Abstract
Alzheimer’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.