Bio21 - Theses

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    Molecular characterisation of the amyloid precursor protein: a key biomolecule in Alzheimer's disease
    Young, Tessa Rose ( 2017)
    The amyloid precursor protein (APP) is strongly implicated in Alzheimer’s disease pathogenesis due to the observed aggregation of its Aβ sequence in the brain of AD patients. Despite being a molecule of intense interest, a sound molecular understanding of the normal functions of APP remains elusive. This work provides molecular insights into two ostensibly significant interactions involving the extracellular domain of APP, namely: (i) copper binding; and (ii) heparin-induced homo-dimerization. Biological copper is available in two oxidation states: Cu(I) and Cu(II). Well-characterised spectroscopic probes for Cu(I) binding studies are available, but reliable probes for Cu(II) binding studies are lacking. This work has developed a new method for in vitro characterisation of Cu(II) binding affinities. This involved the design and characterisation of four fluorescent peptide-based probes which respond to the binding of paramagnetic Cu(II) ions. Each displays a different affinity for the metal and together they are capable of detecting Cu(II) binding from micromolar to femtomolar range. This allows quantification of the Cu(II) binding affinities of individual protein targets on a unified scale. These probes facilitated studies of copper binding in the APP ectodomain. Biological studies performed over the last two decades have suggested that APP has a role in maintaining cellular copper homeostasis and it is commonly cited as a copper-binding protein. Several putative copper binding sites in the extracellular domain have previously been identified using structural techniques (NMR, X-ray crystallography). However, the chemistry of these has never been systematically studied under uniform conditions and the preferred metal coordination site is unknown. This work has characterised the thermodynamics of Cu(I) and Cu(II) binding in the APP ectodomain. The preferred binding site for both metal oxidation states is a (His)4 centre of picomolar affinity in the α-helical E2 domain. Interestingly, copper coordination leads to conformational changes in this flexible subdomain which could be significant in a cellular context. Homo- and hetero-dimeric interactions between APP and its ‘amyloid precursor-like protein’ homologues (APLP1 and APLP2) have been observed in many previous in vivo studies. These interactions are promoted by the binding of heparins (glycosaminoglycan chains abundant in the extracellular space). The dimers are thought to play a critical role in the normal functions of this protein family including trans-cellular adhesion and synaptogenesis. Detailed structural studies of these dimeric assemblies have not previously been performed. This is largely due to the size and heterogeneity of these complexes which present challenges for high-resolution techniques such as NMR and X-ray crystallography. However, homo-dimeric structures have been solved for two isolated extracellular domains (E1 and E2) which each contain heparin-binding regions. In this work, a chemical cross-linking mass spectrometry approach is used to provide structural information about the dimeric assembly of the complete secreted ectodomain sAPPα. The results offer direct proof of a parallel dimeric interaction between two sAPPα monomers. Additionally, this provides a means to evaluate E1 and E2 homo-dimeric models within the context of the overall ectodomain structure. This preliminary study provides a basis for further investigation of homo- and hetero-dimeric interactions involving APP homologues.  
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    The Role of metals and Aβ in excitotoxicity and Alzheimer’s disease
    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.