Pathology - Theses
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ItemIdentifying the key Aβ oligomer species by correlating cell neurotoxicity and binding properties( 2016)Alzheimer’s disease (AD) represents 60-80% of dementia cases and predominantly affects elderly people above 65 years of age. While it is the second most leading cause of death in Australia, there is no cure or effective treatment available. Amyloid beta (Aβ) peptide which aggregates into fibrils and is deposited as extracellular amyloid plaques in the brain, were originally hypothesized as the pathogenic agent causing AD. Today, there is strong agreement that the lower molecular weight Aβ oligomers correlates best with AD clinical symptoms and are most likely the toxic species causing AD. Different oligomeric Aβ species have been claimed as the toxic Aβ species but the true identity of the toxic species is yet to be resolved. The major challenge working with the Aβ peptide oligomers has been its isolation, preparation, or purification of either synthetic material or brain tissue because of its ability to continually self-aggregate into larger species. In addition, previous reports from our laboratory demonstrated that Aβ binding to neurons was critical for Aβ to induce toxicity. Taken together, my hypothesis for this PhD thesis is that there is a specific oligomeric Aβ species that binds to neurons in order to induce neuronal cell death associated with AD. Therefore, the major aim of my thesis was to identify the specific neurotoxic oligomeric Aβ species that will bind and kill neurons. To address this aim, mouse cortical neuronal cultures were treated with soluble monomerised synthetic Aβ40 and Aβ42 peptides. We identified increasing Aβ trimer and tetramer bound to treated neuronal cultures which correlated with increased cell death. Photo induced cross linking of unmodified peptides (PICUP) technique was utilized to prepare individual Aβ 2mer, 3mer and 4mer. The neurotoxicity of these purified Aβ40 3mer and 4mer was up to 50-fold more potent compared to soluble uncross-linked Aβ peptides. Biophysical and biochemical analyses demonstrated that PICUP crosslinking significantly halted further aggregation of these species, they adopted an identical compact physical arrangement and cross-linking occurred via a di-tyrosine bond. Furthermore, purified PICUP cross-linked Aβ40 2mer, 3mer and 4mer but not 1mer, were able to induce significant inhibition of long term potentiation in mouse hippocampal brain slices indicating that these oligomers can disrupt synaptic function. Aβ peptide immunofluorescence staining of treated neurons showed predominant punctate binding along the axonal and neurite outgrowth and the observation of retrograde trafficking of purified Aβ40 3mers and soluble uncross-linked together support the notion that these oligomers directly target the synaptic membrane. Brilliant blue G dye was used in identifying PICUP cross-linked Aβ peptide bands in SDS-PAGE gels and when bound to Aβ peptide, this resulted in diminished Aβ cell binding and neurotoxicity. However, blocking potential Aβ binding receptor sites with chemical antagonists or deleting PrPc expression did not reduce Aβ cellular binding and neurotoxicity. In conclusion, the results presented in this thesis have demonstrated that Aβ 3mer and 4mer are the key toxic Aβ oligomers causing the AD pathogenesis. Based on these findings, future therapeutic strategies that target these oligomeric species and modulate their toxic or cell binding behaviours will greatly assist in the treatment AD.