Thesis (Ph.D.) -- University of Adelaide, Dept. of Pathology, 1999. / Appendum pasted into front end-papers. Bibliography: leaves xiii-xliii.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 113-125). Also available in electronic version. Access restricted to campus users.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 14, 2009) Includes bibliographical references.

Alzheimer’s disease (AD) is the most common form of dementia, with more than 25 million people worldwide suffering this progressive intellectual failure. The disease was first described by the German psychiatrist, Alois Alzheimer in 1907, and is characterised by the appearance of proteinaceous depositions (first isolated in 1984), which are comprised of insoluble amyloid-ß (Aß)-aggregates. Aβ is derived from the β-amyloid precursor protein from which it is generated by the action of two proteases. Initially it was assumed that the insoluble amyloid fibrils, which were easily detectable, mediated the observed toxicity although it was recognised that amyloid plaque number did not correlate well with the severity of dementia. However, further studies with synthetic and human-derived Aß provided strong evidence that soluble prefibrillar aggregates of Aß mediated the synaptic failure and loss of cognitive performance. In 2008 genetic evidence showed that the presence of soluble Aß-oligomers is sufficient to cause an AD-like dementia, which centres the oligomeric Aβ as the probable effector of synapse loss. Although a variety of assemblies have been described their meta-stability and technical limitations caused a controversial debate about aggregate related pathogenesis. Thus, this study aimed to establish a structure-activity relationship comparing different synthetic Aß-aggregates using biophysical methods to follow aggregation and to assess morphology, absolute MW and meta-stability of monomeric, oligomeric, protofibrillar and fibrillar Aß. However, interference with the aggregate equilibrium, by changing the ionic environment, can cause structural conversion of Aß-aggregates. Therefore, different Aß-aggregates were only compared in short-termed physiological settings i.e. neuronal binding and hippocampal neurotransmission. Herein, only prefibrillar aggregates bound to neurons and differentially impaired hippocampal neurotransmission either by inhibition of basal neurotransmission or NMDA-dependent long-term potentiation. In addition, changing the ionic environment provoked a structural conversion, which also changed the pathogenic mode of action. This study provides experimental evidence that different soluble Aß-aggregates are highly potent synaptotoxins, impairing neurotransmission by different mechanisms. Furthermore, solution-based biophysical characterisation and acute biological paradigms are crucial for differential characterisation of Aß-aggregates revealing that virtually similar aggregates can have opponent pathogenic effects; thus, morphology only does not explain observed pathogenicity.

Late-onset Alzheimer’s disease (LOAD) is the most common form of dementia, with an incidence of up to 50% in western populations over the age of 85 and a high heritability (up to 80%). The identification of risk factors for the development of LOAD is imperative for improving our understanding of this disease and for identifying therapeutic targets for treatment or prevention. Currently, the major known risk factors for the development of LOAD are age and the ApoE ε4 genotype. Previous studies have implicated plasma levels of the amyloid beta (Aß) peptide as a LOAD-associated quantitative trait and identification of loci influencing this trait could provide new insights into LOAD. In this thesis, plasma levels of the Aß peptides Aß40 and Aß42 have been measured in two isolated populations and genome-wide linkage and association analyses were performed. The genome-wide association analyses identified a number of promising quantitative trait loci; highlighting both novel and previously reported LOAD genes for further study, whilst also providing an excellent resource for genetic convergence studies with other LOAD related traits. Several studies have reported an association between levels of oxidative stress and levels of Aß such that increasing levels of Aß appear to increase markers for oxidative stress and vice versa. The role of oxidative stress in LOAD and aging was therefore also investigated through analysis of mitochondrial mutational burden and DNA damage respectively, using DNA isolated from both blood and the brain and by carrying out a candidate gene association study of loci involved in mitochondrial function in LOAD cases and controls. Approaches to the investigation of mitochondrial genetics for the study of LOAD are comprehensively reviewed, adapted and tested and the results indicate a need for additional research in this aspect of the disease. This thesis therefore presents a focus on two aspects of genetic research into LOAD, a complex disease with multiple environmental and genetic influences which aims to advance our understanding of the disease and bring us closer to treatment and prevention strategies.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Amyloid formation occurs when normally soluble proteins and peptides misfold and aggregate into intractable threadlike structures called fibrils. There are currently more than 30 proteins associated with this aberrant structure, including the Aβ peptide in Alzheimer’s disease (AD) and transthyretin (TTR) in TTR amyloidosis. TTR is a homotetrameric transporter protein present in both cerebrospinal fluid and plasma. Dissociation of its tetrameric structure is required for the formation of amyloid fibrils. Small molecule ligands able to bind and stabilize the tetrameric structure of TTR thus represent a potential therapeutic intervention. Interestingly, apart from TTR’s role as a toxic agent in TTR amyloidosis, it also has a role as an inhibitor of the Aβ toxicity associated with AD. The work presented in this thesis focused on small molecules that have the potential ability to prevent TTR amyloidosis. We also sought to gain a greater understanding of the interaction between TTR and the Aβ peptide with respect to Aβ fibril formation. The ability of a drug to stabilize TTR is directly correlated to its binding affinity. However, since TTR is a plasma protein, it is of great importance that the drug binds selectively to TTR. In paper I, we used a newly developed urea denaturation assay, in combination with isothermal titration calorimetry, to show that, in a complex environment such as plasma, the enthalpy of binding correlates better with a drug’s ability to stabilize TTR than the binding affinity. In paper II, we modified the highly selective but rapidly degraded TTR ligand luteolin in order to increase its resistance against biotransformation. Using a liver-based microsome assay, in combination with HPLC, we show how the luteolin analogues have gained increased stability. However, using the urea assay, we also show that the analogues have lost much of luteolin’s selectivity. In paper III, we show that tetrabromobisphenol A is a highly selective binder of TTR in plasma and is able to rescue cells from TTR-induced toxicity. In paper IV, we studied the interaction of TTR with Aβ and its effect on Aβ fibril formation. We used a ThT fluorescence-based assay and dot blotting to show that TTR inhibits Aβ amyloid formation and promotes the formation of high molecular weight assemblies with an open N-terminus. Using surface plasmon resonance, we further show how TTR is unable to inhibit fibril elongation and instead targets the nucleation processes, both primary and fibril-catalyzed secondary nucleation. To conclude, we present new molecules with the ability to selectively stabilize TTR that can serve as scaffolds in drug design. We also elucidate TTR’s inhibiting effects on toxic Aβ amyloid formation.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2014. / Alzheimer’s Disease (AD) is characterized by neurofibrillary tangles, senile plaques and neuronal loss. Although the mechanisms underlying Amyloid beta 42 (Aβ42) neurotoxicity have not been firmly established, it is proposed that the neuronal loss is elicited through associations with cell surface receptors. The cellular prion protein (PrPc) has been identified as an Aβ42 receptor and as a regulator of the amyloidogenic cleavage pathway. As Aβ42 shares common binding partners with the 37kDa/67kDa laminin receptor (LRP/LR), including PrPc, we investigated whether these proteins interact and assessed the pathological significance of this association. LRP/LR was found to co-localize with Aβ on the cell surface. The occurrence of FRET suggested that an interaction between LRP/LR and Aβ indeed exists at the cell surface. Furthermore, pull down assays and Aβ-specific ELISAs demonstrated that LRP/LR forms a physical association with endogenously shed Aβ, thereby verifying the physiological relevance of this association. Antibody blockade by IgG1-iS18 and shRNAmediated downregulation of LRP/LR significantly enhanced cell viability and proliferation and decreased apoptosis in cells co-treated with Aβ42 when compared to cells incubated with Aβ42 alone. In addition, antibody blockade and shRNA-mediated downregulation of LRP/LR significantly impeded Aβ42 internalization. These results suggest that LRP/LR acts as an internalization receptor for Aβ42 and may thereby contribute to the cytotoxicity of the neuropeptide by facilitating intracellular Aβ42 accumulation and aggregation - which has consequences for cell proliferation and may promote apoptosis. These findings recommend anti-LRP/LR specific antibodies and shRNAs as potential therapeutic tools for Alzheimer’s Disease treatment.

