Investigating a putative neuroprotective pathway in Alzheimer’s disease.
Attwood, Christopher Michael
Alzheimer’s disease is a debilitating neurological disorder, the worldwide costof 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 inthe brain of amyloid beta (Aβ), a protein product of the Amyloid PrecursorProtein (APP) processing pathway. The increasing concentration of Aβfacilitates aggregation, which generates a number of neurologically harmfulproducts, and causes a collection of symptoms, primarily dementia, which arecollectively known as Alzheimer’s disease. In the normal cellular environment,it is suspected that there are neuroprotective pathways that act to counter thisaccumulation of Aβ. One such pathway is mediated by a soluble product ofAPP processing called sAPPα. sAPPα has been shown to bind to Aβ in vitroand also to rescue early stage Alzheimer’s phenotype in mouse models. It is ofsignificant interest for the development of a therapy against Alzheimer’sdisease in humans. It is, however, not currently known to which Aβ aggregatedstate sAPPα interacts as Aβ can aggregate into a multitude of forms withdiffering levels of pathogenicity. It is therefore crucial to understand howsAPPα might counter Aβ toxicity by characterising this binding relationshipand thereby deucing how it might be used in a future therapy againstAlzheimer’s. In this study I produced recombinant human sAPPα in bacteria as a fusionprotein with a glutathione transferase (GST) N terminal tag and purified it byaffinity 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 andaggregated forms of Aβ. Species within this mixture interacted and co-elutedwith GST-sAPPα after addition of exogenous glutathione. The critical questionthen was which form of Aβ interacts with sAPPα? The mixture of aggregateforms was therefore fractionated, first by centrifugation, and then moredefinitively by FPLC on a size exclusion column. As an initial test for whichform of Aβ bound to sAPPα, nitrocellulose membrane was used to facilitate adetection assay for the ability of different species (monomer, dimer, trimer andsoluble higher-order oligomers) to bind to immobilised GST-sAPPα. Thesebinding membranes were probed with antibodies specific to a sequence sharedby both sAPPα and Aβ, to measure enhancement of signal by addition of theAβ species. Using computer analysis with ImageJ, it was possible to directly compare thelevels of fluorescence, relative to the negative controls, which allowed therelative levels of binding between the different aggregate forms of Aβ to becompared. It was found using these methods that the predominant binding partner ofsAPPα in vitro are the soluble higher-order aggregates, but that monomeric, anddimeric/trimeric forms of Aβ also showed some binding capacity. However, abetter detection assay needs to be developed. This study supports a binding relationship between sAPPα and Aβ and thepotential for the development of a therapy utilising the action of sAPPα toprotect against Alzheimer’s disease.
Advisor: Tate, Warren
Degree Name: Master of Science
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: Alzheimer's; sAPPa; AB
Research Type: Thesis