on β-secretaseHolsinger, Ramsworth Michael Damian ( 2003)Aβ amyloid deposition is the pathognomonic feature of Alzheimer's disease (AD) and cytotoxic Aβ oligomers are considered to be responsible for neuronal degeneration. Aβ is proteolytically derived from the type 1 amyloid precursor protein (APP) by the sequential action of β- and y-secretases. This thesis focuses on the characterization of the recently identified β-secretase, BACE1 in human brain. This molecule has been propelled to the forefront of AD research due to its potential as a therapeutic target. The work presented here describes the first report of increased BACE1 protein and activity in AD brain. The BACE1 gene was cloned from human brain total RNA and used to transfect mammalian cells. Expressed protein was detected using three BACE1-specific antibodies generated during this candidature. Protein samples were then prepared from human brain and analyzed using BACE1 C-terminal antibody that revealed a significant 2.7-fold increase in BACE1 protein level in AD frontal cortex compared to age-matched and neurological controls. Examination of the C-terminal membrane-bound stub resulting from BACE1 cleavage demonstrated an ~2-fold increase in β-CTF levels confirming elevated enzyme activity. A more detailed analysis aimed at elucidating the mechanism by which BACE1 protein levels are increased in AD brain showed that this did not occur at the level of the message since BACE1 mRNA levels did not differ between control and AD groups. Analysis of lipid raft fractions prepared from AD and control brains demonstrated the presence of increased levels of amyloid generating machinery as well as amyloidogenic Aβ in rafts. Furthermore, it was also discovered by Western blotting that multiple BACE1 immunoreactive species were present in human brain and that this immunoreactivity was increased in more dense fractions that also contained lipid rafts. The second area of research describes the generation of recombinant BACE1 protein in a mammalian cell expression system. Cloning and characterization of the putative ectodomain construct revealed that a six amino acid sequence previously believed to be part of the ectodomain was sufficient to retain BACE1 within the cell, associated with the membrane as determined by biochemical, enzymatic and microscopic techniques. Removal of these six amino acids resulted in a secreted product that was purified from culture medium by anion chromatography coupled to FPLC. Using techniques developed in this section, lipid raft fractions were examined for BACE1 enzymatic activity and it was discovered that elevated levels of activity were associated with dense fractions. These results add support to those from Western blotting described above that showed increased BACE1 immunoreactivity coupled with that of other amyloidogenic proteins segregating in these dense fractions suggesting possible compartments where Aβ generation may occur. Work presented in the final section describes the first report of BACE1 enzyme activity measured in cerebrospinal fluid. Using a quenched fluorescence enzyme assay BACE1 activity was found to be elevated 3-fold in post-mortem AD CSF compared to control. This finding suggests the possibility of using BACE1 as a biological tool in the diagnosis of Alzheimer's disease. The work presented in this thesis describes the analysis of the β-secretase enzyme BACE1 in human brain. We have shown that BACE1 protein and activity are increased in AD brain and have identified probable compartments where this may occur. We have also shown that BACE1 activity is increased in AD CSF identifying this enzyme as a potential diagnostic marker of the disease.
ItemTherapeutic effects of copper bis(thiosemicarbazone) complexes in Alzheimer’s and Parkinson’s diseasesHUNG, LIN WAI ( 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.