Familial Alzheimer's disease-linked presenilin mutants and intracellular Ca handling: A single-organelle, FRET-based analysis.

An imbalance in Ca homeostasis represents an early event in the pathogenesis of Alzheimer's disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca content is still debated and the mechanism of action of mutant proteins is unclear. To fulfil the need of a direct investigation of intracellular stores Ca dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca concentration within the main intracellular Ca stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca-ATPase) activity. Moreover, by using a cytosolic Ca probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca influx activated by stores depletion (Store-Operated Ca Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway. Our data indicate that FAD-linked PSs mutants differentially modulate the Ca content of intracellular stores yet leading to a complex dysregulation of Ca homeostasis, which represents a common disease phenotype of AD.