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Abstract

Alzheimer’s disease (AD) is the leading cause of dementia in the elderly. In
countries with aging populations, such as Australia, the prevalence of AD is
projected to increase substantially. AD is characterised by two distinctive
pathological lesions in the brain, amyloid plaques and neurofibrillary tangles. The
major component of amyloid plaques is an aggregating protein termed the betaamyloid
protein (Aβ). Aβ is formed normally from a larger precursor protein,
known as the beta-amyloid precursor protein (APP). Although APP is centrally
involved in the pathogenesis of Alzheimer’s disease and the production of Aβ,
relatively little is known about its normal function. Deciphering the function of
APP in the brain may be essential for the development of effective AD
therapeutics.
APP is a type I transmembrane glycoprotein that can be proteolytically processed
by α, β- and γ-secretases to produce a number of secreted ectodomain fragments
termed sAPPβ, sAPPα, Aβ and p3. Many studies have suggested that sAPPα may
act in the maintenance and development of the central nervous system, by acting
as a paracrine factor. In vitro, sAPPα has been reported to modulate the
proliferation and differentiation of a variety of cell types. However, the
mechanistic basis for these effects is unclear. In part, this uncertainty has arisen
because the cell-surface receptor molecules that interact with sAPPα are not
known.
Previous studies have reported that sAPPα may interact with a novel lipid-raft
type membrane domain in the cell. Furthermore, sAPPα has been reported to bind
to the lipid GM1-ganglioside. On the basis of these reports, the work in this thesis
explored the hypothesis that an interaction of APP with cell surface lipids could
facilitate binding and/or signalling by sAPPα. To determine if sAPPα is able to interact with a sub-group of lipids. The relative
ability of sAPPα to bind to 27 physiological lipids was examined using a proteinlipid
overlay assay. This assay identified that sAPPα could bind selectively to
phosphoinositide lipids (PIPs). Further, a recombinant fragment of APP
corresponding to the E1 N-terminal domain (APP-E1) also bound selectively to
PIPs, suggesting there is a PIP-binding region within the E1 domain of APP.
To investigate whether APP and PIP could interact on the cell surface, it was first
necessary to demonstrate that PIPs are present on the cell surface. A live cell
immunolabelling method was used to examine the location of cell surface PIPs.
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) immunoreactivity was found to
be present on the surface of cells in primary murine hippocampal cultures in
discrete puncta <1 μm in size. This observation was also confirmed using a
recombinant PI(4,5)P2 biosensor protein.
To examine whether APP could interact with cell-surface PIP, studies were
performed to examine the degree of colocalisation of exogenous APP-E1 and cellsurface
PI(4,5)P2. APP-E1 that was added to primary hippocampal cultures bound
to the surface of neurons in discrete puncta <1 μm in size. The cell-bound APPE1
and the cell-surface PI(4,5)P2 were highly co-localised on the surface of
neurons. However, cell-surface PI(4,5)P2 was also present on glial cells in culture
where APP-E1 did not bind. Furthermore the binding of APP-E1 to cells could
not be inhibited using a water soluble analogue of PI(4,5)P2. Therefore, these data
suggested that APP-E1 interacts with cell-surface PI(4,5)P2, but the interaction
was not sufficient to explain why APP-E1 binds to the cell surface.
As the APP E1 domain contains a heparin-binding site, the role of this region was
investigated in the binding of APP-E1 to PIP and also the binding of APP-E1 to
cells. Heparin did not block the binding of APP-E1 to PIP in vitro, suggesting the
heparin-binding region and the PIP-binding region in the APP E1 domain are distinct. However, heparin did inhibit the binding of APP-E1 to cells, suggesting
that the heparin-binding region of APP is required for binding to cells.
Furthermore, heparitinase treatment of cells significantly reduced cell surface
heparan sulfate immunoreactivity, but did not affect the binding of APP-E1 to
cells. These results suggest that APP may interact with PIP on the cell surface
along with another cell surface component that binds to the heparin-binding site,
which is not heparan sulfate.
As PIPs are involved in many aspects of cellular physiology, it was hypothesized
that APP may signal through modulation of levels of PIPs. To address this
hypothesis, levels of PIPs were measured in primary cortical cultures by two
methods. Firstly, a mass-spectroscopy based method was developed to measure
total levels of cellular PIP. No change in total PIP levels upon sAPPα treatment
could be detected using this method. Secondly, levels of cell-surface PIPs were
determined using an array of anti-PIP biosensors and antibodies. Under resting
conditions, only PI(4,5)P2 was present on the surface of cells. However, in the
presence of APP-E1, there was an increase in the level of cell surface PI(3,4,5)P3
and an increase in the level of PI(4,5)P2, indicating that APP binding to cells may
result in an increase level of cell surface PIPs.
The data presented in this thesis demonstrate that APP has a novel N-terminal
PIP-binding domain. This domain may play a role in the normal function of APP,
by facilitating PIP-dependent signalling.

Item Type:	Thesis - PhD
Authors/Creators:	Dawkins, E
Keywords:	Alzheimer's disease, Phosphoinositides, beta-amyloid precursor protein, Phosphatidylinositol, phosphate, heparin.
Additional Information:	Copyright the Author
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