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Chronic inflammation in response to Aß peptide deposits is a pathological hallmark of Alzheimer's disease (AD). The inflammatory environment includes populations of reactive and proliferating microglia and astrocytes and perturbed vasculature. However, the association between activated glial cells and cerebrovascular dysfunction remain largely unknown. This study has used Aß1-42 intrahippocampal injection as an animal model of inflamed AD brain to characterize mechanisms of glial-vasculature responses as a basis for chronic inflammation. Preliminary findings suggested Aß1-42-injected brain demonstrated vascular remodeling including evidence for formation of new blood vessels (angiogenesis). This result led to study of the effects of the anti-angiogenic/anti-inflammatory compound, thalidomide on activated glial cells and perturbations in the vasculature in an Aß1-42 peptide-injected rat model. First, Aß1-42 injection was found to cause perturbations in vasculature including new blood vessel formation and increased BBB leakiness. Second, thalidomide decreased the vascular perturbations and the glial reactivity and conferred neuroprotection. Overall, these results suggest that altered cerebral vasculature is integral to the overall inflammatory response induced by peptide. Experiments then examined the level of parenchymal plasma proteins in brain tissue from AD and nondemented (ND) individuals. AD, but not ND, brain tissue demonstrated high levels of fibrinogen immunoreactivity (ir). Aß1_42 injection into the rat hippocampus increased the level of parenchymal fibrinogen, which was reduced by treatment with the defibrinogenating agent, ancrod. In addition, ancrod also attenuated microglial activation and prevented neuronal injury. Overall, these results demonstrate that extravasation of blood protein and a leaky BBB are important in promoting and amplifying inflammatory responses and causing neuronal damage in inflamed AD brain. Microglial chemotactic responses to VEGF (vascular endothelial growth factor) receptor Flt-1 were next studied. Treatment with a monoclonal antibody to Flt-1 (anti-Flt-1 Ab) in the peptide-injected hippocampus diminished microglial reactivity and provided neuroprotection. Secondly, anti-Flt-1 Ab inhibited the AI3142-induced migration of human microglia. These results suggest critical functional roles for Flt-1 in mediating microglial chemotaxis and inflammatory responses in AD brain. The overall conclusion from my work is that AP deposits induce microglial reactivity which subsequently causes vascular remodeling resulting in an amplified inflammatory microenvironment which is damaging to bystander neurons.

The current landscape of nanotechnology has focussed attention on materials that self-assemble. The search for such materials has unsurprisingly led to the biological world, where functional nanoscale biomolecular assemblies are in abundance. Amyloid fibrils are one such self-assembling biological structure, formed when native proteins misfold into insoluble fibrous quaternary structures. This research has explored the use of amyloid fibrils formed from waste proteins, namely crude crystallin proteins from fish eye lenses, as biological nanowires. The use of amyloid fibrils as nanowires was investigated by examining the ability to control their dimensions and arrangement, along with analysis of their properties, such as stability and conductivity. TEM and AFM studies on the model amyloid forming protein, bovine insulin, showed that a number of fibril length distributions can be achieved, by systematically altering fibril growth and storage conditions. Although the same set of conditions cannot be directly applied to crystallin fibrils, these fibrils can also be produced on a range of length scales. Amyloid fibrils can be manipulated and aligned in a controlled manner by dielectrophoresis; this tool could later be used to incorporate amyloid fibrils into a biosensing or bioelectronics device. Dielectrophoresis was also used to immobilise crystallin fibrils between electrode pairs, in order to investigate the conductivity of small numbers of fibrils. These experiments complemented work carried out on the conductivity of amyloid fibril networks, using fabricated interdigitated electrodes. In the unmodified state, amyloid fibrils formed from bovine insulin, fungal hydrophobins, and crude crystallins were all shown to have low conductivity, with current values in the range of 10⁻⁸–10⁻¹⁰ A recorded at bias voltages of 0–2 V. Amyloid fibrils were used as a template for the synthesis of conductive nanowires, by modification with the conducting polymers polyaniline and polypyrrole, increasing conductivity by one and four orders of magnitude respectively. The functionalisation of fibrils with glucose oxidase enabled the creation of a very simple glucose sensing device. This device, consisting of a gold electrode modified with the glucose oxidase functionalised fibrils, showed an electrochemical response in the presence of glucose and the mediator FcOH. Future work is necessary to optimise the use of amyloid fibrils in this way; however, this study confirms a role for amyloid fibrils from a low cost source in bionanotechnology.

