Modulation of Endoplasmatic Reticulum calcium storage by Amyloid Precursor protein and its cleavage products

The work presented in this thesis is aimed to contribute to the
understanding of the physiological role of APP and its underlying
mechanism controlling the Ca2+ homeostasis. In addition, it aims to
understand the pathophysiological role of Amyloid precursor protein
(APP) and its cleavage products in Alzheimer’s disease (AD)
particularly focussing on the disturbances in normal Ca2+
homeostasis resulting from altered APP processing. A central
hypothesis to the pathogenesis of AD is that the accumulation of
amyloid-β peptide (Aβ) and/or Aβ-containing plaques play an
important role in the development of the disease. Mutations in APP,
Presenilin 1 (PS1), or Presenilin 2 (PS2) are linked to the
inherited forms of the disease and result in an increased
production of Aβ, particularly of the Aβ42 isoform. Most mutations
in APP, PS1, or PS2 that are associated with Familial Alzheimer’s
Disease (FAD) alter the γ-secretase cleavage, resulting in
increased levels of Aβ42 species and affecting the generation of
corresponding C-terminal fragments (CTFs), for instance, the APP
intracellular domain (AICD). Recently, several studies have
indicated that not only altered production of Aβ42 affect neuronal
survival, but also the CTFs, predominantly AICD, have a critical
pathophysiological role by modulating neuronal Ca2+ homeostasis. In
the present study, the mechanism by which APP or its cleavage
products maintains Ca2+ homeostasis, is investigated in more detail
by analyzing free cytosolic Ca2+ concentration in different cell
types under various experimental conditions. In all the cell types
analyzed, it was consistently observed that the loss of γ-secretase
derived APP C-terminal fragment, AICD, enhances the basal resting
cytosolic Ca2+ levels and reduce intracellular endoplasmatic
reticulum (ER) Ca2+ storage. The correlation of enhanced resting
Ca2+ and reduced ER Ca2+ storage strongly indicate an alteration of
the mechanisms involved in Ca2+ buffering by the ER. One of the
most important mechanisms involved in the removal of Ca2+ from
cytosol, to maintain the resting Ca2+ concentration, is mediated by
Calcium ATPases (Ca2+ATPases) that pumps Ca2+ from the cytosol into
the ER lumen. This process is strongly ATP dependent. The
mitochondrial membrane potential (or proton motive force) is the
central bioenergetic parameter that controls ATP synthesis,
therefore both mitochondrial membrane potential using rhodamine-123
fluorescent probe and ATP production using a bioluminescent assay
were determined. Cells lacking AICD fragments were found to exhibit
a lower ATP generation and a higher mitochondrial membrane
potential. These results indicate a dysfunction of ATP synthase as
a consequence of loss of AICD. Indeed blockade of mitochondrial ATP
synthase by oligomycin in AICD expressing cells led to the similar
alterations in Ca2+ storage as in the cells lacking AICD. On the
other hand, administration of mitochondrial substrates on AICD
non-expressing cells exhibited responses analogous to AICD
expressing cells. Collectively, our data suggest that AICD is
critically involved in mitochondrial ATP synthesis through a
γ-secretase dependent signalling pathway controlling ATP dependent
cellular mechanisms such as the cellular Ca2+ storage.