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Hamid, Runa (2006): Modulation of Endoplasmatic Reticulum calcium storage by Amyloid Precursor protein and its cleavage products. Dissertation, LMU München: Faculty of Biology
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Abstract

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.