Alzheimer’s disease (AD) is a progressive dementia affecting cognition, behavior, and functional status and there is no cure which exists for it. In AD, Amyloid Beta (Aβ) peptides form aggregates that are neurotoxic in the brain. Hence, molecules that are able to prevent Aß aggregation could be effective in AD treatment. Gemini surfactant (GS) molecules consist of two hydrophilic heads separated by a covalently bound spacer and two hydrophobic tails. Their structure gives rise to a number of unique properties, including low critical micelle concentrations, the ability to form multiple types of aggregates (governed primarily by the nature of the spacer group) and enhanced ability to bind to polymers. These properties make gemini surfactant a good choice for solubilizing very hydrophobic materials such as Aß. The aim of this study was to examine various GS structures to help us to understand their interaction with Aβ and the influence of spacer group in Aβ disassembly. We employed 12-carbon tail GS with varying spacer groups of different hydrophilicities, such as: (-CH2-CH2-O)m, (-CH2)m, N(CH2)m, OH(CH2)4 and (OH)2(CH2)4. Surface tension measurement, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) have been employed to observe the gemini-Aβ interaction. Surface tension measurements did not show a typical surfactant-polymer interaction; rather, the presence of Aß induced aggregate formation at concentrations well below the cmc. Headgroup areas were observed to decrease for some of the surfactants in the presence of Aß, which may result from partial neutralization of the surfactant headgroups and a relaxation of electrostatic repulsion resulting in decreased head group areas. ITC results suggest substantial reorganization of Aß/gemini surfactant aggregates, with distinct difference seen depending upon the nature of the headgroup. It was observed that in 12-(CH2)n-12 (n=2,3,4,7) shorter spacer gemini surfactants have stronger interaction with Aß than the ones with longer spacers. In the 12-4(OH)n-12 series, a stronger interaction was observed in the GS with 2 hydroxyl groups compared to one hydroxyl group GS. For 12-(EO)n-12 GS, a stronger interaction was observed in that GS with two ethoxy groups. In the 12-XN-12 series, although the 8N spacer is more hydrophilic than 5N, the interaction of 12-5N-12 with Aß was stronger than that of 12-8N-12. The particle size data also revealed that there is an interaction between gemini surfactant and Aß. It appeared that mixed micelles formed when the surfactant concentration increased in the Aß solution. Overall, it was observed that changes in the length and hydrophilic character of the gemini surfactant spacer influenced the type of interaction and gemini-Aβ conformation.

The formation of amyloid fibrils is a key characteristic of many neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. Similarly prion diseases, those associated with the prion protein, are neurodegenerative disorders with characteristic protein aggregates accumulating in the brain of affected individuals. While fibrillar deposits of these disorders have long been associated with end-stage disease pathology, it is currently hypothesized that protein oligomers are the cytotoxic structural form of these systems. Residues 106-126 of the human prion protein have been found to form both amyloid fibrils, as well as toxic amyloid oligomers and thus provide a suitable model system. This thesis aims to describe the structures of the amyloid fibrils and oligomers formed by PrP(106-126), how they are interrelated as well as interaction with model membranes and cytotoxicity. Amyloid fibrils of PrP(106-126) contain long, unbranched filaments that contain β-sheet secondary structure and bind the amyloid-indicating dye, thioflavin-T. These fibrils are comprised of parallel β-sheets, stacked in an antiparallel fashion. The non-fibrillar amyloid oligomers are large, spherical structures that contain β-sheets but do not bind thioflavin-T. It was determined that these oligomers contain parallel β-sheets as well as the same intersheet packing as fibrils of PrP(106-126). Finally, the interaction of PrP(106-126) with lipid bilayers and cells was examined. Oligomers of PrP(106-126) were shown to affect model membranes; with anionic lipids losing integrity and cholesterol-containing lipid mixtures losing domain structure upon peptide addition. Additionally, amyloid oligomers of PrP(106-126) cause cell death across a number of cell lines as well as rat cerebellar slices. Overall, these results indicate that the conversion of oligomers to fibrils may be facilitated due to structural similarities between the two. Additionally, the toxicity of PrP(106-126) oligomers may be attributed to a loss of cholesterol domain structure causing subsequent cell death.

The formation of amyloid fibrils is a key characteristic of many neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. Similarly prion diseases, those associated with the prion protein, are neurodegenerative disorders with characteristic protein aggregates accumulating in the brain of affected individuals. While fibrillar deposits of these disorders have long been associated with end-stage disease pathology, it is currently hypothesized that protein oligomers are the cytotoxic structural form of these systems. Residues 106-126 of the human prion protein have been found to form both amyloid fibrils, as well as toxic amyloid oligomers and thus provide a suitable model system. This thesis aims to describe the structures of the amyloid fibrils and oligomers formed by PrP(106-126), how they are interrelated as well as interaction with model membranes and cytotoxicity. Amyloid fibrils of PrP(106-126) contain long, unbranched filaments that contain β-sheet secondary structure and bind the amyloid-indicating dye, thioflavin-T. These fibrils are comprised of parallel β-sheets, stacked in an antiparallel fashion. The non-fibrillar amyloid oligomers are large, spherical structures that contain β-sheets but do not bind thioflavin-T. It was determined that these oligomers contain parallel β-sheets as well as the same intersheet packing as fibrils of PrP(106-126). Finally, the interaction of PrP(106-126) with lipid bilayers and cells was examined. Oligomers of PrP(106-126) were shown to affect model membranes; with anionic lipids losing integrity and cholesterol-containing lipid mixtures losing domain structure upon peptide addition. Additionally, amyloid oligomers of PrP(106-126) cause cell death across a number of cell lines as well as rat cerebellar slices. Overall, these results indicate that the conversion of oligomers to fibrils may be facilitated due to structural similarities between the two. Additionally, the toxicity of PrP(106-126) oligomers may be attributed to a loss of cholesterol domain structure causing subsequent cell death.