Alzheimer’s disease (AD) has been defined as a type of dementia that causes problems with memory, thinking, and behavior. AD is characterized by tau tangles and Aβ plaques in and around neurons, respectively. The impact this disease has on its victims’ health, both physically and mentally, is unimaginable and the rate of progression is not expected to decrease any time soon. This threat to our minds encourages the importance of understanding AD. Amongst the theories as to what bio mechanisms cause the brain to intertwine is the amyloid cascade hypothesis. The purpose of this thesis is to review the amyloid cascade hypothesis and discuss treatments which utilize this model. We also wish to examine social aspects such as loneliness and socioeconomic factors which are associated with the progression of AD. Research presented provides evidence that targeting the accumulation of Aβ in the brain will prevent further biochemical responses to form neurodegenerative pathology. From the collected data, we observe that therapies targeting the amyloidogenic pathway have received positive feedback in the medical community. Amongst them, an Aβ synthetic peptide vaccine which made history in vaccine development due to their responder rate. The impact of social factors such as loneliness in the advancement of AD is also supported by research. While it is acknowledged that any neurodegenerative disease is far too complex to narrow its cause specifically, this thesis provides an association with multiple aspects that can be understood and applied to future research in this field. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Alzheimer’s disease (AD) is a deleterious neurodegenerative disease caused in major part by the aberrant processing and accumulation of amyloid beta peptides. In this dissertation, we systematically investigated the role of N-terminal region (NTR) residues of amyloid (1-40) (Aβ40) peptide in amyloidogenesis, lipid bilayer membrane interaction and damage, as well as neurotoxicity. Herein, we investigated the role of NTR residues on the aggregation and amyloid fibril formation process, to gain understanding on the electrostatic and hydrophobic constituents of the mechanism. This was achieved by substituting specific charged residues located in the NTR of Aβ40 and investigating their effects through a variety of techniques. We also investigated the role of NTR charged residues in their interaction with supported phospholipid bilayer membranes through the use of Quartz Crystal Microbalance with Dissipation (QCM-D) monitoring to gain insight on the mechanistic details of the interaction. To further understand the implications of substituting charged NTR residues on membrane interaction, pore formation and damage, we utilized a carboxyfluorescein dye leakage assay to quantify the membrane damage caused by Aβ40 and the NTR mutants. We also performed neurotoxicity assay with SH-SY5Y neuroblastoma cells to shed light on the effects of NTR substitutions on cellular toxicity. Finally, we synthesized a polymer, trimethyl chitosan (TMC), and utilized it as a polyelectrolyte monitor of electrostatic interactions occurring between TMC and the NTR of Aβ40. Our results demonstrate that the NTR charged residues of Aβ40 contribute significantly to the aggregation process, amyloidogenesis, and phospholipid membrane interaction and perturbation by means of electrostatic, thermodynamic and hydrophobic forces. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection