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dc.contributor.advisorTate, Warren
dc.contributor.authorAttwood, Christopher Michael
dc.date.available2020-03-26T03:03:24Z
dc.date.copyright2019
dc.identifier.citationAttwood, C. M. (2019). Investigating a putative neuroprotective pathway in Alzheimer’s disease. (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/9994en
dc.identifier.urihttp://hdl.handle.net/10523/9994
dc.description.abstractAlzheimer’s disease is a debilitating neurological disorder, the worldwide cost of which has recently entered into the trillions of dollars ($USD) per annum. The primary cause of Alzheimer’s disease is believed to be the accumulation in the brain of amyloid beta (Aβ), a protein product of the Amyloid Precursor Protein (APP) processing pathway. The increasing concentration of Aβ facilitates aggregation, which generates a number of neurologically harmful products, and causes a collection of symptoms, primarily dementia, which are collectively known as Alzheimer’s disease. In the normal cellular environment, it is suspected that there are neuroprotective pathways that act to counter this accumulation of Aβ. One such pathway is mediated by a soluble product of APP processing called sAPPα. sAPPα has been shown to bind to Aβ in vitro and also to rescue early stage Alzheimer’s phenotype in mouse models. It is of significant interest for the development of a therapy against Alzheimer’s disease in humans. It is, however, not currently known to which Aβ aggregated state sAPPα interacts as Aβ can aggregate into a multitude of forms with differing levels of pathogenicity. It is therefore crucial to understand how sAPPα might counter Aβ toxicity by characterising this binding relationship and thereby deucing how it might be used in a future therapy against Alzheimer’s. In this study I produced recombinant human sAPPα in bacteria as a fusion protein with a glutathione transferase (GST) N terminal tag and purified it by affinity chromatography using the GST tag to bind to glutathione on a column. Pure GST –sAPPα was immobilised on the glutathione column as a solid phase. GST-sAPPα was exposed to a mixture containing non-aggregated and aggregated forms of Aβ. Species within this mixture interacted and co-eluted with GST-sAPPα after addition of exogenous glutathione. The critical question then was which form of Aβ interacts with sAPPα? The mixture of aggregate forms was therefore fractionated, first by centrifugation, and then more definitively by FPLC on a size exclusion column. As an initial test for which form of Aβ bound to sAPPα, nitrocellulose membrane was used to facilitate a detection assay for the ability of different species (monomer, dimer, trimer and soluble higher-order oligomers) to bind to immobilised GST-sAPPα. These binding membranes were probed with antibodies specific to a sequence shared by both sAPPα and Aβ, to measure enhancement of signal by addition of the Aβ species. Using computer analysis with ImageJ, it was possible to directly compare the levels of fluorescence, relative to the negative controls, which allowed the relative levels of binding between the different aggregate forms of Aβ to be compared. It was found using these methods that the predominant binding partner of sAPPα in vitro are the soluble higher-order aggregates, but that monomeric, and dimeric/trimeric forms of Aβ also showed some binding capacity. However, a better detection assay needs to be developed. This study supports a binding relationship between sAPPα and Aβ and the potential for the development of a therapy utilising the action of sAPPα to protect against Alzheimer’s disease.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectAlzheimer's
dc.subjectsAPPa
dc.subjectAB
dc.titleInvestigating a putative neuroprotective pathway in Alzheimer’s disease.
dc.typeThesis
dc.date.updated2020-03-01T01:31:20Z
dc.language.rfc3066en
thesis.degree.disciplineBiochemistry
thesis.degree.nameMaster of Science
thesis.degree.grantorUniversity of Otago
thesis.degree.levelMasters
otago.openaccessOpen
otago.evidence.presentYes
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