Alzheimer??s disease (AD) is the most common form of dementia affecting the elderly. Extracellular deposition of beta amyloid (Abeta plaques), intraneuronal tau accumulation, inflammation (activated astrocytes and microglia), and neuronal loss are all consistent pathological features of the disease. Unlike Abeta plaques, inflammation correlates with neuronal loss and cognitive decline in AD, suggesting it plays an important role in disease progression. Recent research has also identified soluble oligomeric Abeta species in the AD brain, which correlate with disease progression and are proposed to be more neurotoxic than their fibrillar counterpart. The main aims of this thesis were to determine whether oligomeric Abeta is a more powerful stimulator of the inflammatory response compared to fibrillar forms of the protein, to identify potential mediators of this response and to determine downstream neuronal changes. Isolated human primary astrocytic, microglial and neuronal cell cultures were used to assess which Abeta alloform and conformation was more neurotoxic and neuroinflammatory. A total of 17 inflammatory cytokines and chemokines were measured simultaneously using a Multiplex approach and neurotoxicity was assessed by lactate dehydrogenase release. Cell cultures involving all three cell types were also used to examine these inflammatory mediators in a more complex system, and direct changes in tau levels and/or phosphorylation determined by western immunoblotting. Further immunohistochemical analysis determined the localisation of oligomeric Abeta within post-mortem human AD brain tissue. The data clearly shows that oligomeric Abeta was more neurotoxic and neuroinflammatory than fibrillar Abeta, with oligomeric Abeta40 favoured in toxic plaque deposits in AD brain tissue. Cell culture experiments showed that glia clearly mediate neuroprotection against oligomeric Abeta. Soluble TNF-alpha, IL-2, IL-4 and IL-12 mediate this response early in disease, with a decline in their secretion and a sustained increase in IL-1alpha, IFN-gamma, GM-CSF, MIP-1beta, and IL-8 provoking increased tau protein expression. Overall, the data clearly demonstrates oligomeric Abeta more powerfully stimulates a suite of inflammatory mediators affecting neuronal survival and tau pathology, although the contribution of additional glial-derived factors must also be determined. The identification of these inflammatory mediators, in combination with other potential factors, may lead to the development of mechanistic therapeutic interventions that could target these early pathological changes.

Alzheimer??s disease (AD) is the most common form of dementia affecting the elderly. Extracellular deposition of beta amyloid (Abeta plaques), intraneuronal tau accumulation, inflammation (activated astrocytes and microglia), and neuronal loss are all consistent pathological features of the disease. Unlike Abeta plaques, inflammation correlates with neuronal loss and cognitive decline in AD, suggesting it plays an important role in disease progression. Recent research has also identified soluble oligomeric Abeta species in the AD brain, which correlate with disease progression and are proposed to be more neurotoxic than their fibrillar counterpart. The main aims of this thesis were to determine whether oligomeric Abeta is a more powerful stimulator of the inflammatory response compared to fibrillar forms of the protein, to identify potential mediators of this response and to determine downstream neuronal changes. Isolated human primary astrocytic, microglial and neuronal cell cultures were used to assess which Abeta alloform and conformation was more neurotoxic and neuroinflammatory. A total of 17 inflammatory cytokines and chemokines were measured simultaneously using a Multiplex approach and neurotoxicity was assessed by lactate dehydrogenase release. Cell cultures involving all three cell types were also used to examine these inflammatory mediators in a more complex system, and direct changes in tau levels and/or phosphorylation determined by western immunoblotting. Further immunohistochemical analysis determined the localisation of oligomeric Abeta within post-mortem human AD brain tissue. The data clearly shows that oligomeric Abeta was more neurotoxic and neuroinflammatory than fibrillar Abeta, with oligomeric Abeta40 favoured in toxic plaque deposits in AD brain tissue. Cell culture experiments showed that glia clearly mediate neuroprotection against oligomeric Abeta. Soluble TNF-alpha, IL-2, IL-4 and IL-12 mediate this response early in disease, with a decline in their secretion and a sustained increase in IL-1alpha, IFN-gamma, GM-CSF, MIP-1beta, and IL-8 provoking increased tau protein expression. Overall, the data clearly demonstrates oligomeric Abeta more powerfully stimulates a suite of inflammatory mediators affecting neuronal survival and tau pathology, although the contribution of additional glial-derived factors must also be determined. The identification of these inflammatory mediators, in combination with other potential factors, may lead to the development of mechanistic therapeutic interventions that could target these early pathological changes.