Year: 2019
Type: dissertation
Abstract: Alzheimer’s disease is a debilitating neurological disorder, the worldwide cost\nof which has recently entered into the trillions of dollars ($USD) per annum.\nThe primary cause of Alzheimer’s disease is believed to be the accumulation in\nthe brain of amyloid beta (Aβ), a protein product of the Amyloid Precursor\nProtein (APP) processing pathway. The increasing concentration of Aβ\nfacilitates aggregation, which generates a number of neurologically harmful\nproducts, and causes a collection of symptoms, primarily dementia, which are\ncollectively known as Alzheimer’s disease. In the normal cellular environment,\nit is suspected that there are neuroprotective pathways that act to counter this\naccumulation of Aβ. One such pathway is mediated by a soluble product of\nAPP processing called sAPPα. sAPPα has been shown to bind to Aβ in vitro\nand also to rescue early stage Alzheimer’s phenotype in mouse models. It is of\nsignificant interest for the development of a therapy against Alzheimer’s\ndisease in humans. It is, however, not currently known to which Aβ aggregated\nstate sAPPα interacts as Aβ can aggregate into a multitude of forms with\ndiffering levels of pathogenicity. It is therefore crucial to understand how\nsAPPα might counter Aβ toxicity by characterising this binding relationship\nand thereby deucing how it might be used in a future therapy against\nAlzheimer’s.\n\nIn this study I produced recombinant human sAPPα in bacteria as a fusion\nprotein with a glutathione transferase (GST) N terminal tag and purified it by\naffinity chromatography using the GST tag to bind to glutathione on a column.\nPure GST –sAPPα was immobilised on the glutathione column as a solid phase.\nGST-sAPPα was exposed to a mixture containing non-aggregated and\naggregated forms of Aβ. Species within this mixture interacted and co-eluted\nwith GST-sAPPα after addition of exogenous glutathione. The critical question\nthen was which form of Aβ interacts with sAPPα? The mixture of aggregate\nforms was therefore fractionated, first by centrifugation, and then more\ndefinitively by FPLC on a size exclusion column. As an initial test for which\nform of Aβ bound to sAPPα, nitrocellulose membrane was used to facilitate a\ndetection assay for the ability of different species (monomer, dimer, trimer and\nsoluble higher-order oligomers) to bind to immobilised GST-sAPPα. These\nbinding membranes were probed with antibodies specific to a sequence shared\nby both sAPPα and Aβ, to measure enhancement of signal by addition of the\nAβ species.\n\nUsing computer analysis with ImageJ, it was possible to directly compare the\nlevels of fluorescence, relative to the negative controls, which allowed the\nrelative levels of binding between the different aggregate forms of Aβ to be\ncompared.\n\nIt was found using these methods that the predominant binding partner of\nsAPPα in vitro are the soluble higher-order aggregates, but that monomeric, and\ndimeric/trimeric forms of Aβ also showed some binding capacity. However, a\nbetter detection assay needs to be developed.\n\nThis study supports a binding relationship between sAPPα and Aβ and the\npotential for the development of a therapy utilising the action of sAPPα to\nprotect against Alzheimer’s disease.
Author: Christopher Michael Attwood
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3013026253